JP2022534982A - Cellular localization signatures and their uses - Google Patents

Abstract

The present invention provides methods of identifying subjects suitable for immuno-oncology (IO) therapy comprising measuring expression of one or more of STAT1, IFNγ, Nectin2 and CSF1R. In some embodiments, IO therapy comprises administering an anti-PD-1 antibody or antigen-binding portion thereof or an anti-PD-L1 antibody or antigen-binding portion thereof to the subject.

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application benefits from priority to U.S. Provisional Application No. 62/854,883 filed May 30, 2019 and U.S. Provisional Application No. 62/931,724 filed November 6, 2019 and each of which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION The present invention provides methods of treating a subject with a tumor using immunotherapy.

BACKGROUND OF THE INVENTION Human cancers possess numerous genetic and epigenetic alterations that produce potentially new antigens that can be recognized by the immune system (Sjoblom et al., Science (2006) 314(5797):268 -274). The adaptive immune system, which consists of T and B lymphocytes, has broad capabilities and potent anti-cancer capabilities with exquisite specificity in responding to various tumor antigens. In addition, the immune system exhibits considerable flexibility and memory components. The successful exploitation of all these attributes of the adaptive immune system makes immunotherapy unique among all cancer treatment modalities.

In the past decade, intensive efforts to develop specific immune checkpoint pathway inhibitors have begun to offer novel immunotherapeutic approaches to cancer treatment, nivolumab and pembrolizumab ( USAN Council Statement, 2013) and atezolizumab, durvalumab and avelumab that specifically bind to PD-L1 (Topalian et al., 2012a, b; Topalian et al., 2014; Hamid et al., 2013; Hamid and Carvajal, 2013; McDermott and Atkins, 2013).

The immune system and response to immunotherapy have been shown to be complex. Furthermore, anti-cancer agents can vary in efficacy based on patient-specific characteristics. Accordingly, there is a need for targeted therapeutic strategies that improve the clinical outcome of patients diagnosed with cancer by identifying patients who are more likely to respond to specific anti-cancer agents.

SUMMARY OF THE INVENTION One aspect of the invention is a pharmaceutical composition for use in a method of identifying a human subject suitable for an anti-PD-1/PD-L1 antagonist, comprising: wherein the method comprises measuring the expression of a panel of genes in a tumor sample obtained from a subject in need of an anti-PD-1/PD-L1 antagonist, wherein the panel of genes is STAT1, IFNγ, Nectin2 and CSF1R. Containing at least three. In some embodiments, the gene panel comprises all of STAT1, IFNγ, Nectin2, CSF1R and 1 additional gene, 2 additional genes, 3 additional genes, 4 additional genes, 5 additional genes, 6 7 additional genes, 8 additional genes or 10 additional genes. In some embodiments, the gene panel consists of STAT1, IFNγ, Nectin2, CSF1R and 1 additional gene, 2 additional genes or 3 additional genes.

In some embodiments, the tumor sample has (i) increased expression of one or more of STAT1 and IFNγ (“upregulated genes”) in the sample compared to the expression of one or more of STAT1 and IFNγ (“upregulated genes”) in the control sample; (ii) control when exhibiting decreased expression of one or more of Nectin2 and CSF1R in the sample compared to one or more of Nectin2 and CSF1R (“downregulated genes”) in the sample, or (iii) both (i) and (ii); , the subject is identified as suitable.

In some embodiments, the subject should be administered an anti-PD-1/PD-L1 antagonist.

An aspect of the invention is a pharmaceutical composition comprising an anti-PD-1/PD-L1 antagonist for use in a method of treating a human subject with a tumor, wherein a tumor sample obtained from the subject is ( i) increased expression of one or more of STAT1 and IFNγ (“upregulated genes”) in a tumor sample obtained from the subject compared to expression of one or more of STAT1 and IFNγ (“upregulated genes”) in control samples; (ii) in control samples decreased expression of one or more of Nectin2 and CSF1R in a tumor sample obtained from the subject compared to expression of one or more of Nectin2 and CSF1R (“downregulated genes”); or (iii)(i) and ( ii) for those exhibiting both. In some embodiments, a control sample comprises non-tumor tissue from a subject, matched non-tumor tissue from a subject, or matched tissue from a subject without a tumor.

One aspect of the invention comprises measuring in vitro expression of a panel of genes in a tumor sample obtained from a subject in need of an anti-PD-1/PD-L1 antagonist. wherein the gene panel comprises at least three of STAT1, IFNγ, Nectin2 and CSF1R. In some embodiments, the gene panel comprises all of STAT1, IFNγ, Nectin2, CSF1R and 1 additional gene, 2 additional genes, 3 additional genes, 4 additional genes, 5 additional genes, 6 7 additional genes, 8 additional genes or 10 additional genes. In some embodiments, wherein the gene panel consists of STAT1, IFNγ, Nectin2, CSF1R and 1 additional gene, 2 additional genes or 3 additional genes.

In certain embodiments, the tumor sample has (i) increased expression of one or more of STAT1 and IFNγ (“upregulated genes”) in the tumor sample compared to the expression of one or more of STAT1 and IFNγ (“upregulated genes”) in a control sample; (ii) or (iii) (i) and (iii) (i) and A subject is identified as eligible when it exhibits both ii).

In some embodiments, further comprising administration of an anti-PD-1/PD-L1 antagonist.

One aspect of the invention is a method of treating a human subject with a tumor comprising administering an anti-PD-1/PD-L1 antagonist to the subject, wherein a tumor sample obtained from the subject is (i a) increased expression of one or more of STAT1 and IFNγ (“upregulated genes”) in a tumor sample obtained from the subject compared to expression of one or more of STAT1 and IFNγ (“upregulated genes”) in a control sample; (ii) nectin in a control sample; decreased expression of one or more of Nectin2 and CSF1R in a tumor sample obtained from the subject compared to expression of one or more of 2 and CSF1R (“downregulated genes”); or (iii) (i) and (ii) ); in certain embodiments, the control sample comprises non-tumor tissue from a subject, matched non-tumor tissue from a subject, or matched tissue from a subject without a tumor.

In some embodiments, the subject is identified as suitable for an anti-PD-1/PD-L1 antagonist prior to an anti-PD-1/PD-L1 antagonist. In some embodiments, the tumor sample exhibits increased expression of at least two of the upregulated genes. In some embodiments, the tumor sample exhibits decreased expression of at least two of the downregulated genes. In some embodiments, the tumor sample exhibits increased expression of all upregulated genes; and the tumor sample exhibits decreased expression of all downregulated genes.

In some embodiments, the expression of one or more of the upregulated genes is 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. In some embodiments, expression of one or more of the upregulated genes is increased at least about 50% higher than expression of one or more of STAT1 and IFNγ in a control sample. In some embodiments, expression of one or more of the upregulated genes is increased at least about 75% higher than expression of one or more of STAT1 and IFNγ in a control sample.

In some embodiments, expression of one or more of the upregulated genes is at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 15%, 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. In some embodiments, expression of one or more of the upregulated genes is decreased at least about 50% lower than expression of one or more of Nectin2 and CSF1R in a control sample. In some embodiments, the expression of one or more of the upregulated genes is decreased by at least about 75% below the expression of one or more of Nectin2 and CSF1R in a control sample.

In some embodiments, the tumor sample is a tumor tissue biopsy sample. In some embodiments, the tumor sample is formalin-fixed, paraffin-embedded or fresh frozen tumor tissue. In some embodiments, the tumor sample is obtained from the tumor parenchyma.

In some embodiments, gene expression is determined by detecting the presence of gene mRNA, the presence of protein encoded by the gene, or both. In some embodiments, the presence of gene mRNA is determined using reverse transcriptase PCR. In some embodiments, the presence of protein encoded by the gene is determined using an IHC assay. In some embodiments, the IHC assay is an automated IHC assay.

In certain embodiments, the tumor sample has increased expression of one or more of CSF1R and Nectin2 compared to expression of one or more of CSF1R and Nectin2 in a control sample; expression of one or more of STAT1 and IFNγ in a control sample. or not both (i) and (ii). In certain embodiments, the tumor sample has increased expression of 2 or 3 of CSF1R and Nectin2 compared to expression of 2 or 3 of CSF1R and Nectin2 in a control sample; , 2, 3 or 4 decreased expression of STAT1 and IFNγ compared to 3 or 4 expression; or no both (i) and (ii).

In some embodiments, a tumor sample is obtained from the stroma of a tumor.

In certain embodiments, the anti-PD-1/PD-L1 antagonist is selected from programmed death 1 (PD-1; "anti-PD-1 antibody") or programmed death ligand 1 (PD-L1; "anti-PD-L1 antibody) Anti-PD-1/PD-L1 antagonist is an anti-PD-1 antibody In certain embodiments, the anti-PD-1/PD-L1 antagonist is an anti-PD-1 antibody. includes nivolumab or pembrolizumab.

In some embodiments, the anti-PD-1/PD-L1 antagonist is an anti-PD-L1 antibody. In some embodiments, the anti-PD-1 antibody comprises avelumab, atezolizumab or durvalumab.

In some embodiments, the anti-PD-1/PD-L1 antagonist is administered as monotherapy. In some embodiments, an anti-PD-1/PD-L1 antagonist is administered with an anti-cancer agent. In some embodiments, the anti-cancer agent is inducible T cell co-stimulatory molecule (ICOS), CD137 (4-1BB), CD134 (OX40), 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), natural killer cell receptor 2B4 (CD244), CD160, T cell immunoreceptors with Ig and ITIM domains (TIGIT) and derivatives of V-domain Ig suppressors of T cell activation (VISTA), KIR, TGFβ, IL-10, IL-8, B7-H4, Fas ligand, Antibodies that specifically bind proteins of CSF1R, CXCR4, mesothelin, CEACAM-1, CD52, HER2 or any combination thereof are included. In some embodiments, the anti-cancer agent comprises an anti-CSF1R antibody.

In some embodiments, the tumor is hepatocellular carcinoma, gastroesophageal cancer, melanoma, bladder cancer, lung cancer, renal cancer, head and neck cancer, colon cancer, pancreatic cancer, prostate cancer, ovarian cancer, urothelial cancer, colorectal cancer and from a cancer selected from the group consisting of any combination thereof.

In some embodiments, the tumor is recurrent. In some embodiments, the tumor is refractory. In some embodiments, the tumor is locally advanced. In some embodiments, the tumor is metastatic. In some embodiments, administration treats a tumor. In some embodiments, administration reduces tumor size. In some embodiments, tumor size is reduced by at least about 10%, about 20%, about 30%, about 40%, or about 50% compared to tumor size prior to administration. In some embodiments, the subject is at least about 1 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 1 year, at least about 18 months, at least about 2 years, at least about 3 years, at least Shows progression-free survival of about 4 years or at least about 5 years.

In some embodiments, the subject exhibits stable disease after administration. In some embodiments, the subject exhibits a post-administration partial response. In some embodiments, the subject exhibits a complete response after administration.

One aspect of the invention is a kit for treating a subject with a tumor comprising (a) an anti-PD-1/PD-L1 antagonist; and (b) an anti-PD- A kit containing instructions for using a 1/PD-L1 antagonist. In some embodiments, an anti-PD-1/PD-L1 antagonist comprises an anti-PD-1 antibody. In some embodiments, an anti-PD-1/PD-L1 antagonist comprises an anti-PD-L1 antibody.

One aspect of the invention relates to a gene panel comprising at least three of STAT1, IFNγ, Nectin2 and CSF1R for use in identifying subjects suitable for anti-PD-1/PD-L1 antagonists. In some embodiments, the gene panel comprises at least 4, at least 5 or at least 6 of STAT1, IFNγ, Nectin2 and CSF1R. In some embodiments, the gene panel comprises STAT1, IFNγ, Nectin2 and CSF1R. In some embodiments, the gene panel comprises STAT1, IFNγ, Nectin2 and CSF1R and 1 additional gene, 2 additional genes, 3 additional genes, 4 additional genes, 5 additional genes, 6 7 additional genes, 8 additional genes, 9 additional genes or 10 additional genes. In some embodiments, the gene panel comprises STAT1, IFNγ, Nectin2 and CSF1R and 1 additional gene, 2 additional genes, 3 additional genes, 4 additional genes, 5 additional genes, 6 7 additional genes, 8 additional genes, 9 additional genes or 10 additional genes. In some embodiments, the gene panel comprises about 90 genes, about 95 genes or about 100 genes.

A method of preparing a nucleic acid fraction from a tumor of a subject in need of IO therapy comprising: (a) removing a tumor biopsy sample from the subject; (b) isolating the nucleic acid extracted in (a). and (c) analyzing the expression level of one or more genes in a gene panel selected from STAT1, IFNγ, Nectin2 and CSF1R. In some embodiments, the nucleic acid is mRNA.

In some embodiments, one or both of the CSF1R and Nectin2 genes are downregulated. In some embodiments, one or both of the STAT1 and IFNγ genes are upregulated. In some embodiments, CSF1R and Nectin2 are downregulated and STAT1 and IFNγ are upregulated.

In some embodiments, expression levels are analyzed by measuring mRNA levels of one or more genes of the gene panel in the tumor sample. In some embodiments, expression levels are measured using a nuclease protection assay. In some embodiments, expression levels are measured using next generation sequencing. In some embodiments, expression levels are measured using reverse transcriptase polymerase chain reaction (RT-PCR).

In some embodiments, the expression of one or both of STAT1 and IFNγ is 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. In some embodiments, expression of one or both of STAT1 and IFNγ is increased at least about 50% higher than expression of one or both of STAT1 and IFNγ in a control sample. In some embodiments, expression of one or both of STAT1 and IFNγ is increased at least about 75% higher than expression of one or both of STAT1 and IFNγ in a control sample.

In some embodiments, expression of one or both of Nectin2 and CSF1R is at least about 10%, at least about 15%, at least about 20%, at least about 25%, than expression of one or more of Nectin2 and CSF1R in a control sample, 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% lower, at least about 275% lower, or at least about 300% lower. In some embodiments, the expression of one or both of Nectin2 and CSF1R is decreased at least about 50% lower than the expression of one or more of Nectin2 and CSF1R in a control sample. In some embodiments, the expression of one or both of Nectin2 and CSF1R is decreased by at least about 75% below the expression of one or more of Nectin2 and CSF1R in a control sample.

Figures 1A-1B are tumor tissue samples labeled using standard CD8+ immunohistochemistry (IHC; Figure 1A) or images further annotated using artificial intelligence (AI) image analysis tools (Figure 1B). . Arrowheads are examples of CD8+ T cells. Tumor parenchymal and stromal areas are indicated (Fig. 1B).

FIG. 2 is a schematic representation of various CD8 phenotypes.

Figures 3A-3B are scatterplots illustrating the parenchymal versus interstitial abundance cutoffs used to define the immunophenotype. FIG. 3B contains the scatterplot of FIG. 3A with overlays showing cutoffs for inflammation, equilibrium, exclusion and desert phenotypes.

Figures 4A-4C show the immunodesert phenotype (T cells without TME; Figure 4A), the immune exclusion phenotype (T cell accumulation without efficient tumor parenchymal invasion; Figure 4B) and the immune inflammatory phenotype (infiltration in the tumor parenchyma). Images of three tumor samples representing T cells; FIG. 4C). Arrowheads label examples of CD8+ T cells.

Figures 5A-5D are graphical representations showing parenchymal CD8 signatures (Figures 5A-5B) and stromal CD8 signatures (Figures 5C-5D). Figures 5A and 5C are heatmaps showing the relative expression of various genes associated with the parenchymal CD8 signature (Figure 5A) and the stromal CD8 signature (Figure 5C). Figures 5B and 5D are bar graphs summarizing selected representative genes of the parenchymal (Figure 5B) and stromal CD8 signatures (Figure 5D).

Figures 6A-6D are scatter plots showing the correlation between CD8 signature score and CD8 IHC score in melanoma (Figures 6A and 6C) and SCCHN (Figures 6B and 6D) tumor samples. Figures 6A-6B show the correlation between parenchymal AI-based CD8 IHC score (y-axis) and parenchymal CD8 signature score (x-axis) (degrees of freedom adjusted R2 = 0.67). Figures 6C-6D show the correlation between stromal AI-based CD8 IHC score (y-axis) and stromal CD8 signature score (x-axis) (degrees of freedom adjusted R2 = 0.65). The x=y and linear regression line are superimposed on each graph. Freedom-adjusted R2 values are derived from pooled analysis.

FIG. 7A is a graphical representation of overall survival (OS) odds ratios for triple D8, dual CD8, parenchymal CD8, CD8 and CD8.IHC_EMT, as indicated. Figures 7B-7F are ROC curves of the OR of triple D8 (Figure 7B), dual CD8 (Figure 7D, parenchymal CD8 (Figure 7C), CD8 (Figure 7E) and CD8.IHC_EMT (Figure 7F).

FIG. 8A is a graphical representation of the progression-free survival (PFS) odds ratio for triple D8, dual CD8, parenchymal CD8, CD8 and CD8.IHC_EMT, as indicated. FIG. 8B is a graphical representation of the OS odds ratio for triple D8, dual CD8, parenchymal CD8, CD8 and CD8.IHC_EMT, as indicated.

Figures 9A-9B are graphical representations of PFS (Figure 9A) and OS (Figure 9B) stratified by parenchymal signature score. The number of patients at risk in each group is shown below each graph.

DETAILED DESCRIPTION OF THE INVENTION One aspect of the invention relates to methods of identifying human subjects suitable for immuno-oncology (IO) therapy, eg, anti-PD-1/PD-L1 antagonist therapy. In one aspect, the invention provides an anti-PD-1/PD-L1 antagonist comprising measuring the expression of a panel of genes in a tumor sample obtained from a subject in need of an anti-PD-1/PD-L1 antagonist. A method of identifying a human subject suitable for therapy, wherein the gene panel comprises at least one of STAT1, IFNγ, Nectin2 and CSF1R. Gene panels and gene signatures comprising the identified genes of the present invention are particularly suitable for and/or responsive to IO therapy in predicting inflammatory phenotypes in the tumor microenvironment (TME) across multiple tumor types Useful for target identification. Therefore, in some embodiments, gene panels and their use can replace the inconvenience and hassle of CD8+ immunohistochemistry.

I. Terminology In order that the invention may be more easily understood, some terms will first be defined. As used herein, unless otherwise specified herein, each of the following terms has the following meaning. Additional definitions are provided throughout this specification.

It is understood that when embodiments are described herein using the term "comprising", analogous embodiments are also provided other than those described with the term "consisting of" and/or "consisting essentially of". be.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; Biology, Revised, 2000, Oxford University Press, provides the skilled artisan with a general dictionary of many of the terms used herein.

Units, prefixes and symbols are given in the form accepted by the International System of Units (SI). Numeric ranges are inclusive of the numbers defining the range. When numerical ranges are recited, it is to be understood that each numerical value between the recited upper and lower limits of the range, and each fraction thereof, is specifically disclosed, as are each subrange of such numerical value. The upper and lower limits of any range may independently be included in or excluded from the range, and in each range, each range with either limit, neither limit, or both, Included within the scope of the present invention. Thus, ranges recited herein are understood to be shorthand for all values within the range, including the recited endpoints. For example, the range 1-10 is understood to include any number, combination of numbers or subranges of the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10.

When values are explicitly quoted, values that are about the same quantity or quantity as the quoted value should also be understood to be within the scope of the invention. When a combination is disclosed, each subcombination of the elements of the combination is also specifically disclosed and is within the scope of the invention. Conversely, when various elements or groups of elements are disclosed individually, combinations thereof are also disclosed. When any element of the disclosure is disclosed as having multiple options, examples of that disclosure are also disclosed herein, with each option excluded either alone or in any combination with other options; Disclosure of more elements allows such exclusion, and all combinations of elements subject to such exclusion are disclosed herein.

"Administering" refers to the physical introduction of a composition containing a therapeutic agent to a subject using any of a variety of methods and delivery systems known to those of ordinary skill in the art. Preferred routes of administration for immunotherapy, such as anti-PD-1 or anti-PD-L1 antibodies, are intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes, such as by injection or infusion. including. As used herein, the term "parenteral administration" refers to methods of administration other than enteral and topical administration, usually by injection, including intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular. , intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, intrathecal, intrathecal, epidural and intrasternal injections and infusions and in vivo electroporation but not limited to these. Other parenteral routes include oral, topical, epithelial or mucosal routes of administration such as intranasal, vaginal, rectal, sublingual or topical. Administration can also be carried out, for example, once, multiple times and/or over one or more periods.

