Methods of using activated t cells to treat cancer

Abstract

The present invention provides a method of treating a cancer in an individual using activated T cells or PBMCs induced by antigen presenting cells (such as dendritic cells) loaded with a plurality of tumor antigen peptides. The method can also include administering to the individual the antigen presenting cells loaded with the plurality of tumor antigen peptides. The method can be used alone or in combination with an immune checkpoint inhibitor. The present invention provides a precise method of treatment using neoantigen peptides or tailored to the individual based on the mutational load in the individual's tumor. The present invention also discloses methods of making the activated T cells, methods of monitoring the treatment, and methods of cloning tumor specific T cell receptors. The present invention also provides isolated cell populations comprising the activated T cells, as well as compositions and kits useful for cancer immunotherapy.

Description

[0001] This application is a divisional application of the invention patent application filed on March 11, 2016, with application number 201680015221.3 and invention title "Method for Treating Cancer Using Activated T Cells".

[0002] Cross-references to related applications

[0003] This application claims priority to international application PCT / CN2015 / 074227, filed on March 13, 2015, the entire contents of which are incorporated herein by reference.

[0004] Submission of sequence lists on ASCII text files

[0005] The following submission of an ASCII text file is incorporated herein by reference in its entirety: Sequence List in Computer-Readable Form (CRF) (filename: 744852000141SEQLIST.TXT, record date: March 11, 2016, size: 7.83KB). Technical Field

[0006] This invention relates to the field of cancer immunotherapy. More specifically, this invention provides methods, compositions, and kits for treating cancer in an individual using activated T cells. Background Technology

[0007] The human body possesses a complex immune system that protects it from diseases, including malignant tumors. Therefore, stimulating the body's own immunity to treat and prevent cancer has long been a goal in oncology. The innate immune response to tumors is typically triggered by tumor antigens, including mutated proteins specifically expressed in cancer cells and tumor-associated antigens (TAAs) overexpressed in cancer-originating tissues but not fully recognized as "self." Antigen-presenting cells (APCs) (especially dendritic cells (DCs)) encountering tumor antigens process them and present them on their cell surface. Upon maturation, tumor antigen-loaded DCs can elicit a T-cell response to the cancer cells hosting the tumor antigens. This T-cell response involves cytotoxic T cells, helper T cells, and functionally distinct effector T cells and memory T cells. Particularly potent T-cell responses involve the production of cytotoxic T cells, which kill cancer cells by releasing cytokines, enzymes, and cytotoxins, or by triggering a pro-apoptotic signaling cascade via cell-cell interactions.

[0008] Cancer immunotherapy aims to treat cancer using the aforementioned process, but its effectiveness has been quite limited to date. Initial attempts focused on developing cancer vaccines based on specific antigenic peptides, full-length antigenic proteins, or viral vectors encoding tumor antigens. Few cancer vaccines have entered clinical trials, and even fewer have produced any noteworthy clinical results. Unlike traditional cancer therapies such as chemotherapy, radiation therapy, and surgical resection, the body's response to cancer immunotherapy (especially cancer vaccines) is typically significantly delayed because APCs require time to process antigens and present them to T cells, and T cells also need time to mature and trigger an immune response. When a tumor is present in a patient's body, the cancer cells within the tumor already possess mechanisms to evade the immune system's surveillance. Therefore, an effective tumor vaccine must be able to bypass the deficiencies in immune surveillance to elicit a strong immune response. Furthermore, several bottlenecks exist in cancer vaccines that prevent this approach from producing specific and durable clinical efficacy. First, cancer cells, even those with the same histological type, exhibit considerable heterogeneity in their genetic composition and expression profiles among different patients and between different lesions within the same patient—a phenomenon well supported by a wealth of genetic data from recent next-generation sequencing experiments on cancer cells, readily available in the literature and public databases. Therefore, the limited number of one or more tumor antigens in a specific cancer vaccine treatment is unlikely to represent the antigenic profile specific to an individual tumor in all patients. Second, many antigenic fractions in cancer vaccines cannot be effectively loaded onto APCs due to serum half-life and bioavailability issues. Third, even when APCs are appropriately sensitized by the antigens contained in the cancer vaccine, the lack of suitable activation signals and microenvironment can lead to the generation of incorrect subsets of T cells, particularly immunosuppressive regulatory T cells (T cells). REG These substances suppress, rather than stimulate, the immune response against tumors. The last two problems stem from the fact that clinicians have absolutely no control over a patient's actual response to any cancer vaccine after its administration.

[0009] Cell-based cancer immunotherapy alleviates some of the challenges associated with cancer vaccines by administering immune-mediated cells or cell products prepared under relatively defined and controlled conditions to patients. In particular, DC-based methods have attracted considerable attention, especially after FDA approval in April 2010. (sipuleucel-T) is used after advanced prostate cancer. Typical DC-based immunotherapy involves isolating DCs from a cancer patient, loading the DCs with a tumor antigen (or multiple tumor antigens, including tumor cell lysates and total mRNA) in vitro, and then administering the DCs back to the patient to elicit a cancer-killing T-cell response. Examples include exposing a patient’s peripheral blood mononuclear cells (PBMCs) to a fusion protein (including a tumor-derived antigen coupled to a cytokine such as GM-CSF), and then infusing the patient with PBMCs (which may be assumed to contain activated dendritic cells (DCs) that can present the tumor-derived antigen to T cells) (see U.S. Patents 5,976,546, 6,080,409, and 6,210,662). In the pivotal Phase III trial (Kantoff PW, Higano CS et al. (2010) "Sipuleucel-T immunotherapy for castration-resistant prostate cancer." NJ Med 363:411-22), a recombinant protein of prostatic acid phosphatase (PAP) (a prostate cancer-associated antigen) fused to GM-CSF (a cytokine known to attract and induce dendritic cells) was used to prepare... The specific implementation plan. Although compared with the median survival of patients in the control group (21.7 months), It was able to extend the median survival of patients in the experimental group (25.8 months), but the clinical trial results did not show statistically significant signs of delayed tumor progression or tumor size reduction. Even more challenging is the fact that... During treatment, individual patient survival did not appear to be associated with specific T-cell responses to the fusion protein or PAP (Cheever MA, Higano CS (2011) "PROVENGE (Sipuleucel-T) in prostate cancer: the first FDA-approved therapeutic cancer vaccine." Clin. Cancer Res. 17:3520-6).

[0010] The second approach in cell-based immunotherapy is called adoptive lymphocyte therapy, which involves isolating tumor-infiltrating lymphocytes (TILs) from a patient's tumor, expanding the TILs in vitro, and then infusing the TILs back into the patient after removing the patient's native non-myeloid lymphocytes. Significant clinical responses (including complete tumor regression and prolonged disease-free survival) have been reported in the clinical application of adoptive lymphocyte therapy in melanoma patients (Restifo NP, Dudley ME, and Rosenberg SA. (2012) "Adoptive immunotherapy for cancer: harnessing the T cell response." Nat. Rev. Immunol. 12:269-81). Further research indicates that the clinical benefit of TIL is associated with or generated by tumor-specific T cells present in the TIL population (Robbins PF et al. (2013) “Mining exomic sequencing data to identify mutated antigens recognized by adoptively transferred tumor-reactive T cells.” Nature Medicine 19:747-752; and Tran E et al. (2014) “Cancer immunotherapy based on mutation-specific CD4+ T cells in a patient with epithelial cancer” Science 344:641-645). Recently, T cells with engineered T cell receptors that have modified affinity for certain tumor antigens or chimeric antigen receptors (CAR-T) have further expanded the capabilities of adoptive lymphocyte therapy by altering the microenvironment of T cell-tumor interactions.The main problems with current adoptive lymphocyte therapy involve multiple reports of serious adverse events (including CNS toxicity) in clinical trials, which may be related to inappropriate target selection (the so-called on-target / off-tumor effect) and biased expansion of the T cell population. Another problem with this approach is the lack of durable responses in some patients due to the rapid development of immune tolerance to tumor-specific antigens presented on the infused T lymphocytes, as well as immune evasion by cancer cells.

[0011] In addition to elevated levels of immunosuppressive cells such as T cells REG Besides MDSCs (myeloid-derived suppressor cells), immune tolerance and immune evasion are often mediated by checkpoint molecules or co-inhibitory signals on cells that interact with T cells in the tumor site microenvironment. A well-studied pair of checkpoint molecules involves the immunosuppressive PD-1 receptor on T cells and the PD-L1 ligand on APCs (such as DCs), MDSCs, and cancer cells. PD-L1 binding to PD-1 triggers signals that inhibit the production of pro-inflammatory cytokines (e.g., IL-2) and the proliferation of cytotoxic T cells. In many cases, PD-L1 binding to PD-1 induces apoptosis of cytotoxic T cells. On the other hand, PD-1 / PD-L1 signaling induces T cell apoptosis. REGThese cells further suppress T cells, which have the ability to attack tumors. Currently, based on the theory that blocking T cell checkpoints can help overcome immune tolerance and immune evasion in tumor sites, several pharmaceutical companies have developed antibodies against PD-1, PD-L1, and other checkpoint molecules (such as CTLA-4 on T cells) as different approaches in cancer immunotherapy. It is worth noting that the antitumor effect of checkpoint blockade requires the pre-existing presence of tumor-specific T cells in vivo (Boussiotis VA (2014) "Somatic mutations and immunotherapy outcome with CTLA-4 blockade in melanoma" N. Engl. J. Med. 371:2230-2232; Wolchok JD and Chan TA (2014) "Cancer: antitumor immunity gets aboost" Nature 515:496-498).

[0012] Given the prospects and challenges of various cancer immunotherapy methods, there is a desire to provide new cancer immunotherapy methods that combine the advantages of previous approaches while avoiding known drawbacks.

[0013] All publications, patents, patent applications, and published patent applications mentioned herein are incorporated herein by reference in their entirety. Summary of the Invention

[0014] This invention provides methods, compositions, and kits for treating cancer in individuals using activated T cells induced by antigen-presenting cells (such as dendritic cells) loaded with a variety of tumor antigen peptides.

[0015] One aspect of this application provides a method for treating cancer in an individual (such as a human individual), the method comprising administering an effective amount of activated T cells to the individual, wherein the activated T cells are prepared by co-culturing a population of T cells with a population of dendritic cells loaded with a plurality of tumor antigen peptides. In some embodiments, the individual has previously been administered an effective amount of dendritic cells loaded with the plurality of tumor antigen peptides. In some embodiments, the method further comprises administering an effective amount of dendritic cells loaded with the plurality of tumor antigen peptides to the individual. In some embodiments, the dendritic cells are administered prior to the administration of the activated T cells (e.g., approximately 7 to 14 days, approximately 14 to 21 days, or approximately 7 to 21 days prior).

[0016] In some embodiments of any of the methods described above, the method further includes preparing activated T cells by co-culturing a population of T cells with a population of dendritic cells loaded with the plurality of tumor antigen peptides prior to the administration step. In some embodiments, the population of T cells is co-cultured with the population of dendritic cells loaded with the plurality of tumor antigen peptides for about 7 days to about 21 days (e.g., about 7 days to about 14 days or about 14 days to about 21 days).

[0017] In some embodiments of any of the methods described above, the T cell population is contacted with an immune checkpoint inhibitor prior to co-culturing. In some embodiments, in the presence of an immune checkpoint inhibitor, the T cell population is co-cultured with a dendritic cell population loaded with the aforementioned multiple tumor antigen peptides. In some embodiments, the immune checkpoint inhibitor is an inhibitor of an immune checkpoint molecule selected from PD-1, PD-L1, and CTLA-4.

[0018] In some embodiments of any of the above methods, the method further includes preparing a dendritic cell population loaded with the plurality of tumor antigen peptides. In some embodiments, the dendritic cell population loaded with the plurality of tumor antigen peptides is prepared by contacting the dendritic cell population with the plurality of tumor antigen peptides. In some embodiments, the dendritic cell population loaded with the plurality of tumor antigen peptides is prepared by contacting the dendritic cell population with the plurality of tumor antigen peptides in the presence of a composition that facilitates the uptake of the plurality of tumor antigen peptides by the dendritic cells.

[0019] In some embodiments of any of the methods described above, the T cell population and the dendritic cell population are derived from the same individual. In some embodiments, the T cell population and the dendritic cell population are derived from an individual receiving treatment.

[0020] One aspect of this application provides a method for preparing an activated T cell population, the method comprising: (a) inducing a monocyte population to differentiate into a dendritic cell population; (b) contacting the dendritic cell population with a plurality of tumor antigen peptides to obtain a dendritic cell population loaded with the plurality of tumor antigen peptides; and (c) co-culturing the dendritic cell population loaded with the plurality of tumor antigen peptides with a non-adhesive PBMC population to obtain an activated T cell population, wherein the monocyte population and the non-adhesive PBMC population are obtained from an individual's PBMC population. In some embodiments, step b) includes contacting the dendritic cell population with the plurality of tumor antigen peptides in the presence of a composition that facilitates dendritic cell uptake of the plurality of tumor antigen peptides. In some embodiments, step b) further includes contacting the dendritic cell population loaded with the plurality of tumor antigen peptides with a plurality of Toll-like receptor (TLR) agonists (such as polyIC, MALP, R848, or any combination thereof) to induce maturation of the dendritic cell population loaded with the plurality of tumor antigen peptides. In some embodiments, step c) further includes contacting the activated T cell population with a variety of cytokines and optionally an anti-CD3 antibody to induce the proliferation and differentiation of the activated T cell population. In some embodiments, the variety of cytokines includes IL-2, IL-7, IL-15, or IL-21. In some embodiments, the non-adherent PBMC population is contacted with an immune checkpoint inhibitor prior to co-culturing. In some embodiments, step c) includes co-culturing a dendritic cell population loaded with the variety of tumor antigen peptides with the non-adherent PBMC population in the presence of an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is an inhibitor of an immune checkpoint molecule selected from PD-1, PD-L1, and CTLA-4.

[0021] Methods for treating cancer in an individual (such as a human individual) are also provided, comprising administering to the individual an effective amount of a population of activated T cells, which is prepared by any of the methods described in the preceding paragraphs. In some embodiments, the population of PBMCs is obtained from the individual receiving treatment.

[0022] In some embodiments of any of the methods for treating cancer as described above, activated T cells are administered to the individual at least three times. In some embodiments, the interval between each administration of activated T cells is from about 0.5 months to about 5 months (e.g., from about 0.5 months to about 2 months).

[0023] In some embodiments of any of the methods for treating cancer as described above, activated T cells are administered intravenously. In some embodiments, the activated T cells are administered at a dose of at least about 3 × 10⁻⁶. 9 Administered at a dose per cell / individual. In some implementations, activated T cells are administered at approximately 1 × 10⁻⁶. 9 To approximately 1×1010 Apply per cell / individual.

[0024] In some embodiments of any of the methods for treating cancer as described above, dendritic cells loaded with the various tumor antigen peptides are administered at least three times. In some embodiments, the interval between each administration of the dendritic cells is from about 0.5 months to about 5 months (e.g., from about 0.5 months to about 2 months).

[0025] In some embodiments of any of the methods for treating cancer as described above, dendritic cells loaded with the various tumor antigen peptides are administered subcutaneously. In some embodiments, the dendritic cells are administered at a dose of approximately 1 × 10⁻⁶. 6 Approximately 5×10 6 Dosage administration per cell / individual.

[0026] One aspect of this application provides a method for treating cancer in an individual (such as a human individual), the method comprising: a) contacting a population of PBMCs with a plurality of tumor antigen peptides to obtain an activated population of PBMCs, and b) administering an effective amount of activated PBMCs to the individual. In some embodiments, step (a) includes contacting the population of PBMCs with a plurality of tumor antigen peptides in the presence of an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is an inhibitor of an immune checkpoint molecule selected from PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, and LAG-3. In some embodiments, the activated PBMCs are administered at least three times. In some embodiments, the interval between each administration of the activated PBMCs is from about 0.5 months to about 5 months (e.g., from about 0.5 months to about 2 months). In some embodiments, the activated PBMCs are administered intravenously. In some embodiments, the activated PBMCs are administered at a dose of about 1 × 10⁻⁶. 9 To approximately 1×10 10 Dosage administration per cell / individual.

[0027] In some embodiments according to any of the methods described above, each of the plurality of tumor antigen peptides is about 20 to about 40 amino acids in length. In some embodiments, the plurality of tumor antigen peptides includes at least one peptide containing an MHC-I epitope. In some embodiments, the at least one peptide containing an MHC-I epitope further includes additional amino acids located flanking the epitope at an N-terminus, a C-terminus, or both ends.

[0028] In some embodiments of any of the methods described above, the plurality of tumor antigen peptides includes at least one peptide containing an MHC-II epitope. In some embodiments, the at least one peptide containing an MHC-II epitope further includes an additional amino acid located flanking the epitope at an N-terminus, a C-terminus, or both ends.

[0029] In some embodiments of any of the methods described above, the plurality of tumor antigen peptides includes a first core group of common tumor antigen peptides. In some embodiments, the plurality of tumor antigen peptides further includes a second group of cancer type-specific antigen peptides. In some embodiments, the first core group contains about 10 to about 20 common tumor antigen peptides. In some embodiments, the second group contains about 1 to about 10 cancer type-specific antigen peptides.

[0030] In some embodiments of any of the methods described above, the plurality of tumor antigen peptides includes neoantigen peptides. In some embodiments, the neoantigen peptides are selected based on the genetic profile of a tumor sample taken from an individual.

[0031] In some embodiments of any of the methods for treating cancer as described above, the cancer is selected from hepatocellular carcinoma, cervical cancer, lung cancer, colorectal cancer, lymphoma, kidney cancer, breast cancer, pancreatic cancer, gastric cancer, esophageal cancer, ovarian cancer, prostate cancer, nasopharyngeal carcinoma, melanoma, and brain cancer.

[0032] In some embodiments of any of the methods for treating cancer as described above, the method further includes administering an effective amount of an immune checkpoint inhibitor to the individual. In some embodiments, the immune checkpoint inhibitor is an inhibitor of an immune checkpoint molecule selected from PD-1, PD-L1, and CTLA-4.

[0033] In some embodiments of any of the methods for treating cancer as described above, individuals are selected for the treatment based on the mutational burden in the cancer. In some embodiments, the individual has a low mutational burden in the cancer. In some embodiments, the individual has a low mutational burden in one or more MHC genes. In some embodiments, the individual has no more than about 10 mutations in said one or more MHC genes. In some embodiments, said one or more MHC genes are MHC class I genes. In some embodiments, said individual is a human individual, and said one or more MHC genes are selected from HLA-A, HLA-B, HLA-C, and B2M. In some embodiments, the individual does not have mutations in B2M. In some embodiments, the individual does not have mutations in functional regions (such as leader peptide sequences, α1 domains, α2 domains, or α3 domains) of said one or more MHC genes. In some embodiments, the mutational burden of the cancer is determined by sequencing a tumor sample taken from the individual.

[0034] In some embodiments of any of the methods for treating cancer as described above, individuals are selected for the treatment based on having one or more neoantigens in the cancer. In some embodiments, the individual has at least five neoantigens. In some embodiments, the method further includes identifying neoantigens in the cancer and incorporating a neoantigen peptide into the plurality of tumor antigen peptides, wherein the neoantigen peptide contains a novel epitope in the neoantigen. In some embodiments, neoantigens are identified by sequencing a tumor sample taken from the individual. In some embodiments, the sequencing is targeted sequencing of cancer-related genes. In some embodiments, the method further includes determining the affinity of the novel epitope for an MHC molecule. In some embodiments, the method further includes determining the affinity of a complex comprising the novel epitope and an MHC molecule for a T-cell receptor. In some embodiments, the MHC molecule is an MHC class I molecule. In some embodiments, the MHC molecule is derived from the individual.

[0035] In some embodiments of any of the methods for treating cancer as described above, the method further includes monitoring the individual after administration of activated T cells or activated PBMCs. In some embodiments, the monitoring includes determining the number of circulating tumor cells (CTCs) in the individual. In some embodiments, the monitoring includes detecting a specific immune response in the individual against the multiple tumor antigen peptides. In some embodiments, the multiple tumor antigen peptides are modulated based on this specific immune response to provide a multiple customized tumor antigen peptide. In some embodiments, the treatment method is repeated using the multiple customized tumor antigen peptides.

