BR122024003085A2 - USE OF A PD-1 AXIS BINDING ANTAGONIST AND/OR ANTI-GPC3 ANTIBODY AND KIT - Google Patents

Abstract

"USE OF A PD-1 AXIS BINDING ANTAGONIST AND/OR ANTI-GPC3 ANTIBODY AND KIT". The present invention relates to compositions and methods for the treatment of cancers. The methods comprising administering a PD-1 axis binding antagonist and an anti-GPC3 antibody. The compositions comprising a pharmaceutical composition for the treatment of cancer comprising a PD-1 axis binding antagonist and an anti-GPC3 antibody. Also referred to are a pharmaceutical composition to be used in combination with a PD-1 axis binding antagonist for the treatment of cancer comprising an anti-GPC3 antibody as the active ingredient; and a pharmaceutical composition to be used in combination with an anti-GPC3 antibody for the treatment of cancer comprising as the active ingredient a PD-1 axis binding antagonist.

Description

FIELD OF INVENTION

[001] This invention relates to methods of treating cancer by administering a PD-1 axis binding antagonist and an anti-GPC3 antibody.

BACKGROUND OF THE INVENTION

[002] Hepatocellular cancer is reportedly the fifth leading cause of cancer deaths worldwide, accounting for approximately 600,000 deaths each year (Non-Patent Literature 1). Most patients with hepatocellular cancer die within 1 year of being diagnosed with the disease. Unfortunately, hepatocellular cancer cases are often diagnosed at an advanced stage that rarely responds to curative therapies. Furthermore, medical treatments including chemotherapy, chemoembolization, ablation, and proton beam therapy are insufficiently effective for such patients. Most patients exhibit disease recurrence with vascular invasion and multiple intrahepatic metastases, which rapidly progress to advanced stage. Their 5-year survival rates are only 7% (Non-Patent Literature 2). Patients with hepatocellular cancer amenable to local resection have relatively good prognoses, although their 5-year survival rates still remain at a level of 15% and 39% (Non-Patent Literature 3). Thus, there has been a demand in the art for new therapy for such a malignant disease, hepatocellular cancer.

[003] Hepatocellular cancer is reportedly responsible for more than 90% of primary liver cancer cases in Japan. Medical methods for treating such hepatocellular cancer include, for example, chemotherapy-based transcatheter arterial embolization (TAE) therapy, which involves inducing selective necrosis of hepatocellular cancer by injecting a mixture of an oil-based contrast medium (Lipiodol), an anticancer agent, and an occlusion substance (Gelfoam) into the hepatic artery (which functions as a nutrient supply pathway) resulting in occlusion of the nutrient artery. In addition, invasive methods are used, such as percutaneous ethanol injection, percutaneous microwave coagulation therapy, and radiofrequency ablation. Furthermore, clinical trials have been conducted in systemic chemotherapy using chemotherapeutic agents such as fluorouracil (5-FU), uracil-tegafur (UFT), mitomycin C (MMC), mitoxantrone (DHAD), adriamycin (ADR), epirubicin (EPI), and cisplatin (CDDP) alone or in combination with interferon (IFN) (Non-Patent Literature 4).

[004] Meanwhile, an orally active form of sorafenib (Nexavar, BAY43-9006) has been approved, which is more advantageously effective than the above-described chemotherapeutic agents, such that this agent blocks cancer cell growth by inhibiting Raf kinase in Raf/MEK/ERK signal transduction while the agent exerts antiangiogenic effects by targeting VEGFR-2, VEGFR-3, and PDGFR-.beta. tyrosine kinases. The efficacy of sorafenib was studied in two phase III multicenter placebo-controlled trials (Sorafenib Evaluation of HCC Randomized Protocol (SHARP) trial and Asia-Pacific trial) targeting advanced hepatocellular cancer. Sorafenib was confirmed to prolong survival durations, with a HR of 0.68, in both of these trials. In the SHARP trial, sorafenib prolonged the duration of survival to 10.7 months versus 7.9 months with placebo. In the Asian trial, this agent prolonged the duration to 6.5 months versus 4.2 months with placebo. The agent, however, had a low objective response and showed no prolongation of time to symptomatic progression, although the agent prolonged time to tumor progression (5.5 months versus 2.8 months in the European and American trial and 2.8 months versus 1.4 months in the Asian trial) on imaging. The Asian cohorts exhibited a shorter duration of survival prolongation, which is probably because their treatments were initiated at a slightly later stage in the disease process in the Asian region compared with Europe and the United States (Non-Patent Literature 5 and 6).

[005] As liver cancer progresses, its specific symptoms associated with liver dysfunction are usually observed, such as anorexia, weight loss, general malaise, palpable right hypochondrial mass, right hypochondrial pain, sensation of abdominal fullness, fever, and jaundice. Chemotherapeutic agents (e.g., sorafenib), however, have complications to be managed, including their inherent adverse reactions such as diarrhea or constipation, anemia, suppression of the immune system to cause infection or sepsis (with lethal severity), hemorrhage, cardiac toxicity, liver toxicity, renal toxicity, anorexia, and weight loss.

[006] Although particular early stage symptoms are not initially observed in liver cancer, its specific symptoms associated with liver dysfunction, such as anorexia, weight loss, general malaise, palpable right hypochondrial mass, right hypochondrial pain, sensation of abdominal fullness, fever and jaundice, are generally observed with the progression of liver cancer. According to clinical observation, such symptoms are enhanced by the use of chemotherapeutic agents. For example, anorexia in a patient with detectable liver cancer cells and symptoms such as weight loss associated with or independent of anorexia may be more enhanced by the administration of chemotherapeutic agents to the patient than without the use of chemotherapeutic agents. In some cases, the use of chemotherapeutic agents must be discontinued by the patient having such symptoms. These enhanced symptoms are impediments to treatments with chemotherapeutic agents. Thus, there has been a demand for the establishment of excellent therapy from the point of view, for example, of improving therapeutic effects or improving the QOL of patients to be treated.

[007] Glypican 3 (GPC3) is frequently expressed at an elevated level in liver cancer and as such, appears to be useful in identifying its functions in liver cancer or as a therapeutic or diagnostic target for liver cancer.

[008] Under the circumstances described above, drugs under development with GPC3 as a therapeutic target of liver cancer. A liver cancer drug comprising an anti-GPC3 antibody as an active ingredient has been developed, the antibody having antibody-dependent cellular cytotoxicity activity (hereinafter referred to as "ADCC") and/or complement-dependent cytotoxicity activity (hereinafter referred to as "CDC") against GPC3-expressing cells (Patent Literature 1). Furthermore, a GPC3-targeting drug comprising a humanized anti-GPC3 antibody having ADCC activity and CDC activity as an active ingredient has been developed (Patent Literature 2). Furthermore, GPC3-targeting drugs have been developed, which comprise a humanized anti-GPC3 antibody with enhanced ADCC activity (Patent Literatures 3 and 4) or an anti-GPC3 antibody having both ADCC activity and CDC activity as well as improved plasma dynamics (Patent Literature 5). These anti-GPC3 antibodies in combination therapy with chemotherapeutic agents such as sorafenib have been found to attenuate adverse reactions, e.g., caused by sole therapy of the chemotherapeutic agents (e.g., sorafenib) and have also been found to exhibit synergistic effects with these agents (Patent Literature 6). Consequently, excellent methods for the treatment of liver cancer are in the process of being established using GPC3-targeting drugs as the core from the viewpoint of, for example, improving therapeutic effects or improving QOL of patients to be treated.

[009] On the other hand, the provision of two distinct signals to T cells is a widely accepted model for lymphocyte activation of resting T lymphocytes by antigen presenting cells (APCs). Lafferty et al, Aust. J. Exp. Biol. Med. Sci 53: 27-42 (1975). This model also provides discrimination of self and non-self immunological intolerance. Bretscher et al, Science 169: 1042-1049 (1970); Bretscher, P. A., Proc. Nat. Acad. Sci. USA 96: 185-190 (1999); Jenkins et al, J. Exp. Med. 165: 302-319 (1987). The primary, or antigen-specific, signal is transduced by the T cell receptor (TCR) upon recognition of a foreign antigen peptide presented in the context of the major histocompatibility complex (MHC). The secondary, or costimulatory, signal is delivered to T cells by costimulatory molecules expressed on antigen-presenting cells (APCs), inducing T cells to undergo clonal expansion, cytokine secretion, and effector function. Lenschow et al., Ann. Rev. Immunol. 14:233 (1996). In the absence of costimulation, T cells may become refractory to antigen stimulation, fail to mount an effective immune response, and may also result in exhaustion or tolerance to foreign antigen.

[0010] In the two-signal model, T cells receive both positive and negative secondary costimulatory signals. Regulation of such positive and negative signals is critical for maximizing protective host immune responses while maintaining immune tolerance and preventing autoimmunity. Negative secondary signals appear necessary for the induction of T cell tolerance, while positive signals promote T cell activation. While the simple two-signal model provides a valid explanation for naïve lymphocytes, a host immune response is a dynamic process, and costimulatory signals can also be provided to antigen-exposed T cells. The mechanism of costimulation is of therapeutic interest because manipulation of costimulatory signals has been shown to provide a means to enhance or terminate the cell-based immune response. Recently, it has been discovered that T cell dysfunction or anergy occurs concurrently with induced or prolonged expression of the inhibitory receptor, programmed death polypeptide 1 (PD-1). As a result, therapeutic targeting of PD-1 and other molecules that signal through interactions with PD-1, such as programmed death ligand 1 (PD-L1) and programmed death ligand 2 (PD-L2), is an area of intense interest.

[0011] PD-L1 is overexpressed in most cancers and is frequently associated with poor prognosis (Okazaki T et al., Intern. Immun. 2007 19(7):813) (Thompson RH et al., Cancer Res 2006, 66(7):3381). Interestingly, most tumor-infiltrating T lymphocytes predominantly express PD-1, unlike normal tissue T lymphocytes and peripheral blood T lymphocytes, indicating that upregulation of PD-1 on tumor-reactive T cells may contribute to impaired antitumor immune responses (Blood 2009 114(8): 1537). This may be due to exploitation of PD-L1-mediated signaling by PD-L1-expressing tumor cells interacting with PD-1-expressing T cells to result in attenuation of T cell activation and evasion of immune surveillance (Sharpe et al., Nat Rev 2002) (Keir ME et al., 2008 Annu. Rev. Immunol. 26:677). Therefore, inhibition of the PD-L1/PD-1 interaction may enhance CD8+ T cell-mediated killing of tumors.

[0012] Therapeutic targeting of PD-1 and other signaling molecules that interact with PD-1, such as programmed death ligand 1 (PD-L1) and programmed death ligand 2 (PD-L2), is an area of intense interest. Inhibition of PD-L1 signaling has been proposed as a means to enhance T cell immunity to cancer treatment (e.g., tumor immunity) and infection, including both acute and chronic (e.g., persistent) inflammation. An ideal therapeutic treatment would combine blockade of PD-1 ligand/receptor interaction with an agent that directly inhibits tumor growth. There remains a need for an ideal therapy for the treatment, stabilization, prevention, and/or delay of development of various cancers.

[0013] All references cited herein, including patent applications, patent publications, and UniProtKB/Swiss-Prot accession numbers, are incorporated herein by reference in their entirety, as if each individual reference were specifically and individually indicated to be incorporated by reference.

[0014] The present invention provides:

[0015] [1] A pharmaceutical composition for treating or delaying the progression of cancer in a subject for use in combination with a PD-1 axis binding antagonist, said composition comprising an anti-GPC3 antibody as an active ingredient.

[0016] [2] The pharmaceutical composition according to [1], wherein the anti-GPC3 antibody is administered prior to administration of the PD-1 axis binding antagonist, simultaneously with administration of the PD-1 axis binding antagonist, or after administration of the PD-1 axis binding antagonist.

[0017] [3] The pharmaceutical composition according to [1] or [2], wherein the PD-1 axis binding antagonist is selected from the group consisting of a PD-1 binding antagonist, a PD-L1 binding antagonist and a PD-L2 binding antagonist.

[0018] [4] The pharmaceutical composition according to [3], wherein the PD-1 axis binding antagonist is a PD-1 binding antagonist.

[0019] [5] The pharmaceutical composition according to [4], wherein the PD-1 binding antagonist inhibits the binding of PD-1 to its ligand binding partners.

[0020] [6] The pharmaceutical composition according to [5], wherein the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1.

[0021] [7] The pharmaceutical composition according to [5], wherein the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L2.

[0022] [8] The pharmaceutical composition according to [5], wherein the PD-1 binding antagonist inhibits the binding of PD-1 to both PD-L1 and PD-L2.

[0023] [9] The pharmaceutical composition according to [5], wherein the PD-1 binding antagonist is an antibody.

[0024] [10] The pharmaceutical composition according to [5], wherein the PD-1 binding antagonist is selected from the group consisting of nivolumab, lambrolizumab (pembrolizumab), and pidilizumab.

[0025] [11] The pharmaceutical composition according to [2], wherein the PD-1 axis binding antagonist is a PD-L1 binding antagonist.

[0026] [12] The pharmaceutical composition according to [11], wherein the PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1.

[0027] [13] The pharmaceutical composition according to [11], wherein the PD-L1 binding antagonist inhibits the binding of PD-L1 to B7-1.

[0028] [14] The pharmaceutical composition according to [11], wherein the PD-L1 binding antagonist inhibits the binding of PD-L1 to both PD-1 and B7-1.

[0029] [15] The pharmaceutical composition according to any one of [12] to [14], wherein the PD-L1 binding antagonist is an anti-PD-L1 antibody.

[0030] [16] The pharmaceutical composition according to [11], wherein the PD-L1 binding antagonist is selected from the group consisting of: YW243.55.S70, Atezolizumab, MPDL3280A, MDX-1105, avelumab, and MEDI4736 (durvalumab).

[0031] [17] The pharmaceutical composition according to [15], wherein the anti-PD-L1 antibody comprises a heavy chain comprising HVR-H1 sequence of SEQ ID NO:19, HVR-H2 sequence of SEQ ID NO:20, and HVR-H3 sequence of SEQ ID NO:21; and a light chain comprising HVR-L1 sequence of SEQ ID NO:22, HVR-L2 sequence of SEQ ID NO:23, and HVR-L3 sequence of SEQ ID NO:24.

[0032] [18] The pharmaceutical composition according to [15], wherein the anti-PD-L1 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:25 or 26 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:4.

[0033] [19] The pharmaceutical composition according to [3], wherein the PD-1 axis binding antagonist is a PD-L2 binding antagonist.

[0034] [20] The pharmaceutical composition according to [19], wherein the PD-L2 binding antagonist is an antibody.

[0035] [21] The pharmaceutical composition according to [19], wherein the PD-L2 binding antagonist is an immunoadhesin.

[0036] [22] The pharmaceutical composition according to any one of [1] to [21], wherein the anti-GPC3 antibody comprises a heavy chain comprising HVR-H1 sequence of SEQ ID NO:34, HVR-H2 sequence of SEQ ID NO:35, and HVR-H3 sequence of SEQ ID NO:36; and a light chain comprising HVR-L1 sequence of SEQ ID NO:37, HVR-L2 sequence of SEQ ID NO:38, and HVR-L3 sequence of SEQ ID NO:39.

[0037] [23] The pharmaceutical composition according to any one of [1] to [21], wherein the anti-GPC3 antibody is capable of binding to an epitope to which a second antibody can bind, wherein said second antibody comprises a heavy chain comprising HVR-H1 sequence of SEQ ID NO:42, HVR-H2 sequence of SEQ ID NO:43, and HVR-H3 sequence of SEQ ID NO:44; and a light chain comprising HVR-L1 sequence of SEQ ID NO:45, HVR-L2 sequence of SEQ ID NO:46, and HVR-L3 sequence of SEQ ID NO:47.

[0038] [24] The pharmaceutical composition according to any one of [1] to [23] wherein the anti-GPC3 antibody is a humanized antibody.

[0039] [25] The pharmaceutical composition according to [24], wherein the anti-GPC3 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:50 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:52.

[0040] [26] The pharmaceutical composition according to any one of [1] to [25], wherein the cancer is selected from the group consisting of liver cancer, breast cancer, mammary cancer, ovarian cancer, gastric cancer, bladder cancer, pancreatic cancer, endometrial cancer, colon cancer, kidney cancer, esophageal cancer and prostate cancer.

[0041] The present invention also provides:

[0042] [27] A pharmaceutical composition for treating or delaying the progression of cancer in a subject for use in combination with an anti-GPC3 antibody, said composition comprising a PD-1 axis binding antagonist as an active ingredient.

[0043] [28] The pharmaceutical composition according to [27], wherein the anti-GPC3 antibody is administered prior to the administration of the PD-1 axis binding antagonist, simultaneously with the administration of the PD-1 axis binding antagonist, or after the administration of the PD-1 axis binding antagonist.

[0044] [29] The pharmaceutical composition according to [27] or [28], wherein the PD-1 axis binding antagonist is selected from the group consisting of a PD-1 binding antagonist, a PD-L1 binding antagonist and a PD-L2 binding antagonist.

[0045] [30] The pharmaceutical composition according to [29], wherein the PD-1 axis binding antagonist is a PD-1 binding antagonist.

[0046] [31] The pharmaceutical composition according to [30], wherein the PD-1 binding antagonist inhibits the binding of PD-1 to its ligand binding partners.

[0047] [32] The pharmaceutical composition according to [31], wherein the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1.

[0048] [33] The pharmaceutical composition according to [31], wherein the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L2.

[0049] [34] The pharmaceutical composition according to [31], wherein the PD-1 binding antagonist inhibits the binding of PD-1 to both PD-L1 and PD-L2.

[0050] [35] The pharmaceutical composition according to [31], wherein the PD-1 binding antagonist is an antibody.

[0051] [36] The pharmaceutical composition according to [31], wherein the PD-1 binding antagonist is selected from the group consisting of nivolumab, lambrolizumab (pembrolizumab), and pidilizumab.

[0052] [37] The pharmaceutical composition according to [28], wherein the PD-1 axis binding antagonist is a PD-L1 binding antagonist.

[0053] [38] The pharmaceutical composition according to [37], wherein the PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1.

[0054] [39] The pharmaceutical composition according to [37], wherein the PD-L1 binding antagonist inhibits the binding of PD-L1 to B7-1.

[0055] [40] The pharmaceutical composition according to [37], wherein the PD-L1 binding antagonist inhibits the binding of PD-L1 to both PD-1 and B7-1.

[0056] [41] The pharmaceutical composition according to any one of [38] to [40], wherein the PD-L1 binding antagonist is an anti-PD-L1 antibody.

[0057] [42] The pharmaceutical composition according to [37], wherein the PD-L1 binding antagonist is selected from the group consisting of: YW243.55.S70, Atezolizumab, MPDL3280A, MDX-1105, avelumab, and MEDI4736(durvalumab).

[0058] [43] The pharmaceutical composition according to [41], wherein the anti-PD-L1 antibody comprises a heavy chain comprising HVR-H1 sequence of SEQ ID NO:19, HVR-H2 sequence of SEQ ID NO:20, and HVR-H3 sequence of SEQ ID NO:21; and a light chain comprising HVR-L1 sequence of SEQ ID NO:22, HVR-L2 sequence of SEQ ID NO:23, and HVR-L3 sequence of SEQ ID NO:24.

[0059] [44] The pharmaceutical composition according to [41], wherein the anti-PD-L1 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:25 or 26 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:4.

[0060] [45] The pharmaceutical composition according to [29], wherein the PD-1 axis binding antagonist is a PD-L2 binding antagonist.

[0061] [46] The pharmaceutical composition according to [45], wherein the PD-L2 binding antagonist is an antibody.

[0062] [47] The pharmaceutical composition according to [45], wherein the PD-L2 binding antagonist is an immunoadhesin.

[0063] [48] The pharmaceutical composition according to any one of [27] to [47], wherein the anti-GPC3 antibody comprises a heavy chain comprising HVR-H1 sequence of SEQ ID NO:34, HVR-H2 sequence of SEQ ID NO:35, and HVR-H3 sequence of SEQ ID NO:36; and a light chain comprising HVR-L1 sequence of SEQ ID NO:37, HVR-L2 sequence of SEQ ID NO:38, and HVR-L3 sequence of SEQ ID NO:39.

[0064] [49] The pharmaceutical composition according to any one of [27] to [47], wherein the anti-GPC3 antibody is capable of binding to an epitope to which a second antibody can bind, wherein said second antibody comprises a heavy chain comprising HVR-H1 sequence of SEQ ID NO:42, HVR-H2 sequence of SEQ ID NO:43, and HVR-H3 sequence of SEQ ID NO:44; and a light chain comprising HVR-L1 sequence of SEQ ID NO:45, HVR-L2 sequence of SEQ ID NO:46, and HVR-L3 sequence of SEQ ID NO:47.

[0065] [50] The pharmaceutical composition according to any one of [27] to [49] wherein the anti-GPC3 antibody is a humanized antibody.

[0066] [51] The pharmaceutical composition according to [50], wherein the anti-GPC3 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:50 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:52.

[0067] [52] The pharmaceutical composition according to any one of [27] to [51], wherein the cancer is selected from the group consisting of liver cancer, breast cancer, mammary cancer, ovarian cancer, gastric cancer, bladder cancer, pancreatic cancer, endometrial cancer, colon cancer, kidney cancer, esophageal cancer and prostate cancer.

[0068] The present invention also provides:

[0069] [53] A pharmaceutical composition for treating or delaying the progression of cancer in a subject comprising a combination of a PD-1 axis binding antagonist and an anti-GPC3 antibody as an active ingredient.

[0070] [54] The pharmaceutical composition according to [53], wherein the pharmaceutical composition is a preparation combination.

[0071] [55] The pharmaceutical composition according to [53], wherein the anti-GPC3 antibody is administered prior to the administration of the PD-1 axis binding antagonist, simultaneously with the administration of the PD-1 axis binding antagonist, or after the administration of the PD-1 axis binding antagonist.

[0072] [56] The pharmaceutical composition according to [53] or [55], wherein the PD-1 axis binding antagonist is selected from the group consisting of a PD-1 binding antagonist, a PD-L1 binding antagonist and a PD-L2 binding antagonist.

[0073] [57] The pharmaceutical composition according to [56], wherein the PD-1 axis binding antagonist is a PD-1 binding antagonist.

[0074] [58] The pharmaceutical composition according to [57], wherein the PD-1 binding antagonist inhibits the binding of PD-1 to its ligand binding partners.

[0075] [59] The pharmaceutical composition according to [58], wherein the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1.

[0076] [60] The pharmaceutical composition according to [58], wherein the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L2.

