Cancer treatment and diagnostic methods

Abstract

Therapeutic and diagnostic methods and compositions for conditions such as cancer (eg, melanoma) are provided. The present invention provides a pharmaceutical agent for treating melanoma or delaying the progression of melanoma in an individual undergoing or having undergone treatment comprising a PD-1 axis-binding antagonist, the pharmaceutical agent comprising a therapeutically effective amount of a PD-1 axis-binding antagonist, and a method for detecting granzyme B in a blood sample from the individual. + CD4 + T cells and FOXP3 + CD4 + The ratio of granzyme B to T cells in individuals who are not or have not received treatment with a PD-1 axis-binding antagonist is + CD4 + T cells and FOXP3 + CD4 + The PD-1 axis binding antagonist is an anti-PD-1 antibody or an anti-PD-L1 antibody.

Description

[Technical Field]

[0001] Provided herein are therapeutic and diagnostic methods and compositions for conditions such as cancer (e.g., melanoma), and methods using PD-1 axially coupled antagonists. In particular, the present invention provides methods for monitoring the response to treatment, therapeutic methods, manufactured products, and kits. [Background technology]

[0002] Cancer remains one of the most deadly threats to human health. Cancer, or malignant tumors, metastasize and grow rapidly in an uncontrolled manner, making timely detection and treatment extremely difficult.

[0003] Programmed death-ligand 1 (PD-1) binds to ligands including PD-L1 and PD-L2. Programmed death-ligand 1 (PD-L1) is involved in suppressing immune responses in chronic infections, pregnancy, tissue allogeneic transplantation, autoimmune diseases, and cancer. In contrast to T lymphocytes in normal tissues and peripheral blood, the majority of tumor-infiltrating T lymphocytes predominantly express PD-1. The formation of PD-L1 / PD-1 and PD-L1 / B7-1 complexes is thought to negatively regulate T cell receptor signaling, leading to CD8+ T cell activation and subsequent downregulation of CD8+ T cell-mediated tumor cell death. CD8 in the tumor microenvironment + Studies focusing on the T cell compartment have failed to elucidate the mechanism of action of anti-PD-1 immunotherapy, despite clear clinical benefits in a significant portion of treated patients, roughly 30%. + While the focus has been on T cells, recently, tumor-infiltrating CD4 in microsatellite-unstable tumors has been studied. + T cells were observed to express higher levels of PD-1 than in microsatellite-stable tumors (Llosa and Cruise, Cancer discovery 5(1): 43-51 (2015)). In addition, tumor-invasive CD4 was observed in melanoma. +T cells often recognize mutated antigens (Linnemann and van Buuren, Nature medicine 21(1): 81-85 (2015)). Consistent with these studies, in experiments conducted in animal models of colorectal cancer and melanoma, MHC class II-restricted T cell responses can play an important role in tumor control (Kreiter et al., Nature 520(7549): 692-696 (2015)), and tumor-reactive CD4 + T cells can develop cytotoxic capabilities and eradicate large established tumors (Quezada et al., J Exp. Med., 207(3): 637-650 (2010)) has been demonstrated. Prior to the studies reported here, whether PD-1 blocking agents, such as anti-PD-1 antibodies, affect CD4 + T cells in the context of human cancer, and how they do so, was not known.

[0004] There remains a need to monitor and optimize treatment strategies that include blocking PD-1 receptor / ligand interactions for the treatment, stabilization, prevention, and / or delay of progression of various cancers such as melanoma. Specifically, there remains a need to identify patients likely to respond to such treatments, and ways to rapidly monitor the effectiveness of ongoing treatments. SUMMARY OF THE INVENTION

[0005] This invention relates to methods and compositions for the treatment and diagnosis of cancer. In one aspect, the invention is a method for treating cancer or delaying the progression of cancer in an individual, comprising administering to the patient a therapeutically effective amount of a PD-1 axis-binding antagonist, and the granzyme B in a blood sample obtained from the patient + CD4 + T cells and FOXP3 + CD4 + characterized in that the ratio to T cells is determined to be 1 or greater.

[0006] In one embodiment, the present invention relates to a method for determining whether an individual with cancer is responding to a treatment comprising a PD-1 axis-coupled antagonist, wherein granzyme B in a sample obtained from the individual is present. + CD4 + T cells and FOXP3 + CD4 + This includes determining the ratio with T cells, and determining one or more granzyme B cells in the sample. + CD4 + T cells and FOXP3 + CD4 + This relates to a method for indicating that an individual is responding to treatment containing a PD-1 axis-binding antagonist by measuring the ratio of T cells to other cells.

[0007] In some embodiments of the above-described model, the sample is a blood sample. In some embodiments, the sample is a whole blood sample or a peripheral blood mononuclear cell sample. In some embodiments, the blood sample is collected from the individual within two months of the last treatment with a PD-1 antagonist. In some embodiments, the individual is currently receiving or has previously received treatment including a PD-1 axially coupled antagonist. In some embodiments, cancer is selected from the group consisting of bladder cancer, squamous cell carcinoma, small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), lung adenocarcinoma, lung squamous cell carcinoma, peritoneal cancer, hepatocellular carcinoma, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine cancer, salivary gland cancer, kidney cancer, prostate cancer, vulvar cancer, thyroid cancer, hepatic carcinoma, anal cancer, penile cancer, Merkel cell carcinoma, testicular cancer, esophageal cancer, biliary tract tumors, head and neck cancers, and hematological malignancies. In some embodiments, cancer is melanoma. In some embodiments, cancer is urothelial bladder cancer. In some embodiments, urothelial bladder cancer is metastatic urothelial bladder cancer. In some embodiments, urothelial bladder cancer is locally advanced urothelial bladder cancer. In some embodiments, cancer cells in an organism express PD-L1.

[0008] In some embodiments, PD-L1 expression is determined by immunohistochemistry (IHC) assay. In some embodiments, the individual has previously received two or fewer cytotoxic therapy regimens (e.g., 0, 1, or 2 previous cytotoxic therapy regimens) for locally advanced or metastatic cancer. In some embodiments, the individual has not previously received targeted systemic therapy for locally advanced or metastatic cancer. In some embodiments, the individual's response to treatment is complete remission. In some embodiments, the individual's response to treatment is partial response. In some embodiments, the individual's response to treatment is a sustained response after discontinuation of treatment.

[0009] In some embodiments, Granzyme B + CD4 + T cells and FOXP3 + CD4 + The ratio to T cells is 4. In some embodiments, granzyme B + CD4 + T cells and FOXP3 + CD4 + The ratio to T cells is 45.

[0010] In some embodiments of the above-described model, the PD-L1 axis-binding antagonist is selected from a PD-L1-binding antagonist, a PD-1-binding antagonist, and a PD-L2-binding antagonist. In some embodiments, the PD-L1 axis-binding antagonist is a PD-L1-binding antagonist. In some embodiments, the PD-L1-binding antagonist inhibits the binding of PD-L1 to one or more of its ligand-binding partners. In some embodiments, the PD-L1-binding antagonist inhibits the binding of PD-L1 to PD-1. In some embodiments, the PD-L1-binding antagonist inhibits the binding of PD-L1 to B7-1. In some embodiments, the PD-L1-binding antagonist inhibits the binding of PD-L1 to both PD-1 and B7-1. In some embodiments, the PD-L1-binding antagonist is an antibody. In some embodiments, the antibody is selected from the group consisting of atezolizumab (MPDL3280A), YW243.55.S70, MDX-1105, MEDI4736 (durvalumab), and MSB0010718C (avelumab). In some embodiments, the antibody comprises a heavy chain containing the HVR-H1 sequence of SEQ ID NO: 19, the HVR-H2 sequence of SEQ ID NO: 20, and the HVR-H3 sequence of SEQ ID NO: 21; and a light chain containing the HVR-L1 sequence of SEQ ID NO: 22, the HVR-L2 sequence of SEQ ID NO: 23, and the HVR-L3 sequence of SEQ ID NO: 24. In some embodiments, the antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 25 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 4.

[0011] In some embodiments, the PD-L1 axis-binding antagonist is a PD-1 binding antagonist. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to one or more of its ligand-binding partners. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L2. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to both PD-L1 and PD-L2. In some embodiments, the PD-1 binding antagonist is an antibody. In some embodiments, the antibody is selected from the group consisting of MDX-1106 (nivolumab), MK-3475 (pembrolizumab), MEDI-0680 (AMP-514), PDR001, REGN2810, and BGB-108.

[0012] In some embodiments, the PD-1 binding antagonist is an Fc fusion protein. In some embodiments, the Fc fusion protein is AMP-224.

[0013] In any of the earlier embodiments of the above-described aspects, the individual has received or has received an effective dose of a second therapeutic agent. In some embodiments, the second therapeutic agent is selected from the group consisting of cytotoxic agents, proliferation inhibitors, radiotherapeutic agents, anti-angiogenic agents, and combinations thereof. In some embodiments, the cytotoxic agent is a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is a taxane. In some embodiments, the taxane is administered before, concurrently with, or after the PD-1 axial antagonist. In some embodiments, the taxane is nab-paclitaxel (ABRAXANE®), paclitaxel, or docetaxel. In some embodiments, the individual is progressing after treatment with a platinum-based chemotherapeutic agent.

[0014] In any of the earlier embodiments of the above-described aspects, Granzyme B + CD4 + T cells and FOXP3+ CD4 + The ratio to T cells is CD4 expressing granzyme B protein. + T cell count and CD4 expressing FOXP3 protein + This is determined by determining the number of T cells. In some embodiments, the expression level of at least one protein, granzyme B and FOXP3, is determined using a method selected from the group consisting of immunohistochemistry (IHC), immunofluorescence, flow cytometry, and Western blotting. In some embodiments, the expression level of at least one protein, granzyme B and FOXP3, is determined using flow cytometry. In some embodiments, the expression level of at least one of granzyme B and FOXP3 is the mRNA expression level. In some embodiments, the mRNA expression level of at least one of granzyme B and FOXP3 is determined using a method selected from the group consisting of quantitative polymerase chain reaction (qPCR), reverse transcription qPCR (RT-qPCR), RNA sequencing, microarray analysis, insight hybridization, and sequential gene expression analysis (SAGE).

[0015] In another aspect, the present invention relates to a method for predicting the response of a patient with cancer to a treatment comprising a PD-L1 axis-coupled antagonist, wherein granzyme B in a sample obtained from the individual. + CD4 + T cells and FOXP3 + CD4 + This includes determining the ratio with T cells, and one or more granzymes B + CD4 + T cells and FOXP3 + CD4 + The method is characterized by a ratio to T cells that indicates the likelihood that an individual is likely to respond to treatment containing a PD-1 axis-binding antagonist. In some embodiments, samples are obtained from individuals after they have been administered at least one dose of a PD-L1 axis-binding antagonist.

[0016] In another aspect, the present invention relates to a method for selecting a treatment for an individual suffering from cancer, comprising granzyme B in a sample obtained from the individual. + CD4 + T cells and FOXP3 + CD4 + Determine the ratio with T cells, and identify one or more granzyme B cells in the sample. + CD4 + T cells and FOXP3 + CD4 + The method is characterized by selecting a treatment regimen for an individual that includes a PD-L1 axis-binding antagonist based on the ratio with T cells.

[0017] In another aspect, the present invention relates to a PD-1 axially coupled antagonist for use in treating an individual suffering from cancer, wherein granzyme B is present in a blood sample obtained from the individual. + CD4 + T cells and FOXP3 + CD4 + It is characterized by being a PD-1 axis-binding antagonist whose ratio with T cells has been determined to be 1 or greater.

[0018] In another aspect, the present invention relates to the use of an effective amount of PD-1 axially coupled antagonist in the manufacture of a pharmaceutical for use in treating an individual suffering from cancer, wherein granzyme B is present in a blood sample obtained from the individual. + CD4 + T cells and FOXP3 + CD4 + It is characterized by its use, which has been determined to have a ratio of 1 or more to T cells.

[0019] In another aspect, the present invention relates to a composition containing an effective amount of a PD-1 axially coupled antagonist for use in a method of treating an individual with cancer, wherein granzyme B is present in a blood sample obtained from the individual. + CD4 + T cells and FOXP3 + CD4 + The composition is characterized by having a ratio of 1 or more to T cells.

[0020] In another embodiment, the present invention relates to a method for enhancing immune function in an individual having melanoma using a PD-1 axially coupled antagonist, comprising administering an effective amount of the PD-1 axially coupled antagonist to the individual, wherein granzyme B is present in a blood sample obtained from the individual. + CD4 + T cells and FOXP3 + CD4 + The method is characterized by the determination that the ratio to T cells is 1 or greater. In one embodiment, granzyme B is present in a blood sample obtained from an individual. + CD4 + The number of T cells is increased compared to before administration of the PD-1 axis-coupled antagonist. In one embodiment, FOXP3 in blood samples obtained from individuals + CD4 + The number of T cells is reduced compared to before administration of the PD-1 axis-coupled antagonist. In one embodiment, T cell depletion is reduced compared to before administration of the PD-1 axis-coupled antagonist.

[0021] In another embodiment, the present invention relates to a method for selecting an individual for treatment, treating cancer in the individual, or slowing the progression of cancer, comprising: (a) obtaining a blood sample from the individual; and (b) granzyme B in the blood sample obtained from the individual. + CD4 + T cells and FOXP3 + CD4 + (c) Granzyme B + CD4 + T cells and FOXP3 + CD4 + The method comprises (d) selecting an individual for treatment including a PD-1 axis-binding antagonist if the ratio to T cells is 1 or greater; and (f) administering the PD-1 axis-binding antagonist to the individual.

[0022] In another embodiment, the present invention features a pharmaceutical product comprising a PD-1 axially coupled antagonist and an optional pharmaceutically acceptable carrier, and a kit comprising a package insert containing instructions for administering the pharmaceutical product to treat cancer or slow the progression of cancer in an individual, wherein granzyme B in a blood sample obtained from an individual. + CD4 + T cells and FOXP3 + CD4 + The ratio to T cells is determined to be 1 or greater. In some embodiments, the PD-1 axis-binding antagonist is a PD-L1 binding antagonist. In some embodiments, the PD-L1 binding antagonist is MPDL3280A. In some embodiments, the drug contains a dose of MPDL3280A of approximately 840 mg. In some embodiments, the package insert includes instructions for administering the drug to an individual once every two weeks.

[0023] In any of the embodiments described above, the PD-1 axis-binding antagonist may be selected from the group consisting of PD-1 binding antagonists, PD-L1 binding antagonists, and PD-L2 binding antagonists. In some embodiments, the PD-1 axis-binding antagonist is a PD-1 binding antagonist. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to its ligand-binding partner. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L2. In some embodiments, the PD-1 binding antagonist inhibits the binding of PD-1 to both PD-L1 and PD-L2. In some embodiments, the PD-1 binding antagonist is an antibody. In some embodiments, the PD-1 conjugated antagonist is selected from the group consisting of MDX1106 (nivolumab), MK-3475 (pembrolizumab), MEDI-0680 (AMP-514), PDR001, REGN2810, and BGB-108. In some embodiments, the PD-1 axis conjugated antagonist is a PD-L1 conjugated antagonist. In some embodiments, the PD-L1 conjugated antagonist inhibits the binding of PD-L1 to PD-1. In some embodiments, the PD-L1 conjugated antagonist inhibits the binding of PD-L1 to B7-1. In some embodiments, the PD-L1 conjugated antagonist inhibits the binding of PD-L1 to both PD-1 and B7-1. In some embodiments, the PD-L1 conjugated antagonist is an antibody. In some embodiments, the antibody is selected from the group consisting of MPDL3280A (atezolizumab), YW243.55.S70, MDX-1105, MEDI4736 (durvalumab), and MSB0010718C (avelumab). In some embodiments, the antibody comprises a heavy chain containing the HVR-H1 sequence of SEQ ID NO: 19, the HVR-H2 sequence of SEQ ID NO: 20, and the HVR-H3 sequence of SEQ ID NO: 21; and a light chain containing the HVR-L1 sequence of SEQ ID NO: 22, the HVR-L2 sequence of SEQ ID NO: 23, and the HVR-L3 sequence of SEQ ID NO: 24.In some embodiments, the antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 26 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 4. In some embodiments, the antibody comprises a heavy chain variable region containing the amino acid sequence of SEQ ID NO: 25 and a light chain variable region containing the amino acid sequence of SEQ ID NO: 4.

[0024] It should be understood that one, some, or all of the characteristics of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other embodiments of the present invention will be obvious to those skilled in the art. These and other embodiments of the present invention will be further described by the following detailed description. [Brief explanation of the drawing]

[0025] This application document includes at least one drawing drawn in color. A copy of this patent or patent application, including the color drawing, will be provided by the Patent Office upon request and payment of the required fees. [Figure 1] Figures 1A and 1B show PD-1 expression in CD4+ T cells alone (Figure 1A) and CD4+ T cells with allogeneic DCs (Figure 1B) in mMLR assays. The y-axis represents PD-1 expression, and the x-axis represents CFSE expression. Figure 1B shows the PD-1 expression profile in allospecific CD4+ T cells identified as a low CFSE population. [Figure 2] Figure 2 shows that PD-1 blockade increases IFN-γ secretion by CD4+ T cells. Levels of IFN-γ present in the supernatant of CD4+ T cells co-cultured with allogeneic DCs, with or without the addition of anti-PD-1 blocking antibody or isotype control. Each data point (circle, square, or triangle) represents one donor from each independent experiment. [Figure 3A-B] Figures 3A-C show that PD-1 blockade increases granzyme B production by CD4+ T cells. Figures 3A and 3B show representative CD4-gated FACS plots indicating the frequency of granzyme B-positive CFSE-low CD4 T cells after mMLR assay alone (Figure 3A) or after 5 days of PD-1 blockade (Figure 3B). y-axis: granzyme B; x-axis: CFSE. [Figure 3C] Figure 3C shows a graph summarizing the frequency of granzyme B-producing CD4 T cells when co-cultured with allogeneic DCs for 5 days, with or without anti-PD-1 blocking antibody, or with isotype control antibody. Each data point (circle, square, or triangle) represents one donor from each independent experiment. [Figure 4A] Figures 4A and 4B show a comparison of the induction of IFN-γ and granzyme B+CD4+ T cells using anti-PD-1 antibodies 0376, MDX-1106, and MK-3475 or isotype controls against baseline (culture without antibody addition, "-"). Anti-PD-1 antibodies 0376, MDX-1106, and MK-3475 induce similar amounts of IFN-γ secretion (Figure 4A), but 0376 is significantly superior in inducing granzyme B+CD4+ T cells (P=0.02) (Figure 4B). [Figure 4B] the above [Figure 5] Figures 5A and 5B show that anti-PD-L1 treatment induces IFN-γ and granzyme B production. Figure 5A shows the level of IFN-γ in the supernatant of CD4+ T cells co-cultured with allogeneic DCs, with or without the addition of anti-PD-1 blocking antibody or anti-PD-L1, and Figure 5B shows the expression of granzyme B by CD4+ T cells. Each data point (circle, square, or triangle) represents one donor from each independent experiment. Under the test conditions, PD-1 blockade induced more IFN-γ secretion and slightly more granzyme B expression than PDL-1 blockade. [Figure 6] Figures 6A and 6B show the CD4+ T cell profiles in the peripheral blood of melanoma patients treated with nivolumab. Figures 6A and 6B show the frequency of regulatory T cells (FOXP3+) versus granzyme B+ in CD4+ T cells from the same melanoma patient at two different time points: Figure 6A shows the results while the patient was responding to anti-PD-1 (MDX-1106) therapy, and Figure 6B shows the results for the same patient during relapse at 4 months. [Figure 7]Figure 7 shows the ratio of granzyme B+ cells to Treg cells in the peripheral blood CD4 T cell population of melanoma patients treated with nivolumab. The ratio between granzyme B+ and FOXP3+CD4+ T cells (y-axis) highlights two melanoma patients with a higher frequency of cytotoxic CD4+ T cells and proportionally longer disease-free survival with Treg cells, while the other patients experienced continuous disease progression. [Figure 8] Figure 8 shows the frequency of cytotoxic CD4+ T cells in healthy donor (HD), untreated, and anti-PD-1 treated melanoma patients. Each point represents one donor. This figure shows an increase in the frequency of cytotoxic CD4+ T cells in the peripheral blood of anti-PD-1 treated melanoma patients (P=0.04). [Figure 9] Figure 9 shows the frequency of granzyme B+CD4+ T cells from melanoma patients treated with anti-PD-1 therapy relative to the number of days without disease progression. The frequency of cytotoxic CD4+ T cells in peripheral blood correlates with the response of melanoma patients to anti-PD-1 therapy. [Figure 10] Figure 10 shows the granzyme B+CD4+ T cell / Treg ratio versus progression-free survival in melanoma patients treated with anti-PD-1 therapy. The ratio of cytotoxic CD4+ T cell frequency to Treg frequency in peripheral blood correlates with the response of melanoma patients to anti-PD1 therapy. [Figure 11] Figure 11 shows that the ratio of cytotoxic CD4 T+ cell frequency to Treg frequency in peripheral blood identifies patients treated with anti-PD-1 and those responding to treatment. The granzyme B+CD4+ T cell / Treg ratio is increased in melanoma patients treated with anti-PD-1 antagonists and is even higher in the responder group. The untreated melanoma group represents patients who did not receive anti-PD-1, and pre-treatment patients (baseline group) are divided into PD=progressive and PR=responder groups based on clinical outcome. HD indicates a healthy donor. Each point represents one donor. [Modes for carrying out the invention]

[0026] I. Definition Before describing the invention in detail, it should be understood that this invention is not limited to any particular composition or biological system (which may, of course, vary). Furthermore, it should be understood that the terminology used herein is solely for the purpose of describing a particular embodiment and is not intended to limit it.

[0027] As used in this specification and the attached claims, the singular forms "a," "an," and "the" refer to multiple objects unless the content clearly indicates otherwise. For example, a reference to "a molecule" may optionally include two or more such combinations of molecules.

[0028] The term “approximately” as used herein means the normal range of error for each value that is readily known to those skilled in the art. A “approximately” reference herein to a value or parameter includes (and describes) embodiments of that value or parameter itself.

[0029] The aspects and embodiments of the invention described herein include "including," "consisting of," and / or "essentially consisting of" the aspects and embodiments.

[0030] The term "PD-1 axis-binding antagonist" refers to a molecule that inhibits the interaction between a PD-1 axis-binding partner and one or more of its binding partners, resulting in the elimination of T cell dysfunction caused by signaling on the PD-1 signaling axis and the restoration or enhancement of T cell function (e.g., proliferation, cytokine production, and / or target cell death). As used herein, a PD-1 axis-binding antagonist includes PD-1 binding antagonists, PD-L1 binding antagonists, and PD-L2 binding antagonists.

[0031] The term "PD-1 binding antagonist" refers to a molecule that reduces, blocks, inhibits, suppresses, or interferes with signaling resulting from the interaction of PD-1 with one or more of its binding partners, such as PD-L1 and / or PD-L2. In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to one or more of its binding partners. In certain embodiments, a PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1 and / or PD-L2. For example, PD-1 binding antagonists include anti-PD-1 antibodies, their antigen-binding fragments, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, suppress, or interfere with signaling resulting from the interaction of PD-1 with PD-L1 and / or PD-L2. In one embodiment, a PD-1-binding antagonist reduces negative costimulatory signals mediated by or via cell surface proteins expressed on T lymphocytes that mediated PD-1-mediated signaling, thereby reducing the dysfunction of dysfunctional T cells (e.g., enhancing the effector response to antigen recognition). In some embodiments, the PD-1-binding antagonist is an anti-PD-1 antibody. In certain embodiments, the PD-1-binding antagonist is MDX-1106 (nivolumab) as described herein. In other specific embodiments, the PD-1-binding antagonist is MK-3475 (pembrolizumab) as described herein. In other specific embodiments, the PD-1-binding antagonist is MEDI-0680 (AMP-514) as described herein. In other specific embodiments, the PD-1-binding antagonist is PDR001 as described herein. In other specific embodiments, the PD-1-binding antagonist is REGN2810 as described herein. In another specific embodiment, the PD-1-conjugated antagonist is BGB-108, as described herein.

[0032] The term "PD-L1-binding antagonist" refers to a molecule that reduces, blocks, inhibits, suppresses, or interferes with signaling resulting from the interaction of PD-L1 with one or more of its binding partners, such as PD-1 and / or B7-1. In some embodiments, a PD-L1-binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partner. In certain embodiments, a PD-L1-binding antagonist inhibits the binding of PD-L1 to PD-1 and / or B7-1. In some embodiments, a PD-L1-binding antagonist includes anti-PD-L1 antibodies, their antigen-binding fragments, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, suppress, or interfere with signaling resulting from the interaction of PD-L1 with one or more of its binding partners, such as PD-1 and / or B7-1. In one embodiment, a PD-L1-binding antagonist reduces negative costimulatory signals mediated by or via cell surface proteins expressed on T lymphocytes that mediated PD-L1-mediated signaling, thereby reducing the dysfunction of dysfunctional T cells (e.g., enhancing the effector response to antigen recognition). In some embodiments, the PD-L1-binding antagonist is an anti-PD-L1 antibody. In certain embodiments, the anti-PD-L1 antibody is MPDL3280A (atezolizumab) as described herein. In another specific embodiment, the anti-PD-L1 antibody is MDX-1105 as described herein. In yet another specific embodiment, the anti-PD-L1 antibody is YW243.55.S70 as described herein. In yet another specific embodiment, the anti-PD-L1 antibody is MEDI4736 (durvalumab) as described herein. In another specific embodiment, the anti-PD-L1 antibody is MSB0010718C (avelumab) as described herein.

