Using 12 chemokine signature to select sting agonist and til treatments for solid tumors

Abstract

Disclosed is a method for treating a tumor in a subject that involves detecting in a biopsy sample from the subject reduced expression of a 12 Chemokine gene signature; and then administering to the subject a demethylating agent in an amount effective to demethylate STING proteins in the tumor cells. The method can further involve administering to the subject a therapeutically effective amount of a STING agonist. The method can further involve administering to the subject tumor infiltrating lymphocytes (TILs), such as HLAmatched TILs.

Description

USING 12 CHEMOKINE SIGNATURE TO SELECT STING AGONIST AND TIL TREATMENTS FOR SOLID TUMORSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This claims benefit of U.S. Provisional Application No. 63 / 520,125, filed August 17, 2023, and U.S. Provisional Application No. 63 / 520,415, filed August 18, 2023, which are hereby incorporated herein by reference in their entireties.BACKGROUND OF THE INVENTION

[0002] Stimulator of interferon genes (STING) is an endoplasmic reticulum-resident signaling molecule. It is responsible for controlling the transcription of several host defense genes, including type I IFNs and pro-inflammatory cytokines, in response to recognition of cytosolic DNA species or cyclic dinucleotides. Recent studies have indicated that STING signaling is the major innate immune pathway involved in the generation of a spontaneous antitumor T cell response. Mice lacking STING cannot generate efficient antitumor T cell responses and reject melanoma tumor growth. Based on this finding, many STING agonists have been developed to utilize STING activation as a cancer therapy. Such agonists have been found to be experimentally useful in inducing robust tumor control through the host immune cell activation. While STING activation has been extensively investigated in antigen presenting cells (in particular dendritic cells), little is known regarding its activation in tumor cells.SUMMARY OF THE INVENTION

[0003] As disclosed herein, STING expression in tumors positively correlates with a 12 Chemokine gene signature comprising CCL2, CCL3, CCL4, CCL5, CCL8, CCL18, CCL19, CCL21 , CXCL9, CXCL10, CXCL11 , and CXCL13. STING activation leads to more tertiary lymphoid structures (TLSs) which in turn can have an anti-tumor effect. So, adding a demethylating agent to restore and activate STING to get more TLS can inhibit a tumor.

[0004] Therefore, disclosed herein is a method for treating a tumor in a subject that involves detecting in a biopsy sample from the subject reduced expression levels of CCL2, CCL3, CCL4, CCL5, CCL8, CCL18, CCL19, CCL21 , CXCL9, CXCL10, CXCL11 , and CXCL13; and then administering to the subject a demethylating agent in an amount effective to demethylate STING proteins in the tumor cells. The method can further involve administering to the subject a therapeutically effective amount of a STING agonist. The method can further involve administering to the subject tumor infiltrating lymphocytes (TILs), such as HLA-matched TILs.

[0005] Alternatively, the method can involve detecting in a biopsy sample from the subject normal or elevated expression levels of CCL2, CCL3, CCL4, CCL5, CCL8, CCL18, CCL19, CCL21 , CXCL9, CXCL10, CXCL11, and CXCL13, and administering to the subject atherapeutically effective amount of a STING agonist, tumor infiltrating lymphocytes, or a combination thereof, i.e. without the demethylating agent.

[0006] Also disclosed is a method of treating a cancer in a subject that involves detecting in a tumor sample from the subject reduced expression levels of CCL2, CCL3, CCL4, CCL5, CCL8, CCL18, CCL19, CCL21 , CXCL9, CXCL10, CXCL11 , and CXCL13 and then administering to the subject a therapeutically effective amount of enhanced TILs. The enhanced TILs can be produced according to a method that involves culturing a tumor sample from a subject in the presence of i) a demethylating agent in an amount effective to demethylate STING proteins in the tumor cells and ii) a STING agonist in an amount effective to increase the antigenicity of the tumor cells and / or upregulating expression of MHC molecules, and then exposing TILs to these modified tumor cells to enhance their function (i.e. killing and cytokine production) and further expansion for subsequent adoptive transfer to the cancer patient. In preferred embodiments, the TILs are HLA matched to the tumor sample from the subject.

