Claims for Patent: 10,241,115

Summary for Patent: 10,241,115

Title:	Immunohistochemical proximity assay for PD-1 positive cells and PD-ligand positive cells in tumor tissue
Abstract:	The present disclosure describes an IHC assay for detecting and quantifying spatially proximal pairs of PD-1-expressing cells (PD-1+ cells) and PD-Ligand-expressing cells (PD-L+ cells) in tumor tissue, and the use of the assay to generate proximity biomarkers that are predictive of which cancer patients are most likely to benefit from treatment with a PD-1 antagonist. The disclosure also provides methods for testing tumor samples for the proximity biomarkers, as well as methods for treating subjects with a PD-1 antagonist based on the test results.
Inventor(s):	Pierce; Robert H. (San Francisco, CA), Yearley; Jennifer H. (Palo Alto, CA), Turner; Scott P. (Sunnyvale, CA), Dogdas; Belma (Secaucus, NJ), Bagchi; Ansuman (Plainsboro, NJ)
Assignee:	Merck Sharp & Dohme Corp. (Rahway, NJ)
Application Number:	15/103,050
Patent Claims:	1. A method for treating a human patient having a tumor, which comprises: (1) obtaining a PD-1:PD-Ligand proximity score for a tumor sample from the patient; wherein the PD-1:PD-Ligand proximity score has been assigned to the tumor sample by a process which comprises: (a) obtaining an image of tissue that has been removed from the tumor sample and immunohistochemically stained for PD-1 and PD-Ligand expression in a manner that allows stained PD-1 cells to be distinguished from stained PD-Ligand cells; (b) defining in the image one or more regions of interest (ROIs) that comprises neoplastic cells and associated stroma, wherein substantially all of the neoplastic cells and associated stroma in the image are contained in the defined ROIs; (c) randomly creating across each defined ROI a plurality of subregions of substantially the same shape and size, wherein each of the subregions defines an area that is large enough to include a spatially proximal pair of a stained PD-1 cell and a stained PD-Ligand cell and small enough to exclude pairs of stained PD-1 and PD-Ligand cells that are not spatially proximal; and (d) calculating the percent of all of the subregions that are positive for both stained PD-1 cells and stained PD-Ligand cells to generate the PD-1:PD-ligand proximity score for the tumor sample, (2) comparing the PD-1:PD-Ligand proximity score for the tumor sample with a threshold PD-1:PD-Ligand proximity score, (3) classifying the tumor as biomarker positive or biomarker negative, wherein if the proximity score for the tumor sample is equal to or greater than the threshold proximity score, then the tumor is classified as positive for the PD-1:PD-Ligand proximity biomarker, and if the obtained score is less than the threshold score, then the tumor is classified as negative for the PD-1:PD-Ligand proximity biomarker, and (4) administering to the patient a PD-1 antagonist if the tumor is positive for the PD-1:PD-Ligand proximity biomarker and administering to the subject a cancer treatment that does not include a PD-1 antagonist if the tumor is negative for the PD-1:PD-Ligand proximity biomarker. 2. The method of claim 1, wherein the steps (1)-(3) further comprise obtaining a tumor sample from the patient, sending the tumor sample to a diagnostic laboratory, and receiving from the diagnostic laboratory a report that states whether the tumor sample is positive or negative for the PD-1:PD-Ligand proximity biomarker. 3. The method of claim 1, wherein the tumor sample is from a human diagnosed with bladder cancer, breast cancer, clear cell kidney cancer, head/neck squamous cell carcinoma, lung squamous cell carcinoma, malignant melanoma, non-small-cell lung cancer (NSCLC), ovarian cancer, pancreatic cancer, prostate cancer, renal cell cancer, small-cell lung cancer (SCLC) or triple negative breast cancer, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), diffuse large B-cell lymphoma (DLBCL), EBV-positive DLBCL, primary mediastinal large B-cell lymphoma, T-cell/histiocyte-rich large B-cell lymphoma, follicular lymphoma, Hodgkin's lymphoma (HL), mantle cell lymphoma (MCL), multiple myeloma (MM), myeloid cell leukemia-1 protein (Mcl-1), myelodysplastic syndrome (MDS), non-Hodgkin's lymphoma (NHL), or small lymphocytic lymphoma (SLL). 4. The method of claim 1 wherein the tumor sample is from a human diagnosed with melanoma or non small cell lung cancer (NSCLC), the PD-Ligand is PD-L1, and the PD-1 antagonist is nivolumab or an antibody comprising a heavy chain comprising a sequence of amino acids as set forth in SEQ JD NO:21 and alight chain comprising a sequence of amino acids as set forth in SEQ ID NO:22. 