Method of treating cancer using a gene expression signature

Abstract

The present disclosure provides a method of treating a cancer in a subject, the method comprising administering to the subject an effective amount of an immunotherapeutic, wherein the subject has been determined to have a T cell signature (TCS) comprising: (a) a composite expression level A comprising the expression of three or more genes selected from Thymocyte Expressed, Positive Selection Associated 1 (TESPA1), Cluster of Differentiation 28 (CD28), G Protein-Coupled Receptor 183 (GPR183), and Zeta-Chain-Associated Protein Kinase 70 (ZAP70) as measured by log2tpm corresponding to a composite log2tpm value of greater than or equal to 3.2; or (b) a composite expression level B comprising (i) the expression of three or more genes selected from TESPA1, CD28, GPR183 and ZAP70 as measured by log2tpm corresponding to a composite log2tpm value of greater than or equal to 3.2, minus (ii) the expression of Interferon Alpha Inducible Protein 27 (IFI27) gene as measured by log2tpm, wherein the composite expression level B corresponds to a value of greater than or equal to -0.25.

Description

METHOD OF TREATING CANCER USING A GENE EXPRESSION SIGNATURE FIELD OF THE INVENTION

[0001] The present disclosure provides a method of treating a cancer in a subject, the method comprising administering to the subject an effective amount of an immunotherapeutic, e.g., a T cell therapeutic, wherein the subject has been determined to have a T cell signature (TCS) comprising: (a) a composite expression level A comprising the expression of three or more genes selected from Thymocyte Expressed, Positive Selection Associated 1 (TESPA1), Cluster of Differentiation 28 (CD28), G Protein-Coupled Receptor 183 (GPR183), and Zeta- Chain-Associated Protein Kinase 70 (ZAP70) as measured by log2tpm corresponding to a composite log2tpm value of greater than or equal to 3.2; or (b) a composite expression level B comprising (i) the expression of three or more genes selected from TESPA1, CD28, GPR183 and ZAP70 as measured by log2tpm corresponding to a composite log2tpm value of greater than or equal to 3.2, minus (ii) the expression of Interferon Alpha Inducible Protein 27 (IFI27) gene as measured by log2tpm, wherein the composite expression level B corresponds to a value of greater than or equal to -0.25. BACKGROUND

[0002] Detection of gene signatures from genomic data has been discussed in the medical domain during the last two decades. A “gene signature” can be stated as a single or a group of genes in a cell having a unique pattern of gene expression that is the consequence of either changed biological process or phenotypic behavior. Genomic profiling can provide prognostic and predictive information to guide clinical care. Biomarkers that reliably predict subject response to chemotherapy, T cell therapy and immune checkpoint inhibition across different tumor types are not well-understood. Current recommended treatments for cancer such as unresectable or metastatic disease include anti-PD-1-based regimens for subjects which are considered as standard of care (SoC) for initial treatment in many settings. Despite these options, at least half of these subjects will progress within 1 year. Cancer treatment is complex and can vary significantly depending on stage of disease, prior treatments, and tumor type. The significant heterogeneity across individual subjects is often underestimated. Even within a certain type of cancer, or tumor type, subjects can have different underlying complexity, making design of trials and interpretation of results complicated.SUMMARY OF THE INVENTION

[0003] In some aspects, the disclosure provides a method of treating a cancer in a subject, the method comprising: administering to the subject an effective amount of an immunotherapeutic, wherein the subject has been determined to have a T cell signature (TCS) comprising: (a) a composite expression level A comprising the expression of three or more genes selected from Thymocyte Expressed, Positive Selection Associated 1 (TESPA1), Cluster of Differentiation 28 (CD28), G Protein-Coupled Receptor 183 (GPR183), and Zeta- Chain-Associated Protein Kinase 70 (ZAP70) as measured by log2tpm corresponding to a composite log2tpm value of greater than or equal to 3.2; or (b) a composite expression level B comprising (i) the expression of three or more genes selected from TESPA1, CD28, GPR183 and ZAP70 as measured by log2tpm corresponding to a composite log2tpm value of greater than or equal to 3.2, minus (ii) the expression of Interferon Alpha Inducible Protein 27 (IFI27) gene as measured by log2tpm, wherein the composite expression level B corresponds to a value of greater than or equal to -0.25.

[0004] In some aspects, the TCS corresponds to TESPA1, CD28, and GPR183. In some aspects, the TCS corresponds to TESPA1, CD28, GPR183 and ZAP70.

[0005] In some aspects, the composite expression level A or the composite expression level B is determined prior to administration of the immunotherapeutic.

[0006] In some aspects, the subject has been determined to have a composite expression level A of greater than or equal to 3.8. In some aspects, the subject has been determined to have a composite expression level A of greater than or equal to 3.96. In some aspects, the subject has been determined to have a composite expression level A of greater than or equal to 4.2. In some aspects, the subject has been determined to have a composite expression level A of greater than or equal to 4.4.

[0007] In some aspects, the TCS corresponds to TESPA1, CD28, GPR183, and IFI27.

[0008] In some aspects, the subject has been determined to have a composite expression level B of greater than or equal to -0.25. In some aspects, the subject has been determined to have a composite expression level B of greater than or equal to 0. In some aspects, the subject has been determined to have a composite expression level B of greater than or equal to 0.25. In some aspects, the subject has been determined to have a composite expression level B of greater than or equal to 1.25.

[0009] In some aspects, the immunotherapeutic comprises an immune checkpoint inhibitor, a methylating agent, a T cell therapy, an immune activator, a chemotherapeutic agent, or a combination thereof.

[0010] In some aspects, the present disclosure provides a method of treating a cancer in a subject, the method comprising administering to the subject an effective amount of a T cell therapeutic, wherein the subject has been determined to have a T cell signature (TCS) comprising: (a) a composite expression level A comprising the expression of three or more genes selected from Thymocyte Expressed, Positive Selection Associated 1 (TESPA1), Cluster of Differentiation 28 (CD28), G Protein-Coupled Receptor 183 (GPR183), and Zeta- Chain-Associated Protein Kinase 70 (ZAP70) as measured by log2tpm corresponding to a composite log2tpm value of greater than or equal to 3.2; or (b) a composite expression level B comprising (i) the expression of three or more genes selected from TESPA1, CD28, GPR183 and ZAP70 as measured by log2tpm corresponding to a composite log2tpm value of greater than or equal to 3.2, minus (ii) the expression of Interferon Alpha Inducible Protein 27 (IFI27) gene as measured by log2tpm, wherein the composite expression level B corresponds to a value of greater than or equal to -0.25.

[0011] In some aspects, the TCS corresponds to TESPA1, CD28, and GPR183. In some aspects, the TCS corresponds to TESPA1, CD28, GPR183 and ZAP70. In some aspects, the subject has been determined to have a composite expression level A of greater than or equal to 4.2. In some aspects, the TCS corresponds to TESPA1, CD28, GPR183, and IFI27. In some aspects, the subject has been determined to have a composite expression level B of greater than or equal to 0. In some aspects, the composite expression level A or composite expression level B is determined prior to administration of the T cell therapeutic.

[0012] In some aspects, the T cell therapeutic comprises a bi-specific molecule. In some aspects, the bi-specific molecule comprises a bi-specific T cell engager (BiTE), or a Bi- specific antibody (BsAb). In some aspects, the T cell therapeutic comprises a T-cell receptor (TCR) protein. In some aspects, the T cell therapeutic comprises a chimeric antigen receptor (CAR) T cell. In some aspects, the T cell therapeutic targets PRAME. In some aspects, the T cell therapeutic is IMC-F106C. In some aspects, the T cell therapeutic targets gp100. In some aspects, the T cell therapeutic comprises tebentafusp.

[0013] In some aspects, the present disclosure provides a method of determining whether a cancer treatment is suitable for treating cancer in a subject in need thereof, the methodcomprising determining if the subject has a T cell signature (TCS), the TCS comprising: (a) a composite expression level A comprising the expression of three or more genes selected from Thymocyte Expressed, Positive Selection Associated 1 (TESPA1), Cluster of Differentiation 28 (CD28), G Protein-Coupled Receptor 183 (GPR183), and Zeta-Chain-Associated Protein Kinase 70 (ZAP70) as measured by log2tpm; or (b) a composite expression level B comprising (i) the expression of three or more genes selected from TESPA1, CD28, GPR183 and ZAP70 as measured by log2tpm corresponding to a composite log2tpm value of greater than or equal to 3.2, minus (ii) the expression of Interferon Alpha Inducible Protein 27 (IFI27) gene as measured by log2tpm, wherein the subject is suitable for a cancer treatment if, (i) the composite expression level A corresponds to a composite log2tpm value of greater than or equal to 3.2; or (ii) the composite expression level B corresponds to a composite log2tpm value of greater than or equal to -0.25.

[0014] In some aspects, the composite expression level A comprises TESPA1, CD28, and GPR183 and corresponds to a composite log2tpm value of greater than or equal to 3.2. In some aspects, the composite expression level A comprises TESPA1, CD28, GPR183, and ZAP70 and corresponds to a composite log2tpm value of greater than or equal to 3.2.

[0015] In some aspects, the composite expression level of B comprises TESPA1, CD28, GPR183, and IFI27 and corresponds to a composite log2tpm value of greater than or equal to –0.25. In some aspects, the composite expression level of B comprises TESPA1, CD28, GPR183, ZAP70 and IFI27 and corresponds to a composite log2tpm value of greater than or equal to –0.25.

[0016] In some aspects, the cancer treatment is an immunotherapeutic cancer treatment. In some aspects, the cancer treatment is a T cell therapeutic.

[0017] In some aspects, the present disclosure provides a method for identifying and treating a subject suffering from a cancer who is suitable for treatment with an immunotherapeutic, e.g., a T cell therapeutic, comprising: (a) determining from a sample obtained from the subject if the subject has a T cell signature (TCS), using RNASeq methodology to quantify gene expression levels, the TCS comprising: (i) a composite expression level A comprising the expression of three or more genes selected from Thymocyte Expressed, Positive Selection Associated 1 (TESPA1), Cluster of Differentiation 28 (CD28), G Protein-Coupled Receptor 183 (GPR183), and Zeta-Chain-Associated Protein Kinase 70 (ZAP70) as measured by log2tpm; or (ii) a composite expression level B comprising 1) the expression of three or moregenes selected from TESPA1, CD28, GPR183 and ZAP70 as measured by log2tpm corresponding to a composite log2tpm value of greater than or equal to 3.2, minus 2) the expression of Interferon Alpha Inducible Protein 27 (IFI27) gene as measured by log2tpm, (b) identifying the subject as suitable for treatment with a T cell therapeutic if (i) the composite expression level A corresponds to a composite log2tpm value of greater than or equal to 3.2, or (ii) the composite expression level B corresponds to a composite log2tpm value of greater than or equal to -0.25; and (c) administering an effective amount of the immunotherapeutic to the subject identified as suitable for treatment with the immunotherapeutic.

[0018] In some aspects, the T cell therapeutic comprises a bi-specific molecule. In some aspects, the T cell therapeutic comprises a T-cell receptor (TCR). In some aspects, the bi- specific molecule comprises a bi-specific T cell engager (BiTE), or a Bi-specific antibody (BsAb). In some aspects, the T cell therapeutic comprises a chimeric antigen receptor (CAR) T cell. In some aspects, the T cell therapeutic targets PRAME. In some aspects, the T cell therapeutic is IMC-F106C. In some aspects, the T cell therapeutic targets gp100. In some aspects, the T cell therapeutic comprises tebentafusp.

[0019] In some aspects, the present disclosure provides a method for treating a cancer in a subject, the method comprising: (a) determining a log2tpm value of genes in a TCS in a biological sample obtained from the subject, wherein the TCS corresponds to: (i) a composite expression level A comprising the expression of three or more genes selected from Thymocyte Expressed, Positive Selection Associated 1 (TESPA1), Cluster of Differentiation 28 (CD28), G Protein-Coupled Receptor 183 (GPR183), and Zeta-Chain-Associated Protein Kinase 70 (ZAP70) as measured by log2tpm; or (ii) a composite expression level B comprising a) the expression of three or more genes selected from TESPA1, CD28, GPR183 and ZAP70 as measured by log2tpm corresponding to a composite log2tpm value of greater than or equal to 3.2, minus (b) the expression of Interferon Alpha Inducible Protein 27 (IFI27) gene as measured by log2tpm, and (b) administering to the subject a dosing regimen comprising a first dosage amount of a T cell therapeutic; wherein if the composite expression level A is greater than or equal to 3.2 or the composite expression level B is greater than or equal to -0.25, then a second dosage amount is administered, wherein the second dosage amount is the same or less than the first dosage amount, or wherein if the composite expression level A is less than 3.2 or the composite expression level B is less than -0.25, thenthe second dosage amount is greater than the first dosage amount and / or further comprises a second cancer treatment.

[0020] In some aspects, the first dosing regimen comprises a single dose of a T cell therapeutic. In some aspects, the first dosing regimen comprises greater than one dose of a T cell therapeutic. In some aspects, the first dosing regimen comprises administering a T cell therapeutic in greater than one doses for a period of about 1 week to about 1 month.

[0021] In some aspects, a second biological sample is obtained from the subject about 1 week to about 3 months after the first composite expression level A or composite expression level B is determined.

[0022] In some aspects, the second composite expression level A is at least 10% greater than the first composite expression level A. In some aspects, the second composite expression level A is at least 20% greater than the first composite expression level A. In some aspects, if the second composite expression level A is greater than the first composite expression level A, then the second dosage amount is at least 10% less than the first dosage amount. In some aspects, if the second composite expression level A is greater than the first composite expression level A, then the second dosage amount is at least 20% less than the first dosage amount. In some aspects, if the second composite expression level A is the same or less than the first composite expression level A, then the second dosage amount is at least 10% greater amount than first dosage amount. In some aspects, if the second composite expression level A is the same or less than the first composite expression level A, then the second dosage amount is at least 20% greater amount than first dosage amount.

[0023] In some aspects, the second composite expression level B is at least 10% greater than the first composite expression level B. In some aspects, the second composite expression level B is at least 20% greater than the first composite expression level B. In some aspects, if the second composite expression level B is greater than the first composite expression level B, then the second dosage amount is at least 10% less than the first dosage amount. In some aspects, if the second composite expression level B is greater than the first composite expression level B, then the second dosage amount is at least 20% less than the first dosage amount. In some aspects, if the second composite expression level B is the same or less than the first composite expression level B, then the second dosage amount is at least 10% greater amount than first dosage amount. In some aspects, if the second composite expression level Bis the same or less than the first composite expression level B, then the second dosage amount is at least 20% greater amount than first dosage amount

[0024] In some aspects, the TCS corresponds to TESPA1, CD28, and GPR183. In some aspects, the TCS corresponds to TESPA1, CD28, GPR183 and ZAP70. In some aspects, the TCS corresponds to TESPA1, CD28, GPR183, ZAP70 and IFI27.

[0025] In some aspects, the biological sample comprises whole blood.

[0026] In some aspects, the immunotherapeutic, cancer treatment or T cell therapeutic is a first anti-cancer therapeutic agent.

[0027] In some aspects, the immunotherapeutic, cancer treatment or T cell therapeutic is a first-line treatment.

[0028] In some aspects, the subject is a human subject.

[0029] In some aspects, the cancer is positive for an antigen selected from the group consisting of gp100 and PRAME. In some aspects, the cancer is a PRAME-positive cancer. In some aspects, the subject is HLA-A*02 positive. In some aspects, the subject is HLA- A*02:01 positive. In some aspects, the subject has BRAF V600 mutation. In some aspects, the subject has been diagnosed with a life expectancy greater than 3 months.

[0030] In some aspects, the cancer is a melanoma, an ovarian cancer, a non-small cell lung adenocarcinoma, a non-small cell lung squamous cell carcinoma, an endometrial cancer, or a breast cancer. In some aspects, the melanoma is a uveal melanoma or a cutaneous melanoma. In some aspects, the melanoma is a cutaneous melanoma. In some aspects, the breast cancer is a triple-negative breast cancer.

[0031] In some aspects, the method further comprises administering to said subject an effective amount of a second anti-cancer therapeutic agent. In some aspects the second anti- cancer therapeutic agent comprises an immune checkpoint inhibitor, a second T cell therapeutic, an immune activator, chemotherapeutic agent, cancer vaccine, a methylating agent or a combination thereof. In some aspects, the second anti-cancer therapeutic agent is administered prior to administration of the T cell therapeutic. In some aspects, the second anti-cancer therapeutic agent is a chemotherapeutic agent.

[0032] In some aspects, the present disclosure provides a method of treating cutaneous melanoma in a subject, the method comprising: administering to the subject an effective amount of an immunotherapeutic, wherein the subject has been determined to have a T cellsignature (TCS) comprising (i) the expression of three or more genes selected from TESPA1, CD28, GPR183 and ZAP70 as measured by log2tpm corresponding to a composite log2tpm value of greater than or equal to 3.2, minus (ii) the expression of Interferon Alpha Inducible Protein 27 (IFI27) gene as measured by log2tpm, wherein the TCS corresponds to a composite log2tpm value of greater than or equal to -0.25.

[0033] In some aspects, the present disclosure provides a kit comprising a solution for use in processing a biological sample obtained from a subject; wherein the solution comprises nucleotide primers for evaluating variant allele frequency in a panel of mutations comprising TESPA1, CD28, and GPR183. In some aspects, the kit further comprises nucleotide primers for evaluating variant allele frequency in a panel of mutations comprising ZAP70, IFI27, or combinations thereof.

[0034] In some aspects, the present disclosure provides a non-transitory computer readable medium having stored thereon a computer program which, when executed by a computer system operably connected to an assay system configured to measure, by RNAseq methodology, a T cell signature (TCS) comprising expression levels of three or more genes selected from: Thymocyte Expressed, Positive Selection Associated 1 (TESPA1), Cluster of Differentiation 28 (CD28), G Protein-Coupled Receptor 183 (GPR183), Zeta-Chain- Associated Protein Kinase 70 (ZAP70), and Interferon Alpha Inducible Protein 27 (IFI27), in a patient sample from a human subject, causes the computer system to perform a method of identifying a immunotherapeutic dose to be administered to the human subject for the treatment of a cancer, the method comprising: (a) fitting said TCS to a response surface model as a function of immunotherapeutic dose; (b) computing a cost function for said TCS; and (c) selecting a immunotherapeutic dose that minimizes said cost function at a known time interval, thereby identifying the immunotherapeutic dose to be administered to the human subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The following drawings form part of the present specification and are included to further demonstrate exemplary embodiments of certain aspects of the present disclosure.

[0036] Unless otherwise indicated, the TCS referred to in the drawings correspond to the TESPA1, CD28, and GPR183 genes. Within the drawings, “high” refers to values at or above the upper 40% percentile of samples analyzed in the dataset, and “low” refers to values below the upper 40% percentile of samples analyzed in the dataset.

[0037] FIG.1 shows the scaled log2tpm output from an RNASeq gene expression analysis in whole blood samples from HLA-A*0201 positive adult subjects with previously untreated advanced uveal melanoma and receiving tebentafusp, revealing genes whose higher expression levels were associated with longer overall survival (OS) and tumor reduction, as well as genes whose higher expression levels were associated with shorter OS and tumor growth.

[0038] FIG.2A shows a waterfall plot of the change in tumor size (TS) in individual HLA- A*0201 positive adult subjects with advanced uveal melanoma who received treatment with tebentafusp. The patterned bars represent those subjects having a high TCS (determined from TESPA1, CD28, and GPR183) corresponding in this case to a TCS log2tpm value of greater than or equal to 4.4. The solid bars represent a “low” value, which is a TCS log2tpm value of less than 4.4. Similar waterfall plots show the change in where the patterned and solid bars indicate whether subjects had high or low levels of effector T cells (FIG.2B) or high or low levels of exhausted T cells (FIG.2C). As indicated by the plots in FIG.2, there was a strong association between the TCS and change in TS in contrast to effector T cell level or exhausted T cell level.