[0033] The term "PD-L2-binding antagonist" refers to a molecule that reduces, blocks, inhibits, suppresses, or interferes with signaling resulting from the interaction of PD-L2 with one or more of its binding partners, such as PD-1. In some embodiments, a PD-L2-binding antagonist is a molecule that inhibits the binding of PD-L2 to one or more of its binding partners. In certain embodiments, a PD-L2-binding antagonist inhibits the binding of PD-L2 to PD-1. In some embodiments, a PD-L2 antagonist includes anti-PD-L2 antibodies, their antigen-binding fragments, immunoadhesins, fusion proteins, oligopeptides, and other molecules that reduce, block, inhibit, suppress, or interfere with signaling resulting from the interaction of PD-L2 with one or more of its binding partners, such as PD-1. In one embodiment, a PD-L2-binding antagonist reduces negative costimulatory signals mediated by or via cell surface proteins expressed on T lymphocytes that are mediated by PD-L2-mediated signaling, thereby reducing the dysfunction of dysfunctional T cells (e.g., enhancing the effector response to antigen recognition). In some embodiments, the PD-L2-binding antagonist is an immunoadhesin.

[0034] The “taxane” as used herein is a diterpene capable of binding to tubulin, promoting microtubule assembly and stabilization, and / or inhibiting microtubule depolymerization. Taxanes included herein include taxoid 10-deacetylbaccatin III and / or its derivatives. Exemplary taxanes include, but are not limited to, paclitaxel (i.e., TAXOL®, CAS#33069-62-4), docetaxel (i.e., TAXOTERE®, CAS#114977-28-5), larotaxel, cabazitaxel, mirataxel, tesetaxel, and / or orataxel. In some embodiments, the taxane is albumin-coated nanoparticles (e.g., nano-albumin-bound (nab)-paclitaxel, i.e., ABRAXANE® and / or nab-docetaxel, ABI-008). In some embodiments, the taxane is nab-paclitaxel (ABRAXANE®). In some embodiments, the taxane is formulated in CREMAPHOR® (e.g., TAXOL®) and / or Tween, e.g., polysorbate 80 (e.g., TAXOTERE®). In some embodiments, the taxane is a taxane encapsulated in liposomes. In some embodiments, the taxane is a prodrug form and / or conjugate form of the taxane (e.g., paclitaxel, paclitaxel polygrumex, and / or DHA covalently conjugated to linoleyl carbonate-paclitaxel). In some embodiments, paclitaxel is formulated substantially without surfactants (e.g., -TOCOSOL® paclitaxel in the absence of CREMAPHOR and / or Tween).

[0035] In the context of immunodeficiency, the term "dysfunction" refers to a state of reduced immune responsiveness to antigenic stimulation. This term encompasses both "exhaustion" and / or "anergy," where antigen recognition may occur, but the subsequent immune response is ineffective in controlling infection or tumor growth.

[0036] As used herein, the term "dysfunction" also includes resistance or non-responsiveness to antigen recognition, particularly the inability to translate antigen recognition into downstream T cell effector functions such as proliferation, cytokine production (e.g., IL-2) and / or target cell killing.

[0037] As used herein, "cytotoxic CD4" + T cells refer to cytotoxic granzyme B + CD4 + T cells. "Cytotoxic CD4" + T cells, "CD4" + cytotoxic T cells, and "cytotoxic granzyme B" + CD4 + T cells are used synonymously. These cells can be identified by methods known in the art, e.g., staining cells with fluorescently labeled antibodies against CD4 and granzyme B and using fluorescence-activated cell sorting to gate on CD4-positive and granzyme B double-positive cells.

[0038] As used herein, regulatory T cells or Tregs refer to FOXP3 + CD4 + T cells. "Regulatory T cells", "Tregs", and "FOXP3" + CD4 + T cells are used synonymously. These cells can be identified by methods known in the art, e.g., staining cells with fluorescently labeled antibodies against CD4 and FOXP3 and using fluorescence-activated cell sorting to gate on CD4-positive and FOXP3 double-positive cells.

[0039] As used herein, "the ratio of granzyme B" + CD4 + T cells to FOXP3 + CD4 + T cells to" refers to the frequency of granzyme B + CD4 + T cells divided by FOXP3 + CD4 + T cells within the CD4-positive cell population.

[0040] "T-cell enhancement" means inducing, acting upon, or stimulating T cells to have sustained or amplified biological function, or to regenerate or reactivate depleted or inactive T cells. Examples of T-cell enhancement include increased secretion of CD8+ T-cell-derived gamma-interferon, increased proliferation, and increased antigen responsiveness (e.g., viral, pathogen, or tumor clearance) compared to similar levels before therapeutic intervention. In one embodiment, the level of enhancement is at least 50%, or 60%, 70%, 80%, 90%, 100%, 120%, 150%, or 200%. Methods for measuring this enhancement are known to those skilled in the art.

[0041] "T-cell dysfunction" is a disorder or condition of T cells characterized by reduced responsiveness to antigen stimulation. In certain embodiments, T-cell dysfunction is a disorder specifically associated with an inappropriate increase in PD-1 signaling. In other embodiments, T-cell dysfunction is a disorder in which T cells are anergistic or have reduced ability to secrete cytokines, proliferate, or achieve cytolytic activity. In certain embodiments, reduced responsiveness leads to ineffective control of immunogen-expressing pathogens or tumors. Examples of T-cell dysfunction characterized by T-cell dysfunction include unexplained acute infections, chronic infections, and tumor immunity.

[0042] "Tumor immunity" refers to the process by which tumors evade immune recognition and clearance. Therefore, as a therapeutic concept, tumor immunity is "treated" when this evasion is reduced, and the tumor is recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor reduction, and tumor clearance.

[0043] "Immunogenicity" refers to the ability of a particular substance to trigger an immune response. Tumors are immunogenic, and enhancing tumor immunogenicity helps the immune response to clear tumor cells. Examples of enhancing tumor immunogenicity include treatment with PD-L1 axis-linked antagonists and taxanes.

[0044] "Sustained response" refers to a sustained effect on reducing tumor growth after discontinuation of treatment. For example, tumor size may be maintained at the same level as or smaller than the size at the start of the treatment phase. In some embodiments, the sustained response has a duration of at least the same duration as the treatment period, or at least 1.5 ×, 2.0 ×, 2.5 ×, or 3.0 × the treatment period.

[0045] Where used herein, “reduce or inhibit cancer recurrence” means reduce or inhibit the recurrence or progression of a tumor or cancer. Where disclosed herein, cancer recurrence and / or progression of cancer includes, but is not limited to, metastasis of cancer.

[0046] In this context, "complete remission" or "CR" refers to the disappearance of all target lesions.

[0047] Where used herein, “partial response” or “PR” refers to a reduction of at least 30% of the total longest diameter (SLD) of the target lesion, with reference to baseline SLD.

[0048] As used herein, “stable disease” or “SD” refers to the smallest SLD since the start of treatment, with no significant reduction in target lesion size that would qualify for a partial response (PR) and no significant increase that would qualify for a progressive disease (PD).

[0049] Where used herein, “progressive disease” or “PD” refers to at least a 20% increase in the SLD of the target lesion, with reference to the presence of the smallest SLD or one or more new lesions recorded since the start of treatment.

[0050] As used herein, "progression-free survival" (PFS) refers to the length of time during and after treatment in which the treated disease (e.g., cancer) does not worsen. Progression-free survival may include the amount of time a patient experiences complete remission or partial response, as well as the amount of time a patient experiences stable disease.

[0051] In this context, "overall response rate" or "objective response rate" (ORR) refers to the sum of the complete remission (CR) rate and the partial response (PR) rate.

[0052] When used in this context, "overall survival" (OS) refers to the proportion of individuals in a group that are likely to survive after a specific period of time.

[0053] The term "pharmaceutical preparation" refers to a preparation that is in a form that enables the biological activity of the active ingredient and does not contain additional ingredients that are unacceptably toxic to the subject to which the preparation is administered. Such preparations are sterile. A "pharmaceutically acceptable" excipient (vehicle, additive) is one that can be reasonably administered to the target mammal to deliver an effective dose of the active ingredient used.

[0054] A "pharmaceutically acceptable carrier" refers to a component in a pharmaceutical preparation other than the active ingredient that is non-toxic to the target substance. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0055] As used herein, the term “treatment” refers to a clinical intervention designed to alter the natural course of an individual or cells being treated during the course of clinicopathology. Desired effects of treatment include a slowing of disease progression, reduction or mitigation of symptoms, and remission or improved prognosis. For example, an individual is successfully “treated” if one or more cancer-related symptoms are reduced or eliminated, including, but not limited to, a reduction (or destruction) of cancerous cell proliferation, a reduction in symptoms caused by the disease, an improvement in the quality of life of the diseased person, a reduction in the dosage of other drugs required to treat the disease, and / or an extension of the individual’s survival time.

[0056] As used herein, “slowing the progression” of a disease means delaying, preventing, slowing, postponing, stabilizing, and / or postponing the onset of a disease (e.g., cancer). This delay can be of varying lengths depending on the disease history and / or the individual being treated. As will be apparent to those skilled in the art, a sufficient or significant delay can, in practice, encompass prevention of the individual developing the disease. For example, it can delay the progression of terminal cancer, such as the development of metastases.

[0057] An effective dose or therapeutic dose is the minimum amount required to produce at least a measurable improvement or prevention of a particular disorder. The effective dose may vary depending on factors such as the patient's disease state, age, sex, and weight, as well as the drug's ability to produce the desired response in the individual. The effective dose is also the amount at which the therapeutically beneficial effects outweigh any toxic or adverse effects of the treatment. In the case of prophylactic use, beneficial or desirable outcomes include eliminating or reducing risk, reducing severity, or delaying the onset of the disease, including its biochemical, histological, and / or behavioral symptoms, its complications, and intermediate pathological phenotypes presented during the onset of the disease. In the case of therapeutic use, beneficial or desirable outcomes include clinical outcomes such as reduction of one or more symptoms caused by the disease, improvement of the quality of life of the affected person, reduction of the dose of other drugs required to treat the disease, enhancement of the effect of another drug, e.g., by targeting, delay of disease progression, and / or extension of survival. In the case of cancer or tumors, an effective dose of a drug may be effective in reducing the number of cancer cells; shrinking the tumor size; inhibiting (i.e., delaying, and preferably stopping) the invasion of cancer cells into peripheral organs; inhibiting (i.e., delaying, and preferably stopping) tumor metastasis; inhibiting tumor growth to some extent; and / or alleviating to some extent one or more symptoms associated with the disorder. An effective dose may be administered in one or more doses. For the purposes of the present invention, an effective dose of a drug, compound, or pharmaceutical composition is sufficient to achieve a prophylactic or therapeutic treatment, directly or indirectly. As understood in a clinical context, an effective dose of a drug, compound, or pharmaceutical composition may or may not be achieved in combination with another drug, compound, or pharmaceutical composition. Thus, “effective dose” may be considered in a situation in which one or more therapeutic agents are administered, and a single agent may be considered administered in an effective dose if the desired result can or does not occur when combined with one or more other agents.

[0058] In this context, "in conjunction with..." refers to the administration of one treatment method in addition to another treatment method. Furthermore, "in conjunction with..." refers to the administration of one treatment method to an individual before, during, or after the administration of another treatment method.

[0059] "Disability" is any condition that would benefit from treatment, including but not limited to chronic and acute disabilities or diseases, including the pathological conditions that cause a mammal to suffer from such disability.

[0060] The terms "proliferative disorder" and "proliferative disorder" refer to disorders that involve some degree of abnormal cell proliferation. In one embodiment, the proliferative disorder is cancer. In one embodiment, the proliferative disorder is a tumor.

[0061] As used herein, the term “tumor” refers to all neoplastic cell growth and proliferation, whether malignant or benign, as well as all precancerous and cancerous cells and tissues. The terms “cancer,” “cancerous,” “proliferative disorder,” “proliferative disorder,” and “tumor” are not mutually exclusive when used herein.

[0062] The terms “cancer” and “malignant” refer to or describe a physiological condition in mammals typically characterized by uncontrolled cell proliferation. This definition includes both benign and malignant cancers. “Early cancer” or “early tumor” means cancer that is non-invasive or non-metastatic or of stage 0, 1, or 2. Examples of cancer include, but are not limited to, carcinomas, lymphomas, blastomas (including medulloblastoma and retinoblastoma), sarcomas (including liposarcoma and synovial cell sarcoma), neuroendocrine tumors (including carcinoid tumors, gastrin-producing tumors, and islet cell carcinomas), mesothelioma, schwann cell tumors (including acoustic neuromas), meningiomas, adenocarcinomas, melanomas, and leukemias or lymphoid tumors. More specific examples of such cancers include bladder cancer (e.g., urothelial bladder cancer (e.g., transitional or urothelial carcinoma, nonmuscle-invasive bladder cancer, muscle-invasive bladder cancer, and metastatic bladder cancer) and non-urothelial bladder cancer)), squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer including small cell lung cancer (SCLC), non-small cell lung cancer (NSCLC), adenocarcinoma of the lung, and squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular carcinoma, and gastric cancer including gastrointestinal cancer. or include stomach cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, hepatoma, breast cancer (including metastatic 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, Merkel cell carcinoma, mycosis fungoides, testicular cancer, esophageal cancer, bile duct tumors, and head and neck cancers and hematological malignancies. In some embodiments, the cancer is triple-negative metastatic breast cancer, which includes any histologically confirmed triple-negative (ER, PR, HER2) breast cancer with locally recurrent or metastatic disease (locally recurrent disease cannot be removed for therapeutic purposes). In some embodiments, the cancer is bladder cancer. In certain embodiments, the bladder cancer is urothelial bladder cancer.

[0063] As used herein, the term “sample” refers to a composition obtained from or derived from the subject and / or individual of interest, including cells and / or other molecular entities characterized and / or identified, for example, based on physical, biochemical, chemical, and / or physiological properties. For example, the phrase “disease sample” and its variations refer to any sample obtained from the subject of interest that is expected or known to contain characterized cells and / or molecular entities. Samples include, but are not limited to, tissue samples, primary or cultured cells or cell lines, cell supernatants, cell solubilites, platelets, serum, plasma, vitreous fluid, lymph, synovial fluid, follicular fluid, semen, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, tumor solubilites, and tissue culture media, tissue extracts, such as homogenized tissue, tumor tissue, cell extracts, and combinations thereof.

[0064] A “tissue sample” or “cell sample” means a collection of similar cells obtained from the tissue of a subject or individual. Sources of tissue or cell samples may be solid tissues derived from fresh, frozen, and / or preserved organs, tissue samples, biopsies, and / or aspirates; blood or any blood component, e.g., plasma; body fluids, e.g., cerebrospinal fluid, amniotic fluid, ascites, or interstitial fluid; or cells at any point in time during the subject's pregnancy or development. A tissue sample may also be primary or cultured cells or cell lines. In some cases, a tissue or cell sample may be obtained from diseased tissue / organ. For example, a “tumor sample” is a tissue sample obtained from a tumor or other cancerous tissue. A tissue sample may contain a mixed population of cell types (e.g., tumor cells and non-tumor cells, cancerous cells and non-cancerous cells). A tissue sample may contain compounds that do not naturally mix with tissue, such as preservatives, anticoagulants, buffers, fixatives, nutrients, and antibiotics.

[0065] The term "detection" includes any detection means, including direct and indirect detection.

[0066] As used herein, the term “biomarker” refers to indicators that can be detected in a sample, such as predictive indicators, diagnostic indicators, and / or prognostic indicators. Biomarkers can function as indicators of specific subtypes of a disease or disorder (e.g., cancer) characterized by certain molecular, pathological, histological, and / or clinical features. In some embodiments, biomarkers are genes. Biomarkers include, but are not limited to, polynucleotides (e.g., DNA and / or RNA), polynucleotide copy number modifications (e.g., DNA copy number), polypeptides, polypeptide and polynucleotide modifications (e.g., post-translational modifications), carbohydrates, and / or glycolipid-based molecular markers.

[0067] The term "cytotoxic agent" as used herein refers to any agent that is harmful to cells (e.g., causes cell death, inhibits proliferation, or otherwise interferes with cellular function). Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At). 211 , I 131 , I 125 , Y 90 Re 186 Re 188 Sm 153 , Bi 212 , P 32 Pb 212The following are examples of cytotoxic agents: radioisotopes of Lu; chemotherapeutic agents; growth inhibitors; enzymes and their fragments, e.g., nucleases; and toxins, e.g., small molecule toxins, or enzyme-active toxins (including their fragments and / or variants) derived from bacteria, fungi, plants, or animals. Exemplary cytotoxic agents can be selected from antimicrotubule agents, platinum coordination complexes, alkylating agents, antibiotic preparations, topoisomerase II inhibitors, antimetabolites, topoisomerase I inhibitors, hormones and hormone analogs, signaling pathway inhibitors, non-receptor tyrosine kinase angiogenesis inhibitors, immunotherapeutic agents, apoptosis promoters, LDH-A inhibitors, fatty acid biosynthesis inhibitors, cell cycle signaling inhibitors, HDAC inhibitors, proteasome inhibitors, and cancer metabolism inhibitors. In one embodiment, the cytotoxic agent is a platinum-based chemotherapeutic agent. In one embodiment, the cytotoxic agent is an EGFR antagonist. In one embodiment, the cytotoxic agent is N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazoline-4-amine (e.g., erlotinib, TARCEVA). TM In one embodiment, the cytotoxic agent is an RAF inhibitor. In one embodiment, the RAF inhibitor is a BRAF and / or CRAF inhibitor. In one embodiment, the RAF inhibitor is vemurafenib. In one embodiment, the cytotoxic agent is a PI3K inhibitor.

[0068] As used herein, the term "chemotherapeutic agent" includes compounds useful in the treatment of cancer. Examples of chemotherapeutic agents include erlotinib (TARCEVA®, Genentech / OSI Pharm.), bortezomib (VELCADE®, Millennium Pharm.), disulfiram, epigallocatechin gallate, salinosporamide A, carfilzomib, 17-AAG (geldanamycin), radicicol, lactate dehydrogenase A (LDH-A), fulvestrant (FASLODEX®, AstraZeneca), sunitib (Sutent®, Pfizer / Sugen), letrozole (FEMARA®, Novartis), and mesylate. Matinib (GLEEVEC®, Novartis), finasunate (VATALANIB®, Novartis), oxaliplatin (ELOXATIN®, Sanofi), 5-FU (5-fluorouracil), leucovorin, rapamycin (Sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016, Glaxo Smith Kline), lonafamib (SCH66336), sorafenib (NEXAVAR®, Bayer) Labs), gefitinib (IRESSA®, AstraZeneca), AG1478, alkylating agents, e.g., thiotepa and cytoxane® cyclophosphamide; alkyl sulfonates, e.g., busulfan, improsulfan and pigosulfan; aziridines, e.g., benzodopa, carbocone, metsuredopa and uredopa; ethyleneimine and methylamelamine (including altoretamine), triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomellamine; acetogenins (especially bratacin and bratacinone); camptothecin (including topotecan and irinotecan); bryostatin; calistatin; CC-1065 (including its adzeresin, carzelsin and bizeresin synthetic analogs); cryptophycin (especially cryptophycin 1 and cryptophycin 8);Adrenocortical steroids (including prednisone and prednisolone); cyproterone acetate; 5α-reductase including finasteride and dutasteride; vorinostat, romidepsin, panobinostat, valproic acid, mosetinostat, dorastatin; aldesleukin, talcus, ocarmycin (including synthetic analogs, KW-2189 and CB1-TM1); eryuterobin; pancratistatin; sarcodiciin; spongistatin; nitrogen mustard, e.g., chlorambucil, chlomaphazine, chlorophosphamide D, estramustine, ifosfamide, mechloretamine, mechloretamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trophosphamide, uracil mustard; nitrosoureas, e.g., carmustine, chlorozotosine, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, e.g., engine antibiotics (e.g., calicheamicin, especially calicheamicin γ1I and calicheamicin ω1I (Angew Chem. Intl. Ed. Engl. 33:183-186 (1994)); dinemycin including dinemycin A; bisphosphonates, e.g., clodronate; esperamicin;(and neocardinostatin chromophores and related pigment proteins, enediin antibiotic chromophores), acrasinomycin, actinomycin, anthramycin, azaserin, bleomycin, kactinomycin, carabicin, caminomycin, cardinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, Adriamycin (registered trademark) (doxorubicin), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcelomycin, mitomycin, e.g., mitomycin C, mycophenolic acid, nogaramycin, olibomycin, peplomycin, porphyromycin, puromycin, queramycin , rhodorubicin, streptonigrin, streptozocin, tubercidine, ubenimex, dinostatin, zolubicin; antimetabolites, e.g., methotrexate and 5-fluorouracil (5-FU); folate analogs, e.g., denopterin, methotrexate, pteropterin, trimethrexate; purine analogs, e.g., fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs, e.g., anthracite Tabin, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens, e.g., carsterone, dromostanolone propionate, epithiostanol, mepitiostane, testolactone; anti-adrenal agents, e.g., aminoglutethimide, mitotane, trilostane; folic acid supplements, e.g., folinic acid acid); acegraton; aldofhamide glucoside; aminolevulinic acid; enyluracil; amsacrin; bestrabusil; bisanthren; edatraxate; defofamine; demecolsin; diazicone; elfomithine; eriptinium acetate; epotilon; etogluside; gallium nitrate; hydroxyurea; lentinan; lonidainine;Maytansinoids, e.g., maytansine and anthamitosine; mitogwazone; mitoxantrone; mopidamnol; nitraerine; pentostatin; fenamet; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK (registered trademark) polysaccharide complex (JHS Natural Products, Eugene, Oreg.); Lazoxane; Rhizoxin; Schizophyllan; Spirogermanium; Tenuazonic Acid; Triadiquan; 2,2',2''-Trichlorotriethylamine; Trichothecene (especially T-2 toxin, verracurin A, loridine A and anguidin); Urethane; Vindesine; Dacarbazine; Mannomustine; Mitobronitol; Mitractol; Pipobroman; Gacytosine; Arabinoside ("Ara-C"); Cyclophosphamide; Thiotepa; Taxane; Chlorambucil; GEMZAR (registered trademark) This includes gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE® (vinorelbine); novantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (Xeloda®); ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; and any pharmaceutically acceptable salts, acids, and derivatives of the above.

[0069] Chemotherapy agents also include "platinum-based" chemotherapeutic agents, which are organic compounds containing platinum as an essential part of the molecule. Typically, platinum-based chemotherapeutic agents are platinum coordination complexes. Platinum-based chemotherapeutic agents are sometimes referred to as "platins" in the art. Examples of platinum-based chemotherapeutic agents include, but are not limited to, carboplatin, cisplatin, and oxaliplatin.

[0070] Chemotherapy agents include (i) antihormones that act to modulate or inhibit hormonal effects on tumors, such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (NOLVADEX®; including tamoxifen citrate), raloxifen, droloxifen, iodoxifen, 4-hydroxytamoxifen, trioxyfen, keoxyfen, LY117018, onapristone, and Fareston® (toremifine citrate). (ii) Aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal gland, such as 4(5)-imidazole, aminoglutethimide, MEGASE® (megestrol acetate), AROMASIN® (exemestane; Pfizer), formestanie, fadrozol, RIVISOR® (borozol), FEMARA® (letrozole; Novartis), and ARIMIDEX® (anastrozole; AstraZeneca). (iii) Antiandrogens, e.g., flutamide, nilutamide, bicalutamide, leuprolide and goserelin; buserelin, tripterelin, medroxyprogesterone acetate, diethylstilbestrol, premarin, fluoxymesterone, all-trans-retinoic acid, fenretinide, and troxacitabine (1,3-dioxolane nucleoside cytosine analog); (iv) Protein kinase inhibitors; (v) Lipid kinase inhibitors; (vi) Antisense oligonucleotides, in particular, e.g., PKC - Inhibitors of gene expression in signaling pathways involved in abnormal cell proliferation, such as alpha, Ralf, and H-Ras; (vii) Ribozymes, e.g., VEGF expression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors; (viii) Vaccines such as gene therapy vaccines, e.g., ALLOVECTIN®, LEUVECTIN®, and VAXID®; PROLEUKIN®, rIL-2; Topoisomerase 1 inhibitors, e.g., LURTOTECAN®;ABARELIX® rmRH; and (ix) any pharmaceutically acceptable salts, acids, and derivatives of any of the above;

[0071] Chemotherapy agents also include antibodies such as alemtuzumab (Campath), bevacizumab (Avastin®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab (VECTIBIX®, Amgen); rituximab (RITUXAN®, Genentech / Biogen Idec); pertuzumab (OMNITARG®, 2C4, Genentech); trastuzumab (Herceptin®, Genentech); tositumomab (Bexxar, Corixia); and antibody-drug conjugates such as gemtuzumab ozogamicin (Mylotarg®, Wyeth).Additional humanized monoclonal antibodies that have therapeutic potential as agents when combined with the compounds of the present invention include apolizumab, aselizumab, atlizumab, bapinuzumab, vivacuzumab meltansine, cantuzumab meltansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab, fontrizumab, gemtuzumab ozogamicin, and inotuzumab ozogamicin. ipilimumab, rabetsumab, lintuzumab, matuzumab, mepolizumab, motabizumab, motobizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab, pectuzumab, pexelizumab, ra Rivizumab, ranivizumab, reslivizumab, reslizumab, resyvizumab, rovelizumab, ruplizumab, sibrotuzumab, ciprizumab, sontuzumab, tacuzumab tetraxetan, tadocizumab, taliz These include mab, tefivazumab, tocilizumab, tralizumab, tucotzumab cermoloykin, tucusituzumab, umavizumab, urtoxazumab, ustekinumab, bicilizumab, and anti-interleukin-12 (ABT-874 / J695, Wyeth Research and Abbott Laboratories), which is a full-length IgG1λ antibody of a recombinant, exclusive human sequence genetically modified to recognize the interleukin-12p40 protein.