[0077] [61] The pharmaceutical composition according to [58], wherein the PD-1 binding antagonist inhibits the binding of PD-1 to both PD-L1 and PD-L2.

[0078] [62] The pharmaceutical composition according to [58], wherein the PD-1 binding antagonist is an antibody.

[0079] [63] The pharmaceutical composition according to [58], wherein the PD-1 binding antagonist is selected from the group consisting of nivolumab, lambrolizumab (pembrolizumab), and pidilizumab.

[0080] [64] The pharmaceutical composition according to [55], wherein the PD-1 axis binding antagonist is a PD-L1 binding antagonist.

[0081] [65] The pharmaceutical composition according to [64], wherein the PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1.

[0082] [66] The pharmaceutical composition according to [64], wherein the PD-L1 binding antagonist inhibits the binding of PD-L1 to B7-1.

[0083] [67] The pharmaceutical composition according to [64], wherein the PD-L1 binding antagonist inhibits the binding of PD-L1 to both PD-1 and B7-1.

[0084] [68] The pharmaceutical composition according to any one of [65] to [67], wherein the PD-L1 binding antagonist is an anti-PD-L1 antibody.

[0085] [69] The pharmaceutical composition according to [64], wherein the PD-L1 binding antagonist is selected from the group consisting of: YW243.55.S70, Atezolizumab, MPDL3280A, MDX-1105, avelumab, and MEDI4736(durvalumab).

[0086] [70] The pharmaceutical composition according to [68], wherein the anti-PD-L1 antibody comprises a heavy chain comprising HVR-H1 sequence of SEQ ID NO:19, HVR-H2 sequence of SEQ ID NO:20, and HVR-H3 sequence of SEQ ID NO:21; and a light chain comprising HVR-L1 sequence of SEQ ID NO:22, HVR-L2 sequence of SEQ ID NO:23, and HVR-L3 sequence of SEQ ID NO:24.

[0087] [71] The pharmaceutical composition according to [68], wherein the anti-PD-L1 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:25 or 26 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:4.

[0088] [72] The pharmaceutical composition according to [56], wherein the PD-1 axis binding antagonist is a PD-L2 binding antagonist.

[0089] [73] The pharmaceutical composition according to [72], wherein the PD-L2 binding antagonist is an antibody.

[0090] [74] The pharmaceutical composition according to [72], wherein the PD-L2 binding antagonist is an immunoadhesin.

[0091] [75] The pharmaceutical composition according to any one of [53] to [74], wherein the anti-GPC3 antibody comprises a heavy chain comprising HVR-H1 sequence of SEQ ID NO:34, HVR-H2 sequence of SEQ ID NO:35, and HVR-H3 sequence of SEQ ID NO:36; and a light chain comprising HVR-L1 sequence of SEQ ID NO:37, HVR-L2 sequence of SEQ ID NO:38, and HVR-L3 sequence of SEQ ID NO:39.

[0092] [76] The pharmaceutical composition according to any one of [53] to [74], wherein the anti-GPC3 antibody is capable of binding to an epitope to which a second antibody can bind, wherein said second antibody comprises a heavy chain comprising HVR-H1 sequence of SEQ ID NO:42, HVR-H2 sequence of SEQ ID NO:43, and HVR-H3 sequence of SEQ ID NO:44; and a light chain comprising HVR-L1 sequence of SEQ ID NO:45, HVR-L2 sequence of SEQ ID NO:46, and HVR-L3 sequence of SEQ ID NO:47.

[0093] [77] The pharmaceutical composition according to any one of [53] to [76] wherein the anti-GPC3 antibody is a humanized antibody.

[0094] [78] The pharmaceutical composition according to [77], wherein the anti-GPC3 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:50 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:52.

[0095] [79] The pharmaceutical composition according to any one of [53] to [78], wherein the cancer is selected from the group consisting of liver cancer, breast cancer, mammary cancer, ovarian cancer, gastric cancer, bladder cancer, pancreatic cancer, endometrial cancer, colon cancer, kidney cancer, esophageal cancer and prostate cancer.

[0096] The present invention also provides:

[0097] [80] A pharmaceutical composition for enhancing immune responses against tumor cells in subjects for use in combination with a PD-1 axis binding antagonist, said composition comprising an anti-GPC3 antibody as an active ingredient.

[0098] [81] A pharmaceutical composition for enhancing immune responses against tumor cells in individuals for use in combination with an anti-GPC3 antibody, said composition comprising a PD-1 axis binding antagonist as an active ingredient.

[0099] [82] A pharmaceutical composition for enhancing immune responses against tumor cells in individuals comprising a combination of a PD-1 axis binding antagonist and an anti-GPC3 antibody as an active ingredient.

[00100] [83] The pharmaceutical composition according to any one of [80] to [82], wherein the anti-GPC3 antibody is administered prior to administration of the PD-1 axis binding antagonist, simultaneously with administration of the PD-1 axis binding antagonist, or after administration of the PD-1 axis binding antagonist.

[00101] [84] The pharmaceutical composition according to any one of [80] to [83], wherein the cancer is selected from the group consisting of liver cancer, breast cancer, mammary cancer, ovarian cancer, gastric cancer, bladder cancer, pancreatic cancer, endometrial cancer, colon cancer, kidney cancer, esophageal cancer and prostate cancer.

[00102] The present invention also provides:

[00103] [85] A method for treating or delaying the progression of cancer in a subject comprising administering to the subject an effective amount of a PD-1 axis binding antagonist and an anti-GPC3 antibody.

[00104] [86] The method according to [85], wherein the anti-GPC3 antibody is administered prior to administration of the PD-1 axis binding antagonist, simultaneously with administration of the PD-1 axis binding antagonist, or after administration of the PD-1 axis binding antagonist.

[00105] [87] The method according to [85] or [86], wherein the PD-1 axis binding antagonist is selected from the group consisting of a PD-1 binding antagonist, a PD-L1 binding antagonist and a PD-L2 binding antagonist.

[00106] [88] The method according to [87], wherein the PD-1 axis binding antagonist is a PD-1 binding antagonist.

[00107] [89] The method according to [88], wherein the PD-1 binding antagonist inhibits the binding of PD-1 to its ligand binding partners.

[00108] [90] The method according to [89], wherein the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1.

[00109] [91] The method according to [89], wherein the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L2.

[00110] [92] The method according to [89], wherein the PD-1 binding antagonist inhibits the binding of PD-1 to both PD-L1 and PD-L2.

[00111] [93] The method according to [89], wherein the PD-1 binding antagonist is an antibody.

[00112] [94] The method according to [89], wherein the PD-1 binding antagonist is selected from the group consisting of nivolumab, lambrolizumab (pembrolizumab) and pidilizumab.

[00113] [95] The method according to [81], wherein the PD-1 axis binding antagonist is a PD-L1 binding antagonist.

[00114] [96] The method according to [95], wherein the PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1.

[00115] [97] The method according to [95], wherein the PD-L1 binding antagonist inhibits the binding of PD-L1 to B7-1.

[00116] [98] The method according to [95], wherein the PD-L1 binding antagonist inhibits the binding of PD-L1 to both PD-1 and B7-1.

[00117] [99] The method according to any one of [96] to [98], wherein the PD-L1 binding antagonist is an anti-PD-L1 antibody.

[00118] [100] The method according to [95], wherein the PD-L1 binding antagonist is selected from the group consisting of: YW243.55.S70, Atezolizumab, MPDL3280A, MDX-1105, avelumab, and MEDI4736(durvalumab).

[00119] [101] The method according to [99], wherein the anti-PD-L1 antibody comprises a heavy chain comprising HVR-H1 sequence of SEQ ID NO:19, HVR-H2 sequence of SEQ ID NO:20, and HVR-H3 sequence of SEQ ID NO:21; and a light chain comprising HVR-L1 sequence of SEQ ID NO:22, HVR-L2 sequence of SEQ ID NO:23, and HVR-L3 sequence of SEQ ID NO:24.

[00120] [102] The method according to [99], wherein the anti-PD-L1 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:25 or 26 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:4.

[00121] [103] The method according to [87], wherein the PD-1 axis binding antagonist is a PD-L2 binding antagonist.

[00122] [104] The method according to [103], wherein the PD-L2 binding antagonist is an antibody.

[00123] [105] The method according to [103], wherein the PD-L2 binding antagonist is an immunoadhesin.

[00124] [106] The method according to any one of [85] to [105], wherein the anti-GPC3 antibody comprises a heavy chain comprising HVR-H1 sequence of SEQ ID NO:34, HVR-H2 sequence of SEQ ID NO:35, and HVR-H3 sequence of SEQ ID NO:36; and a light chain comprising HVR-L1 sequence of SEQ ID NO:37, HVR-L2 sequence of SEQ ID NO:38, and HVR-L3 sequence of SEQ ID NO:39.

[00125] [107] The method according to any one of [85] to [105], wherein the anti-GPC3 antibody is capable of binding to an epitope to which a second antibody can bind, wherein said second antibody comprises a heavy chain comprising HVR-H1 sequence of SEQ ID NO:42, HVR-H2 sequence of SEQ ID NO:43, and HVR-H3 sequence of SEQ ID NO:44; and a light chain comprising HVR-L1 sequence of SEQ ID NO:45, HVR-L2 sequence of SEQ ID NO:46, and HVR-L3 sequence of SEQ ID NO:47.

[00126] [108] The method according to any one of [85] to [107] wherein the anti-GPC3 antibody is a humanized antibody.

[00127] [127] [109] The method according to [108], wherein the anti-GPC3 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:50 and a light chain variable region comprising the amino acid sequence of SEQ ID NO:52.

[00128] [110] The method according to any one of [85] to [109], wherein the cancer is selected from the group consisting of liver cancer, breast cancer, mammary cancer, ovarian cancer, gastric cancer, bladder cancer, pancreatic cancer, endometrial cancer, colon cancer, kidney cancer, esophageal cancer, and prostate cancer.

[00129] The present invention also provides:

[00130] [111] A method for enhancing immune responses against tumor cells in subjects for use in combination with a PD-1 axis binding antagonist, said composition comprising an anti-GPC3 antibody as an active ingredient.

[00131] [112] A method for enhancing immune responses against tumor cells in subjects for use in combination with an anti-GPC3 antibody, said composition comprising a PD-1 axis binding antagonist as an active ingredient.

[00132] [113] A method for enhancing immune responses against tumor cells in individuals comprising a combination of a PD-1 axis binding antagonist and an anti-GPC3 antibody as an active ingredient.

[00133] [114] The method according to any one of [111] to [113], wherein the anti-GPC3 antibody is administered prior to administration of the PD-1 axis binding antagonist, concurrently with administration of the PD-1 axis binding antagonist, or after administration of the PD-1 axis binding antagonist.

[00134] [115] The method composition according to any one of [111] to [114], wherein the cancer is selected from the group consisting of liver cancer, breast cancer, mammary cancer, ovarian cancer, gastric cancer, bladder cancer, pancreatic cancer, endometrial cancer, colon cancer, kidney cancer, esophageal cancer, and prostate cancer.

[00135] The present invention also provides:

[00136] [116] A composition for treating or delaying the progression of cancer in a subject comprising a PD-1 axis binding antagonist and an anti-GPC3 antibody.

[00137] [117] The combination according to [116], wherein the anti-GPC3 antibody is administered prior to administration of the PD-1 axis binding antagonist, simultaneously with administration of the PD-1 axis binding antagonist, or after administration of the PD-1 axis binding antagonist.

[00138] The combination according to [116] or [117], wherein the cancer is selected from the group consisting of liver cancer, breast cancer, mammary cancer, ovarian cancer, gastric cancer, bladder cancer, pancreatic cancer, endometrial cancer, colon cancer, kidney cancer, esophageal cancer and prostate cancer.

[00139] The present invention also provides:

[00140] [119] A kit comprising (1) a pharmaceutical composition comprising an anti-GPC3 antibody as an active ingredient, (2) a container, and (3) a label or package insert comprising instructions for administration of the pharmaceutical composition in combination with a PD-1 axis binding antagonist for the treatment or delay of progression of a cancer in a subject.

[00141] [120] A kit comprising a first pharmaceutical composition comprising a PD-1 axis binding antagonist as an active ingredient and a second pharmaceutical composition comprising an anti-GPC3 antibody as an active ingredient.

[00142] [121] The kit according to [119] or [120], wherein the cancer is selected from the group consisting of liver cancer, breast cancer, mammary cancer, ovarian cancer, gastric cancer, bladder cancer, pancreatic cancer, endometrial cancer, colon cancer, kidney cancer, esophageal cancer, and prostate cancer.

[00143] The present invention also provides:

[00144] [122] Use of a PD-1 axis binding antagonist in the manufacture of a medicament for the treatment or delay of progression of cancer in a subject, wherein the medicament comprises the PD-1 axis binding antagonist and the optional pharmaceutically acceptable carrier, and wherein the treatment comprises administration of the medicament in combination with a composition comprising an anti-GPC3 antibody and the optional pharmaceutically acceptable carrier.

[00145] [123] Use of an anti-GPC3 antibody in the manufacture of a medicament for the treatment or delay of progression of cancer in a subject, wherein the medicament comprises the anti-GPC3 antibody and the optional pharmaceutically acceptable carrier, and wherein the treatment comprises administration of the medicament in combination with a composition comprising a PD-1 axis binding antagonist and the optional pharmaceutically acceptable carrier.

SUMMARY OF THE INVENTION

[00146] The inventors have discovered that by combining an anti-GPC3 antibody with a human PD-1 axis binding antagonist, better therapeutic effects can be obtained in a cancer patient than when such an anti-GPC3 antibody or a human PD-1 axis binding antagonist is used alone.

[00147] In one aspect, provided herein is a method for treating or delaying the progression of cancer in a subject comprising administering to the subject an effective amount of a human PD-1 axis binding antagonist and an anti-GPC3 antibody.

[00148] In another aspect, provided herein is a method of enhancing immune responses against tumor cells in a subject having cancer comprising administering an effective amount of a PD-1 axis binding antagonist and an anti-GPC3 antibody. For example, enhanced immune response against tumor cells includes infiltration of immune cells including macrophages and multinucleated giant cells into tumor tissues. For another example, enhanced immune response against tumor cells includes increasing CD45-positive lymphocytes, CD3ε-positive lymphocytes, and CD8-positive T lymphocytes in tumor infiltrating lymphocytes (TILs).

[00149] In another aspect, provided herein is use of a human PD-1 axis binding antagonist in the manufacture of a medicament for treating or delaying the progression of cancer in a subject, wherein the medicament comprises the human PD-1 axis binding antagonist and the optional pharmaceutically acceptable carrier, and wherein the treatment comprises administering the medicament in combination with a composition comprising an anti-GPC3 antibody and the optional pharmaceutically acceptable carrier.

[00150] In another aspect, provided herein is use of an anti-GPC3 antibody in the manufacture of a medicament for treating or delaying the progression of cancer in a subject, wherein the medicament comprises the anti-GPC3 antibody and the optional pharmaceutically acceptable carrier, and wherein the treatment comprises administering the medicament in combination with a composition comprising a human PD-1 axis binding antagonist and the optional pharmaceutically acceptable carrier.

[00151] In another aspect, provided herein is a composition comprising a human PD-1 axis binding antagonist and the optional pharmaceutically acceptable carrier for use in treating or delaying the progression of cancer in a subject, wherein the treatment comprises administering said composition in combination with a second composition, wherein the second composition comprises an anti-GPC3 antibody and the optional pharmaceutically acceptable carrier.

[00152] In another aspect, provided herein is a composition comprising an anti-GPC3 antibody and the optional pharmaceutically acceptable carrier for use in treating or delaying the progression of cancer in a subject, wherein the treatment comprises administering said composition in combination with a second composition, wherein the second composition comprises a human PD-1 axis binding antagonist and the optional pharmaceutically acceptable carrier.

[00153] In another aspect, provided herein is a kit comprising a medicament comprising a PD-1 axis binding antagonist and the optional pharmaceutically acceptable carrier, and a package insert comprising instructions for administering the medicament in combination with a composition comprising an anti-GPC3 antibody and the optional pharmaceutically acceptable carrier for treating or delaying the progression of cancer in a subject.

[00154] In another aspect, provided herein is a kit comprising a first medicament comprising a PD-1 axis binding antagonist and the optional pharmaceutically acceptable carrier, and a second medicament comprising an anti-GPC3 antibody and the optional pharmaceutically acceptable carrier. In some embodiments, the kit also comprises a package insert comprising instructions for administration of the first medicament and the second medicament for treating or delaying the progression of cancer in a subject.

[00155] In another aspect, provided herein is a kit comprising a medicament comprising an anti-GPC3 antibody and the optional pharmaceutically acceptable carrier, and a package insert comprising instructions for administering the medicament in combination with a composition comprising a PD-1 axis binding antagonist and the optional pharmaceutically acceptable carrier for treating or delaying the progression of cancer in a subject.

[00156] In some embodiments of the methods, use, compositions, and kits described above and herein, the PD-1 axis binding antagonist is selected from the group consisting of a PD-1 binding antagonist, a PD-L1 binding antagonist, and a PD-L2 binding antagonist. In some embodiments, the PD-1 axis binding antagonist is an antibody. In some embodiments, the antibody is a humanized antibody, chimeric antibody, or human antibody. In some embodiments, the antibody is an antigen-binding fragment. In some embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, scFv, and Fv.

[00157] 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 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 PD-1 binding antagonist is selected from the group consisting of MDX-1106 (nivolumab), MK-3475 (pembrolizumab, lambrolizumab), CT-011 (pidilizumab), PDR001, REGN2810, BGB A317, SHR-1210, AMP-514 (MEDI0680), and AMP-224.

[00158] In some embodiments, the PD-1 axis binding antagonist is a PD-L1 binding antagonist. 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 PD-L1 binding antagonist is selected from the group consisting of: YW243.55.S70, Atezolizumab, MPDL3280A, MDX-1105, avelumab, and MEDI4736 (durvalumab). In some embodiments, the anti-PD-L1 antibody comprises a heavy chain comprising HVR-H1 sequence of SEQ ID NO:19, HVR-H2 sequence of SEQ ID NO:20, and HVR-H3 sequence of SEQ ID NO:21; and/or a light chain comprising HVR-L1 sequence of SEQ ID NO:22, HVR-L2 sequence of SEQ ID NO:23, and HVR-L3 sequence of SEQ ID NO:24. In some embodiments, the anti-PD-L1 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:25 or 26 and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO:4. In some embodiments, the anti-PD-L1 antibody comprises the three antibody heavy chain HVR sequences YW243.55.S70 and/or the three antibody light chain HVR sequences YW243.55.S70 described in WO2010/077634 and U.S. Patent No. 8,217,149 , which are incorporated herein by reference. In some embodiments, the anti-PD-L1 antibody comprises the antibody heavy chain variable region sequence YW243.55.S70 and/or the antibody light chain variable region sequence YW243.55.S70. In some embodiments, the anti-PD-L1 antibody is Atezolizumab.

[00159] In some embodiments, the PD-1 axis binding antagonist is a PD-L2 binding antagonist. In some embodiments, the PD-L2 binding antagonist is an antibody. In some embodiments, the PD-L2 binding antagonist is an immunoadhesion.

[00160] In some embodiments of the methods, uses, compositions, and kits described above and herein, the anti-GPC3 antibody is a humanized antibody, chimeric antibody, or a human antibody. In some embodiments, the antibody is an antigen-binding fragment. In some embodiments, the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, scFv, and Fv.

[00161] In some embodiments, the anti-GPC3 antibody is GC33 or codrituzumab. In some embodiments, the anti-GPC3 antibody comprises a heavy chain comprising HVR-H1 sequence of SEQ ID NO:42, HVR-H2 sequence of SEQ ID NO:43, and HVR-H3 sequence of SEQ ID NO:44; and/or a light chain comprising HVR-L1 sequence of SEQ ID NO:45, HVR-L2 sequence of SEQ ID NO:46, and HVR-L3 sequence of SEQ ID NO:47. In some embodiments, the anti-GPC3 antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:50 and/or a light chain variable region comprising the amino acid sequence of SEQ ID NO:51. In some embodiments, the anti-GPC3 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:50 and/or a light chain comprising the amino acid sequence of SEQ ID NO:52. In some embodiments that can be combined with any other embodiments, the anti-GPC3 antibody is not GC33 or codrituzumab.

[00162] In some embodiments, the antibody described herein (e.g., a PD-1 axis binding antagonist antibody or an anti-GPC3 antibody) comprises an aglycosylation site mutation. In some embodiments, the aglycosylation site mutation is a substitution mutation. In some embodiments, the substitution mutation is at an amino acid residue N297, L234, L235, and/or D265 (EU numbering). In some embodiments, the substitution mutation is selected from the group consisting of N297G, N297A, L234A, L235A, and D265A. In some embodiments, the substitution mutation is a D265A mutation and an N297G mutation. In some embodiments, the aglycosylation site mutation reduces the effector function of the antibody. In some embodiments, the PD-1 axis binding antagonist (e.g., an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-PD-L2 antibody) is a human IgG1 having an Asn to Ala substitution at position 297 according to EU numbering.

[00163] In some embodiments of the methods, uses, compositions, and kits described above and herein, the cancer is a GPC3-positive cancer. In some embodiments, the cancer is liver cancer, breast cancer, ovarian cancer, gastric cancer, bladder cancer, pancreatic cancer, endometrial cancer, colon cancer, kidney cancer, esophageal cancer, prostate cancer, or other cancers described herein. In some embodiments, the subject has cancer or has been diagnosed with cancer. In some embodiments, the cancer cells in the subject express PD-L1.

[00164] In some embodiments of the methods, uses, compositions, and kits described above and herein, treatment or administration of the human PD-1 axis binding antagonist and the anti-GPC3 antibody results in a prolonged response in the subject after cessation of treatment. In some embodiments, the anti-GPC3 antibody is administered before the PD-1 axis binding antagonist, simultaneous with the PD-1 axis binding antagonist, or after the PD-1 axis binding antagonist. In some embodiments, the PD-1 axis binding antagonist and the anti-GPC3 antibody are in the same composition. In some embodiments, the PD-1 axis binding antagonist and the anti-GPC3 antibody are in separate compositions.

[00165] In some embodiments of the methods, uses, compositions, and kits described above and herein, the PD-1 axis binding antagonist and/or the anti-GPC3 antibody is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments of the methods, uses, compositions, and kits described above and herein, the treatment also comprises administering a chemotherapeutic agent for treating or delaying the progression of cancer in a subject. In some embodiments, the subject has been treated with a chemotherapeutic agent prior to the combination treatment with the PD-1 axis binding antagonist and the anti-GPC3 antibody. In some embodiments, the subject treated with the combination of the PD-1 axis binding antagonist and/or the anti-GPC3 antibody is refractory to treatment with a chemotherapeutic agent. Some embodiments of the methods, uses, compositions, and kits described throughout the application also comprise administering a chemotherapeutic agent for treating or delaying the progression of cancer.