As used herein, an "adverse event" (AE) is any unfavorable, generally unintended or undesirable symptom (including abnormal laboratory findings) associated with the use of medical treatment. For example, adverse events can be associated with immune system activation or expansion of immune system cells (eg, T cells) in response to treatment. A medical treatment may be accompanied by one or more AEs, and each AE may have the same or different levels of severity. A reference to a method that can "modify an adverse event" means a treatment regimen that reduces the incidence and/or severity of one or more AEs associated with the use of a different treatment regimen.

An "antibody" (Ab) is a glycoprotein immunoglobulin or immunoglobulin that specifically binds to an antigen and comprises at least two heavy (H) chains and at least two light (L) chains interconnected by disulfide bonds. Including, but not limited to, antigen binding portions. Each H chain comprises a heavy chain variable region (abbreviated herein as _VH ) and a heavy chain constant region. The heavy chain constant region comprises three constant domains, C _H1 , C _H2 and C _H3 . Each light chain comprises a light chain variable region (abbreviated herein as _VL ) and a light chain constant region. The light chain constant region contains one constant domain, _CL . The V _H and V _L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each _VH and _VL contains three CDRs and four FRs, arranged in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The heavy and light chain variable regions contain the binding domains that interact with antigen. The constant regions of antibodies can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (C1q) of the classical complement system. Thus, the term "anti-PD-1 antibody" includes intact antibodies and antigen-binding portions of intact antibodies having two heavy chains and two light chains that specifically bind PD-1. Non-limiting examples of antigen binding moieties are provided elsewhere herein.

Immunoglobulins can be derived 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 skilled in the art and include, but are not limited to, human IgG1, IgG2, IgG3 and IgG4. "Isotype" refers to the antibody class or subclass (eg, IgM or IgG1) that is encoded by the heavy chain constant region genes. The term "antibody" includes, by way of example, both naturally occurring and non-naturally occurring antibodies; monoclonal and polyclonal antibodies; chimeric and humanized antibodies; human or non-human antibodies; fully synthetic antibodies; . Non-human antibodies can be humanized by recombinant methods to reduce immunogenicity in humans. Unless otherwise specified and the context indicates otherwise, the term "antibody" also includes antigen-binding fragments or portions of any of the above immunoglobulins, including monovalent and bivalent fragments or portions and single-chain antibodies. include.

An "isolated antibody" refers to an antibody that is substantially free of other antibodies with different antigenic specificities (e.g., an isolated antibody that specifically binds to PD-1 is specific for an antigen other than PD-1). (substantially free of antibodies that specifically bind). An isolated antibody that specifically binds PD-1 may, however, have cross-reactivity to other antigens, such as PD-1 molecules from different species. Moreover, an isolated antibody may be substantially free of other cellular material and/or chemicals.

The term "monoclonal antibody" (mAb) refers to an antibody molecule of single molecular composition, i.e., a naturally occurring antibody molecule that is essentially identical in primary sequence and displays a single binding specificity and affinity for a particular epitope. refers to preparations that are not present in 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 of skill in the art.

A "human antibody" (HuMAb) 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 is also derived from human germline immunoglobulin sequences. Human antibodies of the invention may comprise amino acid residues not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences from the germline of other mammalian species, such as mice, have been grafted onto human framework sequences. The terms "human antibody" and "fully human antibody" are used interchangeably.

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 have been replaced with corresponding amino acids derived from human immunoglobulin. In certain embodiments of humanized forms of antibodies, some, most, or all of the amino acids outside the CDRs are replaced with amino acids from a human immunoglobulin, while some, most, or all of the amino acids within one or more CDRs are replaced. Or everything is immutable. Minor additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they do not impair the antibody's ability to bind to the specific antigen. A "humanized antibody" retains similar antigen specificity as the original antibody.

A "chimeric antibody" refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species, such as an antibody in which the variable region is derived from a murine antibody and the constant region is derived from a human antibody. .

"Anti-antigen antibody" refers to an antibody that specifically binds to an antigen. For example, an anti-PD-1 antibody specifically binds to PD-1, an anti-PD-L1 antibody specifically binds to PD-1, and an anti-CTLA-4 antibody specifically binds to CTLA-4.

An "antigen-binding portion" (also referred to as an "antigen-binding fragment") of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to the antigen bound by the whole antibody. It has been shown that the antigen-binding function of antibodies can be performed by fragments of full-length antibodies. Examples of binding fragments included in the term "antigen-binding portion" of an antibody, eg, an anti-PD-1 antibody or an anti-PD-L1 antibody described herein are (i) Fab fragments (fragments from papain cleavage) or V (ii) an _F ( _ab ')2 fragment (fragment from pepsin cleavage) or an analogous double fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of the _VH and CH1 domains; (iv) an Fv fragment consisting of the _VL and _VH domains of a single arm of the antibody; (v) a dAb fragment consisting of the _VH domain (Ward et al. ., (1989) Nature 341:544-546); (vi) an isolated complementarity determining region (CDR) and (vii) a combination of two or more isolated CDRs that can optionally be joined by synthetic linkers. In addition, although the two domains of the Fv fragment, V _L and V _H , are encoded by separate genes, using recombinant methods, the V _L and V _H regions pair to form a monovalent molecule. They can be joined by synthetic linkers that allow them to be produced as protein chains (known as single-chain Fvs (scFvs); see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be included within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies. Antigen-binding portions may 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 inducible T cell co-stimulatory molecule (ICOS), CD137 (4-1BB), CD134 (OX40), 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 (CD244), CD160, T cell immunoreceptor with Ig and ITIM domains (TIGIT) and derivative of V-domain Ig suppressor of T cell activation (VISTA), KIR, The group consisting of TGFβ, IL-10, IL-8, IL-2, B7-H4, Fas ligand, CXCR4, CSF1R, mesothelin, CEACAM-1, CD52, HER2, MICA, MICB, CSF1R and any combination thereof Antibodies and antigen-binding portions thereof that specifically bind to proteins selected from, but are not limited to.

"Cancer" refers to a wide variety of diseases characterized by the uncontrolled growth of abnormal cells in the body. Uncontrolled cell division and proliferation leads to the formation of malignant tumors that invade neighboring tissues and may also metastasize to distant sites via the lymphatic system or bloodstream.

The term "immunotherapy" refers to the treatment of a subject who has or is at risk of developing or recurring disease by methods that involve induction, enhancement, suppression or other modification of an immune response. "Treatment" or "treatment" of a subject means reversing, reducing, ameliorating, arresting, slowing or preventing the onset, progression, evolution, severity or recurrence of symptoms, complications or conditions or biochemical manifestations associated with a disease. Any type of intervention or process or administration of an active agent performed on a subject for that purpose.

"Programmed death-1" (PD-1) refers to an immunoinhibitory receptor belonging to the CD28 family. PD-1 is expressed in vivo primarily on activated T cells and binds two ligands, PD-L1 and PD-L2. As used herein, the term "PD-1" 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.

"Programmed death ligand-1" (PD-L1) is one of two cell surface glycoprotein ligands to PD-1 (the other being PD-L2), and binding to PD-1 promotes T cell activation and Downregulates cytokine secretion. As used herein, the term "PD-L1" includes human PD-L1 (hPD-L1), variants, isoforms and species homologues of hPD-L1 and analogs that share at least one common epitope with hPD-L1. The complete hPD-L1 sequence can be found under GenBank Accession No. Q9NZQ7. Human PD-L1 protein is encoded by the human CD274 gene (NCBI Gene ID: 29126).

As used herein, a PD-1 or PD-L1 "inhibitor" blocks, reduces or otherwise limits the interaction of PD-1 and PD-L1 and/or the activity of PD-1 and/or PD-L1 , refers to any molecule. In some embodiments, the inhibitor is an antibody or antigen-binding fragment of an antibody. In certain other embodiments, the inhibitor comprises a small molecule.

As used herein, "signaling and transcriptional activator 1-alpha/beta" or "STAT1" refers to signaling that transduces cellular responses to interferon (IFN), cytokines KITLG/SCF and other cytokines and other growth factors. Refers to substances and transcriptional activators. After binding of type I IFNs (IFN-alpha and IFN-beta) to cell surface receptors, signaling through protein kinases results in activation of Jak kinases (TYK2 and JAK1) and tyrosine phosphorylation of STAT1 and STAT2. Phosphorylated STAT dimerizes and binds ISGF3G/IRF-9 to form a complex called the ISGF3 transcription factor, which enters the nucleus. ISGF3 binds to the IFN-stimulated response element (ISRE) and activates the IFN-stimulated gene (ISG), which renders the cell antiviral. In response to type II IFN (IFN-gamma), STAT1 is tyrosine- and serine-phosphorylated. A homodimer termed IFN-gamma-activating factor (GAF) is then formed, translocates to the nucleus and forms with the IFN-gamma activating sequence (GAS) to drive expression of target genes, Induces a cellular antiviral state. STAT1 is activated in response to KITLG/SCF and KIT signaling. STAT1 may also mediate cellular responses to activate FGFR1, FGFR2, FGFR3 and FGFR4. The complete human STAT1 amino acid sequence can be found under UniProtKB identification number P42224. Human STAT1 protein is encoded by the human STAT1 gene (NCBI Gene ID: 6772).

"Interferon gamma", "IFN-gamma" or "IFNγ" as used herein refers to cytokines involved in innate and adaptive immunity to infection (UniProtKB-P01579). IFNγ is the only member of the type II class of interferons and is therefore sometimes referred to as "type II interferons". IFNγ acts to activate macrophages and induce MHC class II expression. IFNγ is produced mostly by natural killer (NK) and natural killer T (NKT) cells as part of the innate immune response and, once immunity is acquired, by CD4 Th1 (T helper) cells and CD8 cytotoxic T lymphocytes. (CTL) Expressed by effector T cells.

As used herein, "nectin-2" is a modulator of T-cell signaling that acts as both a co-stimulatory and co-inhibitor of T-cell function, depending on the binding of nectin-2 to its receptor. (UniProtKB-Q92692). Binding of Nectin-2 to CD226 stimulates T cell proliferation and production of various cytokines including IL2, IL5, IL10, IL13 and IFNγ. Conversely, binding of Nectin-2 to PVRIG inhibits T cell proliferation.

As used herein, "CSF1R" refers to the gene encoding macrophage colony-stimulating factor 1 receptor (CSF-1-R; UniProtKB-P07333), a multifunctional tyrosine-protein kinase. In particular, CSF-1-R acts as a cell surface receptor for CSF1 and IL34 and plays an essential role in regulating the survival, proliferation and differentiation of hematopoietic progenitor cells, particularly mononuclear phagocytes such as macrophages and monocytes. have. Furthermore, binding of IL34 or CSF1 to CSF-1-R promotes the release of proinflammatory chemokines involved in innate immune and inflammatory processes.

A "subject" includes any human or non-human animal. The term "non-human animal" includes, but is not limited to, vertebrate animals such as non-human primates, sheep, dogs and rodents such as mice, rats and guinea pigs. In preferred embodiments, the subject is 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 one that, when used alone or in combination with other therapeutic agents, reduces the severity of disease symptoms, increases the frequency and duration of disease symptom-free periods, or Any amount of drug that protects a subject against disease development or promotes disease regression as evidenced by prevention of impairment or disability due to disease affliction. The ability of a therapeutic agent to promote disease regression can be determined by a variety of methods known to the skilled practitioner, such as assaying the agent's activity in human subjects during clinical trials, animal model systems predictive of efficacy in humans, or in vitro assays. can be used and evaluated.

By way of example, an "anti-cancer agent" promotes cancer regression in a subject. In preferred embodiments, a therapeutically effective amount of the drug promotes cancer regression to the point of eliminating the cancer. "Promote cancer regression" means that administration of an effective amount of a drug, alone or in combination with an anti-neoplastic agent, results in reduced tumor growth or size, tumor necrosis, reduced severity of at least one disease symptom, no disease symptom It is meant to result in prevention of impairment or disability due to increased frequency and duration of periods or morbidity. Furthermore, the terms "efficacy" and "efficacy" in relation to treatment include both pharmacological efficacy and physiological safety. Pharmacological efficacy refers to the ability of a drug to promote cancer regression in a patient. Physiological safety refers to the level of toxicity or other adverse physiological effects (adverse effects) at the cellular, organ and/or organismal level resulting from drug administration.

As used herein, “immuno-oncology” therapy or “IO” therapy refers to therapy that involves harnessing an immune response against a target to treat a tumor in a subject. Thus, IO therapy as used herein is one type of anti-cancer therapy. In some embodiments and IO therapy comprises administering an antibody or antigen-binding fragment thereof to the subject. In some embodiments, the IO therapy administers to the subject immune cells, e.g., T cells, e.g., modified T cells, e.g., chimeric antigen receptors or T cells that have been modified to express a particular T cell receptor. including doing In some embodiments, IO therapy comprises administering a therapeutic vaccine to the subject. In some embodiments, IO therapy comprises administration of cytokines or chemokines to the subject. In some embodiments, the IO therapy comprises administering an interleukin to the subject. In some embodiments, the IO therapy comprises administration of interferon to the subject. In some embodiments, IO therapy comprises administering colony stimulating factors to the subject.

As an example of tumor treatment, a therapeutically effective amount of an anti-cancer agent preferably reduces cell proliferation or tumor growth by at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and even more preferably at least about 80% inhibition. In other preferred embodiments of the invention, tumor regression can be observed and continued 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. Regardless of these ultimate measures of therapeutic efficacy, evaluation of immunotherapeutic drugs must take into account immune-related response patterns.

"Immune response" is as understood in the art, and generally refers to the biological response within a vertebrate to foreign agents or abnormalities, such as cancer cells, which directs the organism to these agents and thereby Protects against disease caused. The immune response is directed to one or more cells of the immune system (e.g., T lymphocytes, B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells or neutrophils) and these cells or In the case of invasive pathogens, pathogen-infected cells or tissues, cancer or other abnormal cells or autoimmune or pathological inflammation mediated by the action of soluble macromolecules produced by the liver (including antibodies, cytokines and complement) , resulting in selective targeting, binding, damage, destruction and/or elimination from the vertebrate body of normal human cells or tissues. The immune response may be, for example, activation or inhibition of T cells, such as effector T cells, Th cells, CD4 ⁺ cells, CD8 ⁺ T cells or Treg cells, or activation of some other cell of the immune system, such as NK cells. or includes inhibition.

"Immune-related response pattern" refers to the clinical response pattern often observed in cancer patients upon treatment with immunotherapeutic agents that produce anti-tumor effects through induction of cancer-specific immune responses or modification of innate immune processes. This pattern of response is classified as disease progression in the evaluation of traditional chemotherapeutic agents and is characterized by a beneficial therapeutic effect after an initial increase in tumor burden or the appearance of new lesions that are synonymous with drug failure. Proper evaluation of immunotherapeutic agents may therefore require long-term monitoring of the effects of these agents on the target disease.

As used herein, the terms "treatment" and "treating" refer to reversing, reducing, ameliorating, arresting, slowing or slowing the progression, evolution, severity or recurrence of symptoms, complications or conditions or biochemical manifestations associated with a disease. Any type of intervention or process or administration of an active agent administered to a subject for the purpose of prevention or overall survival enhancement. Treatment can be for subjects with or without disease (eg, for prophylactic purposes).

The term "effective dose" or "effective dosage" is defined as an amount sufficient to achieve or at least partially achieve the desired effect. A "therapeutically effective amount" or "therapeutically effective dosage" of a drug or therapeutic agent, when used alone or in combination with other therapeutic agents, reduces the severity of disease symptoms, increases the frequency and duration of disease symptom-free periods, A disease that is evidenced by an increase in overall survival (length of time a patient with a disease, such as cancer, is still alive from the date of diagnosis or the start of treatment) or prevention of impairment or disability due to disease affliction. Any amount of drug that promotes regression. A therapeutically effective amount or dosage of a drug is any amount that, alone or in combination with other therapeutic agents, prevents the development or recurrence of disease when administered to a subject at risk of developing or recurring disease. Includes "prophylactically effective amount" or "prophylactically effective dosage." The ability of a therapeutic agent to promote disease regression or prevent disease onset or recurrence is determined by skilled practice, such as assaying the agent's activity in human subjects during clinical trials, in animal model systems or in vitro assays predictive of efficacy in humans. can be evaluated using various methods known to those skilled in the art.

As an example, an anti-cancer agent is a drug that promotes cancer regression in a subject. In some embodiments, a therapeutically effective amount of the drug promotes cancer regression to the point of eliminating the cancer. "Promoting cancer regression" means reduction in tumor growth or size, tumor necrosis, reduction in severity of at least one disease symptom, no disease symptom, when the drug is administered in an effective amount, either alone or in combination with an anti-neoplastic agent. It means increasing the frequency and duration of periods, increasing overall survival, preventing impairment or disability due to disease, or otherwise ameliorating disease symptoms in a patient. Furthermore, the terms "efficacy" and "efficacy" in relation to treatment include both pharmacological efficacy and physiological safety. Pharmacological efficacy refers to the ability of a drug to promote cancer regression in a patient. Physiological safety refers to the level of toxicity or other adverse physiological effects (adverse effects) at the cellular, organ and/or organismal level resulting from drug administration.

As an example of tumor treatment, a therapeutically effective amount or dosage of a drug reduces cell proliferation or tumor growth by at least about 20%, at least about 40%, at least about 60%, or at least about 80% compared to untreated subjects. impede. In some embodiments, a therapeutically effective amount or dosage of a drug completely inhibits cell or tumor growth, ie, inhibits cell or tumor growth by 100%. The ability of compounds to inhibit tumor growth can be evaluated using the assays described herein. Alternatively, this property of a composition can be evaluated by testing the ability of a compound to inhibit cell proliferation, and such inhibition can be measured in vitro by assays known to the skilled practitioner. In certain embodiments described herein, tumor regression can be observed and continued for a period of at least about 20 days, at least about 40 days, or at least about 60 days.

As used herein, the term "biological sample" refers to biological material isolated from a subject. A biological sample can include any biological material suitable for determining target gene expression, e.g., by sequencing nucleic acid of a tumor (or circulating tumor cells) and identifying genetic alterations in the sequenced nucleic acid. . A biological sample can be any suitable biological tissue or fluid, such as, for example, tumor tissue, blood, plasma and serum. In some embodiments, the sample is a tumor sample. In some embodiments, a tumor sample can be obtained from a tumor tissue biopsy sample, such as formalin-fixed, paraffin-embedded (FFPE) tumor tissue or fresh frozen tumor tissue. In certain other embodiments, the biological sample is, in certain embodiments, a liquid biopsy sample comprising one or more of blood, serum, plasma, circulating tumor cells, exoRNA, ctDNA and cfDNA.

As used herein, "tumor sample" refers to a biological sample that contains tumor tissue. In some embodiments, the tumor sample is a tumor biopsy sample. In some embodiments, the tumor sample comprises tumor cells and one or more non-tumor cells present in the tumor microenvironment (TME). For the purposes of the present invention, a TME consists of at least two regions. A tumor "parenchyma" is an area of the TME that primarily contains tumor cells, or a portion (or portions) of the TME that contains a mass of tumor cells. The tumor parenchyma does not necessarily consist exclusively of tumor cells; rather, other cells such as stromal cells and/or lymphocytes may also be present in the parenchyma. The "stromal" region of the TME contains adjacent non-tumor cells. In some embodiments, the tumor sample comprises all or part of the tumor parenchyma and one or more cells of the stroma. In some embodiments, the tumor sample is obtained from the parenchyma. In some embodiments, the tumor sample is obtained from stroma. In certain other embodiments, tumor samples are obtained from parenchyma and stroma.

The use of alternatives (eg, “or”) should be understood to mean either one, both, or any combination thereof. It should be understood that any singular expression used herein refers to "one or more" of any stated or listed ingredient.

