Method of treating solid tumors

Abstract

To provide a system and a method effective for treating solid tumors.SOLUTION: Solid cancerous tumors are treated with a system having an antigen presenting cell agent, a T cell activating neo-antigen vaccine, and an immunosuppression inhibitor. Another aspect of the invention is a method of treating a solid cancerous tumor (SCT) in a subject by administering an antigen presenting cell agent; a T cell activating vaccine; and an immunosuppression inhibitor.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 880,614, filed July 30, 2019, U.S. Provisional Patent Application No. 62 / 881,887, filed August 1, 2019, U.S. Provisional Patent Application No. 62 / 886,946, filed August 14, 2019, and U.S. Provisional Patent Application No. 62 / 943,155, filed December 3, 2019, the disclosures of each of which are incorporated herein by reference in their entirety.

[0002] Incorporating References This application contains a "Sequence Listing" which has been filed electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy, created on July 30, 2020, has the filename 056705_502001WO_SequenceListing_07302020 and is 8,221 bytes in size.

[0003] FIELD OF THE INVENTION The present invention relates to systems and methods for treating solid tumors. [Background technology]

[0004] Background of the Invention Cancers involving solid tumors are the most common cancer in the United States (D. Wang et al., Cancer J (2013) 19(6):502-10). Current treatments for solid tumors include surgery, chemotherapy, radiation therapy, and immunotherapy. While surgical intervention can be effective when these tumors are detected at an early stage, in many patients, the cancer is not detected until it is already at an advanced stage.

[0005] For example, colorectal cancer (CRC) is a complex disease, and other classification systems have been proposed to better describe its different aspects and phases. One such system is the Immunoscore, which classifies tumors according to the presence of CD3+ and CD8+ cells (T cells) in the tumor center and invasive margin. Scores range from 10 (few or no CD3+ or CD8+ cells in both the center and margin) to 14 (high immune cell density in both locations). The Immunoscore consensus has been comprehensively validated in colon cancer and has superior relative prognostic value than other measures such as lymphovascular invasion, tumor differentiation, and microsatellite stability (MSI) status. CRC tumors are often referred to as either "hot" (14, showing a vigorous cytotoxic lymphocyte (CTL) response) or "cold" (10, showing little or no response). Tumors are classified as either "hot" (I4, showing a vigorous cytotoxic lymphocyte (CTL) response) or "cold" (I0, showing little or no response). Intermediate tumors can be classified as "immune-excluded" and "immunosuppressed." In "immune-excluded" tumors, CTLs and dendritic cells (APCs) are found at the tumor periphery but are prevented from invading. In "immunosuppressed" tumors, CTLs and APCs are found within the tumor but are ineffective and inactive. See, e.g., J. Galon et al., Nat Rev Drug Discov (2019) 18:197-218.

[0006] Tumors can form dense masses containing malignant cells, tumor-associated macrophages (TAMs), tumor- or cancer-associated fibroblasts (TAFs or CAFs), extracellular matrix, and collagen. As tumors grow, they become hypoxic, sometimes with a lower pH than normal tissue. Tumor cells and other cells within tumors can inappropriately express proteins, such as overexpression of CD73, which leads to immunosuppressive levels of adenosine, and overexpression of PD-L1, which can lead to T cell anergy. Underexpression of other proteins, such as interferon receptors and MHC-I, can lead to CTL-mediated immunity, as in "immunosuppressed" tumors. The tumor margins become impenetrable to CTLs, as in "immune-compromised" tumors. See, for example, J.A. Joyce et al., Science (2015) 348(6230):74-80; R. Levayer, Seminars Cancer Biol (2019) https: / / doi.org / 10.1016 / j.semcancer.2019.05.004. This tumor environment can induce CD8+ T cell differentiation toward an inhibitory / regulatory phenotype and induce macrophage shift toward an M2 immunosuppressive phenotype. Consequently, solid tumors such as CRC are more difficult to treat than many other cancers. Furthermore, CRC encompasses at least three subtypes with unique immunological characteristics and altered immune evasion mechanisms (J. Guinney et al., Nat Med (2015) 21(11):1350-56; M.A. Komor et al., J Pathol (2018) 246(3):266-276).

[0007] There are over 2,000 cancer immunological drugs currently being tested or used, addressing over 60 different targets (J. Galon et al., supra). These drugs are taken alone and provide complete responses in only a small number of cases. There are numerous clinical trials underway testing the efficacy of specific pairs of such drugs: only individual targets can be considered and tested. 2There are many more pairs, or over 3,600 possible pairs. To date, pairs of immuno-oncology agents have improved response rates but still ultimately failed in the majority of cases. For example, clinical trial results showing improvement when combining CTLA-4 inhibitors (e.g., ipilimumab) with PD-1 inhibitors (e.g., nivolumab) suggest that addressing only one immune checkpoint is not sufficient and that most or all significant checkpoints must be addressed for a complete response. A single category of treatment, immune checkpoint blockade alone, is unlikely to be completely effective because other pathways of tumor evasion exist, so it is most likely that some or all categories of targets must be addressed. Drug combinations addressing three different targets have been shown to be more effective than 60% of the time. 3 =216,000 possible combinations. 4 different targets: 12.9 million combinations. 5 different targets: 7.776 million combinations. These figures do not include the more than 100 chemotherapy drugs that could be used in combination, nor do they account for other agents such as COX-2 inhibitors (e.g., aspirin) or angiotensin II receptor type 1 inhibitors (e.g., losartan) that could also be combined, yet still undiscovered oncogenic pathways. Clearly, the global economy cannot support the systematic study of all possible immuno-oncology drug combinations.

[0008] The question of choice is usually addressed by animal models. Most basic cancer research is performed using mouse models of cancer development. In some cases, tumors are induced directly in mice using chemical agents. These provide tumors that superficially mimic human tumors but are not necessarily related in any other way. In other cases, human tumor cells (often derived from a single, standardized cell line) are implanted into mice, and the activity of candidate drugs is tested. However, the human immune system (and cancerous tissues in humans) differs from the mouse system, making these models less predictive. This lack of predictability is evidenced by the large number of drugs that show promise in mouse (and other animal) models but fail when they reach clinical trials. This is also evidenced by unforeseen outcomes such as the severe cytokine release syndrome that occurred in the first clinical trials of TGN1412, an anti-CD28 monoclonal antibody, which resulted in fatal outcomes despite the administration of an initial dose that was 0.2% of the dose considered safe in non-human primates (see, e.g., EW St. Clair, J Clin Invest (2008) 118(4):1344-47). Drug combinations predicted to work in humans based on successful animal models have failed to show reproducible efficacy in humans for some cancers, such as cobimetinib and atezolizumab for metastatic colorectal cancer (PJR Ebert et al., Immunity (2016) 44(3):609-21; C. Eng et al., Lancet Oncol (2019) 20(6):849-61, https: / / doi.org / 10.1016 / S1470-2045(19)30027-0), and have not proven to be a reliable way to model the human tumor microenvironment and predict the efficacy or safety of immunotherapy. Commonly used mouse models for testing drugs for mCRC, such as CT26, likely use tumor cells that represent a small subset of patients and therefore provide insight into the response of only those patients' tumor cells to increased immune activity (JC Castle et al., BMC Genomics (2014) 15(1):190-201).In CRC, different patients likely have different mutations, and therefore cancer cells from one patient may not necessarily represent cancer cells in all patients. Furthermore, when tumor cells are tested in mouse models (typically without the addition of stromal cells), this does not necessarily reproduce the immunosuppression produced by human patients, such as that caused by interactions between stromal cells and tumor cells. Therefore, better methods for predicting the efficacy of combined immunotherapies are needed.

[0009] The present disclosure provides correlations of gene expression (by measuring RNA) in patient tumors that may provide a much better way to predict the efficacy of combination immunotherapies across many patients. The use of gene expression data involves correlating genetic biomarkers in patient tissue simultaneously with respect to multiple druggable targets of immunotherapeutic combinations. This also involves correlating the combined expression of one, two, or more genes in patient tumor tissue to determine which gene expression combinations cause immunosuppression and which drug combinations may counteract this cause of immunosuppression. [Brief explanation of the drawings]

[0010] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] Figure 1 shows two radiological scans of the subject described in Example 4 below. The right panel shows the subject's status on September 9, 2019, while the left panel shows the treatment progress on November 26, 2019. Summary of the Invention

[0011] Brief Summary of the Invention In general, the present disclosure determines the shortcomings of prior therapies, identifies target categories to be addressed, and invents effective systems and methods for treating solid tumors based on relevant human data. The results herein are based in part on data generated by the TCGA Research Network, https: / / www.cancer.gov / tcga.

[0012] An aspect of the present invention is, inter alia, a system for treating solid cancer tumors (colorectal, pancreatic, prostate, head and neck, melanoma, lung, liver, stomach, and breast) in a subject, the system comprising: an antigen-presenting cell agent; a T-cell activating vaccine; and an immunosuppressive inhibitor.

[0013] Another aspect of the invention is a method of treating a solid cancer tumor (SCT) in a subject by administering an antigen-presenting cell agent; a T cell activating vaccine; and an immunosuppressive inhibitor.

[0014] Another aspect of the present invention is a T cell activating vaccine having multiple neoantigens or one or more nucleic acids encoding multiple neoantigens; and a pharmaceutically acceptable carrier.

[0015] Another aspect of the invention is a method of supporting a subject's immune response to a neoplastic disease by administering an antigen-presenting cell agent; a T-cell activating vaccine; and an immunosuppressive inhibitor. DETAILED DESCRIPTION OF THE INVENTION

[0016] Detailed Description of the Invention A. System An aspect of the present invention is a system comprising at least the following elements: an antigen-presenting cell agent, a T cell activating vaccine; and an immunosuppressive inhibitor. An embodiment is a system in which the antigen-presenting cell agent is a CD40 agonist, a Toll-like receptor agonist, an adjuvant, FLT3L, or any combination thereof. An embodiment of the present invention is a system in which the immunosuppressive inhibitor is a CD73 inhibitor, a PD-L1 inhibitor, a PD-1 inhibitor, an A2a receptor inhibitor, a multikinase inhibitor, cyclophosphamide, a COX-2 inhibitor, a prostaglandin E2 inhibitor, or a combination thereof. An embodiment is a system further comprising an angiotensin II receptor type 1 antagonist.

[0017] 1. Antigen-presenting cell drugs Most types of cells can present antigens to immune cells. However, "professional" antigen-presenting cells (APCs) are a rare and heterogeneous population of cells with unique morphology and widespread tissue distribution. See, e.g., R.M. Steinman, Annu Rev Immunol (1991) 9:271-96. APCs include dendritic cells (DCs), macrophages, and B cells. DCs display an unusual cell surface phenotype and are characterized by the expression of cell surface markers CD1, CD86, CD11c, CD-205, CD40, and MHC-II, and the lack of CD14 and other lineage markers. APCs can sensitize MHC-restricted T cells and provide an efficient route for the in situ presentation of antigens to T cells, both self-antigens during T cell development and foreign antigens during immune responses.

[0018] Before encountering a foreign antigen, APCs express very low levels of MHC-II molecules and costimulatory molecules. APCs continuously sample the surrounding tissue and environment and endocytose and process the antigens they encounter. When APC pattern recognition receptors (see Toll-like receptors below) recognize pathogen-associated molecular patterns or damage-associated molecular patterns, APCs phagocytose the antigen and become activated, upregulating the expression of MHC-II molecules and costimulatory molecules necessary for T cell activation, such as CD40 and B7. APCs then fully mature and migrate from the tissue to lymph nodes, where they encounter and activate T cells.

[0019] a. FLT3L FMS-like tyrosine kinase 3 ligand (FLT3L, also known as FLT3LG) can be used to stimulate the production of downstream or intermediate cells, such as myeloid precursor cells, monocytes, macrophages, B cells, and dendritic cells, from CD34+ myeloid progenitor and stem cells. It can also be used to mobilize antigen-presenting cells in vivo and expand them ex vivo, e.g., for activation with a selected antigen and reintroduction into a subject. FLT3L and derivative polypeptides are described in U.S. Patent No. 5,554,512, WO 94 / 28391, and US 20060292166, all of which are incorporated herein by reference.

[0020] FLT3L and its derivatives may be made and administered by the methods described in U.S. Patent No. 5,554,512, WO 94 / 28391, and US 20060292166, and may also be administered as nucleic acids encoding the proteins and polypeptides described therein. An embodiment is a system comprising FLT3L or a derivative thereof. Another embodiment is a system comprising the FDA-approved leukocyte growth factor pegfilgrastim (Neulasta, Amgen) (https: / / clinicaltrials.gov / ct2 / show / NCT03789097), which, like FLT3L, has been shown to generate new dendritic cells in patients that support immunogenic responses (Bonanno et al., J Transl Med. 2010; 8:114).

[0021] b. Toll-like receptor agonists Toll-like receptor (TLR) agonists activate TLRs and thus APCs that express TLRs. TLR3 is mediated by poly(I:C) and its derivatives (e.g., AmpliGen®, Hiltonol®), poly-ICLC, poly(IC-R), poly(I:C 12 It can be activated by TLR3 (TLR3), TLR4 (TLR5), and non-CpG bacterial DNA and RNA. Poly(I:C) is a dimer of polyinosinic acid and polycytidylic acid. This double-stranded RNA structure stimulates TLR3. Poly-ICLC (Hiltonol®) is poly-(I:C) plus poly-L-lysine and carboxymethylcellulose. TLR agonists are generally administered by intradermal or intramuscular injection and may be combined with a specific antigen. One embodiment of the present invention is a system comprising a TLR agonist. An embodiment of the present invention is a system in which the agonist comprises poly(I:C) or a derivative thereof. Another embodiment of the present invention is a system in which the agonist comprises poly-ICLC.

