Transcriptional biomarkers for response to innate immune activators

Abstract

As used herein, the expression level of the vidutolimod core signature, and / or high expression of ELF2, and / or low expression of the GABA B cell signature, and / or T cell signature in the tumor are indicative of a marker of progression-free survival. reg or macrophage / monocyte low levels.Also provided herein are methods and kits for diagnosing cancer patients likely to respond to treatment with TLR9 agonists or other innate immune activators or cancer immunotherapy based on detection of low levels of TLR9 agonists or macrophages / monocytes.Also provided herein are methods for treating cancer, comprising administering a CpG oligonucleotide formulated in a virus-like particle, and optionally further administering a checkpoint inhibitor, such as an anti-PD-1 antibody, an anti-PD-L1 antibody, after determining enrichment of a vidutolimod response signature.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 296,098, filed January 3, 2022, the entire disclosure of which is incorporated herein by reference.

[0002] INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY The computer readable sequence listing, filed concurrently herewith and identified as follows, is incorporated by reference in its entirety: Filename: RGN9-009PC-SL26.xml, Size: 2,125 bytes, Created: December 20, 2022. [Background technology]

[0003] Cancer immunotherapy has generated a considerable amount of excitement, based primarily on the success of immune checkpoint blockade, such as inhibitors of the PD-1 / PD-L1 pathway. Despite this excitement, most patients do not respond to PD-1 blockade, particularly those whose tumors lack an IFN signature. This leads to the evaluation of approaches designed to induce IFN responses, such as innate immune activators, including, for example, intratumoral (IT) delivery of agents that can activate tumor-infiltrating plasmacytoid dendritic cells (pDCs) and thereby enhance tumor-specific T cell responses.

[0004] Synthetic unmethylated CG-rich CpG oligodeoxynucleotides (ODNs) mimic prokaryotic DNA and activate TLR9. Previously, PCT / US2015 / 067269 (published as WO / 2016 / 109310) disclosed the combination of CMP-001 (vidutolimod), a CpG-A ODN formulated in VLPs, with checkpoint inhibitors (CPIs). In patients with early-stage melanoma receiving neoadjuvant treatment with PD-1 blockade, the response rate, including pathological cure, was approximately doubled by the addition of IT vidutolimod (Davar, Diwakar, et al. “303 Phase II trial of neoadjuvant nivolumab (Nivo) and intra-tumoral (IT) CMP-001 in high-risk resectable melanoma (Neo-C-Nivo): final results.” (SITC 2020). In patients with metastatic melanoma resistant to PD-1 blockade, IT vidutolimod induced RECIST v1.1 objective responses in 8 / 40 (20%) patients and in 23 / 98 (23%) patients with the combination of IT vidutolimod and PD-1 blockade (Kirkwood, John, et al. “950 Final analysis: phase 1b study investigating intratumoral injection of toll-like receptor 9 agonist vidutolimod ± pembrolizumab in patients with PD-1 blockade-refractory melanoma.”(SITC 2021).

[0005] There remains a need to improve the efficacy of cancer therapy by identifying patients who are likely to respond to a particular anti-cancer therapy prior to the administration of the therapy.For example, in the field of cancer immunotherapy, it is well established that PD-1 blockade is most effective in treating "hot" or inflamed tumors, as determined by standard assays such as i) IHC for PD-L1 (total positive score; CPS), ii) immunoscore, iii) TIDE (doi.org / 10.1038 / s41591-018-0136-1), iv) IFN18 signature (Ayers, M., et al., J Clin Invest., 2017, 127(8):2930-2940; and WO / 2016 / 094377), and other assays known to those skilled in the art, and that treatment is much less likely to be effective in patients whose tumors are cold, i.e., not inflamed at baseline. These assays for predicting response to PD-1 blockade do not predict response to vidutolimod or other innate immune activators (Luke, Jason John, et al. “Abstract CT032:CMP-001 demonstrates improved response in noninflamed anti-PD-1 refractory melanoma and response is associated with serum CXCL10.” (SITC 2021):CT032-CT032.). Therefore, there is a need for assays to predict response to vidutolimod and other TLR9 agonists, or other TLR agonists (including, for example, TLR4, TLR7, and TLR8 agonists), and / or other innate immune activators, ideally from a baseline biopsy of the patient's tumor or from a liquid biopsy, such as a blood sample, that contains tumor cells.Such an assay would make it possible to significantly improve response rates to vidutolimod and other TLR agonists or innate immune activators by identifying patients prior to treatment who are most likely to respond to such therapies, and by offering other treatment options to patients whose baseline biopsies indicate that they are unlikely to respond to vidutolimod or other TLR agonists or innate immune activators. Summary of the Invention

[0006] The present disclosure provides formulations, compositions, and methods for treating cancer. As described herein, in one embodiment, a method for identifying a subject likely to benefit from a cancer immunotherapy comprising an innate immune activator is provided, the method comprising: (i) obtaining a tumor sample from a subject having cancer; and (ii) detecting in the sample the following: SEC16B, AREG, COL7A1, SEC24D, CNIH2, MAPK15, TGFA, CUL3, SAR1B, USO1, TRAPPC6A, SEC24A, TMED2, SEC31A, BET1, GOLGA2, RAB1A, NAPA, LMAN1, TRAPPC3, PREB, SCFD1, SEC2 and detecting expression levels of at least five vidutolimod responsive core signature gene transcripts selected from the group consisting of: 3A, CTSC, TRAPPC1, ANKRD28, SEC24B, MCFD2, CD59, TMED10, STX5, TRAPPC6B, PPP6R3, SEC13, TRAPPC9, CEP19, ARFGAP3, GOSR1, SEC23B, MIA3, and VAPA, wherein said detecting enriched expression levels identifies subjects who are likely to benefit from cancer immunotherapy comprising an innate immune activator.

[0007] In another embodiment of the present disclosure, there is provided a method for identifying a subject likely to benefit from a cancer immunotherapy comprising an innate immune activator, the method comprising: (i) obtaining a tumor sample from a subject having cancer; (ii) confirming that the tumor is not "hot"; and (iii) detecting in the sample any of the following: SEC16B, AREG, COL7A1, SEC24D, CNIH2, MAPK15, TGFA, CUL3, SAR1B, USO1, TRAPPC6A, SEC24A, TMED2, SEC31A, BET1, GOLGA2, RAB1A, NAPA, LMAN1, TRAPPC3, PREB, SCFD1, SEC23A, CTSC, TRA and detecting expression levels of at least five vidutolimod responsive core signature gene transcripts selected from the group consisting of PPC1, ANKRD28, SEC24B, MCFD2, CD59, TMED10, STX5, TRAPPC6B, PPP6R3, SEC13, TRAPPC9, CEP19, ARFGAP3, GOSR1, SEC23B, MIA3, and VAPA, wherein said detection of enriched expression levels of at least five vidutolimod core signature gene transcripts in tumors that are not "hot" identifies subjects who are likely to benefit from cancer immunotherapy comprising an innate immune activator.

[0008] In yet another embodiment, the disclosure provides a method for identifying a subject likely to benefit from a cancer immunotherapy comprising an innate immune activator, the method comprising: (i) obtaining a tumor sample from the subject; and (ii) detecting in the sample one or more of: SEC16B, AREG, COL7A1, SEC24D, CNIH2, MAPK15, TGFA, CUL3, SAR1B, USO1, TRAPPC6A, SEC24A, TMED2, SEC31A, BET1, GOLGA2, RAB1A, NAPA, LMAN1, TRAPPC3, PREB, SCFD1, SEC23A, CTSC, TRAPPC1, ANKR and (iii) detecting elevated expression levels of at least five vidutolimod responsive core signature gene transcripts selected from the group consisting of D28, SEC24B, MCFD2, CD59, TMED10, STX5, TRAPPC6B, PPP6R3, SEC13, TRAPPC9, CEP19, ARFGAP3, GOSR1, SEC23B, MIA3, and VAPA, wherein said detection identifies a subject who is likely to benefit from a cancer immunotherapy comprising an innate immune activator.

[0009] In yet another embodiment, the disclosure provides a method for identifying a subject likely to benefit from a cancer immunotherapy comprising an innate immune activator, the method comprising: (i) obtaining a tumor sample from the subject; and (ii) detecting in the sample one or more of: SEC16B, AREG, COL7A1, SEC24D, CNIH2, MAPK15, TGFA, CUL3, SAR1B, USO1, TRAPPC6A, SEC24A, TMED2, SEC31A, BET1, GOLGA2, RAB1A, NAPA, LMAN1, TRAPPC3, PREB , SCFD1, SEC23A, CTSC, TRAPPC1, ANKRD28, SEC24B, MCFD2, CD59, TMED10, STX5, TRAPPC6B, PPP6R3, SEC13, TRAPPC9, CEP19, ARFGAP3, GOSR1, SEC23B, MIA3, and VAPA; and (iii) detecting elevated expression levels of at least five vidutolimod core signature gene transcripts selected from the group consisting of: (a) elevated expression of ELF2, and / or (b) elevated expression of T reg and (c) detecting one or more of: (a) an underexpression of a GABA B cell signature; and / or (b) detecting one or more of: (a) an underexpression of a GABA B cell signature; and / or (c) detecting one or more of: (a) an underexpression of a GABA B cell signature;

[0010] In yet another embodiment, the present disclosure provides a method for identifying a tumor that is characterized by obtaining a tumor sample from a subject, and detecting in the tumor sample (a) a low presence of macrophage and / or monocyte cell populations, (b) high expression of ELF2, and / or (c) T reg and (d) detecting one or more of: (a) a low presence of a B cell population; and (b) a low expression of a GABA B cell signature, wherein said detecting identifies the subject who is likely to benefit from the cancer therapy comprising an innate immune activator.

[0011] In some embodiments, the present disclosure provides a method for identifying a subject who is likely to benefit from a cancer therapy that includes a natural immune activator, where the natural immune activator is vidutolimod. In some embodiments, the present disclosure provides a method for identifying a subject who is likely to benefit from a vidutolimod cancer therapy.

[0012] In another embodiment, the present disclosure provides a method of treating a subject suffering from cancer with a cancer immunotherapy comprising an innate immune activator, the method comprising: (i) obtaining a tumor sample from a subject suffering from cancer; and (ii) detecting in the sample any of the following: SEC16B, AREG, COL7A1, SEC24D, CNIH2, MAPK15, TGFA, CUL3, SAR1B, USO1, TRAPPC6A, SEC24A, TMED2, SEC31A, BET1, GOLGA2, RAB1A, NAPA, LMAN1, TRAPPC3, PREB, SCFD1, SEC23A, CTSC, CTSC-C, CTSC-D, CTSC-E, CTSC-F ... detecting expression levels of at least five vidutolimod responsive core signature gene transcripts selected from the group consisting of TRAPPC1, ANKRD28, SEC24B, MCFD2, CD59, TMED10, STX5, TRAPPC6B, PPP6R3, SEC13, TRAPPC9, CEP19, ARFGAP3, GOSR1, SEC23B, MIA3, and VAPA, where enriched expression levels are detected; and (iv) administering to the subject a cancer immunotherapy comprising an innate immune activator.

[0013] In yet another embodiment, the present disclosure provides a method of treating a subject afflicted with cancer with a cancer immunotherapy comprising an innate immune activator, the method comprising: (i) obtaining a tumor sample from a subject afflicted with cancer; (ii) confirming that the tumor is not "hot"; and (iii) detecting in the sample any of the following: SEC16B, AREG, COL7A1, SEC24D, CNIH2, MAPK15, TGFA, CUL3, SAR1B, USO1, TRAPPC6A, SEC24A, TMED2, SEC31A, BET1, GOLGA2, RAB1A, NAPA, LMAN1, TRAPPC3, PREB, S detecting expression levels of at least five vidutolimod responsive core signature gene transcripts selected from the group consisting of CFD1, SEC23A, CTSC, TRAPPC1, ANKRD28, SEC24B, MCFD2, CD59, TMED10, STX5, TRAPPC6B, PPP6R3, SEC13, TRAPPC9, CEP19, ARFGAP3, GOSR1, SEC23B, MIA3, and VAPA, where enriched expression levels are detected; and (iv) administering to the subject a cancer immunotherapy comprising an innate immune activator.

[0014] In yet another embodiment, the present disclosure provides a method of treating a subject afflicted with cancer with a cancer immunotherapy comprising an innate immune activator, the method comprising: (i) obtaining a tumor sample from the subject; (ii) detecting in the sample an innate immune activator that is one of the following: SEC16B, AREG, COL7A1, SEC24D, CNIH2, MAPK15, TGFA, CUL3, SAR1B, USO1, TRAPPC6A, SEC24A, TMED2, SEC31A, BET1, GOLGA2, RAB1A, NAPA, LMAN1, TRAPPC3, PREB, SCFD1, SEC23A, CTSC, TRAPPC1, ANKRD28, SEC24B, MCFD2, CD59, TMED10, STX5, detecting elevated expression levels of at least five vidutolimod responsive core signature gene transcripts selected from the group consisting of TRAPPC6B, PPP6R3, SEC13, TRAPPC9, CEP19, ARFGAP3, GOSR1, SEC23B, MIA3, and VAPA, and / or (iii) detecting an under-presence of macrophage and / or monocyte cell populations, and (iv) administering to the subject a cancer immunotherapy comprising an innate immune activator if enriched expression levels are detected in (ii) and / or an under-presence of macrophage and / or monocyte cell populations is detected.

