WO2021205274A1

WO2021205274A1 - Methods for assessing chemokine activity

Info

Publication number: WO2021205274A1
Application number: PCT/IB2021/052505
Authority: WO
Inventors: Christopher W. SZETO; Saihitha VEERAPANENI
Original assignee: Immunitybio, Inc.
Priority date: 2020-04-08
Filing date: 2021-03-26
Publication date: 2021-10-14

Abstract

Provided herein are methods assessing the expression of chemokine ligands in a tumor microenvironment. The method involves quantifying expression of mRNAs coding for chemokine ligands and assessing the amount and type of chemokine ligands present in the tumor micro-environment from the expression level of mRNAs that code for chemokine ligands. The results of this method would be useful for providing immune therapy to a patient having a tumor.

Description

METHODS FOR ASSESSING GHEMOKTNE ACTIVITY

[0001] This application claims the benefit of the co-pending U.S. provisional application 62/007,316, filed April 8, 2020, which is incorporated by reference herein in its entirety.

Field

[0002] The present disclosure concerns genetic analysis of tumor tissue, especially as it relates to immune cells signatures.

Background

[0003] The background description includes information that may be useful in understanding the present disclosure. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art.

[0004] All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Where a definition or use of a term in an incorporated reference is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

[0005] Chemotactic cytokines (chemokines) control cell migration between tissues and the positioning and interactions of cells within tissue. The chemokine superfamily consists of ~50 endogenous chemokine ligands and ~20 receptors. Chemokines mediate the host-response to cancer by directing leukocyte trafficking into the tumor microenvironment. Leukocyte surfaces may comprise chemokine receptors, and these chemokine receptors bind chemokines in the tumor micro environment. Thus, the chemokine-chemokine receptor binding directs the leukocytes into the tumor microenvironment.

[0006] Chemokine receptors are G protein-coupled receptors containing seven transmembrane domains. They are all composed of about 350 amino acids, divided into a short and acidic N-terminal end, seven helical transmembrane domains with three intracellular and three extracellular hydrophilic loops, and an intracellular C-terminus. The C-terminus contains serine and threonine phosphorylation sites.

[0007] Nagarsheth etal. (2017) Nat. Rev. Immunol. 17(9): 559-72 list some of the main chemokines that are found in the human tumor microenvironment. While the roles of chemokines and chemokine receptors with respect to autoimmune diseases and cancer are disclosed, the targeting of chemokines and chemokine receptors has so far failed to yield any viable results. There is presently no accurate tool to assess the amount of each cytokine in a tumor microenvironment.

[0008] Complex interactions between tumor microenvironment and cytokines remain to be elucidated. Furthermore, there remains a need in the art for accurate and rapid assessment of the type and amount of each cytokine present in a tumor microenvironment.

Summary

[0009] The present disclosure describes various methods of assessing chemokine production in a tumor micro-environment. The methods comprise obtaining or having obtained whole transcriptomic sequencing data from a single tumor of a patient having cancer. The normalized expression level of each mRNA coding for a plurality of chemokine ligands expressed in the tumor is then determined. This normalized expression level is adjusted by comparing the expression level of chemokine ligands in the tumor cells with the expression level of chemokine ligands in a matched normal cell, to thereby obtain scaled expression values. The scaled expression values for each chemokine ligand is then grouped by their cognate chemokine receptor group. For each chemokine receptor group, the expression values are averaged, and Z score is determined. From the Z score, the chemokine production for each chemokine receptor group can be assessed.