[0022] c. CD40 agonist CD40 is a costimulatory protein found on APCs and is required for their activation. Expression of CD40L (also known as CD154) on CD4+ T cells and its binding to CD40 activates APCs and induces or "licenses" antigen-presenting cells to mature, thereby triggering T cell activation and differentiation. CD40 agonists (including CD40L mimetics and fragments) can be used to trigger APC maturation and migration, resulting in the expansion of APC populations, including APCs within and around tumors. CD40 agonists include CD40L (either membrane-bound or soluble), CD40 agonists (e.g., those described in US2019071509 and US7338660, both of which are incorporated herein by reference), anti-CD40 antibodies such as lucatumumab and dacetuzumab, and CD40 agonist peptides (e.g., those described in US9161976, which is incorporated herein by reference).

[0023] CD40 agonists can be administered by known methods appropriate for the form of the agonist. Protein-based CD40 agonists can also be administered in the form of nucleic acids encoding the agonist, such as in the form of viral vectors or gene therapy vehicles. Another embodiment is a system including the APC cell maturation agent Maravairoc (https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC6685512 / ).

[0024] d. Adjuvants Other compounds generally useful in modulating immune responses include adjuvants, such as Montanide™, squalene, muramyl di- and tri-peptides, saponins, and the like, which may be used in combination with each other and with the other APC agents mentioned above. Montanide™ is an oil-in-water emulsion that uses a mannide monooleate derivative as an emulsifier and was developed as a vaccine adjuvant. Montanide™ can contain mineral oil, squalene, or other oils. Most adjuvants have an oil or emulsion base and are combined with the vaccine antigen before administration.

[0025] e. Combination In addition, the aforementioned APC agents can be combined, co-administered, or otherwise used together in the methods of the present invention. One embodiment of the present invention is a system comprising FLT3L or a derivative thereof and poly(I:C) or a derivative thereof. Another embodiment of the present invention is a system comprising FLT3L and a CD40 agonist. Another embodiment of the present invention is a system comprising FLT3L, a CD40 agonist, and poly(I:C) or a derivative thereof. Another embodiment of the present invention is a system comprising a CD40 agonist and poly(I:C) or a derivative thereof. An embodiment is a system comprising FLT3L and poly-ICLC. An embodiment is a system comprising FLT3L, poly-ICLC, and Montanide™.

[0026] 2. T cell-activating vaccines Overall survival and progression-free survival are associated with the presence of tumor-infiltrating lymphocytes (TILs), particularly activated effector T cells (CTLs), within the tumor (J. Galon et al., Science (2006) 313:1960-64). Obtaining such CTLs requires exposure to appropriate tumor antigens and activation by APCs. SBRT and related ablative therapies also rely on T cell activity for the abscopal effect (tumor killing outside the area directly exposed to radiation).

[0027] a. Neoantigens and neoepitopes Early evidence indicates that neoantigen-based vaccination or neoantigen vaccines can induce T cell responses, and that neoantigen-targeted T cells can cause tumor regression in select patients under certain circumstances. To recognize tumor cells, T cells must bind tumor-specific antigens with high affinity, and these antigens must be sufficiently distinct from normal proteins to avoid tolerance. Because tumor cells accumulate numerous mutations during their evolution toward malignancy, some mutations result in alterations in the amino acid sequences of proteins expressed in tumors. These distinct alterations may also be immunogenic. Antigens expressed by tumor cells but not by normal cells are called neoantigens.

[0028] Tumor cells are characteristically dysregulated and heterogeneous, so it is unlikely that all cells within a tumor will express the same set of neoantigens. Furthermore, treatments targeting a single antigen may pressure tumor cells to downregulate expression of that antigen, or even MHC protein expression, such that most or all antigens are not presented. In the absence of neoantigens presented on MHC-I proteins on the surface of tumor cells, CTLs are unable to recognize and kill tumor cells. As a result, multiple neoantigens are required to kill as many tumor cells as possible.

[0029] Candidate neoantigens can be identified by immunological testing, sequencing (e.g., deep RNA sequencing of biopsied tissue), and prediction, or a combination thereof. Sequencing biopsied or excised tumor tissue obtained from a subject can determine which proteins are mutated and thus candidate "personalized" neoantigens for that subject. Sequencing such tissue from many subjects can be used to determine which proteins are most frequently mutated and therefore can be combined into a general neoantigen formulation. As a further refinement, neoantigen binding to specific MHC-I and MHC-II alleles can be correlated based on the subject's personal alleles to determine or predict which neoantigens are likely to work well for a particular subject. See, e.g., M. Rajasagi et al., Blood (2014) 124(3):453-62; Y. Chu et al., Theranostics (2018) 8(15):4238-46; RE Hollingsworth et al., npj Vaccines (2019) 4:7-17; and PA Ott et al., Nature (2017) 547(7662):217-21. For illustrative technology, see Carreno et al., WO2016 / 040900; Rooney, WO2017 / 173321; and Yelensky et al., WO2019 / 050994 (and their published corresponding U.S. patents), each of which is incorporated herein by reference.

[0030] The multiple neoantigen peptides and / or multiantigen polypeptides can be divided into multiple different pools containing two or more different combinations of neoantigens. For example, the vaccine can be provided in the form of 1, 2, 3, 4, 5, 6, 7, 8, 9, or about 10 distinct neoantigen combinations, which may or may not be unique, and which are administered to a patient at separate injection sites. These injection sites can be selected to target different lymph nodes, i.e., the lymph nodes that ultimately receive the neoantigen combinations can be different. The targeted lymph nodes can be selected based on being draining lymph nodes for one or more tumor sites.

[0031] Destruction of tumor cells that bear a particular antigen creates selective pressure favoring surviving cells that do not express that antigen. Thus, the best neoantigen(s) for treating a particular tumor may develop over time as a response to treatment. See, e.g., G. Rospo et al., Genome Med (2019) 11:42-64, https: / / doi.org / 10.1186 / s13073-019-0654-6. To counter this, the system of the present invention involves administering multiple neoantigens, reanalyzing the neoantigens present in the tumor(s) as treatment progresses, and administering updated neoantigens.

[0032] In one embodiment, the vaccine contains about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 20, 25, 30, 35, 40, 45, or 50 neoantigens, which can be provided as individual peptides or linked into several longer multiantigen polypeptides with about 12, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids per multiantigen polypeptide. The neoantigens and multiantigen polypeptides can be synthesized and stored by standard methods, such as suspension in a buffer solution and lyophilization.

[0033] Vaccines or vaccine subset combinations are formulated for administration by subcutaneous or intradermal injection. Generally, such formulations contain a neoantigen or neoantigen polypeptide in an aqueous vehicle, which may further include buffers and suspending agents, such as saline, phosphate-buffered saline, surfactants, and the like. Vaccines are often formulated in oil-based or emulsion-based adjuvant compositions. In embodiments, the neoantigen peptide or polypeptide is formulated in an oil-in-water or water-in-oil emulsion. In embodiments of the present invention, the neoantigen or multiantigen polypeptide formulation further comprises an APC agent. In embodiments, the APC agent is FLT3L, poly-ICLC, poly-ICLC, or Montanide™. In the event that the neoantigen peptide or multiantigen polypeptide is not sufficiently soluble in aqueous solution, it can be formulated directly into the oil phase of an oil-in-water or water-in-oil emulsion. In an embodiment, the neoantigen peptide and / or multiantigen polypeptide is formulated directly into an adjuvant solution, such as Montanide™.

[0034] 3. Immunosuppressant inhibitors The immune system contains regulatory mechanisms that modulate or suppress immune responses, including immune checkpoints, adenosine receptor A2aR regulation, CTLA-4, and cytokine factors such as TGF-β. These endogenous mechanisms are often used by tumor cells to evade cytotoxic T cell responses.

[0035] a. CD73 and adenosine The adenosine 2a receptor (A2aR) is expressed primarily on cells of hematopoietic origin, particularly activated CTL cells and CD4+ T cells. H It is a G protein-coupled receptor expressed on T cells. Activation of this receptor leads to T cell anergy, inhibition of CTLs, and the production of immunosuppressive T cells. regA2aR stimulation leads to the differentiation of CD8+ CTLs into T cells. Stimulation of A2aR during antigen presentation leads to immune tolerance (PE Zerk et al., Blood (2008) 111:251-59). Extracellular adenosine is produced by CD73+ cells, which is found in many cancer types, including glioblastoma, breast cancer, CRC, ovarian cancer, gastric cancer, and gallbladder cancer (ZW. Gao et al., BioMed Rsch. Intl. (2014) 2014:460654). High expression levels of CD73 (also known as ecto-5'-nucleotidase) correlate with poor prognosis in CRC and gastric cancer. CD73 expression can be driven by hypoxic conditions, which are frequently observed in advanced tumors. Therefore, antagonists of A2aR, CD73, or both can reduce or prevent T cell anergy.

[0036] A2aR inhibitors include anti-A2aR antibodies and derivatives, as well as small molecule inhibitors such as CPI-444, PBF-509, MK-3814, and AZD4635, which are currently in clinical trials. CD73 antagonists include anti-CD73 antibodies and their derivatives, such as oleclumab and BMS-986179, both of which are currently in clinical trials. Extracellular adenosine can also be reduced by administration of Adagen® (PEGylated adenosine deaminase), which is currently prescribed for the treatment of severe combined immunodeficiency due to adenosine deaminase deficiency. Adenosine activity at the A2aR can also be antagonized or blocked by caffeine, which competes for binding to the A2aR without activating it. An embodiment of the present invention is a system comprising an A2aR inhibitor, a CD73 antagonist, or a combination thereof. An embodiment is a system comprising a CD73 antagonist and caffeine.

[0037] b. Multikinase inhibitors Regorafenib is a small molecule drug that inhibits multiple kinases and targets angiogenic, stromal, and oncogenic receptor tyrosine kinases. The compound's tumor-reducing activity was initially attributed to its inhibition of raf kinase and VEGFR2, but it was later shown to inhibit CSF1R, TIE2, VEGFR1, VEGFR3, PDGFR-β, FGFR, KIT, RET, and BRAF. See, e.g., SM Wilhelm et al., Int J Cancer (2011) 129:245-55. It also inhibits soluble epoxide hydrolase (sEH). Inhibition of multiple angiogenic pathways and oncogenic enzymes provides broad anti-tumor effects.

[0038] For example, data from CRC tumors show that CD4 (i.e., the presence of CD4+ T cells within the tumor) is highly correlated with the expression of many regorafenib targets within the tumor: CSF1R (92%), VEGFR1 (also known as FLT1) (58%), VEGFR2 (also known as KDR) (62%), VEGFR3 (also known as FLT4) (66%), FGFR1 (66%), PDGFR-α (62%), and PDGFR-β (68%). These representative checkpoints, just like PD-1 and PD-L1, are induced in response to T cell engagement with tumors, and they may also be correlated with CD4. A similar correlation exists for CD8A (representing the presence of CTLs).

[0039] The synthesis and use of regorafenib is described in US 7,351,834 and US 9,957,232, which are incorporated herein by reference. An embodiment of the present invention is a system comprising regorafenib, sorafenib, fruquintinib, axitinib, lenvatinib, or related compounds.

[0040] c. PD-1 and PD-L1 inhibitors PD-1 (programmed cell death protein 1), also known as CD279, is an immune checkpoint protein expressed on the surface of activated T cells, B cells, and macrophages. When PD-1 binds to PD-L1 (CD274, or B7-H1) or PD-L2, the T cell receptor is downregulated, which reduces the proliferation of antigen-specific T cells and leads to immunosuppression. At the same time, the T reg Binding to cells reduces their apoptosis and further enhances immunosuppression. PD-L1 expression is stimulated by interferon-γ (IFN-γ) on T cells, NK cells, macrophages, bone marrow DCs, B cells, epithelial cells, and vascular endothelial cells. PD-L1 is highly expressed on some tumor cells, giving them the ability to induce anergy and avoid attack by CTLs. Inhibition of either or both PD-1 and PD-L1 reduces or prevents anergy and restores anti-tumor immune responses.

[0041] PD-1 can be inhibited or antagonized by anti-PD-1 antibodies and their derivatives, such as nivolumab, pembrolizumab, cemiplimab, pidilizumab, AMP-224, AMP-514, and PDR001. PD-L1 can be inhibited or antagonized by anti-PD-L1 antibodies and their derivatives, such as durvalumab, atezolizumab, avelumab, BMS-936559, and CK-301. The expression and function of PD-1 and PD-L1 are controlled by receptor tyrosine kinases (TRKs) and can be modulated by TRK inhibitors. An embodiment of the present invention is a system comprising a PD-1 inhibitor, a PD-L1 inhibitor, or both. An embodiment of the present invention is a system comprising nivolumab. An embodiment of the present invention is a system comprising nivolumab and atezolizumab. An embodiment of the invention is a system comprising: nivolumab, pembrolizumab, cemiplimab, or pidilizumab; and durvalumab, atezolizumab, or avelumab.

[0042] d. CTLA-4 inhibitors CTLA-4 (cytotoxic T lymphocyte-associated protein 4), CD152, is another protein that functions as an immune checkpoint and downregulates T cell function. reg While CTLA-4 is constitutively expressed on some T cells, it is expressed only after activation in other T cells. CTLA-4 binds with higher affinity to CD80 (B7-1) and CD86 (B7-2) than CD28 does and can completely compete with CD28 for binding, thereby inhibiting CD28-derived stimulatory signals. CTLA-4 antagonism can reduce immunosuppression. Suitable CTLA-4 inhibitors include anti-CTLA-4 antibodies, such as ipilimumab and tremelimumab. An embodiment is a system comprising ipilimumab or tremelimumab.

