Therapeutic compositions and methods combining multiple immunotherapy and cancer vaccines for the treatment of cancer

Abstract

Therapeutic compositions and methods are provided that combine multiple immunotherapy and cancer vaccines for the treatment of cancer. The present invention relates to a therapeutic composition comprising i) at least two immune checkpoint inhibitors, ii) at least one drug selected from a cytokine, a cytotoxic or cytostatic chemotherapeutic drug, and combinations thereof, and iii) a cancer vaccine prepared from tumor or cancer cells, or derivatives thereof, prepared by an ex vivo treatment that generates necrotic and / or necroptotic cancer cells while minimizing destruction of cancer antigens.The present invention also relates to a method of treating a tumor or cancer in a patient, comprising administering the therapeutic composition to a patient in need thereof in an amount effective to treat the tumor or cancer.

Description

[Technical field]

[0001] This application claims priority to U.S. Provisional Application No. 63 / 148,922, filed February 12, 2021, which is incorporated by reference in its entirety.

[0002] The present invention relates to therapeutic compositions that combine immunological and chemotherapeutic methods for the treatment of cancer with cancer vaccines. [Background technology]

[0003] Cancer is the second leading cause of death in the United States, claiming the lives of 580,000 Americans each year, more than 1,500 people every day. The National Institutes of Health (NIH) estimates the total annual cost of cancer care to be more than $227 billion (2007); this includes $89 billion in direct medical costs. Much of the total medical cost of cancer treatment comes from managing the harmful side effects of radiation and conventional chemotherapy. Several therapies are used for metastatic solid tumors, depending on the site and histology of the tumor (e.g., squamous cell carcinoma, urothelial carcinoma, adenocarcinoma, sarcoma, lymphoma, melanoma, etc.), including chemotherapy, immunotherapy, surgery, manipulative therapies such as external beam radiation therapy, brachytherapy, and cryosurgery, and hormonal therapy (for certain tumors). Many of these therapies confer survival benefits, but these must be considered in conjunction with the incidence of significant adverse events in this population. For example, immunological cancer treatments are poised to completely change the landscape of oncological therapeutics. Checkpoint inhibitors such as CTLA-4 and PD-1 have already had a major impact on the treatment of metastatic melanoma and non-small cell lung cancer. These drugs are currently being used in combination to improve their efficacy. These drugs are typically delivered intravenously, which can lead to severe and sometimes fatal systemic toxicity as a result of the non-specific distribution of these cytocidal agents in the body, which can adversely affect treatment regimens and patient outcomes by killing both cancer cells and normal cells. Cryosurgical freezing is a surgical technique used to selectively damage or destroy cells, organs, or abnormal growths (such as cancer). Cryosurgical freezing is known to induce an immune response in patients through the presentation of a unique set of tumor-associated antigens to the patient's antigen-presenting cells and dendritic cells. However, this "cryoimmunological effect" is known to be variable and even harmful in some cases. WO 2017 / 123981 relates to pharmaceutical compositions comprising at least two immune checkpoint inhibitors and at least one cytokine, as well as combinations thereof with engineering processes. Cytokines are naturally occurring proteins secreted by cells of the immune system or non-immune cells (e.g., epithelial cells) in response to several stimuli and help regulate the development of immune effector cells. Cytokines are immunomodulators that act through mechanisms that ultimately alter gene expression in target cells. The combination of two immune checkpoint inhibitors and a cytokine is within the scope of immunotherapy regimens by using exclusive immunological agents.

[0004] Many cancer immunotherapy efforts to date have focused on the mechanism of action of drugs, with little consideration given to the process and mechanisms of cancer antigen release and presentation. Cancer vaccines often focus on a single antigen without considering the possibility that cancers may adapt to the threat posed by an attack against only one antigen. When the entire payload of tumor-associated antigens (TAA) is released from cancer cells, nearby immature dendritic cells are activated and begin the process of identifying other intact cancer cells elsewhere in the body that express the same antigen, which can subsequently recruit cytotoxic T cells that surround and destroy the cancer. While the adaptive immune system is a major mediator of tumor rejection, it also plays a protective role in the development of human cancers. As a result, immunotherapeutic antibodies can be used to target T RegEfforts have been made to suppress or blunt this response by inactivating cells and prolonging this inactivation to allow maximum immune destruction of cancer cells, ensuring that cancer cell destruction by cytotoxic T cells does not self-terminate prematurely. However, these therapeutic approaches focus on only one of the cancer antigens.

[0005] In addition to the subjects who are diagnosed with cancer at a later stage, the subjects who fail the first-line therapy for localized cancer may progress, and in some cases reach metastatic state.For example, in the case of metastatic prostate cancer, androgen deprivation therapy is the standard treatment, and based on serum PSA level and X-ray evaluation, temporary control or regression is achieved in the majority of subjects.However, many subjects with metastatic prostate cancer will eventually experience disease progression while undergoing this therapy, thereby reaching the state of metastatic androgen-independent prostate cancer.

[0006] Several therapeutic cancer vaccines are available or are being evaluated in clinical trials, including autologous dendritic cells pulsed with prostatic acid phosphatase (see Harzstark et al., "Immunotherapy for prostate cancer using antigen-loaded antigen-presenting cells: APC8015 (Provenge)," Expert Opin Biol Ther. 7(8):1275-1280 (2007)); fully allogeneic cell lines transfected to secrete GM-CSF (see Small et al., "Granulocyte macrophage colony-stimulating factor--secreting allogeneic cellular immunotherapy for hormone-refractory prostate cancer," Clin Cancer Res. 13(13):3883-3891 (2007)); and recombinant attenuated vaccinia viruses engineered to express prostate-specific antigen and three immune co-stimulatory molecules, ICAM-1, B7.1, and lymphocyte function-associated antigen-3 (Doehn et al., "Immunotherapy for prostate cancer using antigen-loaded antigen-presenting cells: APC8015 (Provenge)," Expert Opin Biol Ther. 7(8):1275-1280 (2007)). al., “Drug evaluation: Therion's rV-PSA-TRICOM + rF-PSA-TRICOM prime-boost prostate cancer vaccine,” Curr Opin Mol Ther. 9(2):183-189 (2007)).Several therapeutic vaccine approaches have been reviewed (Sonpavde et al., “Vaccine therapy for prostate cancer. Urol Oncol. 25(6):451-459 (2007); Slovin, “Emerging role of immunotherapy in the management of prostate cancer,” Oncology (Williston Park) 21(3):326-333; discussion 334, 338, 346-348 (2004); Karnes et al., “Immunotherapy for prostate cancer,” Curr Pharm Des. 2(7):807-817 (2006); Ragde et al., “Dendritic cell based vaccines: progress in immunotherapy studies for prostate cancer,” J Urol. 172(6 Pt 2):2532-2538 (see, e.g., Friedrichs, et al., 2004). For example, for prostate cancer, sipuleucel-T clinical trials used dendritic cells as cellular adjuvants loaded with recombinant antigens - PAP and PSMA, respectively - to induce a mature phenotype prior to administration. Results from these and other dendritic cell immunotherapy trials have resulted in specific immune responses and an average 4-month extension of life (e.g., for sipuleucel-T). However, these therapies typically cost over $100,000, and patients may still die shortly thereafter. These findings indicate that current immunotherapy treatments are of limited value for the majority of patients.

[0007] Adjuvants or other modalities have been added to cancer vaccines to enhance antigen recognition and T cell activation, which may include one or more of the following: 1) genetic or chemical modification of cell-based vaccines; 2) cross-priming of T cells to tumor-associated antigens by engaging dendritic cells; 3) adoptive T cell therapy; 4) stimulation of cytotoxic inflammation with non-specific immunomodulators, toll-like receptor agonists, cytokines, chemokines or hormones; 5) reduction of immune suppression and / or stimulation of anti-tumor effector cells using antibodies, small molecules; and 6) various cytostatic or cytoreductive modalities, including chemotherapy. However, current techniques combining cancer vaccines with these modalities have not reached their full clinical potential, at least in part due to their toxicity and the difficulty in mounting a significant anti-cancer response in subjects due to existing tolerance mechanisms that actively turn off immune recognition and / or disable effector T cells in the tumor microenvironment. Thus, there remains a need in the art to develop improved methods that not only reduce the toxicity associated with conventional systemic cancer treatments, but also generate optimal cancer immune responses for improved cancer treatment. The present disclosure addresses this need. Summary of the Invention

[0008] One aspect of the present invention relates to a therapeutic composition comprising i) at least two immune checkpoint inhibitors, ii) at least one drug selected from a cytokine, a cytotoxic or cytostatic chemotherapeutic drug, and combinations thereof, and iii) a cancer vaccine prepared from tumor or cancer cells, or derivatives thereof, prepared by ex vivo treatments that generate necrotic and / or necroptotic cancer cells while minimizing destruction of cancer antigens. The therapeutic composition may include a pharmaceutically acceptable carrier. Another aspect of the present invention relates to a method of treating a tumor or cancer comprising administering to a subject a therapeutic composition comprising: i) at least two immune checkpoint inhibitors; ii) at least one drug selected from a cytokine, a cytotoxic or cytostatic chemotherapeutic drug, and combinations thereof; and iii) a cancer vaccine prepared from tumor or cancer cells, or derivatives thereof, prepared by an ex vivo treatment that generates necrotic and / or necroptotic cancer cells while minimizing destruction of cancer antigens, in an amount effective to treat the tumor or cancer. Additional aspects, advantages, and features of the present invention are described herein and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. The invention disclosed in this application is not limited to any particular set or combination of aspects, advantages, and features. It is contemplated that various combinations of the described aspects, advantages, and features constitute the invention disclosed in this application. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] The present disclosure is based, at least in part, on the development of new compositions and methods for eliciting cancer immune responses by combining tumor-directed immunological cancer treatments with cancer vaccines prepared by ex vivo procedures, such as engineering techniques. The inventors have discovered an approach that utilizes additive or synergistic mechanisms of systemic cancer killing while minimizing antagonistic interactions and adverse events by combining cancer vaccines (e.g., autologous) produced ex vivo (e.g., via UV irradiation and / or other manipulations) with multiple checkpoint inhibitors and cytokines or low-dose chemotherapy drugs. The therapeutic compositions and treatment methods described in the present invention can utilize not just one, but all of the cancer antigens present throughout a particular patient's cancer (individualized cancer treatment) by releasing a myriad of antigens while carefully controlling the induction of necrosis and necroptosis to balance the need for cell destruction with the need for structural preservation and release of antigens.

[0010] The term "ex vivo" means that the treatment of the cancer specimen (e.g., by one or more manipulations, which may also include irradiation) is carried out outside the patient's body under more controlled conditions than is possible for in vivo treatments (in the patient), at the expense of altering the "native" environment. The main advantage of using ex vivo tissues or cells is that it allows the carrying out of procedures (e.g., manipulations / irradiation) or tests that would otherwise be considered complex and dangerous (e.g., damage to vital structures and exposure to spinal or general anesthesia) in a living subject. The tissues or cells can be removed in many ways, including in parts, as whole organs, or as larger organ systems.

[0011] Ex vivo treatment of cancer obtained from tissue specimens and subsequent administration of such cancer vaccines produced ex vivo may have significant advantages over conventional systemic delivery of anti-cancer drugs. The therapeutic compositions and treatment methods disclosed herein may allow for administration of smaller doses to subjects than conventional doses (e.g., in embodiments where the therapeutic composition is administered directly to the skin), stimulation of the immune system against tumor antigens, and improved results by placing the drug and tumor antigen in close proximity to the immune inflammatory process. Ex vivo treatment may avoid the need for general (general) anesthesia during treatment, avoid the need for treatment equipment such as cryosurgical or other operating equipment used in contact with the patient (thereby reducing the risk induced by the operation), and reduce the length of time required for each treatment. In comparison, many other methods of cell destruction that use heat, such as microwaves and HIFU (high intensity focused ultrasound), denature antigenic proteins, altering antigens from their native state so that they cannot act as the correct signals to target cancer antigens on other cancer cells.

[0012] This difference in antigen handling provided by the therapeutic compositions and treatment methods described herein can ensure that antigens are adequately exposed and preserved without denaturation, allowing for the targeting and activation of immature dendritic cells with patient-specific and cancer-specific antigens that may be identified at metastatic sites.

[0013] The inventors have surprisingly discovered that by using a combination of immune checkpoint inhibitors, cytokines or cytotoxic or cytostatic chemotherapeutic drugs, and a cancer vaccine derived from ex vivo treated cancer (obtained from a tissue specimen, e.g., biopsy, resection, aspiration, etc.), a treatment method provides advantages including at least the following: (1) soft tissue injection of multiple combination immunotherapies in combination with ex vivo treatment allows for low dose (lower than conventional dose) immunotherapy; (2) the side effect profile of low dose immunotherapy is more favorable than conventional dosing and exposes patients to less risk; (3) cancer neo-antigens are preserved by no or minimal use of thermal destruction (e.g., no or minimal use of thermal manipulation); and (4) lower complications and risks than conventional in vivo treatments (e.g., ex vivo rather than in vivo manipulations).

[0014] Additionally, the approach described herein is beneficial in that it enables: It can ensure that the full, or at least a broader, range of patient-specific and cancer-specific antigens are exposed to the immune system, unlike current cancer vaccines (e.g., Provenge for prostate cancer) which recognize a single, predefined cancer antigen that may or may not be expressed by the patient's cancer. By using special methods to induce necrosis and necroptosis without significantly denaturing these proteins (because they do not function as well when denatured), we can maximize or at least increase the quantity and quality of tumor antigens exposed, preserving the vasculature that allows the entry and exit of immune system dendritic cells and cytotoxic T cells; · The natural range of costimulatory substances in cancer lysates induced by necrosis or necroptosis in combination with an inflammatory environment can be utilized; -Cancer lysates and drugs can be injected intradermally or intratumorally to create proximity of tumor antigens and inflammatory immune processes; Instead of focusing on one or a few preselected antigens, cancer cells can be destroyed to release the entire repertoire, or at least many, of cancer-specific antigens, allowing for targeted, personalized vaccines.

[0015] In one aspect, the present disclosure provides a therapeutic composition for the treatment of tumors or cancers comprising, consisting essentially of, or consisting of a combination of i) at least two immune checkpoint inhibitors, ii) at least one drug selected from cytokines, cytotoxic or cytostatic chemotherapeutic drugs, and combinations thereof, and iii) a cancer vaccine prepared from tumor or cancer cells, or derivatives thereof, prepared by ex vivo treatments that kill cancer cells (e.g., create necrotic and / or necroptotic cancer cells) while minimizing destruction of cancer antigens. The therapeutic composition may include a pharmaceutically acceptable carrier.

[0016] Immune checkpoint inhibitors are a class of drugs that block certain proteins made by several types of immune system cells, including T cells and some cancer cells. These proteins help suppress the immune response and can keep T cells from killing cancer cells. When these proteins are blocked, the "brakes" on the immune system are released, allowing T cells to kill cancer cells more effectively. Thus, checkpoint inhibitors act to activate the immune system to attack tumors and inhibit immune response proteins involved in downregulating the immune system. Such checkpoint inhibitors can include small molecule inhibitors, or can include antibodies or antigen-binding fragments thereof that bind to and block or inhibit immune checkpoint receptors, or antibodies that bind to and block or inhibit immune checkpoint receptor ligands.

[0017] For example, PD-1 and CTLA-4 attenuate T cell activity through independent molecular mechanisms. See Das et al., "Early B cell changes predict autoimmunity following combination immune checkpoint blockade." J Clin Invest. 128(2):715-720 (2018); Das et al., "Combination therapy with anti-CTLA-4 and anti-PD-1 leads to distinct immunologic changes in vivo." J Immunol. 194(3):950-959 (2015), which are incorporated by reference in their entireties. The enhanced benefit of blockade under combined checkpoint inhibitors is more likely mediated by multiple mechanisms distinct from the component monotherapies, rather than an additive contribution of the cellular and molecular mechanisms of each monotherapy. See Wei et al., "Fundamental Mechanisms of Immune Checkpoint Blockade Therapy. Cancer Discov." 8(9):1069-1086 (2018), which is incorporated by reference in its entirety. Positive costimulation by blockade above physiological levels can promote enhanced cytolytic capacity or acquisition of novel properties not exhibited by standard T cell populations, resulting in enhanced efficacy. The relative contribution of each of the several known molecular mechanisms of PD-1 and CTLA-4 blockade to therapeutic efficacy is largely unknown. Combination checkpoint inhibitor blockade therapy can improve therapeutic efficacy compared to monotherapy in both preclinical and clinical trials.See Curran et al., "PD-1 and CTLA-4 combination blockade expands infiltrating T cells and reduces regulatory T and myeloid cells within B16 melanoma tumors." Proc Natl Acad Sci US A. 107(9):4275-4280 (2010); Postow et al., "Immunologic correlates of the abscopal effect in a patient with melanoma." New England Journal of Medicine. 366(10):925-931 (2012); Wolchok et al., "Ipilimumab monotherapy in patients with pretreated advanced melanoma: a randomised, double-blind, multicentre, phase 2, dose-ranging study." Lancet Oncol. 11(2):155-164 (2010), which are incorporated by reference in their entireties. Patients with metastatic melanoma treated with combination therapy with PD-1 and CTLA-4 blockade can achieve a 36% response rate, and in some cases a greater than 50% response rate, with a potential 3-year overall survival rate of 57%. See Larkin et al., "Combined Nivolumab and Ipilimumab or Monotherapy in Untreated Melanoma." N Engl J Med. 373(1):23-34 (2015), which is incorporated by reference in its entirety. Combination therapy may also result in an overall survival advantage in metastatic renal cell carcinoma when compared to standard of care.See Motzer et al., "Nivolumab plus Ipilimumab versus Sunitinib in Advanced Renal-Cell Carcinoma." N Engl J Med. 378(14):1277-1290 (2018), which is incorporated by reference in its entirety.

[0018] Checkpoint inhibitors include inhibitors of, for example, CD137 (4-1BB); CD134; PD-1; KIR; LAG-3; PD-L1; PDL2; CTLA-4; B7 family ligands, for example, B7.1 (or CD80) or B7.2 (or CD86), B7-DC, B7-H1, B7-H2, B7-H3 (or CD276), B7-H4, B7-H5, B7-H6, and B7-H7; BTLA (or CD27 2);LIGHT;HVEM;GAL9;TIM-3;TIGHT;VISTA;2B4;CGEN-15049;CHK1;CHK2;A2aR;TGF-β;PI3Kγ;GITR;ICOS;IDO;TLR;IL-2R;IL-10;PVRIG(B7 / CD28);CCRY;OX-40;CD160;CD20;CD52;CD47;CD73;CD27-CD70;and inhibitors of CD40.

