WO2021177980A1 - Polythérapie contre le cancer comprenant un antagoniste de liaison à l'axe pd-1 et un antagoniste de l'il 6 - Google Patents

Polythérapie contre le cancer comprenant un antagoniste de liaison à l'axe pd-1 et un antagoniste de l'il 6 Download PDF

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WO2021177980A1
WO2021177980A1 PCT/US2020/021738 US2020021738W WO2021177980A1 WO 2021177980 A1 WO2021177980 A1 WO 2021177980A1 US 2020021738 W US2020021738 W US 2020021738W WO 2021177980 A1 WO2021177980 A1 WO 2021177980A1
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antibody
antagonist
cancer
patient
cells
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PCT/US2020/021738
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Mahrukh HUSENI
Joanna KLEMENTOWICZ
Yijin LI
Li-fen LIU
Sanjeev Mariathasan
Mark Merchant
Luciana MOLINERO
Lifen Wang
Nathan West
Patrick Williams
Kobe YUEN
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Genentech, Inc.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • A61K39/001116—Receptors for cytokines
    • A61K39/001119—Receptors for interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001136—Cytokines
    • A61K39/00114—Interleukins [IL]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • C07K16/248—IL-6
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2818—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A61K2039/507—Comprising a combination of two or more separate antibodies

Definitions

  • the invention also relates to combination therapy for cancer comprising a PD-1 axis binding antagonist (e.g., atezolizumab) and an IL6 antagonist (e.g. tocilizumab), optionally with further chemotherapy.
  • a PD-1 axis binding antagonist e.g., atezolizumab
  • an IL6 antagonist e.g. tocilizumab
  • the invention further relates to treating certain cancer patient subpopulations with the combination, including patients with high CRP and/or IL-6 level(s), optionally also having a PD-L1 positive tumor.
  • the invention also relates to methods of reducing or preventing therapeutic resistance to a PD-1 axis binding antagonist (e.g.
  • an anti-PD-L1 antibody such as atezolizumab
  • a cancer patient comprising administering the PD-1 axis binding antagonist in combination with an IL-6 antagonist (such as an anti-IL6 receptor antibody like tocilizumab), optionally where the patient has abnormal CRP and/or IL-6 level(s).
  • the patient’s cancer is PD-L1 positive.
  • BACKGROUND OF THE INVENTION Cancer remains one of the deadliest threats to human health. Cancers, or malignant tumors, metastasize and grow rapidly in an uncontrolled manner, making timely detection and treatment extremely difficult. Worldwide, urothelial carcinoma (UC) is the most common cancer of the urinary system.
  • TCC Transitional cell carcinoma
  • mUC metastatic urothelial carcinoma
  • KPS Karnofsky Performance Status
  • visceral metastasis i.e., lung, liver, or bone.
  • the presence of these unfavorable features was associated with a median survival of 4 months compared with 18 months in patients without these features.
  • the overall 5-year survival rate for mUC is approximately 5.2%.
  • platinum-based chemotherapy has been the standard of care for patients with previously untreated mUC. In an effort to develop a less toxic regimen, the combination of gemcitabine and cisplatin (GC) was subsequently developed.
  • the treatment algorithm for patients with metastatic breast cancer is based on several factors that include clinical, pathologic, and histologic characteristics such as the presence or absence of HER2 amplification, hormone receptor status, PD-L1 status, prior response to and/or failure of hormonal agents, number and specific sites of metastatic disease, and treatment history in both the metastatic and adjuvant settings.
  • cytotoxic chemotherapy agents have shown activity in metastatic breast cancer, including anthracyclines, taxanes, carboplatin, gemcitabine, capecitabine, vinorelbine, eribulin, and ixabepilone.
  • the response rates and progression-free intervals observed with these agents vary depending on the extent and type of prior therapy and the extent of metastatic disease, as well as the biology of the disease.
  • anthracycline-based combination therapy and taxanes are believed to show the greatest activity.
  • taxanes are now the most commonly used chemotherapy agent for patients with locally advanced or metastatic disease, particularly in the front-line setting.
  • Triple-negative breast cancer may be simply defined by the absence of immunostaining for estrogen receptor (ER), progesterone receptor (PR), and HER2. Overall, approximately 15% ⁇ 20% of breast cancers are classified as TNBC.
  • TNBCs are more likely to have aggressive features, such as a high proliferative rate, and exhibit an invasive phenotype.
  • Programmed Death-Ligand 1 (PD-L1)
  • the PD-L1 pathway serves as an immune checkpoint to temporarily dampen immune responses in states of chronic antigen stimulation, such as chronic infection or cancer.
  • PD-L1 is an extracellular protein that downregulates immune responses through binding to its two receptors, PD-1 and B7-1.
  • PD-1 is an inhibitory receptor expressed on T cells following T-cell activation, and expression is sustained in states of chronic stimulation (Blank et al. Interaction of PD-L1 on tumor cells with PD-1 on tumor-specific T cells as a mechanism of immune evasion: implications for tumor immunotherapy. Cancer Immunol Immunother 54:307 ⁇ 14 (2005); Keir et al. PD-1 and its ligands in tolerance and immunity. Annu Rev Immunol 2008;26:677 ⁇ 704 (2008). B7-1 is a molecule expressed on antigen-presenting cells and activated T cells.
  • Atezolizumab is programmed death-ligand 1 (PD-L1) blocking antibody. More specifically, it is an Fc-engineered, humanized, non-glycosylated IgG1 kappa immunoglobulin that has a calculated molecular mass of 145 kDa.
  • Atezolizumab is approved for the following uses in the United States: 1.
  • Urothelial Carcinoma for the treatment of adult patients with locally advanced or metastatic urothelial carcinoma who: a. are not eligible for cisplatin-containing chemotherapy and whose tumors express PD-L1 (PD-L1 stained tumor-infiltrating immune cells [IC] covering ⁇ 5% of the tumor area), as determined by an FDA-approved test, or b. are not eligible for any platinum-containing chemotherapy regardless of PD-L1 status, or o have disease progression during or following any platinum-containing chemotherapy, or within 12 months of neoadjuvant or adjuvant chemotherapy.
  • NSCLC Non-Small Cell Lung Cancer
  • TNBC Triple-Negative Breast Cancer
  • PD-L1 PD-L1 stained tumor-infiltrating immune cells [IC] of any intensity covering ⁇ 1% of the tumor area
  • SCLC Small Cell Lung Cancer
  • Atezolizumab can be administered by various dosing schedules including: 840 mg every 2 weeks, 1200 mg every 3 weeks, and 1680 mg every 4 weeks, as a single agent or with chemotherapy and/or bevacizumab.
  • the following clinical trials involve Atezolizumab: Interleukin 6 (IL-6)
  • IL-6 Receptor Interleukin-6 (IL-6) is a proinflammatory, multifunctional cytokine produced by a variety of cell types. IL-6 is involved in such diverse processes as T-cell activation, B-cell differentiation, induction of acute phase proteins, stimulation of hematopoietic precursor cell growth and differentiation, promotion of osteoclast differentiation from precursor cells, proliferation of hepatic, dermal and neural cells, bone metabolism, and lipid metabolism (Hirano T.
  • IL-6 has been implicated in the pathogenesis of a variety of diseases including autoimmune diseases, osteoporosis, neoplasia, and aging (Hirano, T. (1992), supra; and Keller et al., supra).
  • IL-6 exerts its effects through a ligand-specific receptor (IL-6R) present both in soluble and membrane-expressed forms. Elevated IL-6 levels have been reported in the serum and synovial fluid of RA patients, indicative of production of IL-6 by the synovium (Irano et al. Eur J Immunol.18:1797-1801 (1988); and Houssiau et al. Arthritis Rheum.1988; 31:784-788 (1988)). IL-6 levels correlate with disease activity in RA (Hirano et al. (1988), supra), and clinical efficacy is accompanied by a reduction in serum IL-6 levels (Madhok et al. Arthritis Rheum.33:S154.
  • IL-6R ligand-specific receptor
  • T ocilizumab is a recombinant humanized monoclonal antibody of the immunoglobulin IgG1 subclass which binds to human IL-6R.
  • Tocilizumab is approved for: 1.
  • GCA Giant Cell Arteritis
  • GCA Giant Cell Arteritis
  • pJIA Polyarticular Juvenile Idiopathic Arthritis
  • sJIA Systemic Juvenile Idiopathic Arthritis
  • CRS Cytokine Release Syndrome
  • CAR chimeric antigen receptor T cell-induced severe or life-threatening cytokine release syndrome.
  • Tocilizumab was combined with carboplatin and doxorubicin and interferon- ⁇ 2b in patients with recurrent epithelial ovarian cancer (Dijkgraaf et al.
  • Elevated plasma IL-6 correlated with reduced sensitivity to PD-1 blockade in small cohorts of melanoma patients treated with the anti-PD-1 antibody nivolumab (Tsukamoto et al. Cancer Res 78: 5011–5022 (2016); and Weber et al. Journal of Clinical Oncology 37: 100–100 (2019)).
  • IL-6 anti-PD-1 treatment
  • other indications such as breast cancer, urothelial carcinoma, or renal cell carcinoma
  • an anti-PD-L1 antibody such as atezolizumab
  • an anti-IL6 receptor antibody such as tocilizumab
  • MP5-20F3 anti-IL6 antibody
  • Anti-IL-6 and anti-PD-L1 was evaluated in murine models of pancreatic cancer (Mace et al. IL-6 and PD-L1 antibody blockade combination therapy reduces tumor progression in murine model of pancreatic cancer. Gut 2016; epub ahead of print: doi: 10.1136/gutjnl-2016-311585).
  • the invention concerns a method of treating a cancer patient comprising administering to the patient a combination of an IL-6 antagonist and a PD-1 axis binding antagonist in an amount effective to treat the cancer.
  • cancers to be treated with the combination include, without limitation, breast cancer, such as triple negative breast cancer (TNBC), bladder cancer, urothelial carcinoma, kidney cancer, and renal cell carcinoma.
  • cancer examples include: a liver cancer, a lung cancer, a colorectal cancer, an ovarian cancer, a pancreatic cancer, a gastric carcinoma, an esophageal cancer, a mesothelioma, a melanoma, a head and neck cancer, a thyroid cancer, a sarcoma, a prostate cancer, a glioblastoma, a cervical cancer, a thymic carcinoma, a leukemia, a lymphoma, a myeloma, a mycosis fungoides, a Merkel cell cancer, or a hematologic malignancy.
  • the cancer is not melanoma or not pancreatic cancer.
  • the patient has C-reactive protein (CRP) level above the upper limit of normal.
  • CRP C-reactive protein
  • the patient may have ⁇ 3 mg/L CRP, e.g. ⁇ 10 mg/L CRP.
  • Various assays for measuring CRP are available.
  • the CRP is measured by enzyme-linked immunosorbent assay (ELISA) assay and the sample is a blood sample from the patient.
  • the patient has IL-6 level above the upper limit of normal.
  • the patient may have ⁇ 10 pg/mL IL-6, e.g. ⁇ 15 pg/mL IL-6.
  • Various assays are available for measuring IL-6.
  • IL-6 is measured by enzyme-linked immunosorbent assay (ELISA) assay and the sample is a blood sample from the patient.
  • the patient expresses PD-L1.
  • the patient may have PD-L1 stained tumor cells (TC) and/or tumor-infiltrating immune cells (IC), e.g. where the PD-L1 stained IC cover ⁇ 1% of the tumor area, e.g. ⁇ 5% of the tumor area.
  • the patient has CRP and/or IL-6 above the upper limit of normal and expresses PD-L1.
  • the IL-6 antagonist is an anti-IL6 receptor antibody, e.g.
  • the PD-L1 axis binding antagonist is a PD-L1 binding antagonist, e.g. which inhibits the binding of PD-L1 to both PD-1 and B7-1 and/or is an antibody.
  • PD-L1 binding antibodies contemplated herein include atezolizumab, MDX-1105, MEDI4736 (durvalumab), or MSB0010718C (avelumab), atezolizumab being preferred.
  • the treatment results in an increased abundance of CD8 + T cells in the patient relative to that of a subject who has not been administered the IL-6 antagonist.
  • the treatment reduces or prevents therapeutic resistance to the PD-1 axis binding antagonist.
  • the invention concerns a method of treating a cancer patient comprising administering to the patient a combination of an anti-IL6 receptor antibody and an anti-PD-L1 antibody in an amount effective to treat the cancer.
  • the cancer can be breast cancer, urothelial carcinoma, or renal cell carcinoma.
  • the invention provides a method of treating a cancer patient with C- reactive protein (CRP) level above the upper limit of normal comprising administering to the patient a combination of an anti-IL6 receptor antibody and an anti-PD-L1 antibody in an amount effective to treat the cancer.
  • CRP C- reactive protein
  • the invention concerns a method of treating advanced urothelial carcinoma in a cancer patient comprising administering to the patient a combination of tocilizumab and atezolizumab in an amount effective to treat the cancer.
  • the invention concerns a method of treating triple negative breast cancer (TNBC) in a cancer patient comprising administering to the patient a combination of tocilizumab, atezolizumab, and chemotherapy (e.g.
  • TNBC triple negative breast cancer
  • the invention provides a method of reducing or preventing therapeutic resistance to a PD-1 axis binding antagonist (e.g. an anti-PD-L1 antibody, e.g. atezolizumab) in a cancer patient (e.g. a breast cancer patient, urothelial carcinoma patient, or renal cell carcinoma patient) comprising administering the PD-1 axis binding antagonist to the patient in combination with an IL-6 antagonist (e.g. an anti-IL6 receptor antibody, e.g. tocilizumab) in an amount effective to treat the cancer.
  • a PD-1 axis binding antagonist e.g. an anti-PD-L1 antibody, e.g. atezolizumab
  • a cancer patient e.g. a breast cancer patient, urothelial carcinoma patient, or renal cell carcinoma patient
  • an IL-6 antagonist e.g. an anti-IL6 receptor antibody, e.g. tocilizumab
  • the treatment optionally inhibits CD8+ T cell function.
  • the cancer patient optionally has abnormal CRP and/or IL-6 level(s).
  • the IL-6 antagonist is optionally administered prior to the PD-1 axis binding antagonist.
  • FIGs 1a-i depict plasma IL-6 and clinical outcomes in metastatic triple negative breast cancer (mTNBC), metastatic renal cell carcinoma (mRCC), and metastatic urothelial bladder carcinoma (mUC).
  • FIG. 1b Pearson Correlation of plasma CRP with plasma IL-6 in patients with mTNBC, mRCC, or mUC.
  • FIG. 1c Plasma IL-6 concentration in mTNBC patients who experienced complete response (CR), partial response (PR), stable disease (SD), or progressive disease (PD) following treatment with atezolizumab (compared using Kruskal-Wallis test).
  • Figs.1d–f Association of high baseline plasma IL-6 with poor OS in mTNBC patients from PCD4989g (Fig. 1d), mRCC patients from IMmotion150 (Fig. 1e), and mUC patients from IMvigor210 (Fig. 1f).
  • FIG. 1g Association of high baseline plasma IL-6 with poor OS in mUC patients treated with atezolizumab or with chemotherapy (IMvigor211).
  • Fig.1h Association of OS with on-treatment increase in plasma IL-6 (ratio between week 6 concentration and pre-treatment concentration (cutoff 1.05)) in IMvigor211.
  • all HR values (with 95% CI in parentheses) are corrected in multivariate analysis as follows: ECOG (Eastern Cooperative Oncology Group) performance status, liver metastasis, and line of therapy in mTNBC; ECOG performance status and presence of liver metastasis in mUC; and MSKCC (Memorial Sloan Kettering Cancer Centre) prognostic risk score, previous nephrectomy, and liver metastasis in mRCC.
  • FIG.2a In situ hybridization (ISH) staining of IL6 mRNA in representative histological sections of mRCC tumors. An example of epithelial-restricted expression is shown in the left panel, and mixed epithelial and stromal staining in the right panel.
  • ISH In situ hybridization
  • FIG. 2b Proportion of tumors with low/negative IL6 expression (staining in ⁇ 1% of cells) or positive expression in epithelial cells only (yellow), stromal cells only (blue), or both epithelial and stromal cells (red).
  • FIG.2c Association of high IL6 expression and overall survival (OS) in the atezolizumab (left panel), atezolizumab + bevacizumab (middle panel), and sunitinib (right panel) treatment arms from IMmotion150.
  • FIG. 2d Association of high IL6 expression and OS in patients with high tumor T cell signature expression from IMmotion150.
  • FIG.2c and Fig.2d HR values (with 95% CI in parentheses) are adjusted after multivariate analysis including MSKCC (Memorial Sloan Kettering Cancer Centre) prognostic risk score, previous nephrectomy, and liver metastasis in mRCC.
  • Figures 3a-j depict suppression of CD8 + T cell effector function by IL-6.
  • OT-I splenocytes were activated as shown (Fig.3e) and analyzed on day 7 by flow cytometry, cytotoxicity assay, or RNA-sequencing.
  • Fig. 3f Killing of SIINFEKL-pulsed MC38-GFP cells by OT-I CD8 + T cells (5:1 T cell to target ratio).
  • Figs. 3g–h OT-I cells were activated with or without IL-6, hyper-IL-6, isotype control antibody, or anti-IL6R antibody. CD8 + T cells were then FACS- sorted and analyzed by RNA-sequencing.
  • Fig. 3g Principal components analysis.
  • Fig. 3h Selected differentially expressed genes (FDR ⁇ 0.05) associated with CD8 + T cell effector differentiation.
  • FIGS. 4a-k depict combination blockade of PD-L1 and IL6R in vivo.
  • Figs.4a–c C57BL/6J mice were immunized as shown (panel a). After 7 days, splenocytes were stimulated with PMA (phorbol myristate acetate)/ionomycin and cytokine expression by CD8 + OT-I T cells was assessed by flow cytometry.
  • FIG. 4b Detection of CD90.1 + OT-I cells among total CD8 + T cells (upper row), and expression of IFN- ⁇ and GzmB by OT-I cells after restimulation (bottom row).
  • Fig.4f Composition of CD45 + TIL (representative of 3 independent experiments).
  • FIG.4h IFN- ⁇ , TNF, and GzmB expression in tumor-infiltrating CD8 + T cells.
  • FIG. 4j Volumes of individual tumors from one of three independent experiments. Day 0 indicates start of treatment. CR, complete response; PR, partial response; PD, progressive disease.
  • PFS Progression- free survival
  • FIG. 6a Distribution of plasma IL-6 in healthy adult and mTNBC patients in PCD4989g, mUBC patients in IMvigor 210 and IMvigor 211, and mRCC patients in IMmotion150.
  • FIG. 6b Plasma IL-6 values were transformed into normality using Box-Cox transformation, and pIL-6 values were derived at the stated standard deviations and confidence intervals from the pIL-6 distribution of healthy adults. Based on this analysis, a concentration of ⁇ 10 pg/ml was chosen for downstream analyses as the definition of high pIL-6 status.
  • Figure 7 depicts association of pIL-6 with objective response in mUBC.
  • FIG. 8a-c depict correlation of plasma CRP with OS. Association of high baseline plasma CRP (>3 mg/L) with poor OS in atezolizumab-treated mTNBC patients from PCD4989g (Fig.8a), mUC patients from IMvigor210 (Fig.8b), and mRCC patients from IMmotion150 (Fig.8c).
  • Figures 9a-h depict single-cell RNA-sequencing analysis of PBMCs from mUC patients with high or low plasma IL-6.
  • FIG. 9b Expression of diagnostic lineage genes in the UMAP-organized cell clusters. Co-expression of CD3D and CD8A identify clusters 4 and 7 as CD8+ T cells.
  • Fig. 9c Number of cells in each cluster.
  • Fig.9d Number of transcripts per cell identified in each cluster.
  • FIG.9e Distribution of cells originating from plasma IL-6-low patients (yellow) and plasma IL-6-high patients (blue) across clusters.
  • Fig. 10d IFN- ⁇ expression (left) and viability relative to cytokine-free controls (right) from cells cultured as described for Fig.10c. Data are mean +/ ⁇ s.e.m. of 4 technical replicates and are representative of 4 independent experiments (assessing the effect of IL-2) and one of two independent experiments (assessing the effect of IL-15).
  • FIG. 11a depict effect of IL-6 on na ⁇ ve and memory CD8+ T cell activation.
  • Fig. 11a Na ⁇ ve and memory CD8+ T cells were FACS-purified from splenocytes of wild type C57BL/6J mice, activated with anti-CD3/CD28 antibodies in the presence or absence of IL-6 or hyper-IL-6, and assessed for effector function by flow cytometry on day 3.
  • Fig. 11b Frequency of CD8+ T cells co-expressing IFN- ⁇ , TNF, and GzmB.
  • FIG. 12a depict effect of IL-6 on CD8+ T cell cytotoxicity.
  • OT-I splenocytes were incubated for 2 days with SIINFEKL peptide in the presence or absence of recombinant IL-6 or hyper-IL-6.
  • Cells were then maintained with IL-2 alone for 3 days before co-culture with MC38-GFP cells that express ovalbumin (Fig. 12a) or were pulsed with SIINFEKL peptide (Fig. 12b).
  • Fig. 12a depict effect of IL-6 on CD8+ T cell cytotoxicity.
  • Figures 13a-c depict transcriptomic effects of IL-6 signaling in CD8+ T cells.
  • OT-I splenocytes were incubated for 2 days with SIINFEKL peptide in the presence or absence of recombinant IL-6, hyper-IL-6, isotype control IgG, or anti-IL6R antibody. Cells were then maintained with IL-2 alone for a further 3 days before re-stimulation with anti-CD3 and anti-CD28 antibodies. Live CD8+ T cells were purified by FACS on day 7 and analyzed by RNA-sequencing. (Fig. 13a) Volcano plots of differential gene expression in all possible pairwise comparisons. Numbers of differentially expressed genes refer to those with an absolute fold-change >2 (log2 fold change >1) and FDR (false discovery rate) ⁇ 0.05.
  • FIG.13b Heat maps of genes that were significantly differentially expressed (FDR ⁇ 0.05) between cells treated with anti-IL6R versus IL-6 or hyper-IL-6. Selected genes are organized into separate heat maps according to function.
  • (c) Gene ontology analysis of genes differentially expressed between cells treated with anti-IL6R versus IL-6 or hyper-IL-6. Genes with FDR ⁇ 0.05 and log2 fold change ⁇ 1 or ⁇ 1 were selected for analysis. Significantly enriched GO terms (FDR ⁇ 0.05) are plotted by fold enrichment on the x-axis, and FDR on the y-axis.
  • Figures 14a-c depict impact of IL-6R and PD-L1 blockade on CD8+ T cell activation in vivo.
  • mice were then treated with isotype control, anti-IL6R, or anti-PD-L1 antibodies and immunized intravenously with DEC-OVA (50 ⁇ g/kg) and agonistic anti-CD40 antibody (2.5 mg/kg).
  • Splenocytes were isolated after 7 days, restimulated with PMA/ionomycin, and evaluated for effector function by flow cytometry. Data shown are gated on Thy1.1+ CD8+ T cells (OT-I cells).
  • Fig. 14b Total viable OT-I cells prior to restimulation.
  • Fig. 14c Frequency of OT-I cells co-expressing IFN- ⁇ , TNF, and GzmB after restimulation.
  • Figures 15a-b depict effect of IL-6 on EMT6 cell growth in vitro.
  • Fig. 15a Activation of STAT3 (assessed by detection of p-STAT3 Y705; MSD assay) after 15 minutes of treatment with IL-6 or hyper-IL- 6. Values are normalized to untreated cells.