[0007] In some embodiments, the tumor of the disclosed compositions and methods is a solid tumor. In some cases, the tumor is a melanoma, ovarian cancer, breast cancer, or colorectal cancer. The cancer can be metastatic, recurrent, or a combination thereof. In some embodiments, these TILs are administered back to the subject. In some of these embodiments, the subject is treated with a demethylating agent and a STING agonist.

[0008] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. BRIEF DESCRIPTION OF FIGURES

[0009] FIG. 1 shows STING is positively correlated with the chemokines present in the 12-chemokine gene signature TLS classifier in melanoma. N = 21 .

[0010] FIG. 2 shows STING is positively correlated with the chemokines present in the 12-chemokine gene signature TLS classifier in urothelial bladder carcinoma. N = 129.

[0011] FIG. 3 shows STING is positively correlated with the secondary lymphoid organ-associated chemokines in melanoma. N = 21.

[0012] FIG. 4 shows STING is positively correlated with the lymphoid-associated adhesion molecules in melanoma. N = 21.

[0013] FIG. 5 shows STING is positively correlated with the lymphoid-associated adhesion molecules in urothelial bladder carcinoma. N = 129.

[0014] FIG. 6 shows STING is positively correlated with the markers of B cellmemory and TLS maturity in melanoma. N = 21.

[0015] FIGs. 7A and 7B show the correlation between the methylation level of cGAS (x-axis) and the 12-chemokine score (y-axis) across all TCGA tumors and normal samples(FIG. 7A). There was a negative correlation, r=-0.365, indicating that samples with high STING methylation have a lower 12-chemokine score. This was further confirmed when the gene expression of cGAS was compared with 12-Chemokine score (FIG. 7B). There was a similar degree of correlation, r=0.457 between cGAS expression and 12-chemokine score across the TCGA samples. Taken together this would indicate that samples with low cGAS expression also show a lower 12-chemokine score.

[0016] FIGs. 8A and 8B show the correlation between the methylation level of STING (x-axis) and the 12-chemokine score (y-axis) across all TCGA tumors and normal samples (FIG. 8A). There was a negative correlation, r=-0.37, indicating that samples with high STING methylation have a lower 12-chemokine score. This was especially true for samples with high degree of STING methylation show low 12-chemokine score, marked in rectangle. This was further confirmed when we compare the gene expression of STING with 12-Chemokine score (FIG. 8B). There was a similar degree of correlation, r=0.38 between STING expression and 12-chemokine score across the TCGA samples. Taken together this would indicate that samples with low STING expression also show a lower 12-chemokine score.

[0017] FIG. 9 shows a correlation between melanomas that express STING and the presence of TLSs.

[0018] FIG. 10 shows STING is positively correlated with the chemokines present in the 12-chemokine gene signature TLS classifier in Lung Squamous Cell Carcinoma. N=80.

[0019] FIG. 11 shows STING is positively correlated with the chemokines present in the 12-chemokine gene signature TLS classifier in Prostate Adenocarcinoma. N=65. DETAILED DESCRIPTION

[0020] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0021] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0022] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0023] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed.

[0024] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0025] Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of chemistry, biology, and the like, which are within the skill of the art.

[0026] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the probes disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.

[0027] Before the embodiments of the present disclosure are described in detail, it is to be understood that, unless otherwise indicated, the present disclosure is not limited to particular materials, reagents, reaction materials, manufacturing processes, or the like, as such can vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It is also possible in the present disclosure that steps can be executed in different sequence where this is logically possible.

[0028] It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise.

[0029] The term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0030] The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician.

[0031] The term “therapeutically effective” refers to the amount of the composition used is of sufficient quantity to ameliorate one or more causes or symptoms of a disease or disorder. Such amelioration only requires a reduction or alteration, not necessarily elimination.

[0032] The term “treatment” refers to the medical management of a patient with the intent to cure, ameliorate, stabilize, or prevent a disease, pathological condition, or disorder. This term includes active treatment, that is, treatment directed specifically toward the improvement of a disease, pathological condition, or disorder, and also includes causal treatment, that is, treatment directed toward removal of the cause of the associated disease, pathological condition, or disorder. In addition, this term includes palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disease, pathological condition, or disorder; preventative treatment, that is, treatment directed to minimizing or partially or completely inhibiting the development of the associated disease, pathological condition, or disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the associated disease, pathological condition, or disorder.Chemokine gene signature

[0033] Disclosed herein are Tumor-infiltrating lymphocytes (TILs) with enhanced tumor reactivity produced from donor melanoma cells with tertiary lymphoid structures (TLSs) that express the herein disclosed chemokine gene signature.