[0019] Suitable CD137 (4-1BB) inhibitors include, but are not limited to, utomilumab, urelumab, or combinations thereof.Suitable CD134 inhibitors or OX40 inhibitors include, but are not limited to, OX40-immunoglobulin (OX40-Ig), GSK3174998 (anti-OX40 antibody), 9B12, MOXR 0916, PF-04518600 (PF-8600), MEDI6383, MEDI0562, INCAGN01949, or combinations thereof.Suitable KIR inhibitors include, but are not limited to, IPH4102, 1-7F9 (human monoclonal antibody that binds to KIR2DL1 / 2L3), lirilumab, or combinations thereof. Suitable LAG-3 inhibitors include, but are not limited to, leratolimab, IMP321 (Immuntep®), GSK2831781 (agonist antibody against LAG3), BMS-986016, LAG525, or combinations thereof. Suitable CTLA-4 inhibitors include, but are not limited to, ipilimumab, tremelimumab, or combinations thereof. Suitable PD-1 inhibitors include, but are not limited to, pembrolizumab, nivolumab, pidilizumab, MK-3475, MED 14736 (monoclonal antibody), CT-011, spartalizumab, or combinations thereof. Suitable PD-L1 or PD-L2 inhibitors include, but are not limited to, durvalumab, atezolizumab, avelumab, AMP224, BMS-936559, MPLDL3280A (anti-PD-L1 antibody), MSB0010718C (anti-PD-L1 antibody), or combinations thereof. Suitable B7.1 (or CD80) or B7.2 (or CD86) inhibitors include, but are not limited to, ludex, abatacept, or combinations thereof. Suitable B7-H3 inhibitors include, but are not limited to, enoblituzumab (MGA271), MGD009, 8H9 (monoclonal antibody against B7-H3), or combinations thereof. Suitable CD20 inhibitors include, but are not limited to, rituximab, ofatumumab, or combinations thereof.Suitable CD52 inhibitors include, but are not limited to, alemtuzumab. Suitable CD47 inhibitors include, but are not limited to, Hu5F9-G4, TTI-621 (SIRPαFc), or combinations thereof. Suitable CD73 inhibitors include, but are not limited to, MEDI9447. Suitable CD27-CD70 inhibitors include, but are not limited to, ARGX-110, BMS-936561 (MDX-1203), varlilumab, or combinations thereof. Suitable CD40 inhibitors include, but are not limited to, CP-870893, APX005M, ADC-1013, JNJ-64457107, SEA-CD40, RO7009789, or combinations thereof.

[0020] Suitable BTLA (or CD272) inhibitors include, but are not limited to, 40E4; 40E4 mIgG1; D265A, or a combination thereof. Suitable LIGHT (or CD272) inhibitors include, but are not limited to, T5-39; 17-2589-42 (CD258 (LIGHT) monoclonal antibody), TNFSF14, or a combination thereof. Suitable HVEM inhibitors include, but are not limited to, anti-CD270. Suitable TIM-3 inhibitors include, but are not limited to, MBG453, MEDI9447, or a combination thereof. Suitable TIGHT inhibitors include, but are not limited to, OMP-31M32. Suitable VISTA inhibitors include, but are not limited to, JNJ-61610588, CA-170, or a combination thereof. Suitable CGEN-15049 inhibitors include, but are not limited to, anti-CGEN-15049. Suitable A2aR inhibitors include, but are not limited to, CPI-444.Suitable TGF-β inhibitors include, but are not limited to, travedersen (AP12009), M7824, galusertinib (LY2157299), or combinations thereof.Suitable PI3Kγ inhibitors include, but are not limited to, IPI-549.Suitable GITR inhibitors include, but are not limited to, TRX-518, BMS-986156, AMG 228, MEDI1873, MEDI6469, MK-4166, INCAGN01876, GWN323, or combinations thereof.Suitable ICOS inhibitors include, but are not limited to, JTX-2011, GSK3359609, MEDI-570, or combinations thereof. Suitable IDO inhibitors include, but are not limited to, BMS-986205, indoximod, epacadostat, or combinations thereof.Suitable TLR inhibitors include, but are not limited to, MEDI9197, PG545 (pixatimod, pINN), polyinosinic-polycytidylic polylysine, carboxymethylcellulose (poly-ICLC), or combinations thereof.Suitable IL-2R inhibitors include, but are not limited to, NKTR-214. Suitable IL-10 inhibitors include, but are not limited to, AM0010. Suitable PVRIG (B7 / CD28) inhibitors include, but are not limited to, COM701.

[0021] Additional checkpoint inhibitors suitable for use herein include those described in Marin-Acevedo et al., “Next generation of immune checkpoint therapy in cancer: new developments and challenges,” Journal of Hematology & Oncology 11:39 (2018), which is incorporated by reference in its entirety.

[0022] The therapeutic composition may include any combination of two or more checkpoint inhibitors. They may be the same type of checkpoint inhibitors or different checkpoint inhibitors. In some embodiments, the at least two checkpoint inhibitors include a CTLA-4 inhibitor and a PD-1 inhibitor. In some embodiments, the at least two checkpoint inhibitors include a CTLA-4 inhibitor and a PD-L1 inhibitor. In some embodiments, the CTLA-4 inhibitor is ipilimumab and the PD-1 inhibitor is pembrolizumab.

[0023] The skilled artisan will appreciate that many other combinations of checkpoint inhibitors are suitable for therapeutic compositions. A non-limiting list of combinations includes: a CD137 inhibitor and a CD134 inhibitor; a PD-1 inhibitor and a KIR inhibitor; a LAD-3 inhibitor and a PD-L1 inhibitor; a CTLA-4 inhibitor and a CD40 inhibitor; a CD134 inhibitor and a PD-1 inhibitor; a KIR inhibitor and a LAG-3 inhibitor; a PD-L1 inhibitor and a CTLA-4 inhibitor; a CD40 inhibitor and a CD137 inhibitor; a CTLA-4 inhibitor and a PD-L1 inhibitor; a PD-1 inhibitor and a CD40 inhibitor; or any other combination of two or more of the checkpoint inhibitors known in the art.

[0024] The therapeutic composition can also include at least one drug selected from a cytokine, a cytotoxic or cytostatic chemotherapeutic drug, and combinations thereof. The drug may be a cytokine drug. The term "cytokine" refers to a large group of cellular protein components or drugs that can be secreted by certain cells of the immune system that mediate cell signaling and regulate immunity, inflammation, and hematopoiesis.

[0025] Suitable cytokines include, but are not limited to, erythropoietin, G-CSF, GM-CSF, IL-2, IL-4, IL-6, IL-12, TNF, interferons such as INF-α-2a, INF-α-2b, INF-β, and INF-γ, or combinations thereof. In one embodiment, the cytokine is GM-CSF.

[0026] Alternatively, the drug can be a cytotoxic or cytostatic chemotherapeutic drug. The terms "cytotoxic" or "cytostatic" refer to cellular constituents or drugs that can cause inhibition of the growth and proliferation of, or kill, cancer cells.

[0027] Suitable cytotoxic or cytostatic chemotherapeutic agents include actinomycin, aldesleukin, alemtuzumab, alitretinoin, altretamine, amsacrine, anastrozole, arsenic trioxide, asparaginase, azacitidine, azathioprine, Bacillus Calmette-Guerin vaccine (BCG), bevacizumab, bexarotene, bicalutamide, bleomycin, bortezomib, botulinum toxin (Botox), busulfan, capecitabine, carboplatin, carmustine, cetrorelix acetate, cetuximab, chlorambucil, chloramphenicol, bromocriptine ... Chole, chlormethine hydrochloride, chorionic gonadotropin alpha, cyclosporine, cidofovir, cisplatin, cladribine, clofarabine, chlorambucil, colchicine, crisantaspase, cyclophosphamide, cytarabine, dacarbazine, dactinomycin, danazol, dasatinib, daunorubicin hydrochloride, decitabine, denileukin, dienostrol, diethylstilbestrol, dinoprostone, dithranol-containing products, docetaxel, doxorubicin, dutasteride, epirubicin, ergometrine / Methylergometrine, estradiol, estramustine sodium phosphate, estrogen-progestin combinations, conjugated estrogens, esterified estrogens, estrone, estropipate, etoposide, exemestane, finasteride, floxuridine, fludarabine, fluorouracil, fluoxymesterone, flutamide, fulvestrant, ganciclovir, ganirelix acetate, gemcitabine, gemtuzumab ozogamicin, gonadotropin, chorionic goserelin (Zoladex), hydroxylase Carbamide, ibritumomab tiuxetan, idarubicin, ifosfamide, imatinib mesylate, interferon alpha-2b, interferon-containing products, irinotecan HCl, leflunomide, letrozole, leuprolide acetate, lomustine, lymphoglobulin, medroxyprogesterone, megestrol, melphalan, menotropins, mercaptopurine, mesna, methotrexate, methyltestosterone, mifepristone, mitomycin, mitotane, mitoxantrone HCl, mycophenolate, mofetil,Nafarelin, natalizumab, nilutamide, estrogen-containing products, oxaliplatin, oxytocin (including syntocinone and syntometrine), paclitaxel, paraldehyde, pegaspargase, pemetrexed disodium, pentamidine isethionate, pentostatin, perfosfamide, pipobroman, piritrexim isethionate, plicamycin, podofrillox, podophyllin, podophyllum resin, prednimustine, procarbazine, progesterone-containing Products include, but are not limited to, progestins, raloxifene, raltitrexed, ribavirin, rituximab, sirolimus, streptozocin, tacrolimus, tamoxifen, temozolomide, teniposide, testolactone, testosterone, thalidomide, thioguanine, thiotepa, thymoglobulin, thioguanine, topotecan, toremifene citrate, tositumomab, trastuzumab, treosulfan, tretinoin, trifluridine, trimetrexate glucoronate, triptorelin, uramustine, vaccine (live), valganciclovir, valrubicin, vidarabine, vinblastine sulfate, vincristine, vindesine, vinorelbine tartrate, zidovudine, or combinations thereof.

[0028] Exemplary cytotoxic or cytostatic chemotherapeutic agents include asparaginase, bleomycin, busulfan, carboplatin, cetuximab, cisplatin, cyclophosphamide, BCG, chloramphenicol, colchicine, cyclosporine, dacarbazine, doxorubicin, etoposide, fludarabine, gemcitabine, ifosfamide, irinotecan, lomustine, melphalan, methotrexate, mitomycin, mitoxantrone, paclitaxel, procarbazine, rituximab, temozolomide, thitepa, vinblastine, vincristine, zidovudine, and combinations thereof. In one embodiment, the cytotoxic or cytostatic chemotherapeutic agent is cyclophosphamide.

[0029] The combination of two or more checkpoint inhibitors with a cytokine is different from the combination of two or more checkpoint inhibitors with a chemotherapeutic agent (cytostatic or cytotoxic), such as cyclophosphamide. The combination of two or more checkpoint inhibitors with a chemotherapeutic agent (cytostatic or cytotoxic) is different from the combination of two or more checkpoint inhibitors with another immunotherapeutic agent, such as a cytokine. Essentially, the drug class and mechanism of action of the latter combination polypharmaceutical are different from those of the former combination. In particular, chemotherapeutic agents are usually antimetabolites and synthetic drugs rather than protein drugs, whereas cytokines are naturally occurring proteins and are considered biologics. Both classes of agents exert pleiotropic effects on the immune system, but the repertoire of actions and the mechanism of action that induces these effects are significantly different for these two different classes of agents. Furthermore, the mechanism of cytokine suppression (suppressors of cytokine signaling proteins) is different from that of chemotherapeutic agents.

[0030] Cytokines are small regulatory proteins or glycoproteins that are normally secreted by cells of the immune system or non-immune cells (e.g., epithelial cells) in response to several stimuli and help regulate the development of immune effector cells. Cytokines bind to specific receptors on the membrane of target cells and trigger signaling pathways that ultimately alter gene expression in the target cells. Cytokine action is involved in a wide range of biological processes.

[0031] On the other hand, chemotherapeutic agents can promote cancer immunity by directly or indirectly inducing immunogenic cell death. Direct effects of chemotherapy include induction of necroptosis or autophagy. Indirect effects include altering cell signaling pathways, countering efforts used by cancer to evade immune regulation (see Emens et al., “Chemotherapy: friend or foe to cancer vaccines?” Curr Opin Mol Ther. 3(1):77-84 (2001), which is incorporated by reference in its entirety); enhancing release and presentation of cancer neoantigens and damage-associated molecular patterns (DAMPs), such as when chemokine signaling by CXCL8 stimulates dendritic cell identification and consumption of damaged cancer cells by exposing calreticulin to the cell surface (Sukkurwala et al., “Immunogenic calreticulin exposure occurs through a phylogenetically conserved stress pathway involving the chemokine CXCL8.” Cell Death Differ. 21(1):59-68 (2014), which is incorporated herein by reference in its entirety); upregulating MHC class 1 expression, costimulatory molecules such as B7-1, or cancer neoantigens themselves; or enhancing effector T cell activity by downregulating co-inhibitory molecules such as PD-L1 / B7-H1 or B7-H4 (see Chen et al., "Chemoimmunotherapy: reengineering tumor immunity." Cancer Immunol Immunother. 62:203-216 (2013), which is incorporated herein by reference in its entirety). Chemoimmunotherapy-induced T cell-mediated cancer killing can involve fas-dependent, perforin-dependent, and granzyme B-dependent mechanisms.See Chen et al., "Chemoimmunotherapy: reengineering tumor immunity." Cancer Immunol Immunother. 62:203-216 (2013), which is incorporated by reference in its entirety.

[0032] Cytostatic and cytotoxic chemotherapeutic agents alone have shown dose-dependent effects on the immune system. See Emens, "Chemoimmunotherapy." Cancer J 16:295-303 (2010); Chen et al., "Chemoimmunotherapy: reengineering tumor immunity." Cancer Immunol Immunother 62:203-216 (2013), which are incorporated by reference in their entireties.

[0033] Chemotherapeutic agents have been used to modulate cancer immunity while avoiding the toxicity associated with the higher doses required for direct cell killing. This modulation has been demonstrated with several chemotherapeutic agents, such as cyclophosphamide, paclitaxel, cisplatin, and temozolomide. Cyclophosphamide, for example, has demonstrated pleiotropic immune modulating properties, including, for example, depleting Tregs. See Machiels et al., "Cyclophosphamide, doxorubicin, and paclitaxel enhance the antitumor immune response of granulocyte / macrophage-colony stimulating factor-secreting whole-cell vaccines in HER-2 / neu tolerized mice." Cancer Res. 61(9):3689-3697 (2001), which is incorporated herein by reference in its entirety. Taxanes such as paclitaxel also deplete Tregs, promote dendritic cell maturation, and shift the CD4+ T helper phenotype from type 2 to type 1, resulting in the secretion of proinflammatory cytokines and enhanced priming and lytic activity of CD8+ T cells. Doxorubicin may slow tumor growth and enhance vaccine activity, but the mechanism of this immunomodulation is unclear. The combination of cyclophosphamide and doxorubicin has also shown favorable effects, curing some cancer mice by selective depletion of Tregs and allowing the mobilization of high-avidity cancer-specific T cells. The combination of a breast cancer vaccine secreting HER2p, GM-CSF with immunomodulatory doses of cyclophosphamide and doxorubicin can selectively deplete CD4+ Tregs relative to effector T cells and activate effector T cells.See, "Immediate versus deferred treatment for advanced prostatic cancer: initial results of the Medical Research Council Trial. The Medical Research Council Prostate Cancer Working Party Investigators Group." Br J Urol. 79(2):235-246 (1997), which is incorporated by reference in its entirety. Other chemotherapeutic agents, such as gemcitabine, have also shown effects on the immune system, including induction of apoptosis, promotion of dendritic cell cancer antigen presentation, and promotion of cross-priming of CD8+ T cells. See, Nowak et al., "Induction of tumor cell apoptosis in vivo increases tumor antigen cross-presentation, cross-priming rather than cross-tolerizing host tumor-specific CD8 T cells." J Immunol. 170(10):4905-4913 (2003), which is incorporated by reference in its entirety.

[0034] Combinations of two or more checkpoint inhibitors with cytokines or chemotherapeutic agents (cytostatic or cytotoxic) may benefit from targeting other non-redundant aspects of the cancer immune life cycle, such as novel molecules, tissue sites of action, immune cell populations, and biological processes. For example, VISTA, a molecule from the immunoglobulin superfamily (IgSF), is predominantly expressed on M2 macrophages after ipilimumab (anti-CTLA-4) treatment in patients with metastatic prostate cancer. See Gao et al., "VISTA is an inhibitory immune checkpoint that is increased after ipilimumab therapy in patients with prostate cancer. Nat Med. 2017;23(5):551-555, which is incorporated by reference in its entirety. VISTA and PD-1 have non-redundant inhibitory effects on T cells. See Liu et al., "Immune-checkpoint proteins VISTA and PD-1 nonredundantly regulate murine T-cell responses." Proc Natl Acad Sci US A. 112(21):6682-6687 (2015), which is incorporated by reference in its entirety. As another example, gemcitabine can enhance the efficacy of a dendritic cell-based vaccine in a mouse pancreatic cancer model by increasing T cell trafficking and sensitizing tumor cells to T cell-mediated lysis.See Bauer et al., "Concomitant gemcitabine therapy negatively affects DC vaccine-induced CD8(+) T-cell and B-cell responses but improves clinical efficacy in a murine pancreatic carcinoma model." Cancer Immunol Immunother. 63(4):321-333 (2014), which is incorporated by reference in its entirety. A further example is the use of cyclophosphamide and the multi-peptide vaccine IMA901, in a Phase II clinical trial in patients with metastatic renal cell carcinoma, which can improve survival in patients who generated a multi-peptide immune response, suggesting a diverse tumor-specific immune response generated by multiple antigens. See Walter et al., "Multipeptide immune response to cancer vaccine IMA901 after single-dose cyclophosphamide associates with longer patient survival." Nat Med. 18(8):1254-1261 (2012), which is incorporated by reference in its entirety.

[0035] Certain studies in patients with non-small cell lung cancer and melanoma have shown that the combination of chemotherapy with ipilimumab can be safe (see Weber et al. "Randomized phase I pharmacokinetic study of ipilimumab with or without one of two different chemotherapy regimens in patients with untreated advanced melanoma," Cancer Immunology 13:7 (2013), which is incorporated herein by reference in its entirety). However, there have also been reports of increased certain adverse effects, such as transaminemia, possibly due to intravenous administration (see Robert et al., "Ipilimumab plus dacarbazine for previously untreated metastatic melanoma. New England Journal of Medicine 11;364(26):2517-26 (2011), which is incorporated herein by reference in its entirety).

[0036] Factors such as dose, treatment schedule, and route of administration may contribute to minimizing toxicity. However, data on the efficacy of combining immune checkpoint blockade with cytokines or low-dose chemotherapeutic agents is very limited. Combining two or more checkpoint inhibitors with cytokines or low-dose chemotherapeutic agents (cytotoxic or cytostatic) can exploit additive or synergistic mechanisms of systemic cancer killing while minimizing antagonistic interactions and adverse events.