[0043] e. Cyclophosphamide Cyclophosphamide, (RS)-N,N-bis(2-chloroethyl)-1,3,2-oxazaphosphinan-2-amine 2-oxide, is an alkylating agent used to suppress the immune system. reg These cells have been used in low doses to deplete lymphocytes in cancer patients, with the understanding that they take longer to recover than CTLs. See, e.g., M. Scurr et al., Clin Cancer Res (2017) 23(22):6771-80; M. Scurr et al., JAMA Oncol (2017) 3(10):e172579; V. Radojcic et al., Cancer Immunol Immunother (2010) 59:137-48. Scurr et al. demonstrated that cyclophosphamide 50 mg twice daily (bid) for 7 days, followed by 7 days off, followed by 50 mg bid for another 7 days, significantly improved T lymphocyte depletion. reg found that cyclophosphamide treatment significantly depleted DCs and restored immune responses to mCRC in subjects. Radojcic et al. regIt has been discovered that without induction of cells, they are better able to infiltrate tumors and present tumor antigens.An embodiment of the present invention is a system that includes cyclophosphamide.

[0044] f. PGE2 inhibitors Prostaglandin E2 (PGE2) is a natural prostaglandin that reduces inflammation by downregulating T cell interactions with APCs and altering T cell migratory behavior (AJ Wiemer et al., J Immunol (2011) 187:3663-70). PGE2 is increased in SCT and promotes tumor growth and development, resistance to apoptosis, proliferation, invasion and metastasis, angiogenesis, and drug resistance in SCT. It also promotes fibrosis, which helps establish a dense stroma in the tumor microenvironment and creates a physical barrier to CTL invasion. PGE2 is produced by the enzymes COX-2 and mPGES-2 (encoded by microsomal prostaglandin E synthase 2, PTGES2) and stimulates the expression of more COX-2, leading to a positive feedback loop.

[0045] PGE2 activity can be inhibited by inhibitors of mPGES-2 and / or COX-2, which may take the form of specific antibodies or small molecules. Examples of COX-2 inhibitors are nonsteroidal anti-inflammatory drugs (NSAIDs), such as aspirin, ibuprofen, naproxen, fenoprofen, flurbiprofen, ketoprofen, indomethacin, tolmetin, ketorolac, diclofenac, prioxicam, tenoxicam, phenylbutazone, mefenamic acid, meclofenamic acid, celecoxib, etoricoxib, parecoxib, nimesulide, clonixin, licofelone, and related compounds. See, e.g., A. Grancher et al., Bull Cancer (2018) 105(2):171-80; L. Emilsson et al., Aliment Pharmacol Ther (2017) 45(2):193-204. An embodiment of the invention is a system comprising aspirin, ibuprofen, indomethacin, and / or naproxen.

[0046] g. Combination Because there are multiple pathways that suppress CTL activity, it is advantageous to include countermeasures for more than one inhibitory pathway. Accordingly, an aspect of the present invention is a system comprising a combination of two or more of a PD-1 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, a CD73 inhibitor, an A2aR inhibitor, a multikinase inhibitor, a COX-2 and / or a PGE2 inhibitor. An embodiment of the present invention is a system comprising a PD-1 inhibitor and a PD-L1 inhibitor. An embodiment of the present invention is a system comprising a PD-1 inhibitor, a multikinase inhibitor, and a COX-2 inhibitor. Another embodiment is a system comprising a PD-1 inhibitor, a CD73 inhibitor, and a COX-2 inhibitor. Another embodiment is a system comprising a PD-1 inhibitor, a CD73 inhibitor, a CTLA-4 inhibitor, and a COX-2 inhibitor. An embodiment of the invention is a system comprising a PD-1 inhibitor and one or more inhibitors selected from a PD-L1 inhibitor, a CTLA-4 inhibitor, a CD73 inhibitor, an A2aR inhibitor, a multikinase inhibitor, a COX-2 inhibitor, and a PGE2 inhibitor.An embodiment of the invention is a system comprising a PD-1 inhibitor and a PD-L1 inhibitor and one or more inhibitors selected from a CTLA-4 inhibitor, a CD73 inhibitor, an A2aR inhibitor, a multikinase inhibitor, a COX-2 inhibitor, and a PGE2 inhibitor. An embodiment of the invention is a system comprising a PD-1 inhibitor and a PD-L1 inhibitor, and one or more inhibitors selected from a CTLA-4 inhibitor, a CD73 inhibitor, an A2aR inhibitor, a multikinase inhibitor, a COX-2 inhibitor, and a PGE2 inhibitor.An embodiment of the invention is a system comprising a PD-1 inhibitor and a CTLA-4 inhibitor, and one or more inhibitors selected from a PD-L1 inhibitor, a CD73 inhibitor, an A2aR inhibitor, a multikinase inhibitor, a COX-2 inhibitor, and a PGE2 inhibitor.An embodiment of the invention is a system comprising a PD-1 inhibitor and a CD73 inhibitor, and one or more inhibitors selected from a PD-L1 inhibitor, a CTLA-4 inhibitor, an A2aR inhibitor, a multikinase inhibitor, a COX-2 or a PGE2 inhibitor.

[0047] An embodiment of the invention is a system comprising: a PD-1 inhibitor; a CD73 inhibitor; and a COX-2 or PGE2 inhibitor. An embodiment of the invention is a system comprising: a PD-1 inhibitor; a CD73 inhibitor; a COX-2 or PGE2 inhibitor; and one or more inhibitors selected from a PD-L1 inhibitor, a CTLA-4 inhibitor, an A2aR inhibitor, and a multikinase inhibitor. An embodiment of the invention is a system comprising: a PD-1 inhibitor; a multikinase inhibitor; and a COX-2 or PGE2 inhibitor. An embodiment of the present invention is a system comprising: a PD-1 inhibitor; a multikinase inhibitor; an inhibitor of COX-2 or PGE2; and one or more inhibitors selected from a PD-L1 inhibitor, a CTLA-4 inhibitor, an A2aR inhibitor, and a CD73 inhibitor.

[0048] An embodiment of the invention is a system comprising: a multikinase inhibitor, a PD-1 inhibitor, and one or more inhibitors selected from a COX-2 inhibitor, a PGE2 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, an A2aR inhibitor, and a CD73 inhibitor.An embodiment of the invention is a system comprising: a multikinase inhibitor, a PD-1 inhibitor, and a CTLA-4 inhibitor, and one or more inhibitors selected from a COX-2 inhibitor, a PGE2 inhibitor, a PD-L1 inhibitor, an A2aR inhibitor, and a CD73 inhibitor. An embodiment of the present invention is a system comprising: a multikinase inhibitor; a PD-1 inhibitor; a CTLA-4 inhibitor; a COX-2 inhibitor or a PGE2 inhibitor; and one or more inhibitors selected from a PD-L1 inhibitor, an A2aR inhibitor, and a CD73 inhibitor.

[0049] 4. Angiotensin II receptor type 1 antagonists and stromal factors Antagonists of the angiotensin II receptor type 1 (AT1R), such as losartan, normalize the collagen or interstitial matrix of solid tumors, which promotes tumor distribution and invasion by CTLs and APCs. For example, losartan reduces the level or production of collagen I by cancer-associated fibroblasts (CAFs). This further promotes vascular decompression and normalization, as well as improving intratumoral blood flow and delivery of low-molecular-weight chemotherapeutic agents and oxygen, thereby enhancing the therapeutic effects of cancer therapy and immunotherapy. Other angiotensin inhibitors, such as angiotensin receptor blockers (ARBs) such as candesartan and valsartan, and angiotensin-converting enzyme inhibitors (ACE-Is) such as lisinopril and captopril, can also be used. Each of these agents may be used alone or in combination in the practice of the present invention. See, for example, Y. Zhao et al., Proc Natl Acad Sci USA (2019) 116(6):2210-19; Y. Tang et al., Drug Deliv Transl Res (2019) 9(3):615-24; J. Scott-Emuakpor et al., J Exp Ther Oncol (2017) 11(2):107-15; R. Coulson et al., Oncotarget (2017) 8(12):18640-56; RK Jain et al., US2013-0287688. ARBs do not directly attract CTLs, but when they are attracted to tumors by other elements of the present invention, normalization of the tumor stroma facilitates physical entry of CTLs.

[0050] Many angiotensin II receptor type 1 antagonists have been developed for the treatment of hypertension and / or heart failure. Suitable AT1R antagonists include losartan, candesartan, valsartan, irbesartan, telmisartan, eprosartan, olmesartan, azilsartan, fimasartan, and the like. Typical doses of these compounds used to treat hypertension range from about 4 to about 800 mg for adults. For losartan, the typical dosage range is about 50 to 100 mg, while for candesartan it is about 4 to 32 mg and for eprosartan it is about 400 to 800 mg. However, other doses may be effective in the systems and methods of the present invention and can be determined by standard methods. An aspect of the present invention is the use of an ARB in combination with other components of the system. An embodiment is a system including an angiotensin II receptor type 1 antagonist. An embodiment is a system including losartan.

[0051] 5. Stereotactic body radiation therapy (SBRT) Radiation therapy (RT) is an effective tool for the treatment of tumors and other lesions. Stereotactic body radiation therapy (SBRT) combines the principles of stereotactic (3-D target localization) with multiple cross-fired beams from a high-energy radiation source to precisely deliver radiation to targets within the patient. This technique allows for maximal aggressive dosing of the treatment target, while normal surrounding tissue receives lower, non-damaging doses of radiation. It has long been known that targeted ionizing radiation causes direct, localized cell death but can also induce tumor regression in non-irradiated areas (the "abscopal effect"). Localized radiation therapy induces an immune-stimulatory form of cell death (immunogenic cell death, or "ICD"), which is known to mimic an immune response. This response is currently thought to be caused by the release of damage-associated molecular patterns (DAMPs), which trigger antigen phagocytosis by APCs and presentation to the immune system. Irradiation has also been shown to increase MHC-I expression on tumor cells and improve tumor cell antigen expression. For these reasons, it is sometimes referred to as "in situ vaccination." However, SBRT also upregulates IFN, TNF, IL-1α, and IL-6, induces the expression of CXCL10, which recruits CTLs to tumors, and enhances the priming of effector CD8+ T cells. Thus, improved antigen expression and presentation, as well as enhanced CTL function, provide a plausible potential rationale for an immune-mediated abscopal effect (JYH Lim et al., Cancer Immunol Immunother (2014) 63(3):259-71).

[0052] SBRT differs from previous forms of RT in that RT often delivers relatively low doses of radiation (e.g., 0.5-2 Gy) in multiple exposures (e.g., 5 doses over 5 days), whereas SBRT more often delivers higher doses of radiation (e.g., about 5-50 Gy) fractionated into fewer exposures (e.g., once weekly for 3 weeks). In embodiments of the invention, SBRT is utilized at doses of at least about 1 Gray (Gy), 2 Gy, 3 Gy, 4 Gy, 5 Gy, 6 Gy, 7 Gy, 8 Gy, 9 Gy, 10 Gy, 12 Gy, 15 Gy, 20 Gy, 25 Gy, 30 Gy, 40 Gy, 50 Gy, 60 Gy, or 75 Gy. The total dose is less than about 100 Gy, 90 Gy, 80 Gy, 70 Gy, 60 Gy, 50 Gy, 40 Gy, 30 Gy, 20 Gy, 15 Gy, 14 Gy, 13 Gy, 12 Gy, 11 Gy, 10 Gy, 9 Gy, 8 Gy, 7 Gy, 6 Gy, 5 Gy, 4 Gy, 3 Gy, or 2 Gy. In embodiments of the invention, the dose is divided into about 2, 3, 4, 5, 6, 7, 8, 9, or 10 fractions.

[0053] 6. Interim chemotherapy Because there is a slight lag time between administration of the vaccine and full T cell priming, it may be necessary to begin standard or conventional therapy while the neoantigen is being designed, synthesized, and administered. The particular therapy used is selected so as not to interfere with T cell priming and APC expansion, such as treatment with capecitabine with or without bevacizumab. This treatment is combined with administration of a PGE2 inhibitor (e.g., aspirin or other COX-2 inhibitor) and an angiotensin II receptor antagonist (e.g., losartan). An embodiment is a system that includes treatment with capecitabine with or without bevacizumab prior to administration of the neoantigen. An embodiment is a system that includes treatment with bevacizumab, capecitabine, and a COX-2 inhibitor prior to administration of the neoantigen. An embodiment is a system that includes treatment with bevacizumab, capecitabine, aspirin, and losartan prior to administration of the neoantigen.

[0054] B. Treatment method SCT is effectively treated by using the system of the present invention. The method of the present invention may also be referred to as a method of adjuvant therapy or a method of supporting a subject's immune system. An embodiment is a method in which the SCT is selected from colorectal cancer (CRC), pancreatic cancer, prostate cancer, head and neck cancer, lung cancer, melanoma, breast cancer, liver cancer, esophageal cancer, and gastric cancer. These cancer types share a common response as indicated by RNA expression data of response to treatment. An embodiment is a method in which the SCT is CRC. An embodiment is a method in which the CRC is metastatic CRC (mCRC). An embodiment is a method in which the cancer is microsatellite-stable mCRC (MSS mCRC).

[0055] The methods of the present invention comprise at least the following steps: administering an effective amount of an antigen-presenting cell agent; administering an effective amount of a T cell activating vaccine; and administering an effective amount of an immunosuppressant inhibitor. An embodiment is a method comprising administering an effective amount of an antigen-presenting cell agent; administering an effective amount of a T cell activating vaccine; and administering an effective amount of an immunosuppressant inhibitor. The methods of the present invention optionally comprise administering an angiotensin II receptor type 1 antagonist and / or SBRT. An embodiment of the present invention is a method further comprising administering an effective amount of an angiotensin II receptor type 1 antagonist. An embodiment is a method comprising treatment with SBRT. An embodiment is a method comprising administering an angiotensin II receptor type 1 antagonist and administering SBRT. An illustrative case of a subject receiving the treatment methods described herein is shown in FIG. 1, which shows two radiological scans of the subject described in Example 4 below. The right panel shows the subject's status on September 9, 2019 (highly metastatic prior to receiving the treatment described herein), while the left panel shows the treatment progression on November 26, 2019 (greatly reduced metastasis after receiving the treatment described herein).

