Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
  • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • 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
  • C07K16/2803—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
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
• • 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
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/158—Expression markers
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2800/00—Detection or diagnosis of diseases
  • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

• the present disclosure provides a method for treating a subject afflicted with a tumor using an immunotherapy.
• PD- ⁇ /Programmed Death ligand 1 (PD-L1) pathway such as nivolumab and pembrolizumab (formerly lambrolizumab; USAN Council Statement, 2013) that bind specifically to the PD-1 receptor and atezolizumab, durvalumab, and avelumab that bind specifically to PD-L1
• PD-L1 pathway such as nivolumab and pembrolizumab (formerly lambrolizumab; USAN Council Statement, 2013) that bind specifically to the PD-1 receptor and atezolizumab, durvalumab, and avelumab that bind specifically to PD-L1
• anti-cancer agents can vary in their effectiveness based on the unique patient characteristics. Accordingly, there is a need for targeted therapeutic strategies that identify patients who are more likely to respond to a particular anti-cancer agent and, thus, improve the clinical outcome for patients diagnosed with cancer.
• Certain aspects of the present disclosure are directed to a pharmaceutical composition
• a pharmaceutical composition comprising an anti-PD-l/PD-Ll antagonist for use in a method of identifying a human subject suitable for the anti-PD-l/PD-Ll antagonist, wherein the method comprises measuring expression of a panel of genes in a tumor sample obtained from a subject in need of the anti-PD-l/PD-Ll antagonist, wherein the gene panel comprises at least three of STAT1 , IFNy, NECTIN2, and CSF1R.
• the gene panel comprises all of ST ATI , IFNy, NECTIN2, CSF1R, and an additional gene, two additional genes, three additional genes, four additional genes, five additional genes, six additional genes, seven additional genes, eight additional genes, or ten additional genes.
• the gene panel consists of STAT1 , IFNy, NECTIN2, CSF1R , and an additional gene, two additional genes, or three additional genes.
• the subject is identified as being suitable when the tumor sample exhibits: (i) an increased expression of one or more of STAT1 and IFNy ("upregulated genes") in the sample compared to the expression of the one or more of ST ATI and IFNy in a reference sample; (ii) a decreased expression of one or more of NECTIN2 and CSF1R (“down-regulated genes") in the sample compared to the expression of one or more of NECTIN2 and CSF1R in a reference sample or (iii) both (i) and (ii).
• STAT1 and IFNy upregulated genes
• NECTIN2 and CSF1R down-regulated genes
• the subject is to be administered an anti-PD-l/PD-Ll antagonist.
• Certain aspects of the present disclosure are directed to a pharmaceutical composition comprising an anti-PD-l/PD-Ll antagonist for use in a method of treating a human subject afflicted with a tumor, wherein a tumor sample obtained from the subject exhibits: (i) an increased expression of one or more of STAT1 and IFNy ("upregulated genes") in a tumor sample obtained from the subject compared to the expression of the one or more of ST ATI and IFNy in a reference sample; (ii) a decreased expression of one or more of NECTIN2 and CSF1R (“down- regulated genes”) in a tumor sample obtained from the subject compared to the expression of one or more of NECTIN2 and CSF1R in a reference sample; or (iii) both (i) and (ii).
• STAT1 and IFNy upregulated genes
• NECTIN2 and CSF1R down- regulated genes
• the reference sample comprises a non-tumor tissue of the subject, a corresponding non-tumor tissue of the subject, or the corresponding tissue of subjects without a tumor.
• Certain aspects of the present disclosure are directed to a method of identifying a human subject suitable for an anti-PD-l/PD-Ll antagonist, comprising in vitro measuring expression of a panel of genes in a tumor sample obtained from a subject in need of the anti-PD- l/PD-Ll antagonist, wherein the gene panel comprises at least three of STAT1 , IFNy, NECTIN2, and CSF1R.
• the gene panel comprises all of STAT1 , IFNy, NECTIN2, CSF1R and an additional gene, two additional genes, three additional genes, four additional genes, five additional genes, six additional genes, seven additional genes, eight additional genes, or ten additional genes. In some aspects, wherein the gene panel consists of SI A 77, IFNy, NECTIN2, CSF1R , and an additional gene, two additional genes, or three additional genes.
• the subject is identified as being suitable when the tumor sample exhibits: (i) an increased expression of one or more of STAT1 and IFNy ("upregulated genes") in the tumor sample compared to the expression of the one or more of ST ATI and IFNy in a reference sample; (ii) a decreased expression of one or more of NECTIN2 and CSF1R ("down- regulated genes") in the tumor sample compared to the expression of one or more of NECNN2 and CSF1R in a reference sample; or (iii) both (i) and (ii).
• STAT1 and IFNy upregulated genes
• NECTIN2 and CSF1R down- regulated genes
• Certain aspects of the present disclosure are directed to a method of treating a human subject afflicted with a tumor, comprising administering an anti-PD-l/PD-Ll antagonist to the subject, wherein a tumor sample obtained from the subject exhibits: (i) an increased expression of one or more of STAT1 and IFNy ("upregulated genes") in a tumor sample obtained from the subject compared to the expression of the one or more of ST ATI and IFNy in a reference sample; (ii) a decreased expression of one or more of NECTIN2 and CSF1R (“down-regulated genes") in a tumor sample obtained from the subject compared to the expression of one or more of NECTIN2 and CSF1R in a reference sample; or (iii) both (i) and (ii);
• the reference sample comprises a non-tumor tissue of the subject, a corresponding non-tumor tissue of the subject, or the corresponding tissue of subjects without a tumor.
• the subject is identified as being suitable for the anti-PD-l/PD-
• the tumor sample exhibits increased expression of at least two of the upregulated genes. In some aspects, the tumor sample exhibits decreased expression of at least two of the down-regulated genes. In some aspects, the tumor sample exhibits increased expression of all of the upregulated genes; and the tumor sample exhibits decreased expression of all of the down-regulated genes.
• the expression of one or more of the upregulated genes is increased at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 225%, at least about 250%, at least about 275%, or at least about 300% higher than the expression of one or more of ST ATI and IFNy in the reference sample.
• the expression of one or more of the upregulated genes is increased at least about 50% higher than the expression of one or more of ST ATI and IFNy in the reference sample. In some aspects, the expression of one or more of the upregulated genes is increased at least about 75% higher than the expression of one or more of ST ATI and IFNy in the reference sample.
• the expression of one or more of the upregulated genes is decreased at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 225%, at least about 250%, at least about 275%, or at least about 300% lower than the expression of one or more of NECTIN2 and CSF1R in the reference sample.
• the expression of one or more of the upregulated genes is decreased at least about 50% lower than the expression of one or more of NECTIN2 and CSF1R in the reference sample. In some aspects, the expression of one or more of the upregulated genes is decreased at least about 75% lower than the expression of one or more of NECTIN2 and CSF1R in the reference sample.
• the tumor sample is a tumor tissue biopsy.
• the tumor sample is a formalin-fixed, paraffin-embedded tumor tissue or a fresh-frozen tumor tissue.
• the tumor sample is obtained from a parenchyma of the tumor.
• gene expression is determined by detecting the presence of gene mRNA, the presence of a protein encoded by the gene, or both.
• the presence of gene mRNA is determined using reverse transcriptase PCR.
• the presence of the protein encoded by the gene is determined using an IHC assay.
• the IHC assay is an automated IHC assay.
• the tumor sample does not exhibit: an increased expression of one or more of CSF1R and NECTIN2 compared to the expression of the one or more of CSF1R and NECTIN2 in a reference sample; a decreased expression of one or more of STAT1 and IFNy compared to the expression of one or more of STAT1 and IFNy in a reference sample; or both (i) and (ii).
• the tumor sample does not exhibit: an increased expression of two or three of CSF1R and NECTIN2 compared to the expression of two or three of CSF1R and NECTIN2 in a reference sample; a decreased expression of two, three, or four of STAT1 and IFNy compared to the expression of two, three, or four of STAT1 and IFNy in a reference sample; or both (i) and (ii).
• the tumor sample is obtained from a stroma of the tumor.
• the anti-PD-l/PD-Ll antagonist comprises an antibody or antigen-binding fragment thereof that specifically binds a target protein selected from programmed death 1 (PD-1; an "anti-PD-1 antibody”) or programmed death ligand 1 (PD-L1; an "anti-PD-Ll antibody).
• PD-1 programmed death 1
• PD-L1 programmed death ligand 1
• the anti-PD-l/PD-Ll antagonist is an anti-PD-1 antibody.
• the anti-PD-1 antibody comprises nivolumab or pembrolizumab.
• the anti-PD-l/PD-Ll antagonist is an anti-PD-Ll antibody.
• the anti-PD-1 antibody comprises avelumab, atezolizumab, or durvalumab.
• the anti-PD-l/PD-Ll antagonist is administered as a monotherapy. In some aspects, the anti-PD-l/PD-Ll antagonist is administered with an anti cancer agent.
• the anti-cancer agent comprises an antibody that specifically binds a protein of Inducible T cell Co-Stimulator (ICOS), CD137 (4-1BB), CD134 (0X40), NKG2A, CD27, CD96, Glucocorticoid-Induced TNFR-Related protein (GITR), and Herpes Virus Entry Mediator (HVEM), Programmed Death- 1 (PD-1), Programmed Death Ligand- 1 (PD-L1), CTLA- 4, B and T Lymphocyte Attenuator (BTLA), T cell Immunoglobulin and Mucin domain-3 (TIM- 3), Lymphocyte Activation Gene-3 (LAG-3), adenosine A2a receptor (A2aR), Killer cell Lectin like Receptor G1 (KLRG-1
• the tumor is derived from a cancer selected from the group consisting of hepatocellular cancer, gastroesophageal cancer, melanoma, bladder cancer, lung cancer, kidney cancer, head and neck cancer, colon cancer, pancreatic cancer, prostate cancer, ovarian cancer, urothelial cancer, colorectal cancer, and any combination thereof.
• the tumor is relapsed. In some aspects, the tumor is refractory. In some aspects, the tumor is locally advanced. In some aspects, the tumor is metastatic. In some aspects, the administering treats the tumor. In some aspects, the administering reduces the size of the tumor. In some aspects, the size of the tumor is reduced by at least about 10%, about 20%, about 30%, about 40%, or about 50% compared to the tumor size prior to the administration.
• the subject exhibits progression-free survival of at least about one month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, at least about one year, at least about eighteen months, at least about two years, at least about three years, at least about four years, or at least about five years after the initial administration.
• the subject exhibits stable disease after the administration. In some aspects, the subject exhibits a partial response after the administration. In some aspects, the subject exhibits a complete response after the administration.
• kits for treating a subject afflicted with a tumor comprising: (a) an anti-PD-l/PD-Ll antagonist; and (b) instructions for using the anti-PD-l/PD-Ll antagonist in any method disclosed herein.
• the anti-PD-l/PD-Ll antagonist comprises an anti-PD-1 antibody.
• the anti-PD-l/PD-Ll antagonist comprises an anti-PD-Ll antibody.
• Certain aspects of the present disclosure are directed to a gene panel comprising at least three of STAT1 , IFNy, NECTIN2, and CSF1R, for use in identifying a subject suitable for an anti-PD-l/PD-Ll antagonist.
• the gene panel comprises at least four, at least five, or at least six of ST ATI, IFNy , NECTIN2, and CSF1R.
• the gene panel comprises ST ATI , IFNy, NECTIN2, and CSF1R
• the gene panel consists of STAT1 , IFNy, NECTIN2, and CSF1R and one additional gene, two additional genes, three additional genes, four additional genes, five additional genes, six additional genes, seven additional genes, eight additional genes, nine additional genes, or ten additional genes.
• the gene panel consists of ST ATI , IFNy, NECTIN2, and CSF1R, and one additional gene, two additional genes, three additional genes, four additional genes, five additional genes, six additional genes, seven additional genes, eight additional genes, nine additional genes, or ten additional genes.
• the gene panel comprises about 90 genes, about 95 genes, or about 100 genes.
• a method for preparing a nucleic acid fraction from a tumor of a subject in need of an I/O therapy comprising: (a) extracting a tumor biopsy from the subject; (b) producing a fraction of nucleic acids extracted in (a) by isolating the nucleic acids; and (c) analyzing the expression level of one or more genes in a gene panel selected from ST ATI, IFNy, NECTIN2, and CSF1R.
• the nucleic acids are mRNA.
• CSF1R and NECTIN2 genes are downregulated.
• one or both of ST ATI and IFNy genes are upregulated.
• CSF1R and NECNN2 are downregulated, and ST ATI and IFNy are upregulated.
• the expression level is analyzed by measuring an mRNA level of the one or more genes in the gene panel in the tumor sample. In some aspects, the expression level is measured using a nuclease protection assay. In some aspects, the expression level is measured using next-generation sequencing. In some aspects, the expression level is measured using reverse transcriptase polyermase chain reaction (RT-PCR).
• RT-PCR reverse transcriptase polyermase chain reaction
• the expression of one or both of STAT1 and IFNy is increased at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 225%, at least about 250%, at least about 275%, or at least about 300% higher than the expression of one or both of ST ATI and IFNy in the reference sample.
• the expression of one or both of STAT1 and IFNy is increased at least about 50% higher than the expression of one or both of ST ATI and IFNy in the reference sample. In some aspects, the expression of one or both of STAT1 and IFNy is increased at least about 75% higher than the expression of one or both of ST ATI and IFNy in the reference sample.
• the expression of one or both of NECTIN2 and CSF1R is decreased at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 225%, at least about 250%, at least about 275%, or at least about 300% lower than the expression of one or more of NECTIN2 and CSF1R in the reference sample.
• the expression of one or both of Nl CTIN2 and CSF1R is decreased at least about 50% lower than the expression of one or more of NECTIN2 and CSF1R in the reference sample. In some aspects, the expression of one or both of Nl CTIN2 and CSF1R is decreased at least about 75% lower than the expression of one or more of NECTIN2 and CSF1R in the reference sample.
• FIGs. 1A-1B are images of tumor tissue samples labeled using standard CD8+ immunohistochemistry (IHC; FIG. 1A) or further annotated using an artificial intelligence (AI) image analysis tool (FIG. IB). Arrowheads are examples of CD8+ T cells. Tumor parenchyma and stromal regions are indicated (FIG. IB).
• IHC standard CD8+ immunohistochemistry
• AI artificial intelligence
• FIG. 2 is a schematic illustration of the various CD8 phenotypes.
• FIGs. 3A-3B are scatter plots illustrating the cutoffs for parenchymal vs. stromal abundance used to define immune phenotypes.
• FIG. 3B includes the scatter plot of FIG. 3 A with an overlay indicating the cutoffs for inflamed, balanced, excluded, and desert phenotypes.
• FIGs. 4A-4C are images of three tumor samples, which are representative of an immune desert phenotype (T cells absent from the TME; FIG. 4A), an immune excluded phenotype (accumulated T cells without efficient tumor parenchyma infiltration; FIG. 4B), and an immune inflamed phenotype (infiltrated T cells in the tumor parenchyma; FIG. 4C). Arrowheads label examples of CD8+ T cells.
• FIGs. 5A-5D are graphical representations, illustrating the parenchymal CD8 signature (FIGs. 5A-5B) and the stromal CD8 signature (FIGs. 5C-5D).
• FIGs. 5A and 5C are heat maps showing the relative expressions of various genes that are associated with a parenchymal CD8 signature (FIG. 5A) and the stromal CD8 signature (FIG. 5C).
• FIGs. 5B and 5D are bar graph summaries of select representative genes of the parenchymal CD8 signature (FIG. 5B) and the stromal CD8 signature (FIG. 5D).
• FIGs. 6A-6D are scatter plots, illustrating the correlation of CD8 signature scores with CD8 IHC scores in melanoma (FIGs. 6A and 6C) and SCCHN (FIGs. 6B and 6D) tumor samples.
• FIG. 7A is graphical representation of the overall survival (OS) odds ratios for
• FIGs. 7B-7F are ROC curves for OR for Triple CD8 (FIG. 7B), Dual CD8 (FIG. 7D, Parenchymal CD8 (FIG. 7C), CD8 (FIG. 7E), and CD8.IHC EMT (FIG. 7F).
• FIG. 8A is graphical representation of the progression free survival (PFS) odds ratios for Triple CD8, Dual CD8, Parenchymal CD8, CD8, and CD8.IHC_EMT, as indicated.
• FIG. 8B is graphical representation of the OS odds ratios for Triple CD8, Dual CD8, Parenchymal CD8, CD8, and CD8.IHC_EMT, as indicated.
• PFS progression free survival
• FIGs. 9A-9B are graphical representations of PFS (FIG. 9A) and OS (FIG. 9B) as stratified by parenchymal signature score. The number of patients at risk in each group is shown below each graph.
• Certain aspects of the present disclosure are directed to methods of identifying a human subject suitable for an immune-oncology (I-O) therapy, e.g., an anti-PD-l/PD-Ll antagonist therapy.
• I-O immune-oncology
• the present disclosure is directed to a method of identifying a human subject suitable for an anti-PD-l/PD-Ll antagonist therapy, comprising measuring expression of a panel of genes in a tumor sample obtained from a subject in need of the anti-PD- l/PD-Ll antagonist, wherein the gene panel comprises at least one of STAT1 , IFNy, NECTIN2, and CSF1R.