[0036] One aspect of this application provides a method for cloning a tumor-specific T-cell receptor, the method comprising: (a) treating an individual using any of the methods for treating cancer as described above; (b) isolating T cells from the individual, wherein the T cells specifically recognize a tumor antigen peptide among the plurality of tumor antigen peptides; and (c) cloning a T-cell receptor from the T cells to provide a tumor-specific T-cell receptor. In some embodiments, the individual has a strong specific immune response to the tumor antigen peptide. In some embodiments, the T cells are isolated from a PBMC sample of the individual. In some embodiments, the tumor antigen peptide is a neoantigen peptide.

[0037] Also provided are tumor-specific T-cell receptors cloned using any of the methods described above for cloning tumor-specific T-cell receptors, isolated T cells containing tumor-specific T-cell receptors, and methods for treating cancer in an individual, the method comprising administering an effective amount of isolated T cells to the individual.

[0038] It also provides isolated cell populations (such as activated T cells or activated PBMCs) prepared by any of the preparation methods described above.

[0039] One aspect of this application provides a cell population comprising isolated activated T cells, wherein less than about 1% of the activated T cells are regulatory T (T) cells. REG )cell.

[0040] In some embodiments of the isolated cell population according to any of the above-described methods, the isolated cell population contains about 0.3% to about 0.5% CD4. + CD25 + FoxP3 + Cells. In some embodiments, the isolated cell population contains approximately 65% ​​to approximately 75% CD3. + CD8 + Cells. In some embodiments, the isolated cell population contains approximately 16% to approximately 22% CD3+. + CD4 + Cells. In some embodiments, the isolated cell population contains approximately 13% to approximately 15% CD3. + CD56 + cell.

[0041] In some embodiments of the cell populations isolated according to any of the above descriptions, activated T cells are capable of in vivo or in vitro initiating specific responses to a variety of tumor antigen peptides. In some embodiments, activated T cells express a variety of pro-inflammatory molecules. In some embodiments, the various pro-inflammatory molecules include IFNγ, TNFα, granzyme B, or perforin.

[0042] In some embodiments of the cell population isolated according to any of the above descriptions, the activated T cells either do not express a variety of immunosuppressive cytokines or express a variety of immunosuppressive cytokines at low levels. In some embodiments, the variety of immunosuppressive cytokines includes IL-10 or IL-4.

[0043] In some embodiments based on any of the cell populations isolated above, less than about 5% of the activated T cells express the immunosuppressive molecule PD-1.

[0044] In some embodiments of the isolated cell population described above, at least about 90% of the cells in the isolated cell population are activated T cells.

[0045] One aspect of this application provides a composition comprising at least 10 tumor antigen peptides, wherein each of the at least 10 tumor antigen peptides comprises at least one epitope selected from SEQ ID NO:1-35. In some embodiments, the at least 10 tumor antigen peptides are selected from... Figure 2CThe tumor antigen peptides in the formulation. In some embodiments, each of the at least 10 tumor antigen peptides comprises one or more epitopes encoded by cancer-associated genes, the epitopes being selected from hTERT, p53, Survivin, NY-ESO-1, CEA, CCND1, MET, RGS5, MMP7, VEGFR, AFP, GPC3, HBVc, HBVp, CDCA, KRAS, PARP4, MLL3, and MTHFR.

[0046] Kits, pharmaceuticals, and products containing any of the compositions described above (such as isolated cell populations or tumor antigen peptide compositions) are also provided.

[0047] These and other aspects and advantages of the invention will become apparent from the following detailed description and the appended claims. It should be understood that one, some, or all of the features of the various embodiments described herein can be combined to form other embodiments of the invention. Attached Figure Description

[0048] Figure 1 Two preferred embodiments of the MASCT method are depicted, including the timing of DC and T cell preparation steps and the single or multiple administration of the cell-specific composition. Arrows below the timeline indicate the administration steps.

[0049] Figures 2A to 2C The cell preparation process of the exemplary MASCT method described in Example 1 is depicted. Figure 2A This is a schematic diagram illustrating the cell preparation process of a preferred embodiment of the MASCT method. Figure 2B An exemplary composition of an HCC antigen peptide library loaded in dendritic cells (DCs) for MASCT treatment in HCC patients is shown. Some tumor antigen peptides have been used in clinical trials of cancer immunotherapy; references on such DC vaccines, adoptive cell transfer (ACT), and peptide vaccines are included. OC: Ovarian cancer; BC: Breast cancer; PC: Pancreatic cancer; LC: Lung cancer; RCC: Renal cell carcinoma; HCC: Hepatocellular carcinoma. Figure 2C A list of epitopes contained in the HCC peptide library is shown.

[0050] Figure 3 This illustrates the cellular uptake of tumor antigen peptides by iDCs. Human monocyte-derived iDCs were shock-treated with a fluorescently labeled peptide of survivin (second column from the left, 2.5 μg / ml) for 2 hours, followed by DAPI (first column from the left) and... (Second column from the right) Labels were used to identify the cell nucleus and lysosomes separately. Fluorescence images were recorded using confocal microscopy (Leica TCSST5) with a scale bar of 7.5 μm, and these images represent four independent experiments.

[0051] Figures 4A to 4B Characterization of the mature DC prepared in Example 1 is shown. Figure 4A Flow cytometry results of DCs before (grey peak) and after (black peak) induction of maturation with TLR agonists are shown. Molecular markers targeted by the antibodies used to isolate cells in the flow cytometry experiments are shown above each chromatogram. The percentage of DCs with high expression levels (within the labeled range) of each molecular marker is indicated within each subplot. The results indicate that most mature DCs exhibit cell surface expression signatures to activate cytotoxic T cells. These DCs express MHC class II molecules and co-stimulatory signaling ligands CD86, CD80, and CD83, as well as the maturation receptor CCR7, but at lower levels of CD14, which is typically expressed in immature DCs. Figure 4B The cytokine secretion levels of the mature DCs prepared in Example 1 are shown. As expected of functional mature DCs, the DCs secrete high levels of the pro-inflammatory cytokine IL-12, but low levels of the immunosuppressive cytokine IL-10.

[0052] Figures 5A to 5E Characterization of the activated T cells prepared in Example 1 is shown. Figure 5A Cell counts based on trypan blue rejection assays show T cell proliferation 14 to 17 days later. Median values ​​from samples taken from 10 patients are shown. Figure 5B The percentage of T cell subsets in the co-culture is shown, indicating extremely low levels of T cells among activated T cells. REG Cells (CD4) + CD25 + FoxP3 + (0.4% ± 0.1%). Figure 5C A pie chart is shown, illustrating the percentage of T cell subsets co-expressing cytokines (IFNγ and TNFα) and granzyme B. The mean ± standard error of measurement (SEM) for five patients in each group is shown. Triple producers: dark gray; dual producers: light gray; single producer: black; non-producer: white. Figure 5D Three-dimensional flow cytometry chromatograms of activated T cells prepared from patient-derived PBMCs and co-cultured with shock-treated DCs were shown, followed by further stimulation with phorbol 12-tetradecanoate 13-acetate (PMA) for approximately 4 hours. Data represent five independent experiments. The activated T cells contained a large subset of CD3... + CD8 + Cytotoxic T cells, CD3 + CD4 + Helper T cells and CD3 + CD56+ NK T cells, most of which have high intracellular production of pro-inflammatory cytokines (IFNγ and TNFα) and protease granzyme B. Figure 5E Three-dimensional flow cytometry chromatograms of inactive T cells isolated from a patient after approximately 4 hours of PMA stimulation are shown. The inactive T cells exhibited only low levels of IFNγ, TNFα, and granzyme B.

[0053] Figures 6A to 6F The molecular and functional characterization of the activated T cells prepared in Example 1 is shown. Figure 6A The secretion of various cytokines by activated T cells is shown. Cells derived from HCC patients secrete large amounts of IFNγ and TNFα, but little or no IL10 and IL4. Mean ± SEM values ​​from 6 patients are shown. Figures 6B to 6C The study showed that CD3 levels in T cells isolated from HCC patients were higher than those in healthy donors. + CD8 + ( Figure 6B ) and CD3 + CD4 + ( Figure 6C The expression frequency of PD-1 on the surface of the subgroup was reduced. The expression percentage and statistical values ​​of 7 patients are shown. Figure 6D This shows the CD3+ of T cells after in vitro activation. + CD8 + The frequency of T cells expressing PD-1 decreased in the subset. Figure 6E This shows the CD3+ of T cells after in vitro activation. + CD4 + The frequency of T cells expressing PD-1 was decreased in the subset. The expression percentage and statistical values ​​for 7 patients are shown. Figure 6F This demonstrates HLA (or MHC)-restricted cytotoxicity in activated T cells. From HLA-A2 + Activated T cells generated by the patient's PBMCs (n=7, left group) showed resistance to the HCC cell line HepG2 (white bars, HLA-A2). + The cytotoxic activity level of HLA-A2 cells was greater than that of HuH-7 cells (hashed bars). - The cytotoxic activity level of HLA-A2 was measured. - Activated T cells generated from the patient's PBMCs (n=7, right group) exhibited similar levels of cytotoxicity against both cell lines. The relative ratio (E:T ratio) of effector T cells (the prepared activated T cells) to target cells (HepG2 or HuH-7 cells) in each cell lysis assay was approximately 40:1.

[0054] Figure 7A A flowchart is depicted illustrating the inclusion and exclusion of patients in a retrospective analysis of clinical data on MASCT treatment as described in Example 1.

[0055] Figure 7B A schematic diagram depicts a retrospective analysis of stage B (according to the Barcelona Clinical Hepatocellular Carcinoma Staging) HCC patients who received continuous treatment and were followed up regularly.

[0056] Figure 8A The characteristics, treatment, and RECIST evaluation of patients with hepatocellular carcinoma (stage B) in the control group analyzed in Example 1 are shown.

[0057] Figure 8B This study presents the characteristics, treatment, and RECIST evaluation of patients with hepatocellular carcinoma (stage B) who received only standard therapy during the 1-year period following diagnosis (control group, n=17).

[0058] Figure 9A The characteristics, treatment, and RECIST evaluation of patients with hepatocellular carcinoma (stage B) in the MASCT treatment group analyzed in Example 1 are shown.

[0059] Figure 9B This study presents the characteristics, treatment, and RECIST evaluation of patients with hepatocellular carcinoma (stage B) who received multiple MASCTs within one year of diagnosis (control group + MASCT, n = 15).

[0060] Figure 10A This presents a summary of the patient comparisons between the control group and the MASCT treatment group analyzed in Example 1.

[0061] Figure 10B The characteristics of patients with hepatocellular carcinoma (stage B) included in the retrospective analysis are shown.

[0062] Figures 11A to 11F The immune response induced in HCC patients after one or more MASCT treatments as described in Example 1 is depicted. Figure 11A The image shows the T values ​​in PBMCs of four patients after three MASCT treatments. REG The percentage decreased significantly. The expression percentages and statistical values ​​for four patients are shown. Figure 11B The percentage increase in proliferating T cells is shown in PBMC samples taken from seven different HCC patients who received MASCT treatment. Figure 11C The percentage increase in INFγ-producing cytotoxic T cells (CD8+INFγ+) is shown in PBMC samples taken from seven different HCC patients who received MASCT treatment. Figure 11DFlow cytometry chromatograms of PBMC samples from HCC patients treated with MASCT are shown. These results indicate that INFγ-producing cytotoxic T cells (CD8+INFγ+) co-express CD27 and CD28, thus demonstrating a high potential for acquiring immune memory to elicit HCC-specific T cell responses. Figure 11E The image shows CD8 samples taken from HCC patients after three MASCT treatments. + Intracellular production of IFNγ in T cells was increased. PBMCs were isolated from patients before and after three MASCT treatments to measure T cell responses. Figure 11F This study demonstrates specific T-cell proliferation, with T-cells progressively increasing in patients during multiple MASCT cell therapy treatments. PBMCs were isolated from patients before and after one and three MASCT treatments, respectively. T-cell proliferation in two patients was measured by EdU (5-ethynyl-2'-deoxyuridine) staining. Figures 11B to 11F In this study, specific T cell responses were measured after PBMCs were stimulated with an HCC antigen peptide library (HCC-pep). Control responses were measured after PBMCs were stimulated with an unrelated peptide library (ir-pep, control). All fold changes were calculated by normalizing specific response values ​​to control response values.

[0063] Figures 12A to 12D This demonstrates the specific immune response to the HCC antigen peptide in the patients of Example 1. In HCC patients who had undergone multiple MASCT treatments ( Figure 12A (n=6) and HCC patients who did not receive any MASCT treatment ( Figure 12B (n=5) The average specific immune response to a single HCC antigen peptide. Figure 12C The study shows the specific immune response to each HCC antigen peptide in a patient before (hollow bar) and after (striped bar) 3 MASCT treatments. Figure 12D The diagram shows a progressively enhanced specific immune response to each HCC antigen peptide in another HCC patient during multiple MASCT treatments (white bars: before treatment; gray: after 1 treatment; diagonal stripes: after 3 treatments). IFNγ secretion in patient PBMCs stimulated with individual HCC antigen peptides was calculated using ELISPOT. Results are expressed as mean ± SEM fold change in IFNγ secretion compared to unstimulated PBMCs. These values ​​indicate responding patients / total patients. Higher dashed lines indicate a cutoff value of 1.5-fold increment. W / O: Unstimulated.

[0064] Figures 13A to 13F Clinical data of WJ, a patient with metastatic cervical cancer, who underwent seven MASCT treatments are presented. Figures 13A to 13D This is the patient's ECT result. Figure 13A , Figure 13B Figure 13D shows the patient's ECT results obtained in December 2013 (before any MASCT treatment), June 2014 (after 10 local radiotherapy treatments followed by 3 MASCT treatments), and December 2014 (after a total of 7 MASCT treatments). The arrows and circles indicate metastatic sites on the right sacroiliac joint bone, showing a response to MASCT treatment with shrinkage of the metastatic tumor and no additional metastases. Figure 13E This diagram illustrates the specific immune responses to a cervical cancer antigen peptide library (CC pep library) and each antigen peptide within it after MASCT treatment. PBMCs were isolated from patients before any MASCT treatment and after a total of 6 MASCT treatments, and stimulated with the CC pep library and each individual antigen peptide within it. Each column represents the level of immune response of patients' PBMCs to each antigen peptide (or CC pep library) after MASCT treatment, as measured by the fold change in IFNγ (Y-axis) relative to the corresponding response of patients' PBMCs before MASCT treatment. W / O = Response without stimulation with any antigen peptide. ENV refers to the experiment using unrelated peptides. Dashed lines indicate the threshold of a non-elevated immune response as measured by the fold change in IFNγ. Arrows point to specific antigen peptides that elicit an elevated immune response as measured by the fold change in IFNγ. Figure 13F The ELISPOT results demonstrate that the patient-specific antigen peptide library further enhances the specific response.

[0065] Figure 14 A summary of the patient's treatment history in Example 2 is shown.

[0066] Figure 15 A schematic diagram of an exemplary experimental plan for preparing activated T cells is shown.

[0067] Figure 16A The FACS results of mature dendritic cells obtained using anti-PD-L1 antibody and anti-CD11c antibody are shown. Figure 16B The PD-1 expression levels of T cells were shown in PBMC samples from four different donors before and after activation 8 days prior.

[0068] Figure 17A The results show peptide-specific CD8 in co-culture samples stimulated with the antigen peptide once or twice, with or without the presence of an anti-PD-1 antibody (nivolumab). + The percentage of T cells. Figure 17B This demonstrates the functional peptide-specific CD8 in co-culture samples stimulated with the antigen peptide once or twice, with or without anti-PD-1 antibody. +The percentage of T cells. Figure 17C The results show peptide-specific CD8 in co-culture samples stimulated with the antigen peptide once or twice, with or without anti-PD-1 antibody (SHR-1210 or nivolumab). + The percentage of T cells. Figure 17D The results show peptide-specific CD8 in co-culture samples stimulated with the antigen peptide once or twice, with or without anti-PD-1 antibody (SHR-1210 or nivolumab). + The percentage of T cells.

[0069] Figure 18 A schematic diagram of an exemplary experimental plan for preparing activated T cells is shown.

[0070] Figure 19A The results show peptide-specific CD8 in co-cultured samples after one antigen peptide stimulation and 5 or 10 days of culture, with or without anti-PD-1 antibody (SHR-1210 or nivolumab). + The percentage of T cells. Figure 19B The results show peptide-specific CD8 levels in co-cultured samples, with or without anti-PD-1 antibodies (SHR-1210 or nivolumab), after one antigen peptide stimulation followed by 10 days of culture, or after two antigen peptide stimulations followed by 5 days of culture after the second stimulation. + The percentage of T cells. Figure 19C This demonstrates the functional peptide-specific CD8 in co-cultured samples, with or without anti-PD-1 antibodies (SHR-1210 or nivolumab), after one antigen peptide stimulation followed by 10 days of culture, or after two antigen peptide stimulations followed by 5 days of culture after the second stimulation. + The percentage of T cells.

[0071] Figures 20A to 20B The total T cell count over time is shown in co-cultures of PBMCs from two different donors with or without anti-PD-1 antibodies (SHR-1210 or nivolumab).

[0072] Figures 21A to 21B The percentage of cells expressing PD-1 on the cell surface over time in co-cultures of PBMCs from two different donors, with or without anti-PD-1 antibodies (SHR-1210 or nivolumab), is shown.

[0073] Figure 22 The statistics of next-generation sequencing (NGS) of 333 cancer-related genes in tumor samples are shown, along with the clinical evaluation of 5 patients in Example 5.

[0074] Figures 23A to 23B A DMM classification analysis of 35 tumor samples was described. Figure 23A The optimal number of classification groups was determined. Figure 23B DMM classification maps were plotted for 35 tumor samples. 14 samples were clustered into DMM group 1 (red, in box A), and 21 samples were clustered into DMM group 0 (green, in box B).

[0075] Figures 24A to 24B Cluster analysis of 35 tumor tissue samples was performed based on the mutational load of 333 oncogenes in each sample. Figure 24A A heatmap of 35 tumor samples clustered based on mutational load detected in each of the 333 cancer-related genes is shown, with cancer clinical type, MMR deficiency type (0: MMR deficient, 1: MMR healthy) and DMM group marked. Figure 24B It shows the order with Figure 24A A bar graph showing the HLA-I gene mutation load for each sample sorted in the same order of matching. The black lines mark the six mutations in the HLA-I mutation load.

[0076] Figures 25A to 25B Statistical analysis was performed to depict the HLA-I gene mutation load of each tumor tissue sample within the two DMM groups.

[0077] Figures 26A to 26E The CT scans of patient 3-HJL at five time points were depicted. Figure 26A The CT scan showed sarcoidosis in two lung lobes, the largest of which had a diameter of 2 cm. Figure 26B The study showed similar sarcoidosis after two cycles of chemotherapy. Figure 26C The study showed no improvement in pulmonary sarcoidosis after four cycles of chemotherapy. Figure 26D CT scans depicted the effects of PD-1 inhibitors during 3 cycles. After combination therapy with MASCT, the size of pulmonary sarcoidosis shrank by about 50%. Figure 26E Five cycles of PD-1 inhibitors were shown. After combined therapy with MASCT, sarcoidosis disappeared from both lung lobes.

[0078] Figures 27A to 27D The CT scans of the patient at four time points were depicted. Figure 27A The CT scan indicated a brain metastasis with a tumor size of approximately 3 cm. Figure 27B The image shows tumor shrinkage after radiotherapy. Figure 27C Follow-up CT scans showed tumor shrinkage and reduced cerebral edema. Figure 27D It showed further relief of the tumor and edema condition.

[0079] Figure 28 An overview flowchart of an exemplary precise MASCT is shown, which uses neoantigen peptides predicted based on sequencing results from patient tumor samples and prognostic data based on HLA mutation status.

[0080] Figure 29A The study shows candidate neoantigens for patients obtained from sequencing analysis of patient tumor samples. Figure 29B The results of continuous monitoring of circulating tumor cells (CTCs) in patients before and after MASCT treatment are shown. Figure 29C The results of ELISPOT, obtained by attacking PBMCs from patients with various antigenic peptides, are shown after the patients received three cycles of precise MASCT treatment.