[0056] 1. Treatment phase The methods of the present invention can be conceptually divided into three phases: antigen-presenting cell activation, T cell vaccination, and inhibition of immunosuppression. During all phases, a COX-2 inhibitor and / or an AT1R antagonist may be administered. In an embodiment, a COX-2 inhibitor is administered throughout the treatment period. In an embodiment, the COX-2 inhibitor is aspirin. In an embodiment, an AT1R antagonist is administered throughout the treatment period. In an embodiment, the AT1R antagonist is losartan. In an embodiment, both a COX-2 inhibitor and an AT1R antagonist are administered throughout the treatment period. In an embodiment, the COX-2 inhibitor is aspirin, celecoxib, ibuprofen, or naproxen, and the AT1R antagonist is losartan.

[0057] During the initial treatment phase, standard chemotherapy is used to prevent or delay disease progression before a specific T cell response is obtained and before benefit from the methods of the present invention can be seen. Standard therapies are selected that do not interfere with the proliferation and activation of antigen-presenting cells or T cells. A preferred standard therapy includes bevacizumab in combination with capecitabine. Standard therapy is administered at or shortly after diagnosis and may further include administration of a COX-2 inhibitor and / or an AT1R antagonist. An embodiment of the present invention is a method comprising treating a subject in need thereof with bevacizumab, capecitabine, a COX-2 inhibitor, and an AT1R antagonist. In an embodiment, the COX-2 inhibitor is aspirin, celecoxib, indomethacin, ibuprofen, or naproxen, and the AT1R antagonist is losartan.

[0058] At the start of the patient's SCT treatment, or shortly thereafter, samples are collected from both normal and tumor tissue for sequencing purposes. The latter can be biopsy or resected tumor tissue. Using the normal tissue sample, the patient's HLA (MHC-I and -II) alleles are determined by either sequencing or standard immunoassay techniques. If the patient's HLA alleles are already known, re-determination is unnecessary. The tumor tissue is sequenced, such as by NGS deep sequencing, to determine the neoantigens currently expressed in the tumor tissue. This neoantigen sequence is then analyzed for binding affinity to the patient's MHC-I and -II proteins by either kinetic methods (e.g., by determining affinity constants using labeled peptides representing neoantigens that compete with non-mutated "self" antigens for binding to appropriate patient cell samples) or predictive methods (e.g., computationally or in silico). Weakly binding neoantigens can be improved by known methods, such as by including a strong T cell epitope at one or both ends of the neoantigen peptide and / or by derivatizing the neoantigen peptide to increase its binding affinity to MHC-I and -II.

[0059] Neoantigen peptides are then synthesized. Some mutations occur regularly, especially in cancer (e.g., BRAF V600E in melanoma and CRC): for common mutations, it is known that neoantigen peptides can be synthesized and stocked in advance. See, for example, Z. Liang et al., doi: https: / / doi.org / 10.1101 / 682617 (July 9, 2019). It is possible to record the occurrence of each neoantigen along with the peptide sequence found to bind to a specific HLA allele and maintain a stock of suitable peptides for immediate use.

[0060] APCs endocytose foreign proteins and polypeptides and process them with proteolytic enzymes to break them down into oligopeptides of a size suitable for binding to MHC-I or -II. Thus, neoantigen peptides are provided as longer multiantigen polypeptides containing two or more neoepitopes. In embodiments, neoantigen peptides are administered in the form of multiantigen polypeptides containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more neoantigens, and multiantigen polypeptides can contain 12, 15, 20, 25, 30, 35, 40, 45, 50, or more amino acids, or any number between these values.

[0061] a. Antigen presenting cell activation In one embodiment, FLT3L is administered to a patient to proliferate and mobilize the patient's APCs. FLT3L is administered immediately at the start of treatment, one or two weeks before initiating T cell activating vaccine administration, during the first week or two weeks of vaccine administration, and at the end of vaccine administration and / or one to two weeks thereafter, and combinations thereof. In one embodiment, FLT3L is administered one week before T cell vaccination. In one embodiment, FLT3L is administered at the time of T cell vaccination or within 24 hours before or after vaccination. In one embodiment, FLT3L is administered within one week of the start of treatment and within 24 hours of T cell vaccination. In one embodiment, FLT3L is administered at the time of radiation therapy or within 24 hours before or after radiation therapy.

[0062] The other APC agent is administered at or near the time of T cell vaccination. In embodiments, one or more additional APC agents are combined with the T cell activating vaccine or are formulated for administration with the T cell activating vaccine. In embodiments, the APC agent is Montanide™. In embodiments, the APC agent is Montanide™ ISA51. In embodiments, the APC agent is poly-ICLC. In embodiments, the APC agent is Montanide™ and poly-ICLC.

[0063] b. T cell vaccination As demonstrated herein, T cell-activating vaccines contain neoantigen peptides or multiantigen polypeptides, where neoantigens are selected based on (a) immunogenicity (which depends in part on the degree of difference between the neoantigen and the non-mutated "self" sequence peptide), (b) binding affinity to MHC-I and -II, and (c) the degree of expression in tumor tissue(s). Because tumors are characteristically heterogeneous, the vaccine contains multiple neoantigens. As described herein, the vaccine is administered as a combination of one or more subsets of neoantigens at different injection sites.

[0064] The T cell activating vaccine is administered as soon as practicable, or as soon as possible after time has passed to activate APCs and expand their populations. The vaccine can be administered multiple times, for example, about every 1, 2, 3, 4, 5, or 6 weeks, and each time point can contain a different neoantigen and neoantigen combination, and can be administered at a different injection site. Each administration can be accompanied by administration of an APC agent within about one week before or after vaccine administration. An embodiment is a method in which the T cell activating vaccine is administered within 24 hours of FLT3L administration. An embodiment is a method in which the T cell activating vaccine is administered between 1 and 30 days after FLT3L administration. An embodiment is a method in which the T cell activating vaccine is administered between 7 and 20 days after FLT3L administration. An embodiment is a method in which the T cell activating vaccine is administered in combination with an adjuvant, poly(I:C), or poly-ICLC.

[0065] c. Radiation therapy Radiation therapy (RT) can be used in the method of the present invention when the tumor is of sufficient size and located in a position accessible to RT without causing unacceptable damage to surrounding tissues. In an embodiment of the present invention, stereotactic body radiation therapy (SBRT) is administered before or after the completion of T cell vaccination. In an embodiment, SBRT is administered about 1 day, 2 days, 3 days, 5 days, 1 week, or 2 weeks after the final vaccine administration. In an embodiment, SBRT is administered about 1 day, 2 days, 3 days, 5 days, 1 week, or 2 weeks before the first vaccine administration. In an embodiment, SBRT is administered between the first and last vaccine administrations. In an embodiment, SBRT is administered at an intensity of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 Gray (Gy). In embodiments, SBRT is administered at an intensity not exceeding about 60, 50, 40, 30, 25, 22, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 Gy. In embodiments, SBRT is administered at an intensity between about 5 Gy and about 10 Gy. In embodiments, SBRT is administered in about 2, 3, 4, 5, 6, 7, 8, 9, or 10 fractions. In embodiments, SBRT is administered in about 5 fractions. In embodiments, FLT3L is administered within about 1 week, before, after, or at the time of SBRT administration. In embodiments, FLT3L is administered within 24 hours of the first SBRT administration. In embodiments, FLT3L is administered by intradermal or subcutaneous injection 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 times on consecutive days. In one embodiment, the amount of FLT3L administered in each injection is at least about 10, 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600, 700, 750, 800, 900, or 1000 μg / Kg. In another embodiment, the amount of FLT3L administered in each injection is no more than about 5000, 4000, 3000, 2000, 1500, 1000, 900, 800, 700, 600, 550, 500, 450, 400, 350, 300, 250, or 200 μg / Kg.

[0066] d. Immunosuppression The immunosuppressive inhibitor is administered together with the administration of the vaccine. In an embodiment, the one or more immunosuppressive inhibitors are administered within about one week of the completion of the vaccine administration, before, after, or at the time of the administration of the final dose. In an embodiment, the immunosuppressive inhibitor is a CD73 inhibitor. In an embodiment, the CD73 inhibitor is oleclumab (MED19447). In an embodiment, the CD73 inhibitor is BMS-986179 (Bristol-Myers Squibb), AB680 (Arcus Biosciences), CB-708 (Calithera Biosciences), CPI-006 (Corvus Pharmaceuticals), or α,β-methylene adenosine 5'-diphosphate sodium salt. In an embodiment, the immunosuppressive inhibitor is an A2aR inhibitor. In an embodiment, the immunosuppressive inhibitor is a PD-1 inhibitor. In one embodiment, the immunosuppressive inhibitor is a PD-L1 inhibitor. In one embodiment, the PD-1 inhibitor is nivolumab. In one embodiment, the immunosuppressive inhibitor is a CTLA-4 inhibitor. In one embodiment, the CTLA-4 inhibitor is ipilimumab.

[0067] In an embodiment, the immunosuppressive inhibitor is a combination of inhibitors and can be administered together or separately. In an embodiment, the combination comprises a CD73 inhibitor and a PD-1 inhibitor. In an embodiment, the combination comprises olecurumab and nivolumab. In an embodiment, the combination comprises a CD73 inhibitor, a CTLA-4 inhibitor, and a PD-1 or PD-L1 inhibitor. In an embodiment, the combination comprises nivolumab, ipilimumab, and olecurumab. An embodiment is a method comprising administering a PD-1 inhibitor and one or more inhibitors selected from a PD-L1 inhibitor, a CTLA-4 inhibitor, a CD73 inhibitor, an A2aR inhibitor, a multikinase inhibitor, a COX-2 inhibitor, and a PGE2 inhibitor. An embodiment is a method comprising administering a PD-1 inhibitor and a PD-L1 inhibitor, and one or more inhibitors selected from a CTLA-4 inhibitor, a CD73 inhibitor, an A2aR inhibitor, a multikinase inhibitor, a COX-2 inhibitor, and a PGE2 inhibitor.An embodiment is a method comprising administering a PD-1 inhibitor and a PD-L1 inhibitor, and one or more inhibitors selected from a CTLA-4 inhibitor, a CD73 inhibitor, an A2aR inhibitor, a multikinase inhibitor, a COX-2 inhibitor, and a PGE2 inhibitor. An embodiment is a method comprising administering a PD-1 inhibitor and a CTLA-4 inhibitor, and one or more inhibitors selected from a PD-L1 inhibitor, a CD73 inhibitor, an A2aR inhibitor, a multikinase inhibitor, a COX-2 inhibitor, and a PGE2 inhibitor.An embodiment is a method comprising administering a PD-1 inhibitor and a CD73 inhibitor, and one or more inhibitors selected from a PD-L1 inhibitor, a CTLA-4 inhibitor, an A2aR inhibitor, a multikinase inhibitor, a COX-2 or a PGE2 inhibitor.

[0068] An embodiment is a method comprising the administration of a multikinase inhibitor, a PD-1 inhibitor, and one or more inhibitors selected from a COX-2 inhibitor, a PGE2 inhibitor, a PD-L1 inhibitor, a CTLA-4 inhibitor, an A2aR inhibitor, and a CD73 inhibitor.An embodiment is a method comprising the administration of a multikinase inhibitor, a PD-1 inhibitor, and a CTLA-4 inhibitor, and one or more inhibitors selected from a COX-2 inhibitor, a PGE2 inhibitor, a PD-L1 inhibitor, an A2aR inhibitor, and a CD73 inhibitor. An embodiment is a method comprising administering one or more inhibitors selected from a multikinase inhibitor; a PD-1 inhibitor; a CTLA-4 inhibitor; a COX-2 inhibitor or a PGE2 inhibitor; and a PD-L1 inhibitor, an A2aR inhibitor, and a CD73 inhibitor.

[0069] Immunosuppressive inhibitors are administered according to their approved doses and schedules, and administration is continued throughout the course of treatment unless otherwise specified.

[0070] e. AT1 antagonists and stromal treatment Dense stromal barriers are treated with angiotensin II receptor type 1 (AT1R) antagonists, angiotensin receptor blockers (ARBs), and / or ACE inhibitors. Even tumors that do not exhibit an "immune unattackable" phenotype can be treated with these agents to normalize the stroma and prevent the development of immune unattackable barriers. In an embodiment of the invention, the AT1R antagonist is losartan. In an embodiment of the invention, the AT1R antagonist, ARB, or ACE inhibitor is administered beginning at or near the initiation of treatment. In an embodiment, the AT1R antagonist, ARB, or ACE inhibitor is initially administered within about one week of initiating treatment. In an embodiment, administration of the AT1R antagonist, ARB, or ACE inhibitor continues substantially throughout the course of treatment. An embodiment is a method comprising administration of losartan substantially throughout the course of treatment.

[0071] 2. Timing and Order The timing of this treatment phase may vary depending on the patient's response. In an embodiment, the patient is treated with bevacizumab, capecitabine, losartan, and aspirin, ibuprofen, or naproxen until the T cell-activating vaccine can be administered. The vaccine is administered about 1, 2, or 3 times, at intervals of about 1 week to about 6 weeks, about 2 to about 5 weeks, about 4 weeks, or about 1 month. In an embodiment, the vaccine is administered 1, 2, or 3 times, at intervals of about 1 month. FLT3L is administered up to about 1 week before each T cell vaccination or during one or more T cell vaccinations. In an embodiment, FLT3L is administered with each vaccination.

[0072] SBRT is suitably administered before vaccination, during the vaccination phase, or after completion of the vaccination phase. An embodiment is a method in which SBRT is administered about 1, 2, 3, 4, or 5 weeks after the final vaccination. An embodiment is a method in which SBRT is administered about 1, 2, 3, 4, or 5 weeks before the first vaccination. An embodiment is a method in which SBRT is administered between the first and last vaccination. FLT3L can also be administered together with SBRT. In an embodiment of the invention, SBRT is administered about 1, 2, 3, 4, or 5 weeks after the last vaccination. An embodiment is a method in which SBRT is administered about one week after the last vaccination. An embodiment is a method in which FLT3L is administered within about one week before SBRT and up to about 24 hours after SBRT treatment. In embodiments, SBRT is administered at an intensity of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 Gray (Gy). In embodiments, SBRT is administered at an intensity not exceeding about 60, 50, 40, 30, 25, 22, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 Gy. In embodiments, SBRT is administered at an intensity between about 5 Gy and about 10 Gy. In embodiments, SBRT is administered in about 2, 3, 4, 5, 6, 7, 8, 9, or 10 fractions. In embodiments, SBRT is administered in about 5 fractions.