[0015] In one embodiment, the present disclosure provides a method of treating a subject suffering from cancer with a cancer immunotherapy comprising an innate immune activator, the method comprising: (i) obtaining a tumor sample from the subject; and (ii) detecting in the sample an innate immune activator-dependent mutation in one or more of SEC16B, AREG, COL7A1, SEC24D, CNIH2, MAPK15, TGFA, CUL3, SAR1B, USO1, TRAPPC6A, SEC24A, TMED2, SEC31A, BET1, GOLGA2, RAB1A, NAPA, LMAN1, TRAPPC3, PREB, SCFD1, or IL-16. , SEC23A, CTSC, TRAPPC1, ANKRD28, SEC24B, MCFD2, CD59, TMED10, STX5, TRAPPC6B, PPP6R3, SEC13, TRAPPC9, CEP19, ARFGAP3, GOSR1, SEC23B, MIA3, and VAPA; and (iii) detecting elevated expression levels of at least five vidutolimod core signature gene transcripts selected from the group consisting of: SEC23A, CTSC, TRAPPC1, ANKRD28, SEC24B, MCFD2, CD59, TMED10, STX5, TRAPPC6B, PPP6R3, SEC13, TRAPPC9, CEP19, ARFGAP3, GOSR1, SEC23B, MIA3, and VAPA in the tumor sample. reg (c) detecting one or more of the presence of a low expression of a GABA B cell signature; and (iv) administering to the subject a cancer immunotherapy comprising an innate immune activator.

[0016] In yet another embodiment, the present disclosure provides a method of treating a subject suffering from cancer with a cancer immunotherapy comprising an innate immune activator, the method comprising: obtaining a tumor sample from the subject; detecting in the tumor sample one or more of: (a) a low presence of macrophage and / or monocyte cell populations; (b) high expression of ELF2; (c) a low presence of a Treg cell population; and / or (d) low expression of a GABA B cell signature; and (iv) administering to the subject a cancer immunotherapy comprising an innate immune activator.

[0017] In some embodiments, the present disclosure provides a method of treating a subject suffering from cancer with a cancer immunotherapy comprising a natural immune activator, where the natural immune activator is vidutolimod. In some embodiments, the present disclosure provides a method of treating a subject suffering from cancer with a vidutolimod cancer immunotherapy.

[0018] In some embodiments, the aforementioned method is provided, wherein the innate immune activator is selected from the group consisting of TLR agonists. In one embodiment, the TLR agonist is selected from the group consisting of TLR9, TLR4, TLR7, and TLR8 agonists. In another embodiment, the TLR agonist is a TLR9 agonist. In yet another embodiment, the TLR9 agonist is selected from the group consisting of A class CpG DNA, C class CpG DNA, E class CpG DNA, A / E class CpG DNA, P class CpG DNA, and any combination thereof. In yet another embodiment, the TLR9 agonist is an A class CpG DNA. In one embodiment, the A class CpG DNA comprises the sequence GGGGGGGGGGGACGATCGTCGGGGGGGGGG (SEQ ID NO: 1). In yet another embodiment, the A class CpG DNA is formulated as a virus-like particle (VLP). In another embodiment, the VLP comprises bacteriophage Qβ coat protein. In some embodiments, the present disclosure provides a method of treating a subject suffering from cancer with a cancer immunotherapy comprising an innate immune activator, wherein the innate immune activator is A-class CpG DNA comprising the sequence GGGGGGGGGGGACGATCGTCGGGGGGGGGG (SEQ ID NO: 1) and formulated as a VLP comprising bacteriophage Qβ coat protein. In some embodiments, the innate immune activator recited in any of the aforementioned methods is vidutolimod.

[0019] In another embodiment, the aforementioned method is provided, wherein the tumor is determined to be not "hot" based on one or more of: (a) a TIDE score <-1; (b) low expression of an IFN-γ transcriptional signature comprising five or more of the following gene transcripts: CD3D, IDO1, CIITA, CD3E, CCL5, GZMK, CD2, HLA-DRA, CXCL13, IL2RG, NKG7, HLA-E, LAG3, TAGAP, CXCL10, STAT1, GZMB, CXCR6; (c) a low Immunoscore; and / or (d) a PD-L1 CPS<10.

[0020] In some embodiments, the aforementioned methods are provided, wherein the tumor is resistant to an inhibitor of a checkpoint selected from the group consisting of PD-1 and PD-L1. In yet other embodiments, the aforementioned methods are provided, wherein the subject is a human suffering from one or more cancerous tumors. In one embodiment, the cancerous tumor is a lymphoma or a cancerous tumor of a tissue or organ selected from the group consisting of skin, head and neck, esophagus, stomach, liver, colon, rectum, pancreas, lung, breast, cervix, ovary, kidney, bladder, prostate, thyroid, brain, muscle, and bone. In yet another embodiment, the cancerous tumor is melanoma, NSCLC, and HNSCC. In yet another embodiment, the subject is receiving a therapy selected from the group consisting of radiation therapy, chemotherapy, immunotherapy, therapy including a checkpoint inhibitor, surgery, and hormone therapy.

[0021] In some embodiments, the aforementioned method is provided wherein the detection of gene transcripts is selected from the group consisting of RNA sequencing (RNA-Seq), mRNA sequencing (mRNA-Seq), targeted RNA-Seq, and non-coding RNA-Seq, nanostring, microarray, or other hybridization-based technologies.

[0022] In still other embodiments, the disclosure provides the aforementioned methods for detecting the presence of 10, 15, 20, 25, 30, 35, or more vidutolimod core signature gene transcripts. In some embodiments, the disclosure provides the aforementioned methods for detecting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 41 vidutolimod core signature gene transcripts. In still other embodiments, the disclosure provides the aforementioned methods, wherein the detected vidutolimod core signature gene transcripts have an enrichment score in up to 80% of patient biopsies for a tumor type. In other embodiments, the disclosure provides the aforementioned method, wherein the detected vidutolimod core signature gene transcripts are expressed in one or more cells selected from the group consisting of plasmacytoid dendritic cells (pDCs), plasma cells, mast cells, activated monocytes, macrophages, dendritic cells, and / or T cells.

[0023] In yet another embodiment, the disclosure provides the aforementioned method, wherein the cancer immunotherapy is administered via intratumoral, peritumor, systemic, intravenous, intraperitoneal, enteral, oral, intramuscular, subcutaneous, transmucosal, topical, intravesicular, and / or transdermal routes. In one embodiment, the composition is administered intratumorally. In yet another embodiment, the method further comprises administering to the subject at least one dose of a composition comprising a checkpoint inhibitor (CPI). In yet another embodiment, the CPI is an antibody or antigen-binding fragment thereof that specifically binds to an antigen selected from the group consisting of PD-1, PD-L1, LAG-3, TIM-3, and CTLA-4. In one embodiment, the CPI is an antibody or antigen-binding fragment thereof that specifically binds to PD-1. In yet another embodiment, the TLR9 agonist is vidutolimod and the CPI is a PD-1 inhibitor.

[0024] The present disclosure also provides a kit. In one embodiment, a kit is provided that includes one or more compositions that include reagents capable of detecting at least five vidutolimod core signature gene transcripts selected from the group consisting of SEC16B, AREG, COL7A1, SEC24D, CNIH2, MAPK15, TGFA, CUL3, SAR1B, USO1, TRAPPC6A, SEC24A, TMED2, SEC31A, BET1, GOLGA2, RAB1A, NAPA, LMAN1, TRAPPC3, PREB, SCFD1, SEC23A, CTSC, TRAPPC1, ANKRD28, SEC24B, MCFD2, CD59, TMED10, STX5, TRAPPC6B, PPP6R3, SEC13, TRAPPC9, CEP19, ARFGAP3, GOSR1, SEC23B, MIA3, and VAPA. In another embodiment, the present invention relates to a method for detecting a mutated IL-1 deficiency associated with ... reg The kits include one or more compositions comprising reagents capable of detecting one or more of: (a) the presence of an underexpression of a GABA B cell signature; and / or (b) the presence of an underexpression of a GABA B cell signature. [Brief description of the drawings]

[0025] [Figure 1] Gene set enrichment analysis (GSEA; www.gsea-msigdb.org / gsea / index.jsp; two-arm comparison) performed on RNA-Seq data from baseline "cold tumors" comparing subsequent responders and PDs of clinical trial NCT02680184 against all signatures in the Molecular Signature Database (MSigDB) was shown. A four-gene signature was identified that was enriched in responders and PDs (FDR<0.25, default cutoff). [Diagram 2]We show how we used leading edge analysis to identify core genes in four signatures that were highly enriched in vidutolimod responders with PD-1-resistant melanoma and baseline "cold" tumor biopsies in clinical trial NCT02680184. [Diagram 3] Venn diagram to illustrate how the COPII_COATED_VESICLE_BUDDING and VESICLE_TARGETING_TO_FROM_OR_WITHIN_GOLGI core genes are highly overlapping with each other, including 35 common genes. In contrast, these two signatures share minimal overlap with two other signatures that were originally associated with vidutolimod response. [Figure 4A] 1A-1C are example plots (only one shown for this example) showing that response to vidutolimod as monotherapy or in combination with pembrolizumab was associated with increased expression of the Golgi core signature ("enrichment score") for baseline biopsies of patients with PD-1 resistant melanoma in clinical trial NCT02680184 who had "cold" tumors. Patients transitioning to PD had the lowest enrichment scores, and patients achieving SD had intermediate enrichment scores. [Figure 4B] The same plot of enrichment scores for all of the baseline biopsies, including those characterized as "cold," "medium," or "hot," is shown. [Figure 4C] We show that there is no association between subsequent response to vidutolimod and Golgi core signature enrichment score in patients with a baseline "hot" biopsy. [Diagram 5] Figure 1 shows that there is no significant difference in the overall enrichment of the Golgi core signature between patients with baseline "hot," "intermediate," or "cold" biopsies. [Figure 6]Shown is the combined enrichment of the Golgi core signature by response status in the total baseline population (left panel) and by baseline inflammation status of the biopsy using the IFN18 signature. Because patient data from the baseline "intermediate" biopsy were not used in the original identification of the Golgi signature, these data provide an independent confirmation of the significant correlation between the Golgi signature enrichment score and the probability of response to vidutolimod + pembrolizumab therapy. [Figure 7] We present another independent dataset confirming a significant correlation between Golgi signature enrichment scores and the probability of tumor shrinkage in response to vidutolimod + PD-1 blockade (in this case, anti-PD-L1 atezolizumab) therapy in patients with PD-1 resistant cancer (in this case, NSCLC in clinical trial NCT03438318). [Figure 8] From the published dataset, we show that enrichment scores for the Golgi core signature are not associated with response to or induction by PD-1 or CTLA-4 blockade. [Figure 9] Using publicly available single-cell RNA-Seq datasets from various tumors (in this case, from melanoma), we show that the Golgi signature is most highly expressed in tumor-associated plasma cells, followed by pDCs, malignant cells, macrophages, and immature DCs. [Figure 10] Using a published single-cell RNA-Seq dataset from in vitro-activated purified pDCs, we show that the Golgi signature is most highly expressed within the P1 pDC subset, which was also identified as the major subset producing type I IFN. [Figure 11] Within the TCGA database of RNA-Seq from thousands of tumors, we show that the Golgi signature is highly expressed across most tumor types. [Figure 12]Using publicly available RNA-Seq datasets from irradiated and normal tissues, we show that the Golgi core signature tends to be highly enriched in irradiated normal or malignant cells. [Figure 13] We present the results of an analysis using Qlattice, an AI tool, to identify ELF2 as a potential "partner" gene of the Golgi signature using a novel algorithm that may better predict response to vidutolimod (and other innate immune activators or TLR agonists) from baseline tumor biopsy gene expression data. [Figure 14] We show that ELF2 expression independently correlates with response to vidutolimod, regardless of baseline tumor inflammatory status. [Figure 15] For our RNA-Seq dataset of PD-1-resistant melanoma patients, we show that BRAF V600E mutation status correlates with the published IFN18 signature but not with the Golgi signature, and there is no correlation between expression of the Golgi signature and prior Braf inhibitor treatment. [Figure 16] We show that enrichment of the Golgi core signature does not correlate with baseline LDH or liver metastases, known adverse prognostic factors for melanoma response to immunotherapy. [Figure 17] We show that enrichment of the Golgi core signature does not correlate with baseline tumor burden, another known poor prognostic factor for melanoma response to immunotherapy. [Figure 18] We show that baseline "hot" biopsies with a high signature for macrophages / monocytes are less likely to respond to vidutolimod + pembrolizumab. [Figure 19] We show that low expression of macrophage / monocyte expressed genes, including TLR8, TGF-b, TNFR, C1QC, or PD-L2, predicts clinical response in baseline "hot" tumor biopsies. [Figure 20]We show that low expression of TNF-a or TLR9 predicts clinical response in cold but not hot tumor biopsies. [Figure 21] Among all baseline biopsies, those with a high Treg signature were less likely to respond to vidutolimod plus pembrolizumab. [Figure 22] We show that among baseline hot biopsies, those with a high signature for GABA B-cell activation were less likely to respond to vidutolimod plus pembrolizumab. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] The present disclosure provides biomarkers that respond to cancer therapy, including, for example, cancer immunotherapy. As used herein, the term "cancer immunotherapy" refers to cancer therapy that exerts its effect primarily or predominantly through an impact on immune function, rather than a direct effect on tumor cells. Cancer immunotherapy includes i) checkpoint inhibitors, which essentially function to "remove the brakes" on the immune system, and ii) innate immune activators, which function to stimulate innate and adaptive immunity resulting in anti-tumor immunity. As used herein, innate immune activators relevant to cancer immunotherapy include, for example, various Toll-like receptors (TLRs), particularly TLR3, TLR4, TLR7, TLR8, and TLR9, RIG-I-like receptors (RLRs), cGAS / STING, cytokines, interferons, and oncolytic viruses.

[0027] As used herein, "biomarker(s)" refers to the presence or absence of a gene product (e.g., a transcript or protein), a cell type or population of cell types, and / or a protein, cytokine, or hormone in a sample. The presence or absence of the biomarkers described herein, individually or collectively, is used to determine or identify patients who are likely to benefit from cancer treatments that include innate immune activators, such as TLR9 agonists.