[0072] Chemotherapy agents also include "EGFR inhibitors," also known as "EGFR antagonists," which are compounds that bind to EGFR or otherwise directly interact with it to prevent or reduce its signaling activity. Examples of such drugs include antibodies and small molecules that bind to EGFR. Examples of antibodies that bind to EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB 8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see U.S. Patent No. 4,943,533) and their variants, such as chimeric 225 (C225 or cetuximab; ERBUTIX®) and reshaped human 225 (H225) (e.g., International Publication No. 96 / 40210 (Imclone Systems)). See Inc.), IMC-11F8, fully human EGFR-targeted antibody (Imclone); antibody that binds to type II mutant EGFR (U.S. Patent No. 5212290), humanized and chimeric antibodies that bind to EGFR as described in U.S. Patent No. 5891996, and human antibodies that bind to EGFR, e.g., ABX-EGF or panitumumab (see International Publication No. 98 / 50433 (Abgenix / Amgen)); EMD55900 (Stragliotto et al. Eur. J. Cancer 32A:636-640) (1996)); Humanized EGFR antibody EMD7200 (matsuzumab) (EMD / Merck) produced against EGFR that competes with both EGF and TGF-alpha for EGFR binding; Human EGFR antibody HuMax-EGFR (GenMab); Fully human antibody known as E1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6.3 and E7.6.3, described in U.S. Patent No. 6,235,883; MDX-447 (Medarex Inc); and mAb806 or humanized mAb806 (Johns et al., J. Biol. Chem. 279(29):30375-30384 (2004)). Anti-EGFR antibodies can be conjugated with cytotoxic agents to create immunoconjugates (see, for example, European Patent Application Publication No. 659439A2, Merck Patent GmbH).EGFR antagonists include U.S. Patent Nos. 5616582, 5457105, 5475001, 5654307, 5679683, 6084095, 6265410, 6455534, 6521620, 6596726, 6713484, 5770599, 6140332, 5866572, and 639 This includes small molecules such as compounds described in publications 9602, 6344459, 6602863, 6391874, 6344455, 5760041, 6002008, and 5747498, as well as the following PCT publications: International Publication No. 98 / 14451, International Publication No. 98 / 50038, International Publication No. 99 / 09016, and International Publication No. 99 / 24037.Specific small molecule EGFR antagonists include OSI-774 (CP-358774, erlotinib, TARCEVA® Genentech / OSI Pharmaceuticals); PD183805 (CI1033, 2-propenamide, N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholinyl)propoxy]-6-quinazolinyl]-, dihydrochloride, Pfizer) Inc.); ZD1839, Gefitinib (IRESSA®) 4-(3'-chloro-4'-fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy)quinazoline, AstraZeneca); ZM105180 ((6-amino-4-(3-methylphenyl-amino)-quinazoline, Zeneca); BIBX-1382 (N8-(3-chloro-4-fluorophenyl)-N2-(1-methyl-piperidine-4-yl)-pyrimido[5,4-d]pyrimidine-2,8-diamine, Boehringer Ingelheim); PKI-166((R)-4-[4-[(1-phenylethyl)amino]-1H-pyrrolo[2,3-d]pyrimidine-6-yl]phenol); (R)-6-(4-hydroxyphenyl)-4-[(1-phenylethyl)amino]-7H-pyrrolo[2,3-d]pyrimidine); CL-387785(N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butinamide); EKB-569(N-[4-[(3-chloro-4-fluorophenyl)amino]-3-cyano [-7-ethoxy-6-quinolinyl]-4-(dimethylamino)-2-butinamide) (Wyeth); AG1478 (Pfizer); AG1571 (SU5271; Pfizer); dual EGFR / HER2 tyrosine kinase inhibitors, such as lapatinib (TYKERB®, GSK572016 or N-[3-chloro-4-[(3-fluorophenyl)methoxy]phenyl]-6[5[[[2-methylsulfonyl)ethyl]amino]methyl]-2-furanyl]-4-quinazolinamine).

[0073] Chemotherapy agents also include "tyrosine kinase inhibitors," which are EGFR-targeted drugs as described in the previous paragraph; small molecule HER2 tyrosine kinase inhibitors, e.g., TAK165 available from Takeda; oral selective inhibitors of ErbB2 receptor tyrosine kinase, such as CP-724714 (Pfizer and OSI); dual HER inhibitors such as EKB-569 (available from Wyeth), which preferentially binds to EGFR but inhibits both HER2 and EGFR overexpressing cells; oral HER2 and EGFR tyrosine kinase inhibitors such as lapatinib (GSK572016; available from Glaxo-SmithKline); PKI-166 (available from Novartis); pan-HER inhibitors, e.g., canertinib (CI-1033; Pharmacia); and Raf-1 inhibitors, e.g., ISIS, which inhibits Raf-1 signaling. Antisense agents available from pharmaceutical companies include ISIS-5132; non-HER-targeted TK inhibitors, e.g., imatinib mesylate (GLEEVEC®, available from Glaxo SmithKline); multi-targeted tyrosine kinase inhibitors, e.g., sunitinib (Sutent®, available from Pfizer); and VEGF receptor tyrosine kinase inhibitors, e.g., batalanib (PTK787 / ZK222584, Novartis / Scherin). Available from AG); MAPK extracellular regulatory kinase I inhibitor CI-1040 (available from Pharmacia); quinazolines, e.g., PD153035, 4-(3-chloroanilino)quinazoline; pyridopyrimidine; pyrimidopyrimidine; pyrrolopyrimidine, e.g., CGP59326, CGP60261 and CGP62706; pyrazolopyrimidine, 4-(phenylamino)-7H-pyrrolo[2,3-d]pyrimidine; curcumin (diferuloylmethane, 4,5-bis(4-fluoroanilino)phthalimide); thyrophostin containing nitrothiophene moiety; PD-0183805 (Warner-Lamber); antisense molecules (e.g., those that bind to HER-coding nucleic acids); quinoxaline derivatives (US Patent No. 5804396); tryphostin (US Patent No. 5804396); ZD6474 (Astra Zeneca);PTK-787 (Novartis / Schering AG); pan-HER inhibitors, e.g., CI-1033 (Pfizer); Affinitac (ISIS3521; Isis / Lilly); Imatinib mesylate (GLEEVEC®); PKI166 (Novartis); GW2016 (Glaxo SmithKline); CI-1033 (Pfizer); EKB-569 (Wyeth); Semaxinib (Pfizer); ZD6474 (AstraZeneca); PTK-787 (Novartis / Schering AG); INC-1C11 (Imclone), Rapamycin (Sirolimus, RAPAMUNE®); or the following patent publications: U.S. Patent No. 5804396; International Publication No. 1999 / 09016 (American Cyanamid); International Publication No. 1998 / 43960 (American This includes items described in either International Publication No. 1997 / 38983 (Warner Lambert), International Publication No. 1999 / 06378 (Warner Lambert), International Publication No. 1999 / 06396 (Warner Lambert), International Publication No. 1996 / 30347 (Pfizer, Inc.), International Publication No. 1996 / 33978 (Zeneca), or International Publication No. 1996 / 33980 (Zeneca).

[0074] Chemotherapy agents include dexamethasone, interferon, colchicine, methoprine, cyclosporine, amphotericin, metronidazole, alemtuzumab, alitretinoin, allopurinol, amifostin, arsenic trioxide, asparaginase, BCG vaccine, bevacuzimab, besarotene, cladribine, clofarabine, darbepoetin alfa, denileukin, dexrazoxane, epoetin alfa, elotinib, filgrastim, histrelin acetate, ibritumomab, interferon alfa-2a, and This also includes tarferon alpha-2b, lenalidomide, levamisol, mesna, methoxsalen, nandrolone, nelarabine, nofetumomab, oprelbequin, parifermin, pamidronate, pegademase, pegaspargase, pegfilgrastim, pemetrexed disodium, plicamycin, porfimer sodium, quinacrine, rasburicase, salglamostim, temozolomide, VM-26, 6-TG, toremifene, tretinoin, ATRA, barrubicin, zoledronate, and zoledronic acid, as well as pharmaceutically acceptable salts thereof.

[0075] Chemotherapy agents include hydrocortisone, hydrocortisone acetate, cortisone acetate, thixocortol pivalate, triamcinolone acetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide, desonide, fluocinonide, fluocinolone acetonide, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, fluocortone, hydrocortisone-17-butyrate, and hydrocortisone. n-17-valerate, acromethasone dipropionate, betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasone-17-butyrate, clobetasol-17-propionate, fluocortone caproate, fluocortone pivalate, and flupredniden acetate; immunoselective anti-inflammatory peptides (ImSAIDs), such as phenylalanine-glutamine-glycine (FEG) and its D-isomer (feG) (IMULAN BioTherapeutics, LLC) offers anti-rheumatic drugs such as azathioprine, cyclosporine (cyclosporine A), D-penicillamine, gold salt, hydroxychloroquine, leflunomidominocycline, sulfasalazine, tumor necrosis factor alpha (TNFα) blockers such as etanercept (Enbrel), infliximab (Remicade), adalimumab (Humira), certolizumab pegol (Cimzia), golimumab (Simponi), and interleukin-1 (IL-1) blockers such as anakinra (Kineret). Cellular co-stimulatory blockers, e.g., abatacept (Orencia); interleukin-6 (IL-6) blockers, e.g., tocilizumab (ACTEMRA®); interleukin-13 (IL-13) blockers, e.g., lebrikizumab; interferon-alpha (IFN) blockers, e.g., lontalizumab; beta-7 integrin blockers, e.g., rhuMAb beta-7; IgE pathway blockers, e.g., anti-M1 prime; secretory homotrimer LTa3 and membrane-bound heterotrimer LTa1 / β2 blockers, e.g., anti-lymphotoxin alpha (LTa); radioactive isotopes (e.g., At 211 , I 131 , I 125 , Y 90 Re 186 Re 188 Sm153 , Bi 212 , P 32 Pb 212 and radioactive isotopes of Lu); Various investigational drugs, e.g., thioplatin, PS-341, phenylbutyrate, ET-18-OCH3, or farnesyltransferase inhibitors (L-739749, L-744832); polyphenols, e.g., quercetin, resveratrol, picetanol, epigallocatechin gallate, theaflavin, flavanol, procyanidin, betulinic acid and its derivatives; autophagy inhibitors, e.g., chloroquine; delta-9-tetrahydrocannabinol (Dronabinol, MARINOL®); Beta-Lapacon; Lapachol; Colchicine; Betulic acid; Acetylcamptothecin, Scopolectin, and 9-Aminocamptothecin); Podophyllotoxin; Tegafur (UFTORAL®); Bexarotene (TARGRETIN®); Bisphosphonates, e.g., Clodronate (e.g., BONEFOS® or OSTAC®) Trademarks)), etidronate (DIDROCAL®), NE-58095, zoledronic acid / zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tildronate (SKELID®), or risedronate (ACTONEL®); and epidermal growth factor receptor (EGF-R); vaccines, e.g., THERATOPE® vaccine Tin; Perifosin, COX-2 inhibitors (e.g., celecoxib or etoricoxib), proteosome inhibitors (e.g., PS341); CCI-779; Tipifanib (R11577); Orafenib, ABT510; Bcl-2 inhibitors, e.g., Oblimersen sodium (GENASENSE®); Pixantrone; Farnesyltransferase inhibitors, e.g., Ronafarnib (SCH6636, SARASAR) TM); and any pharmaceutically acceptable salt, acid, or derivative of the above; as well as combinations of two or more of the above, for example, CHOP (abbreviation for combination therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone); and FOLFOX (oxaliplatin in combination with 5-FU and leucovorin (ELOXATIN TM This also includes abbreviations for treatment regimens using ).

[0076] Chemotherapy agents also include nonsteroidal anti-inflammatory drugs (NSAIDs) that have analgesic, antipyretic, and anti-inflammatory effects. NSAIDs include non-selective inhibitors of the enzyme cyclooxygenase. Specific examples of NSAIDs include aspirin, propionic acid derivatives such as ibuprofen, fenoprofen, ketoprofen, flurbiprofen, oxaprozin, and naproxen, acetate derivatives such as indomethacin, sulindac, etodolac, and diclofenac, enolic acid derivatives such as piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, and isoxicam, fenamic acid derivatives such as mefenamic acid, meclofenamic acid, flufenamic acid, and tolfenamic acid, and COX-2 inhibitors such as celecoxib, etoricoxib, lumiracoxib, parecoxib, lofecoxib, and valdecoxib. NSAIDs may be used to relieve symptoms of conditions such as rheumatoid arthritis, osteoarthritis, inflammatory arthritis, ankylosing spondylitis, psoriatic arthritis, Reiter's syndrome, acute gout, dysmenorrhea, metastatic bone pain, headaches and migraines, postoperative pain, mild to moderate pain resulting from inflammation and tissue injury, fever, bowel obstruction, and renal colic.

[0077] As used herein, “proliferation inhibitor” refers to a compound or composition that inhibits cell proliferation either in vitro or in vivo. In one embodiment, a proliferation inhibitor is a growth inhibitory antibody that prevents or reduces the proliferation of cells expressing an antigen to which the antibody binds. In another embodiment, a proliferation inhibitor may be one that significantly reduces the proportion of cells in the S phase. Examples of proliferation inhibitors include drugs that block cell cycle progression (at a point other than the S phase), such as drugs that induce G1 arrest or M phase arrest. Classical M phase blockers include vinca (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors, such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Furthermore, these drugs that cause G1 arrest also have an effect on S phase arrest, such as DNA alkylating agents, e.g., tamoxifen, prednisone, dacarbazine, mechloretamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C (arabinofuranosylcytosine). Further information can be found, for example, on page 13 of Mendelsohn and Israel, eds., The Molecular Basis of Cancer, Chapter 1, Murakami et al., titled "Cell cycle regulation, oncogenes, and antineoplastic drugs" (WB Saunders, Philadelphia, 1995).

[0078] "Radiation therapy" refers to the use of directed gamma or beta rays to induce sufficient damage to cells to limit their ability to function normally or to completely destroy them. It is understood that there are many methods known in the art for determining the dosage and duration of treatment. Typical therapeutic agents are given as a single dose, and typical dosages range from 10 to 200 units (Gray) per day.

[0079] For therapeutic purposes, “individual” or “subject” refers to any animal classified as a mammal, including humans, domesticated animals, and zoo, sport, or pet animals, such as dogs, horses, cats, and cows. The individual or subject may be a patient. In certain embodiments, the individual or patient is a human.

[0080] The term "antibody" as used here is used in its broadest sense, specifically encompassing monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments insofar as they exhibit the desired biological activity.

[0081] "Isolated" antibodies are those identified, separated, and / or recovered from components of their natural environment. These contaminants are substances that interfere with the study, diagnosis, or therapeutic use of the antibody and may include enzymes, hormones, and other protein-like or non-protein-like solutes. In some embodiments, the antibody is purified (1) to more than 95% by weight, and in some embodiments more than 99% by weight, by measurement, e.g., by the Lowry method; (2) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence, e.g., by using a spinning cup sequencer; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions, e.g., using Coomassie blue or silver dye. Isolated antibodies contain antibodies in recombinant cells because they lack at least one component of the antibody's natural environment. However, typically, isolated antibodies are prepared by at least one purification step.

[0082] "Natural antibodies" are typically heterotetrameric glycoproteins with approximately 150,000 daltons, consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain is attached to a heavy chain by a single covalent disulfide bond, and the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly separated intrachain disulfide bridges. Each heavy chain has a variable domain (V) followed by multiple constant domains. HEach light chain has a variable domain (V) at one end. L The other end of the light chain has a constant domain; the constant domain of the light chain aligns with the first constant domain of the heavy chain, and the variable domain of the light chain aligns with the variable domain of the heavy chain. Certain amino acid residues are thought to form the interface between the light chain and the variable domain of the heavy chain.

[0083] The term "constant domain" refers to the portion of immunoglobulin that has more conserved amino acid sequences than the other parts of the immunoglobulin, i.e., the variable domain, which contains the antigen-binding site. The constant domain is the C of the heavy chain. H 1, C H 2 and C H It includes three domains (collectively referred to as CH) and the CHL (or CL) domain of the light chain.

[0084] 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 called "V H It is sometimes called "V". The variable domain of the light chain is "V L These domains are sometimes referred to as "antigen-binding sites." These domains are typically the most variable parts of an antibody and contain the antigen-binding site.

[0085] The term "variable" refers to the fact that certain parts of the variable domain exhibit wide sequence differences between antibodies, and is used to determine the binding and specificity of each particular antibody to its specific antigen. However, variability is not uniformly distributed throughout the antibody's variable domain. It is concentrated in three segments called hypervariable regions (HVRs) in both the light and heavy chain variable domains. The more highly conserved portion of the variable domain is called the framework region (FR). The natural heavy and light chain variable domains largely adopt a beta-sheet configuration linked by three HVRs, each containing four FR regions, forming loops that link beta-sheet structures, and in some cases form parts of them. The HVRs of each chain are held in close proximity to each other by the FR regions and, together with the HVRs of other chains, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, National Institute of Health, Bethesda, Md. (1991)). The constant domain is not directly involved in the binding of antibodies to antigens, but it exhibits various effector functions, such as the involvement of antibodies in antibody-dependent cytotoxicity.

[0086] The "light chain" of an antibody (immunoglobulin) derived from any mammalian species can be assigned to one of two distinct types called kappa (κ) and lambda (λ), based on the amino acid sequence of its constant domain.

[0087] The terms "isotype" or "subclass" of IgG used herein refer to any subclass of immunoglobulin determined by the chemical and antigenic properties of their constant regions.

[0088] Antibodies (immunoglobulins) can be assigned to different classes depending on the amino acid sequence of the constant domain of their heavy chain. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IGA1, and IgA2. The heavy chain constant domains corresponding to 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 generally described, for example, in Abbas et al., Cellular and Mol. Immunology, 4th edition (WB Saunders, Co., 2000). An antibody may also be part of a larger fusion molecule formed by covalent or non-covalent bonding of the antibody to one or more other proteins or peptides.

[0089] The terms “full-length antibody,” “intact antibody,” and “whole antibody” are used interchangeably here to refer to the substantially intact form of the antibody, and not to the antibody fragments defined below. These terms particularly refer to antibodies having a heavy chain containing the Fc region.

[0090] For the purposes of this discussion, a "naked antibody" is an antibody that is not conjugated with a cytotoxic moiety or radiolabeling.

[0091] An "antibody fragment" comprises a portion of an intact antibody, preferably including its antigen-binding region. In some embodiments, the antibody fragment described herein is an antigen-binding fragment. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0092] Papain digestion of antibodies produces two identical antibody-binding fragments called "Fab" fragments, each with a single antigen-binding site, and the remaining "Fc" fragment, whose name reflects its ability to easily crystallize. Papain treatment yields an F(ab')2 fragment, which has two antigen-binding sites and is still capable of crosslinking antigens.

[0093] "Fv" is the smallest antibody fragment containing a complete antigen-binding site. In one embodiment, a double-stranded "Fv" species consists of a dimer in which one heavy chain and one light chain variable domain are firmly bonded non-covalently. In a single-stranded Fv (scFv) species, one heavy chain and one light chain variable domain may be covalently linked by a flexible peptide linker, and the light and heavy chains may bind in a "dimer" structure similar to that of the double-stranded Fv species. It is in this configuration that the three HVRs of each variable domain interact to form 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 the three HVRs specific to the antigen) has the ability to recognize and bind to an antigen, although with lower affinity than the entire binding site.

[0094] The Fab fragment contains heavy and light chain variable domains, as well as a constant domain of the light chain and a primary constant domain (CH1) of the heavy chain. The Fab' fragment differs from the Fab fragment in that several residues are added to the carboxyl terminus of the heavy chain CH1 domain, which contains one or more cysteines from the antibody hinge region. Fab'-SH is here the name for Fab' in which the cysteine ​​residues of the constant domain have free thiol groups. The F(ab')2 antibody fragment was produced as a pair of Fab' fragments with hinge cysteines in between. Other chemical bonds of the antibody fragment are also known.

[0095] A "single-chain Fv" or "scFv" antibody fragment contains the VH and VL domains of the antibody, and these domains are present on a single polypeptide chain. Generally, scFv polypeptides further contain a polypeptide linker between the VH and VL domains, which enables the scFv to form the structure desired for antigen binding. For an overview of scFv, see, for example, Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore (eds.), (Springer-Verlag, New York, 1994), pp. 269–315.

[0096] The term "diabody" refers to an antibody fragment having two antigen-binding sites, the fragment containing a heavy-chain variable domain (VH) linked to a light-chain variable domain (VL) of the same polypeptide chain (VH-VL). A linker that is too short to pair the two domains on the same chain is used to force the domains to pair with complementary domains on other chains, thereby generating two antigen-binding sites. Diabodies may be bivalent or bispecific. Diabodies are well described, for example, in European Patent Application Publication No. 404097; International Publication No. 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).

[0097] The term “monoclonal antibody” as used herein means an antibody obtained from a substantially homogeneous population of antibodies, where, with the exception of possible mutations that may exist in small amounts, such as naturally occurring mutations, the individual antibodies constituting that population are identical. Thus, the modifier “monoclonal” indicates the property of the antibody that it is not a mixture of separate antibodies. In a given embodiment, such a monoclonal antibody typically comprises an antibody containing a target-binding polypeptide sequence, where the target-binding polypeptide sequence is obtained by a process comprising selecting 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 pool of clones, such as a hybridoma clone, a phage clone, or a recombinant DNA clone. It should be understood that by further modifying the selected target-binding sequence, it is possible to improve affinity for the target, humanize the target-binding sequence, improve its generation in cell culture, reduce immunogenicity in vivo, and produce multispecific antibodies, and that antibodies containing modified target-binding sequences are also monoclonal antibodies of the present invention. In contrast to polyclonal antibody preparations, which typically contain different antibodies against different determinants (epitopes), each monoclonal antibody in a monoclonal antibody preparation is against a single determinant on an antigen. In addition to its specificity, monoclonal antibody preparations have the advantage of not being typically contaminated with other immunoglobulins.

[0098] The modifier "monoclonal" indicates that the antibody is obtained from a substantially homogeneous antibody population, and does not mean that the antibody must be produced by any specific method. For example, monoclonal antibodies used in accordance with the present invention include, for example, hybridoma methods (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 edition 1988); Hammerling et al.: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981)), recombinant DNA methods (e.g., see U.S. Patent No. 4816567), phage display technology (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. See 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), as well as techniques for producing human or human-like antibodies in animals that have genes encoding human immunoglobulin sequences or some or all of human immunoglobulin loci (e.g., International Publication No. 1998 / 24893; No. 1996 / 34096; No. 1996 / 33735; No. 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 No. 5545807; No. 5545806; No. 5569825; No. 5625126; No. 5633425; and No. 5661016; 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 et al., Intern. Rev. Immunol. 13: 65-93 It can be manufactured using various techniques, including (see 1995).

[0099] The term "monoclonal antibody" as used herein particularly includes "chimeric" antibodies in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, but the remainder of the chain is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies insofar as they exhibit the desired biological activity (see, for example, U.S. Patent No. 4816567; 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, for example, from an antibody produced by immunizing macaque monkeys with the target antigen.

[0100] The "humanized" form of a non-human (e.g., mouse) antibody is a chimeric antibody containing a minimal sequence derived from a non-human immunoglobulin. In one embodiment, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues derived from the recipient's HVR are replaced by residues derived from the HVR of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, having the desired specificity, affinity, and / or capabilities. In some examples, the FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, the humanized antibody may contain residues not found in the recipient antibody or donor antibody. These modifications may be made to further refine antibody performance. Generally, the humanized antibody substantially contains all of at least one, typically two, variable domains, all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the FRs are human immunoglobulin sequences. The humanized antibody may also contain at least a portion of the immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, for example, 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, for example, 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.