5. The method of claim 1, wherein the tumor sample is from a human diagnosed with metastatic melanoma, the PD-1 antagonist is an antibody comprising a heavy chain comprising a sequence of amino acids as set forth in SEQ ID NO:21 and a light chain comprising a sequence of amino acids as set forth in SEQ ID NO:22, the PD-Ligand is PD-L1 and the threshold PD-1:PD-Ligand proximity score is about 0.2%. 6. The method of claim 1, wherein the image represents the entirety of the stained tissue and only one ROI is defined for the image or wherein the image represents the entirety of the stained tissue and two or more ROIs are defined for the image. 7. The method of claim 1, wherein the tumor sample has also been immunohistochemically stained for expression of a worker protein specific for the type of tumor comprising the tumor sample. 8. The method of claim 7, wherein the tissue has been immunohistochemically stained for the expression of PD-L1, PD-L2 or both PD-L1 and PD-L2. 9. The method of claim 8, wherein the tissue has been counterstained with a nuclear stain. 10. The method of claim 8, wherein the image is obtained by a method comprising: obtaining a tissue section from the tumor sample, contacting the tissue section with an antibody specific for PD-1 (anti-PD-1 Ab) under conditions suitable for forming antibody-antigen complexes, and washing the tissue section to remove unbound antibody and detecting antibody-antigen complexes comprising the PD-1 antibody, contacting the tissue section with an antibody specific for PD-Ligand (anti-PD-Ligand Ab) under conditions suitable for forming antibody-antigen complexes, and washing the tissue section to remove unbound antibody and detecting antibody-antigen complexes comprising the PD-Ligand antibody, wherein steps (b) and (c) are performed before or after steps (d) and (e), or wherein steps (b) and (d) are performed simultaneously and steps (c) and (e) are performed simultaneously. 11. The method of claim 8, wherein the image is obtained by a method comprising: obtaining at least two adjacent tissue sections that have been stained in separate monoplex immunohistochemistry (IHC) assays, wherein one of the tissue sections has been stained for PD-1 using the anti-PD-1 Ab and the other tissue section has been stained for the PD-Ligand using the anti-PD-Ligand Ab, creating a registered digital image of each of the stained tissue sections, and superimposing the registered digital images to generate a composite image. 12. The method of claim 11, wherein the anti-PD-Ligand Ab is specific for PD-L1 or is a bispecific antibody that binds both PD-L1 and PD-L2. 13. The method of claim 12, wherein the immunohistochemically stained tissue is obtained using a direct immunohistochemistry (IHC) assay or an indirect INC assay. 14. The method of claim 1, wherein the PD-1 cells are stained with a first detectable label and the PD-Ligand cells are stained with a second detectable label that is distinguishable from the first label, wherein the first and second detectable labels are first and second colors produced by fluorophores or chromogens. 15. The method of claim 14, wherein the image is a digital image and the process further comprises: segmenting the ROI to extract a first set of pixels for the first color and a second set of pixels for the second color, assigning a positive value to each of the ROI subregions that has a pixel from each of the first and second extracted pixel sets, and assigning a negative value to each of the ROI subregions that lacks a pixel from either of the first and second extracted pixel sets, wherein the segmenting step occurs prior to creating the subregions in the one or more ROIs. 16. The method of claim 15, which further comprises examining the image for fluorescing artifacts and removing any detected artifacts from the image, wherein the examining is performed prior to the segmenting step. 17. The method of claim 1, wherein the area covered by each of the subregions is selected such that it captures physiologic interactions between a single PD-Ligand positive cell and a single PD-1 positive cell. 18. The method of claim 15, wherein the image is magnified 200 times and the subregions are discs with radii in the range of 2-10 pixels.

Details for Patent 10,241,115

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Bristol-myers Squibb Company	OPDIVO	nivolumab	Injection	125554	12/22/2014		2039-03-26
Bristol-myers Squibb Company	OPDIVO	nivolumab	Injection	125554	10/04/2017		2039-03-26
Bristol-myers Squibb Company	OPDIVO	nivolumab	Injection	125554	08/27/2021		2039-03-26