  • FIG.15b Longitudinal measurement of EMT6 cell confluence (Incucyte live-cell analysis).
  • FIG. 16a-b depict immunological features of EMT6 tumor-bearing mice during anti-IL6R and/or anti-PD-L1 therapy.
  • Figures 17a-c depict peripheral assessment of immune activation in EMT6 tumor-bearing mice treated with anti-IL6R and anti-PD-L1.
  • FIG. 17b–c CD8+ T cells were harvested from tumor-draining lymph nodes and re-stimulated ex vivo with PMA/ionomycin before analysis by flow cytometry.
  • FIG. 17b Representative plots depicting expression of GzmB and IFN- ⁇ in CD8+ T cells.
  • Figures 18a-e depict immunostimulatory activity of anti-IL6R/anti-PD-L1 combination therapy in subcutaneous CT26 tumors.
  • BALB/c mice with established (130 ⁇ 250 mm3) CT26 tumors were treated with antibodies against IL6R, PD-L1, a combination of the two, or isotype control antibodies for 11–12 days before sacrifice.
  • Fig. 18a Representative flow cytometry plots depicting GzmB and IFN- ⁇ expression by re-stimulated tumor-infiltrating CD8+ T cells.
  • the PD-1/PD- L1 axis is a major inhibitor of CD8+ T cell activation via repression of TCR (“signal 1”) and CD28 (“signal 2”) signaling.
  • signal 1 CD8+ T cell activation via repression of TCR
  • CD28 CD28
  • PD-1/PD-L1 signaling is limited, T cell activation is efficient, and the ability of IL-6 signaling to inhibit effector function (a form of “signal 3”) is readily apparent.
  • PD-1/PD-L1 signaling is a dominant checkpoint on T cell activation in vivo; in this context, IL-6 has only a modest influence on T cell effector function due to PD-1/PD-L1-mediated blockage of TCR and CD28.
  • FIG. 20 depicts demographic characteristics of patients in the IMvigor210 and IMvigor211 studies.
  • Figure 21 depicts demographic characteristics of the patients in the IMmotion150 study.
  • Figure 22 depicts demographic characteristics of the TNBC patients in the PCD4989g study.
  • Figure 23 depicts schematically how the PD-L1 pathway downregulates the anticancer immune response during two steps within the cancer-immunity cycle.
  • F igure 24 depicts clinical activity associated with atezolizumab monotherapy in patients with PD-L1–positive mTNBC.
  • Figure 25 depicts biomarkers of systemic myeloid inflammation are associated to poor prognostic baseline characteristics in mTNBC.
  • Figure 26 depicts correlation between plasma inflammatory biomarkers.
  • Figure 27 depicts plasma inflammatory biomarkers are associated with increased neutrophils and monocytes in peripheral blood.
  • Figure 28 depicts how atezolizumab monotherapy responders have lower baseline levels of systemic biomarkers of inflammation.
  • Figure 29 depicts improved PFS and OS with atezolizumab monotherapy in patients with reduced inflammatory systemic biomarkers.
  • Figure 30 depicts multivariate analysis: baseline circulating IL-6/CRP axis, but not IL8, is associated with atezolizumab monotherapy OS in TNBC.
  • Figure 31 depicts Increase of IL-6/CRP in mTNBC patients experiencing disease progression.
  • Figure 32 depicts a possible mechanism of action: Systemic inflammation (IL-6/CRP) might reduce atezolizumab-induced T cell proliferation.
  • Figure 33 depicts poor prognosis associated with elevated baseline IL-6/CRP axis regardless of treatment.
  • Figure 34 depicts how dual PD-L1/IL6R blockade controls tumor growth in syngeneic EMT6 TNBC mouse model.
  • Figures 35a-c depict effect of IL-6 conditioning on CD8 + T cell effector function.
  • the present invention provides therapeutic methods and compositions for cancer, including bladder cancer, urothelial carcinoma, kidney cancer, renal cell carcinoma, and breast cancer (e.g. triple- negative breast cancer) with a combination of a PD-1 axis binding antagonist (e.g. an anti-PD-L1 antibody such as atezolizumab) and an IL6 antagonist (e.g. an anti-IL6 receptor monoclonal antibody such as tocilizumab).
  • a PD-1 axis binding antagonist e.g. an anti-PD-L1 antibody such as atezolizumab
  • an IL6 antagonist e.g. an anti-IL6 receptor monoclonal antibody such as tocilizumab
  • the cancer patient has CRP and/or IL-6 above the upper limit of normal and, optionally, also expresses PD-L1.
  • PD-1 axis binding antagonist refers to a molecule that inhibits the interaction of a PD-1 axis binding partner with either one or more of its binding partner, so as to remove T-cell dysfunction resulting from signaling on the PD-1 signaling axis, with a result being to restore or enhance T-cell function (e.g., proliferation, cytokine production, and/or target cell killing).
  • a PD-1 axis binding antagonist includes a PD-L1 binding antagonist, a PD-1 binding antagonist, and a PD-L2 binding antagonist.
  • programmed death ligand 1 and “PD-L1” refer herein to a native sequence PD-L1 polypeptide, polypeptide variants, and fragments of a native sequence polypeptide and polypeptide variants (which are further defined herein).
  • the PD-L1 polypeptide described herein may be that which is isolated from a variety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods.
  • a “native sequence PD-L1 polypeptide” comprises a polypeptide having the same amino acid sequence as the corresponding PD-L1 polypeptide derived from nature.
  • a “PD-L1 polypeptide variant,” or variations thereof, means a PD-L1 polypeptide, generally an active PD-L1 polypeptide, as defined herein having at least about 80% amino acid sequence identity with any of the native sequence PD-L1 polypeptide sequences as disclosed herein.
  • Such PD-L1 polypeptide variants include, for instance, PD-L1 polypeptides wherein one or more amino acid residues are added, or deleted, at the N- or C-terminus of a native amino acid sequence.
  • a PD-L1 polypeptide variant will have at least about 80% amino acid sequence identity, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity, to a native sequence PD-L1 polypeptide sequence as disclosed herein.
  • PD- L1 variant polypeptides are at least about 10 amino acids in length, alternatively at least about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 281, 282, 283, 284, 285, 286, 287, 288, or 289 amino acids in length, or more.
  • PD- L1 variant polypeptides will have no more than one conservative amino acid substitution as compared to a native PD-L1 polypeptide sequence, alternatively no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions as compared to a native PD-L1 polypeptide sequence.
  • T he term “PD-L1 binding antagonist” refers to a molecule that decreases, blocks, inhibits, abrogates, or interferes with signal transduction resulting from the interaction of PD-L1 with either one or more of its binding partners, such as PD-1 and/or B7-1.
  • a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partners.
  • the PD-L1 binding antagonist inhibits binding of PD-L1 to PD-1 and/or B7-1.
  • the 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 resulting from the interaction of PD-L1 with one or more of its binding partners, such as PD-1 and/or B7-1.
  • a PD-L1 binding antagonist reduces the negative co-stimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes mediated signaling through PD-L1 so as to render a dysfunctional T-cell less dysfunctional (e.g., enhancing effector responses to antigen recognition).
  • a PD-L1 binding antagonist is an anti-PD-L1 antibody.
  • an anti-PD-L1 antibody is atezolizumab, marketed as TECENTRIQ® with a WHO Drug Information (International Nonproprietary Names for Pharmaceutical Substances), Proposed INN: List 112, Vol.28, No.4, published January 16, 2015 (see page 485) described herein.
  • an anti-PD-L1 antibody is MDX-1105 described herein. In still another specific aspect, an anti-PD-L1 antibody is YW243.55.S70 described herein. In still another specific aspect, an anti-PD-L1 antibody is MEDI4736 (durvalumab) described herein. In still another specific aspect, an anti-PD-L1 antibody is MSB0010718C (avelumab) described herein.
  • PD-1 binding antagonist refers to a molecule that decreases, blocks, inhibits, abrogates or interferes with signal transduction resulting from the interaction of PD-1 with one or more of its binding partners, such as PD-L1 and/or PD-L2.
  • the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to one or more of its binding partners.
  • the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1 and/or PD-L2.
  • PD-1 binding 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 resulting from the interaction of PD-1 with PD-L1 and/or PD-L2.
  • a PD-1 binding antagonist reduces the negative co-stimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes mediated signaling through PD-1 so as render a dysfunctional T-cell less dysfunctional (e.g., enhancing effector responses to antigen recognition).
  • the PD- 1 binding antagonist is an anti-PD-1 antibody.
  • a PD-1 binding antagonist is MDX- 1106 (nivolumab) described herein.
  • a PD-1 binding antagonist is MK-3475 (pembrolizumab) described herein.
  • a PD-1 binding antagonist is MEDI-0680 (AMP-514) described herein.
  • a PD-1 binding antagonist is PDR001 described herein. In another specific aspect, a PD-1 binding antagonist is REGN2810 described herein. In another specific aspect, a PD-1 binding antagonist is BGB-108 described herein.
  • the term “PD-L2 binding antagonist” refers to a molecule that decreases, blocks, inhibits, abrogates or interferes with signal transduction resulting from the interaction of PD-L2 with either one or more of its binding partners, such as PD-1.
  • 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 binding of PD-L2 to PD-1.
  • the 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 resulting from the interaction of PD-L2 with either one or more of its binding partners, such as PD-1.
  • a PD-L2 binding antagonist reduces the negative co-stimulatory signal mediated by or through cell surface proteins expressed on T lymphocytes mediated signaling through PD-L2 so as render a dysfunctional T-cell less dysfunctional (e.g., enhancing effector responses to antigen recognition).
  • a PD-L2 binding antagonist is an immunoadhesin.
  • human interleukin 6 (abbreviated as “IL-6”) is a cytokine also known as B cell-stimulating factor 2 (BSF-2), or interferon beta-2 (IFNB2), hybridoma growth factor, and CTL differentiation factor.
  • BSF-2 B cell-stimulating factor 2
  • IFNB2 interferon beta-2
  • IL- 6 was discovered as a differentiation factor contributing to activation of B cells (Hirano et al., Nature 324: 73-76 (1986)), and was later found to be a multifunction cytokine which influences the functioning of a variety of different cell types (Akira et al., Adv. in Immunology 54: 1-78 (1993)).
  • Human IL-6 variants are known and included in this definition. Human IL-6 amino acid sequence information has been disclosed, see for example, www.uniprot.org/uniprot/P05231.
  • human interleukin 6 receptor (abbreviated as “IL-6R”) refers to the receptor which binds IL-6, including both membrane-bound IL-6R (mIL-6R) and soluble IL-6R (sIL-6R). IL- 6R can combine with interleukin 6 signal transducer glycoprotein 130 to form an active receptor complex. Alternatively spliced transcript variants encoding distinct isoforms of IL-6 have been reported and are included in this definition.
  • a “neutralizing” anti-IL-6R antibody herein is one which binds to IL-6R and is able to inhibit, to a measurable extent, the ability of IL-6 to bind to and/or active IL-6R.
  • Toclizumab is an example of a neutralizing anti-IL-6R antibody.
  • Tocilizumab or “TCZ” is a recombinant humanized monoclonal antibody that binds to human interleukin-6 receptor (IL-6R).
  • the light chain and heavy chain amino acid sequences of Tocilizumab comprise SEQ ID NOs.32 and 33.
  • the term “biomarker” as used herein refers to an indicator, e.g., predictive, diagnostic, and/or prognostic, which can be detected in a sample, for example, PD-L1, IL-6, and/or CRP biomarker(s).
  • the biomarker may serve as an indicator of a particular subtype of a disease or disorder (e.g., cancer) characterized by certain, molecular, pathological, histological, and/or clinical features.
  • a biomarker is a gene.
  • Biomarkers include, but are not limited to, polynucleotides (e.g., DNA and/or RNA), polynucleotide copy number alterations (e.g., DNA copy numbers), polypeptides, polypeptide and polynucleotide modifications (e.g., post-translational modifications), carbohydrates, and/or glycolipid-based molecular markers.
  • the “amount” or “level” of a biomarker associated with an increased clinical benefit to an individual is a detectable level in a biological sample. These can be measured by methods known to one skilled in the art and also disclosed herein. The expression level or amount of biomarker assessed can be used to determine the response to the treatment.
  • a “level above the upper limit of normal” refers to an amount of a biomarker that is abnormal or atypical in a subject (including a healthy subject) or patient (including a cancer patient, e.g. with breast cancer, urothelial carcinoma, or renal cell carcinoma).
  • Assays for measuring such abnormal amounts of CRP and/or IL-6 are disclosed herein, along with exemplary “cut-offs” or “comparator” amounts of CRP and/or IL-6 for identifying patients eligible for therapy.
  • the terms “level of expression” or “expression level” in general are used interchangeably and generally refer to the amount of a biomarker in a biological sample.
  • “Expression” generally refers to the process by which information (e.g., gene-encoded and/or epigenetic information) is converted into the structures present and operating in the cell. Therefore, as used herein, “expression” may refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and/or polypeptide modifications (e.g., posttranslational modification of a polypeptide).
  • Fragments of the transcribed polynucleotide, the translated polypeptide, or polynucleotide and/or polypeptide modifications shall also be regarded as expressed whether they originate from a transcript generated by alternative splicing or a degraded transcript, or from a post- translational processing of the polypeptide, e.g., by proteolysis.
  • “Expressed genes” include those that are transcribed into a polynucleotide as mRNA and then translated into a polypeptide, and also those that are transcribed into RNA but not translated into a polypeptide (for example, transfer and ribosomal RNAs).
  • sample refers to a composition that is obtained or derived from a subject and/or individual of interest that contains a cellular and/or other molecular entity that is to be characterized and/or identified, for example, based on physical, biochemical, chemical, and/or physiological characteristics.
  • disease sample and variations thereof refers to any sample obtained from a subject of interest that would be expected or is known to contain the cellular and/or molecular entity that is to be characterized.
  • Samples include, but are not limited to, tissue samples, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous fluid, lymph fluid, synovial fluid, follicular fluid, seminal fluid, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebro-spinal fluid, saliva, sputum, tears, perspiration, mucus, tumor lysates, and tissue culture medium, tissue extracts such as homogenized tissue, tumor tissue, cellular extracts, and combinations thereof.
  • the sample is a blood specimen from the patient (e.g. for a CRP and/or IL-6 bioassay).
  • tissue sample or “cell sample” is meant a collection of similar cells obtained from a tissue of a subject or individual.
  • the source of the tissue or cell sample may be solid tissue as from a fresh, frozen and/or preserved organ, tissue sample, biopsy, and/or aspirate; blood or any blood constituents such as plasma; bodily fluids such as cerebral spinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; cells from any time in gestation or development of the subject.
  • the tissue sample may also be primary or cultured cells or cell lines.
  • the tissue or cell sample is obtained from a disease tissue/organ.
  • a “tumor sample” is a tissue sample obtained from a tumor (e.g., a liver tumor) or other cancerous tissue.
  • the tissue sample may contain a mixed population of cell types (e.g., tumor cells and non-tumor cells, cancerous cells and non-cancerous cells).
  • the tissue sample may contain compounds which are not naturally intermixed with the tissue in nature such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like.
  • a “tumor-infiltrating immune cell,” as used herein, refers to any immune cell present in a tumor or a sample thereof.
  • Tumor-infiltrating immune cells include, but are not limited to, intratumoral immune cells, peritumoral immune cells, other tumor stroma cells (e.g., fibroblasts), or any combination thereof.
  • Such tumor-infiltrating immune cells can be, for example, T lymphocytes (such as CD8+ T lymphocytes and/or CD4+ T lymphocytes), B lymphocytes, or other bone marrow-lineage cells, including granulocytes (e.g., neutrophils, eosinophils, and basophils), monocytes, macrophages, dendritic cells (e.g., interdigitating dendritic cells), histiocytes, and natural killer cells.
  • T lymphocytes such as CD8+ T lymphocytes and/or CD4+ T lymphocytes
  • B lymphocytes or other bone marrow-lineage cells, including granulocytes (e.g., neutrophils, eosinophils, and basophils), monocytes, macrophages, dendritic cells (e.g., interdigitating dendritic cells), histiocytes, and natural killer cells.
  • granulocytes e.g., neutrophils,
  • Tumor cells may be distinguished from other cells that may be present in a tumor sample, for example, stromal cells and tumor-infiltrating immune cells, using methods known in the art and/or described herein.
  • the sample comprises “tumor cells and/or tumor-infiltrating immune cells” from the patient (e.g. for a PD-L1 bioassay).
  • a “reference sample,” “reference cell,” “reference tissue,” “control sample,” “control cell,” or “control tissue,” as used herein, refers to a sample, cell, tissue, standard, or level that is used for comparison purposes.
  • a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and/or non-diseased part of the body (e.g., tissue or cells) of the same subject or individual.
  • the reference sample, reference cell, reference tissue, control sample, control cell, or control tissue may be healthy and/or non-diseased cells or tissue adjacent to the diseased cells or tissue (e.g., cells or tissue adjacent to a tumor).
  • a reference sample is obtained from an untreated tissue and/or cell of the body of the same subject or individual.
  • a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from a healthy and/or non-diseased part of the body (e.g., tissues or cells) of an individual who is not the subject or individual.
  • a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained from an untreated tissue and/or cell of the body of an individual who is not the subject or individual.
  • a “section” of a tissue sample is meant a single part or piece of a tissue sample, for example, a thin slice of tissue or cells cut from a tissue sample (e.g., a tumor sample).
  • tumor immunity refers to the process in which tumors evade immune recognition and clearance.
  • tumor immunity is “treated” when such evasion is attenuated, and the tumors are recognized and attacked by the immune system.
  • tumor recognition include tumor binding, tumor shrinkage and tumor clearance.
  • objective response rate or “objective response rate” (ORR) refers to the sum of complete response (CR) rate and partial response (PR) rate.
  • ORR refers to the proportion of patients with a confirmed objective response, either CR or PR, observed on two assessments greater than or equal to 28 days apart per RECIST v1.1, based on investigator assessment.
  • complete response or “CR” refers to disappearance of all target lesions.
  • partial response or “PR” refers to at least a 30% decrease in the sum of the longest diameters (SLD) of target lesions, taking as reference the baseline SLD.
  • stable disease or “SD” refers to neither sufficient shrinkage of target lesions to qualify for PR, nor sufficient increase to qualify for PD, taking as reference the smallest SLD since the treatment started.
  • progressive disease or “PD” refers to at least a 20% increase in the SLD of target lesions, taking as reference the smallest SLD recorded since the treatment started or the presence of one or more new lesions.
  • progression-free survival (PFS) refers to the length of time during and after treatment during which the disease being treated (e.g., cancer) does not get worse. Progression-free survival may include the amount of time patients have experienced a complete response or a partial response, as well as the amount of time patients have experienced stable disease.
  • PFS may be defined as the time from randomization or the beginning of treatment to the first documented disease progression as assessed by RECIST v1.1, or death from any cause, whichever occurs first.
  • PFS of a combination of PD-1 axis binding antagonist and IL6 antagonist can be compared to the PFS without the IL6 antagonist (e.g. compared with PD-1 axis binding antagonist alone).
  • overall survival (OS) refers to the percentage of individuals in a group who are likely to be alive after a particular duration of time.
  • OS of a combination of PD-1 axis binding antagonist and IL6 antagonist can be compared to the OS without the IL6 antagonist (e.g. compared with PD-1 axis binding antagonist alone).
  • the term “duration of response” refers to a length of time from documentation of a tumor response until disease progression or death from any cause, whichever occurs first.
  • treatment refers to clinical intervention designed to alter 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, ameliorating or palliating the disease state, and remission or improved prognosis.
  • an individual is successfully “treated” if one or more symptoms associated with cancer (e.g.,breast cancer, urothelial carcinoma, or renal cell carcinoma) are mitigated or eliminated, including, but are not limited to, reducing the proliferation of (or destroying) cancerous cells, 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 survival of individuals.
  • An “effective amount” or “therapeutically effective amount,” as used interchangeably herein, 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 agent to elicit a desired response in the individual.
  • An effective amount is also one in which any toxic or detrimental effects of the treatment are outweighed by the therapeutically beneficial effects.
  • 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 presenting during development of the disease.
  • beneficial or desired results include clinical results such as decreasing 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, and enhancing effect of another medication such as via targeting, delaying the progression of the disease, and/or prolonging survival.
  • an effective amount of the drug may have the effect in reducing the number of cancer cells; reducing the tumor size; inhibiting (i.e., slow to some extent or desirably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and desirably stop) 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 can be administered in one or more administrations.
  • an effective amount of drug, compound, or pharmaceutical composition is an amount sufficient to accomplish prophylactic or therapeutic treatment either directly or indirectly.
  • an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition.
  • an “effective amount” may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be given in an effective amount if, in conjunction with one or more other agents, a desirable result may be or is achieved.
  • an amount effective to treat the cancer comprises amounts of each of the components of the combination that treat the cancer patient.
  • the “amount effective” of the combination achieves a clinical response greater than treatment with either agent alone, greater than PD-1 axis binding antagonist (e.g. anti-PD-L1 antibody such as atezolizumab) alone, greater than treatment without IL6 antagonist (e.g. without anti-IL6 receptor antibody or without tocilizumab); or greater than treatment with PD-1 axis binding antagonist (e.g. anti-PD- L1 antibody such as atezolizumab) and chemotherapy (without the IL6 antagonist).
  • the amount effective of the combination may inhibit CD8+ T cell function and/or reduce or prevent therapeutic resistance to the PD-1 axis binding antagonist.
  • cancer and “cancerous” refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth.
  • blade cancer includes, but is not limited to, UC, and which may be, for example, locally advanced or metastatic.
  • the methods described herein are suitable for treatment of various stages of cancer, including cancers that are locally advanced and/or metastatic.
  • locally advanced is generally defined as cancer that has spread from a localized area to nearby tissues and/or lymph nodes.
  • locally advanced usually is classified in Stage II or III.
  • Cancer which is metastatic is a stage where the cancer spreads throughout the body to distant tissues and organs (stage IV).
  • stage IV The term “upper tract UC” refers to UC of the renal pelvis or ureter.
  • the upper tract UC may be upper tract metastatic UC. A minority of cases (e.g., about 5-10%) of UC are upper tract UC.
  • the term “lower tract UC” refers to UC of the bladder or urethra.
  • the lower tract UC may be lower tract metastatic UC. The majority of cases (e.g., about 90-95%) of UC are lower tract UC.
  • 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 hinders a cellular function).
  • Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 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, plant or animal origin, including fragments and/or variants thereof.
  • radioactive isotopes e.g., At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and radioactive isotopes of Lu
  • chemotherapeutic agents e.g., At 211 , I 131 , I 125
  • Exemplary cytotoxic agents can be selected from anti-microtubule agents, platinum coordination complexes, alkylating agents, antibiotic agents, topoisomerase II inhibitors, antimetabolites, topoisomerase I inhibitors, hormones and hormonal analogues, signal transduction pathway inhibitors, non-receptor tyrosine kinase angiogenesis inhibitors, immunotherapeutic agents, proapoptotic agents, inhibitors of LDH-A, inhibitors of fatty acid biosynthesis, cell cycle signaling inhibitors, HDAC inhibitors, proteasome inhibitors, and inhibitors of cancer metabolism.
  • the cytotoxic agent is a platinum-based chemotherapeutic agent.
  • the cytotoxic agent is an antagonist of EGFR.
  • the cytotoxic agent is N-(3-ethynylphenyl)-6,7-bis(2- methoxyethoxy)quinazolin-4-amine (e.g., erlotinib, TARCEVA®).