[0101] A “human antibody” is one that has an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human, and / or is prepared using any of the techniques disclosed herein for the preparation of human antibodies. This definition of a human antibody excludes humanized antibodies that contain non-human antigen-binding residues in particular. 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)). Other methods available for the preparation of human monoclonal antibodies are those 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). See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can be prepared by administering an antigen to transgenic animals, such as immunized xenomouse mice, in which the antigen has been modified to produce such antibodies in response to antigen exposure, but its endogenous locus has been rendered inactive (e.g., XENOMOUSE). TM (See U.S. Patents 6,075,181 and 6,150,584, relating to the technology.) See also, for example, Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006), relating to human antibodies produced by human B-cell hybridoma technology.

[0102] In this context, the terms “hypervariable region,” “HVR,” or “HV” refer to regions of antibody variable domains whose sequences are hypervariable and / or form structurally defined loops. Generally, antibodies contain a total of six HVRs: three in VH (H1, H2, H3) and three in VL (L1, L2, L3). In naturally occurring 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 high 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, for example, Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[0103] Multiple HVR descriptions are used and are included here. The Kabat complementarity-determining region (CDR) is based on sequence variability and is the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia, on the other hand, refers to the location of the structural loop (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). The AbM HVR represents a compromise between the Kabat HVR and the Chothia structural loop and is used by Oxford Molecular's AbM antibody modeling software. The "contact" HVR is based on the analysis of available complex crystal structures. The residues from each of these HVRs are listed below.

[0104] TIFF0007843689000001.tif62169

[0105] HVR may include the following "extended HVRs": VL residues 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3), and VH residues 26-35 (H1), 50-65 or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3). Variable domain residues are numbered according to Kabat et al. above for each of these definitions.

[0106] "Framework" or "FR" residues are variable domain residues other than the HVR residues defined herein.

[0107] The terms “Kabat variable domain residue numbering” or “Kabat amino acid position numbering” and their variations refer to the numbering system used for the heavy chain or light chain variable domains of Kabat-etynn antibody editing described above. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to abbreviations or insertions into the FR or HVR of the variable domain. For example, the heavy chain variable domain may contain a single amino acid insertion after H2 residue 52 (residue 52a according to Kabat) and an inserted residue after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat). The Kabat numbering of residues can be determined for a given antibody by alignment in regions where the antibody sequence has homology to a “standard” Kabat numbered sequence.

[0108] The Kabat numbering system is generally used to refer 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 edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). When referring to residues within the constant region of the immunoglobulin heavy chain, the "EU numbering system" or "EU index" is generally used (e.g., the EU index reported by Kabat et al. above). "Kabat's EU index" refers to the residue numbering of human IgG1 EU antibodies.

[0109] The term "linear antibody" refers to antibodies described by Zapata et al. (1995 Protein Eng, 8(10):1057-1062). In short, these antibodies contain a pair of tandem Fd segments (VH-CH1-VH-CH1) that form a pair of antigen-binding regions together with a complementary light chain polypeptide. Linear antibodies can be bispecific or monospecific.

[0110] As used herein, the terms “binding,” “specifically binding to,” or “specific to” refer to measurable and reproducible interactions, such as binding between a target and an antibody, that determine the presence of the target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that binds to or specifically binds to a target (which may be an epitope) is an antibody that binds to this target more readily, and / or for a longer period of time, with higher affinity and binding activity than it binds to other targets. In one embodiment, the degree of binding of an antibody to an unrelated target is less than about 10% of the antibody’s binding to the target, as measured, for example, by radioimmunoassay (RIA). In a given embodiment, an antibody that specifically binds to a target has a dissociation constant (Kd value) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, or ≤0.1 nM. In a given embodiment, the antibody specifically binds to an epitope on a protein that is conserved between proteins of different species. In another embodiment, specific binding may include non-essential but exclusive binding.

[0111] II. PD-1 Axis Coupling Antagonist Provided herein is a method for treating a patient with cancer, comprising administering a therapeutically effective dose of a PD-1 axially coupled antagonist to the patient, wherein granzyme B is present in a blood sample obtained from the patient. + CD4 + T cells and FOXP3 + CD4 +This is a method in which the ratio to T cells is determined to be 1 or greater. Also provided herein is a method for determining whether a patient with cancer is likely to respond to treatment including a PD-1 axis-binding antagonist. For example, PD-1 axis-binding antagonists include PD-1 binding antagonists, PD-L1 binding antagonists, and PD-L2 binding antagonists. PD-1 (programmed death 1) is also referred to in the art as “programmed cell death 1,” “PDCD1,” “CD279,” and “SLEB2.” An exemplary human PD-1 is shown in UniProtKB / Swiss-Prot accession number Q15116. PD-L1 (programmed cell death ligand 1) is also referred to in the art as “programmed cell death 1 ligand 1,” “PDCD1LG1,” “CD274,” “B7-H,” and “PDL1.” An exemplary human PD-L1 is shown in UniProtKB / Swiss-Prot accession number Q9NZQ7.1. PD-L2 (programmed cell death ligand 2) is also referred to in the art as “programmed cell death ligand 12,” “PDCD1LG2,” “CD273,” “B7-DC,” “Btdc,” and “PDL2.” An exemplary human PD-L2 is shown in UniProtKB / Swiss-Prot accession number Q9BQ51. In some embodiments, PD-1, PD-L1, and PD-L2 are human PD-1, PD-L1, and PD-L2.

[0112] In some embodiments, a PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand-binding partner. In certain embodiments, the PD-1 ligand-binding partner is PD-L1 and / or PD-L2. In another embodiment, a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partner. In certain embodiments, the PD-L1 binding partner is PD-1 and / or B7-1. In yet another embodiment, a PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to its binding partner. In certain embodiments, the PD-L2 binding partner is PD-1. The antagonist may be an antibody, its antigen-binding fragment, an immunoadhesin, a fusion protein, or an oligopeptide.

[0113] In some embodiments, the PD-1 conjugated antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). In some embodiments, the anti-PD-1 antibody is selected from the group consisting of MDX1106 (nivolumab), MK-3475 (pembrolizumab), MEDI-0680 (AMP-514), PDR001, REGN2810, and BGB-108. In some embodiments, the PD-1 conjugated antagonist is an immunoadhesin (e.g., an immunoadhesin containing an extracellular or PD-1 binding moiety of PD-L1 or PD-L2 fused to a constant region (e.g., the Fc region of an immunoglobulin sequence)). In some embodiments, the PD-1 conjugated antagonist is AMP-224. In some embodiments, the PD-L1 conjugated antagonist is an anti-PD-L1 antibody. In some embodiments, the anti-PD-L1 antibody is selected from the group consisting of MPDL3280A, YW243.55.S70, MDX-1105, MEDI4736 (durvalumab), and MSB0010718C (avelumab). Antibody YW243.55.S70 is an anti-PD-L1 antibody described in International Publication No. 2010 / 077634. ​​MDX-1105, also known as BMS-936559, is an anti-PD-L1 antibody described in International Publication No. 2007 / 005874. MEDI4736 is an anti-PD-L1 monoclonal antibody described in International Publication No. 2011 / 066389 and U.S. Patent Application Publication No. 2013 / 034559. MDX-1106, also known as MDX-1106-04, ONO-4538, BMS-936558, or nivolumab, is an anti-PD-1 antibody described in International Publication No. 2006 / 121168. MK-3475, also known as lambrolizumab, is an anti-PD-1 antibody described in International Publication No. 2009 / 114335. AMP-224, also known as B7-DCIg, is a PD-L2-Fc fusion soluble receptor described in International Publication Nos. 2010 / 027827 and International Publication Nos. 2011 / 066342.

[0114] In some embodiments, the PD-1 axis-conjugated antagonist is an anti-PD-L1 antibody. 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 humanized antibody. In some embodiments, the anti-PD-L1 antibody is a human antibody.

[0115] Examples of anti-PD-L1 antibodies useful in the methods of this invention, as well as methods for producing them, are described in PCT patent applications International Publication Nos. 2010 / 077634, 2007 / 005874, 2011 / 066389, and U.S. Patent Application Publication No. 2013 / 034559, which are incorporated herein by reference with due attribution. Anti-PD-L1 antibodies useful in this invention (including compositions containing such antibodies) can be used in combination with taxanes to treat cancer.

[0116] [Anti-PD-1 antibody] In some embodiments, the anti-PD-1 antibody is MDX-1106. Other names for "MDX-1106" include MDX-1106-04, ONO-4538, BMS-936558, or nivolumab. In some embodiments, the anti-PD-1 antibody is nivolumab (CAS registry number: 946414-94-4). Further embodiments provide an isolated anti-PD-1 antibody comprising a heavy chain variable region containing the heavy chain variable region amino acid sequence of SEQ ID NO: 1 and / or a light chain variable region containing the light chain variable region amino acid sequence of SEQ ID NO: 2. Further embodiments also provide an isolated anti-PD-1 antibody comprising heavy chain and / or light chain sequences, where, (a) Heavy chain sequence: QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVS SASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPA PEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(Sequence ID:1) It has at least 85%, 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%, at least 99%, or 100% sequence identity, and (b) Light chain sequence: EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID: 2) It has at least 85%, 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%, at least 99%, or 100% sequence identity.

[0117] [Anti-PD-L1 antibody] In some embodiments, the antibody in the formulation contains at least one tryptophan (e.g., at least two, at least three, or at least four) in the heavy chain and / or light chain sequences. In some embodiments, the amino acid tryptophan is located in the HVR region, framework region, and / or constant region of the antibody. In some embodiments, the antibody contains two or three tryptophan residues in the HVR region. In some embodiments, the antibody in the formulation is an anti-PD-L1 antibody. PD-L1 (programmed death 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-PD-L1 antibodies that can be used in the methods herein are described in International Publication No. 2010 / 077634A1 and U.S. Patent No. 8217149 of the PCT Patent Application, which are incorporated herein by attribution.

[0118] In some embodiments, the anti-PD-L1 antibody can inhibit the binding between PD-L1 and PD-1 and / or 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 humanized antibody. In some embodiments, the anti-PD-L1 antibody is a human antibody.

[0119] The anti-PD-L1 antibodies described in International Publication No. 2010 / 077634A1 and U.S. Patent No. 8217149 may be used in the methods described herein. In some embodiments, the anti-PD-L1 antibody comprises the heavy chain variable region sequence of SEQ ID NO: 3 and / or the light chain variable region sequence of SEQ ID NO: 4. Further embodiments provide an isolated anti-PD-L1 antibody comprising the heavy chain variable region and / or the light chain variable region sequence, wherein, (a) Heavy chain sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSA(Sequence ID:3) It has at least 85%, 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%, at least 99%, or 100% sequence identity, and (b) Light chain sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR(Sequence ID: 4) It has at least 85%, 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%, at least 99%, or 100% sequence identity.

[0120] In one embodiment, the anti-PD-L1 antibody comprises a heavy chain variable region polypeptide containing HVR-H1, HVR-H2, and HVR-H3 sequences, where, (a) The HVR-H1 sequence is GFTFSX1SWIH (sequence number: 5); (b) The HVR-H2 sequence is AWIX2PYGGSX3YYADSVKG (sequence number: 6); (c) The HVR-H3 sequence is RHWPGGFDY (sequence number: 7); Furthermore, here X1 is D or G; X2 is S or L; and X3 is T or S. In a particular embodiment, X1 is D; X2 is S, and X3 is T.

[0121] In another embodiment, the polypeptide further comprises a variable-region heavy chain framework sequence juxtaposed between HVRs according to the formula: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4). In yet another embodiment, the framework sequence is derived from a human consensus framework sequence. In yet another embodiment, the framework sequence is a VH subgroup III consensus framework. In yet another embodiment, at least one of the framework sequences is as follows: HC-FR1 is EVQLVESGGGLVQPGGSLRLSCAAS (Sequence ID: 8). HC-FR2 is WVRQAPGKGLEWV (Sequence ID: 9). HC-FR3 is RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (Sequence ID: 10). HC-FR4 is WGQGTLVTVSA (Sequence ID: 11).

[0122] In a further embodiment, the heavy chain polypeptide is further combined with a variable region light chain containing HVR-L1, HVR-L2, and HVR-L3, where, (a) The HVR-L1 sequence is RASQX4X5X6TX7X8A (sequence number: 12); (b) The HVR-L2 array is SASX9LX 10 S, (sequence number: 13); (c) The HVR-L3 sequence is QQX 11 X 12 X 13 X 14 PX 15 T is (sequence number: 14); Here, X4 is D or V; X5 is V or I; X6 is S or N; X7 is A or F; X8 is V or L; X9 is F or T; X 10 is Y or A; X 11 is Y, G, F, or S; X 12 is L, Y, F, or W; X 13 is Y, N, A, T, G, F or I; X 14is H, V, P, T or I; X 15 is A, W, R, P, or T. In a further embodiment, X4 is D; X5 is V; X6 is S; X7 is A; X8 is V; X9 is F; X 10 is Y; X 11 is Y; X 12 is L; X 13 is Y; X 14 H is X 15 It is A.

[0123] In a further embodiment, the light chain further comprises variable-region light chain framework sequences juxtaposed between HVRs according to the formula: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In a further embodiment, the framework sequences are derived from human consensus framework sequences. In a further embodiment, the framework sequences are VL Kappa I consensus frameworks. In a further embodiment, at least one of the framework sequences is as follows: LC-FR1 is DIQMTQSPSSLSASVGDRVTITC (Sequence ID: 15) LC-FR2 is WYQQKPGKAPKLLIY (Sequence ID: 16), LC-FR3 is GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (Sequence ID: 17), LC-FR4 is FGQGTKVEIKR (Sequence ID: 18).

[0124] In another embodiment, an isolated anti-PD-L1 antibody or antigen-binding fragment comprising heavy chain and light chain variable region sequences is provided, where, (a) The heavy chain includes HVR-H1, HVR-H2 and HVR-H3, and further, (i) The HVR-H1 sequence is GFTFSX1SWIH; (Sequence ID: 5), (ii) The HVR-H2 sequence is AWIX2PYGGSX3YYADSVKG (sequence number: 6), (iii) The HVR-H3 sequence is RHWPGGFDY (sequence number: 7), and (b) The light chain includes HVR-L1, HVR-L2 and HVR-L3, and further, (i) The HVR-L1 sequence is RASQX4X5X6TX7X8A (sequence number: 12), (ii) The HVR-L2 array is SASX9LX 10 S (Sequence ID 13), and (iii) The HVR-L3 sequence is QQX 11 X 12 X 13 X 14 PX 15 T (sequence number: 14), Here, X1 is D or G; X2 is S or L; X3 is T or S; X4 is D or V; X5 is V or I; X6 is S or N; X7 is A or F; X8 is V or L; X9 is F or T; X 10 is Y or A; X 11 is Y, G, F, or S; X 12 is L, Y, F, or W; X 13 is Y, N, A, T, G, F or I; X 14 is H, V, P, T or I; X 15 is A, W, R, P, or T. In a particular embodiment, X1 is D; X2 is S, and X3 is T. In another embodiment, X4 is D; X5 is V; X6 is S; X7 is A; X8 is V; X9 is F; X 10 is Y; X 11 is Y; X 12 is L; X 13 is Y; X 14 H is X 15 is A. In another embodiment, X1 is D; X2 is S, X3 is T, X4 is D; X5 is V; X6 is S; X7 is A; X8 is V; X9 is F; X 10 is Y; X 11 is Y; X 12 is L; X 13 is Y; X 14 H is H, and X 15It is A.

[0125] In further embodiments, the heavy chain variable region includes one or more framework sequences juxtaposed between HVRs, such as (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable region includes one or more framework sequences juxtaposed between HVRs, such as (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In further embodiments, the framework sequences are derived from human consensus framework sequences. In further embodiments, the heavy chain framework sequences are derived from Kabat subgroup I, II, or III sequences. In further embodiments, the heavy chain framework sequences are VH subgroup III consensus frameworks. In further embodiments, one or more heavy chain framework sequences are defined as sequence numbers: 8, 9, 10, and 11. In further embodiments, the light chain framework sequence is derived from a Kabat kappa I, II, II, or IV subgroup sequence. In further embodiments, the light chain framework sequence is a VL kappa I consensus framework. In further embodiments, one or more light chain framework sequences are defined as sequence numbers 15, 16, 17, and 18.

[0126] In further specific embodiments, the antibody further comprises a human or mouse constant region. In further specific embodiments, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, and IgG4. In further specific embodiments, the human constant region is IgG1. In further specific embodiments, the mouse constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, and IgG3. In further specific embodiments, the mouse constant region is IgG2A. In further specific embodiments, the antibody has reduced or minimal effector function. In further specific embodiments, minimal effector function is due to an "effector-deficient Fc mutation" or non-glycosylation. In further embodiments, the effector-deficient Fc mutation is an N297A or D265A / N297A substitution in the constant region.

[0127] In another embodiment, an anti-PD-L1 antibody comprising heavy chain and light chain variable region sequences is provided, where, (a) The heavy chain further comprises HVR-H1, HVR-H2, and HVR-H3 sequences having at least 85% sequence identity to GFTFSDSWIH (SEQ ID NO: 19), AWISPYGGSTYYADSVKG (SEQ ID NO: 20), and RHWPGGFDY (SEQ ID NO: 21), or (b) The light chain further comprises HVR-L1, HVR-L2, and HVR-L3 sequences having at least 85% sequence identity with RASQDVSTAVA (SEQ ID NO: 22), SASFLYS (SEQ ID NO: 23), and QQYLYHPAT (SEQ ID NO: 24), respectively. In certain embodiments, sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0128] In another embodiment, the heavy chain variable region includes one or more framework sequences juxtaposed between HVRs, such as (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable region includes one or more framework sequences juxtaposed between HVRs, such as (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet another embodiment, the framework sequences are derived from human consensus framework sequences. In yet another embodiment, the heavy chain framework sequences are derived from Kabat subgroup I, II, or III sequences. In yet another embodiment, the heavy chain framework sequences are VH subgroup III consensus frameworks. In yet another embodiment, one or more heavy chain framework sequences are defined as sequence numbers: 8, 9, 10, and 11. In further embodiments, the light chain framework sequence is derived from a Kabat kappa I, II, II, or IV subgroup sequence. In further embodiments, the light chain framework sequence is a VL kappa I consensus framework. In further embodiments, one or more light chain framework sequences are defined as sequence numbers 15, 16, 17, and 18.

[0129] In further specific embodiments, the antibody further comprises a human or mouse constant region. In further specific embodiments, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, and IgG4. In further specific embodiments, the human constant region is IgG1. In further specific embodiments, the mouse constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, and IgG3. In further specific embodiments, the mouse constant region is IgG2A. In further specific embodiments, the antibody has reduced or minimal effector function. In further specific embodiments, minimal effector function is due to an "effector-deficient Fc mutation" or non-glycosylation. In further embodiments, the effector-deficient Fc mutation is an N297A or D265A / N297A substitution in the constant region.

[0130] In another further embodiment, an isolated anti-PD-L1 antibody comprising heavy chain and light chain variable region sequences is provided, where, (a) Heavy chain sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSS(Sequence ID: 25) It has at least 85% sequence identity with respect to, and / or (b) Light chain sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR(Sequence ID: 4) It has at least 85% sequence identity.

[0131] In certain embodiments, sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In other embodiments, the heavy chain variable region includes one or more framework sequences juxtaposed between HVRs, such as (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable region includes one or more framework sequences juxtaposed between HVRs, such as (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet another embodiment, the framework sequences are derived from human consensus framework sequences. In further embodiments, the heavy chain framework sequence is derived from a Kabat subgroup I, II, or III sequence. In even further embodiments, the heavy chain framework sequence is a VH subgroup III consensus framework. In even further embodiments, one or more heavy chain framework sequences are defined as sequence numbers 8, 9, 10 and WGQGTLVTVSS (sequence number 27).

[0132] In further embodiments, the light chain framework sequence is derived from a Kabat kappa I, II, II, or IV subgroup sequence. In further embodiments, the light chain framework sequence is a VL kappa I consensus framework. In further embodiments, one or more light chain framework sequences are defined as sequence numbers 15, 16, 17, and 18.

[0133] In further specific embodiments, the antibody further comprises a human or mouse constant region. In further specific embodiments, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, and IgG4. In further specific embodiments, the human constant region is IgG1. In further specific embodiments, the mouse constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, and IgG3. In further specific embodiments, the mouse constant region is IgG2A. In further specific embodiments, the antibody has reduced or minimal effector function. In further specific embodiments, minimal effector function is due to production in prokaryotic cells. In further specific embodiments, minimal effector function is due to an "effector-deficient Fc mutation" or non-glycosylation. In further embodiments, the effector-deficient Fc mutation is an N297A or D265A / N297A substitution in the constant region.

[0134] In a further embodiment, the heavy chain variable region includes one or more framework sequences juxtaposed between HVRs, such as (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable region includes one or more framework sequences juxtaposed between HVRs, such as (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In a further embodiment, the framework sequences are derived from human consensus framework sequences. In a further embodiment, the heavy chain framework sequences are derived from Kabat subgroup I, II, or III sequences. In a further embodiment, the heavy chain framework sequences are VH subgroup III consensus frameworks. In a further embodiment, one or more heavy chain framework sequences are as follows: HC-FR1 EVQLVESGGGLVQPGGSLRLSCAASGFTFS (Sequence ID: 29) HC-FR2 WVRQAPGKGLEWVA (Sequence ID: 30) HC-FR3 RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (Sequence ID: 10) HC-FR4 WGQGTLVTVSS (Sequence ID: 27).

[0135] In further embodiments, the light chain framework sequence is derived from a Kabat kappa I, II, II, or IV subgroup sequence. In further embodiments, the light chain framework sequence is a VL kappa I consensus framework. In further embodiments, one or more light chain framework sequences are as follows: LC-FR1 DIQMTQSPSSLSASVGDRVTITC (Sequence ID: 15) LC-FR2 WYQQKPGKAPKLLIY (Sequence number: 16) LC-FR3 GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (Sequence ID: 17) LC-FR4 FGQGTKVEIK (Sequence ID: 28).

[0136] In further specific embodiments, the antibody further comprises a human or mouse constant region. In further specific embodiments, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, and IgG4. In further specific embodiments, the human constant region is IgG1. In further specific embodiments, the mouse constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, and IgG3. In further specific embodiments, the mouse constant region is IgG2A. In further specific embodiments, the antibody has reduced or minimal effector function. In further specific embodiments, minimal effector function is due to an "effector-deficient Fc mutation" or non-glycosylation. In further embodiments, the effector-deficient Fc mutation is an N297A or D265A / N297A substitution in the constant region.

[0137] In another embodiment, an anti-PD-L1 antibody comprising heavy chain and light chain variable region sequences is provided, where, (c) The heavy chain further comprises HVR-H1, HVR-H2, and HVR-H3 sequences having at least 85% sequence identity to GFTFSDSWIH (SEQ ID NO: 19), AWISPYGGSTYYADSVKG (SEQ ID NO: 20), and RHWPGGFDY (SEQ ID NO: 21), and / or (d) The light chain further comprises HVR-L1, HVR-L2, and HVR-L3 sequences having at least 85% sequence identity with RASQDVSTAVA (SEQ ID NO: 22), SASFLYS (SEQ ID NO: 23), and QQYLYHPAT (SEQ ID NO: 24), respectively. In certain embodiments, sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0138] In another embodiment, the heavy chain variable region includes one or more framework sequences juxtaposed between HVRs, such as (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable region includes one or more framework sequences juxtaposed between HVRs, such as (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet another embodiment, the framework sequences are derived from human consensus framework sequences. In yet another embodiment, the heavy chain framework sequences are derived from Kabat subgroup I, II, or III sequences. In yet another embodiment, the heavy chain framework sequences are VH subgroup III consensus frameworks. In a further embodiment, one or more heavy chain framework sequences are defined as sequence numbers 8, 9, 10 and WGQGTLVTVSSASTK (sequence number 31).

[0139] In further embodiments, the light chain framework sequence is derived from a Kabat kappa I, II, II, or IV subgroup sequence. In further embodiments, the light chain framework sequence is a VL kappa I consensus framework. In further embodiments, one or more light chain framework sequences are designated as SEQ ID NOs: 15, 16, 17, and 18. In even more specific embodiments, the antibody further comprises a human or mouse constant region. In even more specific embodiments, the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, and IgG4. In even more specific embodiments, the human constant region is IgG1. In even more specific embodiments, the mouse constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, and IgG3. In even more specific embodiments, the mouse constant region is IgG2A. In even more specific embodiments, the antibody has reduced or minimal effector function. In even more specific embodiments, minimal effector function is due to an "effector-deficient Fc mutation" or non-glycosylation. In further embodiments, the effector-deficient Fc mutation is an N297A or D265A / N297A substitution in the steady-state region.

[0140] In further embodiments, isolated anti-PD-L1 antibodies comprising heavy chain and light chain variable region sequences are provided, where, (a) Heavy chain sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTK(Sequence ID: 26) It has at least 85% sequence identity, or (b) Light chain sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKR(Sequence ID: 4) It has at least 85% sequence identity.