[0028] In some embodiments, cancer immunotherapy may include a combination of a TLR9 agonist and a checkpoint inhibitor, such as an antibody against PD-1. Exemplary TLR9 agonists and checkpoint inhibitors are provided herein. Additionally, as will be appreciated by those skilled in the art, the signatures and methods described herein encompass cancer treatments that include other TLR agonists, including TLR3, TLR4, TLR7, and TLR8 agonists.

[0029] As used herein, a patient who is likely to benefit from cancer therapy is a patient who shows alleviation of one or more cancer symptoms, including, for example, by inducing anti-tumor response.Anti-tumor response, when referring to a cancer patient treated with a therapeutic agent, a TLR9 agonist (optionally in combination with a checkpoint inhibitor, such as an antibody against PD-1), means at least one positive therapeutic effect, such as, for example, a reduction in the number of cancer cells, a reduction in tumor size, a reduction in the rate of cancer cell invasion into peripheral organs, a reduction in tumor metastasis or tumor growth rate, or progression-free survival.Positive therapeutic effect in cancer can be measured in several ways (see WA Weber, J.Null.Med.50:1S-10S (2009); Eisenhauer et al., supra).In some embodiments, anti-tumor response to PD-1 antagonist is evaluated using RECIST1.1 criteria, bidimensional irRC, or unidimensional irRC. In some embodiments, the anti-tumor response is SD (stable), PR (partial response), CR (complete response), PFS (progression-free survival), DFS (disease-free survival), or OS (overall survival). In some embodiments, the transcriptional signature of the present disclosure predicts whether a subject with a solid tumor is likely to achieve PR or CR.

[0030] The transcriptional signatures and signatures or genetic biomarkers obtained using the methods described herein may be useful in identifying cancer patients who are most likely to derive clinical benefit from treatment with a TLR9 agonist, optionally in combination with one or more checkpoint inhibitors, such as antibodies against PD-1. This utility supports the use of such biomarkers in a variety of research and commercial applications, including, but not limited to, clinical trials of innate immune activators such as TLR9 agonists (optionally in combination with checkpoint inhibitors such as antibodies to PD-1) where patients test positive or negative for a genetic signature or genetic biomarker, or are selected based on the genetic signature or genetic biomarker, diagnostic methods and products for determining a patient's genetic signature score or classifying a patient as positive or negative for a genetic signature and / or genetic biomarker, personalized treatment methods that include adjusting a patient's drug therapy based on the patient's genetic signature score or biomarker status, and pharmaceutical compositions and medicaments comprising a TLR9 agonist (optionally in combination with a checkpoint inhibitor such as an antibody to PD-1) for use in treating patients who test positive for a genetic signature biomarker.

[0031] The utility of any of the research and commercial applications claimed herein does not require that 100% of patients who test positive for a gene signature biomarker achieve an anti-tumor response to an innate immune activator, such as a TLR9 agonist (optionally in combination with a checkpoint inhibitor, such as an antibody to PD-1), nor does it require that a diagnostic method or kit have a particular degree of specificity or sensitivity in determining the presence or absence of a biomarker in every subject, nor does it require that the diagnostic method claimed herein be 100% accurate in predicting, for every subject, whether that subject is likely to have a beneficial response to an innate immune activator, such as a TLR9 agonist (optionally in combination with a checkpoint inhibitor, such as an antibody to PD-1). Accordingly, the present disclosure herein provides that the terms "determine," "determining," and "predicting" should not be construed as requiring a definite or particular result, but instead, these terms should be construed to mean either that the claimed method will provide an accurate result for at least a majority of subjects, or that the result or prediction for any given subject is more likely to be accurate than inaccurate.

[0032] Biomarkers associated with response to TLR9 agonist cancer therapy The present disclosure provides biomarkers for responding to cancer therapy. The present disclosure further provides formulations, compositions, and methods for promoting immune activation and reducing immune inhibition to treat cancer, e.g., metaphorically "stepping on the gas" of the immune system while "releasing the brakes" to treat cancer, e.g., after patients who are likely to benefit from cancer treatment have been identified. The present disclosure can be used, for example, to convert "cold" (non-inflamed, treatment-resistant or treatment-refractory) cancers or tumors into "hot" (or inflamed) cancers or tumors suitable for treatment, including treatment with checkpoint inhibition. For example, the TLR9 agonists described herein are used to convert so-called "cold tumors" (i.e., tumors with a low degree of infiltration by activated immune cells), or intermediate tumors, into immunogenic tumors, i.e., tumors with intermediate or high degree of activated immune cell infiltration, "hot tumors." TLR9 agonists can also be used in the treatment of patients with "hot" tumors, either by themselves or in combination with CPIs, e.g., PD-(L)1 blockers.

[0033] The concepts of "cold" and "hot" tumors are well known to those skilled in the art. Cold tumors are often enriched in immunosuppressive cytokines and contain large numbers of T reg Cold tumors may contain activated T cells and / or myeloid-derived suppressor cells (MDSCs). H1 Cells, NK cells, and CD8 + In contrast, hot tumors have relatively few T cells and few functional antigen-presenting cells (APCs) (e.g., dendritic cells / DCs). H1 These cells are relatively enriched in type chemokines and activate effector immune cells (T H1 Cells, NK cells, and CD8 + T cells) and / or functional DCs.

[0034] The degree of immune cell infiltration can be measured, for example, by the so-called "Immunoscore", which is used to predict the clinical outcome of cancer patients.+ and CD8 + It is a scoring system that summarizes the density of T cells and their invasive margins. For example, Immunoscore is + / CD8 + Depending on the T cell density, they may be classified as low, intermediate, or high, with a density of 0-25% being preferably scored as low, a density of 25-70% being preferably scored as intermediate, and a density of 70-100% being preferably scored as high in the reference study (Page F. et al. (2018) Lancet 391(10135):2128-2139). Cold tumors are defined as having a low degree of immune cell infiltration, i.e., preferably a low Immunoscore. Hot tumors may be defined as having intermediate or high immune cell infiltration, i.e., preferably an intermediate or high Immunoscore.

[0035] Some tumor types often belong to hot tumor types, e.g., melanoma, even before treatment. These hot tumors respond more frequently to checkpoint inhibitors than cold tumors. Cold tumors usually do not respond well to checkpoint inhibitors. The present disclosure provides that TLR9 agonists can improve such responsiveness by increasing the infiltration of immune cells into tumors, thereby positively affecting the tumor microenvironment. Thus, cold tumor patients may particularly benefit from treatment with TLR9 agonists. As a result, tumors may show better response to checkpoint inhibitors. Thereby, cold tumors can be transformed into hot tumors.

[0036] Tumor-infiltrating immune cells, which, if present, may indicate a hot tumor, may be involved in T H1 Cells, Natural Killer (NK) cells, CD8 + These include, but are not limited to, T cells, and / or dendritic cells (DCs). Similarly, the presence of inflammatory cytokines, such as interferon, IL-12, IFN-g, and / or type I IFN, is also indicative of a hot tumor.

[0037] TIDE is an algorithm developed to estimate the level of immune dysfunction in tumors as an alternative approach to functionally distinguish tumors that are likely to respond to CPIs from those that are not, based in part on the level of tumor inflammation.

[0038] WO / 2016 / 094377 describes one of several approaches to distinguish between hot / inflamed and cold / non-inflamed tumours, in this case using transcriptional profiling to determine the levels of RNA transcripts of the "IFN18" genes CD3D, IDO1, CIITA, CD3E, CCL5, GZMK, CD2, HLA-DRA, CXCL13, IL2RG, NKG7, HLA-E, LAG3, TAGAP, CXCL10, STAT1, GZMB, CXCR6.

[0039] A transcriptional signature (i.e., a biomarker signature associated with response to TLR9 agonist cancer therapy) is determined in a sample of tumor tissue removed from the subject. The tumor may be primary or recurrent, and may be of any type (as described above), any stage (e.g., stage I, II, III, or IV, or the equivalent of other staging systems), and / or histology. The subject may be of any age, sex, treatment history, and / or extent and duration of remission. Tumor samples may be obtained by a variety of procedures, including, but not limited to, surgical resection, aspiration, or biopsy. In some embodiments, tumor samples may be obtained from "liquid biopsies" or blood samples in which tumor cells can be detected and tested. The tissue sample may be sectioned and assayed as a fresh specimen, or the tissue sample may be frozen for further sectioning. In some embodiments, the tissue sample is preserved by fixing and embedding in paraffin or the like.

[0040] Once a suitable sample of tumor tissue is obtained, in some embodiments, it is analyzed to quantify RNA expression levels for each, or one or more, or five or more, etc., of the vidutolimod response core signature genes or other genes and signatures provided herein, or gene signatures derived therefrom. In some embodiments, the sample is analyzed to quantify RNA expression levels for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or 41 of the vidutolimod response core signature genes or other genes and signatures provided herein, or gene signatures derived therefrom. As used herein, the phrase "determining the RNA expression level of a gene" refers to detecting and quantifying the RNA transcribed from that gene. The term "RNA transcript" includes the mRNA transcribed from a gene, and / or specific splice variants thereof, and / or fragments of such mRNAs and splice variants.

[0041] Those skilled in the art will understand that RNA can be isolated from tissue samples for analysis using several methods. For example, RNA can be isolated from frozen tissue samples by homogenization in guanidinium isothiocyanate and acid phenol-chloroform extraction. Commercial kits are available for isolating RNA from FFPE samples, and many companies provide this service for tumor biopsies. If the tumor sample is a formalin-fixed paraffin-embedded (FFPE) tissue section on a slide, gene expression analysis can be performed on total cell lysate rather than on isolated total RNA.

[0042] Those skilled in the art also recognize several methods that are useful for detecting and quantifying the level of RNA transcripts in isolated RNA or whole cell lysates. Quantitative detection methods include, but are not limited to, arrays (i.e., microarrays), quantitative real-time PCR (RT-PCR), multiplex assays, nuclease protection assays, and Northern blot analysis. In general, such methods use a labeled probe that is complementary to a portion of each transcript to be detected. Probes for use in these methods can be easily designed based on the known sequence of the gene and the transcript expressed therefrom. In some embodiments, the probes for detecting the transcripts of the vidutolimod response core signature genes provided herein are designed to specifically hybridize to the target region of the gene. Suitable labels for probes are well known and include, for example, fluorescent, chemiluminescent, and radioactive labels.

[0043] In some embodiments, assaying tumor samples for expression of the vidutolimod response core signature genes, or transcriptional signatures derived therefrom, provided herein employs detection and quantification of RNA levels in real time using nucleic acid sequence-based amplification (NASBA) in combination with molecular beacon detection molecules. NASBA is described, for example, in Compton J., Nature 350(6313):91-92(1991). NASBA is a single-step, isothermal RNA-specific amplification method. In other embodiments, the assay format is a flap endonuclease-based format, such as the Invader™ assay (Third Wave Technologies). In yet other embodiments, the assay format employs branched DNA direct mRNA capture (QuantiGene™, Panomics) or Hybrid Capture™ (Digene).

[0044] One example of an array technology suitable for use in measuring gene expression in the gene expression platform of the present invention is the ArrayPlate™ assay technology sold by HTG Molecular, Tucson Arizona and described in Martel, RR, et al, Assay and Drug Development Technologies 1(1):61-71, 2002.

[0045] In another embodiment, one assay method for measuring the abundance of transcripts of vidutolimod response core signature genes provided herein utilizes the nCounter® analysis system sold by NanoString® Technologies (Seattle, Washington USA). This system, described in Geiss et al., Nature Biotechnol. 2(3):317-325 (2008), utilizes a pair of probes, a capture probe and a reporter probe, each of which contains a 35-50 base sequence complementary to the transcript to be detected. The capture probe further comprises a short common sequence coupled to an immobilization tag, e.g., an affinity tag, which allows the complex to be immobilized for data collection. The reporter probe further comprises a detectable signal or label, e.g., is attached to a color-coded tag. After hybridization, excess probe is removed from the sample and the hybridized probe / target complexes are aligned and immobilized via affinity tags or other tags in the cartridge. The sample is then analyzed, e.g., using a digital analyzer or other processor adapted for this purpose. Generally, the color-coded tags of each transcript are counted and tabulated for each target transcript to obtain the expression level of each transcript in the sample. Using capture and reporter probes designed by NanoString, the system can measure the expression of hundreds of unique gene transcripts in a single multiplex assay.

[0046] When measuring the expression of the vidutolimod response core signature genes and other genes provided herein, in some embodiments, the absolute expression of each of the genes in the tumor sample is compared to a control, such as one or more housekeeping or constitutively expressed genes of the same tumor sample. This method normalizes the expression levels of the biomarker signature or genes within each sample, allowing the relative levels of expression of the biomarker signature or genes to be compared between samples, thereby defining an enrichment score for a particular sample or set of samples compared to the average expression level for a particular population or data set. To increase the sensitivity of the comparison, the expression level values ​​are preferably transformed in any of several ways. Thus, as used herein, the phrase "detecting enriched levels of expression of ..." refers to an indication that the magnitude of RNA expression of the biomarker signature or genes is relatively greater than that in patients with a low probability of responding to treatment with an innate immune activator, such as a TLR9 agonist, when referring to applications for distinguishing cancer patients who are likely to respond to treatment with an innate immune activator. As will be understood by those skilled in the art, the enrichment score cutoff used as an assay for selecting patients to be treated with a therapy is based on the sensitivity and specificity of the biomarker for predicting response in a particular patient population and is determined using standard methods in the field of biomarkers. Ideally, the enrichment score cutoff includes all patients who will respond to the treatment and excludes all patients who will not respond. Thus, the threshold value may depend on many factors that are believed to provide an individual patient with the greatest chance of tumor treatment success, depending on the patient's personal characteristics and other available treatment options. For example, the enrichment score cutoff can be determined based on the average expression levels in a dataset for at least five vidutolimod response core signature gene transcripts (see, for example, Jiang et al. Nature Medicine, 24 (2018): 1550-1558).In some embodiments, the enrichment score cutoff is determined using any of the statistical methods described in Ayers et al. (WO / 2016 / 094377), which is incorporated by reference in its entirety.