• a gene panel comprising the identified genes of the present disclosure and the gene signature are useful to identify a subject suitable for and/or responsive to an 1-0 therapy, especially in predicting an inflammatory phenotype in the tumor microenvironment (TME) across multiple tumor types. Therefore, in some aspects, the gene panel and its use can replace the inconvenient and burdensome CD8+ immunohistochemistry.
• administering refers to the physical introduction of a composition comprising a therapeutic agent to a subject, using any of the various methods and delivery systems known to those skilled in the art.
• Preferred routes of administration for the immunotherapy include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion.
• parenteral administration means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion, as well as in vivo electroporation.
• Other non-parenteral routes include an oral, topical, epidermal or mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually or topically.
• Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods.
• An "adverse event” as used herein is any unfavorable and generally unintended or undesirable sign (including an abnormal laboratory finding), symptom, or disease associated with the use of a medical treatment.
• an adverse event can be associated with activation of the immune system or expansion of immune system cells (e.g ., T cells) in response to a treatment.
• a medical treatment can have one or more associated AEs and each AE can have the same or different level of severity.
• Reference to methods capable of "altering adverse events” means a treatment regime that decreases the incidence and/or severity of one or more AEs associated with the use of a different treatment regime.
• an "antibody” shall include, without limitation, a glycoprotein immunoglobulin which binds specifically to an antigen and comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding portion thereof.
• Each H chain comprises a heavy chain variable region (abbreviated herein as YH) and a heavy chain constant region.
• the heavy chain constant region comprises three constant domains, Cm, Cm and Cm.
• Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region.
• the light chain constant region is comprises one constant domain, CL.
• the YH and YL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs).
• CDRs complementarity determining regions
• FRs framework regions
• Each YH and YL comprises three CDRs and four FRs, arranged from amino-terminus to carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
• the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
• the constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. Therefore, the term "anti-PD-1 antibody” includes a full antibody having two heavy chains and two light chains that specifically binds to PD-1 and antigen-binding portions of the full antibody. Non limiting examples of the antigen-binding portions are shown elsewhere herein.
• An immunoglobulin can derive from any of the commonly known isotypes, including but not limited to IgA, secretory IgA, IgG and IgM.
• IgG subclasses are also well known to those in the art and include but are not limited to human IgGl, IgG2, IgG3 and IgG4.
• immunotype refers to the antibody class or subclass (e.g ., IgM or IgGl) that is encoded by the heavy chain constant region genes.
• antibody includes, by way of example, both naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human or nonhuman antibodies; wholly synthetic antibodies; and single chain antibodies.
• a nonhuman antibody can be humanized by recombinant methods to reduce its immunogenicity in man.
• antibody also includes an antigen-binding fragment or an antigen binding portion of any of the aforementioned immunoglobulins, and includes a monovalent and a divalent fragment or portion, and a single chain antibody.
• an "isolated antibody” refers to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that binds specifically to PD-1 is substantially free of antibodies that bind specifically to antigens other than PD-1).
• An isolated antibody that binds specifically to PD-1 may, however, have cross-reactivity to other antigens, such as PD-1 molecules from different species.
• an isolated antibody can be substantially free of other cellular material and/or chemicals.
• the term "monoclonal antibody” refers to a non-naturally occurring preparation of antibody molecules of single molecular composition, i.e., antibody molecules whose primary sequences are essentially identical, and which exhibits a single binding specificity and affinity for a particular epitope.
• a monoclonal antibody is an example of an isolated antibody.
• Monoclonal antibodies can be produced by hybridoma, recombinant, transgenic or other techniques known to those skilled in the art.
• a “human antibody” refers to an antibody having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences.
• the human antibodies of the disclosure can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g, mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo).
• the term "human antibody,” as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
• a “humanized antibody” refers to an antibody in which some, most or all of the amino acids outside the CDRs of a non-human antibody are replaced with corresponding amino acids derived from human immunoglobulins. In one aspect of a humanized form of an antibody, some, most or all of the amino acids outside the CDRs have been replaced with amino acids from human immunoglobulins, whereas some, most or all amino acids within one or more CDRs are unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they do not abrogate the ability of the antibody to bind to a particular antigen.
• a "humanized antibody” retains an antigenic specificity similar to that of the original antibody.
• a "chimeric antibody” refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species, such as an antibody in which the variable regions are derived from a mouse antibody and the constant regions are derived from a human antibody.
• an "anti-antigen antibody” refers to an antibody that binds specifically to the antigen.
• an anti-PD-1 antibody binds specifically to PD-1
• an anti-PD-Ll antibody binds specifically to PD-L1
• an anti-CTLA-4 antibody binds specifically to CTLA-4.
• an "antigen-binding portion" of an antibody refers to one or more fragments of an antibody that retain the ability to bind specifically to the antigen bound by the whole antibody. It has been shown that the antigen binding function of an antibody can be performed by fragments of a full-length antibody.
• binding fragments encompassed within the term "antigen-binding portion" of an antibody include (i) a Fab fragment (fragment from papain cleavage) or a similar monovalent fragment consisting of the VL, VH, LC and CHI domains; (ii) a F(ab')2 fragment (fragment from pepsin cleavage) or a similar bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al ., (1989) Nature 341 :544-546), which consists of a VH domain; (vi) an isolated complementarity determining region (CDR
• the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).
• single chain Fv single chain Fv
• Such single chain antibodies are also intended to be encompassed within the term "antigen-binding portion" of an antibody.
• Antigen-binding portions can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.
• Antibodies useful in the methods and compositions described herein include, but are not limited to, antibodies and antigen-binding portions thereof that specifically bind a protein selected from the group consisting of Inducible T cell Co-Stimulator (ICOS), CD137 (4-1BB), CD 134 (0X40), NKG2A, CD27, CD96, Glucocorticoid-Induced TNFR-Related protein (GITR), and Herpes Virus Entry Mediator (HVEM), Programmed Death- 1 (PD-1), Programmed Death Ligand- 1 (PD-L1), Cytotoxic T-Lymphocyte Antigen-4 (CTLA-4), B and T Lymphocyte Attenuator (BTLA), T cell Immunoglobulin and Mucin domain-3 (TIM-3), Lymphocyte Activation Gene-3 (LAG-3), adenosine A2a receptor (A2aR), Killer cell Lectin-like Receptor G1 (KLRG-1), Natural Killer Cell Receptor 2B4 (
• a "cancer” refers a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth divide and grow results in the formation of malignant tumors that invade neighboring tissues and can also metastasize to distant parts of the body through the lymphatic system or bloodstream.
• immunotherapy refers to the treatment of a subject afflicted with, or at risk of contracting or suffering a recurrence of, a disease by a method comprising inducing, enhancing, suppressing or otherwise modifying an immune response.
• Treatment or “therapy” of a subject refers to any type of intervention or process performed on, or the administration of an active agent to, the subject with the objective of reversing, alleviating, ameliorating, inhibiting, slowing down or preventing the onset, progression, development, severity or recurrence of a symptom, complication or condition, or biochemical indicia associated with a disease.
• PD-1 Protein Determination-1
• PD-1 refers to an immunoinhibitory receptor belonging to the CD28 family. PD-1 is expressed predominantly on previously activated T cells in vivo , and binds to two ligands, PD-L1 and PD-L2.
• the term "PD-1 " as used herein includes human PD-1 (hPD-1), variants, isoforms, and species homologs of hPD-1, and analogs having at least one common epitope with hPD-1. The complete hPD-1 sequence can be found under GenBank Accession No. U64863.
• P-L1 Programmed Death Ligand- 1
• PD-L1 is one of two cell surface glycoprotein ligands for PD-1 (the other being PD-L2) that downregulate T cell activation and cytokine secretion upon binding to PD-1.
• the term "PD-L1" as used herein includes human PD-L1 (hPD- Ll), variants, isoforms, and species homologs of hPD-Ll, and analogs having at least one common epitope with hPD-Ll.
• the complete hPD-Ll sequence can be found under GenBank Accession No. Q9NZQ7.
• the human PD-L1 protein is encoded by the human CD274 gene (NCBI Gene ID: 29126).
• a PD-1 or PD-L1 "inhibitor” refers to any molecule capable of blocking, reducing, or otherwise limiting the interaction between PD-1 and PD-L1 and/or the activity of PD-1 and/or PD-L1.
• the inhibitor is an antibody or an antigen binding fragment of an antibody.
• the inhibitor comprises a small molecule.
• Signal transducer and activator of transcription 1-alpha/beta refers to a signal transducer and transcription activator that mediates cellular responses to interferons (IFNs), cytokine KITLG/SCF, and other cytokines and other growth factors.
• IFNs interferons
• cytokine KITLG/SCF cytokine KITLG/SCF
• other cytokines and other growth factors cytokines and other growth factors.
• signaling via protein kinases leads to activation of Jak kinases (TYK2 and JAKl) and to tyrosine phosphorylation of STAT1 and STAT2.
• ISGF3 The phosphorylated STATs dimerize and associate with ISGF3G/IRF-9 to form a complex termed ISGF3 transcription factor, that enters the nucleus.
• ISGF3 binds to the IFN stimulated response element (ISRE) to activate the transcription of IFN- stimulated genes (ISG), which drive the cell in an antiviral state.
• IFN- stimulated response element IFN- stimulated genes
• STAT1 is tyrosine- and serine-phosphorylated.
• IFN- gamma-activated factor migrates into the nucleus and binds to the IFN gamma activated sequence (GAS) to drive the expression of the target genes, inducing a cellular antiviral state.
• GAF IFN- gamma activated sequence
• STAT1 becomes activated in response to KITLG/SCF and KIT signaling. STAT1 may also mediate cellular responses to activated FGFR1, FGFR2, FGFR3, and FGFR4.
• the complete human STAT1 amino acid sequence can be found under UniProtKB identification number P42224.
• the human STAT1 protein is encoded by the human STAT1 gene (NCBI Gene ID: 6772).
• Interferon gamma refers to a cytokine that is involved in innate and adaptive immunity against infection (UniProtKB - P01579). IFNy is the only member of the type II class of interferons, and is therefore sometimes referred to as "type II interferon.” IFNy serves to activate macrophages and induce MHC class II expression. IFNy is largely expressed by natural killer (NK) cells and natural killer T (NKT) cells as part of the innate immune response, and by CD4 Thl (T helper) cells and CD8 cytotoxic T lymphocyte (CTL) effector T cells once immunity develops.
• NK natural killer
• NKT natural killer T
• T helper CD4 Thl
• CTL cytotoxic T lymphocyte
• Nectin-2 refers to the gene encoding Nectin-2, which is a modulator of T cell signaling, serving as both a co-stimulator and a co-inhibitor of T cell function, depending on which receptor that Nectin-2 binds (UniProtKB - Q92692). Binding of Nectin-2 to CD226 stimulates T cell proliferation and production of various cytokines including IL2, IL5, IL10, IL13, and IFNy. Conversely, binding of Nectin-2 to PVRIG inhibits T cell proliferation.
• CSF1R refers to the gene encoding macrophage colony- stimulating factor 1 receptor (CSF-l-R; UniProtKB - P07333), a tyrosine-protein kinase with multiple functions.
• CSF-l-R acts as a cell-surface receptor for CSF1 and IL34 and plays an essential role in the regulation of survival, proliferation, and differentiation of hematopoietic precursor cells, especially mononuclear phagocytes, such as macrophages and monocytes.
• binding of IL34 or CSF1 to CSF-l-R promotes the release of proinflammatory chemokines involved in innate immunity and the inflammatory process.
• a “subject” includes any human or nonhuman animal.
• nonhuman animal includes, but is not limited to, vertebrates such as nonhuman primates, sheep, dogs, and rodents such as mice, rats and guinea pigs.
• the subject is a human.
• the terms, "subject” and “patient” are used interchangeably herein.
• a “therapeutically effective amount” or “therapeutically effective dosage” of a drug or therapeutic agent is any amount of the drug that, when used alone or in combination with another therapeutic agent, protects a subject against the onset of a disease or promotes disease regression evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction.
• the ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.
• an “anti-cancer agent” promotes cancer regression in a subject.
• a therapeutically effective amount of the drug promotes cancer regression to the point of eliminating the cancer.
• “Promoting cancer regression” means that administering an effective amount of the drug, alone or in combination with an anti -neoplastic agent, results in a reduction in tumor growth or size, necrosis of the tumor, a decrease in severity of at least one disease symptom, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction.
• the terms “effective” and “effectiveness” with regard to a treatment includes both pharmacological effectiveness and physiological safety.
• Pharmacological effectiveness refers to the ability of the drug to promote cancer regression in the patient.
• Physiological safety refers to the level of toxicity, or other adverse physiological effects at the cellular, organ and/or organism level (adverse effects) resulting from administration of the drug.
• an "immuno-oncology" therapy or an “I-O” therapy refers to a therapy that comprises utilizing an immune response to target and treat a tumor in a subject.
• an 1-0 therapy is a type of anti-cancer therapy.
• 1-0 therapy comprises administering an antibody or an antigen-binding fragment thereof to a subject.
• an 1-0 therapy comprises administering to a subject an immune cell, e.g ., a T cell, e.g. , a modified T cell, e.g. , a T cell modified to express a chimeric antigen receptor or an particular T cell receptor.
• the 1-0 therapy comprises administering a therapeutic vaccine to a subject. In some aspects, the 1-0 therapy comprises administering a cytokine or a chemokine to a subject. In some aspects, the 1-0 therapy comprises administering an interleukin to a subject. In some aspects, the 1-0 therapy comprises administering an interferon to a subject. In some aspects, the 1-0 therapy comprises administering a colony stimulating factor to a subject.
• a therapeutically effective amount of an anti-cancer agent preferably inhibits cell growth or tumor growth by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80% relative to untreated subjects.
• tumor regression can be observed and continue for a period of at least about 20 days, more preferably at least about 40 days, or even more preferably at least about 60 days. Notwithstanding these ultimate measurements of therapeutic effectiveness, evaluation of immunotherapeutic drugs must also make allowance for immune-related response patterns.
• an "immune response” is as understood in the art, and generally refers to a biological response within a vertebrate against foreign agents or abnormal, e.g., cancerous cells, which response protects the organism against these agents and diseases caused by them.
• An immune response is mediated by the action of one or more cells of the immune system (for example, a T lymphocyte, B lymphocyte, natural killer (NK) cell, macrophage, eosinophil, mast cell, dendritic cell or neutrophil) and soluble macromolecules produced by any of these cells or the liver (including antibodies, cytokines, and complement) that results in selective targeting, binding to, damage to, destruction of, and/or elimination from the vertebrate's body of invading pathogens, cells or tissues infected with pathogens, cancerous or other abnormal cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues.
• a T lymphocyte, B lymphocyte, natural killer (NK) cell for example, a T lymphocyte, B lymphocyte, natural killer (NK) cell, macrophage, eosinophil, mast cell, dendritic cell or neutrophil
• soluble macromolecules produced by any of these cells or the liver (including antibodies, cytokines, and complement) that results
• An immune reaction includes, e.g, activation or inhibition of a T cell, e.g, an effector T cell, a Th cell, a CD4 + cell, a CD8 + T cell, or a Treg cell, or activation or inhibition of any other cell of the immune system, e.g, NK cell.
• a T cell e.g, an effector T cell, a Th cell, a CD4 + cell, a CD8 + T cell, or a Treg cell
• any other cell of the immune system e.g, NK cell.
• an "immune-related response pattern” refers to a clinical response pattern often observed in cancer patients treated with immunotherapeutic agents that produce antitumor effects by inducing cancer-specific immune responses or by modifying native immune processes.
• This response pattern is characterized by a beneficial therapeutic effect that follows an initial increase in tumor burden or the appearance of new lesions, which in the evaluation of traditional chemotherapeutic agents would be classified as disease progression and would be synonymous with drug failure. Accordingly, proper evaluation of immunotherapeutic agents can require long term monitoring of the effects of these agents on the target disease.
• treat refers to any type of intervention or process performed on, or administering an active agent to, the subject with the objective of reversing, alleviating, ameliorating, inhibiting, or slowing down or preventing the progression, development, severity or recurrence of a symptom, complication, condition or biochemical indicia associated with a disease or enhancing overall survival.
• Treatment can be of a subject having a disease or a subject who does not have a disease (e.g, for prophylaxis).
• an effective dose or “effective dosage” is defined as an amount sufficient to achieve or at least partially achieve a desired effect.
• a “therapeutically effective amount” or “therapeutically effective dosage” of a drug or therapeutic agent is any amount of the drug that, when used alone or in combination with another therapeutic agent, promotes disease regression evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, an increase in overall survival (the length of time from either the date of diagnosis or the start of treatment for a disease, such as cancer, that patients diagnosed with the disease are still alive), or a prevention of impairment or disability due to the disease affliction.
• a therapeutically effective amount or dosage of a drug includes a "prophylactically effective amount” or a “prophylactically effective dosage”, which is any amount of the drug that, when administered alone or in combination with another therapeutic agent to a subject at risk of developing a disease or of suffering a recurrence of disease, inhibits the development or recurrence of the disease.
• a therapeutic agent to promote disease regression or inhibit the development or recurrence of the disease can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.
• an anti-cancer agent is a drug that promotes cancer regression in a subject.
• a therapeutically effective amount of the drug promotes cancer regression to the point of eliminating the cancer.
