METHODS FOR IDENTIFYING PATIENTS RESPONSIVE TO ANTI-PD-L1 ANTIBODY THERAPY

Abstract

The disclosure provides methods for treating lung cancer (e.g., non-small cell lung cancer) with an anti-PD-L1 antibody in a patient identified using a polynucleotide or polypeptide marker of the disclosure: CXCL9, KRT8, TRIM29, and/or IFNgamma.

Claims

1. A method of treatment comprising administering an anti-PD-L1 antibody, or an antigen binding fragment thereof, to a patient identified as having a non-small cell lung cancer or a head and neck tumor that expresses one or more markers selected from the group consisting of CXCL9, KRT8, TRIM29, and IFNgamma.

2. The method of claim 1, wherein the anti-PD-L1 antibody is MEDI4736.

3. A method of treatment comprising administering MEDI4736 or an antigen binding fragment thereof to a patient identified as having a non-small cell lung cancer or a head and neck tumor that expresses one or more markers selected from the group consisting of CXCL9, KRT8, TRIM29, and IFNgamma.

4. The method of any of claims 1-3, wherein the patient is identified as responsive to MEDI4736.

5. The method of any of claims 1-4, wherein the patient is further identified as having a tumor expressing PD-L1.

6. The method of any of claims 1-5, wherein the tumor expresses CXCL9 and IFNgamma.

7. The method of any of claims 1-5, wherein the tumor expresses CXCL9 and KRT8.

8. The method of any of claims 1-5, wherein the tumor expresses CXCL9 and TRIM29.

9. The method of any of claims 1-5, wherein the tumor expresses CXCL9, KRT8, TRIM29, and IFNgamma.

10. The method of any of claims 1-5, wherein the tumor expresses CXCL9, KRT8, TRIM29, IFNgamma, and PD-L1.

11. The method of any of claims 1-10, wherein marker gene expression is detected in a Real-Time PCR assay.

12. The method of any of claims 1-10, wherein marker polypeptide expression is detected using immunohistochemistry.

13. The method of claim 12, wherein the tumor cells are formalin fixed and paraffin embedded.

14. The method of any of claims 1-10, wherein the non-small cell lung cancer is selected from the group consisting of squamous cell carcinoma, non-squamous cell carcinoma, adenocarcinoma, large cell carcinoma, adenosquamous carcinoma and sarcomatoid carcinoma.

15. The method of any of claims 1-10, wherein at least about 0.1, about 0.3, about 1, about 3, about 10, or about 15 mg/kg MEDI4736, or an antigen-binding fragment thereof, is administered

16. The method of claim 15, wherein about 1 mg/kg MEDI4736, or an antigen-binding fragment thereof, is administered.

17. The method of claim 16, wherein about 3 mg/kg MEDI4736, or an antigen-binding fragment thereof, is administered.

18. The method of claim 17, wherein about 10 mg/kg MEDI4736 or an antigen-binding fragment thereof is administered.

19. The method of claim 18, wherein about 15 mg/kg MEDI4736, or an antigen-binding fragment, thereof is administered.

20. The method of any of claims 1-10, wherein the administration is repeated about every 14 or 21 days.

21. The method of any of claims 1-10, wherein at least two doses is administered.

22. The method of any of claims 1-10, wherein at least three doses is administered.

23. The method of any of claims 1-10, wherein at least five doses is administered.

24. A method of identifying a subject having non-small cell lung cancer or head and neck cancer responsive to anti-PD-L1 therapy, the method comprising detecting an increase in the level of one or more markers selected from the group consisting of CXCL9, KRT8, TRIM29, and IFNgamma in a non-small cell lung cancer or head and neck tumor of the subject, relative to a reference, thereby identifying said non-small cell lung cancer or head and neck cancer as responsive to anti-PD-L1 therapy.

25. The method of claim 24, further comprising detecting PD-L1 expression in the tumor.

26. The method of any one of claims 1-24, wherein the markers are detected by a method selected from the group consisting of real-time PCR, DNA microarray, immunostaining, ELISA, FACS, radioimmunoassay, immunoblot, Western blot, immunofluorescence, and immunoprecipitation.

27. A set of primers and/or probes for characterizing non-small cell lung cancer (NSCLC) or head and neck cancer wherein the primers and/or probes hybridize to two or more polynucleotide markers selected from the group consisting of CXCL9, KRT8, TRIM29, and IFNgamma.

28. A set of primers and/or probes for characterizing non-small cell lung cancer (NSCLC) wherein the primers and/or probes hybridize to three or more polynucleotide markers selected from the group consisting of CXCL9, KRT8, TRIM29, and IFNgamma.

29. The set of primers and/or probes of claim 27 or 28, comprising markers CXCL9, KRT8, TRIM29, and IFNgamma.

30. A kit comprising the primers and/or probes of any one of claim 27 or 28.

31. The kit of claim 30, further comprising a reagent to measure the level of PD-L1.

32. A method of increasing the efficacy of anti-PD-L1 therapeutic antibody treatment in a lung cancer or head and neck cancer patient comprising administering anti-PD-L1 therapeutic antibody to a lung cancer patient or a head and neck cancer patient identified as expressing a marker selected from CXCL9, KRT8, TRIM29, and IFNgamma.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0049] FIG. 1 shows PD-L1 membrane expression in micrographs at baseline in non-small cell lung cancer (NSCLC) patients treated with anti-PD-L1 antibody (MEDI4736). The micrograph at left shows negligible PD-L1 staining. This patient did not respond to anti-PD-L1 antibody therapy and continued to show progressive disease (PD). In fact, there was a 43% tumor increase at 6 weeks (PD). The micrograph at right shows strong PD-L1 staining. This patient showed a partial response (PR) to anti-PD-L1 antibody therapy, displaying a 90% tumor decrease at 6 weeks (uPR).