[0034] Chemokines, which are small protein molecules involved in immune and inflammatory responses, direct leukocyte trafficking to areas of injury as well as to locations where primary immune responses are initiated (secondary lymphoid tissues such as lymph nodes, spleen, Peyer's patches, and tonsils). There are presently four classes of chemokine molecules (C, CC, CXC, and CX3C) that are named for the number and location of cysteineresidues on the amino terminus of the protein. These molecules communicate with their target cells via G-protein coupled receptors that are pertussis toxin sensitive. Different chemokines act on different leukocyte populations, thereby modulating the influx of immune effector cells to the area in question based on the needs of the particular situation. Chemokines are secreted proteins involved in immunoregulatory and inflammatory processes. The chemokines of the disclosed gene signature are shown in Table 1.

[0035] In some embodiments, the methods include assaying the presence or levels of chemokine mRNA or proteins in the sample. The presence and / or level of a protein can be evaluated using methods known in the art, e.g., using quantitative immunoassay methods. The presence and / or level of an mRNA can be evaluated using methods known in the art, e.g., Northern blotting or quantitative PCR methods, e.g., RT-PCR. In some embodiments, high throughput methods, e.g., protein or gene chips as are known in the art (see, e.g, Ch. 12, Genomics, in Griffiths et al., Eds. Modern genetic Analysis, 1999, W. H. Freeman and Company; Ekins and Chu, Trends in Biotechnology, 1999, 17:217-218; MacBeath and Schreiber, Science 2000, 289(5485): 1760- 1763; Simpson, Proteins and Proteomics: A Laboratory Manual, Cold Spring Harbor Laboratory Press; 2002;Hardiman, Microarrays Methods and Applications: Nuts & Bolts, DNA Press, 2003), can be used to detect the presence and / or level of chemokine proteins as described herein.

[0036] In some embodiments, the methods include assaying levels of one or more control genes or proteins, and comparing the level of expression of the chemokine genes or proteins to the level of the control genes or proteins, to normalize the levels of the chemokine genes or proteins. Suitable endogenous control genes includes a gene whose expression level should not differ between samples, such as a housekeeping or maintenance gene, e.g., 18S ribosomal RNA; beta Actin; Glyceraldehyde-3-phosphatedehydrogenase; Phosphoglycerate kinase 1 ; Peptidylprolyl isomerase A (cyclophilin A); Ribosomal protein L13a; large Ribosomal protein PO; Beta-2-microglobulin; Tyrosine 3- monooxygenase / tryptophan 5-monooxygenase activation protein, zeta polypeptide; Succinate dehydrogenase; Transferrin receptor (p90, CD71); Aminolevulinate, delta-, synthase 1 ; Glucuronidase, beta; Hydroxymethyl-bilane synthase; Hypoxanthine phosphoribosyltransferase 1 ; TATA box binding protein; and / or Tubulin, beta polypeptide.

[0037] Generally speaking, the methods described herein can be performed on cells from a tumor. The cells can be obtained by known methods, e.g., during a biopsy (such as a core needle biopsy), or during a surgical procedure to remove all or part of the tumor. The cells can be used fresh, frozen, fixed, and / or preserved, so long as the mRNA or protein that is to be assayed is maintained in a sufficiently intact state to allow accurate analysis.

[0038] In some embodiments of the methods described herein, the levels of the chemokine genes in the tumor sample can be compared individually to levels in a reference. The reference levels can represent levels in a melanoma that does not have tertiary lymphoid structures (TLSs). Alternatively, reference levels can represent levels in a melanoma that doe shave TLSs. In some embodiments, the reference levels represent a threshold.

[0039] In some embodiments of the methods described herein, values representing the levels of the chemokine genes can be summed to produce a “chemokine gene score” that can be compared to a reference chemokine gene score, wherein a chemokine gene score that is above the reference chemokine gene score indicates that the melanoma will produce TILs with enhanced tumor reactivity, and an chemokine gene score below the reference score indicates that the melanoma will produce TILs that do not have enhanced tumor reactivity.