• "Promoting cancer regression” means that administering an effective amount of the drug, alone or in combination with an antineoplastic agent, results in a reduction in tumor growth or size, necrosis of the tumor, a decrease in severity of at least one disease symptom, an increase in frequency and duration of disease symptom-free periods, an increase in overall survival, a prevention of impairment or disability due to the disease affliction, or otherwise amelioration of disease symptoms in the patient.
• the terms "effective” and “effectiveness” with regard to a treatment includes both pharmacological effectiveness and physiological safety.
• Pharmacological effectiveness refers to the ability of the drug to promote cancer regression in the patient.
• Physiological safety refers to the level of toxicity, or other adverse physiological effects at the cellular, organ and/or organism level (adverse effects) resulting from administration of the drug.
• a therapeutically effective amount or dosage of the drug inhibits cell growth or tumor growth by at least about 20%, by at least about 40%, by at least about 60%, or by at least about 80% relative to untreated subjects.
• a therapeutically effective amount or dosage of the drug completely inhibits cell growth or tumor growth, i.e., inhibits cell growth or tumor growth by 100%.
• the ability of a compound to inhibit tumor growth can be evaluated using an assay described herein. Alternatively, this property of a composition can be evaluated by examining the ability of the compound to inhibit cell growth, such inhibition can be measured in vitro by assays known to the skilled practitioner.
• tumor regression can be observed and continue for a period of at least about 20 days, at least about 40 days, or at least about 60 days.
• the term "biological sample” as used herein refers to biological material isolated from a subject.
• the biological sample can contain any biological material suitable for determining target gene expression, for example, by sequencing nucleic acids in the tumor (or circulating tumor cells) and identifying a genomic alteration in the sequenced nucleic acids.
• the biological sample can be any suitable biological tissue or fluid such as, for example, tumor tissue, blood, blood plasma, and serum.
• the sample is a tumor sample.
• the tumor sample can be obtained from a tumor tissue biopsy, e.g ., a formalin-fixed, paraffin- embedded (FFPE) tumor tissue or a fresh-frozen tumor tissue or the like.
• the biological sample is a liquid biopsy that, in some aspects, comprises one or more of blood, serum, plasma, circulating tumor cells, exoRNA, ctDNA, and cfDNA.
• a tumor sample refers to a biological sample that comprises tumor tissue.
• a tumor sample is a tumor biopsy.
• a tumor sample comprises tumor cells and one or more non-tumor cell present in the tumor microenvironment (TME).
• TME tumor microenvironment
• the TME is made up of at least two regions.
• the tumor "parenchyma” is a region of the TME that includes predominantly tumor cells, e.g. , the part (or parts) of the TME that includes the bulk of the tumor cells.
• the tumor parenchyma does not necessarily consist of only tumor cells, rather other cells such as stromal cells and/or lymphocytes can also be present in the parenchyma.
• the "stromal" region of the TME includes the adjacent non-tumor cells.
• the tumor sample comprises all or part of the tumor parenchyma and one or more cells of the stroma.
• the tumor sample is obtained from the parenchyma.
• the tumor sample is obtained from the stroma.
• the tumor sample is obtained from the parenchyma and the stroma.
• the terms "about” or “comprising essentially of refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system.
• “about” or “comprising essentially of' can mean within 1 or more than 1 standard deviation per the practice in the art.
• “about” or “comprising essentially of' can mean a range of up to 10%.
• the terms can mean up to an order of magnitude or up to 5-fold of a value.
• any concentration range, percentage range, ratio range or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
• Inflammation in the TME can be an indicator of potential responsiveness to an 1-0 therapy.
• contemporary methods for measuring inflammation in a tumor require the laborious process of immunohistochemistry to detect and analyze CD8 expression in a tumor biopsy. It was surprisingly found that the expression pattern of a relatively small number of genes (in some aspects, at least about 4 genes) correlates with inflammation in a tumor microenvironment. In some aspects, the methods described herein can replace the need for time- consuming IHC.
• Some aspects of the present disclosure are directed to methods of identifying a human subject suitable for an 1-0 therapy, e.g., an anti-PD-l/PD-Ll antagonist, comprising measuring expression of a panel of genes in a tumor sample obtained from a subject in need of the anti-PD-l/PD-Ll antagonist, wherein the gene panel comprises at least one of STAT1 , IFNy, NECTIN2, and CSF1R. In some aspects the measuring is conducted in vitro.
• Some aspects of the present disclosure are directed to a method for preparing a nucleic acid fraction from a tumor of a subject in need of an I/O therapy, comprising: (a) extracting a tumor biopsy from the subject; (b) producing a fraction of nucleic acids extracted in (a) by isolating the nucleic acids; and (c) analyzing the expression level of one or more genes in a gene panel selected from ST ATI, IFNy , NECTIN2, and CSF1R.
• the nucleic acids are mRNA.
• the gene panel comprises at least two of STAT1 , IFNy,
• the gene panel comprises STAT1 and IFNy. In some aspects, the gene panel comprises STAT1 and NECNN2. In some aspects, the gene panel comprises STAT1 and CSF1R. In some aspects, the gene panel comprises IFNy and NECTIN2. In some aspects, the gene panel comprises IFNy and CSF1R. In some aspects, the gene panel comprises NECTIN2 and CSF1R.
• the gene panel comprises at least three of STAT1 , IFNy,
• the gene panel comprises STAT1 , IFNy, and NECTIN2. In some aspects, the gene panel comprises STAT1 , IFNy, and CSF1R. In some aspects, the gene panel comprises STAT1 , NECTIN2, and CSF1R. In some aspects, the gene panel comprises IFNy, NECTIN2 , and CSF1R.
• the gene panel comprises ST ATI , IFNy, NECTIN2 , and CSF1R.
• the gene panel further comprises one or more additional genes.
• the gene panel comprises at least 2 to at least about 100 genes.
• the gene panel comprises at least 2 to at least about 95, at least 2 to at least about 90, at least 2 to at least about 85, at least 2 to at least about 80, at least 2 to at least about 75, at least 2 to at least about 70, at least 2 to at least about 65, at least 2 to at least about 60, at least 2 to at least about 55, at least 2 to at least about 50, at least 2 to at least about 45, at least 2 to at least about 40, at least 2 to at least about 35, at least 2 to at least about 30, at least 2 to at least about 25, at least 2 to at least about 20, at least 2 to at least about 15, at least 2 to at least about 10, at least 2 to at least about 9, at least 2 to at least about 8, at least 2 to at least about 7, at least 2 to at least about 6, at least 2 to at least about 5, or at least 2 to at least about 4 genes.
• the gene panel comprises at least 2, at least about 3, at least about
• the gene panel consists of STAT1 , IFNy, NECTIN2, and CSF1R.
• the gene panel consists essentially of STAT1 , IFNy, NECTIN2, and CSF1R. In some aspects, the gene panel consists or consists essentially of STAT1 , IFNy, NECTIN2, and CSF1R and at least 1 additional gene, at least 2 additional genes, at least 3 additional genes, at least 4 additional genes, at least 5 additional genes, at least 6 additional genes, at least 7 additional genes, at least 8 additional genes, at least 9 additional genes, at least 10 additional genes, at least 11 additional genes, at least 12 additional genes, at least 13 additional genes, at least 14 additional genes, at least 15 additional genes, at least 20 additional genes, at least 25 additional genes, at least 30 additional genes, at least 35 additional genes, at least 40 additional genes, at least 45 additional genes, at least 50 additional genes, at least 55 additional genes, at least 60 additional genes, at least 65 additional genes, at least 70 additional genes, at least 75 additional genes, at least 80 additional genes, at least 85 additional genes, at least 90 additional genes, at least 95 additional genes, or at least 100
• Gene expression profiling is a measurement of the combined expression level the genes in a panel disclosed herein, e.g ., comprising, consisting essentially of, or consisting of at least one, at least two, or at least three of SI A 77, IFNy, NECTIN2, and CSF1R.
• the measurement is made using a sample obtained from a subject.
• the sample is a tumor sample. Any biological sample comprising one or more tumor cell can be used in the methods disclosed herein.
• the sample is selected from a tumor biopsy, a blood sample, a serum sample, or any combination thereof.
• the sample is a tumor biopsy collected from the subject prior to administration of a therapy described herein, e.g. , an 1-0 therapy, e.g., an anti-PD-l/PD-Ll agonist.
• a therapy described herein e.g. , an 1-0 therapy, e.g., an anti-PD-l/PD-Ll agonist.
• the sample obtained from the subject is a formalin-fixed tumor biopsy.
• the sample obtained from the subject is a paraffin-embedded tumor biopsy.
• the sample obtained from the subject is a fresh-frozen tumor biopsy.
• any method known in the art for measuring the expression of a particular gene or a panel of genes can be used in the methods of the present disclosure.
• the expression of one or more of the inflammatory genes in the inflammatory gene panel is determined by detecting the presence of mRNA transcribed from the inflammatory gene, the presence of a protein encoded by the inflammatory gene, or both.
• the expression a gene is determined by measuring the level of gene mRNA, e.g., by measuring the level of one or more of STAT1 mRNA, IFNy mRNA, NECTIN2 mRNA, and CSF1R mRNA, in a sample obtained from the subject.
• the gene expression profile is determined by measuring the level of STAT1 mRNA, IFNy mRNA, NECTIN2 mRNA, CSF1R mRNA, or any combination thereof in a sample obtained from the subject. Any method known in the art can be used to measure the level of the gene mRNA.
• the gene mRNA is measured using reverse transcriptase PCR.
• the gene mRNA is measured using a nuclease protection assay.
• the gene mRNA is measured using next-generation sequencing (NGS).
• the gene mRNA is measured using RNA in situ hybridization.
• the expression of a gene is determined by measuring the level of protein encoded by the gene, e.g ., by measuring the level of one or more of STAT1 protein, IFNy protein, NECTIN2 protein, and CSF1R protein, in a sample obtained from the subject.
• the gene expression profile is determined by measuring the level of STAT1 protein, IFNy protein, NECTIN2 protein, CSF1R protein, or any combination thereof in a sample obtained from the subject. Any method known in the art can be used to measure the level of the protein.
• the gene expression profile is measured using an immunohistochemistry (IHC) assay.
• the IHC is an automated IHC.
• the expression of one or more of the genes of the gene panel is normalized relative to the expression of one or more housekeeping genes.
• the one or more housekeeping genes are made up of genes that have relatively consistent expression across various tumor types in various subjects.
• raw gene expression values are normalized following standard gene expression profiling protocols.
• a gene expression profile can be calculated as the median or average of the log2 -transformed normalized and scaled expression values across all of the target genes in the signature, and presented on a linear scale.
• profiles have positive or negative values, depending on whether gene expression is up- or down-regulated under a particular condition.
• increased/decreased expression is characterized by an expression level that is greater/less than the expression of the same gene or genes in a reference sample.
• the reference sample comprises a non-tumor tissue of the same subject.
• the reference sample comprises a corresponding non-tumor tissue of the same subject.
• the reference sample comprises a corresponding tissue in a subject that does not have a tumor.
• the reference sample comprises more than one tumor tissue sample from more than one other subject, e.g. , the increased expression is relative to an average expression level across more than one other tumor samples.
• the increased/decreased expression is characterized by an expression level that is greater/less than a reference expression level.
• the reference expression level is an average expression level.
• the average expression level is determined by measuring the expression of the gene (or the multiple genes) present in the gene panel in tumor samples obtained from a population of subjects, and calculating the average for the population of subjects.
• each member of the population of subjects is afflicted with the same tumor as the subject being administered the 1-0 therapy, e.g., an anti-PD- 1/PD-Ll antagonist.
• upregulated genes e.g., ST ATI or
• IFNy for parenchymal gene signature or NECTIN2 and CSF1R for stromal gene signature is characterized by an expression level that is at least about 5%, at least about 10%, at least about
• increased expression is characterized by an expression level that is at least about 25% higher than an expression level in a reference sample or than an average expression level. In certain aspects, increased expression is characterized by an expression level that is at least about 30% higher than an expression level in a reference sample or than an average expression level. In certain aspects, increased expression is characterized by an expression level that is at least about 35% higher than an expression level in a reference sample or than an average expression level.
• increased expression is characterized by an expression level that is at least about 40% higher than an expression level in a reference sample or than an average expression level. In certain aspects, increased expression is characterized by an expression level that is at least about 45% higher than an expression level in a reference sample or than an average expression level. In certain aspects, increased expression is characterized by an expression level that is at least about 50% higher than an expression level in a reference sample or than an average expression level. In certain aspects, increased expression is characterized by an expression level that is at least about 55% higher than an expression level in a reference sample or than an average expression level. In certain aspects, increased expression is characterized by an expression level that is at least about 60% higher than an expression level in a reference sample or than an average expression level.
• increased expression is characterized by an expression level that is at least about 65% higher than an expression level in a reference sample or than an average expression level. In certain aspects, increased expression is characterized by an expression level that is at least about 70% higher than an expression level in a reference sample or than an average expression level. In certain aspects, increased expression is characterized by an expression level that is at least about 75% higher than an expression level in a reference sample or than an average expression level. In certain aspects, increased expression is characterized by an expression level that is at least about 80% higher than an expression level in a reference sample or than an average expression level. In certain aspects, increased expression is characterized by an expression level that is at least about 85% higher than an expression level in a reference sample or than an average expression level.
• increased expression is characterized by an expression level that is at least about 90% higher than an expression level in a reference sample or than an average expression level. In certain aspects, increased expression is characterized by an expression level that is at least about 95% higher than an expression level in a reference sample or than an average expression level. In certain aspects, increased expression is characterized by an expression level that is at least about 100% higher than an expression level in a reference sample or than an average expression level. In certain aspects, increased expression is characterized by an expression level that is at least about 125% higher than an expression level in a reference sample or than an average expression level. In certain aspects, increased expression is characterized by an expression level that is at least about 150% higher than an expression level in a reference sample or than an average expression level.
• increased expression is characterized by an expression level that is at least about 175% higher than an expression level in a reference sample or than an average expression level. In certain aspects, increased expression is characterized by an expression level that is at least about 200% higher than an expression level in a reference sample or than an average expression level. In certain aspects, increased expression is characterized by an expression level that is at least about 225% higher than an expression level in a reference sample or than an average expression level. In certain aspects, increased expression is characterized by an expression level that is at least about 250% higher than an expression level in a reference sample or than an average expression level. In certain aspects, increased expression is characterized by an expression level that is at least about 275% higher than an expression level in a reference sample or than an average expression level. In certain aspects, increased expression is characterized by an expression level that is at least about 300% higher than an expression level in a reference sample or than an average expression level.
• increased expression of the upregulated genes e.g., ST ATI or
• IFNy for parenchymal gene signature or NECTIN2 and CSF1R for stromal gene signature is characterized by an expression level that is at least about 1.25-fold, at least about 1.30-fold, at least about 1.35-fold, at least about 1.40-fold, at least about 1.45-fold, at least about 1.50-fold, at least about 1.55-fold, at least about 1.60-fold, at least about 1.65-fold, at least about 1.70-fold, at least about 1.75-fold, at least about 1.80-fold, at least about 1.85-fold, at least about 1.90-fold, at least about 1.95-fold, at least about 2-fold, at least about 2.25-fold, at least about 2.50-fold, at least about 2.75-fold, at least about 3-fold, at least about 3.25-fold, at least about 3.50-fold, at least about 3.75-fold, or at least about 400-fold higher than an expression level in a reference sample or than an average expression level.
• increased expression is characterized by an expression level that is at least about 1.25-fold higher than an expression level in a reference sample or than an average expression level. In certain aspects, increased expression is characterized by an expression level that is at least about 1.30-fold higher than an expression level in a reference sample or than an average expression level. In certain aspects, increased expression is characterized by an expression level that is at least about 1.35-fold higher than an expression level in a reference sample or than an average expression level. In certain aspects, increased expression is characterized by an expression level that is at least about 1.40- fold higher than an expression level in a reference sample or than an average expression level.
• increased expression is characterized by an expression level that is at least about 1.45-fold higher than an expression level in a reference sample or than an average expression level. In certain aspects, increased expression is characterized by an expression level that is at least about 1.50-fold higher than an expression level in a reference sample or than an average expression level. In certain aspects, increased expression is characterized by an expression level that is at least about 1.55-fold higher than an expression level in a reference sample or than an average expression level. In certain aspects, increased expression is characterized by an expression level that is at least about 1.60-fold higher than an expression level in a reference sample or than an average expression level.
• increased expression is characterized by an expression level that is at least about 1.65-fold higher than an expression level in a reference sample or than an average expression level. In certain aspects, increased expression is characterized by an expression level that is at least about 1.70-fold higher than an expression level in a reference sample or than an average expression level. In certain aspects, increased expression is characterized by an expression level that is at least about 1.75-fold higher than an expression level in a reference sample or than an average expression level. In certain aspects, increased expression is characterized by an expression level that is at least about 1.80- fold higher than an expression level in a reference sample or than an average expression level.
• increased expression is characterized by an expression level that is at least about 1.85-fold higher than an expression level in a reference sample or than an average expression level. In certain aspects, increased expression is characterized by an expression level that is at least about 1.90-fold higher than an expression level in a reference sample or than an average expression level. In certain aspects, increased expression is characterized by an expression level that is at least about 1.95-fold higher than an expression level in a reference sample or than an average expression level. In certain aspects, increased expression is characterized by an expression level that is at least about 2-fold higher than an expression level in a reference sample or than an average expression level.