[0010] Most typically, the chemokine receptor groups comprise CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, XCR1, CX3CR1, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, and/or CCR10. The chemokine ligand may be associated with CXCR1 comprise CXCL1, CXCL6, CXCL7, and/or CXCL8. The chemokine ligand may be associated with CXCR2 comprise CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, and/or CXCL8. The chemokine ligand associated with CXCR3 may comprise CXCL4, CXCL9, CXCL10, CXCL11, and/or CXCL13. The chemokine ligand associated with CXCR4 comprise CXCL12. The chemokine ligand associated with CXCR5 comprise CXCL13. The chemokine ligand may be associated with CXCR6 comprise CXCL16. The chemokine ligand associated with, CXCR7 comprise CXCL11 and/or CXCL12. The chemokine ligand associated with XCR1 comprise XCL1. The chemokine ligand associated with CX3CR1 comprise CX3CL1. The chemokine ligand associated with CCR1 comprise CCL3, CCL4, CCL5, CCL7, CCL8, CCL13, CCL14, CCL15, CCL16, and/or CCL23. The chemokine ligand associated with CCR2 comprise CCL2, CCL7, CCL8, CCL13, and/or CCL16. The chemokine ligand associated with CCR3 comprise CCL4, CCL5, CCL7, CCL8, CCL11, CCL13, CCL15, CCL16, CCL23, CCL24, CCL26, and/or CCL28. The chemokine ligand associated with CCR4 comprise CCL3, CCL5, CCL17, and/or CCL22. The chemokine ligand associated with CCR5 comprise CCL2, CCL3, CCL4, CCL5, CCL8, CCL11, CCL13, CCL14, and/or CCL16. The chemokine ligand associated with CCR6 comprise CCL20. The chemokine ligand associated with CCR7 comprise CCL19 and/or CCL21. The chemokine ligand associated with CCR8 comprise CCL1, CCL4, CCL16, CCL17, and/or CCL18. The chemokine ligand associated with CCR9 comprise CCL25. The chemokine ligand may be associated with CCR10 comprise CCL27 and/or CCL28.

[0011] A plurality of distinct genes may be used to characterize a tumor, to treat a tumor, or to predict treatment outcome for immune therapy of the tumor, wherein the plurality of distinct genes are associated with respective distinct types of chemokines, and wherein the use comprises a quantification of expression levels of the distinct genes. Once more, suitable genes for such analysis include those noted above, and over-expression or under-expression for each of the distinct genes is preferably determined relative to respective reference ranges, wherein the reference ranges are specific for a specific tumor type and/or a specific patient type.

[0012] Most typically, but not necessarily, the expression level of mRNA is measured via qPCR or RNAseq. In further embodiments, a threshold for determination of over-expression or under expression may be when the quantified expression level exceeds +/- 2SD of the reference range. Most preferably, the reference range is specific for a particular tumor type as classified in ICD10. Furthermore, the mRNA is annotated as expressed if observed in more than two RNAseq reads. [0013] In preferred embodiments, the method disclosed herein may be used to administer a treatment for the tumor. For example, when chemokine ligands associated with CCR6 are overexpressed, a suitable therapy for the tumor would be dendritic cell therapy. In another example, when chemokine ligands associated with CCR6 or CXCR3 are overexpressed, the treatment may be T cell therapy. When chemokine ligands associated with CXCR3 are overexpressed, a suitable treatment is CD8+ T cell therapy. When chemokine ligands associated with CXCR3 are overexpressed, a suitable treatment may be NK cell therapy. Furthermore, when chemokine ligands associated with CCR2 or CCR5 are overexpressed, the treatment may comprise monocyte cell therapy.

[0014] Various types of tumors may be treated using the methods disclosed herein. Non limiting examples include breast cancer, colon cancer, lung cancer, bone and soft tissue cancers including sarcoma, pancreatic cancer, ovarian cancer, brain cancer, prostate cancer, gastric (stomach) cancer, melanoma, esophageal cancer, head and neck cancer, kidney cancer, liver cancer, oral and throat cancer (including thyroid cancer), rectal cancer, bladder cancer,, uterine cancer, soft tissue cancer, cervical cancer, thymic cancer, lymphoma, skin (non-melanoma) cancer, renal pelvis and ureter cancer, mesothelioma, adrenal cancer, biliary tract cancer, testicular cancer, anal cancer, bile duct cancer, vaginal cancer, small intestine cancer, myeloma, vulvar cancer, urethral cancer, ampulla of vater cancer, penile cancer, and/or leukemia.