[00166] In some embodiments of the methods, uses, compositions, and kits described above and herein, CD8 T cells in the subject have priming, activation, proliferation, and/or cytolytic activity relative to before administration of the combination. In some embodiments, the number of CD8 T cells is elevated relative to before administration of the combination. In some embodiments, the CD8 T cell is an antigen-specific CD8 T cell.

[00167] It is to be understood that one, some, or all of the properties of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the invention will become apparent to one of ordinary skill in the art. These and other embodiments of the invention are further described by the detailed description that follows.

BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1]

[00168] Figure 1 is a diagram showing Hepa1-6 expressing human GPC3 tumor volume changes in each of the mice treated three times weekly by vehicle control, 1 mg/kg, or 5 mg/kg mGC33. The arrow indicates the date of injection. Five mice per each group were treated. [Figure 2A]

[00169] Figure 2A is a diagram showing hematoxylin and eosin (HE) staining or F4/80 immunohistochemical (IHC) staining images of tissues isolated from mice treated with either vehicle control or 5 mg/kg mGC33. [Figure 2B]

[00170] Figure 2B is a diagram showing hematoxylin and eosin (HE) staining or PD-L1 histochemical staining (IHC) images of tissues isolated from mice treated with either vehicle control or 5 mg/kg mGC33. The arrow indicates PD-L1 immunoreactivity observed on cell membranes of infiltrated immune cells. [Figure 3A]

[00171] Figure 3A is a diagram showing CT26 expressing human GPC3 tumor volume changes in each of the mice treated by either monotherapy (mGC33 or 10F.9G2 (anti-PD-L1)) or combination (mGC33 + 10F.9G2). The arrow indicates the date of injection. Five mice per group were treated. Mean tumor volume in each group and SD bar were plotted. [Figure 3B]

[00172] Figure 3B is a diagram showing the progression-free survival rate in CT26/hGPC3-bearing mice treated by either monotherapy (mGC33 or 10F.9G2 (anti-PD-L1)) or combination (mGC33 + 10F.9G2). Progression was defined when the tumor size reached more than 100 mm3. [Figure 4A]

[00173] Figure 4A is a diagram showing the CT26 expressing human GPC3 tumor volume changes in each of the mice treated by either monotherapy or combination. The arrow indicates the date of injection. Five mice per group were treated. Mean tumor volume in each group and SD bar were plotted. The tumor growth inhibition values of 5 mg/kg or 25 mg/kg mGC33, 10F.9G2, 5 mg/kg or 25 mg/kg mGC33 + 10F.9G2 were 52%, 58%, 68%, 75% and 85%, respectively. [Figure 4B]

[00174] Figure 4B is a diagram showing individual tumor volume at day 29. Mean (*) tumor volume in each group was also plotted. [Figure 5A]

[00175] Figure 5A is a diagram showing Hepa1-6 expressing human GPC3 tumor volume changes in each of the mice treated with either vehicle control, 1 mg/kg, 5 mg/kg, or 25 mg/kg mGC33, 10F.9G2, or the combination of 5 mg/kg or 25 mg/kg GC33 and 10F.9G2. The arrow indicates the date of injection. Five mice per group were treated. [Figure 5B]

[00176] Figure 5B is a diagram showing the individual tumor area or mean + SD of tumor area in each treated group on day 34. The tumor area (mm2) was calculated by (length (mm) of long axis of tumor tissue) × (length (mm) of short axis of tumor tissue) after HE staining of tumor tissues isolated from each of the mice, and the pathological evaluation results of each of the tumor tissues were added at the bottom of the graphs. Pathological process of disease (pPD) was defined as "no tumor regression was observed". Pathological partial regression (pPR) was defined as "degeneration and/or necrosis of tumor cells with immune cell infiltration was observed". Pathological complete regression (pCR) was defined as "no tumor cells were observed at the tumor implantation site". [Figure 6A]

[00177] Figure 6A is a diagram showing Hepa1-6 expressing human GPC3 tumor volume changes in each of the mice treated with either monotherapy or combination of mGC33 and 6E11 (anti-PD-L1) at various dose levels and schedules indicated. The arrow indicates the date of injection. Five mice per group were treated. [Figure 6B]

[00178] Figure 6B is a diagram showing the mean values of Hepa1-6 expressing human GPC3 tumor volume changes in each group of mice treated by either mGC33 and 6E11 monotherapy or combination. Tumor growth in the group was significantly inhibited compared with that in the groups treated by either GC33 or 6E11 monotherapy. The arrow indicates the date of injection. Five mice per group were treated. *: P < 0.05 by Wilcoxon (SAS Institute Inc.) [Figure 6C]

[00179] Figure 6C is a diagram showing the mean values of body weight changes in each of the treatment groups of mice carrying Hepa1-6 expressing human GPC3. SD bars are added. [Figure 7]

[00180] Figure 7 is a diagram showing photomicrographs of subcutaneous tumor tissues in each group. The gray area indicates the area of tumor or lymphoid/granulation tissues without necrosis. The blue area indicates the area of tumor or granulation tissue with necrosis. The dotted line indicates the edge of the mass. L means lymphoid tissue, G means granulation tissue, pCR means pathologic complete response (no tumor tissue on the histopathology slide), and MOR means moribund sacrifice animal caused by tumor progression. [Figure 8A]

[00181] Figure 8A is a diagram showing representative photomicrographs of HE-stained tumor tissues at the periphery of fitting stroma in each of the treated groups. Tumor necrosis with infiltration of immune cells including macrophages/multinucleated giant cells is observed in all treated groups. The severity of the lesions is in the following order: mGC33 5 mg/kg plain + 6E11 q7d x 3 > mGC33 5 mg/kg plain or 6E11 q7d x 3. S stands for stroma fitting tumor mass, and P stands for periphery of tumor mass. [Figure 8B]

[00182] Figure 8B is a diagram showing representative IHC photomicrographs of F4/80 tumor tissues at the periphery of mass fitting stroma in each of the treated groups. In vehicle, F4/80 positive cells are mainly observed in the stroma. On the other hand, increased severity of F4/80 positive immune cells including macrophages/multinucleated giant cells are mainly observed at the periphery of tumor mass in all treated groups. The severity of positive cell infiltration is in the following order: mGC33 5 mg/kg plain + 6E11 q7d x 3 > mGC33 5 mg/kg plain or 6E11 q7d x 3. S stands for stroma fitting tumor mass, and P stands for periphery of tumor mass. [Figure 8C]

[00183] Figure 8C is a diagram showing representative IHC photomicrographs of PD-L1 from tumor tissues at the periphery of mass fitting stroma in each of the treated groups. In vehicle, PD-L1 positive reactions are mainly observed in the cytoplasm of tumor cells with weak intensity. On the other hand, increased severity of PD-L1 positive reactions in immune cells including macrophages/multinucleated giant cells are observed with weak to medium intensity in all treated groups. S stands for stroma fitting tumor mass, and P stands for periphery of tumor mass. [Figure 9]

[00184] Figure 9 is a diagram showing the percentage of marker positive cells observed in tumor tissues in each of the treated groups. Individual values and means in each of the groups are plotted and mean values are indicated in each of the graphs.

DETAILED DESCRIPTION

[00185] The data in the application show that the combination of an anti-GPC3 antibody with anti-PD-L1 immune therapy resulted in enhanced tumor growth inhibition, increased response rates, and durable responses. The inventors demonstrated the benefit of combining the two therapies: cytotoxic activity against tumor cells expressing PD-1 together with inhibition of T cell suppressive PD-1/PD-L1 signaling results in enhanced therapeutic effects and durable long-term responses.

[00186] In one aspect, provided herein are methods, compositions, and uses for treating or delaying the progression of cancer in a subject comprising administering an effective amount of a human PD-1 axis binding antagonist and an anti-GPC3 antibody.

[00187] In another aspect, provided herein are methods, compositions, and uses for enhancing immune responses against tumor cells in a subject having cancer comprising administering an effective amount of a human PD-1 axis binding antagonist and an anti-GPC3 antibody. For example, enhanced immune response against tumor cells includes infiltration of immune cells including macrophages and multinucleated giant cells into tumor tissues. For another example, enhanced immune response against tumor cells includes increasing CD45-positive lymphocytes, CD3ε-positive lymphocytes, and CD8-positive T lymphocytes in tumor infiltrating lymphocytes (TILs).

Definitions

[00188] Before describing the invention in detail, it should be understood that this invention is not limited to particular compositions or biological systems, which may, of course, vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[00189] As used in the specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless clearly indicated otherwise. Thus, for example, reference to "a molecule" optionally includes a combination of two or more such molecules, and the like.

[00190] The term "about" as used herein refers to the usual error range for the respective value readily known to the person skilled in the art. Reference to "about" a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se.

[00191] It is understood that aspects and embodiments of the invention described herein include "comprising", "consisting", and "consisting essentially of" aspects and embodiments.

[00192] The term "PD-1 axis binding antagonist" refers to a molecule that inhibits the interaction of a PD-1 axis binding partner with one or more of its binding partners, so as to remove T cell dysfunction resulting from PD-1 signaling axis signaling - with one result being to restore or enhance T cell function (e.g., proliferation, cytokine production, target cell killing). As used herein, a PD-1 axis binding antagonist includes a PD-1 binding antagonist, a PD-L1 binding antagonist, and a PD-L2 binding antagonist.

[00193] The term "PD-1 binding antagonist" refers to a molecule that decreases, blocks, inhibits, abrogates, or interferes with signal transduction that results from the interaction of PD-1 with one or more of its binding partners, such as PD-L1, PD-L2. In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to one or more of its binding partners. In a specific aspect, the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1 and/or PD-L2. For example, PD-1 inhibition antagonists include anti-PD-1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that decrease, block, inhibit, abrogate, or interfere with signal transduction that results from the interaction of PD-1 with PD-L1 and/or PD-L2. In one embodiment, a PD-1 binding antagonist reduces the negative costimulatory signal mediated through cell surface expressed T lymphocyte-mediated signaling through PD-1 so as to render a dysfunctional T cell less dysfunctional (e.g., enhancing effective responses to antigen recognition). In some embodiments, the PD-1 binding antagonist is an anti-PD-1 antibody. In a specific aspect, a PD-1 binding antagonist is MDX-1106 (nivolumab) described herein. In another specific aspect, a PD-1 binding antagonist is MK-3475 (lambrolizumab) described herein. In another specific aspect, a PD-1 binding antagonist is CT-011 (pidilizumab) described herein. In another specific aspect, a PD-1 binding antagonist is AMP-224 or AMP-514 (MEDI0680) described herein. In another specific aspect, a PD-1 antagonist is selected from the group consisting of PDR001, REGN2810, BGB A317, and SHR-1210 described herein.

[00194] The term "PD-L1 binding antagonist" refers to a molecule that decreases, blocks, inhibits, abrogates, or interferes with signal transduction that results from the interaction of PD-L1 with one or more of its binding partners, such as PD-1, 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 a specific aspect, the PD-L1 binding antagonist inhibits the binding of PD-L1 to PD-1 and/or B7-1. In some embodiments, PD-L1 binding antagonists include anti-PD-L1 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that decrease, block, inhibit, abrogate, or interfere with signal transduction that results from the interaction of PD-L1 with one or more of its binding partners, such as PD-1, B7-1. In one embodiment, a PD-L1 binding antagonist reduces the negative costimulatory signal mediated by or through cell surface proteins expressed in T lymphocyte-mediated signaling through PD-L1 so as to render a dysfunctional T cell less dysfunctional (e.g., enhancing effective responses to antigen recognition). In some embodiments, a PD-L1 binding antagonist is an anti-PD-L1 antibody. In a specific aspect, an anti-PD-L1 antibody is YW243.55.S70 or Atezolizumab described herein. In another specific aspect, an anti-PD-L1 antibody is MDX-1105 described herein. In another specific aspect, an anti-PD-L1 antibody is avelumab described herein. In yet another specific aspect, an anti-PD-L1 antibody is MPDL3280A described herein. In yet another specific aspect, an anti-PD-L1 antibody is MEDI4736 (durvalumab) described herein.

[00195] The term "PD-L2 binding antagonist" refers to a molecule that decreases, blocks, inhibits, abrogates, or interferes with signal transduction that results 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 a specific aspect, the PD-L2 binding antagonist inhibits the binding of PD-L2 to PD-1. In some embodiments, PD-L2 antagonists include anti-PD-L2 antibodies, antigen-binding fragments thereof, immunoadhesins, fusion proteins, oligopeptides, and other molecules that decrease, block, inhibit, abrogate, or interfere with signal transduction that results 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 the negative costimulatory signal mediated by or through cell surface proteins expressed in T lymphocyte-mediated signaling through PD-L2 so as to render a dysfunctional T cell less dysfunctional (e.g., by enhancing effective responses to antigen recognition). In some embodiments, a PD-L2 binding antagonist is an immunoadhesin.

[00196] The term "dysfunction" in relation to immune dysfunction, refers to a state of reduced responsiveness to antigenic stimulation. The term includes the common elements of both exhaustion and/or anergy in that antigen recognition can occur, but ensuring that the immune response is effective in controlling infection or tumor growth.

[00197] The term "dysfunctional" as used herein also includes refractory or unresponsive to antigen recognition, specifically, impaired ability to translate antigen recognition into effective downstream T cell functions such as proliferation, cytokine (e.g., IL-2) production, and/or target cell killing.

[00198] The term "anergy" refers to the state of unresponsiveness to antigen stimulation resulting from incomplete or insufficient signals delivered through the T cell receptor (e.g., increased intracellular Ca2+ in the absence of ras activation). T cell anergy can also result from antigen stimulation in the absence of costimulation, resulting in the cell becoming refractory to subsequent activation by antigen even in the face of costimulation. The unresponsive state can often be overridden by the presence of interleukin-2. Anergic T cells do not undergo clonal expansion and/or acquire effectors.

[00199] The term "exhaustion" refers to T cell exhaustion as a state of T cell dysfunction that results from prolonged TCR signaling that occurs during very chronic infections and cancer. It is distinguished from anergy in that it does not result from incomplete or deficient signaling, but from prolonged signaling. It is defined by poor effector function, prolonged expansion of inhibitory receptors, and a transcriptional state distinct from that of functional effector or memory T cells. Exhaustion prevents optimal control of infection and tumors. Exhaustion can result from both a series of extrinsic negative regulatory responses (e.g., immunoregulatory cytokines) as well as a series of intrinsic negative regulatory (costimulatory) responses (PD-1, B7-H3, B7-H4, etc.).

[00200] "T cell function enhancement" means to induce, cause, or stimulate a T cell to have an amplified or prolonged biological function, or to renew or reactivate inactive or exhausted T cells. Examples of T cell function enhancement include: increased secretion of interferon Y from CD8+ T cells, increased proliferation, increased antigen responsiveness (e.g., viral, pathogen, or tumor clearance) relative to such levels prior to the invention. In one embodiment, the level of enhancement is at least 50%, alternatively 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%. The manner of measuring this enhancement is known to one of skill in the art.

[00201] A "dysfunctional T cell disorder" is a disorder or condition of T cells characterized by decreased responsiveness to antigenic stimulation. In a particular embodiment, a dysfunctional T cell disorder is a disorder that is especially associated with inappropriate increased signaling through PD-1. In another embodiment, a disorder of T cell dysfunction is one in which T cells are anergic or have a decreased capacity to secrete cytokines, proliferate, or perform cytolytic activity. In a specific aspect, the decreased responsiveness results in ineffective control of a pathogen or tumor expressing an immunogen. Examples of dysfunctional T cell disorders characterized by T cell dysfunction include unresolved acute infection, chronic infection, and tumor immunity.

[00202] "Tumor immunity" refers to the process by which tumors evade immune recognition and clearance. Thus, as a therapeutic concept, tumor immunity is "treated" when such evasion is attenuated, and tumors are recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage, and tumor clearance.

[00203] "Immunogenicity" refers to the ability of a particular substance to elicit an immune response. Tumors are immunogenic and enhance immunogenicity aids in the clearance of tumor cells by the immune response. Examples of enhancing tumor immunogenicity include treatment with a PD-1 axis binding antagonist and an anti-GPC3 antibody.

[00204] "Prolonged response" refers to the prolonged effect of reducing tumor growth after cessation of a treatment. For example, the tumor size may remain the same or smaller compared to the size at the beginning of the administration phase. In some embodiments, the prolonged response has a duration at least equal to the duration of treatment, at least 1.5X, 2.0X, 2.5X, or 3.0X the length of the duration of treatment.

[00205] The term "pharmaceutical formulation" refers to a preparation that is of such a form that it allows the biological activity of the active ingredient to be effective, and that does not contain any additional components that would be unacceptably toxic to an individual to whom the formulation would be administered. Such formulations are sterile. "Pharmaceutically acceptable" excipients (vehicles, additives) are those that can reasonably be administered to a mammalian individual to provide an effective dose of the active ingredient employed.

[00206] As used herein, the term "treatment" refers to clinical intervention planned following the natural course of the individual or cell being treated during the course of clinical pathology. Desirable effects of treatment include decreasing the rate of disease progression, amelioration or palliation of the disease state, remission or improved prognosis. For example, an individual is successfully "treated" if one or more symptoms associated with cancer are mitigated or eliminated, including, but not limited to, reducing the proliferation of (or destruction of) cancer cells, reducing tumor growth, decreasing symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, and/or prolonging the survival of individuals.

[00207] As used herein, "delaying the progression of a disease" means to delay, hamper, delay, retard, stabilize, and/or postpone the development of the disease (such as cancer). This delay may be for varying lengths of time, depending on the history of the disease and/or the individual being treated. As is apparent to one skilled in the art, a sufficient or significant delay may, in fact, encompass prevention, in that the individual does not develop the disease. For example, end-stage cancer, such as metastasis, may be delayed.

[00208] An "effective amount" is at least the minimum amount required to effect a measurable improvement or prevention of a particular disorder. An effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the patient, and the ability of the antibody to elicit a desired response in the individual. An effective amount is also one at which any toxic or harmful effects of the treatment are outweighed by the therapeutically beneficial effects. For prophylactic use, beneficial or desired results include results such as eliminating or reducing the risk, lessening the severity, or delaying the onset of the disease, including biochemical, histological, and/or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes that present during the development of the disease. For therapeutic use, beneficial or desired results include clinical outcomes such as lessening one or more symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, enhancing the effect of other medications such as by targeting, slowing the progression of the disease, and/or prolonging survival. In the case of cancer or tumor, an effective amount of the drug may have the effect of reducing the number of cancer cells; reducing tumor size; inhibiting (i.e., slowing to some extent or desirably stopping) cancer cell infiltration into peripheral organs; inhibiting (i.e., slowing to some extent and desirably stopping) tumor metastasis; inhibiting to some extent tumor growth; and/or relieving to some extent one or more of the symptoms associated with the disorder. An effective amount may be administered in one or more administrations. For purposes of this invention, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to effect prophylactic or therapeutic treatment directly or indirectly. As understood in the clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be obtained in conjunction with another drug, compound, or pharmaceutical composition. Thus, an "effective amount" may be considered in relation to the administration of one or more therapeutic agents, and a single agent may be considered to be provided in an effective amount if, in conjunction with one or more other agents, a desirable result may be or is obtained.

[00209] As used herein, "along with" refers to the administration of one treatment modality in addition to another treatment modality. As such, "along with" refers to the administration of one treatment modality before, during, or after the administration of the other treatment modality to the subject.

[00210] A "disorder" is any condition that would benefit from treatment, including, but not limited to, chronic and acute diseases or disorders including those pathological conditions that predispose the mammal to the disorder in question.

[00211] The terms "cell proliferative disorder" and "proliferative disorder" refer to disorders that are associated with some degree of abnormal cell proliferation. In one embodiment, the cell proliferative disorder is cancer. In one embodiment, the cell proliferative disorder is a tumor.

[00212] "Tumor", as used herein, refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all cancerous and precancerous cells and tissues. The terms "cancer", "cancerous", "cell proliferative disorder", "proliferative disorder" and "tumor" are not mutually exclusive as referred to herein.

[00213] The terms "cancer" and "cancerous" refer to or describe the physiological condition, in mammals, that is typically characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include, but are not limited to, squamous cell cancer (e.g., epithelial squamous cell cancer), lung cancer including small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and squamous cell carcinoma of the lung, peritoneal cancer, hepatocellular cancer, stomach or gastric cancer including gastrointestinal cancer and gastrointestinal stromal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, urinary tract cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial carcinoma or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulvar cancer, thyroid cancer, liver carcinoma, anal carcinoma, penile carcinoma, melanoma, superficial disseminated melanoma, malignant lentiginous melanoma, acral lentiginous melanoma, and other malignant melanomas. melanomas, nodular melanomas, multiple myeloma, and B-cell lymphoma (including low-grade/follicular non-Hodgkin's lymphoma (NHL); small lymphocytic (SL) NHL; intermediate-grade/follicular NHL; diffuse intermediate-grade NHL; high-grade immunoblastic NHL; high-grade lymphoblastic NHL; high-grade small noncleaved cell NHL; bulky disease NHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom macroglobulinemia); chronic lymphocytic leukemia (CLL); acute lymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblastic leukemia; and posttransplant lymphoproliferative disorder (PTLD), as well as abnormal vascular proliferation associated with phakomatosis, edema (such as that associated with brain tumors), Meigs syndrome, brain as well as head and neck cancers, and associated metastases. In certain embodiments, cancers that are amenable to treatment by the antibodies of the invention include breast cancer, colorectal cancer, rectal cancer, non-small cell lung cancer, glioblastoma, Hodgkins lymphoma (NHL), renal cell cancer, prostate cancer, liver cancer, pancreatic cancer, soft tissue sarcoma, Kaposi's sarcoma, carcinoid carcinoma, head and neck cancer, ovarian cancer, mesothelioma, and multiple myeloma. In some embodiments, the cancer is selected from: small cell lung cancer, glioblastoma, neuroblastomas, melanoma, breast carcinoma, gastric cancer, colorectal cancer (CRC), and hepatocellular carcinoma. Further, in some embodiments, the cancer is selected from: non-small cell lung cancer, colorectal cancer, glioblastoma, breast carcinoma, and hepatocellular carcinoma, including metastatic forms of those cancers.