The term "about" or "consisting essentially of" means the allowable range of a particular value or composition as determined by one of skill in the art due, in part, to the method by which the value or composition was measured or determined, i.e., the limits of the measurement system. means the value or composition within the error range For example, "about" or "consisting essentially of" can mean within 1 or 1 or more standard deviations, per practice in the art. Alternatively, "about" or "consisting essentially of" can mean a range of up to 10%. Further, particularly with respect to biological systems or processes, the term can mean one order of magnitude or up to five times the value. When a particular value or composition is provided in the specification and claims, unless otherwise stated, the meaning of "about" or "consisting essentially of" does not mean the error allowed for that particular value or composition. should be assumed to be within range.

Any concentration range, percentage range, ratio range or integer range used herein, unless otherwise specified, is understood to include any integer and, where appropriate, fractional values thereof within the stated range.

Various aspects of the invention are described in further detail in the following subsections.

II. Methods of the Invention Inflammation in the TME can be an indicator of potential responsiveness to IO therapy. However, modern methods of measuring inflammation in tumors require a laborious immunohistochemistry process to detect and analyze CD8 expression in tumor biopsy samples. Surprisingly, expression patterns of a relatively small number of genes (in some embodiments, at least about 4 genes) have been found to correlate with inflammation in the tumor microenvironment. In some embodiments, the methods described herein can replace the need for time-consuming IHC. One aspect of the invention is an IO therapy, such as anti-PD-1/PD, comprising measuring the expression of a panel of genes in a tumor sample obtained from a subject in need of an anti-PD-1/PD-L1 antagonist. - A method of identifying a human subject suitable for an L1 antagonist, wherein the gene panel comprises at least one of STAT1, IFNγ, Nectin2 and CSF1R. In some embodiments, the measurement is performed in vitro.

One aspect of the invention is a method of preparing a nucleic acid fraction from a tumor of a subject in need of IO therapy by (a) removing a tumor biopsy sample from the subject; (a) obtaining a fraction of the nucleic acid extracted in; and (c) analyzing the expression level of one or more genes in a gene panel selected from STAT1, IFNγ, Nectin2 and CSF1R. In some embodiments, the nucleic acid is mRNA.

In some embodiments, the gene panel comprises at least two of STAT1, IFNγ, Nectin2 and CSF1R. In some embodiments, the gene panel includes STAT1 and IFNγ. In some embodiments, the gene panel includes STAT1 and Nectin2. In some embodiments, the gene panel includes STAT1 and CSF1R. In some embodiments, the gene panel includes IFNγ and Nectin2. In some embodiments, the gene panel comprises IFNγ and CSF1R. In some embodiments, the gene panel includes Nectin2 and CSF1R.

In some embodiments, the gene panel comprises at least three of STAT1, IFNγ, Nectin2 and CSF1R. In some embodiments, the gene panel includes STAT1, IFNγ and Nectin2. In some embodiments, the gene panel comprises STAT1, IFNγ and CSF1R. In some embodiments, the gene panel includes STAT1, Nectin2 and CSF1R. In some embodiments, the gene panel includes IFNγ, Nectin2 and CSF1R.

In some embodiments, the gene panel comprises STAT1, IFNγ, Nectin2 and CSF1R. In some embodiments, the gene panel further comprises one or more additional genes. In some embodiments, the gene panel comprises from at least 2 to at least about 100 genes. In some embodiments, 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 1 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 comprising from to at least about 6, from at least 2 to at least about 5 or from at least 2 to at least about 4 genes.

In some embodiments, the gene panel comprises at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10 at least about 11, at least about 12, at least about 13, at least about 14, 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 or at least about 100 genes.

In some embodiments, the gene panel consists of STAT1, IFNγ, Nectin2 and CSF1R. In some embodiments, the gene panel consists essentially of STAT1, IFNγ, Nectin2 and CSF1R. In some embodiments, the gene panel comprises STAT1, IFNγ, 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 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 consists of or consists essentially of 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 additional genes.

II.A. Gene Expression Profiling Gene expression profiling as used herein comprises, consists essentially of or consists of, for example, at least one, at least two or at least three of STAT1, IFNγ, Nectin2 and CSF1R Figure 10 is a measurement of combined expression levels of a panel of genes disclosed herein. In some embodiments, measurements are made using a sample obtained from a subject. In some embodiments, the sample is a tumor sample. Any biological sample containing one or more tumor cells can be used in the methods disclosed herein. In some embodiments, the sample is selected from a tumor biopsy sample, blood sample, serum sample, or any combination thereof. In some embodiments, the sample is a tumor biopsy sample taken from the subject prior to administration of a treatment described herein, eg, IO therapy, eg, an anti-PD-1/PD-L1 agonist. In specific embodiments, the sample obtained from the subject is a formalin-fixed tumor biopsy sample. In some embodiments, the sample obtained from the subject is a paraffin-embedded tumor biopsy sample. In some embodiments, the sample obtained from the subject is a fresh frozen tumor biopsy sample.

Any method known in the art for measuring the expression of a particular gene or panel of genes can be used in the disclosed methods. In some embodiments, expression of one or more of the inflammatory genes of the inflammatory gene panel is determined by detecting the presence of mRNA transcribed from the inflammatory gene, the presence of protein encoded by the inflammatory gene, or both.

In some embodiments, gene expression is determined by measuring gene mRNA, eg, by measuring levels of one or more of STAT1 mRNA, IFNγ mRNA, Nectin2 mRNA and CSF1R mRNA, in a sample obtained from the subject. In some embodiments, the gene expression profile is determined by measuring levels of STAT1 mRNA, IFNγ 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 levels of gene mRNA. In some embodiments, gene mRNA is measured using reverse transcriptase PCR. In some embodiments, gene mRNA is measured using a nuclease protection assay. In some embodiments, gene mRNA is measured using next generation sequencing (NGS). In some embodiments, gene mRNA is measured using RNA in situ hybridization.

In some embodiments, expression of a gene is measured in a sample obtained from a subject by measuring the level of a protein encoded by the gene, e.g., by measuring the level of one or more of STAT1 protein, IFNγ protein, Nectin2 protein and CSF1R protein. ,It is determined. In some embodiments, the gene expression profile is determined by measuring levels of STAT1 protein, IFNγ 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 protein levels. In some embodiments, gene expression profiles are measured using immunohistochemistry (IHC) assays. In some embodiments, the IHC is automated IHC.

In some embodiments, expression of one or more of the genes of the gene panel is normalized to expression of one or more housekeeping genes. In certain embodiments, the one or more housekeeping genes consist of genes whose expression is relatively constant across different tumor types in different subjects.

In some embodiments, raw gene expression values are normalized according to standard gene expression profiling protocols. In these embodiments, the gene expression profile can be calculated as the median or mean of the log2-transformed normalized and adjusted expression values across all of the target genes in the signature and displayed on a linear scale. In some embodiments, the profile is positive or negative depending on whether gene expression is upregulated or downregulated under certain conditions.

In some embodiments, increased/decreased expression is characterized by an expression level that is greater/less than the expression of the same one or more genes in a control sample. In some embodiments, the control sample comprises non-tumor tissue from the same subject. In some embodiments, the control sample comprises matched non-tumor tissue from the same subject. In certain embodiments, control samples comprise matched tissue from non-tumor-bearing subjects. In some embodiments, the control sample comprises more than one tumor tissue sample from more than one other subject, eg, increased expression relative to the average expression level across more than one other tumor sample.

In some embodiments, the increased/decreased expression is characterized by an expression level that is greater/less than the control expression level. In some embodiments, the control expression level is the average expression level. In certain embodiments, the mean expression level is determined by measuring the expression of a gene (or genes) present in a gene panel in tumor samples obtained from a subject population and calculating the mean of the subject population. In some embodiments, each member of the subject population has the same tumor as the subject receiving IO therapy, eg, an anti-PD-1/PD-L1 antagonist.

In certain embodiments, the increased expression of upregulated genes, e.g., STAT1 or IFNγ for parenchymal gene signatures or Nectin2 and CSF1R for stromal gene signatures, is at least about 5%, at least about 10% higher than the expression level or mean expression level of control samples. %, 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. In some embodiments, increased expression is characterized by an expression level that is at least about 25% higher than the expression level or average expression level of a control sample. In some embodiments, increased expression is characterized by an expression level that is at least about 30% higher than the expression level or average expression level of a control sample. In some embodiments, increased expression is characterized by an expression level that is at least about 35% higher than the expression level or average expression level of a control sample. In some embodiments, increased expression is characterized by an expression level that is at least about 40% higher than the expression level or average expression level of a control sample. In some embodiments, increased expression is characterized by an expression level that is at least about 45% higher than the expression level or mean expression level of a control sample. In some embodiments, increased expression is characterized by an expression level that is at least about 50% higher than the expression level or average expression level of a control sample. In some embodiments, increased expression is characterized by an expression level that is at least about 55% higher than the expression level or average expression level of a control sample. In some embodiments, increased expression is characterized by an expression level that is at least about 60% higher than the expression level or mean expression level of a control sample. In some embodiments, increased expression is characterized by an expression level that is at least about 65% higher than the expression level or mean expression level of a control sample. In some embodiments, increased expression is characterized by an expression level that is at least about 70% higher than the expression level or average expression level of a control sample. In some embodiments, increased expression is characterized by an expression level that is at least about 75% higher than the expression level or mean expression level of a control sample. In some embodiments, increased expression is characterized by an expression level that is at least about 80% higher than the expression level or mean expression level of a control sample. In some embodiments, increased expression is characterized by an expression level that is at least about 85% higher than the expression level or mean expression level of a control sample. In some embodiments, increased expression is characterized by an expression level that is at least about 90% higher than the expression level or average expression level of a control sample. In some embodiments, increased expression is characterized by an expression level that is at least about 95% higher than the expression level or average expression level of a control sample. In some embodiments, increased expression is characterized by an expression level that is at least about 100% higher than the expression level or average expression level of a control sample. In some embodiments, increased expression is characterized by an expression level that is at least about 125% higher than the expression level or mean expression level of a control sample. In some embodiments, increased expression is characterized by an expression level that is at least about 150% higher than the expression level or mean expression level of a control sample. In some embodiments, increased expression is characterized by an expression level that is at least about 175% higher than the expression level or mean expression level of a control sample. In some embodiments, increased expression is characterized by an expression level that is at least about 200% higher than the expression level or average expression level of a control sample. In some embodiments, increased expression is characterized by an expression level that is at least about 225% higher than the expression level or mean expression level of a control sample. In some embodiments, increased expression is characterized by an expression level that is at least about 250% higher than the expression level or mean expression level of a control sample. In some embodiments, increased expression is characterized by an expression level that is at least about 275% higher than the expression level or mean expression level of a control sample. In some embodiments, increased expression is characterized by an expression level that is at least about 300% higher than the expression level or average expression level of a control sample.

In some embodiments, the increased expression of an upregulated gene, e.g., STAT1 or IFNγ for parenchymal gene signatures or Nectin2 and CSF1R for stromal gene signatures, is at least about 1.25-fold, or at least about 1.30 times, at least about 1.35 times, at least about 1.40 times, at least about 1.45 times, at least about 1.50 times, at least about 1.55 times, at least about 1.60 times, 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 times, at least about 2.25 times, at least about 2.50 times, at least about 2.75 times, at least about 3 times, at least about 3.25 times, at least about 3.50 times, at least about 3.75 times or at least characterized by about 400 times higher. In some embodiments, increased expression is characterized by an expression level that is at least about 1.25-fold higher than the expression level or mean expression level of a control sample. In some embodiments, increased expression is characterized by an expression level that is at least about 1.30-fold higher than the expression level or mean expression level of a control sample. In some embodiments, increased expression is characterized by an expression level that is at least about 1.35-fold higher than the expression level or mean expression level of a control sample. In some embodiments, increased expression is characterized by an expression level that is at least about 1.40-fold higher than the expression level or mean expression level of a control sample. In some embodiments, increased expression is characterized by an expression level that is at least about 1.45-fold higher than the expression level or mean expression level of a control sample. In some embodiments, increased expression is characterized by an expression level that is at least about 1.50-fold higher than the expression level or mean expression level of a control sample. In some embodiments, increased expression is characterized by an expression level that is at least about 1.55-fold higher than the expression level or mean expression level of a control sample. In some embodiments, increased expression is characterized by an expression level that is at least about 1.60-fold higher than the expression level or mean expression level of a control sample. In some embodiments, increased expression is characterized by an expression level that is at least about 1.65-fold higher than the expression level or mean expression level of a control sample. In some embodiments, increased expression is characterized by an expression level that is at least about 1.70-fold higher than the expression level or mean expression level of a control sample. In some embodiments, increased expression is characterized by an expression level that is at least about 1.75-fold higher than the expression level or mean expression level of a control sample. In some embodiments, increased expression is characterized by an expression level that is at least about 1.80-fold higher than the expression level or mean expression level of a control sample. In some embodiments, increased expression is characterized by an expression level that is at least about 1.85-fold higher than the expression level or mean expression level of a control sample. In some embodiments, increased expression is characterized by an expression level that is at least about 1.90-fold higher than the expression level or mean expression level of a control sample. In some embodiments, increased expression is characterized by an expression level that is at least about 1.95-fold higher than the expression level or mean expression level of a control sample. In some embodiments, increased expression is characterized by an expression level that is at least about 2-fold higher than the expression level or average expression level of a control sample. In some embodiments, increased expression is characterized by an expression level that is at least about 2.25-fold higher than the expression level or mean expression level of a control sample. In some embodiments, increased expression is characterized by an expression level that is at least about 2.50-fold higher than the expression level or mean expression level of a control sample. In some embodiments, increased expression is characterized by an expression level that is at least about 2.75-fold higher than the expression level or mean expression level of a control sample. In some embodiments, increased expression is characterized by an expression level that is at least about 3-fold higher than the expression level or mean expression level of a control sample. In some embodiments, increased expression is characterized by an expression level that is at least about 3.25-fold higher than the expression level or mean expression level of a control sample. In some embodiments, increased expression is characterized by an expression level that is at least about 3.50-fold higher than the expression level or mean expression level of a control sample. In some embodiments, increased expression is characterized by an expression level that is at least about 3.75-fold higher than the expression level or mean expression level of a control sample. In some embodiments, increased expression is characterized by an expression level that is at least about 4-fold higher than the expression level or mean expression level of a control sample.

In certain embodiments, decreased expression of downregulated genes, eg, Nectin2 and CSF1R for a parenchymal gene signature or STAT1 or IFNγ for a stromal gene signature, is characterized by an expression level that is lower than the control expression level. In some embodiments, the decreased expression 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 Characterized by a 275% or at least about 300% lower expression level. In some embodiments, decreased expression is characterized by an expression level that is at least about 25% lower than the expression level or mean expression level of a control sample. In some embodiments, decreased expression is characterized by an expression level that is at least about 30% lower than the expression level or average expression level of a control sample. In some embodiments, decreased expression is characterized by an expression level that is at least about 35% lower than the expression level or mean expression level of a control sample. In some embodiments, decreased expression is characterized by an expression level that is at least about 40% lower than the expression level or mean expression level of a control sample. In some embodiments, decreased expression is characterized by an expression level that is at least about 45% lower than the expression level or mean expression level of a control sample. In some embodiments, decreased expression is characterized by an expression level that is at least about 50% lower than the expression level or mean expression level of a control sample. In some embodiments, decreased expression is characterized by an expression level that is at least about 55% lower than the expression level or mean expression level of a control sample. In some embodiments, decreased expression is characterized by an expression level that is at least about 60% lower than the expression level or average expression level of a control sample. In some embodiments, decreased expression is characterized by an expression level that is at least about 65% lower than the expression level or mean expression level of a control sample. In some embodiments, decreased expression is characterized by an expression level that is at least about 70% lower than the expression level or mean expression level of a control sample. In some embodiments, decreased expression is characterized by an expression level that is at least about 75% lower than the expression level or mean expression level of a control sample. In some embodiments, decreased expression is characterized by an expression level that is at least about 80% lower than the expression level or mean expression level of a control sample. In some embodiments, decreased expression is characterized by an expression level that is at least about 85% lower than the expression level or average expression level of a control sample. In some embodiments, decreased expression is characterized by an expression level that is at least about 90% lower than the expression level or mean expression level of a control sample. In some embodiments, decreased expression is characterized by an expression level that is at least about 95% lower than the expression level or mean expression level of a control sample. In some embodiments, decreased expression is characterized by an expression level that is at least about 100% lower than the expression level or average expression level of a control sample. In some embodiments, decreased expression is characterized by an expression level that is at least about 125% lower than the expression level or mean expression level of a control sample. In some embodiments, decreased expression is characterized by an expression level that is at least about 150% lower than the expression level or mean expression level of a control sample. In some embodiments, decreased expression is characterized by an expression level that is at least about 175% lower than the expression level or mean expression level of a control sample. In some embodiments, decreased expression is characterized by an expression level that is at least about 200% lower than the expression level or mean expression level of a control sample. In some embodiments, decreased expression is characterized by an expression level that is at least about 225% lower than the expression level or mean expression level of a control sample. In some embodiments, decreased expression is characterized by an expression level that is at least about 250% lower than the expression level or mean expression level of a control sample. In some embodiments, decreased expression is characterized by an expression level that is at least about 275% lower than the expression level or mean expression level of a control sample. In some embodiments, decreased expression is characterized by an expression level that is at least about 300% lower than the expression level or average expression level of a control sample.

In some embodiments, the decreased expression of downregulated genes, e.g., Nectin2 and CSF1R for parenchymal gene signatures or STAT1 or IFNγ for stromal gene signatures, is at least about 1.25-fold, or at least about 1.30 times, at least about 1.35 times, at least about 1.40 times, at least about 1.45 times, at least about 1.50 times, at least about 1.55 times, at least about 1.60 times, 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 times, at least about 2.25 times, at least about 2.50 times, at least about 2.75 times, at least about 3 times, at least about 3.25 times, at least about 3.50 times, at least about 3.75 times or at least It is characterized by an approximately 400-fold lower expression level. In some embodiments, decreased expression is characterized by an expression level that is at least about 1.25-fold lower than the expression level or mean expression level of a control sample. In some embodiments, decreased expression is characterized by an expression level that is at least about 1.30-fold lower than the expression level or mean expression level of a control sample. In some embodiments, decreased expression is characterized by an expression level that is at least about 1.35-fold lower than the expression level or mean expression level of a control sample. In some embodiments, decreased expression is characterized by an expression level that is at least about 1.40-fold lower than the expression level or mean expression level of a control sample. In some embodiments, decreased expression is characterized by an expression level that is at least about 1.45-fold lower than the expression level or mean expression level of a control sample. In some embodiments, decreased expression is characterized by an expression level that is at least about 1.50-fold lower than the expression level or mean expression level of a control sample. In some embodiments, decreased expression is characterized by an expression level that is at least about 1.55-fold lower than the expression level or mean expression level of a control sample. In some embodiments, decreased expression is characterized by an expression level that is at least about 1.60-fold lower than the expression level or mean expression level of a control sample. In some embodiments, decreased expression is characterized by an expression level that is at least about 1.65-fold lower than the expression level or mean expression level of a control sample. In some embodiments, decreased expression is characterized by an expression level that is at least about 1.70-fold lower than the expression level or mean expression level of a control sample. In some embodiments, decreased expression is characterized by an expression level that is at least about 1.75-fold lower than the expression level or mean expression level of a control sample. In some embodiments, decreased expression is characterized by an expression level that is at least about 1.80-fold lower than the expression level or mean expression level of a control sample. In some embodiments, decreased expression is characterized by an expression level that is at least about 1.85-fold lower than the expression level or mean expression level of a control sample. In some embodiments, decreased expression is characterized by an expression level that is at least about 1.90-fold lower than the expression level or mean expression level of a control sample. In some embodiments, decreased expression is characterized by an expression level that is at least about 1.95-fold lower than the expression level or mean expression level of a control sample. In some embodiments, decreased expression is characterized by an expression level that is at least about 2-fold lower than the expression level or mean expression level of a control sample. In some embodiments, decreased expression is characterized by an expression level that is at least about 2.25-fold lower than the expression level or mean expression level of a control sample. In some embodiments, decreased expression is characterized by an expression level that is at least about 2.50-fold lower than the expression level or mean expression level of a control sample. In some embodiments, decreased expression is characterized by an expression level that is at least about 2.75-fold lower than the expression level or mean expression level of a control sample. In some embodiments, decreased expression is characterized by an expression level that is at least about 3-fold lower than the expression level or mean expression level of a control sample. In some embodiments, decreased expression is characterized by an expression level that is at least about 3.25-fold lower than the expression level or mean expression level of a control sample. In some embodiments, decreased expression is characterized by an expression level that is at least about 3.50-fold lower than the expression level or mean expression level of a control sample. In some embodiments, decreased expression is characterized by an expression level that is at least about 3.75-fold lower than the expression level or mean expression level of a control sample. In some embodiments, decreased expression is characterized by an expression level that is at least about 4-fold lower than the expression level or mean expression level of a control sample.