• increased expression is characterized by an expression level that is at least about 2.25-fold higher than an expression level in a reference sample or than an average expression level. In certain aspects, increased expression is characterized by an expression level that is at least about 2.50-fold higher than an expression level in a reference sample or than an average expression level. In certain aspects, increased expression is characterized by an expression level that is at least about 2.75-fold higher than an expression level in a reference sample or than an average expression level. In certain aspects, increased expression is characterized by an expression level that is at least about 3-fold higher than an expression level in a reference sample or than an average expression level. In certain aspects, increased expression is characterized by an expression level that is at least about 3.25- fold higher than an expression level in a reference sample or than an average expression level.
• increased expression is characterized by an expression level that is at least about 3.50-fold higher than an expression level in a reference sample or than an average expression level. In certain aspects, increased expression is characterized by an expression level that is at least about 3.75-fold higher than an expression level in a reference sample or than an average expression level. In certain aspects, increased expression is characterized by an expression level that is at least about 4-fold higher than an expression level in a reference sample or than an average expression level.
• decreased expression of the down-regulated genes e.g., a decreased expression of the down-regulated genes, e.g., a decreased expression of the down-regulated genes, e.g., a decreased expression of the down-regulated genes, e.g., a decreased expression of the down-regulated genes, e.g., a decreased expression of the down-regulated genes, e.g., a decreased expression of the down-regulated genes, e.g.,
• NECTIN2 and CSF1R for parenchymal gene signature or ST ATI or IFNy for stromal gene signature is characterized by an expression level that is less than a reference expression level.
• decreased expression is characterized by an expression level that is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 100%, at least about 125%, at least about 150%, at least about 175%, at least about 200%, at least about 225%, at least about 250%, at least about 275%, or at least about 300% lower than an expression level in a reference sample or than an average expression level.
• decreased expression is characterized by an expression level that is at least about 25% lower than an expression level in a reference sample or than an average expression level. In certain aspects, decreased expression is characterized by an expression level that is at least about 30% lower than an expression level in a reference sample or than an average expression level. In certain aspects, decreased expression is characterized by an expression level that is at least about 35% lower than an expression level in a reference sample or than an average expression level. In certain aspects, decreased expression is characterized by an expression level that is at least about 40% lower than an expression level in a reference sample or than an average expression level. In certain aspects, decreased expression is characterized by an expression level that is at least about 45% lower than an expression level in a reference sample or than an average expression level.
• decreased expression is characterized by an expression level that is at least about 50% lower than an expression level in a reference sample or than an average expression level. In certain aspects, decreased expression is characterized by an expression level that is at least about 55% lower than an expression level in a reference sample or than an average expression level. In certain aspects, decreased expression is characterized by an expression level that is at least about 60% lower than an expression level in a reference sample or than an average expression level. In certain aspects, decreased expression is characterized by an expression level that is at least about 65% lower than an expression level in a reference sample or than an average expression level. In certain aspects, decreased expression is characterized by an expression level that is at least about 70% lower than an expression level in a reference sample or than an average expression level.
• decreased expression is characterized by an expression level that is at least about 75% lower than an expression level in a reference sample or than an average expression level. In certain aspects, decreased expression is characterized by an expression level that is at least about 80% lower than an expression level in a reference sample or than an average expression level. In certain aspects, decreased expression is characterized by an expression level that is at least about 85% lower than an expression level in a reference sample or than an average expression level. In certain aspects, decreased expression is characterized by an expression level that is at least about 90% lower than an expression level in a reference sample or than an average expression level. In certain aspects, decreased expression is characterized by an expression level that is at least about 95% lower than an expression level in a reference sample or than an average expression level.
• decreased expression is characterized by an expression level that is at least about 100% lower than an expression level in a reference sample or than an average expression level. In certain aspects, decreased expression is characterized by an expression level that is at least about 125% lower than an expression level in a reference sample or than an average expression level. In certain aspects, decreased expression is characterized by an expression level that is at least about 150% lower than an expression level in a reference sample or than an average expression level. In certain aspects, decreased expression is characterized by an expression level that is at least about 175% lower than an expression level in a reference sample or than an average expression level. In certain aspects, decreased expression is characterized by an expression level that is at least about 200% lower than an expression level in a reference sample or than an average expression level.
• decreased expression is characterized by an expression level that is at least about 225% lower than an expression level in a reference sample or than an average expression level. In certain aspects, decreased expression is characterized by an expression level that is at least about 250% lower than an expression level in a reference sample or than an average expression level. In certain aspects, decreased expression is characterized by an expression level that is at least about 275% lower than an expression level in a reference sample or than an average expression level. In certain aspects, decreased expression is characterized by an expression level that is at least about 300% lower than an expression level in a reference sample or than an average expression level.
• decreased expression of the down-regulated genes e.g., a decreased expression of the down-regulated genes, e.g., a decreased expression of the down-regulated genes, e.g., a decreased expression of the down-regulated genes, e.g., a decreased expression of the down-regulated genes, e.g., a decreased expression of the down-regulated genes, e.g., a decreased expression of the down-regulated genes, e.g.,
• NECTIN2 and CSF1R for parenchymal gene signature or ST ATI or IFNy for stromal gene signature is characterized by an expression level that is at least about 1.25-fold, at least about 1.30-fold, at least about 1.35-fold, at least about 1.40-fold, at least about 1.45-fold, at least about
• decreased expression is characterized by an expression level that is at least about 1.25-fold lower than an expression level in a reference sample or than an average expression level. In certain aspects, decreased expression is characterized by an expression level that is at least about 1.30-fold lower than an expression level in a reference sample or than an average expression level. In certain aspects, decreased expression is characterized by an expression level that is at least about 1.35-fold lower than an expression level in a reference sample or than an average expression level.
• decreased expression is characterized by an expression level that is at least about 1.40- fold lower than an expression level in a reference sample or than an average expression level. In certain aspects, decreased expression is characterized by an expression level that is at least about 1.45-fold lower than an expression level in a reference sample or than an average expression level. In certain aspects, decreased expression is characterized by an expression level that is at least about 1.50-fold lower than an expression level in a reference sample or than an average expression level. In certain aspects, decreased expression is characterized by an expression level that is at least about 1.55-fold lower than an expression level in a reference sample or than an average expression level.
• decreased expression is characterized by an expression level that is at least about 1.60-fold lower than an expression level in a reference sample or than an average expression level. In certain aspects, decreased expression is characterized by an expression level that is at least about 1.65-fold lower than an expression level in a reference sample or than an average expression level. In certain aspects, decreased expression is characterized by an expression level that is at least about 1.70-fold lower than an expression level in a reference sample or than an average expression level. In certain aspects, decreased expression is characterized by an expression level that is at least about 1.75-fold lower than an expression level in a reference sample or than an average expression level.
• decreased expression is characterized by an expression level that is at least about 1.80- fold lower than an expression level in a reference sample or than an average expression level. In certain aspects, decreased expression is characterized by an expression level that is at least about 1.85-fold lower than an expression level in a reference sample or than an average expression level. In certain aspects, decreased expression is characterized by an expression level that is at least about 1.90-fold lower than an expression level in a reference sample or than an average expression level. In certain aspects, decreased expression is characterized by an expression level that is at least about 1.95-fold lower than an expression level in a reference sample or than an average expression level.
• decreased expression is characterized by an expression level that is at least about 2-fold lower than an expression level in a reference sample or than an average expression level. In certain aspects, decreased expression is characterized by an expression level that is at least about 2.25-fold lower than an expression level in a reference sample or than an average expression level. In certain aspects, decreased expression is characterized by an expression level that is at least about 2.50-fold lower than an expression level in a reference sample or than an average expression level. In certain aspects, decreased expression is characterized by an expression level that is at least about 2.75-fold lower than an expression level in a reference sample or than an average expression level. In certain aspects, decreased expression is characterized by an expression level that is at least about 3-fold lower than an expression level in a reference sample or than an average expression level.
• decreased expression is characterized by an expression level that is at least about 3.25- fold lower than an expression level in a reference sample or than an average expression level. In certain aspects, decreased expression is characterized by an expression level that is at least about 3.50-fold lower than an expression level in a reference sample or than an average expression level. In certain aspects, decreased expression is characterized by an expression level that is at least about 3.75-fold lower than an expression level in a reference sample or than an average expression level. In certain aspects, decreased expression is characterized by an expression level that is at least about 4-fold lower than an expression level in a reference sample or than an average expression level.
• Certain aspects of the present disclosure are directed to methods of identifying a subject suitable for a therapy and then administering the therapy to the suitable subject.
• the methods of identifying a suitable subject described herein can be used in advance of any immuno-oncology (I-O) therapy.
• the suitable subject is to be administered and/or subsequently administered an antibody or antigen-binding fragment thereof that specifically binds a protein selected from PD-1, PD-L1, CTLA-4, LAG-3, TIGIT, TIM3, CSF1R, NKG2a, 0X40, ICOS, CD137, KIR, TGFp, IL-10, IL-8, IL-2, CD96, VISTA, B7-H4, Fas ligand, CXCR4, mesothelin, CD27, GITR, MICA, MICB, and any combination thereof.
• a protein selected from PD-1, PD-L1, CTLA-4, LAG-3, TIGIT, TIM3, CSF1R, NKG2a, 0X40, ICOS, CD137, KIR, TGFp, IL-10, IL-8, IL-2, CD96, VISTA, B7-H4, Fas ligand, CXCR4, mesothelin, CD27, GITR,
• the suitable subject is to be administered and/or subsequently administered an antibody or antigen-binding fragment thereof that specifically binds PD-1. In some aspects, the suitable subject is to be administered and/or subsequently administered an antibody or antigen-binding fragment thereof that specifically binds PD-L1. In some aspects, the suitable subject is to be administered and/or subsequently administered an antibody or antigen binding fragment thereof that specifically binds CTLA-4. In some aspects, the suitable subject is to be administered and/or subsequently administered an antibody or antigen-binding fragment thereof that specifically binds LAG-3. In some aspects, the suitable subject is to be administered and/or subsequently administered an antibody or antigen-binding fragment thereof that specifically binds TIGIT.
• the suitable subject is to be administered and/or subsequently administered an antibody or antigen-binding fragment thereof that specifically binds TIM3. In some aspects, the suitable subject is to be administered and/or subsequently administered an antibody or antigen-binding fragment thereof that specifically binds GITR. In some aspects, the suitable subject is to be administered and/or subsequently administered an antibody or antigen-binding fragment thereof that specifically binds MICA. In some aspects, the suitable subject is to be administered and/or subsequently administered an antibody or antigen binding fragment thereof that specifically binds MICB. In some aspects, the suitable subject is to be administered and/or subsequently administered an antibody or antigen-binding fragment thereof that specifically binds CSF1R.
• the suitable subject is to be administered and/or subsequently administered more than one antibody or antigen-binding fragment thereof disclosed herein. In some aspects, the suitable subject is to be administered and/or subsequently administered at least two antibodies or antigen-binding fragments thereof. In some aspects, the suitable subject is to be administered and/or subsequently administered at least three antibodies or antigen-binding fragments thereof. In certain aspects the suitable subject is to be administered and/or subsequently administered an antibody or antigen-binding fragment thereof that specifically binds PD-1 and an antibody or antigen-binding fragment thereof that specifically binds CTLA-4.
• the suitable subject is to be administered and/or subsequently administered an antibody or antigen-binding fragment thereof that specifically binds PD-L1 and an antibody or antigen-binding fragment thereof that specifically binds CTLA-4. In certain aspects the suitable subject is to be administered and/or subsequently administered an antibody or antigen-binding fragment thereof that specifically binds PD-1 and an antibody or antigen-binding fragment thereof that specifically binds CSF1R. In certain aspects the suitable subject is to be administered and/or subsequently administered an antibody or antigen-binding fragment thereof that specifically binds PD-L1 and an antibody or antigen-binding fragment thereof that specifically binds CSF1R.
• the suitable subject is to be administered and/or subsequently administered an antibody or antigen-binding fragment thereof that specifically binds PD-1 and an antibody or antigen-binding fragment thereof that specifically binds LAG-3. In certain aspects the suitable subject is to be administered and/or subsequently administered an antibody or antigen-binding fragment thereof that specifically binds PD-L1 and an antibody or antigen binding fragment thereof that specifically binds LAG-3.
• the therapy is administered to the suitable subject after the gene expression profile has been measured.
• the measuring is in vitro.
• the measuring is in vivo.
• the therapy is administered at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, or at least about 14 days after the gene expression profile has been measured.
• the particular therapy to be administered and/or subsequently administered to the suitable subject is dependent on the gene expression profile.
• the gene expression profile has a parenchymal signature.
• the gene expression profile has a stromal signature.
• the present disclosure is directed to a pharmaceutical composition
• a pharmaceutical composition comprising an 1-0 therapy, e.g., an anti-PD-l/PD-Ll antagonist, for use in a method of identifying a human subject suitable for the anti-PD-l/PD-Ll antagonist, wherein the method comprises measuring expression of a panel of genes in a tumor sample obtained from a subject in need of the anti-PD-l/PD-Ll antagonist, wherein the gene panel comprises at least three or four of STAT1 , IFNy, NECTIN2, and CSF1R.
• the subject is identified as being suitable when the tumor sample exhibits:
• the subject is to be administered an anti-PD-l/PD- Ll antagonist.
• the present disclosure provides, in some aspects, a pharmaceutical composition comprising an 1-0 therapy, e.g., an anti-PD-l/PD-Ll antagonist, for use in a method of treating a human subject afflicted with a tumor, wherein a tumor sample obtained from the subject exhibits:
• an 1-0 therapy e.g., an anti-PD-l/PD-Ll antagonist
• the disclosure provides a method of identifying a human subject suitable for an anti-PD-l/PD-Ll antagonist therapy, comprising measuring expression of a panel of genes in a tumor sample obtained from a subject in need of the anti-PD-l/PD-Ll antagonist, wherein the gene panel comprises at least three of STAT1 , IFNy, NECTIN2, and CSF1R.
• the measuring is in vitro. In other aspects, the measuring is in vivo.
• the subject has a parenchymal signature when the subject exhibits
• a subject having a parenchymal signature can be identified as being suitable for an 1-0 therapy, e.g ., an anti-PD-l/PD-Ll antagonist therapy.
• a subject having a parenchymal signature has a tumor characterized by (i) increased expression of ST ATI and/or IFNy ("upregulated genes"), (ii) decreased expression of NECTIN2 and/or CSF1R (“down- regulated genes”), or (ii) both (i) and (ii).
• the subject is identified as being suitable when the tumor sample exhibits: (i) an increased expression of STAT1 and/or IFNy ("upregulated genes") in the tumor sample compared to the expression of STAT1 and/or IFNy in a reference sample; (ii) a decreased expression of NECTIN2 and/or CSF1R ("down-regulated genes") in the tumor sample compared to the expression of one or more of NECTIN2 and/or CSF1R in a reference sample; or (iii) both (i) and (ii).
• a suitable subject has a tumor characterized by increased expression of STATE
• a suitable subject has a tumor characterized by increased expression of IFNy.
• a suitable subject has a tumor characterized by increased expression of ST ATI and IFNy. In some aspects, a suitable subject has a tumor characterized by decreased expression of NECTIN2. In some aspects, a suitable subject has a tumor characterized by decreased expression of CSF1R. In some aspects, a suitable subject has a tumor characterized by decreased expression of NECTIN2 and CSF1R. In some aspects, a suitable subject has a tumor characterized by increased expression of ST ATI and decreased expression of NECTIN2. In some aspects, a suitable subject has a tumor characterized by increased expression of ST ATI and decreased expression of NECTIN2. In some aspects, a suitable subject has a tumor characterized by increased expression of IFNy and decreased expression of NECTIN2.
• a suitable subject has a tumor characterized by increased expression of IFNy and decreased expression of CSF1R. In some aspects, a suitable subject has a tumor characterized by increased expression of ST ATI and IFNy and decreased expression of NECTIN2 and CSF1R.
• the suitable subject e.g ., the subject having a parenchymal signature described herein
• the suitable subject e.g., the subject having a parenchymal signature described herein
• the suitable subject e.g., the subject having a parenchymal signature described herein, is to be administered and/or subsequently administered an anti-PD-1 antibody.
• the suitable subject e.g, the subject having a parenchymal signature described herein
• the suitable subject e.g, the subject having a parenchymal signature described herein
• the suitable subject e.g, the subject having a parenchymal signature described herein
• the suitable subject e.g, the subject having a parenchymal signature described herein, is to be administered and/or subsequently administered a combination therapy comprising an anti-PD-1 antibody and one or more additional anti-cancer agents (e.g, one or more additional 1-0 therapy described herein, one or more chemotherapy, or any combination thereof).
• a combination therapy comprising an anti-PD-1 antibody and one or more additional anti-cancer agents (e.g, one or more additional 1-0 therapy described herein, one or more chemotherapy, or any combination thereof).
• an anti-PD-l/PD-Ll antagonist is administered to a subject, wherein a tumor sample obtained from the subject exhibits: (i) an increased expression of STAT1 and IFNy ("upregulated genes") in a tumor sample obtained from the subject compared to the expression of ST ATI and IFNy in a reference sample; (ii) a decreased expression of NECNN2 and CSF1R ("down-regulated genes") in a tumor sample obtained from the subject compared to the expression of one or more of NECTIN2 and CSF1R in a reference sample; or (iii) both (i) and (ii).