[0037] In some embodiments, the drugs in the therapeutic composition may include a combination of a cytokine and a cytotoxic or cytostatic chemotherapeutic drug.

[0038] In some embodiments, the drug in the therapeutic composition comprises a cytokine drug, and the therapeutic composition may further comprise a second cytokine, which may be the same as or different from the first cytokine drug.

[0039] In some embodiments, the drug in the therapeutic composition comprises a cytotoxic or cytostatic chemotherapeutic agent, and the therapeutic composition may further comprise a second cytotoxic or cytostatic chemotherapeutic agent, which may be the same as or different from the first cytotoxic or cytostatic chemotherapeutic agent.

[0040] The immune checkpoint inhibitors are present in the therapeutic composition in a therapeutically effective amount. For example, the concentration of each immune checkpoint inhibitor may be in the range of about 0.1 to about 500 mg / ml, for example, about 0.1 to about 300 mg / ml, about 0.1 to about 200 mg / ml, about 0.1 to about 100 mg / ml, about 0.5 to about 100 mg / ml, about 0.5 to about 50 mg / ml, about 0.5 to about 30 mg / ml, about 0.5 to about 20 mg / ml, about 0.5 to about 10 mg / ml, about 1 to about 10 mg / ml, about 1 to about 5 mg / ml, or about 1 to about 2 mg / ml. When present, the cytokine drug or cytotoxic or cytostatic chemotherapeutic agent is also present in the therapeutic composition in a therapeutically effective amount. For example, the concentration of each cytokine drug may be about 1 μg / ml to about 1000 mg / ml, about 1 to about 1000 mg / ml, about 1 to about 500 mg / ml, about 10 to about 500 mg / ml, about 50 to about 500 mg / ml, about 100 to about 500 mg / ml, about 1 μg / ml to about 50 mg / ml, about 1 μg / ml to about 30 mg / ml, about 1 μg / ml to about 20 mg / ml, about 1 μg / ml to about 1 ... It may range from about 5 mg / ml, about 1 μg / ml to about 1 mg / ml, about 1 to about 500 μg / ml, about 1 to about 500 μg / ml, about 1 to about 300 μg / ml, about 1 to about 200 μg / ml, about 1 to about 100 μg / ml, about 1 to about 50 μg / ml, about 1 to about 30 μg / ml, about 1 to about 20 μg / ml, about 5 to about 50 μg / ml, about 5 to about 30 μg / ml, about 5 to about 20 μg / ml, or about 5 to about 10 μg / ml. In some cases, the cytokine drug is GM-CSF and its concentration in the composition may range from about 100 to about 500 mg / ml.

[0041] The concentration of each cytotoxic or cytostatic chemotherapeutic agent is about 1 μg / ml to about 100 mg / ml, about 1 μg / ml to about 50 mg / ml, about 1 μg / ml to about 30 mg / ml, about 1 μg / ml to about 20 mg / ml, about 1 μg / ml to about 10 mg / ml, about 1 μg / ml to about 5 mg / ml, about 1 μg / ml to about 1 mg / ml, about 1 to about 500 μg / ml. The concentration of the cytotoxic or cytostatic chemotherapeutic agent in the composition may range from about 10 to about 500 μg / ml, about 1 to about 300 μg / ml, about 1 to about 200 μg / ml, about 1 to about 100 μg / ml, about 1 to about 50 μg / ml, about 1 to about 30 μg / ml, about 1 to about 20 μg / ml, about 5 to about 50 μg / ml, about 5 to about 30 μg / ml, about 5 to about 20 μg / ml, or about 5 to about 10 μg / ml. In some cases, the cytotoxic or cytostatic chemotherapeutic agent is cyclophosphamide and its concentration in the composition may range from about 10 to about 500 μg / ml.

[0042] In some cases, the therapeutic composition comprises, consists essentially of, or consists of a CTLA-4 inhibitor at a concentration of about 0.5-10 mg / ml, a PD-1 inhibitor at a concentration of about 0.5-20 mg / ml, and either a cytokine drug at a concentration of about 1-1000 mg / ml (e.g., 100-500 mg / ml) or a cytotoxic or cytostatic chemotherapeutic drug at a concentration of about 1-1000 μg / ml (e.g., 10-500 μg / ml). In some cases, the composition comprises a CTLA-4 inhibitor at a concentration of about 1-2 mg / ml, a PD-1 inhibitor at a concentration of about 1-10 mg / ml, and either a cytokine drug at a concentration of about 10 to about 500 mg / ml (e.g., about 250 mg / ml) or a cytotoxic or cytostatic chemotherapeutic drug at a concentration of about 10 to about 500 μg / ml (e.g., about 250 μg / ml). For example, the composition may include a CTLA-4 inhibitor at a concentration of about 3.3 mg / ml, a PD-1 inhibitor at a concentration of about 6.6 mg / ml, and either a cytokine drug at a concentration of about 250 mg / ml or a cytotoxic or cytostatic chemotherapeutic drug at a concentration of about 16.6 μg / ml. In some cases, the composition includes a CTLA-4 inhibitor at a concentration of about 0.5-10 mg / ml, a PD-1 inhibitor at a concentration of about 0.5-20 mg / ml, and either GM-CSF at a concentration of about 100 to about 500 mg / ml (e.g., about 250 mg / ml) or cyclophosphamide at a concentration of about 10 to about 500 μg / ml (e.g., about 250 μg / ml). In some cases, the composition is at least or about 15 ml in volume. In some cases, the composition is at least or about 10 ml in volume. In some cases, the composition is less than about 1.0 ml in volume.

[0043] The combination of at least two checkpoint inhibitors with a cytokine drug or a cytotoxic or cytostatic chemotherapeutic drug is superior to the combination of at least two checkpoint inhibitors (but without a cytokine drug or a cytotoxic or cytostatic chemotherapeutic drug) due to the additive or synergistic effect of these immunostimulatory drugs on the ability to deplete intratumoral regulatory T cells (Tregs). Furthermore, the generation of an efficient systemic adaptive anti-cancer immune response can be achieved by a soft tissue (e.g., intradermal, intramuscular, etc.) immunization strategy that combines Treg depletion with immunogenic tumor cell death and dendritic cell activation. Traditionally, checkpoint inhibitors are administered intravenously, which can cause severe and sometimes fatal systemic toxicity as a result of the non-specific distribution of these cytocidal agents in the body. The non-specific distribution of these agents can kill both cancer cells and normal cells, adversely affecting treatment regimens and patient outcomes. Soft tissue injection methods can reduce systemic toxicity and result in fewer side effects by sequestering drugs in the soft tissue microenvironment and sparing normal cells and tissues from drug toxicity (see Marabelle et al., "Intratumoral Immunization: A New Paradigm for Cancer Therapy" Clin. Cancer Res. 20(7): 1747-56 (2014), which is incorporated herein by reference in its entirety).

[0044] Multiple costimulatory and cohibitory receptors influence the control of T cell activation, proliferation, and the gain or loss of effector functions, including CTLA-4. CTLA4 promotes anti-cancer activity by binding to B7-1 and B7-2 ligands and activating CD8+ cytotoxic T cells and simultaneously depleting CD4+ Tregs. See Selby et al., “Anti-CTLA-4 antibodies of IgG2a isotype enhance antitumor activity through reduction of intratumoral regulatory T cells” Cancer Immunol Res 1:32-42; 2013, which is incorporated by reference in its entirety. These results may explain the systemic anti-cancer immune response generated in a mouse model using localized low-dose delivery of anti-CTLA-4. Low-dose anti-CTLA-4 antibodies injected around established mouse colon cancers could eradicate local tumors and prevent the development of cancer at uninjected distant sites (abscopal effect) by directly enhancing cancer-specific CD8+ T cell responses. See Fransen et al., "Controlled local deliver of CTLA-4 blocking antibody induces CD8+ T-cell-dependent tumor eradication and decreases risk of toxic side effects" Clin Cancer Res 19: 5381-9; 2013, which is incorporated by reference in its entirety.

[0045] Furthermore, by combining approaches that target both cancer cells and the immune system, the therapeutic compositions may be more effective not only in inhibiting cancer, but also in eliciting an effective anti-tumor immune response that may then target metastatic sites and eliminate cancer throughout the subject.

[0046] The therapeutic compositions further include cancer vaccines prepared from tumor or cancer cells, or derivatives thereof, prepared by ex vivo procedures that kill cancer cells (e.g., generate necrotic and / or necroptotic cancer cells) while minimizing destruction of cancer antigens.

[0047] Cancer vaccines are typically vaccines that induce specific immune responses to certain cancers to treat or prevent the onset of the cancer. They can be classified into tumor-associated antigen (TAA)-based vaccines and dendritic cell (DC)-based vaccines. Tumor-associated antigen-based vaccines contain tumor-specific antigens to activate immune cells. Dendritic cell-based vaccines aim to promote antigen presentation by dendritic cells to induce anti-tumor responses. Non-limiting examples of cancer vaccines include tumor cell vaccines, antigen vaccines, dendritic cell vaccines, DNA vaccines, and vector-based vaccines.

[0048] Cancer vaccines prime the cellular immune response by providing tumor-associated antigens (TAA) or antigenic epitopes. A variety of vaccine delivery platforms exist, including MHC-specific synthetic or cancer-purified peptides, whole or partial proteins, RNA and DNA plasmids, recombinant viral and bacterial vectors, direct targeting or ex vivo pulsing of dendritic cells, and injection of cancer-derived whole cell lysates, fragments, apoptotic bodies, or exosomes. See Patel et al., "Next generation approaches for tumor vaccination." Chin Clin Oncol 6:19 (2017); Gonzalez et al., "Tumor cell lysates as immunogenic sources for cancer vaccine design. Hum Vaccin Immunother 10:3261 (2014), which are incorporated by reference in their entireties. Many have limitations that prevent their clinical utility. For example, eradicating cancer and preventing recurrence requires targeting a broad range of TAAs, which is a major drawback of strategies that use preselected peptides or proteins, plasmids, or recombinant vectors. Dendritic cell vaccination requires the ex vivo generation of clinical-grade dendritic cells and subsequent loading with cancer-associated antigens or whole cancer cells, which is a costly, time-consuming, and laborious process that has prevented widespread clinical application of this approach.

[0049] Antigens constitute the immune targets of cancer vaccines. To control cancer, vaccines stimulate the host immune system to eradicate (or prevent) large clinically significant cancer deposits. The antigenic material introduced into human subjects can take the form of "naked" proteins or peptides with or without adjuvants, proteins expressed by viral vectors and introduced as viral particles, whole cancer cells or lysates expressing a wide range of potential antigens, or recombinant or autologous proteins loaded into antigen-presenting cells such as dendritic cells. Thus, cancer vaccines can be made using whole cancer cells, cell fragments, tissue fragments, lysates, cellular component derivatives such as apoptotic blebs or exosomes, or combinations thereof, which can be fresh, irradiated, fixed (e.g., formalin-fixed, ethanol-fixed, or glutaraldehyde-fixed), and manipulated to create lysates.

[0050] Necrotic tumor cells have been shown to induce partial maturation in DCs without the need for adjuvants, likely due to the large amounts of heat shock proteins (HSPs) 70 and 90 released from dying cells following treatment (see Sauter et al., "Consequences of cell death: exposure to necrotic tumor cells, but not primary tissue cells or apoptotic cells, induces the maturation of immunostimulatory dendritic cells," J Exp Med. 191(3):423-34 (2000); Somersan et al, "Primary tumor tissue lysates are enriched in heat shock proteins and induce the maturation of human dendritic cells," J Immunol. 167(9):4844-52 (2001), which are incorporated by reference in their entireties). These HSPs are recognized by Toll-like receptor 4 (TLR4) expressed on DCs, enabling intracellular antigen processing and presentation (see Asea et al. "HSP70 stimulates cytokine production through a CD14-dependant pathway, demonstrating its dual role as a chaperone and cytokine," Nat Med. 6(4):435-42 (2000), which is incorporated herein by reference in its entirety).The proinflammatory factor high mobility group box 1 (HMGB1) is also released during necrotic cell death and interacts with TLR4 on DCs, stimulating antigen processing and presentation (see Scaffidi et al., "Release of chromatin protein HMGB1 by necrotic cells triggers inflammation," Nature 418(6894):191-95 (2002); Apetoh et al., "Molecular interactions between dying tumor cells and the innate immune system determine the efficacy of conventional anticancer therapies," Cancer Res. 68(11):4026-30 (2008), which are incorporated by reference in their entireties). HMGB1 blocks phagosome-lysosome fusion by ligating TLR4, thereby preventing the degradation of antigens and aiding their transport to antigen-presenting cells. Neutralization or knockdown of HMGB1 or knockout of TLR4 abolishes the ability of dying tumor cells to induce antitumor responses both in vitro and in vivo. Uric acid, an endogenous danger signal, accumulates after purine degradation in damaged or dying cells and also induces DC maturation, thereby enhancing vaccination (see Shi et al., "Molecular identification of a danger signal that alerts the immune system to dying cells," Nature 425(6957):516-21 (2003), which is incorporated herein by reference in its entirety).

[0051] Necroptosis, one of multiple mechanisms of programmed cell death, is characterized by rapid membrane permeabilization via mixed lineage kinase-like protein (MLKL) and activation of the RIPK1 / RIPK3 necrosome complex pathway. Unlike apoptosis, necroptosis strongly stimulates immune responses due to the release of intracellular contents such as damage-associated membrane proteins (DAMPs), production of inflammatory chemokines and cytokines, and RIPK3 promotion of antigen loading by APCs. Intratumoral injection of necroptotic cells in mice can result in CD8+ leukocyte-dependent antitumor immunity.

[0052] In one embodiment, the tumor or cancer cells, or derivatives thereof, are fresh whole cells. Tumor cells or cancer cells or their derivatives can be treated ex vivo when preparing cancer vaccines. Treatment or manipulation includes, but is not limited to, removal of non-cancerous tissue by physical or chemical methods to ensure maximum purity of cancer; manipulation or cell modification (e.g., irradiation, cryosurgical freezing, hyperthermia, radiofrequency manipulation, etc.) to release and expose tumor-associated antigens by apoptosis, necroptosis, or necrosis mechanisms to ensure that injected vaccine does not contain viable cancer cells; and / or gene transfection (only in fresh samples) to enable production of preferred drugs such as cytokines (e.g., GM-CSF).

[0053] In one embodiment, the tumor or cancer cells, or derivatives thereof, are a cancer cell lysate prepared by one or more procedures such as dissociation or dissection, fixation, centrifugation, resuspension, concentration, other manipulations, and combinations thereof.

[0054] Cancer vaccines can be made from actual cancer cells taken from a subject. Once taken, the cancer cells can be modified in the laboratory, for example, by ex vivo treatment to create necrotic and necroptotic cancer cells, so that they cannot form more tumors. For example, the cancer cells can be modified by adding chemicals or new genes, which makes the cells more likely to be recognized as foreign by the immune system. The ex vivo treated cells are then injected into the subject. The immune system can recognize antigens on these cells, and through natural physiological processes, seek out and attack / kill cells expressing the intended antigen.

[0055] Biopsy or aspirate samples removed from a subject are typically dissected and enriched to ensure a high yield of cancer cells, which are then rendered necroptotic or necrotic. This process produces cellular material containing cellular fragments of disrupted membranes, intracellular organelles such as mitochondria, and a crude mixture of cellular RNA and DNA.

[0056] In one embodiment, the tumor or cancer cells, or derivatives thereof, are cancer cell lysates fixed, for example, by formalin, ethanol, or glutaraldehyde. Further fixation methods can be found in Wang, et al., "Protective antitumor immunity induced by tumor cell lysates conjugated with diphtheria toxin and adjuvant epitope in mouse breast tumor models" Chin J Cancer 31:295-305 (2012); Baogang et al., "Fixed-tumor vaccine: A practical formulation with cytokine-microspheres for protective and therapeutic antitumor immunity" Chinese-German Journal of Clinical Oncology 2:196-202 (2003), which are incorporated herein by reference in their entireties.

[0057] In one embodiment, cancer whole cells or lysates are used for cancer vaccines. The use of cancer whole cells or lysates provides multiple advantages, including: (1) providing and targeting all potential tumor-associated antigens; (2) avoiding antigen loss; (3) eliminating the need for pre-identification of preferred antigens; (4) targeting all subjects eligible for treatment regardless of HLA type; (5) improving immunogenicity, including that antigen epitopes expressed intracellularly and / or on cell membranes are more likely to produce MHC-dependent immune stimulation to induce higher immunogenicity than the same peptides in soluble unbound form; (6) ensuring inclusion of antigens from histologically identical tumors containing antigenically diverse cells; and (7) providing a wealth of antigens and biomolecules, including proteins, lipids, and glycoproteins, that can simultaneously engage and activate many types of scavenger receptors, such as LOX-1, CD36, and MARCO, and possibly Toll-like receptors on immune cells. See Chiang et al., "Whole Tumor Antigen Vaccines: Where Are We? Vaccines" (Basel) 3:344 (2015); Seledtsov et al., "Clinically feasible approaches to potentiating cancer cell-based immunotherapies." Hum Vaccin Immunother 11:851 (2015), which are incorporated by reference in their entireties.

[0058] Cancer vaccines can be autologous or allogeneic. In one embodiment, cancer vaccines are allogeneic, i.e., tumor cells or cancer cells, or their derivatives, are made from materials derived from another member of the same species as the patient.In other words, cancer cells are taken from a subject, modified, and then injected back into a second, different subject.Commonly used allogeneic materials include the use of established laboratory-grown cancer cell lines that are known to express the TAA of a particular tumor type. In one embodiment, the cancer vaccine is autologous, i.e., the tumor or cancer cells, or derivatives thereof, are produced from material derived from the patient's tumor or cancer, in other words, cancer cells are taken from a subject, modified, and then injected back into the same subject. Autologous vaccines may offer superior survival when compared to allogeneic vaccines (see Dillman et al., “Randomized phase II trial of autologous dendritic cell vaccines versus autologous tumor cell vaccines in metastatic melanoma: 5-year follow up and additional analyses.” J Immunother Cancer 6:19 (2014), which is incorporated herein by reference in its entirety). The use of autologous cells can effectively deliver multivalent patient-specific vaccine treatments containing multiple antigenic epitopes for priming CD4+ and CD8+ T cell responses, thereby creating personalized cancer vaccines. For example, clinical trials in ovarian cancer have successfully utilized whole cancer lysates as an antigen source for loading dendritic cells, and the vaccine has been shown to be safe, feasible, and to generate both cellular and humoral immune responses. Other studies in human melanoma, glioma, renal cell carcinoma, prostate cancer, and pancreatic cancer have used irradiated and / or cryoengineered allogeneic or autologous tumor cells in combination with adjuvant proteins, resulting in modest responses without significant toxicity.Furukawa et al., “A practical approach to pancreatic cancer immunotherapy using resected tumor lysate vaccines processed to express alpha-gal epitopes” PLoS One 12: e0184901 (2017); Plautz et al., “T cell adoptive immunotherapy of newly diagnosed gliomas” Clin Cancer Res 6:2209-2218 (2000); Mehrotra et al., “Vaccination with poly(IC:LC) and peptide-pulsed autologous dendritic cells in patients with pancreatic cancer” J Hematol Oncol 10:82 (2017); Simons et al., “Induction of immunity to prostate cancer antigens: results of a clinical trial of vaccination with irradiated autologous prostate tumor cells engineered to secrete granulocyte-macrophage colony-stimulating factor using ex vivo gene transfer” Cancer Res 59:5160-68 (1999); Jocham et al., “Adjuvant autologous renal tumour cell vaccine and risk of tumour progression in patients with renal-cell carcinoma after radical nephrectomy: phase III, randomised controlled trial. Lancet 363:594-99 (2004), which are incorporated by reference in their entireties.