[0015] Methods of treating a patient having cancer are described herein, comprising tailoring an immune therapy to treat the patient and administering the tailored immune therapy to the patient, wherein generating the tailored immune therapy includes a step of assessing chemokine production in a tumor micro-environment. Most typically, the tailored immune therapy is generated by obtaining whole transcriptomic sequencing data from a single tumor of a patient having cancer; obtaining normalized expression level of each mRNA coding for a chemokine ligand in the tumor; adjusting the normalized expression levels by comparing the expression level of mRNA coding for chemokine ligand in the tumor cells with the expression level of mRNA coding for chemokine ligand in a matched normal cell, to thereby obtain scaled expression values; grouping the scaled expression values, for each chemokine ligand, by their cognate chemokine receptor group; averaging the grouped scaled expression values for each chemokine receptor and determining Z score for each chemokine receptor group; and assessing chemokine production for each chemokine receptor group from the Z score to tailor the immune therapy for the patient.

[0016] Various objects, features, aspects and advantages of the technological advances described herein will become more apparent from the following detailed description of preferred embodiments, along with the accompanying drawing figures in which like numerals represent like components.

Brief Description of the Drawings

[0017] Fig. 1 depicts exemplary chemokine ligand/receptor groups.

[0018] Fig. 2 depicts an exemplary chemokine ligand assessing method disclosed herein.

[0019] Fig. 3 depicts exemplary reference distributions generated after scaling and averaging across ligand for each receptor group. 3(A) CCR10; 3(B) CCR1; 3(C) CCR2; 3(D) CCR3; 3(E) CCR4; 3(F) CCR5; 3(G) CCR6; 3(H) CCR7; 3(1) CCR8; 3(J) CCR9; 3(K) CX3CR1; 3(L) CXCR1; 3(M) CXCR2; 3(N) CXCR3; 3(0) CXCR4; 3(P) CXCR5; 3(Q) CXCR6; 3(R) CXCR7; 3(S) XCR1;

[0020] Fig. 4 illustrates exemplary inferred ligand activities for each receptor sub-grouped by tissue type. [0021] Fig. 5 illustrates exemplary chemokine receptor/ligand pairs associated with tumor destruction.

[0022] Fig. 6 illustrates CXCR3 receptor and its ligands CXCL9, CXCL10, & CXCL11 recruiting CD 8+ T cells.

[0023] Fig. 7 illustrates CXCR3 receptor and its ligands CXCL9, CXCL10, & CXCL11 recruiting TH1 helper cells.

[0024] Fig. 8 illustrates CXCR3 receptor and its ligands CXCL9, CXCL10, & CXCL11 not recruiting NK cells.

[0025] Fig. 9 illustrates CCL20 signaling through CCR6 to recruit Thl7 cells.

[0026] Fig. 10 illustrates chemokine receptor/ligand pairs associated with tumor evasion. CCL, CC- chemokine ligand; CCR, CC-chemokine receptor; CXCL, CXC-chemokine ligand; CXCR, CXC- chemokine receptor.

[0027] Fig. 11 illustrates Tregs recruited through CCR4 & CCR10 receptor activations [0028] Fig. 12 illustrates macrophages recruited by CCL2-CCR2 signaling.

[0029] Fig. 13 is a research report output obtained from the methods disclosed herein.

Detailed Description

[0030] Immune cell signatures can be obtained from a tumor tissue using gene expression signatures that are specific to or at least characteristic for various immune cells. Single-cell experiments defined gene sets that can differentiate between immune-cell types. By observing expression patterns of those gene sets within a tumor sample, it was then possible to determine whether a tumor tissue sample is enriched or suppressed in those cell types.

[0031] Chemokines are secreted in the tumor microenvironment to form a chemoattractant gradient for immune cells to chemotaxically home in on. The present disclosure describes a new tool that uses RNAseq to quantify expression of mRNAs coding for chemokine ligands as a measure of how much chemokine attractant is in the microenvironment. These ligand expression values are grouped by their cognate receptor group. For example, CXCL9 and CXCL10 both bind the CXCR3 receptor, so these two ligands are grouped in the category of CXCR3. In general, there was agreement between this chemokine ligand assessing tool and immune-cell profiling tool. For example, high CXCL9&10 would attract cells that express CXCR3 (i.e. T cells), thus our CXCR3 group should correlate with T cell count. However, the immune cell profiling tool has certain disadvantages that can be overcome by the chemokine assessment tool/method disclosed herein. For example, disconnects between various TME biomarkers such as neoantigen load, immune-cell populations, and checkpoint expression could be explained further by the current disclosure’s method of assessing of chemokine production