[0081] Figure 30A The clinical characteristics of 45 patients with hepatocellular carcinoma (HCC) who underwent MASCT are shown.

[0082] Figure 30B The results of routine blood tests in 45 patients before and after MASCT treatment are shown.

[0083] Figure 30C The liver and kidney function parameters of 45 patients before and after MASCT treatment are shown.

[0084] Figure 30D The ALT and AST levels in eight HCC patients before MASCT treatment and during five MASCT treatments are shown. Detailed Implementation

[0085] This invention discloses novel cell-based immunotherapies, collectively referred to as multiantigen-specific cell therapy (MASCT), which can be used to treat various cancers, delay cancer progression in an individual, prevent cancer recurrence or metastasis in an individual, and / or alleviate cancer symptoms in an individual. In some embodiments, the methods utilize activated T cells induced by dendritic cells (DCs) loaded with multiple tumor antigen peptides. The T cells and DCs may, for example, be derived from the individual's own peripheral blood mononuclear cells (PBMCs). Multiantigen-loaded DCs can be prepared by exposing DCs (such as immature DCs) to multiple tumor antigen peptides, including common tumor antigen peptides and optionally cancer type-specific antigen peptides. Activated T cells can be prepared by co-culturing a population of T cells with multiantigen-loaded DCs. Optionally, the T cell population is contacted with an immune checkpoint inhibitor before and / or during co-culturing. Administration of activated T cells to an individual can thereby induce an adoptive immune response to tumor antigens in vivo. Optionally, multiantigen-loaded DCs can be administered to an individual to induce active immunity to tumor antigens. Alternatively, a PBMC-based MASCT method is provided, the method comprising administering activated PBMCs. Any of the MASCT methods described herein may be used alone or in combination with immune checkpoint inhibitors (such as PD-1 inhibitors) for the treatment of cancer in an individual.

[0086] This invention also provides precise MASCT treatment methods tailored to individuals undergoing treatment, such as the genetic profile of an individual's tumor. For example, individuals can be selected for MASCT treatment based on the mutational load in their tumors (such as in one or more MHC genes). Individuals can also be selected for MASCT treatment based on the number of neoantigens present in their tumors. In some cases, one or more neoantigens can be identified by sequencing a tumor sample taken from the individual. Neoantigen peptides can be designed based on the individual's neoantigens and incorporated into the plurality of tumor antigen peptides to provide precise MASCT to the individual. In some embodiments, the individual's specific immune response to each tumor antigen peptide is monitored after a MASCT treatment cycle, allowing the plurality of tumor antigen peptides to be tailored for future MASCT treatment cycles based on the strength of the specific immune response. Additionally, tumor-specific T-cell receptors (TCRs) can be cloned from the individual after MASCT. These T-cell receptors specifically recognize epitopes in the tumor antigen peptides and elicit strong specific immune responses, which can be used for further precise immunotherapy to the individual.

[0087] The MASCT methods and compositions presented herein (including PBMC-based MASCT and precise MASCT) alleviate many of the technical problems encountered in previous cancer immunotherapy methods discussed in the "Background Art" section. For example, by exposing dendritic cells (DCs) in vitro to a library of tumor antigen peptides, unlike many cancer vaccines or... The library contains a single tumor antigen, with a large number of tumor antigens presented by dendritic cells (DCs), allowing for a broader spectrum of responses to tumors with different antigen expression profiles within the same or different individuals, as long as the tumors share one or more specific tumor antigens from the library. The tumor antigen peptide library can be further customized to each individual's specific circumstances, such as cancer type, viral infection status, and response to individual antigen peptides, to achieve optimal efficacy in each treatment. Unlike cancer vaccines and DC-based therapies, MASCT treatment involves the administration of activated T cells, bypassing the in vivo T cell induction step of previous immunotherapies, which is often associated with weakened responses in cancer patients due to various immunodeficiencies caused by tumor cells; therefore, the MASCT approach can elicit a strong, rapid, and specific T cell response against cancer cells. Furthermore, activated T cells have very low T... REG The levels and expression of PD-1 are reduced, which decreases immunosuppression against cancer-aggressive T cells, thereby delaying cancer immune evasion. In summary, this invention provides an effective, durable, and widely applicable cancer immunotherapy approach to address the significant unmet medical needs of cancer patients, especially when current standard of care is ineffective or unavailable.

[0088] definition

[0089] Unless otherwise defined below, the terms used herein are used in the manner commonly used in the art.

[0090] As used herein, “treatment” is a method for obtaining beneficial or desired outcomes (including clinical outcomes). For the purposes of this invention, beneficial or desired clinical outcomes include, but are not limited to, one or more of the following: alleviating one or more symptoms caused by a disease, weakening the severity of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread of the disease (e.g., metastasis), preventing or delaying the onset or recurrence of the disease, delaying or slowing the progression of the disease, improving the disease state, providing remission of the disease (whether partial or complete), reducing the dosage of one or more other medications required to treat the disease, delaying the progression of the disease, improving quality of life, and / or prolonging survival. “Treatment” also encompasses alleviating the pathological consequences of cancer. The methods of this invention are contemplated to include any or more of these therapeutic aspects.

[0091] The terms "individual" or "patient" are used as synonyms herein to describe mammals, including humans. Individuals include, but are not limited to, humans, bovines, equines, felines, canines, rodents, or primates. In some embodiments, the individual is a human. In some embodiments, the individual suffers from a disease, such as cancer. In some embodiments, the individual requires treatment.

[0092] As used herein, “delayed” cancer development means the postponement, inhibition, delay, slowing, stabilization, and / or postponement of disease progression. This delay can vary in duration, depending on the patient’s history of the disease and / or the individual receiving treatment. As will be apparent to those skilled in the art, adequate or significant delay can effectively encompass prevention, in which case the individual does not develop the disease. A method for “delayed” cancer development is one that, when compared to not using the method, reduces the likelihood of disease development and / or the severity of the disease within a given timeframe. Such comparisons are typically based on clinical studies using statistically significant numbers of individuals. Cancer development can be detected using standard methods, including but not limited to computed tomography (CAT), magnetic resonance imaging (MRI), abdominal ultrasound, coagulation tests, arteriography, or biopsy. Development can also refer to cancer progression that may initially be undetectable, and includes occurrence, recurrence, and onset.

[0093] As understood in the art, an "effective amount" means an amount of composition (e.g., multi-antigen-loaded DCs, activated T cells, activated PMBCs, or isolated T cells), a first therapy, a second therapy, or a combination therapy sufficient to produce the desired therapeutic outcome (e.g., reducing the severity or duration of cancer, stabilizing the severity of cancer, or eliminating one or more symptoms of cancer). For therapeutic use, beneficial or desired outcomes include, for example: alleviating one or more symptoms (biochemical, histological, and / or behavioral) caused by the disease, including its complications and intermediate pathological phenotypes that occur during disease development; improving the quality of life of an individual suffering from the disease; reducing the dosage of other drugs required to treat the disease; enhancing the efficacy of another drug; delaying disease progression; and / or prolonging patient survival.

[0094] The method can be practiced in an adjuvant setting. An adjuvant setting refers to a clinical environment in which an individual has a history of a proliferative disease (particularly cancer) and generally (but not necessarily) responds to therapy, including but not limited to surgery (such as surgical resection), radiation therapy, and chemotherapy. However, due to their history of a proliferative disease (such as cancer), these individuals are considered at risk of disease progression. Treatment or administration in an adjuvant setting refers to subsequent treatment modalities. The level of risk (i.e., when an individual in an adjuvant setting is considered "high-risk" or "low-risk") depends on several factors, most typically the extent of the disease at the time of initial treatment.

[0095] The method described in this paper can also be practiced in a "neoadjuvant setting," that is, before initial / deterministic treatment. In some implementations, the individual has previously received treatment. In some implementations, the individual has not previously received treatment. In some implementations, the treatment is first-line therapy.

[0096] As used herein, the term "combination therapy" means the combined administration of a first agent with another agent. "Combination" refers to administering a treatment modality in addition to another, such as administering activated T cells or PBMCs as described herein in addition to administering another agent (such as an immune checkpoint inhibitor) to the same individual. Therefore, "combination" means administering a treatment modality before, during, or after delivery of another treatment modality to an individual. Such combinations are considered part of a single treatment regimen or decision.

[0097] As used herein, the term "simultaneous administration" means that the first and second therapies in a combination therapy are administered at intervals not exceeding about 15 minutes (such as not exceeding any one of about 10, 5, or 1 minute). When the first and second therapies are administered simultaneously, the first and second therapies may be contained in the same composition (e.g., a composition containing the first and second therapies) or in separate compositions (e.g., the first therapy is contained in one composition and the second therapy is contained in another composition).

[0098] As used herein, the term "sequential administration" means that the first and second therapies in a combination therapy are administered at intervals of more than about 15 minutes (such as more than about 20, 30, 40, 50, 60 or more minutes). The first or second therapy may be administered first. The first and second therapies are contained in separate compositions, which may be contained in the same or different packages or kits.

[0099] As used herein, the term “concurrent administration” means that the administration of the first therapy and the second therapy in a combination therapy overlaps with each other.

[0100] As used herein, "pharmaceutically acceptable" or "pharmaceutically compatible" means a material that is not biologically or otherwise undesirable; for example, the material can be incorporated into a pharmaceutical composition administered to an individual without causing any apparent adverse biological effects or interacting harmfully with any other component of the composition containing the material. Pharmaceutically acceptable carriers or excipients preferably meet the required standards for toxicological and manufacturing testing and / or are listed in the Inactive Ingredient Guide developed by the U.S. Food and Drug Administration.

[0101] As used herein, an "adverse event" or "AE" means any adverse medical event that occurs in an individual after receiving a commercially available drug or, in the case of an individual participating in a clinical trial, after receiving an investigational or non-investigative drug. An AE is not necessarily causally related to an individual's treatment. Therefore, an AE can be any unfavorable and unexpected sign, symptom, or illness temporarily associated with the use of the drug, regardless of whether they are considered to be related to the drug. AEs include, but are not limited to: exacerbation of a pre-existing condition; an increase in the frequency or intensity of a pre-existing event or symptom; a condition that is detected or diagnosed after administration of the study drug, even if the condition existed before the start of the study; and a persistent, chronic illness or symptom that was present at baseline and worsened after the start of the study. AEs generally do not include: medical or surgical procedures (e.g., surgery, endoscopy, tooth extraction, or intravenous infusion); however, the condition that caused the procedure is an adverse event; a pre-existing, non-worsening medical condition, symptom, or laboratory abnormality present or detected at the start of the study; hospitalization or procedures performed for elective purposes not related to an adverse medical event (e.g., hospitalization for cosmetic or elective surgery or admission for social care / facilities needs); the disease being studied or signs / symptoms associated with that disease, unless the individual's condition is more severe than expected; and overdose of a study drug without any clinical signs or symptoms.

[0102] As used herein, a “serious adverse event” or (SAE) means any adverse medical event at any dose, including but not limited to: a) fatal; b) life-threatening (defined as an immediate risk of death at the time of occurrence); c) resulting in permanent or severe disability or loss of mobility; d) requiring hospitalization or prolonging existing hospitalization (exceptions: hospitalization for selective treatment of a pre-existing condition that did not worsen during the study period is not considered an adverse event. Complications occurring during hospitalization are AEs, and if the complication prolongs hospitalization, the event is considered serious); e) congenital abnormalities / birth defects in the offspring of an individual receiving the drug; or f) conditions not included in the foregoing definitions but which may endanger the individual or require intervention to prevent one of the aforementioned outcomes, unless clearly related to the individual’s underlying disease. “Lack of efficacy” (disease progression) is not considered an AE or SAE. Signs and symptoms or clinical sequelae resulting from lack of efficacy should be reported when they meet the definition of an AE or SAE.

[0103] The following definitions can be used to evaluate response based on the target lesion: "Complete response" or "CR" means the disappearance of all target lesions; "Partial response" or "PR" means a reduction of at least 30% in the target lesion's SLD relative to the baseline sum of longest diameters (SLD); "Stable disease" or "SD" means that, relative to the minimum SLD since the start of treatment, the target lesion has neither reduced sufficiently to qualify as PR nor increased sufficiently to qualify as PD; and "Disease progression" or "PD" means an increase of at least 20% in the target lesion's SLD relative to the minimum SLD recorded since the start of treatment, or the appearance of one or more new lesions.

[0104] The following definitions of response assessment can be used to evaluate non-target lesions: "Complete response" or "CR" means the disappearance of all non-target lesions; "Stable disease" or "SD" means the presence of one or more non-target lesions that are not classified as CR or PD; and "Disease progression" or "PD" means that the "clear progression" of one or more existing non-target lesions or the appearance of one or more new lesions is considered disease progression (if an individual's PD is assessed at a specific time point based solely on the progression of one or more non-target lesions, additional criteria must be met).

[0105] "Progression-free survival" (PFS) refers to the length of time during and after treatment when cancer does not grow. PFS includes the amount of time an individual experiences a complete or partial response, as well as the amount of time an individual experiences disease stabilization.

[0106] In this article, "prediction" refers to the likelihood that an individual may respond favorably or unfavorably to a treatment plan.

[0107] As used in this article, “at the start of treatment” or “baseline” refers to the period at or before the first exposure to treatment.

[0108] As used herein, a “sample” refers to a composition containing molecules to be characterized and / or identified (e.g., based on physical, biochemical, chemical, physiological and / or genetic characteristics).

[0109] As used herein, “cell” should be understood not only to a specific individual cell, but also to its offspring or potential offspring. Due to mutations or environmental influences, certain modifications may occur in subsequent generations, so such offspring may not actually be identical to their parent cells, but are still included within the scope of the term as used herein.

[0110] The term "peptide" refers to an amino acid polymer (including fragments of proteins) of no more than about 100 amino acids. This polymer may be linear or branched, may contain modified amino acids, and / or may be interrupted by non-amino acid components. The term also covers naturally occurring or artificially modified amino acid polymers, including, for example, disulfide bond formation, glycosylation, esterification, acetylation, phosphorylation, or any other manipulation or modification. Also included within this term are, for example, one or more analogs of amino acids (including, for example, non-natural amino acids) and other modified polypeptides known in the art. The peptides described herein may be naturally occurring, i.e., derived from or derived from natural sources (e.g., blood), or may be synthetic (e.g., chemically synthesized or synthesized using recombinant DNA technology).

[0111] As used in this article, "multiple tumor antigen peptides", "multiple tumor antigen peptides", "library of tumor antigen peptides" and "tumor antigen peptide library" are used interchangeably and refer to a combination of more than one tumor antigen peptide.

[0112] As used herein, "dendritic cells loaded with multiple tumor antigen peptides" and "dendritic cells loaded with multiple antigens" are used interchangeably and refer to dendritic cells whose presentation of more than one of the multiple tumor antigen peptides has been enhanced. Similarly, "APC loaded with multiple tumor antigen peptides" and "APC loaded with multiple antigens" are used interchangeably and refer to antigen-processing cells whose presentation of more than one of the multiple tumor antigen peptides has been enhanced.

[0113] As used herein, “activated T cells” refers to a population of monoclonal (e.g., encoding the same TCR) or polyclonal (e.g., where clones encode different TCRs) T cells that have a T cell receptor that recognizes at least one tumor antigen peptide. Activated T cells may comprise one or more subtypes of T cells, including but not limited to cytotoxic T cells, helper T cells, natural killer T cells, γδ T cells, regulatory T cells, and memory T cells.

[0114] As used herein, “immune checkpoint inhibitor” refers to a molecule or agent (including antibodies) that inhibits or blocks inhibitory immune checkpoint molecules on immune cells (such as T cells or PBMCs) or tumor cells. “Immune checkpoint molecules” include molecules that upregulate immune signaling against tumor cells (i.e., “co-stimulatory molecules”) or molecules that downregulate immune signaling (i.e., “inhibitory immune checkpoint molecules”).

[0115] As used herein, “mutation burden” refers to the total number of mutations accumulated at one or more loci (such as genes) in the genome of a cell (such as tumor cells). These mutations include, but are not limited to, point mutations, insertions, deletions, frameshift mutations, gene fusions, and copy number variations. These mutations may or may not adversely affect the physical / chemical properties and / or function of the product encoded by the locus.

[0116] As used herein, "T-cell receptor" or "TCR" refers to an endogenous or engineered T-cell receptor containing an extracellular antigen-binding domain that binds to a specific antigenic epitope defined in an MHC molecule. A TCR may contain a TCRα polypeptide chain and a TCRβ polypeptide chain. "Tumor-specific TCR" refers to a TCR that specifically recognizes tumor antigens expressed by tumor cells.

[0117] As used herein, the term “HLA” or “human leukocyte antigen” refers to human genes encoding MHC (major histocompatibility complex) proteins on the cell surface responsible for regulating the immune system. “HLA-I” or “HLA class I” refers to human MHC class I genes, including the HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, and β2-microglobulin loci. “HLA-II” or “HLA class II” refers to human MHC class II genes, including the HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA1, HLA-DRB1, HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DM, HLA-DOA, and HLA-DOB loci.

[0118] The term “antibody” as used in this article is used in the broadest sense and specifically covers monoclonal antibodies (including full-length monoclonal antibodies), multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, provided they exhibit the desired biological activity.

[0119] An "antibody fragment" comprises a portion of a complete antibody, preferably containing its antigen-binding region. In some embodiments, the antibody fragments described herein are antigen-binding fragments. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; biantibodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0120] As used herein, the terms “specifically bind to,” “recognize,” “specifically recognize,” “target,” or “specific to” refer to measurable and reproducible interactions, such as binding between a target and an antibody, a receptor and a ligand, or a receptor and an epitope / MHC complex, which can determine the presence of a target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody that binds to or specifically binds to a target (which may be an epitope) is an antibody that binds to that target with greater affinity, avidity, ease, and / or longer duration than binding to other targets. In one embodiment, as measured, for example, by radioimmunoassay (RIA), the degree of antibody binding to an irrelevant target is less than about 10% of the antibody binding to the target. In some embodiments, antibodies that specifically bind to an antigenic peptide (or epitope) have a dissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, or ≤0.1 nM. In some embodiments, the antibody specifically binds to an epitope on a protein that is conserved across different species of the protein. In another embodiment, specific binding may include, but is not required to be, specific binding.

[0121] It should be understood that the aspects and embodiments of the invention described herein include "consisting of aspects and embodiments" and / or "consisting substantially of aspects and embodiments".

[0122] This article uses the word "about" to refer to a value or parameter, including (and describing) the change that refers to that value or parameter itself. For example, a description of "about X" includes a description of "X".

[0123] The term “about XY” as used in this article has the same meaning as “about X to about Y”.

[0124] As used in this article, mentioning "not" as a value or parameter generally means and describes "other than a certain value or parameter". For example, "This method is not used to treat type X cancer" means that this method is used to treat types of cancer other than X.

[0125] As used herein and in the appended claims, unless the context clearly specifies otherwise, the singular forms “a,” “or,” and “the” include multiple referents.

[0126] MASCT method

[0127] This invention provides a cell-based immunotherapy approach for treating cancer in an individual, collectively referred to as multiantigen-specific cell therapy (MASCT). The method utilizes antigen-presenting cells (APCs, such as dendritic cells) loaded with multiple tumor antigen peptides, and activated T cells induced by multiantigen-loaded APCs. Both the multiantigen-loaded APCs and the activated T cells can elicit tumor antigen-specific T cell responses in vivo and in vitro, including cytotoxic T cell and helper T cell responses, as well as the generation of immune memory through memory T cells. Therefore, in various embodiments of the MASCT method, multiantigen-loaded APCs (such as dendritic cells), activated T cells, co-cultures of APCs and T cells (including activated PBMCs), or any combination thereof, can be administered to an individual to treat cancer or neoplastic conditions, or to prevent tumor recurrence, progression, or metastasis.

[0128] In one aspect, the present invention provides a method for treating cancer in an individual, the method comprising administering an effective amount of activated T cells to the individual, wherein the activated T cells are prepared by co-culturing a population of T cells with a population of antigen-presenting cells (such as dendritic cells) loaded with a variety of tumor antigenic peptides. In some embodiments, the activated T cells are administered intravenously. In some embodiments, the activated T cells are administered at least three times. In some embodiments, the activated T cells and the antigen-presenting cell population are derived from the same individual. In some embodiments, the activated T cells and / or the antigen-presenting cell population are derived from an individual receiving treatment. In some embodiments, the antigen-presenting cell population is a population of dendritic cells, B cells, or macrophages. In some embodiments, the antigen-presenting cells are dendritic cells.