• STAT1 and IFNy upregulated genes
• NECNN2 and CSF1R down-regulated genes
• an anti-PD-l/PD-Ll antagonist is administered to a subject, wherein a tumor sample obtained from the subject does not exhibit: (i) a decreased expression of STAT1 or IFNy ("upregulated genes") in a tumor sample obtained from the subject compared to the expression of STAT1 or IFNy in a reference sample; (ii) an increased expression of NECTIN2 or CSF1R ("down-regulated genes") in a tumor sample obtained from the subject compared to the expression of one or more of NECTIN2 and CSF1R in a reference sample; or (iii) both (i) and (ii).
• STAT1 or IFNy upregulated genes
• NECTIN2 or CSF1R down-regulated genes
• the tumor sample from a subject does not exhibit parenchymal gene signature, but rather exhibits a gene signature showing: (i) an increased expression of one or more of NECTIN2 and CSF1R ("upregulated genes") in the sample compared to the expression of the one or more of NECTIN2 and CSF1R in a reference sample; (ii) a decreased expression of one or more of STAT1 and IFNy ("down-regulated genes") in the sample compared to the expression of one or more of STAT1 and IFNy in a reference sample or (iii) both (i) and (ii), e.g., stromal signature.
• NECTIN2 and CSF1R upregulated genes
• STAT1 and IFNy down-regulated genes
• the subject with a stromal gene signature is not suitable for an 1-0 monotherapy, e.g. an anti-PD-l/PD-Ll antagonist monotherapy.
• the subject with a stromal gene signature is suitable for a combination therapy of an 1-0 therapy and an anti cancer agent.
• the disclosure provides a pharmaceutical composition comprising an anti-PD-l/PD-Ll antagonist for use in a method of identifying a human subject suitable for a combination therapy of the anti-PD-l/PD-Ll antagonist in combination with an anti-cancer agent, wherein the method comprises measuring expression of a panel of genes in a tumor sample obtained from a subject in need of the combination therapy, wherein the gene panel comprises at least three of CSF1R, NECTIN2 , STAT1, and IFNy.
• the subject is identified as being suitable when the tumor sample exhibits: (i) an increased expression of one or more of CSF1R and NEE ⁇ N2 ("upregulated genes") in the sample compared to the expression of the one or more of CSF1R and NECTIN2 in a reference sample; (ii) a decreased expression of one or more of STAT1 and IFNy ("down- regulated genes") in the sample compared to the expression of one or more of STAT1 and IFNy in a reference sample or (iii) both (i) and (ii).
• the subject is to be administered an anti-PD-l/PD-Ll antagonist in combination with an anti-cancer agent.
• the disclosure provides a pharmaceutical composition comprising an anti-PD-l/PD-Ll antagonist in combination with an anti-cancer agent for use in a method of treating a human subject afflicted with a tumor, wherein a tumor sample obtained from the subject exhibits: (i) an increased expression of one or more of CSF1R and NEE ⁇ N2 ("upregulated genes") in a tumor sample obtained from the subject compared to the expression of the one or more of CSF1R and NECTIN2 in a reference sample; (ii) a decreased expression of one or more of STAT1 and IFNy (“down-regulated genes”) in a tumor sample obtained from the subject compared to the expression of one or more of STAT1 and IFNy in a reference sample; or (iii) both (i) and (ii).
• CSF1R and NEE ⁇ N2 upregulated genes
• STAT1 and IFNy down-regulated genes
• the disclosure is directed to a method of identifying a human subject suitable for a combination therapy of an anti-PD-l/PD-Ll antagonist in combination with an anti-cancer agent, comprising measuring expression of a panel of genes in a tumor sample obtained from a subject in need of the anti-PD-l/PD-Ll antagonist, wherein the gene panel comprises at least three of CSF1R , NECTIN2, STAT1 , and IFNy.
• the measuring is in vivo. In some aspects, the measuring is in vitro.
• the subject is identified as being suitable when the tumor sample exhibits: (i) an increased expression of one or more of CSF1R and NECNN2 ("upregulated genes") in the tumor sample compared to the expression of the one or more of CSF1R and NECTIN2 in a reference sample; (ii) a decreased expression of one or more of STAT1 and IFNy ("down-regulated genes") in the tumor sample compared to the expression of one or more of STAT1 and IFNy in a reference sample; or (iii) both (i) and (ii).
• the method further comprises administering the anti-PD-l/PD-Ll antagonist in combination with an anti-cancer agent.
• the subject has a stromal signature when the subject exhibits (i) an increased expression of one or more of CSF1R and NECTIN2 ("upregulated genes") in the tumor sample compared to the expression of the one or more of CSF1R and NECTIN2 in a reference sample; (ii) a decreased expression of one or more of STAT1 and IFNy (“down-regulated genes") in the tumor sample compared to the expression of one or more of STAT1 and IFNy in a reference sample; or (iii) both (i) and (ii).
• CSF1R and NECTIN2 upregulated genes
• STAT1 and IFNy down-regulated genes
• a subject having a stromal signature is identified as being suitable for a combination therapy comprising (i) an 1-0 therapy, e.g., anti-PD-l/PD-Ll antagonist therapy and (ii) an additional anti-cancer therapy.
• an 1-0 therapy e.g., anti-PD-l/PD-Ll antagonist therapy
• an additional anti-cancer therapy e.g., an additional anti-cancer therapy.
• a subject having a stromal signature has a tumor characterized by (i) decreased expression of STAT1 and/or IFNy ("down-regulated genes"), (ii) increased expression of NECTIN2 and/or CSF1R (“upregulated genes”), or (ii) both (i) and (ii).
• the subject is identified as being suitable when the tumor sample exhibits: (i) a decreased expression of STAT1 and/or IFNy ("down- regulated genes") in the tumor sample compared to the expression of STAT1 and/or IFNy in a reference sample; (ii) an increased expression of NECTIN2 and/or CSF1R ("upregulated genes") in the tumor sample compared to the expression of NECTIN2 and/or CSF1R in a reference sample; or (iii) both (i) and (ii).
• a suitable subject has a tumor characterized by decreased expression of STAT1.
• a suitable subject has a tumor characterized by decreased expression of IFNy.
• a suitable subject has a tumor characterized by decreased expression of ST ATI and IFNy. In some aspects, a suitable subject has a tumor characterized by increased expression of NECTIN2. In some aspects, a suitable subject has a tumor characterized by increased expression of CSF1R. In some aspects, a suitable subject has a tumor characterized by increased expression of NECTIN2 and CSF1R. In some aspects, a suitable subject has a tumor characterized by decreased expression of ST ATI and increased expression of NECTIN2. In some aspects, a suitable subject has a tumor characterized by decreased expression of STAT1 and increased expression of NECTIN2. In some aspects, a suitable subject has a tumor characterized by decreased expression of IFNy and increased expression of NECTIN2.
• a suitable subject has a tumor characterized by decreased expression of IFNy and increased expression of CSF1R. In some aspects, a suitable subject has a tumor characterized by decreased expression of STAT1 and IFNy and increased expression of NECNN2 and CSF1R.
• the suitable subject e.g., the subject having a stromal signature described herein
• a combination therapy comprising (i) an 1-0 therapy described herein and (ii) one or more additional anti-cancer agents.
• the suitable subject e.g, the subject having a stromal signature described herein
• a combination therapy comprising (i) an anti-PD-l/PD-Ll antagonist and (ii) one or more additional anti-cancer agents.
• the suitable subject e.g, the subject having a stromal signature described herein
• a combination therapy comprising (i) an anti-PD- 1 antibody and (ii) one or more additional anti-cancer agents.
• the suitable subject e.g, the subject having a stromal signature described herein
• a combination therapy comprising (i) an anti-PD-Ll antibody and (ii) one or more additional anti-cancer agents.
• the suitable subject e.g, the subject having a stromal signature described herein
• a combination therapy comprising (i) an anti-PD-1 antibody and (ii) an anti-CSFIR antibody.
• the suitable subject e.g, the subject having a stromal signature described herein
• a combination therapy comprising (i) an anti-PD-Ll antibody and (ii) an anti-CSFIR antibody.
• a suitable subject exhibits (i) an increased expression of one or more of CSF1R and NECTIN2 ("upregulated genes") in the tumor sample compared to the expression of the one or more of CSF1R and NECTIN2 in a reference sample; (ii) a decreased expression of one or more of STAT1 and IFNy (“down-regulated genes") in the tumor sample compared to the expression of one or more of STAT1 and IFNy in a reference sample; or (iii) both (i) and (ii).
• a subject is identified as being suitable for a combination therapy comprising (i) an 1-0 therapy, e.g., anti-PD-l/PD-Ll antagonist therapy and (ii) an additional anti-cancer therapy.
• a subject suitable for a combination therapy comprising (i) an 1-0 therapy, e.g., anti-PD-l/PD-Ll antagonist therapy has a tumor characterized by (i) decreased expression of STAT1 and/or IFNy ("down-regulated genes"), (ii) increased expression of NECTIN2 and/or CSF1R (“upregulated genes"), or (ii) both (i) and (ii).
• the subject is identified as being suitable when the tumor sample exhibits: (i) a decreased expression of STAT1 and/or IFNy ("down-regulated genes") in the tumor sample compared to the expression of STAT1 and/or IFNy in a reference sample; (ii) an increased expression of NECTIN2 and/or CSF1R ("upregulated genes") in the tumor sample compared to the expression of NECTIN2 and/or CSF1R in a reference sample; or (iii) both (i) and (ii).
• a suitable subject has a tumor characterized by decreased expression of STAT1.
• a suitable subject has a tumor characterized by decreased expression of IFNy.
• a suitable subject has a tumor characterized by decreased expression of STAT1 and IFNy. In some aspects, a suitable subject has a tumor characterized by increased expression of NECFN2. In some aspects, a suitable subject has a tumor characterized by increased expression of CSF1R. In some aspects, a suitable subject has a tumor characterized by increased expression of NECFN2 and CSF1R. In some aspects, a suitable subject has a tumor characterized by decreased expression of STAT1 and increased expression of NECTIN2. In some aspects, a suitable subject has a tumor characterized by decreased expression of STAT1 and increased expression of NECTIN2. In some aspects, a suitable subject has a tumor characterized by decreased expression of IFNy and increased expression of NECTIN2.
• a suitable subject has a tumor characterized by decreased expression of IFNy and increased expression of CSF1R. In some aspects, a suitable subject has a tumor characterized by decreased expression of STAT1 and IFNy and increased expression of Nl CTIN2 and CSF1R.
• the suitable subject is to be administered and/or subsequently administered a combination therapy comprising (i) an 1-0 therapy described herein and (ii) one or more additional anti-cancer agents.
• the suitable subject is to be administered and/or subsequently administered a combination therapy comprising (i) an anti-PD-l/PD-Ll antagonist and (ii) one or more additional anti-cancer agents.
• the suitable subject is to be administered and/or subsequently administered a combination therapy comprising (i) an anti-PD-1 antibody and (ii) one or more additional anti-cancer agents.
• the suitable subject is to be administered and/or subsequently administered a combination therapy comprising (i) an anti-PD-Ll antibody and (ii) one or more additional anti-cancer agents.
• the suitable subject is to be administered and/or subsequently administered a combination therapy comprising (i) an anti-PD-1 antibody and (ii) an anti-CSFIR antibody.
• the suitable subject is to be administered and/or subsequently administered a combination therapy comprising (i) an anti-PD-Ll antibody and (ii) an anti-CSFIR antibody.
• a combination therapy comprising (i) an anti-PD-l/PD-Ll antagonist and (ii) one or more additional anti-cancer agents is administered to a subject, wherein a tumor sample obtained from the subject exhibits: (i) a decreased expression of ST ATI and IFNy ("down-regulated genes") in a tumor sample obtained from the subject compared to the expression of ST ATI and IFNy in a reference sample; (ii) an increased expression of NECTIN2 and CSF1R ("upregulated genes”) in a tumor sample obtained from the subject compared to the expression of one or more of NECTIN2 and CSF1R in a reference sample; or (iii) both (i) and (ii).
• the one or more additional anti-cancer agents comprises an anti-CSFIR antibody.
• a combination therapy comprising (i) an anti-PD-l/PD-Ll antagonist and (ii) one or more additional anti-cancer agents is administered to a subject, wherein a tumor sample obtained from the subject does not exhibit: (i) an increased expression of ST ATI or IFNy ("down-regulated genes") in a tumor sample obtained from the subject compared to the expression of ST ATI or IFNy in a reference sample; (ii) a decreased expression of NECTIN2 or CSF1R ("upregulated genes”) in a tumor sample obtained from the subject compared to the expression of one or more of NECTIN2 and CSF1R in a reference sample; or (iii) both (i) and (ii).
• ST ATI or IFNy down-regulated genes
• NECTIN2 or CSF1R upregulated genes
• the present disclosure are directed to methods of treating a suitable subject, as determined according to a method disclosed herein, using an 1-0 therapy.
• Any 1-0 therapy known in the art can be used in the methods described herein.
• the 1-0 therapy comprises administering to the suitable subject an antibody or an antigen-binding fragment thereof the specifically binds a protein selected from PD-1, PD-L1, CTLA-4, LAG-3, TIGIT, TIM3, CSF1R, NKG2a, 0X40, ICOS, CD137, KIR, TGFp, IL-10, IL-8, IL-2, CD96, VISTA, B7-H4, Fas ligand, CXCR4, mesothelin, CD27, GITR, MICA, MICB, and any combination thereof.
• the subject is administered a single 1-0 therapy, i.e., a monotherapy.
• the subject is administered an anti-PD-1 antibody monotherapy.
• the subject is administered a combination therapy comprising a first 1-0 therapy and a second 1-0 therapy.
• the subject is administered a combination therapy comprising administering a first 1-0 therapy and an additional anti-cancer agent.
• the additional anti-cancer agent comprises a second 1-0 therapy, a chemotherapy, a standard of care therapy, or any combination thereof.
• the subject is administered a combination therapy comprising an anti-PD-1 antibody and a second anti-cancer agent. In certain aspects, the subject is administered a combination therapy comprising an anti-PD-1 antibody and an anti-CTLA-4 antibody. In certain aspects, the subject is administered a combination therapy comprising an anti-PD-1 antibody and an anti-CSFIR antibody.
• the subject is administered a combination therapy comprising an anti-PD-Ll antibody and a second anti-cancer agent.
• the subject is administered a combination therapy comprising an anti-PD-Ll antibody and an anti-CTLA-4 antibody.
• the subject is administered a combination therapy comprising an anti-PD-Ll antibody and an anti-CSFIR antibody.
• Anti-PD-1 antibodies that are known in the art can be used in the presently described compositions and methods.
• Various human monoclonal antibodies that bind specifically to PD-1 with high affinity have been disclosed in U.S. Patent No. 8,008,449.
• Anti-PD-1 antibodies usable in the present disclosure include monoclonal antibodies that bind specifically to human PD-1 and exhibit at least one, in some aspects, at least five, of the preceding characteristics. [0135] Other anti-PD-1 monoclonal antibodies have been described in, for example, U.S.
• the anti-PD-1 antibody is selected from the group consisting of nivolumab (also known as OPDIVO®, 5C4, BMS-936558, MDX-1106, and ONO-4538), pembrolizumab (Merck; also known as KEYTRUDA®, lambrolizumab, and MK-3475; see WO2008/156712), PDR001 (Novartis; see WO 2015/112900), MEDI-0680 (AstraZeneca; also known as AMP-514; see WO 2012/145493), cemiplimab (Regeneron; also known as REGN- 2810; see WO 2015/112800), JS001 (TAIZHOU JUNSHI PHARMA; also known as toripalimab; see Si-Yang Liu et ak, ./.
• nivolumab also known as OPDIVO®, 5C4, BMS-936558, MDX-1106, and ONO-4538
• BGB-A317 Beigene; also known as Tislelizumab; see WO 2015/35606 and US 2015/0079109
• INCSHR1210 Jiangsu Hengrui Medicine; also known as SHR-1210; see WO 2015/085847; Si-Yang Liu et ak, J. Hematol. Oncol. 70: 136 (2017)
• TSR-042 Tesaro Biopharmaceutical; also known as ANBOl l; see WO2014/179664)
• GLS-010 Wangi/Harbin Gloria Pharmaceuticals; also known as WBP3055; see Si-Yang Liu et ak, J.
• the anti-PD-1 antibody is nivolumab.
• Nivolumab is a fully human
• IgG4 S228P PD-1 immune checkpoint inhibitor antibody that selectively prevents interaction with PD-1 ligands (PD-L1 and PD-L2), thereby blocking the down-regulation of antitumor T-cell functions (U.S. Patent No. 8,008,449; Wang et ak, 2014 Cancer Immunol Res. 2(9): 846-56).
• the anti-PD-1 antibody is pembrolizumab.
• Pembrolizumab is a humanized monoclonal IgG4 (S228P) antibody directed against human cell surface receptor PD- 1 (programmed death-1 or programmed cell death-1). Pembrolizumab is described, for example, in U.S. Patent Nos. 8,354,509 and 8,900,587.