[0032] The chemokine receptors and ligands that belong to each of the main chemokine families (namely, the C-, CC-, CXC- and CX3C-chemokine families) are shown in Fig. 1. This figure serves as a basis for grouping ligands into their cognate receptor categories. Categories are in yellow boxes, and their associated ligands used are in black text and/or red text boxes. Groups with no specific receptor and ligands in blue boxes were ignored. It is noted that there is a degree of redundancy in the chemokine superfamily, with many ligands binding different receptors and vice versa (Fig. 1).

[0033] More specifically, chemokine receptor groups slot into the following 19 categories: CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, XCR1, CX3CR1, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, and/or CCR10. Various chemokine ligands associate with each of the above chemokine receptor groups. Exemplary chemokine receptor groups and their associated ligands are shown below: a. chemokine receptor group CXCR1 is associated with CXCL1, CXCL6, CXCL7, and/or CXCL8; b. chemokine receptor group CXCR2 is associated with CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, and/or CXCL8; c. chemokine receptor group CXCR3 is associated with CXCL4, CXCL9, CXCL10, CXCL11, and/or CXCL13; d. chemokine receptor group CXCR4 is associated with CXCL12; e. chemokine receptor group CXCR5 is associated with CXCL13; f. chemokine receptor group CXCR6 is associated with CXCL16; g. chemokine receptor group CXCR7 is associated with CXCL11 and/or CXCL12; h. chemokine receptor group XCR1 is associated with XCL1; i. chemokine receptor group CX3CR1 is associated with CX3CL1; j. chemokine receptor group CCR1 is associated with CCL3, CCL4, CCL5, CCL7, CCL8, CCL13, CCL14, CCL15, CCL16, and/or CCL23; k. chemokine receptor group CCR2 is associated with CCL2, CCL7, CCL8, CCL13, and/or

CCL16; l. chemokine receptor group CCR3 is associated with CCL4, CCL5, CCL7, CCL8, CCL11, CCL13, CCL15, CCL16, CCL23, CCL24, CCL26, and/or CCL28; m. chemokine receptor group CCR4 is associated with CCL3, CCL5, CCL17, and/or CCL22; n. chemokine receptor group CCR5 is associated with CCL2, CCL3, CCL4, CCL5, CCL8, CCL11, CCL13, CCL14, and/or CCL16; o. chemokine receptor group CCR6 is associated with CCL20; p. chemokine receptor group CCR7 is associated with CCL19 and/or CCL21 ; q. chemokine receptor group CCR8 is associated with CCL1, CCL4, CCL16, CCL17, and/or CCL18; r. chemokine receptor group CCR9 is associated with CCL25; s. chemokine receptor group CCR10 is associated with CCL27 and/or CCL28.

[0034] Fig. 2 shows a schematic of the method disclosed herein for assessing chemokine production in a tumor micro-environment. In this example, RNA-Seq, also called RNA sequencing, is used to determine the expression level of various cytokine ligand mRNAs. RNAseq may determine the presence and quantity of RNA in a biological sample at a given moment, analyzing the continuously changing tumor microenvironment and cellular transcriptome.

[0035] First, normalized expression levels (i.e. log2[TPM+l]) were determined for the 40 ligands included (4 shown). Transcripts Per Million (TPM) is a normalization method for RNA-seq, should be read as “for every 1,000,000 RNA molecules in the RNA-seq sample, x came from this gene/transcript.” Next, scales were adjusted by subtracting the min value observed in the reference set and dividing by the range in the reference set. Scaled expression values are then averaged across all ligands in a receptor group. Finally, the Z-score for is provided for each receptor group based on comparison to the reference set. The chemokine production for each chemokine receptor group is then assessed from the Z score.