[0129] In some embodiments, a method for treating cancer in an individual is provided, the method comprising administering an effective amount of activated T cells to the individual, wherein the activated T cells are prepared by co-culturing a population of T cells with a population of antigen-presenting cells (such as dendritic cells) loaded with a variety of tumor antigenic peptides, and wherein the individual has previously been administered an effective amount of antigen-presenting cells loaded with said multiple tumor antigenic peptides. In some embodiments, the interval between the administration of antigen-presenting cells and the administration of activated T cells is about 7 days to about 21 days (e.g., about 7 days to about 14 days, or about 14 days to about 21 days). In some embodiments, antigen-presenting cells are administered subcutaneously. In some embodiments, antigen-presenting cells are administered at least three times. In some embodiments, activated T cells are administered intravenously. In some embodiments, activated T cells are administered at least three times. In some embodiments, the activated T cells and the population of antigen-presenting cells are derived from the same individual. In some embodiments, the activated T cells and / or the population of antigen-presenting cells are derived from an individual receiving treatment. In some embodiments, the population of antigen-presenting cells is a population of dendritic cells, B cells, or macrophages. In some embodiments, the antigen-presenting cells are dendritic cells.

[0130] In some embodiments, a method for treating cancer in an individual is provided, the method comprising: (a) administering to the individual an effective amount of antigen-presenting cells (such as dendritic cells) loaded with the plurality of tumor antigenic peptides; and (b) administering to the individual an effective amount of activated T cells, wherein the activated T cells are prepared by co-culturing a population of T cells with a population of antigen-presenting cells loaded with the plurality of tumor antigenic peptides. In some embodiments, the antigen-presenting cells are administered approximately 7 to approximately 21 days prior to administration of the activated T cells (e.g., approximately 7 to approximately 14 days, or approximately 14 to approximately 21 days). In some embodiments, the antigen-presenting cells are administered at least three times. In some embodiments, the activated T cells are administered intravenously. In some embodiments, the activated T cells are administered at least three times. In some embodiments, the activated T cells and the antigen-presenting cell population are derived from the same individual. In some embodiments, the activated T cells and / or the antigen-presenting cell population are derived from an individual receiving treatment. In some embodiments, the antigen-presenting cell population is a population of dendritic cells, B cells, or macrophages. In some embodiments, the antigen-presenting cells are dendritic cells.

[0131] Any suitable antigen-presenting cells can be used in the MASCT method, including but not limited to dendritic cells, B cells, and macrophages. In some embodiments, the antigen-presenting cells are dendritic cells.

[0132] Therefore, in some embodiments, a method for treating cancer in an individual is provided, the method comprising administering an effective amount of activated T cells to the individual, wherein the activated T cells are prepared by co-culturing a population of T cells with a population of dendritic cells loaded with a plurality of tumor antigen peptides. In some embodiments, the activated T cells are prepared by co-culturing a population of T cells with a population of dendritic cells loaded with the plurality of tumor antigen peptides prior to administration. In some embodiments, the dendritic cells loaded with the plurality of tumor antigen peptides are administered subcutaneously. In some embodiments, the dendritic cells loaded with the plurality of tumor antigen peptides are administered at least three times. In some embodiments, the activated T cells are administered intravenously. In some embodiments, the activated T cells are administered at least three times. In some embodiments, the activated T cells and the dendritic cell population are derived from the same individual. In some embodiments, the activated T cells and / or the dendritic cell population are derived from an individual receiving treatment.

[0133] In some embodiments, a method for treating cancer in an individual is provided, the method comprising administering an effective amount of activated T cells to the individual, wherein the activated T cells are prepared by co-culturing a population of T cells with a population of dendritic cells loaded with a plurality of tumor antigen peptides, and wherein the individual has previously been administered an effective amount of dendritic cells loaded with the plurality of tumor antigen peptides. In some embodiments, the dendritic cells are administered approximately 7 to approximately 21 days prior to the administration of the activated T cells (e.g., approximately 7 to approximately 14 days, or approximately 14 to approximately 21 days). In some embodiments, the dendritic cells loaded with the plurality of tumor antigen peptides are administered subcutaneously. In some embodiments, the dendritic cells loaded with the plurality of tumor antigen peptides are administered at least three times. In some embodiments, the activated T cells are administered intravenously. In some embodiments, the activated T cells are administered at least three times. In some embodiments, the activated T cells and the dendritic cell population are derived from the same individual. In some embodiments, the activated T cells and / or the dendritic cell population are derived from an individual receiving treatment.

[0134] In some embodiments, a method for treating cancer in an individual is provided, the method comprising: (a) administering to the individual an effective amount of dendritic cells loaded with a plurality of tumor antigen peptides; and (b) administering to the individual an effective amount of activated T cells, wherein the activated T cells are prepared by co-culturing a population of T cells with a population of dendritic cells loaded with the plurality of tumor antigen peptides. In some embodiments, the dendritic cells are administered approximately 7 to approximately 21 days prior to the administration of the activated T cells (e.g., approximately 7 to approximately 14 days, or approximately 14 to approximately 21 days). In some embodiments, the dendritic cells loaded with the plurality of tumor antigen peptides are administered subcutaneously. In some embodiments, the dendritic cells loaded with the plurality of tumor antigen peptides are administered at least three times. In some embodiments, the activated T cells are administered intravenously. In some embodiments, the activated T cells are administered at least three times. In some embodiments, the activated T cells and the population of dendritic cells are derived from the same individual. In some embodiments, the activated T cells and / or the population of dendritic cells are derived from an individual receiving treatment.

[0135] In addition to one or more administration steps, some embodiments of the MASCT method include one or both of the following cell preparation steps: 1) preparing a population of antigen-presenting cells (such as dendritic cells) loaded with the plurality of tumor antigen peptides; and 2) preparing activated T cells. In some embodiments, activated T cells are prepared by co-culturing the T cell population with the antigen-presenting cell population loaded with the plurality of tumor antigen peptides prior to administration. In some embodiments, the T cell population is co-cultured with the antigen-presenting cell population loaded with the plurality of tumor antigen peptides for about 7 days to about 21 days (e.g., about 7 days to about 14 days, about 14 days to about 21 days, about 10 days, about 14 days, or about 21 days). In some embodiments, the antigen-presenting cell population loaded with the plurality of tumor antigen peptides is prepared by contacting the antigen-presenting cell population with the plurality of tumor antigen peptides. In some embodiments, the antigen-presenting cell population is contacted with the plurality of tumor antigen peptides in the presence of a composition that facilitates the uptake of the plurality of tumor antigen peptides by the antigen-presenting cells. In some embodiments, the T cell population is contacted with an immune checkpoint inhibitor prior to co-culturing. In some implementations, T cell populations are co-cultured with antigen-presenting cell populations in the presence of immune checkpoint inhibitors. In some implementations, the T cell populations and antigen-presenting cell populations are derived from the same individual. In some implementations, the T cell populations and antigen-presenting cell populations are derived from an individual receiving treatment.

[0136] Therefore, in some embodiments, a method for treating cancer in an individual is provided, the method comprising: (a) co-culturing a population of dendritic cells loaded with multiple tumor antigen peptides with a population of T cells to obtain a population of activated T cells; and (b) administering an effective amount of activated T cells to the individual. In some embodiments, the dendritic cells loaded with the multiple tumor antigen peptides are administered subcutaneously. In some embodiments, the dendritic cells loaded with the multiple tumor antigen peptides are administered at least three times. In some embodiments, the activated T cells are administered intravenously. In some embodiments, the activated T cells are administered at least three times. In some embodiments, the T cell population is co-cultured with the dendritic cell population loaded with the multiple tumor antigen peptides for about 7 days to about 21 days (e.g., about 7 days to about 14 days, about 14 days to about 21 days, about 10 days, about 14 days, or about 21 days). In some embodiments, the T cell population is derived from the non-adhesive portion of a population of peripheral blood mononuclear cells (PBMCs). In some embodiments, co-culture further includes contacting activated T cells with a variety of cytokines (such as IL-2, IL-7, IL-15, IL-21, or any combination thereof) and optionally an anti-CD3 antibody. In some embodiments, the T cell population is contacted with an immune checkpoint inhibitor (such as an inhibitor of PD-1, PD-L1, or CTLA-4) before and / or during co-culture. In some embodiments, a dendritic cell population loaded with the various tumor antigen peptides is prepared by contacting a dendritic cell population with the various tumor antigen peptides. In some embodiments, the T cell population and the dendritic cell population are derived from the same individual. In some embodiments, the T cell population, dendritic cell population, PBMC population, or any combination thereof are derived from a patient receiving treatment.

[0137] In some embodiments, a method for treating cancer in an individual is provided, the method comprising: (a) co-culturing a population of dendritic cells loaded with a plurality of tumor antigen peptides with a population of T cells to obtain a population of activated T cells; and (b) administering an effective amount of activated T cells to an individual, wherein the individual has previously been administered an effective amount of dendritic cells loaded with the plurality of tumor antigen peptides. In some embodiments, the interval between the administration of dendritic cells and the administration of activated T cells is about 7 days to about 21 days (e.g., about 7 days to about 14 days, about 14 days to about 21 days, about 10 days, or about 14 days). In some embodiments, the dendritic cells loaded with the plurality of tumor antigen peptides are administered subcutaneously. In some embodiments, the dendritic cells loaded with the plurality of tumor antigen peptides are administered at least three times. In some embodiments, the activated T cells are administered intravenously. In some embodiments, the activated T cells are administered at least three times. In some embodiments, the population of T cells is co-cultured with the population of dendritic cells loaded with the plurality of tumor antigen peptides for about 7 days to about 21 days (e.g., about 7 days to about 14 days, about 14 days to about 21 days, or about 10 days). In some embodiments, the T cell population is derived from the non-adhesive portion of a peripheral blood mononuclear cell (PBMC) population. In some embodiments, co-culture further includes contacting activated T cells with a variety of cytokines (such as IL-2, IL-7, IL-15, IL-21, or any combination thereof) and optionally an anti-CD3 antibody. In some embodiments, the T cell population is contacted with an immune checkpoint inhibitor (such as an inhibitor of PD-1, PD-L1, or CTLA-4) before and / or during co-culture. In some embodiments, a dendritic cell population loaded with the various tumor antigen peptides is prepared by contacting a dendritic cell population with the aforementioned multiple tumor antigen peptides. In some embodiments, the T cell population and the dendritic cell population are derived from the same individual. In some embodiments, the T cell population, dendritic cell population, PBMC population, or any combination thereof are derived from a patient receiving treatment.

[0138] In some embodiments, a method for treating cancer in an individual is provided, the method comprising: (a) administering to the individual an effective amount of dendritic cells loaded with a plurality of tumor antigen peptides; (b) co-culturing a population of dendritic cells loaded with the plurality of tumor antigen peptides with a population of T cells to obtain a population of activated T cells; and (c) administering to the individual an effective amount of activated T cells. In some embodiments, the interval between the administration of dendritic cells and the administration of activated T cells is about 7 days to about 21 days (e.g., about 7 days to about 14 days, about 14 days to about 21 days, about 10 days, or about 14 days). In some embodiments, the dendritic cells loaded with the plurality of tumor antigen peptides are administered subcutaneously. In some embodiments, the dendritic cells loaded with the plurality of tumor antigen peptides are administered at least three times. In some embodiments, the activated T cells are administered intravenously. In some embodiments, the activated T cells are administered at least three times. In some embodiments, a population of T cells is co-cultured with a population of dendritic cells loaded with the various tumor antigen peptides for approximately 7 to approximately 21 days (e.g., approximately 7 to approximately 10 days, approximately 10 to approximately 15 days, approximately 15 to approximately 21 days, approximately 14 to approximately 21 days, or approximately 10 days). In some embodiments, the T cell population is derived from the non-adhesive portion of a population of peripheral blood mononuclear cells (PBMCs). In some embodiments, co-culture further includes contacting activated T cells with a variety of cytokines (such as IL-2, IL-7, IL-15, IL-21, or any combination thereof) and optionally an anti-CD3 antibody. In some embodiments, the T cell population is contacted with an immune checkpoint inhibitor (such as an inhibitor of PD-1, PD-L1, or CTLA-4) before and / or during co-culture. In some embodiments, a population of dendritic cells loaded with the various tumor antigen peptides is prepared by contacting the dendritic cell population with the various tumor antigen peptides. In some embodiments, the T cell population and the dendritic cell population are derived from the same individual. In some implementations, the T cell population, dendritic cell population, PBMC population, or any combination thereof are derived from the individual receiving treatment.

[0139] In some embodiments, a method for treating cancer in an individual is provided, the method comprising: (a) preparing a dendritic cell population loaded with a plurality of tumor antigen peptides; (b) co-culturing the dendritic cell population loaded with the plurality of tumor antigen peptides with a T cell population to obtain an activated T cell population; and (c) administering an effective amount of activated T cells to the individual. In some embodiments, the dendritic cells loaded with the plurality of tumor antigen peptides are administered subcutaneously. In some embodiments, the dendritic cells loaded with the plurality of tumor antigen peptides are administered at least three times. In some embodiments, the activated T cells are administered intravenously. In some embodiments, the activated T cells are administered at least three times. In some embodiments, the T cell population is co-cultured with the dendritic cell population loaded with the plurality of tumor antigen peptides for about 7 days to about 21 days (e.g., about 7 days to about 14 days, about 14 days to about 21 days, or about 10 days). In some embodiments, the T cell population is derived from the non-adhesive portion of a peripheral blood mononuclear cell (PBMC) population. In some embodiments, co-culture further includes contacting activated T cells with a variety of cytokines (such as IL-2, IL-7, IL-15, IL-21, or any combination thereof) and optionally an anti-CD3 antibody. In some embodiments, the T cell population is contacted with an immune checkpoint inhibitor (such as an inhibitor of PD-1, PD-L1, or CTLA-4) before and / or during co-culture. In some embodiments, a dendritic cell population loaded with the various tumor antigen peptides is prepared by contacting a dendritic cell population with the various tumor antigen peptides. In some embodiments, the T cell population and the dendritic cell population are derived from the same individual. In some embodiments, the T cell population, dendritic cell population, PBMC population, or any combination thereof are derived from a patient receiving treatment.

[0140] In some embodiments, a method for treating cancer in an individual is provided, the method comprising: (a) preparing a population of dendritic cells loaded with a plurality of tumor antigen peptides; (b) co-culturing the population of dendritic cells loaded with the plurality of tumor antigen peptides with a population of T cells to obtain a population of activated T cells; and (c) administering an effective amount of activated T cells to an individual, wherein the individual has previously been administered an effective amount of dendritic cells loaded with the plurality of tumor antigen peptides. In some embodiments, the interval between the administration of dendritic cells and the administration of activated T cells is about 7 days to about 21 days (e.g., about 7 days to about 14 days, about 14 days to about 21 days, about 10 days, or about 14 days). In some embodiments, the dendritic cells loaded with the plurality of tumor antigen peptides are administered subcutaneously. In some embodiments, the dendritic cells loaded with the plurality of tumor antigen peptides are administered at least three times. In some embodiments, the activated T cells are administered intravenously. In some embodiments, the activated T cells are administered at least three times. In some embodiments, a population of T cells is co-cultured with a population of dendritic cells loaded with the various tumor antigen peptides for approximately 7 to approximately 21 days (e.g., approximately 7 to approximately 14 days, approximately 14 to approximately 21 days, or approximately 10 days). In some embodiments, the T cell population is derived from the non-adhesive portion of a population of peripheral blood mononuclear cells (PBMCs). In some embodiments, co-culture further includes contacting activated T cells with a variety of cytokines (such as IL-2, IL-7, IL-15, IL-21, or any combination thereof) and optionally an anti-CD3 antibody. In some embodiments, the T cell population is contacted with an immune checkpoint inhibitor (such as an inhibitor of PD-1, PD-L1, or CTLA-4) before and / or during co-culture. In some embodiments, a population of dendritic cells loaded with the various tumor antigen peptides is prepared by contacting the dendritic cell population with the various tumor antigen peptides. In some embodiments, the T cell population and the dendritic cell population are derived from the same individual. In some embodiments, the T cell population, the dendritic cell population, the PBMC population, or any combination thereof are derived from an individual receiving treatment.

[0141] In some embodiments, a method for treating cancer in an individual is provided, the method comprising: (a) preparing a population of dendritic cells loaded with a plurality of tumor antigen peptides; (b) administering an effective amount of the dendritic cells loaded with the plurality of tumor antigen peptides to the individual; (c) co-culturing the population of dendritic cells loaded with the plurality of tumor antigen peptides with a population of T cells to obtain a population of activated T cells; and (d) administering an effective amount of activated T cells to the individual. In some embodiments, the interval between the administration of dendritic cells and the administration of activated T cells is about 7 days to about 21 days (e.g., about 7 days to about 14 days, about 14 days to about 21 days, about 10 days, or about 14 days). In some embodiments, the dendritic cells loaded with the plurality of tumor antigen peptides are administered subcutaneously. In some embodiments, the dendritic cells loaded with the plurality of tumor antigen peptides are administered at least three times. In some embodiments, the activated T cells are administered intravenously. In some embodiments, the activated T cells are administered at least three times. In some embodiments, a population of T cells is co-cultured with a population of dendritic cells loaded with the various tumor antigen peptides for approximately 7 to approximately 21 days (e.g., approximately 7 to approximately 14 days, approximately 14 to approximately 21 days, or approximately 10 days). In some embodiments, the T cell population is derived from the non-adhesive portion of a population of peripheral blood mononuclear cells (PBMCs). In some embodiments, co-culture further includes contacting activated T cells with a variety of cytokines (such as IL-2, IL-7, IL-15, IL-21, or any combination thereof) and optionally an anti-CD3 antibody. In some embodiments, the T cell population is contacted with an immune checkpoint inhibitor (such as an inhibitor of PD-1, PD-L1, or CTLA-4) before and / or during co-culture. In some embodiments, the T cell population and the dendritic cell population are derived from the same individual. In some embodiments, the dendritic cell population loaded with the various tumor antigen peptides is prepared by contacting the dendritic cell population with the various tumor antigen peptides. In some embodiments, the T cell population and the dendritic cell population are derived from the same individual. In some implementations, the T cell population, dendritic cell population, PBMC population, or any combination thereof are derived from the individual receiving treatment.

[0142] In some embodiments, a method for treating cancer in an individual is provided, the method comprising: (a) inducing a population of monocytes to differentiate into a population of dendritic cells; (b) contacting the dendritic cell population with a plurality of tumor antigen peptides to obtain a population of dendritic cells loaded with said plurality of tumor antigen peptides; (c) co-culturing the population of dendritic cells loaded with said plurality of tumor antigen peptides with a population of non-adhesive PBMCs to obtain a population of activated T cells, wherein the monocyte population and the non-adhesive PBMCs are obtained from a population of PBMCs; and (d) administering an effective amount of activated T cells to the individual. In some embodiments, the dendritic cells loaded with said plurality of tumor antigen peptides are administered subcutaneously. In some embodiments, the dendritic cells loaded with said plurality of tumor antigen peptides are administered at least three times. In some embodiments, the activated T cells are administered intravenously. In some embodiments, the activated T cells are administered at least three times. In some embodiments, the T cell population is co-cultured with the population of dendritic cells loaded with said plurality of tumor antigen peptides for about 7 days to about 21 days (e.g., about 7 days to about 14 days, about 14 days to about 21 days, or about 10 days). In some embodiments, co-culture further includes contacting activated T cells with a variety of cytokines (such as IL-2, IL-7, IL-15, IL-21, or any combination thereof) and optionally an anti-CD3 antibody. In some embodiments, non-adherent PBMCs are contacted with immune checkpoint inhibitors (such as inhibitors of PD-1, PD-L1, or CTLA-4) before and / or during co-culture. In some embodiments, the PBMCs are derived from a patient receiving treatment.