[0050] FIG. 2 is a scatter plot showing a correlation between percent tumor size change from baseline and IHC M-score of PD-L1 defined as the percentage tumor cells with PD-L1 staining in NSCLC patients from CP1108 trial. Clinical response status was measured as best overall response (BOR) indicated by colors at (left) 6 weeks and (right) 12 weeks post dosing with anti-PD-L1. An immunohistochemistry membrane (IHC M) score greater than or equal to 25 is considered positive for PD-L1. In general, patients with an IHC M score greater than or equal to 25 responded to anti-PD-L1. Abbreviations: SD=stable disease; PR=partial responder; PD=progressive disease; ND=no clinical response status assigned.

[0051] FIG. 3 shows a heat map depicting the expression profiling of a panel of 125 candidate genes in NSCLC patients treated for 6 or 12 weeks with anti-PD-L1 antibody. Heatmap shows the scaled expression intensities across all NSCLC patient samples collected to date in CP1108 clinical trial. Red indicates high gene expression and blue indicates low gene expression. Black indicates no measurement taken for that patient/gene.

[0052] FIG. 4 is a scatter plot showing a correlation between percent tumor size change from baseline and baseline gene expression transformed to a final gene expression score. The relative gene expression (RGE) is defined as (20-Δcycle threshold, so that this score is positive overall and a high score represents high expression in the log2 scale. RGE values are provided for CXCL9 in NSCLC patients from the trial. Clinical response status was measured as best overall response (BOR) indicated by colors at (left) 6 weeks and (right) 12 weeks post dosing with anti-PD-L1. SD=stable disease; PR=partial responder; PD=progressive disease; ND=no clinical response status assigned.

[0053] FIG. 5 is a scatter plot showing a correlation between percent tumor size change from baseline and baseline gene expression transformed RGE values for KRT8 in NSCLC patients from trial. Clinical response status was measured as best overall response (BOR) indicated by colors at (left) 6 weeks and (right) 12 weeks post dosing with anti-PD-L1. SD=stable disease; PR=partial responder; PD=progressive disease; ND=no clinical response status assigned.

[0054] FIG. 6 is a scatter plot showing a correlation between percent tumor size change from baseline and baseline gene expression transformed RGE values for TRIM29 in NSCLC patients from trial. Clinical response status measured as best overall response (BOR) indicated by colors at (left) 6 weeks and (right) 12 weeks post dosing with anti-PD-L1. SD=stable disease; PR=partial responder; PD=progressive disease; ND=no clinical response status assigned.

[0055] FIG. 7 is a scatter plot showing a correlation between percent tumor size change from baseline and baseline gene expression transformed RGE values for IFNγ in NSCLC patients from the trial. Clinical response status measured as best overall response (BOR) indicated by colors at (left) 6 weeks and (right) 12 weeks post dosing with anti-PD-L1. SD=stable disease; PR=partial responder; PD=progressive disease; ND=no clinical response status assigned.

[0056] FIG. 8 is a scatter plot showing a correlation between baseline gene expression transformed RGE values for CXCL9 and TRIM29 in NSCLC patients from the trial. Clinical response status measured as best overall response (BOR) indicated by colors at (left) 6 weeks and (right) 12 weeks post dosing with anti-PD-L1. Red box indicates a group of PRs. SD=stable disease; PR=partial responder; PD=progressive disease; ND=no clinical response status assigned.

[0057] FIG. 9 is a scatter plot showing a correlation between percentage change in tumor size from baseline following treatment with MEDI4736 versus CXCL9 and IFNy mRNA in NSCLC squamous cell patient tumors. PR=partial responder; PD=progressive disease; SD=stable disease; NE=not evaluable.

[0058] FIG. 10 is a scatter plot between CXCL9 mRNA versus IFNγ mRNA in NSCLC squamous cell patient tumors. PR=partial responder; PD=progressive disease; SD=stable disease; NE=not evaluable.

[0059] FIG. 11 is a set of boxplots plots of CXCL9 mRNA within each response category in NSCLC squamous cell patient tumors. PR vs. PD p=0.06; PR vs. SD p=0.41; PR=partial responder; PD=progressive disease; SD=stable disease; NE=not evaluable.

[0060] FIG. 12 is a set of boxplots plots of IFNγ mRNA within each response category in NSCLC squamous cell patient tumors. PR vs. PD p=0.01; PR vs. SD p=0.43; PR=partial responder; PD=progressive disease; SD=stable disease; NE=not evaluable.

[0061] FIG. 13 is a scatter plot between percentage change in tumor size from baseline versus IFNγ mRNA in H&N squamous cell patient tumors. PR=partial responder; PD=progressive disease; SD=stable disease; NE=not evaluable.