[0036] Fig. 3 illustrates the reference distributions generated after scaling and averaging across ligand for each receptor group, binned by tumor tissue types (x-axis). Tumor tissue-types are sorted by frequency. These give a sense of differential chemokine activity among various tumor tissues. Fig. 3(a-s) illustrates results on the following chemokine receptor groups: CCR10; CCR1; CCR2; CCR3; CCR4; CCR5; CCR6; CCR7; CCR8; CCR9; CX3CR1 ; CXCR1 ; CXCR2; CXCR3; CXCR4; CXCR5; CXCR6; CXCR7; and XCR1. The tumor tissue results in Fig. 3 comprise breast cancer, colon cancer, lung cancer, bone and soft tissue cancers including sarcoma, pancreatic cancer, ovarian cancer, brain cancer, prostate cancer, gastric (stomach) cancer, melanoma, esophageal cancer, head and neck cancer, kidney cancer, liver cancer, oral and throat cancer (including thyroid cancer), rectal cancer, bladder cancer,, uterine cancer, soft tissue cancer, cervical cancer, thymic cancer, lymphoma, skin (non-melanoma) cancer, renal pelvis and ureter cancer, mesothelioma, adrenal cancer, biliary tract cancer, testicular cancer, anal cancer, bile duct cancer, vaginal cancer, small intestine cancer, myeloma, vulvar cancer, urethral cancer, ampulla of vater cancer, penile cancer, and/or leukemia. [0037] Inferred activity of ligands for each receptor sub-grouped by tumor tissue type are illustrated in Fig. 4. This figure provides a standardized map for which chemokines may be most active in which tissues, which may be helpful for targeting Immuno- Oncology (10) agents to indications.

[0038] Fig. 5 illustrates chemokine receptor/ligand pairs associated with tumor destruction. Specific chemokine receptors are primarily expressed on the surface of specific immune cell types. In this figure, the immune cell types that are recruited to attack and destroy cells producing neoantigens such as tumoral neoplasms are illustrated. CXC-chemokine receptor 3 (CXCR3) and its ligands CXC- chemokine ligand 9 (CXCL9) and CXCL10 have a key role in driving the trafficking of THl cells, CD8+ T cells and NK cells into the tumor microenvironment, whereas CC-chemokine ligand 20 (CCL20) signaling through CC-chemokine receptor 6 (CCR6) promotes the recruitment of THl 7 cells. Antigen-presenting cells (APCs) such as macrophages and dendritic cells are also recruited into the tumor microenvironment, and they can activate and expand the local effector immune cells, thereby promoting tumor regression.

[0039] Immune cells with antitumor effects (e.g., CD8+ T cells, T helper 1 (THl) cells, polyfunctional THl 7 cells, & natural killer (NK) cells) recruited to the tumor microenvironment through chemokine- chemokine receptor signaling pathways. Figs. 6-9 show correlation between chemokine assessment and RNAseq-based immune deconvolution results for these pairs. Fig. 6 shows significant correlation between inferred CD8+ T-cell activity (y-axis) and inferred CXCR3 ligand levels (x-axis). Similarly, Fig. 7 shows significant correlation between inferred Thl activity (y-axis) and inferred CXCR3 ligand levels (x-axis). Fig. 8 shows that CXCR3 receptor and its ligands CXCL9,CXCL10 and CXCL11 do not play an important role in recruiting NK cells. Comparison of inferred NK activity (y-axis) vs. inferred CXCR3 ligand levels (x-axis). While this correlation is significant, the overall correlation is low. Fig 9 shows weak positive correlation between inferred Thl 7 activity (y-axis) and inferred CCR6 ligand levels (x-axis), suggesting other mechanisms of Thl 7 recruitment. [0040] Fig 10 illustrates chemokine receptor/ligand pairs associated with tumor evasion. Immune cell populations such as granulocytic and monocytic myeloid-derived suppressor cells (MDSCs), regulatory T (Treg) cells, IL-22+CD4+ T helper 22 (TH22) cells, IL-22+ innate lymphoid cells (ILCs) and plasmacytoid dendritic cells (pDCs) can promote tumor growth. These cells recruit to the tumor microenvironment in response to different chemokines in the tumor microenvironment. Relevant receptors and ligands are shown in Fig. 10. Pro-tumor immune cells inhibit anti-tumor immune responses and promote cancer sternness and angiogenesis, leading to cancer progression. Specific chemokine receptors are primarily expressed on the surface of specific immune cell types.