[00214] The term "cytotoxic agent" as used herein refers to any agent that is detrimental to cells (e.g., causes cell death, inhibits proliferation, or otherwise impairs a cellular function). Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212, and radioactive isotopes of Lu); chemotherapeutic agents; growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, animal, or plant origin, including fragments and/or variants thereof. Exemplary cytotoxic agents can be selected from antimicrotubule agents, platinum coordination complexes, alkylating agents, antibiotic agents, topoisomerase II inhibitors, antimetabolites, topoisomerase I inhibitors, hormone homonyms and analogs, inhibitors of a series of signal transduction reactions, non-receptor tyrosine kinase angiogenesis inhibitors, immunotherapeutic agents, proapoptotic agents, 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 taxane. In one embodiment, the taxane is paclitaxel or docetaxel. In one embodiment, the cytotoxic agent is a platinum agent. In one embodiment, the cytotoxic agent is an EGFR antagonist. In one embodiment, the EGFR antagonist is N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine (e.g., erlotinib). In one embodiment, the cytotoxic agent is a 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.

[00215] "Chemotherapeutic agent" includes, but is not limited to, nitrogen mustard analogs, alkyl sulfonates, ethylene imines, nitrosoureas, epoxides, other alkylating agents, folic acid analogs, purine analogs, pyrimidine analogs, other antimetabolic agents, vinca alkanoids or analogs, podophyllotoxin derivatives, camptotecan analogs, colchicine derivatives, taxanes, other plant alkanoids or natural products, actinomycins, anthracyclines or related substances, other cytotoxic antibiotics, platinum compounds, methylhydrazines, kinase inhibitors, enzymes, histone deacetylase inhibitors, retinoids, immune checkpoint inhibitors, antibodies, and other molecularly targeted drugs.

[00216] "Chemotherapeutic agent" also includes compounds useful in the treatment of cancer. Examples of chemotherapeutic agents include erlotinib (TARCEVA (registered), Genentech/OSI Pharm.), bortezomib (VELCADE (registered), Millennium Pharm.), disulfiram, epigallocatechin gallate, salinosporamide A, carfilzomib, 17-AAG (geldanamycin), radicicol, lactate dehydrogenase A (LDH-A), fulvestrant (FASLODEX (registered), AstraZeneca), sunitib (SUTENT (registered), Pfizer/Sugen), letrozole (FEMARA (registered), Novartis), imatinib mesylate (GLEEVEC (registered), Novartis), finasunate (VA-TALANIB (registered), Novartis), oxaliplatin (ELOXATIN (registered), Sanofi), 5-FU (5-fluorouracil), leucovorin, rapamycin (Sirolimus, RAPAMUNE (registered), Wyeth), Lapatinib (TYKERB (registered), GSK572016, Glaxo Smith Kline), Lonafamib (SCH 66336), Sorafenib (NEXAVAR (registered), Bayer Labs), Gefitinib (IRESSA (registered), AstraZeneca), AG1478, alkylating agents such as thiotepa and cyclosphosphamide from CYTOXAN (registered); alkyl sulfonates such as busulfan, improsulfan, and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamines, triethylenemelamines, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylomelamines; acetogenins (especially bulatacin and bulatacinone); a camptothecin (including topotecan and irinotecan); bryostatin; calistatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin and bizelesin); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); adrenocorticosteroids (including prednisone and prednisolone); cyproterone acetate; 5α-reductases including finasteride and dutasteride); vorinostat, romidepsin, panobinostat, valproic acid, dolastatin mocetinostat; aldesleukin, duocarmycin talc (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictin; spongistatin; nitrogen mustards such as chlorambucil, clomaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembiquin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediine antibiotics (e.g., calicheamicin, especially calicheamicin Y1I and calicheamicin w1I (Angew Chem. Intl. Ed. Engl. 1994 33: 183-186); dynemycin, including dynemycin A; bisphosphonates such as clodronate; a esperamycin; as well as the statin chromophore neocarzin and related chromoprotein enediine antibiotic chromophores), aclacinomisins, actinomycin, autramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycin, dactinomycin, daunorubicin, detorrubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN (registered) (doxorubicin), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porphyromycin, puromycin, chelamicin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogues such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; antiadrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenishers such as phrolininic acid; aceglaton; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamin; demecolcine; diaziquone; elfomitin; elliptine acetate; an epothilone; etoglucid; gallium nitrate; hydroxourea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamnol; nitraerin; pentostatin; fenamete; pirarubicin; losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK polysaccharide complex (registered) (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"- trichlorotriethylamines; trichothecenes (especially T-2 toxin, verracurin A, roridine A, and anguidin); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobromane; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL (pacli-taxel; Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE (registered) (Cremophor-free), the nanoparticle formulations created with paclitaxel albumin (American Pharmaceutical Partners, Schaumberg, I11.), and TAXOTERE (registered) (docetaxel, doxetaxel; Sanofi-Aventis); chlorambucil; GEMZAR (registered) (gemcitabine); 6- thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE (registered) (vinorelbine); no-vantrone; teniposide; edatrexate; daunomycin; aminopterin; capecitabine (XELODA (registered)); ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylmitine (DMFO); retinoids such as retinoic acid; and pharmaceutically acceptable acids and salts, and derivatives of any of the above.

[00217] The chemotherapeutic agent also includes antibodies such as alemtuzumab (Campath), bevacizumab (AVASTIN (registered), Genentech); cetuximab (ERBITUX (registered), Imclone); pani- tumumab (VECTIBIX (registered), Amgen), rituximab (RITUXAN (registered), Genentech/Biogen Idec), pertuzumab (OMNITARG (registered), 2C4, Genentech), trastuzumab (HERCEPTIN (registered), Genentech), tositumomab (Bexxar, Corixia), and the antibody drug conjugate, gemtuzumab ozogamicin (MYLOTARG (registered), Wyeth). Additional humanized monoclonal antibodies with therapeutic potential as agents in combination with the compounds of the invention include: apolizumab, aselizumab, atlizumab, bapineuzumab, bivatuzumab mertansine, cantuzumab mertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab, fontolizumab, gentuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab, pectuzumab, pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab, resyvizumab, rovelizumab, ruplizumab, sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan , tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab, tucotuzumab celmoleukin, tucusituzumab, umavizumab, urtoxazumab, ustekinumab, visilizumab, and the anti-interleukin-12 (ABT-874/J695, Wyeth Research and Abbott Laboratories) which is a sequence exclusively recombinant human, full-length IgG1 antibody genetically modified to recognize the interleukin-12 p40 protein.

[00218] A "growth inhibitory agent" when used herein, refers to a compound or combination that inhibits growth of a cell in vitro or in vivo. In one embodiment, the growth inhibitory agent is a growth inhibitory antibody that prevents or reduces proliferation of a cell expressing an antigen to which the antibody binds. In another embodiment, the growth inhibitory agent can be one that significantly reduces the percentage of cells in S phase. Examples of growth inhibitory agents include agents that block cell cycle progression (at a site other than S phase), such as agents that induce G1 arrest and M phase arrest. Classical M phase blockers include the vincas (vincristine and vinblastine), taxanes, and topoisomerase II inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Those agents that arrest G1 also spill over into S phase arrest, e.g., DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information can be found in Mendelsohn and Israel, eds., The Molecular Basis of Cancer, Chapter 1, entitled "Cell cycle regulation, oncogenes, and antineoplastic drugs" by Murakami et al., (W.B. Saunders, Philadelphia, 1995), e.g., p. 13. The taxanes (paclitaxel and docetaxel) are anticancer drugs both derived from the yew tree. Docetaxel (TA-XOTERE (registered), Rhone-Poulenc Rorer), derived from the European yew tree, is a semisynthetic analogue of paclitaxel (TAXOL (registered), Bristol-Myers Squibb). Paclitaxel and docetaxel promote microtubule assembly from tubulin dimers and stabilize microtubules by preventing depolymerization, which results in inhibition of mitosis in cells.

[00219] By "radiation therapy" is meant the use of targeted gamma rays or beta rays to induce sufficient damage to a cell so as to limit its ability to function normally or to destroy the cell altogether. It will be appreciated that there will be many ways known in the art to determine the dosage and duration of treatment. Typical treatments are provided as a single administration and typical doses range from 10 to 200 units (Grays) per day.

[00220] A "subject" or an "individual", for purposes of treatment, refers to any animal classified as a mammal, including humans, farm and domestic animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. Preferably, the mammal is a human.

[00221] The term "antibiotic" herein is used in the broadest sense and specifically encompasses monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity.

[00222] An "isolated" antibody is one that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that may interfere with research, diagnostic, or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In some embodiments, an antibody is purified (1) to greater than 95% by weight of the antibody as determined, e.g., by the Lowry method, and in some embodiments, greater than 99% by weight; (2) to a degree sufficient to obtain at least 15 residues of internal or N-terminal amino acid sequence by use of, e.g., a rotary sequencer, or (3) to homogeneity by SDS-PAGE under reducing or under reducing or non-reducing conditions using, e.g., Coomassie blue or silver stain. Isolated antibody includes the antibody in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

[00223] "Native antibodies" are generally heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by a covalent disulfide bond, while the number of disulfide bonds varies among the heavy chains of different immunoglobulin isotypes. Each light and heavy chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain of the light chain is aligned with the variable domain of the heavy chain. Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains.

[00224] The term "constant domain" refers to the portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to the other portion of the immunoglobulin, the variable domain, which contains the antigen-binding site. The constant domain contains the CH1, CH2, and CH3 (collectively, CH) domains of the heavy chain and the CHL (or CL) domain of the light chain.

[00225] The "variable region" or "variable domain" of an antibody refers to the amino terminal domains of the antibody's heavy or light chain. The heavy chain variable domain may be referred to as "VH." The light chain variable domain may be referred to as "VL." These domains are generally the most variable parts of an antibody and contain the antigen binding sites.

[00226] The term "variable" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the capacity is not uniformly distributed throughout the antibody domains. It is concentrated in those segments called hypervariable regions (HVRs) in both the light chain and heavy chain variable domains. The most highly conserved portions of the various domains are the so-called framework regions (FR). The variable domains of native light and heavy chains comprise four FR regions, broadly adopting a configuration, connected by three HVRs, which form loops connecting, and in some cases form part of, the beta-sheet structure. The HVRs in each chain are brought together in close proximity by the FR regions and, with the HVRs of the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not directly involved in the binding of an antibody to an antigen, but they exhibit various effector functions, such as participation of the antibody in cellular toxicity, depending on the antibody.

[00227] The "light chains" of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa ("K") and lambda ("À"), based on the amino acid sequences of their constant domains.

[00228] The term IgG "isotype" or "subclass", as used herein, encompasses any of the subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions.

[00229] Depending on the amino acid sequences of the constant domains of their heavy chains, antibodies (immunoglobulins) can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called α, Y, ε, Y, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and generally described in, e.g., Abbas et al., Cellular and Mol. Immunology, 4th ed. (W.B. Saunders, Co., 2000). An antibody may be part of a larger fusion molecule formed by covalent or non-covalent association of the antibody with one or more other proteins or peptides.

[00230] The terms "full-length antibody", "intact antibody" and "whole antibody" are used interchangeably herein to refer to an antibody in its substantially intact form, antibody fragments, as defined herein below. The terms particularly refer to an antibody with heavy chains that contain an Fc region.

[00231] A "naked antibody" for purposes herein is an antibody that is not conjugated to a cytotoxic moiety or radiolabel.

[00232] "Antibody fragments" comprise a portion of an intact antibody, preferably comprising the antigen-binding region thereof. In some embodiments, the antibody fragment described herein is an antigen-binding fragment. Examples of antibody fragments include Fab, Fab', F(ab')2, scFv, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[00233] Papin digestion of antibodies produces two identical antigen fragments, called "Fab" fragments, each with a unique antigen-binding site, and a residual "Fc" fragment, whose name reflects its ability to crystallize readily. Pepsin treatment produces an F(ab')2 fragment that has two antigen-combining sites and is further capable of cross-linking antigen.

[00234] "Fv" is the minimal antibody fragment that contains a complete antigen-binding site. In one embodiment, a two-chain Fv species consists of a dimer of one light chain and one heavy chain variable domain in tight, non-covalent association. In a single-chain Fv species (scFv), one heavy chain and one light chain variable domain can be covalently linked by a flexible peptide such that the heavy and light chains can associate into a "dimeric" structure analogous to that in a two-chain Fv species. It is in this configuration that the three HVRs of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six HVRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three antigen-specific HVRs) has the ability to recognize and bind antigen, albeit at a lower affinity than the entire binding site.

[00235] The Fab' fragment contains the light and heavy chain variable domains and also contains the light chain constant domain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab' fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear(s) a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments having hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[00236] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of antibodies, wherein these domains are present in a single polypeptide chain. Generally, the scFv polypeptide also comprises a polypeptide linker between the VH and VL domains that allows the scFv to form the desired structure for antigen binding. For a review of scFv, see, e.g., Pluckthuen, in The Pharmacology of Monoclonal Antibodies, volume 113, Rosenburg and Moore, eds. (Springer-Verlag, New York, 1994), pages 269 to 315.

[00237] The term "diabodies" refers to antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) on the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies can be bivalent or bispecific. Diabodies are more fully described in, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. U.S.A. 90: 6444–6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9: 129–134 (2003).

[00238] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible mutations, e.g., naturally occurring mutations, which may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies. In certain embodiments, such a monoclonal antibody typically includes an antibody comprising a polypeptide sequence that binds to a target, wherein the target-binding polypeptide sequence was obtained by a process that includes selecting a unique target-binding polypeptide sequence from multiple polypeptide sequences. For example, the selection process may be selecting a single clone from multiple clones, such as a mixture of hybridoma clones, phage clones, or recombinant DNA clones. It should be understood that a selected target binding sequence may also be altered, for example, to improve the affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to create a multispecific antibody, etc., and that an antibody comprising the altered target binding sequence is also a monoclonal antibody of the invention. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. In addition to their specificity, monoclonal antibody preparations are advantageous in that they are typically uncontaminated by others.

[00239] The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the invention may be made by various techniques, including, for example, the hybridoma method (e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al., Hybridoma, 14 (3):253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed., 1988); Hammerling et al., In: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, for example, U.S. Patent No. 4,816,567), phage display technologies (see, for example, Clackson et al., Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol. 222: 581–597 (1992); Sidhu et al., J. Mol. Biol. 338(2): 299–310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073–1093 (2004); Fellouse, Proc. Natl. Acad. Sci. U.S.A. 101(34): 12467–12472 (2004); and Lee et al., J. Immunol. Methods 284(1–2): 119–132 (2004), and technologies for producing human or human-like antibodies in animals that have parts or all of the human immunoglobulin loci or genes that encode human immunoglobulin sequences (see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits et al., Proc. Natl. Academic. Sci. United States of America 90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993); Bruggemann et al, Year in Immunol. 7:33 (1993); United States Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016; Marks et al., Bio/Technology 10: 779-783 (1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994); Fishwild et al., Nature Biotechnol. 14: 845-851 (1996); Neuberger, Nature Biotechnol. 14: 826 (1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995).

[00240] Monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see, e.g., U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. U.S. 81:6851-6855 (1984)). Chimeric antibodies include PRIMATTZED (registered) antibodies in which the antigen-binding region of the antibody is derived from an antibody produced by, for example, immunization of macaque monkeys with the antigen of interest.

[00241] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In one embodiment, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from an HVR of the recipient are replaced with residues from an HVR of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity, affinity, and/or capacity. In some examples, FR residues of the immunoglobulin are replaced with corresponding non-human residues. Furthermore, humanized antibodies can comprise residues that are not found in the recipient antibody or the donor antibody. These modifications can be made to further refine the performance of the antibody. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, wherein all or substantially all of the corresponding hypervariable loops of a non-human immunoglobulin, and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody will optionally also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see, e.g., Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, e.g., Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1: 105115 (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.

[00242] A "human antibody" is one that has an amino acid sequence that corresponds to that of an antibody produced by a human and/or that was made using any of the techniques for making human antibodies as described herein. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues. Human antibodies can be produced using various skills known in the art, including phage display library. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Also available for the preparation of human monoclonal antibodies are the methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, page 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 the antigen to a transgenic animal that has been engineered to produce such antibodies in response to antigenic challenge but whose endogenous loci have been disabled, e.g., immunized xenomice (see, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 with respect to XENOMOUSE (trade name) technology). See also, e.g., Li et al., Proc. Natl. Acad. Sci. U.S.A., 103:3557-3562 (2006) with respect to human antibodies generated by human B-cell hybridoma technology.

[00243] A "species-dependent antibody" is one that has a stronger binding affinity for an antigen of a first mammalian species than it has for a homologue of that antigen from a second mammalian species. Typically, the species-dependent antibody "binds specifically" to a human antigen (e.g., has a binding affinity (Kd) value of no more than about 1x10-7M, preferably no more than about 1x10-8M, and preferably no more than about 1x10-9M), but has a binding affinity for a homologue of the antigen of a second non-human mammalian species that is at least about 50-fold, or at least about 500-fold, or at least about 1000-fold, weaker than its binding affinity for the human antigen. The species-dependent antibody can be any of the various types of antibodies as defined above, but preferably is a human or humanized antibody.

[00244] The term "hypervariable region", "HVR", or "HV", when used herein, refers to those regions of an antibody variable domain that are hypervariable in sequence and/or form structurally defined loops. Generally, antibodies comprise six HVRs; three in the VH (HI, H2, H3), and three in the VL (L1, L2, L3). In native antibodies, H3 and L3 exhibit the greatest diversity of the six HVRs, and H3, in particular, is believed to play a unique role in providing fine specificity to antibodies. See, e.g., Xu et al., Im-munity 13:37-45 (2000); Johnson and Wu, in Methods in Molecular Bi-ology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003). Indeed, naturally occurring camelid antibodies consisting of a heavy chain only are functional and stable in the absence of a light chain. See, for example, Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct. Biol. 3:733-736 (1996).

[00245] Several HVR delineations are in use and are covered here. Kabat's Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia refers instead to the location of structural loops (Chothia and Lesk J. Mol. Biol. 196:901917 (1987)). AbM HVRs represent a compromise between Kabat HVRs and Chothia structural loops, and are used by Oxford Molecular's AbM antibody modeling software. "Contact" HVRs are based on an analysis of available complex crystal structures. The residuals of each of these HVRs are noted below. Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2 L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1 H31-H35B H26-H35B H26-H H30-H35B

[00246] The HVRs may comprise "extended HVRs" as follows: 24-36 or 24-34 (L1), 46-56 or 50-56 (L2), and 89-97 or 89-96 (L3) in the VL, and 26-35 (H1), 50-65, or 49-65 (H2), and 93-102, 94-102, or 95-102 (H3) in the VH. The variable domain residues are numbered according to Kabat et al., supra, for each of these definitions.

[00247] "Framework" or "FR" residues are those variable domain residues other than the HVR residues as defined herein.

[00248] The terms "Kabat-like variable domain residue numbering" or "Kabat-like amino acid position numbering", and variations thereof, refer to the numbering system used for heavy chain variable domains or light chain variable domains of the antibody compilation in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or more amino acids corresponding to a shortening of, or insertion into, a FR or HVR of the variable domain. For example, a heavy chain variable domain may include a single amino acid insertion (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g., residues 82a, 82b, and 82c, etc. according to Kabat) after residue 82 of the heavy chain FR. Kabat numbering of residues can be determined for a given antibody by aligning regions of homology of the antibody sequence with a "standard" Kabat numbered sequence.

[00249] The Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1 to 107 of the light chain and residues 1 to 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)). The "EU numbering system" or "EU index" is generally used when referring to a residue in an immunoglobulin heavy chain constant region (e.g., the EU index described in Kabat et al., supra). The "EU index as in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

[00250] The term "linear antibodies" refers to the antibodies described in Zapata et al., (1995 Protein Eng, 8(10): 1057-1062). Briefly, these antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) that, together with complementary light chain polypeptides, form a pair of antigen-binding regions. Linear antibodies can be bispecific or monospecific.

[00251] As used herein, the term "binds", "specifically, binds to" or is "specific for" refers to measurable and reproducible interactions, such as binding between a target and an antibody, which is determinative of the presence of the target in the presence of a heterogeneous population of molecules including biological molecules. For example, an antibody that binds to or specifically binds to a target (which may be an epitope) is an antibody that binds to that target with greater affinity, avidity, more readily, and/or for a longer duration than when it binds to other targets. In one embodiment, the extent of binding of an antibody to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured, for example, by a radioimmunoassay (RIA). In certain embodiments, an antibody that specifically binds to a target has a dissociation constant (Kd) of ^1 μM, ^100 nM, ^10 nM, ^1 nM, or ^0.1 nM. In certain embodiments, an antibody specifically binds to an epitope on a protein that is conserved among proteins of different species. In another embodiment, specific binding may include, but does not require, exclusive binding.

II. PD-1 Axis Binding Antagonists

[00252] Provided herein is a method of treating or delaying the progression of cancer in a subject comprising administering to a subject an effective amount of a PD-1 axis binding antagonist and an anti-GPC3 antibody. Also provided herein is a method of enhancing immune responses against tumor cells in a subject having cancer comprising administering to a subject an effective amount of a PD-1 axis binding antagonist and an anti-GPC3 antibody. For example, enhanced immune responses against tumor cells include infiltration of immune cells including macrophages and multinucleated giant cells into tumor tissues. For another example, enhanced immune responses against tumor cells include increased CD45-positive lymphocytes, CD3ε-positive lymphocytes, and CD8-positive T lymphocytes in tumor infiltrating lymphocytes (TILs). For example, a PD-1 axis binding antagonist includes a PD-1 binding antagonist, a PD-L1 binding antagonist, and a PD-L2 binding antagonist. PD-1 (programmed death 1) is also referred to in the art as "programmed cell death 1", PDCD1, CD279, and SLEB2. PD-L1 (programmed death ligand 1) is also referred to in the art as "programmed cell death ligand 1", PDCD1LG1, CD274, B7-H, and PD-L1. PD-L2 (programmed death ligand 2) is also referred to in the art as "programmed cell death 1 ligand 2", PDCD1LG2, CD273, B7-DC, Btdc, and PDL2. In some embodiments, PD-1, PD-L1, and PD-L2 are human PD-1, PD-L1, and PD-L2.

[00253] In some embodiments, the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partners. In a specific aspect, the PD-1 ligand binding partners are 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 a specific aspect, the PD-L1 binding partners are PD-1 and/or B7-1. In another embodiment, the PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to its binding partner. In a specific aspect, a PD-L2 binding partner is PD-1. The antagonist can be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide.