[0062] FIG. 14 is a scatter plot between percentage change in tumor size from baseline versus CXCL9 mRNA in H&N squamous cell patient tumors. PR=partial responder; PD=progressive disease; SD=stable disease; NE=not evaluable.

[0063] FIG. 15 is a set of boxplots plots of IFNγ mRNA within each response category in H&N squamous cell patient tumors. PR vs. PD p=0.01; PR vs. SD p=0.43; PR=partial responder; PD=progressive disease; SD=stable disease; NE=not evaluable.

[0064] FIG. 16 is a set of boxplots plots of CXCL9 mRNA within each response category in H&N squamous cell patient tumors. PR vs. PD p=0.01; PR vs. SD p=0.43; PR=partial responder; PD=progressive disease; SD=stable disease; NE=not evaluable.

LIST OF SEQUENCES

[0065] MEDI4736 light chain variable region amino acid sequence: SEQ ID NO:1

[0066] MEDI4736 heavy chain variable region amino acid sequence: SEQ ID NO:2.

[0067] MEDI4736 heavy chain variable region amino acid sequence of CDR1, CDR2, and CDR3: SEQ ID NOs:3-5.

[0068] MEDI4736 light chain variable region amino acid sequence of CDR1, CDR2, and CDR3: SEQ ID NOs:6-8.

DETAILED DESCRIPTION OF THE INVENTION

[0069] The present invention provides methods for treating lung cancer (e.g., non-small cell lung cancer) with an anti-PD-L1 antibody in a patient identified using a polynucleotide or polypeptide marker of the invention (e.g., CXCL9, KRT8, TRIM29, and/or IFNgamma) In other embodiments, the present invention provides methods for treating lung cancer in a patient identified using any one or more of markers CXCL9, KRT8, TRIM29, and/or IFNgamma in combination with marker PD-L1.

[0070] The invention is based, at least in part, on the discovery that levels of CXCL9, KRT8, TRIM29, IFNgamma, and/or PD-L1 are differentially expressed in tumors (i.e., increased in a tumor sample) obtained from a subject suffering from lung cancer or head and neck cancer relative to a reference, and that this increased expression can be used to identify patients responsive to treatment with an anti-PD-L1 antibody. Accordingly, the invention provides methods for identifying subjects that have lung cancer that are likely to respond to anti-PD-L1 antibody treatment based on the level of CXCL9, KRT8, TRIM29, and/or IFNgamma, optionally in combination with marker PD-L1 expression, in a subject tumor sample.

B7-H1/PD-L1

[0071] B7-H1, also known as PD-L1, is a type I transmembrane protein of approximately 53kDa in size. In humans B7-H1 is expressed on a number of immune cell types including activated and anergic/exhausted T cells, on naive and activated B cells, as well as on myeloid dendritic cells (DC), monocytes and mast cells. It is also expressed on non-immune cells including islets of the pancreas, Kupffer cells of the liver, vascular endothelium and selected epithelia, for example airway epithelia and renal tubule epithelia, where its expression is enhanced during inflammatory episodes. B7-H1 expression is also found at increased levels on a number of tumours including, but not limited to breast, colon, colorectal, lung, renal, including renal cell carcinoma, gastric, bladder, non-small cell lung cancer (NSCLC), hepatocellular cancer (HCC), and pancreatic cancer, as well as melanoma.

[0072] B7-H1 is known to bind two alternative ligands, the first of these, PD-1, is a 50-55 kDa type I transmembrane receptor that was originally identified in a T cell line undergoing activation-induced apoptosis. PD-1 is expressed on activated T cells, B cells, and monocytes, as well as other cells of the immune system and binds both B7-H1 (PD-L1) and the related B7-DC (PD-L2). The second is the B7 family member B7-1, which is expressed on activated T cells, B cells, monocytes and antigen presenting cells.

[0073] Signaling via the PD-1/B7-H1 axis is believed to serve important, non-redundant functions within the immune system, by negatively regulating T cell responses. B7-H1 expression on tumor cells is believed to aid tumors in evading detection and elimination by the immune system. B7-H1 functions in this respect via several alternative mechanisms including driving exhaustion and anergy of tumour infiltrating T lymphocytes, stimulating secretion of immune repressive cytokines into the tumour micro-environment, stimulating repressive regulatory T cell function and protecting B7-H1 expressing tumor cells from lysis by tumor cell specific cytotoxic T cells.

Anti-PD-L1 Antibodies

[0074] Antibodies that specifically bind and inhibit PD-L1 activity (e.g., binding to PD-1 and/or CD80) are useful for the treatment of lung cancer (e.g., non-small cell lung cancer

[0075] MEDI4736 is an exemplary anti-PD-L1 antibody that is selective for B7-H1 and blocks the binding of B7-H1 to the PD-1 and CD80 receptors. MEDI4736 can relieve B7-H1-mediated suppression of human T-cell activation in vitro and inhibits tumor growth in a xenograft model via a T-cell dependent mechanism. Other agents that could be used include agents that inhibit PD-L1 and/or PD-1 (AB or other).

[0076] Information regarding MEDI4736 (or fragments thereof) for use in the methods provided herein can be found in International Application Publication No. WO 2011/066389 A1, the disclosure of which is incorporated herein by reference in its entirety. The fragment crystallizable (Fc) domain of MEDI4736 contains a triple mutation in the constant domain of the IgG1 heavy chain that reduces binding to the complement component C1q and the Fcγ receptors responsible for mediating antibody-dependent cell-mediated cytotoxicity (ADCC).