[0047] The enrichment score may be used in combination with other prognostic factors or biomarkers to make this prediction, including, but not limited to, (a) the presence / absence of low macrophage and / or monocyte cell populations, (b) the expression level of ELF2, (c) low T reg (d) the presence / absence of a GABA B cell signature; and / or (e) the expression level of a GABA B cell signature.

[0048] To standardize enrichment scores across different biopsies and data sets for the purpose of selecting patients to be treated with cancer therapy, standard methods known to those skilled in the art are used to set the desired enrichment level so that signature expression is normalized to a housekeeping or other RNA selected as an internal control. Different enrichment scores may be used to select patients for treatment depending on tumor stage and location, tumor type, history of other treatments, and the presence or absence of other biomarker signatures and genes. In some embodiments, mathematically combining "partner" genes or signatures with biomarker genes or signatures provides a higher level of discrimination in selecting patients with the highest probability of responding to treatment without excluding patients who are likely to be responders. Taking all of these factors into consideration, the enriched expression level for the purpose of predicting responders may be higher than the expression level in patients with the lowest 10% expression (i.e., the enrichment score cutoff may include 90% of patients in a particular population), or the lowest 20% expression, or the lowest 30%, or the lowest 40%, or in some embodiments, the enriched expression level is higher than the average expression level in the entire population. In some embodiments, the enrichment score used to select patients for treatment includes only the highest 10%, or the highest 20%, or the highest 30%, or the highest 40% of expression, or intermediate levels between any of these values.

[0049] Similarly, when detecting or measuring other signatures or genes, the abundance, gene expression, or other markers of a cell population are measured based on known standards or by comparing with a control sample. For example, as used herein, the phrase "elevated expression of ELF2" refers to an expression level that is associated with a higher probability of response to innate immune activators, such as TLR9 agonists, compared to patients with tumors that contain relatively low levels of ELF2 RNA compared to control genes, similar to the use of enrichment scores described above. Some biomarker signatures and genes provided herein are associated with resistance to treatment with innate immune activators or TLR9 agonists, and thus, treatment selection using these biomarkers is based on detecting decreased expression levels or enrichment compared to the entire population, as opposed to the method described above for biomarker signatures and genes that are positively correlated with response.

[0050] Signatures for immune cells described herein are contained within the Molecular Signature Database (MSigDB), e.g., REACTOME_GABA_B_RECEPTOR_ACTIVATION for the GABA B cell signature, T reg For more information, see T_cell_regulatory_(T reg As used herein, the phrase "low T reg "Low expression of a GABA B cell signature," "low expression of a macrophage / monocyte signature," or low expression of any of TLR8, TLR9, TGFB2, TNFA, TNFRSF1A, TNFRSF1B, C1QC, and / or PD-L1 refers to the relative level of expression of any one or more of these signatures and / or genes that have been shown to be associated with an increased likelihood of response to innate immune activators, such as TLR9 agonists, as compared to patients with higher expression levels within a particular patient population as described herein.

[0051] The raw expression values ​​of clinical response genes in the gene expression platforms described herein may be normalized by either: quantile normalization to a common reference distribution by the average RNA levels of a set of housekeeping genes, global normalization relying on percentiles, e.g., the 75th percentile, or other biologically and clinically relevant normalization approaches known to those of skill in the art.

[0052] For example, the expression level of each clinical response gene can be normalized by the average RNA expression level of all genes in the gene expression platform or by the average expression level of a set of normalization genes, e.g., housekeeping genes. Thus, in one embodiment, the genes in the gene expression platform are represented by a set of probes, and the RNA expression level of each of the genes is normalized by the average or median expression level across all of the represented genes, i.e., across all clinical response genes and normalization genes in the gene expression platform described herein. In a particular embodiment, the normalization is performed by dividing the median or average RNA expression level of all genes in the gene expression platform. In another embodiment, the RNA expression level of the clinical response gene is normalized by the average or median expression level of the set of normalization genes. In a particular embodiment, the normalization gene comprises a housekeeping gene. In another particular embodiment, normalization of the measured RNA expression level for the clinical response gene is achieved by dividing the measured level by the median or average expression level of the normalization gene.

[0053] When comparing a subject's tumor sample to a standard or control, the expression value of a particular gene in the sample is compared to the expression value of that gene in the standard or control. In some embodiments, for each gene in the gene signature of the present invention, a log(10) ratio is generated for the expression value in each individual sample to the standard or control. The score of the gene signature is calculated by determining the average log(10) ratio of the genes in the signature. If the gene signature score of the test sample is equal to or greater than the predetermined threshold value of that gene signature, the sample is considered positive for that gene signature biomarker. The predetermined threshold value can also be the mean, median, or percentile of the score of that gene signature in the collection of samples or pooled samples used as the standard or control.

[0054] The signatures obtained herein were defined in patients resistant to PD-1 blockade. That is, patients who originally had tumor signatures predictive of response to PD-1 blockade would usually respond to CPIs and would not be candidates for clinical trials with vidutolimod. If patients who are naive to PD-1 blockade are considering treatment with a combination therapy of innate immune activators in combination with checkpoint inhibitors, separate biomarkers may be used to predict the probability that the patient will respond to checkpoint inhibitors alone, as well as to predict the response to innate immune activators. Patients with biomarkers predictive of response to checkpoint inhibitors (e.g., TIDE, IFN18, CPS, Immunoscore, etc.) may be selected for treatment with the combination if these patients show enrichment of one or more of the biomarkers described herein for predicting response to innate immune activators, even if the enrichment level of those biomarkers responding to innate immune activators is below the threshold that would be used if the patient did not have a signature predictive of response to PD-1 blockade (i.e., if the patient had a "cold" tumor). It is believed that the addition of an innate immune activator to a checkpoint inhibitor may improve the duration and depth of response compared to treatment with a checkpoint inhibitor alone, and the biomarker strategies described herein may aid in the selection of patients most likely to benefit from such a combination.

[0055] One of skill in the art will recognize that other differential expression values ​​in addition to log(10) ratios may be used in calculating the signature score, so long as the value represents an objective measure of gene transcript abundance. Examples include, but are not limited to, xdev, error-weighted log(ratio), and mean-subtracted log(intensity).

[0056] Assaying of tumor samples for expression of genes in the gene expression platform or gene signature described herein may be performed using a kit specifically designed for this purpose. In one embodiment, the kit comprises a set of oligonucleotide probes capable of hybridizing to a set of target transcripts, such as the vidutolimod response core signature gene transcripts described herein. The set of oligonucleotide probes may comprise an ordered array of oligonucleotides on a solid surface, such as a microchip, silica beads (such as the BeadArray technology of Illumina, San Diego, CA), or glass slides (see, e.g., WO98 / 20020 and WO98 / 20019). In some embodiments, the oligonucleotide probes are provided in one or more compositions in liquid or dry form. The kit may also comprise reagents for RNA sequencing (RNA-Seq), mRNA sequencing (mRNA-Seq), targeted RNA-Seq, and non-coding RNA-Seq, nanostring, microarray, or other hybridization-based techniques, in some embodiments. For example, the kits may, in some embodiments, include reagents capable of detecting the presence or determining the expression level of one or more target transcripts, such as the vidutolimod responsive core signature gene transcripts described herein.

[0057] The kit of the present disclosure may also include other reagents, such as hybridization buffers and reagents for detecting when hybridization with a specific target molecule occurs. Detection reagents may include biotin or fluorescent tagged oligonucleotides, and / or enzyme-labeled antibodies, and one or more substrates that generate detectable signals when acted upon by enzymes. Those skilled in the art will understand that a set of oligonucleotides and reagents for performing an assay is provided in a separate receptacle that is placed in the kit container, as appropriate, to preserve biological or chemical activity and allow for appropriate use in the assay.

[0058] Transcriptional biomarkers As described herein, the present disclosure provides transcriptional markers (e.g., mRNA transcripts) that can be used to determine whether a patient will benefit from, for example, a TLR9-based cancer therapy. In some embodiments, the transcriptional markers are associated with genes involved in ER-to-Golgi transport and vesicle trafficking. In some embodiments, these genes include some or all of the following genes, referred to herein as the "vidutolimod responsive core signature" or "vidutolimod core signature": SEC16B, AREG, COL7A1, SEC24D, CNIH2, MAPK15, TGFA, CUL3, SAR1B, USO1, TRAPPC6A, SEC24A, TMED2, SEC31A, BET1, GOLGA2, RAB1A, NAPA, LMAN1, TRAPPC3, PREB, SCFD1, SEC23A, CTSC, TRAPPC1, ANKRD28, SEC24B, MCFD2, CD59, TMED10, STX5, TRAPPC6B, PPP6R3, SEC13, TRAPPC9, CEP19, ARFGAP3, GOSR1, SEC23B, MIA3, VAPA.

[0059] Like the closely related endosomal innate immune receptors TLR3, TLR7, and TLR8, TLR9 is produced in and resides primarily in the endoplasmic reticulum (ER) in resting cells (Chockalingam, A., et al., Immunol. And Cell Biology, 2009, 87, 209-217). Upon stimulation with CpG-DNA, TLR9 redistributes to early endosomes. Endolysosomal localization is important for the TLR9 response to CpG DNA, because CpG-A DNA, which maximally induces IFN-a production, accumulates in early endosomes, whereas CpG-B and CpG-C DNAs with phosphorothioate backbones, which induce more potent inflammatory cytokines and immune cell maturation, accumulate and activate TLR9 in late endosomes and / or lysosomes. A pool of TLR9 is believed to be constitutively transported from the ER through the Golgi complex and present in endolysosomes, and this pool of TLR9 is believed to be involved in signal transduction (Chockalingham et al.). The protein Unc93b1 is required for TLR9 transport from the ER to endolysosomes via the Golgi (reviewed in Ewald, SE, and Barton, GM, Current Opin. Immunol., 2011, 23:3-9). The ectodomain of TLR9 (and TLR7) is cleaved in endolysosomes, presumably by cathepsins such as cathepsin K, and cleavage of the TLR9 ectodomain appears to be essential for responses to CpG DNA (Ewald, SE, et al., Nature Letters, doi.10.1038 / nature07405; Ewald, SE, and Barton, GM, Current Opin. Immunol., 2011, 23:3-9). Furthermore, it has been reported that TLR9 translocation through the Golgi complex occurs in response to CpG DNA and is required for optimal signaling (Chockalingham et al.).

[0060] Confocal microscopy reveals that HMGB1 preassociates with TLR9 and colocalizes with markers of the ER, ERGIC, and Golgi in resting cells (Ivanov, S., et al., Blood, 2007, 110:6, 1970-1981). Upon stimulation with CpG oligodeoxynucleotides (CpG-ODN), HMGB1 and TLR9 colocalize with the early endosome marker EEA1. Ablation or depletion of HMGB1 impaired the redistribution of TLR9 to early endosomes in response to CpG-ODN. As a result, HMGB1-deficient cells showed a substantial reduction in response to CpG-ODN (Ivanov et al). Rab proteins are important regulators of intracellular membrane trafficking. For example, Rab7 regulates trafficking to late endosomes and lysosomes and is important for the biogenesis of lysosomes and phagosomes, as well as the maturation of late autophagic vacuoles, while Rab7b controls vesicle trafficking from endosomes to the trans-Golgi network (TGN) (Bucci, C., et al., Commun. And Integrative Biology, 2010, 3:5, 401-404). Overexpression of RAB7 in mouse B cells upregulates the activity of TRAF6 and enhances the activation of NF-kB (Yan, Hui, et al., The Journal of Immunology, 204.5(2020):1146-1157). High levels of Rab7b expression downregulate TLR4- and TLR9-mediated inflammatory responses, whereas depletion leads to upregulation of these responses (Bucci, C., et al., Commun. And Integrative Biology, 2010, 3:5, 401-404). Proprotein convertase 1 / 3 (PC1 / 3), another key regulator of TLR4- and TLR9-dependent signaling, rapidly colocalizes with TLR9 in CpG-ODN-containing endosomes after CpG stimulation and prevents TLR9 clustering in multivesicular bodies (MVBs) and colocalization with Rab7 (Duhamel, M., et al. “Scientific reports, 6.1 (2016): 1-13).

[0061] The similarities in the basic biological properties of various TLRs and other innate immune receptors suggest that biomarkers for response to TLR9 agonists may have utility in predicting responses not only to other TLR9 agonists, but more generally to other TLR agonists (including, in particular, TLR3, TLR4, TLR7, and TLR8), as well as other innate immune activators known to those of skill in the art, including, for example, cGAS / STING and RIG-I agonists, or activators of LGP2, MDA5, PKR, and / or AIM2.

[0062] Mechanisms regulating TLR9 translocation from the ER to the Golgi network and other intracellular compartments are likely important in regulating immune responses in vitro and in vivo, but until recently, biomarkers associated with these processes had not been identified in tumor biopsies from cancer patients.

[0063] Additionally, the present disclosure provides other transcriptional signatures that can be used, either alone or in combination with the Golgi signature, to determine whether a patient will benefit from, for example, a TLR9-based cancer therapy. For example, high expression of the transcription factor ELF2 has been associated with response to vidutolimod across baseline hot and cold tumors, and ELF2 expression can be combined with enrichment of the Golgi signature to achieve a higher predictive value for response to vidutolimod than either biomarker alone in a population of cancer patients.