[0041] Figs. 11 & 12 show varying levels of correlation between chemokine assessment and RNAseq- based immune deconvolution results for these pairs. Fig 11 shows that Tregs are recruited through CCR4 & CCR10 receptor activations. Comparison of inferred Treg activity (y-axis) vs. inferred CCR4 & CCR10 ligand levels (x-axes) shows high correlation between CCR4 and Treg activity, more so than CCR10. Fig 12 shows that macrophages are recruited by CCL2-CCR2 signaling. Comparison of inferred macrophage activity (y-axis) vs. inferred CCR2 ligand levels (x-axes) shows high correlation between CCR2 and macrophage activity. Table 1 illustrate reference min-max values, for scaling novel samples to the expected space.

Table 1

CXCL13 0 12.22

[0042] Furthermore, reference mean and standard deviation values for scoring novel samples are provided below in Table 2.

[0043] In one embodiment, a panel of 40 ligands that bind to 19 chemokine receptor categories were used as the basis of an analysis using the methods disclosed herein to assess chemokines. Each of the 40 ligands and expression values (as TPMs) are first scaled using reference minimum and maximum values defined from a set of 1479 unselected clinical cases. These scaled ligand expression scores are then condensed to an activity per receptor, by averaging across ligands associated with each receptor. Additionally, these mean average scaled activity scores of the 19 chemokine signatures are compared to similar samples based on ICD10 category, when available, to infer if activation is over or under the expected range for the cancer’s tissue type. Fig. 13 illustrates an output of this method. Sample- specific high/normal/low calls, and z-scores, are given for each receptor group. A shout-out is included in the top of the matter that highlights how many ligand groups are elevated. In this case, it is noted that CCR8 is elevated.

[0044] Immune cell specific gene expression analysis can be used in predictive analysis of immune therapy, particularly for immune therapy targeting chemokines. Methods and analyses herein may also be useful in determination of suitable treatment where location may provide a contributing factor. For example, upper and lower GI tumors are distinct in their tolerated immune cell infiltration. Immune therapies should therefore be tailored based on location to take advantage of the innate immune apparatus present. Specifically, upper GI cancers appear especially fit for checkpoint therapy despite having lower average TMB.

[0045] As used in the description herein and throughout the claims that follow, the meaning of “a,” “an,” and “the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of “in” includes “in” and “on” unless the context clearly dictates otherwise. Unless the context dictates the contrary, all ranges set forth herein should be interpreted as being inclusive of their endpoints, and open-ended ranges should be interpreted to include commercially practical values. Similarly, all lists of values should be considered as inclusive of intermediate values unless the context indicates the contrary.

[0046] Moreover, all methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the presently claimed invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the presently claimed invention. [0047] Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience and/or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all Markush groups used in the appended claims. [0048] Those skilled in the art know that many more modifications besides those already described are possible without departing from the inventive concepts herein. The inventive subject matter, therefore, is not to be restricted except in the scope of the appended claims. Moreover, in interpreting both the specification and the claims, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms “comprises” and “comprising” should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced. Where the specification claims refers to at least one of something selected from the group consisting of A, B, C ... . and N, the text should be interpreted as requiring only one element from the group, not A plus N, or B plus N, etc.

Claims

CLAIMS What is claimed is:

1. A method of assessing chemokine production in a tumor micro-environment, comprising: obtaining whole transcriptomic sequencing data from a single tumor of a patient having cancer; obtaining normalized expression level of each mRNA coding for a plurality of chemokine ligands expressed in the tumor; adjusting the normalized expression levels by comparing the expression level of chemokine ligands in the tumor cells with the expression level of chemokine ligands in a matched normal cell, to thereby obtain scaled expression values; grouping the scaled expression values, for each chemokine ligand, by their cognate chemokine receptor group; averaging the grouped scaled expression values for each chemokine receptor and determining Z score for each chemokine receptor group; and assessing chemokine production for each chemokine receptor group from the Z score.

2. The method of claim 1, wherein the chemokine receptor groups comprise CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, XCR1, CX3CR1, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, and/or CCR10.

3. The method of claim 2, wherein the chemokine ligand associated with CXCR1 comprise CXCL1, CXCL6, CXCL7, and/or CXCL8.

4. The method of claim 2, wherein the chemokine ligand associated with CXCR2 comprise CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, and/or CXCL8.