  • the cytotoxic agent is a RAF inhibitor.
  • the RAF inhibitor is a BRAF and/or CRAF inhibitor.
  • the RAF inhibitor is vemurafenib.
  • the cytotoxic agent is a PI3K inhibitor.
  • the term “chemotherapeutic agent” includes compounds useful in the treatment of cancer, such as bladder cancer (e.g., UC, including locally advanced or metastatic UC).
  • chemotherapeutic agents include erlotinib (TARCEVA®, Genentech/OSI Pharm.), bortezomib (VELCADE®, Millennium Pharm.), disulfiram, epigallocatechin gallate, salinosporamide A, carfilzomib, 17- AAG (geldanamycin), radicicol, lactate dehydrogenase A (LDH-A), fulvestrant (FASLODEX®, AstraZeneca), sunitib (SUTENT®, Pfizer/Sugen), letrozole (FEMARA®, Novartis), imatinib mesylate (GLEEVEC®, Novartis), finasunate (VATALANIB®, Novartis), oxaliplatin (ELOXATIN®, Sanofi), 5-FU (5- fluorouracil), leucovorin, rapamycin (Sirolimus, RAPAMUNE®, Wyeth), lapatinib
  • dynemicin including dynemicin A; bisphosphonates, such as clodronate; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin, 6-diazo-5- oxo-L-norleucine, ADRIAMYCIN® (doxorubicin), morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin,
  • Chemotherapeutic agents also include “platinum-based” chemotherapeutic agents, which comprise an organic compound which contains platinum as an integral part of the molecule. Typically, platinum-based chemotherapeutic agents are coordination complexes of platinum. Platinum-based chemotherapeutic agents are sometimes called “platins” in the art. Examples of platinum-based chemotherapeutic agents include, but are not limited to, cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, lipoplatin, and satraplatin.
  • a “platinum-based chemotherapy,” as used herein, refers to a chemotherapy regimen that includes a platinum-based chemotherapeutic agent.
  • an IL6 antagonist may include a platinum-based chemotherapeutic agent (e.g., cisplatin or carboplatin) in combination with one or more additional chemotherapeutic agents, e.g., a nucleoside analog (e.g., gemcitabine).
  • a platinum-based chemotherapeutic agent e.g., cisplatin or carboplatin
  • additional chemotherapeutic agents e.g., a nucleoside analog (e.g., gemcitabine).
  • Chemotherapeutic agents also include (i) anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX®; tamoxifen citrate), raloxifene, droloxifene, iodoxyfene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and FARESTON® (toremifine citrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrol acetate), AROMASIN® (exemestane; Pfizer), formestanie, fadrozole, RIVISOR® (vorozole), FEMARA® (let
  • C hemotherapeutic agents also include antibodies such as alemtuzumab (Campath), bevacizumab (AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab (VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech/Biogen Idec), pertuzumab (2C4, Genentech), trastuzumab (HERCEPTIN®, Genentech), tositumomab (Bexxar, Corixia), and the antibody drug conjugate, gemtuzumab ozogamicin (MYLOTARG®, Wyeth).
  • antibodies such as alemtuzumab (Campath), bevacizumab (AVASTIN®, Genentech); cetuximab (ERBITUX®, Imclone); panitumumab (VECTIBIX®, Amgen), rituximab (RITUXAN®, Gene
  • 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, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab, motovizumab, natalizumab, nimotuzumab, nolovizum
  • Chemotherapeutic agents also include “EGFR inhibitors,” which refers to compounds that bind to or otherwise interact directly with EGFR and prevent or reduce its signaling activity, and is alternatively referred to as an “EGFR antagonist.”
  • EGFR inhibitors refers to compounds that bind to or otherwise interact directly with EGFR and prevent or reduce its signaling activity
  • Examples of such agents include antibodies and small molecules that bind to EGFR.
  • antibodies which bind to EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507), MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see, US Patent No.
  • EMD7200 a humanized EGFR antibody directed against EGFR that competes with both EGF and TGF-alpha for EGFR binding
  • human EGFR antibody HuMax-EGFR (GenMab)
  • Fully human antibodies known as E1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6.3 and E7.6.3 and described in US 6,235,883; MDX-447 (Medarex Inc); and mAb 806 or humanized mAb 806 (Johns et al., J. Biol. Chem.279(29):30375-30384 (2004)).
  • the anti-EGFR antibody may be conjugated with a cytotoxic agent, thus generating an immunoconjugate (see, e.g., EP659439A2, Merck Patent GmbH).
  • EGFR antagonists include small molecules such as compounds described in US Patent Nos: 5,616,582, 5,457,105, 5,475,001, 5,654,307, 5,679,683, 6,084,095, 6,265,410, 6,455,534, 6,521,620, 6,596,726, 6,713,484, 5,770,599, 6,140,332, 5,866,572, 6,399,602, 6,344,459, 6,602,863, 6,391,874, 6,344,455, 5,760,041, 6,002,008, and 5,747,498, as well as the following PCT publications: WO98/14451, WO98/50038, WO99/09016, and WO99/24037.
  • EGFR antagonists include OSI-774 (CP- 358774, erlotinib, TARCEVA® Genentech/OSI Pharmaceuticals); PD 183805 (CI 1033, 2-propenamide, N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholinyl)propoxy]-6-quinazolinyl]-, dihydrochloride, Pfizer Inc.); ZD1839, gefitinib (IRESSA®) 4-(3’-Chloro-4’-fluoroanilino)-7-methoxy-6-(3- morpholinopropoxy)quinazoline, AstraZeneca); ZM 105180 ((6-amino-4-(3-methylphenyl-amino)- quinazoline, Zeneca); BIBX-1382 (N8-(3-chloro-4-fluoro-phenyl)-N2-(1-methyl-pipe
  • Chemotherapeutic agents also include “tyrosine kinase inhibitors” including the EGFR-targeted drugs noted in the preceding paragraph; small molecule HER2 tyrosine kinase inhibitor such as TAK165 available from Takeda; CP-724,714, an oral selective inhibitor of the ErbB2 receptor tyrosine kinase (Pfizer and OSI); dual-HER inhibitors such as EKB-569 (available from Wyeth) which preferentially binds EGFR but inhibits both HER2 and EGFR-overexpressing cells; lapatinib (GSK572016; available from Glaxo-SmithKline), an oral HER2 and EGFR tyrosine kinase inhibitor; PKI-166 (available from Novartis); pan-HER inhibitors such as canertinib (CI-1033; Pharmacia); Raf-1 inhibitors such as antisense agent ISIS-5132 available from ISIS Pharmaceuticals which inhibit Raf-1 signaling; non-HER-target
  • Patent No.5,804,396 WO 1999/09016 (American Cyanamid); WO 1998/43960 (American Cyanamid); WO 1997/38983 (Warner Lambert); WO 1999/06378 (Warner Lambert); WO 1999/06396 (Warner Lambert); WO 1996/30347 (Pfizer, Inc); WO 1996/33978 (Zeneca); WO 1996/3397 (Zeneca) and WO 1996/33980 (Zeneca).
  • Chemotherapeutic agents also include dexamethasone, interferons, colchicine, metoprine, cyclosporine, amphotericin, metronidazole, alemtuzumab, alitretinoin, allopurinol, amifostine, arsenic trioxide, asparaginase, BCG live, bevacuzimab, bexarotene, cladribine, clofarabine, darbepoetin alfa, denileukin, dexrazoxane, epoetin alfa, elotinib, filgrastim, histrelin acetate, ibritumomab, interferon alfa-2a, interferon alfa-2b, lenalidomide, levamisole, mesna, methoxsalen, nandrolone, nelarabine, nofetumomab, oprelvekin,
  • Chemotherapeutic agents also include hydrocortisone, hydrocortisone acetate, cortisone acetate, tixocortol pivalate, triamcinolone acetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide, desonide, fluocinonide, fluocinolone acetonide, betamethasone, betamethasone sodium phosphate, dexamethasone, dexamethasone sodium phosphate, fluocortolone, hydrocortisone-17-butyrate, hydrocortisone-17-valerate, aclometasone dipropionate, betamethasone valerate, betamethasone dipropionate, prednicarbate, clobetasone-17-butyrate, clobetasol-17-propionate, fluocortolone caproate, fluocortolone pivalate and fluprednidene acetate; immune selective
  • an “anti-angiogenesis agent” or “angiogenesis inhibitor” refers to a small molecular weight substance, a polynucleotide, a polypeptide, an isolated protein, a recombinant protein, an antibody, or conjugates or fusion proteins thereof, that inhibits angiogenesis, vasculogenesis, or undesirable vascular permeability, either directly or indirectly. It should be understood that the anti-angiogenesis agent includes those agents that bind and block the angiogenic activity of the angiogenic factor or its receptor.
  • an anti-angiogenesis agent is an antibody or other antagonist to an angiogenic agent as defined above, e.g., antibodies to VEGF-A or the VEGF-A receptor (e.g., KDR receptor or Flt-1 receptor), anti-PDGFR inhibitors such as GLEEVECTM (imatinib mesylate).
  • Anti-angiogenesis agents also include native angiogenesis inhibitors, e.g., angiostatin, endostatin, etc. See, for example, Klagsbrun and D’Amore, Annu. Rev.
  • a “subject” or “patient” refer to a human subject or human patient.
  • antibody herein is used in the broadest sense and specifically covers 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.
  • “Native antibodies” are usually heterotetrameric glycoproteins of about 150,000 Daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges.
  • 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 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.
  • 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 domains (collectively, CH) of the heavy chain and the CHL (or CL) domain of the light chain.
  • the “variable region” or “variable domain” of an antibody refers to the amino-terminal domains of the heavy or light chain of the antibody.
  • the variable domain of the heavy chain may be referred to as “VH.”
  • the variable domain of the light chain may be referred to as “VL.” These domains are generally the most variable parts of an antibody and contain the antigen-binding sites.
  • 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 variability is not evenly distributed throughout the variable domains of antibodies.
  • variable domains of native heavy and light chains each comprise four FR regions, largely adopting a beta-sheet configuration, connected by three HVRs, which form loops connecting, and in some cases forming part of, the beta-sheet structure.
  • the HVRs in each chain are held together in close proximity by the FR regions and, with the HVRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)).
  • the constant domains are not involved directly in the binding of an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity.
  • the “light chains” of antibodies (immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa (“ ⁇ ”) and lambda (“ ⁇ ”), based on the amino acid sequences of their constant domains.
  • ⁇ kappa
  • ⁇ lambda
  • IgG “isotype” or “subclass” as used herein is meant any of the subclasses of immunoglobulins defined by the chemical and antigenic characteristics of their constant regions.
  • antibodies (immunoglobulins) can be assigned to different classes.
  • immunoglobulins There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.
  • the heavy chain constant domains that correspond to the different classes of immunoglobulins are called ⁇ , ⁇ , ⁇ , ⁇ , and ⁇ , respectively.
  • the subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and described generally in, for example, 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.
  • full-length antibody “intact antibody,” and “whole antibody” are used herein interchangeably to refer to an antibody in its substantially intact form, not antibody fragments as defined below. The terms particularly refer to an antibody with heavy chains that contain an Fc region.
  • a “naked antibody” for the purposes herein is an antibody that is not conjugated to a cytotoxic moiety or radiolabel.
  • “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.
  • antibody fragments include Fab, Fab’, F(ab’)2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
  • monoclonal antibody refers to an antibody obtained from a population of substantially homogeneous antibodies, e.g., the individual antibodies comprising the population are identical except for possible mutations, e.g., naturally occurring mutations, that may be present in minor amounts. Thus, the modifier “monoclonal” indicates the character of the antibody as not being a mixture of discrete antibodies.
  • such a monoclonal antibody typically includes an antibody comprising a polypeptide sequence that binds a target, wherein the target-binding polypeptide sequence was obtained by a process that includes the selection of a single target binding polypeptide sequence from a plurality of polypeptide sequences.
  • the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, or recombinant DNA clones.
  • a selected target binding sequence can be further altered, for example, to improve 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 this invention.
  • an antibody comprising the altered target binding sequence is also a monoclonal antibody of this invention.
  • polyclonal antibody preparations 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.
  • monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins.
  • 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.
  • the monoclonal antibodies to be used in accordance with the invention may be made by a variety of 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, e.g., U.S.
  • phage-display technologies see, e.g., Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol. Biol.222: 581-597 (1992); Sidhu et al., J. Mol. Biol.338(2): 299-310 (2004); Lee et al., J. Mol. Biol.340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol.
  • the monoclonal antibodies herein specifically include “chimeric” antibodies in which a portion of the heavy and/or light chain is identical with 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 with 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. Pat. No.4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).
  • Chimeric antibodies include PRIMATIZED® antibodies wherein the antigen-binding region of the antibody is derived from an antibody produced by, e.g., immunizing macaque monkeys with the antigen of interest.
  • “Humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin.
  • a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from an HVR of the recipient are replaced by residues from an HVR of a non-human species (donor antibody) such as mouse, rat, rabbit, or nonhuman primate having the desired specificity, affinity, and/or capacity.
  • FR residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications may be made to further refine antibody performance.
  • a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin, and all or substantially all of the FRs are those of a human immunoglobulin sequence.
  • the humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • a “human antibody” is one which possesses an amino acid sequence which corresponds to that of an antibody produced by a human and/or has been made using any of the techniques for making human antibodies as disclosed 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 techniques known in the art, including phage-display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Also available for the preparation of human monoclonal antibodies are methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R.
  • Human antibodies can be prepared by administering the antigen to a transgenic animal that has been modified 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. Pat. Nos.6,075,181 and 6,150,584 regarding XENOMOUSE TM technology). See also, for example, Li et al., Proc. Natl. Acad. Sci.
  • HVR hypervariable region
  • VL VL1 L2, L3
  • H3 and L3 display the most diversity of the six HVRs, and H3 in particular is believed to play a unique role in conferring fine specificity to antibodies.
  • the Kabat Complementarity Determining Regions are based on sequence variability and are the most c ommonly used (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia refers instead to the location of the structural loops (Chothia and Lesk J. Mol. Biol.196:901-917 (1987)).
  • the AbM HVRs represent a compromise between the Kabat HVRs and Chothia structural loops, and are used by Oxford Molecular’s AbM antibody modeling software.
  • the “contact” HVRs are based on an analysis of the available complex crystal structures.
  • 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.
  • “Framework” or “FR” residues are those variable domain residues other than the HVR residues as herein defined.
  • variable domain residue numbering as in Kabat or “amino acid position numbering as in Kabat,” and variations thereof, refers to the numbering system used for heavy chain variable domains or light chain variable domains of the compilation of antibodies in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, a FR or HVR of the variable domain.
  • a heavy chain variable domain may include a single amino acid insert (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 heavy chain FR residue 82.
  • the Kabat numbering of residues may be determined for a given antibody by alignment at regions of homology of the sequence of the antibody with a “standard” Kabat numbered sequence.
  • the Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences of Immunological Interest.5th Ed.
  • 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 reported in Kabat et al., supra).
  • EU index as in Kabat refers to the residue numbering of the human IgG1 EU antibody.
  • package insert is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.
  • pharmaceutical formulation and “pharmaceutical composition” are used interchangeably herein and refer to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.
  • Such formulations are sterile.
  • the pharmaceutical composition or pharmaceutical formulation is administered to a human subject.
  • a “sterile” pharmaceutical formulation is aseptic or free or essentially free from all living microorganisms and their spores.
  • pharmaceutically acceptable carrier refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject.
  • a pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.
  • “in combination with” or “in conjunction with” refers to administration of one treatment modality in addition to another treatment modality, for example, a treatment regimen that includes administration of a PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody such as atezolizumab) and a IL6 antagonist (e.g., an anti-IL6 receptor antibody such as tocilizumab).
  • a PD-1 axis binding antagonist e.g., an anti-PD-L1 antibody such as atezolizumab
  • a IL6 antagonist e.g., an anti-IL6 receptor antibody such as tocilizumab
  • “in conjunction with” refers to administration of one treatment modality before, during, or after administration of the other treatment modality to the individual.
  • a treatment regimen comprising an effective amount of combination of a PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody, such as atezolizumab) and an IL6 antagonist (e.g. an anti-IL6 receptor antibody, such as tocilizumab).
  • a PD-1 axis binding antagonist e.g., an anti-PD-L1 antibody, such as atezolizumab
  • an IL6 antagonist e.g. an anti-IL6 receptor antibody, such as tocilizumab
  • the invention concerns a method of treating a cancer patient comprising administering to the patient a combination of an IL-6 antagonist and a PD-1 axis binding antagonist in an amount effective to treat the cancer.
  • the cancer is a breast cancer, a bladder cancer, a kidney cancer, a liver cancer, a lung cancer, a colorectal cancer, an ovarian cancer, a pancreatic cancer, a gastric carcinoma, an esophageal cancer, a mesothelioma, a melanoma, a head and neck cancer, a thyroid cancer, a sarcoma, a prostate cancer, a glioblastoma, a cervical cancer, a thymic carcinoma, a leukemia, a lymphoma, a myeloma, a mycosis fungoides, a Merkel cell cancer, or a hematologic malignancy.
  • the cancer is not melanoma or pancreatic cancer.
  • the cancer is breast cancer, such as triple negative breast cancer (TNBC).
  • the cancer is bladder cancer.
  • the cancer is urothelial carcinoma. I n one embodiment, the cancer is kidney cancer.
  • the cancer is renal cell carcinoma.
  • the cancer is hepatocellular carcinoma.
  • the patient to be treated herein may have been subjected to one or more assays, more detail about such assays being provided in Section VII. below.
  • the patient has C-reactive protein (CRP) level above the upper limit of normal.
  • the patient may have ⁇ 3 mg/L CRP, e.g. ⁇ 10 mg/L CRP.
  • the CRP is measured by enzyme-linked immunosorbent assay (ELISA) assay and the sample is a blood sample from the patient.
  • the patient has IL-6 level above the upper limit of normal.
  • the patient may have ⁇ 10 pg/mL IL-6, e.g. ⁇ 15 pg/mL IL-6.
  • IL-6 is measured by enzyme-linked immunosorbent assay (ELISA) assay and the sample is a blood sample from the patient.
  • the patient expresses PD-L1.
  • the patient may have PD-L1 stained tumor cells (TC) and/or tumor-infiltrating immune cells (IC), e.g. where the PD-L1 stained TC and/or IC cover ⁇ 1% of the tumor area, e.g. ⁇ 5% of the tumor area.
  • the patient has CRP above the upper limit of normal and the patient’s tumor expresses PD-L1.
  • the patient has IL-6 above the upper limit of normal and the patient’s tumor expresses PD-L1.
  • the patient has both CRP and IL-6 above the upper limit of normal.
  • the patient has both CRP and IL-6 above the upper limit of normal and the patient’s tumor expresses PD-L1.
  • the patient’s tumor expresses PD-L1 (e.g. mTNBC).
  • CRP and/or IL-6 is evaluated prior to treatment and the patient has elevated CRP and/or IL-6 prior to treatment.
  • CRP and/or IL-6 is evaluated during treatment or following treatment and the patient has elevated CRP and/or IL-6.
  • the patient fails to respond or has unacceptable toxicity to a prior therapy e.g. where the prior therapy is therapy with a PD-L axis binding antagonist (e.g. an anti-PD-L1 antibody such as atezolizumab) without the IL-6 antagonist (e.g.
  • a PD-L axis binding antagonist e.g. an anti-PD-L1 antibody such as atezolizumab
  • the IL-6 antagonist e.g.
  • the IL-6 antagonist is administered to the patient prior to the administration of the PD-1 axis binding antagonist.
  • the patient does not have cytokine release syndrome (CRS).
  • the IL-6 antagonist is an anti-IL6 receptor antibody, e.g. tocilizumab, satralizumab, sarilumab, NI-1201, or vobarilizumab, preferably tocilizumab.
  • the IL-6 antagonist binds IL-6, e.g.
  • the PD-L1 axis binding antagonist is a PD-L1 binding antagonist, a PD-1 binding antagonist, or a PD-L2 binding antagonist.
  • the PD-L1 axis binding antagonist is a PD-L1 binding antagonist, e.g. which inhibits the binding of PD-L1 to both PD-1 and B7-1 and/or is an antibody.
  • PD-L1 binding antibodies contemplated herein include atezolizumab, MDX-1105, MEDI4736 (durvalumab), or MSB0010718C (avelumab), atezolizumab being preferred.
  • the PD-L1 axis binding antagonist is a PD-1 binding antagonist, examples of which include: MDX-1106 (nivolumab), MK-3475 (pembrolizumab), MEDI-0680 (AMP-514), PDR001, REGN2810, BGB-108, and AMP-224. More detail about PD-1 axis binding antagonists is provided in Section IV. below.
  • the IL-6 antagonist is an IL-6 receptor binding antibody (e.g.
  • tocilizumab is administered by intravenous (iv) infusion at a dose of 8 mg/kg every 4 weeks (Q4w) on Day 1 of each 28-day cycle, e.g. administered until disease progression or unacceptable toxicity.
  • atezolizumab is administered intravenously (iv) at a fixed dose of 840 mg every 2 weeks (Q2W) on Days 1 and 15 of each 28-day cycle, e.g. until disease progression or unacceptable toxicity.
  • the tocilizumab is administered first and atezolizumab is administered after the tocilizumab administration.
  • the atezolizumab may be administered about two hours after the conclusion of the tocilizumab administration.
  • treatment achieves an objective response rate (ORR), including a complete response (CR) and/or a partial response (PR).
  • ORR objective response rate
  • the treatment extends progression free survival (PFS) and/or overall survival (OS), e.g. to a greater extent than treatment without the IL-6 antagonist.
  • PFS progression free survival
  • OS overall survival
  • the treatment results in an increased abundance of CD8 + T cells in the patient relative to that of a subject who has not been administered the IL-6 antagonist.
  • the treatment reduces or prevents therapeutic resistance to the PD-1 axis binding antagonist.
  • the invention concerns a method of treating a cancer patient comprising administering to the patient a combination of an anti-IL6 receptor antibody and an anti-PD-L1 antibody in an amount effective to treat the cancer.
  • the cancer can be breast cancer, bladder cancer, or renal cell carcinoma.
  • the invention provides a method of treating a cancer patient with high C-reactive protein (CRP) level comprising administering to the patient a combination of an anti-IL6 receptor antibody and an anti-PD-L1 antibody in an amount effective to treat the cancer.
  • CRP C-reactive protein
  • the invention concerns a method of treating advanced urothelial carcinoma in a cancer patient comprising administering to the patient a combination of tocilizumab and atezolizumab in an amount effective to treat the cancer.
  • the invention concerns a method of treating triple negative breast cancer (TNBC) in a cancer patient comprising administering to the patient a combination of tocilizumab, atezolizumab, and chemotherapy (e.g. a taxane such as Nab paclitaxel) in an amount effective to treat the cancer.
  • TNBC triple negative breast cancer
  • exemplary dosages for tocilizumab include 8mg/kg or 4mg/kg (by intravenous infusion) and, alternatively, 162 mg administered by subcutaneous administration.
  • each dosing cycle may have any suitable length, e.g., about 7 days, about 14 days, about 21 days, about 28 days, or longer. In one embodiment, each dosing cycle is about 28 days.
  • dosing cycles Any suitable number of dosing cycles may be used, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, or more dosing cycles. In some embodiments, 10 or fewer dosing cycles may be used. In some embodiments, 20 or fewer dosing cycles are used. In some embodiments, 25 or fewer dosing cycles are used. In one embodiment, the combination is administered until disease progression or unacceptable toxicity.