[0064] Other biomarkers In some embodiments, identifying subjects who are likely to benefit from cancer immunotherapy comprising natural immune activators comprises detecting vidutolimod core signature gene transcripts in combination with one or more other prognostic factors or biomarkers.Transcription signatures represent an example of a prognostic factor.These other prognostic factors or biomarkers can be combined with one or more of the Golgi signatures to more accurately predict the probability of responding to vidutolimod alone or in combination with other immunotherapy such as PD-1 blockade.

[0065] Other transcriptional signatures or biomarkers that can be used to determine whether a patient is likely to benefit from an innate immune activator, such as a TLR9-based cancer therapy, included in the present invention, include, for example, a low baseline frequency of macrophages / monocytes (using any of several published transcriptional signatures), a low baseline T reg Signature (specifically, T_cell_regulatory_(T regs )_CIBERSORT), and an intermediate / low B cell-derived GABA signature. Individual genes that may form part of the biomarkers for this purpose include TLR8, TGFB2, TNFRSF1A, TNFRSF1B, C1QC, and / or PD-L1, particularly in baseline "hot" tumors, or TLR9 and / or TNFA, particularly in baseline "cold" tumors, with relatively low levels of expression of one or more of these genes being used to identify patients likely to respond to treatment with an innate immune activator.

[0066] Methods of Treating Patients Presenting with Transcriptional or Other Biomarkers Vidutolimod (CMP-001) is a first-in-class CpG-A TLR9 agonist in a virus-like particle that activates plasmacytoid dendritic cells (pDCs), linking innate and adaptive immunity. Intratumoral (IT) injection of vidutolimod serves as in situ immunization and induces antitumor CD8+ with systemic responses to neoantigens. + The tumor microenvironment can be reprogrammed to promote T cell generation. Vidutolimod has shown evidence of antitumor activity alone and / or in combination with PD-1 blockade in PD-1 resistant and / or neoadjuvant melanoma (NCT02680184 and NCT03618641), NSCLC (NCT03438318), and HNSCC (NCT02554812), and is also being evaluated in MSS CRC (NCT03507699).

[0067] In some embodiments, the CpG ODN of the present disclosure is characterized, at least in part, by its tendency to induce large amounts of type I IFN.Exemplary CpG ODNs are the CpG-A class molecules described in PCT / US2015 / 067269 (incorporated herein by reference in its entirety).In various embodiments of the present disclosure, one or more CpG ODNs are formulated using virus-like particles (VLPs).PCT / US2015 / 067269 describes the use of VLPs with A class CpGs, including CMP-001, which is specifically contemplated herein.

[0068] Viral capsids provide a protective shell of various sizes for most naturally occurring viruses (Mannige, RV, and Brooks, CL, PLoSONE, 5(3):e9423 (2010); Perlmutter, JD, and Hagan, MF, J. Mol. Biol., 427(15):2451-2467 (2015). Structural information is also available for the ssRNA phage Qβ (Gorzlenik, KV, et al., PNAS, 113(41):11519-11524 (2016). Various groups have focused on stabilizing VLPs in formulations (Lang, r., and Winter, G., Drug Devt. And Industr. Pharm., 35:83-97 (2009); Mertens, BS, and Velev, OD, Soft Matter, 11(44):8621-8631(2015); and Shi, L., et al., J.Pharm.Sci.,94(7):1538-1551(2005)).

[0069] As used herein, the term "therapeutically effective amount" refers to an amount that, when administered to a mammal, preferably a human, mediates a detectable therapeutic response compared to the response detected in the absence of the compound. Therapeutic responses, such as, but not limited to, inhibition and / or reduction of tumor growth (including stasis of tumor size), tumor size, metastasis, etc., can be readily assessed by a number of art-recognized methods, including, for example, those disclosed herein.

[0070] A "therapeutically effective amount" is intended to define the amount of agent required to detectably alleviate to some extent one or more symptoms of a neoplastic disorder, including, but not limited to, 1) a reduction in the number of cancer cells, 2) a reduction in tumor size, 3) inhibition (i.e., slowing to some extent, and preferably halting) cancer cell invasion into peripheral organs, 4) inhibition (i.e., slowing to some extent, and preferably halting) tumor metastasis, 5) inhibition to some extent of tumor growth, 6) alleviation or reduction to some extent of one or more symptoms associated with the disorder, and / or 7) alleviation or reduction of side effects associated with administration of anticancer drugs.

[0071] Certain preferred CpG ODNs induce high or large amounts of type I IFN. Assays for measuring type I IFN are well known in the art, including in vitro enzyme-linked immunosorbent assays (ELISAs) and cell-based assays, such as those described herein. Without intending to be limiting, high or large amounts of type I IFN may refer to about 1000 pg / mL or more of IFN-α as measured by such in vitro assays. In certain embodiments, high or large amounts of type I IFN may refer to about 2000 pg / mL or more of IFN-α as measured by such in vitro assays. In certain embodiments, high or large amounts of type I IFN may refer to about 3000 pg / mL or more of IFN-α as measured by such in vitro assays. In certain embodiments, high or large amounts of type I IFN may refer to about 4000 pg / mL or more of IFN-α as measured by such in vitro assays. In certain embodiments, high or massive type I IFN may refer to greater than or equal to about 5,000 pg / mL of IFN-α as measured by such in vitro assays.

[0072] Unless otherwise noted, the terms "patient" or "subject" are used interchangeably and refer to mammals, such as human patients and non-human primates, as well as veterinary subjects, such as rabbits, rats, and mice, and other animals. Preferably, a "patient" or "subject" refers to a human. In certain embodiments, the subject is an adult human. In certain embodiments, the subject is a child. In certain embodiments, the subject is less than about 18 years of age. In certain embodiments, the subject is less than about 12 years of age.

[0073] As used herein, "treating" means reducing the frequency with which a patient experiences symptoms of a disease (i.e., tumor growth and / or metastasis, or other effects mediated by immune cell numbers and / or activity, etc.). Treatment may be prophylactic (preventing or delaying the onset of a disease or preventing the development of clinical or asymptomatic symptoms thereof) or may be therapeutic suppression or alleviation of symptoms after the onset of a disease. The term "treating" includes administering a compound or agent of the present disclosure to (i) prevent or delay the onset of symptoms, complications, or biochemical signs of a disease, condition, or disorder, (ii) alleviate symptoms, and / or (iii) suppress or arrest further development.

[0074] CpG oligonucleotides contain at least one unmethylated CpG dinucleotide. An oligonucleotide containing at least one unmethylated CpG dinucleotide is an oligonucleotide molecule that contains a cytosine-guanine dinucleotide sequence (i.e., "CpG DNA", i.e., DNA containing a 5' cytosine linked to a 3' guanine by a phosphate bond). The entire CpG oligonucleotide can be unmethylated, or it can be partially unmethylated, but at least the C of the 5' CG3' must be unmethylated.

[0075] CpG ODNs are generally about 8 to 100 nucleotides in length. In certain embodiments, CpG ODNs are about 8 to 50 nucleotides in length, about 8 to 40 nucleotides in length, about 8 to 30 nucleotides in length, about 8 to 24 nucleotides in length, about 8 to 20 nucleotides in length, or about 8 to 16 nucleotides in length.

[0076] As described herein, in some embodiments, the CpG ODNs of the present disclosure are characterized, at least in part, by their propensity to induce high amounts of type I IFN. Exemplary CpG ODNs are the CpG-A class of molecules, as described, for example, in PCT / US2015 / 067269, which is incorporated herein by reference in its entirety. Specifically, the entire PO ODN G10 (SEQ ID NO: 82, designated "CYT003" in Figures 6 and 7 of PCT / US2015 / 067269) is contemplated herein: GGGGGGGGGGGACGATCGTCGGGGGGGGGG (SEQ ID NO: 1)

[0077] As described herein, the methods described herein may, in various embodiments, include administration of radiation therapy, chemotherapy, surgery, and / or cyclophosphamide, small molecule inhibitors, and / or checkpoint inhibitors, including but not limited to CTLA-4, 4-1BB (CD137), 4-1BBL (CD137L), PDL1 (PD-L1), PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, TIM3, B7H3, B7H4, VISTA, KIR, BTLA, SIGLEC9, and 2B4, as well as pembrolizumab, avelumab, atezolizumab, cetrelimab, dostarimab, cemiplimab, spartalizumab, camrelizumab, durvalumab, or nivolumab.

[0078] In one embodiment, the present disclosure relates to combination tumor immunotherapy comprising local administration of CpG ODN in or near a cancerous tumor and systemic administration of a checkpoint inhibitor, such as an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-CTLA-4 antibody, to treat cancer.

[0079] The combination of high IFN-inducible CpG ODN with anti-PD-1, anti-PD-L1, or anti-CTLA-4 is useful for the treatment of primary and secondary (i.e., metastatic) cancers. More specifically, among many potential treatment options, a combination therapy of CpG ODN and anti-checkpoints can be used to treat cancer. In certain embodiments, the cancer to be treated is or includes a cancerous tumor. As used herein, "cancerous tumor" refers to an abnormal swelling or macroscopic collection of cells, including abnormal cells, characterized by their growth or proliferation not regulated by normal external signals. In certain embodiments, the cancerous tumor is a carcinoma, sarcoma, or adenocarcinoma, which may also be referred to as a solid tumor. In certain embodiments, the cancerous tumor excludes hematological malignancies. In certain embodiments, the cancerous tumor includes certain hematological malignancies, such as lymphoma.

[0080] Representative cancers that can be treated by the method of the present disclosure (e.g., by administering one or more CpG-ODNs alone or in combination with one or more CPIs to treat cancer) include, but are not limited to, skin, head and neck, esophagus, stomach, liver, colon, rectum, pancreas, lung, breast, cervix, ovary, kidney, bladder, prostate, thyroid, brain, muscle, and bone cancers.Some cancers that can be treated by the method of the present invention also include melanoma, renal cell carcinoma, and non-small cell lung cancer (NSCLC).Some cancers that can be treated by the method of the present invention also include lymphoma, bone marrow cancer, carcinoid tumor, and neuroblastoma.

[0081] While in some embodiments the aforementioned cancers are preferred, the present disclosure also provides for the treatment of cancers including Kaposi's sarcoma, synovial sarcoma, mesothelioma, hepatobiliary (hepatic and bile duct), primary or secondary brain tumors, lung cancer (NSCLC and SCLC), bone cancer, skin cancer, head and neck cancer, cutaneous or intraocular melanoma, anal cancer, stomach (gastric) cancer, gastrointestinal (gastric, colorectal, and duodenal) cancer, colon cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, prostate cancer, penile cancer, testicular cancer, bladder cancer, kidney or ureter cancer, renal sac cancer, pancreatic cancer, central nervous system (CNS) cancer, The present invention relates to the treatment of a wide variety of malignant cell proliferative disorders, including, but not limited to, neoplasms (including primary or secondary CNS tumors), spinal axis tumors, brain stem gliomas, glioblastomas, meningiomas, myoblastomas, astrocytomas, pituitary adenomas, adrenocortical carcinomas, gallbladder carcinomas, cholangiocarcinomas, fibrosarcomas, neuroblastomas, and retinoblastomas, and in some embodiments, non-Hodgkin's lymphoma (NHL, including low-grade and aggressive), Hodgkin's lymphoma, cutaneous T-cell lymphoma, lymphocytic lymphoma, primary CNS lymphoma, chronic or acute myeloid leukemia, chronic or acute lymphocytic leukemia, erythrocytoma, and multiple myeloma, or a combination of two or more of the foregoing cancers.

[0082] "Tumor" as applied to a subject diagnosed with or suspected of having cancer refers to a malignant or potentially malignant neoplasm or mass of tissue of any size, including primary tumors and secondary neoplasms. A solid tumor is an abnormal growth or mass of tissue that usually does not contain cysts or liquid areas. Different types of solid tumors are named for the cell type that forms the tumor. Examples of solid tumors are sarcomas, carcinomas, and lymphomas. Leukemias (cancers of the blood) generally do not form solid tumors (National Cancer Institute, Dictionary of Cancer Terms).

[0083] "Tumor volume", also referred to as "tumor burden", refers to the total amount of tumor material distributed throughout the body. Tumor volume refers to the total number of cancer cells or the total size of the tumor(s) throughout the body, including lymph nodes and bone stenoses. Tumor volume can be determined by various methods known in the art, such as by measuring the dimensions of the tumor(s) upon removal from the subject, e.g., with a caliper, or while in the body, with an imaging technique, e.g., ultrasound, bone scan, computed tomography (CT), or magnetic resonance imaging (MRI) scan.

[0084] The term "tumor size" refers to the total size of a tumor, which can be measured as the length and width of the tumor. Tumor size may be determined by various methods known in the art, such as by measuring the dimensions of the tumor(s) when removed from a subject, for example, using a caliper, or while in the body, using imaging techniques, such as bone scan, ultrasound, CT, or MRI scan.

[0085] The cancer to be treated may be a resistant cancer, for example, a cancer resistant to PD-1. As used herein, a resistant cancer is a cancer that is resistant to the usual standard treatment prescribed. These cancers may appear to respond to treatment at first (and then recur), or may not respond to treatment at all. The usual standard treatment varies depending on the type of cancer in the subject and the degree of progression. The standard treatment may be chemotherapy, immunotherapy, surgery, radiation therapy, or a combination thereof. Those skilled in the art will recognize such standard treatments. Thus, subjects treated by the present invention for resistant cancer may have already been exposed to another treatment for their cancer. Alternatively, if the cancer is likely to be resistant (e.g., if analysis of the subject's cancer cells or medical history is possible), the subject may not have been exposed to another treatment yet.