[0141] In some embodiments, an isolated anti-PD-L1 antibody is provided comprising heavy chain and light chain variable region sequences, wherein the light chain variable region 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%, at least 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 4. In some embodiments, an isolated anti-PD-L1 antibody is provided comprising heavy chain and light chain variable region sequences, wherein the heavy chain variable region 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%, at least 99%, or 100% sequence identity with respect to the amino acid sequence of SEQ ID NO: 26. In some embodiments, an isolated anti-PD-L1 antibody is provided comprising heavy chain and light chain variable region sequences, wherein the light chain variable region 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%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 26, and the heavy chain variable region 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%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 26. In some embodiments, amino acid residues 1, 2, 3, 4, or 5 at the N-terminus of the heavy chain and / or light chain may be deleted, substituted, or modified.

[0142] In further embodiments, isolated anti-PD-L1 antibodies comprising heavy chain and light chain sequences are provided, where, (a) Heavy chain sequence: EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP PCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (Sequence code: 32) It has at least 85% sequence identity with respect to, and / or (b) Light chain sequence: DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID: 33) It has at least 85% sequence identity.

[0143] In some embodiments, an isolated anti-PD-L1 antibody is provided, comprising a heavy chain and a light chain sequence, 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: 33. In some embodiments, an isolated anti-PD-L1 antibody is provided, comprising a heavy chain and a light chain sequence, 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: 32. In some embodiments, an isolated anti-PD-L1 antibody is provided comprising a heavy chain and a light chain sequence, 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: 33, 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: 32.

[0144] In some embodiments, the isolated anti-PD-L1 antibody is non-glycosylated. Antibody glycosylation is typically either N-linked or O-linked. N-linking refers to the binding of a carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine (where X is any amino acid other than proline) are recognition sequences for the enzymatic binding of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in the polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the binding of one of the sugars, N-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used. Removal of glycosylation sites from antibodies is easily achieved by modifying the amino acid sequence so that one of the above tripeptide sequences (the N-linked glycosylation site) is removed. Modifications can be made by substituting an asparagine, serine, or threonine residue within the glycosylation site with another amino acid residue (e.g., glycine, alanine, or a conservative substitution).

[0145] In any of the embodiments described herein, the isolated anti-PD-L1 antibody can bind to human PD-L1, for example, human PD-L1 represented by UniProtKB / Swiss-Prot accession number Q9NZQ7.1 or a variant thereof.

[0146] Further embodiments provide isolated nucleic acids encoding any of the antibodies described herein. In some embodiments, the nucleic acid further comprises a vector suitable for the expression of the nucleic acid encoding any of the aforementioned anti-PD-L1 antibodies. In even more specific embodiments, the vector is located in a host cell suitable for nucleic acid expression. In even more specific embodiments, the host cell is a eukaryotic or prokaryotic cell. In even more specific embodiments, the eukaryotic cell is a mammalian cell, such as a Chinese hamster ovary (CHO) cell.

[0147] Antibodies or their antigen-binding fragments can be produced using methods known in the art, for example, by culturing host cells containing nucleic acids encoding either the aforementioned anti-PD-L1 antibody or antigen-binding fragment in a form suitable for expression under conditions suitable for producing such antibodies or fragments, and then recovering the antibodies or fragments.

[0148] III. Antibody preparation The antibodies described herein were prepared using techniques available in the art for antibody production, and exemplary methods thereof are described in more detail in the following sections.

[0149] The antibody is against the target antigen (e.g., PD1 (e.g., human PD-1), PD-L1 (e.g., human PD-L1), PD-L2 (e.g., human PD-L2), etc.). Preferably, administration of the antibody to a mammal suffering from the disorder may provide a therapeutic benefit to that mammal.

[0150] In a given embodiment, the antibody provided herein has a concentration of ≤1 μM, ≤150 nM, ≤100 nM, ≤50 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (for example, 10 -8 M or less, for example, 10 -8 M to 10 -13 M, for example, 10 -9 M to 10 -13 It has a dissociation constant (Kd) of M.

[0151] In one embodiment, Kd is measured by a radiolabeled antigen-binding assay (RIA) performed using the Fab type of the antibody of interest and its antigen, as described by the following assay: the lowest concentration of the unlabeled antigen in the presence of the titration series. 125I) The solution binding affinity of Fab to the antigen is measured by equilibrating Fab with a labeled antigen and then capturing the bound antigen with an anti-Fab antibody coated plate (see, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999)). To establish assay conditions, a MICROTITER® multiwell plate (Thermo Scientific) is coated overnight with 5 μg / ml of capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and then blocked with 2% (w / v) bovine serum albumin in PBS for 2-5 hours at room temperature (approximately 23°C). In a non-adsorbent plate (Nunc#269620), 100 pM or 26 pM [ 125 Mix the antigen of [I] with the serially diluted target Fab. Then incubate the target Fab overnight, but incubation may be continued for a longer time (e.g., about 65 hours) to ensure that equilibrium is reached. Then transfer the mixture to a capture plate and incubate at room temperature (e.g., 1 hour). Then remove the solution and wash the plate eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate is dry, add 150 μl / well of flashing material (MICROSCINT-20) TM Add Packard and set the plate to TOPCOUNT TM The gamma assay (Packard) is performed for 10 minutes. The Fab concentration that yields less than 20% of the maximum binding is selected for use in competitive binding assays.

[0152] According to another embodiment, Kd is measured using a surface plasmon resonance assay with a fixed antigen CM5 chip at 25°C using BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) at approximately 10 response units (RUs). Briefly, a carboxymethylated dextran biosensor chip (CM5, BIACORE, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / ml (approximately 0.2 μM) in 10 mM sodium acetate (pH 4.8) and then injected at a flow rate of 5 μl / min so that the response units (RUs) of the binding protein are approximately 10. After antigen injection, 1 M ethanolamine is injected to block unresponsive groups. For dynamic measurement, Fab was subjected to two-fold serial dilutions (0.78 nM to 500 nM) at a flow rate of approximately 25 μl / min at 25°C in 0.05% polysorbate 20 (TWEEN-20). TM The sample is injected into PBS containing a surfactant (PBST). The association rate (k) is calculated using a simple one-to-one Langmuir coupling model (BIACORE® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. on ) and dissociation rate (k off ) is calculated. The equilibrium dissociation constant (Kd) is k off / k on Calculate as a ratio. For example, see Chen et al., J. Mol. Biol. 293:865-881 (1999). If the association rate by the surface plasmon resonance assay described above is 10 6 M -1 s -1 If it exceeds this, the association rate is measured using a spectrometer, for example, a spectrophotometer with stop flow (Aviv Instruments) or an 8000 series SLM-AMINCO with a stirring cuvette. TMThis can be measured using fluorescence quenching techniques, which involve measuring the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, band-pass = 16 nm) of a 20 nM anti-antigen antibody (Fab type) in PBS (pH 7.2) at 25°C in the presence of gradually increasing antigen concentrations.

[0153] (i) Antigen preparation Soluble antigens or fragments thereof, which may be conjugated to other molecules in some cases, can be used as immunogens for antibody production. In the case of transmembrane molecules such as receptors, these fragments (e.g., the extracellular domain of the receptor) can be used as immunogens. Alternatively, cells expressing transmembrane molecules can be used as immunogens. Such cells may be derived from natural sources (e.g., cancer cell lines) or may be cells transformed by recombinant technology to express transmembrane molecules. Other antigens and their forms useful for antibody preparation will be apparent to those skilled in the art.

[0154] (ii) Prescribed antibody-based method Polyclonal antibodies are preferably produced in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and adjuvant. The relevant antigen is converted into a protein that is immunogenic in the immunized species, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soy trypsin inhibitor, with a bifunctional or derivatizing agent, such as maleimide benzoyl sulfosuccinimide (conjugated by a cysteine ​​residue), N-hydroxysuccinimide (conjugated by a lysine residue), glutaraldehyde, succinic anhydride, SOCl2, or R 1 N=C=NR(where R and R 1 It may be useful to conjugate it to a different alkyl group.

[0155] Animals are immunized to an antigen, immunogenic conjugate, or derivative by intradermal injection of a solution containing, for example, 100 μg or 5 μg of protein or conjugate (for rabbits or mice, respectively) in combination with 3 volumes of complete Freund's adjuvant at multiple sites. After one month, animals are additionally immunized by subcutaneous injection at multiple sites with a peptide or conjugate in an amount of 1 / 5 to 1 / 10 of the original amount of complete Freund's adjuvant. After 7 to 14 days, animals are induced to bleed, and serum is assayed for antibody titer. Animals are further immunized until the titer reaches a plateau. Preferably, animals are additionally immunized with a conjugate of the same antigen, but with a different protein and / or a different crosslinking agent. The conjugate can also be prepared in recombinant cell culture as a protein fusion. A coagulant such as alum may also be used to enhance the immune response.

[0156] The monoclonal antibodies of the present invention were first described by Kohler et al., Nature, 256:495 (1975), and can be further produced using hybridoma methods described, for example, Hongo et al., Hybridoma, 14 (3): 253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd edition 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, NY, 1981), and Ni, Xiandai Mianyixue, 26(4):265-268 (2006). Further methods include, for example, the method described in U.S. Patent No. 7,189,826, relating to the production of monoclonal human native IgM antibodies from hybridoma cell lines. Human hybridoma technology (trioma technology) is described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0157] For various other hybridoma technologies, see, for example, U.S. Patent Application Publications 2006 / 258841; 2006 / 183887 (Full Human Antibodies), 2006 / 059575; 2005 / 287149; 2005 / 100546; and 2005 / 026229; and U.S. Patents 7,078492 and 7153507. An exemplary protocol for the production of monoclonal antibodies using the hybridoma method is described as follows: In one embodiment, a mouse or other suitable host animal such as a hamster is immunized to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization. Antibodies are produced in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the polypeptide or fragment thereof of the present invention and an adjuvant, such as monophosphoryl lipid A (MPL) / diclinomycolate trehalose (TDM) (Ribi Immunochem. Research, Inc., Hamilton, MT). The polypeptide (e.g., antigen) or fragment thereof of the present invention can be prepared using methods well known in the art, such as some recombinant methods further described herein. Serum from immunized animals is assayed for anti-antigen antibodies, and additional immunizations are performed if necessary. Lymphocytes from animals producing anti-antigen antibodies are isolated. Alternatively, lymphocytes may be immunized in vitro.

[0158] Next, lymphocytes are fused with myeloma cells using a suitable fusion agent such as polyethylene glycol to form hybridoma cells. See, for example, Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986). Myeloma cells that fuse efficiently, support stable high-level antibody production by selected antibody-producing cells, and are sensitive to media such as HAT medium may be used. Exemplary myeloma cell lines include, but are not limited to, mouse myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from Salk Institute Cell Distribution Center (San Diego, Calif., USA) and SP-2 or X63-Ag8-653 cells available from American Type Culture Collection (Rockville, Md., USA). Human myeloma and mouse-human heterozygous myeloma cell lines for the production of human monoclonal antibodies have also been described (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0159] The hybridoma cells thus prepared are seeded and cultured in a suitable medium, for example, a medium containing one or more substances that inhibit the growth or survival of unfused parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine-guanine phosphoribosyl transferase (HGPRT or HPRT), the medium for the hybridoma typically contains hypoxanthine, aminopterin, and thymidine (HAT medium), and these substances prevent the growth of HGPRT-deficient cells. Preferably, for example, as described in Even et al., Trends in Biotechnology, 24(3), 105-108 (2006), a serum-free hybridoma cell culture method is used to reduce the use of animal-derived sera such as fetal bovine serum.

[0160] Oligopeptides as tools for improving the productivity of hybridoma cell culture are described in Franek, Trends in Monoclonal Antibody Research, 111-122 (2005). Specifically, standard media are enriched with predetermined amino acids (alanine, serine, asparagine, proline) or fractions of protein hydrolysates, and apoptosis can be significantly inhibited by synthetic oligopeptides consisting of 3 to 6 amino acid residues. The peptides are present at millimolar or higher concentrations.

[0161] The medium in which the hybridoma cells grow can be assayed for the production of monoclonal antibodies that bind to the antibody of the present invention. The binding specificity of the monoclonal antibodies produced by the hybridoma cells can be determined by immunoprecipitation or by in vitro binding assays such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of the monoclonal antibodies can be determined, for example, by Scatchard analysis. See, for example, Munson et al., Anal. Biochem., 107:220 (1980).

[0162] After hybridoma cells producing antibodies of desired specificity, affinity, and / or activity are identified, clones can be subcloned by limiting the dilution procedure and grown by standard methods. See, for example, Goding above. Suitable media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells may be grown in vivo as ascites tumors in animals. Monoclonal antibodies secreted by subclones are appropriately isolated from the medium, ascites, or serum by common immunoglobulin purification procedures, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. One procedure for isolating proteins from hybridoma cells is described in U.S. Patent Application Publication 2005 / 176122 and U.S. Patent No. 6919436. The method includes using a small amount of salt, such as an excipient salt, in the binding step and preferably a small amount of organic solvent in the elution step.

[0163] (iii) Library-derived antibodies The antibodies of the present invention can be isolated by screening a combinatorial library of antibodies having a desired activity or group of activity. For example, various methods are known in the art for creating phage display libraries and screening such libraries of antibodies having a desired binding property. Further methods are outlined, for example, in Hoogenboom et al., in Methods in Molecular Biology 178:1-37 (edited by O'Brien et al., Human Press, Totowa, NJ, 2001), and further, for example, McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (edited by Lo, Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): This is described in 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).

[0164] In a prescribed phage display method, the VH and VL gene repertoires can be separately cloned by polymerase chain reaction (PCR), randomly recombined in a phage library, and then screened for antigen-binding phages, as described by Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). The phages typically present antibody fragments as either single-chain Fv (scFv) or Fab fragments. Libraries from immunized sources provide high-affinity antibodies against immunogens without the need to construct hybridomas. Alternatively, as described by Griffiths et al., EMBO J, 12: 725-734 (1993), a naive repertoire can be cloned (e.g., from humans) to provide a single-origin antibody against a wide range of non-self and self-antigens without any immunosensitization. Finally, naive libraries can also be synthetically constructed by cloning an unrearranged V gene segment derived from stem cells, encoding a highly variable CDR3 region using PCR primers containing random sequences, and achieving in vitro rearrangement as described by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373, and U.S. Patent Application Publications 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0165] Antibodies or antibody fragments isolated from a human antibody library are considered human antibodies or human antibody fragments in this context.

[0166] (iv) Chimeras, humanizations and human antibodies In a given embodiment, the antibody provided herein is a chimeric antibody. A given chimeric antibody is described, for example, in U.S. Patent No. 4,816,567 and in Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In one example, the chimeric antibody includes a non-human variable region (e.g., a variable region derived from a non-human primate such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region. In a further example, the chimeric antibody is a “class-switched” antibody in which the class or subclass is changed from that of the parent antibody. The chimeric antibody includes its antigen-binding fragment.

[0167] In certain embodiments, the chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized while retaining the specificity and affinity of the parent non-human antibody in order to reduce its immunogenicity against humans. Generally, a humanized antibody contains one or more variable domains in which the HVR, e.g., CDR (or a portion thereof), is derived from the non-human antibody and the FR (or a portion thereof) is derived from the human antibody sequence. The humanized antibody may also contain at least a portion of the human constant region. In some embodiments, some FR residues of the humanized antibody are replaced with corresponding residues derived from the non-human antibody (e.g., the antibody from which the HVR residues are derived) to restore or improve antibody specificity or affinity, for example.

[0168] Humanized antibodies and their production methods are outlined, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further, for example, Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Patents 5821337, 7527791, 6982321, and 7087409; Kashmiri et al., Methods 36:25-34 (2005) (SDR(a-CDR) grafting is described); Padlan, Mol. Immunol. 28:489-498 (1991) ("Resurfacing" is described); Dall'Acqua et al., Methods This is described in 36:43-60 (2005) ("FR shuffling" is described); and in Osbourn et al., Methods 36:61-68 (2005); and Klimka et al., Br. J. Cancer, 83:252-260 (2000) ("Induction selection" method for FR shuffling is described).

[0169] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best fit" method (see, e.g., Sims et al., J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of specific subgroups of light chain or heavy chain variable regions (see, e.g., Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al., J. Immunol., 151:2623 (1993)); human maturation (somatic mutation) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening of FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. This includes (see Biol. Chem. 271:22611-22618 (1996)).

[0170] In a given embodiment, the antibody provided herein is a human antibody. Human antibodies can be produced using various techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20: 450-459 (2008).

[0171] Human antibodies can be prepared by administering immunogens to transgenic animals modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of human immunoglobulin loci that either replace endogenous immunoglobulin loci or are extrachromosomal or randomly incorporated into the animal's chromosomes. In such transgenic mice, endogenous immunoglobulin loci are generally inactivated. For an overview of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Also, see, for example, XENOMOUSE. TM See U.S. Patent Nos. 6,075,181 and 6,150,584 describing the technology, U.S. Patent No. 5,770,429 describing the HUMAB® technology, U.S. Patent No. 7,041,870 describing the KM MOUSE® technology, and U.S. Patent Application Publication 2007 / 0061900 describing the VELOCIMOUSE® technology. Human variable regions derived from intact antibodies produced by such animals can be further modified, for example, by combining them with different human constant regions.

[0172] Human antibodies can also be produced by hybridoma-based methods. Human myeloma and mouse-human xenomyeloma cell lines for the production of human monoclonal antibodies have been described. (See, for example, Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991)). Human antibodies produced by human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Further methods include, for example, those described in U.S. Patent No. 7189826 (describes the production of monoclonal human IgM antibodies derived from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describes human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0173] Human antibodies can also be produced by isolating selected Fv clone variable domain sequences from human-derived phage display libraries. These variable domain sequences may then be combined with desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0174] (v)Antibody fragment Antibody fragments can be produced by conventional methods such as enzymatic digestion or by recombinant technology. Under certain circumstances, using antibody fragments is advantageous over using whole antibodies. Smaller fragments allow for faster clearance, which can lead to improved access to solid tumors. For an overview of specific antibody fragments, see Hudson et al. (2003) Nat. Med. 9:129-134.

[0175] Various techniques have been developed to generate antibody fragments. Traditionally, these fragments were induced by the proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be generated directly by recombinant host cells. Fab, Fv, and ScFv antibody fragments can all be expressed in and secreted from E. coli, thus facilitating the large-scale production of these fragments. Antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab'-SH fragments can be recovered directly from E. coli and chemically bound to form F(ab')2 fragments (Carter et al., Bio / Technology 10:163-167 (1992)). Alternatively, the F(ab')2 fragment can be isolated directly from recombinant host cell cultures. Fab and F(ab')2 fragments with increased in vivo half-life due to the presence of a salvage receptor-binding epitope residue are described in U.S. Patent No. 5,869,046. Other techniques for generating antibody fragments will be apparent to those skilled in the art. In certain embodiments, the antibody is a single-stranded Fv fragment (scFv). See, for example, International Publication No. 93 / 16185; U.S. Patent No. 5,571,894; and U.S. Patent No. 5,587,458. Fv and scFv are the only species with intact binding sites lacking a constant region; therefore, they may be suitable for nonspecific binding reduction during in vivo use. scFv fusion proteins can be constructed to result in the fusion of an effector protein to either the amino or carboxyl terminus of the scFv. See Antibody Engineering, edited by Borrebaeck, cited above. The antibody fragment may also be a “linear antibody,” as described, for example, in U.S. Patent No. 5641870. Such a linear antibody may be monospecific or bispecific.

[0176] (vi) Multispecific antibodies Multispecific antibodies have binding specificity to at least two different epitopes, which are typically derived from different antigens. While such molecules usually bind to only two different epitopes (i.e., bispecific antibodies, BsAb), the term, when used here, also includes antibodies with further specificity, such as tripspecific antibodies. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies).

[0177] Methods for producing bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, where the two chains have different specificities (see, e.g., Millstein et al., Nature, 305:537-539 (1983)). Because the heavy and light chains of the immunoglobulins are randomly combined, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, usually by affinity chromatography, is quite cumbersome and results in low product yields. Similar procedures are disclosed in International Publication No. 93 / 08829 and Traunecker et al., EMBO J., 10:3655-3659 (1991).

[0178] One method known in the art for producing bispecific antibodies is the “knob-into-hole” or “protuberance-into-cavity” method (see, for example, U.S. Patent No. 5,731,168). In this method, two immunoglobulin polypeptides (e.g., heavy-chain polypeptides) each contain an interface. The two immunoglobulin polypeptides associate by the interaction of the interface of one immunoglobulin polypeptide with the corresponding interface of the other immunoglobulin polypeptide. These interfaces can be modified so that a “knob” or “protrusion” (these terms may be used interchangeably here) located at the interface of one immunoglobulin polypeptide corresponds to a “hole” or “cavity” (these terms may be used interchangeably here) located at the interface of the other immunoglobulin polypeptide. In some embodiments, the hole is the same size as or similar to the knob and is appropriately positioned so that when the two interfaces interact, the knob of one interface can be placed in the corresponding hole of the other interface. While we do not wish to be constrained by theory, this suggests that it stabilizes heteromultimers and promotes the formation of heteromultimers more readily than other species, such as homomultimers. In some embodiments, this technique can be used to promote the heteromultimerization of two different immunoglobulin polypeptides, generating a bispecific antibody containing two immunoglobulin polypeptides with binding specificity to different epitopes.

[0179] In some embodiments, knobs can be constructed by replacing a small amino acid side chain with a larger side chain. In some embodiments, holes can be constructed by replacing a large amino acid side chain with a smaller side chain. Knobs or holes may be present at the original interface or may be introduced synthetically. For example, knobs or holes can be introduced synthetically by modifying the nucleic acid sequence encoding the interface such that at least one “original” amino acid residue is replaced with at least one “imported” amino acid residue. Methods for modifying nucleic acid sequences may include standard molecular biological techniques well known in the art. The side chain volumes of various amino acid residues are shown in the following table. In some embodiments, the original residues have small side chain volumes (e.g., alanine, asparagine, aspartic acid, glycine, serine, threonine, or valine), and the imported residues for forming the knob are naturally occurring amino acids, including arginine, phenylalanine, tyrosine, and tryptophan. In some embodiments, the original residues have larger side-chain volumes (e.g., arginine, phenylalanine, tyrosine, and tryptophan), and the transfer residues for forming the hole are naturally occurring amino acids, which may include alanine, serine, threonine, and valine.

[0180] TIFF0007843689000002.tif158170

[0181] In some embodiments, the original residues for forming the knob or hole are identified based on the three-dimensional structure of the heteromultimer. Techniques known in the art for obtaining the three-dimensional structure can include X-ray crystallography and NMR. In some embodiments, the interface is the CH3 domain of the immunoglobulin constant domain. In these embodiments, the CH3 / CH3 interface of human IgG1 contains 16 residues in each domain located on four anti-parallel β-strands. Without wishing to be bound by theory, the mutated residues are preferably located on two central anti-parallel β-strands to minimize the risk that the knob is accommodated in the surrounding solvent rather than in the complementary hole within the partner CH3 domain. In some embodiments, the mutations forming the knob and hole corresponding to two immunoglobulin polypeptides correspond to one or more of the pairs provided in the following table.

[0182] TIFF0007843689000003.tif87170

[0183] In some embodiments, the immunoglobulin polypeptide comprises a CH3 domain comprising one or more of the amino acid substitutions listed in Table 2 above. In some embodiments, the bispecific antibody comprises a first immunoglobulin polypeptide comprising a CH3 domain comprising one or more of the amino acid substitutions listed in the left column of Table 2 and a second immunoglobulin polypeptide comprising a CH3 domain comprising one or more of the corresponding amino acid substitutions listed in the right column of Table 2.

[0184] Following the DNA mutations discussed above, polynucleotides encoding immunoglobulin polypeptides modified with one or more corresponding knob or hole-forming mutations can be expressed and purified using standard recombination techniques and cell lines known in the art. See, for example, U.S. Patent Nos. 5,731,168; 5,807,706; 5,821,333; 7,642,228; 7,695,936; 8,216,805; U.S. Patent Application Publication No. 2013 / 0089553; and Spiess et al., Nature Biotechnology 31: 753-758, 2013. The modified immunoglobulin polypeptides can be produced using prokaryotic host cells such as Escherichia coli, or eukaryotic host cells such as CHO cells. The immunoglobulin polypeptides having the corresponding knobs and holes may be expressed in host cells in a co-culture and purified together as a heteromultimer, or expressed in a single culture, purified separately, and assembled in vivo. In some embodiments, two strains of bacterial host cells (one expressing the immunoglobulin polypeptide with the knob and the other expressing the immunoglobulin polypeptide with the hole) are co-cultured using standard bacterial culture techniques known in the art. In some embodiments, the two strains may be mixed in a specific ratio to achieve equal expression levels in the culture, for example. In some embodiments, the two strains may be mixed in a ratio of 50:50, 60:40, or 70:30. After polypeptide expression, the cells may be lysed together and the proteins may be extracted. Standard techniques known in the art that enable the measurement of the homomultimer vs. heteromultimer ratio may include size exclusion chromatography. In some embodiments, each modified immunoglobulin polypeptide may be expressed separately using standard recombination techniques and assembled together in vitro. Assembly can be achieved, for example, by purifying each modified immunoglobulin polypeptide, mixing and incubating them together in equal masses, reducing the disulfide (e.g., by treatment with dithiothreitol), concentrating, and re-oxidizing the polypeptides.The formed bispecific antibodies can be purified using standard techniques, including cation exchange chromatography, and measured using standard techniques, including size exclusion chromatography. For a more detailed description of these methods, see Spiess et al., Nat Biotechnol 31:753-8, 2013. In some embodiments, modified immunoglobulin polypeptides can be expressed separately in CHO cells and assembled in vitro using the methods described above.