[0077] MEDI4736 and antigen-binding fragments thereof for use in the methods provided herein comprises a heavy chain and a light chain or a heavy chain variable region and a light chain variable region. In a specific aspect, MEDI4736 or an antigen-binding fragment thereof for use in the methods provided herein comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO:1 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:2. In a specific aspect, MEDI4736 or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the Kabat-defined CDR1, CDR2, and CDR3 sequences of SEQ ID NOs:3-5, and wherein the light chain variable region comprises the Kabat-defined CDR1, CDR2, and CDR3 sequences of SEQ ID NOs:6-8. Those of ordinary skill in the art would easily be able to identify Chothia-defined, Abm-defined or other CDR definitions known to those of ordinary skill in the art. In a specific aspect, MEDI4736 or an antigen-binding fragment thereof for use in the methods provided herein comprises the variable heavy chain and variable light chain CDR sequences of the 2.14H9OPT antibody as disclosed in U.S. Pat. No. 8,779,108; U.S. Patent Application Publication No. US2013034559; and in WO 2011/066389 A1, which are herein incorporated by reference in their entirety.

Characterizing Responsiveness to Anti-PD-L1 Antibody Therapy

[0078] In characterizing the responsiveness of lung cancer in a subject to anti-PD-L1 antibody treatment, the level of CXCL9, KRT8, TRIM29, and/or IFNgamma expression, optionally in combination with PD-L1, is measured in different types of biologic samples (e.g., tumor sample).

[0079] CXCL9, KRT8, TRIM29, IFNgamma, and/or PD-L1 polynucleotide or polypeptide expression is higher in a tumor sample obtained from a subject that is responsive to anti-PD-L1 antibody treatment than the level of expression in a non-responsive subject (e.g., a subject with progressive disease). In one embodiment, an alteration in expression is calculated using cycle threshold (Ct) values. For example, the Ct value of a gene of interest (e.g., CXCL9, KRT8, TRIM29, IFNgamma, and/or PD-L1) is obtained and from that value the Ct value of a reference gene (e.g., B2M, ACTB, GAPDH) is subtracted from the mean Ct value for each gene to obtain a Delta-Ct value. The final gene expression score is defined as (20-ΔCt). In evaluating increased polypeptide expression (e.g., PD-L1, CXCL9, KRT8, TRIM29, IFNgamma), an immunohistochemical (IHC) score or IHC-membrane (IHC-M) score may be used. In other embodiments, expression of a marker of the invention is increased by at least about 2, 3, 4, 5 or 10-fold in a responsive patient relative to the level in a non-responsive subject (e.g., a subject with progressive disease). CXCL9, KRT8, TRIM29, IFNgamma, and/or PD-L1 polynucleotide or polypeptide fold change values are determined using any method known in the art, including but not limited to quantitative PCR, RT-PCR, Northern blotting, Western blotting, flow cytometry, immunocytochemistry, binding to magnetic and/or antibody-coated beads, in situ hybridization, fluorescence in situ hybridization (FISH), flow chamber adhesion assay, ELISA, microarray analysis, colorimetric assays, mass spectrometry (e.g., laser desorption/ionization mass spectrometry), fluorescence (e.g. sandwich immunoassay), surface plasmon resonance, ellipsometry, and atomic force microscopy.

[0080] In particular embodiments, the responsiveness of lung cancer in a subject to anti-PD-L1 antibody treatment, is assayed using one of the following combinations of polynucleotide markers: CXCL9 and KRT8; CXCL9 and TRIM29; CXCL9 and IFNgamma; KRT8 and TRIM29; KRT8 and IFNgamma; TRIM29 and IFNgamma; CXCL9, KRT8, and TRIM29; KRT8, TRIM29, and IFNgamma; CXCL9, KRT8, TRIM29, and IFNgamma. PD-L1 may be added to any of the preceding groups of markers (e.g., CXCL9, KRT8, TRIM29, IFNgamma and PD-L1).

Selection of a Treatment Method

[0081] Subjects suffering from lung cancer (e.g., non-small cell lung cancer) or head and neck cancer may be tested for CXCL9, KRT8, TRIM29, IFNgamma, and/or PD-L1 polynucleotide or polypeptide expression in the course of selecting a treatment method. Patients characterized as having high expression (e.g., as defined by Ct or IHC-M score) or increased expression relative to a reference level are identified as responsive to anti-PD-L1 treatment.

Treatment with an Anti-PD-L1 Antibody

[0082] Patients identified as having tumors that express CXCL9, KRT8, TRIM29, and/or IFNgamma, particularly at high levels, are likely to be responsive to anti-PD-L1 antibody therapy. Such patients are administered an anti-PD-L1 antibody, such as MEDI4736, or an antigen-binding fragment thereof. MEDI4736 or an antigen-binding fragment thereof can be administered only once or infrequently while still providing benefit to the patient. In further aspects the patient is administered additional follow-on doses. Follow-on doses can be administered at various time intervals depending on the patient's age, weight, clinical assessment, tumor burden, and/or other factors, including the judgment of the attending physician.