[0039] FIG.3 shows line graphs plotting overall survival (OS) against time in subjects with advanced uveal melanoma treated with tebentafusp having either high (solid lines) or low (dotted lines) TCS , where the TCS was analysed from whole blood samples. (FIG.3A). The high and low TCS values correspond to those as described in FIG.2A. The number of individuals at risk at every 6 month timepoint is noted in the table underneath the graph. Graphical plots of similar evaluation measuring levels of effector T cells (FIG.3B) or exhausted T cells (FIG.3C) are also shown.

[0040] FIG.4 shows waterfall plots showing the change in TS in subjects who received tebentafusp where each bar represents an individual subject and where the TCS status (high or low) of each subject is indicated (FIG.4A) or where traditional histological scoring assessment (H-score) is indicated (FIG.4B). The high and low TCS values correspond to those as described in FIG.2A.

[0041] FIG.5 shows line graphs where OS is plotted against time in subjects receiving tebentafusp where the lines correspond to patients having either high or low TCS (FIG.5A), or high or low H-score from a tumor biopsy (FIG.5B). The high and low TCS valuescorrespond to those as described in FIG.2A. The TCS was found to have a superior association with OS as compared to H scores.

[0042] FIG.6 shows line graphs plotting OS (FIG.6A) or progression-free survival (PFS) (FIG.6B) against time in subjects treated with IMCF106C, where the two lines represent subjects having either high (i.e., TCS log2tpm value of greater than or equal to 3.96) or low (TCS log2tpm value of less than 3.96) TCS. A high TCS was seen to be associated with higher OS and PFS relative to low TCS.

[0043] FIG.7 shows a waterfall plot of change in tumor size in subjects treated with IMCF106C. The high and low TCS values correspond to those as described in FIG.6. PRAME negative subjects are noted with the asterisks (*). The TCS exhibited a strong association with change in TS.

[0044] FIG.8 shows spider plots of the change in tumor size over time in TCS-high (FIG. 8A) or TCS-low (FIG.8B) subjects with different tumor types who were treated with IMCF106C. The high and low TCS values correspond to those as described in FIG.6. TCS- high subjects showed a relatively greater and more durable reduction in tumor size as compared to TCS-low subjects.

[0045] FIG.9A shows a waterfall plot of TS in subjects where the TCS level (high or low) is indicated (FIG.9A). Line graphs plotting PFS (FIG.9B) or OS (FIG.9C) against time for subjects with uveal melanoma who were treated with IMCF106C are indicated. The high and low TCS values correspond to those as described in FIG.6.

[0046] FIG.10 shows a box and whisker graph plotting tumor type of patients treated with IMCF106C against TCS levels (log2tpm). As can be seen in the Figure, TCS log2tpm values by tumor type range from 3.2 to 5.0, with a high number of data points falling within the range of 3.8 to 4.8, and a median value of all evaluated samples across the multiple tumor types corresponding to just below 4.0 at 3.96, with ovarian and cutaneous melanoma patients indicating a mean TCS log2tpm value of about 4.4. The majority of melanoma and ovarian cancers have a mean TCS value at or higher than the median log2tpm value of 3.96, or about 4. A TCS log2tpm value of 3.2 corresponds to the minimum cutoff value across all tumor indications.

[0047] FIG.11 shows box and whisker graphs of neutrophil-to-lymphocyte ratio (NLR), absolute lymphocyte count (ALC), and fibrinogen (FIBRINO), identified from a multivariate analysis of forty baseline clinical factors. NLR, ALC and fibrinogen levels had a strong correlation with TCS.

[0048] FIG.12 shows a spider plot of the change in target lesion against survival time, portraying the association of disease control rate (DCR) (change in target lesion from baseline over time) with fibrinogen levels (high fibrinogen = dashed line; normal fibrinogen = solid line) in uveal melanoma subjects treated with IMCF106C.

[0049] FIG.13 shows a line graph plotting change in target lesion against survival time, portraying the association of disease control rate (DCR) (change in target lesion from baseline over time) in ovarian cancer subjects treated with IMCF106C.

[0050] FIG.14 shows a waterfall plot of change in TS (FIG.14A), and line graphs plotting PFS (FIG.14B) or OS (FIG.14C) against time where the levels of IFI27 (either high or low) are indicated. IFI27 gene expression analyses were performed as described in Example 3 using whole blood samples from melanoma subjects treated with IMC-F106C. Below median IFI27 expression was associated with better outcomes in all melanomas tested.

[0051] FIG.15 shows a waterfall plot of change in TS (FIG.15A), and line graphs plotting PFS (FIG.15B) or OS (FIG.15C) against time where the levels of IFI27 (either high or low) are indicated. IFI27 gene expression analyses were performed as described in Example 3 using whole blood samples from the subset of cutaneous melanoma subjects treated with IMC-F106C. Below median IFI27 expression was associated with better outcomes in cutaneous melanoma.

[0052] FIG.16 shows a waterfall plot showing the change in TS from baseline in subjects having either high or low IFI27 levels (FIG.16A). Line graphs plotting PFS (FIG.16B) or OS (FIG.16C) over time in uveal melanoma subjects treated with tebentafusp. IFI27 levels were measured as described in Example 4 from whole blood samples taken from subjects at baseline. Below median IFI27 expression from the analyses was associated with better outcomes in these subjects.DETAILED DESCRIPTION

[0053] Unless otherwise defined herein, scientific and technical terms used in the present disclosure shall have meanings that are commonly understood by one of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0054] The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element.

[0055] The use of the term “or” in the claims is used to mean “and / or,” unless explicitly indicated to refer only to alternatives or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and / or.”

[0056] As used herein, the terms “comprising” (and any variant or form of comprising, such as “comprise” and “comprises”), “having” (and any variant or form of having, such as “have” and “has”), “including” (and any variant or form of including, such as “includes” and “include”) or “containing” (and any variant or form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited, elements or method steps.

[0057] The use of the term “for example” and its corresponding abbreviation “e.g.” means that the specific terms recited are representative examples and embodiments of the disclosure that are not intended to be limited to the specific examples referenced or cited unless explicitly stated otherwise.

[0058] As used herein, “about” can mean plus or minus 10% of the provided value. Where ranges are provided, they are inclusive of the boundary values. “About” can additionally or alternately mean either within 10% of the stated value, or within 5% of the stated value, or in some cases within 2.5% of the stated value; or “about” can mean rounded to the nearest significant digit.

[0059] As used herein, “between” is a range inclusive of the ends of the range. For example, a number between x and y explicitly includes the numbers x and y and any numbers that fall within x and y.T Cell Signature

[0060] In some embodiments, the disclosure provides identification of specific genes that are differentially expressed in cells in a subject, e.g., a cancer subject, wherein the differential expression of the specific genes can correlate with increased efficacy of an immunotherapeutic, e.g., a T cell therapy, when administered to the subject. The specific groups of differentially expressed genes are denoted herein as a T cell signature, or TCS. In some embodiments, the specific genes are highly expressed in cells of the subject. However, in some embodiments, the disclosure provides that one or more genes, i.e., TCS secondary factors, can exhibit reduced expression in the subject that correlates with increased efficacy of an immunotherapeutic.

[0061] In some embodiments, gene expression levels in the TCS can be quantified by RNA sequencing, i.e., RNA-Seq. RNA-seq is a technique that can examine the quantity and sequences of RNA in a sample using next-generation sequencing (NGS). It analyzes the transcriptome, indicating which of the genes are turned on or off and to what extent. RNA- seq can be performed using means known to the skilled artisan. For example, in some embodiments, RNA-seq can include the following steps: (1) RNA extraction, (2) reverse transcription into cDNA, (3) adapted ligation, (4) amplification, and (5) sequencing. Thus, in some embodiments, the methods described herein can include any of the above steps.

[0062] In some embodiments, in order to determine the gene expression levels of the TCS, RNA is extracted from the biological material of choice (e.g., cells, tissues). In some embodiments, subsets of RNA molecules are isolated using a specific protocol, such as the poly-A selection protocol to enrich for polyadenylated transcripts or a ribo-depletion protocol to remove ribosomal RNAs. In some embodiments, the RNA is converted to complementary DNA (cDNA) by reverse transcription and sequencing adaptors are ligated to the ends of the cDNA fragments. Following amplification by PCR, the RNA-Seq library is ready for sequencing. In specific embodiments, whole blood samples are collected in PAXgene RNA tubes and RNA is isolated from the samples. RNA libraries from isolated RNA are prepared using, e.g., an Illumina TruSeq® stranded mRNA prep kit, and then sequenced using 100bp paired-end sequencing at 50 million reads per sample. To map sequences against a reference genome, the resulting reads can be first aligned using STAR aligner (version 2.6.1), and then mapped to the GRCh38 primary assembly provided by the European BioinformaticsInstitute’s Ensembl Genomes database. Gene expressions can be quantified using RSEM (version 1.3.1).

[0063] In some embodiments, gene expression levels of the TCS are indicated by values of log2tpm where “tpm” corresponds to transcripts per million and log2tpm represents a measure of the frequency of a gene transcripts, or set of gene transcripts, if applicable, within a population of transcripts in a sample. For example, a log2tpm of 9 for gene A means that for every million transcripts in the sample, 29of them are from gene A.

[0064] If more than one gene is included in the TCS, then the TCS is a composite expression level of the one or more genes, corresponding to a composite log2tpm value. In some aspects, the term “composite” is defined as the mean or average of two or more values. In some aspects, the term “composite” is defined as the mean or average of two or more values, minus one or more values. For example, a composite expression level comprising three or more genes selected from Thymocyte Expressed, Positive Selection Associated 1 (TESPA1), Cluster of Differentiation 28 (CD28), G Protein-Coupled Receptor 183 (GPR183), and Zeta- Chain-Associated Protein Kinase 70 (ZAP70) as measured by log2tpm, is the mean value of the individual log2tpm values of each gene.

[0065] A “composite expression level,” e.g., composite expression level A, with respect to a TCS comprising TESPA1, CD28, GPR183, and / or ZAP70 corresponds to the mean of the log2tpm values of TESPA1, CD28, GPR183, and / or ZAP70. For example, if a TCS comprises TESPA1, CD28, and GPR183, and TESPA1 has a log2tpm of 4.1, CD28 has a log2tpm of 5.3, and GPR183 has a log2tpm of 5.9, then the TCS, or composite log2tpm value, is the mean of the log2tpm values, i.e. (4.1 +5.3+5.9) / 3, or 5.1.

[0066] A “composite expression level,” e.g., composite expression level B, with respect to a TCS as described above that further comprises IFI27 is determined as follows: first, the mean of the log2tpm values of TESPA1, CD28, GPR183, and / or ZAP70 is calculated; second, the log2tpm value of IFI27 is subtracted from the mean log2tpm value from the first step. The resulting value corresponds to the value of a TCS that further comprises IFI27. For example, if a TCS comprises TESPA1, CD28, GPR183, and IFI27, and TESPA1 has a log2tpm of 4.1, CD28 has a log2tpm of 5.3, GPR183 has a log2tpm of 5.9, and IFI27 has a log2tpm value of 2.1, then the TCS, or composite log2tpm value, is the mean of the log2tpm values ofTESPA1, CD28, GPR183 (i.e. (4.1 +5.3+5.9) / 3, or 5.1) minus the log2tpm value of IFI27 (2.1), resulting in a final calculation of 5.1-2.1, or 3.

[0067] Transcripts per million (tpm) is a unit of measure used to facilitate comparisons across samples. A tpm value represents a relative gene expression level that, in principle, should be comparable between samples, for example multiple samples from the same subject. In some embodiments when multiple samples are used, the median log2tpm value across all genes is calculated per subject. In some embodiments when multiple samples are used, the mean log2tpm value across all genes is calculated per subject. In some embodiments when multiple samples are used, the median TCS log2tpm value is calculated per subject. In some embodiments when multiple samples are used, the mean TCS log2tpm value is calculated per subject.

[0068] In some embodiments, the TCS log2tpm value can be determined from the blood sample of a subject. In some embodiments, the TCS log2tpm value can be determined from genes expressed in an immune cell or a progenitor or precursor thereof. In some embodiments, the immune cell is a primary immune cell. In some embodiments, the immune cell is a neutrophil, eosinophil, basophil, mast cell, monocyte, macrophage, dendritic cell, natural killer cell, or a lymphocyte. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a naïve T cell, a memory T cell, an effector T cell, a memory T cell, or a helper T cell. In some embodiments, the immune cell is a Th1, Th2, Th9, Th17, or Th22 cell. In some embodiments, the immune cell is a T-follicular helper (Tfh) cell. In some embodiments, the immune cell is a regulatory (Treg) T cell, a cytotoxic T lymphocyte (CTL) or killer T cell, a central memory T cell, an effector memory T cell (TEMor TEMR), a tissue resident memory (Trm) T cell, a virtual memory T cell, an innate memory T cell, or a memory stem cell (Tscm) T cell. In some embodiments, the immune cell is a γδ T cell. In some embodiments, the immune cell is a natural killer (NKT) T cell. In some embodiments, the immune cell is an inflammatory T cell. In some embodiments, the immune cell is a CD4+ or CD8+ T cell. In some embodiments, the immune cell is a mucosal associated invariant T cell.

[0069] In some embodiments, the TCS log2tpm value of the subject in the present disclosure is determined at baseline. In some embodiments, “baseline” means an initial measurement of a condition or gene expression. As the determination of TCS log2tpm value includes the steps of obtaining a sample, preparing the sample (e.g., RNA extraction, reverse transcription,amplification, sequencing, etc.), and analyzing the results, the determination occurs over a period of time. Thus, as used herein, the phrase "TCS log2tpm value is determined" refers to the timepoint in which the sample is taken from the subject, independently of whether the actual RNA-seq analysis is performed immediately or processed at a later time. For example, if the sample of the subject is taken one month before administration of the first dosage of an immunotherapeutic, but the RNA-seq analysis is not fully completed until after administration of the first dosage of the immunotherapeutic, the “baseline” date is considered to be one month before administration of the first dosage of an immunotherapeutic.

[0070] In some embodiments, the TCS log2tpm value is determined at an initial point and then determined again later, wherein the second TCS log2tpm value determination is used for comparison over time to look for changes in gene expression levels over time. In some embodiments, the TCS log2tpm value is determined before administration of the first dosage of an immunotherapeutic, e.g., 1 month, 1 week or 1 day before administration of the first dosage. In some embodiments, the TCS log2tpm value is determined the same day as administration of the first dosage of an immunotherapeutic. In some embodiments, the TCS log2tpm value is determined shortly after administration of the first dosage of an immunotherapeutic, e.g., 1 day or 1 week after administration of the first dosage of an immunotherapeutic. In some embodiments, the TCS log2tpm value is determined before the start of a new cancer treatment, e.g., the subject could have been administered one or more anti-cancer treatments previously, wherein the term “baseline” refers to administration of the most recent therapeutic. In some embodiments, the TCS log2tpm value baseline is determined from samples collected from subjects just prior to or at the time of first treatment, i.e., baseline.

[0071] In some embodiments, a TCS log2tpm value can be determined multiple times, e.g., before administration and then at timepoints after administration. For example, the TCS log2tpm value can be determined one or more times throughout the duration of the treatment regimen. In some embodiments, the TCS log2tpm value is determined after treatment ends. In some embodiments, the TCS log2tpm value is determined at multiple time points before, during and after the treatment regimen.

[0072] In some embodiments, the disclosure provides at least five differentially expressed genes can correlate with improved efficacy of an immunotherapeutic, e.g., a T cell therapeutic. Combinations of these differentially expressed genes are referred to herein as theT cell signature, or TCS. Genes that can be included in the TCS include: Thymocyte Expressed, Positive Selection Associated 1 (TESPA1), Cluster of Differentiation 28 (CD28), G Protein-Coupled Receptor 183 (GPR183), zeta-chain-associated protein kinase (ZAP70), and interferon alpha inducible protein 27 (IFI27). In the case of TESPA1, CD28, GPR183, and ZAP70, the disclosure provides that expression of these genes above a certain threshold, i.e., high expression, correlates with increased efficacy of an immunotherapeutic, e.g., a T cell therapeutic. In the case of IFI27, the disclosure provides that expression of this gene below a certain threshold, i.e., low expression, correlates with increased efficacy of an immunotherapeutic, e.g., a T cell therapeutic.

[0073] TESPA1 encodes a protein exclusively expressed in T and B lymphocytes and is known to be one the best indicators of naïve T cells. CD28 is important for T cell survival and responsiveness. CD28 is also known to be high on naïve T cells, becoming downregulated following activation. Furthermore, the absence of CD28 on T cells is known to be associated with exhaustion / senescence and susceptibility to AICD. While GPR183 has a slightly broader scope of expression, it is known to be an important receptor for chemotaxis of T cells. ZAP70 encodes an enzyme belonging to the protein tyrosine kinase family, and it plays a role in T-cell development and lymphocyte activation. IFI27 participates in varieties of biological processes, including innate immunity and apoptosis, and has been shown to be a key gene in several types of cancer.

[0074] In some embodiments, the TCS corresponds to gene expression of one of the following genes: TESPA1, CD28, GPR183, or ZAP70, wherein the log2tpm is greater than or equal to 3.2, 3.4, 3.6, 3.8, 3.96, 4.0, 4.2, or 4.4. In some embodiments, the TCS corresponds to gene expression of IFI27, wherein the log2tpm is less than or equal to 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, or 4.8. In some embodiments, the TCS corresponds gene expression of two or more of the following genes: TESPA1, CD28, GPR183, or ZAP70 as follows: Combination # TCS Combination # TCS combination combination 1 TESPA1, CD28 4 CD28, GPR183 2 TESPA1, 5 CD28, ZAP70 GPR1833 TESPA1, 6 GPR183, ZAP70 ZAP70 wherein the composite of the two log2tpm values is greater than or equal to 3.2, 3.4, 3.6, 3.8, 3.96, 4.0, 4.2, or 4.4

[0075] In some embodiments, the TCS comprises a composite expression level A comprising expression of three or more of the following genes: TESPA1, CD28, GPR183, or ZAP70 as follows. Combination # TCS Combination # TCS combination combination 7 TESPA1, 9 TESPA1, CD28, GPR183 GPR183, ZAP70 8 TESPA1, 10 CD28, GPR183, CD28, ZAP70 ZAP70, wherein the composite log2tpm value of the three log2tpm is greater than or equal to 3.2, 3.4, 3.6, 3.8, 3.96, 4.0, 4.2, or 4.4.

[0076] In some embodiments, the TCS comprises a composite expression level B comprising (i) expression of any of the gene combinations found in combinations 7, 8, 9 or 10 as measured by log2tpm with a value of greater than or equal to 3.2, 3.4, 3.6, 3.8, 3.96, 4.0, 4.2, or 4.4, minus (ii) the expression of IFI27 gene as measured by log2tpm, wherein the composite expression level B corresponds to a value of greater than or equal to -0.25.

[0077] In some embodiments, the TCS comprises a composite expression level A comprising expression of all four of the following genes: TESPA1, CD28, GPR183, and ZAP70, wherein the composite log2tpm value of the four genes is greater than or equal to 3.2, 3.4, 3.6, 3.8, 3.96, 4.0, 4.2, or 4.4.

[0078] In some embodiments, the TCS comprises a composite expression level B comprising (i) expression of all four of TESPA1, CD28, GPR183, and ZAP70 as measured by log2tpm with a value of greater than or equal to 3.2, 3.4, 3.6, 3.8, 3.96, 4.0, 4.2, or 4.4, minus (ii) the expression of IFI27 gene as measured by log2tpm, wherein the composite expression level B corresponds to a value of greater than or equal to -0.25.