II.B. Methods of Treatment Certain aspects of the invention relate to methods of identifying a subject suitable for treatment and then administering the treatment to the suitable subject. The methods of identifying suitable subjects described herein may be used prior to any immuno-oncology (IO) therapy. In certain embodiments, suitable subjects are PD-1, PD-L1, CTLA-4, LAG-3, TIGIT, TIM3, CSF1R, NKG2a, OX40, ICOS, CD137, KIR, TGFβ, IL-10, IL-8, An antibody or antigen binding fragment thereof that specifically binds to a protein selected from IL-2, CD96, VISTA, B7-H4, Fas ligand, CXCR4, mesothelin, CD27, GITR, MICA, MICB and any combination thereof is administered and/or subsequently administered.

In certain embodiments, suitable subjects are administered and/or subsequently administered an antibody or antigen-binding fragment thereof that specifically binds to PD-1. In certain embodiments, suitable subjects are administered and/or subsequently administered an antibody or antigen-binding fragment thereof that specifically binds to PD-L1. In certain embodiments, suitable subjects are administered and/or subsequently administered an antibody or antigen-binding fragment thereof that specifically binds CTLA-4. In certain embodiments, suitable subjects are administered and/or subsequently administered an antibody or antigen-binding fragment thereof that specifically binds LAG-3. In certain embodiments, suitable subjects are administered and/or subsequently administered an antibody or antigen-binding fragment thereof that specifically binds to TIGIT. In certain embodiments, suitable subjects are administered and/or subsequently administered an antibody or antigen-binding fragment thereof that specifically binds to TIM3. In certain embodiments, suitable subjects are administered and/or subsequently administered an antibody or antigen-binding fragment thereof that specifically binds to GITR. In certain embodiments, suitable subjects are administered and/or subsequently administered an antibody or antigen-binding fragment thereof that specifically binds to MICA. In certain embodiments, suitable subjects are administered and/or subsequently administered an antibody or antigen-binding fragment thereof that specifically binds MICB. In certain embodiments, suitable subjects are administered and/or subsequently administered an antibody or antigen-binding fragment thereof that specifically binds CSF1R.

In certain embodiments, suitable subjects are administered and/or subsequently administered more than one antibody or antigen-binding fragment thereof disclosed herein. In certain embodiments, suitable subjects are administered and/or subsequently administered at least two antibodies or antigen-binding fragments thereof. In certain embodiments, suitable subjects are administered and/or subsequently administered at least three antibodies or antigen-binding fragments thereof. In certain embodiments, a suitable subject is administered and/or subsequently administered an antibody or antigen-binding fragment thereof that specifically binds to PD-1 and an antibody or antigen-binding fragment thereof that specifically binds to CTLA-4. be. In certain embodiments, a suitable subject is administered and/or subsequently administered an antibody or antigen-binding fragment thereof that specifically binds to PD-L1 and an antibody or antigen-binding fragment thereof that specifically binds to CTLA-4. be. In certain embodiments, a suitable subject is administered and/or subsequently administered an antibody or antigen-binding fragment thereof that specifically binds to PD-1 and an antibody or antigen-binding fragment thereof that specifically binds to CSF1R. In certain embodiments, a suitable subject is administered and/or subsequently administered an antibody or antigen-binding fragment thereof that specifically binds to PD-L1 and an antibody or antigen-binding fragment thereof that specifically binds to CSF1R. In certain embodiments, a suitable subject is administered and/or subsequently administered an antibody or antigen-binding fragment thereof that specifically binds to PD-1 and an antibody or antigen-binding fragment thereof that specifically binds to LAG-3. be. In certain embodiments, a suitable subject is administered and/or subsequently administered an antibody or antigen-binding fragment thereof that specifically binds to PD-L1 and an antibody or antigen-binding fragment thereof that specifically binds to LAG-3. be.

In some embodiments, therapeutic agents are administered to a suitable subject after gene expression profiles have been measured. In some embodiments, the measuring is in vitro. In certain other embodiments, the measuring is in vivo. In some embodiments, 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 the gene expression profile was measured. , 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 later, the therapeutic agent is administered.

In certain embodiments, the particular therapeutic agent administered to and/or subsequently administered to a suitable subject is by gene expression profile. In some embodiments, the gene expression profile has a substantial signature. In certain other embodiments, the gene expression profile has a stromal signature.

II.B.1. Substantial Signatures In certain embodiments, the present invention provides IO therapy, such as anti-PD-1, for use in methods of identifying human subjects suitable for anti-PD-1/PD-L1 antagonists. /PD-L1 antagonist, the method comprising measuring the expression of a panel of genes in a tumor sample obtained from a subject in need of an anti-PD-1/PD-L1 antagonist, wherein the gene panel comprises at least three or four of STAT1, IFNγ, Nectin2 and CSF1R. In some embodiments, the tumor sample is
(i) increased expression of one or more of STAT1 and IFNγ in a sample compared to expression of one or more of STAT1 and IFNγ (“upregulated genes”) in a control sample;
(ii) decreased expression of one or more of Nectin2 and CSF1R in a sample compared to one or more of Nectin2 and CSF1R (“downregulated genes”) in a control sample; or
(iii) Subjects are identified as eligible when they exhibit both (i) and (ii). In certain other embodiments, the subject should be administered an anti-PD-1/PD-L1 antagonist.

The invention provides, in one aspect, a pharmaceutical composition comprising an IO therapy, e.g., an anti-PD-1/PD-L1 antagonist, for use in a method of treating a human subject with a tumor, wherein , the tumor sample obtained from the subject is
(i) increased expression of one or more of STAT1 and IFNγ in a tumor sample obtained from the subject compared to expression of one or more of STAT1 and IFNγ (“upregulated genes”) in control samples;
(ii) decreased expression of one or more of Nectin2 and CSF1R in a tumor sample obtained from the subject compared to expression of one or more of Nectin2 and CSF1R (“downregulated genes”) in a control sample; or
(iii) Show both (i) and (ii).

In certain other embodiments, the invention provides an anti-PD-1/PD-L1 antagonist comprising measuring the expression of a panel of genes in a tumor sample obtained from a subject in need of an anti-PD-1/PD-L1 antagonist. A method is provided for identifying a human subject suitable for L1 antagonist therapy, wherein the gene panel comprises at least three of STAT1, IFNγ, Nectin2 and CSF1R. In some embodiments, the measuring is in vitro. In certain other embodiments, the measuring is in vivo.

A subject, as used herein, means that the subject (i) expresses one or more of STAT1 and IFNγ in a tumor sample obtained from the subject compared to the expression of one or more of STAT1 and IFNγ (“upregulated genes”) in a control sample. (ii) decreased expression of one or more of Nectin2 and CSF1R in a tumor sample obtained from the subject compared to the expression of one or more of Nectin2 and CSF1R (“downregulated genes”) in control samples. or (iii) have a substantial signature when exhibiting both (i) and (ii).

That is, subjects with a parenchymal signature can be identified as suitable for IO therapy, eg, anti-PD-1/PD-L1 antagonist treatment. A subject with a parenchymal signature as used herein is defined as (i) increased expression of STAT1 and/or IFNγ (“upregulated genes”), (ii) decreased expression of Nectin2 and/or CSF1R (“downregulated genes”) or (ii) have a tumor characterized by both (i) and (ii); Thus, in certain embodiments, the subject determines that the tumor sample (i) has increased expression of STAT1 and/or IFNγ in a tumor sample compared to expression of STAT1 and/or IFNγ (an "upregulated gene") in a control sample; ii) decreased expression of Nectin2 and/or CSF1R in tumor samples compared to expression of one or more of Nectin2 and/or CSF1R (“downregulated genes”) in control samples; or (iii) (i) and It is identified as suitable when it exhibits both (ii). In certain embodiments, suitable subjects have a tumor characterized by increased expression of STAT1. In certain embodiments, suitable subjects have tumors characterized by increased expression of IFNγ. In certain embodiments, suitable subjects have tumors characterized by increased expression of STAT1 and IFNγ. In certain embodiments, suitable subjects have a tumor characterized by decreased expression of Nectin-2. In certain embodiments, suitable subjects have a tumor characterized by decreased expression of CSF1R. In certain embodiments, suitable subjects have tumors characterized by decreased expression of Nectin2 and CSF1R. In certain embodiments, suitable subjects have tumors characterized by increased expression of STAT1 and decreased expression of Nectin2. In certain embodiments, suitable subjects have tumors characterized by increased expression of STAT1 and decreased expression of Nectin2. In certain embodiments, suitable subjects have tumors characterized by increased expression of IFNγ and decreased expression of Nectin-2. In certain embodiments, suitable subjects have tumors characterized by increased expression of IFNγ and decreased expression of CSF1R. In certain embodiments, suitable subjects have tumors characterized by increased expression of STAT1 and IFNγ and decreased expression of Nectin2 and CSF1R.

In certain embodiments, a suitable subject, eg, a subject having a parenchymal signature described herein, is administered and/or subsequently administered an IO therapy described herein. In certain embodiments, a suitable subject, eg, a subject having a parenchymal signature described herein, is administered and/or subsequently administered an anti-PD-1/PD-L1 antagonist. In certain embodiments, a suitable subject, eg, a subject having a parenchymal signature described herein, is administered and/or subsequently administered an anti-PD-1 antibody. In certain embodiments, a suitable subject, eg, a subject with a parenchymal signature described herein, is administered and/or subsequently administered an anti-PD-L1 antibody. In certain embodiments, a suitable subject, eg, a subject having a parenchymal signature described herein, is administered and/or subsequently administered anti-PD-1 antibody monotherapy. In certain embodiments, a suitable subject, eg, a subject having a parenchymal signature described herein, is administered and/or subsequently administered anti-PD-L1 antibody monotherapy. In certain embodiments, a suitable subject, eg, a subject with a parenchymal signature described herein, is administered an anti-PD-1 antibody and one or more additional anti-cancer agents (eg, one or more additional IO therapies described herein, one are administered and/or subsequently administered combination therapy, including chemotherapy (above chemotherapy or any combination thereof).

In some embodiments, an anti-PD-1/PD-L1 antagonist is administered to a subject, wherein a tumor sample obtained from the subject is (i) compared to the expression of STAT1 and IFNγ (“upregulated genes”) in a control sample. (ii) expression of one or more of Nectin2 and CSF1R (“downregulated genes”) in a tumor sample from the subject compared to the expression in the tumor sample from the subject; Decreased expression of Nectin2 and CSF1R; or (iii) both (i) and (ii).

In some embodiments, an anti-PD-1/PD-L1 antagonist is administered to a subject, wherein a tumor sample obtained from the subject is (i) compared to the expression of STAT1 or IFNγ (“upregulated gene”) in a control sample. (ii) expression of one or more of Nectin2 and CSF1R (“downregulated genes”) in a tumor sample from the subject compared to a control sample; Increased expression of Nectin2 or CSF1R; or (iii) not both (i) and (ii).

II.B.2. Stromal Signature In some embodiments, a sample that does not show a parenchymal gene signature, but rather (i) compared to expression of one or more of Nectin2 and CSF1R (“upregulated genes”) in a control sample. (ii) expression of one or more of STAT1 and IFNγ in a sample compared to expression of one or more of STAT1 and IFNγ (“downregulated genes”) in a control sample; Shows a genetic signature, eg, a stromal signature, showing a decrease or (iii) both (i) and (ii). In some embodiments, a subject with a stromal gene signature is not suitable for IO monotherapy, eg, anti-PD-1/PD-L1 antagonist monotherapy. In certain other embodiments, subjects with a stromal gene signature are suitable for combination therapy with IO therapy and anti-cancer agents.

In one aspect, the invention provides a pharmaceutical composition comprising an anti-PD-1/PD-L1 antagonist for use in a method of identifying human subjects suitable for combination therapy with a combination of a PD-1/PD-L1 antagonist and 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 combination therapy, wherein the panel of genes comprises at least three of CSF1R, Nectin2, STAT1 and IFNγ Including pcs.

In certain embodiments, the tumor sample has (i) increased expression of one or more of CSF1R and Nectin2 (“upregulated genes”) in the sample compared to expression of one or more of CSF1R and Nectin2 (“upregulated genes”) in a control sample; (ii) a) reduced expression of one or more of STAT1 and IFNγ in a sample compared to expression of one or more of STAT1 and IFNγ (“downregulated genes”) in a control sample or (iii) both (i) and (ii); Subjects are identified as eligible when indicated. In certain other embodiments, the subject is administered a combination of an anti-PD-1/PD-L1 antagonist and an anti-cancer agent.

In one aspect, the invention provides a pharmaceutical composition comprising a combination of an anti-PD-1/PD-L1 antagonist and an anti-cancer agent for use in a method of treating a human subject with a tumor, wherein from the subject (i) expression of one or more of CSF1R and Nectin2 (“upregulated genes”) in a tumor sample obtained from a subject compared to expression of one or more of CSF1R and Nectin2 (“upregulated genes”) in a control sample; (ii) decreased expression of one or more of STAT1 and IFNγ (“downregulated genes”) in a tumor sample obtained from the subject compared to expression of one or more of STAT1 and IFNγ (“downregulated genes”) in a control sample; or (iii) Both (i) and (ii) are shown.

In certain other aspects, the invention provides a method of identifying a human subject suitable for combination therapy with an anti-PD-1/PD-L1 antagonist in combination with an anti-cancer agent, which method requires an anti-PD-1/PD-L1 antagonist. measuring the expression of a panel of genes in a tumor sample obtained from a subject, wherein the panel of genes comprises at least three of CSF1R, Nectin2, STAT1 and IFNγ. In certain other embodiments, the measuring is in vivo. In some embodiments, the measuring is in vitro. In certain embodiments, the subject has the expression of one or more of CSF1R and Nectin2 in a tumor sample, where the tumor sample is (i) compared to the expression of one or more of CSF1R and Nectin2 (“upregulated genes”) in a control sample; (ii) decreased expression of one or more of STAT1 and IFNγ (“downregulated genes”) in tumor samples compared to expression of one or more of STAT1 and IFNγ (“downregulated genes”) in control samples; or (iii) (i) and A subject is identified as eligible when both (ii) are indicated. In some embodiments, the method further comprises administering an anti-PD-1/PD-L1 antagonist in combination with an anti-cancer agent.

A subject as used herein means that the subject (i) has increased expression of one or more of CSF1R and Nectin2 (“upregulated genes”) in a tumor sample compared to expression of one or more of CSF1R and Nectin2 (“upregulated genes”) in a control sample. (ii) decreased expression of one or more of STAT1 and IFNγ in a tumor sample compared to expression of one or more of STAT1 and IFNγ (“downregulated genes”) in a control sample; or (iii) (i) and ( A subject has a stromal signature when exhibiting both ii). In certain embodiments, a subject with a stromal signature is identified as suitable for combination therapy comprising (i) an IO therapy, e.g., an anti-PD-1/PD-L1 antagonist therapy and (ii) an additional anti-cancer therapy. . A subject with a stromal signature as used herein has (i) decreased expression of STAT1 and/or IFNγ (“downregulated genes”), (ii) increased expression of Nectin2 and/or CSF1R (“upregulated genes”). or (ii) have a tumor characterized by both (i) and (ii). Thus, in certain embodiments, a subject determines that the tumor sample has (i) reduced expression of STAT1 and/or IFNγ in a tumor sample compared to expression of STAT1 and/or IFNγ (a “downregulated gene”) in a control sample; a) increased expression of Nectin2 and/or CSF1R in tumor samples compared to expression of Nectin2 and/or CSF1R (“upregulated genes”) in control samples; or (iii) both (i) and (ii). Subjects are identified as eligible when indicated. In certain embodiments, suitable subjects have a tumor characterized by decreased expression of STAT1. In certain embodiments, suitable subjects have a tumor characterized by decreased expression of IFNγ. In certain embodiments, suitable subjects have tumors characterized by decreased expression of STAT1 and IFNγ. In certain embodiments, suitable subjects have a tumor characterized by increased expression of Nectin-2. In certain embodiments, suitable subjects have tumors characterized by increased expression of CSF1R. In certain embodiments, suitable subjects have tumors characterized by increased expression of Nectin2 and CSF1R. In certain embodiments, suitable subjects have tumors characterized by decreased expression of STAT1 and increased expression of Nectin2. In certain embodiments, suitable subjects have tumors characterized by decreased expression of STAT1 and increased expression of Nectin2. In certain embodiments, suitable subjects have tumors characterized by decreased expression of IFNγ and increased expression of Nectin2. In certain embodiments, suitable subjects have tumors characterized by decreased expression of IFNγ and increased expression of CSF1R. In certain embodiments, suitable subjects have tumors characterized by decreased expression of STAT1 and IFNγ and increased expression of Nectin2 and CSF1R.

In certain embodiments, a suitable subject, e.g., a subject with a stromal signature described herein, is administered a combination therapy comprising (i) an IO therapy described herein and (ii) one or more additional anti-cancer agents and /or subsequently administered. In certain embodiments, a suitable subject, eg, a subject with a stromal signature described herein, is administered a combination therapy comprising (i) an anti-PD-1/PD-L1 antagonist and (ii) one or more additional anti-cancer agents. and/or subsequently administered. In certain embodiments, a suitable subject, e.g., a subject having a stromal signature described herein, is administered a combination therapy comprising (i) an anti-PD-1 antibody and (ii) one or more additional anti-cancer agents and/or subsequently administered to In certain embodiments, a suitable subject, e.g., a subject with a stromal signature described herein, is administered a combination therapy comprising (i) an anti-PD-L1 antibody and (ii) one or more additional anti-cancer agents and/or thereafter administered to In certain embodiments, a suitable subject, e.g., a subject with a stromal signature described herein, is administered and/or subsequently administered a combination therapy comprising (i) an anti-PD-1 antibody and (ii) an anti-CSF1R antibody be done. In certain embodiments, a suitable subject, e.g., a subject with a stromal signature described herein, is administered and/or subsequently administered a combination therapy comprising (i) an anti-PD-L1 antibody and (ii) an anti-CSF1R antibody be done.

Suitable subjects for use herein are (i) increased expression of one or more of CSF1R and Nectin2 (“upregulated genes”) in a tumor sample compared to expression of one or more of CSF1R and Nectin2 (“upregulated genes”) in a control sample; (ii) decreased expression of one or more of STAT1 and IFNγ in a tumor sample compared to expression of one or more of STAT1 and IFNγ (“downregulated genes”) in a control sample; or (iii) (i) and (ii) ). In some embodiments, the subject is identified as suitable for combination therapy comprising (i) an IO therapy, eg, an anti-PD-1/PD-L1 antagonist therapy and (ii) an additional anti-cancer therapy. As used herein, (i) patients suitable for combination therapy, including IO therapy, e.g., anti-PD-1/PD-L1 antagonist therapy, must (i) express STAT1 and/or IFNγ (“downregulated genes”) (ii) increased expression of Nectin2 and/or CSF1R (“upregulated genes”) or (ii) both (i) and (ii). Thus, in certain embodiments, a subject determines that the tumor sample has (i) reduced expression of STAT1 and/or IFNγ in a tumor sample compared to expression of STAT1 and/or IFNγ (a “downregulated gene”) in a control sample; a) increased expression of Nectin2 and/or CSF1R in tumor samples compared to expression of Nectin2 and/or CSF1R (“upregulated genes”) in control samples; or (iii) both (i) and (ii). Identified as suitable when indicated. In certain embodiments, suitable subjects have a tumor characterized by decreased expression of STAT1. In certain embodiments, suitable subjects have a tumor characterized by decreased expression of IFNγ. In certain embodiments, suitable subjects have tumors characterized by decreased expression of STAT1 and IFNγ. In certain embodiments, suitable subjects have a tumor characterized by increased expression of Nectin-2. In certain embodiments, suitable subjects have tumors characterized by increased expression of CSF1R. In certain embodiments, suitable subjects have tumors characterized by increased expression of Nectin2 and CSF1R. In certain embodiments, suitable subjects have tumors characterized by decreased expression of STAT1 and increased expression of Nectin2. In certain embodiments, suitable subjects have tumors characterized by decreased expression of STAT1 and increased expression of Nectin2. In certain embodiments, suitable subjects have tumors characterized by decreased expression of IFNγ and increased expression of Nectin2. In certain embodiments, suitable subjects have tumors characterized by decreased expression of IFNγ and increased expression of CSF1R. In certain embodiments, suitable subjects have tumors characterized by decreased expression of STAT1 and IFNγ and increased expression of Nectin2 and CSF1R.