[0143] In some embodiments, a method for treating cancer in an individual is provided, the method comprising: (a) inducing a population of monocytes to differentiate into a population of dendritic cells; (b) contacting the dendritic cell population with a plurality of tumor antigen peptides to obtain a population of dendritic cells loaded with said plurality of tumor antigen peptides; (c) co-culturing the population of dendritic cells loaded with said plurality of tumor antigen peptides with a population of non-adhesive PBMCs to obtain a population of activated T cells; and (d) administering an effective amount of activated T cells to the individual, wherein the monocyte population and the non-adhesive PBMC population are obtained from a population of PBMCs, and wherein the individual has previously been administered an effective amount of dendritic cells loaded with said plurality of tumor antigen peptides. In some embodiments, the interval between the administration of dendritic cells and the administration of activated T cells is about 7 days to about 21 days (e.g., about 7 days to about 14 days, about 14 days to about 21 days, about 10 days, or about 14 days). In some embodiments, the dendritic cells loaded with said plurality of tumor antigen peptides are administered subcutaneously. In some embodiments, the dendritic cells loaded with said plurality of tumor antigen peptides are administered at least three times. In some embodiments, the activated T cells are administered intravenously. In some embodiments, activated T cells are administered at least three times. In some embodiments, co-culture lasts from about 7 days to about 21 days (e.g., from about 7 days to about 14 days, from about 14 days to about 21 days, or about 10 days). In some embodiments, co-culture also includes contacting activated T cells with a variety of cytokines (such as IL-2, IL-7, IL-15, IL-21, or any combination thereof) and optionally an anti-CD3 antibody. In some embodiments, non-adhesive PBMC populations are contacted with immune checkpoint inhibitors (such as inhibitors of PD-1, PD-L1, or CTLA-4) before and / or during co-culture. In some embodiments, the PBMC populations and / or dendritic cell populations are obtained from the individual receiving treatment.

[0144] In some embodiments, a method for treating cancer in an individual is provided, the method comprising: (a) inducing a population of monocytes to differentiate into a population of dendritic cells; (b) contacting the dendritic cell population with a plurality of tumor antigen peptides to obtain a population of dendritic cells loaded with said plurality of tumor antigen peptides; (c) administering an effective amount of the dendritic cells loaded with said plurality of tumor antigen peptides to the individual; (d) co-culturing the population of dendritic cells loaded with said plurality of tumor antigen peptides with a population of non-adhesive PBMCs to obtain a population of activated T cells; and (e) administering an effective amount of activated T cells to the individual, wherein the monocyte population and the non-adhesive PBMC population are obtained from a population of PBMCs. In some embodiments, the interval between the administration of dendritic cells and the administration of activated T cells is about 7 days to about 21 days (e.g., about 7 days to about 14 days, about 14 days to about 21 days, about 10 days, or about 14 days). In some embodiments, the dendritic cells loaded with said plurality of tumor antigen peptides are administered subcutaneously. In some embodiments, the dendritic cells loaded with said plurality of tumor antigen peptides are administered at least three times. In some embodiments, the activated T cells are administered intravenously. In some embodiments, activated T cells are administered at least three times. In some embodiments, co-culture lasts from about 7 days to about 21 days (e.g., from about 7 days to about 14 days, from about 14 days to about 21 days, or about 10 days). In some embodiments, co-culture also includes contacting the activated T cells with a variety of cytokines (such as IL-2, IL-7, IL-15, IL-21, or any combination thereof) and optionally an anti-CD3 antibody. In some embodiments, the non-adhesive PBMC population is contacted with an immune checkpoint inhibitor (such as an inhibitor of PD-1, PD-L1, or CTLA-4) before and / or during co-culture. In some embodiments, the PBMC population is obtained from a patient receiving treatment.

[0145] The methods described herein are applicable to the treatment of various cancers, such as those described herein, including cancers selected from the following: hepatocellular carcinoma, cervical cancer, lung cancer, colorectal cancer, lymphoma, kidney cancer, breast cancer, pancreatic cancer, gastric cancer, esophageal cancer, ovarian cancer, prostate cancer, nasopharyngeal carcinoma, melanoma, and brain cancer. The methods are applicable to cancers at all stages, including early, advanced, and metastatic cancers. In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a liquid-filled tumor.

[0146] In some embodiments, the method reduces the severity of one or more cancer-related symptoms by at least one percentage point, approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%, compared to corresponding symptoms in the same individual before treatment or compared to corresponding symptoms in other individuals who did not receive the treatment. In some embodiments, the method delays cancer progression.

[0147] Examples of cancers treatable by the methods described herein include, but are not limited to, adrenocortical carcinoma, idiopathic medullary metaplasia, anal cancer, appendiceal cancer, astrocytoma (e.g., cerebellum and cerebrum), basal cell carcinoma, bile duct cancer (e.g., extrahepatic), bladder cancer, bone cancer (osteosarcoma and malignant fibrous histiocytoma), brain tumors (e.g., glioma, brainstem glioma, cerebellar or cerebral astrocytoma (e.g., pilocytic astrocytoma, diffuse astrocytoma, anaplastic (malignant) astrocytoma), malignant glioma, ependymoma, oligodendroglioma, meningioma, craniopharyngioma, hemangioblastoma, medulloblastoma, supratentorial primitive neuroectodermal tumor, optic pathway and hypothalamic glioma. Gastrointestinal stromal tumors (including glioblastomas), breast cancer, bronchial adenoma / carcinoid tumors, carcinoid tumors (e.g., gastrointestinal carcinoid tumors), metastatic cancers of unknown primary origin, central nervous system lymphomas, cervical cancer, colon cancer, colorectal cancer, chronic myeloproliferative disorders, endometrial cancer (e.g., uterine cancer), ependymoma, esophageal cancer, Ewing family tumors, eye cancer (e.g., intraocular melanoma and retinoblastoma), gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), germ cell tumors (e.g., extracranial, extragonadal, ovarian), gestational trophoblastic tumors, head and neck cancer, hepatocellular carcinoma (e.g., liver cancer). Cancers include: heptaphylax, hypopharyngeal cancer, pancreatic islet cell carcinoma (endocrine pancreas), laryngeal cancer, leukemia (excluding T-cell leukemia), lip and oral cavity cancer, oral cancer, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), lymphoma (excluding T-cell lymphoma), medulloblastoma, melanoma, mesothelioma, metastatic squamous neck cancer, mouth cancer, and multiple endocrine adenoma syndrome. Myelodysplastic syndrome, myelodysplastic / myeloproliferative disorders, nasal cavity and sinus carcinoma, nasopharyngeal carcinoma, neuroblastoma, neuroendocrine carcinoma, oropharyngeal carcinoma, ovarian cancer (e.g., ovarian epithelial carcinoma, ovarian germ cell tumor, low-grade malignant potential ovarian tumor), pancreatic cancer, parathyroid carcinoma, penile cancer, peritoneal cancer, pharyngeal cancer, pheochromocytoma, pineal blastoma and supratentorial primitive neuroectodermal tumor, pituitary adenoma, pleural pulmonary blastoma, primary central nervous system lymphoma (small Glioma, pulmonary lymphangioleiomyomatosis, rectal cancer, renal cancer, renal pelvis and ureter cancer (transitional cell carcinoma), rhabdomyosarcoma, salivary gland cancer, skin cancer (e.g., non-melanoma (e.g., squamous cell carcinoma), melanoma and Merkel cell carcinoma), small bowel cancer, squamous cell carcinoma, testicular cancer, laryngeal cancer, thyroid cancer, tuberous sclerosis, urethral cancer, vaginal cancer, vulvar cancer, Wilms' tumor, abnormal angiogenesis associated with scarring nevus, edema (such as that associated with brain tumors), and Megs syndrome.

[0148] Therefore, in some embodiments, a method for treating hepatocellular carcinoma (HCC) in an individual is provided, the method comprising administering an effective amount of activated T cells to the individual, wherein the activated T cells are prepared by co-culturing a population of T cells with a population of antigen-presenting cells (such as dendritic cells) loaded with multiple tumor antigen peptides. In some embodiments, the individual has previously been administered an effective amount of antigen-presenting cells loaded with said multiple tumor antigen peptides. In some embodiments, the method further comprises administering an effective amount of antigen-presenting cells loaded with said multiple tumor antigen peptides to the individual prior to the administration of the activated T cells. In some embodiments, HCC is early HCC, non-metastatic HCC, primary HCC, advanced HCC, locally advanced HCC, metastatic HCC, HCC in remission, or recurrent HCC. In some implementations, HCC is defined as localized resectable (i.e., a tumor confined to a portion of the liver that can be surgically removed), localized unresectable (i.e., a localized tumor that may be unresectable due to involvement of important vascular structures or liver damage), or unresectable (i.e., a tumor involving all lobes of the liver and / or that has spread to other organs (e.g., lungs, lymph nodes, bones). In some implementations, according to the TNM classification, HCC is defined as stage I tumor (a single tumor without vascular invasion), stage II tumor (a single or multiple tumors with vascular invasion, all no larger than 5 cm), stage III tumor (any tumor with vascular invasion). Multiple tumors larger than 5 cm, or tumors involving the portal vein or main branches of the hepatic vein; stage IV tumors (tumors directly invading adjacent organs other than the gallbladder or with perforation of the visceral peritoneum); N1 tumors (regional lymph node metastases); or M1 tumors (distant metastases). In some implementations, HCC is T1, T2, T3, or T4 stage HCC according to the AJCC (American Joint Committee on Cancer) staging criteria. In some implementations, HCC is any of hepatocellular carcinoma, fibrolamellar variant of HCC, and mixed hepatocellular carcinoma-cholangiocarcinoma. In some implementations, HCC is caused by hepatitis B virus (HBV) infection.

[0149] In some embodiments, a method for treating lung cancer in an individual is provided, the method comprising administering an effective amount of activated T cells to the individual, wherein the activated T cells are prepared by co-culturing a population of T cells with a population of antigen-presenting cells (such as dendritic cells) loaded with multiple tumor antigen peptides, such as in the presence of an immune checkpoint inhibitor. In some embodiments, the individual has previously been administered an effective amount of antigen-presenting cells loaded with said multiple tumor antigen peptides. In some embodiments, the method further comprises administering an effective amount of antigen-presenting cells loaded with said multiple tumor antigen peptides to the individual prior to the administration of the activated T cells. In some embodiments, the lung cancer is non-small cell lung cancer (NSCLC). Examples of NSCLC include, but are not limited to, large cell carcinoma (e.g., large cell neuroendocrine carcinoma, complex large cell neuroendocrine carcinoma, basal cell-like carcinoma, lymphoepithelioma-like carcinoma, clear cell carcinoma, and large cell carcinoma with rhabdomyomorph phenotype), adenocarcinoma (e.g., acinar, papillary (e.g., bronchioloalveolar carcinoma, non-mucinous, mucinous, mixed mucinous and non-mucinous, and undetermined cell types), solid adenocarcinoma with mucinous involvement, adenocarcinoma with mixed subtypes, well-differentiated fetal adenocarcinoma, mucinous (colloid) adenocarcinoma, mucinous cystadenocarcinoma, signet ring adenocarcinoma, and clear cell adenocarcinoma), pulmonary neuroendocrine tumors, and squamous cell carcinoma (e.g., papillary, clear cell, small cell, and basal cell-like). In some embodiments, according to the TNM classification, NSCLC may be a T-stage tumor (primary tumor), an N-stage tumor (regional lymph node), or an M-stage tumor (distant metastasis).

[0150] In some embodiments, lung cancer is a carcinoid tumor (typical or atypical), adenosquamous carcinoma, cylindrical tumor, or salivary gland carcinoma (e.g., adenoid cystic carcinoma or mucoepidermoid carcinoma). In some embodiments, lung cancer is a carcinoma with pleomorphic, sarcomatoid, or sarcomatous components (e.g., carcinoma with spindle and / or giant cells, spindle cell carcinoma, giant cell carcinoma, carcinosarcoma, or pulmonary blastoma). In some embodiments, lung cancer is small cell lung cancer (SCLC; also known as oat cell carcinoma). Small cell lung cancer can be localized, extensive-stage, or recurrent. In some implementations, an individual may be a human with a gene, genetic mutation, or polymorphism suspected or proven to be associated with lung cancer (e.g., SASH1, LATS1, IGF2R, PARK2, KRAS, PTEN, Kras2, Krag, Pas1, ERCC1, XPD, IL8RA, EGFR, α1-AD, EPHX, MMP1, MMP2, MMP3, MMP12, IL1β, RAS, and / or AKT) or with one or more additional copies of a gene associated with lung cancer.

[0151] In some embodiments, a method for treating cervical cancer in an individual is provided, the method comprising administering an effective amount of activated T cells to the individual, wherein the activated T cells are prepared by co-culturing a population of T cells with a population of antigen-presenting cells (such as dendritic cells) loaded with multiple tumor antigen peptides (e.g., in the presence of an immune checkpoint inhibitor). In some embodiments, the individual has previously been administered an effective amount of antigen-presenting cells loaded with said multiple tumor antigen peptides. In some embodiments, the method further comprises administering an effective amount of antigen-presenting cells loaded with said multiple tumor antigen peptides to the individual prior to the administration of the activated T cells. In some embodiments, the cervical cancer is early-stage cervical cancer, non-metastatic cervical cancer, locally advanced cervical cancer, metastatic cervical cancer, cervical cancer in remission, unresectable cervical cancer, cervical cancer in an adjuvant setting, or cervical cancer in a neoadjuvant setting. In some embodiments, the cervical cancer is caused by human papillomavirus (HPV) infection. In some embodiments, according to the TNM classification, the cervical cancer may be a T-stage tumor (primary tumor), an N-stage tumor (regional lymph node), or an M-stage tumor (distant metastasis). In some implementations, cervical cancer is any of the following stages: stage 0, stage I (Tis, N0, M0), stage IA (T1a, N0, M0), stage IB (T1b, N0, M0), stage IIA (T2a, N0, M0), stage IIB (T2b, N0, M0), stage IIIA (T3a, N0, M0), stage IIIB (T3b, N0, M0, or T1-3, N1, M0), stage IVA (T4, N0, M0), or stage IVB (T1-T3, N0-N1, M1). In some implementations, cervical cancer is cervical squamous cell carcinoma, cervical adenocarcinoma, or adenosquamous carcinoma.

[0152] In some embodiments, a method for treating breast cancer in an individual is provided, the method comprising administering an effective amount of activated T cells to the individual, wherein the activated T cells are prepared by co-culturing a population of T cells with a population of antigen-presenting cells (such as dendritic cells) loaded with multiple tumor antigen peptides (e.g., in the presence of an immune checkpoint inhibitor). In some embodiments, the individual has previously been administered an effective amount of antigen-presenting cells loaded with said multiple tumor antigen peptides. In some embodiments, the method further comprises administering an effective amount of antigen-presenting cells loaded with said multiple tumor antigen peptides to the individual prior to the administration of the activated T cells. In some embodiments, the breast cancer is early-stage breast cancer, non-metastatic breast cancer, locally advanced breast cancer, metastatic breast cancer, hormone receptor-positive metastatic breast cancer, remission-stage breast cancer, breast cancer in an adjuvant setting, ductal carcinoma in situ (DCIS), invasive ductal carcinoma (IDC), or breast cancer in a neoadjuvant setting. In some embodiments, the breast cancer is hormone receptor-positive metastatic breast cancer. In some embodiments, the breast cancer (which may be HER2-positive or HER2-negative) is advanced breast cancer. In some embodiments, the breast cancer is ductal carcinoma in situ. In some implementations, an individual may be a human with a gene, genetic mutation, or polymorphism associated with breast cancer (e.g., BRCA1, BRCA2, ATM, CHEK2, RAD51, AR, DIRAS3, ERBB2, TP53, AKT, PTEN, and / or PI3K) or with one or more additional copies of a gene associated with breast cancer (e.g., one or more additional copies of the HER2 gene).

[0153] In some embodiments, a method for treating pancreatic cancer in an individual is provided, the method comprising administering an effective amount of activated T cells to the individual, wherein the activated T cells are prepared by co-culturing a population of T cells with a population of antigen-presenting cells (such as dendritic cells) loaded with multiple tumor antigen peptides, such as in the presence of an immune checkpoint inhibitor. In some embodiments, the individual has previously been administered an effective amount of antigen-presenting cells loaded with said multiple tumor antigen peptides. In some embodiments, the method further comprises administering an effective amount of antigen-presenting cells loaded with said multiple tumor antigen peptides to the individual prior to the administration of the activated T cells. In some embodiments, pancreatic cancer includes, but is not limited to, serous microcystic adenoma, intraductal papillary myxoma, myxoid cystic tumor, solid pseudopapillary tumor, pancreatic cancer, pancreatic ductal carcinoma, or pancreatoblastoma. In some implementations, pancreatic cancer is any of the following: early-stage pancreatic cancer, non-metastatic pancreatic cancer, primary pancreatic cancer, post-resectable pancreatic cancer, advanced pancreatic cancer, locally advanced pancreatic cancer, metastatic pancreatic cancer, unresectable pancreatic cancer, pancreatic cancer in remission, recurrent pancreatic cancer, pancreatic cancer in an adjuvant setting, or pancreatic cancer in a neoadjuvant setting.

[0154] In some embodiments, a method for treating ovarian cancer in an individual is provided, the method comprising administering an effective amount of activated T cells to the individual, wherein the activated T cells are prepared by co-culturing a population of T cells with a population of antigen-presenting cells (such as dendritic cells) loaded with multiple tumor antigenic peptides, such as in the presence of an immune checkpoint inhibitor. In some embodiments, the individual has previously been administered an effective amount of antigen-presenting cells loaded with said multiple tumor antigenic peptides. In some embodiments, the method further comprises administering an effective amount of antigen-presenting cells loaded with said multiple tumor antigenic peptides to the individual prior to the administration of the activated T cells. In some embodiments, the ovarian cancer is ovarian epithelial cancer. Exemplary histological classifications of ovarian epithelial cancer include: serous cystomas (e.g., benign serous cystadenomas, serous cystadenomas with proliferative activity and nuclear abnormalities but no invasive, destructive growth, or serous cystadenocarcinoma), mucinous cystomas (e.g., benign mucinous cystadenomas, mucinous cystadenomas with proliferative activity and nuclear abnormalities but no invasive, destructive growth, or mucinous cystadenocarcinoma), endometrioid tumors (e.g., benign endometrioid cysts, endometrioid tumors with proliferative activity and nuclear abnormalities but no invasive, destructive growth, or endometrioid adenocarcinoma), clear cell (mesonephric) tumors (e.g., benign clear cell tumors, clear cell tumors with proliferative activity and nuclear abnormalities but no invasive, destructive growth, or clear cell cystadenocarcinoma), unclassified tumors that cannot be classified into any of the above groups, or other malignant tumors. In various embodiments, the ovarian epithelial cancer is stage I (e.g., stage IA, IB, or IC), stage II (e.g., stage IIA, IIB, or IIC), stage III (e.g., stage IIIA, IIIB, or IIIC), or stage IV. In some embodiments, the individual may be a human having a gene, genetic mutation, or polymorphism associated with ovarian cancer (e.g., BRCA1 or BRCA2) or having one or more additional copies of a gene associated with ovarian cancer (e.g., one or more additional copies of the HER2 gene). In some embodiments, the ovarian cancer is an ovarian germ cell tumor. Exemplary histological subtypes include dysgerminomas or other germ cell tumors (e.g., endodermal sinus tumors, such as hepatoid or intestinal tumors, embryonal carcinomas, polyembryomas, choriocarcinomas, teratomas, or mixed-form tumors). Exemplary teratomas are immature teratomas, mature teratomas, solid teratomas, and cystic teratomas (e.g., dermoid cysts, such as mature cystic teratomas, and dermoid cysts with malignant transformation). Some teratomas are monoblastic and highly specialized, such as ovarian goiter, carcinoid tumor, ovarian goiter and carcinoid tumor or others (e.g., malignant neuroectodermal and ependymoma).In some implementations, the ovarian germ cell tumor is stage I (e.g., IA, IB, or IC), stage II (e.g., IIA, IIB, or IIC), stage III (e.g., IIIA, IIIB, or IIIC), or stage IV.

[0155] In some implementations, the MASCT method described herein is not applicable to patients with T-cell-origin cancers, such as T-cell lymphoma.