[0040] For example, in some embodiments, the expression levels of each of the evaluated genes can be assigned a value (e.g., a value that represents the expression level of the gene, e.g., normalized to an endogenous control gene as described herein). That value (optionally weighted to increase or decrease its effect on the final score) can be summed to produce an immune-related gene score. One of skill in the art could optimize such a method to determine an optimal algorithm for determining an immune-related gene score.

[0041] The methods described herein can include determining levels (or scores) for all of the 12 chemokines. In some embodiments all of the genes are evaluated, but in some embodiments a subset of one or all of the sets is evaluated.Demethylating Agents

[0042] In some embodiments, the disclosed methods involve treating the subject with a demethylating agent. Cytidine analogs with demethylating activity are known.Examples of cytidine analogs with demethylating activity include, but are not limited to, 5- Azacytidine, 5,6-dihydro-5-azacytidine, 1-p-D-arabinofuranosyl-5-azacytidine, 5-Aza-2'- deoxycytidine (decitabine) or 1-(beta-D-ribofuranosyl)-1 ,2dihydropropyrimindin-2-one (zebularine). Additional examples of demethylating agents include 5-aza-cytidine (5-aza-C), 5-aza-2’-deoxycytidine (5-aza-dC or decitabine), 5-fluoro-2’-deoxycytidine (5-F-dC), Pseudoisocytidine, 2-H pyrimidinone-1-b-D (2’-deoxyriboside) (Zebularine), Guadecitabine (SGI-110), and Disulfiram.STING Agonists

[0043] In some aspects, the disclosed methods involve treating the subject with a cyclic GMP-AMP Synthase (cGAS)ZStimulator of Interferon Genes (STING) pathway agonist. In some aspects, the cGAS / STING pathway agonist is 2'3'-cyclic-GM -AM (2'3'-cGAMP). Additional STING agonists are known in the art, including those disclosed in U.S. Patent Publication No. 2016 / 0287623 and U.S. Patent Publication No. 2017 / 0044206, which are incorporated by reference herein for these agonists. In some aspects, the STING agonist is a natural cyclic dinucleotide (CDN), such as 2’3’-cGAMP, 3’3’-cGAMP, c-di-AMP, or c-di- GMP. In some aspects, the STING agonist is a cAIM-derived CDN, such as cAIMP, cAIMP Difluor, or cAIM(PS)2 Difluor (Rp / Sp). In some aspects, the STING agonist is a cGAMP- derived CDN, such as 2’2’-cGAMP, 2’3’-cGAM(PS)2 (Rp / Sp), or 3'3'-cGAMP Fluorinated. In some aspects, the STING agonist is a c-di-AMP-derived CDN, such as c-di-AMP Fluorinated, 2'3'-c-di-AMP, or 2’3’-c-di-AM(PS)2 (Rp,Rp). In some aspects, the STING agonist is a c-di-GMP-derived CDN, such as c-di-GMP Fluorinated or 2’3’-c-di-GMP. In some aspects, the STING agonist is a c-di-IMP-derived CDN, such as c-di-IMP. In some aspects, the STING agonist is a xanthenone analog, such as DMXAA. In some aspects, the STING agonist is ADU-S100 (MIW815, Aduro Biotech). In some aspects, the STING agonist is MK-1454 (Merck).

[0044] A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.EXAMPLESExample 1: STING and Tertiary Lymphoid Structures

[0045] FIG. 1 shows STING is positively correlated with the chemokines present in the 12-chemokine gene signature TLS classifier in melanoma. N = 21 .

[0046] FIG. 2 shows STING is positively correlated with the chemokines present in the 12-chemokine gene signature TLS classifier in urothelial bladder carcinoma. N = 129.

[0047] FIG. 3 shows STING is positively correlated with the secondary lymphoid organ-associated chemokines. N = 21.

[0048] FIG. 4 shows STING is positively correlated with the lymphoid-associated adhesion molecules. N = 21.

[0049] FIG. 5 shows STING is positively correlated with the lymphoid-associated adhesion molecules. N = 129.

[0050] FIG. 6 shows STING is positively correlated with the markers of B cellmemory and TLS maturity. N = 21.