[00254] In some embodiments, the PD-1 binding 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 MDX-1106 (nivolumab), MK-3475 (lambrolizumab), and CT-011 (pidilizumab). In some embodiments, the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising a PD-1 or extracellular binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence)). In some embodiments, the PD-1 binding antagonist is AMP-224 or AMP-514 (MEDI0680). In some embodiments, the PD-1 binding antagonist is selected from the group consisting of PDR001, REGN2810, BGB A317, and SHR-1210. In some embodiments, the PD-L1 binding antagonist is an anti-PD-L1 antibody. In some embodiments, the anti-PD-L1 binding antagonist is selected from the group consisting of YW243.55.S70, Atezolizumab, MPDL3280A, MDX-1105, avelumab, and MEDI4736 (durvalumab). The YW243.55.S70 antibody is an anti-PD-L1 described in WO2010/077634. MDX-1105, also known as BMS-936559, is an anti-PD-L1 antibody described in WO2007/005874. Avelumab is an anti-PDL1 antibody described in WO2013079174. ME-DI4736 (durvalumab), is an anti-PD-L1 monoclonal antibody described in WO2011/066389 and US2013/034559. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538, BMS-936558, and OP-DIVO (registered), is an anti-PD-1 antibody described in WO2006/121168. Pembrolizumab, also known as MK-3475, Merck-3475, lambrolizumab, KEYTRUDA (registered), and SCH-900475, is an anti-PD-1 antibody described in WO2009/114335. CT-011, also known as hBAT, hBAT-1 or pidilizumab, is an anti-PD-1 antibody described in WO2009/101611. AMP-224, also known as B7-DCIg, is a soluble PD-L2-Fc fusion receptor described in WO2010/027827 and WO2011/066342.

[00255] In some embodiments, the PD-1 axis binding antagonist is an anti-PD-L1 antibody. In some embodiments, the anti-PD-L1 antibody is capable of inhibiting 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.

[00256] Examples of anti-PD-L1 antibodies useful in the methods of the invention, and methods for preparing the same, are described in PCT patent applications WO2010/077634, WO2007/005874, WO2011/066389, and US2013/034559, which are incorporated herein by reference. Anti-PD-L1 antibodies useful in the invention, including compositions containing such antibodies, can be used in combination with an anti-GPC3 antibody to treat cancer.

Anti-PD1 antibodies

[00257] In some embodiments, the anti-PD-1 antibody is MDX1106. Alternative 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). In yet another embodiment, an isolated anti-PD-1 antibody comprising a heavy chain variable region comprising the heavy chain variable region amino acid sequence of SEQ ID NO:1 and/or a light chain variable region comprising the light chain variable region amino acid sequence of SEQ ID NO:2 is provided. In yet another embodiment, an isolated anti-PD-1 antibody comprising a heavy chain and/or a light chain sequence is provided, wherein: (a) the heavy chain sequence 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 to the heavy chain sequence set forth in SEQ ID NO:1, and (b) the light chain sequence 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 to the heavy chain sequence set forth in SEQ ID NO:1. sequence with the light chain sequence mentioned in SEQ ID NO:2.

Anti-PD-L1 antibody

[00258] In some embodiments, the antibody in the formulation comprises at least one tryptophan (e.g., at least two, at least three, or at least four) in the heavy and/or light chain sequence. In some embodiments, the tryptophan amino acid is in the CDR arrays, framework regions, and/or constant regions of the antibody. In some embodiments, the antibody comprises two or three tryptophan residues in the CDR arrays. In some embodiments, the antibody in the formulation is an anti-PD-L1 antibody. PD-L1 (programmed death ligand 1), also known as PD-L1, B7-H1, B7-4, CD274, and B7-H, is a transmembrane protein, and its interaction with PD-1 inhibits T cell activation and cytokine production. In some embodiments, the anti-PD-L1 antibody described herein binds to human PD-L1. Examples of anti-PD-L1 antibodies that may be used in the methods described herein are Atezolizumab, or anti-PD-L1 antibodies described in PCT patent application WO 2010/077634 A1 and US 8,217,149, which are incorporated herein by reference.

[00259] In some embodiments, the anti-PD-L1 antibody is capable of inhibiting 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.

[00260] The anti-PD-L1 antibodies described in WO 2010/077634 A1 and US 8,217,149 can be used in the methods described herein. In some embodiments, the anti-PD-L1 antibody comprises a heavy chain variable region sequence of SEQ ID NO:3 and/or a light chain variable region sequence of SEQ ID NO:4. In yet another embodiment, an isolated anti-PD-L1 antibody comprising a heavy chain and/or a light chain sequence is provided, wherein: (a) the heavy chain sequence 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 to the heavy chain sequence set forth in SEQ ID NO:3, and (b) the light chain sequence 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 with the light chain sequence mentioned in SEQ ID NO:4.

[00261] In one embodiment, the anti-PD-L1 antibody comprises a heavy chain variable region polypeptide comprising a sequence of HVR-H1, HVR-H2, and HVR-H3, wherein: (a) the sequence of HVR-H1 is GFTFSX1SWIH (SEQ ID NO:5); (b) the sequence of HVR-H2 is AWIX2PYGGSX3YYADSVKG (SEQ ID NO:6); (c) the sequence of HVR-H3 is RHWPGGFDY (SEQ ID NO:7);

[00262] Also wherein: X1 is D or G; X2 is S or L; X3 is T or S. In a specific aspect, X1 is D; X2 is S; and X3 is T.

[00263] In another aspect, the polypeptide further comprises variable region heavy chain framework sequences juxtaposed between the HVRs according to the formula: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4). In yet another aspect, the framework sequences are derived from human consensus framework sequences. In another aspect, the framework sequences are VH subgroup III consensus framework. In yet another aspect, at least one of the structural sequences is as follows: HC-FR1 is EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO:8) HC-FR2 is WVRQAPGKGLEWV (SEQ ID NO:9) HC-FR3 is RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO:10) HC-FR4 is WGQGTLVTVSA (SEQ ID NO:11).

[00264] In yet another aspect, the heavy chain polypeptide - wherein: 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; X10 is Y or A; X11 is Y, G, F, or S; X12 is L, Y, F, or W; X13 is Y, N, A, T, G, F, or I; X14 is H, V, P, T, or I; X15 is A, W, R, P, or T. In yet another aspect, X4 is D; X5 is V; X6 is S; X7 is A; X8 is V; X9 is F; X10 is Y; X11 is Y; X12 is L; X13 is Y; X14 is H; X15 is A.

[00265] In yet another aspect, the light chain also comprises variable region light chain framework sequences juxtaposed between the HVRs according to the formula: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet another aspect, the framework sequences are derived from human consensus framework sequences. In yet another aspect, the framework sequences are the VL kappa I consensus structure. In yet another aspect, at least one of the framework sequences is as follows: LC-FR1 is DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO:15) LC-FR2 is WYQQKPGKAPKLLIY (SEQ ID NO:16) LC-FR3 is GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO:17) LC-FR4 is FGQGTKVEIKR (SEQ ID NO:18).

[00266] In another embodiment, an isolated anti-PD-L1 antibody or antigen binding fragment comprising a heavy chain and a light chain variable region sequence is provided, wherein: 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; X10 is Y or A; X11 is Y, G, F, or S; X12 is L, Y, F, or W; X13 is Y, N, A, T, G, F, or I; X14 is H, V, P, T, or I; X15 is A, W, R, P, or T. In a specific aspect, X1 is D; X2 is S, and X3 is T. In another aspect, X4 is D; X5 is V; X6 is S; X7 is A; X8 is V; X9 is F; X10 is Y; X11 is Y; X12 is L; X13 is Y; X14 is H; X15 is A. In yet another aspect, X1 is D; X2 is S and X3 is T, X4 is D; X5 is V; X6 is S; X7 is A; X8 is V; X9 is F; X10 is Y; X11 is Y; X12 is L; X13 is Y; X14 is H and X15 is A.

[00267] In another aspect, the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs such as: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable regions comprise one or more framework sequences juxtaposed between the HVRs such as: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet another aspect, the framework sequences are derived from human consensus framework sequences. In yet another aspect, the heavy chain framework sequences are derived from a Kabat subgroup I, II, or III sequence. In yet another aspect, the heavy chain framework sequence is a VH subgroup III framework sequence. In yet another aspect, one or more of the heavy chain framework sequences are set forth as SEQ ID NOs:8, 9, 10, and 11. In yet another aspect, the light chain framework sequences are derived from a Kabat kappa subgroup I, II, III, or IV sequence. In yet another aspect, the light chain framework sequences are VL kappa I consensus framework. In yet another aspect, one or more of the light chain framework sequences are set forth as SEQ ID NOs:15, 16, 17, and 18.

[00268] In yet another specific aspect, the antibody further comprises a human or murine constant region. In yet another aspect, the human constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, IgG3, IgG4. In yet another specific aspect, the human constant region is IgG1. In yet another aspect, the murine constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In yet another aspect, the murine constant region is IgG2A. In yet another specific aspect, the antibody has reduced or minimal effector function. In yet another specific aspect, the minimal effector function results from a "less effector Fc mutation" or aglycosylation. In yet another embodiment, the less effector Fc mutation is a N297A or D265A/N297A substitution in the constant region.

[00269] In yet another embodiment, there is provided an anti-PD-L1 antibody comprising a heavy chain and a light chain variable region sequence, wherein: (a) the heavy chain further comprises a sequence of HVR-H1, HVR-H2, and HVR-H3 having at least 85% sequence identity to GFTFSDSWIH (SEQ ID NO:19), AWIS-PYGGSTYYADSVKG (SEQ ID NO:20), and RHWPGGFDY (SEQ ID NO:21), respectively, or (b) the light chain further comprises a sequence of HVR-L1, HVR-L2, and HVR-L3 having at least 85% sequence identity to RASQDVSTAVA (SEQ ID NO:22), SASFLYS (SEQ ID NO:23), and QQYLYHPAT (SEQ ID NO:24), respectively.

[00270] In a specific aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[00271] In another aspect, the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs such as: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable regions comprise one or more framework sequences juxtaposed between the HVRs such as: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet another aspect, the framework sequences are derived from human consensus framework sequences. In yet another aspect, the heavy chain framework sequences are derived from a Kabat subgroup I, II, or III sequence. In yet another aspect, the heavy chain framework sequence is a VH subgroup III framework sequence. In yet another aspect, one or more of the heavy chain framework sequences are set forth as SEQ ID NOs:8, 9, 10, and 11. In yet another aspect, the light chain framework sequences are derived from a Kabat kappa subgroup I, II, III, or IV sequence. In yet another aspect, the light chain framework sequences are VL kappa I consensus framework. In yet another aspect, one or more of the light chain framework sequences are set forth as SEQ ID NOs:15, 16, 17, and 18.

[00272] In yet another specific aspect, the antibody further comprises a human or murine constant region. In yet another aspect, the human constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, IgG3, IgG4. In yet another specific aspect, the human constant region is IgG1. In yet another aspect, the murine constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In yet another aspect, the murine constant region is IgG2A. In yet another specific aspect, the antibody has reduced or minimal effector function. In yet another specific aspect the minimal effector function results from a "less effector Fc mutation" or aglycosylation. In yet another embodiment, the less effector Fc mutation is a N297A or D265A/N297A substitution in the constant region.

[00273] In another embodiment, an isolated anti-PD-L1 antibody comprising a heavy chain and a light chain variable region sequence is provided, wherein: (a) the heavy chain sequence has at least 85% sequence identity to the heavy chain sequence set forth in SEQ ID NO:25, and/or (b) the light chain sequence has at least 85% sequence identity to the light chain sequence set forth in SEQ ID NO:4.

[00274] In a specific aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In another aspect, the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs such as: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable regions comprise one or more framework sequences juxtaposed between the HVRs such as: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet another aspect, the framework sequences are derived from human consensus framework sequences. In another aspect, the heavy chain framework sequences are derived from a Kabat subgroup I, II, or III sequence. In yet another aspect, the heavy chain framework sequence is a VH subgroup III framework sequence. In yet another aspect, one or more of the heavy chain framework sequences are mentioned as SEQ ID NOs: 8, 9, 10 and WGQGTLVTVSS (SEQ ID NO:27).

[00275] In yet another aspect, the light chain framework sequences are derived from a Kabat kappa subgroup I, II, III or IV sequence. In yet another aspect, the light chain framework sequences are VL kappa I consensus structure. In yet another aspect, one or more of the light chain framework sequences are referred to as SEQ ID NOs:15, 16, 17 and 18.

[00276] In yet another specific aspect, the antibody further comprises a human or murine constant region. In yet another aspect, the human constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, IgG3, IgG4. In yet another specific aspect, the human constant region is IgG1. In yet another aspect, the murine constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In yet another aspect, the murine constant region is IgG2A. In yet another specific aspect, the antibody has reduced or minimal effector function. In yet another specific aspect, the minimal effector function results from production in prokaryotic cells. In yet another specific aspect, the minimal effector function results from a "less effector Fc mutation" or aglycosylation. In yet another embodiment, the less effector Fc mutation is a N297A or D265A/N297A substitution in the constant region.

[00277] In another aspect, the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs such as: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable regions comprise one or more framework sequences juxtaposed between the HVRs such as: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet another aspect, the framework sequences are derived from human consensus framework sequences. In yet another aspect, the heavy chain framework sequences are derived from a Kabat subgroup I, II, or III sequence. In yet another aspect, the heavy chain framework sequence is a VH subgroup III framework sequence. In yet another aspect, one or more of the heavy chain framework sequences is as follows: HC-FR1 is EVQLVESGGGLVQPGGSLRLSCAASGFTFS (SEQ ID NO:29) HC-FR2 is WVRQAPGKGLEWVA (SEQ ID NO:30) HC-FR3 is RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO:10) HC-FR4 is WGQGTLVTVSS (SEQ ID NO:27).

[00278] In yet another aspect, the light chain framework sequences are derived from a Kabat kappa subgroup I, II, III, or IV sequence. In yet another aspect, the light chain framework sequences are VL kappa I consensus structure. In yet another aspect, one or more of the light chain framework sequences is as follows: LC-FR1 is DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 15) LC-FR2 is WYQQKPGKAPKLLIY (SEQ ID NO: 16) LC-FR3 is GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 17) LC-FR4 is FGQGTKVEIK (SEQ ID NO: 28).

[00279] In yet another specific aspect, the antibody further comprises a human or murine constant region. In yet another aspect, the human constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, IgG3, IgG4. In yet another specific aspect, the human constant region is IgG1. In yet another aspect, the murine constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In yet another aspect, the murine constant region is IgG2A. In yet another specific aspect, the antibody has reduced or minimal effector function. In yet another specific aspect, the minimal effector function results from a "less effector Fc mutation" or aglycosylation. In yet another embodiment, the less effector Fc mutation is a N297A or D265A/N297A substitution in the constant region.

[00280] In yet another embodiment, there is provided an anti-PD-L1 antibody comprising a heavy chain and a light chain variable region sequence, wherein: (a) the heavy chain further comprises a sequence of HVR-H1, HVR-H2, and HVR-H3 having at least 85% sequence identity to GFTFSDSWIH (SEQ ID NO:19), AWIS-PYGGSTYYADSVKG (SEQ ID NO:20), and RHWPGGFDY (SEQ ID NO:21), respectively, and/or (b) the light chain further comprises a sequence of HVR-L1, HVR-L2, and HVR-L3 having at least 85% sequence identity to RASQDVSTAVA (SEQ ID NO:22), SASFLYS (SEQ ID NO:23), and QQYLYHPAT (SEQ ID NO:24), respectively.

[00281] In a specific aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[00282] In another aspect, the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs such as: (HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and the light chain variable regions comprise one or more framework sequences juxtaposed between the HVRs such as: (LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yet another aspect, the framework sequences are derived from human consensus framework sequences. In yet another aspect, the heavy chain framework sequences are derived from a Kabat subgroup I, II, or III sequence. In yet another aspect, the heavy chain framework sequence is a VH subgroup III framework sequence. In yet another aspect, one or more of the heavy chain framework sequences are set forth as SEQ ID NOs: 8, 9, 10 and WGQGTLVTVS-SASTK (SEQ ID NO:31).

[00283] In yet another aspect, the light chain framework sequences are derived from a Kabat kappa subgroup I, II, III, or IV sequence. In yet another aspect, the light chain framework sequences are VL kappa I consensus framework. In yet another aspect, one or more of the light chain framework sequences are set forth as SEQ ID NOs: 15, 16, 17, and 18. In yet another specific aspect, the antibody further comprises a human or murine constant region. In yet another aspect, the human constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, IgG3, IgG4. In yet another specific aspect, the human constant region is IgG1. In yet another aspect, the murine constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In yet another aspect, the murine constant region is IgG2A. In yet another specific aspect, the antibody has reduced or minimal effector function. In yet another specific aspect, the minimal effector function results from a "less effector Fc mutation" or aglycosylation. In yet another embodiment, the less effector Fc mutation is a N297A or D265A/N297A substitution in the constant region.

[00284] In yet another embodiment, there is provided an isolated anti-PD-L1 antibody comprising a heavy chain and a light chain variable region sequence, wherein: (a) the heavy chain sequence has at least 85% sequence identity to the heavy chain sequence set forth in SEQ ID NO:26, or (b) the light chain sequence has at least 85% sequence identity to the light chain sequence set forth in SEQ ID NO:4.

[00285] In some embodiments, an isolated anti-PD-L1 antibody comprising a heavy chain and a light chain variable region sequence is provided, 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:4. In some embodiments, an isolated anti-PD-L1 antibody comprising a heavy chain and a light chain variable region sequence is provided, 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 to the amino acid sequence of SEQ ID NO: 26. In some embodiments, an isolated anti-PD-L1 antibody comprising a heavy chain and a light chain variable region sequence is provided, wherein the light ...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. 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:4 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:4 NO:26. In some embodiments, one, two, three, four, or five amino acid residues at the N-terminus of the heavy and/or light chain can be deleted, substituted, or modified.

[00286] In yet another embodiment, an isolated anti-PD-L1 antibody comprising a heavy chain and a light chain sequence is provided, wherein: (a) the heavy chain sequence has at least 85% sequence identity to the heavy chain sequence set forth in SEQ ID NO:32, and/or (b) the light chain sequence has at least 85% sequence identity to the light chain sequence set forth in SEQ ID NO:33.

[00287] In yet another embodiment, an isolated anti-PD-L1 antibody comprising a heavy chain and a light chain sequence is provided, wherein: (a) the heavy chain sequence has at least 85% sequence identity to the heavy chain sequence set forth in SEQ ID NO:55, and/or (b) the light chain sequence has at least 85% sequence identity to the light chain sequence set forth in SEQ ID NO:33.

[00288] In some embodiments, an isolated anti-PD-L1 antibody comprising a heavy chain and a light chain sequence is provided, 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 comprising a heavy chain and a light chain sequence is provided, 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 or 55. In some embodiments, an isolated anti-PD-L1 antibody comprising a heavy chain and a light chain sequence is provided, 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 or 55.

[00289] In some embodiments, the isolated anti-PD-L1 antibody is aglycosylated. Glycosylation of antibodies is typically N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the site chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the site chain of asparagine. Thus, the presence of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment 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 forms an antibody that is conveniently accompanied by alteration of the amino acid sequence such that one of the tripeptide sequences described above (for N-linked glycosylation sites) is removed. The alteration may be made by replacing an asparagine, serine, or threonine residue within the glycosylation site with another amino acid residue (e.g., glycine, alanine, or a conservative substitution).

[00290] In any of the embodiments herein, the isolated anti-PD-L1 antibody can bind to a human PD-L1, e.g., a human PD-L1 as shown in UniProtKB/Swiss-Prot accession no. Q9NZQ7.1, or a variant thereof.

[00291] In yet another embodiment, an isolated nucleic acid encoding any of the antibodies described herein is provided. In some embodiments, the nucleic acid also comprises a vector suitable for expression of the nucleic acid encoding any of the previously described anti-PD-L1 antibodies. In yet another specific aspect, the vector is in a host cell suitable for expression of the nucleic acid. In yet another specific aspect, the host cell is a eukaryotic cell or a prokaryotic cell. In yet another specific aspect, the eukaryotic cell is a mammalian cell, such as Chinese Hamster Ovary (CHO) cells.

[00292] The antibody, or antigen-binding fragment thereof, can be made using methods known in the art, for example, by a process comprising culturing a host cell containing nucleic acid encoding any of the previously described anti-PD-L1 antibodies or antigen-binding fragment in a form suitable for expression, under conditions suitable for producing such antibody or fragment, and recovering the antibody or fragment.

III. Anti-GPC3 antibodies

[00293] Provided herein is a method of treating or delaying the progression of cancer in a subject comprising administering to a subject an effective amount of a PD-1 axis binding antagonist and an anti-GPC3 antibody. Also provided herein is a method of enhancing immune responses against tumor cells in a subject having cancer comprising administering to a subject an effective amount of a PD-1 axis binding antagonist and an anti-GPC3 antibody. For example, enhanced immune responses against tumor cells include infiltration of immune cells including macrophages and multinucleated giant cells into tumor tissues. For another example, enhanced immune responses against tumor cells include increased CD45-positive lymphocytes, CD3ε-positive lymphocytes, and CD8-positive T lymphocytes in tumor infiltrating lymphocytes (TILs).

[00294] Provided herein are antibodies that bind to a human glypican 3 (GPC3). Alternative names for "GPC3" include SGB, DGSX, MXR7, SDYS, SGBS, OCI-5, SGBS1, and GTR2-2. The term "GPC3" as used herein refers to any native GPC3 from any human source. The term encompasses unprocessed, "full-length" GPC3 as well as any form of GPC3 that results from processing in the cell (e.g., mature protein), including, but not limited to, a C-terminal peptide of GPC3. The term also encompasses naturally occurring variants and isoforms of GPC3, e.g., splice variants or allelic variants. For example, descriptions and sequences of GPC3 are provided in UniProtKB/Swiss-Prot accession no. P51654.1.

[00295] In some embodiments, the anti-GPC3 antibody binds to GPC3 and inhibits cell proliferation or growth of cancer cells. In some embodiments, the anti-GPC3 antibody is co-drituzumab.

[00296] In some embodiments, an anti-GPC3 antibody can include an antibody drug conjugate (ADC) (WO2007/137170) comprising a 1G12 antibody (WO2003/100429) (sold under catalog no. B0134R by BioMosaics Inc.) conjugated to a cytotoxic substance.

[00297] In some embodiments, an anti-GPC3 antibody is a humanized anti-GPC3 antibody described in WO2006/006693 or WO2007/047291.