[0079] In some embodiments, the TCS corresponds to the combination of TESPA1, CD28, GPR183, ZAP70, and IFI27 with the composite expression level B as determined herein. In some embodiments, the TCS corresponds to the combination of TESPA1, CD28, GPR183, and IFI27 with the composite expression level B as determined herein. In some embodiments, the TCS corresponds to the combination of TESPA1, CD28, and GPR183 with the composite expression level B as determined herein.

[0080] In some embodiments, the TCS described herein can include other T cell-associated genes (e.g., IL7R). However, in some embodiments, the inclusion of these other gene(s) do not improve the overall correlation with the improved efficacy of the immunotherapeutic as described herein.

[0081] In some embodiments, the subject has been determined to have a T cell signature (TCS) as follows: two or more genes selected from Thymocyte Expressed, Positive Selection Associated 1 (TESPA1), Cluster of Differentiation 28 (CD28), G Protein-Coupled Receptor 183 (GPR183), and zeta-chain-associated protein kinase (ZAP70) having a composite log2tpm value as found in ranges 1-6 as found below in Table 1. Table 1 Combo TCS Composite log2tpm # combination range 1 range 2 range 3 range 4 range 5 range 6 1 TESPA1, 2 to 10 3 to 6 ≥3.2 ≥3.4 ≥3.8 ≥4.2 CD28 2 TESPA1, 2 to 10 3 to 6 ≥3.2 ≥3.4 ≥3.8 ≥4.2 GPR183 3 TESPA1, 2 to 10 3 to 6 ≥3.2 ≥3.4 ≥3.8 ≥4.2 ZAP70 4 CD28, 2 to 10 3 to 6 ≥3.2 ≥3.4 ≥3.8 ≥4.2 GPR183 5 CD28, 2 to 10 3 to 6 ≥3.2 ≥3.4 ≥3.8 ≥4.2 ZAP70 6 GPR183, 2 to 10 3 to 6 ≥3.2 ≥3.4 ≥3.8 ≥4.2 ZAP70

[0082] In some embodiments, the subject has been determined to have a T cell signature (TCS) as follows: three or more genes selected from Thymocyte Expressed, Positive Selection Associated 1 (TESPA1), Cluster of Differentiation 28 (CD28), G Protein-Coupled Receptor 183 (GPR183), and zeta-chain-associated protein kinase (ZAP70) having a log2tpm value as found in ranges 7-10 as found below in Table 2. Table 2 Combo TCS combination Composite log2tpm # range 1 range 2 range 3 range 4 range 5 range 6 7 TESPA1, CD28, 2 to 10 3 to 6 ≥3.2 ≥3.4 ≥3.8 ≥4.2 GPR183, 8 TESPA1, CD28, 2 to 10 3 to 6 ≥3.2 ≥3.4 ≥3.8 ≥4.2 ZAP70, 9 TESPA1, 2 to 10 3 to 6 ≥3.2 ≥3.4 ≥3.8 ≥4.2 GPR183, ZAP70, 10 CD28, GPR183, 2 to 10 3 to 6 ≥3.2 ≥3.4 ≥3.8 ≥4.2 ZAP70,

[0083] In some embodiments, the subject has been determined to have a T cell signature (TCS) as follows: four genes selected from Thymocyte Expressed, Positive Selection Associated 1 (TESPA1), Cluster of Differentiation 28 (CD28), G Protein-Coupled Receptor 183 (GPR183), and zeta-chain-associated protein kinase (ZAP70) having a log2tpm value as found in ranges 1-6 as found below in Table 3. Table 3 Combo TCS combination Composite log2tpm # range 1 range 2 range 3 range 4 range 5 range 6 11 TESPA1, CD28, 2 to 10 3 to 6 ≥3.2 ≥3.4 ≥3.8 ≥4.2 GPR183, ZAP70 12 TESPA1, CD28, -0.25 to -0.25 to ≥-0.25 ≥0 ≥0.25 ≥1.25 GPR183, IFI27 10 5

[0084] In some embodiments, the subject has been determined to have a T cell signature (TCS) as follows: the interferon alpha inducible protein 27 (IFI27) gene has a log2tpm value as found in one of ranges 1-6 in Table 4. Table 4 TCS gene log2tpm range 1 range 2 range 3 range 4 range 5 range 6 IFI27 0 to 3.0 to ≤(3.4) ≤(3.8) ≤(4.2) ≤(5.0) 10.0 10.0

[0085] In some embodiments, the subject has been determined to have a T cell signature (TCS) as follows: - any of the gene combinations as described in any one of Table 1, Table 2 or Table 3 in one of the composite log2tpm ranges 1 to 6, and - The IFI27 gene in Table 4 in one of the log2tpm ranges 1 to 6.

[0086] In some embodiments, the association between high TCS and disease control is observed to be applicable across multiple tumor types. In some embodiments, optimized log2tpm cutoffs can be utilized for specific tumor types. In some embodiments, the TCS comprises a composite expression level A cutoff of log2tpm for a TCS combination as described in any one of Table 1, Table 2 or Table 3 can be as follows in Table 5. Table 5 Cancer type Composite log2tpm range 1 range 2 range 3 range 4 range 5 range 6 cutaneous 2 to 6 3 to 6 ≥3 ≥3.5 ≥3.8 ≥3.86 melanoma (CM) ovarian cancer 2 to 6 3 to 6 ≥3 ≥3.9 ≥4 ≥4.04 endometrial cancer 2 to 6 3 to 6 ≥3 ≥3.2 ≥3.3 ≥3.36 breast cancer 2 to 6 3 to 6 ≥3 ≥3.4 ≥3.5 ≥3.57 non-small cell lung 2 to 6 3 to 6 ≥3 ≥3.2 ≥3.3 ≥3.35 cancer (NSCLC)

[0087] In some embodiments, the TCS comprises a composite expression level B for a TCS combination of any of combinations 1-11 in Table 1, 2 and 3 as described, minus the expression level of IFI27 as found in Table 4, wherein the cutoff log2tpm can be as follows in Table 6. Table 6 Cancer type Composite log2tpm range 1 range 2 range 3 range 4 range 5 range 6 cutaneous (-0.3) to 6 (-0.25) to 5 ≥0.2 ≥0 ≥(-0.10) ≥(-0.25) melanoma (CM) ovarian cancer (-0.3) to 6 (-0.25) to 5 ≥0.2 ≥0 ≥(-0.10) ≥(-0.25) endometrial cancer (-0.3) to 6 (-0.25) to 5 ≥0.2 ≥0 ≥(-0.10) ≥(-0.25) breast cancer (-0.3) to 6 (-0.25) to 5 ≥0.2 ≥0 ≥(-0.10) ≥(-0.25) non-small cell (-0.3) to 6 (-0.25) to 5 ≥0.2 ≥0 ≥(-0.10) ≥(-0.25) lung cancer (NSCLC)

[0088] In some embodiments, the T cell signature (TCS) comprises (a) a composite expression level A comprising the expression of three or more genes selected from Thymocyte Expressed, Positive Selection Associated 1 (TESPA1), Cluster of Differentiation 28 (CD28), G Protein-Coupled Receptor 183 (GPR183), and Zeta-Chain-Associated Protein Kinase 70 (ZAP70) as measured by log2tpm corresponding to a composite log2tpm value of greater than or equal to 3.2; or (b) a composite expression level B comprising (i) the expression of three or more genes selected from TESPA1, CD28, GPR183 and ZAP70 as measured by log2tpm corresponding to a composite log2tpm value of greater than or equal to 3.2, minus (ii) the expression of Interferon Alpha Inducible Protein 27 (IFI27) gene as measured by log2tpm, wherein the composite expression level B corresponds to a value of greater than or equal to -0.25.

[0089] In some embodiments, composite expression level A comprises ≥3.8, ≥3.96, ≥4.2, or ≥4.4. In some embodiments, composite expression level B comprises ≥-0.25, ≥0, ≥0.25, ≥1.25Method of Treating

[0090] The present disclosure provides methods of determining the T-cell signature of a given subject suffering from cancer to determine the best course of action to treat the cancer. In some embodiments, identifying a T cell signature as described herein represents a prognostic and predictive biomarker to guide the clinical care of subjects suffering from cancer. Thus, in some embodiments, the disclosure provides for methods of treating a cancer, wherein the TCS described herein is determined before or shortly after a treatment begins, or before a new treatment begins, to determine whether to continue with the given treatment, or alternatively, to adjust the dosage amount or regimen of the given treatment.

[0091] The term “treating” refers to administering an active agent with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect a cancer, the symptoms of the cancer, or to prevent or delay the onset of the symptoms, complications, biochemical indicia of a cancer, or otherwise arrest or inhibit further development of the cancer in a statistically significant manner.

[0092] In some embodiments, the TCS can be used to determine how likely and / or to what extent a subject will respond to a treatment, e.g., an immunotherapeutic treatment for cancer. The efficacy and / or effectiveness of a treatment can be determined by methods known in the art. In some embodiments, the efficacy is determined by correlation of TCS with longer overall survival (OS) and tumor shrinkage (TS) and / or progression-free survival (PFS).

[0093] In some embodiments, the TCS is used to evaluate efficacy and predict responses to administered immunotherapeutics, e.g., T cell therapeutics. In some embodiments, a subject having a TCS with the disclosed log2tpm value correlates with improved potency or benefit of the administered immunotherapeutic. In some embodiments the TCS of the subject corresponds to genes whose differential, e.g., higher, expression levels are associated with longer overall survival (OS) and tumor shrinkage (TS). In some embodiments, consideration of a gene’s (or set of genes) utility is undertaken based on whether its biological mechanism is related to T cell biology, and whether any particular combination of genes exhibits a relatively stronger association with longer OS and / or TS. In some embodiments, the subset of higher expression genes associated with longer OS and / or TS is associated with the presence of multiple genes having an association with T cells. In some embodiments, theTCS, or some parts thereof, is not T cell-associated. In some embodiments, the TCS is used to evaluate activation states of T cells.

[0094] In some embodiments, the present disclosure provides a method of treating a cancer in a subject, the method comprising: administering to the subject an effective amount of an immunotherapeutic, , e.g., a T cell therapeutic, wherein the subject has been determined to have a T cell signature (TCS) comprising: a. a composite expression level A comprising the expression of three or more genes selected from Thymocyte Expressed, Positive Selection Associated 1 (TESPA1), Cluster of Differentiation 28 (CD28), G Protein-Coupled Receptor 183 (GPR183), and Zeta-Chain- Associated Protein Kinase 70 (ZAP70) as measured by log2tpm corresponding to a composite log2tpm value of greater than or equal to 3.2; or b. a composite expression level B comprising (i) the expression of three or more genes selected from TESPA1, CD28, GPR183 and ZAP70 as measured by log2tpm corresponding to a composite log2tpm value of greater than or equal to 3.2, minus (ii) the expression of Interferon Alpha Inducible Protein 27 (IFI27) gene as measured by log2tpm, wherein the composite expression level B corresponds to a value of greater than or equal to -0.25.

[0095] In some embodiments, the present disclosure provides a method of treating a cancer in a subject, the method comprising: administering to the subject an effective amount of a T cell therapeutic, wherein the subject has been determined to have a T cell signature (TCS) comprising: a. a composite expression level A comprising the expression of three or more genes selected from Thymocyte Expressed, Positive Selection Associated 1 (TESPA1), Cluster of Differentiation 28 (CD28), G Protein-Coupled Receptor 183 (GPR183), and Zeta-Chain- Associated Protein Kinase 70 (ZAP70) as measured by log2tpm corresponding to a composite log2tpm value of greater than or equal to 3.2; or b. a composite expression level B comprising (i) the expression of three or more genes selected from TESPA1, CD28, GPR183 and ZAP70 as measured by log2tpm corresponding to a composite log2tpm value of greater than or equal to 3.2, minus (ii) the expression of Interferon Alpha Inducible Protein 27 (IFI27) gene as measured by log2tpm,wherein the composite expression level B corresponds to a value of greater than or equal to -0.25.

[0096] In some embodiments, the TCS of the above methods corresponds to TESPA1, CD28, and GPR183. In some embodiments, the TCS corresponds to TESPA1, CD28, GPR183 and ZAP70. In some embodiments, the TCS corresponds to TESPA1, CD28, GPR183, ZAP70 and IFI27.

[0097] In some embodiments of the methods of treatment described herein, when the log2tpm value is high of one or more of the subject’s TCS combinations in Tables 1 to 3, then TS, OS and PFS can increase relative to a subject who has a low log2tpm value. In some embodiments, when the log2tpm value is high of one or more of the TCS combinations in Tables 1 to 3 is high, then tumor size decreases relative to a subject who has a low log2tpm value.

[0098] In some embodiments of the methods of treatment described herein, when the log2tpm value is low in the TCS gene in Table 4, then TS, OS and PFS can increase relative to a subject who has a high log2tpm value. In some embodiments, when the log2tpm value is low in TCS gene in Table 4, then tumor size decreases relative to a subject who has a high log2tpm value. Methods of Identifying Suitable Subjects for Treatment

[0099] In some embodiments, the disclosure provides a method of determining whether a cancer treatment is suitable for treating cancer in a subject in need thereof, the method comprising determining if the subject has a T cell signature (TCS), the TCS comprising: a. a composite expression level A comprising the expression of three or more genes selected from Thymocyte Expressed, Positive Selection Associated 1 (TESPA1), Cluster of Differentiation 28 (CD28), G Protein-Coupled Receptor 183 (GPR183), and Zeta-Chain- Associated Protein Kinase 70 (ZAP70) as measured by log2tpm; or b. a composite expression level B comprising (i) the expression of three or more genes selected from TESPA1, CD28, GPR183 and ZAP70 as measured by log2tpm corresponding to a composite log2tpm value of greater than or equal to 3.2, minus (ii) the expression of Interferon Alpha Inducible Protein 27 (IFI27) gene as measured by log2tpm, wherein the subject is suitable for a cancer treatment if :(i) the composite expression level A corresponds to a composite log2tpm value of greater than or equal to 3.2; or (ii) the composite expression level B corresponds to a composite log2tpm value of greater than or equal to -0.25.

[0100] As used herein, a subject is “suitable for treatment” if the subject has a higher likelihood of a successful outcome. In some embodiments, the subject is suitable for treatment if the log2tpm of the TCS of the subject in any of the log2tpm values in Table 1-4 for the listed TCS combination, e.g., ranges 4, 5 or 6 in Tables 1-4. In some embodiments, a successful outcome is defined as any increase in TS, OS, and / or PFS, and / or decrease in tumor size in the subject. Thus, in some embodiments, the use of RNA-seq methodology to determine the log2tpm of genes in a select TCS of a given subject can be tested before the first administration of the cancer treatment, thereby reducing or eliminating the administration of a given cancer treatment to a subject who exhibits a low likelihood of success with the cancer treatment. In some embodiments, the cancer treatment for which suitability is determined is an immunotherapeutic. In some embodiments, the cancer treatment for which suitability is determined is a T cell therapeutic.

[0101] In some embodiments, the disclosure provides both a means for method for identifying and treating a subject suffering from a cancer who is suitable for treatment with an immunotherapeutic, e.g., a T cell therapeutic, comprising: a. determining from a sample obtained from the subject if the subject has a T cell signature (TCS), using RNASeq methodology to quantify gene expression levels, the TCS comprising: (i) a composite expression level A comprising the expression of three or more genes selected from Thymocyte Expressed, Positive Selection Associated 1 (TESPA1), Cluster of Differentiation 28 (CD28), G Protein-Coupled Receptor 183 (GPR183), and Zeta- Chain-Associated Protein Kinase 70 (ZAP70) as measured by log2tpm; or (ii) a composite expression level B comprising a) the expression of three or more genes selected from TESPA1, CD28, GPR183 and ZAP70 as measured by log2tpm corresponding to a composite log2tpm value of greater than or equal to 3.2, minus (b) the expression of Interferon Alpha Inducible Protein 27 (IFI27) gene as measured by log2tpm,b. identifying the subject as suitable for treatment with the immunotherapeutic, e.g., the T cell therapeutic, if (i) the composite expression level A corresponds to a composite log2tpm value of greater than or equal to 3.2, or (ii) the composite expression level B corresponds to a composite log2tpm value of greater than or equal to -0.25; and c. administering an effective amount of the immunotherapeutic, e.g., the T cell therapeutic, to the subject identified as suitable for treatment with the immunotherapeutic, e.g., the T cell therapeutic.

[0102] In some embodiments, the present disclosure provides a reference log2tpm. The term “reference log2tpm” refers to a log2tpm level for gene expression associated with a subject which was previously determined to not likely to respond from treatment with a T cell therapeutic. The reference log2tpm can vary according to the specific TCS, or the type of cancer. In some embodiments, the reference log2tpm can include any of ranges 3, 4 or 5 in Tables 1 – 4, depending on which TCS combination is used. In some embodiments, the reference log2tpm can include any of ranges 3, 4 or 5 as found in Table 5, depending on which cancer is being treated. In some embodiments, the reference log2tpm can include any of ranges 3, 4 or 5 as found in Table 6, depending on which cancer is being treated. In some embodiments, the reference log2tpm is -0.25, 3.2 or 4.2.

[0103] By way of example only, if the log2tpm value of genes in a TCS (e.g., TESPA1, CD28, GPR183) in a biological sample obtained from the subject using RNASeq methodology is determined to be 4.1, and the reference log2tpm is 3.8, then the subject’s TCS log2tpm value is higher than the first reference log2tpm and the subject is identified as being suitable for treatment with a T cell therapeutic, and administered the T cell therapeutic.

[0104] In a separate example, if the log2tpm value of genes in a TCS (e.g., TESPA1, CD28, GPR183) in a biological sample obtained from the subject using RNASeq methodology is determined to be 3.5, and the reference log2tpm is 3.8, then the subject’s TCS log2tpm value is lower than the reference log2tpm. Since the subject’s TCS log2tpm value is lower than the reference log2tpm, then the subject is identified as not being suitable for treatment with a T cell therapeutic. Thus, the T cell therapeutic is not administered to the subject until the subject’s TCS log2tpm value is greater than the reference log2tpm. In some embodiments, the disclosure provides that if the subject’s TCS log2tpm value (eithercomposite expression level A, or composite expression level B) is below a reference log2tpm, then dosage amounts and / or frequency can be adjusted, e.g., the dosage amount can be increased or the frequency increased, such that that the immunotherapeutic will work more effectively in the treatment of the cancer in the subject.

[0105] In some embodiments, the method of identifying and treating a subject suffering from a cancer who is suitable for treatment with a an immunotherapeutic, e.g., a T cell therapeutic, uses a TCS comprising TESPA1, CD28, and GPR183 having a log2tpm value of greater than or equal to 3.2. In some embodiments, the method of identifying and treating a subject suffering from a cancer who is suitable for treatment with an immunotherapeutic, e.g., a T cell therapeutic, uses a TCS comprising TESPA1, CD28, GPR183, and IFI27 having a log2tpm value of greater than or equal to -0.25. In some embodiments, the methods of identifying and treating a subject suffering from a cancer who is suitable for treatment with an immunotherapeutic, e.g., a T cell therapeutic, use a TCS comprising TESPA1, CD28, GPR183, and ZAP70 having a log2tpm value of greater than or equal to 3.2. In some embodiments, the methods of identifying and treating a subject suffering from a cancer who is suitable for treatment with an immunotherapeutic, e.g., a T cell therapeutic, use a TCS comprising TESPA1, CD28, GPR183, ZAP70 and IFI27 having a log2tpm value of greater than or equal to 3-0.25. Optimization of Methods of Treatment

[0106] In some embodiments, the TCS disclosed herein can be determined more than one time, and used to determine dosing amounts and / or dosing regimens when administering an immunotherapeutic, e.g., a T cell therapeutic, to a subject. Thus, in some embodiments, the TCS of the subject can be used to adjust dosage amounts for future regimens of the immunotherapeutic, thereby providing an optimized dosing or treatment regimen.