  • the PD-L1 binding antagonist is an anti-PD-L1 antibody. Any suitable anti- PD-L1 antibody described herein or known in the art may be used.
  • the anti-PD-L1 antibody is selected from the group consisting of atezolizumab (TECENTRIQ®), MDX-1105, MEDI4736 (durvalumab), and MSB0010718C (avelumab).
  • Atezolizumab may be administered to the subject at any suitable dosage.
  • atezolizumab is administered to the subject intravenously at a dose of about 840 mg every 2 weeks, about 1200 mg every 3 weeks, or about 1680 mg of every 4 weeks.
  • atezolizumab is administered to the subject intravenously at a dose of about 840 mg every 2 weeks.
  • atezolizumab is administered to the subject in a 28-day dosing cycle.
  • the PD-1 axis binding antagonist is a PD-1 binding antagonist.
  • the PD-1 binding antagonist inhibits the binding of PD-1 to one or more of its ligand binding partners.
  • the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1, PD- L2, or both PD-L1 and PD-L2.
  • the PD-1 binding antagonist is an anti-PD-1 antibody.
  • the anti-PD-1 antibody is selected from the group consisting of: MDX-1106 (nivolumab), MK-3475 (pembrolizumab), MEDI-0680 (AMP-514), PDR001, REGN2810, and BGB-108.
  • the PD-1 binding antagonist is an Fc fusion protein.
  • the Fc fusion protein is AMP-224.
  • the therapeutically effective amount of a PD-1 axis binding antagonist e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody
  • administered to a human will be in the range of about 0.01 to about 50 mg/kg of patient body weight, whether by one or more administrations.
  • the antagonist e.g., a PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody)
  • a PD-1 axis binding antagonist e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody
  • the antagonist is administered in a dose of about 0.01 to about 45 mg/kg, about 0.01 to about 40 mg/
  • the antagonist e.g., a PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody) and/or a VEGF antagonist (e.g., an anti-VEGF antibody (e.g., bevacizumab)) is administered at 15 mg/kg.
  • a PD-1 axis binding antagonist e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody
  • a VEGF antagonist e.g., an anti-VEGF antibody (e.g., bevacizumab)
  • other dosage regimens may be useful.
  • a PD-1 axis binding antagonist e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody
  • a human 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, about 1400 mg, or about 1500 mg.
  • the antagonist e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody
  • the antagonist may be administered at a dose of about 1000 mg to about 1400 mg every three weeks (e.g., about 1100 mg to about 1300 mg every three weeks, e.g., about 1150 mg to about 1250 mg every three weeks).
  • the antagonist e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody
  • the antagonist e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody
  • 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 combination treatment may be reduced as compared to a single treatment.
  • the treatment regimen comprises administering intravenously to the subject about 1200 mg of atezolizumab every three weeks.
  • the PD-1 axis binding antagonist e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody
  • the IL6 antagonist e.g., anti-IL6 receptor antibody, e.g. tocilizumab
  • the PD-1 axis binding antagonist e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti- PD-1 antibody
  • the IL6 antagonist e.g., anti-IL6 receptor antibody, e.g.
  • the PD-1 axis binding antagonist e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody
  • the IL6 antagonist e.g., anti-IL6 receptor antibody, e.g. tocilizumab
  • the PD-1 axis binding antagonist is administered prior to the IL6 antagonist (e.g., anti-IL6 receptor antibody, e.g. tocilizumab).
  • the PD-1 axis binding antagonist is administered after the IL6 antagonist (e.g., anti-IL6 receptor antibody, e.g. tocilizumab). In yet other embodiments, the PD-1 axis binding antagonist is administered concurrently with the IL6 antagonist (e.g., anti-IL6 receptor antibody, e.g. tocilizumab). In some embodiments, the PD- 1 axis binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody) is in a separate composition as the IL6 antagonist (e.g., anti-IL6 receptor antibody, e.g. tocilizumab).
  • the IL6 antagonist e.g., anti-IL6 receptor antibody, e.g. tocilizumab
  • the PD- 1 axis binding antagonist e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody
  • the PD-1 axis binding antagonist e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody
  • the IL6 antagonist e.g., anti-IL6 receptor antibody, e.g. tocilizumab.
  • the PD-1 axis binding antagonist e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti- PD-1 antibody
  • the IL6 antagonist e.g., anti-IL6 receptor antibody, e.g. tocilizumab
  • the PD-1 axis binding antagonist e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody
  • IL6 antagonist e.g., anti-IL6 receptor antibody, e.g. tocilizumab
  • An effective amount of the PD-1 axis binding antagonist e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti- PD-1 antibody
  • IL6 antagonist e.g., anti-IL6 receptor antibody, e.g. tocilizumab
  • the appropriate dosage of the PD-1 axis binding antagonist e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody
  • the IL6 antagonist e.g., anti- IL6 receptor antibody, e.g.
  • tocilizumab may be determined based on the type of disease to be treated, the type of the PD-1 axis binding antagonist and the IL6 antagonist (e.g., anti-IL6 receptor antibody, e.g. tocilizumab), the severity and course of the disease, the clinical condition of the individual, the individual’s clinical history and response to the treatment, and the discretion of the attending physician.
  • the PD-1 axis binding antagonist e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody
  • the IL6 antagonist e.g., anti-IL6 receptor antibody, e.g. tocilizumab
  • the PD-1 axis binding antagonist e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody
  • the IL6 antagonist e.g., anti-IL6 receptor antibody, e.g. tocilizumab
  • the treatment may further comprise an additional therapy, such as chemotherapy and/or anti-angiogenic therapy (e.g. bevacizumab) discussed in more detail in Section VI. below.
  • additional therapy may be radiation therapy, surgery (e.g., transurethral bladder tumor resection (TURBT) or cystectomy (including a partial or radical cystectomy)), 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.
  • the additional therapy is the administration of small molecule enzymatic inhibitor or anti-metastatic agent.
  • the additional therapy is the administration of side-effect limiting agents (e.g., agents intended to lessen the occurrence and/or severity of side effects of treatment, such as anti-nausea agents, and the like).
  • the additional therapy is radiation therapy.
  • the additional therapy is surgery.
  • the additional therapy is a combination of radiation therapy and surgery.
  • the additional therapy is gamma irradiation.
  • the additional therapy is therapy targeting PI3K/AKT/mTOR pathway, HSP90 inhibitor, tubulin inhibitor, apoptosis inhibitor, and/or chemopreventative agent.
  • the additional therapy may be one or more of the chemotherapeutic agents described herein.
  • IV. PD-1 Axis Binding Antagonists the methods herein involve treating cancer with a PD-1 axis binding antagonist.
  • a PD-1 axis binding antagonist includes a PD-L1 binding antagonist, a PD-1 binding antagonist, and a PD-L2 binding antagonist.
  • PD-L1 (programmed death ligand 1) is also referred to in the art as “programmed cell death 1 ligand 1,” “PDCD1LG1,” “CD274,” “B7-H,” and “PDL1.”
  • An exemplary human PD-L1 is shown in UniProtKB/Swiss-Prot Accession No. Q9NZQ7.1.
  • PD-1 (programmed death 1) is also referred to in the art as “programmed cell death 1,” “PDCD1,” “CD279,” and “SLEB2.”
  • An exemplary human PD-1 is shown in UniProtKB/Swiss-Prot Accession No. Q15116.
  • 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.”
  • An exemplary human PD-L2 is shown in UniProtKB/Swiss-Prot Accession No. Q9BQ51.
  • PD-L1, PD-1, and PD-L2 are human PD-L1, PD-1, and PD-L2.
  • the PD-1 axis binding antagonist is an anti-PD-L1 antibody.
  • the anti-PD-L1 antibody is atezolizumab, YW243.55.S70, MDX-1105, MEDI4736 (durvalumab), or MSB0010718C (avelumab).
  • Antibody YW243.55.S70 is an anti-PD-L1 antibody described in WO 2010/077634.
  • MDX-1105 also known as BMS-936559, is an anti-PD-L1 antibody described in WO2007/005874.
  • MEDI4736 is an anti-PD-L1 monoclonal antibody described in WO2011/066389 and US2013/034559.
  • the anti-PD-L1 antibody is capable of inhibiting binding between PD-L1 and PD-1 and/or between PD-L1 and B7-1.
  • the anti-PD-L1 antibody is a monoclonal antibody.
  • the anti-PD-L1 antibody is an antibody fragment selected from the group consisting of Fab, Fab’-SH, Fv, scFv, and (Fab’)2 fragments.
  • the anti-PD-L1 antibody is a humanized antibody.
  • the anti- PD-L1 antibody is a human antibody. Examples of anti-PD-L1 antibodies useful for the methods of this invention, and methods for making thereof are described in PCT Patent Application Nos.
  • the anti-PD-L1 antibodies useful in this invention may be used as a monotherapy or in combination with one or more additional therapeutic agents, e.g., an IL6 antagonist.
  • the PD-1 binding antagonist is a molecule that inhibits the binding of PD-1 to its ligand binding partners.
  • the PD-1 ligand binding partners are PD-L1 and/or PD- L2.
  • a PD-L1 binding antagonist is a molecule that inhibits the binding of PD-L1 to its binding partners.
  • PD-L1 binding partners are PD-1 and/or B7-1.
  • the PD-L2 binding antagonist is a molecule that inhibits the binding of PD-L2 to its binding partners.
  • a PD-L2 binding partner is PD-1.
  • the antagonist may be an antibody, an antigen-binding fragment thereof, an immunoadhesin, a fusion protein, or oligopeptide. Any suitable anti-PD-L1 antibody may be used in the methods and compositions provided herein.
  • Anti-PD-L1 antibodies described in WO 2010/077634 A1 and US 8,217,149 may be used in the methods and compositions provided herein.
  • 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.
  • an isolated anti-PD-L1 antibody comprising a heavy chain variable region and/or a light chain variable region sequence, 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: (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 light chain sequence:
  • the anti-PD-L1 antibody comprises a heavy chain variable region comprising an HVR-H1, HVR
  • the polypeptide further comprises variable region heavy chain framework sequences juxtaposed between the HVRs according to the formula: (FR-H1)-(HVR-H1)-(FR-H2)-(HVR- H2)-(FR-H3)-(HVR-H3)-(FR-H4).
  • the framework sequences are derived from human consensus framework sequences.
  • the framework sequences are VH subgroup III consensus framework.
  • the heavy chain polypeptide is further combined with a variable region light chain comprising an HVR-L1, HVR-L2 and HVR-L3, wherein: 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.
  • the light chain further comprises variable region light chain framework sequences juxtaposed between the HVRs according to the formula: (FR-L1)-(HVR-L1)-(FR-L2)-(HVR-L2)- (FR-L3)-(HVR-L3)-(FR-L4).
  • the framework sequences are derived from human consensus framework sequences.
  • the framework sequences are VL kappa I consensus framework.
  • at least one of the framework sequence is the following: FR-L1 is DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 15)
  • FR-L2 is WYQQKPGKAPKLLIY (SEQ ID NO: 16)
  • FR-L3 is GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 17)
  • FR-L4 is FGQGTKVEIKR (SEQ ID NO: 18).
  • an isolated anti-PD-L1 antibody or antigen binding fragment comprising a heavy chain and a light chain variable region sequence
  • the heavy chain comprises an HVR-H1, HVR-H2 and HVR-H3, wherein further: (i) the HVR-H1 sequence is GFTFSX1SWIH; (SEQ ID NO: 5)
  • the HVR-H2 sequence is AWIX2PYGGSX3YYADSVKG (SEQ ID NO: 6)
  • the HVR-H3 sequence is RHWPGGFDY
  • the light chain comprises an HVR-L1, HVR-L2 and HVR-L3, wherein further: (i) the HVR-L1 sequence is RASQX4X5X6TX7X8A (SEQ ID NO: 12)
  • the HVR-L2 sequence is SASX9LX10S; and (SEQ ID NO: 13) (iii) the
  • the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs as: (FR-H1)-(HVR-H1)-(FR-H2)-(HVR-H2)-(FR-H3)-(HVR-H3)-(FR-H4)
  • the light chain variable regions comprises one or more framework sequences juxtaposed between the HVRs as: (FR-L1)-(HVR-L1)-(FR-L2)-(HVR-L2)-(FR-L3)-(HVR-L3)-(FR-L4).
  • the framework sequences are derived from human consensus framework sequences.
  • the heavy chain framework sequences are derived from a Kabat subgroup I, II, or III sequence. In a still further aspect, the heavy chain framework sequence is a VH subgroup III consensus framework. In a still further aspect, one or more of the heavy chain framework sequences are set forth as SEQ ID NOs:8, 9, 10, and 11. In a still further aspect, the light chain framework sequences are derived from a Kabat kappa I, II, II or IV subgroup sequence. In a still further aspect, the light chain framework sequences are VL kappa I consensus framework. In a still further aspect, one or more of the light chain framework sequences are set forth as SEQ ID NOs: 15, 16, 17, and 18.
  • the antibody further comprises a human or murine constant region.
  • the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, and IgG4.
  • the human constant region is IgG1.
  • the murine constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, and IgG3.
  • the antibody has reduced or minimal effector function.
  • the minimal effector function results from an “effector-less Fc mutation” or aglycosylation.
  • the effector-less Fc mutation is an N297A or D265A/N297A substitution in the constant region.
  • an anti-PD-L1 antibody comprising a heavy chain and a light chain variable region sequence, wherein: (a) the heavy chain further comprises an HVR-H1, HVR-H2 and an HVR-H3 sequence having at least 85% sequence identity to GFTFSDSWIH (SEQ ID NO: 19), AWISPYGGSTYYADSVKG (SEQ ID NO: 20) and RHWPGGFDY (SEQ ID NO: 21), respectively, or (b) the light chain further comprises an HVR-L1, HVR-L2 and an HVR-L3 sequence having at least 85% sequence identity to RASQDVSTAVA (SEQ ID NO: 22), SASFLYS (SEQ ID NO: 23) and QQYLYHPAT (SEQ ID NO: 24), respectively.
  • the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
  • the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs as: (FR-H1)-(HVR-H1)-(FR-H2)-(HVR-H2)-(FR-H3)-(HVR-H3)-(FR-H4)
  • the light chain variable regions comprises one or more framework sequences juxtaposed between the HVRs as: (FR-L1)-(HVR-L1)-(FR-L2)-(HVR-L2)-(FR-L3)-(HVR-L3)-(FR-L4).
  • the framework sequences are derived from human consensus framework sequences.
  • the heavy chain framework sequences are derived from a Kabat subgroup I, II, or III sequence.
  • the heavy chain framework sequence is a VH subgroup III consensus framework.
  • one or more of the heavy chain framework sequences are set forth as SEQ ID NOs: 8, 9, 10, and 11.
  • the light chain framework sequences are derived from a Kabat kappa I, II, II, or IV subgroup sequence.
  • the light chain framework sequences are VL kappa I consensus framework.
  • the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs as: (FR-H1)-(HVR-H1)-(FR-H2)-(HVR-H2)-(FR-H3)-(HVR-H3)-(FR-H4)
  • the light chain variable regions comprises one or more framework sequences juxtaposed between the HVRs as: (FR-L1)-(HVR-L1)-(FR-L2)-(HVR-L2)-(FR-L3)-(HVR-L3)-(FR-L4).
  • the framework sequences are derived from human consensus framework sequences.
  • the heavy chain framework sequences are derived from a Kabat subgroup I, II, or III sequence.
  • the heavy chain framework sequence is a VH subgroup III consensus framework.
  • one or more of the heavy chain framework sequences is the following:
  • the light chain framework sequences are derived from a Kabat kappa I, II, II or IV subgroup sequence.
  • the light chain framework sequences are VL kappa I consensus framework.
  • the antibody further comprises a human or murine constant region.
  • the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, and IgG4.
  • the human constant region is IgG1.
  • the murine constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, and IgG3.
  • the antibody has reduced or minimal effector function.
  • the minimal effector function results from an “effector-less Fc mutation” or aglycosylation.
  • the effector-less Fc mutation is an N297A or D265A/N297A substitution in the constant region.
  • an anti-PD-L1 antibody comprising a heavy chain and a light chain variable region sequence, wherein: (c) the heavy chain further comprises an HVR-H1, HVR-H2 and an HVR-H3 sequence having at least 85% sequence identity to GFTFSDSWIH (SEQ ID NO: 19), respectively, and/or (d) the light chain further comprises an HVR-L1, HVR-L2 and an HVR-L3 sequence having at least 85% sequence identity to RASQDVSTAVA (SEQ ID NO: 22), SASFLYS (SEQ ID NO: 23) and QQYLYHPAT (SEQ ID NO: 24), respectively.
  • the sequence identity is 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.
  • the heavy chain variable region comprises one or more framework sequences juxtaposed between the HVRs as: (FR-H1)-(HVR-H1)-(FR-H2)-(HVR-H2)-(FR-H3)-(HVR-H3)-(FR-H4)
  • the light chain variable regions comprises one or more framework sequences juxtaposed between the HVRs as: (FR-L1)-(HVR-L1)-(FR-L2)-(HVR-L2)-(FR-L3)-(HVR-L3)-(FR-L4).
  • the framework sequences are derived from human consensus framework sequences.
  • the heavy chain framework sequences are derived from a Kabat subgroup I, II, or III sequence.
  • the heavy chain framework sequence is a VH subgroup III consensus framework.
  • one or more of the heavy chain framework sequences are set forth as SEQ ID NOs: 8, 9, 10, and WGQGTLVTVSSASTK (SEQ ID NO: 29).
  • the light chain framework sequences are derived from a Kabat kappa I, II, II or IV subgroup sequence.
  • the light chain framework sequences are VL kappa I consensus framework.
  • the antibody further comprises a human or murine constant region.
  • the human constant region is selected from the group consisting of IgG1, IgG2, IgG2, IgG3, and IgG4.
  • the human constant region is IgG1.
  • the murine constant region is selected from the group consisting of IgG1, IgG2A, IgG2B, and IgG3.
  • the antibody has reduced or minimal effector function.
  • the minimal effector function results from an “effector-less Fc mutation” or aglycosylation.
  • the effector-less Fc mutation is an N297A or D265A/N297A substitution in the constant region.
  • 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: (b) the light chain sequences has at least 85% sequence identity to the light chain sequence: ( )
  • an isolated anti-PD-L1 antibody comprising a heavy chain and a light chain variable region sequence, 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.
  • an isolated anti-PD-L1 antibody comprising a heavy chain and a light chain variable region sequence, 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 s equence of SEQ ID NO: 25.
  • an isolated anti-PD-L1 antibody comprising a heavy chain and a light chain variable region sequence, 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 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: 25.
  • one, two, three, four, or five amino acid residues at the N-terminal of the heavy and/or light chain may be deleted, substituted or modified.
  • an isolated anti-PD-L1 antibody comprising a heavy chain and a light chain sequence, wherein: (a) the heavy chain sequence has at least 85% sequence identity to the heavy chain sequence: QQG SCS Q S S S G (S Q O 30), a d/o (b) the light chain sequences has at least 85% sequence identity to the light chain sequence: ( )
  • an isolated anti-PD-L1 antibody comprising a heavy chain and a light chain sequence, wherein the light chain sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino
  • an isolated anti-PD-L1 antibody comprising a heavy chain and a light chain sequence, wherein the heavy chain sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 30.
  • an isolated anti-PD-L1 antibody comprising a heavy chain and a light chain sequence, wherein the light chain sequence has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 31 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: 30.
  • an isolated anti- PD-L1 antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:30 and a light chain sequence comprising the amino acid sequence of SEQ ID NO:31.
  • the isolated anti-PD-L1 antibody is aglycosylated. Glycosylation of antibodies is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue.
  • the tripeptide sequences asparagine-X-serine and asparagine-X- threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain.
  • O-linked glycosylation refers to the attachment of one of the sugars N-aceylgalactosamine, 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 form an antibody is conveniently accomplished by altering the amino acid sequence such that one of the above-described tripeptide sequences (for N-linked glycosylation sites) is removed.
  • the alteredation may be made by substitution of an asparagine, serine or threonine residue within the glycosylation site another amino acid residue (e.g., glycine, alanine or a conservative substitution).
  • the isolated anti-PD-L1 antibody can bind to a human PD-L1, for example a human PD-L1 as shown in UniProtKB/Swiss-Prot Accession No. Q9NZQ7.1, or a variant thereof.
  • provided is an isolated nucleic acid encoding any of the antibodies described herein.
  • the nucleic acid further comprises a vector suitable for expression of the nucleic acid encoding any of the previously described anti-PD-L1 antibodies.
  • the vector is in a host cell suitable for expression of the nucleic acid.
  • the host cell is a eukaryotic cell or a prokaryotic cell.
  • the eukaryotic cell is a mammalian cell, such as Chinese hamster ovary (CHO) cell.
  • the antibody or antigen binding fragment thereof may 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 fragments in a form suitable for expression, under conditions suitable to produce such antibody or fragment, and recovering the antibody or fragment.
  • the PD-1 axis binding antagonist is a PD-1 binding antagonist.
  • the PD-1 binding antagonist is an anti-PD-1 antibody (e.g., a human antibody, a humanized antibody, or a chimeric antibody). Any suitable anti-PD-1 antibody may be used in the context of the invention.
  • the anti-PD-1 antibody is selected from the group consisting of MDX- 1106 (nivolumab), MK-3475 (pembrolizumab), MEDI-0680 (AMP-514), PDR001, REGN2810, and BGB- 108.
  • the PD-1 binding antagonist is an immunoadhesin (e.g., an immunoadhesin comprising an extracellular or PD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g., an Fc region of an immunoglobulin sequence).
  • the PD-1 binding antagonist is AMP- 224.
  • the PD-L1 binding antagonist is anti-PD-L1 antibody.
  • MDX-1106 also known as MDX-1106-04, ONO-4538, BMS-936558, or nivolumab, is an anti-PD-1 antibody described in WO2006/121168.
  • MK-3475 also known as lambrolizumab
  • AMP-224 also known as B7-DCIg
  • PD-L2-Fc fusion soluble receptor described in WO2010/027827 and WO2011/066342.
  • the anti-PD-1 antibody is MDX-1106.
  • Alternative names for “MDX-1106” include MDX-1106-04, ONO-4538, BMS-936558, and nivolumab.
  • the anti-PD-1 antibody is nivolumab (CAS Registry Number: 946414-94-4).
  • an isolated anti-PD-1 antibody comprising a heavy chain variable region comprising the heavy chain variable region amino acid sequence from SEQ ID NO: 1 and/or a light chain variable region comprising the light chain variable region amino acid sequence from SEQ ID NO: 2.
  • an isolated anti-PD-1 antibody comprising a heavy chain and/or a light chain sequence, 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: (SEQ ID NO: 1), and (b) the light chain sequences 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 light chain sequence: G C (S Q ID NO: 2).
  • nucleic acid encoding any of the antibodies described herein.
  • nucleic acid further comprises a vector suitable for expression of the nucleic acid encoding any of the previously described anti-PD-1 antibodies.
  • the vector is in a host cell suitable for expression of the nucleic acid.
  • the host cell is a eukaryotic cell or a prokaryotic cell.
  • the eukaryotic cell is a mammalian cell, such as Chinese hamster ovary (CHO) cell.
  • the antibody or antigen-binding fragment thereof may 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-1 antibodies in a form suitable for expression, under conditions suitable to produce such antibody or fragment, and recovering the antibody or fragment, or according to any method described below.