[00298] In some embodiments, an anti-GPC3 antibody includes a humanized anti-GPC3 antibody described in WO2006/006693 or WO2009/041062. In some embodiments, a humanized anti-GPC3 antibody comprising a heavy chain and a light chain variable region sequence is provided, wherein: (a) the heavy chain comprises HVR-H1, HVR-H2, and HVR-H3, wherein also: (i) the sequence of HVR-H1 is DYSMH (SEQ ID NO:34) (ii) the sequence of HVR-H2 is WINTETGEPTYADDFKG (SEQ ID NO:35) (iii) the sequence of HVR-H3 is LY (SEQ ID NO:36) (b) the light chain comprises HVR-L1, HVR-L2, and HVR-L3, wherein also: (i) a sequence of HVR-L1 is KSSQSLLHSDGKTFLN (SEQ ID NO:37) (ii) a sequence of HVR-L2 is LVSRLDS (SEQ ID NO:38) (iii) a sequence of HVR-L3 is CQGTHFPRT (SEQ ID NO:39).

[00299] In a specific aspect, the anti-GPC3 antibody is humanized. The humanized anti-GPC3 antibody can be prepared using, as templates for humanization, appropriately selected human frameworks having high sequence identity to a heavy chain fragment sequence represented by SEQ ID NO:40 or a light chain framework sequence represented by SEQ ID NO:41.

[00300] In some embodiments, provided herein is a chimeric anti-GPC3 antibody or a humanized anti-GPC3 antibody comprising a heavy chain and a light chain variable region sequence, wherein: (a) the heavy chain comprises HVR-H1, HVR-H2, and HVR-H3, wherein also: (i) the sequence of HVR-H1 is DYEMH (SEQ ID NO:42) (ii) the sequence of HVR-H2 is ALDPKTGDTAYSQKFKG (SEQ ID NO:43) (iii) the sequence of HVR-H3 is FYSYTY (SEQ ID NO:44) (b) the light chain comprises HVR-L1, HVR-L2, and HVR-L3, wherein also: (i) a sequence of HVR-L1 is RSSQSLVHSNRNTYLH (SEQ ID NO:45) (ii) a sequence of HVR-L2 is KVSNRFS (SEQ ID NO:46) (iii) a sequence of HVR-L3 is SQNTHVPPT (SEQ ID NO:47).

[00301] The humanized anti-GPC3 antibody can be prepared using, as templates for humanization, appropriately selected human framework sequences having high sequence identity to a heavy chain fragment sequence represented by SEQ ID NO:48 or a light chain framework sequence represented by SEQ ID NO:49.

[00302] In another embodiment, provided herein is a humanized anti-GPC3 antibody capable of binding to an epitope to which a second antibody can bind, wherein said second antibody comprising a heavy chain and a light chain variable region sequence, wherein: (a) the heavy chain comprises HVR-H1, HVR-H2, and HVR-H3, wherein also: (i) the sequence of HVR-H1 is DYEMH (SEQ ID NO:42) (ii) the sequence of HVR-H2 is ALDPKTGDTAYSQKFKG (SEQ ID NO:43) (iii) the sequence of HVR-H3 is FYSYTY (SEQ ID NO:44) (b) the light chain comprises HVR-L1, HVR-L2, and HVR-L3, wherein also: (i) a sequence of HVR-L1 is RSSQSLVHSNRNTYLH (SEQ ID NO:45) (ii) a sequence of HVR-L2 is KVSNRFS (SEQ ID NO:46) (iii) a sequence of HVR-L3 is SQNTHVPPT (SEQ ID NO:47).

[00303] In another embodiment, there is provided a humanized anti-GPC3 antibody comprising a heavy chain variable region selected from the group consisting of heavy chain variable regions represented by SEQ ID NOs:50 and a light chain variable region represented by SEQ ID NO:51. In another alternative non-limiting aspect, there is provided a humanized anti-GPC3 antibody comprising a heavy chain variable region selected from the group of heavy chain variable regions represented by SEQ ID NO:50 and a light chain variable region selected from the group of light chain variable regions represented by SEQ ID NO:52.

[00304] In yet another embodiment, a humanized anti-GPC3 antibody comprising a heavy chain variable region represented by SEQ ID NO:53 and a light chain variable region represented by SEQ ID NO:54 is provided.

[00305] Alternative examples of the anti-GPC3 antibody of the present invention include an anti-GPC3 antibody having cytotoxic activity. In the present invention, non-limiting examples of cytotoxic activity include antibody-dependent cell-mediated cytotoxicity or antibody-dependent cellular cytotoxic activity (ADCC), complement-dependent cytotoxicity (CDC) activity, and T-cell-based cytotoxic activity. In the present invention, CDC activity means cytotoxic activity elicited by the complement system. On the other hand, ADCC activity means target cell damaging activity by, for example, immunocytes, through the binding of the immunocytes via Fc receptors expressed on the immunocytes to the Fc regions of antigen-binding molecules comprising antigen-binding domains capable of binding to the molecules expressed on the cell membranes of the target cells. Whether or not the antigen binding molecule of interest has ADCC activity or has CDC activity can be determined by a method known in the art (e.g., Current protocols in Immunology, Chapter 7. Immunologic studies in humans, Coligan et al., 1993 edition)).

[00306] In some embodiments, alternative examples of the anti-GPC3 antibody of the present invention include an anti-GPC3 antibody conjugated to a cytotoxic substance. Such an anti-GPC3 antibody-drug conjugate (ADC) is specifically described in, for example, WO2007/137170, although the conjugate of the present invention is not limited to those described herein. Specifically, the cytotoxic substance can be any of the chemotherapeutic agents listed below or can be a compound described in Alley et al., (Curr. Opin. Chem. Biol. (2010) 14, 529-537) or WO2009/140242. Antigen binding molecules are conjugated to these compounds via appropriate linkers or the like.

[00307] In some embodiments, alternative examples of the anti-GPC3 antibody of the present invention include an anti-GPC3 antibody comprising an FCYR-binding modified Fc region having greater binding activity against FCY receptors than that of the native human IgG Fc region against Fcy receptors. The modification may include amino acid modification at any position, so long as the resulting Fc region has greater binding activity against Fcy receptors than that of the native human IgG Fc region against Fcy receptors. When the antigen binding molecules contain a human IgG1 Fc region as a human Fc region, the modification preferably allows the Fc region to contain a fucose-containing sugar chain as a sugar chain linkage at position 297 (EU numbering) and is effective for producing greater binding activity against Fcy receptors than that of the native human IgG Fc region against Fcy receptors. Such an amino acid modification has been reported, for example, in International Publication Nos. WO2007/024249, WO2007/021841, WO2006/031370, WO2000/042072, WO2004/029207, WO2004/099249, WO2006/105338, WO2007/041635, WO2008/092117, WO2005/070963, WO2006/020114, WO2006/116260, WO2006/023403 and WO2014/097648.

[00308] In some embodiments, the Fc region contained in the anti-GPC3 antibody provided by the present invention may also include an Fc region modified such that a higher proportion of fucose-deficient sugar chains are linked to the Fc region or a higher proportion of sectioning N-acetylglucosamines are added to the Fc region in the composition of sugar chains linked to the Fc region. WO2006/046751 and WO2009/041062 describe specific examples of the anti-GPC3 antibody comprising the Fc region modified such that a higher proportion of fucose-deficient sugar chains are linked to the Fc region or a higher proportion of sectioning N-acetylglucosamines are added to the Fc region in the composition of sugar chains linked to the Fc region.

[00309] In some embodiments, the anti-GPC3 antibody that can be used in the present invention includes an anti-GPC3 antibody having an amino acid residue modified to alter its isoelectric point (pI). Preferred examples of the "altering the electrical shift of an amino acid residue" in the anti-GPC3 antibody are described in WO2009/041062.

[00310] In some embodiments, the anti-GPC3 antibody includes a modified form of the antibody that has received a post-translational modification of the polypeptide that constitutes the primary structure of the anti-GPC3 antibody. Post-translational modification of a polypeptide refers to the chemical modification provided to the translated polypeptide during polypeptide biosynthesis. For example, an anti-GPC3 antibody that has received the modification of N-terminal glutamines to pyroglutamic acid by pyroglutamylation is also included in the anti-GPC3 antibody of the present invention, as a matter of course. Further, for example, a post-translationally modified anti-GPC3 antibody comprising heavy and light chains or heavy chains linked via a "disulfide bond", meaning that a covalent bond formed between two sulfur atoms is included in the anti-GPC3 antibody of the present invention. A thiol group contained in an amino acid cysteine can form a disulfide bond or crosslink with a second thiol group. In general IgG molecules, the CH1 and CL regions are linked via a disulfide bond, and two polypeptides constituting heavy chains are linked via a disulfide bond between cysteine residues at positions 226 and 229 based on EU numbering. A post-translationally modified anti-GPC3 antibody having such a linkage via a disulfide bond is also included in the anti-GPC3 antibody of the present invention.

[00311] In yet another embodiment, an isolated nucleic acid encoding any of the antibodies described herein is provided. In some embodiments, the nucleic acid also comprises a vector suitable for expression of the nucleic acid encoding any of the previously described anti-GPC3 antibodies. In yet another specific aspect, the vector is in a host cell suitable for expression of the nucleic acid. In yet another specific aspect, the host cell is a eukaryotic cell or a prokaryotic cell. In yet another specific aspect, the eukaryotic cell is a mammalian cell, such as a Chinese Hamster Ovary (CHO) cell.

[00312] The antibody, or antigen-binding fragment thereof, can be made using methods known in the art, for example, by a process comprising culturing a host cell containing nucleic acid encoding any of the previously described anti-GPC3 antibodies or antigen-binding fragments in a form suitable for expression, under conditions suitable for producing such antibody or fragment, and recovering the antibody or fragment.

IV. Antibody Preparation

[00313] The antibody described herein is prepared using skills available in the art for generating antibodies, exemplary methods of which are described in greater detail in the following sections.

[00314] The antibody is directed against an antigen of interest (i.e., PD-L1 (such as human PD-L1) or GPC3 (such as human glypican 3)). Preferably, the antigen is a biologically important polypeptide and administration of the antibody to a mammal suffering from a disorder may result in a therapeutic benefit in that mammal.

[00315] In certain embodiments, an antibody provided herein has a dissociation constant (Kd) of ^1μM, ^150nM, ^100nM, ^50nM, ^10nM, ^1nM, ^0.1nM, ^0.01nM, or ^0.001nM (e.g., 10-8M or less, e.g., from 10-8M to 10-13M, e.g., from 10-9M to 10-13M).

[00316] In one embodiment, Kd is measured by a radiolabeled antigen binding assay (RIA) performed with the Fab' version of an antibody of interest and its antigen as described by the following assay. The binding affinity of the Fabs solution for antigen is measured by equilibrating the Fab' with a minimum concentration of (125I)-labeled antigen in the presence of a titration series of unlabeled antigen, then capturing the bound antigen with an anti-Fab antibody coating plate (see, e.g., Chen et al., J. Mol. Biol. 293:865-881(1999)). To establish assay conditions, MICROTITER (registered) multiwell plates (Scientific Term) are coated overnight with 5 μg/ml of a capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23°C). On a nonabsorbent plate (Nunc #269620), 100 pM or 26 pM [125I]-antigen is mixed with serial dilutions of a Fab’ of interest. The Fab’ of interest is then incubated overnight; however, incubation may continue for a longer period (e.g., approximately 65 hours) to ensure that equilibrium is achieved. The mixtures are then transferred to the capture plate for incubation at room temperature (e.g., for one hour). The solution is then removed and the plate washed eight times with 0.1% polysorbate 20 (TWEEN-20 (proprietary)) in PBS. When the plates are dry, 150 μl/well of scintillant (MICROSCINT-20 (trademark); Packard) is added, and the plates are counted in a TOPCOUNT (trademark) gamma counter (Packard) for ten minutes. Concentrations of each Fab’ that provide less than or equal to 20% maximal binding are chosen for use in competitive binding assays.

[00317] According to another embodiment, Kd is measured using surface plasmon resonance assays using a BIACO-RE (registered)-2000 or a BIACORE (registered)-3000 (BIAcore, Inc., Piscataway, NJ) at 25°C with immobilized CM5 antigen chips at ~10 response units (RU). Briefly, carboxymethylated dextran (CM5, BIACORE, Inc.) biosensor chips are activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (~0.2 μM) prior to injection at a flow rate of 5 μl/min to obtain approximately 10 response units (RU) of coupled protein. Following antigen injection, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20 (TWEEN-20 (trade name) surfactant) (PBST) at 25°C at a flow rate of approximately 25 μl/min. Association rates (kon) and dissociation rates (k0ff) are calculated using a simple one-to-one Langmuir binding model (BIACORE (registered) Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (Kd) is calculated as the ratio k0ff/kon. See, for example, Chen et al., J. Mol. Biol. 293:865–881 (1999). If the association rate exceeds 106 M-1 s-1 by the above surface plasmon resonance assay, then the association rate can be determined using a fluorescent quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) at 25°C of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen as measured on a spectrometer such as a spectrophotometer equipped with a flow stop (Aviv Instruments) or an SLM-AMINCO (trade name) 8000 series spectrophotometer (ThermoSpectronic) with a stirred cuvette. (i) Preparation of antigen

[00318] Soluble antigens or fragments thereof, optionally conjugated to other molecules, can be used as immunogens for generating antibodies. For transmembrane molecules, such as receptors, fragments thereof (e.g., the extracellular domain of a receptor) can be used as the immunogen. Alternatively, cells expressing the transmembrane molecule can be used as the immunogen. Such cells can be derived from a natural source (e.g., cancer cell lines) or can be cells that have been transformed by recombinant techniques to express the transmembrane molecule. Other antigens and forms thereof useful for preparing antibodies will be apparent to those skilled in the art. (ii) Certain Antibody-Based Methods

[00319] Monoclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It may be useful to conjugate the relevant antigen to a protein that is immunogenic in the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor using a bifunctional or derivatizing agent, e.g., maleimidobenzoyl sulfosuccinimide ester (conjugation via cysteine residues), N-hydroxysuccinimide (via lysine residues), glutaraldehyde, succinic anhydride, SOCl2, or R1N=C=NR, where R and R1 are different alkyl groups.

[00320] Animals are immunized against the antigen-immunogenic conjugates, immunogenic conjugates, or derivatives by combining, for example, 100 μg or 5 μg of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of complete Freund's adjuvant and injecting the solution intradermally at multiple sites. One month later, the animals are boosted with 1/5 to 1/10 the original amount of peptide or conjugate in complete Freund's adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later, the animals are bled and the serum is assayed for antibody titer. The animals are boosted to plateau titer. Preferably, the animal is boosted with the conjugate of the same antigen, but conjugated to a different protein and/or through a different crosslinking reagent. Conjugates can also be made in recombinant cell culture as protein fusions. Additionally, aggregating agents such as alum are suitably used to enhance the immune response.

[00321] Monoclonal antibodies of the invention can be prepared using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), and also described, for example, in Hongo et al., Hybridoma, 14(3):253-260 (1995), Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981), and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) considering human-human hybridomas. Additional methods include those described, for example, in U.S. Patent No. 7,189,826 regarding the production of natural human monoclonal 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).

[00322] For other hybridoma techniques, see, e.g., US2006/258841; US2006/183887 (fully human antibodies), US2006/059575; US2005/287149; US2005/100546; US2005/026229; and U.S. Patent Nos. 7,078,492 and 7,153,507. 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 elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Antibodies are raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of a polypeptide of the invention or a fragment thereof, and an adjuvant, such as monophosphoryl lipid A (MPL)/trehalose dicrinomycolate (TDM) (Ribi Immunochem. Research, Inc., Hamilton, Mont.). A polypeptide of the invention (e.g., antigen) or a fragment thereof can be prepared using methods known in the art, such as recombinant methods, some of which are also described herein. Serum from immunized animals is screened for anti-antigen antibodies, and booster immunizations are optionally administered. Lymphocytes from animals producing anti-antigen antibodies are isolated. Alternatively, lymphocytes can be immunized in vitro.

[00323] The lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell. See, for example, Goding, Monoclonal Antibodies: Principles and Practice, pages 59 to 103 (Academic Press, 1986). Myeloma cells can be used that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. Exemplary myeloma cells include, but are not limited to, murine myeloma lines, such as those derived from mouse tumors of MOPC-21 and MPC-11 available from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Md. USA. Mouse-human heteromyeloma and human myeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al, Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[00324] The hybridoma cells thus prepared are seeded and cultured in a suitable culture medium, e.g., a medium containing one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme, hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas will typically include hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells. Preferably, serum-free hybridoma culture methods are used to reduce the use of animal-derived serum such as fetal bovine serum, as described, e.g., in Even et al., Trends in Biotechnology, 24(3), 105-108 (2006).

[00325] Oligopeptides as tools for improving the productivity of hybridoma cell cultures are described in Franek, Trends in Monoclonal Antibody Research, 111-122 (2005). Specifically, standard culture media are enriched with certain amino acids (alanine, serine, asparagine, proline), or with protein hydrolysate fractions, and apoptosis can be significantly suppressed by synthetic oligopeptides consisting of three to six amino acid residues. The peptides are present in millimolar or higher concentrations.

[00326] Culture medium in which hybridoma cells are grown can be assayed for the production of monoclonal antibodies that bind to an antibody of the invention. The binding specificity of monoclonal antibodies produced by hybridoma cells can be determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of the monoclonal antibody can be determined, for example, by Scatchard analysis. See, for example, Munson et al., Anal. Biochem., 107:220 (1980).

[00327] After hybridoma cells are identified that produce antibodies of the desired specificity, affinity, and/or activity, the clones can be subcloned by limiting dilution procedures and cultured by standard methods. See, e.g., Goding, supra. Suitable culture media for this purpose include, e.g., D-MEM or RPMI-1640 medium. In addition, hybridoma cells can be cultured in vivo as ascites tumors in an animal. Monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluids, or serum by conventional immunoglobulin purification procedures such as, e.g., protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. A procedure for isolating proteins from hybridoma cells is described in US2005/176122 and US Patent No. 6,919,436. The method includes using minimal salts, such as lyotropic salts, in the binding process and preferably also using small amounts of organic solvents in the elution process. (iii) Library-Derived Antibodies

[00328] Antibodies of the invention can be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening for antibodies having the desired binding characteristics. Additional methods are reviewed, e.g., in Hoogenboom et al., in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001) and also described, e.g., in 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 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5):1073-1093 (2004); Fellouse, Proc. Natl. Academic. Sci USA 101(34):12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2):119-132(2004).

[00329] In certain phage display methods, VH and VL gene repertoires are separately cloned by polymerase chain reaction (PCR) and randomly recombined into phage libraries, which can then be screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol, 12:433455 (1994). The phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of hybridoma construction. Alternatively, the naïve repertoire can be cloned (e.g., from human) to provide a simple source of antibodies to a wide variety of non-self and also self-antigens without any immunization as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be prepared synthetically by cloning unrearranged V gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to perform the rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol, 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example: United States Patent No. 5,750,373, and United States Patent Publication Nos. 2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598, 2007/0237764, 2007/0292936, and 2009/0002360.

[00330] Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein. (iv) Chimeric, Humanized, and Human Antibodies

[00331] In certain embodiments, an antibody provided herein is a chimeric antibody. Certain chimeric antibodies are described, for example, in U.S. Pat. Nos. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). In one example, the chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, non-human primate, such as a monkey) and a human constant region. In another example, the chimeric antibody is a "class-swapped" antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[00332] In certain embodiments, the chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans, while maintaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody will optionally also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[00333] Humanized antibodies and methods for preparing them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are also described, e.g., in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Na- t'lAcad. Sci. USA 86:10029-10033 (1989); U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing SDR (a-CDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "refacement"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR scrambling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing the "guided selection" method for FR scrambling).

[00334] Human framework regions that may 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 the human antibody consensus sequence of a particular subgroup of light 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)); mature human (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from analysis of FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[00335] In certain embodiments, an antibody provided herein is a human antibody. Human antibodies can be produced using various techniques known in the art. Human antibodies are described generally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5:368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[00336] Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been engineered to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or randomly integrated into the chromosomes of the animal. In such transgenic mice, the endogenous immunoglobulin loci have generally been inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See also, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 describing XENOMOUSE technology (trade name); U.S. Patent No. 5,770,429 describing HUMAB technology (registered); U.S. Patent No. 7,041,870 describing K-M MOUSE technology (registered); and U.S. Patent Application Publication No. US2007/0061900 describing VELOCIMOUSE technology (registered). Human variable regions of intact antibodies generated by such animals can also be modified, for example, by combining with a different human constant region.

[00337] Human antibodies can also be prepared by hybridoma-based methods. Mouse-human heteromyeloma and human myeloma cell lines for the production of human monoclonal antibodies have been described. (See, e.g., 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 generated by human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Additional methods include those described, for example, in U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mi- anyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histo-pathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[00338] Human antibodies can also be generated by isolating selected Fv clone variable domain sequences from human-derived Fv display libraries. Such variable domain sequences can then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.

(v) Antibody Fragments

[00339] Antibody fragments can be generated by traditional methods, such as enzymatic digestion, or by recombinant techniques. In certain circumstances, there are advantages to using antibody fragments as opposed to whole antibodies. The smaller size of the fragments allows for rapid clearance, and may lead to improved access to solid tumors. For a review of certain antibody fragments, see Hudson et al., (2003) Nat. Med. 9:129-134.

[00340] Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments have been derived by 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 produced directly by recombinant host cells. Fab, Fv, and ScFv antibody fragments can all be expressed in and secreted from E. coli, thereby allowing for the facile production of large quantities of these fragments. Antibody fragments can be isolated from the antibody phage libraries described above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab') 2 fragments (Carter et al., Bio/Technology 10:163-167 (1992)). According to another method, F(ab') 2 fragments can be isolated directly from recombinant host cell culture. Fab and F(ab') 2 fragments with increased in vivo half-lives comprising recovering receptor binding epitope residues are described in U.S. Patent No. 5,869,046. Other techniques for producing antibody fragments will be apparent to the skilled artisan. In certain embodiments, an antibody is a single chain Fv (scFv) fragment. See WO93/16185; U.S. Patent Nos. 5,571,894; and 5,587,458. Fv and scFv are the only species with intact combining sites that lack constant regions; thus, they may be suitable for reduced nonspecific binding during in vivo use. scFv fusion proteins can be constructed to produce the fusion of an effector protein to the amino or carboxy terminus of an scFv. See Antibody Engineering, ed. Borrebaeck, supra. The antibody fragment may also be a "linear antibody," e.g., as described in U.S. Patent No. 5,641,870, for example. Such linear antibodies may be monospecific or bispecific.

(vi) Single Domain Antibodies

[00341] In some embodiments, an antibody of the invention is a single domain antibody. A single domain antibody is a single polypeptide chain comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Patent No. 6,248,516). In one embodiment, a single domain antibody consists of all or a portion of the heavy chain variable domain of an antibody.