[0059] An autologous vaccine made from advanced non-small cell lung cancer cells harvested from pleural effusion can be administered intradermally to generate specific antibody responses against TAAs without significant immunotoxicity (see Sanborn et al., "A pilot study of an autologous tumor-derived autophagosome vaccine with docetaxel in patients with stage IV non-small cell lung cancer" J Immunother Cancer 5:103 (2017), which is incorporated by reference in its entirety). Autologous intradermal cancer vaccines for Ewing's sarcoma can provide survival benefit with low and acceptable adverse event levels (no toxicity above grade 3) (see Ghisoli et al., "Three-year Follow up of GMCSF / bi-shRNA(furin) DNA-transfected Autologous Tumor Immunotherapy (Vigil) in Metastatic Advanced Ewing's Sarcoma" Mol Ther 24:1478-83 (2016), which is incorporated by reference in its entirety).

[0060] In one embodiment, the cancer vaccine is autologous and prepared by fixation, such as formalin fixation.

[0061] Autologous formalin-fixed tumor vaccines (AFTVs) are made to order from the patient's own surgically removed paraffin-embedded cancer tissue and injected intradermally, providing a convenient source of personalized tumor-associated antigens. See Ishikawa et al., "Prospect of Immunotherapy for Glioblastoma: Tumor Vaccine, Immune Checkpoint Inhibitors and Combination Therapy" Neurol Med Chir (Tokyo) 57: 321-330; 2017, which is incorporated herein by reference in its entirety.

[0062] Formalin fixation preserves the antigenicity of cancer cells, allowing the use of preserved surgical tissue to generate antitumor immune responses. Cancer-specific autologous cytotoxic T cells can be generated by injection of formalin-fixed sections with activity and specificity comparable to those induced by cultured cells. See Liu et al., "Induction of human autologous cytotoxic T lymphocytes on formalin-fixed and paraffin-embedded tumour sections" Nat Med 1995;1: 267-271, 1995, which is incorporated herein by reference in its entirety.

[0063] AFTV differs from vaccines made from predefined molecular targeted agents such as peptides in that the autologous cancer serves as a source of diverse, unidentified, patient-specific antigens. Thus, AFTV-induced cytotoxic T cells and dendritic cells are polyclonal in nature and provide specific cellular immune responses against TAAs (tumor-associated antigens) (e.g., glypican-3, a protein frequently expressed in hepatocellular carcinoma). See Kawashima et al., “Suppression of postsurgical recurrence of hepatocellular carcinoma treated with autologous formalin-fixed tumor vaccine, with special reference to glypican-3” Clin Case Rep 3: 444-447, 2015, which is incorporated herein by reference in its entirety.

[0064] AFTV can increase white blood cells and lymphocytes, the number of CD3+ T cells, the proportion of Th1 in CD4+ T cells, and the ratio of Th1 to regulatory T cells. See Kuranishi et al., “Rate of Clinical Complete Response for 1 Year or More in Bone-Metastatic Breast Cancer after Comprehensive Treatments including Autologous Formalin-Fixed Tumor Vaccine” Int J Breast Cancer 4879406, 2018, which is incorporated by reference in its entirety. AFTV is also easier and cheaper to prepare and handle than other vaccines, facilitating outpatient clinic-based treatment. The efficacy of AFTV has been demonstrated preclinically in rodent brain tumors and mouse liver cancer, and clinically confirmed in patients with breast cancer, glioblastoma multiforme, hepatocellular carcinoma, malignant fibrous histiocytoma, recurrent peritoneal serous carcinoma, small cell carcinoma of the cervix, upper tract urothelial carcinoma, cholangiocarcinoma, and colon cancer.

[0065] However, AFTV immunotherapy has been limited when tissue sources such as biopsies contain small amounts of cancer tissue, and when a heterogeneous cancer or mixture of abundant benign cells is present, modifications are required to ensure that a minimum amount of cancer antigen is included in each vaccine. In some embodiments of the present invention, when an autologous formalin-fixed tumor vaccine is used in a cancer vaccine, the AFTV source should contain a majority, if not exclusively, malignant cells and be as representative as possible of the entire target cancer.

[0066] Other adjuvants or modalities are often added to cancer vaccines to enhance antigen recognition and T cell activation, including: 1) genetic or chemical modification of cell-based vaccines; 2) cross-priming of T cells with tumor-associated antigens by engaging dendritic cells; 3) adoptive T cell therapy; 4) stimulation of cytotoxic inflammation with non-specific immunomodulators, toll-like receptor agonists, cytokines, chemokines or hormones; 5) reduction of immune suppression using antibodies, small molecules and / or stimulation of anti-tumor effector cells; and 6) various cytostatic or cytoreductive modalities including chemotherapy. See Patel et al., "Next generation approaches for tumor vaccination." Chin Clin Oncol 6:19 (2017), which is incorporated by reference in its entirety. Combining vaccines with immunomodulatory agents has improved outcomes, but their full clinical potential has not been achieved, likely due to difficulties in mounting significant anti-cancer responses in subjects due to pre-existing tolerance mechanisms that actively turn off immune recognition and / or disable effector T cells in the tumor microenvironment. See Chiang et al., "Whole Tumor Antigen Vaccines: Where Are We? Vaccines" (Basel) 3:344 (2015); Seledtsov et al., "Clinically feasible approaches to potentiating cancer cell-based immunotherapies." Hum Vaccin Immunother 11:851 (2015); Godoy-Calderon et al., "Autologous tumor cells / bacillus Calmette-Guerin / formalin-based novel breast cancer vaccine induces an immune antitumor response" Oncotarget 9: 20222-38 (2018), all of which are incorporated herein by reference in their entirety.

[0067] In some embodiments, the cancer lysate can be produced from the original starting tissue and includes about 1 million to about 1 billion whole cancer cells or derivatives thereof. The lysate can be delivered in a cumulative volume of about 0.1 to about 10 ml, or about 0.25 to about 5 ml. The lysate can be delivered to multiple sites simultaneously. In some cases, ex vivo treatment for preparing tumor or cancer cells or derivatives thereof comprises, consists essentially of, or consists of one or more steps of ex vivo irradiation and / or manipulation.

[0068] As used herein, the terms "manipulation" or "manipulating" refer to minimally invasive surgical methods for damaging or destroying cells, and may be interchangeable with the terms "ablation" or "ablating." As used herein, the term "cryosurgical freezing" refers to a process of destroying cancer cells or tissue using freezing temperatures, and may be interchangeable with the terms "cryoablation" or "cryoablation."

[0069] Ex vivo irradiation and / or manipulation can be performed by using various irradiation and / or manipulation methods known in the art or a combination thereof. Suitable manipulation methods include cryogenic manipulation, such as cryosurgical freezing; thermal manipulation, such as radio frequency (RF), microwave, laser, light, or plasma manipulation, ultrasound, high intensity focused ultrasound (HIFU), or steam manipulation; electrical manipulation, such as reversible electroporation (RE), irreversible electroporation (IRE), radio frequency electrical membrane disruption (RF-EMB), RF-EMB type manipulation, manipulation with ultrashort electrical pulses; manipulation using photodynamic therapy; mechanical or physical manipulation, such as manipulation using non-thermal shock waves, cavitation, or other mechanical or physical means that result in cell destruction; chemical manipulation, such as manipulation by injection of chemicals, e.g., alcohol, hypertonic saline, acetic acid, etc.; manipulation with biological agents, such as oncolytic viruses; or any combination thereof. Suitable irradiation or radiation methods include, but are not limited to, laser radiation (e.g., ultraviolet or near infrared laser radiation), X-ray radiation, or gamma radiation. Suitable radiation includes both solid and liquid. For example, the radiation source can be a radionuclide such as I-125, I-131, Yb-169, Ir-192 as a solid source, I-125 as a solid source, or other radionuclides that emit photons, beta particles, gamma radiation, or other therapeutic radiation. The radioactive material can also be a fluid made from any solution of the radionuclide, for example, a solution of I-125 or I-131, or the radioactive fluid can be made using a slurry of a suitable fluid containing small particles of a solid radionuclide, such as Au-198, Y-90, etc. Furthermore, the radionuclide can be incorporated into a gel or radioactive microspheres.

[0070] A pharmaceutical composition containing at least two checkpoint inhibitors and a cytokine drug or a cytotoxic or cytostatic chemotherapeutic drug can be combined with a method of ex vivo treated (e.g., engineered) cancer vaccine composition to provide systemic, durable, and reproducible cancer immunity. When used alone, engineering techniques such as cryotherapy and radiation therapy result in the inhibition of regulatory T cells, activation of effector T cells and B cells, and release of cancer-associated antigens (see Maia et al., "A comprehensive review of immunotherapies in prostate cancer." Crit Rev Oncol Hematol. 113:292-303 (2017), which is incorporated herein by reference in its entirety), effectively creating an adjuvant effect that stimulates cytotoxic T lymphocyte responses. For example, cells that have been rendered necrotic by freeze-thawing have immunostimulatory activity when injected in vivo to enhance T cell responses to co-injected antigens. See Shi et al., "Cell injury releases endogenous adjuvants that stimulate cytotoxic T cell responses." Proc Natl Acad Sci US A. 97(26):14590-14595 (2000), which is incorporated by reference in its entirety.

[0071] Injection of a combination of immunotherapeutic drugs and ex vivo engineered cancer compositions can enhance the immune response, likely by taking advantage of different mechanisms of action. The ex vivo engineering described herein affects at least two factors known to affect immune responses to engineered tumors: the first is the effect of the engineering process on protein structure, i.e., the antigenicity of tumor proteins; the second is the mechanism of cell death associated with the engineering modality.

[0072] Necrosis (immediate cell death) can, under certain conditions, rupture the cell membrane, releasing cell membrane fragments and various cellular contents from devitalized cells into the extracellular environment, which triggers costimulation of dendritic cells, leading to T-cell proliferation and activation. In contrast, apoptosis (programmed cell death) is another form of irreversible damage, in which the cell shrivels and dies over time (usually within a few days). Apoptosis leaves the cell intact, trapping the cellular contents and preventing costimulation. This exposure of the cellular contents and lack of costimulation suppresses immunological effects by preventing T-cell activation and proliferation. Thus, necrosis induces large immunogenic stimuli, whereas apoptosis usually induces little or no immune response.

[0073] Ex vivo manipulation of cancer cells and subsequent administration to soft tissue induces the body's defense and healing mechanisms to attempt to eliminate them. This creates an opportunity for the patient to essentially undergo autoimmune treatment against potential cancer neo-antigens (i.e., against the patient's own cancer) by utilizing the body's immune defense mechanisms (see Veenstra et al., "In situ immunization via non-surgical manipulation to prevent local and distant tumor recurrence" Oncoimmunology 4(3): e989762 (2015), which is incorporated herein by reference in its entirety). Furthermore, by stimulating the immune system against cancer cell antigens, the therapeutic approaches disclosed herein can (i) treat primary tumors; (ii) activate immune responses against cancer cell antigens; and (iii) induce immune system targeting of metastatic lesions.

[0074] Different types of manipulation methods can have different results on protein structure and cell death mechanisms. For example, thermal manipulation destroys structure due to denaturing proteins, which also destroys the collagen matrix underlying the tissue. This destruction of proteins and tissues makes a robust immune response less likely. Low temperature manipulation, for example, cryosurgical freezing, can denature proteins and destroy both protein and tissue structure. Irreversible electroporation (IRE) and non-thermal manipulation modalities, for example, RF-EMB, preserve structure and can be used to treat cancers in other areas such as the pancreas, central liver, and head and neck. IRE is a technique that applies an electric field to cells to increase the permeability of their membranes. The high voltage of IRE destroys the target cells, while neighboring cells are unaffected. Radiofrequency electrical membrane disruption (RF-EMB) is another non-thermal method that produces necrosis by completely electrically disrupting the cell membrane (see WO 2015 / 085162, which is incorporated herein by reference in its entirety). Under certain conditions, RF-EMB can also be used to deliver DNA plasmids. Reversible electroporation (RE) can also be used to deliver DNA plasmids. RE is similar to IRE, but the electricity applied to the target cells is below the electric field threshold of the target cells. Thus, the cells can recover when the electric field is removed, reconstruct the cell membrane, and continue cellular function. RE can be used as a tool for gene therapy, since the reversible element allows the entry of nucleic acids (e.g., DNA plasmids) into living cells. Exemplary ex vivo manipulation treatment methods and a brief description of their mechanisms are summarized in Table 1.

[0075] [Table 1]

[0076] For ex vivo treatment of cancer cells to generate cancer vaccines, any of the manipulation methods described herein can be used alone or in combination with one or more other manipulation methods. Two or more manipulation methods can be applied sequentially or simultaneously. In some cases, the combination of manipulation methods can have a synergistic effect on tissue. A non-limiting list of combinations includes, for example, thermal manipulation and RF-EMB, cryosurgical freezing and RF-EMB, IRE and RF-EMB, RE and RF-EMB, IRE and cryosurgical freezing, thermal manipulation and cryosurgical freezing, thermal manipulation and IRE, RE and IRE, thermal manipulation and RE, and any combination in which two or more methods are used. In some cases, the ex vivo manipulation method described herein uses a combination of RF-EMB and cryosurgical freezing techniques to produce RF-EMB-type lesions. This combination of manipulation methods can produce a synergistic effect on tissue. The synergistic effect can be that RF-EMB-type lesions are created that require less energy input than other means. As a result, for example, in liver tissue, in areas adjacent to sterile non-inflammatory coagulation necrosis, there are changes in liver architecture, including dilation of bile duct canaliculi, and characteristic diffuse changes in cytoplasmic organelles, including distortion of mitochondrial cristae and vacuolization of endoplasmic reticulum.

[0077] Those skilled in the art will understand that the manipulation methods described herein can be adapted according to the particular aspects of the ex vivo application and cancer, such as the size of the cancer specimen, the amount of non-cancerous tissue mixed in, etc. Those skilled in the art will understand that the parameters of each of the various manipulation methods are known and described in the art (see, e.g., Percutaneous Prostate Cryoablation (Edited by Onik, Rubinsky, Watson, and Ablin. Quality Medical Publishing, St Louis, MO, 1995), which is incorporated herein by reference in its entirety). As an example of the variability and diversity of operational parameters, the process of cryosurgical freezing includes adjustable variables such as the number of freeze-thaw cycles, the rate of freezing, and the thawing portion of the cycle to affect the outcome of the operation, e.g., the size of the cancer specimen and the immune response to the lesion. Similarly, the process of RF-EMB includes adjustable variables such as the strength, frequency, polarity, shape, duration, number, and interval of the electric field, which can also affect the outcome of the operation. The proximity of the tumor cells to the electric pulse dictates the strength and outcome of the RF-EMB for any particular cell. For example, as the electric field strength is decreased from the administration site (e.g., the probe), the cells furthest from the administration site are treated with a lower strength electric field and therefore may not be manipulated but rather reversibly electroporated.

[0078] In some cases, a first portion or all of the tumor cells or their derivatives are manipulated / irradiated ex vivo using a first manipulation / irradiation method, and a second portion or all of the tumor cells or their derivatives are manipulated / irradiated ex vivo using a second manipulation / irradiation method. The first and second manipulation / irradiation methods may be the same or different. The first and second portions of the tumor or cancer cells or their derivatives may be the same or different portions of the tumor or cancer cells or their derivatives.

[0079] In some embodiments, the ex vivo manipulation is performed using both RF-EMB and cryosurgical freezing. In some cases, the ex vivo manipulation / irradiation step is performed, at least in part, using cryosurgical freezing. As discussed above, cryosurgical freezing is a process that uses low temperatures to destroy tissue and cause necrosis through dehydration and ice formation. Cryosurgical freezing induces necroptotic and necrotic cancer cell death, releasing the full set of tumor-associated antigens (TAA) required for the induction of anti-tumor immunity. However, the "cryoimmunological effect" varies.

[0080] Cryosurgical freezing as used herein is significantly different from conventional cryosurgical freezing: whereas typical cryosurgery is used to create complete and immediate destruction of cancer (coagulation necrosis) and negative surgical margins, cryosurgical freezing herein is used to ensure intact presentation of cancer antigens to the immune system to induce the abscopal effect (both necroptosis and necrosis). Furthermore, one objective of the present invention is to take advantage of one of the three mechanisms of cell death of cryosurgical freezing (rupture of cell and nuclear membranes and cytoplasmic organelles) and minimize the other two mechanisms (denaturation of proteins and destruction of local microvasculature). This can be achieved by setting the freezing temperature at -40°C (temperature limit) and using ex vivo cryosurgical freezing of fresh cancerous tissue or cells. This combination therapy can reduce the systemic toxicity associated with conventional systemic cancer treatments and provide antigen-specific stimulation of the immune system, resulting in personalized tumor-targeted immune responses.

[0081] The cryosurgical freezing technique typically involves inserting a hollow needle (cryoprobe) into tissue, followed by supplying cryogen to the tip of the cryoprobe.Cryosurgical freezing can be performed using two or more cryoprobes.Cryosurgical freezing can also be performed using any of the multi-purpose probes described herein. Tissue temperature is reduced to a temperature that correlates with complete coagulation necrosis. Common cryosurgical freezing techniques involve the use of high pressure (e.g., about 80 psi) liquid nitrogen systems or high pressure (e.g., 3000-4500 psi) argon gas systems. Freezing of tissue is usually followed by its thawing (usually using helium gas or resistive heating), which results in the disruption of cell membranes and induces cell destruction. Cell destruction is further accelerated by repeated freeze-thaw cycles. In some cases, ex vivo cryosurgical freezing can include, consist essentially of, or consist of at least one freeze-thaw cycle. For example, cryosurgical freezing can include 1-4 freeze-thaw cycles. The freezing portion of the freeze-thaw cycle can be, for example, at least or about 30 seconds long. The freezing portion of the freeze-thaw cycle can range from about 30 seconds to about 15 minutes, about 30 seconds to about 12 minutes, about 30 seconds to about 10 minutes, or about 30 seconds to about 5 minutes. The thawing time can be at least or about 30 seconds long. For example, thawing times can range from about 30 seconds to about 15 minutes, from about 30 seconds to about 12 minutes, from about 30 seconds to about 10 minutes, or from 30 seconds to about 5 minutes. In some embodiments, the entire cryosurgical freezing process lasts for 30 minutes or less, 25 minutes or less, 20 minutes or less, 15 minutes or less, 10 minutes or less, 5 minutes or less, or 1 minute or less.