[0073] The immunosuppressive inhibitor can be administered at any time during the method. An embodiment is a method in which a PD-1 inhibitor and / or a PD-L1 inhibitor is administered and a CD73 inhibitor is administered. An embodiment is a method in which a PD-1 inhibitor and / or a PD-L1 inhibitor is administered and a multikinase inhibitor is administered. An embodiment is a method in which nivolumab and regorafenib are administered. An embodiment is a method in which administration of the immunosuppressive inhibitor is initiated between the last vaccine administration and about six weeks after the last vaccination. An embodiment is a method in which administration of the immunosuppressive inhibitor is initiated within one week of the last vaccination.

[0074] 3. Measurement of Responses Tumor response is monitored. If progression occurs, the combination of immunosuppressive inhibitors is changed. In an embodiment, the administration of nivolumab and regorafenib is changed to nivolumab and a CD73 inhibitor. In an embodiment, the administration of nivolumab and a CD73 inhibitor is changed to nivolumab and regorafenib. If progression continues, a new neoantigen is designed, and this vaccination phase is repeated, followed by administration of SBRT and an immunosuppressive inhibitor and measurement of response.

[0075] A patient's response to a therapy can be measured in several ways. For example, the effect on the patient's tumor(s) can be determined by x-ray measurement of tumor regression. The tumor can be biopsied or excised, and the tissue can be examined histologically for tumor cell death and CTL and APC infiltration. Biopsied or excised tissue can be examined by deep sequencing for changes in tumor markers, changes in heterogeneity, etc. Blood can be examined for a decrease in circulating tumor cells, tumor DNA, and / or tumor antigens, for an increase in circulating CTLs and migrating APCs, and for changes in cytokine levels or other biomarkers. Such diagnostic results can be used to determine when to progress to the next phase (e.g., when the APC population has sufficiently expanded, when sufficient CTLs have been produced, when immunosuppression has appeared or increased, etc.). Diagnostic results can also be used to determine whether a particular drug lacks effectiveness against the patient's tumor(s) and when to switch to another drug. Tumor response can also be measured by measurements of biomarkers.

[0076] In an embodiment of the invention, the activation state, population size, or distribution of T cells is determined between about 5 days and about 30 days after administration of a T cell activating vaccine. In an embodiment of the invention, the tumor response to T cell vaccination is determined prior to repeat vaccination. [Example]

[0077] Example The following examples are provided as a guide for those skilled in the art and are not intended to limit the scope of the claimed invention in any way. When gene expression levels are provided for tumor tissue samples, unless otherwise noted, this is understood to refer to the RNA expression (RNASeq) of that gene in the tissue sample, which has been shown to produce comparable results to microarray technology for expression profiling (Guo et al., PLoS One (2013) 8(8):e71462).

[0078] Example 1: T cell costimulatory proteins The expression of CD4, CD3D (which forms part of the TCR-CD3 antigen receptor complex), and CD8A in tumor-infiltrating immune cells was compared with the expression of the T cell costimulatory proteins CD80 and CD86 in SCTs. The results are shown in Table 1. [Table 1]

[0079] These results demonstrate that the expression of these characteristic T cell antigens correlates well with the expression of the T cell activation proteins CD80 and CD86 in the SCT described above. This suggests that APC-activating agents that increase the number of mature APCs in CRC tumors (which express CD80 and CD86) promote T cell infiltration and provide synergistic results with T cell-activating vaccines. This is consistent with the finding that a greater number of mature APCs in the invasive margin of CRC tumors predicts better survival in CRC (A. Pryczynicz et al., Gastroenterol Res Pract (2016) 2016:2405437).

[0080] Example 2: Correlation of PTGES2 with CD4, CD83, and CD86 Expression of PTGES2 in CRC tumors vs. CD4(T HThe expression of CD83 (an APC maturation marker), CD86 (also known as B7-2, an APC marker) was examined. The results are shown in Table 2. [Table 2]

[0081] These results indicate that expression of PTGES2 (which increases PGE2 production) is associated with downregulation of T cells and APCs in colorectal, pancreatic, prostate, head and neck, melanoma, lung, and esophageal cancers. See also T. Seo et al., Virchows Archiv (2009) 454(6):667-76, in which the authors report that PGE synthase is overexpressed in CRC tumors and correlates with poor prognosis.

[0082] The use of aspirin to inhibit COX-2 and consequently PGE2 synthesis has been reported to improve survival in CRC patients (see, for example, Y. Cao et al., Gastroenterol (2016) 151(5):879-92, which reported a reduced risk of CRC with low TILs in patients who regularly used aspirin; and T. Hamada et al., J Clin Oncol (2017) 35(16):1836-44, which reported that aspirin use was positively correlated with cancer-specific survival and overall survival in CRC patients with low PD-L1 expression, but no benefit was observed in patients with high PD-L1 expression).

[0083] Example 3: Correlation of FLT3L expression with CD4 and CD8 The correlation between FLT3LG expression and the expression of CD4, CD8A, CD8B, and CD86 in SCT was compared with the correlation between CD40 expression and the expression of CD4, CD8A, CD8B, and CD86. The results are shown in Table 3. [Table 3]

[0084] These results demonstrate that expression of CD4, CD8, and CD86 strongly correlates with expression of FLT3LG in colorectal, prostate, head and neck, melanoma, lung, esophageal, liver, stomach, breast, and kidney (renal cell carcinoma) cancers, as well as CD40 in pancreatic cancer.

[0085] In a paper by MA Morse et al., J Clin Oncol (2000) 18(23):3883-93, it was reported that administration of FLT3L (20 μg / kg / day) for 14 days (1 to 3 cycles at 1-month intervals) to patients with mCRC resulted in a significant reduction in peripheral blood white blood cell count (5,900 / mm 3 from 11,200 / mm 3 reported that treatment with IFN-γ significantly increased the number of APCs in PBMCs (from 2.4% to 8.8%), the percentage of APCs in PBMCs (from 2.4% to 8.8%), and the number of APCs found in the periphery of the tumor.

[0086] Example 4 - Clinical Trials The patient was diagnosed with stage IV mCRC and was treated with standard-of-care chemotherapy (FOLFOX - folinic acid, fluorouracil, and oxaliplatin) followed by FOLFIRI (folinic acid, fluorouracil, and irinotecan), during which neoantigens were analyzed. Fresh-frozen tumor samples from the primary tumor resection and normal tissue samples were sent to Avera Institute for Human Genetics (Sioux Falls, SD) for whole-exome sequencing and HLA typing. These results were analyzed using Vaxrank and MHCflurry (OpenVax, Mt. Sinai, New York, NY). See, e.g., A. Rubinsteyn et al., Front Immunol (2017) 8:1807.

[0087] This software identified mutations in MT-CO2 (cytochrome C oxidase subunit 2) and predicted a series of peptides that were predicted to bind strongly to the MHC protein of interest. The top candidates are shown in Table 4. [Table 4]

[0088] The top two peptides (SEQ ID NOs: 1-2) were selected for use, along with 28 other peptides based on neoantigens from the following genes: NONO, TANGO6, ADAM19, HLA-DRA, DMKN, ELL, SMURF2, ARID4A, HACL1, BRIX1, NTRK2, CDC42, LPCAT3, NRAS, and NUP85. These peptides ranged in size from 9 to 25 amino acids in length. These peptides were commercially synthesized, purified, and lyophilized for storage until administration.

[0089] At the time of administration, these peptides (60 μg each) were combined in six groups of five and suspended in 1.0 mL of water for injection containing 10% DMSO. The peptide mixture was then combined with Montanide™ ISA51 (Seppic, France) in a 1:1.4 ratio. The six formulations were then administered intradermally to the upper arm. These peptide formulation groups were administered three times, approximately one month apart. The third-administered formulation also contained poly:ICLC (Hiltonol®, Oncovir, 1.8 mg / mL) in a peptide:Hiltonol® ratio of 3:1.

[0090] Therapy included FOLFIRI with bevacizumab when the vaccine was administered. After the third dose in January 2019, FOLFIRI was discontinued, and the subject was treated with capecitabine (2,000 mg daily for 1 week on and 1 week off), bevacizumab (5 mg / kg every 2 weeks), aspirin (750 mg / day), celecoxib (a COX-2 inhibitor, 200 mg / day), and losartan (100 mg / day).

[0091] Approximately 6 months after the final dose of the primary vaccine, a new set of 29 neoantigen peptides was designed and prepared (Table 5). The peptides were suspended in water for injection containing 10% DMSO in three separate formulations (60 μg of each peptide). These three peptide suspensions (290 μL each) were then individually combined with Hirutonol® (110 μL) and Montanide™ (400 μL), and the resulting formulations were injected subcutaneously in the thigh. The subject then received two doses of the humanized anti-CD73 IgG1 antibody CPI-006 (18 mg / Kg, Corvus Pharmaceuticals) in late July and mid-August 2019, and one dose of the humanized anti-PD-1 antibody pembrolizumab (100 mg, Keytruda, Merck) in late August 2019, followed by four daily doses of the multikinase inhibitor regorafenib (40 mg, Bayer) in late August 2019. [Table 5]

[0092] result Before the first vaccination, the subject showed growing disease despite FOLFOX treatment. After the third vaccination, the subject showed stable disease over a 5-month period on capecitabine, bevacizumab, aspirin, losartan, and celecoxib, which was unexpected for a patient heavily pretreated with a combination of aspirin, capecitabine, and bevacizumab (Giampieri et al., Clinical Colorectal Cancer, Vol. 16, No. 1, 38-43). Five months later, the subject stopped bevacizumab, received a CD73 inhibitor, and developed suspicion of progression based on a slight rise in CEA and lymph node metastases visible on a PET (positron emission tomography) scan. This subject received one dose of a novel 29-peptide vaccine and two doses of a CD73 inhibitor along with one dose of anti-PD-1, and a PET scan in September 2019 showed dramatic progression of metastases and new lesions, as shown in Figure 1 (right panel). As a result, the subject discontinued CPI-006 and resumed FOLFIRI in combination with bevacizumab and Neulasta while maintaining aspirin, celecoxib, and losartan. Two weeks after the first biweekly dose of FOLFIRI, bevacizumab, and Neulasta, the CEA level did not decline but rose slightly to 18.6; therefore, maraviroc was added to FOLFIRI, bevacizumab, and Neulasta while continuing aspirin, celecoxib, and losartan.

[0093] Two weeks after the second dose of FOLFIRI, the CEA decreased to 8.4, and the subject was switched from FOLFIRI to oral capecitabine, which was more easily tolerated and administered. From this point onward, the subject continued on a treatment regimen of capecitabine (2,000 mg twice daily for 1 week on and 1 week off), bevacizumab (every 2 weeks), Neulasta (6 mg every 2 weeks), maraviroc (150 mg twice daily), one dose of pembrolizumab (anti-PD-1, 100 mg, Keytruda, Merck), aspirin, celecoxib, and losartan. Approximately two and a half months later, the metastases had significantly decreased, as shown in Figure 1 (left panel) and described below in a comparison of PET scans between September 9, 2019, and November 26, 2019, and confirmed by a decrease in the blood marker CEA from 17.2 ng / mL to 2.9 ng / mL over the same period. The radiologist who performed the PET-CT scan on November 26, 2019, noted the following impression in his report: "From the FDG PET / CT on September 9, 2019, there was a general significant decrease in the hypermetabolic malignant soft tissue metastases, described as residual disease."

[0094] The radiologist's detailed comments are included below: In particular, the findings of a sharp decrease in SUV (standardized uptake value) and resolution ("resolved") were highly unexpected for a pre-treated subject with end-stage MSS mCRC such as DS.

[0095] " lung : No suspicious hypermetabolic findings at this time. Previous scattered hypermetabolic pulmonary metastases have largely resolved; with a few scattered residual mildly FDG-avid reduced pulmonary nodules, e.g.: *Left lower lobe pulmonary nodule, CT image 94, PET image 90 (PET-CT fusion image - allow for some imprecision in localization imaging); 0.7 cm, previously 0.9 cm, SUV 2.9, previously 4.9.

[0096] Pleura / pericardium : The exudate that was previously traced has dissipated.

[0097] Thoracic lymph nodesCT evaluation was limited by lack of IV contrast and low CT mA / dose. Previous hypermetabolic mediastinal / hilar adenopathy has largely resolved; minimal adenopathy remains; e.g., *Subcarinal focus, SUV 2.7, previously 6.4.

[0098] hepatobiliary Hypermetabolic capsular / subcapsular liver implants are significantly reduced in size scintigraphically with significant residual visible disease; e.g., *PET image 104, SUV 10.8, previously 19.6.

[0099] spleen : The previous hypermetabolic perisplenole implant is no longer scintigraphically evident.

[0100] pancreas : No abnormal uptake.

[0101] Adrenal glands : No abnormal uptake.

[0102] Kidneys / Ureters / Bladder :Excretory activity is present.

[0103] abdominopelvic tubercle : The preceding hypermetabolic abdominopelvic adenopathy resolved.