[0156] Several viruses are associated with cancers in humans. For example, hepatitis B virus (HBV) can cause chronic liver infection, increasing an individual's chance of developing liver cancer or hepatocellular carcinoma (HCC). Human papillomavirus (HPV) is a group of over 150 related viruses that cause papillomas or warts when they infect and grow in the skin or mucous membranes (such as the mouth, throat, or vagina). Several types of HPV (including types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, and 6) are known to cause cervical cancer. HPV also plays a role in inducing or causing other genital cancers and is associated with some cancers of the mouth and throat. Epstein-Barr virus (EBV) is a herpesvirus that can infect B lymphocytes for a long time and remain latent in them. EBV infection increases an individual's risk of developing nasopharyngeal carcinoma and certain types of rapidly growing lymphomas, such as Burkitt lymphoma. EBV is also associated with Hodgkin's lymphoma and some cases of stomach cancer. In addition to causing cancer or increasing the risk of developing cancer, viral infections such as HBV, HPV, and EBV can also cause tissue or organ damage, which can increase the disease burden in individuals with cancer and promote cancer progression.

[0157] It is known in the art that effective and specific immune responses, including cytotoxic T-cell responses, can be induced in the human body against several cancer-associated viruses, such as HBV, HPV, and EBV, including their various subtypes. Therefore, in some embodiments, a method for treating virus-associated cancer in an individual is provided, the method comprising administering an effective amount of activated T cells to the individual, wherein the activated T cells are prepared by co-culturing a population of T cells with a population of antigen-presenting cells (such as dendritic cells) loaded with multiple tumor antigen peptides. In some embodiments, the individual has previously been administered an effective amount of antigen-presenting cells loaded with said multiple tumor antigen peptides. In some embodiments, the method further comprises administering an effective amount of antigen-presenting cells loaded with multiple tumor antigen peptides to the individual. In some embodiments, the virus is HBV, HPV, or EBV. In some embodiments, the cancer is HBV-associated hepatocellular carcinoma, HPV-associated cervical cancer, or EBV-associated nasopharyngeal carcinoma.

[0158] The methods described herein may be used for any one or more of the following purposes: relieving one or more symptoms of cancer, delaying cancer progression, reducing cancer tumor size, disrupting (e.g., destroying) cancer stroma, inhibiting cancer tumor growth, prolonging overall survival, prolonging disease-free survival, prolonging time to cancer progression, preventing or delaying cancer metastasis, reducing (e.g., eradicating) previous cancer metastasis, reducing the incidence or burden of previous cancer metastasis, preventing cancer recurrence, and / or improving clinical benefit from cancer.

[0159] APC, T cells, and tumor antigen peptides

[0160] In some embodiments, the methods described herein utilize antigen-presenting cells (APCs) and activated T cells. APCs are immune system cells capable of activating T cells. APCs include, but are not limited to, certain macrophages, B cells, and dendritic cells (DCs). Dendritic cells are members of a diverse population of morphologically similar cell types found in lymphatic or non-lymphatic tissues. These cells are characterized by their distinctive morphology and high expression levels of class I and II MHC molecules on their surface, which are proteins that present antigenic peptides to T cells. DCs, other APCs, and T cells can be isolated from or derived from (e.g., differentiated from) various tissue sources, conveniently from peripheral blood, such as peripheral blood mononuclear cells (PBMCs) derived from peripheral blood.

[0161] T cells, or T lymphocytes, play a crucial role in cell-mediated immunity. Each clone of an activated T cell expresses a unique T cell receptor (TCR) on its surface, which is responsible for recognizing antigens that bind to MHC molecules on APCs and target cells (such as cancer cells). T cells are subdivided into several types, each expressing a unique combination of surface proteins and each type having a unique function.

[0162] Cytotoxic T cells (TCs) participate in the immune response and destruction of tumor cells and other infected cells, such as those infected by viruses. Generally, TCs function by recognizing class I MHC antigens presented on APCs or any target cells. Stimulation of the TCR, along with co-stimulatory factors (e.g., CD28 on T cells that bind to B7 on APCs, or stimulation by helper T cells), induces TC cell activation. Activated TCs then proliferate and release cytotoxins, thereby destroying APCs or target cells (such as cancer cells). Mature TCs generally express the surface proteins CD3 and CD8. Cytotoxic T cells belong to the CD3 group. + CD8 + T cells.

[0163] Helper T cells (TH) are T cells that promote the activity of other immune cells by releasing T cell cytokines. These cytokines can regulate or suppress immune responses, induce cytotoxic T cells, and maximize the cytotoxic activity of macrophages. Generally, TH cells function by recognizing class II MHC antigens presented on APCs. Mature TH cells express the surface proteins CD3 and CD4. Helper T cells belong to the CD3 group. + CD4 + T cells.

[0164] Natural killer (NK) T cells are a heterogeneous group of T cells that possess characteristics of both T cells and natural killer cells. Activation of NK T cells leads to the production of pro-inflammatory cytokines, chemokines, and other cytokines. They express CD56, a surface molecule commonly expressed on natural killer cells. NK T cells belong to the CD3 group. + CD56 + T cells.

[0165] Regulatory T cells (T cells) REG Cells typically regulate the immune system by increasing their tolerance to self-antigens, thereby limiting autoimmune activity. In cancer immunotherapy, T cells... REG This allows cancer cells to evade the immune response. (T) REG Cells typically express CD3, CD4, CD7, CD25, CTLA4, GITR, GARP, FOXP3, and / or LAP. CD4 + CD25 + FoxP3 + T cells are a type of T cell. REG cell.

[0166] Memory T cells (Tm) are T cells that have previously encountered and responded to their specific antigens, or T cells differentiated from activated T cells. Although tumor-specific Tm constitutes a small proportion of the total T cell count, they play a crucial role in monitoring tumor cells throughout a person's lifespan. If a tumor-specific Tm encounters a tumor cell expressing its specific tumor antigen, the Tm immediately activates and expands clonally. The activated and expanded T cells differentiate into effector T cells to efficiently kill tumor cells. Memory T cells are important for establishing and maintaining a long-term tumor antigen-specific response in T cells.

[0167] Typically, T cell antigens are protein molecules or linear fragments of protein molecules that can be recognized by T cell receptors (TCRs) and elicit a specific T cell response. Antigens can be exogenous (such as proteins encoded by viruses) or endogenous (such as proteins expressed inside or on the cell surface). The smallest fragment of an antigen that directly participates in the interaction with a specific TCR is called an epitope. Multiple epitopes can exist within a single antigen, each of which is recognized by a unique TCR encoded by a specific clone of the T cell.

[0168] To be recognized by the TCR, antigenic peptides or fragments can be processed into epitopes by APCs (such as dendritic cells) and then bound in an extended conformation to major histocompatibility (MHC) molecules, thereby forming an MHC-peptide complex on the surface of the APCs (such as dendritic cells). Human MHC molecules are also known as human leukocyte antigens (HLA). MHC provides an enlarged binding surface for strong association between the TCR and the epitope, while the unique combination of amino acid residues within the epitope ensures the specificity of the interaction between the TCR and the epitope. Based on their structural characteristics, particularly the length of the epitope bound to the corresponding MHC complex, human MHC molecules are classified into two types—MHC class I and MHC class II. MHC-I epitopes are epitopes that bind to and are represented by MHC class I molecules. MHC-II epitopes are epitopes that bind to and are represented by MHC class II molecules. MHC-I epitopes are typically about 8 to about 11 amino acids long, while MHC-II epitopes are about 13 to about 17 amino acids long. Due to genetic polymorphism, various subtypes of MHC class I and MHC class II molecules exist in the population. The T cell response to specific antigenic peptides presented by MHC class I or MHC class II molecules on APCs or target cells is called the MHC-restricted T cell response.

[0169] Tumor antigen peptides are derived from tumor antigen proteins (also referred to herein as “tumor antigens”) that are overexpressed in cancer cells but have low or no expression levels in normal cells (such as less than about 10, 100, 1000, or 5000 copies / cell). Some tumor antigen peptides are derived from tumor-specific antigens (TSA), differentiation antigens, or overexpressed antigens (also referred to as tumor-associated antigens or TAAs). Some tumor antigen peptides are derived from mutant protein antigens that are present only in cancer cells and not in normal cells.

[0170] Antigen loading of dendritic cells

[0171] This invention provides a method for preparing a dendritic cell population loaded with multiple tumor antigen peptides, said dendritic cell population being used to elicit an MHC-restricted T cell response in an individual. The method includes contacting the dendritic cell population with multiple tumor antigen peptides. The dendritic cells prepared by this method can be used in any embodiment of the MASCT method described herein, or for preparing activated T cells or co-cultures of dendritic cells and T cells, as described in the following section.

[0172] In some embodiments of the method for preparing multi-antigen-loaded dendritic cells, a population of dendritic cells is contacted with any number of tumor antigen peptides selected from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 25, 30, 40, or 50. In some embodiments, the population of dendritic cells is contacted with multiple tumor antigen peptides comprising any number of epitopes selected from at least about 1, 5, 10, 15, 20, 25, 30, 35, or 40 of SEQ ID NO: 1-40. In some embodiments, the population of dendritic cells is contacted with any number of tumor antigen peptides selected from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more of tumor antigen peptides selected from... Figure 2C and Figure 29A The tumor antigen peptides are present in the form of a group of cells. In some embodiments, the dendritic cell population is contacted with any number of tumor antigen peptides selected from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more, said tumor antigen peptides being derived from proteins selected from hTERT, p53, survivin, NY-ESO-1, CEA, CCND1, MET, RGS5, MMP7, VEGFR, AFP, GPC3, HBVc, HBVp, CDCA1, KRAS, PARP4, MLL3 and MTHFR.

[0173] In some embodiments, the dendritic cells are mature dendritic cells that present one or more of the aforementioned multiple tumor antigen peptides. Mature dendritic cells prepared by any of the methods described herein can present any number of tumor antigen peptides selected from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 25, 30, 40, or 50. Multi-antigen-loaded dendritic cells can have an enhanced presentation level of more than any number of tumor antigen peptides selected from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 25, 30, 40, or 50. In some embodiments, mature dendritic cells have enhanced presentation levels of any number of epitopes selected from at least about 1, 5, 10, 15, 20, 25, 30, 35, or 40 of SEQ ID NO:1-40. In some embodiments, mature dendritic cells have enhanced presentation levels of more than ten tumor antigen peptides. In some embodiments, mature dendritic cells have enhanced presentation levels of any number of tumor antigen peptides selected from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more, such as... Figure 2C and Figure 29A As shown in the illustration. In some embodiments, mature dendritic cells have enhanced presentation levels of any number of tumor antigen peptides selected from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more, said tumor antigen peptides being derived from proteins selected from hTERT, p53, survivin, NY-ESO-1, CEA, CCND1, MET, RGS5, MMP7, VEGFR, AFP, GPC3, HBVc, HBVp, CDCA1, KRAS, PARP4, MLL3 and MTHFR.

[0174] Exemplary embodiments of contacting dendritic cell populations with multiple tumor antigen peptides include shocking a population of dendritic cells, such as immature dendritic cells, or dendritic cells contained in or derived from (such as differentiated from) PBMCs. As is known in the art, shocking refers to the process of mixing cells, such as dendritic cells, with a solution containing the antigen peptides, and optionally subsequently removing the antigen peptides from the mixture. The dendritic cell population may be contacted with multiple tumor antigen peptides for seconds, minutes, or hours, such as any of the following durations: about 30 seconds, 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 1 hour, 5 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, or longer. The concentration of each tumor antigen peptide used in the contacting step may be any of the following concentrations: about 0.1, 0.5, 1, 2, 3, 5, or 10 μg / mL. In some embodiments, the concentration of the tumor antigen peptide is about 0.1-200 μg / mL, including, for example, any of the concentrations of about 0.1-0.5, 0.5-1, 1-10, 10-50, 50-100, 100-150, or 150-200 μg / mL.

[0175] In some embodiments, a dendritic cell population is contacted with the multiple tumor antigen peptides in the presence of a composition that facilitates uptake of the multiple tumor antigen peptides by dendritic cells. In some embodiments, compounds, materials, or compositions may be added to the solution of the multiple tumor antigen peptides to facilitate uptake of the peptides by dendritic cells. Compounds, materials, or compositions that facilitate uptake of the multiple tumor antigen peptides by dendritic cells include, but are not limited to, lipid molecules and peptides having a plurality of positively charged amino acids. In some embodiments, the dendritic cell population uptakes more than any percentage of the tumor antigen peptides selected from about 50%, 60%, 70%, 80%, 90%, or 95%. In some embodiments, more than any percentage of the dendritic cells in the population selected from about 50%, 60%, 70%, 80%, 90%, or 95% uptake at least one tumor antigen peptide.

[0176] In some embodiments, a method is provided for preparing a population of dendritic cells loaded with multiple tumor antigen peptides, the method comprising contacting an immature dendritic cell population with multiple tumor antigen peptides. In some embodiments, the method further comprises inducing the maturation of the immature dendritic cell population with multiple Toll-like receptor (TLR) agonists. In some embodiments, the method comprises contacting an immature dendritic cell population with multiple TLR agonists and multiple tumor antigen peptides to obtain a mature dendritic cell population loaded with said multiple tumor antigen peptides. Exemplary TLR agonists include, but are not limited to, polyIC, MALP, and R848. Cytokines and other suitable molecules may be further added to the culture medium during the maturation step. The immature dendritic cell population may be induced by a TLR agonist for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 days to mature. In some embodiments, the immature dendritic cell population is induced for approximately 8 days to mature.

[0177] Dendritic cells (such as immature dendritic cells) can be obtained from various sources, including autologous sources, i.e., from an individual receiving treatment. A convenient source of dendritic cells is peripheral blood mononuclear cells (PBMCs). For example, monocytes (a type of white blood cell) are abundant in PBMCs, comprising approximately 10%–30% of the total PBMCs. Cytokines can be used to induce monocytes to differentiate into dendritic cells, such as immature dendritic cells. In some embodiments, immature dendritic cells are prepared by obtaining a PBMC population, obtaining a monocyte population from the PBMC population, and contacting the monocyte population with a variety of cytokines to obtain an immature dendritic cell population. Exemplary cytokines that can be used to induce monocyte differentiation include, but are not limited to, GM-CSF and IL-4, and are performed under conditions known in the art (such as concentration, temperature, CO2 level, etc.). The adhesion fraction of PBMCs contains the majority of monocytes in the PBMCs. In some embodiments, monocytes from the PBMC adhesion fraction are contacted with cytokines to obtain an immature dendritic cell population. PBMCs can be conveniently obtained by centrifuging peripheral blood samples or by collecting them from individuals using plasma separation methods. In some embodiments, PBMC populations are obtained by density gradient centrifugation of human peripheral blood samples. In some embodiments, samples are taken from individuals receiving multiantigen-loaded dendritic cells, activated T cells, or other immunotherapeutic compositions prepared using multiantigen-loaded dendritic cells.

[0178] In some embodiments, a method is provided for preparing a dendritic cell population loaded with multiple tumor antigen peptides, said dendritic cell population being used to elicit an MHC-restricted T cell response in an individual. The method includes the steps of: obtaining a peripheral blood mononuclear cell (PBMC) population from the individual; obtaining a mononuclear cell population from the PBMC population; obtaining a dendritic cell population from the mononuclear cell population; and contacting the dendritic cell population with multiple tumor antigen peptides to obtain a dendritic cell population loaded with said multiple tumor antigen peptides. In some embodiments, a method is provided for preparing a dendritic cell population loaded with multiple tumor antigen peptides, said dendritic cell population being used to elicit an MHC-restricted T cell response in an individual. This method includes the steps of: obtaining a PBMC population from an individual (such as the individual); obtaining a mononuclear cell population from the PBMC population; contacting the mononuclear cell population with multiple cytokines (such as GM-CSF and IL-4) to obtain an immature dendritic cell population; and contacting the immature dendritic cell population with multiple TLR agonists and multiple tumor antigen peptides to obtain a dendritic cell population loaded with said multiple tumor antigen peptides.

[0179] This invention also provides isolated dendritic cell populations prepared by any embodiment of the methods described herein. In some embodiments, the isolated dendritic cell populations are capable of eliciting MHC-restricted T cell responses in vivo or in vitro. In some embodiments, the MHC-restricted T cell responses are mediated by both MHC class I and MHC class II molecules. In some embodiments, the isolated dendritic cell populations are capable of inducing the differentiation and proliferation of tumor antigen-specific T cells.

[0180] Preparation of activated T cells

[0181] This invention also provides a method for preparing a population of activated T cells that can be used to treat cancer in an individual. The method includes co-culturing the T cell population with a population of antigen-presenting cells (such as dendritic cells) loaded with multiple tumor antigenic peptides. Any embodiment of the multi-antigen-loaded dendritic cells described in the preceding sections can be used to prepare activated T cells. In some embodiments, the T cell population and the dendritic cell population are derived from the same individual, such as an individual with cancer (e.g., low- to intermediate-grade cancer). In some embodiments, the T cell population, the dendritic cell population, or both are derived from an autologous source, i.e., from an individual receiving activated T cells, multi-antigen-loaded dendritic cells, or both.

[0182] In some embodiments, T cell populations and dendritic cell populations loaded with the various tumor antigen peptides are co-cultured for at least about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30 days. In some embodiments, T cell populations and dendritic cell populations loaded with the various tumor antigen peptides are co-cultured for about 7 to about 21 days (e.g., about 7 to about 14 days, about 7 to about 10 days, about 10 to about 15 days, about 14 to about 21 days, about 10 days, 14 days, 16 days, 18 days, or 21 days). In some embodiments, T cell populations and dendritic cell populations loaded with the various tumor antigen peptides are co-cultured for about 10 days. In some embodiments, T cell populations and dendritic cell populations loaded with the various tumor antigen peptides are co-cultured for about 14 days.

[0183] The T cell population used in any embodiment of the methods described herein can be derived from a variety of sources. A convenient source of T cells is PBMCs from human peripheral blood. The T cell population can be isolated from PBMCs, or alternatively, a T cell-rich PBMC population (e.g., by adding T cell-specific antibodies and cytokines) can be used in co-cultures. In some embodiments, the T cell population used in co-cultures is obtained from the non-adhesive fraction of peripheral blood mononuclear cells (PBMCs). In some embodiments, PBMCs are obtained by density gradient centrifugation of a peripheral blood sample. In some embodiments, the T cell population is obtained by culturing the non-adhesive fraction of PBMCs with at least one cytokine (such as IL-2) in the presence or absence of an anti-CD3 antibody (such as OKT3) (this process is referred to herein as "maintaining T cells"). In some embodiments, the non-adhesive fraction of PBMCs is cultured in the presence of an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is an inhibitor of an immune checkpoint molecule selected from PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, and LAG-3. The non-adhesive fraction of PBMCs can be cultured for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 days or longer. In some embodiments, activated T cell populations are prepared by obtaining a non-adhesive PBMC population and (e.g., in the presence of at least one cytokine (such as IL-2) and optionally an anti-CD3 antibody and optionally an immune checkpoint inhibitor) co-culturing the non-adhesive PBMC population with a dendritic cell population loaded with multiple tumor antigen peptides.

[0184] The co-culture may also include cytokines and other compounds to promote T cell activation, maturation, and / or proliferation, and to sensitize T cells for subsequent differentiation into memory T cells. Exemplary cytokines that may be used in this step include, but are not limited to, IL-7, IL-15, IL-21, etc. Certain cytokines may help suppress T cells in the activated T cell population within the co-culture. REG Percentage. For example, in some embodiments, high doses (such as any one of at least about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, or 1500 U / ml) of cytokines (such as IL-2) are used to co-culture T cell populations and dendritic cell populations loaded with the aforementioned multiple tumor antigen peptides to obtain T cells with a low percentage of tumor antigen peptides. REG Activation of the T cell population.

[0185] The co-culture may also include one or more immune checkpoint inhibitors (such as any combination of 1, 2, 3, or more). In some embodiments, the T cell population is contacted with the immune checkpoint inhibitor prior to co-culture. For example, the T cell population may be isolated T cells or T cells present in a cell mixture (such as a non-adhesive fraction of PBMCs). In some embodiments, the non-adhesive PBMC population is contacted with the immune checkpoint inhibitor prior to co-culture. In some embodiments, the T cell population or the non-adhesive PBMC population is contacted with the immune checkpoint inhibitor for at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14 days or more. In some embodiments, the T cell population or the non-adhesive PBMC population is contacted with the immune checkpoint inhibitor for about 5 days to about 14 days. In some embodiments, the PBMCs are contacted with the immune checkpoint inhibitor for about 8 days.