[0082] As discussed above, one advantage of the therapeutic compositions and treatment methods provided herein is the induction of immune-stimulated necrosis by ex vivo manipulation. In some embodiments, the ex vivo manipulation is performed by the insertion of a single probe (e.g., a cryosurgical needle probe); the manipulation procedure step lasts for 5 minutes or less to achieve the desired temperature and effect. The freezing portion of the freeze-thaw cycle can be performed at a temperature of, for example, about -30°C to about -196°C, e.g., about -30 to about -80°C, about -35 to about -45°C, about -35 to about -40°C, about -40 to about -50°C, about -40 to about -45°C, or about -40°C. As discussed above, one advantage of the therapeutic compositions and treatment methods provided herein is that cancer neoantigens are preserved by using minimal thermal manipulation. Cancer neoantigens are unique foreign proteins present on the inner and outer surfaces of cell membranes. These neoantigens are immunodeterminants that can impact immunotherapy treatment for early cancer recognition and destruction by antigen-specific T cells. See Desrichard et al., “Cancer neoantigens and applications for immunotherapy” Clin. Cancer Res. 22: 807-12 (2016), which is incorporated by reference in its entirety. Preservation of neoantigens is necessary for immune activation. The immune system can control cancer development and mediate its regression by generating and activating cancer neoantigen-specific dendritic cells and cytotoxic CD8+ T cells. This allows immune cells to recognize and target neoantigens on cancer cells at metastatic sites such as lymph nodes and bone.

[0083] Most cancer engineering methods induce necrosis but often fail to preserve the three-dimensional protein structure of cancer neoantigens (see Onik et al., "Electrical membrane breakdown (EMB): Preliminary findings of a new method of non-thermal tissue ablation" J. Clin. Exp. Pathol. 7:5-11 (2017), which is incorporated herein by reference in its entirety). This can be undesirable as it prevents immune cells from identifying neoantigens.

[0084] Thus, in some embodiments, ex vivo cryosurgical freezing is used at a relatively low temperature of about -40°C instead of the usual -80°C to preserve the three-dimensional structure of neoantigens. Cryosurgical freezing at about -40°C avoids or minimizes thermal destruction of protein neoantigen destruction while causing immune-stimulated necrosis by exceeding the threshold for cell death. See Larson et al., "In vivo interstitial temperature mapping of the human prostate during cryosurgery with correlation to histopathologic outcomes" Urology 55:547-52 (2000), which is incorporated by reference in its entirety.

[0085] In some cases, the ex vivo treatment of tumor or cancer cells or derivatives thereof further comprises, consists essentially of, or consists of administering a train of electric pulses. In some cases, the administration of the electric pulses occurs simultaneously with the manipulation. In some cases, the administration of the electric pulses occurs prior to the manipulation. In some cases, the administration of the electric pulses occurs after the manipulation. The electric pulses can be administered via a cryoprobe. In some cases, the train of electric pulses includes approximately 1-1000 pulses and / or includes a frequency of 100-500 kHz. In some cases, the train of electric pulses includes approximately 1-4000 pulses and / or includes a frequency of 100-500 kHz. In some cases, the train of electric pulses includes approximately 1-4000 pulses. In some cases, the train of electric pulses includes a frequency of 100-500 kHz. The electric pulses may be, for example, bipolar and / or have a momentary charge reversal. Ex vivo manipulation of at least a portion of the tumor or cancer cells, or derivatives thereof, can be performed using RF-EMB, for example, using a probe. The probe can be any probe disclosed herein. In some cases, the probe administers a train of electrical pulses, thereby causing the manipulation. In some cases, the train of electrical pulses includes approximately 1-1000 pulses. In some cases, the train of electrical pulses includes approximately 1-4000 pulses. In some cases, the electrical pulses include a frequency between 100-500 kHz. The electrical pulses can be bipolar. The electrical pulses can also have a momentary charge reversal.

[0086] In some cases, certain ex vivo manipulation methods can produce a unique tissue necrosis characterized by the destruction of cell membranes, including many thermal manipulations (e.g., cryosurgical freezing) and RF-EMB. When cell membranes are destroyed, intracellular components and cell membrane components are dispersed into the extracellular space, thereby enhancing immunological identification and response. This is different from other types of manipulation methods (e.g., IRE) that produce tissue apoptosis.

[0087] As used herein, the term "RF-EMB type manipulation" refers to any manipulation technique or combination of techniques that, when performed, produces essentially the same result as an RF-EMB manipulation. Such ex vivo manipulation provides at least one of the following advantages: the intracellular components and membrane antigens of the cells are not or only minimally denatured by the manipulation; the amount of exposed intracellular components and membrane antigens of the cells is sufficient to stimulate the immune system; and / or the amount of exposed intracellular components and membrane antigens of the cells does not or only minimally result in immune tolerance. In one embodiment, ex vivo manipulation preserves the structure of the cancer neo-antigens such that the antigens stimulate the immune system. In certain embodiments, the cancer vaccine is prepared by ex vivo treatment, including one or more of irradiation and manipulation (such as cryosurgical freezing), and exposed to at least one drug (a cytokine, a cytotoxic or cytostatic chemotherapeutic drug, or a combination thereof).

[0088] In some cases, the cancer vaccine is prepared by ex vivo procedures including UV irradiation and cryosurgical freezing, and exposed to low doses of at least one cytokine (e.g., GM-CSF) or cytotoxic or cytostatic chemotherapeutic agent (e.g., cyclophosphamide). In one embodiment, cryosurgical freezing is performed at a temperature ranging from about -35 to about -100°C, e.g., about -40°C. In one embodiment, cryosurgical freezing can be performed using a single probe with a total operation time of 10 minutes or less. In one embodiment ... GM-CSF (e.g., at a dose of 250 mg) or cyclophosphamide (e.g., at a dose of 200 to 300 mg / m 2 The drug is suspended in a low-dose solution of 0.01g / kg (at a dosage of 0.01g / kg). The therapeutic composition may or may not be administered to a subject in need in a single dose. The various components of the therapeutic composition may be administered to a subject simultaneously or sequentially. For example, the checkpoint inhibitor, the cytokine drug or the cytotoxic or cytostatic chemotherapeutic drug, and the ex vivo treated cancer vaccine may be administered to a subject simultaneously. Alternatively, the administration of each of the checkpoint inhibitors may be performed, for example, before, simultaneously, and / or after the administration of each of the cytokine drug or the cytotoxic or cytostatic chemotherapeutic drug, and / or the ex vivo treated cancer vaccine. The administration of each of the cytokine drug or the cytotoxic or cytostatic chemotherapeutic drug may be performed, for example, before, simultaneously, and / or after the administration of each of the checkpoint inhibitors and / or the ex vivo treated cancer vaccine. The administration of the ex vivo treated cancer vaccine may be performed, for example, before, simultaneously, and / or after the administration of each of the checkpoint inhibitors and / or the cytokine drug or the cytotoxic or cytostatic chemotherapeutic drug.

[0089] In one embodiment, a cytokine drug (e.g., GM-CSF) or a cytotoxic or cytostatic chemotherapeutic drug (e.g., cyclophosphamide) is administered during cancer vaccination. This can be accomplished, for example, by formulating the cancer vaccine in a suspension of the cytokine drug or cytotoxic or cytostatic chemotherapeutic drug. In one embodiment, a cytokine drug (e.g., GM-CSF) or a cytotoxic or cytostatic chemotherapeutic drug (e.g., cyclophosphamide) can be administered (e.g., subcutaneously or orally) following cancer vaccination. In one embodiment, a cytokine drug (e.g., GM-CSF) or a cytotoxic or cytostatic chemotherapeutic drug (e.g., cyclophosphamide) is administered at the time of cancer vaccination, and an additional administration of the same or a different cytokine drug (e.g., subcutaneous) or cytotoxic or cytostatic chemotherapeutic drug (e.g., oral) is administered following cancer vaccination.

[0090] Regulatory T lymphocytes (T Reg ) population is sensitive to subclinical doses of the chemotherapy drug cyclophosphamide. Low doses of cyclophosphamide suppress circulating T Reg It has been shown that sub-cytotoxic doses of cyclophosphamide or GM-CSF do not result in immune enhancement. Cyclophosphamide or GM-CSF have been used as immune enhancers in several clinical trials of active specific immunotherapy (e.g., cancer vaccine) trials for advanced cancer, but at low doses (typically 250-300 mg / m 2 ) are typically administered 3-4 days prior to the vaccine regimen. Here, administration of cyclophosphamide or GM-CSF at the time of cancer vaccination can promote recruitment of T cells and other immune stimulatory cells at the time of vaccination; additional administration after, during, or following the vaccine regimen can promote recruitment of T cells and other immune stimulatory cells.Reg To address any "rebound" of the vaccine, a continued but limited regimen can be introduced after the vaccine therapy.

[0091] In certain embodiments, the cancer vaccine is prepared by ex vivo UV irradiation of cancer tissue samples, exposure to low dose cyclophosphamide or GM-CSF, and temperature-restricted freezing surgical freezing to create personalized vaccine lysates of necrotic and necroptotic cells and intact tumor-associated antigens, which when injected (e.g., intradermally or intratumorally) can prime the immune system to target cancer cells. In one embodiment, after injection of such a cancer vaccine lysate, two immunotherapeutic drugs (e.g., PD-1 inhibitory monoclonal antibody nivolumab (or pembrolizumab) and anti-CTLA-4 monoclonal antibody ipilimumab) are immediately injected sequentially, preferably at the same site (e.g., intradermally or intratumorally). Furthermore, low dose GM-CSF may be administered thereafter for a period of time (e.g., 6 weeks) to prolong the immune response; oral low dose cyclophosphamide may be administered thereafter for a period of time (e.g., 6 weeks) to prolong the immune response.

[0092] One exemplary therapeutic composition and treatment method for treating metastatic solid cancer involves the following three therapeutic components and treatment regimens: (a) ex vivo treatment of fresh cancer cells from a biopsy or needle aspirate with UV irradiation, low dose cyclophosphamide or GM-CSF, and temperature restricted cryosurgical freezing to: a. Creating personalized vaccine lysates of necrotic and necrotic cancer cells while minimizing the destruction of cancer antigens, thereby ensuring a good immune response; b. Providing a full set of tumor-associated antigens and associated cytokines and other immune stimulants released by cancer cells; and c. Initiating the process of suppressing inhibitory signals from regulatory T cells; Following this, (b) Sequential intradermal or intratumoral injection of a cancer lysate vaccine (autologous) and two checkpoint inhibitors with complementary roles targeting different pathways to produce synergistic or enhanced anti-cancer effects, such as: a. CTLA-4 inhibitors to suppress inhibitory signals from regulatory T cells and prolong anti-tumor cytotoxic T cell responses; and b. PD-1 inhibitors to reverse T cell exhaustion and enhance antitumor activity by "unmasking" cancer neoantigens, thereby exposing cancer cell antigens to dendritic cells and cytotoxic (killer) T cells, while promoting the antitumor activity of cytotoxic T cells; Following this, (c) Subsequent administration of low-dose cyclophosphamide (eg, orally) or GM-CSF (eg, subcutaneously) to prolong immune cell mobilization.

[0093] Without being bound by theory, the cancer neo-antigens and immune stimulants, e.g., cytokines, contained in the cancer lysate vaccine may be available in the dermis at the site of skin injection, which is a rich source of immature dendritic cells and other immune cells, or in the tumor. Immature antigen-presenting cells (APCs) (e.g., dendritic cells), T cells, and other immune cells resident at the injection site then take up the neo-antigens and are activated to recognize the cancer-specific antigen protein. The activated dendritic cells are drained to nearby lymph nodes, where they activate T cells that target the cancer-specific neo-antigens and recruit other cytotoxic T lymphocytes to destroy cancer cells throughout the body that carry the correct antigen epitope, thereby stimulating the abscopal (bystander) effect. In this way, dendritic cells can be combined with cytotoxic T cells to initiate a cell-mediated systemic anti-tumor immune response. Regulatory T lymphocytes, which are involved in dampening or terminating cell-mediated antigen-specific immune responses, can be selectively depleted by anti-CTLA-4 monoclonal antibodies and low doses of cyclophosphamide or GM-CSF. Intradermal or intratumoral injection of drugs induces fewer side effects than systemic therapy.

[0094] The therapeutic composition may further comprise a therapeutically effective amount of one or more of the therapeutic and / or biological agents known in the art to be effective in treating cancer, i.e., anti-cancer agents, or agents known in the art to be effective in stimulating the immune system, i.e., immunostimulatory or immunomodulatory agents. Such therapeutic compositions may be used to treat cancer as described herein. The therapeutic composition may also include one or more therapeutically effective amounts of nucleic acid medicines. The nucleic acid medicine may be, for example, DNA, DNA plasmid, nDNA, mtDNA, gDNA, RNA, siRNA, miRNA, mRNA, piRNA, antisense RNA, snRNA, snoRNA, vRNA, etc. For example, the nucleic acid medicine may be a DNA plasmid. In some cases, the DNA plasmid may comprise, consist essentially of, or consist of a nucleotide sequence encoding a gene selected from the group consisting of GM-CSF, IL-12, IL-6, IL-4, IL-12, TNF, IFNy, IFNa, and / or combinations thereof. The nucleic acid medicine may have clinical utility, for example, in enhancing the therapeutic effect of a cell or providing a therapeutic agent to a patient. In other cases, the nucleic acid medicine may function as a marker or resistance gene. The nucleotide sequence may encode a gene that may or may not be secreted from a cell. The nucleic acid medicine may encode a gene and a promoter sequence for increasing expression of the gene.

[0095] The therapeutic composition may also include one or more therapeutically effective amounts of a toll-like receptor (TLR). The toll-like receptor may be selected from the group consisting of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12, TLR13, and / or combinations thereof. In some embodiments, the toll-like receptor may be TLR3. In some embodiments, the therapeutic composition may include a first TLR and a second TLR. In some cases, the first and second TLR are the same; in other cases, they are different. The TLR may be administered at a concentration of about 0.01 to about 5 mg / m 2 The compound may be delivered at a concentration of One of skill in the art will appreciate that therapeutic compositions may be adapted according to individual aspects of the cancer and / or subject, e.g., tumor size, tumor location, subject, clinical evidence of drug response, etc.

[0096] The therapeutic composition may include a delivery agent or a pharma- ceutically acceptable carrier or excipient. As used herein, the term "pharma-ceutically acceptable carrier or excipient" includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., that are compatible with pharmaceutical administration. These carriers or excipients do not impair the pharmacological activity and are non-toxic when administered in a dose sufficient to deliver a therapeutic amount of the composition. Supplementary active compounds may also be incorporated into the formulation for the therapeutic composition containing the antibody or antigen-binding fragment thereof described herein. Therapeutic compositions can be formulated for a variety of routes of administration, including, but not limited to, oral or parenteral, e.g., intravenous, intramuscular, subcutaneous, intratumoral, intraorbital, intracapsular, intraperitoneal, intrarectal, intracapsular, intravascular, intradermal (e.g., through the skin or soft tissue); by passive or facilitated absorption through the skin, e.g., using a skin patch or transdermal iontophoresis, respectively; by intravenous or intraarterial administration to the site of a pathological condition, e.g., into a blood vessel supplying a tumor; or combinations thereof.

[0097] Methods for formulating suitable therapeutic / pharmaceutical compositions are known in the art (e.g., Troy, "Remington: The Science and Practice of Pharmacy" (21st Ed., Lippincott Williams & Wilkins, 2006); Willig, "Drugs and the Pharmaceutical Sciences: a Series of Textbooks and Monographs" (M. Dekker, 1975), both of which are incorporated herein by reference in their entirety. For example, solutions or suspensions used for parenteral, intradermal, or subcutaneous application can contain the following components: a sterile diluent, such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents, such as benzyl alcohol or methylparabens; antioxidants, such as ascorbic acid or sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, citrates, or phosphates, and agents for adjusting tonicity, such as sodium chloride or dextrose. The pH value can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. Parenteral formulations can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0098] In some embodiments, the therapeutic composition and its components can be formulated for intradermal or subcutaneous administration and injected intradermally or subcutaneously. For example, the components of the therapeutic composition can be formulated for intradermal administration, such as through the skin or soft tissue, and injected intradermally through the skin or soft tissue. The therapeutic composition or various components of the therapeutic composition (e.g., checkpoint inhibitors, cytokine drugs or cytotoxic or cytostatic chemotherapeutic drugs, cancer vaccines, nucleic acid medicines, and / or combinations thereof) can be formulated for soft tissue delivery. For example, the therapeutic composition or various components of the therapeutic composition can be delivered intradermally via an injection device. The injection device can be part of a probe. The probes described herein can be configured for various manipulation methods. Furthermore, the probes can be configured to combine the methods described herein, for example, a cryoprobe can be configured to administer electrical pulses, cryoagents, chemical or biological manipulation agents, and / or drug compositions.

[0099] The combination of at least two checkpoint inhibitors and a cytokine drug or a cytotoxic or cytostatic chemotherapeutic agent administered to benign or cancerous soft tissue produces fewer adverse side effects and / or immune-related adverse events than the combination of two checkpoint inhibitors administered intravenously (without a cytokine drug or a cytotoxic or cytostatic chemotherapeutic agent). The combination of these three or more immunostimulatory drugs delivered to benign or cancerous soft tissue can be sufficient to induce a systemic CD4+ and CD8+ T cell-mediated antitumor immune response that can eradicate distant metastatic tumor sites, including the central nervous system of mice. This local combination strategy can also generate a better CD8+ memory antitumor immune response to prevent late-stage tumor recurrence in contrast to the systemic delivery of antibodies.