In certain embodiments, suitable subjects are administered and/or subsequently administered a combination therapy comprising (i) an IO therapy described herein and (ii) one or more additional anti-cancer agents. In certain embodiments, suitable subjects are administered and/or subsequently administered a combination therapy comprising (i) an anti-PD-1/PD-L1 antagonist and (ii) one or more additional anti-cancer agents. In certain embodiments, suitable subjects are administered and/or subsequently administered a combination therapy comprising (i) an anti-PD-1 antibody and (ii) one or more additional anti-cancer agents. In certain embodiments, suitable subjects are administered and/or subsequently administered a combination therapy comprising (i) an anti-PD-L1 antibody and (ii) one or more additional anti-cancer agents. In certain embodiments, suitable subjects are administered and/or subsequently administered a combination therapy comprising (i) an anti-PD-1 antibody and (ii) an anti-CSF1R antibody. In certain embodiments, suitable subjects are administered and/or subsequently administered a combination therapy comprising (i) an anti-PD-L1 antibody and (ii) an anti-CSF1R antibody.

In certain embodiments, a combination therapy comprising (i) an anti-PD-1/PD-L1 antagonist and (ii) one or more additional anticancer agents is administered to a subject, wherein a tumor sample obtained from the subject is (i) a control (ii) decreased expression of STAT1 and IFNγ in tumor samples obtained from the subject compared to expression of STAT1 and IFNγ (“downregulated genes”) in the samples; (ii) Nectin2 and CSF1R (“upregulated genes”) in control samples; or (iii) both (i) and (ii). In some embodiments, the one or more additional anti-cancer agents comprise an anti-CSF1R antibody.

In certain embodiments, a combination therapy comprising (i) an anti-PD-1/PD-L1 antagonist and (ii) one or more additional anticancer agents is administered to a subject, wherein a tumor sample obtained from the subject is (i) a control (ii) increased expression of STAT1 or IFNγ in a tumor sample obtained from the subject compared to expression of STAT1 or IFNγ (“downregulated genes”) in the sample; (ii) nectin2 and CSF1R (“upregulated genes”) in control samples; Decreased expression of Nectin2 or CSF1R in a tumor sample obtained from the subject compared to expression of one or more; or (iii) not both (i) and (ii).

II.C. Antibodies Certain aspects of the invention relate to methods of treating suitable subjects as determined by the methods disclosed herein using IO therapy. Any IO therapy known in the art can be used in the methods described herein. In some embodiments, the IO therapy is PD-1, PD-L1, CTLA-4, LAG-3, TIGIT, TIM3, CSF1R, NKG2a, OX40, ICOS, CD137, KIR, TGFβ, IL-10, IL- 8, an antibody or antigen thereof that specifically binds to a protein selected from IL-2, CD96, VISTA, B7-H4, Fas ligand, CXCR4, mesothelin, CD27, GITR, MICA, MICB and any combination thereof Including administering the binding fragment to a suitable subject.

In some embodiments, the subject is administered a single IO therapy, ie, monotherapy. In some embodiments, the subject is administered anti-PD-1 antibody monotherapy. In some embodiments, the subject is administered a combination therapy comprising a first IO therapy and a second IO therapy. In some embodiments, the subject is administered a combination therapy comprising administering a first IO therapy and an additional anti-cancer agent. In some embodiments, the additional anti-cancer agent comprises a second IO therapy, chemotherapy, standard therapy, or any combination thereof.

In some embodiments, the subject is administered a combination therapy comprising an anti-PD-1 antibody and a second anti-cancer agent. In some embodiments, the subject is administered a combination therapy comprising an anti-PD-1 antibody and an anti-CTLA-4 antibody. In some embodiments, the subject is administered a combination therapy comprising an anti-PD-1 antibody and an anti-CSF1R antibody.

In some embodiments, the subject is administered a combination therapy comprising an anti-PD-L1 antibody and a second anti-cancer agent. In some embodiments, the subject is administered a combination therapy comprising an anti-PD-L1 antibody and an anti-CTLA-4 antibody. In some embodiments, the subject is administered a combination therapy comprising an anti-PD-L1 antibody and an anti-CSF1R antibody.

II.C.1. Anti-PD-1 Antibodies Useful in the Present Invention Anti-PD-1 antibodies known in the art can be used in the methods and compositions described herein. Various human monoclonal antibodies that specifically bind PD-1 with high affinity are disclosed in US Pat. No. 8,008,449. The anti-PD-1 human antibodies disclosed in US Pat. No. 8,008,449 exhibit one or more of the following characteristics. (a) binds human PD-1 with a K _D of 1×10 ⁻⁷ M or less as determined by surface plasmon resonance using a Biacore biosensor system; (c) increases T cell proliferation in a mixed lymphocyte reaction (MLR) assay; (d) increases interferon-γ production in an MLR assay; (e) increases IL-2 secretion in an MLR assay. (f) binds to human PD-1 and cynomolgus monkey PD-1; (g) inhibits the binding of PD-L1 and/or PD-L2 to PD-1; (h) induces an antigen-specific memory response (i) stimulate an antibody response; and (j) inhibit tumor cell growth in vivo. Anti-PD-1 antibodies useful in the present invention include monoclonal antibodies that specifically bind to human PD-1 and exhibit at least one, and in some embodiments at least five, of the above characteristics.

Other anti-PD-1 monoclonal antibodies include, for example, US Pat. ＷＯ２００８／１５６７１２、ＷＯ２０１５／１１２９００、ＷＯ２０１２／１４５４９３、ＷＯ２０１５／１１２８００、ＷＯ２０１４／２０６１０７、ＷＯ２０１５／３５６０６、ＷＯ２０１５／０８５８４７、ＷＯ２０１４／１７９６６４、ＷＯ２０１７／０２０２９１、ＷＯ２０１７／０２０８５８、ＷＯ２０１６／１９７３６７、ＷＯ２０１７／０２４５１５、ＷＯ２０１７／ ０２５０５１、ＷＯ２０１７／１２３５５７、ＷＯ２０１６／１０６１５９、ＷＯ２０１４／１９４３０２、ＷＯ２０１７／０４０７９０、ＷＯ２０１７／１３３５４０、ＷＯ２０１７／１３２８２７、ＷＯ２０１７／０２４４６５、ＷＯ２０１７／０２５０１６、ＷＯ２０１７／１０６０６１、ＷＯ２０１７／１９８４６、ＷＯ２０１７／０２４４６５、ＷＯ２０１７／０２５０１６、 WO2017/132825 and WO2017/133540, each of which is incorporated herein by reference in its entirety.

In some embodiments, the anti-PD-1 antibody is nivolumab (also known as Opdivo®, 5C4, BMS-936558, MDX-1106 and ONO-4538), pembrolizumab (Merck; Keytruda®, lambrolizumab and Also known as MK-3475; see WO2008/156712), PDR001 (Novartis; see WO2015/112900), MEDI-0680 (AstraZeneca; also known as AMP-514; see WO2012/145493), cemiplimab (Regeneron; REGN- 2810; see WO2015/112800), JS001 (TAIZHOU JUNSHI PHARMA; also known as tripalimab; see Si-Yang Liu et al., J. Hematol. Oncol. 10:136 (2017)), BGB-A317 (Beigene; also known as Tislelizumab; see WO2015/35606 and US2015/0079109), INCSHR1210 (Jiangsu Hengrui Medicine; also known as SHR-1210; WO2015/085847; Si-Yang Liu et al., J. Hematol. Oncol 10:136 (2017)), TSR-042 (Tesaro Biopharmaceutical; also known as ANB011; see WO2014/179664), GLS-010 (Wuxi/Harbin Gloria Pharmaceuticals; also known as WBP3055; Si-Yang Liu et al. al., J. Hematol. Oncol. 10:136 (2017)), AM-0001 (Armo), STI-1110 (Sorrento Therapeutics; see WO2014/194302), AGEN2034 (Agenus; see WO2017/040790), MGA012 ( Macrogenics, WO2017/19846), BCD-100 (Biocad; Kaplon et al., mAbs 10(2):183-203 (2018)) and IBI308 (Innovent; WO2017/024465, WO2017/025016, WO2017 /132825 and WO2017/133540).

In some embodiments, the anti-PD-1 antibody is nivolumab. Nivolumab selectively blocks interaction with PD-1 ligands (PD-L1 and PD-L2), thereby blocking downregulation of antitumor T-cell function. 1 immune checkpoint inhibitory antibody (US Pat. No. 8,008,449; Wang et al., 2014 Cancer Immunol Res. 2(9):846-56).

In certain other embodiments, the anti-PD-1 antibody is pembrolizumab. Pembrolizumab is a humanized monoclonal IgG4 (S228P) antibody against the human cell surface receptor PD-1 (programmed death-1 or programmed cell death-1). Pembrolizumab is described, for example, in US Pat. Nos. 8,354,509 and 8,900,587.

Anti-PD-1 antibodies useful in the compositions and methods disclosed herein specifically bind to human PD-1 and either human PD-1 or the anti-PD-1 antibodies disclosed herein, e.g., nivolumab (see, eg, US Pat. Nos. 8,008,449 and 8,779,105; WO2013/173223). In some embodiments, the anti-PD-1 antibody binds to the same epitope as an anti-PD-1 antibody described herein, eg, 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 inhibit the binding of other cross-competing antibodies to that particular epitope region. . These cross-competing antibodies are expected to have functional properties very similar to control antibodies, eg, nivolumab, due to binding to the same epitopic regions 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, eg, WO2013/173223).

In some embodiments, the human PD-1 antibody, an antibody that cross-competes for binding to nivolumab and human PD-1 or binds to the same epitope region, is a monoclonal antibody. For administration to human subjects, these cross-competing antibodies are chimeric, engineered 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 useful in the compositions and methods of the invention also include antigen-binding portions of the antibodies described above. It has been well demonstrated that the antigen-binding function of antibodies can be performed by fragments of full-length antibodies.

Suitable anti-PD-1 antibodies for use in the compositions and methods of the invention bind PD-1 with high specificity and affinity, block the binding of PD-L1 and or PD-L2, 1 signaling pathway that inhibits immunosuppressive effects. In any of the compositions or methods disclosed herein, the anti-PD-1 "antibody" binds to the PD-1 receptor and exhibits functional properties similar to intact antibodies in inhibiting ligand binding and upregulating the immune system. Includes antigen-binding portions or fragments as indicated. In some embodiments, the anti-PD-1 antibody or antigen-binding portion thereof cross-competes with nivolumab for binding to human PD-1.

In certain embodiments, the anti-PD-1 antibody is 0.1 mg/kg to 20.0 mg/kg body weight once every 2, 3, 4, 5, 6, 7 or 8 weeks, e.g. Dosages ranging from 0.1 mg/kg to 10.0 mg/kg body weight once every 2, 3 or 4 weeks are administered. In certain other embodiments, the anti-PD-1 antibody is 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 two weeks. In certain other embodiments, the anti-PD-1 antibody is 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 three weeks. In some embodiments, the anti-PD-1 antibody is administered at a dose of about 5 mg/kg body weight once every three weeks. In certain other embodiments, the anti-PD-1 antibody, eg, nivolumab is administered at a dose of about 3 mg/kg body weight once every two weeks. In certain other embodiments, the anti-PD-1 antibody, eg, pembrolizumab, is administered at a dose of about 2 mg/kg body weight once every three weeks.

Anti-PD-1 antibodies useful in the present invention can be administered at a fixed dose. In some embodiments, the anti-PD-1 antibody is about 100 mg to about 1000 mg, about 100 mg to about 900 mg, about 100 mg to about 800 mg, about 100 mg to about 700 mg, about 100 mg to about 600 mg, about 100 mg to about 500 mg, about 200 mg to administered at a fixed dose of 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 200 mg to about 500 mg, about 200 mg to about 480 mg, or about 240 mg to about 480 mg. In some embodiments, the anti-PD-1 antibody is 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 for about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks; It is administered in fixed doses at 9-week or 10-week dosing intervals. In certain other embodiments, the anti-PD-1 antibody is 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 for about 1 week, 2 weeks, 3 weeks or 4 weeks. is administered at fixed dose intervals of

In some embodiments, the anti-PD-1 antibody is administered in a flat dose of about 200 mg about once every three weeks. In certain other embodiments, the anti-PD-1 antibody is administered in a flat dose of about 200 mg about once every two weeks. In certain other embodiments, the anti-PD-1 antibody is administered in a flat dose of about 240 mg about once every two weeks. In some embodiments, the anti-PD-1 antibody is administered in a flat dose of about 480 mg about once every four weeks.

In certain embodiments, nivolumab is administered at a fixed dose of about 240 mg about once every two weeks. In certain embodiments, nivolumab is administered in a flat dose of about 240 mg about once every three weeks. In certain embodiments, nivolumab is administered in a flat dose of about 360 mg about once every three weeks. In certain embodiments, nivolumab is administered at a fixed dose of about 480 mg about once every 4 weeks.

In certain embodiments, pembrolizumab is administered at a fixed dose of about 200 mg about once every two weeks. In certain embodiments, pembrolizumab is administered at a fixed dose of about 200 mg about once every three weeks. In certain embodiments, pembrolizumab is administered at a constant dose of about 400 mg about once every four weeks.

In some embodiments, the PD-1 inhibitor is a small molecule. In some embodiments, the PD-1 inhibitor comprises miramolecule. In some embodiments, PD-1 inhibitors comprise macrocyclic peptides. In some embodiments, the PD-1 inhibitor comprises BMS-986189. In some embodiments, PD-1 inhibitors include inhibitors disclosed in International Publication No. WO2014/151634, which is incorporated herein by reference in its entirety. In some embodiments, PD-1 inhibitors include INCMGA00012 (Insight Pharmaceuticals). In some embodiments, the PD-1 inhibitor comprises a combination of an anti-PD-1 antibody disclosed herein and a PD-1 small molecule inhibitor.

II.C.2. Anti-PD-L1 Antibodies Useful in the Invention In some embodiments, an anti-PD-L1 antibody is replaced with an anti-PD-1 antibody in any of the methods disclosed herein. Anti-PD-L1 antibodies known in the art can be used in the compositions and methods of the invention. Examples of anti-PD-L1 antibodies useful in the compositions and methods of the invention include those disclosed in US Pat. No. 9,580,507. The anti-PD-L1 human monoclonal antibodies disclosed in US Pat. No. 9,580,507 exhibit one or more of the following characteristics. (a) binds human PD-L1 with a K _D of 1×10 ⁻⁷ M or less as determined by surface plasmon resonance using a Biacore biosensor system; (b) T in a mixed lymphocyte reaction (MLR) assay (c) increases interferon-γ production in the MLR assay; (d) increases IL-2 secretion in the MLR assay; (e) stimulates antibody responses; and (f) T cell effectors. Reversing the effects of T regulatory cells on cells and/or dendritic cells. Anti-PD-L1 antibodies useful in the present invention include monoclonal antibodies that specifically bind to human PD-L1 and exhibit at least one, and in some embodiments at least five, of the above characteristics.

In certain embodiments, the anti-PD-L1 antibody is BMS-936559 (12A4, also known as MDX-1105; see, eg, US Pat. No. 7,943,743 and WO2013/173223), atezolizumab (Roche; Tecentriq®) MPDL3280A, also known as RG7446; see US 8,217,149; see also Herbst et al. (2013) J Clin Oncol 31(suppl):3000), durvalumab (AstraZeneca; ^ImfinziTM , also known as MEDI-4736) see WO2011/066389), avelumab (Pfizer; BAVENCIO®, also known as MSB-0010718C; see WO2013/079174), STI-1014 (Sorrento; see WO2013/181634), CX-072 (Cytomx; see WO2016 /149201), KN035 (3D Med/Alphamab; Zhang et al., Cell Discov. 7:3 (March 2017)), LY3300054 (Eli Lilly Co.; see, for example, WO2017/034916), BGB-A333 (Beigene Desai et al., JCO 36 (15suppl): TPS3113 (2018)) and CK-301 (Checkpoint Therapeutics; see Gorelik et al., AACR: Abstract 4606 (Apr 2016)).

In some embodiments, the PD-L1 antibody is atezolizumab (Tecentriq®). Atezolizumab is a fully humanized IgG1 monoclonal anti-PD-L1 antibody.

In some embodiments, the PD-L1 antibody is durvalumab (Imfinzi ^™ ). Durvalumab is a human IgG1 kappa monoclonal anti-PD-L1 antibody.

In some embodiments, the PD-L1 antibody is avelumab (Bavencio®). Avelumab is a human IgG1 lambda monoclonal anti-PD-L1 antibody.

Anti-PD-L1 antibodies useful in the compositions and methods of the invention specifically bind to human PD-L1 and include human PD-L1 and any of the anti-PD-L1 antibodies disclosed herein, e.g., atezolizumab, Also included are isolated antibodies that cross-compete with durvalumab and/or avelumab binding. In some embodiments, an anti-PD-L1 antibody binds to the same epitope as an anti-PD-L1 antibody described herein, eg, atezolizumab, durvalumab and/or avelumab. The ability of antibodies to cross-compete for binding to an antigen indicates that these antibodies bind to the same epitope region of the antigen and sterically inhibit the binding of other cross-competing antibodies to that particular epitope region. These cross-competing antibodies are expected to have functional properties very similar to control antibodies, eg, atezolizumab and/or avelumab, due to binding to the same epitopic regions 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. WO2013/173223). ).

In some embodiments, an antibody that cross-competes for binding to human PD-L1 or binds to the same epitope region with a human PD-L1 antibody such as atezolizumab, durvalumab and/or avelumab is a monoclonal antibody. For administration to human subjects, these cross-competing antibodies are chimeric, engineered 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-L1 antibodies useful in the compositions and methods of the invention also include antigen-binding portions of the antibodies described above. It has been well demonstrated that the antigen-binding function of antibodies can be performed by fragments of full-length antibodies.

Anti-PD-L1 antibodies suitable for use in the compositions and methods of the invention bind PD-L1 with high specificity and affinity, block PD-1 binding, and immunize the PD-1 signaling pathway. It is an antibody that inhibits inhibitory action. In any of the compositions or methods disclosed herein, an anti-PD-L1 "antibody" binds to PD-L1 and exhibits functional properties similar to intact antibodies in receptor binding inhibition and immune system upregulation. , including antigen-binding portions or fragments. In certain embodiments, the anti-PD-L1 antibody or antigen-binding portion thereof cross-competes with atezolizumab, durvalumab and/or avelumab for binding to human PD-L1.

Anti-PD-L1 antibodies useful in the present invention include antibodies that cross-compete for binding to human PD-1 with any PD-L1 antibody that specifically binds PD-L1, such as durvalumab, avelumab or atezolizumab, such as , durvalumab, avelumab or atezolizumab. In certain embodiments, the anti-PD-L1 antibody is durvalumab. In certain other embodiments, the anti-PD-L1 antibody is avelumab. In some embodiments, the anti-PD-L1 antibody is atezolizumab.

In some embodiments, the anti-PD-L1 antibody is 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 at doses ranging about once every 2, 3, 4, 5, 6, 7 or 8 weeks administered.

In some embodiments, the anti-PD-L1 antibody is administered at a dose of about 15 mg/kg body weight about once every three weeks. In certain other embodiments, the anti-PD-L1 antibody is administered at a dose of about 10 mg/kg body weight about once every two weeks.