  • V. IL6 Antagonists IL6 antagonists contemplated herein include antagonists that bind to IL6 or IL6 receptor.
  • the IL6 antagonist is an antibody.
  • the IL6 antagonist is an antibody that binds IL6 receptor.
  • Antibodies that bind IL-6R include tocilizumab (including intravenous, iv, and subcutaneous sc formulations thereof) (Chugai, Roche, Genentech), satralizumab (Chugai, Roche, Genentech), sarilumab (Sanofi, Regeneron), NI-1201 ( Novimmune and Tiziana), and vobarilizumab (Ablynx).
  • the IL6 antagonist is tocilizumab.
  • Tocilizumab also named Myeloma Receptor Antibody (MRA) is a recombinant humanized monoclonal antibody that selectively binds to human interleukin-6 receptor (IL-6R).
  • MRA Myeloma Receptor Antibody
  • the tocilizumab molecule is composed of two heterodimers. Each of the heterodimers is composed of a heavy (H) and a light (L) polypeptide chain. The four polypeptide chains are linked intra- and inter-molecularly by disulfide linkages.
  • the molecular formula and theoretical molecular weight of the tocilizumab antibody are as follows: Molecular formula: C6428H9976N1720O2018S42 (polypeptide moiety only) Molecular weight: 144,985 Da (polypeptide moiety only).
  • the amino acid sequence of the light chain deduced from complimentary deoxyribonucleic acid (cDNA) sequences and confirmed by liquid chromatography mass-spectrometry (LC-MS) peptide mapping is in SEQ ID Nos.32 and 33.
  • the five light chain cysteine residues of each heterodimer are involved in two intrachain disulfide linkages and one interchain disulfide linkage: Intrachain linkages: CysL23-CysL88 and CysL134-CysL194 Linkage between heavy and light chain: CysL214 and CysH222 Assignments of the disulfide linkages are based on sequence homology to other IgG1 antibodies and were confirmed by liquid chromatography mass-spectrometry (LC-MS) peptide mapping performed using material from the fourth generation (G4) process.
  • LC-MS liquid chromatography mass-spectrometry
  • CysLx and CysHx denote cysteine residues at position x of the light and heavy chains, respectively.
  • S EQ ID NO. 32 Amino Acid Sequence of the L Chain of the Tocilizumab Molecule Note: The entire sequence has been determined by LC-MS peptide mapping. The amino acid sequence of the heavy chain deduced from complimentary deoxyribonucleic acid (cDNA) sequences and confirmed by amino acid sequencing is in SEQ ID NO. x.
  • the eleven heavy chain cysteine residues of each heterodimer are involved in four intrachain disulfide linkages, two interchain disulfide linkages between the two heavy chains and the third interchain disulfide linkage between the heavy chain and the light chain of each of the heterodimers: Intrachain linkages: CysH22-CysH96, CysH146-CysH202, CysH263-CysH323 and CysH369-CysH427 Linkages between the two heavy chains: CysH228-CysH228 and CysH231-CysH231 Linkage between heavy and light chain: CysL214-CysH222 Assignments of the disulfide linkages are based on sequence homology to other IgG1 antibodies and were confirmed by LC-MS peptide mapping performed using material from the G4 process.
  • the IL6 antagonist is satralizumab.
  • Satralizumab also called SA237) is a humanized monoclonal antibody that binds IL6 receptor. See US Patent No. US 8,562,991.
  • the IL6 antagonist is a monoclonal antibody that binds IL6.
  • Antibodies that bind IL-6 include sirukumab (Centecor, Janssen), olokizumab (UCB), clazakizumab (BMS and Alder), siltuximab (Janssen), EBI-031 (Eleven Biotherapeutics and Roche).
  • the IL6 antagonist is olamkicept.
  • Olamkicept is a recombinant protein that fuses the extracellular domain of the signal transducing subunit of the IL-6 receptor, IL-6R ⁇ (glycoprotein 130, gp130), to a human IgG Fc fragment.
  • the full construct is a dimer of covalently linked identical peptide chains.
  • Mechanistically olamkicept acts as an inhibitor of the IL-6 signalling pathway. Olamkicept inhibits trans signalling by the soluble IL-6 receptor (sIL-6R).
  • chemotherapeutic agent(s) and/or anti-antiogenic agents include taxoids (such as paclitaxel and docetaxel and modified forms thereof such as nanoparticle albumin-bound paclitaxel (“Nab-paclitaxel”).
  • chemotherapeutic agents that can be further combined include platinum-containing chemotherapy, e.g. cisplatin, and the combination of gemcitabine and cisplatin (GC).
  • chemotherapeutic agents that can be further combined include platinum-containing chemotherapy, e.g. cisplatin, and the combination of gemcitabine and cisplatin (GC).
  • GC gemcitabine and cisplatin
  • further drugs to combine with the combination include: bevacizumab, paclitaxel, and/or carboplatin; paclitaxel (e.g. Nab-paclitaxel) and/or carboplatin.
  • chemotherapeutic agents include carboplatin and/or etoposide.
  • methods for treating cancer in a subject comprising administering to the subject a treatment regimen comprising an effective amount of a PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody) and/or an IL6 antagonist (e.g. an anti- IL6 receptor antibody (e.g. tocilizumab)) in conjunction with another anti-cancer agent or cancer therapy.
  • a PD-1 axis binding antagonist e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody
  • an IL6 antagonist e.g. an anti- IL6 receptor antibody (e.g. tocilizumab)
  • the methods comprise administering to the individual a PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody), an IL6 antagonist (e.g. an anti-IL6 receptor antibody (e.g. tocilizumab)), and an additional therapeutic agent.
  • a PD-1 axis binding antagonist e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody
  • an IL6 antagonist e.g. an anti-IL6 receptor antibody (e.g. tocilizumab)
  • a PD-1 axis binding antagonist e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody) and/or an IL6 antagonist (e.g. an anti-IL6 receptor antibody (e.g. tocilizumab)) may be administered in conjunction with a radiation therapy or radiotherapeutic agent.
  • a PD-1 axis binding antagonist e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody
  • an IL6 antagonist e.g. an anti-IL6 receptor antibody (e.g.
  • a PD-1 axis binding antagonist e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody
  • a IL6 antagonist e.g. an anti-IL6 receptor antibody (e.g. tocilizumab)
  • an immunotherapy or immunotherapeutic agent for example, a monoclonal antibody.
  • a PD-1 axis binding antagonist e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody
  • an IL6 antagonist e.g. an anti-IL6 receptor antibody (e.g. tocilizumab)
  • an activating co-stimulatory molecule may include CD40, CD226, CD28, OX40, GITR, CD137, CD27, HVEM, or CD127.
  • the agonist directed against an activating co-stimulatory molecule is an agonist antibody that binds to CD40, CD226, CD28, OX40, GITR, CD137, CD27, HVEM, or CD127.
  • a PD-1 axis binding antagonist e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody
  • an IL6 antagonist e.g. an anti-IL6 receptor antibody (e.g.
  • an inhibitory co-stimulatory molecule may include CTLA-4 (also known as CD152), PD-1, TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO, TIGIT, MICA/B, or arginase.
  • the antagonist directed against an inhibitory co-stimulatory molecule is an antagonist antibody that binds to CTLA-4, PD-1, TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO, TIGIT, MICA/B, or arginase.
  • a PD-1 axis binding antagonist e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody) and/or an IL6 antagonist (e.g. an anti-IL6 receptor antibody (e.g. tocilizumab)) may be administered in conjunction with an antagonist directed against CTLA-4 (also known as CD152), for example, a blocking antibody.
  • a PD-1 axis binding antagonist e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody
  • an IL6 antagonist e.g. an anti-IL6 receptor antibody (e.g.
  • a PD-1 axis binding antagonist e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody
  • an IL6 antagonist e.g. an anti-IL6 receptor antibody (e.g. tocilizumab)
  • tremelimumab also known as ticilimumab or CP-675,206
  • a PD-1 axis binding antagonist e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody) and/or an IL6 antagonist (e.g. an anti-IL6 receptor antibody (e.g. tocilizumab)) may be administered in conjunction with an antagonist directed against B7-H3 (also known as CD276), for example, a blocking antibody.
  • a PD-1 axis binding antagonist e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody
  • an IL6 antagonist e.g. an anti-IL6 receptor antibody (e.g.
  • a PD-1 axis binding antagonist e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody
  • an IL6 antagonist e.g. an anti-IL6 receptor antibody (e.g. tocilizumab)
  • TGF beta for example, metelimumab (also known as CAT-192), fresolimumab (also known as GC1008), or LY2157299.
  • a PD-1 axis binding antagonist e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody
  • an IL6 antagonist e.g. an anti-IL6 receptor antibody (e.g. tocilizumab)
  • a treatment comprising adoptive transfer of a T cell (e.g., a cytotoxic T cell or CTL) expressing a chimeric antigen receptor (CAR).
  • a T cell e.g., a cytotoxic T cell or CTL
  • CAR chimeric antigen receptor
  • a PD-1 axis binding antagonist e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody
  • an IL6 antagonist e.g. an anti-IL6 receptor antibody (e.g. tocilizumab)
  • a treatment comprising adoptive transfer of a T cell comprising a dominant-negative TGF beta receptor, e.g., a dominant-negative TGF beta type II receptor.
  • a PD-1 axis binding antagonist e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody) and/or an IL6 antagonist (e.g. an anti-IL6 receptor antibody (e.g. tocilizumab)) may be administered in conjunction with a treatment comprising a HERCREEM protocol (see, e.g., ClinicalTrials.gov Identifier NCT00889954).
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with an agonist directed against CD137 (also known as TNFRSF9, 4-1BB, or ILA), for example, an activating antibody.
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with urelumab (also known as BMS-663513). In some embodiments, a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with an agonist directed against CD40, for example, an activating antibody. In some embodiments, a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with CP-870893. In some embodiments, a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with an agonist directed against OX40 (also known as CD134), for example, an activating antibody.
  • OX40 also known as CD134
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with an anti-OX40 antibody (e.g., AgonOX).
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with an agonist directed against CD27, for example, an activating antibody.
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with CDX-1127.
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with an antagonist directed against indoleamine-2,3- dioxygenase (IDO).
  • IDO indoleamine-2,3- dioxygenase
  • the IDO antagonist is 1-methyl-D-tryptophan (also known as 1-D-MT).
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with an antibody-drug conjugate.
  • the antibody-drug conjugate comprises mertansine or monomethyl auristatin E (MMAE).
  • MMAE monomethyl auristatin E
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with and anti-NaPi2b antibody-MMAE conjugate (also known as DNIB0600A or RG7599).
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with trastuzumab emtansine (also known as T-DM1, ado-trastuzumab emtansine, or KADCYLA®, Genentech).
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with DMUC5754A.
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with an antibody-drug conjugate targeting the endothelin B receptor (EDNBR), for example, an antibody directed against EDNBR conjugated with MMAE.
  • EDNBR endothelin B receptor
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with an angiogenesis inhibitor.
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with an antibody directed against angiopoietin 2 (also known as Ang2).
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with MEDI3617.
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with an antineoplastic agent.
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with an agent targeting CSF-1R (also known as M-CSFR or CD115).
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with anti-CSF-1R (also known as IMC-CS4).
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with an interferon, for example interferon alpha or interferon gamma.
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with Roferon- A (also known as recombinant Interferon alpha-2a).
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with GM-CSF (also known as recombinant human granulocyte macrophage colony stimulating factor, rhu GM-CSF, sargramostim, or LEUKINE®).
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with IL-2 (also known as aldesleukin or PROLEUKIN®).
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with IL-12.
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with an antibody targeting CD20.
  • the antibody targeting CD20 is obinutuzumab (also known as GA101 or GAZYVA®) or rituximab.
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with an antibody targeting GITR.
  • the antibody targeting GITR is TRX518.
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with a cancer vaccine.
  • the cancer vaccine is a peptide cancer vaccine, which in some embodiments is a personalized peptide vaccine.
  • the peptide cancer vaccine is a multivalent long peptide, a multi-peptide, a peptide cocktail, a hybrid peptide, or a peptide-pulsed dendritic cell vaccine (see, e.g., Yamada et al., Cancer Sci, 104:14-21 (2013)).
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with an adjuvant.
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with a treatment comprising a TLR agonist, for example, Poly-ICLC (also known as HILTONOL®), LPS, MPL, or CpG ODN.
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with tumor necrosis factor (TNF) alpha.
  • TNF tumor necrosis factor
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with IL-1.
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with HMGB1.
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with an IL-10 antagonist. In some embodiments, a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with an IL-4 antagonist. In some embodiments, a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with an IL-13 antagonist. In some embodiments, a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with an HVEM antagonist.
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with an ICOS agonist, e.g., by administration of ICOS-L, or an agonistic antibody directed against ICOS.
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with a treatment targeting CX3CL1.
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with a treatment targeting CXCL9.
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with a treatment targeting CXCL10.
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with a treatment targeting CCL5.
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with an LFA-1 or ICAM1 agonist.
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with a Selectin agonist.
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with a targeted therapy.
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with an inhibitor of B-Raf.
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with vemurafenib (also known as ZELBORAF®). In some embodiments, a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with dabrafenib (also known as TAFINLAR®). In some embodiments, a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with erlotinib (also known as TARCEVA®).
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with an inhibitor of a MEK, such as MEK1 (also known as MAP2K1) or MEK2 (also known as MAP2K2).
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with cobimetinib (also known as GDC-0973 or XL-518).
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with trametinib (also known as MEKINIST®).
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with an inhibitor of K-Ras. In some embodiments, a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with an inhibitor of c-Met. In some embodiments, a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with onartuzumab (also known as MetMAb). In some embodiments, a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with an inhibitor of Alk.
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with AF802 (also known as CH5424802 or alectinib).
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with an inhibitor of a phosphatidylinositol 3-kinase (PI3K).
  • PI3K phosphatidylinositol 3-kinase
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with BKM120.
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with idelalisib (also known as GS- 1101 or CAL-101).
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with perifosine (also known as KRX-0401). In some embodiments, a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with an inhibitor of an Akt. In some embodiments, a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with MK2206. In some embodiments, a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with GSK690693. In some embodiments, a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with GDC-0941.
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with an inhibitor of mTOR.
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with sirolimus (also known as rapamycin).
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with temsirolimus (also known as CCI-779 or TORISEL®).
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with everolimus (also known as RAD001).
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with ridaforolimus (also known as AP-23573, MK-8669, or deforolimus).
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with OSI-027.
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with AZD8055.
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with INK128.
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with a dual PI3K/mTOR inhibitor. In some embodiments, a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with XL765. In some embodiments, a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with GDC-0980. In some embodiments, a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with BEZ235 (also known as NVP-BEZ235). In some embodiments, a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with BGT226.
  • BEZ235 also known as NVP-BEZ235
  • a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with GSK2126458. In some embodiments, a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with PF-04691502. In some embodiments, a PD-1 axis binding antagonist and/or an IL6 antagonist may be administered in conjunction with PF-05212384 (also known as PKI-587). In any of the preceding embodiments, the PD-1 axis binding antagonist may be a human PD-1 axis binding antagonist.
  • the PD-1 axis binding antagonist is an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody.
  • the platinum-based chemotherapy includes a platinum- based chemotherapeutic agent (e.g., cisplatin or carboplatin). In some embodiments, the platinum-based chemotherapy includes cisplatin. In some embodiments, the platinum-based chemotherapy includes carboplatin. In some embodiments, the platinum-based chemotherapy further includes one or more additional chemotherapeutic agents, e.g., a nucleoside analog. In some embodiments, the nucleoside analog is gemcitabine.
  • the platinum-based chemotherapy includes cisplatin and gemcitabine. In other embodiments, the platinum-based chemotherapy includes carboplatin and gemcitabine.
  • VII. PD-L1, IL6, and CRP Biomarker Assessment the patient treated herein has been subjected to an assay which has found the patient or his or her tumor to have one or more of the following biomarker measurements: 1. C-reactive protein (CRP) level above the upper limit of normal; 2. ⁇ 3 mg/L CRP; 3. ⁇ 10 mg/L CRP; 4. ⁇ 3 mg/L CRP as measured by enzyme-linked immunosorbent assay (ELISA); 5. ⁇ 10 mg/L CRP as measured by enzyme-linked immunosorbent assay (ELISA); 6.
  • CRP C-reactive protein
  • PD-L1 stained tumor cells or tumor-infiltrating immune cells (IC) covering ⁇ 1% of the tumor area; 15. PD-L1 expression as determined by PD-L1 IHC 22C3 pharmDx (Merck); 16. PD-L1 expression as determined by PD-L1 (SP142) Assay (Ventana); and/or 17. PD-L1 expression as determined by PD-L1 (SP263) Assay (Ventana).
  • the patient has C-reactive protein (CRP) level above the upper limit of normal.
  • the patient has IL-6 level above the upper limit of normal.
  • the patient’s cancer expresses PD-L1.
  • the patient has C-reactive protein (CRP) level above the upper limit of normal and expresses PD-L1. In one embodiment, the patient has C-reactive protein (CRP) and IL-6 levels above the upper limit of normal. In one embodiment, the patient has C-reactive protein (CRP) and IL-6 levels above the upper limit of normal and expresses PD-L1. I n one embodiment, the patient has IL-6 levels above the upper limit of normal and expresses PD- L1. In one embodiment, the assay (measuring CRP and/or IL-6 and/or PD-L1) is performed on a sample from the patient obtained from the patient prior to administration of an anti-cancer therapy.
  • the assay is performed on a sample from the patient obtained from the patient after administration of an anti-cancer therapy, including after administration of the PD-1 axis binding antagonist and IL-6 antagonist.
  • the sample is a blood sample from the patient.
  • the sample from the patient is a whole blood sample, a plasma sample, a serum sample, or a combination thereof.
  • the sample is an archival sample, a fresh sample, or a frozen sample.
  • the sample from the patient is a tumor tissue sample, e.g.
  • the expression level of IL-6 in a sample from the individual has been determined to be above a reference IL-6 expression level, e.g. wherein the reference IL-6 expression level is a pre-assigned IL-6 expression level.
  • the expression level of IL-6 in the sample is an expression level of IL-6 that is at least four standard deviations above the reference IL-6 expression level.
  • the expression level of IL-6 in the sample is a protein expression level of IL-6.
  • the expression level of IL-6 is an mRNA expression level of IL-6.
  • Assays for measuring mRNA expression level of IL-6 include in situ hybridization (ISH) (e.g. using a probe targeting nucleotides 2-1082 of an IL-6 mRNA), RNA-seq, RT-qPCR, qPCR, multiplex qPCR or RT-qPCR, microarray analysis, SAGE, MassARRAY technique, FISH, or a combination thereof.
  • ISH in situ hybridization
  • the reference IL-6 expression level is between about 10 pg/mL to about 15 pg/mL.
  • the reference IL-6 expression level is 10 pg/mL.
  • the reference IL-6 expression level is an expression level of IL-6 in a reference population of healthy individuals.
  • the reference IL-6 expression level is an expression level of IL-6 in a reference population of individuals with the tumor type being treated.
  • CRP tests that measure markedly high levels of the CRP protein are available in the art. Such tests can measure CRP in the range from 10 to 1000 mg/L.
  • the CRP assay is a highly sensitive CRP (hsCRP) assay.
  • the CRP assay is an ELISA assay.
  • the CRP assay is a Luminex CRP assay. Normal CRP levels are below 3.0 mg/L. Levels of CRP > 3.0 mg/L can put a patient at a higher than average risk for heart disease. Levels of CRP > 10.0 mg/L signify infection or an inflammatory condition.
  • the expression level of CRP in a sample from the patient has been determined to be above a reference CRP expression level, e.g.3 mg/L or 10 mg/L.
  • the reference CRP expression level is a pre-assigned CRP expression level.
  • the expression level of CRP in the sample is a protein expression level of CRP or an mRNA expression level of CRP. I n one embodiment, the protein expression level of CRP is measured using nephelometry.
  • the reference CRP expression level is an expression level of CRP in a reference population of healthy individuals.
  • the reference CRP expression level is an expression level of CRP in a reference population of individuals with the tumor type being treated.
  • the expression of PD-L1 may be assessed in a subject treated according to any of the methods and compositions for use described herein.
  • the method includes determining the expression level of PD-L1 in a biological sample (e.g., a tumor sample) obtained from the subject.
  • the expression level of PD-L1 in a biological sample (e.g., a tumor sample) obtained from the subject has been determined prior to initiation of treatment.
  • the expression level of PD-L1 in a biological sample (e.g., a tumor sample) obtained from the subject may be determined after initiation of treatment.
  • a tumor sample obtained from the subject has been determined to have a detectable expression level of PD-L1 in tumor-infiltrating immune cells that comprise about 1% or more (e.g., about 1% or more, 2% or more, 3% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 16% or more, 17% or more,18% or more, 19% or more, 20% or more, 21% or more, 22% or more, 23% or more, 24% or more, 25% or more, 26% or more, 27% or more, 28% or more, 29% or more, 30% or more, 31% or more, 32% or more, 33% or more, 34% or more, 35% or more, 36% or more, 37% or more, 38% or more, 39% or more, 40% or more, 41% or more, 42% or more, 43% or
  • a tumor sample obtained from the subject has been determined to have a detectable expression level of PD-L1 in tumor-infiltrating immune cells that comprise from about 1% to less than about 5% (e.g., from 1% to 4.9%, from 1% to 4.5%, from 1% to 4%, from 1% to 3.5%, from 1% to 3%, from 1% to 2.5%, or from 1% to 2%) of the tumor sample.
  • a detectable expression level of PD-L1 in tumor-infiltrating immune cells that comprise from about 1% to less than about 5% (e.g., from 1% to 4.9%, from 1% to 4.5%, from 1% to 4%, from 1% to 3.5%, from 1% to 3%, from 1% to 2.5%, or from 1% to 2%) of the tumor sample.
  • a tumor sample obtained from the subject has been determined to have a detectable expression level of PD-L1 in about 1% or more (e.g., about 1% or more, 2% or more, 3% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 16% or more, 17% or more,18% or more, 19% or more, 20% or more, 21% or more, 22% or more, 23% or more, 24% or more, 25% or more, 26% or more, 27% or more, 28% or more, 29% or more, 30% or more, 31% or more, 32% or more, 33% or more, 34% or more, 35% or more, 36% or more, 37% or more, 38% or more, 39% or more, 40% or more, 41% or more, 42% or more, 43% or more, 44% or more, 45% or more, 10% or
  • a tumor sample obtained from the subject has been determined to have a detectable expression level of PD-L1 in from about 1% to less than about 5% (e.g., from 1% to 4.9%, from 1% to 4.5%, from 1% to 4%, from 1% to 3.5%, from 1% to 3%, from 1% to 2.5%, or from 1% to 2%) of the tumor-infiltrating immune cells in the tumor sample.
  • a tumor sample obtained from the subject has been determined to have a detectable expression level of PD-L1 in tumor-infiltrating immune cells that comprise about 5% or more of the tumor sample.
  • a tumor sample obtained from the subject has been determined to have a detectable expression level of PD-L1 in tumor-infiltrating immune cells that comprise from about 5% to less than about 10% (e.g., from 5% to 9.5%, from 5% to 9%, from 5% to 8.5%, from 5% to 8%, from 5% to 7.5%, from 5% to 7%, from 5% to 6.5%, from 5% to 6%, from 5% to 5.5%, from 6% to 9.5%, from 6% to 9%, from 6% to 8.5%, from 6% to 8%, from 6% to 7.5%, from 6% to 7%, from 6% to 6.5%, from 7% to 9.5%, from 7% to 9%, from 7% to 7.5%, from 8% to 9.5%, from 8% to 9%, or from 8% to 8.5%) of the tumor sample.