[0100] Skin is a preferred portal for the delivery of anti-cancer vaccines, serving as a convenient injection site that allows cancer cell lysates and adjuvants to be safely administered without the need for and risks associated with intravenous injection or surgical intratumoral and drug manipulation. See Patel et al., "Next generation approaches for tumor vaccination." Chin Clin Oncol 6:19 (2017), which is incorporated herein by reference in its entirety. Intradermal drug injections can reduce systemic toxicity and cause fewer side effects by isolating the drug in the skin microenvironment (see Marabelle et al., "Intratumoral Immunization: A New Paradigm for Cancer Therapy" Clin. Cancer Res. 20(7): 1747-56 (2014), which is incorporated herein by reference in its entirety). Intradermal injection delivers antigen directly to the skin, an anatomical site that contains a dense and diverse population of immune cells, including antigen-presenting dendritic cells and effector T cells, and is more likely to be immunogenic than intramuscular injection of a given amount of antigen (see Kenney et al., "Dose Sparing with Intradermal Injection of Influenza Vaccine" N Eng J Med 351, 2295-301 (2004); Patel et al., "Next generation approaches for tumor vaccination." Chin Clin Oncol 6:19 (2017), which are incorporated by reference in their entireties). For example, the Bacillus Calmette-Guerin vaccine is routinely injected intradermally.

[0101] Intradermal administration of cancer lysate products enhances uptake by five subsets of lymph node-migrating skin dendritic cells (DCs) (Langerhans cells, Langerin, CD103-, CD103+, CD11b+, and CD11b-) (see Vardam et al., "Langerhans Cells Orchestrate TFH-Dependent Humoral Immunity" J Invest Dermatol 137:1826-1828 (2017), which is incorporated by reference in its entirety). Following recruitment and activation, these DCs process tumor-associated antigens (TAA) into peptides that bind to major histocompatibility complex 1 (MHC-I) for CD8+ T cells or MHC-II for CD4+ T cells, and subsequently migrate from the skin to draining lymph nodes to cross-present TAAs to their cognate T cells, stimulating the priming and expansion of effector T cells and the induction of differentiation of follicular center T cells. See Ruben et al., "In situ loading of skin dendritic cells with apoptotic bleb-derived antigens for the induction of tumor-directed immunity" Oncoimmunol 3; 7: e946360 (2014), which is incorporated by reference in its entirety.Thus, immune responses in lymph nodes are triggered by a combination of antigen recognition (presentation of TAAs in the context of MHC molecules and T cell receptors on T lymphocytes by DCs) and immune activation (generation of type 1 helper T cell responses that generate cytotoxic T lymphocytes, as well as type 2 helper T cell responses that result in the production of antigen-specific antibodies to induce cell-mediated cell death) (see Dillman et al., “Randomized phase II trial of autologous dendritic cell vaccines versus autologous tumor cell vaccines in metastatic melanoma: 5-year follow up and additional analyses.” J Immunother Cancer 6:19 (2014), which is incorporated herein by reference in its entirety). Subsequently, activated antigen-specific T cells, likely guided by chemokine gradients and other inflammatory cues, migrate to the cancer site, where they recognize tumor cells through interactions between T cell receptors on T lymphocytes and antigens in the presence of MHC molecules on the cancer cells. Cytotoxic T cells release enzymes such as perforin and granzyme B, resulting in cell necrosis, and this lytic effect on tumor cells triggers a positive feedback loop that causes more antigens to be released, restarting the "cancer-immune cycle." The release of other antigens also leads to an expanded response, referred to as "antigen spreading." See Patel et al., "Next generation approaches for tumor vaccination." Chin Clin Oncol 6:19 (2017), which is incorporated herein by reference in its entirety.

[0102] Combined with dendritic cell activation, intradermal cancer vaccination also targets the 1 million / cm2 population of normal human skin that contains two subsets of long-lived memory CD8C T cells that confer long-lasting protective adaptive and innate immunity against foreign antigens (pathogens, TAAs, etc.). 2It also stimulates many of the T cells in the bloodstream: (1) resident memory cells (Trm) that serve as sentinels in numerous nonlymphoid tissues (including the skin, gastrointestinal tract, brain, and lungs) that constitutively express CD69 and CD103; and (2) effector memory cells (Tem) that circulate between the blood and nonlymphoid tissues and express tissue-homing receptors. A third subset of memory CD8C T cells, central memory cells (Tcm), circulate between the blood and lymphoid tissues and express CD62L and CCR7. Upon antigen rechallenge, all of these T cells are activated and proliferate, but Trm cells provide the first and strongest local defense, secreting abundant effector cytokines and chemokines (e.g., IFN-g, granzyme B) that lead to targeted engagement of Tcm, Tems, B cells, and other immune cells. The intradermal route of vaccination can stimulate the production of antigen-specific Trm cells that accumulate locally and in the unvaccinated skin. See Galvez-Cancino et al., "Vaccination-induced skin-resident memory CD8(+) T cells mediate strong protection against cutaneous melanoma" Oncoimmunology 7:e1442163 (2018), which is incorporated by reference in its entirety.

[0103] Cutaneous injection of immunostimulatory drugs can reduce systemic toxicity and cause fewer side effects by preventing them from circulating in high concentrations in the blood. This delivery route also results in a much higher concentration of the immunostimulatory product in the microenvironment than in the case of systemic injection, thereby enhancing better efficacy. On the other hand, this delivery route can also reduce the amount of administered composition required for the treatment to be effective. For example, a cancer lysate vaccine applied to the skin produces a strong immunotherapeutic response that is comparable to or better than other vaccinations by needle, such as intramuscular injection, despite a 5-10 times smaller dose of antigen. See Depelsenaire et al., “Colocalization of cell death with antigen deposition in skin enhances vaccine immunogenicity” J Invest Dermatol 134:2361-2370 (2014), which is incorporated by reference in its entirety.

[0104] Preclinical intradermal (ID) delivery of cancer vaccines has proven safe and effective in a number of diverse cancers. Chen et al. found that mice injected with a whole-cell cancer lysate vaccine combined with GM-CSF stimulated and activated intradermal DCs, resulting in cancer phagocytosis, presentation of cancer-specific tumor antigens, DC migration, and stimulation of other immune cells. See Chen et al., “Leveraging Engineering of Cells for Drug Delivery” Acc Chem Res 51:668-77 (2018), which is incorporated by reference in its entirety. In one mouse model, ID administration of an antigen-specific Trm cell-stimulating vaccine resulted in suppression of invasion and melanoma growth, which was independent of circulating CD83 T cells. See Galvez-Cancino et al., "Vaccination-induced skin-resident memory CD8(+) T cells mediate strong protection against cutaneous melanoma" Oncoimmunology 7: e1442163 (2018), which is incorporated by reference in its entirety. Injection of mice with breast cancer cell lysates combined with heat shock protein fragments and diphtheria toxin resulted in good humoral and cellular immune responses and protective antitumor immunity. See Wang, et al., "Protective antitumor immunity induced by tumor cell lysates conjugated with diphtheria toxin and adjuvant epitope in mouse breast tumor models" Chin J Cancer 31:295-305 (2012), which is incorporated by reference in its entirety. In one mouse model, half of all colon cancers were eradicated by ID immunization with a cancer vaccine composed of baculovirus, CT26 colon cancer lysate, and cytotoxic T cell epitope peptides; the antitumor effect correlated with tumor antigen-specific responses of CD8C T cells.See Kawahara, et al. "A tumor lysate is an effective vaccine antigen for the stimulation of CD4(+) T-cell function and subsequent induction of antitumor immunity mediated by CD8(+) T cells" Cancer Biol Ther 16:1616-25 (2015), which is incorporated by reference in its entirety. ID injection of autologous B-cell lymphoma cell membrane fragments in combination with IL-2 and GM-CSF into dogs produced specific cell-mediated immunity and delayed-type hypersensitivity responses without significant toxicity. See Henson et al., "Immunotherapy with autologous tumour antigen-coated microbeads (large multivalent immunogen), IL-2 and GM-CSF in dogs with spontaneous B-cell lymphoma" Vet Comp Oncol 9:95-105 (2011), which is incorporated by reference in its entirety. Vaccination with irradiated GM-CSF-producing cancer cells improved survival of mice bearing tumors implanted in the brain. See Sampson et al., "Immunotherapy for Brain Tumors" J Clin Oncol 356:2450-56 (2017), which is incorporated by reference in its entirety. Vaccination with irradiated Lewis lung carcinoma cell lines transfected to produce IL-18 and GMCSF produced anti-cancer responses in mouse models, including proliferation of cytotoxic T cells and prolonged survival.See Tian et al., "Cellular immunotherapy using irradiated lung cancer cell vaccine co-expressing GM-CSF and IL-18 can induce significant antitumor effects" BMC Cancer 14:48 (2014), which is incorporated by reference in its entirety.

[0105] Similar to preclinical findings, treatment with ID cancer vaccination in patients has also proven safe, well tolerated, and effective in multiple cancers. Mehrotra et al. delivered a combination of Poly-ICLC and an autologous DC vaccine pulsed with HLA-A2 restricted peptides hTERT, CEA, and survivin intradermally to eight patients with pancreatic cancer. See Mehrotra et al., “Vaccination with poly(IC:LC) and peptide-pulsed autologous dendritic cells in patients with pancreatic cancer” J Hematol Oncol 10:82 (2017), which is incorporated by reference in its entirety. Treatment was well tolerated, with common symptoms being fatigue and / or self-limited flu-like symptoms. Four patients had stable disease, while four patients progressed; median overall survival was 7.7 months. MHC class I tetramer analysis before and after vaccination revealed effective generation of antigen-specific T cells in three patients with stable disease. Treatment of patients with metastatic cancer at various sites with intradermal injection of dendritic cells loaded with autologous cancer cell lysates was well tolerated with limited grade 1 and 2 adverse events such as fever, asthenia, and pain at the injection site. See Alfaro et al., “Pilot clinical trial of type 1 dendritic cells loaded with autologous tumor lysates combined with GM-CSF, pegylated IFN, and cyclophosphamide for metastatic cancer patients” J Immunol 187:6130-42 (2011), which is incorporated herein by reference in its entirety. Treatment with intradermal polyvalent vaccination with irradiated whole cells from multiple melanoma cell lines combined with BCG resulted in a 26% response over 19 months in high-stage patients with minimal adverse events.See Vilella et al., "Treatment of patients with progressive unresectable metastatic melanoma with a heterologous polyvalent melanoma whole cell vaccine" Int J Cancer 106:626-31 (2003), which is incorporated by reference in its entirety. Use of an autologous hapten-modified melanoma vaccine resulted in regression of pulmonary metastases. See Berd et al., "Induction of cell-mediated immunity to autologous melanoma cells and regression of metastases after treatment with a melanoma cell vaccine preceded by cyclophosphamide" Cancer Res 46: 2572-77 (1986), which is incorporated by reference in its entirety. Treatment with polyvalent vaccines in high-stage melanoma improved 5-year survival rates compared with other treatments that involved complete resection of metastases (often to multiple sites) prior to vaccine therapy. See Vilella et al., "Treatment of patients with progressive unresectable metastatic melanoma with a heterologous polyvalent melanoma whole cell vaccine" Int J Cancer 106:626-31 (2003), which is incorporated by reference in its entirety.

[0106] In one embodiment, the therapeutic composition is formulated for intratumoral administration and can be injected into the tumor. Intratumoral injection of immune stimulating drugs in humans can significantly reduce the incidence of adverse events of systemic immunotherapy when compared with intravenous injection. Systemic administration of combinations of immunotherapy drugs such as PD1 / PD-L1 (e.g., Keytruda, Opdivo) and anti-CTLA-4 (e.g., Yervoy) has a high incidence of adverse events. Intratumoral injection allows the use of combinations of drugs with different mechanisms of action, avoiding many adverse events. Studies in animal models have shown that local costimulation using agonist antibodies can produce systemic antitumor effects and induce T cell-dependent antitumor immune memory with less toxicity than expected from systemic treatment. Intratumoral or juxtatumoral injection helps produce systemic tumor effects. For example, intratumoral or juxtatumoral injection increases accumulation in tumor-draining lymph nodes and reduces Cmax (maximum serum concentration) compared to systemic administration.

[0107] Therapeutic compositions suitable for injection can include sterile aqueous solutions (where water soluble), dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, sterile purified water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). The composition should be sterile and fluid to the extent that easy syringability exists. Therapeutic compositions should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0108] Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is desirable to include isotonic agents, such as sugars, polyalcohols, such as mannitol, sorbitol, and sodium chloride, in the therapeutic composition. Prolonged absorption of the injectable compositions can be brought about by including in the therapeutic composition an agent that delays absorption, such as aluminum monostearate and gelatin.

[0109] Sterile injectable solutions can be prepared by incorporating the required amount of active compound into a suitable solvent with one or a combination of the above-listed components as required, followed by filtration sterilization.Generally, dispersions are prepared by incorporating active compound into a sterile vehicle that contains a basic dispersion medium and other required components from the above-listed components.For sterile powders for preparing sterile injectable solutions, the preferred preparation method is vacuum drying and freeze-drying, which obtains a powder of active ingredient plus any additional desired components from the previously sterilized filtered solution.In some embodiments, therapeutic compositions can be prepared with carriers that protect the active compound against rapid elimination from the body, such as controlled release formulations, including implants and microencapsulated delivery systems.

[0110] The therapeutic compositions can be included in a container, pack, cartridge, or dispenser together with instructions for administration. The terms "administering" or "administration" with respect to a method include not only the act of prescribing and / or ordering from a medical professional, but also the act of a patient receiving the prescription and / or ordering, as well as the act of a patient actually receiving the composition or treatment step. Another aspect of the present invention provides a method for treating a tumor or cancer in a subject in need thereof. The method can comprise, consist essentially of, or consist of administering to a subject in need thereof a therapeutic composition comprising: i) at least two immune checkpoint inhibitors; ii) at least one drug selected from a cytokine, a cytotoxic or cytostatic chemotherapeutic agent, and combinations thereof; and iii) a cancer vaccine prepared from tumor or cancer cells, or derivatives thereof, prepared by an ex vivo treatment that produces necrotic and / or necroptotic cancer cells while minimizing destruction of cancer antigens. The composition may contain a pharmaceutically acceptable carrier. For example, the administered composition may be a therapeutic composition described herein.

[0111] All the above embodiments relating to aspects of the therapeutic composition, including suitable immune checkpoint inhibitors, suitable cytokine drugs or suitable cytotoxic or cytostatic chemotherapeutic drugs, cancer vaccines and their preparations by suitable ex vivo treatment methods, optionally suitable pharma- ceutically acceptable carriers, effective amounts thereof for treating tumors or cancer, as well as formulations of the therapeutic composition for various routes of administration, are applicable to this aspect of the method of treating tumor or cancer in a subject. In some embodiments, the method further comprises producing a cancer vaccine by ex vivo treatment of tumor or cancer cells, or derivatives thereof. The ex vivo treatment of tumor or cancer cells, or derivatives thereof for the preparation of a cancer vaccine is described herein in the aspect of a therapeutic composition and is applicable to this aspect of the method of treating a tumor or cancer. In one embodiment, the method further comprises dissociating cells or cellular components from the tumor or cancer specimen ex vivo using mechanical, enzymatic, or other methods known to one of skill in the art, and centrifuging the cells and resuspending them in a sterile fluid, such as buffered saline, prior to further manipulation, such as manipulation.

[0112] In some cases, the method comprises, consists essentially of, or consists of administering the therapeutic composition intradermally, intratumorally to the patient. In some cases, the method comprises, consists essentially of, or consists of administering the therapeutic composition to the skin or soft tissue of the patient. Administration can be performed by using an injection device.

[0113] In some embodiments, the method further comprises: i) detecting one or more of the following: CD137, CD134, PD-1, KIR, LAG-3, PD-L1, PDL2, CTLA-4, B7.1, B7.2, B7-DC, B7-H1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, B7-H7, BTLA, LIGHT, HVEM, GAL9, TIM-3, TIGHT, VISTA, 2B4, CGEN-15049, CHK, or any combination thereof; The method comprises, consists essentially of, or consists of administering to a patient, in an amount effective to treat a tumor or cancer, a composition comprising at least two different immune checkpoint inhibitors, the immune checkpoint inhibitors being selected from the group consisting of 1, CHK2, A2aR, TGF-β, PI3Kγ, GITR, ICOS, IDO, TLR, IL-2R, IL-10, PVRIG, CCRY, OX-40, CD160, CD20, CD52, CD47, CD73, CD27-CD70, and CD40, ii) at least one cytokine drug or cytotoxic or cytostatic chemotherapeutic drug, and iii) a cancer vaccine prepared from tumor or cancer cells, or derivatives thereof, prepared by an ex vivo process that generates necrotic and / or necroptotic cancer cells while minimizing destruction of cancer antigens. In some embodiments, the at least two checkpoint inhibitors comprise a CTLA-4 inhibitor, a PD-1 inhibitor. In some embodiments, the at least two checkpoint inhibitors comprise a CTLA-4 inhibitor and a PD-L1 inhibitor.

[0114] In some embodiments, the method comprises the step of combining i) at least two immune checkpoint inhibitors; ii) at least one cytokine selected from the group consisting of erythropoietin, G-CSF, GM-CSF, IL-2, IL-4, IL-6, IL-12, TNF, interferons, e.g., INF-α-2a, INF-α-2b, INF-β, INF-γ, and combinations thereof; or asparaginase, bleomycin, busulfan, carboplatin, cetuximab, cisplatin, cyclophosphamide, BCG, chloramphenicol, colchicine, cyclosporine, dacarbazine, doxorubicin, etoposide, fludarabine, gemcitabine, ifosfamide, irinotecan, lomustine, The method comprises, consists essentially of, or consists of administering to a patient, in an amount effective to treat a tumor or cancer, a composition comprising at least one cytotoxic or cytostatic chemotherapeutic agent selected from the group consisting of melphalan, methotrexate, mitomycin, mitoxantrone, paclitaxel, procarbazine, rituximab, temozolomide, thitepa, vinblastine, vincristine, zidovudine, and combinations thereof; and iii) a cancer vaccine prepared from tumor or cancer cells, or derivatives thereof, prepared by ex vivo treatments that generate necrotic and / or necroptotic cancer cells while minimizing destruction of cancer antigens. In one embodiment, the composition comprises at least one cytokine, at least one of which is GM-CSF. In one embodiment, the composition comprises at least one cytotoxic or cytostatic chemotherapeutic agent, at least one of which is cyclophosphamide.

[0115] As discussed above, the therapeutic composition may or may not be administered to a subject in need thereof in a single dose. Methods of administration of the therapeutic composition and / or various components of the therapeutic composition are described herein in the context of the therapeutic composition and are applicable to this aspect of the method of treating a tumor or cancer.

[0116] In one embodiment, the method comprises simultaneously administering to a subject a checkpoint inhibitor, a cytokine, or a cytotoxic or cytostatic chemotherapeutic agent, and an ex vivo administered cancer vaccine. In one embodiment, the method comprises: i) administering each of the checkpoint inhibitors; ii) administering prior to, concomitantly with, and / or subsequent to i), a cytokine agent or a cytotoxic or cytostatic chemotherapeutic agent, respectively; iii) administering an ex vivo treated cancer vaccine prior to, simultaneously with, and / or after i) or ii); Includes.