[0107] In some embodiments, the present disclosure provides a method of increasing the activity of an immune cell, comprising administering to a subject in need thereof an effective amount of an immunotherapeutic, e.g., a T cell therapeutic, and further comprising measuring T cell activation ex vivo in a sample derived from the subject, wherein an increase in T cell activity indicates that the treatment should be continued. In other embodiments, the disclosure comprises a method of increasing the activity of an immune cell, comprising administering to a subject in need thereof an effective amount of an immunotherapeutic, andfurther comprising measuring T cell activation ex vivo in a sample derived from the subject, wherein an increase in T cell activity predicts the likelihood that the treatment will be successful. In some embodiments, an increase in T cell activity increases or enhances the potency of the immunotherapeutic. In some embodiments, an increase in T cell activity by administering the immunotherapeutic results in improvement of cancer symptoms, including OS, PFS and TS.

[0108] In some embodiments, the disclosure provides a method for treating a cancer in a subject, the method comprising: a. determining a log2tpm value of genes in a TCS in a biological sample obtained from the subject, wherein the TCS corresponds to: (i) a composite expression level A comprising the expression of three or more genes selected from Thymocyte Expressed, Positive Selection Associated 1 (TESPA1), Cluster of Differentiation 28 (CD28), G Protein-Coupled Receptor 183 (GPR183), and Zeta-Chain-Associated Protein Kinase 70 (ZAP70) as measured by log2tpm; or (ii) a composite expression level B comprising a) the expression of three or more genes selected from TESPA1, CD28, GPR183 and ZAP70 as measured by log2tpm corresponding to a composite log2tpm value of greater than or equal to 3.2, minus (b) the expression of Interferon Alpha Inducible Protein 27 (IFI27) gene as measured by log2tpm, and b. administering to the subject a dosing regimen comprising a first dosage amount of a T cell therapeutic; wherein if the composite expression level A is greater than or equal to 3.2 or the composite expression level B is greater than or equal to -0.25, then a second dosage amount is administered, wherein the second dosage amount is the same or less than the first dosage amount, or wherein if the composite expression level A is less than 3.2 or the composite expression level B is less than -0.25, then the second dosage amount is greater than the first dosage amount and / or further comprises a second cancer treatment.

[0109] In some embodiments, the first dosing regimen comprises a single dose of a T cell therapeutic. In some embodiments, the first dosing regimen comprises greater than one dose of a T cell therapeutic. In some embodiments, the first dosing regimen comprisesadministering a T cell therapeutic in greater than one doses for a period of about 1 week to about 1 month. In some embodiments, a second biological sample is obtained from the subject about 1 week to about 3 months after the first composite expression level A or composite expression level B is determined. In some embodiments, the second composite expression level A is at least 10% greater than the first composite expression level A. In some embodiments, the second composite expression level A is at least 20% greater than the first composite expression level A. In some embodiments, if the second composite expression level A is greater than the first composite expression level A, then the second dosage amount is at least 10% less than the first dosage amount. In some embodiments, if the second composite expression level A is greater than the first composite expression level A, then the second dosage amount is at least 20% less than the first dosage amount. In some embodiments, if the second composite expression level A is the same or less than the first composite expression level A, then the second dosage amount is at least 10% greater amount than first dosage amount. In some embodiments, if the second composite expression level A is the same or less than the first composite expression level A, then the second dosage amount is at least 20% greater amount than first dosage amount. In some embodiments, the second composite expression level B is at least 10% greater than the first composite expression level B. In some embodiments, the second composite expression level B is at least 20% greater than the first composite expression level B. In some embodiments, if the second composite expression level B is greater than the first composite expression level B, then the second dosage amount is at least 10% less than the first dosage amount. In some embodiments, if the second composite expression level B is greater than the first composite expression level B, then the second dosage amount is at least 20% less than the first dosage amount. In some embodiments, if the second composite expression level B is the same or less than the first composite expression level B, then the second dosage amount is at least 10% greater amount than first dosage amount. In some embodiments, if the second composite expression level B is the same or less than the first composite expression level B, then the second dosage amount is at least 20% greater amount than first dosage amount.

[0110] The treatments described herein can be used as known to the skilled artisan. For example, the first dosing regimen can include any of the dosing regimens described herein for the immumotherapeutics described herein. In some embodiments, the dosing regimen can include weekly, monthly, bi-monthly, etc., administration of a specific dosing amount. According to some embodiments, a first log2tpm value of genes in a TCS is determined,followed by administering a first dosing regimen comprising a first dosage amount of a T cell therapeutic. After the first dosing regimen is complete, then the second log2tpm value of genes in a TCS is determined. The value of the second log2tpm can determine whether the same dosing amount is used in the second dosing regimen, or whether a different dosage amount is used. Thus, the second (or subsequent) log2tpm provides information and guidance as to the dosing amount of the second (or subsequent) amount in future dosing regimens. If the second log2tpm value is greater than the first log2tpm value, then the second dosage amount is the same or less than the first dosage amount. If the second log2tpm value is the same or less than the first log2tpm value, then the second dosage amount can be increased, i.e., is a greater than the first dosage amount. In some embodiments, a subsequent log2tpm value, e.g., a third, fourth, fifth, sixth, etc., log2tpm value can be determined after a third, fourth, fifth, sixth, etc., dosing regimen, respectively, and the respective dosing amounts can be adjusted accordingly.

[0111] In some embodiments, the first dosing regimen can include a single dose of a T cell therapeutic. In some embodiments, the first dosing regimen comprises greater than one dose of a T cell therapeutic, e.g., two, three, four, five, six, or greater than six doses. In some embodiments, the first dosing regimen comprises administering a T cell therapeutic in greater than one doses for a period of about 1 week to about 6 months, about 1 week to about 3 months, or about 1 week to about 1 month.

[0112] In some embodiments, after the first dosing regimen is complete, a second (or subsequent) biological sample is obtained from the subject, and the second log2tpm is determined. The second (or subsequent) biological sample can be obtained 1 day to 6 months, about 1 day to about 3 months, about 1 day to about 1 month, or about 1 day to about 1 week after the first (or previous) dosing regimen is complete. In some embodiments, the second (or subsequent) biological sample can be obtained 1 day to 6 months, about 1 day to about 3 months, about 1 day to about 1 month, or about 1 day to about 1 week after the first (or previous) dosing regimen is complete, about 1 week to about 3 months after the first (or previous) log2tpm value is determined.

[0113] Using the optimization of treatments described herein, the dosage amount is adjusted when the second TCS log2tpm value is at least 10% greater than the first TCS log2tpm value. In some embodiments, the dosage amount is adjusted when the second TCS log2tpm value is at least 20% greater than the first TCS log2tpm value.

[0114] In some embodiments, the amount that the second dosage amount is adjusted relative to the first dosage amount can vary. In some embodiments, when the second log2tpm value is greater than the first log2tpm value, then the second dosage amount is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, or at least 75% less than the first dosage amount. In some embodiments, when the second log2tpm value is greater than the first log2tpm value, then the second dosage amount is the same as the first dosage amount.

[0115] In some embodiments, when the second log2tpm value is less than the first log2tpm value, then the second dosage amount is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, or at least 75% greater than the first dosage amount.

[0116] In some embodiments, the optimization methods herein use a TCS corresponding to TESPA1, CD28, and GPR183. In some embodiments, the the optimization methods use a TCS corresponding TESPA1, CD28, GPR183 and ZAP70. In some embodiments, the optimization methods herein use a TCS corresponding to TESPA1, CD28, GPR183, and IFI27. Administration and Immunotherapeutics

[0117] In some embodiments, the disclosure provides a method of treating a cancer in a subject, the method comprising administering to the subject an effective amount of an immunotherapeutic, , e.g., a T cell therapeutic, based on the log2tpm of the TCS of the subject.

[0118] The term “administration” or “administering” refers to routes of introducing a compound or composition provided herein to a subject to perform its intended function. An example of a route of administration that can be used includes, but is not limited to, e.g., intravenous fusion (e.g., intravenous cannula (IVC) or central venous access device (CVAD)), intramuscular, subcutaneous, oral, sublingual, intranasal, transdermal administration, or any combination thereof. In some embodiments, the term “administration” or “administering” refers intravenous fusion (e.g., intravenous cannula (IVC) or central venous access device (CVAD)). In some embodiments, the term “administration” or “administering” refers oral delivery.

[0119] The term “subject” means any subject, particularly a mammalian subject, in need of treatment. In some embodiments, the term “subject” refers to a human subject. In some embodiments, the term “subject” refers to an adult human subject. In some embodiments, the term “subject” refers to a male human subject. In some embodiments, the term “subject” refers to a female human subject. In some embodiments, the term “subject” refers to administration to a subject in need thereof, i.e., a subject having cancer. As used herein, a “subject in need thereof” can refer to the subject for whom it is desirable to treat, e.g., a subject being diagnosed with cancer as described herein. In some embodiments, the term “subject in need thereof” can refer to a subject having one or more symptoms associated with cancer, e.g., a subject having the following symptoms: coughing that gets worse or doesn't go away, chest pain, shortness of breath, wheezing, coughing up blood, abnormal vaginal discharge or bleeding after menopause, bloating or swollen feeling in the stomach, feeling full very soon after starting to eat, new urinary frequency, new constipation or other changes in bowel movements, discomfort or pain in the pelvic area, abdomen or lower back, fatigue, unexplained weight loss etc. In some embodiments, the term “subject in need thereof” can refer to a subject at high risk for suffering from a gp100-positive and / or a PRAME-positive cancer suitable to treatment with a T cell therapy as described herein, independently of whether the subject has physical manifestations of such condition. In some embodiments, the subject is an adult, i.e., at least 18 years old. In some embodiments, the subject is 12-18 years old. In some embodiments, the subject is less than 12 years old. In some embodiments, the subject has serum LDH of between about 1.1 x to about 2.0 x ULN. In some embodiments, the subject is under about 65 years old. In some embodiments, the subject is at least about 65 years old.

[0120] In some embodiments, the subject can have at least one (e.g., at least any of 2, 3, 4, 5, 6, or 7) of the following characteristics: (1) ≥ 18 years of age, inclusive; (2) Eastern Cooperative Oncology Group (ECOG) status of 0 or 1 at start of treatment. (3) HLA- A*02:01-positive (testing by central laboratory); (4) meet tumor gp100 and / or PRAME testing requirements (testing by central laboratory); (5) possess a recent biopsy (e.g., performed during screening) or an archival tumor biopsy or an adequate tumor biopsy with high tumor PRAME and / or gp100 expression; (6) have an evaluable disease, (at least one target or non-target lesion), and measurable disease (at least one target lesion) according to RECIST v1.1; (7) have a disease that is amenable to biopsy and consent to undergo tumor biopsies during screening and during treatment; (8) documented EGFR exon 19 deletion,exon 21 L858R mutation, or T790M mutation determined by a locally approved diagnostic test and / or (9) have one of the following advanced (metastatic or unresectable) solid tumors: lung cancer, NSCLC, SCLC, melanoma, ovarian carcinoma, includes fallopian tube and primary peritoneal cancers, uterine carcinoma, endometrioid carcinoma (endometrial clear cell, endometrial serous carcinoma, and endometrial carcinosarcoma), triple-negative breast cancer, germ cell tumors (choriocarcinoma, seminoma, and spermatocytic tumors), sarcomas (myxoid liposarcoma, round cell liposarcoma, and synovial sarcoma), adenoid cystic carcinoma, basal cell carcinoma of the skin, thymic carcinoma, peripheral nerve sheath tumor, Merkel cell carcinoma / skin neuroendocrine carcinoma, head and neck squamous cell carcinoma, squamous cervical carcinoma, and urothelial carcinoma. In some embodiments, the subject does not have history or current evidence of brain metastases, including leptomeningeal involvement. In some embodiments, the subject does not have pre-existing peripheral neuropathy of NCI CTCAE Scale of Grade > 2.

[0121] In some embodiments, the subject having cancer has a mutation in BRAF. In some embodiments, the subject has a BRAF V600 mutation. In some embodiments, the subject has a BRAF V600E mutation. In some embodiments, the subject does not comprise a mutation in BRAF (the cancer comprises wild-type BRAF). In some embodiments, the subject does not comprise BRAF mutant such as a BRAF mutant with increased activity (for example, increased kinase activity, and / or increased activity as compared to wild-type BRAF) or a BRAF gain-of-function mutant. In some embodiments, the subject does not comprise a constitutive active BRAF mutant. In some embodiments, the subject does not comprise BRAF V600E mutation (the cancer comprises wild-type BRAF). In some embodiments, the subject comprises wild-type BRAF. In some embodiments, the subject comprises a BRAF mutant such as a BRAF mutant with increased activity (for example, increased kinase activity, and / or increased activity as compared to wild-type BRAF) or a BRAF gain-of-function mutant. In some embodiments, the subject comprises a constitutive active BRAF mutant.

[0122] In some embodiments, the subject has elevated serum lactate dehydrogenase (“LDH”) level. In some embodiments, the subject has serum LDH of less than about 0.8 χ upper limit of normal (“ULN”). In some embodiments, the subject has serum LDH at about 0.8 χ to about 1.1 x ULN. In some embodiments, the subject has serum LDH of between greater than about 1.1 x to about 2.0 x ULN.

[0123] In some embodiments, the subject is treated when the subject has pre-cancerous lesions, or when the subject is first diagnosed. In some embodiments, the methods described herein can be used when the subject has been diagnosed with a life expectancy of greater than 1 year, greater than 6 months, greater than 3 months, or greater than 1 month. In some embodiments, the methods described herein can be used when the subject has been diagnosed with a life expectancy of greater than 3 months. In some embodiments, the methods described herein can be used when the subject has been diagnosed with a life expectancy of less than 5 years, less than 3 years, less than 2 years, less than 1 year, or less than 6 months. The skilled artisan can appreciate that determining “life expectancy” is an estimate that can be determined by skilled artisans using known methods in the art, e.g., age of subject, stage of cancer, thickness and size of tumor, speed of growth, malignancy, contributory factors, etc.

[0124] In some embodiments, the subject is HLA-A*02 positive. The term “HLA-A*02 positive” refers to a subject having T cell receptors (TCRs) that bind to the SLLQHLIGL- HLA-A*02 complex. In some embodiments, the subject is HLA-A*02:01 positive. The term “HLA-A*02:01 positive refers to a subject that expresses the HLA-A*02 genotype with the HLA-A*02:01 allele.

[0125] In some embodiments, the methods described herein are directed to PRAME and / or gp100 positive subjects. In some embodiments, the cancer is metastatic. In some embodiments, the cancer is metastatic malignant. In some embodiments, the cancer to be treated in the subject is stage 0, stage I, stage II, stage III, or stage IV. In some embodiments, the cancer to be treated is stage 0, stage IA, stage IB, stage IIA, stage IIB, stage IIC, stage IIIA, stage IIIB, stage IIIC, or stage IV. In some embodiments, the cancer to be treated is stage III. In some embodiments, the cancer to be treated is stage IV. In some embodiments, the stage of cancer, e.g., stage IV cancer, is histologically confirmed. Histological confirmation refers to the use of visual means, e.g., microscope, to look at a tissue to determine whether it has signs of the cancer, disease or other abnormalities associated with cancer. In some embodiments, the tissue used in the histological examination has been excised by biopsy or surgical section. Staging of cancers can be based on a method known to one skilled in the art. Staging of can be determined according to the criteria in the TNM or AJCC staging system (the contents disclosed therein is incorporated by reference in its entirety) created by the American Joint Committee on Cancer (AJCC) and the InternationalUnion Against Cancer (UICC). The TNM staging system is used to describe most types of cancer.

[0126] In some embodiments, an “effective amount” corresponds to a therapeutically effective amount. A therapeutically effective amount means an amount that is effective in therapy, or an amount sufficient to provide a therapeutic effect. In some embodiments, an amount that is effective in therapy is an amount which produces a biological activity and will depend, among other things, on the individual. In some embodiments, the typical daily dose of the active substance varies and will depend on various factors such as the individual requirements of the subjects, the mode of administration and disease. In some embodiments, the effective amount of an immunotherapeutic, e.g., a T cell therapeutic, can be adjusted based on the log2tpm value of the TCS of the subject, and can be readjusted if the log2tpm value of the TCS of the subject changes over time.

[0127] The terms “immunotherapeutic,” or “immunotherapy” as used herein when referring to cancer treatments refers to a therapy that uses substances to stimulate or suppress the immune system to help the body fight cancer. Immunotherapies can include, but are not limited to antibodies, e.g., monoclonal antibodies, a checkpoint inhibitor, a chimeric antigen receptor (CAR) T-cell therapy, a cytokine, an immunomodulator, a cancer vaccine, an oncolytic virus, a chemotherapeutic agent, a methylating agent, a T cell therapy or any combination thereof. In some embodiments, the immunotherapies can include a monoclonal antibody or T-cell therapy.

[0128] In some embodiments, the term immunotherapeutic can treat the cancer by activating or suppressing the immune system. Immunotherapies designed to elicit or amplify an immune response are classified as activation immunotherapies, while immunotherapies that reduce or suppress are classified as suppression immunotherapies.

[0129] In some embodiments, the immunotherapeutic is a T cell therapeutic. In some embodiments, the T cell therapeutic comprises a checkpoint inhibitor, a chimeric antigen receptor (CAR) T-cell therapy, a cytokine, an immunomodulator, a cancer vaccine, a monoclonal antibody, an oncolytic virus, a chemotherapeutic agent, a methylating agent, a T cell therapy or any combination thereof.

[0130] In some embodiments, the T cell therapeutic comprises at least one T cell receptor (TCR) bispecific protein. In some embodiments, the bispecific protein comprises a bi-specific T cell engager (BiTE), a bispecific antibody (BsAb), or a combination thereof. In specificembodiments, the TCR bispecific protein targets CD3 and PRAME. In specific embodiments, the TCR bispecific protein is IMC-F106C. In specific embodiments, the TCR bispecific protein targets CD3 and gp100. In specific embodiments, the TCR bispecific protein is tebentafusp. In some embodiments, the immunotherapeutic comprises tebentafusp and IMC- F106C. In some embodiments, the TCS log2tpm value is determined prior to administration of the T cell therapeutic.

[0131] In specific embodiments, the TCR bispecific protein is IMC-F106C. In specific embodiments, the TCR bispecific protein targets CD3 and gp100. In specific embodiments, the TCR bispecific protein is tebentafusp. In some embodiments, the immunotherapeutic comprises tebentafusp and IMC-F106C.

[0132] In some embodiments, administering the T cell therapeutic results in the activation of T cells and elevates the TCS profile. As used herein, the term “activation of T cells” refers to a population of monoclonal (for example, encoding the same TCR) or polyclonal (for example, having clones encoding different TCRs) T cells that have T cell receptors recognizing at least one tumor antigen peptide. Activated T cells may include one or more subtypes of T cells, including, but not limited to, one or more selected from the group consisting of cytotoxic T cells, helper T cells, natural killer T cells, γδT cells, regulatory T cells, naïve T cells and memory T cells. T cell activation refers to a process in which T cells can express antigen-specific T cell receptors on their surface to recognize their cognate antigens and respond by entering the cell cycle, secreting cytokines or lytic enzymes, and initiating the cell-based functions of the immune system. Tebentafusp / KIMMTRAK

[0133] In some embodiments, the present disclosure relates to the treatment of gp100 positive melanoma by using the log2tpm value of the TCS of a subject. Glycoprotein 100 (gp100) is a transmembrane glycoprotein, highly expressed in normal melanocytes and melanoma cells. Gp100 is a 100 kDa glycosylated transmembrane protein specific to the melanocyte cells of the skin, mucosa, and retina and involved in melanosome maturation; which are the organelles that transport melanin. The RNA transcripts of gp100 are highly expressed in melanoma tissue at all stages, even in amelanotic lesions, whereas, in normal melanocytes, they are weakly detected. Uveal and cutaneous melanoma differ in their gp100 tissue expression (80% of UM lesions express gp-100 compared to 63% in CM). Severalapproaches have been developed to exploit its immunogenic potential, such as vaccines, adoptive cell therapy, and a new class of molecules, named ImmTACs. Gp100 has recently been effectively targeted by tebentafusp, a first-in-class bispecific protein of the immune- mobilizing monoclonal T cell receptors against cancer (ImmTACs) family. ImmTACs consist of a modified T cell receptor (TCR) that detects a particular peptide bound to a specific human leukocyte antigen (denominated p-HLA complex) on the surface of cells. The TCR is combined with a single-chain antibody against CD3, which is a known co-receptor involved in activating T cells. Therefore, ImmTACs detect tumor cells that present a specific p-HLA, recruiting T cells to lyse the targeted cells. Tebentafusp is an ImmTAC directed to a gp100 peptide united to HLA-A*0201 that prolongs overall survival in subjects. Tebentafusp targets tumor cells that express a peptide of gp100 presented by HLA*A0201, creating an immune synapse that kills targeted tumor cells. The specific p-HLA complex detected by tebentafusp is YLEPGPVTA-HLA-A*0201, which is an immunogenic peptide derived from gp100. (Martinez-Perez et al. “Gp-100 as a Novel Therapeutic Target in Uveal Melanoma,” Cancers (Basel) 13(23):5968 (2021)).