(vii) Antibody Variants

[00342] In some embodiments, amino acid sequence modification(s) of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate changes into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions of, and/or insertions into, and/or substitutions of, residues in the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution may be made to arrive at the final construct, as long as the final construct possesses the desired characteristics. The amino acid changes may be introduced into the subject antibody amino acid sequence at the same time that the sequence is made.

(viii) Substitution, Insertion and Deletion Variants

[00343] In certain embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the HVRs and FRs. Conservative substitutions are shown in Table 1 under the heading "Preferred Substitutions." More substantial changes are provided in Table 1 under the heading "Exemplary Substitutions," and as described below with reference to amino acid chain classes. Amino acid substitutions can be introduced into an antibody of interest and the products analyzed for a desired activity, e.g., maintained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC. Table 1 Table 1- Exemplary Substitutions. Table legends: Table 1: Exemplary substitutions - original residue - exemplary substitutions - preferred substitutions - norleucine.

[00344] Amino acids can be grouped according to common side chain properties: a. hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; b. neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; c. acidic: Asp, Glu; d. basic: His, Lys, Arg; e. residues that influence chain orientation: Gly, Pro; f. aromatic: Trp, Tyr, Phe.

[00345] Non-conservative substitutions will involve exchanging a member of one of these classes for another class.

[00346] One type of substitutional variant involves the replacement of one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have modifications (e.g., improvements) in certain biological properties (e.g., increased affinity, reduced immunogenicity) relative to the parent compound and/or will have certain biological properties substantially maintained from the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which can be conveniently generated, for example, using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies displayed on phage and analyzed for a particular biological activity (e.g., binding affinity).

[00347] Changes (e.g., substitutions) can be made to HVRs, for example, to improve antibody affinity. Such changes can be made to HVR "hotspots," i.e., residues encoded by codons that are mutated at high frequency during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and/or SDRs (a-CDRs), with the resulting variant VH or VL being tested for binding affinity. Affinity maturation by construction and reselection of secondary libraries has been described, for example, in Hoogenboom et al., in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001).) In some embodiments of affinity maturation, diversity is introduced into the variable genes for maturation by any of a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method for introducing this involves HVR-targeted methods, 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 modeling or alanine scanning mutagenesis. CDR-H3 and CDR-L3 in particular are frequently targeted.

[00348] In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, so long as such changes do not substantially reduce the ability of the antibody to bind antigen. For example, conservative changes (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made to HVRs. Such changes may be outside of SDRs or HVR "hotspots." In certain embodiments of the variant VH and VL sequences provided above, each HVR is unchanged, or contains no more than one, two, or three amino acid substitutions.

[00349] A useful method for identifying residues or regions of an antibody 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 target residue or group of residues (e.g., charged residues such as arg, asp, his, lys, and glu) are identified and replaced with a neutrally or negatively charged amino acid (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with antigen is affected. Other substitutions can be introduced at the amino acid sites demonstrating functional sensitivity to the initial substitutions. Alternatively, or additionally, a crystal structure of an antigen-antibody complex is used to identify the points of contact between the antibody and antigen. Such contact residues and neighboring residues can be targeted or eliminated as candidates for replacement. Variants can be analyzed to determine whether they contain the desired properties.

[00350] Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include fusion to the N- or C-terminus of the antibody to an enzyme (e.g., to ADEPT) or a polypeptide that increases the serum half-life of the antibody.

(ix) Glycosylation variants

[00351] In certain embodiments, an antibody is provided herein altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody can conveniently be accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[00352] Where the antibody comprises an Fc region, the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally linked by an N-bond to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in an antibody of the invention may be made in order to create antibody variants with certain improved properties.

[00353] In one embodiment, antibody variants are provided comprising an Fc region, wherein a carbohydrate structure linked to the Fc region has reduced fucose or lacks fucose, which can improve ADCC function. Specifically, antibodies are contemplated herein that have reduced fucose relative to the amount of fucose in the same antibody produced in a wild-type CHO cell. That is, they are characterized by having a lower amount of fucose than they would otherwise have if produced by native CHO cells (e.g., a CHO cell that produces a native glycosylation partner, such as, a CHO cell containing a native FUT8 gene). In certain embodiments, the antibody is one in which less than about 50%, 40%, 30%, 20%, 10%, or 5% of the N-linked glycans thereon comprise fucose. For example, the amount of fucose in such an antibody can be from 1% to 80%, from 1% to 65%, from 5% to 65%, or from 20% to 40%. In certain embodiments, the antibody is one in which none of the N-linked glycans thereon comprise fucose, i.e., wherein the antibody is completely fucose-free, or has no fucose, or is afucosylated. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures linked to Asn 297 (e.g., complex, hybrid, and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO2008/077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues); however, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, for example, United States Patent Publication Nos. US2003/0157108 (Presta, L.); US2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include: US2003/0157108; WO2000/61739; WO2001/29246; US2003/0115614; US2002/0164328; US2004/0093621; US2004/0132140; US2004/0110704; US2004/0110282; US2004/0109865; WO2003/085119; WO2003/084570; WO2005/035586; WO2005/035778; WO2005/053742; WO2002/031140; 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 deficient in protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249:533545 (1986); United States Patent Publication No. US2003/0157108, Presta, L; and WO2004/056312, Adams et al., especially Example 11), and knockout cell lines, such as alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

[00354] Antibody variants are also provided with bisected oligosaccharides, e.g., wherein a bi-antennary oligosaccharide linked to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, e.g., in WO2003/011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); US2005/0123546 (Umana et al.), and Ferrara et al., Biotechnology and Bioengineering, 93(5):851-861 (2006). Antibody variants with at least one galactose residue in the oligosaccharide linked to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO1997/30087 (Patel et al.); WO1998/58964 (Raju, S.); and WO1999/22764 (Raju, S.).

[00355] In certain embodiments, antibody variants comprising an Fc region described herein are capable of binding to an FcyRIII. In certain embodiments, antibody variants comprising an Fc region described herein have ADCC activity in the presence of human effector cells or have increased ADCC activity in the presence of human effector cells compared to the same antibody otherwise comprising a human wild-type IgG1 Fc region.

(x) Fc region variants

[00356] In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. The Fc region variant can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions.

[00357] In certain embodiments, the invention contemplates an antibody variant that possesses some, but not all, of the effector functions that make it a desirable candidate for applications where the in vivo half-life of the antibody is important even if certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays can be conducted to confirm reduction/depletion of CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FCYR binding (hence likely no ADCC activity) but retains FcRn binding capability. The primary cells for mediating ADCC, NK cells, express FCYRIII only, while monocytes express FCYRI, FCYRII, and FCYRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Nonlimiting examples of in vitro assays for evaluating ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362 (see, e.g., Hellstrom, I. 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); 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, nonradioactive assay methods may be employed (see, e.g., ACTI (trade name) nonradioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc., Mountain View, CA; and CytoTox 96 (registered) nonradioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells). Alternatively, or additionally, the ADCC activity of the molecule of interest may be assayed in vivo, e.g., in an animal model such as that described in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652–656 (1998). C1q binding assays may also be performed to confirm that the antibody is unable to bind to C1q and therefore lacks CDC activity. See, e.g., C1q and C3c binding ELISA in WO2006/029879 and WO2005/100402. To assess complement activation, a CDC assay can be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M.S. et al., Blood 101:1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo half-life/clearance determinations can also be performed using methods known in the art (see, e.g., Petkova, S.B. et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[00358] Antibodies with reduced effector function include those with substitution of one or more Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants with substitutions at one or more amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant with substitution of residues 265 and 297 with alanine (U.S. Patent No. 7,332,581).

[00359] Certain antibody variants with improved or decreased binding to FcRs are described. (See, e.g., U.S. Patent No. 6,737,056; WO2004/056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001).)

[00360] In certain embodiments, an antibody variant comprises an Fc region with one or more amino acid substitutions that improve ADCC, e.g., substitutions at positions 298, 333, and/or 334 of the Fc region (EU residue numbering). In the exemplary embodiment, the antibody comprising the following amino acid substitutions in its Fc region: S298A, E333A, and K334A.

[00361] In some embodiments, alterations are made to the Fc region that result in altered (i.e., enhanced or decreased) C1q binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Patent No. 6,194,551, WO99/51642, and Idusogie et al., J. Immunol. 164:4178-4184 (2000).

[00362] Antibodies with increased half-lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in US2005/0014934 (Hinton et al.)). Those antibodies comprise an Fc region with one or more substitutions therein that improve the binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more 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, e.g., substitution of Fc region residue 434 (U.S. Patent No. 7,371,826). See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO94/29351 involving further examples of Fc region variants.

(xi) Antibody Derivatives

[00363] Antibodies of the invention may also be modified to contain additional non-proteinaceous moieties that are known in the art and readily available. In certain embodiments, suitable moieties for antibody derivation are water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, propylpropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they may be the same or different molecules. In general, the number and/or type of polymers used for derivatization may be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.

(xii) Vectors, Host Cells and Recombinant Methods

[00364] Antibodies can also be produced using recombinant methods. For recombinant production of anti-antigen antibodies, nucleic acid encoding the antibody is isolated and inserted into a replicable vector for further cloning (DNA amplification) or for expression. DNA encoding the antibody can be readily isolated and sequenced using conventional procedures (e.g., using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of the antibody). Many vectors are available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription determination sequence. (a) Signal Sequence Component

[00365] An antibody of the invention may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which is preferably a signal sequence or another polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. The selected heterologous signal sequence preferably is one that is recognized and processed (e.g., cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process a native antibody signal sequence, the signal sequence is replaced by a prokaryotic signal sequence selected, for example, from the group of alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders. For yeast secretion, the native signal sequence can be replaced, for example, by the yeast invertase leader, a leader factor (including α-factor leaders from Saccharomyces and Kluyveromyces), or acid phosphatase leader, the C. albicans glucoamylase leader, or the signal described in WO90/13646. In mammalian cell expression, mammalian signal sequences as well as viral secretory leaders, for example the herpes simplex gD signal, are available. (b) Origin of Replication

[00366] Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Typically, in cloning vectors, this sequence is one that enables the vector to replicate independently of chromosomal DNA, and includes origins of replication or autonomously replicating sequences. Such sequences are well known for a variety of bacteria, yeast, and viruses. The pBR322 plasmid origin of replication is suitable for most Gram-negative bacteria, the 2μ plasmid origin is suitable for yeast, and several viral origins (SV40, polyoma, adenovirus, VSV, or BPV) are useful for cloning vectors in mammalian cells. Typically, the origin of replication component is not required for mammalian expression vectors (the SV40 origin can typically be used simply because it contains the upstream promoter). (c) Gene Component Selection

[00367] Expression and cloning vectors may contain a selection gene, also called a selection marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) auxotrophic complement deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.

[00368] An example of a selection scheme uses a drug to stop the growth of a host cell. Those cells that are successfully transformed with a heterologous gene produce a protein conferring resistance to the drug and thus survive the selection regime. Examples of dominant selection use the drugs: neomycin, mycophenolic acid, and hygromycin.

[00369] Another example of selectable markers for mammalian cells are those that allow the identification of cells competent to take up antibody-encoding nucleic acid, such as DHFR, glutamine synthetase (GS), thymidine kinase, metallothionein-I and -II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc.

[00370] For example, cells transformed with the DHFR gene are identified by culturing the transformants in a culture medium containing methotrexate (Mtx), a competitive antagonist of DHFR. Under these conditions, the DHFR gene is amplified along with any other cotransformed nucleic acid. A Chinese hamster ovary (CHO) cell line deficient in endogenous DHFR activity (e.g., ATCC CRL-9096) can be used.

[00371] Alternatively, cells transformed with the GS gene are identified by culturing the transformants in a culture medium containing L-methionine sulfoximine (Msx), a GS inhibitor. Under these conditions, the GS gene is amplified along with any other cotransformed nucleic acids. The GS selection/amplification system can be used in combination with the DHFR selection/amplification system described above.

[00372] Alternatively, host cells (particularly, wild-type hosts containing endogenous DHFR) transformed or co-transformed with DNA sequences encoding an antibody of interest, wild-type DHFR gene, and another selectable marker such as aminoglycoside 3'-phosphotransferase (APH) can be selected by growing the cell in medium containing a selection agent for the selectable marker such as an aminoglycoside antibiotic, e.g., kanamycin, neomycin, or G418. See, U.S. Pat. No. 4,965,199.

[00373] A suitable selection gene for use in yeast is the trp 1 gene present on the yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979)). The trp 1 gene provides a selection marker for a mutant strain of yeast lacking the ability to grow on tryptophan, e.g., ATCC No. 44076 or PEP4-1. Jones, Genetics, 85:12 (1977). The presence of the trp 1 lesion in the yeast host cell genome then provides an effective environment to detect transformation by growth in the absence of tryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or 38,626) are complemented by plasmids transforming the Leu2 gene.

[00374] In addition, vectors derived from the 1.6 μm circular plasmid pKD1 can be used for transformation of Kluyveromyces yeasts. Alternatively, an expression system for large-scale production of recombinant calf chymosin has been reported for K. lactis. Van den Berg, Bio/Technology, 8:135 (1990). Stable multicopy expression vectors for secretion of mature recombinant human serum albumin by industrial strains of Kluyveromyces have also been described. Fleer et al., Bio/Technology, 9:968-975 (1991). (d) Promoter component

[00375] Expression and cloning vectors generally contain a promoter that is recognized by the host organism and is operably linked to the nucleic acid encoding an antibody. Promoters suitable for use in prokaryotic hosts include the phoA promoter, β-lactamase and lactose promoter systems, alkaline phosphatase promoter, a tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter. However, other known bacterial promoters are suitable. Promoters for use in bacterial systems will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding an antibody.

[00376] Promoter sequences are known to eukaryotes. Virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream of the site where transcription is initiated. Another sequence found 70 to 80 bases upstream of the transcription start of many is a CNCAAT region where N can be any nucleotide. At the 3' end of most eukaryotic genes is an AATAAA sequence which may be the signal for addition of the poly A tail to the 3' end of the coding sequence. All of these sequences are suitable for insertion into eukaryotic expression vectors.

[00377] Examples of promoter sequences suitable for use with yeast hosts include the promoters for 3-phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.

[00378] Other yeast promoters, which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Vectors and promoters suitable for use in yeast expression are also described in EP73,657. Yeast enhancers are also advantageously used with yeast promoters.

[00379] Antibody transcription from vectors in mammalian host cells can be controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, contagious epithelioma virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis B virus, Simian Virus 40 (SV40), or from heterologous mammalian promoters, for example, the actin promoter or an immunoglobulin promoter, from heat shock promoters, provided such promoters are compatible with the host cell systems.

[00380] The SV40 virus early and late promoters are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication. The human cytomegalovirus immediate early promoter is conveniently obtained as a HindIII E restriction fragment. A system for DNA expression in mammalian hosts using human papillomavirus as a vector is described in U.S. Patent No. 4,419,446. A modification of this system is described in U.S. Patent No. 4,601,978. See also Reyes et al., Nature 297:598-601 (1982) for expression of human β-interferon cDNA in mouse cells under the control of a herpes simplex virus thymidine kinase promoter. Alternatively, Rous Sarcoma virus together with the terminal repeat can be used as the promoter. (e) Enhancer Element Component

[00381] Transcription of a DNA encoding an antibody of this invention by higher eukaryotes is often increased by insertion of an enhancer sequence into the vector. Many enhancer sequences are currently known from mammalian genes (globin, elastase, albumin, α-fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18 (1982) on enhancement elements for activation of eukaryotic promoters. The enhancer may be ligated into the vector at a position 5' or 3' to the antibody-coding sequence, but is preferably located at a site 5' to the promoter. (f) Transcription Termination Component

[00382] Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells of other multicellular organisms) will also contain sequences necessary for transcription termination and for stabilization of the mRNA. Such sequences are commonly available from the 5' untranslated regions and occasionally from eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the antibody-encoding mRNA. A useful transcription termination component is the bovine growth hormone polyadenylation region. See WO94/11026 and the expression vector described therein. (g) Selection and Transformation of Host Cells

[00383] Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryotic, yeast, or higher eukaryotic cells described above. Prokaryotes suitable for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, e.g., Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B. licheniformis (e.g., B. licheniformis 41P described in DD 266,710 published April 12, 1989), Pseudomonas such as P. aeruginosa, and Streptomyces. A preferred E. coli cloning host is E. coli 294 (ATCC 31,446), although other strains such as E. coli B, E. coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable. These examples are illustrative rather than limiting.

[00384] Full-length antibodies, antibody fusion proteins, and antibody fragments can be produced in bacteria, in particular, when glycosylation and Fc effector function are not required, such as when the therapeutic antibody is conjugated to a cytotoxic agent (e.g., a toxin) that itself shows efficacy in killing tumor cells. Full-length antibodies have a longer half-life in the circulation. Production in E. coli is faster and cheaper. For expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent No. 5,648,237 (Carter et al.), U.S. Patent No. 5,789,199 (Joly et al.), U.S. Patent No. 5,840,523 (Simmons et al.), which describe translation initiation region (TIR) and signal sequences to optimize expression and secretion. See also Charlton, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Human Press, Totowa, N.J., 2003), pp. 245-254, describing expression of antibody fragments in E. coli. After expression, the antibody can be isolated from the E. coli cell paste as a soluble fraction and can be purified by, for example, a protein A or G column depending on the isotype. Final purification can be accomplished by the process of purification of antibody expressed, for example, in CHO cells.

[00385] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among the lower eukaryotic host microorganisms. However, several other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe; Kluyveromyces hosts such as, for example, K. lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. nudes; yarrowia (EP402,226); Pichia pastoris (EP183,070); Candida; Trichoderma reesia (EP244,234); Neurospora crassa; Schwanniomyces such as Schwanniomyces occidentalis; and filamentous fungi such as, for example, Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger. For a review describing the use of yeasts and filamentous fungi for the production of therapeutic proteins, see, for example, Gerngross, Nat. Biotech. 22:1409–1414 (2004).

[00386] Certain strains of yeast and fungi can be selected in which the glycosylation pathway has been "humanized," resulting in the production of an antibody with a partially or fully human glycosylation pattern. See, e.g., Li et al., Nat. Biotech. 24:210-215 (2006) (describing humanization of the glycosylation pathway in Pichia pastoris); and Gerngross et al., supra.

[00387] Suitable host cells for the expression of glycosylated antibody are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (midge), Drosophila melanogaster (fruit fly), and Bombyx mori have been identified. A variety of viral strains for transfection are publicly available, for example, the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses can be used as the virus herein according to the invention, particularly for the transfection of Spodoptera frugiperda cells.

[00388] Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, lentil (Leninaceae), alfalfa (M. truncatula), and tobacco may also be used as hosts. See, for example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTICORPOS (trade name) technology for producing antibodies in transgenic plants).

[00389] Vertebrate cells can be used as hosts, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure. Examples of useful mammalian host cell lines are the SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney 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 carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al, Annals NY. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma cell line (Hep G2). Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub et al, Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as NS0 and Sp2/0. For a review of certain mammalian host cell lines suitable for antibody production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Human Press, Totowa, N.J., 2003), pp. 255-268.

[00390] Host cells are transformed with the above-described expression or cloning vectors for antibody production and cultured in conventional nutrient media modified as appropriate for induction of promoters, selection of transformants, or amplification of genes encoding the desired sequences. (h) Culturing Host Cells

[00391] Host cells used to produce an antibody of this invention may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimum Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM, Sigma)) are suitable for culturing the host cells. In addition, any of the media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal. Biochem. 102:255 (1980), U.S. Patent Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO90/03430; WO87/00195; or U.S. Patent Re. 30,985 can be used as culture media for the cells. host cells. Any of these media may be supplemented as needed with other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as the drug GENTAMICIN (trade name)), trace elements (defined as inorganic compounds usually present in final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. Culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the skilled artisan.

(xiii) Antibody Purification

[00392] When using recombinant techniques, the antibody may be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If the antibody is produced intracellularly, as a first step, particulate debris, or host cell or lysed fragments, are removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio/Technology 10:163167 (1992) describe a procedure for isolating antibodies that are secreted into the periplasmic space of E. coli. Briefly, the cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) for about 30 min. Cell debris can be removed by centrifugation. Where the antibody is secreted into the medium, supernatants from such expression systems are usually first concentrated using a commercially available protein concentration filter, e.g., an Amicon or Millipore ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the above steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.

[00393] The antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being among the typically preferred purification steps. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies that are based on human γ1, Y2, or Y4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and for human Y3 (Guss et al., EMBO J. 5:1567-1575 (1986)). The matrix to which the affinity ligand is bound is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled-pore glass or poly(styrenedivinyl)benzene allow faster flow rates and shorter processing times than can be obtained with agarose. Where the antibody comprises a CH3 domain, Bakerbond ABX (trade name) resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification. Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, reversed-phase HPLC, silica chromatography, heparin SEPHAROSE (trade name) chromatography, chromatography on an anion- or cation-exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.

[00394] In general, various methodologies for preparing antibodies for research, testing, and clinical use are well established in the art, consistent with the methodologies described above and/or as considered appropriate by one of skill in the art for a particular antibody of interest.

C. Selection of Biologically Active Antibodies

[00395] Antibodies produced as described above may be subjected to one or more "biological activity" assays to select an antibody with beneficial properties from a therapeutic perspective or select formulations and conditions that maintain the biological activity of the antibody. The antibody may be tested for its ability to bind the antigen against which it was raised. For example, methods known in the art (such as ELISA, Western Blot, etc.) may be used.

[00396] For example, for an anti-PD-L1 antibody, the antigen binding properties of the antibody can be assessed in an assay that detects the ability to bind to PD-L1. In some embodiments, binding of the antibody can be determined by saturation binding; ELISA; and/or competition assays (e.g., RI-As), for example. Additionally, the antibody can be subjected to other biological activity assays, for example, in order to assess its efficacy as a therapeutic product. Such assays are known in the art and depend on the target antigen and intended use for the antibody. For example, the biological effects of PD-L1 blockade by the antibody can be assessed in CD8+ T cells, a lymphocytic choriomeningitis virus (LCMV) mouse model, and/or a syngeneic tumor model, for example, as described in U.S. Patent 8,217,149.

[00397] To analyze antibodies that bind to a particular epitope on the antigen of interest (e.g., those that block the binding of the example anti-PD-L1 antibody to PD-L1), a routine 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, e.g., as described in Champe et al., J. Biol. Chem. 270:1388-1394 (1995), can be performed to determine whether the antibody binds an epitope of interest.

D. Pharmaceutical Formulations and Compositions

[00398] Also provided herein are pharmaceutical formulations and compositions comprising a PD-1 axis binding antagonist and/or an antibody described herein (such as an anti-PD-L1 antibody or an anti-GPC3 antibody) as an active ingredient and a pharmaceutically acceptable carrier.