[0185] According to different methods, an antibody variable domain (antibody-antigen binding site) with desired binding specificity is fused to an immunoglobulin constant domain sequence. The fusion preferably occurs with an immunoglobulin heavy chain constant domain that includes at least a portion of the hinge, CH2, and CH3 regions. Typically, the first heavy chain constant region (CH1), which contains the site necessary for light chain binding, is present in at least one of the fusions. The DNA encoding the immunoglobulin heavy chain fusion and, optionally, the immunoglobulin light chain, is inserted into separate expression vectors and co-transfected into a suitable host organism. This provides great flexibility in adjusting the relative proportions of the three polypeptide fragments in embodiments where unequal ratios of the three polypeptide chains used in construction yield the optimal yield. However, when yield is higher with equal expression of at least two polypeptide chains, or when the ratio is not particularly important, it is possible to insert the coding sequences of two or all three polypeptide chains into a single expression vector.

[0186] In one embodiment of this method, a bispecific antibody is composed of a hybrid immunoglobulin heavy chain having a first binding specificity in one arm and a hybrid immunoglobulin heavy-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure has been found to facilitate the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as it provides an easy separation method when only half of the bispecific molecule has an immunoglobulin light chain. This method is disclosed in International Publication No. 94 / 04690. For further details on the production of bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology 121:210 (1986).

[0187] According to another method described in International Publication No. 96 / 27011, the proportion of heterodimers recovered from recombinant cell cultures can be maximized by manipulating the interface between a pair of antibody molecules. One interface is the C of the antibody constant domain. H This method involves at least a portion of three domains. In this method, one or more smaller amino acid side chains originating from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Complementary "cavities" having the same or similar size as the larger side chains are formed on the interface of the second antibody molecule by replacing the larger amino acid side chains with smaller side chains (e.g., alanine or threonine). This provides a mechanism that increases the yield of heterodimers compared to other undesirable end products such as homodimers.

[0188] Bispecific antibodies include crosslinked or "heteroconjugated" antibodies. For example, in a heteroconjugated antibody, one side may be bound to avidin and the other to biotin. Such antibodies have been proposed, for example, to target immune system cells against undesirable cells (U.S. Patent No. 4676980) and for the treatment of HIV infection (International Publication No. 91 / 00360, International Publication No. 92 / 200373, and European Patent Application Publication No. 03089). Heteroconjugated antibodies can be prepared using any simple crosslinking method. Suitable crosslinking agents are well known in the art and are disclosed in U.S. Patent No. 4676980 along with many crosslinking techniques.

[0189] Techniques for producing bispecific antibodies from antibody fragments are also described in the literature. For example, bispecific antibodies can be prepared using chemical bonding. Brennan et al., Science, 229: 81 (1985) describes a procedure for producing F(ab')2 fragments by proteolytically cleaving intact antibodies. These fragments are reduced in the presence of sodium arsenite, a dithiol complexing agent, to stabilize adjacent dithiols and prevent intermolecular disulfide formation. The resulting Fab' fragments are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to a Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab'-TNB derivative to form a bispecific antibody. The resulting bispecific antibody can be used as an agent for selective enzyme immobilization.

[0190] Recent advances have made it easier to directly recover Fab'-SH fragments from E. coli that can be chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) described the generation of the fully humanized bispecific antibody F(ab')2 molecule. Each Fab' fragment is secreted separately from E. coli and subjected to directional chemical coupling in vitro to form bispecific antibodies.

[0191] Various techniques for directly producing and isolating bispecific antibody fragments from recombinant cell cultures have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992). Leucine zipper peptides derived from Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimer was reduced at the hinge region to form a monomer, and then reoxidized to form an antibody heterodimer. This method can also be used for the production of antibody homodimers. The "diabody" technique described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993) provided an alternative mechanism for producing bispecific antibody fragments. The fragments have light chain variable domains (V) linked by linkers that are too short to allow pairing between two domains on the same chain. L ) and linked heavy chain variable domain (V H ) includes. Therefore, V of one fragment H and V L The domain is the complementary V of another fragment. L and V H The domain is forced to pair with another, thereby forming two antigen-binding sites. Another strategy for producing bispecific antibody fragments using single-chain Fv(sFv) dimers has also been reported. See Gruber et al., J. Immunol, 152:5368 (1994).

[0192] Another technique for producing bispecific antibody fragments is the “bispecific T cell engager” or BiTE® method (see, for example, International Publications 2004 / 106381, 2005 / 061547, 2007 / 042261, and 2008 / 119567). This method utilizes two antibody variable domains located on a single polypeptide. For example, a single polypeptide chain is a variable heavy chain (V) separated by a polypeptide linker of sufficient length to allow intramolecular association between the two domains. H) and variable light chain (V L The single polypeptide comprises two single-chain Fv(scFv) fragments having a domain. This single polypeptide further includes a polypeptide spacer sequence between the two scFv fragments. Each scFv recognizes a different epitope, and these epitopes may be specific to different cell types such that when each scFv binds to its homologous epitope, cells of two different cell types are brought into close proximity or linked. One particular embodiment of this technique includes an scFv that recognizes a cell surface antigen expressed by immune cells, such as a CD3 polypeptide on a T cell, bound to another scFv that recognizes a cell surface antigen expressed by a target cell, such as a malignant or tumor cell.

[0193] As a single polypeptide, the bispecific T cell engager can be expressed using any prokaryotic or eukaryotic cell expression system known in the art, such as the CHO cell line. However, specific purification techniques (see, for example, European Patent Application Publication No. 1691833) may be required to separate the monomeric bispecific T cell engager from other multimeric species, as it may possess biological activities other than those intended for the monomer. In an exemplary purification scheme, a solution containing the secreted polypeptide is first subjected to metal affinity chromatography, and the polypeptide is eluted using an imidazole concentration gradient. This eluate is further purified using anion exchange chromatography, and the polypeptide is eluted using a sodium chloride concentration gradient. Finally, this eluate is subjected to size exclusion chromatography to separate the monomer from the multimeric species.

[0194] Antibodies with more than two valent nucleotides should also be considered. For example, triplicate antibodies can be prepared. See, for example, Tuft et al. J. Immunol. 147: 60 (1991).

[0195] (vii) Single-domain antibody In some embodiments, the antibody of the present invention is a single-domain antibody. A single-domain antibody is a single polypeptide chain comprising all or part of the heavy chain variable domains or all or part of the light chain variable domains of an antibody. In certain embodiments, the single-domain antibody is a human single-domain antibody (see, for example, U.S. Patent No. 6,248,516,B1; Domantis, Inc., Waltham, Mass.). In one embodiment, the single-domain antibody consists of all or part of the heavy chain variable domains of an antibody.

[0196] (viii) Antibody variant In some embodiments, the amino acid sequence modifications of antibodies described herein are considered. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of antibodies can be prepared by introducing appropriate modifications to the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from and / or insertions of residues in the amino acid sequence of the antibody, and / or substitutions thereof. Any combination of deletions, insertions, and substitutions can be performed to reach the final construct, as long as the final construct has the desired properties. Amino acid modifications can be introduced into the amino acid sequence of the antibody in question when the sequence is constructed.

[0197] (ix) Substitution, insertion, and deletion variants In a given embodiment, an antibody variant having one or more amino acid substitutions is provided. The target sites for substitutional mutagenesis include HVR and FR. Preferred substitutions are shown in Table 3 under the heading "Preferred Substitutions." More substantial changes are shown in Table 3 under the heading "Exemplary Substitutions" and are further described below with reference to amino acid side chain classes. The amino acid substitutions can be introduced into the antibody of interest, and the product is screened for desired activity, e.g., retention / improvement of antigen binding, decreased immunogenicity, or improvement of ADCC or CDC.

[0198] TIFF0007843689000004.tif168170

[0199] Amino acids can be grouped according to their common side-chain characteristics: a. Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; b. Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln; c. Acidic: Asp, Glu; d. Basicity: His, Lys, Arg; e. Residues that affect chain orientation: Gly, Pro; f. Aromatic: Trp, Tyr, Phe.

[0200] Non-conservative substitutions require replacing a member of one of these classes with one of another.

[0201] One type of substitution variant involves the substitution of one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant selected for further study will have modifications (e.g., improvements) to a predetermined biological property (e.g., increased affinity, decreased immunogenicity) compared to the parent antibody, and / or substantially retain the predetermined biological property of the parent antibody. An exemplary substitution variant is an affinity-matured antibody, which can be readily produced using phage display-based affinity maturation techniques, such as those described herein. In short, one or more HVR residues are mutated, the mutated antibody is presented on a phage, and it is screened for specific biological activity (e.g., binding affinity).

[0202] Modifications (e.g., substitutions) can be made in HVR, for example, to improve antibody affinity. Such modifications can be made in HVR "hot spots," i.e., residues encoded by codons that are frequently mutated during somatic cell maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or in SDR(a-CDR), and the resulting variant VH or VL is tested for binding affinity. Affinity maturation by reconstructing and reselecting from a secondary library is described, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., Human Press, Totowa, NJ, (2001)). In some embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by one of various methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then constructed. The library is then screened to identify antibody variants with the desired affinity. Another method for introducing diversity involves an HVR-directed approach in which several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3 are often targeted in particular.

[0203] In a given embodiment, substitutions, insertions, or deletions may occur within one or more HVRs, provided that such modifications do not substantially reduce the antibody's ability to bind to the antigen. For example, conservative modifications (e.g., the conservative substitutions provided herein) that do not substantially reduce binding affinity can be made within an HVR. Such modifications may be outside the HVR "hotspot" or SDR. In a given embodiment of the variant VH or VL sequence given above, each HVR is either immutable or contains at most one, two, or three amino acid substitutions.

[0204] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and replaced with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the antigen-antibody interaction is affected. Further substitutions may be introduced at amino acid positions that exhibit functional sensitivity to the initial substitution. Alternatively or in addition, the crystal structure of the antigen-antibody complex may be used to identify the antibody-antigen contact sites. Such contact residues and adjacent residues may be targeted as candidates for substitution or excluded. Variants may be screened to determine whether they contain the desired properties.

[0205] Amino acid sequence insertions include amino- and / or carboxyl-terminated fusions ranging from one residue to the length of a polypeptide having 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody having an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusions to the N-terminus or C-terminus of an antibody against a polypeptide or enzyme (e.g., for ADEPT) that extends the serum half-life of the antibody.

[0206] (x) Glycosylated variant In a given embodiment, the antibody provided herein is modified to increase or decrease the degree to which the antibody is glycosylated. The addition or deletion of glycosylation sites to the antibody can be easily achieved by modifying the amino acid sequence so that one or more glycosylation sites are created or removed.

[0207] If an antibody contains an Fc region, the glycans bound to it can be modified. Natural antibodies produced by mammalian cells typically contain branched oligosaccharides commonly bound to Asn297 of the CH2 domain of the Fc region via N-bonding. See, for example, Wright et al. TIBTECH 15:26-32 (1997). Oligosaccharides can include various glycans, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose bound to the GlcNAc of the "stem" of the branched oligosaccharide structure. In some embodiments, the modification of oligosaccharides in the antibodies of the present invention may be made to produce antibody variants having predetermined improved properties.

[0208] In one embodiment, an antibody variant is provided in which the glycosylation structure bound to the Fc region includes an Fc region with reduced or no fucose, which may enhance ADCC function. Specifically, here we consider antibodies having reduced fucose compared to the amount of fucose on the same antibody produced in wild-type CHO cells. That is, they are characterized by having less fucose than would be present if produced by natural CHO cells (e.g., CHO cells that produce a natural glycosylation pattern, e.g., CHO cells containing the natural FUT8 gene). In a given embodiment, the antibody contains fucose in about 50%, 40%, 30%, 20%, 10%, or less than 5% of the N-linked glycan on it. For example, the amount of fucose in such an antibody is 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. In a given embodiment, the antibody is one in which none of the N-linked glycans on which it is attached contain fucose, i.e., the antibody is completely fucose-free, fucose-free, or afucosylated. The amount of fucose is determined by calculating the average amount of fucose in Asn297 in the glycan relative to the sum of all sugar structures (e.g., complex, hybrid, and high-mannose structures) bound to Asn297, measured by MALDI-TOF mass spectrometry, as described, for example, in International Publication No. 2008 / 077546. Asn297 refers to the asparagine residue located approximately 297th position in the Fc region (Eu numbering of Fc region residues); however, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations of the antibody. Such fucosylated variants may have improved ADCC function. For example, see U.S. Patent Application Publication No. 2003 / 0157108 (Presta, L.); U.S. Patent Application Publication No. 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.).Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include U.S. Patent Application Publication 2003 / 0157108; International Publication 2000 / 61739; International Publication 2001 / 29246; U.S. Patent Application Publication 2003 / 0115614; International Publication 2002 / 0164328; International Publication 2004 / 0093621; International Publication 2004 / 013 This includes publications No. 2140; No. 2004 / 0110704; No. 2004 / 0110282; No. 2004 / 0109865; International Publication No. 2003 / 085119; International Publication No. 2003 / 084570; No. 2005 / 035586; No. 2005 / 035778; No. 2005 / 053742; No. 2002 / 031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells lacking protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application Publication No. 2003 / 0157108A1; and International Publication No. 2004 / 056312A1, particularly Example 11), and knockout cell lines, e.g., alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and International Publication No. 2003 / 085107).

[0209] For example, antibody variants having a bifid oligosaccharide are provided, in which a bifid oligosaccharide bound to the Fc region of the antibody is bifid by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in International Publication 2003 / 011878; U.S. Patent No. 6602684; U.S. Patent Application Publication 2005 / 0123546; and Ferrara et al., Biotechnology and Bioengineering, 93(5): 851-861 (2006). Antibody variants having at least one galactose residue in the oligosaccharide bound to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in International Publication 1997 / 30087; 1998 / 58964; and 1999 / 22764.

[0210] In a given embodiment, the antibody variant containing the Fc region described herein can bind to FcγRIII. In a given embodiment, the antibody variant containing the Fc region described herein has ADCC activity in the presence of human effector cells, or has increased ADCC activity in the presence of human effector cells compared to an antibody that is otherwise identical except for containing the human wild-type IgG1 Fc region.

[0211] (xi)Fc region variant In a given embodiment, one or more amino acid modifications can be introduced into the Fc region of the antibody provided herein to create an Fc region variant. The Fc region variant may include a human Fc region sequence (e.g., human IgG1, IgG2, IgG3, or IgG4 Fc region) that includes amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0212] In certain embodiments, the present invention intends for antibody variants possessing some, but not all, effector functions, making them desirable candidates for applications where the in vivo antibody half-life is important, but certain effector functions (e.g., complement and ADCC) are unnecessary or detrimental. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / deficient CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to confirm that an antibody lacks FcγR binding (and therefore likely lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression in hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for evaluating the ADCC activity of a target molecule are described in U.S. Patent No. 5500362 (see, e.g., Hellstrom et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); No. 5821337 (see Bruggemann et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be used (e.g., ACTI for flow cytometry). TMNon-radioactive cytotoxicity assays (see CellTechnology, Inc., Mountain View, CA; and CytoTox96® non-radioactive cytotoxicity assay (Promega, Madison, WI)). Effector cells useful for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively, or in addition, the ADCC activity of the molecule of interest can be evaluated in vivo in animal models, such as those disclosed in Clynes et al., Proc. Natl. Acad. Sci. USA 95:652-656 (1998). C1q binding assays can also be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in International Publication Nos. 2006 / 029879 and 2005 / 100402. To evaluate complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg et al., Blood 101:1045-1052 (2003); and Cragg et al., Blood 103:2738-2743 (2004)). Determination of FcRn binding and in vivo clearance / half-life can also be performed using methods known in the field (see, e.g., Petkova et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0213] Antibodies with reduced effector function include those with one or more substitutions among Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc variants include the so-called "DANA" Fc variant with alanine substitutions at residues 265 and 297, as well as Fc variants with two or more substitutions among amino acid positions 265, 269, 270, 297, and 327 (U.S. Patent No. 7,332,581).

[0214] Certain antibody variants exhibiting improved or reduced binding to FcR are described. (See, for example, U.S. Patent No. 6,737,056; International Publication No. 2004 / 056312; and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)).

[0215] In a given embodiment, the antibody variant includes an Fc region containing one or more amino acid substitutions that improve ADCC, for example, substitutions at positions 298, 333, and / or 334 (EU numbering of residues) in the Fc region. In an exemplary embodiment, the antibody includes the following amino acid substitutions in its Fc region: S298A, E333A, and K334A.

[0216] In some embodiments, modifications are made in the Fc region to produce modified (i.e., improved or reduced) C1q binding and / or complement-dependent cell injury (CDC), as described, for example, in U.S. Patent No. 6,194,551, International Publication No. 99 / 51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

[0217] Antibodies with extended half-lives and improved binding affinity to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in U.S. Patent Application Publication No. 2005 / 0014934A1 (Hinton et al.). These antibodies contain an Fc region with one or more substitutions that improve the binding of the Fc region to FcRn. Such Fc variants include substitutions in one or more of the Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, for example, substitutions in Fc region residue 434 (U.S. Patent No. 7,371,826). For other examples of Fc region variants, see also Duncan and Winter, Nature 322:738-40 (1988); U.S. Patent Nos. 5,648,260; 5,624,821; and International Publication No. 94 / 29351.

[0218] (xii) Antibody derivatives The antibodies of the present invention may be further modified to include additional non-protein moieties known and readily available in the art. In certain embodiments, the moiety suitable for derivatization of the antibody is a water-soluble polymer. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxolane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, propylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have manufacturing advantages due to its stability in water. The polymers may have any molecular weight and may be branched or unbranched. The number of polymers bound to the antibody may vary, and if more than one polymer is bound, they may be the same molecule or different molecules. In general, the number and / or types of polymers used in derivatization are not limited but can be determined based on considerations including the specific properties or functions of the antibody to be improved and whether the antibody derivative will be used for therapeutic purposes under specific conditions.

[0219] (xiii) Vectors, host cells, and recombinant methods Antibodies can also be produced using recombinant methods. In recombinant production of anti-antigen antibodies, the nucleic acid encoding the antibody is isolated and inserted into a replicable vector for further cloning (DNA amplification) or expression. The DNA encoding the antibody can be readily isolated and sequenced using standard procedures (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the antibody). Many vectors are available. The components of a vector generally include, but are not limited to, one or more of the following: a signal sequence, a replication start site, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0220] (a) Signal sequence component The antibodies of the present invention can be produced not only by direct recombinant production, but also as fusion peptides with heterologous polypeptides, which are signal sequences or other polypeptides having a specific cleavage site at the N-terminus of a mature protein or polypeptide. The selected heterologous signal sequence is preferably one that is recognized and processed (e.g., cleaved by a signal peptidase) by the host cell. In the case of prokaryotic host cells that do not recognize and process the native antibody signal sequence, the signal sequence is replaced by a prokaryotic signal sequence selected from, for example, alkaline phosphatase, penicillinase, lpp, or a heat-stable enterotoxin II leader. In the case of yeast secretion, the native signal sequence can be replaced by, for example, a yeast invertase leader, a factor leader (including Saccharomyces and Cliveromyces α-factor leaders), or an acid phosphatase leader, a Candida albicans glucoamylase leader, or a signal described in International Publication No. 90 / 13646. For expression in mammalian cells, mammalian signal sequences and viral secretion leaders, such as the herpes simplex gD signal, are available.

[0221] (b) Replication starting point Both expression and cloning vectors contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells. Generally, in cloning vectors, these sequences enable the vector to replicate independently of the host's chromosomal DNA and include replication origins or self-replicating sequences. Such sequences are well known for various bacteria, yeasts, and viruses. Replication origins from plasmid pBR322 are suitable for most Gram-negative bacteria, 2μ plasmid origins are suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV, or BPV) are useful for cloning vectors in mammalian cells. Generally, replication origin components are not required for mammalian expression vectors (SV40 origins are commonly used only because they contain early promoters).

[0222] (c) Selected gene components Expression and cloning vectors may include select genes, also known as selectable markers. Typical select genes encode proteins that (a) confer resistance to antibiotics or other toxins, such as ampicillin, neomycin, methotrexate, or tetracycline; (b) compensate for nutritional deficiencies; or (c) supply the gene-coding D-alanine racemase to essential nutrients not available from complex media, such as basilicum.

[0223] One example of a selection scheme involves using drugs that halt the proliferation of host cells. Cells successfully transformed by heterologous genes produce proteins that confer drug resistance, thus surviving the selection regimen. Examples of such dominant selection use the drugs neomycin, mycophenolate, and hygromycin.

[0224] Another example of a suitable selectable marker for mammalian cells is one that allows for the identification of cells capable of taking up antibody-coding nucleic acids, such as DHFR, glutamine synthetase (GS), thymidine kinase, metallothionein-I and -II, preferably primate metallothionein genes, adenosine deaminase, and ornithine decarboxylase.

[0225] For example, cells transformed with the DHFR gene are identified by culturing the transformants in a medium containing methotrexate (Mtx), a competitive antagonist of DHFR. Under these conditions, the DHFR gene is amplified together with any other co-transforming nucleic acid. Chinese hamster ovary (CHO) cell lines lacking endogenous DHFR activity (e.g., ATCC CRL-9096) can be used.

[0226] Alternatively, cells transformed with the GS gene are identified by culturing the transformants in a medium containing L-methionine sulfoximine (Msx), a GS inhibitor. Under these conditions, the GS gene is amplified together with any other cotransforming nucleic acid. The GS selection / growth system may be used in combination with the DHFR selection / growth system described above.

[0227] Alternatively, host cells transformed or co-transformed with the DNA sequence encoding the antibody of interest, the wild-type DHFR gene, and another selectable marker such as aminoglycoside 3'-phosphotransferase (APH) (in particular, wild-type hosts containing endogenous DHFR) may be selected by cell growth in a medium containing a selector for the selectable marker, such as kanamycin, neomycin, or an aminoglycoside antibiotic such as G418. See U.S. Patent No. 4,965,199.

[0228] The trp1 gene, located in the yeast plasmid YRp7, is a suitable select gene for use in yeast (Stinchcomb et al., Nature, 282:39 (1979)). The trp1 gene provides a select marker for yeast mutants lacking the ability to grow in tryptophan, such as ATCC number 44076 or PEP4-1 (Jones, Genetics, 85:12 (1977)). Subsequently, the presence of trp1 damage in the yeast host cell genome provides an effective environment for detecting transformation by growth in the absence of tryptophan. Similarly, Leu2-deficient yeast strains (ATCC20622 or 38626) are complemented by known plasmids containing the Leu2 gene.

[0229] In addition, vectors derived from the 1.6 μm circular plasmid pKD1 can be used for the transformation of Cliveromyces yeast. Alternatively, an expression system for the large-scale production of recombinant bovine chymosin has been reported for Cliveromyces lactis. Van den Berg, Bio / Technology, 8:135 (1990). A stable multicopy expression vector for the secretion of mature recombinant human serum albumin by industrial strains of Cliveromyces has also been disclosed. Fleer et al., Bio / Technology, 9:968-975 (1991).

[0230] (d) Promoter component Expression and cloning vectors generally contain a promoter that is recognized by the host organism and ligated to the nucleic acid encoding the antibody. Promoters suitable for use with prokaryotic hosts include the phoA promoter, β-lactamase and lactose promoter systems, alkaline phosphatase promoters, tryptophan (trp) promoter systems, and hybrid promoters such as the tac promoter. However, other known bacterial promoters are also suitable. Promoters used in bacterial systems would also likely contain a Shine-Dalgarno (SD) sequence ligated to the antibody-encoding DNA.

[0231] Promoter sequences for eukaryotes are known. Almost all eukaryotic genes have an AT-rich region located approximately 25 to 30 base pairs upstream from the transcription initiation site. Another sequence found 70 to 80 base pairs upstream from the transcription initiation of many genes is the CNCAAT region, where N can be any nucleotide. The 3' end of most eukaryotic genes contains an AATAAA sequence, which can be a signal for the addition of a poly(A) tail to the 3' end of the coding sequence. All of these sequences are appropriately inserted into eukaryotic expression vectors.

[0232] Examples of suitable promoter sequences for use with a yeast host include promoters for 3-phosphoglycerate kinase or other glycoseptic enzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triose phosphate isomerase, phosphoglucose isomerase, and glucokinase.

[0233] Other yeast promoters are inducible promoters that have the further advantage of transcription being regulated by growth conditions, and include promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatases, nitrogen metabolism-related degrading enzymes, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes that contribute to maltose and galactose utilization. Suitable vectors and promoters used in yeast expression are further described in European Patent Application Publication No. 73657. Yeast enhancers can also be advantageously used in conjunction with yeast promoters.

[0234] Antibody transcription from vectors in mammalian host cells can be controlled by promoters derived from viral genomes such as polyomaviruses, fowlpox virus, adenoviruses (e.g., adnovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis B virus, and simian virus 40 (SV40), or from heterologous mammalian promoters such as actin promoters or immunoglobulin promoters, or from heat shock promoters, as long as such promoters are compatible with the host cell system.