In certain other embodiments, the anti-PD-L1 antibodies useful in the invention are fixed dose. In some embodiments, the anti-PD-L1 antibody is 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 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 is administered at a fixed dose of In some embodiments, the anti-PD-L1 antibody is 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 about 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, in fixed doses at dosing intervals of about 1, 2, 3, or 4 weeks. In some embodiments, the anti-PD-L1 antibody is administered in a flat dose of about 1200 mg about once every three weeks. In certain other embodiments, the anti-PD-L1 antibody is administered in a flat dose of about 800 mg about once every two weeks. In certain other embodiments, the anti-PD-L1 antibody is administered in a flat dose of about 840 mg about once every two weeks.

In certain embodiments, atezolizumab is administered at a constant dose of about 1200 mg about once every three weeks. In certain embodiments, atezolizumab is administered at a fixed dose of about 800 mg about once every two weeks. In certain embodiments, atezolizumab is administered at a fixed dose of about 840 mg about once every two weeks.

In certain embodiments, avelumab is administered at a fixed dose of about 800 mg about once every two weeks.

In some embodiments, durvalumab is administered at a dose of about 10 mg/kg about once every two weeks. In certain embodiments, durvalumab is administered at a constant dose of about 800 mg/kg about once every two weeks. In certain embodiments, durvalumab is administered at a constant dose of about 1200 mg/kg about once every three weeks.

In some embodiments, the PD-L1 inhibitor is a small molecule. In some embodiments, the PD-L1 inhibitor comprises miramolecule. In some embodiments, PD-L1 inhibitors comprise macrocyclic peptides. In some embodiments, the PD-L1 inhibitor comprises BMS-986189.

In certain embodiments, the PD-L1 inhibitor is of Formula (I)

wherein R ¹ -R ¹³ are amino acid side chains, R ^a -R ⁿ are hydrogen, methyl or form a ring with the adjacent R group, and R ¹⁴ is -C(O)NHR ¹⁵ ; , where R ¹⁵ is hydrogen or a glycine residue optionally substituted with additional glycine residues and/or tails that may improve pharmacokinetic properties. ]
contains miramolecule, which has the formula shown in In certain embodiments, PD-L1 inhibitors include compounds disclosed in International Publication WO2014/151634, which is incorporated herein by reference in its entirety. In some embodiments, the PD-L1 inhibitor is a /085750, WO2018/237153 or WO2019/070643, each of which is hereby incorporated by reference in its entirety.

In some embodiments, the PD-L1 inhibitor is disclosed in International Publications WO2015/034820, WO2015/160641, WO2018/044963, WO2017/066227, WO2018/009505, WO2018/183171, WO2018/118848, WO2019/1472129 or of small molecule PD-L1 inhibitors, each of which is incorporated herein by reference in its entirety.

In some embodiments, the PD-L1 inhibitor comprises a combination of an anti-PD-L1 antibody disclosed herein and a PD-L1 small molecule inhibitor disclosed herein.

II.C.3. Anti-CTLA-4 Antibodies Anti-CTLA-4 antibodies known in the art can be used in the compositions and methods of the invention. The anti-CTLA-4 antibodies of the invention bind to human CTLA-4 so as to interfere with the interaction of CTLA-4 with the human B7 receptor. Since the interaction between CTLA-4 and B7 transmits signals that activate CTLA-4 receptor-bearing T cells, blocking the interaction effectively induces and enhances the activation of such T cells. or prolong, thereby inducing, enhancing or prolonging an immune response.

A human monoclonal antibody that specifically binds CTLA-4 with high affinity is disclosed in US Pat. No. 6,984,720. Other anti-CTLA-4 monoclonal antibodies are disclosed, for example, in US Pat. 196237 and WO2000/037504, each of which is incorporated herein by reference in its entirety. The anti-CTLA-4 human monoclonal antibodies disclosed in US Pat. No. 6,984,720 exhibit one or more of the following characteristics. (a) reflected by a parallel binding 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 more, as determined by Biacore analysis; (b) a dynamic binding constant (k _a ) of at least about 10 ³ , about 10 ⁴ or about 10 ⁵ m ⁻¹ s ⁻¹ (c) at least Dynamic dissociation constants (k _d ) of about 10 ³ , about 10 ⁴ or about 10 ⁵ m ⁻¹ s ⁻¹ and (d) binding of CTLA-4 to B7-1 (CD80) and B7-2 (CD86) impede Anti-CTLA-4 antibodies useful in the present invention include monoclonal antibodies that specifically bind to human CTLA-4 and exhibit at least one, at least two, or at least three of the above characteristics.

In certain embodiments, the CTLA-4 antibody is ipilimumab (also known as Yervoy®, MDX-010, 10D1; see US Pat. No. 6,984,720), MK-1308 (Merck), AGEN-1884 (Agenus Inc.; see WO2016/196237) and tremelimumab (AstraZeneca; ticilimumab, also known as CP-675,206; see WO2000/037504 and Ribas, Update Cancer Ther. 2(3): 133-39 (2007)). selected from the group. In a specific embodiment, the anti-CTLA-4 antibody is ipilimumab.

In a specific embodiment, the CTLA-4 antibody is ipilimumab for use in the compositions and methods disclosed herein. Ipilimumab is a fully human, IgG1 monoclonal antibody that blocks the binding of CTLA-4 to its B7 ligand, thereby stimulating T-cell activation and improving overall survival (OS) in patients with advanced melanoma .

In a specific embodiment, the CTLA-4 antibody is tremelimumab.

In a specific embodiment, the CTLA-4 antibody is MK-1308.

In a specific embodiment, the CTLA-4 antibody is AGEN-1884.

Anti-CTLA-4 antibodies that can be used in the compositions and methods of the invention specifically bind to human CTLA-4 and include human CTLA-4 and any of the anti-CTLA-4 antibodies disclosed herein, such as ipilimumab. and/or an isolated antibody that cross-competes with tremelimumab binding. In some embodiments, an anti-CTLA-4 antibody binds to the same epitope as an anti-CTLA-4 antibody described herein, eg, ipilimumab and/or tremelimumab. The ability of antibodies to cross-compete for binding to an antigen indicates that these antibodies bind to the same epitope region of the antigen and sterically inhibit the binding of other cross-competing antibodies to that particular epitope region. These cross-competing antibodies are expected to have functional properties very similar to control antibodies, eg, ipilimumab and/or tremelimumab, due to binding to the same epitopic regions 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. WO2013/173223). ).

In some embodiments, an antibody that cross-competes for binding to human CTLA-4 or binds to the same epitope region as a human CTLA-4 antibody, such as ipilimumab and/or tremelimumab, is a monoclonal antibody. For administration to human subjects, these cross-competing antibodies are chimeric, engineered 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 that can be used in the compositions and methods of the invention also include the antigen-binding portion of the antibodies described above. It has been well demonstrated that the antigen-binding function of antibodies can be performed by fragments of full-length antibodies.

Anti-CTLA-4 antibodies suitable for use in the methods or compositions of the invention bind CTLA-45 with high specificity and affinity, block the activity of CTLA-4, and inhibit the activity of CTLA-4 and the human B7 receptor. An antibody that interferes with the interaction. In any of the compositions or methods disclosed herein, the anti-CTLA-4 "antibody" binds to CTLA-4 and inhibits the interaction of CTLA-4 with the human B7 receptor and upregulates the immune system. It includes antigen-binding portions or fragments that exhibit functional properties similar to antibodies. In some embodiments, the anti-CTLA-4 antibody or antigen-binding portion thereof cross-competes with ipilimumab and/or tremelimumab for binding to human CTLA-4.

In certain embodiments, the anti-CTLA-4 antibody or antigen-binding portion thereof is administered at 0.1 mg/kg to 10.0 mg/kg body weight for 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks or 8 weeks. It is administered in a single range of doses. In some embodiments, 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 some embodiments, the anti-CTLA-4 antibody or antigen-binding portion thereof is administered at a dose of 3 mg/kg body weight once every two weeks. In certain other embodiments, the anti-PD-1 antibody or antigen-binding portion thereof is administered at a dose of 1 mg/kg body weight once every six weeks.

In some embodiments, the anti-CTLA-4 antibody or antigen-binding portion thereof is administered as a fixed dose. In some embodiments, the anti-CTLA-4 antibody is about 10 to about 1000 mg, about 10 mg to about 900 mg, about 10 mg to about 800 mg, about 10 mg to about 700 mg, about 10 mg to about 600 mg, about 10 mg to about 500 mg, about 100 mg to administered at a fixed dose of about 1000 mg, about 100 mg to about 900 mg, about 100 mg to about 800 mg, about 100 mg to about 700 mg, about 100 mg to about 100 mg, about 100 mg to about 500 mg, about 100 mg to about 480 mg or about 240 mg to about 480 mg. be. In some embodiments, the anti-CTLA-4 antibody or antigen-binding portion thereof is 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; A fixed dose of 700 mg or at least about 720 mg is administered. In certain other embodiments, the anti-CTLA-4 antibody or antigen-binding portion thereof is administered as a fixed dose about once every 1, 2, 3, 4, 5, 6, 7 or 8 weeks. be.

In certain embodiments, ipilimumab is administered at a dose of about 3 mg/kg about once every three weeks. In certain embodiments, ipilimumab is administered at a dose of about 10 mg/kg about once every three weeks. In certain embodiments, ipilimumab is administered at a dose of about 10 mg/kg about once every 12 weeks. In some embodiments, ipilimumab is administered four times.

II.C.4. Anti-LAG-3 Antibodies The anti-LAG-3 antibodies of the invention bind to human LAG-3. Antibodies that bind LAG-3 are disclosed in International Publication WO/2015/042246 and US Publications 2014/0093511 and 2011/0150892, each of which is incorporated herein by reference in its entirety.

An example of a LAG-3 antibody useful in the present invention is 25F7 (described in US Publication 2011/0150892). A further example of a LAG-3 antibody useful in the present invention is BMS-986016. In some embodiments, anti-LAG-3 antibodies useful in the compositions cross-compete with 25F7 or BMS-986016. In certain other embodiments, the anti-LAG-3 antibodies useful in the compositions bind to the same epitope as 25F7 or BMS-986016. In certain other embodiments, the anti-LAG-3 antibody comprises the 6 CDRs of 25F7 or BMS-986016. In certain other embodiments, 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. These and other anti-LAG-3 antibodies useful in the present invention are, for example, ＷＯ２０１５／２００１１９、ＷＯ２０１７／０１９８４６、ＷＯ２０１７／１９８７４１、ＷＯ２０１７／２２０５５５、ＷＯ２０１７／２２０５６９、ＷＯ２０１８／０７１５００、ＷＯ２０１７／０１５５６０、ＷＯ２０１７／０２５４９８、ＷＯ２０１７／０８７５８９、ＷＯ２０１７／０８７９０１、ＷＯ２０１８／０８３０８７、ＷＯ２０１７／１４９１４３、ＷＯ２０１７／ 219995, US2017/0260271, WO2017/086367, WO2017/086419, WO2018/034227 and WO2014/140180, each of which is incorporated herein by reference in its entirety.

II.C.5. Anti-CD137 Antibodies Anti-CD137 antibodies specifically bind to and activate CD137-expressing immune cells and stimulate immune responses against tumor cells, particularly in cytotoxic T cell responses. Antibodies that bind to CD137 are disclosed in CD137, each of which is incorporated herein by reference in its entirety.

In some embodiments, the anti-CD137 antibody is Urelumab (BMS-663513), which is described in US Pat. No. 7,288,638 (20H4.9-IgG4 [10C7 or BMS-663513]). In some embodiments, the anti-CD137 antibody is BMS-663031 (20H4.9-IgG1), described in US Pat. No. 7,288,638. In some embodiments, the anti-CD137 antibody is 4E9 or BMS-554271, described in US Pat. No. 6,887,673. In some embodiments, the anti-CD137 antibody is an antibody disclosed in US Patents 7,214,493; 6,303,121; 6,569,997; 6,905,685; In some embodiments, the anti-CD137 antibody is 1D8 or BMS-469492; 3H3 or BMS-469497; or 3E1 as described in US Pat. No. 6,362,325. In some embodiments, the anti-CD137 antibody is an antibody disclosed in issued US Pat. No. 6,974,863 (eg, 53A2). In some embodiments, the anti-CD137 antibody is an antibody disclosed in issued US Pat. No. 6,210,669 (eg, 1D8, 3B8 or 3E1). In some embodiments, the antibody is Pfizer's PF-05082566 (PF-2566). In certain other embodiments, the anti-CD137 antibodies useful in the methods disclosed herein cross-compete with the anti-CD137 antibodies disclosed herein. In some embodiments, an anti-CD137 antibody binds to the same epitope as an anti-CD137 antibody disclosed herein. In certain other embodiments, anti-CD137 antibodies useful in the invention comprise the 6 CDRs of the anti-CD137 antibodies disclosed herein.

II.C.6. Anti-KIR Antibodies Antibodies that specifically bind to KIR block the interaction between killer cell immunoglobulin-like receptors (KIR) on NK cells and their ligands. Blockade of these receptors promotes NK cell activation and, possibly, tumor cell destruction. Examples of anti-KIR antibodies are International Publications WO/2014/055648, WO2005/003168, WO2005/009465, WO2006/072625, WO2006/072626, WO2007/042573, WO2008/084106, WO2010/0612/072140/939, WO20 160448, each of which is incorporated herein by reference in its entirety.

One anti-KIR antibody useful in the present invention is Lililumab (also referred to as BMS-986015, the S241P variant of IPH2102 or 1-7F9), first described in WO2008/084106. A further anti-KIR antibody useful in the present invention is 1-7F9 (also referred to as IPH2101) disclosed in International Publication WO2006/003179. In some embodiments, the anti-KIR antibody of the composition cross-competes with lililumab or I-7F9 for binding to KIR. In certain other embodiments, the Anti-KIR antibody binds to the same epitope as lililumab or I-7F9. In certain other embodiments, the Anti-KIR antibody comprises the 6 CDRs of Lililumab or I-7F9.

II.C.7. Anti-GITR Antibodies An anti-GITR antibody useful in the methods of the invention is any anti-GITR that specifically binds to a human GITR target and activates the glucocorticoid-induced tumor necrosis factor receptor (GITR). Contains antibodies. 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 antibody"). In particular, GITR activation increases effector T cell proliferation and function and abrogates suppression induced by activated T regulatory cells. Furthermore, GITR stimulation promotes antitumor immunity by increasing the activity of other immune cells such as NK cells, antigen presenting cells and B cells. Examples of anti-GITR antibodies are International Publications WO/2015/031667, WO2015/184,099, WO2015/026,684, WO11/028683 and WO/2006/105021, US Patents 7,812,135 and 8,388,967 and US Publications 2009/0136494, 2014/0220002, 2013/0183321 and 2014/0348841, each of which is incorporated herein by reference in its entirety.

In certain embodiments, an anti-GITR antibody useful in the present invention is TRX518 (described, for example, in Schaer et al. Curr Opin Immunol. (2012) Apr;24(2):217-224 and WO/2006/105021). . In certain other embodiments, anti-GITR antibodies are described in MK4166, MK1248 and WO 11/028683 and US 8,709,424, e.g., VH chains comprising SEQ ID NO: 104 and VL chains comprising SEQ ID NO: 105 (wherein SEQ ID NOs are selected from antibodies, including from WO 11/028683 or US 8,709,424). In certain embodiments, the anti-GITR antibody is an anti-GITR antibody disclosed in WO2015/031667, such as VH CDR1-3 comprising SEQ ID NOs:31, 71 and 63 of WO2015/031667 and SEQ ID NOs:5, 14 and SEQ ID NOs:5, 14 and WO2015/0316677, respectively. Antibodies containing VL CDRs 1-3, including 30. In certain embodiments, the anti-GITR antibody is an anti-GITR antibody disclosed in WO2015/184099, eg, antibody Hum231#1 or Hum231#2 or CDRs thereof or derivatives thereof (eg, pab1967, pab1975 or pab1979). In certain embodiments, the anti-GITR antibody is an anti-GITR antibody disclosed in JP2008278814, WO09/009116, WO2013/039954, US20140072566, US20140072565, US20140065152 or WO2015/026684 or INBRX-110 (INDIKHIBRx) or INBRX-110 (INDIKHIBRx), -1873 (MedImmune). In some embodiments, the anti-GITR antibody is an anti-GITR antibody described in PCT/US2015/033991 (eg, an antibody comprising the variable regions of 28F3, 18E10 or 19D3).

In some embodiments, the anti-GITR antibody cross-competes with an anti-GITR antibody described herein, eg, TRX518, MK4166 or an antibody comprising the VH domain and VL domain amino acid sequences described herein. In some embodiments, the anti-GITR antibody binds to the same epitope as an anti-GITR antibody described herein, eg, TRX518 or MK4166. In some embodiments, the anti-GITR antibody comprises the 6 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. In certain embodiments, the anti-TIM3 antibody is disclosed in WO2018013818, WO/2015/117002 (e.g., MGB453, Novartis), WO/2016/161270 (e.g., TSR-022, Tesaro/AnaptysBio), WO2011155607, WO2016/144803 (e.g., 、ＳＴＩ－６００、Sorrento Therapeutics)、ＷＯ２０１６／０７１４４８、ＷＯ１７０５５３９９；ＷＯ１７０５５４０４、ＷＯ１７１７８４９３、ＷＯ１８０３６５６１、ＷＯ１８０３９０２０(例えば、Ｌｙ－３２２１３６７、Eli Lilly)、ＷＯ２０１７２０５７２１、ＷＯ１７０７９１１２；ＷＯ１７０７９１１５；ＷＯ１７０７９１１６、ＷＯ１１１５９８７７、ＷＯ１３００６４９０、ＷＯ２０１６０６８８０２、ＷＯ２０１６０６８８０３、ＷＯ２０１６ /111947 and anti-TIM3 antibodies disclosed in WO/2017/031242, each of which is incorporated herein by reference in its entirety.

II.C.9. Anti-OX40 Antibodies Any antibody or antigen binding fragment thereof that specifically binds to OX40 (also known as CD134, TNFRSF4, ACT35 and/or TXGP1L) can be used in the methods disclosed herein. In one aspect, the anti-OX40 antibody is BMS-986178 (Bristol-Myers Squibb Company), which is described in International Publication WO20160196228.ある態様において、抗ＯＸ４０抗体は、国際公開ＷＯ９５０１２６７３、ＷＯ１９９９４２５８５、ＷＯ１４１４８８９５、ＷＯ１５１５３５１３、ＷＯ１５１５３５１４、ＷＯ１３０３８１９１、ＷＯ１６０５７６６７、ＷＯ０３１０６４９８、ＷＯ１２０２７３２８、ＷＯ１３０２８２３１、ＷＯ１６２００８３６、ＷＯ１７０６３１６２、ＷＯ１７１３４２９２、ＷＯ１７０９６１７９、ＷＯ１７０９６２８１およびＷＯ１７０９６１８２に記載される抗ＯＸ４０抗体からselected, each of which is incorporated herein by reference in its entirety.

II.C.10. Anti-NKG2A Antibodies Any antibody or antigen-binding fragment thereof that specifically binds to NKG2A can be used in the methods disclosed herein. NKG2A is a member of the C-type lectin receptor family expressed on natural killer (NK) cells and a subset of T lymphocytes. In particular, NKG2A is expressed primarily on tumor-infiltrating innate immune effector NK cells and some CD8+ T cells. The natural ligand, human leukocyte antigen E (HLA-E), is expressed on solid and hematological tumors. NKG2A is an inhibitory receptor that binds HLA-E.

In some embodiments, the anti-NKG2A antibody can be BMS-986315, a human monoclonal antibody that blocks the interaction of NKG2A with its ligand HLA-E, thus allowing activation of an anti-tumor immune response. In some embodiments, the anti-NKG2A antibody is a checkpoint inhibitor that activates T cells, NK cells and/or tumor-infiltrating immune cells. In certain embodiments, the anti-NKG2A antibody is, for example, WO2006/070286 (Innate Pharma S.A.; University of Genova); U.S. Pat. No. 8,993,319 (Innate Pharma S.A.; University of Genova); Novo Nordisk A/S; University of Genova); U.S. Patent 9,447,185 (Innate Pharma S/A; Novo Nordisk A/S; University of Genova); 8,206,709; 8,901,283; 9,683,041 (Novo Nordisk A/S); WO2009/092805 (Novo Nordisk A/S); Novo Nordisk A/S); WO2016/134371 (Ohio State Innovation Foundation); WO2016/032334 (Janssen); WO2016/041947 (Innate); and anti-NKG2A antibodies described in WO2016/041945 (Innate Pharma), each of which is hereby incorporated by reference in its entirety.