• Anti-PD-1 antibodies usable in the disclosed compositions and methods also include isolated antibodies that bind specifically to human PD-1 and cross-compete for binding to human PD-1 with any anti-PD-1 antibody disclosed herein, e.g., nivolumab (see, e.g, U.S. Patent No.
• the anti-PD-1 antibody binds the same epitope as any of the anti-PD-1 antibodies described herein, e.g, nivolumab.
• the ability of antibodies to cross-compete for binding to an antigen indicates that these monoclonal antibodies bind to the same epitope region of the antigen and sterically hinder the binding of other cross-competing antibodies to that particular epitope region.
• These cross-competing antibodies are expected to have functional properties very similar those of the reference antibody, e.g, nivolumab, by virtue of their binding to the same epitope region of PD-1.
• Cross-competing antibodies can be readily identified based on their ability to cross-compete with nivolumab in standard PD-1 binding assays such as Biacore analysis, ELISA assays or flow cytometry (see, e.g, WO 2013/173223).
• the antibodies that cross-compete for binding to human PD-1 with, or bind to the same epitope region of human PD-1 antibody, nivolumab are monoclonal antibodies.
• these cross-competing antibodies are chimeric antibodies, engineered antibodies, or humanized or human antibodies.
• Such chimeric, engineered, humanized or human monoclonal antibodies can be prepared and isolated by methods well known in the art.
• Anti-PD-1 antibodies usable in the compositions and methods of the disclosed disclosure also include antigen-binding portions of the above antibodies. It has been amply demonstrated that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
• Anti-PD-1 antibodies suitable for use in the disclosed compositions and methods are antibodies that bind to PD-1 with high specificity and affinity, block the binding of PD-L1 and or PD-L2, and inhibit the immunosuppressive effect of the PD-1 signaling pathway.
• an anti-PD-1 "antibody” includes an antigen binding portion or fragment that binds to the PD-1 receptor and exhibits the functional properties similar to those of whole antibodies in inhibiting ligand binding and up-regulating the immune system.
• the anti-PD-1 antibody or antigen-binding portion thereof cross- competes with nivolumab for binding to human PD-1.
• the anti-PD-1 antibody is administered at a dose ranging from 0.1 mg/kg to 20.0 mg/kg body weight once every 2, 3, 4, 5, 6, 7, or 8 weeks, e.g, 0.1 mg/kg to 10.0 mg/kg body weight once every 2, 3, or 4 weeks. In other aspects, the anti-PD-1 antibody is administered at a dose of about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, or 10 mg/kg body weight once every 2 weeks.
• the anti-PD-1 antibody is administered at a dose of about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, or 10 mg/kg body weight once every 3 weeks.
• the anti-PD-1 antibody is administered at a dose of about 5 mg/kg body weight about once every 3 weeks.
• the anti-PD-1 antibody e.g., nivolumab
• the anti-PD-1 antibody e.g, pembrolizumab
• the anti-PD-1 antibody useful for the present disclosure can be administered as a flat dose.
• the anti-PD-1 antibody is administered at a flat dose of from about 100 to about 1000 mg, from about 100 mg to about 900 mg, from about 100 mg to about 800 mg, from about 100 mg to about 700 mg, from about 100 mg to about 600 mg, from about 100 mg to about 500 mg, from about 200 mg to about 1000 mg, from about 200 mg to about 900 mg, from about 200 mg to about 800 mg, from about 200 mg to about 700 mg, from about 200 mg to about 600 mg, from about 200 mg to about 500 mg, from about 200 mg to about 480 mg, or from about 240 mg to about 480 mg,
• the anti-PD-1 antibody is administered as a flat dose of at least about 200 mg, at least about 220 mg, at least about 240 mg, at least about 260 mg, at least about 280 mg, at least about 300 mg, at least about 320 mg, at least about 340 mg, at least about 360 mg, at least about
• the anti-PD-1 antibody is administered as a flat dose of about 200 mg to about 800 mg, about 200 mg to about 700 mg, about 200 mg to about 600 mg, about 200 mg to about 500 mg, at a dosing interval of about 1, 2, 3, or 4 weeks.
• the anti-PD-1 antibody is administered as a flat dose of about 200 mg at about once every 3 weeks. In other aspects, the anti-PD-1 antibody is administered as a flat dose of about 200 mg at about once every 2 weeks. In other aspects, the anti-PD-1 antibody is administered as a flat dose of about 240 mg at about once every 2 weeks. In certain aspects, the anti-PD-1 antibody is administered as a flat dose of about 480 mg at about once every 4 weeks. [0146] In some aspects, nivolumab is administered at a flat dose of about 240 mg once about every 2 weeks. In some aspects, nivolumab is administered at a flat dose of about 240 mg once about every 3 weeks. In some aspects, nivolumab is administered at a flat dose of about 360 mg once about every 3 weeks. In some aspects, nivolumab is administered at a flat dose of about 480 mg once about every 4 weeks.
• pembrolizumab is administered at a flat dose of about 200 mg once about every 2 weeks. In some aspects, pembrolizumab is administered at a flat dose of about 200 mg once about every 3 weeks. In some aspects, pembrolizumab is administered at a flat dose of about 400 mg once about every 4 weeks.
• the PD-1 inhibitor is a small molecule. In some aspects, the PD-1 inhibitor comprises a millamolecule. In some aspects, the PD-1 inhibitor comprises a macrocyclic peptide. In certain aspects, the PD-1 inhibitor comprises BMS-986189. In some aspects, the PD-1 inhibitor comprises an inhibitor disclosed in International Publication No. WO2014/151634, which is incorporated by reference herein in its entirety. In some aspects, the PD-1 inhibitor comprises INCMGA00012 (Insight Pharmaceuticals). In some aspects, the PD-1 inhibitor comprises a combination of an anti -PD-1 antibody disclosed herein and a PD-1 small molecule inhibitor.
• an anti-PD-Ll antibody is substituted for the anti-PD-1 antibody in any of the methods disclosed herein.
• Anti-PD-Ll antibodies that are known in the art can be used in the compositions and methods of the present disclosure.
• Examples of anti-PD-Ll antibodies useful in the compositions and methods of the present disclosure include the antibodies disclosed in US Patent No. 9,580,507.
• 9,580,507 have been demonstrated to exhibit one or more of the following characteristics: (a) bind to human PD-L1 with a KD of 1 x 10 7 M or less, as determined by surface plasmon resonance using a Biacore biosensor system; (b) increase T-cell proliferation in a Mixed Lymphocyte Reaction (MLR) assay; (c) increase interferon-g production in an MLR assay; (d) increase IL-2 secretion in an MLR assay; (e) stimulate antibody responses; and (f) reverse the effect of T regulatory cells on T cell effector cells and/or dendritic cells.
• MLR Mixed Lymphocyte Reaction
• Anti- PD-Ll antibodies usable in the present disclosure include monoclonal antibodies that bind specifically to human PD-L1 and exhibit at least one, in some aspects, at least five, of the preceding characteristics.
• the anti-PD-Ll antibody is selected from the group consisting of BMS-936559 (also known as 12A4, MDX-1105; see, e.g., U S. Patent No. 7,943,743 and WO 2013/173223), atezolizumab (Roche; also known as TECENTRIQ®; MPDL3280A, RG7446; see US 8,217,149; see, also, Herbst et al.
• the PD-L1 antibody is atezolizumab (TECENTRIQ®).
• Atezolizumab is a fully humanized IgGl monoclonal anti-PD-Ll antibody.
• the PD-L1 antibody is durvalumab (IMFINZITM)
• Durvalumab is a human IgGl kappa monoclonal anti-PD-Ll antibody.
• the PD-L1 antibody is avelumab (BAVENCIO®).
• Avelumab is a human IgGl lambda monoclonal anti-PD-Ll antibody.
• Anti-PD-Ll antibodies usable in the disclosed compositions and methods also include isolated antibodies that bind specifically to human PD-L1 and cross-compete for binding to human PD-L1 with any anti-PD-Ll antibody disclosed herein, e.g, atezolizumab, durvalumab, and/or avelumab.
• the anti-PD-Ll antibody binds the same epitope as any of the anti-PD-Ll antibodies described herein, e.g, atezolizumab, durvalumab, and/or avelumab.
• antibodies to cross-compete for binding to an antigen indicates that these antibodies bind to the same epitope region of the antigen and sterically hinder the binding of other cross- competing antibodies to that particular epitope region.
• These cross-competing antibodies are expected to have functional properties very similar those of the reference antibody, e.g, atezolizumab and/or avelumab, by virtue of their binding to the same epitope region of PD-L1.
• Cross-competing antibodies can be readily identified based on their ability to cross-compete with atezolizumab and/or avelumab in standard PD-L1 binding assays such as Biacore analysis, ELISA assays or flow cytometry (see, e.g., WO 2013/173223).
• the antibodies that cross-compete for binding to human PD-L1 with, or bind to the same epitope region of human PD-L1 antibody as, atezolizumab, durvalumab, and/or avelumab are monoclonal antibodies.
• these cross-competing antibodies are chimeric antibodies, engineered antibodies, or humanized or human antibodies.
• Such chimeric, engineered, humanized or human monoclonal antibodies can be prepared and isolated by methods well known in the art.
• Anti-PD-Ll antibodies usable in the compositions and methods of the disclosed disclosure also include antigen-binding portions of the above antibodies. It has been amply demonstrated that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
• Anti-PD-Ll antibodies suitable for use in the disclosed compositions and methods are antibodies that bind to PD-L1 with high specificity and affinity, block the binding of PD-1, and inhibit the immunosuppressive effect of the PD-1 signaling pathway.
• an anti-PD-Ll "antibody” includes an antigen-binding portion or fragment that binds to PD-L1 and exhibits the functional properties similar to those of whole antibodies in inhibiting receptor binding and up-regulating the immune system.
• the anti-PD-Ll antibody or antigen-binding portion thereof cross-competes with atezolizumab, durvalumab, and/or avelumab for binding to human PD-L1.
• the anti-PD-Ll antibody useful for the present disclosure can be any PD-L1 antibody that specifically binds to PD-L1, e.g., antibodies that cross-compete with durvalumab, avelumab, or atezolizumab for binding to human PD-1, e.g, an antibody that binds to the same epitope as durvalumab, avelumab, or atezolizumab.
• the anti-PD-Ll antibody is durvalumab.
• the anti-PD-Ll antibody is avelumab.
• the anti-PD-Ll antibody is atezolizumab.
• the anti-PD-Ll antibody is administered at a dose ranging from about 0.1 mg/kg to about 20.0 mg/kg body weight, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg, about 5 mg/kg, about 6 mg/kg, about 7 mg/kg, about 8 mg/kg, about 9 mg/kg, about 10 mg/kg, about 11 mg/kg, about 12 mg/kg, about 13 mg/kg, about 14 mg/kg, about 15 mg/kg, about 16 mg/kg, about 17 mg/kg, about 18 mg/kg, about 19 mg/kg, or about 20 mg/kg, about once every 2, 3, 4, 5, 6, 7, or 8 weeks.
• the anti-PD-Ll antibody is administered at a dose of about 15 mg/kg body weight at about once every 3 weeks. In other aspects, the anti-PD-Ll antibody is administered at a dose of about 10 mg/kg body weight at about once every 2 weeks.
• the anti-PD-Ll antibody useful for the present disclosure is a flat dose.
• the anti-PD-Ll antibody is administered as a flat dose of from about 200 mg to about 1600 mg, about 200 mg to about 1500 mg, about 200 mg to about 1400 mg, about 200 mg to about 1300 mg, about 200 mg to about 1200 mg, about 200 mg to about 1100 mg, about 200 mg to about 1000 mg, about 200 mg to about 900 mg, about 200 mg to about 800 mg, about 200 mg to about 700 mg, about 200 mg to about 600 mg, about 700 mg to about 1300 mg, about 800 mg to about 1200 mg, about 700 mg to about 900 mg, or about 1100 mg to about 1300 mg.
• the anti-PD-Ll antibody is administered as a flat dose of at least about 240 mg, at least about 300 mg, at least about 320 mg, at least about 400 mg, at least about 480 mg, at least about 500 mg, at least about 560 mg, at least about 600 mg, at least about 640 mg, at least about 700 mg, at least 720 mg, at least about 800 mg, at least about 840 mg, at least about 880 mg, at least about 900 mg, at least 960 mg, at least about 1000 mg, at least about 1040 mg, at least about 1100 mg, at least about 1120 mg, at least about 1200 mg, at least about 1280 mg, at least about 1300 mg, at least about 1360 mg, or at least about 1400 mg, at a dosing interval of about 1, 2, 3, or 4 weeks.
• the anti-PD-Ll antibody is administered as a flat dose of about 1200 mg at about once every 3 weeks. In other aspects, the anti-PD-Ll antibody is administered as a flat dose of about 800 mg at about once every 2 weeks. In other aspects, the anti-PD-Ll antibody is administered as a flat dose of about 840 mg at about once every 2 weeks.
• Atezolizumab is administered as a flat dose of about 1200 mg once about every 3 weeks. In some aspects, atezolizumab is administered as a flat dose of about 800 mg once about every 2 weeks. In some aspects, atezolizumab is administered as a flat dose of about 840 mg once about every 2 weeks.
• avelumab is administered as a flat dose of about 800 mg once about every 2 weeks.
• durvalumab is administered at a dose of about 10 mg/kg once about every 2 weeks. In some aspects, durvalumab is administered as a flat dose of about 800 mg/kg once about every 2 weeks. In some aspects, durvalumab is administered as a flat dose of about 1200 mg/kg once about every 3 weeks.
• the PD-L1 inhibitor is a small molecule. In some aspects, the PD-L1 inhibitor is a small molecule.
• the L1 inhibitor comprises a millamolecule.
• the PD-L1 inhibitor comprises a macrocyclic peptide.
• the PD-L1 inhibitor comprises BMS-986189.
• the PD-L1 inhibitor comprises a millamolecule having a formula set forth in formula (I): wherein R'-R 1 ’ are amino acid side chains, R a -R n are hydrogen, methyl, or form a ring with a vicinal R group, and R 14 is -C(0)NHR 15 , wherein R 15 is hydrogen, or a glycine residue optionally substituted with additional glycine residues and/or tails which can improve pharmacokinetic properties.
• the PD-L1 inhibitor comprises a compound disclosed in International Publication No. WO2014/151634, which is incorporated by reference herein in its entirety.
• the PD-L1 inhibitor comprises a compound disclosed in International Publication No. WO2016/039749, WO2016/149351, WO2016/077518, W02016/100285, WO2016/100608, WO2016/126646, WO2016/057624, W02017/151830, WO2017/176608, W02018/085750, WO2018/237153, or WO2019/070643, each of which is incorporated by reference herein in its entirety.
• the PD-L1 inhibitor comprises a small molecule PD-L1 inhibitor disclosed in International Publication No. WO2015/034820, WO2015/160641, WO2018/044963, WO2017/066227, W02018/009505, WO2018/183171, WO2018/118848, WO2019/147662, or
• the PD-L1 inhibitor comprises a combination of an anti-PD-Ll antibody disclosed herein and a PD-L1 small molecule inhibitor disclosed herein.
• Anti-CTLA-4 antibodies that are known in the art can be used in the compositions and methods of the present disclosure.
• Anti-CTLA-4 antibodies of the instant disclosure bind to human CTLA-4 so as to disrupt the interaction of CTLA-4 with a human B7 receptor. Because the interaction of CTLA-4 with B7 transduces a signal leading to inactivation of T-cells bearing the CTLA-4 receptor, disruption of the interaction effectively induces, enhances or prolongs the activation of such T cells, thereby inducing, enhancing or prolonging an immune response.
• 6,984,720 have been demonstrated to exhibit one or more of the following characteristics: (a) binds specifically to human CTLA-4 with a binding affinity reflected by an equilibrium association constant (K a ) of at least about 10 7 M 1 , or about 10 9 M 1 , or about 10 10 M 1 to 10 11 M 1 or higher, as determined by Biacore analysis; (b) a kinetic association constant (U) of at least about 10 3 , about 10 4 , or about 10 5 m 1 s 1 ; (c) a kinetic disassociation constant (L/) of at least about 10 3 , about 10 4 , or about 10 5 m 1 s 1 ; and (d) inhibits the binding of CTLA-4 to B7-1 (CD80) and B7-2 (CD86).
• Anti-CTLA-4 antibodies useful for the present disclosure include monoclonal antibodies that bind specifically to human CTLA-4 and exhibit at least one, at least two, or at least three of the preceding characteristics.
• the CTLA-4 antibody is selected from the group consisting of ipilimumab (also known as YERVOY®, MDX-010, 10D1; see U.S. Patent No. 6,984,720), MK- 1308 (Merck), AGEN-1884 (Agenus Inc.; see WO 2016/196237), and tremelimumab (AstraZeneca; also known as ticilimumab, CP-675,206; see WO 2000/037504 and Ribas, Update Cancer Ther. 2(3): 133-39 (2007)).
• the anti-CTLA-4 antibody is ipilimumab.
• the CTLA-4 antibody is ipilimumab for use in the compositions and methods disclosed herein.
• Ipilimumab is a fully human, IgGl monoclonal antibody that blocks the binding of CTLA-4 to its B7 ligands, thereby stimulating T cell activation and improving overall survival (OS) in patients with advanced melanoma.
• the CTLA-4 antibody is tremelimumab.
• the CTLA-4 antibody is MK-1308.