5. The method of claim 2, wherein the chemokine ligand associated with CXCR3 comprise CXCL4, CXCL9, CXCL10, CXCL11, and/or CXCL13.

6. The method of claim 2, wherein the chemokine ligand associated with CXCR4 comprise CXCL12.

7. The method of claim 2, wherein the chemokine ligand associated with CXCR5 comprise CXCL13.

8. The method of claim 2, wherein the chemokine ligand associated with CXCR6 comprise CXCL16.

9. The method of claim 2, wherein the chemokine ligand associated with, CXCR7 comprise CXCL11 and/or CXCL12.

10. The method of claim 2, wherein the chemokine ligand associated with XCR1 comprise XCL1.

11. The method of claim 2, wherein the chemokine ligand associated with CX3CR1 comprise CX3CL1.

12. The method of claim 2, wherein the chemokine ligand associated with CCR1 comprise CCL3, CCL4, CCL5, CCL7, CCL8, CCL13, CCL14, CCL15, CCL16, and/or CCL23.

13. The method of claim 2, wherein the chemokine ligand associated with CCR2 comprise CCL2, CCL7, CCL8, CCL13, and/or CCL16.

14. The method of claim 2, wherein the chemokine ligand associated with CCR3 comprise CCL4, CCL5, CCL7, CCL8, CCL11, CCL13, CCL15, CCL16, CCL23, CCL24, CCL26, and/or CCL28.

15. The method of claim 2, wherein the chemokine ligand associated with CCR4 comprise CCL3, CCL5, CCL17, and/or CCL22.

16. The method of claim 2, wherein the chemokine ligand associated with CCR5 comprise CCL2, CCL3, CCL4, CCL5, CCL8, CCL11, CCL13, CCL14, and/or CCL16.

17. The method of claim 2, wherein the chemokine ligand associated with CCR6 comprise CCL20.

18. The method of claim 2, wherein the chemokine ligand associated with CCR7 comprise CCL19 and/or CCL21.

19. The method of claim 2, wherein the chemokine ligand associated with CCR8 comprise CCL1, CCL4, CCL16, CCL17, and/or CCL18.

20. The method of claim 2, wherein the chemokine ligand associated with CCR9 comprise CCL25.

21. The method of claim 2, wherein the chemokine ligand associated with CCR10 comprise CCL27 and/or CCL28.

22. The method of any one of the preceding claims, wherein the expression level of mRNA is determined by RNAseq.

23. The method of any one of the preceding claims, wherein the mRNA is annotated as expressed if observed in more than two RNAseq reads.

24. The method of any one of the preceding claims, further comprising a step of administering a treatment for the tumor.

25. The method of claim 24, wherein the treatment is dendritic cell therapy when chemokine ligands associated with CCR6 are overexpressed.

26. The method of claim 24, wherein the treatment is T cell therapy when chemokine ligands associated with CCR6 or CXCR3 are overexpressed.

27. The method of claim 24, wherein the treatment is CD8+ T cell therapy when chemokine ligands associated with CXCR3 are overexpressed.

28. The method of claim 24, wherein the treatment is NK cell therapy when chemokine ligands associated with CXCR3 are overexpressed.

29. The method of claim 24, wherein the treatment is monocyte cell therapy when chemokine ligands associated with CCR2 or CCR5 are overexpressed.

30. The method of claim 24, wherein the tumor is breast cancer, colon cancer, lung cancer, bone and soft tissue cancers including sarcoma, pancreatic cancer, ovarian cancer, brain cancer, prostate cancer, gastric (stomach) cancer, melanoma, esophageal cancer, head and neck cancer, kidney cancer, liver cancer, oral and throat cancer (including thyroid cancer), rectal cancer, bladder cancer,, uterine cancer, soft tissue cancer, cervical cancer, thymic cancer, lymphoma, skin (non melanoma) cancer, renal pelvis and ureter cancer, mesothelioma, adrenal cancer, biliary tract cancer, testicular cancer, anal cancer, bile duct cancer, vaginal cancer, small intestine cancer, myeloma, vulvar cancer, urethral cancer, ampulla of vater cancer, penile cancer, and/or leukemia.