[0104] Intestine / Peritoneum / MesenteryImage 158, SUV 8.6, previously 22.4. *Subcutaneous hepatic arterial pump reservoir with catheter in predicted perihepatic region; where catheter penetrates the anterior abdominal musculature, where focal hypermetabolic activity is again visible - likely infection / inflammation versus tumor; unchanged; SUV 22.7, previously 20.0. *Subcutaneous hepatic arterial pump reservoir with catheter in predicted perihepatic region; where catheter penetrates the anterior abdominal musculature, where focal hypermetabolic activity is again visible - diminished. SUV 4.6, previously 12.7. *Hypermetabolic nodular focus within midline recovery wound in anterior abdominal wall. Image 158, SUV 8.6, previously 22.4. Previous hypermetabolic peritoneum and other abdominal implants have largely resolved with residual disease; e.g., *inseparable from colonic anastomosis, image 203, SUV 11.7, previously 36.7. Previous small amount of ascites has resolved.

[0105] pelvic organs : No abnormal uptake.

[0106] bone / soft tissue : No suspicious bone lesions. Diffuse hypermetabolic activity in the red marrow regions of the skeleton, consistent with uptake of a physiological variant.

[0107] Example 5: Regorafenib Treatment High expression of PD-L1 or PD-1, along with high expression of CD8 in CRC, indicates that T cells actively bind to tumors, and as a result, anti-PD-1 or anti-PD-L1 therapy helps reduce immunosuppression (APR Bally et al., J Immunol (2016) 196:2431-37; AM Valentini et al., Oncotarget (2018) 9:8584-96). For example, PD-1 expression on T cells reflects avidity and anti-tumor reactivity (S. Simon et al., Oncoimmunol (2018) 7(1):e1364828). Also referring to the paper by JH Park et al., J Clin Oncol (2018) 36(4 supp):631, the authors reported that PD-L1 expression did not correlate with clinicopathological or tumor microenvironmental characteristics in patients with mismatched repair-competent CRC (grade TNM I-III CRC) after tumor resection. TIL PD-1 expression was not associated with clinicopathological characteristics, but was associated with a higher Clintrup-Makinen grade (P<0.001), a higher immunoscore (P<0.001), a lower tumor stromal percentage (TSP, P=0.068), and a lower Glasgow microenvironment score (P<0.001). Multivariate survival analysis showed that high TIL PD-1 expression was associated with improved cancer-specific survival (HR 0.60, P = 0.016), but not with immunoscore (HR 0.74, P = 0.03) or TSP (HR 1.91, P = 0.027). PD-L1 expression was not associated with CSS in univariate or multivariate analyses.

[0108] Recent clinical trial results showed that the multikinase inhibitor regorafenib, when combined with anti-PD-1 nivolumab, demonstrated synergistic therapeutic results, providing objective tumor responses in 40% of pretreated patients with advanced colorectal or gastric cancer and stable disease in 88% (S. Fukuoka et al., J Clin Oncol (2019) 37:(suppl; abst 2522)). Regorafenib also demonstrated synergistic therapeutic results when combined with anti-PD-L1 avelumab, providing a significant increase in CD8+ T cell infiltration in 60% of patients with advanced colorectal cancer and stable disease in 57.3% (S. Cousin et al., J Clin Oncol (2020) 38:(15_suppl; abst 4019)). This combination compares favorably with the modest clinical activity of regorafenib as a single agent in the lung (MS Kies et al., https: / / ascopubs.org / doi / abs / 10.1200 / jco.2010.28.15_suppl.7585), pancreas (S. Bozzarelli et al., Ann Oncol (2016) 27(sup 6):692P; JS Salmon et al., https: / / ascopubs.org / doi / abs / 10.1200 / JCO.2017.35.15_suppl.e15751), and its FDA-approved use in colorectal, liver (hepatocellular carcinoma), and gastric cancer. Regorafenib blocks multiple protein kinases involved in tumor angiogenesis (VEGFR1-3, TIE2), oncogenesis (KIT, RET, RAF-1, BRAF), metastasis (VEGFR3, PDGFR, FGFR), and tumor immunity (CSF1R). Blockade of CSF1R inhibits macrophage generation, resulting in a decrease in the number of tumor-associated macrophages (TAMs), which exert immunosuppressive effects on CTLs as well as Treg cells. Blockade of angiogenesis prevents tumors from obtaining oxygen and nutrients from the patient's vasculature.A similar drug, axitinib, blocks some of the same protein kinases involved in tumor angiogenesis (VEGFR1-3) and metastasis (VEGFR3, PDGFR), and achieved modest clinical activity as monotherapy in colorectal cancer (C. Gravalos et al., Clin Colorectal Cancer (2018) 17(2):e323-29). However, when combined with the anti-PD-L1 avelumab in kidney (renal cell carcinoma), it achieved breakthrough therapy status. Another similar drug, lenvatinib, also blocks some of the same protein kinases involved in tumor angiogenesis (VEGFR1-3) and metastasis (VEGFR3, PDGFR) (S. Sarcognato et al., Clinical Liver Disease, (2019) 14 (2):62-65), and achieved modest clinical activity as monotherapy for colorectal cancer (H. Shoji et al., J Clin Oncol (2019) 37(15): 3538-3538). However, when combined with the anti-PD-1 agent pembrolizumab in advanced gastric cancer, it achieved a 69% objective response rate (A. Kawazoe et al., Lancet Oncol (2020) June 23, 2020, first published online, https: / / doi.org / 10.1016 / S1470-2045(20)30271-0).

[0109] Data from SCT tumors show that CD4 expression is highly correlated with the expression of many of the aforementioned targets. Tables 6 and 7 show the correlation between many of the regorafenib targets and the expression of CD4 and CD8 in CRC tumors. [Table 6] [Table 7]

[0110] Correlations between T cell markers (CD4 and CD8A) and the listed target genes indicate tumor defense against tumor angiogenesis, carcinogenesis, and metastasis, as well as potent stimulation of tumor immunity from T cells in colorectal, pancreatic, head and neck, and lung cancers (M. Kissel et al., Oncotarget (2017) 8(63):107096-108). These correlations indicate that regorafenib is synergistic with nivolumab, which suggests that it may also be synergistic with T cell-activating vaccines.

[0111] Data from solid tumors also show that for each cancer, survival is closely correlated with the expression of proteins corresponding to one of the target receptors. Table 8 shows the "COX proportional hazard ratios" comparing the overall survival of patients in the top quartile of expression of proteins that bind to regorafenib targets in a strong relationship with those in the bottom quartile for each cancer. VEGF-B binds to VEGFR1, VEGFA binds to VEGFR1 and VEGFR2, and VEGFC binds to VEGFR3 (https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC3411125 / ). These hazard ratios in Table 8 indicate that regorafenib may have beneficial activity against angiogenesis in cancers other than colorectal and gastric cancers, and, together with the correlations in Table 8, suggest that it may also be synergistic with T cell-activating vaccines. These findings presented here are consistent with results reported by S. Zong et al., Clin Chim Acta (2016) 458:106-14 (CRC); Z. Zhang et al., PLoS ONE (2016) 11(11):e0165725 (breast); H. Xia et al., Cancer Biomark (2016) 17(2):165-70 (esophagus); W. Cao et al., Tumor Biol (2014) 35(4):3377-83 (stomach); and H. Jiang et al., Clin Chim Acta (2014) 427:94-99 (non-small cell lung cancer). [Table 8]

[0112] Example 6: Bevacizumab Treatment Tumor cells can upregulate VEGFR1 and VEGFR2, which reduces T cell infiltration due to vascular and endothelial dysregulation. For example, in patients with renal cell carcinoma, anti-VEGF treatment with bevacizumab increased intratumoral T cell infiltration, potentially through vascular normalization and endothelial cell activation (Wallin et al., Nat Commun 2016 Aug 30;7:12624).

[0113] Similar to regorafenib, recent clinical trial results found that treatment with the monoclonal antibody bevacizumab and the chemotherapy drug capecitabine, when combined with the anti-PD-L1 atezolizumab, demonstrated synergistic therapeutic results, providing an 8.5% objective tumor response rate and an 8.5% progressive response rate at publication in patients with pretreated metastatic colorectal cancer (N. Mettu et al., Annals of Oncology (2019) 30 (suppl_5): v198-v252.). This triple combination in pretreated patients was associated with an ongoing response rate of 2.2%, which compares favorably with a more modest 4.4% objective response rate in the same trial of bevacizumab and capecitabine in pretreated patients with metastatic colorectal cancer. Bevacizumab blocks multiple protein kinases (VEGFR1-2) associated with tumor angiogenesis by binding to circulating VEGF-A (Pandey et al., Hypertension. 2018;71:e1-e8). Blocking angiogenesis prevents tumors from obtaining oxygen and nutrients from the patient's vasculature.

[0114] The data from colorectal cancer tumors in Table 6 above show that CD4 and CD8A are closely correlated with the expression of the VEGF-A target receptors, VEGFR1 and VEGFR2, in colorectal cancer. Clinical data from Example 4 showed that when bevacizumab was removed from maintenance therapy with capecitabine, bevacizumab, aspirin, celecoxib, and losartan, CEA and scanning showed signs of progression. In contrast, when antigen-presenting cell agents (Neulasta and Maraviroc) and anti-PD-1 were added to capecitabine, bevacizumab, aspirin, celecoxib, and losartan, PET scanning and CEA showed a dramatic reduction in tumor burden.

[0115] Example 7: Cetuximab Treatment Similar to regorafenib, recent clinical trial results found that the monoclonal antibody cetuximab and chemotherapy FOLFOX, when combined with anti-PD-L1 avelumab, demonstrated synergistic therapeutic results, providing an 80% overall response rate in previously untreated metastatic colorectal patients with RAS wild-type tumors (jnccn360.org / colorectal / news / avelumab-plus-folfox-and-cetuximab-in-metastatic-colorectal-cancer / ). This triple combination compares favorably with the more modest 66% objective response predicted with cetuximab and FOLFOX in metastatic colorectal cancer in a similar setting (www.ncbi.nlm.nih.gov / pmc / articles / PMC6324088 / ). In both patient and preclinical models, cetuximab acts on the EGFR receptor and induces immunogenic cell death, attracting dendritic cells and T-cells to tumors (www.ncbi.nlm.nih.gov / pubmed / 27135741; www.ncbi.nlm.nih.gov / pmc / articles / PMC5378263 / ).

[0116] Data from colorectal cancer tumors show that EGFR expression correlates poorly with specific markers of T cell infiltration, including CD8A; IFNG (interferon gamma, Kosmidis et al., J Cancer. 2018; 9(2): 232-238, www.ncbi.nlm.nih.gov / pmc / articles / PMC5771329 / ) correlates 83% with CD8A; and the supporting antigen presentation mechanisms described by TAP1 and B2M both correlate 56% with CD8A (Ozcan et al., Oncoimmunology. 2018; 7(7): e1445453, www.ncbi.nlm.nih.gov / pmc / articles / PMC5993484 / ). This indicates that some patients with low T cell infiltration, low levels of interferon gamma, and dysfunctional antigen presentation machinery may express EGFR and benefit from cetuximab treatment. [Table 9]

[0117] Implementation An embodiment of the present invention is a system for treating a solid cancer tumor (SCT) in a subject, the system comprising: an antigen-presenting cell agent; a T cell-activating vaccine; and an immunosuppressive inhibitor, wherein the antigen-presenting cell agent is a CD40 agonist, a Toll-like receptor agonist, an adjuvant, FLT3L, or any combination thereof. An embodiment is a system wherein the immunosuppressive inhibitor is selected from the group consisting of a CD73 inhibitor, a PD-L1 inhibitor, a PD-1 inhibitor, an A2a receptor inhibitor, a multikinase inhibitor, cyclophosphamide, a COX-2 inhibitor, a prostaglandin E2 inhibitor, and any combination thereof. An embodiment is a system further comprising an angiotensin II receptor type 1 antagonist. An embodiment is a system wherein the immunosuppressive inhibitor is a PD-1 inhibitor, a PD-L1 inhibitor, or a multikinase inhibitor. An embodiment is a system in which the immunosuppressive inhibitor is a PD-1 inhibitor, a PD-L1 inhibitor, or a multikinase inhibitor. An embodiment is a system in which the immunosuppressive inhibitor is a CD73 inhibitor and a PD-L1 inhibitor. An embodiment is a system in which the immunosuppressive inhibitor is a COX-2 inhibitor, a multikinase inhibitor, and a PD-1 inhibitor or a PD-L1 inhibitor. An embodiment is a system in which the multikinase inhibitor is regorafenib, sorafenib, fruquintinib, axitinib, or lenvatinib. An embodiment is a system in which the PD-1 inhibitor is nivolumab. An embodiment is a system in which the PD-L1 inhibitor is durvalumab.

[0118] In an embodiment is a system further comprising radiation therapy. In an embodiment is a system wherein the radiation therapy is stereotactic body radiation therapy (SBRT). In an embodiment is a system wherein the SCT is selected from colorectal cancer, pancreatic cancer, prostate cancer, head and neck cancer, lung cancer, melanoma, breast cancer, liver cancer, esophageal cancer, and gastric cancer.

[0119] In one embodiment, the T cell activating vaccine comprises a neoantigen vaccine. In another embodiment, the neoantigen vaccine comprises a plurality of neoantigen peptides or multiantigen polypeptides, or a nucleic acid encoding a plurality of neoantigen peptides or multiantigen polypeptides. In another embodiment, the plurality of neoantigen peptides or multiantigen polypeptides comprises about 3 to about 50 polypeptides. In another embodiment, the plurality of neoantigen peptides or multiantigen polypeptides comprises about 5 to about 40 polypeptides. In another embodiment, the plurality of neoantigen peptides or multiantigen polypeptides comprises about 10 to about 30 polypeptides. In another embodiment, the plurality of neoantigen peptides or multiantigen polypeptides comprises one or more short neoantigen polypeptides. In another embodiment, the short neoantigen comprises about 6 to about 12 amino acids in length. In another embodiment, the short neoantigen comprises about 8 to about 10 amino acids in length. In another embodiment, the plurality of neoantigen peptides or multiantigen polypeptides comprises one or more long neoantigens. An embodiment is a system in which the long neoantigen is about 12 to about 30 amino acids in length. An embodiment is a system in which the long neoantigen is about 15 to about 24 amino acids in length.