[0134] Tebentafusp is composed of an alpha chain and a beta chain. The alpha chain comprises a TCR alpha chain variable region domain and a constant region domain. The beta chain comprises a TCR beta chain variable region domain and a constant region domain where the TCR beta chain is linked to a single-chain variable fragment (“scFv”) anti-CD3 antibody at its N-terminus. The two chains are covalently bonded via a disulfide bond between the cysteine at position 157 on the alpha chain (al 57) amino acid sequence of SEQ ID NO: 1 and the cysteine in position 427 on the anti-CD3 scFv beta chain (P427) of SEQ ID NO:2 (indicated with asterisks, *, in the amino acid sequences below).

[0135] The amino acid sequence of tebentafusp is provided below and tebentafusp is further described in International Patent Publication No. WO 2011 / 001152 and U.S. Patent Nos.8,519,100 and 9,068,178, each of which is hereby incorporated by reference in its entirety. The alpha chain is composed of 195 amino acid residues and the beta chain is composed of 500 amino acid residues. The linker sequence between the two variable domains of the scFv antibody (24 amino acids) and the linker sequence between the scFv and TCR P chain variable region (5 amino acids) are underlined. The initiator methionine residues are post- translationally removed from both alpha and beta chains.

[0136] Amino acid sequence of tebentafusp-tebn:

[0137] Alpha chain: AQQGEEDPQALSIQEGENATMNCSYKTSINNLQWYRQNSGRGLVHLILIRSNEREKH SGRLRVTLDTSKKSSSLLITASRAADTASYFCATDGSTPMQFGKGTRLSVIANIQKPD PAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKC*VLDMRSMDFKSNSA VAWSNKSDFACANAFNNSIIPEDT (SEQ ID NO: 1)

[0138] Wild type gp100-specific TCR alpha chain with K113 substituted for N113: SQQGEEDPQALSIQEGENATMNCSYKTSINNLQWYRQNSGRGLVHLILIRSNEREKH SGRLRVTLDTSKKSSSLLITASRAADTASYFCATDGSTPMQFGKGTRLSVIANIQKPD PAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKTVLDMRSMDFKSNSA VAWSNKSDFACANAFNNSIIPEDTFFPSPESS (SEQ ID NO: 2)

[0139] Wild-type gp100-specific TCR beta chain DGGITQSPKYLFRKEGQNVTLSCEQNLNHDAMYWYRQDPGQGLRLIYYSWAQGDF QKGDIAEGYSVSREKKESFPLTVTSAQKNPTAFYLCASSWGAPYEQYFGPGTRLTVT EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGV STDPQPLKEQPALNDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQD RAKPVTQIVSAEAWGRAD (SEQ ID NO: 3)

[0140] Anti-CD3 scFv Beta chain: AIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLESGV PSRFSGSGSGTDYTLTISSLOPEDFATYYCOQGNTLPWTFGOGTKVEIKGGGGSGGG GSGGGGSGGGGSGGGSEVOLVESGGGLVQPGGSLRLSCAASGYSFTGYTMNWVRO APGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQMNSLRAEDTAVYY CARSGYYGDSDWYFDVWGQGTLVTVSSGGGGSDGGITQSPKYLFRKEGQNVTLSC EQNLNHDAMYWYRQDPGQGLRLIYYSWAQGDFQKGDIAEGYSVSREKKESFPLTV TSAQKNPTAFYLCASSWGAPYEQYFGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHT QKATLVCLATGFYPDHVELSWWVNGKEVHSGVC*TDPQPLKEQPALNDSRYALSSR LRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQIVSAEAWGRAD (SEQ ID NO: 4)

[0141] Beta chain: DGGITQSPKYLFRKEGQNVTLSCEQNLNHDAMYWYRQDPGQGLRLIYYSWAQGDF QKGDIAEGYSVSREKKESFPLTVTSAQKNPTAFYLCASSWGAPYEQYFGPGTRLTVT EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVC*TDPQPLKEQPALNDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQ DRAKPVTQIVSAEAWGRAD (SEQ ID NO: 5)

[0142] CDR sequences of tebentafusp is as follows: Alpha Alpha Alpha chain Beta chain Beta chain Beta chain CDR3 chain chain CDR3 CDR1 CDR2 (SEQ ID NO: 11) CDR1 CDR2 (SEQ ID NO: 8) (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 9) NO: 10) NO: 6) NO: 7) TSINN IRS ATDGSTPMQ LNHDA SWAQGD ASSWGAPYEQY

[0143] Framework region sequences of tebentafusp: Alpha chain Alpha Alpha chain FR3 Alpha chain FR4 (SEQ ID NO: 15) FR1 chain (SEQ ID NO: 14) (SEQ ID FR2 NO: 12) (SEQ ID NO: 13) QQGEEDP LQWY NEREKHSGRL GKGTRLSVIANIQKPDPAVYQLRDSKSS QALSIQE RQNS RVTLDTSKKSS DKSVCLFTDFDSQTNVSQSKDSDVYITD GENATM GRGL SLLITASRAAD KC*VLDMRSMDFKSNSAVAWSNKSDFA NCSYK VHLIL TASYFC CANAFNNSIIPEDT Beta Beta Beta chain Beta chain FR4 (SEQ ID NO: 19) chain chain FR3 (SEQ ID FR1 FR2 NO: 18) (SEQ ID (SEQ NO: 16) ID NO: 17) DGGITQ MYW FQKGDIAE FGPGTRLTVTEDLKNVFPPEVAVFEPSEAEISHT SPKYLF YRQ GYSVSREK QKATLVCLATGFYPDHVELSWWVNGKEVHSG RKEGQ DPGQ KESFPLTV VC*TDPQPLKEQPALNDSRYALSSRLRVSATFW NVTLSC GLRL TSAQKNPT QDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQ EQN IYY AFYLC IVSAEAWGRAD

[0144] Additional linker sequences referred to herein: GGGSG (SEQ ID NO: 120), GGSGG (SEQ ID NO: 121), GSGGG (SEQ ID NO: 122), GSGGGP (SEQ ID NO: 123), GGEPS (SEQ ID NO: 124), GGEGGGP (SEQ ID NO: 125), and GGEGGGSEGGGS (SEQ ID NO: 126).

[0145] In some embodiments, the present disclosure provides for methods of treating a cancer by determining log2tpm of the TCS of a subject, and then administering a T cell therapeutic, wherein the T cell therapeutic comprises an isolated or non-naturally occurring or engineered T cell receptor (TCR) having the property of binding to YLEPGPVTA (SEQ ID NO: 20) HLA-A2 complex and comprising a TCR alpha variable domain and / or a TCR beta variable domain.

[0146] In some embodiments, the disclosure provides a TCR alpha chain amino acid sequence of SEQ ID NO:1 or a TCR alpha chain amino acid sequence that has at least 90%, at least 95% or 100% identity to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the disclosure provides a TCR beta chain amino acid sequence of SEQ ID NO: 5 or a TCR beta chain amino acid sequence that has at least 90%, at least 95% or 100% identity to the amino acid sequence of SEQ ID NO: 5. In some embodiments, the TCR alpha chain variable domain comprises CDRs 1, 2 and 3 having the amino acid sequences of SEQ ID NOs: 6, 7 and 8 respectively and the TCR beta chain variable domain comprises CDRs 1 , 2 and 3 having the amino acid sequences of SEQ ID NOs: 9, 10, and 11 respectively.

[0147] “Isolated” or “Non-naturally occurring” or “Engineered” TCRs refers to TCRs encoded by a sequence that is at least substantially free from at least one other component that the sequence is naturally associated and found in nature. Isolated or non-naturally occurring or engineered means that the sequence is at least substantially free from at least one other component that the sequence is naturally associated and found in nature, e.g., genomic sequences or has been modified in any manner from its natural state. e.g., genomic sequences have been modified in any manner from its natural state, including by isolation. Non- naturally occurring has the meaning ascribed to it in Diamond v. Chakrabarty, 447 U.S.303, 206 U.S.P.Q. (BNA) 193 (1980) and MPEP 2105, i.e. a product of human ingenuity.

[0148] In some embodiments, the methods described herein provide to treatment of treating gp100 positive cancer in a subject comprising administering a T cell therapeutic or immunotherapeutic to said subject when suitable as determined by the log2tpm profile of the TCS of the subject, wherein each dose is administered every 5-10 days, at least the first and second doses are below 40 μg and the second dose is higher than the first dose.

[0149] In some embodiments, tebentafusp is administered when suitable as determined by the log2tpm profile of the TCS of the subject in an intra-subject dose escalation regimenas described in U.S. Pat. No.11,827,688, which is herein incorporated by reference in its entirety.

[0150] In some embodiments, tebentafusp is administered in multiple treatment cycles without any off treatment weeks between treatment cycles.

[0151] All combinations of the embodiments pertaining to the T cell bispecific therapeutic for use and method for treating gp100 positive cancer are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. Heterodimeric TCR-anti-CD3 antibody fusion molecules are described in, e.g., US 8,519,100, US 11,827,688 and WO 2020 / 157211, and are incorporated by reference in their entirety.

[0152] Treatment of cancer with administration of tebentafusp relates to the treatment of gp100 positive cancers. One such cancer is melanoma. The melanoma may be cutaneous melanoma (CM) or uveal melanoma (also known as ocular melanoma). Other non-melanoma gp100 positive cancers include clear cell sarcoma, and neurologic cancers such as gliomas. IMC-F106C

[0153] In some embodiments, the present disclosure relates to the treatment of cancer by administering an immunotherapeutic that targets PRAME. PRAME refers to a PReferentially expressed Antigen in MElanoma (i.e., PRAME) and was first identified as an antigen that is over expressed in melanoma (Ikeda et al. Immunity.1997 Feb;6(2):199-208); it is also known as CT130, MAPE, OIP-4 and has Uniprot accession number P78395. The protein functions as a repressor of retinoic acid receptor signaling (Epping et al., “The human tumor antigen PRAME is a dominant repressor of retinoic acid receptor signaling,” Cell 122(6): 835-47 (2005)). PRAME belongs to the family of germline-encoded antigens known as cancer testis antigens. Cancer testis antigens are attractive targets for immunotherapeutic intervention since they typically have limited or no expression in normal adult tissues. PRAME is expressed in a number of solid tumors as well as in leukemias and lymphomas. PRAME targeting therapies of the disclosure may be particularly suitable for treatment of cancers including, but not limited to, melanoma, lung cancer, breast cancer, ovarian cancer, endometrial cancer, esophageal cancer, bladder cancer, head and neck cancer, uterine cancer, Acute myeloid leukemia, chronic myeloid leukemia, and Hodgkin’s lymphoma.

[0154] The peptide SLLQHLIGL (SEQ ID NO: 101) corresponds to amino acids 425-433 of the full length PRAME protein and is presented on the cell surface in complex with HLA- A*02 (Kessler et al., “Efficient identification of novel HLA-A(*)0201-presented cytotoxic T lymphocyte epitopes in the widely expressed tumor antigen PRAME by proteasome-mediated digestion analysis,” J Exp Med.193(1):73-88 (2001)). This peptide-HLA complex provides a useful target for TCR-based immunotherapeutic intervention.

[0155] WO 2018 / 234319, and corresponding US Pat. Nos.11,427,624 and 11,718,657, describes TCRs that bind to the SLLQHLIGL-HLA-A*02 complex, each of which is incorporated by reference herein in their entirety. The TCRs are mutated relative to a native PRAME TCR alpha and / or beta variable domains to have improved binding affinities for, and / or binding half-lives, for the complex, and can be associated (covalently or otherwise) with a therapeutic agent. One such therapeutic agent is an anti-CD3 antibody, or a functional fragment or variant of said anti-CD3 antibody such as a single chain variable fragment (scFv). The anti-CD3 antibody or fragment may be covalently linked to the C- or N- terminus of the alpha or beta chain of the TCR.

[0156] Treatment with IMCF106C relates to treatment of a PRAME positive cancer. A PRAME positive cancer is a cancer associated with PRAME expression. In some embodiments, the cancer is known to be associated with expression of PRAME. For example, in some embodiments, the prevalence of PRAME expression is known to be elevated in a cancer and thus PRAME expression is not assessed or is assessed retrospectively. Alternatively, PRAME expression can be assessed using any method known in the art, including, for example, histological methods or other quantitative or qualitative measurements, including PCR, RNA expression analysis, and / or kits or sequence panels designed to measure the expression level of PRAME. However, the disclosure is not intended to be limited to the treatment of cancers for which PRAME expression can be detected by histological methods. In particular, the disclosure is not intended to be limited to the treatment of subject in whom PRAME expression can be detected, for example by histological methods. Rather, the disclosure is useful for the treatment of cancers and tumor types which are considered to be PRAME positive. In some embodiments, a PRAME positive subject can have a cutaneous melanoma. In some embodiments, a PRAME positive subject does not have cutaneous melanoma, but has another typey of PRAME positivecancer. In some embodiments, the PRAME positive cancer, e.g., cutaneous melanoma, can have relapsed from, be refractory to, or be intolerant of standard treatment regimens.

[0157] PRAME expression, when detected by histological methods like immunohistochemistry (IHC), can be quantified using an H-score. Expression of PRAME in subject cells or their sub-cellular compartments within a tumor is first detected and classified as either positive or negative. The positive cells can be further classified into high, medium, or low based on the IHC signal intensity. The H-score captures both the intensity and the proportion of the biomarker of interest from the IHC image and comprises values between 0 and 300, thereby offering a dynamic range to quantify abundance or a particular marker or gene.

[0158] Various cancers and tumour types are considered to be PRAME positive. PRAME positive cancers include, but are not limited to, melanoma, lung cancer, breast cancer, ovarian cancer, endometrial cancer, oesophageal cancer, bladder cancer, head and neck cancer, uterine cancer, Acute myeloid leukemia, chronic myeloid leukemia, and Hodgkin’s lymphoma. For example, the PRAME positive cancer may be melanoma. The melanoma may be uveal melanoma or cutaneous melanoma. The lung cancer may be non-small cell lung carcinoma (NSCLC) or small cell lung cancer (SCLC). The breast cancer may be triple- negative breast cancer (TNBC) The bladder cancer may be urothelial carcinoma. The oesophageal cancer may be gastroesophageal junction (GEJ) adenocarcinoma. The ovarian cancer may be epithelial ovarian cancer, such as high grade serous ovarian cancer. The cancer may have relapsed from, be refractory to, or be intolerant of standard treatment regimens.

[0159] In some embodiments, the methods described herein can comprise administration of T cell therapeutic when suitable as determined by the log2tpm profile of the TCS of the subject, wherein the T cell therapeutic is a heterodimeric TCR-anti-CD3 antibody fusion molecules. The heterodimeric TCR-anti-CD3 antibody fusion molecules can comprise two domains: – a heterodimeric TCR; and – an anti-CD3 antibody.

[0160] Heterodimeric TCR-anti-CD3 antibody fusion molecules are described in, e.g., WO 2023 / 099622, incorporated by reference in its entirety.

[0161] Heterodimeric TCR can comprise a TCR comprising an alpha chain (i.e., TCR alpha chain) and a beta chain (i.e., TCR beta chain). Each chain can comprise variable, joining and constant regions, and the beta chain also usually contains a short diversity region between the variable and joining regions, but this diversity region can be considered as part of the joining region. Each variable region can comprise three CDRs (Complementarity Determining Regions) embedded in a framework sequence, one being the hypervariable region named CDR3. There are several types of alpha chain variable (Vα) regions and several types of beta chain variable (Vβ) regions distinguished by their framework, CDR1 and CDR2 sequences, and by a partly defined CDR3 sequence. The Vα types are referred to in IMGT nomenclature by a unique TRAV number, Vβ types are referred to by a unique TRBV number.

[0162] In some embodiments, the disclosure provides a TCR alpha chain amino acid sequence of SEQ ID NO: 114 or a TCR alpha chain amino acid sequence that has at least 90%, at least 95% or 100% identity to the amino acid sequence of SEQ ID NO: 114. In some embodiments, the disclosure provides a TCR beta chain-anti-CD3 amino acid sequence of SEQ ID NO: 116 or a TCR beta chain-anti-CD3 amino acid sequence that has at least 90%, at least 95% or 100% identity to the amino acid sequence of SEQ ID NO: 116. In some embodiments, the TCR alpha chain variable domain comprises CDRs 1, 2 and 3 having the amino acid sequences of SEQ ID NOs: 103, 104 and 105 respectively and the TCR beta chain variable domain comprises CDRs 1 , 2 and 3 having the amino acid sequences of SEQ ID NOs: 109, 110 and 111 respectively.

[0163] In some embodiments, the TCR can bind the sequence SLLQHLIGL (SEQ ID NO: 101). The peptide of SEQ ID NO: 101 corresponds to amino acids 425-433 of the full length PRAME protein and is presented on the cell surface in complex with HLA-A*02 (Kessler et al., “Efficient identification of novel HLA-A(*)0201-presented cytotoxic T lymphocyte epitopes in the widely expressed tumor antigen PRAME by proteasome-mediated digestion analysis,” J Exp Med 193(1):73-88 (2001)). This peptide-HLA complex provides a useful target for TCR-based immunotherapeutic intervention. WO 2018 / 234319 describes TCRs that bind to the SLLQHLIGL-HLA-A*02 complex and is incorporated herein in its entirety.

[0164] In some embodiments, the heterodimeric TCR is fused with an antibody to CD3 to have improved binding affinities for, and / or binding half-lives. One such antibody is an anti-CD3 antibody, or a functional fragment or variant of said anti-CD3 antibody such as a single chain variable fragment (scFv). The anti-CD3 antibody or fragment may be covalently linked to the C- or N- terminus of the alpha or beta chain of the TCR. The resulting molecule is a TCR bispecific.

[0165] TCR bispecific proteins redirect polyclonal T cells to target peptides derived from intra- or extra-cellular disease associated antigens and presented on the cell surface in complex with an HLA molecule. This approach has been tested clinically in the context of a different antigen with a TCR bispecific protein targeting an HLA-A*02 restricted peptide from gp100 and CD3 (tebentafusp). Administration of this molecule provided an OS benefit in uveal melanoma (Nathan et al., 2021).

[0166] The term “antibody” includes, but is not limited to, genetically engineered or otherwise modified forms of immunoglobulins, such as intrabodies, chimeric antibodies, fully human antibodies, humanized antibodies (e.g. generated by “CDR-grafting”), antibody fragments, and heteroconjugate antibodies (e.g., bispecific antibodies, diabodies, triabodies, tetra-bodies, etc.). The term “antibody” includes cys-diabodies and minibodies. Thus, each and every embodiment provided herein in regard to “antibodies”, or “antibody like constructs” is also envisioned as, bi-specific antibodies, diabodies, scFv fragments, chimeric antibody receptor (CAR) constructs, diabody and / or minibody embodiments, unless explicitly denoted otherwise. The term “antibody” includes a polypeptide of the immunoglobulin family or a polypeptide comprising fragments of an immunoglobulin that is capable of non- covalently, reversibly, and in a specific manner binding a corresponding antigen, as disclosed herein. An exemplary antibody structural unit comprises a tetramer. In some embodiments, a full-length antibody can be composed of two identical pairs of polypeptide chains, each pair having one “light” and one “heavy” chain (connected through a disulfide bond). The term “antibody” also comprises immunoglobulins (Ig's) of different classes (i.e., IgA, IgG, IgM, IgD and IgE) and subclasses (such as IgG1, IgG2 etc.).