[0117] In one embodiment, the method includes administering a cytokine drug (e.g., GM-CSF) or a cytotoxic or cytostatic chemotherapeutic drug (e.g., cyclophosphamide) during the cancer vaccination. This can be accomplished, for example, by formulating the cancer vaccine in a suspension of the cytokine drug or cytotoxic or cytostatic chemotherapeutic drug. In one embodiment, the method includes administering a cytotoxic or cytostatic chemotherapeutic drug (e.g., cyclophosphamide, oral) or administering a cytokine drug (e.g., GM-CSF) after cancer vaccination. In one embodiment, the method includes administering a cytokine drug (e.g., GM-CSF) at the time of cancer vaccination and administering the same or a different cytokine drug (e.g., intradermally) after cancer vaccination. In one embodiment, the method includes administering a cytotoxic or cytostatic chemotherapeutic agent (e.g., cyclophosphamide) at the time of cancer vaccination, and administering the same or a different cytotoxic or cytostatic chemotherapeutic agent after cancer vaccination.

[0118] In some cases, the method further includes administering a therapeutically effective amount of the nucleic acid pharmaceutical to the tumor or cancer. In some cases, the method further comprises administering to the subject a cytokine drug (e.g., GM-CSF) or a cytotoxic or cytostatic chemotherapeutic drug (e.g., cyclophosphamide) after administration of the therapeutic composition. The cytokine drug or cytotoxic or cytostatic chemotherapeutic drug can be administered subcutaneously. In certain embodiments, the method includes: 1) preparing a cancer vaccine by ex vivo UV irradiation of a cancer tissue sample, exposure to low-dose GM-CSF or cyclophosphamide, and temperature restricted freezing surgical freezing; 2) injecting the cancer vaccine lysate into a subject (e.g., intradermally); 3) injecting two immunotherapeutic agents (e.g., pembrolizumab (or nivolumab), a PD-1 inhibitor monoclonal antibody, and ipilimumab, an anti-CTLA-4 monoclonal antibody) into a subject, preferably at the same site (e.g., intradermally or intratumorally); and 4) administering low-dose GM-CSF (e.g., subcutaneously) or low-dose cyclophosphamide (e.g., orally) to the subject for a period of time (e.g., 6 weeks).

[0119] As discussed above, administration of the therapeutic composition or components thereof can be performed via a variety of routes, including, but not limited to, oral or parenteral, e.g., intravenous, intramuscular, subcutaneous, intratumoral, intraorbital, intracapsular, intraperitoneal, intrarectal, intracapsular, intravascular, intradermal; administration by passive or enhanced absorption through the skin, e.g., using a skin patch, transdermal iontophoresis, or coated microneedles, respectively; intravenous or intraarterial administration to the site of the pathological condition, e.g., into the blood vessels supplying a tumor; or combinations thereof.

[0120] The therapeutic composition or its components can be administered in effective amounts, dosages, and for periods of time necessary to achieve the desired results. An effective amount can be administered in one or more administrations, applications, or dosages. The therapeutically effective amount (i.e., effective dosage) of the therapeutic composition will depend on the therapeutic composition selected. The composition can be administered from one or more times per day to one or more times per week, including once every other day. One of skill in the art will appreciate that certain factors, including, but not limited to, the severity of the disease or disorder, previous treatments, the overall health and / or age of the subject, and other diseases present, can affect the dosage and timing required to effectively treat a subject. Furthermore, treatment of a subject with a therapeutically effective amount of a therapeutic composition described herein can include a single treatment or a series of treatments. In some cases, the therapeutic composition is administered to the patient using an injection device. The injection device may include multiple tines or a single tine. The composition may be administered using a probe (which serves a variety of purposes) as described herein. In some embodiments, the therapeutic compositions described herein may be administered in one or more doses, which may be the same or different administration methods as those described herein, e.g., subcutaneous, intravenous, intramuscular, intratumoral, intradermal, or any combination thereof.

[0121] In some embodiments, the first composition (or a component thereof) is administered intratumorally and the second composition (or a component thereof) is administered subcutaneously. In some embodiments, the first and second compositions are administered simultaneously, sequentially, or in a series of treatments. In some embodiments, the first and second compositions are the same, different, or partially the same. In some embodiments, the treatment method comprises two or more administrations. In some embodiments, the first administration is an intradermal administration of at least two checkpoint inhibitors (e.g., a PD-1 inhibitor and a CTLA-4 inhibitor), at least one cytokine or a cytotoxic or cytostatic chemotherapeutic agent, and a cancer cell vaccine following ex vivo manipulation.

[0122] Dosage regimens can be adjusted to provide the desired therapeutic response. For example, several divided doses can be administered daily, or the dose can be proportionally reduced as indicated by the exigencies of the therapeutic situation. Those skilled in the art will be aware of suitable dosages and dosing regimens for administering the novel monoclonal antibodies or antigen-binding fragments thereof disclosed herein to a subject. See, for example, Physicians' Desk Reference 2008 (62nd Ed., Thomson Reuters, 2008), which is incorporated herein by reference in its entirety. For example, dosage, toxicity, and therapeutic efficacy of therapeutic compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, for example, to determine the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, which can be expressed as the LD50 / ED50 ratio. Compounds that exhibit high therapeutic indices are preferred. Compounds that exhibit toxic side effects may be used, but care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to non-infected cells, thereby reducing side effects.

[0123] Data obtained from cell culture assays and animal studies can be used in formulating a range of dosages for use in humans. The dosage of such compounds is preferably within a range of circulating concentrations that includes the ED50 with little or no toxicity. Dosages can vary within this range depending on the dosage form used and the route of administration utilized. For any compound used in the treatment method, a therapeutically effective dose can be estimated initially from cell culture assays. Dosages can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves half-maximal inhibition of symptoms) determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

[0124] The composition can be administered in a single dose, or in more than two doses. The dosage ranges for immune checkpoint inhibitors, cytokine drugs, or cytotoxic or cytostatic chemotherapeutic drugs, and cancer vaccines are discussed herein in the therapeutic composition aspect and are applicable to this aspect of the method of treating a tumor or cancer in a subject. In some embodiments, the composition is administered in a volume of less than about 1.0 ml. In some embodiments, the composition is administered in a volume of about 15 ml. In some embodiments, the dosage of the immune checkpoint inhibitor, when measured based on the subject's body weight, may be in the range of about 0.01 to about 10 mg / kg, e.g., about 0.05 to about 10 mg / kg, about 0.1 to about 10 mg / kg, about 0.1 to about 5 mg / kg, about 0.1 to about 3 mg / kg, about 0.1 to about 2 mg / kg, about 0.1 to about 1 mg / kg, or about 0.5 to about 1 mg / kg.

[0125] In some embodiments, the dosage of the cytokine drug, when measured based on the subject's body weight, may be in the range of about 1 mg / kg to about 10 mg / kg, e.g., about 1 mg / kg to about 10 mg / kg, about 2 mg / kg to about 10 mg / kg, about 2 mg / kg to about 5 mg / kg, about 2 mg / kg to about 3 mg / kg, about 2 mg / kg to about 2 mg / kg, about 2 mg / kg to about 1 mg / kg, about 2 to about 500 mg / kg, about 2 to about 100 mg / kg, about 2 to about 50 mg / kg, or about 2 to about 10 mg / kg. In some embodiments, the dosage of a cytotoxic or cytostatic chemotherapeutic agent, as measured based on the subject's body weight, may be in the range of about 1 μg / kg to about 10 mg / kg, e.g., about 1 μg / kg to about 10 mg / kg, about 2 μg / kg to about 10 mg / kg, about 2 μg / kg to about 5 mg / kg, about 2 μg / kg to about 3 mg / kg, about 2 μg / kg to about 2 mg / kg, about 2 μg / kg to about 1 mg / kg, about 2 to about 500 μg / kg, about 2 to about 100 μg / kg, about 2 to about 50 μg / kg, or about 2 to about 10 μg / kg.

[0126] In some cases, the cytokine drug or cytotoxic or cytostatic chemotherapeutic drug contained in the therapeutic composition is at a concentration of about 0.1 to about 1000 mg / m 2 , for example, about 10 to about 600 mg / m 2 In one embodiment, the cytokine drug or cytotoxic or cytostatic chemotherapeutic drug contained in the therapeutic composition can be administered at a low dose, for example about 500 mg / m 2 Less than 400 mg / m 2 Less than or about 300 mg / m 2 is administered in less than In one embodiment, the dose of the cytokine drug or cytotoxic or cytostatic chemotherapeutic drug contained in the therapeutic composition in each administration is about 0.25% to about 75% of its maximum tolerated dose after a conventional dosing regimen. For example, the cytokine drug or cytotoxic or cytostatic chemotherapeutic drug contained in the therapeutic composition is administered at a low dose, with the dose per administration being about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, or about 75% of the maximum tolerated dose.

[0127] In some embodiments, soft tissue administration of the therapeutic compositions described herein results in fewer adverse side effects and / or immune-related adverse events when compared to conventional IV administration of the same compositions, including gastrointestinal, respiratory, neurological, endocrine, dermatological, fatigue, renal, and hepatic effects. In some embodiments, administration of a therapeutic composition described herein results in fewer adverse side effects and / or immune-related adverse events in vivo when compared to administration of the same therapeutic composition comprising at least two immune checkpoint inhibitors and a cytokine drug or a cytotoxic or cytostatic chemotherapeutic drug, but not a cancer vaccine processed ex vivo, or when compared to administration of the same therapeutic composition comprising at least two immune checkpoint inhibitors and a cancer vaccine processed ex vivo, but not a cytokine drug or a cytotoxic or cytostatic chemotherapeutic drug.

[0128] In some cases, the method further comprises inspecting the position of the probe for soft tissue administration prior to administering the composition. Inspecting the position of the probe may include administering a test injection through the probe and measuring the soft tissue pressure during administration of the test injection. In some cases, the method includes repositioning the probe if an increase or decrease in soft tissue pressure is detected during the test injection compared to the pressure of the surrounding tumor tissue. For example, an increase in pressure may indicate that the probe is in scar tissue, and a decrease in pressure may indicate that the probe is in a blood vessel. During treatment, the skilled professional can use a system, such as a computer system, a computing unit, software and / or algorithm, to plan, target, position, deliver, monitor, adjust, image and / or test the treatment protocol. The skilled professional will understand that each injection method includes several parameters and variables that can be adjusted, and an algorithm can be used to control and design the injection. Any algorithm known in the art can be used in the methods described herein. Examples of computer systems, computing units, software and / or algorithms for use in the operation methods are known in the art.

[0129] The ex vivo manipulation / irradiation step can be carried out by manipulation / irradiation techniques and systems known in the art, depending on the manipulation / irradiation method used. The following discussion provides non-limiting examples of various manipulation methods and devices. For example, cryosurgical freezing can be performed by the methods and devices described in PCT Publication Nos. WO 2004 / 086936 and WO 2008 / 142686; U.S. Patent Nos. 6,074,412; 6,579,287; 6,648,880; 6,875,209; 7,220,257; and 7,001,378, all of which are incorporated by reference herein in their entireties. Exemplary devices include the Endocare™ CryoCare™ series, e.g., CryoCare™ and CryoCare CN2 (HealthTronics, Inc., Austin, TX); CryoCor™ Cardiac Cryoablation System (CryoCor Inc.); Arctic Front™ Cardiac CryoAblation Catheter System (Medtronic, Minneapolis, MN); Cryo Painblocker™ (Epi-Med, Dallas, TX).

[0130] Radio frequency (RF) manipulation can be performed by methods and devices described in U.S. Patent Nos. 5,246,438; 5,540,681; 5,573,533; 5,693,078; 6,932,814; and 8,152,801, all of which are incorporated herein by reference in their entireties. Microwave processing can be carried out by the methods and devices described in U.S. Pat. Nos. 6,325,796, 6,471,696, 7,160,292, 7,226,446, and 7,301,131, and U.S. Patent Application Publication No. 2003 / 0065317, all of which are incorporated herein by reference in their entireties. Laser, light, or plasma manipulation may be performed by methods and devices described in U.S. Patent Nos. 4,785,806; 5,231,047; 5,487,740; 6,132,424; 8,088,126; 9,204,918; and 10,023,858; and U.S. Patent Application Publication No. 2007 / 0129712, all of which are incorporated herein by reference in their entireties.

[0131] Ultrasonic manipulation can be performed by the methods and devices described in U.S. Pat. Nos. 5,342,292; 6,821,274; 7,670,335; and 8,974,446; and U.S. Patent Application Publication Nos. 2006 / 0052706 and 2009 / 00184, all of which are incorporated herein by reference in their entireties. High Intensity Focused Ultrasound (HIFU) operations can be performed by the methods and devices described in U.S. Patent Nos. 6,488,639; 6,936,046; 7,311,701; and 7,706,882; and U.S. Patent Application Publication No. 2008 / 0039746, all of which are incorporated herein by reference in their entireties. Steam operations can be carried out by the methods and devices described in U.S. Pat. Nos. 6,813,520 and 9,345,532, and U.S. Patent Application Publication No. 2013 / 0178910, all of which are incorporated herein by reference in their entireties.

[0132] Reversible electroporation (RE) operations can be performed by the methods and devices described in U.S. Patent Application Publication Nos. 2010 / 0023004 and 2012 / 0109122, which are incorporated by reference in their entireties. Irreversible electroporation (IRE) operations can be performed by the methods and devices described in U.S. Patent Nos. 7,655,004 and 8,048,067; PCT Publication No. WO2012071526; and U.S. Patent Publication Nos. 2012 / 0109122 and 2013 / 0253415, all of which are incorporated by reference in their entireties.

[0133] The high frequency electrical membrane rupture operation can be performed by the methods and devices described in U.S. Patent Application No. 2015 / 0150618, PCT Publication No. WO 2015 / 085162, WO 2016 / 123608, WO 2016 / 127162, WO 2016 / 126905, WO 2016 / 126778, and WO 2016 / 126811, which are incorporated herein by reference in their entireties. The method of manipulation with ultrashort electric pulses can be carried out by the methods and devices described in U.S. Patent No. 8,926,606; and U.S. Patent Application Publication Nos. 2006 / 0056480, 2010 / 0261994, and 2018 / 015414, all of which are incorporated herein by reference in their entirety. Exemplary devices include the Nano-Pulse Stimulation™ device (Pulse Biosciences, Inc., Hayward, Calif.).

[0134] Methods of operation using photodynamic therapy can be carried out by the methods and devices described in U.S. Patent Nos. 6,811,562; 7,996,078; and 8,057,418, all of which are incorporated by reference herein in their entireties. Methods of operation using non-thermal shock waves can be carried out by the methods and devices described in U.S. Pat. Nos. 5,524,620 and 8,556,813; U.S. Patent Application Publication No. 2016 / 0008016; and Japanese Patent Application JP2009061083, all of which are incorporated herein by reference in their entireties. Chemical and / or biological engineering may be accomplished using methods described in U.S. Pat. No. 6,428,968; PCT Publication No. WO 2004 / 035110; PCT Publication No. WO 2006 / 095330, PCT Publication No. WO 2007 / 093036, and PCT Publication No. WO 2008 / 093037. No. 2014 / 070820; and the methods and devices described in U.S. Patent Publication Nos. 2004 / 0002647, 2005 / 0255039, 2009 / 0192505, 2010 / 0178684, 2010 / 0145304, 2012 / 0253192; 2012 / 0046656, 2016 / 0310200, and 2016 / 0074626, all of which are incorporated by reference herein in their entireties.

[0135] Those skilled in the art will appreciate that any of the parameters of the above manipulation techniques and devices can be modified as necessary for ex vivo use and combined to achieve the desired manipulation. For example, if it is desired to combine cryosurgical freezing and RF-EMB manipulation, the methods and devices can be modified or combined.

[0136] Further description of various devices that can combine cryosurgical freezing, electroporation, and / or RF-EMB is described in detail in PCT Publication No. WO 2017 / 123981, which is incorporated herein by reference in its entirety. More detailed description of the use of multi-purpose probes as cryoprobes and / or electrodes is also described in WO 2017 / 123981. As discussed above, with respect to laser irradiation (e.g., ultraviolet or near-infrared laser irradiation), X-ray irradiation, or gamma-ray irradiation, techniques or systems for performing ex vivo irradiation or irradiation are well known in the art. As used herein, the term "nucleic acid drug" or "therapeutic nucleic acid" refers to a nucleotide, nucleoside, oligonucleotide, or polynucleotide used to achieve a desired therapeutic effect. Exemplary nucleic acid drugs include, for example, DNA, nDNA, mtDNA, gDNA, RNA, siRNA, miRNA, mRNA, piRNA, antisense RNA, snRNA, snoRNA, vRNA, etc. For example, the nucleic acid drug can be a DNA plasmid.

[0137] The term "subject" is used throughout the specification to describe an animal, human or non-human, to which treatment by the method of the invention is provided. Veterinary applications are expressly contemplated by the present invention. This term includes, but is not limited to, birds, reptiles, amphibians, and mammals, such as humans, other primates, pigs, rodents such as mice and rats, rabbits, guinea pigs, hamsters, cows, horses, cats, dogs, sheep and goats. Preferred subjects are humans, livestock, and domestic pets such as cats and dogs. The term "treat" is used herein to refer to delaying, inhibiting, reducing the effects of, or extending the life span of a patient suffering from a condition, such as cancer.

[0138] An "effective amount" is an amount sufficient to produce a beneficial or desired result. For example, a therapeutically effective amount is an amount that achieves a desired therapeutic effect or promotes a desired physiological response. Effective amounts of the compositions described herein for use in the present invention include, for example, an amount that enhances an immune response against tumors and / or tumor cells to improve outcomes for patients suffering from or at risk of cancer, and improve outcomes of other cancer treatments. For cancer patients who show clinical response, an effective amount is sufficient to alleviate, ameliorate, stabilize, reverse or delay the progression of the disease, or otherwise reduce the pathological consequences of the disease. An effective amount can be administered in one or multiple administrations, applications or dosages. The therapeutically effective amount (i.e., effective amount) of a therapeutic composition depends on the therapeutic composition selected. The therapeutically effective amount of a therapeutic composition depends on the method of administration selected.