[0083] In some embodiments, at least two doses of MEDI4736 or an antigen-binding fragment thereof are administered to the patient. In some embodiments, at least three doses, at least four doses, at least five doses, at least six doses, at least seven doses, at least eight doses, at least nine doses, at least ten doses, or at least fifteen doses or more can be administered to the patient. In some embodiments, MEDI4736 or an antigen-binding fragment thereof is administered over a two-week treatment period, over a four-week treatment period, over a six-week treatment period, over an eight-week treatment period, over a twelve-week treatment period, over a twenty-four-week treatment period, or over a one-year or more treatment period. In some embodiments, MEDI4736 or an antigen-binding fragment thereof is administered over a three-week treatment period, a six-week treatment period, over a nine-week treatment period, over a twelve-week treatment period, over a twenty-four-week treatment period, or over a one-year or more treatment period. In some embodiments, MEDI4736 or an antigen-binding fragment thereof is administered over a two-month treatment period, over a four-month treatment period, or over a six-month or more treatment period (e.g., during a maintenance phase).

[0084] The amount of MEDI4736 or an antigen-binding fragment thereof to be administered to the patient will depend on various parameters, such as the patient's age, weight, clinical assessment, tumor burden and/or other factors, including the judgment of the attending physician.

[0085] In certain aspects the patient is administered one or more doses of MEDI4736 or an antigen-binding fragment thereof wherein the dose is about 0.1 mg/kg. In certain aspects the patient is administered one or more doses of MEDI4736 or an antigen-binding fragment thereof wherein the dose is about 0.3 mg/kg. In certain aspects the patient is administered one or more doses of MEDI4736 or an antigen-binding fragment thereof wherein the dose is about 1 mg/kg.

[0086] In certain aspects the patient is administered one or more doses of MEDI4736 or an antigen-binding fragment thereof wherein the dose is about 3 mg/kg. In certain aspects the patient is administered one or more doses of MEDI4736 or an antigen-binding fragment thereof wherein the dose is about 10 mg/kg. In certain aspects the patient is administered one or more doses of MEDI4736 or an antigen-binding fragment thereof wherein the dose is about 15 mg/kg.

[0087] In certain aspects the patient is administered at least two doses of MEDI4736 or an antigen-binding fragment thereof wherein the dose is about 0.1 mg/kg. In certain aspects the patient is administered at least two doses of MEDI4736 or an antigen-binding fragment thereof wherein the dose is about 0.3 mg/kg. In certain aspects the patient is administered at least two doses of MEDI4736 or an antigen-binding fragment thereof wherein the dose is about 1 mg/kg. In certain aspects the patient is administered at least two doses of MEDI4736 or an antigen-binding fragment thereof wherein the dose is about 3 mg/kg. In certain aspects the patient is administered at least two doses of MEDI4736 or an antigen-binding fragment thereof wherein the dose is about 10 mg/kg. In certain aspects the patient is administered at least two doses of MEDI4736 or an antigen-binding fragment thereof wherein the dose is about 15 mg/kg. In some embodiments, the at least two doses are administered about two weeks apart. In some embodiments, the at least two doses are administered about three weeks apart.

[0088] In certain aspects the patient is administered at least three doses of MEDI4736 or an antigen-binding fragment thereof wherein the dose is about 0.1 mg/kg. In certain aspects the patient is administered at least three doses of MEDI4736 or an antigen-binding fragment thereof wherein the dose is about 0.3 mg/kg. In certain aspects the patient is administered at least three doses of MEDI4736 or an antigen-binding fragment thereof wherein the dose is about 1 mg/kg. In certain aspects the patient is administered at least three doses of MEDI4736 or an antigen-binding fragment thereof wherein the dose is about 3 mg/kg. In certain aspects the patient is administered at least three doses of MEDI4736 or an antigen-binding fragment thereof wherein the dose is about 10 mg/kg. In certain aspects the patient is administered at least three doses of MEDI4736 or an antigen-binding fragment thereof wherein the dose is about 15 mg/kg. In some embodiments, the at least three doses are administered about two weeks apart. In some embodiment, the at least three doses are administered about three weeks apart.

[0089] In certain aspects, administration of MEDI4736 or an antigen-binding fragment thereof according to the methods provided herein is through parenteral administration. For example, MEDI4736 or an antigen-binding fragment thereof can be administered by intravenous infusion or by subcutaneous injection. In some embodiments, the administration is by intravenous infusion.

[0090] In certain aspects, MEDI4736 or an antigen-binding fragment thereof is administered according to the methods provided herein in combination or in conjunction with additional cancer therapies. Such therapies include, without limitation, chemotherapeutic agents such as Vemurafenib, Erlotinib, Afatinib, Cetuximab, Carboplatin, Bevacizumab, Erlotinib, or Pemetrexed, or other chemotherapeutic agents, as well radiation or any other anti-cancer treatments.

[0091] The methods provided herein can decrease tumor size, retard tumor growth or maintain a steady state. In certain aspects the reduction in tumor size can be significant based on appropriate statistical analyses. A reduction in tumor size can be measured by comparison to the size of patient's tumor at baseline, against an expected tumor size, against an expected tumor size based on a large patient population, or against the tumor size of a control population. In certain aspects provided herein, the administration of MEDI4736 can reduce a tumor size by at least 25%. In certain aspects provided herein, the administration of MEDI4736 can reduce a tumor size by at least 25% within about 6 weeks of the first treatment. In certain aspects provided herein, the administration of MEDI4736 can reduce a tumor size by at least 50%. In certain aspects provided herein, the administration of MEDI4736 can reduce a tumor size by at least 50% within about 10 weeks of the first treatment. In certain aspects provided herein, the administration of MEDI4736 can reduce a tumor size by at least 75%. In certain aspects provided herein, the administration of MEDI4736 can reduce a tumor size by at least 75% within about 10 weeks of the first treatment.