II.C.11. Anti-ICOS Antibodies Any antibody or antigen-binding fragment thereof that specifically binds ICOS can be used in the methods disclosed herein. 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 in T cells after T cell activation and co-stimulates T cell activation after binding to its ligand, ICOS-L (B7H2). ICOS is also known as inducible T cell co-stimulatory factor, CVID1, AILIM, inducible co-stimulatory factor, CD278, activation-inducible lymphocyte immune mediation molecule and CD278 antigen.

In one aspect, the anti-ICOS antibody is BMS-986226, a humanized IgG monoclonal antibody that binds to and stimulates human ICOS. In some embodiments, the anti-ICOS antibody is, for example, WO2016/154177 (Jounce Therapeutics, Inc.), WO2008/137915 (MedImmune), WO2012/131004 (INSERM, French National Institute of Health and Medical Research), EP3147297 (INSERM, French National Institute of Health and Medical Research), WO2011/041613 (Memorial Sloan Kettering Cancer Center), EP2482849 (Memorial Sloan Kettering Cancer Center), WO1999/15553 (Robert Koch Institute), US Pat. 872 (Robert Kotch Institute); WO1998/038216 (Japan Tobacco Inc.), U.S. Pat. and 9,771,424 (GlaxoSmithKline) and anti-ICOS antibodies described in WO2017/220988 (Kymab Limited), each of which is incorporated herein by reference in its entirety.

II.C.12. Anti-TIGIT Antibodies Any antibody or antigen-binding fragment thereof that specifically binds to TIGIT can be used in the methods disclosed herein. In some embodiments, the anti-TIGIT antibody is BMS-986207. In one aspect, the anti-TIGIT antibody is clone 22G2 as described in WO2016/106302. In some embodiments, the anti-TIGIT antibody is TIG7192A/RG6058/RO7092284 or clone 4.1D3 as described in WO2017/053748. In some embodiments, the anti-TIGIT antibody is selected, for example, from anti-TIGIT antibodies described in WO2016/106302 (Bristol-Myers Squibb Company) and WO2017/053748 (Genentech).

II.C.13. Anti-CSF1R Antibodies Any antibody or antigen-binding fragment thereof that specifically binds to CSF1R can be used in the methods disclosed herein. In certain embodiments, anti-CSF1R antibodies cabilarizumab, RG7155 (emactuzumab), AMG820, SNDX 6352 (UCB 6352), CXIIG6, IMC-CS4, JNJ-40346527, MCS110, etc. International Publications WO2013/132044, WO2009/026303, WO4202419/ The anti-CSF1R antibody in this method is an antibody species disclosed in any of WO2009/112245 or is BLZ-945, pexidartinib (PLX3397, PLX108-01), AC-708, PLX-5622, PLX7486, ARRY-382 or Replaced by anti-CSF1 inhibitors such as PLX-73086.

II.D. Additional Anti-Cancer Therapies In certain aspects of the invention, the methods disclosed herein administer an IO therapy, such as an anti-PD-1 antibody or an anti-PD-L1 antibody and one or more additional anti-cancer therapies. further comprising: In some embodiments, the method comprises (i) first an IO therapy, such as an anti-PD-1 antibody or an anti-PD-L1 antibody), (ii) then an IO therapy, such as an anti-CTLA-4 antibody or an anti-PD-L1 antibody. administering a CSF1R antibody and (iii) one or more additional anti-cancer therapies.

Additional anti-cancer therapies may include any therapy and/or any standard therapy known in the art of treating tumors in subjects disclosed herein. In some embodiments, the additional anti-cancer therapy comprises surgery, radiotherapy, chemotherapy, immunotherapy, or any combination thereof. In some embodiments, the additional anti-cancer therapy comprises chemotherapy, including any chemotherapy disclosed herein.

Any chemotherapy known in the art can be used in the methods disclosed herein. In some embodiments, the chemotherapy is platinum-based chemotherapy. Platinum-based chemotherapy is a coordination complex of platinum. In some embodiments, the platinum-based chemotherapy is platinum doublet chemotherapy. In some embodiments, chemotherapy is administered at doses approved for specific indications. In certain other embodiments, chemotherapy is administered at any dose disclosed herein. In some embodiments, the platinum-based chemotherapy is cisplatin, carboplatin, oxaliplatin, satraplatin, picoplatin, nedaplatin, tripplatin, lipoplatin, or combinations thereof. In some embodiments, the platinum-based chemotherapy is any other platinum-based chemotherapy known in the art. In some embodiments, the chemotherapy is the nucleotide analog gemcitabine. In some embodiments, the chemotherapy is an antifolate. In some embodiments, the antifolate is pemetrexed. In some embodiments, the chemotherapy is a taxane. In certain other embodiments, the taxane is paclitaxel. In some embodiments, the chemotherapy is any other chemotherapy known in the art. In some embodiments, at least one, at least two or more chemotherapeutic agents are administered in combination with IO therapy. In some embodiments, IO therapy is administered in combination with gemcitabine and cisplatin. In some embodiments, IO therapy is administered in combination with pemetrexed and cisplatin. In some embodiments, IO therapy is administered in combination with gemcitabine and pemetrexed. In some embodiments, IO therapy is administered in combination with paclitaxel and carboplatin. In some embodiments, IO therapy is additionally administered.

In some embodiments, the additional anti-cancer therapy comprises immunotherapy. In some embodiments, the additional anti-cancer therapy is LAG-3, TIGIT, TIM3, NKG2a, CSF1R, OX40, ICOS, MICA, MICB, CD137, KIR, TGFβ, IL-10, IL-8, B7-H4, Fas ligand , CXCR4, mesothelin, CD27, GITR or any combination thereof, or an antibody or antigen-binding portion thereof.

II.E. Tumors In some embodiments, the tumor is selected from the group consisting of hepatocellular carcinoma, gastroesophageal carcinoma, melanoma, bladder cancer, lung cancer, renal cancer, head and neck cancer, colon cancer and any combination thereof. It is derived from cancer. In some embodiments, the tumor is from hepatocellular carcinoma, wherein the tumor has a high inflammatory signature score. In some embodiments, the tumor is from gastroesophageal cancer, wherein the tumor has a high inflammatory signature score. In some embodiments, the tumor is from melanoma, wherein the tumor has a high inflammatory signature score. In some embodiments, the tumor is from bladder cancer, wherein the tumor has a high inflammatory signature score. In some embodiments, the tumor is derived from lung cancer, wherein the tumor has a high inflammatory signature score. In some embodiments, the tumor is from renal cancer, wherein the tumor has a high inflammatory signature score. In some embodiments, the tumor is from head and neck cancer, wherein the tumor has a high inflammatory signature score. In some embodiments, the tumor is from colon cancer, wherein the tumor has a high inflammatory signature score.

In some embodiments, the subject has had 1, 2, 3, 4, 5 or more pre-cancer treatments. In certain other embodiments, the subject is treatment naive. In some embodiments, the subject is progressing with another cancer treatment. In some embodiments, the pre-cancer treatment included immunotherapy. In certain other embodiments, the prior cancer treatment included chemotherapy. In some embodiments, the tumor has recurred. In some embodiments, the tumor is metastatic. In certain other embodiments, the tumor is not metastatic. In some embodiments, the tumor is locally advanced.

In some embodiments, the subject has received prior therapy for treatment of a tumor, and the tumor is relapsed or refractory. In some embodiments, the at least one prior therapy comprises standard therapy. In some embodiments, at least one prior therapy comprises surgery, radiotherapy, chemotherapy, immunotherapy, or any combination thereof. In some embodiments, at least one pretreatment comprises chemotherapy. In some embodiments, the subject has received prior immuno-oncology (IO) therapy for treatment of a tumor, and the tumor is relapsed or refractory. In some embodiments, the subject has received more than one prior therapy to treat the tumor and the subject is relapsed or refractory. In certain other embodiments, the subject is undergoing anti-PD-1 or anti-PD-L1 antibody therapy.

In some embodiments, prior treatment options include chemotherapy. In some embodiments, chemotherapy comprises platinum-based therapy. In some embodiments, the platinum-based therapy is a platinum-based antineoplastic selected from the group consisting of cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, satraplatin, and any combination thereof. including. In some embodiments, the platinum-based therapy comprises cisplatin. In certain embodiments, the platinum-based therapy comprises carboplatin.

In some embodiments, the at least one pretreatment is a platinum agent (e.g., cisplatin, carboplatin), a taxane agent (e.g., paclitaxel, albumin-bound paclitaxel, docetaxel), vinorelbine, vinblastine, etoposide, pemetrexed, gemcitabine, bevacizumab (Avastin®). (trademark)), erlotinib (Tarceva®), crizotinib (Zarkori®), cetuximab (Erbitux®) and any combination thereof. is selected from In some embodiments, at least one prior treatment comprises platinum-based doublet chemotherapy.

In some embodiments, the subject has experienced disease progression after at least one prior treatment. In some embodiments, the subject has received at least 2 pretreatments, at least 3 pretreatments, at least 4 pretreatments, or at least 5 pretreatments. In some embodiments, the subject has received at least two prior treatments. In some embodiments, the subject has experienced disease progression after at least two prior treatments. In certain embodiments, the at least two pretreatments comprise a first pretreatment and a second pretreatment, wherein the subject has experienced disease progression after the first pretreatment and/or the second pretreatment. wherein the first prior therapy comprises surgery, radiation therapy, chemotherapy, immunotherapy or any combination thereof; and the second prior therapy comprises surgery, radiation therapy, chemotherapy, immunotherapy or Including any combination of these. In some embodiments, the first pretreatment comprises platinum-based doublet chemotherapy and the second pretreatment comprises single agent chemotherapy. In some embodiments, the single agent chemotherapy comprises docetaxel.

II.F. Pharmaceutical Compositions and Dosages The therapeutic agents of the present invention may constitute compositions, eg, pharmaceutical compositions, comprising antibodies and/or cytokines and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are physiologically compatible. . Preferably, the carrier for the composition comprising the antibody is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epithelial administration (e.g. by injection or infusion), while the composition comprising the antibody and/or cytokine The carrier for the article is suitable for parenteral, eg oral administration. In one embodiment, the subcutaneous injection is based on Halozyme Therapeutics' ENHANZE® drug delivery technology (see US Pat. No. 7,767,429, incorporated herein by reference in its entirety). ENHANZE® uses a co-formulation of an antibody and a recombinant human hyaluronidase enzyme (rHuPH20) that removes traditional limitations on the volume of biologics and drugs that can be delivered subcutaneously by the extracellular matrix (U.S. Patent 7,767,429). Pharmaceutical compositions of the invention may contain one or more pharmaceutically acceptable salts, antioxidants, aqueous and non-aqueous carriers and/or adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Therefore, in certain embodiments, the pharmaceutical composition of the invention may further comprise a recombinant human hyaluronidase enzyme, eg, rHuPH20.

Although high nivolumab monotherapy up to 10 mg/kg every 2 weeks has been achieved without reaching the maximum tolerated dose (MTD), substantial toxicity has been observed in other trials of checkpoint inhibitors plus antiangiogenic therapy. (eg Johnson et al., 2013; Rini et al., 2011), supporting the choice of nivolumab doses lower than 10 mg/kg.

Treatment is continued as long as clinical benefit is observed or until unacceptable toxicity or disease progression occurs. Nevertheless, in some embodiments, the antibodies disclosed herein are administered at doses well below the approved, ie, sub-therapeutic, doses of the drug. Antibodies are administered at doses that have been shown to produce the best efficacy as monotherapy in clinical trials, e.g., about 3 mg/kg of nivolumab every three weeks (Topalian et al., 2012a; al., 2012) or at significantly lower, ie, sub-therapeutic, doses.

Dosage and frequency will vary depending on the half-life of the antibody in the subject. In general, human antibodies show the longest half-life, followed by humanized, chimeric and non-human antibodies. Dosage and frequency of administration may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively low doses are generally administered infrequently over long periods of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, relatively high dosages at relatively short intervals may be required until disease progression is reduced or halted, preferably until the patient shows partial or complete amelioration of disease symptoms. The patient may then be administered a prophylactic regimen.

The actual dosage level of the pharmaceutical composition in the pharmaceutical composition of the present invention will be effective to achieve the desired therapeutic response without being unduly toxic to the patient for a particular patient, composition and method of administration. It may be varied to obtain a certain amount of active ingredient. The dosage level selected will depend on the activity of the particular composition of the invention employed, the route of administration, the time of administration, the excretion rate of the particular compound employed, the duration of treatment, other drugs used in combination with the particular composition employed. , the compound and/or substance, the age, sex, weight, condition, general health and prior medical history of the patient being treated and similar factors well known in the pharmaceutical arts. A composition of the present invention 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 recognized by those skilled in the art, the route and/or method of administration will vary depending on the desired result.

III. Kits Also within the scope of the invention are kits for therapeutic use comprising (a) anti-PD-1 antibodies or anti-PD-L1 antibodies. Kits generally include a label indicating the intended use of the contents of the kit and directions for use. The term label includes any written or recorded medium on or with the kit or otherwise associated with the kit. Accordingly, the present invention provides a kit for treating a subject with a tumor comprising (a) a single dose ranging from 0.1 to 10 mg/kg body weight of an anti-PD-1 antibody or 0 dose of an anti-PD-L1 antibody; and (b) instructions for using the anti-PD-1 antibody or anti-PD-L1 antibody in the methods disclosed herein. The present invention provides a kit for treating a subject with a tumor comprising (a) a single dose of an anti-PD-1 antibody ranging from about 4 mg to about 500 mg or an anti-PD-L1 antibody ranging from about 4 mg to about 2000 mg; and (b) instructions for using the anti-PD-1 antibody or anti-PD-L1 antibody in the methods disclosed herein. In one aspect, the invention provides a kit for treating a subject with a tumor, comprising (a) a single dose of anti-PD-1 antibody in the range of 200 mg to 800 mg or an anti-PD-L1 antibody in the range of 200 mg to 1800 mg; and (b) instructions for using the anti-PD-1 antibody or anti-PD-L1 antibody in the methods disclosed herein.

In certain embodiments for treatment of human patients, the kits include an anti-human PD-1 antibody disclosed herein, eg, nivolumab or pembrolizumab. In certain embodiments for treatment of human patients, the kits comprise an anti-human PD-L1 antibody disclosed herein, eg, atezolizumab, durvalumab or avelumab.

In some embodiments, the kit further comprises an anti-CTLA-4 antibody. In certain embodiments for the treatment of human patients, the kit includes an anti-human CTLA-4 antibody disclosed herein, eg ipilimumab, tremelimumab, MK-1308 or AGEN-1884.

In some embodiments, the kit further comprises a gene panel assay disclosed herein. In some embodiments, the kit further comprises instructions for administering the anti-PD-1 antibody or anti-PD-L1 antibody to a subject more suitable for the methods disclosed herein.

All references cited above and all references cited herein are hereby incorporated by reference in their entirety.

The following examples are provided for illustrative purposes and not for purposes of limitation.

Example 1
Inflammation of the tumor microenvironment (TME), indicated by infiltration of 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 of immuno-oncology (IO) treatments, and intratumoral localization also influences outcomes, highlighting the importance of spatial analysis of CD8+ T cells within the TME. be. CD8+ T-cell patterns within tumors, as assessed by immunohistochemistry (IHC), are variable and include (i) immune desert (minimal T-cell infiltration); or (iii) immune inflammation (T cells infiltrating the tumor parenchyma, located close to the tumor cells).

Emerging data suggest that artificial intelligence (AI)-based image analysis can be used to characterize tumor parenchyma and stromal compartments in TME. Pathology data can be quantitative, making IHC assays amenable to future in vitro diagnostic development; however, the ability for multiplexing within IHC assays is limited.

Gene expression profiling (GEP) offers another approach for TME inflammation assessment. Correlations between inflammatory gene signature scores and response to IO therapy have been demonstrated in multiple tumor types.

Using an inflammatory gene panel containing 95 genes for GEP combined with AI-based image analysis to analyze T-cell infiltration patterns in tumor parenchyma and stromal compartments, potential biomarkers for IO therapy The assay was evaluated.

Objectives The primary objective of this study is the quantification of regional CD8+ T cell localization using AI-based image analysis. A secondary objective is to identify the genetic signatures that define CD8+ T cell infiltration and localization to parenchymal and stromal compartments in TME.

Method

IHC for CD8 expression (CD8 IHC) and GEP were performed on commercially produced melanoma (n=158) and squamous cell carcinoma of the head and neck (SCCHN; n=250) tumor samples. CD8 IHC was performed by Mosaic Laboratories using a monoclonal CD8 (clone C8/144B) antibody (Dako, Agilent Technologies Co, Santa Clara, Calif.). Convolutional neural networks (PathAI Inc, Boston, Mass.) and AI-based image analysis algorithms were used to quantify CD8+ T cell abundance in tumor parenchymal and stromal regions (FIGS. 1A-1B; Table 1).

GEP was performed by next generation sequencing (NGS) using an inflammation panel. The inflammation panel contains 95 genes, including genes associated with tumor inflammation and other IO processes, housekeeping genes and control genes. This inflammation panel is indicative of the mRNA expression levels of all 95 genes on the panel.

A generalized constrained regression model was used to predict CD8+ T cell abundance in tumor parenchyma and stroma using specific gene signatures (FIG. 2). Repeated cross-validation (100 replicates of 5-fold cross-validation) was performed to estimate the generalization of the model.

Correlations between CD8+ T cell abundance and tumor parenchymal and stromal CD8 signatures were analyzed using the degrees of freedom adjusted coefficient of determination (R2).

Results AI-based quantification of CD8+ T cells was used to define and characterize the immunophenotype of melanoma and SCCHN samples (Figures 1B and 4A-4C). Using an inflammation panel to analyze melanoma and SCCHN samples, we derive gene signatures that correlate with parenchymal and stromal CD8+ T cell abundance, showing that different gene signatures can be developed using the same assay. (Figures 2 and 5A-5D).

The 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; the best predictors of parenchymal CD8+ T cell abundance included upregulation of STAT1 and IFNγ and downregulation of Nectin2 and CSF1R (Fig. 5B). ).

The 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. The best predictors of tumor stromal CD8+ T cell abundance included upregulation of CSF1R and Nectin2 and downregulation of STAT1 and IFNγ.

Limited overlap was observed between the two sets of genes. Some genes correlated with stromal CD8+ T cell localization showed an inverse correlation with parenchymal CD8+ T cell localization (FIGS. 5A-5D). Upregulation of STAT1 and IFNγ correlated with parenchymal CD8+ T cell abundance, while downregulation of these genes correlated with stromal CD8+ T cell abundance. Downregulation of CSF1R and Nectin2 correlated with parenchymal CD8+ T cell abundance, while upregulation of these genes correlated with stromal CD8+ T cell abundance.

Gene signature scores were derived from tumor parenchymal and stromal CD8 signatures. For both tumor-specific and pooled analyzes of melanoma and SCCHN samples, gene signature scores were highly consistent with AI-based quantification of CD8+ T cells, depending on local CD8+ T cell infiltration (FIGS. 6A-6D). Freedom-adjusted R2 of parenchymal CD8 signature score (pooled analysis) = 0.67 (P < 0.01). Freedom-adjusted R2 of stromal CD8 signature score (pooled analysis) = 0.65 (P < 0.01).

This example describes a GEP-based, exploratory inflammation assay that may be used prospectively in a clinical trial setting. This panel was used to further derive a gene signature predictive of CD8+ T cell abundance in the tumor parenchyma and stroma. Parenchymal and stromal CD8 signature scores correlate with CD8+ T cell abundance determined by IHC in melanoma and SCCHN samples (individually and in aggregate), and these gene signatures can be utilized to assess T cell abundance. is shown. A combination of GEP and AI-based image analysis can be developed as an analytical tool to characterize immune cell infiltration in TME.

Example 2
CD8 IHC using the gene expression signature of CD8+ T cell infiltration (CD8 signature, CD8 topological signature) and EMT gene expression (CD8.IHC_EMT) and correlation of response to nivolumab were compared in patients with urothelial carcinoma (UC) .