  • a detectable expression level of PD-L1 in tumor-infiltrating immune cells that comprise from about 5% to less than about 10% (e.g., from 5%
  • a tumor sample obtained from the subject has been determined to have a detectable expression level of PD-L1 in about 5% or more of the tumor-infiltrating immune cells in the tumor sample.
  • a tumor sample obtained from the subject has been determined to have a detectable expression level of PD-L1 in from about 5% to less than about 10% (e.g., from 5% to 9.5%, from 5% to 9%, from 5% to 8.5%, from 5% to 8%, from 5% to 7.5%, from 5% to 7%, from 5% to 6.5%, from 5% to 6%, from 5% to 5.5%, from 6% to 9.5%, from 6% to 9%, from 6% to 8.5%, from 6% to 8%, from 6% to 7.5%, from 6% to 7%, from 6% to 6.5%, from 7% to 9.5%, from 7% to 9%, from 7% to 7.5%, from 8% to 9.5%, from 8% to 8.5
  • a tumor sample obtained from the subject has been determined to have a detectable expression level of PD-L1 in tumor-infiltrating immune cells that comprise about 10% or more (e.g., 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 16% or more, 17% or more,18% or more, 19% or more, 20% or more, 21% or more, 22% or more, 23% or more, 24% or more, 25% or more, 26% or more, 27% or more, 28% or more, 29% or more, 30% or more, 31% or more, 32% or more, 33% or more, 34% or more, 35% or more, 36% or more, 37% or more, 38% or more, 39% or more, 40% or more, 41% or more, 42% or more, 43% or more, 44% or more, 45% or more, 46% or more, 47% or more, 48% or more, 49% or more, 50% or more, 60% or more, 70%
  • a tumor sample obtained from the subject has been determined to have a detectable expression level of PD-L1 in about 10% or more (e.g., 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 16% or more, 17% or more, 18% or more, 19% or more, 20% or more, 21% or more, 22% or more, 23% or more, 24% or more, 25% or more, 26% or more, 27% or more, 28% or more, 29% or more, 30% or more, 31% or more, 32% or more, 33% or more, 34% or more, 35% or more, 36% or more, 37% or more, 38% or more, 39% or more, 40% or more, 41% or more, 42% or more, 43% or more, 44% or more, 45% or more, 46% or more, 47% or more, 48% or more, 49% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or
  • a tumor sample obtained from the subject has been determined to have a detectable expression level of PD-L1 in about 50% or more (e.g., about 50% or more, 51% or more, 52% or more, 53% or more, 54% or more, 55% or more, 56% or more, 57% or more, 58% or more, 59% or more, 60% or more, 61% or more, 62% or more, 63% or more, 64% or more, 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 9
  • the percentage of the tumor sample comprised by tumor-infiltrating immune cells may be in terms of the percentage of tumor area covered by tumor-infiltrating immune cells in a section of the tumor sample obtained from the subject, for example, as assessed by IHC using an anti-PD-L1 antibody (e.g., the SP142 antibody).
  • an anti-PD-L1 antibody e.g., the SP142 antibody
  • Any suitable anti-PD-L1 antibody may be used, including, e.g., SP142 (Ventana), SP263 (Ventana), 22C3 (Dako), 28-8 (Dako), E1L3N (Cell Signaling Technology), 4059 (ProSci, Inc.), h5H1 (Advanced Cell Diagnostics), and 9A11.
  • the anti-PD-L1 antibody is SP142. In some embodiments, the anti-PD-L1 antibody is SP263. In some embodiments, a tumor sample obtained from the subject has been determined to have a detectable expression level of PD-L1 in about 1% or more (e.g., about 1% or more, 2% or more, 3% or more, 5% or more, 6% or more, 7% or more, 8% or more, 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, 14% or more, 15% or more, 16% or more, 17% or more,18% or more, 19% or more, 20% or more, 21% or more, 22% or more, 23% or more, 24% or more, 25% or more, 26% or more, 27% or more, 28% or more, 29% or more, 30% or more, 31% or more, 32% or more, 33% or more, 34% or more, 35% or more, 36% or more, 37% or more, 38% or
  • a tumor sample obtained from the subject has been determined to have a detectable expression level of PD-L1 in from about 1% to less than about 5% (e.g., from 1% to 4.9%, from 1% to 4.5%, from 1% to 4%, from 1% to 3.5%, from 1% to 3%, from 1% to 2.5%, or from 1% to 2%) of the tumor cells in the tumor sample.
  • a tumor sample obtained from the subject has been determined to have a detectable expression level of PD-L1 in less than about 1% of the tumor cells in the tumor sample.
  • a tumor sample obtained from the subject has been determined to have a detectable expression level of PD-L1 in about 5% or more of the tumor cells in the tumor sample.
  • a tumor sample obtained from the subject has been determined to have a detectable expression level of PD-L1 in from about 5% to less than 50% (e.g., from 5% to 49.5%, from 5% to 45%, from 5% to 40%, from 5% to 35%, from 5% to 30%, from 5% to 25%, from 5% to 20%, from 5% to 15%, from 5% to 10%, from 5% to 9%, from 5% to 8%, from 5% to 7%, from 5% to 6%, from 10% to 49.5%, from 10% to 40%, from 10% to 35%, from 10% to 30%, from 10% to 25%, from 10% to 20%, from 10% to 15%, from 15% to 49.5%, from 15% to 45%, from 15% to 40%, from 15% to 35%, from 15% to 30%, from 15% to 25%
  • a tumor sample obtained from the subject has been determined to have a detectable expression level of PD-L1 in about 50% or more (e.g., about 50% or more, 51% or more, 52% or more, 53% or more, 54% or more, 55% or more, 56% or more, 57% or more, 58% or more, 59% or more, 60% or more, 61% or more, 62% or more, 63% or more, 64% or more, 65% or more, 66% or more, 67% or more, 68% or more, 69% or more, 70% or more, 71% or more, 72% or more, 73% or more, 74% or more, 75% or more, 76% or more, 77% or more, 78% or more, 79% or more, 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 9
  • a tumor sample obtained from the subject has been determined to have a detectable expression level of PD-L1 in from about 50% to about 99% (e.g., from 50% to 99%, from 50% to 95%, from 50% to 90%, from 50% to 85%, from 50% to 80%, from 50% to 75%, from 50% to 70%, from 50% to 65%, from 50% to 60%, from 50% to 55%, from 55% to 99%, from 55% to 95%, from 55% to 90%, from 55% to 85%, from 55% to 80%, from 55% to 75%, from 55% to 70%, from 55% to 65%, from 55% to 60%, from 60% to 99%, from 60% to 95%, from 60% to 90%, from 60% to 85%, from 60% to 80%, from 60% to 75%, from 60% to 70%, from 60% to 65%, from 65% to 99%, from 65% to 95%, from 65% to 90%, from 65% to 85%, from 65% to 80%, from 65% to 75%, from 65% to 70%, from
  • the tumor sample is a formalin-fixed and paraffin-embedded (FFPE) tumor sample, an archival tumor sample, a fresh tumor sample, or a frozen tumor sample.
  • FFPE formalin-fixed and paraffin-embedded
  • the presence and/or expression level of any of the biomarkers described above may be assessed qualitatively and/or quantitatively based on any suitable criterion known in the art, including but not limited to DNA, mRNA, cDNA, proteins, protein fragments, and/or gene copy number.
  • the expression level of a biomarker may be a protein expression level.
  • the method comprises contacting the sample with antibodies that specifically bind to a biomarker described herein under conditions permissive for binding of the biomarker, and detecting whether a complex is formed between the antibodies and biomarker.
  • a biomarker described herein may be an in vitro or in vivo method.
  • an antibody is used to select subjects eligible for treatment with an anti-cancer therapy that includes a PD-1 axis binding antagonist, e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody, e.g., a biomarker for selection of subjects.
  • a PD-1 axis binding antagonist e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody, e.g., a biomarker for selection of subjects.
  • an antibody is used to select subjects eligible for treatment with an anti-cancer therapy that includes a PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody) and an IL6 antagonist (e.g., anti-IL6 receptor antibody such as tocilizumab), e.g., a biomarker for selection of subjects.
  • a PD-1 axis binding antagonist e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody
  • an IL6 antagonist e.g., anti-IL6 receptor antibody such as tocilizumab
  • a protein expression level of a biomarker is determined using a method selected from the group consisting of immunohistochemistry (IHC), flow cytometry (e.g., fluorescence-activated cell sorting (FACSTM)), Western blot, enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, immunofluorescence, radioimmunoassay, dot blotting, immunodetection methods, HPLC, surface plasmon resonance, optical spectroscopy, mass spectrometry, and HPLC.
  • the protein expression level of the biomarker e.g., PD-L1 is determined in tumor-infiltrating immune cells.
  • the protein expression level of the biomarker is determined in tumor cells. In some embodiments, the protein expression level of the biomarker is determined in tumor-infiltrating immune cells and/or in tumor cells. In some embodiments, the protein expression level of the biomarker is determined in peripheral blood mononuclear cells (PBMCs). In certain embodiments, the presence and/or expression level/amount of a biomarker protein (e.g., PD-L1) in a sample is examined using IHC and staining protocols. IHC staining of tissue sections has been shown to be a reliable method of determining or detecting the presence of proteins in a sample.
  • a biomarker protein e.g., PD-L1
  • the biomarker is one or more of the protein expression products of PD-L1.
  • an expression level of biomarker is determined using a method comprising: (a) performing IHC analysis of a sample (such as a tumor sample obtained from a subject) with an antibody; and (b) determining expression level of a biomarker in the sample.
  • IHC staining intensity is determined relative to a reference.
  • the reference is a reference value.
  • the reference is a reference sample (e.g., a control cell line staining sample, a tissue sample from non-cancerous subject, or a tumor sample that is determined to be negative for the biomarker of interest).
  • the protein expression level of PD-L1 is determined using IHC.
  • the protein expression level of PD-L1 is detected using an anti-PD-L1 antibody. Any suitable anti-PD-L1 antibody may be used, including, e.g., SP142, SP263, 22C3, 28-8, E1L3N, 4059, h5H1, and 9A11.
  • the anti-PD-L1 antibody is SP142.
  • the anti-PD-L1 antibody is SP263.
  • IHC may be performed in combination with additional techniques such as morphological staining and/or in situ hybridization (e.g., ISH).
  • additional techniques such as morphological staining and/or in situ hybridization (e.g., ISH).
  • ISH in situ hybridization
  • direct and indirect assays According to the first assay, binding of antibody to the target antigen is determined directly.
  • This direct assay uses a labeled reagent, such as a fluorescent tag or an enzyme-labeled primary antibody, which can be visualized without further antibody interaction.
  • unconjugated primary antibody binds to the antigen and then a labeled secondary antibody binds to the primary antibody.
  • a chromogenic or fluorogenic substrate is added to provide visualization of the antigen.
  • Signal amplification occurs because several secondary antibodies may react with different epitopes on the primary antibody.
  • the primary and/or secondary antibody used for IHC typically will be labeled with a detectable moiety.
  • radioisotopes such as 35 S, 14 C, 125 1, 3 H, and 131 I
  • colloidal gold particles such as a) fluorescent labels including, but are not limited to, rare earth chelates (europium chelates), Texas Red, rhodamine, fluorescein, dansyl, lissamine, umbelliferone, phycocrytherin, phycocyanin, or commercially-available fluorophores such as SPECTRUM ORANGE® and SPECTRUM GREEN® and/or derivatives of any one or more of the above; (d) various enzyme-substrate labels are available and U.S.
  • Patent No.4,275,149 provides a review of some of these.
  • enzymatic labels include luciferases (e.g., firefly luciferase and bacterial luciferase; see, e.g., U.S.
  • Patent No.4,737,456 luciferin, 2,3- dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, ⁇ -galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g., glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like.
  • HRPO horseradish peroxidase
  • alkaline phosphatase alkaline phosphatase
  • ⁇ -galactosidase glucoamylase
  • lysozyme saccharide oxidases
  • enzyme-substrate combinations include, for example, horseradish peroxidase (HRPO) with hydrogen peroxidase as a substrate; alkaline phosphatase (AP) with para-Nitrophenyl phosphate as chromogenic substrate; and ⁇ -D-galactosidase ( ⁇ -D-Gal) with a chromogenic substrate (e.g., p-nitrophenyl- ⁇ -D-galactosidase) or fluorogenic substrate (e.g., 4-methylumbelliferyl- ⁇ - D-galactosidase).
  • HRPO horseradish peroxidase
  • AP alkaline phosphatase
  • ⁇ -D-galactosidase ⁇ -D-Gal
  • a chromogenic substrate e.g., p-nitrophenyl- ⁇ -D-galactosidase
  • fluorogenic substrate e.g., 4-methylumbelliferyl- ⁇ - D-gal
  • Specimens may be prepared, for example, manually, or using an automated staining instrument (e.g., a Ventana BenchMark XT or Benchmark ULTRA instrument). Specimens thus prepared may be mounted and coverslipped. Slide evaluation is then determined, for example, using a microscope, and staining intensity criteria, routinely used in the art, may be employed. In one embodiment, it is to be understood that when cells and/or tissue from a tumor is examined using IHC, staining can be determined or assessed in tumor cell(s) and/or tissue (as opposed to stromal or surrounding tissue that may be present in the sample). In other embodiments, staining can be determined or assessed in stromal or surrounding tissue that may be present in the sample.
  • an automated staining instrument e.g., a Ventana BenchMark XT or Benchmark ULTRA instrument. Specimens thus prepared may be mounted and coverslipped. Slide evaluation is then determined, for example, using a microscope, and staining intensity criteria, routinely used in
  • staining includes determining or assessing in tumor-infiltrating immune cells, including intratumoral or peritumoral immune cells.
  • the presence of a biomarker is detected by IHC in >0% of the sample, in at least 1% of the sample, in at least 5% of the sample, in at least 10% of the sample, in at least 15% of the sample, in at least 15% of the sample, in at least 20% of the sample, in at least 25% of the sample, in at least 30% of the sample, in at least 35% of the sample, in at least 40% of the sample, in at least 45% of the sample, in at least 50% of the sample, in at least 55% of the sample, in at least 60% of the sample, in at least 65% of the sample, in at least 70% of the sample, in at least 75% of the sample, in at least 80% of the sample, in at least 85% of the sample, in at least 90% of the sample, in at least 95%
  • the biomarker is detected by immunohistochemistry using a diagnostic antibody (i.e., primary antibody).
  • the diagnostic antibody specifically binds human antigen.
  • the diagnostic antibody is a non-human antibody.
  • the diagnostic antibody is a rat, mouse, or rabbit antibody.
  • the diagnostic antibody is a rabbit antibody.
  • the diagnostic antibody is a monoclonal antibody.
  • the diagnostic antibody is directly labeled. In other embodiments, the diagnostic antibody is indirectly labeled (e.g., by a secondary antibody).
  • the expression level of a biomarker may be a nucleic acid expression level (e.g., a DNA expression level or an RNA expression level (e.g., an mRNA expression level)). Any suitable method of determining a nucleic acid expression level may be used. In some embodiments, the nucleic acid expression level is determined using RNAseq, RT-qPCR, qPCR, multiplex qPCR or RT-qPCR, microarray analysis, SAGE, MassARRAY technique, ISH, or a combination thereof.
  • Methods for the evaluation of mRNAs in cells include, for example, serial analysis of gene expression (SAGE), whole genome sequencing (WGS), hybridization assays using complementary DNA probes (such as in situ hybridization using labeled riboprobes specific for the one or more genes, Northern blot and related techniques) and various nucleic acid amplification assays (such as RT-PCR (e.g., qRT-PCR) using complementary primers specific for one or more of the genes, and other amplification type detection methods, such as, for example, branched DNA, SISBA, TMA and the like).
  • SAGE serial analysis of gene expression
  • WGS whole genome sequencing
  • hybridization assays using complementary DNA probes such as in situ hybridization using labeled riboprobes specific for the one or more genes, Northern blot and related techniques
  • various nucleic acid amplification assays such as RT-PCR (e.g., qRT-PCR) using complementary primers specific for one or more of the genes
  • such methods can include one or more steps that allow one to determine the levels of target mRNA in a biological sample (e.g., by simultaneously examining the levels a comparative control mRNA sequence of a “housekeeping” gene such as an actin family member).
  • the sequence of the amplified target cDNA can be determined.
  • Optional methods include protocols which examine or detect mRNAs, such as target mRNAs, in a tissue or cell sample by microarray technologies. Using nucleic acid microarrays, test and control mRNA samples from test and control tissue samples are reverse transcribed and labeled to generate cDNA probes. The probes are then hybridized to an array of nucleic acids immobilized on a solid support.
  • the array is configured such that the sequence and position of each member of the array is known. For example, a selection of genes whose expression correlates with increased or reduced clinical benefit of treatment comprising an immunotherapy and a suppressive stromal antagonist may be arrayed on a solid support. Hybridization of a labeled probe with a particular array member indicates that the sample from which the probe was derived expresses that gene.
  • the sample may be obtained from the subject at any suitable time. For example, in some embodiments, the sample is obtained from the subject prior to (e.g., minutes, hours, days, weeks (e.g., 1, 2, 3, 4, 5, 6, or 7 weeks), months, or years prior to) administration of the treatment regimen.
  • the sample from the subject is obtained about 2 to about 10 weeks (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks) following administration of the treatment regimen. In some embodiments, the sample from the subject is obtained about 4 to about 6 weeks following administration of the treatment regimen.
  • the expression level or number of a biomarker is detected in a tissue sample, a primary or cultured cells or cell line, a cell supernatant, a cell lysate, platelets, serum, plasma, vitreous fluid, lymph fluid, synovial fluid, follicular fluid, seminal fluid, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebro-spinal fluid, saliva, sputum, tears, perspiration, mucus, tumor lysates, and tissue culture medium, tissue extracts such as homogenized tissue, tumor tissue, cellular extracts, or any combination thereof.
  • a biomarker e.g., PD-L1
  • the sample is a tissue sample (e.g., a tumor tissue sample), a cell sample, a whole blood sample, a plasma sample, a serum sample, or a combination thereof.
  • the tumor tissue sample wherein the tumor tissue sample includes tumor cells, tumor-infiltrating immune cells, stromal cells, or a combination thereof.
  • the tumor tissue sample is a formalin-fixed and paraffin-embedded (FFPE) sample, an archival sample, a fresh sample, or a frozen sample.
  • FFPE formalin-fixed and paraffin-embedded
  • the expression level of a biomarker is detected in tumor-infiltrating immune cells, tumor cells, PBMCs, or combinations thereof using known techniques (e.g., IHC, immunofluorescence microscopy, or flow cytometry).
  • Tumor-infiltrating immune cells include, but are not limited to, intratumoral immune cells, peritumoral immune cells or any combinations thereof, and other tumor stroma cells (e.g., fibroblasts).
  • Such tumor infiltrating immune cells may be T lymphocytes (such as CD8 + T lymphocytes (e.g., CD8 + T effector (Teff) cells) and/or CD4 + T lymphocytes (e.g., CD4 + Teff cells), B lymphocytes, or other bone marrow-lineage cells including granulocytes (neutrophils, eosinophils, basophils), monocytes, macrophages, dendritic cells (e.g., interdigitating dendritic cells), histiocytes, and natural killer (NK) cells.
  • the staining for a biomarker is detected as membrane staining, cytoplasmic staining, or combinations thereof.
  • the absence of a biomarker is detected as absent or no staining in the sample, relative to a reference sample.
  • the expression level of a biomarker is assessed in a sample that contains or is suspected to contain cancer cells.
  • the sample may be, for example, a tissue biopsy or a metastatic lesion obtained from a subject suffering from, suspected to suffer from, or diagnosed with cancer (e.g., bladder cancer (e.g., UC, including locally advanced or metastatic UC).
  • the sample is a sample of tissue (e.g., renal pelvis, ureter, urinary bladder, and/or urethral tissue), a biopsy of a tumor (e.g., a locally advanced or metastatic UC tumor, including a pelvis, ureter, urinary bladder, and/or urethral tumor), a known or suspected metastatic bladder cancer (e.g., metastatic UC) lesion or section, or a blood sample, e.g., a peripheral blood sample, known or suspected to comprise circulating cancer cells, e.g., bladder cancer cells (e.g., UC cells, including locally advanced or metastatic UC cells).
  • tissue e.g., renal pelvis, ureter, urinary bladder, and/or urethral tissue
  • a biopsy of a tumor e.g., a locally advanced or metastatic UC tumor, including a pelvis, ureter, urinary bladder, and/or urethral tumor
  • a known or suspected metastatic bladder cancer e
  • the sample may comprise both cancer cells, i.e., tumor cells, and non-cancerous cells (e.g., lymphocytes, such as T cells or NK cells), and, in certain embodiments, comprises both cancerous and non-cancerous cells.
  • cancer cells i.e., tumor cells
  • non-cancerous cells e.g., lymphocytes, such as T cells or NK cells
  • Methods of obtaining biological samples including tissue resections, biopsies, and body fluids, e.g., blood samples comprising cancer/tumor cells, are well known in the art.
  • the subject may have an advanced, refractory, recurrent, and/or chemotherapy-resistant form of the cancer.
  • the presence and/or expression levels/amount of a biomarker in a first sample is increased or elevated as compared to presence/absence and/or expression levels/amount in a second sample.
  • the presence/absence and/or expression levels/amount of a biomarker in a first sample is decreased or reduced as compared to presence and/or expression levels/amount in a second sample.
  • the second sample is a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue.
  • a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a single sample or combined multiple samples from the same subject that are obtained at one or more different time points than when the test sample is obtained.
  • a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is obtained at an earlier time point from the same subject than when the test sample is obtained.
  • Such reference sample, reference cell, reference tissue, control sample, control cell, or control tissue may be useful if the reference sample is obtained during initial diagnosis of cancer and the test sample is later obtained when the cancer becomes metastatic.
  • a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a combined multiple samples from one or more healthy individuals who are not the subject.
  • a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is a combined multiple samples from one or more individuals with a disease or disorder (e.g., cancer) who are not the subject.
  • a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is pooled RNA samples from normal tissues or pooled plasma or serum samples from one or more individuals who are not the subject.
  • a reference sample, reference cell, reference tissue, control sample, control cell, or control tissue is pooled RNA samples from tumor tissues or pooled plasma or serum samples from one or more individuals with a disease or disorder (e.g., cancer) who are not the subject.
  • the method further includes administering an effective amount of a treatment regimen described herein (e.g., a treatment regimen comprising a PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody) and/or an IL6 antagonist (e.g. an anti-IL6 receptor antibody (e.g. tocilizumab)) to the subject, for example, based on the expression level of one or more biomarkers (e.g., PD-L1).
  • a treatment regimen described herein e.g., a treatment regimen comprising a PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody) and/or an IL6 antagonist (e.g. an anti-IL6 receptor antibody (e.g. tocilizumab)) to the subject, for example, based on the expression level of one or more biomark
  • compositions and formulations comprising a PD-1 axis binding antagonist and/or an antibody described herein (such as an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody) and, optionally, a pharmaceutically acceptable carrier.
  • an IL6 antagonist e.g., an anti-IL6 receptor antibody such as tocilizumab
  • a pharmaceutically acceptable carrier optionally, a pharmaceutically acceptable carrier.