[0120] In one embodiment, the neoantigen peptides or multiantigen polypeptides are designed to correspond to antigens expressed by the subject's SCT. In another embodiment, the neoantigen peptides or multiantigen polypeptides are provided as subgroups, each subgroup containing at least one neoantigen that is not present in at least one other subgroup. In another embodiment, the subgroups comprise from about 2 to about 10 subgroups. In another embodiment, the subgroups comprise from about 3 to about 8 subgroups. In another embodiment, each subgroup comprises from about 3 to about 20 neoantigens. In another embodiment, each subgroup comprises from about 5 to about 10 neoantigens.

[0121] In one embodiment, the antigen-presenting cell agent is FLT3L; and the immunosuppressive inhibitor is selected from a CD73 inhibitor, a multikinase inhibitor, a PD-1 inhibitor, a PD-L1 inhibitor, aspirin, and celecoxib, or a combination thereof. In another embodiment, the antigen-presenting cell agent comprises FLT3L; and the immunosuppressive inhibitor comprises regorafenib, nivolumab, and aspirin.

[0122] An aspect of the present invention is a T cell activation vaccine for treating SCT in a subject, the vaccine comprising multiple neoantigens or one or more nucleic acids encoding multiple neoantigens, wherein the multiple neoantigens include at least one short neoantigen. An embodiment is a T cell activation vaccine comprising at least one long neoantigen; and a pharmaceutically acceptable carrier. An embodiment is a vaccine wherein the multiple neoantigens comprise about 3 to about 50 neoantigens. An embodiment is a vaccine wherein the multiple neoantigens comprise about 5 to about 40 neoantigens. An embodiment is a vaccine wherein the multiple neoantigens comprise about 10 to about 30 neoantigen peptides and / or multiantigen polypeptides. An embodiment is a vaccine wherein the short neoantigens are about 6 to about 12 amino acids in length. An embodiment is a vaccine wherein the short neoantigens are about 8 to about 10 amino acids in length. An embodiment is a vaccine wherein the multiple neoantigens comprise one or more long neoantigens. An embodiment is a vaccine in which the long neoantigen is about 12 to about 30 amino acids in length. An embodiment is a vaccine in which the long neoantigen is about 15 to about 24 amino acids in length. An embodiment is a vaccine in which the neoantigen peptide or multiantigen polypeptide is designed to correspond to an antigen expressed by the subject's SCT.

[0123] An embodiment is a vaccine in which multiple neoantigens are provided as multiple subgroups, where each subgroup comprises at least one neoantigen that is not present in at least one other subgroup. An embodiment is a vaccine in which the multiple subgroups consist of about 2 to about 10 subgroups. An embodiment is a vaccine in which the multiple subgroups consist of about 3 to about 8 subgroups. An embodiment is a vaccine in which each subgroup comprises about 3 to about 20 neoantigens. An embodiment is a vaccine in which each subgroup comprises about 5 to about 10 neoantigens. An embodiment is a vaccine further comprising an adjuvant. An embodiment is a vaccine further comprising an antigen-presenting cell agent.

[0124] An aspect of the invention is a method of treating SCT in a subject, comprising: a) administering an effective amount of an antigen-presenting cell agent selected from the group consisting of: a CD40 agonist, a Toll-like receptor agonist, an adjuvant, FLT3L, and any combination thereof; b) administering an effective amount of a T cell-activating vaccine; and c) administering an effective amount of an immunosuppressant inhibitor selected from the group consisting of a CD73 inhibitor, a PD-1 inhibitor, a PD-L1 inhibitor, an A2a receptor inhibitor, a multikinase inhibitor, cyclophosphamide, a COX-2 inhibitor, a prostaglandin E2 inhibitor, and any combination thereof. An embodiment is a method further comprising: d) administering an effective amount of an angiotensin II receptor type 1 antagonist. An embodiment is a method wherein the SCT is selected from colorectal cancer, pancreatic cancer, prostate cancer, head and neck cancer, lung cancer, melanoma, breast cancer, liver cancer, esophageal cancer, and gastric cancer.

[0125] An embodiment is a method in which an antigen-presenting cell agent is administered prior to administration of a T cell activating vaccine and an immunosuppressive inhibitor. An embodiment is a method in which at least one antigen-presenting cell agent is administered about 1 day to about 30 days prior to administration of a T cell activating vaccine. An embodiment is a method in which the activation state, population size, or distribution of antigen-presenting cells is measured prior to administration of a T cell activating vaccine. An embodiment is a method in which the T cell activating vaccine is administered only after the activation state, population size, or distribution of antigen-presenting cells reaches a predetermined value. An embodiment is a method in which the Toll-like receptor agonist is poly(I:C) or poly-ICLC. An embodiment is a method in which the adjuvant is Montanide™ or Depovax™. An ​​embodiment is a method in which the COX-2 inhibitor is aspirin. An embodiment is a method in which the COX-2 inhibitor is ibuprofen. An embodiment is a method wherein the COX-2 inhibitor is naproxen.An embodiment is a method wherein the COX-2 inhibitor is indomethacin.An embodiment is a method wherein the COX-2 inhibitor is celecoxib.

[0126] An embodiment is a method wherein step a) comprises administering an effective amount of FLT3L, poly-ICLC, or a CD40 agonist, or a combination thereof; and administering an effective amount of a COX-2 inhibitor. An embodiment is a method wherein each agent is administered independently. An embodiment is a method wherein two or more antigen-presenting cell agents are combined in a single formulation.

[0127] In an embodiment, the T cell activating vaccine comprises a neoantigen vaccine. In an embodiment, the neoantigen vaccine comprises a plurality of neoantigen peptides or multiantigen polypeptides, or one or more nucleic acids encoding a plurality of neoantigen peptides or multiantigen polypeptides. In an embodiment, the plurality of neoantigens comprises about 3 to about 50 neoantigens. In an embodiment, the plurality of neoantigens comprises about 5 to about 40 neoantigens. In an embodiment, the plurality of neoantigens comprises about 10 to about 30 polypeptides.

[0128] In an embodiment, the method comprises administering multiple neoantigens by injection at multiple injection sites. In an embodiment, the multiple injection sites are selected to deliver the neoantigens to different lymph nodes. In an embodiment, the multiple injection sites include about 2 to about 10 different injection sites. In an embodiment, the multiple injection sites include about 3 to about 7 different injection sites. In an embodiment, the method comprises administering a subset of the multiple neoantigens to each injection site. In an embodiment, the subset of the multiple neoantigens includes about 2 to about 7 neoantigens. In an embodiment, the subset of the multiple neoantigens includes about 5 neoantigens.

[0129] An embodiment is a method in which multiple neoantigen subsets together comprise a T cell activating vaccine, wherein at least one subset comprises at least two neoantigens that are not present in at least one of the other subsets. An embodiment is a method in which the neoantigens are designed to correspond to antigens expressed by the subject's SCT but not by normal tissue.

[0130] In some embodiments, the method further comprises identifying one or more neoantigens expressed in the subject's SCT and preparing a T cell activating vaccine using neoantigen peptides and / or multiantigen polypeptides corresponding to the neoantigens expressed in the subject's SCT. In some embodiments, the neoantigens expressed in the subject's SCT are identified by whole exome sequencing. In some embodiments, the plurality of neoantigens comprises one or more short neoantigens. In some embodiments, the short neoantigen is about 6 to about 12 amino acids in length. In some embodiments, the short neoantigen is about 8 to about 10 amino acids in length. In some embodiments, the plurality of neoantigens comprises one or more long neoantigens. In some embodiments, the long neoantigen is about 12 to about 30 amino acids in length. In some embodiments, the long neoantigen is about 15 to about 24 amino acids in length.

[0131] An embodiment is a method in which administration of the T cell activating vaccine is repeated one, two, or three times. An embodiment is a method in which the T cell activating vaccine is administered with an adjuvant or a Toll-like receptor agonist. An embodiment is a method in which the activation state, population size, or distribution of T cells is determined after administration of the T cell activating vaccine. An embodiment is a method in which the activation state, population size, or distribution of T cells is determined about 5 to about 30 days after administration of the T cell activating vaccine. An embodiment is a method in which the T cell activating vaccine is administered again if the activation state, population size, or distribution of T cells does not reach a predetermined value. An embodiment is a method in which the T cell activating vaccine is administered a third time if the activation state, population size, or distribution of T cells does not reach a predetermined value.

[0132] An embodiment is a method in which a second T cell activating vaccine is administered if the activation state, population size, or distribution of T cells does not reach a predetermined value, wherein the second T cell activating vaccine comprises at least one antigen not present in the first T cell activating vaccine. An embodiment is a method in which an immunosuppressive inhibitor is administered about 1 day to about 30 days after the final administration of the T cell activating vaccine. An embodiment is a method in which step c) comprises administering a CD73 inhibitor and a PD-L1 inhibitor. An embodiment is a method in which the immunosuppressive inhibitor comprises a multikinase inhibitor, a PD-1 inhibitor, or a PD-L1 inhibitor. An embodiment is a method in which the immunosuppressive inhibitor comprises a multikinase inhibitor and a PD-1 inhibitor. An embodiment is a method in which the multikinase inhibitor comprises regorafenib, sorafenib, fruquintinib, axitinib, or lenvatinib. In an embodiment is a method wherein the PD-1 inhibitor comprises nivolumab. In an embodiment is a method wherein the PD-L1 inhibitor comprises durvalumab.

[0133] In an embodiment, the prostaglandin E2 inhibitor is a PTGES2 inhibitor. In an embodiment, the angiotensin II receptor type 1 antagonist comprises losartan or a pharmaceutically acceptable salt thereof. In an embodiment, the administration of the antigen-presenting cell agent is continued for about 1 day to about 30 days. In an embodiment, the administration of the T cell activating vaccine is continued for about 1 day to about 60 days. In an embodiment, the administration of the immunosuppressive inhibitor is continued for about 1 day to about 90 days. In an embodiment, the SCT response to the treatment is measured. In an embodiment, the administration of the antigen-presenting cell agent is continued until the SCT response to the treatment reaches a predetermined value. In an embodiment, the administration of the T cell activating vaccine is continued until the SCT response to the treatment reaches a predetermined value. In an embodiment, the administration of the immunosuppressive inhibitor is continued until the SCT response to the treatment reaches a predetermined value.

[0134] An embodiment is a method further comprising irradiating the SCT. An embodiment is a method wherein the irradiation is stereotactic body radiation therapy (SBRT). An embodiment is a method wherein the SCT comprises metastatic CRC (mCRC). An embodiment is a method wherein the cancer comprises microsatellite-stable mCRC (MSS mCRC). An embodiment is a method wherein steps (a), (b), and (c) are performed in the following order: (b), (a), (c); (b), (c), (a), (c); (a), (b), (c); or (a), (b), (a), (c). An embodiment is a method wherein two or more of steps (a), (b), and (c) are performed simultaneously.

Claims

1. 1. A system for treating a solid cancer tumor (SCT) in a subject, comprising: (a) an antigen-presenting cell agent; (b) a T cell activating vaccine; and (c) immunosuppressant inhibitors Including, the system.

2. The system of claim 1 , wherein the antigen-presenting cell agent is selected from the group consisting of a CD40 agonist, a Toll-like receptor agonist, an adjuvant, FLT3L, and any combination thereof.

3. The system of claim 1 or 2, wherein the immunosuppressive inhibitor is selected from the group consisting of a CD73 inhibitor, a PD-L1 inhibitor, a PD-1 inhibitor, an A2a receptor inhibitor, a multikinase inhibitor, cyclophosphamide, a COX-2 inhibitor, a prostaglandin E2 inhibitor, and any combination thereof.

4. The system according to any one of claims 1 to 3, further comprising (d) an angiotensin II receptor type 1 antagonist.

5. The system according to any one of claims 1 to 4, wherein the immunosuppressive inhibitor comprises a PD-1 inhibitor, a PD-L1 inhibitor, or a multikinase inhibitor.

6. The system according to any one of claims 1 to 5, wherein the multikinase inhibitor comprises regorafenib, sorafenib, fruquintinib, axitinib, or lenvatinib.

7. The system of any one of claims 1 to 6, wherein the PD-1 inhibitor comprises nivolumab.

8. The system of any one of claims 1 to 7, wherein the PD-L1 inhibitor comprises durvalumab.

9. The system of any one of claims 1 to 8, further comprising (e) therapeutic radiation.

10. The system of any one of claims 1 to 9, wherein the SCT is selected from colorectal cancer, pancreatic cancer, prostate cancer, head and neck cancer, lung cancer, melanoma, breast cancer, liver cancer, esophageal cancer, and gastric cancer.

11. The system of any one of claims 1 to 10, wherein the T cell activating vaccine comprises a neoantigen vaccine.

12. The system of claim 11 , wherein the neoantigen vaccine comprises multiple neoantigen peptides or multiantigen polypeptides, or a nucleic acid encoding multiple neoantigen peptides or multiantigen polypeptides.

13. The system of claim 12, wherein the plurality of neoantigen peptides or multiantigen polypeptides consists of about 3 to about 50 neoantigens.

14. The system of claim 13, wherein the plurality of neoantigen peptides or multiantigen polypeptides consists of about 5 to about 40 neoantigens.

15. The system of claim 14, wherein the plurality of neoantigen peptides or multiantigen polypeptides consists of about 10 to about 30 neoantigens.

16. The system of any one of claims 11 to 15, wherein the multiple neoantigen peptides or multiantigen polypeptides comprise one or more short neoantigens.

17. 17. The system of claim 16, wherein the short neoantigen is about 6 to about 12 amino acids in length.

18. 18. The system of claim 17, wherein the short neoantigen is about 8 to about 10 amino acids in length.

19. The system of any one of claims 11 to 18, wherein the multiple neoantigen peptides or multiantigen polypeptides comprise one or more long neoantigens.

20. 20. The system of claim 19, wherein the long neoantigen is about 12 to about 30 amino acids in length.

21. 21. The system of claim 20, wherein the long neoantigen is about 15 to about 24 amino acids in length.