[0139] In some cases, intratumoral administration of the composition reduces the therapeutically effective amount of the composition compared to intravenous administration (e.g., conventional IV administration). Those skilled in the art will recognize that certain factors, including but not limited to the severity of the disease or disorder, previous treatments, the overall health and / or age of the subject, and other diseases present, may affect the dosage and timing required to effectively treat a subject. Furthermore, treatment of a subject with a therapeutically effective amount of the therapeutic composition described herein may include a single treatment or a series of treatments. A "cell line" or "cell culture" refers to a culture or higher eukaryotic cell maintained in vitro. It should be understood that the progeny can be identical (or morphologically, genotypically, or phenotypically) to the parent cell. The term "tumor cells" or "cancerous cells", used in the singular or plural form, refers to malignant transformation such that they are pathological cells of the host organism. Primary cancer cells (i.e., cells near the site of malignant transformation obtained) are obtained and easily distinguished from non-cancerous cells by well-established techniques, particularly histochemical examination. Cancer as defined herein includes not only primary cancer cells, but also any cells derived from a cancer cell ancestor. This includes cancer cells and metastatic cancer cells derived from in vitro cultures and cell lines.

[0140] The treatment methods described herein can be used alone or in combination with other methods to treat cancer in patients.Thus, in some cases, the methods described herein can further include treating the patient using surgery (e.g., to remove part of the tumor), chemotherapy, immunotherapy, gene therapy, and / or radiation therapy.The compositions and methods described herein can be administered to the patient at any time, for example, before, during, and / or after surgery, chemotherapy, immunotherapy, gene therapy, and / or radiation therapy.

[0141] The therapeutic compositions and treatment methods described herein are particularly useful for treating cancer in a subject. The term "cancer" refers to cells that have the ability to grow autonomously. Examples of such cells include cells that have an abnormal state or condition characterized by rapidly proliferating cell growth. The term is meant to include cancerous growths, e.g., tumors; metastatic tissues, and malignantly transformed cells, tissues, or organs, regardless of histopathological type or stage of invasiveness. It also includes malignant tumors of various organ systems, such as the respiratory system, cardiovascular system, renal system, reproductive system, hematological system, nervous system, hepatic system, gastrointestinal system, and endocrine system; and adenocarcinomas, including many malignant tumors, such as colon cancer, renal cell carcinoma, prostate cancer, and / or testicular cancer, non-small cell carcinoma of the lung, cancer of the small intestine, and cancer of the esophagus. The therapeutic compositions and treatment methods described herein can be used to treat naturally occurring cancers in a subject. A "naturally occurring" cancer includes any cancer that is not experimentally induced by the implantation of cancer cells into a subject, including, for example, spontaneously occurring cancers, cancers caused by exposure of a patient to a carcinogen, cancers resulting from the insertion of a transgenic cancer gene or the knockout of a tumor suppressor gene, and cancers caused by infection, e.g., viral infection. Cancers treated by the therapeutic compositions and treatment methods described herein also include carcinomas, adenocarcinomas, sarcomas, and hematological cancers. The term "carcinoma" is art-recognized and refers to malignant lesions of epithelial or endocrine tissues. The term also includes carcinosarcomas, which include malignant tumors composed of carcinoma and sarcoma tissues. "Adenocarcinoma" refers to cancers derived from glandular tissue or in which the tumor cells form recognizable glandular structures. The term "sarcoma" is art-recognized and refers to malignant tumors of mesenchymal origin. The term "lymphoma" refers to malignant tumors of hematological origin.

[0142] Cancers or tumors that can be treated using the treatment methods and therapeutic compositions described herein include, for example, cancers or tumors of the stomach, colon, rectum, mouth / pharynx, esophagus, larynx, liver, pancreas, lung, breast, cervix, uterine body, ovary, prostate, testes, bladder, skin, bone, kidney, brain / central nervous system, head, neck, thyroid, throat, and blood, among others; sarcoma, choriocarcinoma, and lymphoma. Exemplary tumors or cancers to be treated are cancers or tumors of the prostate, pancreas, skin, colon, lung, and bladder.

[0143] Metastatic tumors or metastatic cancers (stage IV) can be treated using the treatment methods and pharmaceutical compositions described herein. For example, the treatment methods described herein can be performed on a tumor or cancer (e.g., a primary tumor) located at one site in the subject's body to stimulate the subject's immune defense against the tumor or cancer, leading to an immune attack against the same or even different type of tumor or cancer (e.g., a metastatic tumor) at another site in the subject's body. Metastatic tumors or metastatic cancers can arise from a number of primary tumor or primary cancer types, including, but not limited to, those of brain, prostate, colon, lung, breast, bone, peritoneum, adrenal gland, muscle, and liver origin. Metastasis occurs, for example, when tumor cells shed from a primary tumor attach to vascular endothelium, infiltrate surrounding tissues, and grow to form an independent tumor at a site separate from the primary tumor. The skilled practitioner will appreciate that the treatment methods and therapeutic compositions described herein can also be used to treat other stages of cancer or tumors, such as carcinoma in situ (Stage 0), localized early cancer (Stage I), and larger tumors or cancers (Stage II and Stage III).

[0144] A skilled practitioner will appreciate that the therapeutic compositions and treatment methods described herein can also be used to treat non-cancerous growths, such as non-cancerous tumors. Exemplary non-cancerous growths include, for example, benign tumors, adenomas, breast adenomyoepitheliomas, ductal or lobular hyperplasia, fibroadenomas, fibromas, fibroses, and simple cysts, glandular tumors, hematomas, hamartomas, intraductal papillomas, papillomas, granular cell tumors, hemangiomas, lipomas, meningiomas, fibroids, nevi, osteochondroma, phyllodes tumors, neuromas (e.g., acoustic neuromas, neurofibromas, and pyogenic granulomas), or warts (e.g., plantar warts, genital warts, flat warts, periungual warts, and filiform warts). A skilled practitioner will understand that a subject may be diagnosed by a physician (or, depending on the subject being diagnosed, a veterinarian) as suffering from or at risk for a condition described herein, e.g., cancer, by any method known in the art, such as by evaluating the patient's medical history, performing diagnostic tests, and / or using imaging techniques.

[0145] As described herein, one exemplary method of treating a tumor in a patient includes: (i) obtaining a representative sample of the tumor or cancer in the patient's body by biopsy or surgical resection; (ii) ex vivo manipulation / irradiation of at least a portion of the tumor or cancer; (iii) soft tissue administration of a therapeutic composition described herein (e.g., a therapeutic composition comprising at least two immune checkpoint inhibitors and at least one cytokine or cytotoxic or cytostatic chemotherapeutic agent in combination with an ex vivo engineered autologous cancer composition) to the tumor or cancer; and (iv) optionally, administering a therapeutically effective amount of a nucleic acid pharmaceutical to the tumor.

[0146] Also provided are kits that include one or more of the therapeutic compositions described herein. The kits generally include the following major components: packaging, reagents that include the binding composition as described above, optionally controls, and instructions for use. The packaging can be a box-like structure to hold a vial (or vials) that contains the binding composition, a vial (or vials) that contains a control, and / or instructions for use in the methods described herein. In some cases, the packaging contains a cartridge that can be controlled by a digital device according to systematic instructions. Those skilled in the art can easily modify the packaging to fit individual needs. In some embodiments, the kits provided herein can include at least one (e.g., one, two, three, four, five, or more) composition containing at least one (e.g., one, two, three, four, five, or more) of the compositions described herein, and at least one (e.g., one, two, three, four, five, or more) other composition in a separate vial containing a therapeutic or biological agent known in the art to be effective in treating cancer.

[0147] The compositions and kits provided herein can be used according to any of the methods (e.g., treatment methods) described above. For example, the compositions and kits can be used to treat cancer or tumors. Those skilled in the art will recognize other suitable uses of the compositions and kits provided herein, and it is contemplated that the compositions and kits can be used for such uses. EXAMPLES

[0148] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention in any way.

[0149] Example 1 Treatment regimen Treatment regimes using cytokine drugs Each treatment cycle with a cytokine drug includes the following exemplary treatments: Autologous cancer vaccine: 250mg / m 2 Intradermal (upper arm) or intratumoral injection of UV-irradiated autologous cancer lysate vaccine containing a minimum of 1 million lysed cancer cells diluted in GM-CSF (Leukine) at a concentration of 0.01 mg / kg, total volume 1.0 ml. Pembrolizumab (Keytruda), a PD-1 blocking antibody: Intradermal or intratumoral injection at the same site, 0.5 ml. Ipilimumab (Yervoy), an anti-CTLA-4 antibody: Intradermal or intratumoral injection at the same site, 0.5 ml. GM-CSF: Subcutaneous administration, 250 mg per day for a total of approximately 6 weeks. If necessary, one or two additional treatment cycles can be administered. Treatment regimes using cytotoxic or cytostatic chemotherapeutic agents Each treatment cycle containing a cytotoxic or cytostatic chemotherapeutic agent includes the following exemplary treatments: Autologous cancer vaccine: 250mg / m 2 Intradermal (upper arm) or intratumoral injection of UV-irradiated autologous cancer lysate vaccine containing a minimum of 1 million lysed cancer cells diluted in cyclophosphamide (Cytoxan) at a concentration of 0.01 mg / kg, total volume 1.0 ml. Nivolumab (Opdivo), a PD-1 blocking antibody: Intradermal or intratumoral injection at the same site, 0.5 ml. Ipilimumab (Yervoy), an anti-CTLA-4 antibody: Intradermal or intratumoral injection at the same site, 0.5 ml. Cyclophosphamide: Orally administered, 25 mg twice daily or 50 mg once daily (2 weeks on, 2 weeks off, then 2 weeks back on, starting on day 1 after treatment, for a total of approximately 6 weeks). If necessary, one or two additional treatment cycles can be administered.

[0150] Example 2 Preparation of autologous cancer lysate vaccine Representative tumor tissue specimens (eg, biopsies) and fine needle cytological aspirates are routinely obtained by local treating physicians, soaked in saline, and sealed in sterile containers. All procedures for the preparation of the cancer lysate vaccine will be performed in accordance with Good Manufacturing Practices (GMP). Routine handling methods will be used for each patient sample (e.g., standard operating procedures will be applied for transport, receipt, and identification of laboratory specimens, processing requirements, handling of hazardous waste, etc.). The following processing steps are carried out in the sterile preparation room: The fluid is decanted from the tissue specimen and examined to determine the presence and extent of cancer. The tissue is then dissected with a sterile scalpel to maximize the amount of cancer (by removing much of the non-cancerous tissue during dissection) and the relative proportions of cancer and non-cancerous cells are estimated and recorded. The tissue is then minced and a small portion is placed into a test tube and vortexed for 30 seconds. These steps are repeated to cover the entire specimen. The tissue is then divided into 10 mg aliquots (approximate weight) and placed into test tubes to ensure that there are at least 1 million cancer cells in each.

[0151] Fine needle aspirates are rapidly examined by light microscopy and the relative proportions of cancerous and noncancerous cells are estimated and recorded. The aspirates are then gently centrifuged at 1000 rpm for 1 minute to produce a pellet of intact cells, the supernatant is decanted, and the pellet is divided into 10 mg aliquots (approximate weight) into test tubes to ensure that at least 1 million cancer cells are present.

[0152] Depending on the treatment regimen in Example 1, each of the aliquots is diluted with GM-CSF (in 0.9% sterile sodium chloride) if the treatment regimen involves a cytokine drug, or with cyclophosphamide (reconstituted to a concentration of 2% (20 mg / ml)) if the treatment regimen involves a cytotoxic or cytostatic chemotherapeutic drug. The diluted aliquots were then crosslinked with a Stratalinker UV Crosslinker 1800 (Stratagene) at 300 mJ / cm 2 The cells were treated with 1000 μl of UV radiation to induce necroptosis (immunogenic cell death) and incubated in 5% CO 2 The cells are incubated at 37° C. for 1 hour in 50° C. to allow necroptosis to proceed. Necrosis is then induced by two repeated cycles of freezing (-40° C. dry ice / ethanol bath) and thawing (37° C. water bath). For the third cycle, the aliquots are shipped frozen on dry ice back to the treating physician, where a final thawing (to room temperature) takes place just prior to injection into the patient in combination with an immunotherapeutic agent.

[0153] Example 3 Vaccines and Drug Delivery Intratumoral injection The operator draws the vial contents (autologous cancer vaccine previously thawed and brought to room temperature immediately prior to injection; 1.0 ml total) into a sterile syringe fitted with an 18 gauge, 1.5 inch draw-up needle. The operator then attaches an 18 gauge, 20 cm Chiba® biopsy needle (Cook Medical, Inc., Bloomington, Indiana) to the syringe. The operator inserts the needle into the tumor under image guidance until the tip of the needle reaches the desired location and then deposits the vaccine. Leaving the needle in place, the operator removes the emptied syringe and replaces it with a second syringe containing 1.0 ml of the first immunotherapy agent (anti-PD-1 antibody), depositing the drug over the entire area of ​​the previous injection, with copious overflow to include surrounding tissue, in a manner identical to the administration of the first injection. The operator then repeats this procedure with a third syringe containing 1.0 ml of a second immunotherapy (an anti-CTLA-4 antibody) to deposit the third drug.

[0154] intradermal injection To prepare an intradermal injection of cancer vaccine, anti-PD-1 antibody, or anti-CTLA-4 antibody, the operator draws the vial contents (autologous cancer vaccine previously thawed and brought to room temperature immediately prior to injection; 0.5 ml total) into a sterile syringe fitted with an 18 gauge, 1.5 inch draw-up needle. The operator then attaches a ¼ to ½ inch long, 26 or 27 gauge thick needle to the syringe. Alternatively, the operator may attach an FDA approved microneedle (e.g., Micronjet 600, Nano Pass Technologies, Rehovat, Israel) to the syringe. To administer the cancer vaccine, the operator inserts a needle or microneedle into the skin. For regular-sized needles, the Mantoux procedure is used, the needle is positioned at an administration angle of 5 to 15 degrees, and the syringe contents are slowly injected in their entirety. The microneedle attached to the cancer vaccine syringe is placed directly into the skin. The injection method is the same for anti-PD-1 and anti-CTLA-4 antibodies.

[0155] For administration of anti-PD-1 and anti-CTLA-4 antibodies, the operator inserts a needle or microneedle into the skin at the edge of the first wheal to allow intradermal contact of this fluid with the previously administered cancer vaccine and antibody fluids. The method of injection is the same for anti-PD-1 and anti-CTLA-4 antibodies.

[0156] Subcutaneous administration of low-dose GM-CSF For the treatment regimen involving cytokine drugs described in Example 1, a subcutaneous regimen of 250 mg GM-CSF(Cy) per day is continued for 30 days for vaccine treatment.

[0157] Low-dose oral metronomic administration of cyclophosphamide For treatment regimens involving cytotoxic or cytostatic chemotherapeutic agents described in Example 1, an oral regimen of cyclophosphamide (Cy) at 25 mg bid (twice daily) or 50 mg qd (once daily) is continued for 14 days immediately following each treatment, followed by 14 days without drug, after which the oral dosing is repeated for another 14 days.

Claims

1. A therapeutic composition for use in a method of treating a tumor or cancer in a subject in need thereof, comprising: The therapeutic composition comprises: i) two immune checkpoint inhibitors, including a CTLA-4 inhibitor and a PD-1 inhibitor or a PD-L1 inhibitor; ii) cyclophosphamide; and iii) a cancer vaccine that produces necrotic cancer cells with minimal destruction of cancer antigens in an amount effective to treat a tumor or cancer, the cancer vaccine being prepared from a cancer cell lysate prepared by ex vivo treatment consisting of UV irradiation and cryosurgical freezing, and suspended in a low dose solution of cyclophosphamide; It consists of: The cancer cell lysate is prepared by one or more procedures selected from the group consisting of dissociation or dissection, fixation, centrifugation, resuspension, concentration, and combinations thereof; The method comprises administering the therapeutic composition intradermally to a subject. The therapeutic composition.

2. 10. The therapeutic composition of claim 1, which is administered to the skin or soft tissue of a subject using an injection device.

3. 2. The therapeutic composition of claim 1, wherein the concentration of the CTLA-4 inhibitor ranges from about 0.5 to about 10 mg / ml and the concentration of the PD-1 inhibitor or PD-L1 inhibitor ranges from about 0.5 to about 20 mg / ml.

4. The therapeutic composition described in claim 1, wherein the cancer cell lysate is a cancer cell lysate fixed with formalin, ethanol, or glutaraldehyde.

5. The therapeutic composition of claim 1, wherein the cryosurgical freezing is performed using a single probe with a total operation time of 10 minutes or less.

6. The therapeutic composition of claim 1, wherein the cryosurgical freezing is performed at a temperature of about -35 to about -100°C.

7. 2. The therapeutic composition of claim 1, wherein the cancer vaccine is autologous and the cancer cell lysate is derived from the subject to be treated.

8. 2. The therapeutic composition of claim 1, wherein the cancer vaccine is allogeneic and the cancer cell lysate is derived from a cancer cell line.

9. A therapeutic composition for use in a method of treating a tumor or cancer in a subject in need thereof, comprising: The therapeutic composition comprises: i) two immune checkpoint inhibitors, including a CTLA-4 inhibitor and a PD-1 inhibitor or a PD-L1 inhibitor; ii) cyclophosphamide; and iii) a cancer vaccine that produces necrotic cancer cells with minimal destruction of cancer antigens in an amount effective to treat a tumor or cancer, the cancer vaccine being prepared from a cancer cell lysate prepared by ex vivo treatment consisting of UV irradiation and cryosurgical freezing, and suspended in a low dose solution of cyclophosphamide; It consists of: The cancer cell lysate is prepared by one or more procedures selected from the group consisting of dissociation or dissection, fixation, centrifugation, resuspension, concentration, and combinations thereof; The method includes administering the therapeutic composition intradermally to a subject; following administration of the therapeutic composition, administering to the subject daily a drug selected from a cytokine drug, a cytotoxic or cytostatic chemotherapeutic drug, and combinations thereof.

10. The therapeutic composition of claim 9, wherein the drug is GM-CSF or cyclophosphamide.

11. 10. The therapeutic composition of claim 9, wherein the drug is a cytokine drug administered subcutaneously or a cytotoxic or cytostatic chemotherapeutic drug administered orally.

12. The therapeutic composition of claim 9, administered to the skin or soft tissue of a subject using an injection device.

13. The therapeutic composition of claim 9, wherein the concentration of the CTLA-4 inhibitor is in the range of about 0.5 to about 10 mg / ml, and the concentration of the PD-1 inhibitor or PD-L1 inhibitor is in the range of about 0.5 to about 20 mg / ml.

14. The therapeutic composition of claim 9, wherein the cancer cell lysate is a cancer cell lysate fixed with formalin, ethanol, or glutaraldehyde.

15. The therapeutic composition of claim 9, wherein the cryosurgical freezing is performed using a single probe with a total operation time of 10 minutes or less.

16. The therapeutic composition of claim 9, wherein the cryosurgical freezing comprises cryosurgical freezing performed at a temperature of about -35 to about -100°C.

17. The therapeutic composition of claim 9, wherein the cancer vaccine is autologous and the cancer cell lysate is derived from the subject to be treated.

18. The therapeutic composition of claim 9, wherein the cancer vaccine is allogeneic and the cancer cell lysate is derived from a cancer cell line.