[0235] Early and late promoters of the SV40 virus can be readily obtained as SV40 restriction fragments that also contain the replication origin of the SV40 virus. The pre-early promoter of human cytomegalovirus can be readily obtained as a HindIII E restriction fragment. A system for expressing DNA in a mammalian host using bovine papillomavirus as a vector is disclosed in U.S. Patent No. 4419446. Modifications of this system are described in U.S. Patent No. 4601978. For the expression of human β-interferon cDNA in mouse cells under the control of a herpes simplex virus-derived thymidine kinase promoter, see Reyes et al., Nature 297:598-601 (1982). Alternatively, Rous sarcoma virus terminal repeat sequences can be used as promoters.

[0236] (e) Enhancer element components Transcription of the DNA encoding the antibodies of the present invention by higher eukaryotes is often enhanced by inserting enhancer sequences into the vector. Many enhancer sequences from mammalian genes are now known (globin, elastase, albumin, α-fetoprotein, and insulin). However, typically, enhancers from eukaryotic viruses will be used. Examples include the SV40 enhancer (100-270 base pairs) at the late end of the origin of replication, the cytomegalovirus early promoter enhancer, the polyoma enhancer at the late end of the origin of replication, and the adenovirus enhancer. See also Yaniv, Nature 297:17-18 (1982) for enhancing elements of eukaryotic promoter activation. The enhancer may be spliced ​​into the vector at the 5' or 3' position of the antibody-coding sequence, but is preferably located at the 5' site from the promoter.

[0237] (f) Transcription termination component Expression vectors used in eukaryotic host cells (nucleated cells derived from yeast, fungi, insects, plants, animals, humans, or other multicellular organisms) also contain sequences necessary for transcription termination and mRNA stabilization. Such sequences are commonly obtained from the 5' and sometimes 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments that are transcribed as polyadenylated fragments into the untranslated portion of antibody-encoding mRNA. One useful transcription termination component is the bovine growth hormone polyadenylated region. See International Publication No. 94 / 11026 and the expression vectors disclosed therein.

[0238] (g) Selection and transformation of host cells Suitable host cells for DNA cloning or expression in the vectors here are the prokaryotic cells, yeasts, or higher eukaryotic cells mentioned above. Suitable prokaryotes for this purpose include eubacteria such as Gram-negative or Gram-positive organisms, e.g., Enterobacteriaceae such as Escherichia coli, e.g., Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella e.g., Salmonella typhi, Serratia e.g., Serratia and Sigella, as well as rod-shaped bacteria e.g., Bacillus subtilis and Bacillus licheniformis (e.g., Bacillus licheniformis 41P disclosed in DD266710 published April 12, 1989), Pseudomonas e.g., Pseudomonas aeruginosa, and Streptomyces. One preferred E. coli cloning host is E. coli 294 (ATCC31446), but other strains such as E. coli B, E. coli X1776 (ATCC31537), and E. coli W3110 (ATCC27325) are also suitable. These examples are illustrative and not limiting.

[0239] Full-length antibodies, antibody fusion proteins, and antibody fragments can be produced in bacteria, particularly when glycosylation and Fc effector function are not required, for example, when a therapeutic antibody is conjugated to a cytotoxic drug (e.g., a toxin) that itself exhibits efficacy in tumor cell destruction. Full-length antibodies have a longer circulating half-life. Production in E. coli is faster and most cost-effective. For bacterial expression of antibody fragments and polypeptides, see, for example, U.S. Patent No. 5648237 (Carter et al.), No. 5789199 (Joly et al.), and No. 5840523 (Simmons et al.), which describe translation initiation regions (TIRs) and signal sequences for optimizing expression and secretion. Also see Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes antibody fragment expression in E. coli. After expression, the antibody can be isolated from the E. coli cell paste in the soluble fraction and purified, for example, by a protein A or G column depending on the isotype. Final purification can be carried out in a manner similar to that used to purify antibodies expressed in CHO cells.

[0240] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors. Saccharomyces cerevisiae, or common baker's yeast, are among the most commonly used lower eukaryotic host microorganisms. However, for example, fission yeast; Cliberomyces hosts, such as Cliberomyces lactis, Cliberomyces fragilis (ATCC12424), Cliberomyces bulgaricus (ATCC16045), Cliberomyces wickeramii (ATCC24178), Cliberomyces waltii (ATCC56500), Cliberomyces drosophilarum (ATCC36906), Cliberomyces thermotolerance and Cliberomyces marxianus; Yarrowia (EP402226); Pichia pastris (EP183070); Candida; Trichoderma ligia Many other genera, species, and strains are commonly available and useful here, including those of *Neisseria reesia* (EP244234); *Cornus crassis*; the genera *Schwanniomyces*, e.g., *Schwanniomyces occidentalis*; and filamentous fungi, e.g., *Cornus crassis*, *Penicillium*, *Trypocladium*, and *Aspergillus* hosts, e.g., *Aspergillus nidurans* and *Aspergillus niger*. For an overview of the use of yeasts and filamentous fungi for the production of therapeutic proteins, see, for example, Gerngross, Nat. Biotech. 22:1409-1414 (2004).

[0241] Certain fungal and yeast strains with "humanized" glycosylation pathways can be selected, and antibodies with partial or complete human glucosylation patterns can be produced. See, for example, Li et al., Nat. Biotech. 24:210-215 (2006) (describes the humanization of the glycosylation pathway in Pichia pastris); and Gerngross et al. cited above.

[0242] Host cells suitable for the expression of glycosylated antibodies can also be derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains and variants, as well as corresponding tolerant insect host cells, have been identified from hosts such as the armyworm (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and silkworm. Various virus strains for transfection, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of silkworm NPV, are publicly available, and such viruses are used as the viruses according to the present invention, particularly for the transfection of armyworm cells.

[0243] Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, duckweed (Lemnaceae), alfalfa (Medicago truncatula), and tobacco can also be used as hosts. For example, U.S. Patents 5,959177, 6040498, 6420548, 7125978, and 6417429 (PLANTIBODIES for generating antibodies in transgenic plants) TM See (Technical details) for more information.

[0244] Vertebrate cells may be used as hosts, and the proliferation of vertebrate cells in culture (tissue culture) is a standard procedure. Examples of useful mammalian host cell lines include: SV40-transformed monkey kidney CV1 cell line (COS-7, ATCC CRL 1651); human fetal kidney cell line (293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36: 59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23: 243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (HepG2, HB 8065); mouse mammary tumor cells (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N. Y Acad. Sci. 383: 44-68 (1982)); MRC5 cells; FS4 cells; and human liver cell strain (HepG2). Other useful mammalian host cell lines include DHFR - This includes Chinese hamster ovary (CHO) cells, including CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines, such as NS0 and Sp2 / 0. For an overview of specific mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, Humana Press, Totowa, NJ, 2003), pp. 255-268.

[0245] Host cells are transformed using the aforementioned expression or cloning vectors for antibody production and cultured in a general nutrient medium appropriately modified for promoter induction, transformant selection, or amplification of genes encoding desired sequences.

[0246] (h) Culture of host cells The host cells used to produce the antibodies of this invention can be cultured in various media. Commercial media such as Ham's F10 (Sigma), Minimum Essential Medium ((MEM),(Sigma), RPMI-1640 (Sigma), and Dulbecco's Improved Eagle Medium ((DMEM),Sigma) are suitable for culturing host cells. In addition, Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 Any of the culture media described in (1980), U.S. Patent No. 4,767,704; No. 4,657,866; No. 4,927,762; No. 4,560,655; or No. 5,122,469; International Publication No. 90 / 03430; International Publication No. 87 / 00195; or U.S. Reissued Patent No. 30985 can be used as a culture medium for host cells. Any of these media may contain hormones and / or other growth factors (e.g., insulin, transferrin, or epidermal growth factor), salts (e.g., sodium chloride, calcium, magnesium, and phosphate), buffers (e.g., HEPES), nucleotides (e.g., adenosine and thymidine), or antibiotics (e.g., GENTAMYCIN). TM Drugs, trace elements (defined as inorganic compounds typically present in the micromolar range at final concentrations), and glucose or equivalent energy sources may be supplemented as needed. Any other necessary supplements may also be included in appropriate concentrations known to those skilled in the art. Culture conditions such as temperature and pH are those previously used with the selected host cells for expression and will be obvious to those skilled in the art.

[0247] Purification of (xiv) antibody When recombinant technology is used, antibodies can be produced intracellularly, in the perimembranous space, or secreted directly into the culture medium. If antibodies are produced intracellularly, the first step is to remove particulate debris, which is either host cells or lysed fragments, by, for example, centrifugation or ultrafiltration. Carter et al., Bio / Technology 10: 163-167 (1992) describe a procedure for isolating antibodies secreted into the perimembranous space of E. coli. Briefly, the cell paste is thawed for about 30 minutes in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF). Cell debris can be removed by centrifugation. If antibodies are secreted into the culture medium, the supernatant from such an expression system is generally first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. Protease inhibitors such as PMSF can be included in any of the aforementioned steps to inhibit protein degradation, and antibiotics can be included to prevent the growth of unspecified contaminants.

[0248] Antibody compositions prepared from cells can be purified using, for example, hydroxyl apatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being typically the preferred purification step. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and human γ3 (Guss et al., EMBO J. 5:15671575 (1986)). The matrix to which the affinity ligand binds is usually agarose, but other matrices are also available. Mechanically stable matrices such as pore-controlled glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Antibodies are C H If it contains 3 domains, use bakecarbon ABX for purification. TM Resin (JT Baker, Phillipsburg, NJ) is useful. Other techniques for protein purification include fragmentation with ion exchange columns, ethanol precipitation, reverse-phase HPLC, chromatography with silica, and heparin Sepharose. TMで Chromatography, chromatography with anion or cation exchange resins (e.g., polyaspartate columns), chromatographic focusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody being recovered.

[0249] In general, various methods for preparing antibodies for research, testing, and clinical use are established in the art, are compatible with the above methods, and / or are considered appropriate by those skilled in the art for the specific antibody in question.

[0250] (xv) Selection of biologically active antibodies As described above, the antibodies produced can be subjected to one or more "biological activity" assays to select antibodies with therapeutically advantageous properties, or to select formulations and conditions that preserve the biological activity of the antibodies. The antibodies can be tested for their ability to bind to the antigen on which they were produced. For example, methods known in the art (e.g., ELISA, Western blotting, etc.) can be used.

[0251] For example, the antigen-binding properties of an anti-PD-L1 antibody can be evaluated in an assay that detects its ability to bind to PD-L1. In some embodiments, antibody binding can be determined, for example, by saturated binding; ELISA; and / or competitive assays (e.g., RIA). The antibody may also be subjected to other biological activity assays to evaluate its efficacy, for example, as a therapeutic agent. Such assays are known in the art and depend on the target antigen and the intended purpose of the antibody. For example, the biological effects of PD-L1 blockade by an antibody can be evaluated in CD8+ T cells, lymphocytic choriomeningitis virus (LCMV) mouse models, and / or syngeneic tumor models, as described, for example, in U.S. Patent No. 8,217,149.

[0252] To screen for antibodies that bind to specific epitopes on the target antigen (for example, those that block the binding of the anti-PD-L1 antibody in the example to PD-L1), a standard 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 can be performed, for example, as described in Champe et al., J. Biol. Chem. 270:1388-1394 (1995), to determine whether the antibody binds to the target epitope.

[0253] IV. Pharmaceutical Compositions and Formulations Also provided herein are pharmaceutical compositions and formulations comprising the PD-1 axis-conjugated antagonist and / or antibody described herein (e.g., an anti-PD-1 antibody or an anti-PD-L1 antibody) and optionally a pharmaceutically acceptable carrier.

[0254] The pharmaceutical compositions and formulations described herein can be prepared in the form of lyophilized formulations or aqueous solutions by mixing an active ingredient of desired purity (e.g., a PD-1 axis-binding antagonist) with one or more optionally pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosage and concentration used and are not limited to, but include: buffers, e.g., phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens, e.g., methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); and low molecular weight (less than approximately 10 residues). Polypeptides; proteins, e.g., serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, e.g., polyvinylpyrrolidone; amino acids, e.g., glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, e.g., EDTA; sugars, e.g., sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions, e.g., sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, e.g., polyethylene glycol (PEG). exemplified pharmaceutically acceptable carriers herein further include intervening drug dispersants, e.g., soluble neutrally active hyaluronidase glycoprotein (sHASEGP), e.g., rHuPH20 (HYLENEX®, Baxter International, Inc.). A specified exemplary sHASEGP and its use, including rHuPH20, is described in U.S. Patent Application Publications 2005 / 0260186 and 2006 / 0104968. In one embodiment, sHASEGP is combined with one or more further glycosaminoglycans, such as chondroitinase.

[0255] An example of a lyophilized antibody preparation is described in U.S. Patent No. 6,267,958. Aqueous antibody preparations include those described in U.S. Patent No. 6,171,586 and International Publication No. 2006 / 044908, the latter of which contains a histidine-acetate buffer.

[0256] The compositions and formulations herein may also contain more than one active ingredient necessary for the specific indication being treated, preferably having complementary activities that do not adversely affect each other. Such active ingredients are appropriately present in a combination of amounts effective for the intended purpose.

[0257] The active ingredient can be encapsulated in microcapsules prepared, for example, by coacervation technology or interfacial polymerization, such as hydroxymethylcellulose microcapsules, gelatin microcapsules, and poly-(methyl methacrylate) microcapsules, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16th edition, edited by Osol, A. (1980).

[0258] Sustained-release formulations may be prepared. A preferred example of a sustained-release formulation is one comprising a semipermeable matrix of a solid hydrophobic polymer containing an antibody, such as an anti-PD1 antibody or an anti-PD-L1 antibody, where such a matrix is ​​in the form of a molded article, such as a film or microcapsule. Formulations used for in vivo administration are generally sterile. Sterility can be easily achieved, for example, by filtration through a sterile filtration membrane.

[0259] IV. Treatment Methods Provided herein is a method for treating or delaying the progression of cancer (e.g., melanoma) in an individual, comprising administering an effective amount of a PD-1 axis-coupled antagonist to the individual, wherein granzyme B in a blood sample obtained from the patient.+ CD4 + T cells and FOXP3 + CD4 + The ratio to T cells is determined to be 1 or greater. In some embodiments, the treatment elicits a response in the individual after treatment. In some embodiments, the response is a partial response. In some embodiments, the response is complete remission. In some embodiments, the treatment elicits a sustained response (e.g., a sustained partial response or complete remission) in the individual after discontinuation of treatment. The methods described herein may find applications in the treatment of conditions where enhancement of immunogenicity, for example, increased tumor immunogenicity for the treatment of cancer, is desired. Also provided herein is a method for enhancing immune function in an individual with melanoma, comprising administering an effective amount of a PD-1 axis-binding antagonist (e.g., nivolomab, pembrolizumab, atezolizumab, or avelumab) to the individual. Any of the PD-1 axis-binding antagonists known in the art or described herein may be used in this method.

[0260] In some examples, the methods provided herein involve administering an effective dose of a PD-1 axis-binding antagonist selected from the group consisting of PD-1-binding antagonists, PD-L1-binding antagonists, and PD-L2-binding antagonists. In some examples, the PD-L1-binding antagonist is an antibody, for example, an antibody that can inhibit the binding of PD-L1 to PD-1 and B7.1 but does not interfere with the binding of PD-1 to PD-L2. In some examples, the PD-L1-binding antagonist antibody is MPDL3280A, which may be administered in doses of approximately 700 mg to approximately 900 mg every two weeks (e.g., approximately 750 mg to approximately 900 mg every two weeks, e.g., approximately 800 mg to approximately 850 mg every two weeks). In some embodiments, MPDL3280A is administered in doses of approximately 840 mg every two weeks.

[0261] As a general proposition, the therapeutically effective dose of a PD-1 axis-conjugated antagonist (e.g., anti-PD-L1 antibody, e.g., MPDL3280A) that can be administered to humans will be in the range of approximately 0.01 to approximately 50 mg per kg of the patient's body weight, regardless of whether the administration is one dose or multiple doses. In some embodiments, for example, the antagonist (e.g., anti-PD-L1 antibody, e.g., MPDL3280A) is administered in doses of approximately 0.01 to approximately 45 mg / kg, approximately 0.01 to approximately 40 mg / kg, approximately 0.01 to approximately 35 mg / kg, approximately 0.01 to approximately 30 mg / kg, approximately 0.01 to approximately 25 mg / kg, approximately 0.01 to approximately 20 mg / kg, approximately 0.01 to approximately 15 mg / kg, approximately 0.01 to approximately 10 mg / kg, approximately 0.01 to approximately 5 mg / kg, or approximately 0.01 to approximately 1 mg / kg daily. In some embodiments, the antagonist (e.g., anti-PD-L1 antibody, e.g., MPDL3280A) is administered at 15 mg / kg. However, other dosage regimens may also be useful. In one embodiment, the PD-1 axis-conjugated antagonist (e.g., anti-PD-L1 antibody, e.g., MPDL3280A) is administered to humans in doses of approximately 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, or 1500 mg. In some embodiments, a PD-1 axis-conjugated antagonist (e.g., an anti-PD-L1 antibody, e.g., MPDL3280A) is administered every two weeks at a dose of approximately 800 mg to approximately 850 mg. In some embodiments, a PD-1 axis-conjugated antagonist (e.g., an anti-PD-L1 antibody, e.g., MPDL3280A) is administered every two weeks at a dose of approximately 840 mg. The dose may be administered as a single dose or multiple doses (e.g., two or three doses), for example, by intravenous infusion. The dose of antibody administered in combination therapy may be reduced compared to that of monotherapy.In some embodiments, for example, a method for treating or slowing the progression of locally advanced or metastatic breast cancer in an individual comprises a drug regimen comprising a treatment cycle, in which the individual is administered a dose of approximately 840 mg of a human PD-1 axis-conjugated antagonist (e.g., an anti-PD-L1 antibody, e.g., MPDL3280A) on days 1 and 15 of each cycle, with each cycle lasting 28 days (i.e., each cycle is repeated every 28 days).

[0262] The progression of this treatment is monitored by the method described herein. In some embodiments, CD4 + Cytotoxic granzyme B in T cell populations + CD4 + The size of the T cell population predicts the persistence of the response. For example, a patient observed to be disease-free for three months may have granzyme B + CD4 + The ratio of T cells to Treg cells was 4, but the ratio of patients observed to be disease-free for 6 months was 45. Therefore, B + CD4 + Larger T cell population sizes were associated with longer disease-free responses.

[0263] In some embodiments, the method further includes administering an effective amount of at least one additional therapeutic agent.

[0264] In some embodiments, further therapeutic agents are agents targeting the PI3K / AKT / mTOR pathway, HSP90 inhibitors, tubulin inhibitors, apoptosis inhibitors, and / or chemopreventive agents. Further therapeutic agents may be one or more of the chemotherapeutic agents described herein. In some examples, the chemotherapeutic agent is a platinum-based chemotherapeutic agent such as carboplatin.

[0265] In some cases, the chemotherapy agent is a taxane (e.g., nab-paclitaxel (ABRAXANE®), paclitaxel, or docetaxel). In some cases, the taxane is nab-paclitaxel (ABRAXANE®). In some cases, nab-paclitaxel (ABRAXANE®) is administered at approximately 100 mg / m² weekly. 2 ~about 125mg / m 2 It is administered to the individual at the following dose. In some cases, nab-paclitaxel (ABRAXANE®) is administered weekly at approximately 100 mg / m². 2 It is administered to the individual at the following dose. As a general suggestion, the therapeutically effective dose of a taxane administered to humans (e.g., nab-paclitaxel (ABRAXANE®)) is approximately 25 to approximately 300 mg / m², whether administered in one or multiple doses. 2 (For example, approximately 25 mg / m²) 2 , about 50mg / m 2 , about 75mg / m 2 , about 100mg / m 2 , about 125mg / m 2 , about 150mg / m 2 , about 175mg / m 2 , about 200mg / m 2 , about 225mg / m 2 , about 250mg / m 2 , about 275mg / m 2 , or approximately 300 mg / m² 2 ) would be within the range of ). For example, in some embodiments, it is about 100 mg / m². 2 Nab-paclitaxel (ABRAXANE®) is administered. In some embodiments, nab-paclitaxel (ABRAXANE®) is administered once a week at a dose of 100 mg / m². 2 It is administered at approximately 125 mg / m². In some embodiments, it is administered at approximately 125 mg / m². 2 Paclitaxel is administered. In some embodiments, paclitaxel is administered at a dose of 200 mg / m² every three weeks. 2It is administered as follows. In some embodiments, taxanes (e.g., nab-paclitaxel (ABRAXANE®)) may be administered weekly, every two weeks, every three weeks, every four weeks, on days 1, 8 and 15 of each 21-day cycle, or on days 1, 8 and 15 of each 28-day cycle.

[0266] In some cases, a PD-1 axis-binding antagonist (e.g., anti-PD-L1 antibody, e.g., MPDL3280A) and a taxane (e.g., nab-paclitaxel (ABRAXANE®)) are administered in a single-dose regimen. The administration of these drugs may be simultaneous or separate within the context of the drug regimen. For example, in some cases, the method provided herein includes a drug regimen comprising a treatment cycle, in which the individual receives a dose of approximately 840 mg of a human PD-1 axis-binding antagonist on days 1 and 15 of each cycle, and approximately 100 mg / m² on days 1, 8, and 15 of each cycle. 2 Taxanes are administered at the specified dose, and each cycle is repeated every 28 days.

[0267] In some embodiments, the individual is human. In some embodiments, the individual has melanoma. In some embodiments, the individual has lung cancer, for example, non-small cell lung cancer. In some embodiments, the individual is human. In some embodiments, the individual has pancreatic cancer. In some embodiments, the individual is human. In some embodiments, the individual has gastric cancer. In some embodiments, the individual is human. In some embodiments, the individual has colorectal cancer. In some embodiments, the individual is human. In some embodiments, the individual has renal cell carcinoma. In some embodiments, the individual is human. In some embodiments, the individual has pancreatic cancer. In some embodiments, the individual has locally advanced or metastatic breast cancer. In some embodiments, metastatic breast cancer is mTNBC. In some embodiments, the individual has previously received two or fewer cytotoxic therapy regimens for cancer. In some embodiments, the individual has not previously received targeted systemic therapy for cancer. In some embodiments, the individual has cancer that is resistant to one or more cancer therapies. In some embodiments, resistance to cancer therapy includes recurrence of cancer or refractory cancer. Recurrence may mean the reappearance of cancer at the original site or a new site after treatment. In some embodiments, resistance to cancer therapy includes progression of cancer during treatment with anti-cancer therapy. In some embodiments, resistance to cancer therapy includes cancer that does not respond to treatment. Cancer may become resistant at the start of treatment or during treatment. In some embodiments, the cancer is in an early or late stage.

[0268] A PD-1 axially coupled antagonist and a second therapeutic agent, such as a taxane (e.g., nab-paclitaxel (ABRAXANE®)), may be administered in any preferred manner known in the art. For example, the PD-1 axially coupled antagonist and the second therapeutic agent, such as a taxane, may be administered sequentially (at different time points) or simultaneously (at the same time point). In some embodiments, the PD-1 axially coupled antagonist is present in a separate composition from the second therapeutic agent. In some embodiments, the PD-1 axially coupled antagonist is present in the same composition as the second therapeutic agent.

[0269] In some embodiments, the PD-1 axially coupled antagonist and a second therapeutic agent, such as a chemotherapeutic agent, such as a taxane, may be administered by the same route of administration or by different routes of administration. In some embodiments, the PD-1 axially coupled antagonist is administered intravenously, intramuscularly, subcutaneously, topically, orally, percutaneously, intraperitoneally, or orbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments, the taxane is administered intravenously, intramuscularly, subcutaneously, topically, orally, percutaneously, intraperitoneally, or orbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. An effective dose of the PD-1 axially coupled antagonist and, optionally, a second therapeutic agent may be administered for the prevention or treatment of the disease. The appropriate dosage of PD-1 axial antagonists and / or secondary therapies may be determined based on the type of disease being treated, the type of PD-1 axial antagonist and secondary therapy, the severity and course of the disease, the individual's clinical condition, the individual's clinical history and response to treatment, and the discretion of the attending physician.

[0270] In some embodiments, the method may further include additional therapeutic agents. These additional therapeutic agents may be radiotherapy, surgery (e.g., mammary tumor removal and mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination thereof. The additional therapy may be in the form of adjuvant therapy or neoadjuvant therapy. In some embodiments, the additional therapy is the administration of small molecule enzyme inhibitors or anti-metastatic agents. In some embodiments, the additional therapy is the administration of side effect limiting agents (e.g., drugs intended to reduce the occurrence and / or severity of side effects of treatment, e.g., antiemetics). In some embodiments, the additional therapy is radiotherapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiotherapy and surgery. In some embodiments, the additional therapy is gamma irradiation.