[0086] In certain embodiments, resistant cancers include cancers that are resistant to treatment with checkpoint inhibitors. This type of cancer may be referred to as "cold". The methods of the present invention can be used to treat such "cold" cancers or tumors, converting them into "hot" cancers or tumors, i.e., cancers or tumors that respond to treatment, including treatment with checkpoint inhibitors, and even the same checkpoint inhibitors.

[0087] Examples of resistant cancers include, but are not limited to, melanoma, renal cell carcinoma, colon cancer, liver (hepatic) cancer, pancreatic cancer, non-Hodgkin's lymphoma, other lymphomas, lung cancer, prostate cancer, breast cancer, and leukemia.

[0088] In accordance with the methods of the present disclosure, the cancer therapy described herein is administered locally to the cancerous tumor, i.e., by intratumoral or peritumoral administration, in various embodiments. Alternatively or additionally, in certain embodiments, the cancer therapy comprising a TLR9 agonist, such as CMP-001, is administered locally to the cancerous tumor, for example, by intraperitoneal injection or infusion or intravesicular instillation. As will be appreciated by those skilled in the art, the cancer therapy comprising administration of a TLR9 agonist, such as CMP-001, described herein, in preferred embodiments, comprises the dosage and administration regimen for administration via a VLP, e.g., Qβ, and CMP-001, as described herein. Thus, the VLP comprises a quantity of bacteriophage Qβ coat protein of about 1 mg to about 100 mg. In other embodiments, the VLP comprises a quantity of bacteriophage Qβ coat protein of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 25, 50, or 100 mg. Other embodiments contemplate about 1.56 mg, about 3.125 mg, about 6.25 mg, about 12.5 mg, about 25 mg, about 50 mg, or about 100 mg.

[0089] In some embodiments, about 80 oligonucleotides, such as G10 or other CpG ODNs described herein, or CpG ODN aggregates of approximately 760 kDa molecular weight, are packaged into the VLP. In some embodiments, 1, 5, 10, 50, 80, 100, 120, 150, or 200 or more oligos are packaged.

[0090] The immune effects of XRT prior to cancer therapy including administration of a TLR9 agonist such as CMP-001 disrupt the inhibitory mechanisms that normally limit the effectiveness of CpG-induced responses, increasing the likelihood of clinical response. Furthermore, intratumoral production of IFN-α is associated with and required for improved responses to XRT (Burnette et al, Cancer Res. 2011 71:2488-2496), providing further evidence that tumors benefit from the use of intratumoral high IFN CpG after XRT. The timing of administration of XRT and TLR9 agonist can vary. In some embodiments, XRT is administered at least one month, or at least two weeks, or at least one week prior to the TLR9 agonist. In other embodiments, XRT is administered to new lesions that arise during TLR9 agonist therapy, and the TLR9 agonist is injected into the newly irradiated lesions before, after, or during XRT.

[0091] Methods for intratumoral or peritumoral delivery of cancer therapies that include TLR9 agonists such as CMP-001 can include direct injection as well as localized delivery, e.g., intraperitoneal delivery for abdominal tumors such as ovarian, pancreatic, colon, or stomach, intraocular delivery for ocular malignancies, oral administration for gastric and intestinal cancers, and intravesicular administration for bladder cancer. For intratumoral administration of cancer therapies that include TLR9 agonists such as CMP-001, systemic delivery is also contemplated using tumor delivery vehicles such as tumor-targeting aptamers, antibody conjugates, nanoparticles, ISCOMS, VLPs, multilamellar vesicles, pH-sensitive peptides, and cationic peptides.

[0092] In certain embodiments, the subject's dose of CpG ODN for intratumoral and peritumoral delivery is typically in the range of about 10 μg to about 100 mg per administration, which may be given daily, weekly, or monthly, and any other time period therebetween, depending on the application. In certain embodiments, the subject's dose of cancer therapy including a TLR9 agonist such as CMP-001 for intratumoral and peritumoral delivery is typically in the range of about 100 μg to about 100 mg per administration, which may be given daily, weekly, or monthly, and any other time period therebetween, depending on the application. In certain embodiments, the subject's dose of cancer therapy including a TLR9 agonist such as CMP-001 for intratumoral and peritumoral delivery is typically in the range of about 1 mg to about 100 mg per administration, which may be given daily, weekly, or monthly, and any other time period therebetween, depending on the application. In certain embodiments, subject doses of cancer therapy including a TLR9 agonist such as CMP-001 for intratumoral and peritumoral delivery typically range from about 10 mg to about 100 mg per administration, which may be given daily, weekly, or monthly, and any other time period in between, depending on the application.

[0093] Certain commercially available anti-PD-1 antibodies are currently approved in the United States to be administered as an intravenous infusion at 2 mg / kg body weight once every 3 weeks. Other commercially available anti-PD-1 antibodies are currently approved in the United States to be administered as an intravenous infusion at 3 mg / kg body weight once every 2 weeks. Commercially available anti-CTLA-4 antibodies are currently approved in the United States to be administered as an intravenous infusion at 3 mg / kg body weight once every 3 weeks.

[0094] According to the methods of the present disclosure, in certain embodiments, the CPI antibody is administered at least partially systemically, for example, intravenously.

[0095] Unless otherwise defined herein, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by those skilled in the art.Furthermore, unless otherwise required by context, singular terms shall include plural terms and plural terms shall include singular terms.In general, the nomenclature and techniques used in connection with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry, and hybridization described herein are those well known and commonly used in the art.

[0096] The methods and techniques of the present disclosure are generally carried out according to methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification, unless otherwise indicated. Such references include, for example, Sambrook and Russell, Molecular Cloning, A Laboratory Approach, Cold Spring Harbor Press, Cold Spring Harbor, NY (2001); Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, NY (2002); and Harlow and Lane Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990), which are incorporated herein by reference. Enzymatic reactions and purification techniques are carried out according to manufacturer's specifications as commonly accomplished in the art or as described herein. The nomenclatures used in connection with analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein, as well as the laboratory procedures and techniques thereof, are well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0097] As used herein, each of the following terms has the meaning associated with it in this section.

[0098] The articles "a" and "an" are used herein to refer to one or more (i.e., at least one) of the grammatical object of the article. As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology--A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. Conventional notation is used herein to depict polypeptide sequences: the left-hand end of a polypeptide sequence is the amino-terminus, and the right-hand end of a polypeptide sequence is the carboxyl-terminus. A "conservative amino acid substitution" is an amino acid substitution that replaces an amino acid residue with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially change the functional properties of a protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percentage of sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitutions.Methods for making this adjustment are well known to those skilled in the art.See, for example, Pearson, Methods Mol.Biol.243:307-31 (1994).

[0099] Having now described the present disclosure in detail, it will be more clearly understood by reference to the following examples, which are included herein for illustrative purposes only and are not intended to limit the invention. EXAMPLES

[0100] PCT / US2015 / 067269 discloses several A-class CpG ODNs, each of which is contemplated and incorporated herein by reference. As used herein, CMP-001 refers to an A-class CpG-ODN formulated in a VLP and designated G10 and SEQ ID NO: 1. CMP-001 is also known as CYT003 and QbG10, and has been previously studied under these names in mice and humans for non-oncology indications, e.g., Klimek, L., et al., Clinical & Experimental Allergy, 41.9(2011):1305-1312, and Casale, TB, et al., Allergy, 70.9(2015):1160-1168.

[0101] Example 1 A. A vesicle-Golgi trafficking signature ("Golgi signature") is associated with clinical response to vidutolimod (CMP-001) in PD-1-resistant melanoma The design and outcome metrics of the clinical trial NCT02680184 of intratumoral vidutolimod in PD-1-resistant melanoma, including both monotherapy and pembrolizumab treatment combination arms, have been described previously (Kirkwood, John, et al. “950 Final analysis: phase 1b study investigating intratumoral injection of toll-like receptor 9 agonist vidutolimod ± pembrolizumab in patients with PD-1 blockade-refractory melanoma,” SITC 2021). Vidutolimod therapy was either undiluted (Kirkwood et al., “PS20A” in SITC 2021) or diluted in 0.9% saline (Kirkwood et al., “PS20B” in SITC 2021). RNA-seq data from archival or screening baseline / pre-treatment tumor biopsies of patients in this clinical trial were processed and normalized using standard methods. The dataset included in this study's analysis included 98 baseline biopsies (Table A) from patients who subsequently received vidutolimod monotherapy or in combination with pembrolizumab, classified by subsequent responder status after this treatment, as assessed using RECIST v1.1 (CR=complete response, PR=partial response, SD=stable, PD=progressive disease). Responders (R) demonstrated CR or PR, while non-responders (NR) demonstrated SD or PD. [Table 1]

[0102] In early attempts to identify genes or gene expression signatures associated with response, portions of the complete RNA-Seq dataset were analyzed using standard bioinformatics approaches to identify baseline predictors of response. Although a number of differences in gene expression between responders and non-responders were found, including the identification of candidate response signatures, no candidate signatures or gene expression differences were identified in the complete dataset to significantly distinguish responders from non-responders.

[0103] These analyses showed that gene signatures thought to reflect "hot" tumors, i.e., "inflamed" tumors, that had previously been reported to be associated with response to PD-1 blockade, such as the IFN18 signature of Ayers et al. (WO / 2016 / 094377), or TIDE, or biopsy IHC combined pathological score (CPS) for PD-L1, or tumor mutations, were not associated with vidutolimod response in the PD-1-resistant melanoma dataset (Luke, Jason John, et al. "Abstract CT032:CMP-001 demonstrates improved response in noninflamed anti-PD-1 refractory melanoma and response is associated with serum CXCL10." (SITC 2021):CT032-CT032). This result was expected based on vidutolimod's unique mechanism of action. As described herein, in one embodiment, a tumor is determined to be "not hot" using one or more of the techniques described above and herein, including TIDE, Immunoscore, the IFN18 signature of Ayers et al. (WO / 2016 / 094377), and / or biopsy IHC combined pathology score (CPS) for PD-L1.

[0104] These studies using published markers of tumor "inflammation" such as TIDE and IFN18 signatures revealed a high degree of heterogeneity within the patient population examined, with most of the patients having "cold" or non-inflamed tumors, others having "hot" or inflamed tumors, and the rest intermediate at baseline. Interestingly, there have been no reports of any signatures in baseline "cold" tumors that are associated with response to immunotherapy drugs. To identify potential signatures that may be unique to baseline "cold", "hot", or "intermediate" tumors, the above baseline biopsy set was divided into three subsets using published tools including TIDE, IFN18, and CPS.

[0105] First, we selected only the baseline "cold" and "hot" biopsy datasets and calculated enrichment scores for all signatures from the Molecular Signature Database (MSigDB; N=18,494) and for signatures developed by Checkmate (N=555) using ssGSEA (single-sample GSEA) in each sample and that identified dysregulation between responders (CR and PR) and non-responders (PD or SD) using Limma. The results of this analysis, using IFN18 to select baseline cold biopsies, are shown in Table B below, highlighting signatures related to vesicular and Golgi transport or function. [Table 2]

[0106] Among the top gene signatures, those most highly enriched in responders and PD were signatures involving vesicle budding or targeting and Golgi trafficking (e.g., genes associated with COPII vesicle budding and / or vesicle trafficking to or from the Golgi apparatus).

[0107] Additional analyses between responders and PD against signatures in MSigDB were performed on the data from “cold tumors” using GSEA (www.gsea-msigdb.org / gsea / index.jsp; two-group comparison) (Figure 1). Four gene signatures enriched in responders and PD (FDR<0.25, default cutoff) were identified, including COPII_COATED_VESICLE_BUDDING (73 genes) (FDR=0.115), VESICLE_TARGETING_TO_FROM_OR_WITHIN_GOLGI (74 genes) (FDR=0.156), HALLMARK_PROTEIN_SECRETION (96 genes) (FDR=0.173), and DITTMER_PTHLH_TARGETS_DN (70 genes) (FDR=0.234) (Figure 1).

[0108] To identify the core gene subset that contributes most to the enrichment signal of this four-gene signature, we performed Leading edge analysis (a tool within GSEA; Subramanian, A., et al., PNAS, 2005, 102(43):15545-50) (Figure 2).

[0109] These core gene lists were then examined using a Venn diagram to detect common genes among the four core gene signatures. The Venn diagram (Figure 3) showed that there was very little overlap between the Dittmer and Hallmark protein secretion gene lists, so they were excluded from further analysis.

[0110] By this means, we reduced the original number of genes and defined a novel highly overlapping “core gene set” (herein referred to as the “common core”) derived from COPII_COATED_VESICLE_BUDDING and VESICLE_TARGETING_TO_FROM_OR_WITHIN_GOLGI that contained 35 common genes, plus three additional unique genes for each signature, totaling 41 genes ( Figure 3 ), including SEC16B, AREG, COL7A1, SEC24D, CNIH2, MAPK15, TGFA, CUL3, SAR1B, USO1, TRAPPC6A, SEC24A, TMED2, and SEC3. 1A, BET1, GOLGA2, RAB1A, NAPA, LMAN1, TRAPPC3, PREB, SCFD1, SEC23A, CTSC, TRAPPC1, ANKRD28, SEC24B, MCFD2, CD59, TMED10, STX5, TRAPPC6B, PPP6R3, SEC13, TRAPPC9, CEP19, ARFGAP3, GOSR1, SEC23B, MIA3, VAPA (six unique genes are shown last), which together comprise the Golgi vidutolimod response signature (also referred to herein as the "Golgi signature" or "vidutolimod response core signature").

[0111] We reran the ssGSEA analysis on all 19,000+ gene signatures in the CMP-001-001 baseline samples, plus the four newly identified core signatures and the "common core." The top three signatures that were dysregulated between responders and PD for baseline cold tumors in patients treated with vidutolimod monotherapy or in combination with pembrolizumab were VESICLE_TARGETING_TO_FROM_OR_WITHIN_GOLGI_core (38 genes) (adjusted P=0.059), COPII_COATED_VESICLE_BUDDING_core (38 genes) (adjusted P=0.059), and common_core (35 genes) (adjusted P=0.085). The following additional evaluation focused on these three core signatures.