• the CTLA-4 antibody is AGEN-1884.
• Anti-CTLA-4 antibodies usable in the disclosed compositions and methods also include isolated antibodies that bind specifically to human CTLA-4 and cross-compete for binding to human CTLA-4 with any anti-CTLA-4 antibody disclosed herein, e.g. , ipilimumab and/or tremelimumab.
• the anti-CTLA-4 antibody binds the same epitope as any of the anti-CTLA-4 antibodies described herein, e.g., ipilimumab and/or tremelimumab.
• antibodies to cross-compete for binding to an antigen indicates that these antibodies bind to the same epitope region of the antigen and sterically hinder the binding of other cross- competing antibodies to that particular epitope region.
• These cross-competing antibodies are expected to have functional properties very similar those of the reference antibody, e.g, ipilimumab and/or tremelimumab, by virtue of their binding to the same epitope region of CTLA-4.
• Cross-competing antibodies can be readily identified based on their ability to cross- compete with ipilimumab and/or tremelimumab in standard CTLA-4 binding assays such as Biacore analysis, ELISA assays or flow cytometry (see, e.g, WO 2013/173223).
• these cross- competing antibodies are chimeric antibodies, engineered antibodies, or humanized or human antibodies.
• Such chimeric, engineered, humanized or human monoclonal antibodies can be prepared and isolated by methods well known in the art.
• Anti-CTLA-4 antibodies usable in the compositions and methods of the disclosed disclosure also include antigen-binding portions of the above antibodies. It has been amply demonstrated that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody.
• Anti-CTLA-4 antibodies suitable for use in the disclosed methods or compositions are antibodies that bind to CTLA-4 with high specificity and affinity, block the activity of CTLA-4, and disrupt the interaction of CTLA-4 with a human B7 receptor.
• an anti-CTLA-4 "antibody” includes an antigen binding portion or fragment that binds to CTLA-4 and exhibits the functional properties similar to those of whole antibodies in inhibiting the interaction of CTLA-4 with a human B7 receptor and up-regulating the immune system.
• the anti-CTLA-4 antibody or antigen binding portion thereof cross-competes with ipilimumab and/or tremelimumab for binding to human CTLA-4.
• the anti-CTLA-4 antibody or antigen-binding portion thereof is administered at a dose ranging from 0.1 mg/kg to 10.0 mg/kg body weight once every 2, 3, 4, 5, 6, 7, or 8 weeks. In some aspects, the anti-CTLA-4 antibody or antigen-binding portion thereof is administered at a dose of 1 mg/kg or 3 mg/kg body weight once every 3, 4, 5, or 6 weeks. In one aspect, the anti-CTLA-4 antibody or antigen-binding portion thereof is administered at a dose of 3 mg/kg body weight once every 2 weeks. In another aspect, the anti-PD-1 antibody or antigen binding portion thereof is administered at a dose of 1 mg/kg body weight once every 6 weeks.
• the anti-CTLA-4 antibody or antigen-binding portion thereof is administered as a flat dose.
• the anti-CTLA-4 antibody is administered at a flat dose of from about 10 to about 1000 mg, from about 10 mg to about 900 mg, from about 10 mg to about 800 mg, from about 10 mg to about 700 mg, from about 10 mg to about 600 mg, from about 10 mg to about 500 mg, from about 100 mg to about 1000 mg, from about 100 mg to about 900 mg, from about 100 mg to about 800 mg, from about 100 mg to about 700 mg, from about 100 mg to about 100 mg, from about 100 mg to about 500 mg, from about 100 mg to about 480 mg, or from about 240 mg to about 480 mg.
• the anti-CTLA-4 antibody or antigen binding portion thereof is administered as a flat dose of at least about 60 mg, at least about 80 mg, at least about 100 mg, at least about 120 mg, at least about 140 mg, at least about 160 mg, at least about 180 mg, at least about 200 mg, at least about 220 mg, at least about 240 mg, at least about 260 mg, at least about 280 mg, at least about 300 mg, at least about 320 mg, at least about 340 mg, at least about 360 mg, at least about 380 mg, at least about 400 mg, at least about 420 mg, at least about 440 mg, at least about 460 mg, at least about 480 mg, at least about 500 mg, at least about 520 mg at least about 540 mg, at least about 550 mg, at least about 560 mg, at least about 580 mg, at least about 600 mg, at least about 620 mg, at least about 640 mg, at least about 660 mg, at least about 680 mg, at least about 700 mg, or at least about 720 mg
• ipilimumab is administered at a dose of about 3 mg/kg once about every 3 weeks. In some aspects, ipilimumab is administered at a dose of about 10 mg/kg once about every 3 weeks. In some aspects, ipilimumab is administered at a dose of about 10 mg/kg once about every 12 weeks. In some aspects, the ipilimumab is administered for four doses. II.C.4. Anti-LA G-3 Antibodies
• Anti-LAG-3 antibodies of the instant disclosure bind to human LAG-3.
• An exemplary LAG-3 antibody useful in the present disclosure is 25F7 (described in U.S. Publ. No. 2011/0150892).
• An additional exemplary LAG-3 antibody useful in the present disclosure is BMS-986016.
• an anti -LAG-3 antibody useful for the composition cross-competes with 25F7 or BMS-986016.
• an anti -LAG-3 antibody useful for the composition binds to the same epitope as 25F7 or BMS-986016.
• an anti- LAG-3 antibody comprises six CDRs of 25F7 or BMS-986016.
• the anti-LAG- 3 antibody is IMP731 (H5L7BW), MK-4280 (28G-10), REGN3767, humanized BAP050, IMP- 701 (LAG-5250), TSR-033, BI754111, MGD013, or FS-118.
• anti-LAG-3 antibodies useful in the claimed invention can be found in, for example: WO2016/028672, W02017/106129, WO2017/062888, W02009/044273, WO2018/069500, WO2016/126858, WO2014/ 179664, WO2016/200782, WO2015/200119, WO2017/019846, WO2017/198741, WO2017/220555, WO2017/220569, WO2018/071500, W02017/015560, WO2017/025498, WO2017/087589, WO2017/087901, W02018/083087, WO2017/149143, WO2017/219995, US2017/0260271, WO2017/086367, WO2017/086419, WO2018/034227, and W02014/140180, each of which is incorporated by reference herein in its entirety.
• Anti-CD137 antibodies specifically bind to and activate CD137-expressing immune cells, stimulating an immune response, in particular a cytotoxic T cell response, against tumor cells.
• Antibodies that bind to CD137 have been disclosed in U.S. Publ. No. 2005/0095244 and U.S. Pat. Nos. 7,288,638, 6,887,673, 7,214,493, 6,303,121, 6,569,997, 6,905,685, 6,355,476, 6,362,325, 6,974,863, and 6,210,669, each of which is incorporated by reference herein in its entirety.
• the anti-CD137 antibody is urelumab (BMS-663513), described in U.S. Pat. No. 7,288,638 (20H4.9-IgG4 [10C7 or BMS-663513]).
• the anti- CD137 antibody is BMS-663031 (20H4.9-IgGl), described in U.S. Pat. No. 7,288,638.
• the anti-CD137 antibody is 4E9 or BMS-554271, described in U.S. Pat. No. 6,887,673.
• the anti-CD137 antibody is an antibody disclosed in U.S. Pat. Nos.
• the anti-CD137 antibody is 1D8 or BMS-469492; 3H3 or BMS-469497; or 3E1, described in U.S. Pat. No. 6,362,325.
• the anti-CD137 antibody is an antibody disclosed in issued U.S. Pat. No. 6,974,863 (such as 53A2).
• the anti-CD137 antibody is an antibody disclosed in issued U.S. Pat. No. 6,210,669 (such as 1D8, 3B8, or 3E1).
• the antibody is Pfizer's PF- 05082566 (PF-2566).
• an anti-CD137 antibody useful for the methods disclosed herein cross-competes with the anti-CD137 antibodies disclosed herein.
• an anti- CD137 antibody binds to the same epitope as the anti-CD137 antibody disclosed herein.
• an anti-CD137 antibody useful in the disclosure comprises six CDRs of the anti-CD137 antibodies disclosed herein.
• KIR Killer-cell immunoglobulin-like receptors
• NK cells Blocking these receptors facilitates activation of NK cells and, potentially, destruction of tumor cells by the latter.
• Examples of anti-KIR antibodies have been disclosed in Int'l Publ. Nos. WO/2014/055648, WO 2005/003168, WO 2005/009465, WO 2006/072625, WO 2006/072626, WO 2007/042573, WO 2008/084106, WO 2010/065939, WO 2012/071411 and WO/2012/160448, each of which is incorporated by reference herein in its entirety.
• One anti-KIR antibody useful in the present disclosure is lirilumab (also referred to as BMS-986015, IPH2102, or the S241P variant of 1-7F9), first described in Int'l Publ. No. WO 2008/084106.
• An additional anti-KIR antibody useful in the present disclosure is 1-7F9 (also referred to as IPH2101), described in Int'l Publ. No. WO 2006/003179.
• an anti- KIR antibody for the present composition cross competes for binding to KIR with lirilumab or I- 7F9.
• an anti-KIR antibody binds to the same epitope as lirilumab or I-7F9.
• an anti-KIR antibody comprises six CDRs of lirilumab or I-7F9.
• Anti-GITR antibodies useful in the methods disclosed herein include any anti-GITR antibodies
• GITR antibody that binds specifically to human GITR target and activates the glucocorticoid- induced tumor necrosis factor receptor (GITR).
• GITR is a member of the TNF receptor superfamily that is expressed on the surface of multiple types of immune cells, including regulatory T cells, effector T cells, B cells, natural killer (NK) cells, and activated dendritic cells ("anti-GITR agonist antibodies").
• GITR activation increases the proliferation and function of effector T cells, as well as abrogating the suppression induced by activated T regulatory cells.
• GITR stimulation promotes anti-tumor immunity by increasing the activity of other immune cells such as NK cells, antigen presenting cells, and B cells.
• anti-GITR antibodies examples have been disclosed in Int'l Publ. Nos. WO/2015/031667, WO2015/184,099, WO2015/026,684, WOl 1/028683 and WO/2006/105021, U.S. Pat. Nos. 7,812,135 and 8,388,967 and U.S. Publ. Nos. 2009/0136494, 2014/0220002, 2013/0183321 and 2014/0348841, each of which is incorporated by reference herein in its entirety.
• an anti-GITR antibody useful in the present disclosure is TRX518
• the anti-GITR antibody is selected from MK4166, MK1248, and antibodies described in WOl 1/028683 and U.S. 8,709,424, and comprising, e.g., a VTl chain comprising SEQ ID NO: 104 and a VL chain comprising SEQ ID NO: 105 (wherein the SEQ ID NOs are from WOl 1/028683 or U.S. 8,709,424).
• an anti-GITR antibody is an anti-GITR antibody that is disclosed in WO2015/031667, e.g., an antibody comprising VH CDRs 1-3 comprising SEQ ID NOs: 31, 71 and 63 of WO2015/031667, respectively, and VL CDRs 1-3 comprising SEQ ID NOs: 5, 14 and 30 of WO2015/031667.
• an anti-GITR antibody is an anti-GITR antibody that is disclosed in WO2015/184099, e.g., antibody Hum231#l or Hum231#2, or the CDRs thereof, or a derivative thereof (e.g., pabl967, pabl975 or pabl979).
• an anti-GITR antibody is an anti- GITR antibody that is disclosed in JP2008278814, W009/009116, WO2013/039954, US20140072566, US20140072565, US20140065152, or WO2015/026684, or is INBRX-110 (INEQBRx), LKZ-145 (Novartis), or MEDI-1873 (Medlmmune).
• an anti-GITR antibody is an anti-GITR antibody that is described in PCT/US2015/033991 (e.g., an antibody comprising the variable regions of 28F3, 18E10 or 19D3).
• the anti-GITR antibody cross-competes with an anti-GITR antibody described herein, e.g., TRX518, MK4166 or an antibody comprising a VH domain and a VL domain amino acid sequence described herein.
• the anti-GITR antibody binds the same epitope as that of an anti-GITR antibody described herein, e.g., TRX518 or MK4166.
• the anti-GITR antibody comprises the six CDRs of TRX518 or MK4166. II.C.8. Anti-TIM3 antibodies
• any anti-TIM3 antibody or antigen binding fragment thereof known in the art can be used in the methods described herein.
• the anti-TIM3 antibody is be selected from the anti-TIM3 antibodies disclosed in Int'l Publ. Nos.W02018013818, WO/2015/117002 (e.g., MGB453, Novartis), WO/2017/161270 (e.g., TSR-022, Tesaro/AnaptysBio),
• WO2011155607, WO2016/144803 e.g., STI-600, Sorrento Therapeutics
• WO2016/071448, WO17055399; W017055404, W017178493, WO18036561, W018039020 e.g., Ly-3221367, Eli Lilly
• the anti-OX40 antibody is BMS-986178 (Bristol-Myers Squibb Company), described in Int'l Publ. No. WO20160196228. In some aspects, the anti- 0X40 antibody is selected from the anti-OX40 antibodies described in Int'l Publ. Nos.
• NKG2A is a member of the C-type lectin receptor family that is expressed on natural killer (NK) cells and a subset of T lymphocytes. Specifically, NKG2A primarily expressed on tumor infiltrating innate immune effector NK cells, as well as on some CD8+ T cells. Its natural ligand human leukocyte antigen E (HLA-E) is expressed on solid and hematologic tumors. NKG2A is an inhibitory receptor that blinds HLA-E.
• NK natural killer
• HLA-E human leukocyte antigen E
• the anti-NKG2A antibody may be BMS-986315, a human monoclonal antibody that blocks the interaction of NKG2A to its ligand HLA-E, thus allowing activation of an anti-tumor immune response.
• the anti-NKG2A antibody is a checkpoint inhibitor that activates T cells, NK cells, and/or tumor-infiltrating immune cells.
• the anti-NKG2A antibody is selected from the anti-NKG2A antibodies described in, for example, WO 2006/070286 (Innate Pharma S.A.; University of Genova); U.S. Patent No.
• ICOS is an immune checkpoint protein that is a member of the CD28-superfamily.
• ICOS is a 55-60 kDa type I transmembrane protein that is expressed on T cells after T cell activation and co-stimulates T-cell activation after binding its ligand, ICOS-L (B7H2).
• ICOS is also known as inducible T-cell co-stimulator, CVIDl, AILIM, inducible costimulator, CD278, activation-inducible lymphocyte immunomediatory molecule, and CD278 antigen.
• the anti-ICOS antibody is BMS-986226, a humanized IgG monoclonal antibody that binds to and stimulates human ICOS.
• the anti-ICOS antibody is selected from anti-ICOS antibodies described in, for example, WO 2016/154177 (Jounce Therapeutics, Inc.), WO 2008/137915 (Medlmmune), WO 2012/131004 (INSERM, French National Institute of Health and Medical Research), EP3147297 (INSERM, French National Institute of Health and Medical Research), WO 2011/041613 (Memorial Sloan Kettering Cancer Center), EP 2482849 (Memorial Sloan Kettering Cancer Center), WO 1999/15553 (Robert Koch Institute), U.S.
• Patent Nos. 7,259,247 and 7,722,872 Robot Kotch Institute
• WO 1998/038216 Japanese Tobacco Inc.
• US. Patents. Nos. 7,045,615; 7,112,655, and 8,389,690 Japan Tobacco Inc.
• U.S. Patent Nos. 9,738,718 and 9,771,424 GaxoSmithKline
• WO 2017/220988 Kymab Limited
• any antibody or antigen-binding fragment thereof that specifically binds TIGIT can be used in the methods disclosed herein.
• the anti-TIGIT antibody is BMS- 986207.
• the anti-TIGIT antibody is clone 22G2, as described in WO 2016/106302.
• the anti-TIGIT antibody is MTIG7192A/RG6058/RO7092284, or clone 4.1D3, as described in WO 2017/053748.
• the anti-TIGIT antibody is selected from the anti-TIGIT antibodies described in, for example, WO 2016/106302 (Bristol- Myers Squibb Company) and WO 2017/053748 (Genentech).
• the anti-CSFIR antibody is an antibody species disclosed in any of international publications WO2013/132044, W02009/026303, WO2011/140249, or W02009/112245, such as cabiralizumab, RG7155 (emactuzumab), AMG820, SNDX 6352 (UCB 6352), CXIIG6, IMC-CS4, JNJ-40346527, MCS110, or the anti-CSFIR antibody in the methods is replaced with an anti-CSFIR inhibitor or anti-CSFl inhibitor such as BLZ-945, pexidartinib (PLX3397, PLX108-01), AC-708, PLX-5622, PLX7486, ARRY-382, or PLX-73086.
• an anti-CSFIR inhibitor or anti-CSFl inhibitor such as BLZ-945, pexidartinib (PLX3397, PLX108-01), AC-708, PLX-5622, PLX7486
• the methods disclosed herein further comprise administering an 1-0 therapy, e.g ., an anti-PD-1 antibody or an anti-PD-Ll antibody, and one or more additional anti-cancer therapies.
• an 1-0 therapy e.g ., an anti-PD-1 antibody or an anti-PD-Ll antibody
• the method comprising administering (i) a first 1-0 therapy, e.g. , an anti-PD-1 antibody or an anti-PD-Ll antibody), (ii) a second 1-0 therapy, e.g. , an anti-CTLA-4 antibody or an anti-CSFIR antibody, and (iii) one or more additional anti-cancer therapies.