[0271] In some embodiments, the method further includes administering a platinum-based chemotherapeutic agent together with a PD-1 axis-coupled antagonist and a taxane. In some embodiments, the platinum-based chemotherapeutic agent is carboplatin. The dosage and administration of carboplatin are well known in the art. An exemplary dosage of carboplatin is administered at a target area under the curve (AUC) of 6 mg / ml. In some embodiments, carboplatin is administered intravenously every three weeks.

[0272] While we do not wish to be bound by theory, it is thought that tumor cell death may be promoted by enhancing T cell stimulation by promoting activating costimulatory molecules or inhibiting negative costimulatory molecules, thereby treating cancer or delaying cancer progression. In some embodiments, a PD-1 axis-coupled antagonist (e.g., anti-PD-1, e.g., MDX-1106 (nivolumab, Bristol-Myers Squibb) or MK-3475 (pembrolizumab, Merck), or anti-PD-L1 antibody, e.g., MPDL3280A (atezolizumab) or avelumab) may be administered in combination with an agonist against the activating costimulatory molecule. In some embodiments, the activating costimulatory molecule may include CD40, CD226, CD28, OX40, GITR, CD137, CD27, HVEM, or CD127. In some embodiments, the agonist against the activating costimulatory molecule is an agonist antibody that binds to CD40, CD226, CD28, OX40, GITR, CD137, CD27, HVEM, or CD127. In some embodiments, a PD-1 axis-conjugated antagonist (e.g., anti-PD-1, e.g., MDX-1106 or MK-3475, or anti-PD-L1 antibody, e.g., MPDL3280A) may be administered in combination with an antagonist against the inhibitory costimulatory molecule. In some embodiments, the inhibitory costimulatory molecule may include CTLA-4 (also known as CD152), PD-1, TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO, TIGIT, MICA / B, or arginase. In some embodiments, the antagonist to the inhibitory costimulatory molecule is CTLA-4, PD-1, TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO, TIGIT, MICA / B, or an antagonist antibody that binds to arginase.

[0273] In some embodiments, a PD-1 axially coupled antagonist (e.g., anti-PD-1, e.g., MDX-1106 or MK-3475, or anti-PD-L1 antibody, e.g., MPDL3280A) may be administered in combination with an antagonist against CTLA-4 (also known as CD152), e.g., a blocking antibody. In some embodiments, a PD-1 axially coupled antagonist may be administered in combination with ipilimumab (also known as MDX-010, MDX-101, or YERVOY®). In some embodiments, a PD-1 axially coupled antagonist may be administered in combination with tremelimumab (also known as tisilimucob or CP-675206). In some embodiments, a PD-1 axially coupled antagonist may be administered in combination with an antagonist against B7-H3 (also known as CD276), e.g., a blocking antibody. In some embodiments, the PD-1 axially coupled antagonist may be administered in combination with MGA271. In some embodiments, the PD-1 axially coupled antagonist may be administered in combination with an antagonist for TGF beta, such as meterimumab (also known as CAT-192), fresolimmab (also known as GC1008), or LY2157299.

[0274] In some embodiments, PD-1 axis-coupled antagonists may be administered in combination with therapies including adoptive transplantation of T cells expressing chimeric antigen receptors (CARs) (e.g., cytotoxic T cells or CTLs). In some embodiments, PD-1 axis-coupled antagonists may be administered in combination with therapies including adoptive transplantation of T cells containing dominant-negative TGF beta receptors, such as dominant-negative TGF beta type II receptors. In some embodiments, PD-1 axis-coupled antagonists may be administered in combination with therapies including the HERCREEM protocol (see, for example, ClinicalTrials.gov Identifier NCT00889954).

[0275] In some embodiments, the PD-1 axially coupled antagonist may be administered in combination with an agonist against CD137 (also known as TNFRSF9, 4-1BB, or ILA), such as an activating antibody. In some embodiments, the PD-1 axially coupled antagonist may be administered in combination with urelumab (also known as BMS-663513). In some embodiments, the PD-1 axially coupled antagonist may be administered in combination with an agonist against CD40, such as an activating antibody. In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with CP-870893. In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with an agonist against OX40 (also known as CD-134), such as an activating antibody. In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with an anti-OX40 antibody (e.g., AgonOX). In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with an agonist against CD27, e.g., an activating antibody. In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with CDX-1127. In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with an antagonist against indoleamine-2,3-dioxygenase (IDO). In some embodiments, the IDO antagonist is 1-methyl-D-tryptophan (also known as 1-D-MT).

[0276] In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with an antibody-drug conjugate. In some embodiments, the antibody-drug conjugate includes meltansine or monomethyl auristatin E (MMAE). In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with an anti-NaPi2b antibody-MMAE conjugate (also known as DNIB0600A or RG7599). In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with trastuzumab emtansine (T-DM1, ad-trastuzumab emtansine, or KADCYLA®, also known as Genentech). In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with DMUC5754A. In some embodiments, the PD-1 axis-conjugated antagonist and taxane may be administered in combination with an antibody-drug conjugate targeting the endothelin B receptor (EDNBR), such as an antibody against EDNBR conjugated with MMAE.

[0277] In some embodiments, PD-1 axially coupled antagonists and taxanes may be administered in combination with angiogenesis inhibitors. In some embodiments, PD-1 axially coupled antagonists and taxanes may be administered in combination with antibodies against VEGF, such as VEGF-A. In some embodiments, PD-1 axially coupled antagonists (e.g., anti-PD-L1 antibodies, e.g., MPDL3280A) and taxanes (e.g., nab-paclitaxel (ABRAXANE®)) may be administered in combination with bevacizumab (AVASTIN®, also known as Genetech). In some embodiments, PD-1 axially coupled antagonists and taxanes may be administered in combination with antibodies against angiopoietin 2 (also known as Ang2). In some embodiments, PD-1 axially coupled antagonists and taxanes may be administered in combination with MEDI3617.

[0278] In some embodiments, PD-1 axially coupled antagonists and taxanes may be administered in combination with anti-cancer drugs. In some embodiments, PD-1 axially coupled antagonists and taxanes may be administered in combination with agents targeting CSF-1R (also known as M-CSFR or CD115). In some embodiments, PD-1 axially coupled antagonists and taxanes may be administered in combination with anti-CSF-1R (also known as IMC-CS4). In some embodiments, PD-1 axially coupled antagonists and taxanes may be administered in combination with interferons, such as interferon alpha or interferon gamma. In some embodiments, PD-1 axially coupled antagonists and taxanes may be administered in combination with Loferon-A (also known as recombinant interferon alpha-2a). In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with GM-CSF (recombinant human granulocyte-macrophage colony-stimulating factor, also known as rhu GM-CSF, salglamostim, or LEUKINE®). In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with IL-2 (aldesleukin or PROLEUKIN®). In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with IL-12. In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with a CD20-targeting antibody. In some embodiments, the CD20-targeting antibody is obinutuzumab (also known as GA101 or GAZYVA®) or rituximab. In some embodiments, the PD-1 axis-conjugated antagonist and taxane may be administered in combination with an antibody that targets GITR. In some embodiments, the antibody that targets GITR is TRX518.

[0279] In some embodiments, PD-1 axis-conjugated antagonists and taxanes may be administered in combination with a cancer vaccine. In some embodiments, the cancer vaccine is a peptide cancer vaccine, which in some embodiments is a personalized peptide vaccine. In some embodiments, the peptide cancer vaccine is a polyvalent long-chain peptide, a multipeptide, a peptide cocktail, a hybrid peptide, or a peptide-pulsed dendritic cell vaccine (see, e.g., Yamada et al., Cancer Sci, 104:14-21, 2013). In some embodiments, PD-1 axis-conjugated antagonists and taxanes may be administered in combination with an adjuvant. In some embodiments, PD-1 axis-conjugated antagonists and taxanes may be administered in combination with a treatment including a TLR agonist, e.g., poly-ICLC (also known as HILTONOL®), LPS, MPL, or CpG ODN. In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with tumor necrosis factor (TNF) alpha. In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with IL-1. In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with HMGB1. In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with an IL-10 antagonist. In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with an IL-4 antagonist. In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with an IL-13 antagonist. In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with an HVEM antagonist. In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with an ICOS agonist, such as ICOS-L, or an agonist antibody against ICOS. In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with a CX3CL1-targeted therapy. In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with a CXCL9-targeted therapy.In some embodiments, PD-1 axially coupled antagonists and taxanes may be administered in combination with CXCL10-targeted therapy. In some embodiments, PD-1 axially coupled antagonists and taxanes may be administered in combination with CCL5-targeted therapy. In some embodiments, PD-1 axially coupled antagonists and taxanes may be administered in combination with LFA-1 or ICAM1 agonists. In some embodiments, PD-1 axially coupled antagonists and taxanes may be administered in combination with selectin agonists.

[0280] In some embodiments, PD-1 axial antagonists and taxanes may be administered in combination with targeted therapy. In some embodiments, PD-1 axial antagonists and taxanes may be administered in combination with B-Raf inhibitors. In some embodiments, PD-1 axial antagonists and taxanes may be administered in combination with vemurafenib (also known as ZELBORAF®). In some embodiments, PD-1 axial antagonists and taxanes may be administered in combination with dabrafenib (also known as TAFINLAR®). In some embodiments, PD-1 axial antagonists and taxanes may be administered in combination with erlotinib (also known as TARCEVA®). In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with an inhibitor of MEK, such as MEK1 (also known as MAP2K1) or MEK2 (also known as MAP2K2). In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with cobimetinib (also known as GDC-0973 or XL-518). In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with trametinib (also known as MEKINIST®). In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with an inhibitor of K-Ras. In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with an inhibitor of c-Met. In some embodiments, the PD-1 axial antagonist and taxane may be administered in combination with onartuzumab (also known as MetMAb). In some embodiments, the PD-1 axial antagonist and taxane may be administered in combination with an Alk inhibitor. In some embodiments, the PD-1 axial antagonist and taxane may be administered in combination with AF802 (also known as CH5424802 or alectinib). In some embodiments, the PD-1 axial antagonist and taxane may be administered in combination with a phosphatidylinositol 3-kinase (PI3K) inhibitor.In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with BKM120. In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with idelalisib (also known as GS-1101 or CAL-101). In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with perifosin (also known as KRX-0401). In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with an Akt inhibitor. In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with MK2206. In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with GSK690693. In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with GDC-0941. In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with an mTOR inhibitor. In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with sirolimus (also known as rapamycin). In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with temsirolimus (also known as CCI-779 or TORISEL®). In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with everolimus (also known as RAD001). In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with ridafololimus (also known as AP-23573, MK-8669, or defololimus). In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with OSI-027. In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with AZD8055. In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with INK128. In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with a dual PI3K / mTOR inhibitor.In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with XL765. In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with GDC-0980. In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with BEZ235 (also known as NVP-BEZ235). In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with BGT226. In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with GSK2126458. In some embodiments, the PD-1 axially coupled antagonist and taxane may be administered in combination with PF-04691502. In some embodiments, PD-1 axially coupled antagonists and taxanes may be administered in combination with PF-05212384 (also known as PKI-587).

[0281] The novel methods described herein can be used to determine the effectiveness of treatments containing PD-L1 axis-linked antagonists before tumor reduction can be detected. Furthermore, the novel methods described herein can be used to determine the effectiveness of treatments containing PD-L1 axis-linked antagonists by differentiating between progressive disease and pseudoprogression during treatment. In some cases, tumor size initially increases after treatment with PD-L1 axis-linked antagonists. This increase is not actually due to an increase in tumor volume, but rather the transient increase in tumor size is a result of the treatment, for example, an influx and / or proliferation of immune cells, commonly referred to as "pseudoprogression." The methods described herein can distinguish between such pseudoprogression and progressive disease.

[0282] VI. Products or kits In another embodiment of the present invention, a PD-1 axis-coupled antagonist (e.g., an anti-PD-L1 antibody, e.g., MPDL3280A) and granzyme B in a blood sample obtained from an individual are used. + CD4 + T cells and FOXP3 + CD4+ Granzyme B in individuals with a T cell ratio of 1 or more, or in blood samples obtained from such individuals. + CD4 + T cells and FOXP3 + CD4 + A manufactured product or kit is provided that includes a package insert containing instructions for using a PD-1 axis-linked antagonist to enhance the immune function of an individual with a T cell ratio of 1 or greater. Any of the PD-1 axis-linked antagonists described herein may be included in the manufactured product or kit.

[0283] Suitable containers for the product or kit include, for example, bottles, vials, bags, and syringes. Containers may be formed from a variety of materials such as glass, plastic (e.g., polyvinyl chloride or polyolefin), or metal alloys (e.g., stainless steel or Hastelloy). In some embodiments, the container holds the formulation, and labels on or attached to the container may provide instructions for use. The product or kit may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and accompanying documentation with instructions for use. In some embodiments, the product further includes one or more other agents (e.g., chemotherapeutic agents and antitumor agents). Suitable containers for these one or more agents include, for example, bottles, vials, bags, and syringes.

[0284] This specification is considered sufficient to enable those skilled in the art to carry out the invention. In addition to those shown and described herein, various modifications of the invention will be apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. [Examples]

[0285] The present invention will be better understood by reference to the following embodiments. However, these embodiments should not be considered to limit the scope of the invention. It should be understood that the embodiments and models described herein are for illustrative purposes only, and that various variations or modifications in light thereof will be suggested to those skilled in the art and will be included in the spirit and scope of this application and the appended claims.

[0286] Example 1 CD4 in an mMLR assay + T cells and allogeneic DCs To elucidate the underlying mechanism of PD-1 blockade, we investigated CD4 + CD4 can recognize allogeneic MHCII and initiate a detectable immune response. + We developed an in vitro functional assay focused on T cells. In this assay, called minimal MLR (mMLR), we co-cultured selected CD4 T cells obtained from healthy donors with monocyte-derived mature dendritic cells from unrelated donors.

[0287] [Minimal mixed lymphocyte reaction] We used freshly purified CD4 +We developed an assay for co-culturing T cells in the presence of monocyte-derived allogeneic mature dendritic cells (mDCs) for 5 days. Monocytes were isolated from fresh PBMCs one week prior to co-culturing by plastic adhesion followed by removal of non-adherent cells. We then cultured the monocytes in a medium containing GM-CSF (50 ng / ml) and IL-4 (100 ng / ml) for 5 days to generate immature DCs (iDCs). To induce iDC maturation, we added TNF-α, IL-1β, and IL-6 (50 ng / ml each) to the culture medium for an additional 2 days. We then evaluated the DC maturation by measuring the surface expression of major histocompatibility class II antigen (MHCII, eBioscience), CD80, CD83, and CD86 (all from BD Biosciences) by flow cytometry (LSR Fortessa, BD Biosciences). On the day of the minimal mixed lymphocyte response (mMLR), CD4 was measured using a microbead kit (Miltenyi Biotec) from 108 PBMCs obtained from an unrelated donor. + T cells were enriched. In the previous culture, CD4 + T cells were labeled with 5 μM carboxyfluorescein succinimidyl ester (CFSE). Then, 105 CD4 T cells were plated together with mature allogeneic DCs (5:1) in the presence or absence of a blocking anti-PD-1 antibody (either 0376 or MDX-1106 at a concentration of 10 μg / ml) in a 96-well U-bottom plate. By mMLR, allospecific CD4 was identified by day 5 of co-culture. + It became possible to induce high PD-1 expression levels in most T cells (Figure 1). [Statistics]** corresponds to P<0.01 calculated using one-way ANOVA (GraphPad Prism), and *** corresponds to P<0.001.

[0288] Example 2 PD-1 blockade is CD4 + Increases IFN-γ secretion and granzyme B production by T cells. Next, we determined the effect of the anti-PD-1 blocking antibody added to the mMLR culture described in Example 1. [Intracellular Cytokine Staining and ELISA] On day 5 of mMLR co-culture, we collected the cell culture supernatant and used it for IFN-γ level measurement by ELISA (R&D Systems, following manufacturer's instructions). The cells were left at 37°C for a further 5 hours in the presence of Golgi plugs (Brefelzin A, BD Bioscience) and Golgi stop (Monensin, BD Bioscience). The cells were then washed and the surface was stained with anti-human CD4 antibody and Live / Dead immobilization dye Aqua (Invitrogen) before fixation / permeabilization with Fix / Perm buffer (BD Bioscience). Next, intracellular staining was performed for granzyme B (BD Bioscience), IFN-γ, and IL-2 (both antibodies from eBioscience). By adding anti-PD-1 blocking antibody (Roche 0376) on day 1 after co-culture, the amount of IFN-γ released into the supernatant by day 5 could be increased (Figure 2), and surprisingly, compared to co-culture alone and in the presence of isotype controls, CD4 + We were able to induce significant production of granzyme B by T cells (Figure 3).

[0289] TIFF0007843689000005.tif102170TIFF0007843689000006.tif81170

[0290] Example 3 Cytotoxic CD4 in response to various anti-PD-1 axis blocking antibodies + T cell induction The anti-PD-1 antibodies 0376, MDX-1106 (nivolumab, Bristol-Myers Squibb), and MK-3475 (pembrolizumab, Merck) were compared in an mMLR assay essentially performed as described in Example 2. All antibodies tested were CD4 in the minimal MLR assay. + While they were comparable in inducing IFN-γ secretion by T cells (Figure 4A), 0376 was equivalent in cytotoxic granzyme B + CD4 + It showed excellent effects in inducing T cell proliferation and / or their appearance (Figure 4B). We also found that blocking the PD-1 ligand, PD-L1, with an anti-PD-L1 antibody (PD-L1 mulgG1 DAPG in Table 5) resulted in CD4 in minimal MLR assays. + We evaluated whether they had similar effects on T cells. All anti-PD-1 antibodies (0376, MDX-1106, and MK-3475) and anti-PD-L1 antibodies were CD4 + The anti-PD-L1 antibody induced IFN-γ secretion by T cells (Figure 4A, Figure 5A). + CD4 + This induced the emergence of T cells (Figures 4B and 5B).

[0291] Example 4 CD4 in peripheral blood of melanoma patients treated with nivolumab + T cell profile [Ex vivo phenotypic and functional characterization of PBMCs from melanoma patients] After thawing, PBMCs were subjected to 2 × 10⁶ per ml in the presence of Golgi plugs (Brefelzin A, BD Bioscience) and Golgi stop (Monensin, BD Bioscience). 5 ~10 6 The cells were resuspended in RPMI 10% FBS at concentrations within the specified range and incubated at 37°C for 12 hours. The PBMCs were then washed, and the surface was stained with anti-CD3 (BD ​​Horizon, BD Biosciences), anti-CD8 (BD Biosciences), and anti-CD4 antibodies (BioLegend). Subsequently, permeabilization / fixation was performed using a FOXP3 / transcription factor buffer set (eBioscience). The cells were intracellularly stained for FOXP3 (eBioscience), granzyme B (BD Bioscience), IFN-γ, IL-2 (both from eBioscience), and Ki67 (BD Pharmingen), and acquired using a flow cytometer (LSRFortessa, BD Biosciences). cytotoxic CD4 +T cells are present at very low frequencies in the peripheral blood of healthy individuals, and their numbers have been described as increasing during viral infections, in which they play a crucial role in the antiviral immune response (Appay, Clin. Exp. Immunol. 138(1):10-13 (2004)).

[0292] To confirm our in vitro findings in vivo, we studied cytotoxic CD4 + T cells (granzyme B + CD4 + We evaluated the functional and phenotypic profiles of PBMCs from a small cohort of melanoma patients treated with or not treated with anti-PD-1 antibody (MDX-1106) in search of T cells. After blocking cytokine secretion overnight, we stained cells with serial markers and performed intracellular staining for granzyme B. We evaluated cytotoxic CD4 in PBMCs collected in the last two months after the last treatment in patients treated with anti-PD-1 antibody (MDX-1106). + The T cell population was detected significantly (P=0.04). This CD4 + Cytotoxic T cell populations are virtually absent or present at very low frequencies in healthy donors (Figure 8).

[0293] In a specific case, we found that cytotoxic granzyme B was present in one melanoma patient (HG15443). + CD4 + Although we were able to detect T cells in the patient, the patient remained disease-free. However, we found that once the tumor evaded the immune surveillance mechanism, granzyme B was detected in the same patient (HG15952) at a later stage. + CD4 + We were unable to detect any T cell subpopulations (Figures 6A and 6B).

[0294] We investigated cytotoxic granzyme B in patients treated with anti-PD-1 antibodies. + CD4 + In parallel with the increase in the T cell population, regulatory T cells (Treg, FOXP3) + CD4 +We also observed a decrease in T cell frequency (Figure 6A). Therefore, we observed a decrease in cytotoxic CD4 + We calculated the T cell to Treg ratio. We found that two anti-PD-1 antibody-treated patients with disease-free survival periods of 3 months and 5.6 months received 4 and 45 granzyme B25 cells, respectively. + : Granzyme B having a Treg ratio greater than 1 + CD4 + We found a positive correlation between the T cell-to-Treg ratio and the clinical benefit of anti-PD-1 therapy. In contrast, healthy donors and untreated melanoma patients had a granzyme B:Treg ratio of ≤1 (Figure 7). Furthermore, granzyme B in relation to the Treg population... + CD4 + The size of the T cell population seemed to predict the persistence of the response. Patients with a ratio of 4 were observed to be disease-free for 3 months, while patients with a ratio of 45 were observed to be disease-free for 6 months (Figure 7). Therefore, granzyme B + CD4 + A larger population size of T cells predicted a longer disease-free response.

[0295] To the applicant's knowledge, this is the first time the mechanism of PD-1 blockade has been elucidated. The findings presented here have significant implications for therapies using anti-PD-1 axis-binding molecules, such as anti-PD1 and anti-PD-L1 antibodies. Granzyme B + CD4 + T-cell evaluation can serve as a biomarker for long-term monitoring of a patient's response to anti-PD-1 therapy. Importantly, cells can be isolated from peripheral blood, eliminating the need to isolate cells or tissues through invasive biopsy. Granzyme B + CD4 + The evaluation of T cells, more specifically the calculation of the granzyme B:Treg ratio described herein, may be applied by physicians to rapidly monitor whether a patient is responding to treatment with PD-1 axis-binding molecules, such as anti-PD-1 or anti-PD-L1 antibodies.

[0296] Cytotoxic CD4 as a factor in progression-free survival (PFS)+ The plot of T cell frequencies shows cytotoxic CD4 + A significant correlation (P=0.0163) was revealed between the size of the T cell population and the duration of disease stabilization or regression (Figure 9). This finding relates to cytotoxic CD4 + This indicates a positive correlation between the presence of T cells and the clinical benefit of anti-PD-1 therapy.

[0297] Surprisingly, in patients treated with anti-PD-1 antibodies, we found that cytotoxic CD4 + Alongside the increase in T cells, a decrease in the frequency of regulatory T cells (Tregs) was also observed. Therefore, we found that cytotoxic CD4 + We calculated the ratio of T cells to Treg cells and showed its relationship to disease-free survival. Interestingly, we found that cytotoxic CD4 + We found a very significant correlation between the T cell / Treg ratio and the delay in disease progression (P=0.0013) (Figure 10).

[0298] Patient stratification based on treatment and response to treatment showed that anti-PD-1 therapy compared to a pre-treatment time, indicated as baseline, with cytotoxic CD4 + We revealed that T cells versus Treg cells induced a significant increase (P=0.02) compared to the responder group. In addition, we found that Treg cells were significantly increased in patients responding to anti-PD-1 therapy compared to patients with progressive disease. loss Cytotoxic CD4 + There is a clear trend toward an increase in T cells (Figure 11).

[0299] This finding not only sheds light on the underlying mechanisms of PD-1 blockade but also provides a useful biomarker for long-term monitoring of patient responses to anti-PD-1 therapy by simply taking small amounts of peripheral blood. This tool will ultimately enable physicians to track and estimate in a timely manner whether patients are responding to anti-PD-1 treatment and to select specific combination strategies to increase the likelihood of patients initiating an effective tumor-specific immune response.

[0300] [Other embodiments] Although the aforementioned invention has been described in some detail by examples and embodiments for the purpose of clarifying understanding, the description and embodiments should not be construed as limiting the scope of the invention. All patent and scientific document disclosures cited herein are expressly incorporated in their entirety by attribution.

Claims

1. A method for determining whether an individual suffering from melanoma is responding to treatment including a PD-1 axis-binding antagonist, Granzyme B in samples obtained from the individual before and after the aforementioned treatment. ＋ CD4 ＋ T cells and FOXP3 ＋ CD4 ＋ This includes determining the ratio with T cells, An increase in the ratio after the treatment is performed compared to the ratio before the treatment is performed indicates that the individual is responding to treatment including a PD-1 axis-coupled antagonist. A method in which the PD-1 axis-conjugated antagonist is an anti-PD-1 antibody.

2. The method according to claim 1, wherein the increase is four times or more.

3. The method according to claim 1, wherein the increase is 45 times or more.

4. The method according to any one of claims 1 to 3, wherein the sample is a blood sample.

5. The method according to any one of claims 1 to 3, wherein the sample is a whole blood sample or a peripheral blood mononuclear cell sample.

6. The method according to any one of claims 1 to 5, wherein the individual's response to treatment is complete remission.

7. The method according to any one of claims 1 to 5, wherein the individual's response to the treatment is a partial response.

8. The method according to any one of claims 1 to 5, wherein the individual's response to treatment is a sustained response after discontinuation of treatment.

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