  • compositions and formulations as described herein can be prepared by mixing the active ingredients (e.g., a PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody) and/or an IL6 antagonist (e.g. an anti-IL6 receptor antibody (e.g. tocilizumab)) having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (see, e.g., Remington’s Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), e.g., in the form of lyophilized formulations or aqueous solutions.
  • active ingredients e.g., a PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody) and/or an IL6 antagonist (e.g. an anti-IL6 receptor antibody (e.g. tocilizumab)
  • 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 (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arg
  • Exemplary pharmaceutically acceptable carriers herein further include insterstitial drug dispersion agents such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.).
  • sHASEGP soluble neutral-active hyaluronidase glycoproteins
  • rHuPH20 HYLENEX®, Baxter International, Inc.
  • Certain exemplary sHASEGPs and methods of use, including rHuPH20 are described in U.S. Patent Publication Nos.2005/0260186 and 2006/0104968.
  • a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.
  • additional glycosaminoglycanases such as chondroitinases.
  • Exemplary lyophilized antibody formulations are described in U.S.
  • 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.
  • the compositions and formulations 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 purpose intended.
  • Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • Sustained-release preparations may be prepared.
  • 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.
  • the formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g., by filtration through sterile filtration membranes. IX.
  • an article of manufacture or a kit comprising a PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody) and/or an IL6 antagonist (e.g., anti-IL6 receptor antibody such as tocilizumab).
  • a PD-1 axis binding antagonist e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody
  • an IL6 antagonist e.g., anti-IL6 receptor antibody such as tocilizumab
  • the article of manufacture or kit further comprises package insert comprising instructions for using the PD-1 axis binding antagonist to treat cancer, e.g. breast or urothelial carcinoma.
  • the article of manufacture or kit further comprises package insert comprising instructions for using the PD-1 axis binding antagonist in combination with an IL6 antagonist (e.g.
  • an anti-IL6 receptor antibody e.g. tocilizumab
  • an anti-IL6 receptor antibody e.g. tocilizumab
  • the PD-1 axis binding antagonist e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody
  • the IL6 antagonist e.g., anti-IL6 receptor antibody such as tocilizumab
  • Suitable containers include, for example, bottles, vials, bags 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).
  • 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 further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.
  • the article of manufacture further includes one or more of another agent (e.g., an additional chemotherapeutic agent or anti- neoplastic agent).
  • Suitable containers for the one or more agent include, for example, bottles, vials, bags and syringes.
  • a kit for treating breast cancer (e.g. TNBC) or urothelial carcinoma or renal cell carcinoma in a subject in need thereof comprising a PD-1 axis binding antagonist (e.g., an anti-PD-L1 antibody (e.g., atezolizumab) or an anti-PD-1 antibody) and/or an IL6 antagonist (e.g. an anti-IL6 receptor antibody (e.g.
  • Example 1 Correlations between biomarkers of systemic inflammation and outcome in patients with metastatic triple-negative breast cancer (mTNBC) treated with atezolizumab monotherapy
  • IL-6 and IL-8 myeloid inflammation is linked to poor prognosis in cancer patients treated with chemotherapy, but its association with single agent atezolizumab-treated patients with mTNBC remains unknown.
  • BM biomarkers
  • TNBC has the worst outcomes.
  • chemotherapy has been the typical treatment for metastatic or advanced disease.
  • Estimates of median OS for mTNBC vary but are generally around 18 months or less.
  • Pre-existing tumor immune biology has been associated with clinical activity in mTNBC in patients treated with PD-L1/PD-1–targeting agents, including the anti–PD-L1 checkpoint inhibitor atezolizumab.
  • Single-agent anti–PD-L1/PD-1 mAbs are active in mTNBC but to a lower extent vs ORRs with standard-of-care chemotherapy.
  • Atezolizumab + nab-paclitaxel was the first CIT combination demonstrating clinical benefit (PFS/OS) in the 1L setting in patients with TNBC expressing PD-L1 on IC leading to FDA accelerated approval in this setting.
  • Proof-of-concept combination of atezolizumab + (nab)paclitaxel + ipatasertib shows confirmed ORR of 73% (19/26 patients) in biomarker un-selected 1L mTNBC.
  • a deep dive into the biology of atezolizumab monotherapy–treated patients with TNBC may lead to new CIT combination options in this difficult-to-treat patient population.
  • IL-6, IL-8 and CRP were tested by Luminex assays and their levels were assessed for association with baseline clinical demographic characteristics and atezolizumab clinical activity for response rate (ORR), progression free (PFS) and overall survival (OS).
  • ORR response rate
  • PFS progression free
  • OS overall survival
  • IL-6, IL-8, and CRP levels were positively associated with the clinical prognostic traits ECOG performance status (>0), presence of liver metastases, large size of target lesions ( ⁇ 6.5cm), and increased LDH ( ⁇ 1.5xULN) (Fig.25). Elevated baseline IL-6 and CRP, but not IL-8, were linked with later lines of therapy ( ⁇ 2) (Fig.26). Univariate analyses showed that IL-6 ( ⁇ 15pg/ml), IL-8 ( ⁇ 11.4pg/ml) and CRP ( - 3mg/L) were associated with reduced OS and PFS, but only CRP was associated to reduced ORR (Fig. .
  • IL-6 (HR 2.00 [1.16-3.38]); and CRP (HR 2.74 [1.49-5.28]), but not IL-8 (HR 1.07 [0.78-1.74]), were associated with OS (Fig.30).
  • the IL-6/CRP inflammatory axis is an independent factor linked with poor outcomes of mTNBC patients treated with atezolizumab monotherapy and may play unique roles in affecting the anti- tumor activities.
  • the Objective of this exploratory, signal seeking analysis based on data from a phase I monotherapy study was to identify new potential drug targets that might be relevant for future drug combinations in CIT.
  • TNBC tumor samples for this analysis were collected from PCD4989g (NCT01375842), a single-arm Phase I study that evaluated the clinical activity of atezolizumab in patients with locally advanced or metastatic malignancies, including TNBC.
  • Bladder cancer tumor samples were collected in IMvigor210, a single-arm Phase 2 study investigating atezolizumab in mUC patients (NCT02951767, NCT02108652) and in Phase 3 mUC trial IMvigor211 (NCT02302807) in which patients were treated with either chemotherapy (taxane or vinflunine) or atezolizumab as a second-line or higher treatment. Tumor tissues were taken from all patients two years prior to study entry. RCC samples were collected from IMmotion150 (NCT01984242), a phase II multicenter, randomized, open-label study investigating activity of atezolizumab and atezolizumab+bevacizumab versus sunitinib in metastatic clear cell renal carcinoma.
  • Plasma IL-6 assay EDTA plasma samples were collected from patients before (PCD4989g, IMmotion150, IMvigor210, IMvigor211) and on cycle 3 day 1 after treatment (IMvigor211) and stored at - 80°C. Plasma IL-6 was evaluated by previously qualified immunoassays on a novel multi-analyte platform Simple Plex Ella (Gupta et al. Bioanalysis 8: 2415-2428 (2016)). The samples were diluted twofold in sample diluent and loaded onto the cartridge for data acquisition.
  • RNAseq gene expression profiling Whole-transcriptome profiles were generated using TruSeq RNA Access technology (Illumina).
  • PBMC collection and isolation PBMCs from patients were isolated using 50 ml Leucosep TM tubes (Greiner Bio-One International, Germany) and Ficoll-Paque TM PLUS (GE Healthcare, Sweden).
  • PBMC scRNAseq library preparation Frozen PBMC samples from mUC patients containing at least 1 million cells were thawed for 1 minute at 37 ⁇ C and washed twice with RPMI complete media (10% FBS with glutamate and Pen/Strep). Samples with >50% red blood cells were treated with RBC Lysis buffer for 3 minutes in room temperature to remove red blood cells and then washed one more time with RPMI complete media. The cell density and viability of the single-cell suspension were then determined by Vi- CELL XR cell counter (Beckman Coulter, Pasadena, CA). All of the samples had >80% viable cells.
  • RNA-seq Analysis of mUC PBMC: Seurat (Butler et al.
  • Immunophenotyping of PBMCs was inferred from the annotation of cluster-specific genes; Total T cells (CD3D, CD3E), CD8 + T cells (CD3E, CD8A), B cells (CD79A), CD14 monocytes (CD14), and NK cells (NKG7-positive and CD3E-negative).
  • Differential gene expression analysis for IL-6-high versus IL-6-low cell subsets used raw counts of the samples and was performed by edgeR in R (version 2.13.0,) using the generalized linear model workflow described in the edgeR manual.
  • edgeR version 2.13.0,
  • IL6 in situ hybridization For the detection of IL6 mRNA expression in RCC tumors, in situ hybridization was performed on 4um thick formalin-fixed, paraffin-embedded tissue sections mounted on glass slides.
  • the process was automated on the Leica BOND Rx platform (Buffalo Grove, IL).
  • a 20 base- pair probe to the target region of IL6 (2-1082) was used (Advanced Cell Diagnostics, Inc., Newark, CA).
  • Tissue sections were pre-treated with heat and protease before hybridization with oligonucleotide probes. Detection and amplification was performed with the RNAscope 2.5 LSx Reagent Kit in Red (Advanced Cell Diagnostics, Inc., Newark, CA). Tumor sections were analyzed by a qualified histopathologist and considered IL6 positive if at least 1% of either tumor cell area or stromal area showed IL6 stain.
  • Time-to-event outcomes were estimated using the Kaplan- Meier method, which was used to estimate the probability of overall survival (OS) and median overall survival time, and Kaplan-Meier curves.
  • OS overall survival
  • Kaplan-Meier curves The OS was compared by the log-rank test.
  • For OS analysis data for patients who were alive were censored at the time of the last contact.
  • the hazard ratios and 95% confidence intervals for OS were estimated by a Cox regression model. Cox proportional hazards and linear regression model was performed to conduct univariate and multivariate analysis.
  • Figures and tables were generated using the following packages and versions in R: RColorBrewer, 1.1-2; ggplot2, 3.1.1; gridExtra, 2.3; ComplexHeatmap, 2.0.0 ; superheat, 1.0.0 ; colorspace, 1.3-2; dplyr, 0.7.8; and data for external datasets were obtained using GenomicDataCommons, 1.4.3 ; GEOquery, 2.48.0 .
  • R packages depended secondarily on the following support packages: Matrix, 1.2-17; Biobase, 2.40.0; BiocGenerics, 0.26.0; cowplot, 0.9.3; DDRTree, 0.1.5; edgeR, 2.13.0; irlba, 2.3.2; limma, 3.38.2; magrittr, 1.5; Matrix, 1.2- 15; ranger, 0.10.1; and VGAM, 1.0-6.
  • the EMT6 murine mammary carcinoma cell line was obtained from American Type Culture Collection (ATCC; Manassas, VA), then screened and stored by common cell repository at Genentech.
  • RNA-seq analysis Cell lines are routinely screened and EMT6 cells used in this study were negative for mycoplasma and authenticated by RNA-seq analysis.
  • Cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium plus 2 mM L-glutamine with 10% fetal bovine serum (FBS; Hyclone, Waltham, MA).
  • FBS fetal bovine serum
  • FBS Hyclone, Waltham, MA
  • HBSS Hank’s balanced salt solution
  • BD Biosciences San Jose, CA
  • mice Female BALB/c mice (8–10 weeks old) were obtained from Charles River Laboratories (Hollister, CA) and housed at Genentech in standard rodent microisolator cages. Mice were acclimated for at least 3 days before cell injection. Animals used in this study appeared to be healthy and free of obvious abnormalities. Mice were inoculated in the left #5 mammary fat pad with 1x10 5 EMT6 cells in 100 ⁇ l of HBSS/Matrigel mixture. When tumors reached a volume of 130–230 mm 3 (approximately 8 days after inoculation), animals were distributed into treatment groups such that variance in tumor sizes between treatment groups was minimized.
  • mice were treated with isotype control antibodies, anti-PD-L1 (mouse IgG1 clone 6E11, 10 mg/kg first dose followed by 5 mg/kg thereafter), anti-IL6R (mouse IgG2a clone MR16-1, 15 mg/kg), or a combination of anti-PD-L1 and anti-IL6R.
  • Anti-PD-L1, anti-IL6R, and isotype control antibodies are produced in-house and free of endotoxin contamination. Mice were euthanized after 10–12 days (after 3 doses of treatment) and tumors collected for flow cytometry analysis or IHC.
  • tumors were administered 2 times per week for 3 weeks (intravenously for the first dose and intraperitoneally thereafter). Tumors were measured 2 times per week using digital calipers, and tumor volumes calculated using the modified ellipsoid formula, 1/2 x (length x width 2 ). When tumor volumes fell below 32 mm 3 (lower limit of detection) they were considered a complete response (CR; 100% tumor growth inhibition). Tumors that initially regressed but eventually recurred were considered partial responders (PR), and tumors that never regressed were considered to be progressive disease (PD). For time-to-progression analysis, the disease progression endpoint was defined as a 5x increase in tumor volume compared to the volume at the time of treatment initiation.
  • PR partial responders
  • PD progressive disease
  • mice were euthanized if tumor volumes exceeded 2000 mm 3 or if tumor ulceration occurred. No mice met criteria for euthanasia due to body weight loss or adverse clinical signs.
  • the CT26 murine colon carcinoma cell line was obtained from American Type Culture Collection (ATCC; Manassas, VA), then screened, cultured, tested, and stored as described above. CT26 cells used in this study were free of mycoplasma.
  • Female BALB/c mice were obtained and housed as described above. Mice were inoculated subcutaneously in the right flank with 1x10 5 CT26 cells in 100 ⁇ l of HBSS/Matrigel mixture.
  • mice When tumors reached a volume of 130–230 mm 3 (approximately 8 days after inoculation), animals were distributed into treatment groups such that variance in tumor sizes between treatment groups was minimized. Mice were treated with antibodies as described for the EMT6 model, euthanized after 10–12 days (after 3 doses of treatment), and tumors collected for flow cytometry analysis. Sample size in the mouse studies is based on the number of mice routinely needed to establish statistical significance based on variability within study arms. Treatment arms were not blinded. All animal studies herein were approved by the Genentech Institutional Animal Care and Use Committee.
  • ⁇ cell stimulation media composed as follows: RPMI 1640 medium with 10% FBS (Hyclone, Waltham, MA), 100 U/ml penicillin / 100 ⁇ g/ml streptomycin (Gibco, Thermo Fisher Scientific, Waltham, MA), 55 ⁇ M ⁇ -mercaptoethanol (Gibco, Thermo Fisher Scientific, Waltham, MA), 2 mM L-glutamine (Gibco, Thermo Fisher Scientific, Waltham, MA), 1 mM sodium pyruvate (Gibco, Thermo Fisher Scientific, Waltham, MA), 0.1 mM non-essential amino acids (Gibco, Thermo Fisher Scientific, Waltham, MA), 10 mM HEPES (Gibco, Thermo Fisher Scientific, Waltham, MA), and 1x Cell Stimulation Cocktail with protein transport inhibitors (containing phorbol
  • cells were first incubated with anti-CD16/CD32 Fc block (5 ⁇ g/ml; BD Biosciences, San Jose, CA; clone 2.4G2) and LIVE/DEAD Fixable dead cell stain (APC-efluor780; Invitrogen, Carlsbad, CA) in PBS for 20 minutes at 4–8 °C.
  • anti-CD16/CD32 Fc block 5 ⁇ g/ml; BD Biosciences, San Jose, CA; clone 2.4G2
  • LIVE/DEAD Fixable dead cell stain APC-efluor780; Invitrogen, Carlsbad, CA
  • CD45-BV510 (2 ⁇ g/ml; BD Biosciences, San Jose, CA; clone 30-F11
  • Thy1.2-efluor450 (2 ⁇ g/ml; eBioscience, Thermo Fisher Scientific, Waltham, MA; clone 53-2.1), Thy1.2-alexafluor700 (5 ⁇ g/ml; BioLegend, San Diego, CA; clone 53-2.1), Thy1.1-alexafluor488 (2.5 ⁇ g/ml; BioLegend, San Diego, CA; clone OX-7), CD4-BUV395 (2 ⁇ g/ml; BD Biosciences, San Jose, CA, clone GK1.5), CD8a-BB515 (2 ⁇ g/ml; BD Biosciences, San Jose, CA, clone 53-6.7), CD8a-PE (2 ⁇ g/ml; BioLegend, San Diego, CA;
  • Flow cytometry data were collected with a BD LSRFortessa or BD FACSymphony analyzer (BD Biosciences, San Jose, CA) and analyzed using FlowJo software (Version 10.5, FlowJo LLC, Ashland, OR).
  • FlowJo software Version 10.5, FlowJo LLC, Ashland, OR.
  • In vivo T cell priming C57BL/6J.OT-I.Thy1.1 TCR transgenic mice were bred and housed at Genentech under specific pathogen free (SPF) conditions. Wild type C57BL/6J mice were obtained from the Jackson Laboratory (Sacramento, CA).
  • Na ⁇ ve OT-I T cells were isolated from spleens and lymph nodes of C57BL/6J.OT-I.Thy1.1 mice by first mashing through 70 ⁇ m pore filters using the sterile blunt end of a plunger from a 1 ml syringe. Na ⁇ ve CD8+ T cells were then isolated using the EasySep Mouse Na ⁇ ve CD8+ T cell Isolation Kit (STEMCELL Technologies, Cambridge, MA). Cells were resuspended at 1x10 7 cells/ml in sterile HBSS and 1x10 6 cells (0.1 ml) were injected intravenously via the lateral tail vein into wild type C57BL/6J recipient mice.
  • mice were then treated with isotype control antibodies, anti-PD-L1 (mouse IgG1 clone 6E11, 10 mg/kg first dose followed by 5 mg/kg thereafter), anti-IL6R (mouse IgG2a clone MR16-1, 15 mg/kg), or a combination of anti-PD-L1 and anti-IL6R via intraperitoneal injection.
  • mice were injected intravenously with a mixture of 50 ⁇ g/kg DEC-OVA (ovalbumin fused to anti-DEC205 antibody; produced in-house) and 2.5 mg/kg anti-CD40 antibody (produced in-house; clone FGK4.5).
  • DEC-OVA ovalbumin fused to anti-DEC205 antibody; produced in-house
  • anti-CD40 antibody produced in-house; clone FGK4.5
  • mice were given a second intraperitoneal dose of anti-PD-L1, anti-IL6R, or isotype control antibodies after 3 days.
  • mice were sacrificed, splenocytes were isolated as described above, viable cells were counted using a Vi- CELL XR (Beckman Coulter, Brea, CA), and cells were stimulated for 3 hours with 1x Cell Stimulation Cocktail with protein transport inhibitors (containing phorbol 12-myristate 13-acetate (PMA), ionomycin, brefeldin A, and monensin; eBioscience, Thermo Fisher Scientific, Waltham, MA), as described in the preceding section. Cells were then prepared for flow cytometry analysis as described above.
  • PMA phorbol 12-myristate 13-acetate
  • monensin eBioscience, Thermo Fisher Scientific, Waltham, MA
  • mice were bred and housed at Genentech under specific pathogen free (SPF) conditions. Wild type C57BL/6J mice were obtained from the Jackson Laboratory (Sacramento, CA). Mouse spleens and/or lymph nodes were isolated and mashed through 70 ⁇ m pore filters using the sterile blunt end of a plunger from a 1 ml syringe.
  • splenocytes were seeded in Falcon flat bottom 96 well plates (Corning Life Sciences, Corning, NY) in T cell media as described above (minus cell stimulation cocktail). Cells were pulsed with 100 ng/ml SIINFEKL peptide (AnaSpec, Fremont, CA). After 2 days, cells were analyzed or transitioned to T cell media (without SIINFEKL) containing 10 ng/ml recombinant human IL-2 and incubated for a further 3 days before use in cytotoxicity assays or re-stimulation with anti-CD3 and anti-CD28 antibodies and flow cytometry analysis.
  • SIINFEKL 10 ng/ml recombinant human IL-2
  • Anti-CD28 antibody was added to culture medium at a concentration of 2.5 ⁇ g/ml (BD Biosciences, San Jose, CA, clone 37.51). Unless specified otherwise, recombinant human IL-2 (R&D Systems, Minneapolis, MN) was added to cultures at 10 ng/ml to promote T cell viability and expansion. To facilitate tracking of cell division, cells were labelled in some experiments with Cell Trace Violet-421 (Molecular Probes, Thermo Fisher Scientific, Waltham, MA) according to manufacturer instructions prior to plating.
  • T cells were activated in the presence of recombinant mouse IL-6 (10 ng/ml; R&D Systems, Minneapolis, MN), mouse hyper-IL-6 (20 ng/ml; R&D Systems, Minneapolis, MN), mouse IL-15/IL-15R ⁇ complex (10 ng/ml; eBioscience, Thermo Fisher Scientific, Waltham, MA), isotype control mouse IgG2a antibody (5 ⁇ g/ml), or mouse IgG2a anti-IL6R antibody (5 ⁇ g/ml; clone MR16-1).
  • Fc blocking reagent and viability dye APC-efluor780
  • Fc-efluor780 Fc blocking reagent and viability dye
  • Cells were then surface stained for 20 minutes at 4–8 °C with the following antibodies: CD8a-BB515 (2 ⁇ g/ml; BD Biosciences, San Jose, CA, clone 53-6.7) and CD4-BUV395 (2 ⁇ g/ml; BD Biosciences, San Jose, CA, clone GK1.5).
  • Flow cytometry data were collected with a BD LSRFortessa or BD FACSymphony analyzer (BD Biosciences, San Jose, CA) and analyzed using FlowJo software (Version 10.5, FlowJo LLC, Ashland, OR).
  • T cell cytotoxicity assays OT-I CD8 + T cells were activated with SIINFEKL peptide (AnaSpec, Fremont, CA) as described above in the presence or absence of recombinant mouse IL-6 (10 ng/ml) or recombinant mouse hyper-IL-6 (20 ng/ml). Cells were used in cytotoxicity assays after 5–6 days.
  • MC38-GFP or MC38-GFP-OVA cells (engineered to express ovalbumin) were plated in Falcon flat-bottom 96-well plates (Corning Life Sciences, Corning, NY) at 5,000 cells per well.
  • Parental MC38 cells were originally acquired from ATCC (Manassas, VA). Cells were characterized and maintained as described for EMT6 cells, and were free of mycoplasma contamination.
  • MC38-GFP cells were then pulsed with 10 ng/ml SIINFEKL peptide for 1 hour at 37 °C, washed with PBS, and activated T cells added in complete T cell medium at various ratios (0:1, 1:1, 5:1, 10:1, or 20:1).
  • T cells were added directly without additional SIINFEKL peptide to test killing in the setting of endogenous antigen presentation.
  • MC38 cell killing was quantified over time using IncuCyte Live Cell Analysis (Essen Bioscience, Ann Arbor, MI). Data were collected from the phase contrast and GFP channels using the 10x objective. GFP+ area (which is directly proportional to the number of viable MC38-GFP cells) was quantified every hour and normalized to matched timepoints of MC38 cells cultured in the absence of T cells.
  • OT-I CD8 + T cells were activated with SIINFEKL peptide as described above (see Analysis of T cell activation ex vivo).
  • Experimental treatment conditions were as follows: (1) control (no treatment); (2) recombinant mouse IL-6, 10 ng/ml; (3) recombinant mouse hyper-IL-6, 20 ng/ml; (4) mouse IgG2a isotype control antibody, 5 ⁇ g/ml; and (5) mouse IgG2a anti-IL6R antibody (clone MR16-1), 5 ⁇ g/ml.