[0051] FIGs. 7A and 7B show the correlation between the methylation level of cGAS (x-axis) and the 12-chemokine score (y-axis) across all TOGA tumors and normal samples (FIG. 7A). There was a negative correlation, r=-0.365, indicating that samples with high STING methylation have a lower 12-chemokine score. This was further confirmed when the gene expression of cGAS was compared with 12-Chemokine score (FIG. 7B). There was a similar degree of correlation, r=0.457 between cGAS expression and 12-chemokine score across the TCGA samples. Taken together this would indicate that samples with low cGAS expression also show a lower 12-chemokine score.

[0052] FIGs. 8A and 8B show the correlation between the methylation level of STING (x-axis) and the 12-chemokine score (y-axis) across all TCGA tumors and normal samples (FIG. 8A). There was a negative correlation, r=-0.37, indicating that samples with high STING methylation have a lower 12-chemokine score. This was especially true for samples with high degree of STING methylation show low 12-chemokine score, marked in rectangle. This was further confirmed when we compare the gene expression of STING with 12-Chemokine score (FIG. 8B). There was a similar degree of correlation, r=0.38 between STING expression and 12-chemokine score across the TCGA samples. Taken together this would indicate that samples with low STING expression also show a lower 12-chemokine score.

[0053] FIG. 9 shows a correlation between melanomas that express STING and the presence of TLSs.

[0054] FIG. 10 shows STING is positively correlated with the chemokines present in the 12-chemokine gene signature TLS classifier in lung squamous cell carcinoma. N = 80.

[0055] FIG. 11 shows STING is positively correlated with the chemokines present in the 12-chemokine gene signature TLS classifier in prostate adenocarcinoma. N = 65.

[0056] Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosedinvention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

[0057] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

CLAIMS1. A method for treating a solid tumor in a subject, comprising(b) detecting in a biopsy sample from the subject elevated expression levels of CCL2, CCL3, CCL4, CCL5, CCL8, CCL18, CCL19, CCL21 , CXCL9, CXCL10, CXCL11 , and CXCL13, and administering to the subject a therapeutically effective amount of a STING agonist, tumor infiltrating lymphocytes, or a combination thereof; OR(b) detecting in a biopsy sample from the subject reduced expression levels of CCL2, CCL3, CCL4, CCL5, CCL8, CCL18, CCL19, CCL21 , CXCL9, CXCL10, CXCL11 , and CXCL13, and administering to the subject a therapeutically effective amount of a demethylating agent, and administering to the subject a therapeutically effective amount of a STING agonist, tumor infiltrating lymphocytes, or a combination thereof.

2. The method of claim 1 , wherein the demethylating agent comprises 5-aza-2’- deoxycytidine.

3. The method of claim 1 or 2, wherein the STING agonist comprises 2'3'-cyclic-GMP- AMP (2'3'-cGAMP).

4. The method of any one of claims 1 to 3, further comprising administering to the subject a therapeutically effective amount of a composition comprising HLA-matched tumor infiltrating lymphocytes (TILs).

5. The method of any one of claims 1 to 4, wherein the solid tumor is a melanoma.

6. A method for treating a solid tumor in a subject, comprising(a) detecting in a biopsy sample from the subject reduced expression levels of CCL2, CCL3, CCL4, CCL5, CCL8, CCL18, CCL19, CCL21 , CXCL9, CXCL10, CXCL11 , and CXCL13; and(b) administering to the subject a therapeutically effective amount of tumor infiltrating lymphocytes (TILs) produced according to the method comprising:(i) culturing tumor cells from a subject in the presence of 1) a demethylating agent in an amount effective to demethylate STING proteins in the tumor cells and 2) a STING agonist in an amount effective to increase the antigenicity of the tumor cells, and(ii) co-culturing TILs with these treated tumor cells and further expanding the TILs after their exposure.

7. The method of claim 6, wherein the subject has a cancer and wherein the method treats the cancer in the subject.

8. The method of claims 7, wherein the cancer comprises a bladder cancer, breast cancer, prostate cancer, lung cancer, colorectal cancer, melanoma, or ovarian cancer.

9. The method of any one of claims 6 to 8, wherein the solid tumor is a prostate cancer.

10. The method of any one of claims 6 to 8, wherein the solid tumor is a lung cancer.11 . The method of any one of claims 6 to 8, wherein the solid tumor is a melanoma.

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