[0092] In certain aspects, use of the methods provided herein, i.e., administration of MEDI4736 or an antigen-binding fragment thereof can decrease tumor size within 6 weeks, within 7 weeks, within 8 weeks, within 9 weeks, within 10 weeks, within 12 weeks, within 16 weeks, within 20 weeks, within 24 weeks, within 28 weeks, within 32 weeks, within 36 weeks, within 40 weeks, within 44 weeks, within 48 weeks, or within 52 weeks of the first treatment.

[0093] In some embodiments, administration of 1 mg/kg of MEDI4736 or an antigen-binding fragment thereof (e.g., at least one dose, at least two doses, at least three doses, at least four doses, at least five doses, at least six doses, at least seven doses, at least eight doses, at least nine doses, at least ten doses, or more every two weeks or every three weeks) can be sufficient to reduce tumor size. However, as provided herein, larger doses can also be administered, for example, to optimize efficacy, number of doses necessary, or certain pharmacokinetic parameters.

[0094] The methods provided herein can decrease or retard tumor growth. In some aspects the reduction or retardation can be statistically significant. A reduction in tumor growth can be measured by comparison to the growth of patient's tumor at baseline, against an expected tumor growth, against an expected tumor growth based on a large patient population, or against the tumor growth of a control population.

[0095] In certain aspects, a patient achieves disease control (DC). Disease control can be a complete response (CR), partial response (PR), or stable disease (SD).

[0096] A “complete response” (CR) refers to the disappearance of all lesions, whether measurable or not, and no new lesions. Confirmation can be obtained using a repeat, consecutive assessment no less than four weeks from the date of first documentation. New, non-measurable lesions preclude CR.

[0097] A “partial response” (PR) refers to a decrease in tumor burden ≧50% relative to baseline. Confirmation can be obtained using a consecutive repeat assessment at least 4 weeks from the date of first documentation

[0098] “Progressive disease” (PD) refers to an increase in tumor burden ≧25% relative to the minimum recorded (nadir). Confirmation can be obtained by a consecutive repeat assessment at least 4 weeks from the date of first documentation. New, non-measurable lesions do not define PD.

[0099] “Stable disease” (SD) refers to not meeting the criteria for CR, PR, or PD.

[0100] In certain aspects, administration of MEDI4736 or an antigen-binding fragment thereof can increase progression-free survival (PFS).

[0101] In certain aspects, administration of MEDI4736 or an antigen-binding fragment thereof can increase overall survival (OS).

[0102] According to the methods provided herein, administration of MEDI4736 or an antigen-binding fragment thereof can result in desirable pharmacokinetic parameters. Total drug exposure can be estimated using the “area under the curve” (AUC). “AUC (tau)” refers to AUC until the end of the dosing period, whereas “AUC (inf)”refers to the AUC until infinite time. The administration can produce AUC (tau) of about 100 to about 2,500 d.Math.μg/mL. The administration can produce a maximum observed concentration (Cmax) of about 15 to about 350 μg/mL. The half-life of the MEDI4736 or the antigen-binding fragment thereof can be about 5 to about 25 days. In addition, the clearance of the MEDI4736 or the antigen-binding fragment thereof can be about 1-10 ml/day/kg.

[0103] As provided herein, MEDI4736 or an antigen-binding fragment thereof can also decrease free B7-H1 levels. Free B7-H1 refers to B7-H1 that is not bound (e.g., by MEDI4736). In some embodiments, B7-H1 levels are reduced by at least 80%. In some embodiments, B7-H1 levels are reduced by at least 90%. In some embodiments, B7-H1 levels are reduced by at least 95%. In some embodiments, B7-H1 levels are reduced by at least 99%. In some embodiments, B7-H1 levels are eliminated following administration of MEDI4736 or an antigen-binding fragment thereof. In some embodiments, administration of MEDI4736 or an antigen-binding fragment thereof reduces the rate of increase of B7-H1 levels as compared, e.g., to the rate of increase of B7-H1 levels prior to the administration of MEDI4736 or an antigen-binding fragment thereof.

Kits

[0104] The invention provides kits for characterizing the responsiveness of a subject to anti-PD-L1 antibody treatment. In one embodiment, the kit includes a therapeutic composition containing an effective amount of an antibody that specifically binds a PD-L1 polypeptide in unit dosage form.

[0105] A diagnostic kit of the invention provides a reagent (e.g., TaqMan primers/probes for CXCL9, KRT8, TRIM29, IFNgamma, and/or PD-L1 polynucleotide and housekeeping reference genes) for measuring relative expression of CXCL9, KRT8, TRIM29, IFNgamma, and/or PD-L1 polynucleotide. In other embodiments, the kit further includes reagents suitable for PD-L1 immunohistochemistry (e.g., anti-PD-L1 antibody).