[0167] The terms "anti-CD3 antibody" and "anti-CD3 antibody fragment," as used herein, mean antibodies or antibody fragments which recognize or bind to CD3.The TCR-anti-CD3 fusion molecule for use in the present disclosure can include one or more conservative substitutions which have a similar amino acid sequence and / or which retain the same function (i.e. are phenotypically silent as defined above). The skilled person is aware that various amino acids have similar properties and thus substitutions between them are “conservative”.One or more such amino acids of a protein, polypeptide or peptide can often be substituted by one or more other such amino acids without eliminating a desired activity of that protein, polypeptide, or peptide.

[0168] In some embodiments, the TCR-anti-CD3 fusion molecule for use in the disclosure comprises an anti-CD3 scFv covalently linked to the N-terminus of the beta chain of a TCR via a linker. This type of molecule is known as an ImmTAC® (Immune Mobilizing Monoclonal TCRs Against Cancer). ImmTAC® molecules are engineered to activate a potent T cell response to specifically kill target cancer cells. In some embodiments, the heterodimeric TCR-anti-CD3 antibody fusion molecules for use in the disclosure are ImmTAC molecules (i.e., ImmTACs targeting PRAME) as described in WO 2018 / 234319, which is incorporated by reference herein in its entirety.

[0169] In some embodiments, the heterodimeric TCR-anti-CD3 antibody fusion molecules is IMC-F106C. IMC-F106C is a T cell redirecting bispecific therapeutic agent comprising a soluble affinity enhanced TCR that binds to the SLLQHLIGL peptide-HLA- A*02 complex, fused to an anti-CD3 scFv. The targeting end of IMC-F106C (the soluble TCR) binds to a peptide fragment of the PRAME antigen presented by HLA-A*02 on the surface of cancer cells. HLA molecules are polymorphic; approximately 47% of Caucasian individuals in the US and European countries express the HLA-A*02 genotype with the HLA-A*02:01 allele detected in more than 95% of HLA-A*02-positive individuals. The effector end of IMC-F106C (anti-CD3 scFv) can bind to CD3 on any T cell, redirecting the T cell to produce effector cytokines and / or kill the cell presenting the target. In addition, IMC- F106C-mediated tumor lysis may prime an endogenous anti-tumor immune response. ImmTAC® (Immune Mobilizing Monoclonal TCRs Against Cancer) molecules such as IMC-F106C are highly potent molecules, with redirection of T-cell activity observed against tumor cell lines presenting as few as 10 to 50 target peptide:HLA complexes. IMC-F106C has been shown to selectively redirect T cell activity in the presence of HLA-A*02:01- positive / PRAME-positive cell lines, leading to T cell activation and killing of PRAME- positive cancer cells, at concentrations as low as 1 pM to 10 pM. As described above, the HLA-A*02 restricted peptide SLLQHLIGL (SEQ ID NO: 101) is derived from the germline cancer antigen PRAME. IMC-F106C has a TCR alpha chain amino acid sequence of SEQ ID NO: 114 and a TCR beta chain-anti-CD3 amino acid sequence of SEQ ID NO: 116.

[0170] PRAME is a cancer-testis antigen that is frequently highly expressed in a range of solid and hematologic malignancies including melanoma, ovarian carcinoma, uterine carcinoma, small-cell and non-small cell lung cancer, triple-negative breast cancer, and several rare tumor types.

[0171] The sequences referred to herein are as follows:

[0172] SEQ ID NO: 101 HLA-A*02 restricted peptide: SLLQHLIGL

[0173] SEQ ID NO: 102 Amino acid sequence of the TCR alpha chain variable domain of the ImmTAC designated as IMC-F106C. CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 103, 104 and 105 respectively, framework regions (FR1 , FR2, FR3 and FR4) are in italics and are designated SEQ ID NO: 127, 106, 107 and 128 respectively. Mutations with respect to native alpha chain are in bold. GDAKTTQPNSMESNEEEPVHLPCNHSTISGTDYIHWYRQLPSQGPEYVIHGLTSNVNNR MASLAIAEDRKSSTLILHRATLRDAAVYYCILILGHSRLGNYIATFGKGTKLSVIP

[0174] SEQ ID NO: 108 Amino acid sequence of the TCR beta chain variable domain of the ImmTAC designated as IMC-F106C. CDRs (CDR1 , CDR2 and CDR3) are underlined and are designated SEQ ID NO: 109, 110 and 111 respectively, framework regions (FR1, FR2, FR3 and FR4) are in italics and are designated SEQ ID NO: 129, 112, 113 and 130 respectively. Mutations with respect to native beta chain are in bold. DGGITQSPKYLFRKEGQNVTLSCEQNLNHDAMYWYRQDPGQGLRUYYSQIMGDEQKG DIAEGYSVSREKKESFPLTVTSAQKNPTAFYLCASSWWTGGASPIRFGPGTRLTVT

[0175] SEQ ID NO: 114 Amino acid sequence of the TCR alpha chain of the ImmTAC designated as IMC-F106C. CDRs (CDR1, CDR2 and CDR3) are underlined and are designated SEQ ID NO: 103, 104 and 105 respectively, framework regions (FR1 , FR2, FR3 and FR4) are in italics and are designated SEQ ID NO: 127, 106, 107 and 128 respectively. The constant region is shown in bold and is designated SEQ ID NO: 115. Within the constant region, the nonnative cysteine residue is double underlined (at position 48 of constant region). GDAKTTQPNSMESNEEEPVHLPCNHSTISGTDYIHWYRQLPSQGPEYVIHGLTSNVNNR MASLAIAEDRKSSTLILHRATLRDAAVYYCILILGHSRLGNYIATFGKGTKLSVIPNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNVSQSKDSDVYITDKCVLDMRSMDFKSN SAVAWSNKSDFACANAFNNSIIPEDT

[0176] SEQ ID NO: 116 Amino acid sequence of the TCR beta chain-anti-CD3 of the ImmTAC designated as IMC-F106C. Anti-CD3 scFv (amino acids 1-253) is shown in bold and underline and is designated SEQ ID NO: 117. The linker (GGGGS) appears immediately after the scFv, is shown in paler text and is designated SEQ ID NO: 118. CDRs (CDR1, CDR2 and CDR3) are underlined and are designated SEQ ID NO: 109, 110 and 111 respectively, framework regions (FR1, FR2, FR3 and FR4) are in italics and are designated SEQ ID NO: 129, 112, 113 and 130 respectively. Constant region is shown in bold (no underline) and is designated SEQ ID NO: 119. Within the constant region, the nonnative cysteine residue is double underlined (at position 57 of constant region). Additional non- native amino acids at position 75 and position 89 of the constant region are also double underlined. AIQMTQSPSSLSASVGDRVTITCRASQDIRNYLNWYQQKPGKAPKLLIYYTSRLE SGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQGNTLPWTFGQGTKVEIKGG GGSGGGGSGGGGSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGYSFTG YTMNWVRQAPGKGLEWVALINPYKGVSTYNQKFKDRFTISVDKSKNTAYLQM NSLRAEDTAVYYCARSGYYGDSDWYFDVWGQGTLVTVSSGGGGSDGGITQSPKY LFRKEGQNVTLSCEQNLNHDAMYWYRQDPGQGLRLIYYSQIMGDEQKGDIAEGYSVSRE KKESFPLTVTSAQKNPTAFYLCASSWWTGGASPIRFGPGTRLTVTEDLKNVFPPEVAVF EPSEAEISHTQKATLVCLATGFYPDHVELSWWVNGKEVHSGVCTDPQPLKEQP ALNDSRYALSSRLRVSATFWQDPRNHFRCQVQFYGLSENDEWTQDRAKPVTQI VSAEAWGRAD

[0177] CDR sequences of the ImmTAC designated as IMC-F106C as follows: Alpha Alpha chain Alpha chain Beta chain Beta chain Beta chain chain CDR2 CDR3 CDR1 CDR2 CDR3 CDR1 TISGTDY GLTSN CILILGHSR LNHDA SQIMGDE CASSWWT (SEQ ID (SEQ ID LGNYIATF (SEQ ID (SEQ ID GGASPIRF NO:103) NO: 104) (SEQ ID NO: 109) NO: 110) (SEQ ID NO: 105) NO: 111)

[0178] Framework region sequences of the ImmTAC designated as IMC-F106C: Alpha chain FR1 Alpha chain Alpha chain FR3 Alpha chain FR4 FR2 GDAKTTQPNSMES IHWYRQLP VNNRMASLAIA GKGTKLSVIP NEEEPVHLPCNHS SQGPEYVIH EDRKSSTLILHR (SEQ ID NO: 128) (SEQ ID NO:127) (SEQ ID NO: ATLRDAAVYY 106) (SEQ ID NO: 107) Beta chain FR1 Beta chain FR2 Beta chain FR3 Beta chain FR4 DGGITQSPKYLFRK MYWYRQDP QKGDIAEGYSV GPGTRLTVT EGQNVTLSCEQN GQGLRLIYY SREKKESFPLTV (SEQ ID NO: 130) (SEQ ID NO:129) (SEQ ID NO: TSAQKNPTAFYL 112) (SEQ ID NO: 113)

[0179] Additional linker sequences referred to herein: GGGSG (SEQ ID NO: 120), GGSGG (SEQ ID NO: 121), GSGGG (SEQ ID NO: 122), GSGGGP (SEQ ID NO: 123), GGEPS (SEQ ID NO: 124), GGEGGGP (SEQ ID NO: 125), and GGEGGGSEGGGS (SEQ ID NO: 126)

[0180] In some embodiments, the TCR-anti-CD3 fusion molecule for use in the methods herein comprises: a TCR alpha chain amino acid sequence of SEQ ID NO: 114 or a TOR alpha chain amino acid sequence that has at least 90%, at least 95% or 100% identity to the amino acid sequence of SEQ ID NO: 114, and a TCR beta chain-anti-CD3 amino acid sequence of SEQ ID NO: 116 or a TOR beta chain-anti-CD3 amino acid sequence that has at least 90%, at least 95% or 100% identity to the amino acid sequence of SEQ ID NO: 116, wherein the TOR alpha chain variable domain comprises CDRs 1, 2 and 3 having the amino acid sequences of SEQ ID NOs: 103, 104 and 105 respectively and the TOR beta chain variable domain comprises CDRs 1, 2 and 3 having the amino acid sequences of SEQ ID NOs: 109, 110 and 111, respectively.

[0181] In other words, although the molecule may have some variation in the TCR alpha chain amino acid sequence compared to the sequence of SEQ ID NO: 114 (as long as the TCR alpha chain amino acid sequence has at least 90% identity to SEQ ID NO: 114) and / or some variation in the TCR beta chain-anti-CD3 amino acid sequence compared to thesequence of SEQ ID NO: 116 (as long as the TCR beta chain-anti-CD3 amino acid sequence has at least 90% identity to the amino acid sequence of SEQ ID NO: 116), the CDRs of the TCR alpha chain must have the amino acid sequences of SEQ ID NOs: 103, 104 and 105 respectively and the CDRs of TCR beta chain must have the amino acid sequences of SEQ ID NOs: 109, 110 and 111 respectively. The requirement for the TCR alpha chain variable domain to comprise CDRs 1, 2 and 3 having the amino acid sequences of SEQ ID NOs: 103, 104 and 105 respectively and the requirement for the TCR beta chain variable domain to comprise CDRs 1, 2 and 3 having the amino acid sequences of SEQ ID NOs: 109, 110 and 111 respectively thus applies to all aspects and embodiments of the disclosure described herein. The TCR alpha chain variable domain thus comprises CDRs 1, 2 and 3 having 100% identity to the amino acid sequences of SEQ ID NOs: 103, 104 and 105 respectively and the TCR beta chain variable domain comprises CDRs 1, 2 and 3 having 100% identity to the amino acid sequences of SEQ ID NOs: 109, 110 and 111 respectively.

[0182] The amino acid sequence of IMC-F106C is further described in International Patent Publication No. WO 2023 / 099622 and U.S. Patent No.11,427,624, each of which is hereby incorporated by reference in its entirety.

[0183] The dosing regimen of IMC-F106C is further described in International Patent Publication No. WO 2023 / 099622 and US Application No.63 / 516,914, each of which is hereby incorporated by reference in its entirety. Combination therapies

[0184] In some embodiments, the methods described herein are suitable for use with a first line treatment. The term “first line treatment” refers to the initial, or first treatment recommended for a disease or illness. In some embodiments, this can also be referred to as primary treatment, initial treatment, or induction therapy. In some embodiments, the first line treatment can include a combination treatment, e.g., treatment with another method, e.g., another active agent and / or surgery.

[0185] In some embodiments, the methods described herein are suitable for use with a second line treatment. In some embodiments, the methods described herein are third line, fourth line, fifth line, sixth line, etc. treatment. Second line or further lines of therapy (third line, fourth line, seventh line, etc.) may be used for a few different reasons, e.g., the first-line treatment doesn't work, the first-line treatment worked but has since stopped working, thefirst-line treatment has side effects that are not tolerated, and / or new treatment becomes available that appears to be more effective than the present treatment

[0186] In some embodiments, the methods described herein are a first line mono-therapy treatment when suitable as determined by the log2tpm profile of the TCS of the subject, e.g., the only active agent used for the treatment of the cancer is a heterodimeric TCR-anti-CD3 antibody fusion molecule..

[0187] In some embodiments, the methods described herein are not a monotherapy, i.e., the method comprises administering an immunotherapeutic or a T cell therapeutic, e.g., a heterodimeric TCR-anti-CD3 antibody fusion molecule, and one or more additional active agents, e.g., second immunotherapeutics or anti-cancer therapeutic agents.

[0188] The combination therapies are described in US Application No.63 / 564,812, which is hereby incorporated by reference in its entirety.

[0189] In some embodiments, the disclosure provides administering the T cell therapeutic, e.g., heterodimeric TCR-anti-CD3 antibody fusion molecule, described herein when suitable as determined by the log2tpm profile of the TCS of the subject with a second active agent (wherein the heterodimeric TCR-anti-CD3 antibody fusion molecule is considered herein as the first active agent). The terms “active” and “therapeutic” agents are used interchangeably throughout the application. In some embodiments, the disclosure provides dosing regimen further comprises administering a third active agent, a fourth active agent, or greater than four active agents. In some embodiments, the dosing regimen comprises administering a second active agent. In some embodiments, the second active agent, a third active agent, a fourth active agent, or greater than four active agents can be an anti-cancer therapeutic agent. While not being bound by any particular theory, the disclosure provides that in some embodiments, administration of a second active agent can enhance the activity of the heterodimeric TCR-anti-CD3 antibody fusion molecule. In some embodiments, the effect of the second active agent is not additive, but synergistic.

[0190] In some embodiments, the T cell therapeutic, e.g., heterodimeric TCR-anti-CD3 antibody fusion molecule, and the second active agent are administered in a regimen, wherein the heterodimeric TCR-anti-CD3 antibody fusion molecule is administered as described herein. In some instances, the second active agent is administered prior to starting the treatment of the heterodimeric TCR-anti-CD3 antibody fusion molecule. In someembodiments, the administration of the second active agent is completed before the first doing of the heterodimeric TCR-anti-CD3 antibody fusion molecule. In some embodiments, the second active agent is administered prior to the start of the treatment the heterodimeric TCR-anti-CD3 antibody fusion molecule and then continues while the heterodimeric TCR- anti-CD3 antibody fusion molecule is also being administered. The second active agent and the heterodimeric TCR-anti-CD3 antibody fusion molecule can be administered concurrently, e.g., during the same regimens. In some embodiments, the second active agent follows a regimen when being administered the heterodimeric TCR-anti-CD3 antibody fusion molecule. In some the second active agent and the heterodimeric TCR-anti-CD3 antibody fusion molecule are administered on the same day (when the dosing regimen aligns that both active agents are administered on the same day). For example, the second active agent and the heterodimeric TCR-anti-CD3 antibody fusion molecule can be administered sequentially on the same day.

[0191] In some instances, administration of the second active agent starts at the same time as administration of the heterodimeric TCR-anti-CD3 antibody fusion molecule. In some embodiments, administration of the second active agent ceases before starting the regimen of the heterodimeric TCR-anti-CD3 antibody fusion molecule. In some embodiments, administration of the second active agent ceases at some time during the regimen of the heterodimeric TCR-anti-CD3 antibody fusion molecule. In some embodiments, administration of the second active agent and administration of the heterodimeric TCR-anti-CD3 antibody fusion molecule cease at the same time.

[0192] In some embodiments second active or therapeutic agents include, but are not limited to, radioactive compounds, prodrug activating enzymes (DT-diaphorase (DTD) or Biphenyl hydrolase-like protein (BPHL) for example), chemotherapeutic agents (cis-platin for example), toxins (Pseudomonas exotoxin such as PE38, calcimycin or diphtheria toxin for example), immune-modulating antibody fragments such as anti-CD3 or anti-CD16 for example, or immune-modulating cytokines (IL-2 for example)..

[0193] Other suitable therapeutic agents include but are not limited to: small molecule cytotoxic agents, i.e. compounds with the ability to kill mammalian cells having a molecular weight of less than 700 Daltons. Such compounds could also contain toxic metals capable of having a cytotoxic effect. Furthermore, it is to be understood that these small molecule cytotoxic agents also include pro-drugs, i.e. compounds that decay or are converted underphysiological conditions to release cytotoxic agents. Examples of such agents include cis- platin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodiumphotofrin II, temozolomide, topotecan, trimetreate glucuronate, auristatin E vincristine and doxorubicin; peptide cytotoxins, i.e. proteins or fragments thereof with the ability to kill mammalian cells. In some embodiments, such agents include ricin, diphtheria toxin, pseudomonas bacterial exotoxin A, DNase and RNase; radio-nuclides, i.e. unstable isotopes of elements which decay with the concurrent emission of one or more of α or β particles, or γ rays.. In some embodiments, such agents include iodine 131, rhenium 186, indium 111, yttrium 90, bismuth 210 and 213, actinium 225 and astatine 213; chelating agents may be used to facilitate the association of these radio-nuclides to the high affinity TCRs, or multimers thereof; immuno- stimulants, i.e. immune effector molecules which stimulate immune response. In some embodiments, such agents include cytokines such as IL-2 and IFN-γ, Superantigens and mutants thereof; TCR-HLA fusions, wherein the HLA defines an immunogenic antigen; chemokines such as IL-8, platelet factor 4, melanoma growth stimulatory protein, etc; antibodies or fragments thereof, including anti-T cell or NK-cell determinant antibodies (e.g. anti-CD3 or anti-CD28 or anti-CD16); complement activators; xenogeneic protein domains, allogeneic protein domains, viral / bacterial protein domains, viral / bacterial peptides.

[0194] In some embodiments, the second active agent comprises a chemotherapy agent, a check point inhibitor, a second, i.e., distinct, heterodimeric TCR-anti-CD3 antibody fusion molecule, a targeted therapeutic, hypomethylating agent, and / or multi-modal therapy. In some embodiments, the second active agent comprises a checkpoint inhibitor. In some embodiments, the second active agent comprises a checkpoint inhibitor. In some embodiments, the second active agent comprises a hypomethylating agent. In some embodiments, the second active agent comprises a second TCR-anti-CD3 antibody fusion molecule.