  • Viable CD8 + T cells were sorted to >99% purity on day 7 using a BD FACS Aria Fusion cell sorter (BD Biosciences, San Jose, CA). Cells were then lysed in RLT buffer and RNA was extracted using the RNEasy mini kit (Qiagen, Germantown, MD). Quality control of total RNA was done to determine sample quantity and quality. The concentration of RNA samples was determined using NanoDrop 8000 (Thermo Fisher Scientific, Waltham, MA) and the integrity of RNA was determined by Fragment Analyzer (Agilent Technologies, Santa Clara, CA). 0.1 ⁇ g of total RNA was used as an input material for library preparation using TruSeq Stranded Total RNA Library Prep Kit (Illumina, San Diego, CA).
  • RNA-sequencing data were analyzed using HTSeqGenie (Reeder & Pau, G. HTSeqGenie: a NGS analysis pipeline. R package version 3.14.0 (2012) in BioConductor (Huber et al. Nat.
  • nRPKM size factor
  • PCA Principal components analysis
  • Example 3 Plasma IL-6 is associated with poor clinical outcome to atezolizumab (anti-PD- L1) and reduced CD8 + T cell activation in cancer patients
  • mTNBC metastatic triple negative breast cancer
  • mRCC metastatic renal cell carcinoma
  • mUC metastatic urothelial bladder carcinoma
  • PCD4989g was a single-arm Phase I study that evaluated atezolizumab in patients with locally advanced or metastatic malignancies, including mTNBC (Emens et al. JAMA Oncol 5: 74–82 (2019)).
  • IMvigor210 was a single-arm Phase II study of atezolizumab in mUC (Rosenberg et al. Atezolizumab in patients with locally advanced and metastatic urothelial carcinoma who have progressed following treatment with platinum-based chemotherapy: a single-arm, multicentre, phase 2 trial. Lancet 387: 1909–1920 (2016); Balar et al. Lancet 389: 67–76 (2017)).
  • IMvigor211 was a randomized Phase III mUC trial in which patients with prior platinum-based chemotherapy were treated with either chemotherapy (taxanes or vinflunine) or atezolizumab (Powles et al. Lancet 391: 748–757 (2016)).
  • IMmotion150 was a randomized Phase II trial that investigated atezolizumab with or without bevacizumab versus the anti-angiogenic tyrosine kinase inhibitor sunitinib in patients with untreated mRCC (McDermott et al. Nature Med 24: 749–757 (2016)). Demographic characteristics of biomarker-evaluable patients with high or low levels of plasma IL-6 are presented in Figs.20-23. Multivariate analyses (co-variates defined in figure legends and Methods) were conducted to identify associations with clinical outcomes, reported here as adjusted hazard ratios (HR).
  • HR adjusted hazard ratios
  • IL-6 levels were significantly higher in patients with mTNBC, mRCC, or mUC (Fig.1a) and correlated closely with plasma CRP, an IL-6-inducible biomarker of systemic inflammation (Fig. 1b).
  • high IL-6 concentration as ⁇ 10 pg/ml ( ⁇ 4 standard deviations above the mean concentration in healthy adults; see Example 1 and Figs.6a-b).
  • PCD4989g mTNBC
  • OS overall survival
  • mRCC IMmotion150
  • high plasma IL-6 was also associated with poor OS, but this did not reach statistical significance after adjustment for baseline prognostic factors (Fig.1e).
  • IMvigor210 In IMvigor210 (mUC), high plasma IL-6 was associated with poor OS (HR: 2.16, 95% CI: 1.59, 2.93; P ⁇ 0.0001) and reduced objective response rates (Fig.1f, Fig.7).
  • mUC In the randomized IMvigor211 trial (mUC), patients with high plasma IL-6 had significantly worse OS in both the atezolizumab (HR: 2.42, 95% CI: 1.92, 3.07; P ⁇ 0.001) and chemotherapy arms (HR: 1.89, 95% CI: 1.50, 2.38, P ⁇ 0.001) (Fig. 1g), indicating that high plasma IL-6 is associated with worse prognosis in mUC.
  • atezolizumab improved OS in patients whose plasma IL-6 did not increase during therapy (low ratio) (HR: 0.59, 95% CI: 0.44, 0.79; P ⁇ 0.001) as opposed to patients whose plasma IL-6 increased during therapy (Fig. 1h).
  • This result shows that an on-treatment reduction in plasma IL-6 may be associated with anti-PD-L1-mediated improvement in survival.
  • scRNAseq single cell RNA sequencing
  • PBMCs peripheral blood mononuclear cells
  • UMAP Uniform Manifold Approximation and Projection
  • T cell GES intratumoral CD8 + T cell gene expression signature
  • IL-6 and hyper-IL-6 a fusion protein of IL-6 and IL6R that elicits trans signaling through direct engagement of gp130
  • IL-6 did so for Il6r ⁇ / ⁇ cells
  • Treatment of isolated CD8 + T cells with IL-6 had little effect on cell proliferation following anti-CD3/CD28 stimulation (Figs. 10a–b), but significantly diminished cytokine expression, indicating selective regulation of effector function (Fig. 3c).
  • cytokines IL-2 and IL-15 are well known to promote optimal expansion and effector differentiation of CD8 + T cells
  • IL-6 inhibited effector function with or without the addition of IL-2 or IL-15/IL-15R ⁇ complex (Figs. 10c–d).
  • IL-6-driven suppression of cytokine production was strictly dependent on the transcription factor STAT3 (signal transducer and activator of transcription 3; Fig. 3d).
  • STAT3 signal transducer and activator of transcription 3; Fig. 3d
  • splenocytes from OT-I T cell receptor (TCR)-transgenic mice with SIINFEKL peptide (a high-affinity ovalbumin epitope recognized by the OT-I TCR) and assessed their functional properties after one week.
  • Exposure to IL-6 or hyper-IL-6 during activation caused a 5–10-fold reduction in polyfunctionality, defined as co-expression of IFN- ⁇ , TNF, and GzmB (Fig. 3e).
  • IL-6-conditioned OT-I cells failed to efficiently kill SIINFEKL- pulsed or ovalbumin-expressing target cells, indicating impaired cytotoxicity (Fig.3f and Fig.12a-b).
  • IL-6 and hyper-IL-6 drove a similar gene expression profile that was highly distinct from cells activated in the presence of IL6R blocking antibody, while control conditions (basic culture conditions or isotype control antibody) induced an intermediate phenotype (Fig. 3g, Fig. 13a, Fig. 23).
  • Inhibition of IL-6 signaling promoted high expression of cytotoxic factors, cytokines, chemokines, and transcription factors that are critical for effector differentiation (e.g. Tbx21 and Eomes).
  • IL-6 In contrast, exogenous IL-6 promoted expression of factors that oppose T cell activation and effector differentiation (e.g. Ctla4, Foxo1, Bach2, Batf) and genes associated with na ⁇ ve or central memory cells, including Ccr7 and Sell (Fig. 3h and Fig. 13b).
  • Ctla4, Foxo1, Bach2, Batf factors that oppose T cell activation and effector differentiation
  • genes associated with na ⁇ ve or central memory cells including Ccr7 and Sell
  • Ccr7 and Fig. 13b Gene ontology (GO) analysis confirmed that IL-6 blockade promoted cytotoxic effector polarization, whereas IL-6 treatment promoted repression of cytokine production (Fig.13c and Fig.24).
  • IL-6 potently suppressed the acquisition of an Eomes + Tbet + CD62L ⁇ effector phenotype, while expression of the stem cell memory marker TCF1 was largely unaffected (Fig.3i–j).
  • Example 5 Combined blockade of IL6R and PD-L1 enhances CD8 + T cell activation and promotes tumor control Although PD1/PD-L1 signaling had little effect on CD8 + T cell priming in our in vitro culture conditions (Fig.14a), this axis is known to regulate T cell priming and differentiation in vivo (Goldberg et al. Blood 110:186–192 (2007); Ahn et al. Proc. Natl. Acad. Sci. U.S.A.115: 4749–4754 (2018).
  • naive CD8 + OT-I T cells Thy1.1/CD90.1
  • wild type mice Thy1.2/CD90.2
  • DEC-OVA ovalbumin conjugated to anti- DEC205 antibody
  • Fig.4a agonistic anti-CD40 antibody
  • TIL tumor-infiltrating leukocytes
  • combination treatment promoted a polyfunctional cytotoxic phenotype (GzmB + IFN- ⁇ + TNF + ) in CD8 + T cells (Fig.4h–i), consistent with the DEC-OVA immunization model.
  • GzmB + IFN- ⁇ + TNF + CD8 + T cells
  • Fig.4h–i CD8 + T cells
  • Fig.16b CD4 + T-helper cells
  • Example 6 Discussion of Examples 2 to 5 The data presented here indicate that IL-6 can potentially drive resistance to a PD-1 axis binding antagonist. In this comprehensive evaluation of large clinical studies, it is shows that plasma and intratumoral IL-6 are associated with worse outcome to atezolizumab monotherapy in mTNBC, mUC and mRCC, even in patients whose tumors harboured pre-existing CD8 + T cells. This effect was independent of clinical prognostic factors. Moreover, increases in plasma IL-6 concentration during therapy correlated with worse clinical outcome to atezolizumab, but not to chemotherapy.
  • baseline and on-treatment levels of IL-6 and its target gene CRP may be valuable biomarkers of clinical resistance to a PD-1 axis binding antagonist that can be assessed routinely in clinical laboratories.
  • the mechanisms by which IL-6 impairs anti-PD-L1 efficacy are likely diverse. For example, previous preclinical studies reported that IL-6 inhibits anti-tumor Th1 responses by CD4 + T cells (Tsukamonto (2016), supra; Tsukamoto et al. Cancer Res 77: 2279–2291 (2017)). These data from multiple preclinical models indicate that IL-6 can additionally attenuate the effector function of CD8 + T cells.
  • scRNA-seq analysis of PBMCs from cancer patients indicated reduced CD8+ T cell activation in the presence of elevated plasma IL-6.
  • These data contrast with the well-established pro- inflammatory role of IL-6 in diseases characterized by hyperactive Th17 responses, such as rheumatoid arthritis, emphasizing the context-dependent nature of immune regulation by IL-6 (Hunter & Jones Nat Immunol 16: 448–457 (2015); Schaper & Rose-John Cytokine and Growth Factor Reviews 26: 475–487 (2015)).
  • IL-6 Although expression of IL-6 by multiple cell types in mRCC tumors was observed, IL-6 produced outside of the tumor bed may also potentially influence CD8 + T cell function and anti-tumor responses.
  • lymph node fibroblastic reticular cells were recently shown to regulate CD8 + T cell metabolism and survival via production of IL-6 (Brown et al. Nat Immunol 20: 1668–1680 (2019)).
  • recent analyses of T cell clonality in tumors and peripheral blood have shown that expanded clonotypes found in the tumor are also present in peripheral blood (Wu, T. et al. Peripheral T cell expansion predicts tumor infiltration and clinical response. Nature, In press (2019)), and that in check point inhibitor-treated tumors, T cell expansion in response to therapy may be driven by clones that are newly recruited to the tumor bed (Yost et al. Nature Med 25: 1251–1259 (2019)).
  • circulating IL6 may also contribute to reduced activation potential of intratumoral T cells recruited from the periphery.
  • the findings herein show that IL-6 is an additional factor that limits the potency of anti-tumor CD8 + T cell responses through selective inhibition of effector function (Fig.19).
  • IL6R blockade only affected CD8 + T cell responses in vivo in the context of anti-PD-L1 treatment, the PD-1/PD-L1 axis is likely dominant over IL-6 signaling.
  • Combined blockade of PD-1/PD-L1 and IL-6 signaling thus permits both efficient TCR/CD28 signaling and effector polarization, promoting effective anti-tumor responses (Fig.19).
  • the precise molecular mechanism by which IL-6/STAT3 signaling restricts effector function remains to be defined.
  • the STAT3-driven inhibitory effect of IL-6 on CD8 + T cell effector function makes it an attractive therapeutic target for combination with PD-1 axis binding antagonists, and represents a distinct mechanism of action compared to other factors that restrict PD-1 axix binding antagonist efficacy through indirect means, such as suppression of intratumoral T cell infiltration by TGF ⁇ and VEGF, and recruitment of inhibitory myeloid cells by VEGF, IL-1 ⁇ , and IL-8.
  • the combination of IL-6 blockade and PD-1 axis blockade warrants further clinical investigation in cancer patients, with potential for improved therapeutic efficacy in diverse forms of cancer characterized by elevated IL-6 and/or CRP.
  • Example 7 Effect of IL-6 conditioning on CD8+ T cell effector function
  • Bulk splenocytes or spleen-derived CD8 + T cells from wild type C57BL/6J mice were cultured in base RPMI 1640 medium with 10% fetal bovine serum (control), or in medium supplemented with 10 ng/ml recombinant mouse IL-6 or 20 ng/ml recombinant mouse hyper-IL-6 (IL-6/IL-6R fusion protein). After 24 hours (the “pretreatment” period), cells were centrifuged, medium was discarded, and cells were cultured with anti-CD3 and anti-CD28 antibodies in the presence of 10 ng/ml human IL-2 for 3 days to activate T cells (the “activation” period).
  • IL-6 or hyper-IL-6 was added to cultures as indicated. Control conditions correspond to cells cultured without IL-6 or hyper-IL-6 for the entire experiment.
  • CD8 + T cells were evaluated for cytokine expression by flow cytometry after incubation for 4 hours with brefeldin A. T he results are shown in Figs. 35 a-c.
  • Fig. 35a Representative flow cytometry plots of IFN- ⁇ and TNF expression in CD8 + T cells from bulk splenocytes.
  • IFN- ⁇ mean fluorescence intensity and frequency of IFN- ⁇ /TNF co-expression in CD8 + T cells from bulk splenocytes.
  • IFN- ⁇ mean fluorescence intensity and frequency of IFN- ⁇ /TNF co-expression in CD8 + T cells cultured in isolation.
  • IL-6 can potentially act on resting T cells as well as on stimulated T cells. This supports administering anti-IL6 receptor therapy prior to anti- PD-L1, to provide sufficient time to relieve IL-6-mediated repression of resting cells prior to treatment with anti-PD-L1 antibodies.
  • Example 8 Tocilizumab Combined with Atezolizumab for Urothelial Carcinoma (UC) This is a Phase Ib/II, open-label, multicenter, randomized, umbrella study in patients with locally advanced or metastatic UC who have progressed during or following a platinum-containing regimen.
  • Atezolizumab is a humanized immunoglobulin G1 (IgG1) monoclonal antibody that targets programmed death-ligand 1 (PD-L1) and inhibits the interaction between PD-L1 and its receptors, programmed death-1 (PD-1) and B7-1 (also known as CD80), both of which function as inhibitory receptors expressed on T cells.
  • Therapeutic blockade of PD-L1 binding by atezolizumab has been shown to enhance the magnitude and quality of tumor-specific T-cell responses, resulting in improved anti-tumor activity.
  • Atezolizumab has minimal binding to Fc receptors, thus eliminating detectable Fc-effector function and associated antibody-mediated clearance of activated effector T cells.
  • Atezolizumab shows anti-tumor activity in both nonclinical models and cancer patients and is being investigated as a potential therapy in a wide variety of malignancies. Atezolizumab is being studied as a single agent in the advanced cancer and adjuvant therapy settings, as well as in combination with chemotherapy, targeted therapy, and cancer immunotherapy. Atezolizumab has been generally well tolerated. Adverse events with potentially immune-mediated causes consistent with an immunotherapeutic agent, including rash, influenza-like illness, endocrinopathies, hepatitis or transaminitis, pneumonitis, colitis, hypophysitis, myocarditis, and myasthenia gravis, have been observed. To date, these events have been manageable with treatment.
  • Tocilizumab (TCZ) is a recombinant humanized, anti-human monoclonal antibody of the IgG1 subclass directed against the soluble and membrane-bound interleukin 6 receptor (IL-6R). Tocilizumab binds specifically to both soluble IL-6R (sIL-6R) and membrane-bound IL-6R (mIL-6R) and has been shown to inhibit sIL-6R and mIL-6R ⁇ mediated signaling.
  • Interleukin 6 (IL-6) is a pleiotropic pro- inflammatory, multifunctional, cytokine produced by a variety of cell types.
  • T-cell activation has been shown to be involved in such diverse physiological processes as T-cell activation; induction of acute phase proteins; stimulation of hematopoietic precursor cell growth and differentiation; proliferation of hepatic, dermal and neural cells; bone metabolism; lipid metabolism; hepatoprotection; and fibrosis.
  • Elevated tissue and serum levels of IL-6 have been implicated in the disease pathology of several inflammatory and autoimmune disorders, including rheumatoid arthritis, Castleman’s disease, systemic juvenile idiopathic arthritis, polyarticular juvenile idiopathic arthritis, giant cell arteritis, Takayasu arteritis, systemic sclerosis, and cytokine release syndrome.
  • Atezolizumab is administered at a fixed dose of 840 mg every 2 weeks (Q2W) (840 mg on Days 1 and 15 of each 28-day cycle).
  • Q2W 840 mg on Days 1 and 15 of each 28-day cycle
  • the average concentration following the 840 mg Q2W dosage is expected to be equivalent to that of 1200 mg every 3 weeks (Q3W), the approved dosage for atezolizumab.
  • the atezolizumab drug product will be supplied by the Sponsor as a sterile liquid in a single-use, 20-mL glass vial.
  • the vial contains approximately 20 mL (1200 mg) of atezolizumab solution.
  • Atezolizumab injection for intravenous use is a sterile, preservative-free, colorless to slightly yellow solution in single-dose vials.
  • Each 20 mL vial contains 1200 mg of atezolizumab and is formulated in glacial acetic acid (16.5 mg), L- histidine (62 mg), polysorbate 20 (8 mg), and sucrose (821.6 mg), with a pH of 5.8.
  • Tocilizumab will be administered by iv infusion at a dose of 8 mg/kg every 4 weeks (Q4W) on Day 1 of each 28-day cycle, the approved dose for tocilizumab for the treatment of RA.
  • Tocilizumab will be supplied by the Sponsor as a sterile solution at a concentration of 20 mg/mL in single-use vials containing 4.0, 10.0, or 20.0 mL.
  • Tocilizumab injection is a sterile, clear, colorless to pale yellow, preservative-free solution for further dilution prior to intravenous infusion with a pH of approximately 6.5.
  • Each single-dose vial, formulated with a disodium phosphate dodecahydrate/sodium dihydrogen phosphate dihydrate buffered solution is available at a concentration of 20 mg/mL containing 80 mg/4 mL, 200 mg/10 mL, or 400 mg/20 mL of Tocilizumab.
  • Each mL of solution contains polysorbate 80 (0.5 mg), sucrose (50 mg), and Water for Injection, USP.
  • Patients in the Atezo ⁇ TCZ arm will receive treatment as outlined in the following table until unacceptable toxicity or loss of clinical benefit.
  • Tocilizumab will be administered first. Atezolizumab will be administered 2 hours after the conclusion of the tocilizumab infusion. Treatment Regimen for Atezo ⁇ TCZ Arm Atezo ⁇ TCZ ⁇ atezolizumab plus tocilizumab. Tocilizumab will be administered by IV infusion at a dose of 8 mg/kg Q4W on Day 1 of each 28-day cycle. Each patient will receive 8 mg/kg tocilizumab (or 4 mg/kg in certain circumstances), with a maximum dose of 800 mg tocilizumab (for patients weighing ⁇ 100 kg). The last recorded body weight of a patient should be used for calculating tocilizumab volumes for each infusion. The dose administered should be within 10% of the calculated dose.
  • Atezolizumab will be administered by IV infusion at a fixed dose of 840 mg on Days 1 and 15 of each 28-day cycle. Atezolizumab should be administered 2 hours after the completion of the tocilizumab infusion.
  • Inclusion Criteria ⁇ Histologically documented, locally advanced (T4b, any N; or any T, N2 ⁇ N3) or metastatic UC (M1, Stage IV) (also termed TCC or urothelial cell carcinoma of the urinary tract; including renal pelvis, ureters, urinary bladder, and urethra) Patients with mixed histologies are required to have a dominant transitional cell pattern.
  • Locally advanced bladder cancer must be inoperable on the basis of involvement of pelvic sidewall or adjacent viscera (clinical Stage T4b) or bulky nodal metastasis (N2 ⁇ N3).
  • ⁇ Disease progression during or following treatment with no more than one platinum-containing regimen e.g., GC, MVAC, CarboGem
  • a regimen is defined as patients receiving at least two cycles of a platinum-containing regimen. Patients who received prior adjuvant/neoadjuvant chemotherapy and progressed within 12 months of treatment with a platinum-containing adjuvant/neoadjuvant regimen will be considered as second-line patients.
  • Patients may have received no more than two prior regimens of treatment (including the required platinum-based regimen) for their advanced or metastatic UC. Patients must have demonstrated disease progression during or following all prior regimen(s). Patients with disease progression following chemoradiotherapy must demonstrate progression outside the prior radiotherapy port. Fifteen patients have been randomly assigned to a control arm (atezolizumab [Atezo]) or Atezo in combination with tocilizumab (TCZ). The objectives and corresponding endpoints of the study are summarized in the table below.
  • Example 9 Tocilizumab Combined with Atezolizumab for metastatic Triple Negative Breast Cancer (mTNBC) This study will evaluate the efficacy, safety, and pharmacokinetics of Atezolizumab in combination with Tocilizumab in patients with metastatic triple-negative breast cancer (TNBC). Nab-Paclitaxel chemotherapy will also be administered. Patients are PD-L1 positive. Patients will receive treatment as outlined in the table below. The Atezo and TCZ formulations are as described in Example 8. Treatment Regimen for Atezo + Nab-Pac + TCZ a Atezolizumab should be administered 2 hours after completion of the tocilizumab infusion on Day 1 of the first two cycles.
  • TNBC metastatic Triple Negative Breast Cancer
  • Atezolizumab can be administered after completion of the tocilizumab infusion.
  • b Nab-paclitaxel will be administered after completion of the atezolizumab infusion.
  • HER2 negativity is defined as either of the following by local laboratory assessment: In situ hybridization non-amplified (ratio of HER2 to CEP17 ⁇ 2.0 or single-probe average HER2 gene copy number ⁇ 4 signals/cell) or Immunohistochemistry (IHC) 0 or IHC 1+; and ER and PR negativity are defined as ⁇ 1% of cells expressing hormonal receptors via IHC analysis - For patients in the 1L PD-L1+ cohort: no prior systemic treatment for metastatic or
  • nab-paclitaxel may be reduced by 25 mg/m 2 (one dose level) up to two times and the dose of tocilizumab may be reduced by 4 mg/m 2 (one dose level) up to one time, as outlined in the following table.

Abstract

Cette invention concerne des procédés et des compositions destinés à être utilisés dans le traitement du cancer, y compris le cancer du sein (tel qu'un cancer du sein métastatique triple négatif, mTNBC), le carcinome urothélial et le carcinome des cellules rénales, avec la combinaison d'un antagoniste de liaison de l'axe PD-1 (par ex., un anticorps de liaison PD-L1 tel que l'atézolizumab) et un antagoniste de L'IL 6 (par ex., un anticorps de récepteur anti-IL6 tel que le tocilizumab). Eventuellement, le patient a une protéine C réactive (CRP) et/ou un ou des niveaux d'IL-6 au-dessus de la limite supérieure de la normale. Eventuellement, le cancer est PD-L1-positif.
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