[0106] In some embodiments, the kit comprises a sterile container which contains a therapeutic and/or diagnostic composition; such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.

[0107] In one embodiment, a kit of the invention comprises reagents for measuring CXCL9, KRT8, TRIM29, IFNgamma, and/or PD-L1 polynucleotide or polypeptide expression and a therapeutic anti-PD-L1 antibody. If desired, the kit further comprises instructions for measuring CXCL9, KRT8, TRIM29, IFNgamma, and/or PD-L1 polynucleotide or polypeptide expression and/or instructions for administering the anti-PD-L1 antibody to a subject having a lung cancer (e.g., non-small cell lung cancer, small cell lung cancer) selected as responsive to anti-PD-L1 antibody treatment. In particular embodiments, the instructions include at least one of the following: description of the therapeutic agent; dosage schedule and administration for treatment or prevention of lung cancer (e.g., non-small cell lung cancer, small cell lung cancer) or symptoms thereof; precautions; warnings; indications; counter-indications; over dosage information; adverse reactions; animal pharmacology; clinical studies; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.

[0108] The practice of the present invention employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, “Molecular Cloning: A Laboratory Manual”, second edition (Sambrook, 1989); “Oligonucleotide Synthesis” (Gait, 1984); “Animal Cell Culture” (Freshney, 1987); “Methods in Enzymology” “Handbook of Experimental Immunology” (Weir, 1996); “Gene Transfer Vectors for Mammalian Cells” (Miller and Calos, 1987); “Current Protocols in Molecular Biology” (Ausubel, 1987); “PCR: The Polymerase Chain Reaction”, (Mullis, 1994); “Current Protocols in Immunology” (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

[0109] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the invention, and are not intended to limit the scope of what the inventors regard as their invention.

EXAMPLES

Example 1

RNA Extraction and Real-time PCR analysis

[0110] Total RNA was extracted from snap-frozen tissue specimens collected from patients with non-small cell lung cancer (NSCLC) using the ZR RNA MicroPrep kit (Zymo Research, Orange, CA). RNA purity and concentration were determined spectrophotometrically (260/280>1.9). RNA quality was assessed on an Agilent 2100 Bioanalyzer using the RNA 6000 Nano LabChip®.

[0111] TaqMan Gene Expression assays were purchased from Applied Biosystems/Life Technologies. The assays include: CD274 (Assay ID: Hs01125301_ml); CXCL9 (Assay ID: Hs00171065_ml); IFNG (Assay ID: Hs00989291_ml); KRTS (Assay ID: Hs00361185_ml); KRT8 (Assay ID: Hs01595539_gl), and TRIM29 (Assay ID: Hs00232590_ml), as well as reference genes: ACTB (Hs01060665_gl), GAPDH (Assay ID: Hs02758991_gl) and B2M (Assay ID:Hs00187842_ml).

[0112] The BioMark™ Dynamic Array (Fluidigm, San Francisco, Calif.) microfluidics system allows for high throughput real-time PCR, producing high quality data with low variability. Single stranded cDNA was generated from 50 ng total RNA using the SuperScript® III First-Strand Synthesis SuperMix (Life Technologies). cDNA samples were pre-amplified using TaqMan Gene Expression Assays and TaqMan Pre-Amp Master Mix, according to the manufacturer's instructions. Reactions contained 5 μL of cDNA, 10 μL Pre-Amp Master Mix and 5 μL of 0.2X gene expression assay mix (comprised of all primer/probes to be assayed) for a final volume of 20 μL. Pre-amplified cDNA was assayed by Real-Time PCR with TaqMan Gene Expression Assays specific for target genes of interest and TaqMan Universal Master Mix (Life Technologies) using the BioMark™ instrument (Fluidigm).

[0113] To prepare samples for loading into 96×96 dynamic array chips (Fluidigm), the reaction mix contained 2.5μL 2X Universal Master Mix (Life Technologies), 0.25 μL Sample Loading Buffer (Fluidigm), and 2.25 μL pre-amplified cDNA. To prepare the primer/probes, the reaction mix contained 2.5 μL 20X TaqMan Gene Expression Assay and 2.5 μL Assay Loading Buffer (Fluidigm). Prior to loading the samples and assay reagents into the inlets, the chip was primed in the IFC Controller. Five microliters of sample prepared as described was loaded into each sample inlet of the dynamic array chip, and 5 μL of 10X gene expression assay mix was loaded into each detector inlet. Upon completion of the IFC priming and load/mixing steps, the chip was loaded on the BioMark™ Real-Time PCR System for thermal cycling.

[0114] Cycle threshold (Ct) values were generated using BioMark analysis software (Fluidigm Corporation). Ct values above 25 were excluded from calculations. Delta-Ct values were calculated by subtracting the mean Ct of 3 reference genes (B2M, ACTB, and GAPDH) from the mean Ct value for each gene evaluated. The final gene expression score (see the figures) is defined as (20-ΔCt), so that this score is positive overall and high score represents high expression in the log2 scale.

Example 2

Strong PD-L1 Expression Correlates with a Subject's Responsiveness to Anti-PD-L1 Antibody Treatment

[0115] PD-L1 membrane expression can be measured using immunohistochemistry (FIG. 1). Non-small cell lung cancer (NSCLC) patients with strong PD-L1 membrane expression typically respond to anti-PD-L1 antibody treatment. Whereas patients having little or undetectable levels of PD-L1 membrane expression are less responsive to anti-PD-L1 antibody treatment.