[0195] In some embodiments, the second active agent is a chemotherapy agent. In some embodiments, the second active agent is a biologic agent, e.g., a PD-1 inhibitor, a T-cell inactivator, a BRAF inhibitor, or a MEK inhibitor. In some embodiments, the second active agent comprises a checkpoint inhibitor. In some embodiments, the second active agent is a hypomethylating agent. In some embodiments, the second active agent is a second TCR-anti- CD3 antibody fusion molecule.

[0196] In some embodiments, the second active agent is a chemotherapy agent. In some embodiments, the chemotherapy agent comprises gemcitabine, nab-paclitaxel, PEGylated liposomal doxorubicin (PLD), docetaxel, carboplatin, paclitaxel, doxorubicin, pemetrexed, or a combination thereof..

[0197] Any of the listed chemotherapy agents can be administered, e.g., in the amounts and regimens, as outlined in their respective Prescribing Information.

[0198] In some embodiments, the second active agent comprises a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor comprises: atezolizumab (TECENTRIQ®), avelumab, bevacizumab (Avastin®), binimetinib, braftovi (Encorafenib), cobimetinib fumarate (Cotellic), dabrafenib mesylate (Tafinlar), dacarbazine, durvalumab, encorafenib, galunisertib, gemcitabine, interferon, talimogene laherparepvec (Imlygic), recombinant interferon alfa-2b (Intron A), ipilimumab, pembrolizumab (Keytruda), tebentafusp-tebn (Kimmtrak), trametinib dimethyl sulfoxide (Mekinist), binimetinib (Mektovi), merestinib, nivolumab (Opdivo), nivolumab and relatlimab-rmbw (Opdualag), osimertinib, aldesleukin (Proleukin), tremelimumab, vemurafenib, Ipilimumab (Yervoy), vemurafenib (Zelboraf) or a combination thereof. In some embodiments, the checkpoint inhibitor comprises pembrolizumab. In some embodiments, the checkpoint inhibitor comprises osimertinib. In some embodiments, the checkpoint inhibitor comprises bevacizumab. Any of the listed checkpoint inhibitors can be administered as outlined in their Prescribing Information.

[0199] In some embodiments, the second active agent comprises a hypomethylating agent. In some embodiments, the hypomethylating agent comprises: azacitidine, decitabine, cytidine, cedazuridine, guadecitabine, 5-fluro-2’-deoxycytidine, zebularine, CP-4200, RG108, nanaomycin A or any combination or modification thereof. In some embodiments, the checkpoint inhibitor comprises decitabine. Any of the listed hypomethylating agent can be administered as outlined in their Prescribing Information.

[0200] In some embodiments, the second active agent comprises a “targeted therapeutic.” In some embodiments, the targeted therapeutic is an antibody, e.g., a monoclonal antibody. In some embodiments, the targeted therapeutic comprises osimertinib, bevacizumab, dabrafenib, and trametinib, or combinations thereof, e.g., dabrafenib / trametinib. In some embodiments, the targeted therapeutic recognizes VEGF, e.g., VEGF-A or ANGPT2. In some embodiments, the targeted therapeutic blocks pro-angiogenic pathways, either directlyor indirectly. Any of the listed commercial targeted therapeutics can be administered as outlined in their Prescribing Information. Clinical Assessment

[0201] In some embodiments, the overall survival rate at 1 year for a subject following initial administration of T cell therapy is at least about 60%. In some embodiments, the overall survival rate at 1 year is about 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, wherein the subject has been determined to have a T cell signature (TCS) as follows: a. a composite expression level A comprising the expression of three or more genes selected from Thymocyte Expressed, Positive Selection Associated 1 (TESPA1), Cluster of Differentiation 28 (CD28), G Protein-Coupled Receptor 183 (GPR183), and Zeta-Chain-Associated Protein Kinase 70 (ZAP70) as measured by log2tpm corresponding to a composite log2tpm value of greater than or equal to 3.2; or b. a composite expression level B comprising (i) the expression of three or more genes selected from TESPA1, CD28, GPR183 and ZAP70 as measured by log2tpm corresponding to a composite log2tpm value of greater than or equal to 3.2, minus (ii) the expression of Interferon Alpha Inducible Protein 27 (IFI27) gene as measured by log2tpm, wherein the composite expression level B corresponds to a value of greater than or equal to -0.25.

[0202] In some embodiments, the overall survival rate at 1 year is greater than about 90%.

[0203] In some embodiments of the disclosure, subjects using the methods described herein can have stable disease (SD) according to RECIST vl.l and / or irRECIST criteria following four (4) or more weeks of weekly treatment with T cell therapy. In some embodiments, subject can have stable disease (SD) according to RECIST vl.l and / or irRECIST criteria following at least 5 weeks of weekly treatment with T cell therapy. In some embodiments, subjects can have stable disease (SD) according to RECIST vl. l and / or irRECIST criteria following weekly treatment with T cell therapy for 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 weeks. In some embodiments, subject can have stable disease (SD) according to RECIST vl. l and / or irRECIST criteria following weekly treatment with T cell therapy for greater than 24 weeks.

[0204] In some embodiments of the disclosure, subjects using the methods described herein can have progressive disease (PD) according to RECIST vl.l and / or irRECIST criteria following four (4) or more weeks of weekly treatment with T cell therapy. In some embodiments, subjects can have progressive disease (PD) according to RECIST vl. l and / or irRECIST criteria following at least 5 weeks of weekly treatment with T cell therapy. In some embodiments, subjects have progressive disease (PD) according to RECIST vl. l and / or irRECIST criteria following weekly treatment with T cell therapy for 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 weeks. In some embodiments, subjects have progressive disease (PD) according to RECIST vl. l and / or irRECIST criteria following weekly treatment with T cell therapy for greater than 24 weeks.

[0205] In some embodiments of the disclosure, subjects using the methods described herein achieve a partial response (PR) according to RECIST vl.l and / or irRECIST criteria following four (4) or more weeks of weekly treatment with T cell therapy. In some embodiments, subjects achieve a partial response (PR) according to RECIST vl. l and / or irRECIST criteria following at least 5 weeks of weekly treatment with T cell therapy. In some embodiments, subjects achieve a partial response (PR) according to RECIST vl. l and / or irRECIST criteria following weekly treatment with T cell therapy for 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 weeks. In some embodiments, subjects achieve a partial response (PR) according to RECIST vl.l and / or irRECIST criteria following weekly treatment with T cell therapy for greater than 24 weeks.

[0206] In some embodiments of the disclosure, subjects using the methods described herein treated with T cell therapeutic experience progression-free survival (PFS) at least about one (1) month. In some embodiments, subjects treated with T cell therapy experience PFS at least about 1.5 months. In some embodiments, subjects treated with T cell therapy experience PFS at least about 2 months, 2.2 months, 2.4 months, 2.6 months, 2.8 months, 3 months, 3.2 months, 3.4 months, 3.6 months, 3.8 months, 4 months, 4.2 months, 4.4 months, 4.6 months, 4.8 months, 5 months, 5.2 months, 5.4 months, 5.6 months, 5.8 months, or 6 months. In some embodiments, subjects treated with tebentafusp and / or IMC-F106C experience PFS greater than 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months or 18 months.

[0207] In some embodiments, subjects treated with T cell therapy experience tumor shrinkage. In some embodiments, the change in tumor size from baseline is a decrease of at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, 90%, 95%, 99%, or greater than 99%.In some embodiments, subjects treated with T cell therapy experience tumor growth. In some embodiments, the change in tumor size from baseline is an increase of at least about 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90%.

[0208] Immunohistochemistry (IHC)-stained tissue imaging is the most common approach to characterize the expression of a specific protein across tissues. Initially, the target protein fixed on the IHC slide is recognized by a specific antibody, forming an antigen- antibody complex, and is then visualized through a colored histochemical reaction with reporter molecules such as diaminobenzidine (DAB).1,2 Additionally, a counterstain, typically hematoxylin, is applied to contrast the staining of the target protein. Due to its essential role in demonstrating the distribution and expression of protein biomarkers within tissue sections, IHC staining has become a routine method in basic research and diagnostic pathological examination. In IHC images, DAB intensity serves as an indicator of protein expression and can be effectively quantified using the H-score.

[0209] The H-score is a reliable metric calculated as follows: (1 × percentage of weak staining) + (2 × percentage of moderate staining) + (3 × percentage of strong staining) within the target region, ranging from 0 to 300.6 IHC staining and H-score provide superior protein detection capabilities compared with other methods (e.g., western blot) because they can measure protein expression levels within specific cell regions, which is especially valuable for quantifying proteins within specific types of cells of interest. H-score calculation has been widely employed to establish links between proteins and tumors, playing crucial roles in diagnosis, prognosis, and therapeutic decision-making. For example, over 60% of patients with non–small cell lung cancer (NSCLC) exhibit overexpression of epidermal growth factor receptor (EGFR),14 and a higher EGFR H-score is strongly associated with shorter overall survival.15 Similarly, patients with breast cancer can be stratified into different treatment groups based on the expression levels of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 (HER2) measured using H-scores.Composition / kit

[0210] In some embodiments, the present disclosure provides a kit comprising all components necessary to conduct RNA-seq on a sample taken from a subject to determine the log2tpm for the genes of a specific TCS combination as found in Tables 1-4. In some embodiments, the kit comprises a solution for use in processing a biological sample obtained from a subject; wherein the solution comprises nucleotide primers for evaluating variant allele frequency in a panel of mutations comprising TESPA1, CD28, and GPR183.

[0211] In some embodiments, the kit further comprises nucleotide primers for evaluating variant allele frequency in a panel of mutations comprising ZAP70, IFI27, or combinations thereof.

[0212] Various modifications of the disclosure in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. Preferred features of each aspect of the disclosure are as for each of the other aspects mutatis mutandis. The documents referred to herein are incorporated by reference to the fullest extent permitted by law. Computer Readable Medium

[0213] In some embodiments, the present disclosure provides a method of assessing T cell activity in a patient sample, said method comprising: (a) measuring a T cell signature (TCS) in said sample; (b) applying, by a processor, an algorithm to assess said T cell activity in said patient based on said levels of said TCS in said sample; said TCS comprising three or more genes selected from TESPA1, CD28, GPR183, ZAP70 and IFI27.

[0214] In some embodiments, the present disclosure provides a method of assessing T cell activity in a patient sample, said method comprising (a) measuring three or more gene expression levels selected from TESPA1, CD28, GPR183, ZAP70 and IFI27 in said sample; and (b) applying, by a processor, an algorithm to assess said T cell activity level in said patient based on said gene expression levels in said sample.

[0215] In some embodiments, the preset disclosure provides a method of determining a T cell therapeutic or immunotherapeutic dose for treatment of cancer comprising: (a) measuring a level of TCS, wherein one or more genes of said TCS are altered relative to a normal control in a patient; (b) fitting, by a processor, said measured level of TCS to a response surface model as a function of a T cell therapeutic or immunotherapeutic dose; (c) computing a cost function for a level of TCS; and (d) identifying a T cell therapeutic or immunotherapeutic dose that minimizes said cost function at a known time interval.

[0216] In some embodiments, the present disclosure provides a computer readable medium having stored thereon a computer program which, when executed by a computer system operably connected to an assay system configured to measure a TCS level of three or more genes in a patient sample, causes the computer system to perform a method of calculating a T cell therapeutic dose by a method comprising: (a) fitting said measured TCS level to a response surface model as a function of a T cell therapeutic dose; (b) computing a cost function for TCS level; and (c) identifying T cell therapeutic dose that minimizes said cost function at a known time interval. EXAMPLES

[0217] The present disclosure has been described with respect to representative examples that are to be considered illustrative embodiments that do not limit the scope of the disclosure which is defined solely by the claims. All references to publications, including scientific publications, treatises, textbooks, patent applications and issued patents are hereby incorporated by reference for all purposes.Example 1 - Discovery of a T Cell Signature (TCS) from a Tebentafusp Phase 2 Clinical Trial

[0218] A randomized, controlled Phase 2 trial to determine the overall survival of HLA- A*0201 positive adult subjects with previously untreated advanced uveal melanoma receiving tebentafusp, as compared to receiving an investigator’s choice of dacarbazine, ipilimumab, or pembrolizumab, referred to as IMCgp100-102, was conducted. (Carvajal, et al. Nat Med 28, 2364–2373 (2022). https: / / doi.org / 10.1038 / s41591-022-02015-7). A comprehensive gene expression analysis was performed using samples collected from subjects at baseline (just prior to or at time of first treatment).

[0219] Whole blood samples were collected in PAXgene RNA tubes and RNA was isolated from the samples. RNA libraries from isolated RNA was prepared using the Illumina TruSeq® stranded mRNA prep kit, and then sequenced using 100bp paired-end sequencing at 50 million reads per sample. To map sequences against a reference genome, the resulting reads were first aligned using STAR aligner (version 2.6.1), and then mapped to the GRCh38 primary assembly provided by the European Bioinformatics Institute’s Ensembl Genomes database. Gene expression was quantified using RSEM (version 1.3.1).

[0220] The resulting expression levels were indicated by values of log2tpm where tpm corresponds to transcripts per million and log2tpm represents a measure of the frequency of a gene, or set of genes if applicable, within a population. For example, a log2tpm of 9 for gene A means that for every million transcripts in the sample, 29of them are from gene A. TPM values were log2 transformed in R (version 4.1.0). A pseudo-count value of 1 was added to each TPM value prior to transformation. For multiple samples collected from the same subject, the median expression value across all samples was calculated per subject. The terms “values”, “value”, “levels”, and “level” are used interchangeably in reference to TCS log2tpm and are understood to mean the same thing.

[0221] Survival analysis was carried out using the R package survminer (version 0.4.9), and the Cox likelihood ratio test was used to assess differences between the survival curves. Univariate Cox proportional hazards methods (R package survival version 3.2.11) were used to model the prognostic importance of potential predictors of survival. The Fisher exact test and the Wilcoxon rank sum test were used to assess associations between high and low TCS expression levels and clinically derived patient groupings (tests were two sided and werecarried out using R stats package 4.1). Euclidean distance was applied per subject to generate a distance matrix and complete-linkage clustering was carried out. Heatmaps were generated using R Bioconductor (version 3.16) package ComplexHeatmap (version 2.14.0).

[0222] As represented in FIG.1, output from the RNASeq gene expression analysis indicated that there were genes whose higher expression levels were associated with longer overall survival (OS) and tumor shrinkage (TS), as well as genes whose higher expression levels were associated with shorter OS and tumor growth.

[0223] Further consideration of a gene’s (or set of genes) utility was undertaken based on whether its biological mechanism was related to T cell biology, and whether any particular combination of genes exhibited a relatively stronger association with longer OS and greater TS. Within the subset of higher expression genes associated with longer OS and greater TS, the presence of multiple genes having an association with T cells was observed. Furthermore, there was an enrichment for genes more highly expressed in naïve T cells compared to more differentiated effector T cells.

[0224] The TCS combination of TESPA1, CD28 and GPR183 stood out in particular based on this analysis. Other genes of interest included ZAP70 and IFI27. TESPA1 is known to be one the best indicators of naïve T cells. CD28 is important for T cell survival and responsiveness. CD28 is also known to be high on naïve T cells, becoming downregulated following activation. Furthermore, the absence of CD28 on T cells is known to be associated with exhaustion / senescence and susceptibility to AICD. While GPR183 has a slightly broader scope of expression, it is known to be an important receptor for chemotaxis of T cells. While a number of other naïve T cell-associated genes (e.g., IL7R) also demonstrated an association with OS, the addition of these gene(s) to the TCS combination of TESPA1, CD28 and GPR183 did not appear to improve the overall association with longer OS and greater TS as compared to the TESPA1 / CD28 / GPR183 gene signature itself. From these analyses, the T Cell Signature (TCS) corresponding to the combination of TESPA / CD28 / GPR183 was established.

[0225] The analyses further established that a strong association between the TCS and longer OS and greater TS was observed when stratifying at the 40thpercentile of TCS (high TCS), which equated to 4.36 (i.e. a log2tpm value of 4.36 or greater) in the IMCgp100-102 trial for uveal melanoma subjects.

[0226] In particular, the analysis of samples demonstrated a statistically significant association (p value = 0.005) between the TCS and TS (represented as tumor reduction in the Figure) as compared to the association between effector T cells and TS (p value = 0.58) (FIG. 2B), or exhausted T cells and TS (p value =0.86) (FIG.2C). In the majority of subjects where the TCS was high, the tumor size was decreased relative to baseline. The inverse was also the case, i.e., when the TCS was low (not meeting the threshold for high), tumor size was often unchanged or increased as compared to baseline (FIG.2A).

[0227] Evaluation of the samples also demonstrated a strong association between the TCS and OS. When comparing subjects with high and low TCS with OS, a greater number of subjects with TCS had increased survival as compared to subjects with low TCS (FIG. 3A). A similar evaluation looking at effector T cells or exhausted T cells showed no meaningful difference or association between these characteristics and OS (FIG.3B and FIG. 3C).

[0228] The TCS was compared to other current methods, such as H score, to ascertain its strength in identifying subjects who would be likely to respond to treatment by exhibiting tumor reduction and / or overall survival. With respect to tumor reduction, 45% of subjects having greater than 10% tumor reduction had a high TCS score (FIG.4A) as compared to 37% of subjects having a high H-score (FIG.4B). A percentage of subjects exhibiting a greater than 10% tumor reduction did have a low TCS, but this percentage was less than half of that as compared to those with a low H-score (FIG.4A and FIG.4B). Overall, the TCS was thus a significantly better marker than H score for identifying subjects who would have tumor reduction following treatment (FIG.4A and FIG.4B) and for subjects who would have increased overall survival following treatment (FIG.5A and FIG.5B). Example 2. Application of TCS for Treatment of Cancer with T Cell Therapeutics

[0229] To ascertain whether the TCS was potentially applicable to other T cell therapeutics, RNA sequencing (as described in Example 1) was performed on samples from subjects participating in a trial using a T cell therapeutic against a different target. This trial was a Phase 1 / 2 study of the safety and efficacy of a bispecific T cell therapeutic targeting PRAME (IMC-F106C) in advanced cancers, where IMC-F106C is administered as either a monotherapy, or in combination with other agents such as checkpoint inhibitors or chemotherapy (https: / / classic.clinicaltrials.gov / ct2 / show / NCT04262466).

[0230] RNASeq was again performed utilizing whole blood samples collected at baseline (at or just prior to first treatment) as described in Example 1, from subjects receiving an active dose of IMCF106C. Samples were from subjects who had different types of cancer including uveal melanoma, cutaneous melanoma, lung cancer (incl. non-small cell lung cancer (NSCLC)), endometrial cancer, ovarian cancer, and breast cancer.

[0231] From the analyses across evaluable monotherapy subjects, it was determined that high TCS was associated with longer OS (FIG.6A) and better progression free survival (PFS) (FIG.6B). High TCS in evaluable monotherapy subjects was also associated with tumor reduction (FIG.7). As represented in spider plot graphs showing the change in tumor size plotted against survival time for individual subjects (also referred to as disease control), TCS high subjects showed a relatively greater reduction in tumor size and a relatively longer survival time as compared to TCS low subjects (FIG.8A and FIG.8B).

[0232] Individual subjects represented in the spider plot were being treated for different cancers (see legend of FIG.8A and 8B). From the analyses, the association between high TCS and disease control was therefore observed to be applicable across multiple tumor types.

[0233] Looking at uveal melanoma subjects specifically, the association of high TCS with disease control replicated the data from the IMCgp100-102 trial, where high TCS was associated with tumor reduction, as well as longer PFS and longer OS (FIG.9).