31. A method of assessing chemokine production in a tumor micro-environment, comprising: quantifying expression of mRNAs coding for chemokine ligands; and assessing the amount and type of chemokine ligands present in the tumor micro environment from the expression level of mRNAs that code for chemokine ligands, and wherein the mRNA expression is quantified by the steps of: obtaining whole transcriptomic sequencing data from a single tumor of a patient having cancer; identifying, from the whole transcriptomic sequencing data, presence and/or activity of mRNAs coding for a plurality of chemokine ligands in the tumor; identifying, from the whole transcriptomic sequencing data, expression level of mRNAs coding for chemokine ligands in the tumor; grouping the chemokine ligand expression values by their cognate chemokine receptor group

32. The method of claim 31, wherein the chemokine receptor groups comprise CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, XCR1, CX3CR1, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, and/or CCR10.

33. The method of claim 32, wherein the chemokine ligand associated with CXCR1 comprise CXCL1, CXCL6, CXCL7, and/or CXCL8; wherein the chemokine ligand associated with CXCR2 comprise CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL7, and/or CXCL8; wherein the chemokine ligand associated with CXCR3 comprise CXCL4, CXCL9, CXCL10, CXCL11, and/or CXCL13; wherein the chemokine ligand associated with CXCR4 comprise CXCL12; wherein the chemokine ligand associated with CXCR5 comprise CXCL13; wherein the chemokine ligand associated with CXCR6 comprise CXCL16; wherein the chemokine ligand associated with, CXCR7 comprise CXCL11 and/or CXCL12; wherein the chemokine ligand associated with XCR1 comprise XCL1; wherein the chemokine ligand associated with CX3CR1 comprise CX3CL1; wherein the chemokine ligand associated with CCR1 comprise CCL3, CCL4, CCL5, CCL7, CCL8, CCL13, CCL14, CCL15, CCL16, and/or CCL23; wherein the chemokine ligand associated with CCR2 comprise CCL2, CCL7, CCL8, CCL13, and/or CCL16; wherein the chemokine ligand associated with CCR3 comprise CCL4, CCL5, CCL7, CCL8, CCL11, CCL13, CCL15, CCL16, CCL23, CCL24, CCL26, and/or CCL28; wherein the chemokine ligand associated with CCR4 comprise CCL3, CCL5, CCL17, and/or CCL22; wherein the chemokine ligand associated with CCR5 comprise CCL2, CCL3, CCL4, CCL5, CCL8, CCL11, CCL13, CCL14, and/or CCL16; wherein the chemokine ligand associated with CCR6 comprise CCL20; wherein the chemokine ligand associated with CCR7 comprise CCL19 and/or CCL21; wherein the chemokine ligand associated with CCR8 comprise CCL1, CCL4, CCL16, CCL17, and/or CCL18; wherein the chemokine ligand associated with CCR9 comprise CCL25; and/or wherein the chemokine ligand associated with CCR10 comprise CCL27 and/or CCL28.

34. The method of any one of claims 31-33, wherein the expression level of mRNA is determined by RNAseq.

35. The method of any one of claims 31-34, wherein the mRNA is annotated as expressed if observed in more than two RNAseq reads.

36. The method of any one of claims 31-35, further comprising a step of administering a treatment for the tumor.

37. The method of claim 36, wherein the tumor is breast cancer, colon cancer, lung cancer, bone and soft tissue cancers including sarcoma, pancreatic cancer, ovarian cancer, brain cancer, prostate cancer, gastric (stomach) cancer, melanoma, esophageal cancer, head and neck cancer, kidney cancer, liver cancer, oral and throat cancer (including thyroid cancer), rectal cancer, bladder cancer, uterine cancer, soft tissue cancer, cervical cancer, thymic cancer, lymphoma, skin (non melanoma) cancer, renal pelvis and ureter cancer, mesothelioma, adrenal cancer, biliary tract cancer, testicular cancer, anal cancer, bile duct cancer, vaginal cancer, small intestine cancer, myeloma, vulvar cancer, urethral cancer, ampulla of vater cancer, penile cancer, and/or leukemia.