[0116] Immunohistochemical (IHC) results of PD-L1 membrane (M) expression can be expressed numerically as an IHC-M score. FIG. 2 shows the correlation between percent tumor size change from baseline and IHC M-score of PD-L1. The IHC M score is defined as the percentage of tumor cells with PD-L1 staining in NSCLC patients from the trial. Clinical response status measured as best overall response (BOR) indicated by colors at (left) 6 weeks and (right) 12 weeks post treatment with anti-PD-L1.

Example 3

PD-L1 Antibody Therapy Alters Gene Expression

[0117] A real time gene express assay (i.e., TaqMan assay) was used to determine how anti-PD-L1 antibody therapy altered the expression of candidate genes in patients treated with an anti-PD-L1 antibody. More specifically, the expression of T cell subtype transcripts, cytokine/chemokine transcripts, known IMT (immune modulatory therapy) transcripts, NSCLC subtype transcripts, and other immune-specific transcripts was measured. A complete list of assayed gene is provided below.

[0118] In the heatmap, genes that are highly expressed are indicated in red. Genes with low expression are indicated in blue. Highly expressed genes CXCL9, KRT8, TRIM29, and IFNgamma were selected for further analysis.

[0119] FIGS. 4, 5, 6, and 7 show results of an analysis of tumor size change relative to CXCL9, KRT8, TRIM29, and IFNgamma score, respectively. As indicated, each of CXCL9, KRT8, TRIM29, and IFNgamma are useful markers for identifying patients that are responsive to treatment with an anti-PD-L1 antibody.

[0120] FIG. 8 shows results with CXCL9 score relative to TRIM29. Subjects that were particularly responsive to treatment with anti-PD-L1 antibody had tumors showing high expression of CXCL9 and TRIM29.

[0121] Expression of CXCL9 and INFgamma correlates strongly with response to anti-PD-L1 antibody therapy by showing significant reductions in NSCLC tumor size versus baseline (FIGS. 9) and response (FIG. 10). The correlation between the response to anti-PD-L1 treatment and expression of CXCL9 (FIG. 11) and IFNgamma (FIG. 12) was also demonstrated, where patients with partial response or stable disease upon treatment had tumors that expressed higher levels of these markers.

Example 4

Head and Neck Cancer Response to Anti-PD-L1 Therapy Correlates with Gene Expression

[0122] The relationship between gene expression patterns and tumor response to anti-PD-L1 therapy was investigated. Tumor samples from Head and Neck cancer patients undergoing anti-PD-L1 therapy (MEDI4736) were collected and gene expression patterns were analyzed. As shown in FIGS. 13 and 14, expression of IFNgamma (FIG. 13) and CXCL9 (FIG. 14) correlated with response rate and percentage tumor size change in response to anti-PD-L1 (MEDI4736) treatment. The correlation between the response to anti-PD-L1 treatment and expression of IFNgamma (FIG. 15) and CXCL9 (FIG. 16) was also demonstrated, where patients with partial response or stable disease upon treatment had tumors that expressed higher levels of these markers. These data demonstrate that IFNgamma and/or CXCL9 expression can be used to identify Head and Neck cancer patients that are more likely to benefit from anti-PD-L1 therapy. These markers can also, therefore, be used to increase the efficacy of treatment by selectively targeting patients having tumors that express these markers.

Other Embodiments

[0123] From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

[0124] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

[0125] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.

TABLE-US-00007 SEQUENCE LISTING > PCT/US2010/058007_77 Sequence 77 from  PCT/US2010/058007 Organism: Homo sapiens SEQ ID NO: 1 EIVLTQSPGTLSLSPGERATLSCRASQRVSSSYLAWYQQKPGQAPRLL IYDASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSLP WTFGQGTKVEIK > PCT/US2010/058007_72 Sequence 72 from PCT/US2010/058007 Organism: Homo sapiens SEQ ID NO: 2 EVQLVESGGGLVQPGGSLRLSCAASGFTFSRYWMSWVRQAPGKGLEWV ANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYC AREGGWFGELAFDYWGQGTLVTVSS VH CDR1 > PCT/US2010/058007_73 Sequence 73 from PCT/US2010/058007 Organism: Homo sapiens SEQ ID NO: 3 RYWMS VH CDR2 > PCT/US2010/058007_74 Sequence 74 from PCT/US2010/058007 Organism: Homo sapiens SEQ ID NO: 4 NIKQDGSEKYYVDSVKG VH CDR3 > PCT/US2010/058007_75 Sequence 75 from  PCT/US2010/058007 Organism: Homo sapiens SEQ ID NO: 5 EGGWFGELAFDY VL CDR1 > PCT/US2010/058007_78 Sequence 78 from  PCT/US2010/058007 Organism: Homo sapiens SEQ ID NO: 6 RASQRVSSSYLA VL CDR2 > PCT/US2010/058007_79 Sequence 79 from  PCT/US2010/058007 Organism: Homo sapiens SEQ ID NO: 7 DASSRAT VL CDR3 > PCT/US2010/058007_80 Sequence 80 from  PCT/US2010/058007 Organism: Homo sapiens SEQ ID NO: 8 QQYGSLPWT