[0234] Taking into account results from both the IMCgp100 trial as described in Example 1, as well as the IMC-F106C trial as described in this Example, an overall cutoff of 3.96 log2tpm applicable across multiple tumor types was established. When TCS results were evaluated by tumor type, results showed that optimized log2tpm cutoffs could be utilized for individual tumor types (FIG.10). More specifically, it was found that the log2tpm cutoffs could be optimized as follows: a cutoff of 3.86 log2tpm for cutaneous melanoma (CM), a cutoff of 4.04 log2tpm for ovarian cancer, a cutoff of 3.36 log2tpm for endometrial cancer, a cutoff of 3.57 log2tpm for breast cancer, and a cutoff of 3.35 log2tpm for non-small cell lung cancer (NSCLC). Example 3 - Clinical Correlates of TCS

[0235] An investigation was performed to ascertain what clinical factors may be potentially associated with the TCS. Forty baseline clinical factors were examined includingthe following: cancer type, H-score, lines of therapy ECOG status, smoking status, sum of diameters for target lesions (SOD), maximum target lesion size, age, sex, sites of metastasis, BMI, prior platinum treatment, tumor stage at initial diagnosis, prior PD(L)1 response, medical history of fatigue, day 1 steroid premedication, months since end of last line, months since metastasis, months since diagnosis,, months since PD on last line, hemoglobin, albumin, neutrophil-to-lymphocyte ratio (NLR), platelets, creatinine clearance, white blood cell counts, absolute lymphocyte count (ALC), D-dimer, fibrinogen, INR, ALP, D-dimer-to- fibrinogen ratio, and fibrinogen-to-albumin ratio.

[0236] Multivariate analysis was conducted. Following this analysis, it was observed that clinical factors having the strongest association with TCS were identified as neutrophil-to- lymphocyte ratio (NLR), absolute lymphocyte count (ALC), and fibrinogen (FIG.11). Further analyses studying the association of disease control rate (DCR) with fibrinogen levels in uveal melanoma subjects (FIG.12) or ovarian cancer subjects (FIG.13) demonstrated that normal levels of fibrinogen were associated with an improved DCR. Example 4 Melanoma TCS

[0237] Further analysis of differential gene expression indicated that lower expression of a gene, IFI27, was associated with better outcomes in melanoma subjects treated with tebentafusp or with IMC-F106C. As shown in FIG.14, a waterfall plot of melanoma subjects treated with IMC-F106C indicates that a greater proportion of subjects with low IFI27 have a reduction in tumor size (FIG.14A). In addition, as summarized in the Table below, low IFI27 levels were also associated with better outcomes in the form of a partial response (PR) or stable disease (SD). Low IFI27 levels were also associated with better progression free survival (PFS) (FIG.14B) and longer OS (FIG.14C). IFI27 Non-Evaluable Progressive Partial Stable Disease Expression Subjects Disease (PD) Response (PR) (SD) Level High 3 14 8 Low 2 4 5 14

[0238] Similar results were found when analyzing the subset of cutaneous melanoma subjects treated with IMC-F106C. Low IFI27 was associated with a reduction in tumor size (FIG.15A), better PFS (FIG.15B) and longer OS (FIG.15C).IFI27 Non-Evaluable Progressive Partial Stable Disease Expression Subjects Disease (PD) Response (PR) (SD) Level High 1 9 6 Low 2 3 2 9

[0239] An analysis of subjects treated with tebentafusp in a Phase I / II clinical study of participants with metastatic uveal melanoma (mUM) also showed that low IFI27 was associated with a reduction in tumor size (FIG.16A), better PFS (FIG.16B), and longer OS (FIG.16C). IFI27 Non-Evaluable Progressive Partial Stable Disease Expression Subjects Disease (PD) Response (PR) (SD) Level High 1 39 2 24 Low 2 22 5 37

[0240] Based on the above results, an optimized or alternative signature for melanoma includes the three gene TCS as described in Examples 1 and 2 above, and additionally the determination of whether there is high or low IFI27 gene expression.

Claims

CLAIMS What is claimed is:

1. A method of treating a cancer in a subject, the method comprising: administering to the subject an effective amount of an immunotherapeutic, wherein the subject has been determined to have a T cell signature (TCS) comprising: a. a composite expression level A comprising the expression of three or more genes selected from Thymocyte Expressed, Positive Selection Associated 1 (TESPA1), Cluster of Differentiation 28 (CD28), G Protein-Coupled Receptor 183 (GPR183), and Zeta-Chain- Associated Protein Kinase 70 (ZAP70) as measured by log2tpm corresponding to a composite log2tpm value of greater than or equal to 3.2; or b. a composite expression level B comprising (i) the expression of three or more genes selected from TESPA1, CD28, GPR183 and ZAP70 as measured by log2tpm corresponding to a composite log2tpm value of greater than or equal to 3.2, minus (ii) the expression of Interferon Alpha Inducible Protein 27 (IFI27) gene as measured by log2tpm, wherein the composite expression level B corresponds to a value of greater than or equal to -0.

25.

2. The method of claim 1, wherein the TCS corresponds to TESPA1, CD28, and GPR183.

3. The method of claim 1, wherein the TCS corresponds to TESPA1, CD28, GPR183 and ZAP70.

4. The method of any one of claims 1 to 3, wherein the composite expression level A or the composite expression level B is determined prior to administration of the immunotherapeutic.

5. The method of any one of claims 1 to 4, wherein the subject has been determined to have a composite expression level A of greater than or equal to 3.

8.

6. The method of any one of claims 1 to 5, wherein the subject has been determined to have a composite expression level A of greater than or equal to 3.

96.

7. The method of any one of claims 1 to 6, wherein the subject has been determined to have a composite expression level A of greater than or equal to 4.

2.

8. The method of any one of claims 1 to 7, wherein the subject has been determined to have a composite expression level A of greater than or equal to 4.4.

9. The method of claim 1, wherein the TCS corresponds to TESPA1, CD28, GPR183, and IFI27.

10. The method of any one of claims 1 to 9, wherein the subject has been determined to have a composite expression level B of greater than or equal to -0.

25.

11. The method of any one of claims 1 to 10, wherein the subject has been determined to have a composite expression level B of greater than or equal to 0.

12. The method of any one of claims 1 to 11, wherein the subject has been determined to have a composite expression level B of greater than or equal to 0.

25.

13. The method of any one of claims 1 to 12, wherein the subject has been determined to have a composite expression level B of greater than or equal to 1.

25.

14. The method of any one of claims 1 to 13, wherein the immunotherapeutic comprises an immune checkpoint inhibitor, a methylating agent, a T cell therapy, an immune activator, a chemotherapeutic agent, or a combination thereof.

15. A method of treating a cancer in a subject, the method comprising: administering to the subject an effective amount of a T cell therapeutic, wherein the subject has been determined to have a T cell signature (TCS) comprising: a. a composite expression level A comprising the expression of three or more genes selected from Thymocyte Expressed, Positive Selection Associated 1 (TESPA1), Cluster of Differentiation 28 (CD28), G Protein-Coupled Receptor 183 (GPR183), and Zeta-Chain- Associated Protein Kinase 70 (ZAP70) as measured by log2tpm corresponding to a composite log2tpm value of greater than or equal to 4.0; or b. a composite expression level B comprising (i) the expression of three or more genes selected from TESPA1, CD28, GPR183 and ZAP70 as measured by log2tpm corresponding to a composite log2tpm value of greater than or equal to 4.0, minus (ii) the expression of Interferon Alpha Inducible Protein 27 (IFI27) gene as measured by log2tpm, wherein the composite expression level B corresponds to a value of greater than or equal to -0.

25.

16. The method of claim 15, wherein the TCS corresponds to TESPA1, CD28, and GPR183.

17. The method of claim 15, wherein the TCS corresponds to TESPA1, CD28, GPR183 and ZAP70.

18. The method of any one of claims 15 to 17, wherein the subject has been determined to have a composite expression level A of greater than or equal to 4.

2.

19. The method of claim 15, wherein the TCS corresponds to TESPA1, CD28, GPR183, and IFI27.

20. The method of any one of claims 15 to 19, wherein the subject has been determined to have a composite expression level B of greater than or equal to 0.

21. The method of any one of claims 15 to 20, wherein the composite expression level A or composite expression level B is determined prior to administration of the T cell therapeutic.

22. The method of any one of claims 15 to 21, wherein the T cell therapeutic comprises a bi- specific molecule.

23. The method of claim 22, wherein the bi-specific molecule comprises a bi-specific T cell engager (BiTE), or a Bi-specific antibody (BsAb).

24. The method of any of claims 15 to 23, wherein the T cell therapeutic comprises a T-cell receptor (TCR) protein.

25. The method of any one of claims 15 to 21, wherein the T cell therapeutic comprises a chimeric antigen receptor (CAR) T cell.

26. The method of any one of claims 15 to 24, wherein the T cell therapeutic targets PRAME.

27. The method of any of claims 15 to 24 or claim 26, wherein the T cell therapeutic is IMC- F106C.

28. The method of any one of claims 15 to 24, wherein the T cell therapeutic targets gp100.

29. The method of any of claims 15 to 24 or claim 28, wherein the T cell therapeutic comprises tebentafusp.

30. A method of determining whether a cancer treatment is suitable for treating cancer in a subject in need thereof, the method comprising determining if the subject has a T cell signature (TCS), the TCS comprising: a. a composite expression level A comprising the expression of three or more genes selected from Thymocyte Expressed, Positive Selection Associated 1 (TESPA1), Cluster of Differentiation 28 (CD28), G Protein-Coupled Receptor 183 (GPR183), and Zeta-Chain- Associated Protein Kinase 70 (ZAP70) as measured by log2tpm; or b. a composite expression level B comprising (i) the expression of three or more genes selected from TESPA1, CD28, GPR183 and ZAP70 as measured by log2tpm corresponding to a composite log2tpm value of greater than or equal to 3.2, minus (ii) the expression of Interferon Alpha Inducible Protein 27 (IFI27) gene as measured by log2tpm, wherein the subject is suitable for a cancer treatment if : (i) the composite expression level A corresponds to a composite log2tpm value of greater than or equal to 3.2; or (ii) the composite expression level B corresponds to a composite log2tpm value of greater than or equal to -0.

25.

31. The method of claim 30, wherein the composite expression level A comprises TESPA1, CD28, and GPR183 and corresponds to a composite log2tpm value of greater than or equal to 3.

2.

32. The method of claim 30, wherein the composite expression level A comprises TESPA1, CD28, GPR183, and ZAP70 and corresponds to a composite log2tpm value of greater than or equal to 3.

2.

33. The method of claim 30, wherein the composite expression level of B comprises TESPA1, CD28, GPR183, and IFI27 and corresponds to a composite log2tpm value of greater than or equal to –0.

25.

34. The method of claim 30, wherein the composite expression level of B comprises TESPA1, CD28, GPR183, ZAP70 and IFI27 and corresponds to a composite log2tpm value of greater than or equal to –0.

25.

35. The method of any one of claims 30 to 34, wherein the cancer treatment is an immunotherapeutic cancer treatment.

36. The method of any one of claims 30 to 35, wherein the cancer treatment is a T cell therapeutic.

37. A method for identifying and treating a subject suffering from a cancer who is suitable for treatment with an immunotherapeutic comprising: a. determining from a sample obtained from the subject if the subject has a T cell signature (TCS), using RNASeq methodology to quantify gene expression levels, the TCS comprising: (i) a composite expression level A comprising the expression of three or more genes selected from Thymocyte Expressed, Positive Selection Associated 1 (TESPA1), Cluster of Differentiation 28 (CD28), G Protein-Coupled Receptor 183 (GPR183), and Zeta-Chain-Associated Protein Kinase 70 (ZAP70) as measured by log2tpm; or (ii) a composite expression level B comprising a) the expression of three or more genes selected from TESPA1, CD28, GPR183 and ZAP70 as measured by log2tpm corresponding to a composite log2tpm value of greater than or equal to 3.2, minus (b) the expression of Interferon Alpha Inducible Protein 27 (IFI27) gene as measured by log2tpm, b. identifying the subject as suitable for treatment with a T cell therapeutic if (i) the composite expression level A corresponds to a composite log2tpm value of greater than or equal to 3.2, or (ii) the composite expression level B corresponds to a composite log2tpm value of greater than or equal to -0.25; and c. administering an effective amount of the immunotherapeutic to the subject identified as suitable for treatment with the immunotherapeutic.

38. The method of claim 37, wherein the T cell therapeutic comprises a bi-specific molecule.

39. The method of claim 37 or 38, wherein the T cell therapeutic comprises a T-cell receptor (TCR).

40. The method of claim 38, wherein the bi-specific molecule comprises a bi-specific T cell engager (BiTE), or a Bi-specific antibody (BsAb).

41. The method of any one of claims 34 to 37, wherein the T cell therapeutic comprises a chimeric antigen receptor (CAR) T cell.

42. The method of any one of claims 37 to 40, wherein the T cell therapeutic targets PRAME.

43. The method of claim 42, wherein the T cell therapeutic is IMC-F106C.

44. The method of any one of claims 37 to 40, wherein the T cell therapeutic targets gp100.

45. The method of claim 44, wherein the T cell therapeutic comprises tebentafusp.

46. A method for treating a cancer in a subject, the method comprising: a. determining a log2tpm value of genes in a TCS in a biological sample obtained from the subject, wherein the TCS corresponds to: (i) a composite expression level A comprising the expression of three or more genes selected from Thymocyte Expressed, Positive Selection Associated 1 (TESPA1), Cluster of Differentiation 28 (CD28), G Protein-Coupled Receptor 183 (GPR183), and Zeta-Chain-Associated Protein Kinase 70 (ZAP70) as measured by log2tpm; or (ii) a composite expression level B comprising a) the expression of three or more genes selected from TESPA1, CD28, GPR183 and ZAP70 as measured by log2tpm corresponding to a composite log2tpm value of greater than or equal to 3.2, minus (b) the expression of Interferon Alpha Inducible Protein 27 (IFI27) gene as measured by log2tpm, and b. administering to the subject a dosing regimen comprising a first dosage amount of a T cell therapeutic; wherein if the composite expression level A is greater than or equal to 3.2 or the composite expression level B is greater than or equal to -0.25, then a second dosage amount is administered, wherein the second dosage amount is the same or less than the first dosage amount, or wherein if the composite expression level A is less than 3.2 or the composite expression level B is less than -0.25, then the second dosage amount is greater than the first dosage amount and / or further comprises a second cancer treatment.

47. The method of claim 46, wherein the first dosing regimen comprises a single dose of a T cell therapeutic.

48. The method of claim 46, wherein the first dosing regimen comprises greater than one dose of a T cell therapeutic.

49. The method of claim 46, wherein the first dosing regimen comprises administering a T cell therapeutic in greater than one doses for a period of about 1 week to about 1 month.

50. The method of any one of claims 46 to 49, wherein a second biological sample is obtained from the subject about 1 week to about 3 months after the first composite expression level A or composite expression level B is determined.

51. The method of any one of claims 46 to 50, wherein the second composite expression level A is at least 10% greater than the first composite expression level A.

52. The method of any one of claims 46 to 50, wherein the second composite expression level A is at least 20% greater than the first composite expression level A.

53. The method of any one of claims 46 to 50, wherein if the second composite expression level A is greater than the first composite expression level A, then the second dosage amount is at least 10% less than the first dosage amount.

54. The method of any one of claims 46 to 50, wherein if the second composite expression level A is greater than the first composite expression level A, then the second dosage amount is at least 20% less than the first dosage amount.

55. The method of any one of claims 46 to 50, wherein if the second composite expression level A is the same or less than the first composite expression level A, then the second dosage amount is at least 10% greater amount than first dosage amount.

56. The method of any one of claims 46 to 50, wherein if the second composite expression level A is the same or less than the first composite expression level A, then the second dosage amount is at least 20% greater amount than first dosage amount.

57. The method of any one of claims 46 to 50, wherein the second composite expression level B is at least 10% greater than the first composite expression level B.

58. The method of any one of claims 46 to 50, wherein the second composite expression level B is at least 20% greater than the first composite expression level B.

59. The method of any one of claims 46 to 50, wherein if the second composite expression level B is greater than the first composite expression level B, then the second dosage amount is at least 10% less than the first dosage amount.

60. The method of any one of claims 46 to 50, wherein if the second composite expression level B is greater than the first composite expression level B, then the second dosage amount is at least 20% less than the first dosage amount.

61. The method of any one of claims 46 to 50, wherein if the second composite expression level B is the same or less than the first composite expression level B, then the second dosage amount is at least 10% greater amount than first dosage amount.

62. The method of any one of claims 46 to 50, wherein if the second composite expression level B is the same or less than the first composite expression level B, then the second dosage amount is at least 20% greater amount than first dosage amount 63. The method of any one of claims 46 to 62, wherein the TCS corresponds to TESPA1, CD28, and GPR183.

64. The method of any one of claims 46 to 62, wherein the TCS corresponds to TESPA1, CD28, GPR183 and ZAP70.

65. The method of any one of claims 46 to 64, wherein the TCS corresponds to TESPA1, CD28, GPR183, ZAP70 and IFI27.

66. The method of any one of claims 46 to 65, wherein the biological sample comprises whole blood.

67. The method of any one of claims 1 to 66, wherein the immunotherapeutic, cancer treatment or T cell therapeutic is a first anti-cancer therapeutic agent.

68. The method of any one of claims 1 to 67, wherein the immunotherapeutic, cancer treatment or T cell therapeutic is a first-line treatment.

69. The method of any one of claims 1 to 68, wherein the subject is a human subject.

70. The method of any one of claims 1 to 69, wherein the cancer is positive for an antigen selected from the group consisting of gp100 and PRAME.

71. The method of any one of claims 1 to 70, wherein the cancer is a PRAME-positive cancer.

72. The method of any one of claims 1 to 71, wherein the subject is HLA-A*02 positive.

73. The method of any one of claims 1 to 72, wherein the subject is HLA-A*02:01 positive.

74. The method of any one of claims 1 to 73, wherein the subject has BRAF V600 mutation.

75. The method of any one of claims 1 to 74, wherein the subject has been diagnosed with a life expectancy greater than 3 months.

76. The method of any one of claims 1 to 75, wherein the cancer is a melanoma, an ovarian cancer, a non-small cell lung adenocarcinoma, a non-small cell lung squamous cell carcinoma, an endometrial cancer, or a breast cancer.

77. The method of claim 76, wherein the melanoma is a uveal melanoma or a cutaneous melanoma.

78. The method of claim 77, wherein the melanoma is a cutaneous melanoma.

79. The method of claim 76, wherein the breast cancer is a triple-negative breast cancer.

80. The method of any one of claims 1 to 79, further comprising administering to said subject an effective amount of a second anti-cancer therapeutic agent.

81. The method of claim 80, wherein the second anti-cancer therapeutic agent comprises an immune checkpoint inhibitor, a second T cell therapeutic, an immune activator, chemotherapeutic agent, cancer vaccine, a methylating agent or a combination thereof.

82. The method of claim 80 or 81, wherein the second anti-cancer therapeutic agent is administered prior to administration of the T cell therapeutic.

83. The method of any one of claims 80 to 82, wherein the second anti-cancer therapeutic agent is a chemotherapeutic agent.

84. A method of treating cutaneous melanoma in a subject, the method comprising: administering to the subject an effective amount of an immunotherapeutic, wherein the subject has been determined to have a T cell signature (TCS) comprising (i) the expression of three or more genes selected from TESPA1, CD28, GPR183 and ZAP70 as measured by log2tpm corresponding to a composite log2tpm value of greater than or equal to 3.2, minus (ii) the expression of Interferon Alpha Inducible Protein 27 (IFI27) gene asmeasured by log2tpm, wherein the TCS corresponds to a composite log2tpm value of greater than or equal to -0.

25.

85. The method of claim 84, wherein the immunotherapeutic is a T cell therapeutic.

86. A kit comprising: a solution for use in processing a biological sample obtained from a subject; wherein the solution comprises nucleotide primers for evaluating variant allele frequency in a panel of mutations comprising TESPA1, CD28, and GPR183.

87. The kit of claim 86, wherein the kit further comprises nucleotide primers for evaluating variant allele frequency in a panel of mutations comprising ZAP70, IFI27, or combinations thereof.

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