[00399] "Combination" as described herein, refers to a combination of active ingredients for combination use, and includes both modes where separate substances are used in combination in administration or where they are supplied as a mixture (combination preparation).

[00400] Pharmaceutical formulations and compositions as described herein can be prepared by mixing the active ingredients (such as an anti-PD-L1 antibody and/or an anti-GPC3 antibody) having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th Edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight polypeptides (less than about 10 residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein also include interstitial drug dispersion agents such as soluble, neutral, active hyaluronidase glycoproteins (sHASEGP), e.g., soluble human hyaluronidase PH-20 glycoproteins, such as rHuPH20 (HYLENEX (registered), Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in United States Patent Publication Nos. 2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.

[00401] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO2006/044908, the latter formulations including a histidine acetate buffer. In some embodiments, the anti-PD-L1 antibody described herein is in a formulation comprising the antibody at a concentration of about 60 mg/mL, histidine acetate at a concentration of about 20 mM, sucrose at a concentration of about 120 mM, and polysorbate (e.g., polysorbate 20) at a concentration of 0.04% (w/v), and the formulation has a pH of about 5.8. In some embodiments, the anti-PD-L1 antibody described herein is in a formulation comprising the antibody at a concentration of about 125 mg/mL, histidine acetate at a concentration of about 20 mM, sucrose at a concentration of about 240 mM, and polysorbate (e.g., polysorbate 20) at a concentration of 0.02% (w/v), and the formulation has a pH of about 5.5.

[00402] The composition and formulation herein may also contain more than one active ingredient as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such active ingredients are suitably present in combination in amounts that are effective for the intended purpose.

[00403] Active ingredients can be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions. Such techniques are described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[00404] Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes.

IV. Treatment Methods

[00405] Provided herein are methods for treating, preventing, or delaying the progression of cancer in a subject comprising administering to the subject an effective amount of a PD-1 axis binding antagonist and an anti-GPC3 antibody. In some embodiments, the treatment results in a prolonged response in the subject after cessation of treatment. The methods described herein may find use in treating conditions where enhanced immunogenicity is desired such as increasing tumor immunogenicity for the treatment of cancer. Also provided herein are methods of enhancing immune responses against tumor cells in a subject having cancer comprising administering to the subject an effective amount of a PD-1 axis binding antagonist and an anti-GPC3 antibody. For example, the enhanced immune response against tumor cells includes infiltration of immune cells including macrophages and multinucleated giant cells into tumor tissues. For another example, enhanced immune response against tumor cells includes the increase of CD45 positive lymphocytes, CD3ε positive lymphocytes, and CD8 positive T lymphocytes in tumor infiltrating lymphocytes (TILs). Any of the PD-1 axis binding antagonists and anti-GPC3 antibodies known in the art or described herein can be used in the methods.

[00406] In some embodiments, the individual is a human. In some embodiments, the individual has GPC3 positive cancer. In some embodiments, the GPC3 positive cancer is liver cancer, breast cancer, mammary cancer, ovarian cancer, gastric cancer, bladder cancer, pancreatic cancer, endometrial cancer, colon cancer, kidney cancer, esophageal cancer, or prostate cancer. In some embodiments, the liver cancer is hepatocellular carcinoma. In some embodiments, the breast cancer is breast carcinoma or breast adenocarcinoma. In some embodiments, the breast carcinoma is invasive ductal carcinoma. In some embodiments, the lung cancer is lung adenocarcinoma. In some embodiments, the colon cancer is colorectal adenocarcinoma. In some embodiments, the cancer cells in the individual express PD-L1. In some embodiments, the cancer cells in the subject express GPC3 protein at a level that is detectable (e.g., detectable using methods known in the art).

[00407] In some embodiments, the subject has been treated with a GPC3-targeted therapy prior to combination treatment with a PD-1 axis binding antagonist and an anti-GPC3 antibody. In some embodiments, the GPC3-targeted therapy includes treatment with one or more small molecules, e.g., sorafenib.

[00408] In some embodiments, the combination therapy of the invention comprises administration of a PD-1 axis binding antagonist and an anti-GPC3 antibody. The PD-1 axis binding antagonist and the anti-GPC3 antibody can be administered in any manner known in the art. For example, the PD-1 axis binding antagonist and the anti-GPC3 antibody can be administered sequentially (at different times) or concurrently (at the same time). In some embodiments, the PD-1 axis binding antagonist is in a separate composition as the anti-GPC3 antibody. In some embodiments, the PD-1 axis binding antagonist is in the same composition as the anti-GPC3 antibody.

[00409] The PD-1 axis binding antagonist and the anti-GPC3 antibody can be administered by the same administration routine or by different administration routines. In some embodiments, the PD-1 axis binding antagonist is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. In some embodiments, the anti-GPC3 antibody is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intraventricularly, or intranasally. An effective amount of the PD-1 axis binding antagonist and the anti-GPC3 antibody can be administered for preventing or treating disease. The appropriate dosage of the PD-1 axis binding antagonist and/or anti-GPC3 antibody may be determined based on the type of disease being treated, the type of PD-1 axis binding antagonist and anti-GPC3 antibody, 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 treating physician.

[00410] As a general ratio, the therapeutically effective amount of the antibody administered to the human will be in the range of about 0.01 to about 50 mg/kg of the patient's body weight if by one or more administrations. In some embodiments, the antibody used is about 0.01 to about 45 mg/kg, about 0.01 to about 40 mg/kg, about 0.01 to about 35 mg/kg, about 0.01 to about 30 mg/kg, about 0.01 to about 25 mg/kg, about 0.01 to about 20 mg/kg, about 0.01 to about 15 mg/kg, about 0.01 to about 10 mg/kg, about 0.01 to about 5 mg/kg, or about 0.01 to about 1 mg/kg administered daily, for example. In some embodiments, the antibody is administered at 15 mg/kg. However, other dosing regimens may be useful. In one embodiment, an anti-PD-L1 antibody described herein is administered to a human at a dose of about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, or about 1400 mg on day 1 of 21-day cycles. The dose may be administered as a single dose or as multiple doses (e.g., 2 or 3 doses), such as infusions. The dose of the antibody administered in a composition treatment may be reduced compared to a single treatment. The process of this therapy is readily monitored by conventional techniques.

[00411] In some embodiments, the methods may also comprise an additional therapy. The additional therapy may be radiation therapy, surgery (e.g., lumpectomy and a mastectomy), chemotherapy, gene therapy, DNA therapy, viral therapy, RNA therapy, immunotherapy, bone marrow transplantation, nanotherapy, monoclonal antibody therapy, or a combination of the foregoing. The additional therapy may be in the form of adjuvant or neoadjuvant therapy. In some embodiments, the additional therapy is the administration of a small molecule enzyme inhibitor or anti-metastatic agent. In some embodiments, the additional therapy is the administration of side effect limiting agents (e.g., agents intended to decrease the occurrence and/or severity of treatment side effects, such as anti-nausea agents, etc.). In some embodiments, the additional therapy is radiation therapy. In some embodiments, the additional therapy is surgery. In some embodiments, the additional therapy is a combination of radiation therapy and surgery. In some embodiments, the additional therapy is gamma irradiation. In some embodiments, the additional therapy is therapy targeting the PI3K/AKT/mTOR pathway, HSP90 inhibitor, tubulin inhibitor, apoptosis inhibitor, and/or chemopreventive agent. The additional therapy can be one or more of the chemotherapeutic agents described herein.

V. Manufactured Articles or Kits

[00412] In another embodiment of the invention, an article of manufacture or a kit is provided comprising a PD-1 axis binding antagonist and/or an anti-GPC3 antibody. In some embodiments, the article of manufacture or kit also comprises the package insert comprising instructions for use of the PD-1 axis binding antagonist in conjunction with an anti-GPC3 antibody to treat or delay the progression of cancer in a subject or to enhance immune responses against tumor cells of a subject having cancer. For example, enhanced immune response against tumor cells includes infiltration of immune cells including macrophages and multinucleated giant cells into tumor tissues. For another example, enhanced immune response against tumor cells includes the increase in CD45 positive lymphocytes, CD3ε positive lymphocytes, and CD8 positive T lymphocytes in tumor infiltrating lymphocytes (TILs). Any of the PD-1 axis binding antagonists and/or anti-GPC3 antibodies described herein may be included in the manufacturing article or kits.

[00413] In some embodiments, the PD-1 axis binding antagonist and the anti-GPC3 antibody are in the same container or separate containers. Suitable containers include, for example, bottles, vials, pouches, and syringes. The container may be formed from a variety of materials such as glass, plastic (such as polyvinyl chloride or polyolefin), or metal alloy (such as stainless steel or hastelloy). In some embodiments, the container holds the formulation and the label on, or associated with, the container may indicate directions for use. The article of manufacture or kit may also include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. In some embodiments, the article of manufacture also includes one or more of the other agents (e.g., a chemotherapeutic agent, and antineoplastic agent). Suitable containers for one or more agents include, for example, bottles, vials, bags and syringes.

[00414] The specification is considered to be sufficient to enable one to practice the invention. Various modifications of the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes. Example 1 Mouse Cell Lines Expressing Human Glypican-3 (GPC3)

[00415] Mouse cancer cell lines, Hepa1-6 (ATCC No. CRL-1830) and CT26 (ATCC No. CRL-2638) were transfected with human GPC3 expression vector, pCXND2/hGPC3(FL) [Ishiguro T. et al., Cancer Res. 2008;68:9832-9838] using Fu-GENE6 (Roche Diagnostics Corp) and selected with 1 mg/mL G418 (Invitrogen). Cells that grew even in the presence of G418 were harvested, and colonies were isolated by limiting dilution. Expression of human GPC3 was confirmed using FACS using anti-human GPC3 antibody, GC33 [Ishiguro T. et al., Cancer Res. 2008; 68: 9832-9838]. Representative clones were selected and used for the experiments. Example 2 Antitumor activity of anti-GPC3 antibody in syngeneic mouse model using Hepa1-6 cell line expressing human GPC3

[00416] Hepa1-6/hGPC3 cells were cultured using cell culture flasks in an incubator (set at 37°C and 5% CO2). Cells were detached from the flasks with trypsin and washed with D-MEM containing 10% (v/v) FBS, 0.6 mg/mL G418. Then, cells were resuspended in D-MEM (2 × 108 cells/mL), and an equal volume of Matrigel was added. Cell concentrations for implantation were 1 × 108 cells/mL. Cells were inoculated subcutaneously into the right flank of each C57BL/6J mouse (Charles River Laboratories, Japan) (1 × 107 cells/mouse). Once palpable tumors were stabilized, animals were randomized into test groups so that each group had similar mean tumor volumes at study initiation. 1 or 5 mg/kg of human anti-GPC3 mononoclonal antibody mouse GC33 [WO2006/006693] diluted in PBS, or PBS as a vehicle control was injected intravenously on day 14, 21, and 28 after tumor inoculation. Mouse GC33 showed dose-dependent inhibition of tumor growth compared with vehicle control (Figure 1). Example 3 Pathological changes induced by anti-GPC3 antibody in a syngeneic mouse model using Hepa1-6 cell line expressing human GPC3

[00417] To evaluate the changes in tumor tissue of Hepa1-6 tumor by mouse GC33 treatment, tumor tissue isolated after 3 or 7 days from single injection of mouse GC33 antibody or vehicle control was used for pathological examination. Tumor tissues were fixed by 4% paraformaldehyde (PFA) and embedded in paraffin by AMeX method [Suzuki et al, J Toxicol Sci. 2002; 27:165-172, Watanabe et al, J Toxicol Pathol. 2015; 28:43-49]. Three micrometer paraffin sections were stained with hematoxylin and eosin (HE) or immunohistochemically (IHC). IHC staining was performed according to the labeled streptavidin-biotin (LSAB) method (streptavidin horseradish RTU). Antibodies against F4/80 (murine macrophage marker antigen; A3-1, BioLegend), PD-L1 (mouse B7-H1/PD-L1 marker antigen; AF1019, R&D Systems) were used as the primary antibodies. Positive signals were visualized by the peroxidase-diaminobenzidine reaction, and the sections were counterstained with hematoxylin.

[00418] Upon injection of mouse GC33, immune cell infiltration and tumor cell death were observed in the peripheral regions of the tumor tissues. Furthermore, increased infiltration of F4/80 positive macrophage cells was observed in the area where tumor cell death was observed in the tumor tissues treated by mouse GC33, while F4/80 positive cells were mainly observed in the stromal regions in the vehicle control tumor tissue (Figure 2A). Subsequently, PD-L1 expression was increased by mouse GC33 especially in the infiltrated immune cells, compared to vehicle control, which suggested that PD-L1 could be induced to suppress the antitumor activity by mouse GC33 (Figure 2B). Example 4

[00419] Antitumor activity in combination with anti-GPC3 (GC33) and anti-PD-L1 (10F.9G2) antibodies in a syngeneic mouse model using CT26 cell line expressing human GPC3

[00420] To evaluate the antitumor activities of anti-GPC3 or anti-PD-L1 monotherapies or combination in mouse CT26/hGPC3 model, or mouse GC33 antibody and/or 500 μg of rat anti-mouse PD-L1 antibody, 10F.9G2 (purchased from BioXCell) were injected starting on day 3 (early treatment model) or day 15 (established model) after subcutaneous inoculation of 1x106 CT26/hGPC3 cells. In the early treatment model, 5 or 25 mg/kg mouse GC33 was injected on day 3, 6, 10, and 13 intravenously, and 500 μg of anti-PD-L1 antibody, 10F.9G2, was injected on the same schedule as single agent or combination. In the establishment model, 5 or 25 mg/kg mouse GC33 was injected on day 15 and 18 intravenously, and 500 μg anti-PD-L1 antibody, 10F.9G2, was injected on the same schedule as a single agent or combination.

[00421] In both models, the combination of 25 mg/kg GC33 and 10F.9G2 showed more potent antitumor activity compared to that by either monotherapy (Figures 3 and 4). Example 5

[00422] Antitumor activity in combination with the anti-GPC3 (GC33) and anti-PD-L1 antibodies (10F.9G2) in a syngeneic mouse model using Hepa1-6 cell line expressing human GPC3

[00423] 1, 5, or 25 mg/kg mouse GC33 antibody once weekly for 3 weeks or 200 μg rat anti-mouse PD-L1 antibody, 10F.9G2 followed by 100 μg weekly for 2 weeks as a single agent or combination were injected intravenously to mice bearing He-pa1-6 as above. Pathological examination was conducted with HE-stained sections that were prepared with conventional methods described above. As for IHC, the 1st antibodies listed in Table 2 were used for each of the markers and visualized by LSAB methods described above or ENV+ method. IHC was conducted for 3 representative animals from each group. [Table 2] Table2: List of antibodies used in the IHC staining ' Biotinylated Rabbit Anti-Goat IgS Antibody (Vector Laboratoties, Inc.) LSAB, Streptavidin, Horseradish Peroxidase, RTU (Vector Laboratoties, Inc. or Dakoj ENV+ EnVisionr System- HRP. Labelled Polymer. Anti-Rabbit (Dako) Table legends: Table 2: List of antibodies used in IHC staining - marker - first antibody - clone - isotype - source - visualization system - rat - rabbit - mouse - hamster - biotinylated rabbit anti-goat IgG antibody p streptavidin - horseradish peroxidase - system - Labeled polymer - Anti-rabbit.

[00424] While mouse GC33 or 10F.9G2 showed tumor growth inhibition compared to vehicle control, the combination of mouse GC33 and 10F.9G2 showed the strongest antitumor activity (Figure 5A). Five days after the 3rd injection, all mice were necropsied and tumor tissues were pathologically evaluated. In the tissues examined, no viable tumor cells were observed in all mice treated with 25 mg/kg mouse GC33 in combination with PD-L1 antibody and in 4 of 5 mice treated with 5 mg/kg mouse GC33 in combination with PD-L1 antibody (Figure 5B).

[00425] Pathological examination of each of the treated tumor tissues revealed increase in number of F4/80 positive cells, PD-L1 expression on multinucleated giant cells (MNGC) and CD3 positive cells infiltrating in tumor tissues and decrease in number of CD206, CD163, and CD11b positive cells and PD-L1 expression on mononuclear cells (MNC) by each of the treatment compared to vehicle control. By combination, CD3 positive T cell infiltration was increased than each monotherapy which tended to be related to antitumor activities (Table 3). Table 3 Table legends: Table 3: Pathological evaluations of treated tumors - vehicle - tumor cell - immune cell infiltration in - vasculature - mononuclear cells - multinucleated giant cell - lesion weakness - very mild - mild - moderate - marked. Example 6 Antitumor activity in combination with anti-GPC3 (GC33) and anti-PD-L1 (6E11) antibodies in a syngeneic mouse model using Hepa1-6 cell line expressing human GPC3

[00426] Mouse GC33 at 1 mg/kg or 5 mg/kg was administered intravenously once or three times weekly (q7d x 3) to C57BL/6J mice bearing established Hepa1-6/hGPC3 tumors. Anti-mouse PD-L1 mAb (clone 6E11, mIgG2A, D265A, and N297A; Genentech [WO2015/095418]) at 10 mg/kg intravenously once followed by 5 mg/kg intraperitoneally twice weekly (q7d x 3) were administered.

[00427] Combination therapy increased tumor growth inhibition compared to monotherapy treatment (Figure 6A, B). Final measurements of tumor growth inhibition values of 1 mg/kg mGC33 plain, 5 mg/kg mGC33 plain, 1 mg/kg mGC33 q7d x 3, 5 mg/kg mGC33 q7d x 3, 6E11 q7d x 3, 6E11 q7d x 3 + 1 mg/kg mGC33 plain, 6E11 q7d x 3 + 5 mg/kg mGC33 plain, and 6E11 q7d x 3 + 1 mg/kg mGC33 q7d x 3, 6E11 q7d x 3 + 5 mg/kg mGC33 q7d x 3 were 73%, 84%, 73%, 80%, 88%, 85%, 95%, 88%, and 104%, respectively. One mouse in the 5 mg/kg mGC33 q7d x 3 group, one mouse in the 1 mg/kg mGC33 q7d x 3 group, one mouse in the 5 mg/kg mGC33 plain group, and two mice in the 1 mg/kg mGC33 plain group were euthanized for tumor progression. None of the mice died, and no severe weight loss was observed during the dosing period (Figure 6C).

[00428] From the histopathology result, accompanying decreased tumor area with or without increased severity of necrosis was observed upon administration of mGC33, 6E11 and the combination (Figure 7). In the 6E11 q7d x 3 + 5 mg/kg mGC33 single or q7d x 3 groups, pathological complete responses (pCR; no tumor tissue on the histopathology slide) were observed in one or two of five mice. In addition, tumor necrosis with immune cell infiltration including macrophages/multinucleated giant cells was observed in all treated groups; the severity of lesions were in the following order: mGC33 5 mg/kg single or q7d x 3, 6E11 q7d x 3 < mGC33 5mg/kg single or q7d x 3 + 6E11 q7d x 3 (Figure 8A). As shown in Figure 8B, F4/80-positive cells observed in the periphery of tumor mass were most abundant in combination cases, followed by mGC33 or 6E11 q7d x 3 and vehicle in that order (Figure 8B). In vehicle, PD-L1 positive reactions were mainly observed in the cytoplasm of tumor cells with weak intensity. On the other hand, increased intensity (weak to medium) and frequencies of PD-L1 positive reactions in immune cells including macrophages/multinucleated giant cells were observed in all treated groups (Figure 8C). Example 7 Induction of cytotoxic T cells in a syngeneic mouse model using Hepa1-6 cell line expressing human GPC3

[00429] In addition to innate immunity by NK cells and macrophages, the induction of acquired immunity by cytotoxic T cells is important for killing tumor cells. To assess the induction of acquired immunity in the treated mice, T cells infiltrated into tumor tissues were evaluated. Hepa1-6 cells expressing human GPC3 were inoculated subcutaneously into C57BL/6J mice. Once palpable tumors were established, the mice were allocated into 12 groups; 3 groups were as control, 3 groups were treated by 5 mg/kg mouse GC33 for 2 times on day 13 and 20 after tumor inoculation, 3 groups were treated by 10 mg/kg 6E11 on day 13 and 5 mg/kg 6E11 on day 20 after tumor inoculation, or 3 groups were treated in combination with mouse GC33 (2 times on day 13 and 20 after tumor inoculation) and 6E11 (10 mg/kg 6E11 on day 13 and 5 mg/kg 6E11 on day 20 after tumor inoculation). After 1, 3 and 8 days from the 2nd administration, tumor tissues were isolated and minced. After digestion by gentleMACS (trade name) Octo Dissociator, cells were washed and used for flow cytometry to quantify CD45, CD3ε, CD4 or CD8 positive tumor infiltrating lymphocytes (TILs).

[00430] As shown in Figure 9, in mice treated with mouse GC33 or 6E11, increases in CD45-positive, CD3ε-positive, and CD8-positive T lymphocytes, but not CD4-positive lymphocytes, were observed. Similar to the anti-tumor activities described above, in combination with mouse GC33 and 6E11, stronger induction of lymphocytes including CD8-positive TILs is observed with each monotherapy.

Industrial Applicability

[00431] The present invention contributes to improving the efficacy of a PD-1 axis binding antagonist or an anti-GPC3 antibody by combining with each other and improving the QOL of a patient to be treated, and is useful in the treatment of cancer including liver cancer.

Claims (4)

1. Use of a PD-1 axis binding antagonist and/or anti-GPC3 antibody, characterized by the fact that it is in the manufacture of a composition or medicine for the treatment or delay of progression of cancer in an individual. 2. A kit comprising a composition or medicament comprising a PD-1 axis binding antagonist and the optional pharmaceutically acceptable carrier, and a package insert comprising instructions for administering the medicament in combination with a composition comprising an anti-GPC3 antibody and the optional pharmaceutically acceptable carrier for treating or delaying the progression of cancer in a subject. 3. A kit comprising a first composition or medicament comprising a PD-1 axis binding antagonist and the optional pharmaceutically acceptable carrier, and a second composition or medicament comprising an anti-GPC3 antibody and the optional pharmaceutically acceptable carrier, optionally further comprising a package insert comprising instructions for administration of the first medicament and the second medicament for treating or delaying the progression of cancer in a subject. 4. A kit comprising a composition or medicament comprising an anti-GPC3 antibody and the optional pharmaceutically acceptable carrier, and a package insert comprising instructions for administering the medicament in combination with a composition comprising a PD-1 axis binding antagonist and the optional pharmaceutically acceptable carrier for treating or delaying the progression of cancer in a subject.