22. The system of any one of claims 11 to 21, wherein the neoantigen peptide or multiantigen polypeptide is designed to correspond to an antigen expressed by the subject's SCT but not by normal tissue.

23. The system of any one of claims 12 to 22, wherein the plurality of neoantigen peptides or multiantigen polypeptides are provided as a plurality of subgroups, wherein each subgroup comprises at least one neoantigen that is not present in at least one other subgroup.

24. 24. The system of claim 23, wherein the plurality of subgroups consists of about 2 to about 10 subgroups.

25. 25. The system of claim 24, wherein the plurality of subgroups consists of about 3 to about 8 subgroups.

26. The system of any one of claims 23 to 25, wherein each subgroup comprises from about 3 to about 20 neoantigens.

27. 27. The system of claim 26, wherein each subgroup comprises about 5 to about 10 neoantigens.

28. the antigen-presenting cell agent is FLT3L; and The system of any one of claims 1 to 27, wherein the immunosuppressive inhibitor is selected from a COX-2 inhibitor, a prostaglandin E2 inhibitor, a CD73 inhibitor, a multikinase inhibitor, a PD-1 inhibitor, and a PD-L1 inhibitor, or a combination thereof.

29. the antigen-presenting cell agent comprises FLT3L; and The system of claim 28, wherein the immunosuppressive inhibitors include regorafenib, nivolumab, and aspirin.

30. 1. A T cell activating vaccine for the treatment of SCT in a subject, comprising: a plurality of neoantigen peptides or multiantigen polypeptides, or one or more nucleic acids encoding a plurality of neoantigen peptides or multiantigen polypeptides, wherein the plurality of neoantigen peptides or multiantigen polypeptides comprises at least one short neoantigen; and Pharmaceutically acceptable carrier A T cell activating vaccine comprising:

31. The T cell activating vaccine of claim 30, wherein the multiple neoantigen peptides or multiantigen polypeptides comprise at least one long neoantigen.

32. The T cell activating vaccine of claim 30 or 31, wherein the multiple neoantigen peptides or multiantigen polypeptides comprise from about 3 to about 50 neoantigens.

33. The T cell activating vaccine of claim 32, wherein the plurality of neoantigen peptides or multiantigen polypeptides comprises about 5 to about 40 neoantigens.

34. The T cell activating vaccine of claim 33, wherein the plurality of neoantigen polypeptides consists of about 10 to about 30 polypeptides.

35. 35. The T cell activating vaccine of any one of claims 30 to 34, wherein the short neoantigen is about 6 to about 12 amino acids in length.

36. 36. The T cell activating vaccine of claim 35, wherein the short neoantigen is about 8 to about 10 amino acids in length.

37. The T cell activating vaccine of any one of claims 30 to 36, wherein the multiple neoantigen peptides or multiantigen polypeptides comprise one or more long neoantigens.

38. 38. The T cell activating vaccine of claim 37, wherein the long neoantigen is about 12 to about 30 amino acids in length.

39. 39. The T cell activating vaccine of claim 38, wherein the long neoantigen is about 15 to about 24 amino acids in length.

40. The T cell activating vaccine of any one of claims 30 to 39, wherein the neoantigen peptide or multiantigen polypeptide is designed to correspond to a neoantigen expressed by the subject's SCT.

41. The T cell activating vaccine of any one of claims 30 to 40, wherein the plurality of neoantigen peptides or multiantigen polypeptides are provided as a plurality of subgroups, wherein each subgroup comprises at least one neoantigen that is not present in at least one other subgroup.

42. 42. The T cell activating vaccine of claim 41, wherein the plurality of subgroups consists of about 2 to about 10 subgroups.

43. 43. The T cell activating vaccine of claim 42, wherein said plurality of subgroups consists of about 3 to about 8 subgroups.

44. 44. The T cell activating vaccine of any one of claims 41 to 43, wherein each subgroup comprises from about 3 to about 20 neoantigen or multiantigen polypeptides.

45. 45. The T cell activating vaccine of claim 44, wherein each subgroup comprises about 5 to about 10 neoantigen peptides or multiantigen polypeptides.

46. The T cell activating vaccine according to any one of claims 30 to 45, further comprising an adjuvant.

47. The T cell activating vaccine according to any one of claims 30 to 45, further comprising an antigen-presenting cell agent.

48. 1. A method of treating SCT in a subject, comprising: (a) administering an effective amount of an antigen-presenting cell agent selected from the group consisting of a CD40 agonist, a Toll-like receptor agonist, an adjuvant, FLT3L, and any combination thereof; (b) administering an effective amount of a T cell activating vaccine; and (c) administering an effective amount of an immunosuppressive inhibitor selected from the group consisting of a CD73 inhibitor, a PD-1 inhibitor, a PD-L1 inhibitor, an A2a receptor inhibitor, a multikinase inhibitor, cyclophosphamide, a COX-2 inhibitor, a prostaglandin E2 inhibitor, and any combination thereof. A method comprising:

49. 49. The method of claim 48, further comprising (d) administering an effective amount of an angiotensin II receptor type 1 antagonist.

50. 50. The method of claim 48 or 49, wherein the SCT is selected from colorectal cancer, pancreatic cancer, prostate cancer, head and neck cancer, lung cancer, melanoma, breast cancer, liver cancer, esophageal cancer, and gastric cancer.

51. The method of any one of claims 48 to 50, wherein the antigen-presenting cell agent is administered prior to administration of a T cell activating vaccine and an immunosuppressive inhibitor.

52. 52. The method of claim 51, wherein the at least one antigen-presenting cell agent is administered between about 1 day and about 30 days prior to administration of the T cell activating vaccine.

53. 52. The method of claim 51, wherein the activation state, population size, or distribution of antigen-presenting cells is measured prior to administration of the T cell activating vaccine.

54. 54. The method of claim 53, wherein the T cell activating vaccine is administered only after the activation state, population size, or distribution of antigen-presenting cells reaches a predetermined value.

55. 55. The method of any one of claims 48 to 54, wherein the Toll-like receptor agonist comprises poly(I:C) or poly-ICLC.

56. 56. The method of any one of claims 48 to 55, wherein the adjuvant comprises Montanide™ or Depovax™.

57. 57. The method of any one of claims 48 to 56, wherein the COX-2 inhibitor comprises aspirin.

58. wherein step a) comprises: administering an effective amount of FLT3L, poly-ICLC, or a CD40 agonist, or a combination thereof; and administering an effective amount of a COX-2 inhibitor 58. The method of any one of claims 48 to 57, comprising:

59. 59. The method of claim 58, wherein each of the aforementioned agents is administered independently.

60. 59. The method of any one of claims 48 to 58, wherein said two or more antigen-presenting cell agents are combined in a single formulation.

61. 60. The method of any one of claims 48 to 59, wherein the T cell activating vaccine comprises a neoantigen vaccine.

62. 62. The method of claim 61, wherein the neoantigen vaccine comprises multiple neoantigen peptides or multiantigen polypeptides, or one or more nucleic acids encoding multiple neoantigen peptides or multiantigen polypeptides.

63. The method of claim 62, wherein the plurality of neoantigen peptides or multiantigen polypeptides comprises from about 3 to about 50 neoantigens.

64. 64. The method of claim 63, wherein the plurality of neoantigen polypeptides comprises about 5 to about 40 neoantigens.

65. 65. The method of claim 64, wherein the plurality of neoantigen polypeptides comprises about 10 to about 30 neoantigens.

66. 66. The method of any one of claims 62 to 65, wherein the multiple neoantigen peptides or multiantigen polypeptides are administered by injection at multiple injection sites.

67. 67. The method of claim 66, wherein the multiple injection sites are selected to deliver the neoantigen peptide or multiantigen polypeptide to different lymph nodes.

68. 68. The method of claim 66 or 67, wherein the multiple injection sites comprise from about 2 to about 10 different injection sites.

69. 69. The method of claim 68, wherein the multiple injection sites comprise from about 3 to about 7 different injection sites.

70. 70. The method of any one of claims 66-69, wherein a subset of the plurality of neoantigen polypeptides is administered at each injection site.

71. The method of claim 70, wherein the subset of the plurality of neoantigen peptides or multiantigen polypeptides comprises from about 2 to about 7 neoantigen peptides or multiantigen polypeptides.

72. 72. The method of claim 71, wherein the subset of the plurality of neoantigen peptides or multiantigen polypeptides comprises about five neoantigens.

73. The method of any one of claims 62 to 72, wherein subsets of the plurality of neoantigen peptides or multiantigen polypeptides together comprise a T cell activating vaccine, and wherein at least one of the subsets comprises at least two neoantigen polypeptides that are not present in at least one of the other subsets.

74. The method of any one of claims 61 to 73, wherein the neoantigen peptide or multiantigen polypeptide is designed to correspond to a neoantigen expressed by the subject's SCT.

75. The method of any one of claims 61 to 73, further comprising (e) identifying one or more neoantigens expressed in the subject's SCT and preparing a T cell activating vaccine using neoantigen peptides or multiantigen polypeptides corresponding to neoantigens expressed in the subject's SCT but not in normal tissues.

76. 76. The method of claim 75, wherein the neoantigens expressed in the subject's SCT are identified by whole exome sequencing.

77. The method of any one of claims 62 to 76, wherein the multiple neoantigen peptides or multiantigen polypeptides comprise one or more short neoantigens.

78. 78. The method of claim 77, wherein the short neoantigen is about 6 to about 12 amino acids in length.

79. 79. The method of claim 78, wherein the short neoantigen is about 8 to about 10 amino acids in length.

80. The method of any one of claims 62 to 79, wherein the multiple neoantigen peptides or multiantigen polypeptides comprise one or more long neoantigens.

81. 81. The method of claim 80, wherein the long neoantigen is about 12 to about 30 amino acids in length.

82. 82. The method of claim 81, wherein the long neoantigen is about 15 to about 24 amino acids in length.

83. 83. The method of any one of claims 48 to 82, wherein the administration of the T cell activating vaccine is repeated 1, 2 or 3 times.

84. 84. The method of any one of claims 48 to 83, wherein the T cell activating vaccine is administered together with an adjuvant or a Toll-like receptor agonist.

85. 85. The method of any one of claims 48 to 84, wherein said activation state, population size or distribution of T cells is determined after administration of a T cell activating vaccine.

86. 86. The method of claim 85, wherein the activation state, population size, or distribution of the T cells is determined from about 5 days to about 30 days after administration of the T cell activating vaccine.

87. 87. The method of claim 85 or 86, wherein the T cell activating vaccine is re-administered if the activation state, population size, or distribution of T cells does not reach a predetermined value.

88. 88. The method of any one of claims 85 to 87, wherein step (e) is repeated.

89. 89. The method of any one of claims 85 to 88, wherein a second T cell activating vaccine is administered if the activation state, population size, or distribution of the T cells does not reach a predetermined value, wherein the second T cell activating vaccine comprises at least one antigen that is not present in the first T cell activating vaccine.

90. 90. The method of any one of claims 48-89, wherein the immunosuppressive inhibitor is administered from about 1 day to about 30 days after the last administration of a T cell activating vaccine.

91. The method of any one of claims 48 to 90, wherein step c) comprises administering a CD73 inhibitor and a PD-L1 inhibitor.

92. 92. The method of any one of claims 48 to 91, wherein the immunosuppressive inhibitor comprises a multikinase inhibitor, a PD-1 inhibitor, or a PD-L1 inhibitor.

93. 93. The method of claim 92, wherein the immunosuppressive inhibitor comprises a multikinase inhibitor and a PD-1 inhibitor.

94. 94. The method of claim 93, wherein the multikinase inhibitor comprises regorafenib, sorafenib, fruquintinib, axitinib, or lenvatinib.

95. The method of any one of claims 92 to 94, wherein the PD-L1 inhibitor comprises nivolumab.

96. 95. The method of any one of claims 92-94, wherein the PD-1 inhibitor comprises durvalumab.

97. 97. The method of any one of claims 48 to 96, wherein the prostaglandin E2 inhibitor is a PTGES2 inhibitor.

98. 98. The method of any one of claims 49 to 97, wherein the angiotensin II receptor type 1 antagonist comprises losartan or a pharmaceutically acceptable salt thereof.

99. 99. The method of any one of claims 48 to 98, wherein administration of the antigen-presenting cell agent continues for about 1 day to about 30 days.

100. 100. The method of any one of claims 48 to 99, wherein administration of the T cell activating vaccine continues for about 1 day to about 60 days.

101. 101. The method of any one of claims 48-100, wherein administration of the immunosuppressive inhibitor continues for about 1 day to about 90 days.

102. The method of any one of claims 48 to 101, wherein the SCT response to treatment is measured.

103. 103. The method of claim 102, wherein administration of the antigen-presenting cell agent is continued until the SCT response to the treatment reaches a predetermined value.

104. 104. The method of claim 102 or 103, wherein administration of the T cell activating vaccine is continued until the SCT response to the treatment reaches a predetermined value.

105. The method of any one of claims 102 to 104, wherein administration of the immunosuppressive inhibitor is continued until the SCT response to the treatment reaches a predetermined value.

106. The method of any one of claims 48 to 105, further comprising (f) irradiating with SCT.

107. 107. The method of claim 106, wherein the irradiation comprises stereotactic body radiation therapy (SBRT).

108. The method of any one of claims 48 to 107, wherein the SCT comprises metastatic CRC (mCRC).

109. 109. The method of claim 108, wherein the cancer comprises microsatellite-stable mCRC (MSS mCRC).

110. The aforementioned steps (a), (b), and (c) are: (i) (b), (a), (c); (ii) (b), (c), (a), (c); (iii) (a), (b), (c); or (iv) (a), (b), (a), (c) The method of any one of claims 48, 49, and 55 to 109, wherein the steps are performed in the order of:

111. 111. The method of any one of claims 48, 49, and 55-110, wherein two or more of the preceding steps (a), (b), (c), and (d) are carried out simultaneously.

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