Methods Patients Nivolumab was administered to patients with metastatic UC previously treated with platinum and objective response (OR) was assessed by blinded central imaging review.

CD8 IHC was performed on evaluable baseline samples using monoclonal anti-CD8 (C8/144B) by Mosaic Laboratories (Lake Forest, Calif.); Defined as immune desert, immune exclusion or immune inflammatory phenotypes (FIGS. 4A-4C). EMT gene expression was measured using HTG EdgeSeq Biomarker Panels (HTG Molecular Diagnostics, Tucson, AZ) and EMT signature scores were calculated by arithmetic mean of 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 the tumor parenchyma and stroma (CD8 topological signature).

Biomarker Models and Statistical Analysis A biomarker model was developed that included synergistic interactions between single signatures and two- or three-gene signatures (Table 2). The dual and triple D8 signatures evaluated take into account CD8+ T cell inflammation in the TME and in specific tumor regions within the TME.

A Cox proportional hazards regression model was used to assess the dependence of progression-free survival (PFS) or overall survival (OS) on biomarker score hazard ratio (HR) with two-sided 95% confidence intervals (CI; Wald test statistics). ) represents the difference between the 75th and 25th biomarker percentiles; graphs were adjusted to compare log2(HR) values. Kaplan-Meier plots based on classification of biomarker scores by tertile (high, medium, low)) were used to show correlation with PFS and OS. A logistic regression model was used to assess the dependence or dependence on the biomarker score HR, with a two-sided 95% CI (calculated by Wald's test statistic) representing the difference between the 75th and 25th biomarker percentiles. graphs were adjusted to compare log2(HR) values. The receiver operating characteristic (ROC) curve and area under the ROC curve (AUC) were used to evaluate the model's performance as a predictor of OR.

Results GEP-derived CD8 topological signature and CD8.IHC_EMT were evaluable in 205 of 270 (76%) and 187 of 270 (69%) patients, respectively, in clinical trials. Baseline characteristics and clinical outcomes were similar in cohorts for the entire study population (Table 3).

The CD8 signature biomarker models (CD8, parenchymal CD8, dual CD8 and triple D8) were significantly lower than 0.55 (95% CI, 0.45-0.66) for PFS and OS and 0 for CD8.IHC_EMT signature for triple D8. It showed a similar correlation with CD8.IHC_EMT HR with a PFS range of up to .65 (95% CI, 0.55-0.76) (Fig. 8A). The HR for OS ranged from 0.47 (95% CI, 0.37-0.60) for the CD8 signature to 0.53 (95% CI, 0.44-0.66) for the triple D8 signature. There was (Fig. 8B)

Kaplan-Meier plots show patterns of correlation between PFS or OS and triple D8 signatures (FIGS. 9A-9B), consistent with observations by Cox model analysis (FIGS. 8A-8B). PFS and OS with nivolumab were longer in patients with higher tertile scores than in the two lower tertiles.

CD8 signature biomarker models (CD8, parenchymal CD8, dual CD8 and triple D8) and CD8.IHC_EMT showed similar correlations with OR (Fig. 7A). Odds ratios ranged from 1.46 (95% CI, 1.08-1.97) for the parenchymal CD8 signature to 1.64 (95% CI, 1.12-2.41) for triple D8. The ROC curves for OR were similar for each biomarker (Figures 7B-7F). AUC values ranged from 67.6% (95% CI, 57.9-77.3) for dual CD8 signature to 72.1% (95% CI, 62.8-81.4) for triple D8. rice field.

This example shows that the CD8 IHC-derived score (CD8.IHC_EMT) combined with the CD8 gene signature biomarker and EMT gene expression is comparable to the correlation of response and survival in patients with UC treated with nivolumab. Gene expression signatures correlated with CD8+ T cell localization in TME may facilitate selection of patients likely to benefit from IO therapy. These data demonstrate the potential of combinatorial biomarkers to assess tumor inflammation in response to IO therapy in patients with cancer.

Claims (79)

A pharmaceutical composition comprising an anti-PD-1/PD-L1 antagonist for use in a method of identifying a human subject suitable for an anti-PD-1/PD-L1 antagonist, wherein the method comprises anti-PD-1 A pharmaceutical composition comprising measuring the expression of a panel of genes in a tumor sample obtained from a subject in need of a /PD-L1 antagonist, wherein the panel of genes comprises at least three of STAT1, IFNγ, Nectin2 and CSF1R. thing. The gene panel includes all of STAT1, IFNγ, Nectin2, CSF1R and 1 additional gene, 2 additional genes, 3 additional genes, 4 additional genes, 5 additional genes, 6 additional genes, 2. A pharmaceutical composition for use according to claim 1, comprising 7 additional genes, 8 additional genes or 10 additional genes. 2. The pharmaceutical composition for use according to claim 1, wherein the gene panel consists of STAT1, IFNγ, Nectin2, CSF1R and 1 additional gene, 2 additional genes or 3 additional genes. tumor sample
(i) increased expression of one or more of STAT1 and IFNγ in a sample compared to expression of one or more of STAT1 and IFNγ (“upregulated genes”) in a control sample;
(ii) decreased expression of one or more of Nectin2 and CSF1R in a sample compared to one or more of Nectin2 and CSF1R (“downregulated genes”) in a control sample; or
(iii) a subject is identified as suitable when exhibiting both (i) and (ii);
A pharmaceutical composition for use according to any of claims 1-3. The pharmaceutical composition for use according to any of claims 1-5, wherein the subject is to be administered an anti-PD-1/PD-L1 antagonist. A pharmaceutical composition comprising an anti-PD-1/PD-L1 antagonist for use in a method of treating a human subject having a tumor, wherein a tumor sample obtained from the subject is
(i) increased expression of one or more of STAT1 and IFNγ in a tumor sample obtained from the subject compared to expression of one or more of STAT1 and IFNγ (“upregulated genes”) in control samples;
(ii) decreased expression of one or more of Nectin2 and CSF1R in a tumor sample obtained from the subject compared to expression of one or more of Nectin2 and CSF1R (“downregulated genes”) in a control sample; or
(iii) A pharmaceutical composition exhibiting both (i) and (ii). 7. The pharmaceutical composition of any of claims 4-6, wherein the control sample comprises non-tumor tissue from a subject, matched non-tumor tissue from a subject, or matched tissue from a subject without tumor. Identifying human subjects suitable for anti-PD-1/PD-L1 antagonists comprising measuring in vitro the expression of a panel of genes in a tumor sample obtained from a subject in need of an anti-PD-1/PD-L1 antagonist A method, wherein the gene panel comprises at least three of STAT1, IFNγ, Nectin2 and CSF1R. The gene panel includes all of STAT1, IFNγ, Nectin2, CSF1R and 1 additional gene, 2 additional genes, 3 additional genes, 4 additional genes, 5 additional genes, 6 additional genes, 9. The method of claim 8, comprising 7 additional genes, 8 additional genes or 10 additional genes. 9. The method of claim 8, wherein the gene panel consists of STAT1, IFNγ, Nectin2, CSF1R and 1 additional gene, 2 additional genes or 3 additional genes. tumor sample
(i) increased expression of one or more of STAT1 and IFNγ (“upregulated genes”) in tumor samples compared to expression of one or more of STAT1 and IFNγ (“upregulated genes”) in control samples;
(ii) decreased expression of one or more of Nectin2 and CSF1R in a tumor sample compared to expression of one or more of Nectin2 and CSF1R (“downregulated genes”) in a control sample; or
(iii) A subject is identified as suitable when exhibiting both (i) and (ii). 12. The method of claim 11, further comprising administering an anti-PD-1/PD-L1 antagonist. A method of treating a human subject with a tumor comprising administering an anti-PD-1/PD-L1 antagonist to the subject, wherein a tumor sample obtained from the subject is
(i) increased expression of one or more of STAT1 and IFNγ in a tumor sample obtained from the subject compared to expression of one or more of STAT1 and IFNγ (“upregulated genes”) in control samples;
(ii) decreased expression of one or more of Nectin2 and CSF1R in a tumor sample obtained from the subject compared to expression of one or more of Nectin2 and CSF1R (“downregulated genes”) in a control sample; or
(iii) A method that exhibits both (i) and (ii). 14. The method of any of claims 11-13, wherein the control sample comprises non-tumor tissue from a subject, matched non-tumor tissue from a subject, or matched tissue from a subject without tumor. A pharmaceutical composition for use according to claim 6 or 7 or claim 13 or 14 ways. A pharmaceutical composition for use according to any one of claims 1-7 and 15 or a method according to any one of claims 8-15, wherein the tumor sample exhibits increased expression of at least two of the upregulated genes. A pharmaceutical composition for use according to any of claims 1-7, 15 and 16 or a method according to any of claims 8-16, wherein the tumor sample exhibits decreased expression of at least two of the down-regulated genes. A pharmaceutical composition for use according to any of claims 1-7 and 15-17 or claim wherein the tumor sample exhibits increased expression of all upregulated genes; and the tumor sample exhibits decreased expression of all downregulated genes Any method of 8 to 17. expression of one or more of the upregulated genes is 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. 19. A pharmaceutical composition for use according to any of claims 1-7 and 15-18, wherein expression of one or more of the upregulated genes is increased at least about 50% higher than expression of one or more of STAT1 and IFNγ in a control sample. The article or method of any of claims 11-18. 19. A pharmaceutical composition for use according to any of claims 1-7 and 15-18, wherein expression of one or more of the upregulated genes is increased at least about 75% higher than expression of one or more of STAT1 and IFNγ in a control sample. The article or method of any of claims 11-18. expression of one or more of the upregulated genes is 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 A pharmaceutical composition for use according to any of claims 1-7 and 15-21 or a method according to any of claims 11-21, wherein the reduction is about 275% or at least about 300% lower. 22. A medicament for use according to any of claims 1-7 and 15-21, wherein the expression of one or more of the upregulated genes is reduced by at least about 50% below the expression of one or more of Nectin2 and CSF1R in a control sample. The composition or method of any of claims 11-21. 22. The medicament for use according to any of claims 1-7 and 15-21, wherein the expression of one or more of the upregulated genes is reduced by at least about 75% below the expression of one or more of Nectin2 and CSF1R in a control sample. The composition or method of any of claims 11-21. A pharmaceutical composition for use according to any of claims 1-7 and 15-24 or a method according to any of claims 11-24, wherein the tumor sample is a tumor tissue biopsy sample. A pharmaceutical composition for use according to any of claims 1-7 and 15-25 or a method according to any of claims 11-25, wherein the tumor sample is formalin-fixed, paraffin-embedded or fresh-frozen tumor tissue. . A pharmaceutical composition for use according to any one of claims 1-7 and 15-26 or a method according to any one of claims 11-26, wherein the tumor sample is obtained from the tumor parenchyma. A pharmaceutical composition for use according to any of claims 1-7 and 15-27 or claim wherein gene expression is determined by detecting the presence of gene mRNA, the presence of protein encoded by said gene or both 11-27. 29. A pharmaceutical composition or method for use according to claim 28, wherein the presence of gene mRNA is determined using reverse transcriptase PCR. 29. A pharmaceutical composition or method for use according to claim 27 or 28, wherein the presence of the protein encoded by said gene is determined using an IHC assay. 30. A pharmaceutical composition or method for use according to claim 29, wherein the IHC assay is an automated IHC assay. tumor sample
(i) increased expression of one or more of CSF1R and Nectin2 compared to the expression of one or more of CSF1R and Nectin2 in a control sample;
(ii) decreased expression of one or more of STAT1 and IFNγ compared to expression of one or more of STAT1 and IFNγ in a control sample; or
(iii) A pharmaceutical composition for use according to any of claims 1-7 and 15-31 or a method according to any of claims 11-31, which does not exhibit both (i) and (ii). tumor sample
(iv) increased expression of 2 or 3 of CSF1R and Nectin2 compared to the expression of 2 or 3 of CSF1R and Nectin2 in control samples;
(v) decreased expression of 2, 3 or 4 of STAT1 and IFNγ compared to the expression of 2, 3 or 4 of STAT1 and IFNγ in a control sample; or
(vi) A pharmaceutical composition or method for use according to claim 32, which does not exhibit both (i) and (ii). 34. A pharmaceutical composition or method for use according to claim 32 or 33, wherein the tumor sample is obtained from the stroma of a tumor. anti-PD-1/PD-L1 antagonist to a target protein selected from programmed death 1 (PD-1; "anti-PD-1 antibody") or programmed death ligand 1 (PD-L1; "anti-PD-L1 antibody) A pharmaceutical composition for use according to any of claims 1-7 and 15-34 or a method according to any of claims 11-34, comprising a specifically binding antibody or antigen-binding fragment thereof. A pharmaceutical composition or method for use according to claim 35, wherein the anti-PD-1/PD-L1 antagonist is an anti-PD-1 antibody. 37. A pharmaceutical composition or method for use according to claim 36, wherein the anti-PD-1 antibody comprises nivolumab or pembrolizumab. A pharmaceutical composition or method for use according to claim 35, wherein the anti-PD-1/PD-L1 antagonist is an anti-PD-L1 antibody. 37. A pharmaceutical composition or method for use according to claim 36, wherein the anti-PD-1 antibody comprises avelumab, atezolizumab or durvalumab. A pharmaceutical composition for use according to any of claims 1-7 and 15-39 or a method according to any of claims 11-39, wherein the anti-PD-1/PD-L1 antagonist is administered as monotherapy. A pharmaceutical composition for use according to any of claims 1-7 and 15-39 or a method according to any of claims 11-39, wherein the anti-PD-1/PD-L1 antagonist is administered with an anti-cancer agent. The anti-cancer agent is inducible T cell costimulatory molecule (ICOS), CD137 (4-1BB), CD134 (OX40), 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), natural killer cell receptor 2B4 (CD244), CD160, Ig and ITIM domains T cell immunoreceptor (TIGIT) and derivative of V-domain Ig suppressor of T cell activation (VISTA), KIR, TGFβ, IL-10, IL-8, B7-H4, Fas ligand, CSF1R, CXCR4, mesothelin 42. A pharmaceutical composition or method for use according to claim 41, comprising an antibody that specifically binds to a protein of , CEACAM-1, CD52, HER2 or any combination thereof. 42. A pharmaceutical composition or method for use according to claim 41, wherein the anti-cancer agent comprises an anti-CSF1R antibody. The tumor is hepatocellular carcinoma, gastroesophageal cancer, melanoma, bladder cancer, lung cancer, renal cancer, head and neck cancer, colon cancer, pancreatic cancer, prostate cancer, ovarian cancer, urothelial cancer, colorectal cancer, or any of these A pharmaceutical composition for use according to any one of claims 1-7 and 15-43 or a method according to any one of claims 11-43, from a cancer selected from the group consisting of a combination of: A pharmaceutical composition for use according to any of claims 1-7 and 15-44 or a method according to any of claims 11-44, wherein the tumor is recurrent. A pharmaceutical composition for use according to any of claims 1-7 and 15-44 or a method according to any of claims 11-44, wherein the tumor is refractory. A pharmaceutical composition for use according to any of claims 1-7 and 15-44 or a method according to any of claims 11-44, wherein the tumor is locally advanced. A pharmaceutical composition for use according to any of claims 1-7 and 15-44 or a method according to any of claims 11-44, wherein the tumor is metastatic. A pharmaceutical composition for use according to any of claims 5-7 and 15-48 or a method according to any of claims 12-48, wherein administration treats a tumor. A pharmaceutical composition for use according to any of claims 5-7 and 15-48 or a method according to any of claims 12-48, wherein administration reduces tumor size. 51. The pharmaceutical composition or method of claim 50, wherein tumor size is reduced by at least about 10%, about 20%, about 30%, about 40%, or about 50% compared to tumor size prior to administration. at least about 1 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 1 year, at least about 18 months, at least about 2 years, at least about 3 years, at least about 4 years or at least about A pharmaceutical composition for use according to any of claims 5-7 and 15-51 or a method according to any of claims 12-51 that exhibits 5 years of progression-free survival. A pharmaceutical composition for use according to any of claims 5-7 and 15-51 or a method according to any of claims 12-51, wherein the subject exhibits stable disease after administration. A pharmaceutical composition for use according to any of claims 5-7 and 15-51 or a method according to any of claims 12-51, wherein the subject exhibits a partial response after administration. A pharmaceutical composition for use according to any of claims 5-7 and 15-51 or a method according to any of claims 12-51, wherein the subject shows a complete response after administration. A kit for treating a subject with a tumor comprising:
(a) an anti-PD-1/PD-L1 antagonist; and
(b) a pharmaceutical composition for the use of any of claims 1-7 and 15-55 or instructions for use of an anti-PD-1/PD-L1 antagonist in the method of any of claims 11-55; kit, including 57. The kit of Claim 56, wherein the anti-PD-1/PD-L1 antagonist comprises an anti-PD-1 antibody. 57. The kit of Claim 56, wherein the anti-PD-1/PD-L1 antagonist comprises an anti-PD-L1 antibody. A gene panel comprising at least three of STAT1, IFNγ, Nectin2 and CSF1R for use in identifying subjects suitable for anti-PD-1/PD-L1 antagonists. 60. A gene panel for use according to claim 59, comprising at least 4, at least 5 or at least 6 of STAT1, IFNγ, Nectin2 and CSF1R. 60. A gene panel for use according to claim 59, comprising STAT1, IFNγ, Nectin2 and CSF1R. STAT1, IFNγ, Nectin2 and CSF1R and 1 additional gene, 2 additional genes, 3 additional genes, 4 additional genes, 5 additional genes, 6 additional genes, 7 additional genes , 8 additional genes, 9 additional genes or 10 additional genes. STAT1, IFNγ, Nectin2 and CSF1R and 1 additional gene, 2 additional genes, 3 additional genes, 4 additional genes, 5 additional genes, 6 additional genes, 7 additional genes, 8 additional genes, 9 60. A gene panel for use according to claim 59, consisting of four additional genes or ten additional genes. 63. A gene panel for use according to any of claims 59-62, comprising about 90 genes, about 95 genes or about 100 genes. A method of preparing a nucleic acid fraction from a tumor of a subject in need of I/O therapy, comprising:
(a) removing a tumor biopsy sample from the subject;
(b) isolating the nucleic acid to produce a fraction of the nucleic acid extracted in (a); and
(c) a method comprising analyzing the expression level of one or more genes in a gene panel selected from STAT1, IFNγ, Nectin2 and CSF1R. 66. The method of claim 65, wherein the nucleic acid is mRNA. 67. The method of claim 65 or 66, wherein one or both of the CSF1R and Nectin2 genes are downregulated. 68. The method of any of claims 65-67, wherein one or both of STAT1 and IFNγ are upregulated. 69. The method of any of claims 65-68, wherein CSF1R and Nectin2 are downregulated and STAT1 and IFNγ are upregulated. 70. The method of any of claims 65-69, wherein expression levels are analyzed by measuring mRNA levels of one or more genes of the gene panel in the tumor sample. 71. The method of any of claims 65-70, wherein the expression level is measured using a nuclease protection assay. 71. The method of any of claims 65-70, wherein the expression level is measured using next generation sequencing. 73. The method of any of claims 65-72, wherein the expression level is measured using reverse transcriptase polymerase chain reaction (RT-PCR). expression of one or both of STAT1 and IFNγ is 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 74. The method of any of claims 65-73, wherein the increase is 275% or at least about 300% higher. 75. The method of any of claims 65-74, wherein expression of one or both of STAT1 and IFNγ is increased at least about 50% higher than expression of one or both of STAT1 and IFNγ in a control sample. 76. The method of any of claims 65-75, wherein expression of one or both of STAT1 and IFNγ is increased at least about 75% higher than expression of one or both of STAT1 and IFNγ in a control sample. expression of one or both of Nectin2 and CSF1R is at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, than expression of one or more of Nectin2 and CSF1R in a control sample; 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%, 77. The method of any of claims 65-76, wherein the reduction is at least about 275% or at least about 300% lower. 78. The method of any of claims 65-77, wherein expression of one or both of Nectin2 and CSF1R is decreased at least about 50% below the expression of one or more of Nectin2 and CSF1R in a control sample. 79. The method of any of claims 65-78, wherein expression of one or both of Nectin2 and CSF1R is decreased by at least about 75% below the expression of one or more of Nectin2 and CSF1R in a control sample.

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