[0112] B. Enrichment of Golgi signatures in baseline cold responders to both combination therapy (vidutolimod + pembrolizumab) and vidutolimod monotherapy. Initial identification of a correlation between Golgi signature expression and response was performed on pooled data from all patients with baseline cold tumors treated with undiluted vidutolimod, including patients who received combination or monotherapy. Separate analysis of these subsets showed a similar degree of correlation with response in both subsets, consistent with the hypothesis that this correlation reflects the biology of vidutolimod rather than just the combination treatment (Figure 4A). When the entire population of baseline biopsies was analyzed as a group, there was a similar strong trend for the correlation between Golgi core signature and response (Figure 4B).

[0113] C. Lack of enrichment for the common core Golgi signature in baseline "hot" biopsies For example, the biological properties of baseline "hot" and "cold" tumors are quite different, as shown by the fact that responses to PD-1 blockade are much more frequent in baseline hot tumors compared to cold tumors. Based on this well-established fact, we hypothesized that different baseline factors may predict response to vidutolimod in baseline "cold" and "hot" tumors. Indeed, baseline "hot" tumors showed no correlation between Golgi signature and response (Figure 4C), consistent with the unique biological properties of these tumors. The enrichment score of the Golgi signature was comparable across all baseline "cold" tumors, "intermediate" tumors, and "hot" tumors, indicating that this signature is not a surrogate indicator of any inherent biological difference between "cold" and "hot" tumors (Figure 5).

[0114] D. Validation of correlation between the common core and Golgi vidutolimod response signatures in independent datasets. To determine whether enrichment of the Golgi signature in baseline "cold" undiluted tumors was unique to that dataset, we assessed Golgi signature enrichment scores in an independent subset of baseline biopsy RNA-Seq data from patients with PD-(L)-1 resistant melanoma (clinical trial NCT02680184) or NSCLC (clinical trial NCT03438318) treated with vidutolimod ± anti-PD-(L)1. These data were not used for the initial identification of the Golgi signature and therefore served as a validation dataset to test broader correlations of the signature with response.

[0115] In PD-1-resistant melanoma patients with baseline "intermediate" tumors treated with vidutolimod + pembrolizumab, the Golgi signature was significantly enriched in responders and PD to similar levels as baseline "cold" tumors, with SD patients being intermediate (Figure 6; only one signature shown; comparable significant correlations were observed for other signatures and the common core).

[0116] To independently validate these findings in PD-1-resistant melanoma, Golgi signature enrichment was also assessed in baseline biopsies from PD-1-resistant NSCLC patients treated with vidutolimod + atezolizumab. Two patients in that study had tumor shrinkage that remained stable long-term after treatment. These same two patients also had the highest Golgi signature enrichment in their baseline tumors, which far exceeded the scores of non-responder patients (Figure 7). These data provide an independent confirmation of the validity of the Golgi signature for predicting response to vidutolimod therapy from baseline tumor biopsies.

[0117] E. Scoring method for defining enrichment scores for Golgi signatures In the above analysis, the scoring method is ssGSEA. Also, although ssGSEA is more robust than expressionByMean / expressionByMedian, in various embodiments, both expressionByMean and expressionByMedian scoring methods are contemplated and provided herein.

[0118] F. Methods for grouping cold / hot tumors We evaluated different ways to group cold / hot tumors: (1) IFNg_18 gene signature (Ayers, M., et al., J Clin Invest., 2017, 127(8):2930-2940), low IFNg_18 score (<-0.5 in the current data and analysis) for "cold" tumors, high IFNg_18 score (>0.5) for "hot" tumors, others are "intermediate" tumors. (2) TIDE score (https: / / pubmed.ncbi.nlm.nih.gov / 30127393 / ): high TIDE score (>1, predicted not to respond to CPIs) for "cold" tumors, low TIDE score (<-1, predicted to respond to CPIs) for "hot" tumors, others are "intermediate" tumors. (3) CPS score (PD-L1 IHC assay): cold: CPS<1; intermediate: 1 <CPS<10;ウォーム:CPS> 10.

[0119] G. Lack of correlation between the Golgi vidutolimod response signature and response to CPIs Enrichment of the Golgi signature was also assessed in baseline samples from publicly available CPI-treated RNA-Seq datasets. In one study of anti-CTLA-4 monotherapy (ipilimumab) (Nathanson, T., et al., Cancer Immunol Res., 2017, 5(1):84-91), there was no correlation of the Golgi signature with response, nor induction / reduction of the Golgi signature after anti-CTLA4 treatment (Figure 8). In another study with anti-PD-1 monotherapy (Gide, TN, et al., Cancer Cell, 2019, 35(2):238-255), there was again no correlation of the Golgi core signature with clinical response, nor induction of the signature after treatment, indicating that this signature is not a general marker predicting response to any cancer immunotherapy (Figure 8).

[0120] H. Enrichment of Golgi signatures in plasma cells, IFN-secreting pDCs, and other immune cells It was possible that the Golgi signature may reflect contributions from many different cells, and that a single cell may not express the entire signature. To evaluate the enrichment of the Golgi signature in different individual immune cells, the signature was evaluated in unique publicly available single-cell RNA-Seq datasets. In multiple publicly available single-cell datasets, expression of the Golgi signature is highest in plasma cells, followed by pDCs, macrophages, and several other immune cell populations (Figure 9 is an example of representative data). Furthermore, in published data on purified pDCs activated in vitro into three distinct subsets (P1, P2, P3), the Golgi signature is most highly expressed in the P1 subset, the pDC subset that produces the most type I IFN. (Figure 10) These studies further link the biology of the Golgi signature to the biology of TLR9 activation and vidutolimod's MOA, which rely on the induction of type I IFN to drive antitumor immunity.

[0121] I. The Golgi vidutolimod response signature is highly expressed in many different tumor indications Using the publicly available TCGA, enrichment of the Golgi signature was evaluated in various tumor indications (Figure 11). The analysis showed that expression of the signature was highly prevalent among most tumor types, but not limited to melanoma (SKCM) or NSCLC, indicating great potential for widespread use of this signature alone or in combination with other biomarkers to select cancer patients likely to respond to vidutolimod monotherapy or in combination with other agents.

[0122] J. Golgi signature can be induced by combined standard of care (SOC) chemotherapy and radiation therapy (XRT) To evaluate the effect of XRT and SOC treatment on the Golgi signature, we analyzed public datasets in CRC, which revealed that the signature was enriched in irradiated tissues (both normal and tumor) and separate controls 5 weeks after XRT treatment (Figure 12). In this study, each patient received 50 Gy (delivered in 25 fractions of 2 Gy) over a 5-week period. In addition, patients received daily capecitabine (Xeloda®, Roche) 2500 mg / ml throughout the entire period. Rectal resections were performed 4-6 weeks after preoperative radiotherapy.

[0123] K. Identification of potential "partners" with Golgi signatures to extend prediction of vidutolimod response to baseline "hot" and increase overall discrimination between responders and non-responders The QLattice algorithm (from www.abzu.ai / ) was applied to identify one or more potential "partner" genes or signatures that could potentially be used in combination with the Golgi signature to predict treatment response in all baseline tumors derived from patients treated with vidutolimod. The best model, with an AUC of 0.93 (95% CI: 0.82 to 1), is the gene "partner" ELF2, a transcription factor known to be important in immune regulation (Figure 13). Expression of ELF2 shows a strong independent correlation with response across our dataset of baseline biopsies of PD-1 resistant melanomas, including hot and cold (Figure 14). Other genes / signatures that interact with the Golgi signature with slightly lower AUCs (e.g., MOOTHA_GLUCONEGENESIS, BOLL, BAZ2A, INSL6, etc.) were also identified.

[0124] L. Signature correlation with Golgi signature in melanoma To clarify the potential function of the Golgi signature in the tumor microenvironment, all gene signatures were evaluated for correlation with the Golgi signature in TCGA melanoma (SKCM). The top (positive) correlated signatures are related to vesicle-Golgi trafficking (Table C), which is consistent with our expectations, while the top anti-correlated signatures are more involved in immune cells or their status (Table D), including myeloid cells, especially macrophages, DCs, and pDCs. These data indicate that the presence of the Golgi signature may reflect the activation or functional status of myeloid cells suitable for activation by vidutolimod. Furthermore, these anti-correlated signatures may provide an independent approach to the identification of novel gene signatures related to the response to innate immune activators such as vidutolimod in cancer immunotherapy. [Table 3] [Table 4]

[0125] Correlation of M. Golgi signature with BRAF mutation status and baseline risk factors In the PD-1 resistant melanoma dataset, Golgi signature expression did not correlate with risk factors such as BRAF mutation status or prior BRAFi / MEKi treatment (Figure 15), baseline LDH levels or liver metastases (Figure 16), or baseline tumor burden (Figure 17), providing further evidence that Golgi signature expression is not a surrogate for other known prognostic factors.

[0126] N. High baseline macrophages / monocytes or T reg is an independent predictor of non-response or PD to vidutolimod plus pembrolizumab The macrophage / monocyte signature was found to be associated with PD, particularly in baseline "hot" tumors from the CMP-001-001 data (Figure 18). High intratumoral myeloid cells are known to correlate with resistance to immunotherapy. A signature or other indicator of the presence of high myeloid cells in baseline tumor biopsies may, in some embodiments, be a component of a signature that can be used to select patients for vidutolimod therapy, either alone or in conjunction with the Golgi vidutolimod response signature. For example, individual genes expressed in macrophages and / or monocytes that show a strong inverse correlation with response to vidutolimod, particularly in baseline hot tumors, include TLR8, TGFB2, TNFRSF1A, TNFRSF1B, C1QC, and PD-L1 (Figure 19). The RNA levels of one or more of these genes can form part of the biomarkers for this purpose, with relatively low levels of expression of these genes indicating low levels of immunosuppressive macrophages or monocytes, thereby predicting that patients with relatively low expression of these genes will have a higher probability of responding to innate immune activators compared to patients with higher expression levels of these genes in the patient's baseline tumor biopsy. As used herein, "detecting the presence of a low macrophage and / or monocyte cell population" means detecting either a relatively low macrophage signature, such as Macrophage / Monocyte_MCPCOUNTER, or a relatively low expression level of one or more of the myeloid-related genes TLR8, TGFB2, TNFRSF1A, TNFRSF1B, C1QC, and PD-L1.

[0127] Conversely, low expression of TLR9 and TNFA in baseline "cold" tumors (but not hot tumors) correlates with a higher likelihood of response to innate immune activators compared to patients with higher expression levels of these genes in their baseline tumor biopsies (Figure 20).

[0128] Furthermore, the controllability T (T regHigh expression of the T α -dependent β-amyloid marker was found to correlate with PD in all baseline samples (Figure 21). reg The signatures or other indicators of the presence of cells may in some embodiments be components of a signature that may be used, alone or in conjunction with a Golgi vidutolimod response signature, to select patients for vidutolimod therapy.

[0129] Finally, a signature associated with GABA-producing B cells (REACTOME_GABA_B_RECEPTOR_ACTIVATION) was found to correlate with PD in baseline "hot" tumors (Figure 22). A signature or other indicator of the presence of high levels of GABA-producing B cells in baseline tumor biopsies may, in some embodiments, be a component of a signature that may be used to select patients for vidutolimod therapy, either alone or in conjunction with the Golgi vidutolimod response signature.

[0130] In summary, this example demonstrates the potency of vidutolimod, as well as collectively other TLR9 agonists, other TLR agonists, and other innate immune activators.

[0131] 1. High expression of some or all of the genes in the Golgi vidutolimod response signature is an independent predictor of response to vidutolimod, particularly in patients whose baseline biopsy is not "hot."

[0132] 2. High expression of ELF2 is an independent predictor of response to vidutolimod, and this may be used in combination with one or more of the Golgi signatures to more accurately predict the probability of responding to vidutolimod alone or in combination with other immunotherapies such as PD-1 blockade.

[0133] 3. T reg, and / or low expression of macrophage / monocyte-correlated signatures or genes are independent predictors of response to vidutolimod ± PD-1 blockade, which may be used in combination with one or more of the Golgi signatures to more accurately predict the probability of response to vidutolimod alone or in combination with other immunotherapies such as PD-1 blockade.

[0134] 4. Low expression of the B cell GABA signature is an independent predictor of response to vidutolimod ± PD-1 blockade in patients with baseline "hot" tumors, and this may be combined with one or more of the Golgi signatures to more accurately predict the probability of responding to vidutolimod alone or in combination with other immunotherapies such as PD-1 blockade.

Claims

1. 1. A method for identifying a subject likely to benefit from a cancer immunotherapy comprising an innate immune activator, the method comprising: (i) obtaining a tumor sample from a subject suffering from cancer; (ii) SEC16B, AREG, COL7A1, SEC24D, CNIH2, MAPK15, TGFA, CUL3, SAR1B, USO1, TRAPPC6A, SEC24A, TMED 2, SEC31A, BET1, GOLGA2, RAB1A, NAPA, LMAN1, TRAPPC3, PREB, SCFD1, SEC23A, CTSC, TRAPPC1, ANKRD28, detecting expression levels in the sample of at least five vidutolimod responsive core signature gene transcripts selected from the group consisting of SEC24B, MCFD2, CD59, TMED10, STX5, TRAPPC6B, PPP6R3, SEC13, TRAPPC9, CEP19, ARFGAP3, GOSR1, SEC23B, MIA3, and VAPA; The method of identifying, wherein said detection of enriched expression levels identifies subjects who are likely to benefit from cancer immunotherapy comprising an innate immune activator.