• the additional anti-cancer therapy can comprise any therapy known in the art for the treatment of a tumor in a subject and/or any standard-of-care therapy, as disclosed herein.
• the additional anti-cancer therapy comprises a surgery, a radiation therapy, a chemotherapy, an immunotherapy, or any combination thereof.
• the additional anti-cancer therapy comprises a chemotherapy, including any chemotherapy disclosed herein.
• the chemotherapy is a platinum based-chemotherapy.
• Platinum-based chemotherapies are coordination complexes of platinum.
• the platinum-based chemotherapy is a platinum-doublet chemotherapy.
• the chemotherapy is administered at the approved dose for the particular indication.
• the chemotherapy is administered at any dose disclosed herein.
• the platinum-based chemotherapy is cisplatin, carboplatin, oxaliplatin, satraplatin, picoplatin, Nedaplatin, Triplatin, Lipoplatin, or combinations thereof.
• the platinum-based chemotherapy is any other platinum-based chemotherapy known in the art.
• the chemotherapy is the nucleotide analog gemcitabine.
• the chemotherapy is a folate antimetabolite.
• the folate antimetabolite is pemetrexed.
• the chemotherapy is a taxane.
• the taxane is paclitaxel.
• the chemotherapy is any other chemotherapy known in the art.
• at least one, at least two or more chemotherapeutic agents are administered in combination with the 1-0 therapy.
• the 1-0 therapy is administered in combination with gemcitabine and cisplatin.
• the 1-0 therapy is administered in combination with pemetrexed and cisplatin.
• the 1-0 therapy is administered in combination with gemcitabine and pemetrexed.
• the 1-0 therapy is administered in combination with paclitaxel and carboplatin.
• an I- O therapy is additionally administered.
• the additional anti-cancer therapy comprises an immunotherapy.
• the additional anti-cancer therapy comprises administration of an antibody or antigen-binding portion thereof that specifically binds LAG-3, TIGIT, TIM3, NKG2a, CSF1R, 0X40, ICOS, MICA, MICB, CD137, KIR, TGFp, IL-10, IL-8, B7-H4, Fas ligand, CXCR4, mesothelin, CD27, GITR, or any combination thereof.
• the tumor is derived from a cancer selected from the group consisting of hepatocellular cancer, gastroesophageal cancer, melanoma, bladder cancer, lung cancer, kidney cancer, head and neck cancer, colon cancer, and any combination thereof.
• the tumor is derived from a hepatocellular cancer, wherein the tumor has a high inflammatory signature score.
• the tumor is derived from a gastroesophageal cancer, wherein the tumor has a high inflammatory signature score.
• the tumor is derived from a melanoma, wherein the tumor has a high inflammatory signature score.
• the tumor is derived from a bladder cancer, wherein the tumor has a high inflammatory signature score.
• the tumor is derived from a lung cancer, wherein the tumor has a high inflammatory signature score. In certain aspects, the tumor is derived from a kidney cancer, wherein the tumor has a high inflammatory signature score. In certain aspects, the tumor is derived from a head and neck cancer, wherein the tumor has a high inflammatory signature score. In certain aspects, the tumor is derived from a colon cancer, wherein the tumor has a high inflammatory signature score.
• the subject has received one, two, three, four, five or more prior cancer treatments.
• the subject is treatment-naive.
• the subject has progressed on other cancer treatments.
• the prior cancer treatment comprised an immunotherapy.
• the prior cancer treatment comprised a chemotherapy.
• the tumor has reoccurred.
• the tumor is metastatic.
• the tumor is not metastatic.
• the tumor is locally advanced.
• the subject has received a prior therapy to treat the tumor and the tumor is relapsed or refractory.
• the at least one prior therapy comprises a standard-of-care therapy.
• the at least one prior therapy comprises a surgery, a radiation therapy, a chemotherapy, an immunotherapy, or any combination thereof.
• the at least one prior therapy comprises a chemotherapy.
• the subject has received a prior immuno-oncology (I-O) therapy to treat the tumor and the tumor is relapsed or refractory.
• I-O immuno-oncology
• the subject has received more than one prior therapy to treat the tumor and the subject is relapsed or refractory.
• the subject has received either an anti-PD-1 or anti-PD-Ll antibody therapy.
• the previous line of therapy comprises a chemotherapy.
• the chemotherapy comprises a platinum-based therapy.
• the platinum-based therapy comprises a platinum-based antineoplastic selected from the group consisting of cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, satraplatin, and any combination thereof.
• the platinum-based therapy comprises cisplatin.
• the platinum-based therapy comprises carboplatin.
• the at least one prior therapy is selected from a therapy comprising administration of an anti-cancer agent selected from the group consisting of a platinum agent (e.g ., cisplatin, carboplatin), a taxanes agent (e.g, paclitaxel, albumin-bound paclitaxel, docetaxel), vinorelbine, vinblastine, etoposide, pemetrexed, gemcitabine, bevacizumab (AVASTIN®), erlotinib (TARCEVA®), crizotinib (XALKORI®), cetuximab (ERBITUX®), and any combination thereof.
• the at least one prior therapy comprises a platinum-based doublet chemotherapy.
• the subject has experienced disease progression after the at least one prior therapy.
• the subject has received at least two prior therapies, at least three prior therapies, at least four prior therapies, or at least five prior therapies.
• the subject has received at least two prior therapies.
• the at least two prior therapies comprises a first prior therapy and a second prior therapy, wherein the subject has experienced disease progression after the first prior therapy and/or the second prior therapy, and wherein the first prior therapy comprises a surgery, a radiation therapy, a chemotherapy, an immunotherapy, or any combination thereof; and wherein the second prior therapy comprises a surgery, a radiation therapy, a chemotherapy, an immunotherapy, or any combination thereof.
• the first prior therapy comprises a platinum-based doublet chemotherapy
• the second prior therapy comprises a single-agent chemotherapy.
• the single-agent chemotherapy comprises docetaxel.
• Therapeutic agents of the present disclosure can be constituted in a composition, e.g ., a pharmaceutical composition containing an antibody and/or a cytokine and a pharmaceutically acceptable carrier.
• a pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
• the carrier for a composition containing an antibody is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g, by injection or infusion), whereas the carrier for a composition containing an antibody and/or a cytokine is suitable for non-parenteral, e.g, oral, administration.
• the subcutaneous injection is based on Halozyme Therapeutics’ ENHANZE® drug-delivery technology (see U.S. Patent No. 7,767,429, which is incorporated by reference herein in its entirety).
• ENHANZE® uses a co formulation of an antibody with recombinant human hyaluronidase enzyme (rHuPH20), which removes traditional limitations on the volume of biologies and drugs that can be delivered subcutaneously due to the extracellular matrix (see U.S. Patent No. 7,767,429).
• a pharmaceutical composition of the disclosure can include one or more pharmaceutically acceptable salts, anti oxidant, aqueous and non-aqueous carriers, and/or adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Therefore, in some aspects, the pharmaceutical composition for the present disclosure can further comprise recombinant human hyaluronidase enzyme, e.g., rHuPH20.
• nivolumab monotherapy dosing up to 10 mg/kg every two weeks has been achieved without reaching the maximum tolerated does (MTD)
• MTD maximum tolerated does
• the significant toxi cities reported in other trials of checkpoint inhibitors plus anti-angiogenic therapy support the selection of a nivolumab dose lower than 10 mg/kg.
• the antibodies disclosed herein are administered at doses that are significantly lower than the approved dosage, i.e., a subtherapeutic dosage, of the agent.
• the antibody can be administered at the dosage that has been shown to produce the highest efficacy as monotherapy in clinical trials, e.g. , about 3 mg/kg of nivolumab administered once every three weeks (Topalian et al., 2012a; Topalian et al., 2012), or at a significantly lower dose, i.e., at a subtherapeutic dose.
• Dosage and frequency vary depending on the half-life of the antibody in the subject.
• human antibodies show the longest half-life, followed by humanized antibodies, chimeric antibodies, and nonhuman antibodies.
• the dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic.
• a relatively low dosage is typically administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives.
• a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.
• compositions of the present disclosure can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being unduly toxic to the patient.
• the selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present disclosure employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
• a composition of the present disclosure can be administered via one or more routes of administration using one or more of a variety of methods well known in the art. As will be appreciated by the skilled artisan, the route and/or mode of administration will vary depending upon the desired results.
• kits comprising (a) an anti-PD-
• Kits typically include a label indicating the intended use of the contents of the kit and instructions for use.
• the term label includes any writing, or recorded material supplied on or with the kit, or which otherwise accompanies the kit. Accordingly, this disclosure provides a kit for treating a subject afflicted with a tumor, the kit comprising: (a) a dosage ranging from 0.1 to 10 mg/kg body weight of an anti-PD-1 antibody or a dosage ranging from 0.1 to 20 mg/kg body weight of an anti-PD-Ll antibody; and (b) instructions for using the anti-PD-1 antibody or the anti-PD-Ll antibody in the methods disclosed herein.
• kits for treating a subject afflicted with a tumor comprising: (a) a dosage ranging from about 4 mg to about 500 mg of an anti-PD-1 antibody or a dosage ranging from about 4 mg to about 2000 mg of an anti-PD-Ll antibody; and (b) instructions for using the anti-PD-1 antibody or the anti-PD-Ll antibody in the methods disclosed herein.
• this disclosure provides a kit for treating a subject afflicted with a tumor, the kit comprising: (a) a dosage ranging from 200 mg to 800 mg of an anti-PD-1 antibody or a dosage ranging from 200 mg to 1800 mg of an anti-PD-Ll antibody; and (b) instructions for using the anti-PD-1 antibody or the anti-PD-Ll antibody in the methods disclosed herein.
• the kit comprises an anti-human
• the kit comprises an anti-human PD-L1 antibody disclosed herein, e.g. , atezolizumab, durvalumab, or avelumab.
• the kit further comprises an anti-CTLA-4 antibody.
• the kit comprises an anti-human CTLA-4 antibody disclosed herein, e.g., ipilimumab, tremelimumab, MK-1308, or AGEN-1884.
• the kit further includes a gene panel assay disclosed herein.
• the kit further includes instructions to administer the anti -PD- 1 antibody or the anti-PD-Ll antibody to a suitable subject according to the methods disclosed herein.
• CD8+ T cells has been associated with improved clinical outcomes across multiple tumor types. Parenchymal infiltration of CD8+ T cells has been associated with improved survival with immuno-oncology (I-O) treatment, and intratumoral localization also affects outcome, highlighting the importance of spatial analysis of CD8+ T cells within the TME.
• CD8+ T-cell patterns within tumors are variable and may be classified as: (i) immune desert (minimal T-cell infiltrate); (ii) immune excluded (T cells confined to tumor stroma or invasive margin); or (iii) Immune inflamed (T cells infiltrating tumor parenchyma, positioned in proximity to tumor cells).
• GEP Gene expression profiling
• An inflammation gene panel comprising 95 genes for GEP, in combination with
• AI-based image analysis was used to analyze patterns of T-cell infiltration in tumor parenchymal and stromal compartments and to evaluate potential biomarker assays for 1-0 therapy.
• the first objection of the present study is to quantify regional CD8+ T-cell localization using AI-based image analysis.
• the second objective is to identify gene signatures that define CD8+ T-cell infiltration and localization to parenchymal and stromal compartments in the TME.
• CD8 IHC was performed by Mosaic Laboratories using a monoclonal CD8 (clone C8/144B) antibody (Dako, an Agilent Technologies Co, Santa Clara, CA).
• a convolutional neural network (PathAI Inc, Boston, MA) and AI-based image analysis algorithms were used to quantify the abundance of CD8+ T cells in tumor parenchymal and stromal regions (FIGs. 1A-1B; Table 1)
• Table 1 AI-based quantification of CD8+ T cells in Tumor Parenchymal and Stromal Regions
• GEP was performed by next-generation sequencing (NGS) using an inflammation panel.
• the inflammation panel comprises 95 genes, including genes related to tumor inflammation and other 1-0 processes, housekeeping genes, and control genes. This inflammation panel measures mRNA expression levels of all 95 genes on the panel.
• a parenchymal CD8 gene signature correlated with tumor parenchymal CD8+ T- cell abundance (FIGs. 5A-5B). Of the 23 genes in the signature, 10 were upregulated and 13 were downregulated; top predictors of parenchymal CD8+ T-cell abundance included: upregulation of STAT1 and IFNy, and downregulation of NECTIN2 and CSF1R (FIG. 5B).
• a stromal CD8 gene signature correlated with tumor stromal CD8+ T-cell abundance (FIGs. 5C-5D). Of the 38 genes in the signature, 19 were upregulated and 19 were downregulated. Top predictors of tumor stromal CD8+ T-cell abundance included: upregulation of CSF1R and NECTIN2, and downregulation of STAT1 and IFNy.
• the present example describes a GEP-based, investigational inflammation assay with the potential to be utilized prospectively in a clinical trial setting.
• gene signatures were further derived that predict CD8+ T-cell abundance in the tumor parenchyma and stroma. Parenchymal and stromal CD8 signature scores were concordant with CD8+ T-cell abundance determined by IHC in melanoma and SCCHN samples (individually and pooled), indicating that these gene signatures may be utilized to assess T-cell abundance.
• Combining GEP with AI-based image analysis could be developed as an analytical tool to characterize immune cell infiltration in the TME.
• CD8 signature CD8+ T-cell infiltration
• CD8 topology signatures CD8 topology signatures
• CD8 IHC EMT EMT gene expression
• CD8 IHC was performed using monoclonal anti-
• CD8 C8/144B by Mosaic Laboratories (Lake Forest, CA); CD8+ T-cell infiltration in parenchymal and stromal areas was quantified, and tumors were defined as immune-desert, immune excluded, or immune-inflamed phenotypes (FIGs. 4A-4C).
• EMT gene expression was measured using the HTG EdgeSeq Biomarker Panels (HTG Molecular Diagnostics, Arlington, A Z), and an EMT signature score was calculated by the arithmetic mean of the EMT gene expression levels.
• GEP by next-generation sequencing using an inflammation panel was performed on evaluable UC samples from patients. Scores were derived from previously identified gene expression signatures that assess CD8+ T-cell abundance (CD8 signature) and localization to tumor parenchyma and stroma (CD8 topology signatures).
• Biomarker models were developed that included single signatures and the multiplicative interactions among 2 or 3 gene signatures (Table 2) The dual and triple CD8 signatures evaluated consider CD8+ T-cell inflammation in the TME as well as in specific tumor regions within the TME.
• Cox proportional hazards regression models were used to assess the dependence of progression-free survival (PFS) or overall survival (OS) on the biomarker scores
• Hazard ratios (HRs) and two-sided 95% confidence intervals (CIs; calculated based on Wald test statistics) represent the difference between the 75th and 25th biomarker percentiles; graphs were scaled to compare log2(HR) values.
• Kaplan-Meier plots based on categorization of the biomarker scores by tertile (high, medium, low) were used to illustrate associations with PFS and OS.
• GEP-derived CD8 topology signatures and CD8.IHC_EMT were evaluable in 205 of 270 (76%) and 187 of 270 (69%) patients, respectively, in the clinical trial. Baseline characteristics and clinical outcomes were similar in the cohorts to the overall study population (Table 3).
• Table 3 Baseline characteristics and outcomes for overall study population and biomarker cohorts.
• CR complete response
• NE nonevaluable
• PD progressive disease
• PR partial response
• SD stable disease.
• CD8 signature biomarker models (CD8, parenchymal CD8, dual CD8, and triple CD8) showed similar associations with PFS and OS as CD8.IHC_EMT HRs for PFS ranged from 0.55 (95% Cl, 0.45-0.66) for triple CD8 to 0.65 (95% Cl, 0.55-0.76) for the CD8.IHC EMT signature (FIG. 8A).
• HRs for OS ranged from 0.47 for the CD8 signature (95% Cl, 0.37-0.60) to 0.53 (95% Cl, 0.44-0.66) for the triple CD 8 signature (FIG. 8B)
• Kaplan-Meier plots illustrate patterns of association between PFS or OS and the triple CD8 signature (FIGs. 9A-9B) that are consistent with those observed by Cox model analyses (FIGs. 8A-8B).
• PFS and OS with nivolumab were longer for patients with scores in the upper tertile than those in the lower 2 tertiles.
• CD8 signature biomarker models CD8, parenchymal CD8, dual CD8, and triple CD8
• CD8.IHC EMT showed similar associations with OR (FIG 7A). Odds ratios ranged from 1.46 (95% Cl, 1.08-1.97) for the parenchymal CD8 signature to 1.64 (95% Cl, 1.12-2.41) for triple CD8.
• ROC curves for OR were similar for each biomarker (FIGs. 7B-7F).
• AUC values ranged from 67.6% (95% Cl, 57.9-77.3) for the dual CD8 signature to 72.1% (95% Cl, 62.8-81.4) for triple CD8.
• CD8 gene signature biomarkers and a CD8 IHC- derived score combined with EMT gene expression are comparable for the association with response and survival in patients with UC treated with nivolumab.
• CD8.IHC EMT EMT gene expression
• Gene expression signatures associated with CD8+ T-cell localization in the TME may facilitate the selection of patients who are more likely to benefit from 1-0 therapy.

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