[0186] In some embodiments, in the presence of an immune checkpoint inhibitor, a population of T cells is co-cultured with a population of dendritic cells loaded with the aforementioned multiple tumor antigen peptides. In some embodiments, the immune checkpoint inhibitor is an inhibitor of an inhibitory checkpoint molecule selected from PD-1, PD-L1, PD-L2, CTLA-4, BLTA, TIM-3, and LAG-3. In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-1. In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody, such as nivolumab (e.g., ), pembrolizumab (e.g., ) or SHR-1210. In some embodiments, the immune checkpoint inhibitor is a PD-L1 inhibitor. In some embodiments, the immune checkpoint inhibitor is an anti-PD-L1 antibody. In some embodiments, the immune checkpoint inhibitor is a CTLA-4 inhibitor. In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody, such as ipilimumab (e.g., ).

[0187] The T cell population can be stimulated any number of times, such as 1, 2, 3, or more, using a population of dendritic cells (DCs) loaded with the various tumor antigen peptides. In some embodiments, the T cell population is stimulated once. In some embodiments, the T cell population is stimulated at least twice. In some embodiments, for each stimulation, a population of DCs loaded with the various tumor antigen peptides is added to a co-culture. The DCs can be freshly prepared and pulsed with the various tumor antigen peptides, or they can be obtained from a batch of DCs prepared for the initial stimulation.

[0188] Therefore, a method for preparing an activated T cell population is provided, the method comprising: (a) preparing a dendritic cell population loaded with a variety of tumor antigen peptides; and (b) co-culturing the dendritic cell population loaded with the various tumor antigen peptides with a non-adhesive PBMC population to obtain an activated T cell population, wherein the dendritic cell population and the non-adhesive PBMC population are obtained from a PBMC population taken from an individual. In some embodiments, the co-culturing is performed in the presence of a variety of cytokines (such as IL-2, IL-7, IL-15, IL-21, or any combination thereof). In some embodiments, the co-culturing is performed in the presence of an anti-CD3 antibody (such as OKT3) and a variety of cytokines (such as IL-2, IL-7, IL-15, IL-21, or any combination thereof). In some embodiments, the non-adhesive PBMC population is contacted with an immune checkpoint inhibitor (such as an inhibitor of PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3) before and / or during co-culturing. In some embodiments, the method further includes obtaining the PBMC population from an individual.

[0189] In some embodiments, a method for preparing an activated T cell population is provided, the method comprising: (a) inducing a monocyte population to differentiate into a dendritic cell population (e.g., in the presence of GM-CSF and IL-4); (b) contacting the dendritic cell population with a plurality of tumor antigen peptides to obtain a dendritic cell population loaded with said plurality of tumor antigen peptides; and (c) co-culturing the dendritic cell population loaded with said plurality of tumor antigen peptides with a non-adhesive PBMC population to obtain an activated T cell population, wherein the monocyte population and the non-adhesive PBMC population are obtained from an individual's PBMC population. In some embodiments, the dendritic cell population loaded with said plurality of tumor antigen peptides is contacted with a plurality of TLR agonists to induce maturation of the dendritic cell population loaded with said plurality of tumor antigen peptides. In some embodiments, the co-culture is performed in the presence of a plurality of cytokines (such as IL-2, IL-7, IL-15, IL-21, or any combination thereof). In some embodiments, co-culture is performed in the presence of anti-CD3 antibodies (such as OKT3) and multiple cytokines (such as IL-2, IL-7, IL-15, IL-21, or any combination thereof). In some embodiments, the non-adherent PBMC population is contacted with an immune checkpoint inhibitor (such as an inhibitor of PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3) before and / or during co-culture. In some embodiments, the method further includes any one or a combination of the following steps: (i) obtaining a PBMC population from an individual; (ii) obtaining a monocyte population from the PBMC population; and (iii) obtaining a non-adherent PBMC population from the PBMC population.

[0190] In some embodiments, a method for preparing an activated T cell population is provided, the method comprising obtaining a peripheral blood mononuclear cell (PBMC) population from an individual, obtaining a mononuclear cell population from the PBMC population, inducing the mononuclear cell population to differentiate into a dendritic cell population (e.g., in the presence of GM-CSF and IL-4), contacting the immature dendritic cell population with a variety of Toll-like receptor (TLR) agonists and a variety of tumor antigen peptides to obtain a mature dendritic cell population loaded with said multiple tumor antigen peptides, obtaining a non-adhesive PBMC population from the PBMC population, and co-culturing the mature dendritic cell population loaded with said multiple tumor antigen peptides with the non-adhesive PBMC population in the presence of a variety of cytokines (such as IL-2, IL-7, IL-15, IL-21 or any combination thereof), optionally an anti-CD3 antibody (such as OKT3), and optionally an immune checkpoint inhibitor (such as an inhibitor of PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3) to obtain an activated T cell population.

[0191] This invention also provides isolated activated T cell populations prepared by any embodiment of the methods described herein. This invention also provides co-cultures for use in treating cancer in an individual, comprising a population of T cells and a population of dendritic cells loaded with multiple tumor antigen peptides. In some embodiments of the co-culture, the T cell population and the dendritic cell population loaded with the multiple tumor antigen peptides are derived from the same individual, such as an individual receiving treatment. In some embodiments of the co-culture, the multi-antigen-loaded dendritic cell population is prepared by any embodiment of the preparation methods described in the preceding sections, such as bombarding the dendritic cell population with multiple tumor antigen peptides, or contacting the dendritic cell population with multiple tumor antigen peptides in the presence of a composition (such as lipid molecules or peptides having multiple positively charged amino acids) that facilitates the uptake of the multiple tumor antigen peptides by dendritic cells. The isolated activated T cell populations and co-cultures described in this section can be used in any embodiment of the MASCT method. This invention provides immunotherapeutic compositions for treating cancer, preventing tumor progression or metastasis, or reducing cancer immune evasion, comprising isolated activated T cell populations or co-cultures. The isolated activated T cell populations and co-cultures can also be used to manufacture agents for treating cancer, preventing tumor progression or metastasis, or reducing cancer immune evasion.

[0192] The intent of this invention is that any steps and parameters described herein for preparing dendritic cell populations loaded with multiple tumor antigen peptides or for preparing activated T cell populations can be combined with any steps and parameters described herein for the MASCT method, as if each combination were described separately.

[0193] For example, in some embodiments, a segregated activated T cell population is provided, prepared by co-culturing a T cell population with a dendritic cell population loaded with multiple tumor antigen peptides. In some embodiments, the dendritic cell population is prepared by contacting the dendritic cell population with multiple tumor antigen peptides (e.g., in the presence of a composition that facilitates the uptake of said multiple tumor antigen peptides by the dendritic cells). In some embodiments, the T cell population is contacted with an immune checkpoint inhibitor (such as an inhibitor of PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3) before and / or during co-culturing. In some embodiments, the dendritic cell population or the T cell population is derived from the same source (e.g., an individual receiving treatment with activated T cells).

[0194] In some embodiments, an isolated activated T cell population is provided prepared by: (a) inducing a monocyte population to differentiate into a dendritic cell population (e.g., in the presence of GM-CSF and IL-4); (b) contacting the dendritic cell population with a plurality of tumor antigen peptides to obtain a dendritic cell population loaded with said plurality of tumor antigen peptides; and (c) co-culturing the dendritic cell population loaded with said plurality of tumor antigen peptides with a non-adhesive PBMC population, wherein the monocyte population and the non-adhesive PBMC population are obtained from an individual's PBMC population. In some embodiments, the dendritic cell population loaded with said plurality of tumor antigen peptides is contacted with a plurality of TLR agonists to induce maturation of the dendritic cell population loaded with said plurality of tumor antigen peptides. In some embodiments, the co-culture is performed in the presence of a plurality of cytokines (such as IL-2, IL-7, IL-15, IL-21 or any combination thereof) and optionally an anti-CD3 antibody (such as OKT3). In some embodiments, the non-adhesive PBMC population is contacted with an immune checkpoint inhibitor (such as an inhibitor of PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3) before and / or during co-culture. In some embodiments, the method further includes any one or a combination of the following steps: (i) obtaining a PBMC population from an individual; (ii) obtaining a monocyte population from the PBMC population; and (iii) obtaining a non-adhesive PBMC population from the PBMC population.

[0195] MASCT based on PBMC

[0196] A variant of the MASCT method (called PBMC-based MASCT) directly uses PBMCs containing APCs and T cells without isolating or deriving APCs (such as dendritic cells) or T cells for the treatment of cancer in an individual.

[0197] Therefore, in some embodiments, a method for treating cancer in an individual is provided, the method comprising contacting a population of peripheral blood mononuclear cells (PBMCs) with a plurality of tumor antigen peptides to obtain an activated population of PBMCs, and administering an effective amount of the activated PBMCs to the individual. In some embodiments, the PBMC population is contacted with the plurality of tumor antigen peptides in the presence of a composition that facilitates the uptake of the plurality of tumor antigen peptides by antigen-presenting cells (such as dendritic cells) in the PBMCs. In some embodiments, the PBMC population is contacted with the plurality of tumor antigen peptides in the presence of an inhibitor of immune checkpoint inhibitors such as PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, and LAG-3. In some embodiments, the activated PBMC population is contacted with IL-2. In some embodiments, the activated PBMCs are administered at least three times. In some embodiments, the interval between each administration of the activated PBMCs is from about 2 weeks to about 5 months (e.g., about 3 months). In some embodiments, the activated PBMCs are administered intravenously. In some embodiments, the PBMC population is obtained from the individual receiving treatment.

[0198] The PBMC-based MASCT method is applicable to the treatment of any cancer (including different types or stages) that can be treated with other implementations of the MASCT method as described in the previous sections. In some implementations of the PBMC-based MASCT method, the cancer is selected from hepatocellular carcinoma, cervical cancer, lung cancer, colorectal cancer, lymphoma, renal cell carcinoma, breast cancer, pancreatic cancer, gastric cancer, esophageal cancer, ovarian cancer, prostate cancer, nasopharyngeal carcinoma, melanoma, and brain cancer.

[0199] In some embodiments, the PBMCs are autologous, i.e., obtained from an individual receiving treatment. In some embodiments, the peripheral blood taken from the individual has a low number of dendritic cells or T cells. In some embodiments, the PBMCs are contacted with cytokines such as IL-2, GM-CSF, etc., to induce differentiation, maturation, or proliferation of certain cells (such as dendritic cells, T cells, or combinations thereof) in the PBMCs simultaneously with or after the contact step. In some embodiments, the multiple tumor antigen peptides are removed after the contact step. In some embodiments, the PBMCs are contacted with the multiple tumor antigen peptides for at least about 10 minutes, 15 minutes, 20 minutes, 30 minutes, 1 hour, 5 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 10 days, or longer. In some embodiments, the PBMCs are exposed to cytokines for at least any one of the following durations: about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30 days. In some embodiments, the PBMCs are exposed to cytokines for about 14-21 days. In some embodiments, the PBMCs are exposed to cytokines for about 14 days.

[0200] In any of the PBMC-based MASCT methods described above, the PBMCs are contacted with one or more immune checkpoint inhibitors. In some embodiments, in the presence of immune checkpoint inhibitors, the PBMC population is contacted with the various tumor antigen peptides. In some embodiments, the PBMCs are contacted with immune checkpoint inhibitors for at least about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or 30 days. In some embodiments, the PBMCs are contacted with immune checkpoint inhibitors for about 14 days to about 21 days.

[0201] Combination therapy with immune checkpoint inhibitors

[0202] The methods described herein for treating cancer can be used in monotherapy and in combination therapy with another agent. For example, any MASCT method described herein (including PBMC-based MASCT methods) can be combined with the administration of one or more immune checkpoint inhibitors (such as 1, 2, 3, 4 or more).

[0203] Therefore, in some embodiments, a method for treating cancer in an individual is provided, the method comprising: (a) optionally administering to the individual an effective amount of dendritic cells loaded with a plurality of tumor antigen peptides; (b) administering to the individual an effective amount of activated T cells, wherein the activated T cells are prepared by co-culturing a population of T cells with a population of dendritic cells loaded with the plurality of tumor antigen peptides; and (c) administering to the individual an effective amount of an immune checkpoint inhibitor. In some embodiments, the activated T cells and the immune checkpoint inhibitor are administered simultaneously, such as in the same composition. In some embodiments, the activated T cells and the immune checkpoint inhibitor are administered sequentially. In some embodiments, the interval between the administration of dendritic cells and the administration of activated T cells is from about 7 days to about 21 days (e.g., from about 7 days to about 14 days, from about 14 days to about 21 days, from about 10 days, or from about 14 days). In some embodiments, the dendritic cells loaded with the plurality of tumor antigen peptides are administered subcutaneously. In some embodiments, the dendritic cells loaded with the plurality of tumor antigen peptides are administered at least three times. In some embodiments, the activated T cells are administered intravenously. In some embodiments, the activated T cells are administered at least three times. In some embodiments, a population of T cells is co-cultured with a population of dendritic cells loaded with the various tumor antigen peptides for approximately 7 to approximately 21 days (e.g., approximately 7 to approximately 14 days, approximately 14 to approximately 21 days, or approximately 10 days). In some embodiments, the T cell population is derived from the non-adhesive portion of a population of peripheral blood mononuclear cells (PBMCs). In some embodiments, co-culture further includes contacting activated T cells with a variety of cytokines (such as IL-2, IL-7, IL-15, IL-21, or any combination thereof) and optionally an anti-CD3 antibody. In some embodiments, the T cell population is contacted with an immune checkpoint inhibitor (such as an inhibitor of PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3) before and / or during co-culture. In some embodiments, a population of dendritic cells loaded with the various tumor antigen peptides is prepared by contacting the dendritic cell population with the various tumor antigen peptides. In some embodiments, the T cell population and the dendritic cell population are derived from the same individual. In some implementations, the T cell population, dendritic cell population, PBMC population, or any combination thereof are derived from the individual receiving treatment.

[0204] In some embodiments, a method for treating cancer in an individual is provided, the method comprising: (a) inducing a population of monocytes to differentiate into a population of dendritic cells; (b) contacting the dendritic cell population with a plurality of tumor antigen peptides to obtain a population of dendritic cells loaded with said plurality of tumor antigen peptides; (c) optionally administering to the individual an effective amount of dendritic cells loaded with said plurality of tumor antigen peptides; (d) co-culturing the population of dendritic cells loaded with said plurality of tumor antigen peptides with a population of non-adhesive PBMCs to obtain a population of activated T cells; (e) administering to the individual an effective amount of activated T cells; and (f) administering to the individual an effective amount of an immune checkpoint inhibitor, wherein the monocyte population and the non-adhesive PBMC population are obtained from a population of PBMCs. In some embodiments, activated T cells and immune checkpoint inhibitors are administered simultaneously, such as in the same composition. In some embodiments, activated T cells and immune checkpoint inhibitors are administered sequentially. In some embodiments, the interval between the administration of dendritic cells and the administration of activated T cells is about 7 days to about 21 days (such as about 7 days to about 14 days, about 14 days to about 21 days, about 10 days, or about 14 days). In some embodiments, dendritic cells loaded with the various tumor antigen peptides are administered subcutaneously. In some embodiments, dendritic cells loaded with the various tumor antigen peptides are administered at least three times. In some embodiments, activated T cells are administered intravenously. In some embodiments, activated T cells are administered at least three times. In some embodiments, co-culture lasts from about 7 days to about 21 days (e.g., from about 7 days to about 14 days, from about 14 days to about 21 days, or about 10 days). In some embodiments, co-culture further includes contacting the activated T cells with a variety of cytokines (such as IL-2, IL-7, IL-15, IL-21, or any combination thereof) and optionally an anti-CD3 antibody. In some embodiments, the non-adhesive PBMC population is contacted with an immune checkpoint inhibitor (such as an inhibitor of PD-1, PD-L1, or CTLA-4) before and / or during co-culture. In some embodiments, the PBMC population is obtained from a patient receiving treatment.

[0205] In some embodiments, a method for treating cancer in an individual is provided, the method comprising: (a) contacting a population of PBMCs with a plurality of tumor antigen peptides to obtain an activated population of PBMCs; (b) administering an effective amount of the activated PBMCs to the individual; and (c) administering an effective amount of an immune checkpoint inhibitor to the individual. In some embodiments, the activated PBMCs and the immune checkpoint inhibitor are administered simultaneously, such as in the same composition. In some embodiments, the activated PBMCs and the immune checkpoint inhibitor are administered sequentially. In some embodiments, the PBMCs are contacted with the plurality of tumor antigen peptides in the presence of a composition that favors the uptake of the plurality of tumor antigen peptides by antigen-presenting cells (such as dendritic cells) in the PBMCs. In some embodiments, the activated PBMCs are further contacted with IL-2. In some embodiments, the PBMCs are contacted with the plurality of tumor antigen peptides in the presence of an immune checkpoint inhibitor such as an inhibitor of PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, and LAG-3. In some embodiments, the activated PBMCs are administered at least three times. In some implementations, the interval between each administration of activated PBMCs is approximately 2 weeks to approximately 5 months (e.g., approximately 3 months). In some implementations, the activated PBMCs are administered intravenously. In some implementations, the PBMC group is obtained from the individual receiving treatment.

[0206] In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-1. In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody. Exemplary anti-PD-1 antibodies include, but are not limited to, nivolumab, pembrolizumab, pidilizumab, BMS-936559, and atezolizumab, pembrolizumab, MK-3475, AMP-224, AMP-514, STI-A1110, and TSR-042. In some embodiments, the immune checkpoint inhibitor is nivolumab (e.g., In some implementations, the immune checkpoint inhibitor is pembrolizumab (e.g., In some implementations, the immune checkpoint inhibitor is SHR-1210.

[0207] Therefore, in some embodiments, a method for treating cancer in an individual is provided, the method comprising: (a) optionally administering to the individual an effective amount of dendritic cells loaded with a plurality of tumor antigen peptides; (b) administering to the individual an effective amount of activated T cells, wherein the activated T cells are prepared by co-culturing a population of T cells with a population of dendritic cells loaded with said plurality of tumor antigen peptides; and (c) administering to the individual an effective amount of an inhibitor of PD-1. In some embodiments, the inhibitor of PD-1 is an anti-PD-1 antibody. In some embodiments, the inhibitor of PD-1 is selected from nivolumab, pembrolizumab, and SHR-1210. In some embodiments, the activated T cells and the inhibitor of PD-1 are administered simultaneously, such as in the same composition. In some embodiments, the activated T cells and the inhibitor of PD-1 are administered sequentially. In some embodiments, the interval between the administration of dendritic cells and the administration of activated T cells is about 7 days to about 21 days (such as about 7 days to about 14 days, about 14 days to about 21 days, about 10 days, or about 14 days). In some embodiments, the dendritic cells loaded with said plurality of tumor antigen peptides are administered subcutaneously. In some embodiments, dendritic cells loaded with the multiple tumor antigen peptides are administered at least three times. In some embodiments, activated T cells are administered intravenously. In some embodiments, activated T cells are administered at least three times. In some embodiments, a population of T cells is co-cultured with a population of dendritic cells loaded with the multiple tumor antigen peptides for about 7 days to about 21 days (e.g., about 7 days to about 14 days, about 14 days to about 21 days, or about 10 days). In some embodiments, the T cell population is derived from the non-adhesive portion of a population of peripheral blood mononuclear cells (PBMCs). In some embodiments, co-culture further includes contacting the activated T cells with a variety of cytokines (such as IL-2, IL-7, IL-15, IL-21, or any combination thereof) and optionally an anti-CD3 antibody. In some embodiments, the T cell population is contacted with an immune checkpoint inhibitor (such as an inhibitor of PD-1) before and / or during co-culture. In some embodiments, a population of dendritic cells loaded with the multiple tumor antigen peptides is prepared by contacting the dendritic cell population with the multiple tumor antigen peptides. In some implementations, the T cell population and dendritic cell population are derived from the same individual. In some implementations, the T cell population, dendritic cell population, PBMC population, or any combination thereof are derived from the individual receiving treatment.