2. 1. A method for identifying a subject likely to benefit from a cancer immunotherapy comprising an innate immune activator, the method comprising: (i) obtaining a tumor sample from a subject suffering from cancer; (ii) confirming that the tumor is not "hot"; and (iii) SEC16B, AREG, COL7A1, SEC24D, CNIH2, MAPK15, TGFA, CUL3, SAR1B, USO1, TRAPPC6A, SEC24A, TME D2, SEC31A, BET1, GOLGA2, RAB1A, NAPA, LMAN1, TRAPPC3, PREB, SCFD1, SEC23A, CTSC, TRAPPC1, ANKRD28 detecting in the sample the expression levels of at least five vidutolimod responsive core signature gene transcripts selected from the group consisting of: SEC24B, MCFD2, CD59, TMED10, STX5, TRAPPC6B, PPP6R3, SEC13, TRAPPC9, CEP19, ARFGAP3, GOSR1, SEC23B, MIA3, and VAPA; The method of identifying, wherein said detection of enriched expression levels of at least five vidutolimod core signature gene transcripts in non-"hot" tumors identifies subjects who are likely to benefit from cancer immunotherapy comprising an innate immune activator.

3. 1. A method for identifying a subject likely to benefit from a cancer immunotherapy comprising an innate immune activator, the method comprising: (i) obtaining a tumor sample from a subject; (ii) SEC16B, AREG, COL7A1, SEC24D, CNIH2, MAPK15, TGFA, CUL3, SAR1B, USO1, TRAPPC6A, SEC24A, TMED 2, SEC31A, BET1, GOLGA2, RAB1A, NAPA, LMAN1, TRAPPC3, PREB, SCFD1, SEC23A, CTSC, TRAPPC1, ANKRD28, detecting elevated expression levels in the sample of at least five vidutolimod responsive core signature gene transcripts selected from the group consisting of SEC24B, MCFD2, CD59, TMED10, STX5, TRAPPC6B, PPP6R3, SEC13, TRAPPC9, CEP19, ARFGAP3, GOSR1, SEC23B, MIA3, and VAPA; (iii) detecting the presence of low macrophage and / or monocyte cell populations; The method of identification, wherein the detection identifies a subject who is likely to benefit from cancer immunotherapy comprising an innate immune activator.

4. 1. A method for identifying a subject likely to benefit from a cancer immunotherapy comprising an innate immune activator, the method comprising: (i) obtaining a tumor sample from a subject; (ii) SEC16B, AREG, COL7A1, SEC24D, CNIH2, MAPK15, TGFA, CUL3, SAR1B, USO1, TRAPPC6A, SEC24A, TMED 2, SEC31A, BET1, GOLGA2, RAB1A, NAPA, LMAN1, TRAPPC3, PREB, SCFD1, SEC23A, CTSC, TRAPPC1, ANKRD28 , SEC24B, MCFD2, CD59, TMED10, STX5, TRAPPC6B, PPP6R3, SEC13, TRAPPC9, CEP19, ARFGAP3, GOSR1, SEC23B, MIA3, and VAPA; and detecting elevated expression levels in the sample of at least five vidutolimod core signature gene transcripts selected from the group consisting of: (iii) in said tumor sample, (a) increased expression of ELF2, and / or (b) Low T ｒｅｇ the presence of a cell population; and / or (c) Low expression of the GABA B cell signature and detecting one or more of: The method of identification, wherein the detection identifies a subject who is likely to benefit from cancer immunotherapy comprising an innate immune activator.

5. 1. A method for identifying a subject likely to benefit from a cancer therapy comprising an innate immune activator, comprising obtaining a tumor sample from the subject; (a) the presence of low macrophage and / or monocyte cell populations; (b) high expression of ELF2, and / or (c) Low T ｒｅｇ The presence of cell populations, (d) Low expression of the GABA B cell signature and detecting one or more of: The method of identifying, wherein the detection identifies a subject who is likely to benefit from a cancer therapy comprising an innate immune activator.

6. 1. A method for treating a subject suffering from cancer with cancer immunotherapy comprising an innate immune activator, the method comprising: (i) obtaining a tumor sample from a subject suffering from cancer; (ii) SEC16B, AREG, COL7A1, SEC24D, CNIH2, MAPK15, TGFA, CUL3, SAR1B, USO1, TRAPPC6A, SEC24A, TMED2, SEC31A , BET1, GOLGA2, RAB1A, NAPA, LMAN1, TRAPPC3, PREB, SCFD1, SEC23A, CTSC, TRAPPC1, ANKRD28, SEC24B, MCFD2, CD5 detecting expression levels in the sample of at least five vidutolimod-responsive core signature gene transcripts selected from the group consisting of: 9, TMED10, STX5, TRAPPC6B, PPP6R3, SEC13, TRAPPC9, CEP19, ARFGAP3, GOSR1, SEC23B, MIA3, and VAPA, wherein enriched expression levels are detected; (iii) administering to the subject a cancer immunotherapy comprising a natural immune activator.

7. 1. A method for treating a subject suffering from cancer with cancer immunotherapy comprising an innate immune activator, the method comprising: (i) obtaining a tumor sample from a subject suffering from cancer; (ii) confirming that the tumor is not "hot"; and (iii) SEC16B, AREG, COL7A1, SEC24D, CNIH2, MAPK15, TGFA, CUL3, SAR1B, USO1, TRAPPC6A, SEC24A, TMED2, SEC31 A, BET1, GOLGA2, RAB1A, NAPA, LMAN1, TRAPPC3, PREB, SCFD1, SEC23A, CTSC, TRAPPC1, ANKRD28, SEC24B, MCFD2, CD 59, TMED10, STX5, TRAPPC6B, PPP6R3, SEC13, TRAPPC9, CEP19, ARFGAP3, GOSR1, SEC23B, MIA3, and VAPA, wherein enriched expression levels are detected; and (iv) administering to the subject a cancer immunotherapy comprising a natural immune activator.

8. 1. A method for treating a subject suffering from cancer with cancer immunotherapy comprising an innate immune activator, the method comprising: (i) obtaining a tumor sample from a subject; (ii) SEC16B, AREG, COL7A1, SEC24D, CNIH2, MAPK15, TGFA, CUL3, SAR1B, USO1, TRAPPC6A, SEC24A, TMED2 , SEC31A, BET1, GOLGA2, RAB1A, NAPA, LMAN1, TRAPPC3, PREB, SCFD1, SEC23A, CTSC, TRAPPC1, ANKRD28, SE detecting elevated expression levels in said sample of at least five vidutolimod-responsive core signature gene transcripts selected from the group consisting of C24B, MCFD2, CD59, TMED10, STX5, TRAPPC6B, PPP6R3, SEC13, TRAPPC9, CEP19, ARFGAP3, GOSR1, SEC23B, MIA3, and VAPA; and / or (iii) detecting the presence of low macrophage and / or monocyte cell populations; and (iv) if an enriched expression level is detected in (ii), and / or if a low macrophage and / or monocyte cell population is detected, the method comprising administering to the subject a cancer immunotherapy comprising an innate immune activator.

9. 1. A method for treating a subject suffering from cancer with cancer immunotherapy comprising an innate immune activator, the method comprising: (i) obtaining a tumor sample from a subject; (ii) SEC16B, AREG, COL7A1, SEC24D, CNIH2, MAPK15, TGFA, CUL3, SAR1B, USO1, TRAPPC6A, SEC24A, TMED 2, SEC31A, BET1, GOLGA2, RAB1A, NAPA, LMAN1, TRAPPC3, PREB, SCFD1, SEC23A, CTSC, TRAPPC1, ANKRD28 , SEC24B, MCFD2, CD59, TMED10, STX5, TRAPPC6B, PPP6R3, SEC13, TRAPPC9, CEP19, ARFGAP3, GOSR1, SEC23B, MIA3, and VAPA; and detecting elevated expression levels in the sample of at least five vidutolimod core signature gene transcripts selected from the group consisting of: (iii) in said tumor sample, (a) increased expression of ELF2, and / or (b) Low T ｒｅｇ the presence of a cell population; and / or (c) Low expression of the GABA B cell signature and detecting one or more of: (iv) administering to the subject a cancer immunotherapy comprising a natural immune activator.

10. 1. A method of treating a subject suffering from cancer with cancer immunotherapy comprising a natural immune activator, the method comprising obtaining a tumor sample from the subject; (a) the presence of low macrophage and / or monocyte cell populations; (b) high expression of ELF2; (c) Low T ｒｅｇ the presence of cell populations, and / or (d) Low expression of the GABA B cell signature and detecting one or more of: and administering to the subject a cancer immunotherapy comprising a natural immune activator.

11. 2. The method of any one of the preceding claims, wherein the innate immune activator is selected from the group consisting of TLR agonists.

12. 12. The method of claim 11, wherein the TLR agonist is selected from the group consisting of a TLR9, TLR4, TLR7, and TLR8 agonist.

13. The method of claim 12, wherein the TLR agonist is a TLR9 agonist.

14. 14. The method of claim 13, wherein the TLR9 agonist is selected from the group consisting of A class CpG DNA, C class CpG DNA, E class CpG DNA, A / E class CpG DNA, P class CpG DNA, and any combination thereof.

15. The method of claim 14 , wherein the TLR9 agonist is A class CpG DNA.

16. 16. The method of claim 15, wherein the A class CpG DNA comprises the sequence GGGGGGGGGGGACGATCGTCGGGGGGGGGG (SEQ ID NO: 1).

17. 17. The method of claim 15 or 16, wherein the A class CpG DNA is formulated as a virus-like particle (VLP).

18. The method of claim 17, wherein the VLP comprises a bacteriophage Qβ coat protein.

19. The tumor is (a) TIDE score <-1; (b) low expression of an IFN-γ transcriptional signature that includes five or more of the following gene transcripts: CD3D, IDO1, CIITA, CD3E, CCL5, GZMK, CD2, HLA-DRA, CXCL13, IL2RG, NKG7, HLA-E, LAG3, TAGAP, CXCL10, STAT1, GZMB, CXCR6; (c) low immunoscore, and / or (d) PD-L1 CPS<10 4. The method of claim 1, wherein the determination that the signal is not "hot" is based on one or more of:

20. 2. The method of any one of the preceding claims, wherein the tumor is resistant to a checkpoint inhibitor selected from the group consisting of PD-1 and PD-L1.

21. 2. The method of any one of the preceding claims, wherein the subject is a human suffering from one or more cancerous tumors.

22. 22. The method of claim 21, wherein the cancerous tumor is a lymphoma or a cancerous tumor of a tissue or organ selected from the group consisting of skin, head and neck, esophagus, stomach, liver, colon, rectum, pancreas, lung, breast, cervix, ovary, kidney, bladder, prostate, thyroid, brain, muscle, and bone.

23. 22. The method of claim 21, wherein the cancerous tumor is melanoma, NSCLC, and HNSCC.

24. 22. The method of claim 21, wherein the subject is undergoing a therapy selected from the group consisting of radiation therapy, chemotherapy, immunotherapy, therapy comprising a checkpoint inhibitor, surgery, and hormone therapy.

25. 2. The method of any one of the preceding claims, wherein said detection of gene transcripts is selected from the group consisting of RNA sequencing (RNA-Seq), mRNA sequencing (mRNA-Seq), targeted RNA-Seq, and non-coding RNA-Seq, nanostring, microarray, or other hybridization-based technologies.

26. 13. The method of any preceding claim, wherein the presence of 10, 15, 20, 25, 30, 35 or more vidutolimod core signature gene transcripts is detected.

27. 2. The method of any preceding claim, wherein the detected vidutolimod core signature gene transcripts have an enrichment score in up to 80% of patient biopsies for said tumor type.

28. 2. The method of any of the preceding claims, wherein the detected vidutolimod core signature gene transcripts are expressed in one or more cells selected from the group consisting of plasmacytoid dendritic cells (pDCs), plasma cells, mast cells, activated monocytes, macrophages, dendritic cells, and / or T cells.

29. 20. The method of any of the preceding claims, wherein the cancer immunotherapy is administered via intratumoral, peritumor, systemic, intravenous, intraperitoneal, intraintestinal, oral, intramuscular, subcutaneous, transmucosal, topical, intravesicular, and / or transdermal routes.

30. 30. The method of claim 29, wherein the composition is administered intratumorally.

31. 31. The method of any one of claims 29-30, further comprising administering to the subject at least one dose of a composition comprising a checkpoint inhibitor (CPI).

32. 32. The method of claim 31 , wherein the CPI is an antibody or antigen-binding fragment thereof that specifically binds to an antigen selected from the group consisting of PD-1, PD-L1, LAG-3, TIM-3, and CTLA-4.

33. 33. The method of claim 32, wherein the CPI is an antibody or antigen-binding fragment thereof that specifically binds to PD-1.

34. 32. The method of claim 31 , wherein the TLR9 agonist is vidutolimod and the CPI is a PD-1 inhibitor.

35. SEC16B, AREG, COL7A1, SEC24D, CNIH2, MAPK15, TGFA, CUL3, SAR1B, USO1, TRAPPC6A, SEC24A, TMED2, SE C31A, BET1, GOLGA2, RAB1A, NAPA, LMAN1, TRAPPC3, PREB, SCFD1, SEC23A, CTSC, TRAPPC1, ANKRD28, SEC2 4B, MCFD2, CD59, TMED10, STX5, TRAPPC6B, PPP6R3, SEC13, TRAPPC9, CEP19, ARFGAP3, GOSR1, SEC23B, MIA3, and VAPA.

36. (a) the presence of low macrophage and / or monocyte cell populations; (b) high expression of ELF2, and / or (c) Low T ｒｅｇ the presence of a cell population; and / or (d) Low expression of the GABA B cell signature A kit comprising one or more compositions comprising reagents capable of detecting one or more of the following:

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