[0208] In some embodiments, a method for treating cancer in an individual is provided, the method comprising: (a) inducing a population of monocytes to differentiate into a population of dendritic cells; (b) contacting the dendritic cell population with a plurality of tumor antigen peptides to obtain a population of dendritic cells loaded with said plurality of tumor antigen peptides; (c) optionally administering to the individual an effective amount of dendritic cells loaded with said plurality of tumor antigen peptides; (d) co-culturing the population of dendritic cells loaded with said plurality of tumor antigen peptides with a population of non-adhesive PBMCs to obtain a population of activated T cells; (e) administering to the individual an effective amount of activated T cells; and (f) administering to the individual an effective amount of a PD-1 inhibitor, wherein the monocyte population and the non-adhesive PBMC population are obtained from a population of PBMCs. In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody. In some embodiments, the PD-1 inhibitor is selected from nivolumab, pembrolizumab, and SHR-1210. In some embodiments, activated T cells and a PD-1 inhibitor are administered simultaneously, such as in the same composition. In some embodiments, activated T cells and a PD-1 inhibitor are administered sequentially. In some embodiments, the interval between the administration of dendritic cells and the administration of activated T cells is approximately 7 to approximately 21 days (e.g., approximately 7 to approximately 14 days, approximately 14 to approximately 21 days, approximately 10 days, or approximately 14 days). In some embodiments, dendritic cells loaded with the various tumor antigen peptides are administered subcutaneously. In some embodiments, dendritic cells loaded with the various tumor antigen peptides are administered at least three times. In some embodiments, activated T cells are administered intravenously. In some embodiments, activated T cells are administered at least three times. In some embodiments, co-culture lasts approximately 7 to approximately 21 days (e.g., approximately 7 to approximately 14 days, approximately 14 to approximately 21 days, or approximately 10 days). In some embodiments, co-culture further includes contacting activated T cells with a variety of cytokines (e.g., IL-2, IL-7, IL-15, IL-21, or any combination thereof) and optionally an anti-CD3 antibody. In some embodiments, non-adhesive PBMCs are contacted with an immune checkpoint inhibitor (e.g., an inhibitor of PD-1) before and / or during co-culture. In some implementations, the PBMC group is obtained from individuals who are receiving treatment.

[0209] In some embodiments, a method for treating cancer in an individual is provided, the method comprising: (a) contacting a population of PBMCs with a plurality of tumor antigen peptides to obtain an activated population of PBMCs; (b) administering an effective amount of activated PBMCs to the individual; and (c) administering an effective amount of a PD-1 inhibitor to the individual. In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody. In some embodiments, the PD-1 inhibitor is selected from nivolumab, pembrolizumab, and SHR-1210. In some embodiments, the activated PBMCs and the PD-1 inhibitor are administered simultaneously, such as in the same composition. In some embodiments, the activated PBMCs and the PD-1 inhibitor are administered sequentially. In some embodiments, the PBMCs are contacted with the plurality of tumor antigen peptides in the presence of a composition that facilitates the uptake of the plurality of tumor antigen peptides by antigen-presenting cells (such as dendritic cells) in the PBMCs. In some embodiments, the activated PBMC population is further contacted with IL-2. In some embodiments, the PBMC population is contacted with the plurality of tumor antigen peptides in the presence of an immune checkpoint inhibitor (such as a PD-1 inhibitor). In some embodiments, the activated PBMCs are administered at least three times. In some implementations, the interval between each administration of activated PBMCs is approximately 2 weeks to approximately 5 months (e.g., approximately 3 months). In some implementations, the activated PBMCs are administered intravenously. In some implementations, the PBMC group is obtained from the individual receiving treatment.

[0210] In some embodiments, a method for treating cancer in an individual is provided, the method comprising: (a) optionally administering to the individual an effective amount of dendritic cells loaded with a plurality of tumor antigen peptides; (b) administering to the individual an effective amount of activated T cells, wherein the activated T cells are prepared by co-culturing a population of T cells with a population of dendritic cells loaded with said plurality of tumor antigen peptides; and (c) administering to the individual an effective amount of pembrolizumab (such as... In some embodiments, activated T cells and pembrolizumab are administered simultaneously, such as in the same composition. In some embodiments, activated T cells and pembrolizumab are administered sequentially. In some embodiments, pembrolizumab is administered intravenously (e.g., by infusion over about 30 minutes). In some embodiments, pembrolizumab is administered at about 2 mg / kg. In some embodiments, pembrolizumab is administered approximately every 3 weeks. In some embodiments, the interval between the administration of dendritic cells and the administration of activated T cells is about 7 days to about 21 days (e.g., about 7 days to about 14 days, about 14 days to about 21 days, about 10 days, or about 14 days). In some embodiments, dendritic cells loaded with the multiple tumor antigen peptides are administered subcutaneously. In some embodiments, dendritic cells loaded with the multiple tumor antigen peptides are administered at least three times. In some embodiments, activated T cells are administered intravenously. In some embodiments, activated T cells are administered at least three times. In some embodiments, a population of T cells is co-cultured with a population of dendritic cells loaded with the various tumor antigen peptides for approximately 7 to approximately 21 days (e.g., approximately 7 to approximately 14 days, approximately 14 to approximately 21 days, or approximately 10 days). In some embodiments, the T cell population is derived from the non-adhesive portion of a population of peripheral blood mononuclear cells (PBMCs). In some embodiments, co-culture further includes contacting activated T cells with a variety of cytokines (such as IL-2, IL-7, IL-15, IL-21, or any combination thereof) and optionally an anti-CD3 antibody. In some embodiments, the T cell population is contacted with an immune checkpoint inhibitor (such as an inhibitor of PD-1) before and / or during co-culture. In some embodiments, a population of dendritic cells loaded with the various tumor antigen peptides is prepared by contacting the dendritic cell population with the various tumor antigen peptides. In some embodiments, the T cell population and the dendritic cell population are derived from the same individual. In some embodiments, the T cell population, the dendritic cell population, the PBMC population, or any combination thereof are derived from an individual receiving treatment.

[0211] In some embodiments, a method for treating cancer in an individual is provided, the method comprising: (a) inducing a population of monocytes to differentiate into a population of dendritic cells; (b) contacting the dendritic cell population with a plurality of tumor antigen peptides to obtain a population of dendritic cells loaded with said plurality of tumor antigen peptides; (c) optionally administering to the individual an effective amount of dendritic cells loaded with said plurality of tumor antigen peptides; (d) co-culturing the population of dendritic cells loaded with said plurality of tumor antigen peptides with a population of non-adhesive PBMCs to obtain a population of activated T cells; (e) administering to the individual an effective amount of activated T cells; and (f) administering to the individual an effective amount of pembrolizumab (such as... The PBMC population and non-adhesive PBMC population are obtained from the PBMC population. In some embodiments, activated T cells and pembrolizumab are administered simultaneously, such as in the same composition. In some embodiments, activated T cells and pembrolizumab are administered sequentially. In some embodiments, pembrolizumab is administered intravenously (e.g., by infusion over about 30 minutes). In some embodiments, pembrolizumab is administered at about 2 mg / kg. In some embodiments, pembrolizumab is administered approximately every 3 weeks. In some embodiments, the interval between the administration of dendritic cells and the administration of activated T cells is about 7 days to about 21 days (e.g., about 7 days to about 14 days, about 14 days to about 21 days, about 10 days, or about 14 days). In some embodiments, dendritic cells loaded with the multiple tumor antigen peptides are administered subcutaneously. In some embodiments, dendritic cells loaded with the multiple tumor antigen peptides are administered at least three times. In some embodiments, activated T cells are administered intravenously. In some embodiments, activated T cells are administered at least three times. In some embodiments, co-culture lasts from approximately 7 days to approximately 21 days (e.g., from approximately 7 days to approximately 14 days, from approximately 14 days to approximately 21 days, or from approximately 10 days). In some embodiments, co-culture also includes contacting activated T cells with a variety of cytokines (such as IL-2, IL-7, IL-15, IL-21, or any combination thereof) and optionally an anti-CD3 antibody. In some embodiments, non-adhesive PBMCs are contacted with an immune checkpoint inhibitor (such as an inhibitor of PD-1) before and / or during co-culture. In some embodiments, the PBMCs are obtained from a patient receiving treatment.

[0212] In some implementations, a method for treating cancer in an individual is provided, the method comprising: (a) contacting a group of PBMCs with a variety of tumor antigen peptides to obtain activated PBMCs; (b) administering an effective amount of activated PBMCs to the individual; and (c) administering an effective amount of pembrolizumab (such as...) to the individual. In some embodiments, activated PBMCs and pembrolizumab are administered simultaneously, such as in the same composition. In some embodiments, activated PBMCs and pembrolizumab are administered sequentially. In some embodiments, pembrolizumab is administered intravenously (e.g., by infusion over approximately 30 minutes). In some embodiments, pembrolizumab is administered at approximately 2 mg / kg. In some embodiments, pembrolizumab is administered approximately every 3 weeks. In some embodiments, PBMCs are contacted with the multiple tumor antigen peptides in the presence of a composition that favors the uptake of the multiple tumor antigen peptides by antigen-presenting cells (such as dendritic cells) in the PBMCs. In some embodiments, the activated PBMC population is further contacted with IL-2. In some embodiments, the PBMC population is contacted with the multiple tumor antigen peptides in the presence of an immune checkpoint inhibitor (such as an inhibitor of PD-1). In some embodiments, activated PBMCs are administered at least three times. In some embodiments, the interval between each administration of activated PBMCs is approximately 2 weeks to approximately 5 months (e.g., approximately 3 months). In some embodiments, activated PBMCs are administered intravenously. In some embodiments, the PBMC population is obtained from a treated individual.

[0213] In some embodiments, the immune checkpoint inhibitor is a PD-L1 inhibitor. In some embodiments, the immune checkpoint inhibitor is an anti-PD-L1 antibody. Exemplary anti-PD-L1 antibodies include, but are not limited to, KY-1003, MCLA-145, RG7446, BMS935559, MPDL3280A, MEDI4736, Avelumab, or STI-A1010.

[0214] Therefore, in some embodiments, a method for treating cancer in an individual is provided, the method comprising: (a) optionally administering to the individual an effective amount of dendritic cells loaded with a plurality of tumor antigen peptides; (b) administering to the individual an effective amount of activated T cells, wherein the activated T cells are prepared by co-culturing a population of T cells with a population of dendritic cells loaded with the plurality of tumor antigen peptides; and (c) administering to the individual an effective amount of a PD-L1 inhibitor. In some embodiments, the PD-L1 inhibitor is an anti-PD-L1 antibody. In some embodiments, the activated T cells and the PD-L1 inhibitor are administered simultaneously, such as in the same composition. In some embodiments, the activated T cells and the PD-L1 inhibitor are administered sequentially. In some embodiments, the interval between the administration of dendritic cells and the administration of activated T cells is from about 7 days to about 21 days (e.g., from about 7 days to about 14 days, from about 14 days to about 21 days, from about 10 days, or from about 14 days). In some embodiments, the dendritic cells loaded with the plurality of tumor antigen peptides are administered subcutaneously. In some embodiments, the dendritic cells loaded with the plurality of tumor antigen peptides are administered at least three times. In some embodiments, activated T cells are administered intravenously. In some embodiments, activated T cells are administered at least three times. In some embodiments, a population of T cells is co-cultured with a population of dendritic cells loaded with the various tumor antigen peptides for approximately 7 to approximately 21 days (e.g., approximately 7 to approximately 14 days, approximately 14 to approximately 21 days, or approximately 10 days). In some embodiments, the T cell population is derived from the non-adhesive portion of a population of peripheral blood mononuclear cells (PBMCs). In some embodiments, co-culture further includes contacting the activated T cells with a variety of cytokines (such as IL-2, IL-7, IL-15, IL-21, or any combination thereof) and optionally an anti-CD3 antibody. In some embodiments, the T cell population is contacted with an immune checkpoint inhibitor (such as an inhibitor of PD-L1) before and / or during co-culture. In some embodiments, a population of dendritic cells loaded with the various tumor antigen peptides is prepared by contacting the dendritic cell population with the various tumor antigen peptides. In some embodiments, the T cell population and the dendritic cell population are derived from the same individual. In some implementations, the T cell population, dendritic cell population, PBMC population, or any combination thereof are derived from the individual receiving treatment.

[0215] In some embodiments, a method for treating cancer in an individual is provided, the method comprising: (a) inducing a population of monocytes to differentiate into a population of dendritic cells; (b) contacting the dendritic cell population with a plurality of tumor antigen peptides to obtain a population of dendritic cells loaded with said plurality of tumor antigen peptides; (c) optionally administering to the individual an effective amount of dendritic cells loaded with said plurality of tumor antigen peptides; (d) co-culturing the population of dendritic cells loaded with said plurality of tumor antigen peptides with a population of non-adhesive PBMCs to obtain a population of activated T cells; (e) administering to the individual an effective amount of activated T cells; and (f) administering to the individual an effective amount of a PD-L1 inhibitor, wherein the monocyte population and the non-adhesive PBMC population are obtained from a population of PBMCs. In some embodiments, the PD-L1 inhibitor is an anti-PD-L1 antibody. In some embodiments, activated T cells and a PD-L1 inhibitor are administered simultaneously, such as in the same composition. In some embodiments, activated T cells and a PD-L1 inhibitor are administered sequentially. In some embodiments, the interval between the administration of dendritic cells and the administration of activated T cells is approximately 7 to approximately 21 days (e.g., approximately 7 to approximately 14 days, approximately 14 to approximately 21 days, approximately 10 days, or approximately 14 days). In some embodiments, dendritic cells loaded with the various tumor antigen peptides are administered subcutaneously. In some embodiments, dendritic cells loaded with the various tumor antigen peptides are administered at least three times. In some embodiments, activated T cells are administered intravenously. In some embodiments, activated T cells are administered at least three times. In some embodiments, co-culture lasts approximately 7 to approximately 21 days (e.g., approximately 7 to approximately 14 days, approximately 14 to approximately 21 days, or approximately 10 days). In some embodiments, co-culture further includes contacting activated T cells with a variety of cytokines (e.g., IL-2, IL-7, IL-15, IL-21, or any combination thereof) and optionally an anti-CD3 antibody. In some embodiments, non-adhesive PBMCs are contacted with an immune checkpoint inhibitor (e.g., an inhibitor of PD-L1) before and / or during co-culture. In some implementations, the PBMC group is obtained from individuals who are receiving treatment.

[0216] In some embodiments, a method for treating cancer in an individual is provided, the method comprising: (a) contacting a population of PBMCs with a plurality of tumor antigen peptides to obtain an activated population of PBMCs; (b) administering an effective amount of activated PBMCs to the individual; and (c) administering an effective amount of a PD-L1 inhibitor to the individual. In some embodiments, the PD-L1 inhibitor is an anti-PD-L1 antibody. In some embodiments, the activated PBMCs and the PD-L1 inhibitor are administered simultaneously, such as in the same composition. In some embodiments, the activated PBMCs and the PD-L1 inhibitor are administered sequentially. In some embodiments, the PBMCs are contacted with the plurality of tumor antigen peptides in the presence of a composition that facilitates the uptake of the plurality of tumor antigen peptides by antigen-presenting cells (such as dendritic cells) in the PBMCs. In some embodiments, the activated PBMC population is further contacted with IL-2. In some embodiments, the PBMC population is contacted with the plurality of tumor antigen peptides in the presence of an immune checkpoint inhibitor (such as a PD-L1 inhibitor). In some embodiments, the activated PBMCs are administered at least three times. In some embodiments, the interval between each administration of the activated PBMCs is from about 2 weeks to about 5 months (such as about 3 months). In some implementations, the activated PBMCs are administered intravenously. In some implementations, the PBMC group is obtained from the individual receiving treatment.

[0217] In some embodiments, the immune checkpoint inhibitor is an inhibitor of CTLA-4. In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody. Exemplary anti-CTLA-4 antibodies include, but are not limited to, ipilimumab, trimemumab, and KAHR-102. In some embodiments, the immune checkpoint inhibitor is ipilimumab (e.g., ).

[0218] Therefore, in some embodiments, a method for treating cancer in an individual is provided, the method comprising: (a) optionally administering to the individual an effective amount of dendritic cells loaded with a plurality of tumor antigen peptides; (b) administering to the individual an effective amount of activated T cells, wherein the activated T cells are prepared by co-culturing a population of T cells with a population of dendritic cells loaded with said plurality of tumor antigen peptides; and (c) administering to the individual an effective amount of an inhibitor of CTLA-4. In some embodiments, the inhibitor of CTLA-4 is an anti-CTLA-4 antibody, such as ipilimumab. In some embodiments, the activated T cells and the inhibitor of CTLA-4 are administered simultaneously, such as in the same composition. In some embodiments, the activated T cells and the inhibitor of CTLA-4 are administered sequentially. In some embodiments, the interval between the administration of dendritic cells and the administration of activated T cells is about 7 days to about 21 days (such as about 7 days to about 14 days, about 14 days to about 21 days, about 10 days, or about 14 days). In some embodiments, the dendritic cells loaded with said plurality of tumor antigen peptides are administered subcutaneously. In some embodiments, dendritic cells loaded with the multiple tumor antigen peptides are administered at least three times. In some embodiments, activated T cells are administered intravenously. In some embodiments, activated T cells are administered at least three times. In some embodiments, a population of T cells is co-cultured with a population of dendritic cells loaded with the multiple tumor antigen peptides for about 7 days to about 21 days (e.g., about 7 days to about 14 days, about 14 days to about 21 days, or about 10 days). In some embodiments, the T cell population is derived from the non-adhesive portion of a population of peripheral blood mononuclear cells (PBMCs). In some embodiments, co-culture further includes contacting the activated T cells with a variety of cytokines (such as IL-2, IL-7, IL-15, IL-21, or any combination thereof) and optionally an anti-CD3 antibody. In some embodiments, the T cell population is contacted with an immune checkpoint inhibitor (such as an inhibitor of CTLA-4) before and / or during co-culture. In some embodiments, a population of dendritic cells loaded with the multiple tumor antigen peptides is prepared by contacting the dendritic cell population with the multiple tumor antigen peptides. In some implementations, the T cell population and dendritic cell population are derived from the same individual. In some implementations, the T cell population, dendritic cell population, PBMC population, or any combination thereof are derived from the individual receiving treatment.

[0219] In some embodiments, a method for treating cancer in an individual is provided, the method comprising: (a) inducing a population of monocytes to differentiate into a population of dendritic cells; (b) contacting the dendritic cell population with a plurality of tumor antigen peptides to obtain a population of dendritic cells loaded with said plurality of tumor antigen peptides; (c) optionally administering to the individual an effective amount of dendritic cells loaded with said plurality of tumor antigen peptides; (d) co-culturing the population of dendritic cells loaded with said plurality of tumor antigen peptides with a population of non-adhesive PBMCs to obtain a population of activated T cells; (e) administering to the individual an effective amount of activated T cells; and (f) administering to the individual an effective amount of an inhibitor of CTLA-4, wherein the monocyte population and the non-adhesive PBMC population are obtained from a population of PBMCs. In some embodiments, the inhibitor of CTLA-4 is an anti-CTLA-4 antibody, such as ipilimumab. In some embodiments, activated T cells and an inhibitor of CTLA-4 are administered simultaneously, such as in the same composition. In some embodiments, activated T cells and an inhibitor of CTLA-4 are administered sequentially. In some embodiments, the interval between the administration of dendritic cells and the administration of activated T cells is approximately 7 to approximately 21 days (e.g., approximately 7 to approximately 14 days, approximately 14 to approximately 21 days, approximately 10 days, or approximately 14 days). In some embodiments, dendritic cells loaded with the various tumor antigen peptides are administered subcutaneously. In some embodiments, dendritic cells loaded with the various tumor antigen peptides are administered at least three times. In some embodiments, activated T cells are administered intravenously. In some embodiments, activated T cells are administered at least three times. In some embodiments, co-culture lasts approximately 7 to approximately 21 days (e.g., approximately 7 to approximately 14 days, approximately 14 to approximately 21 days, or approximately 10 days). In some embodiments, co-culture further includes contacting activated T cells with a variety of cytokines (e.g., IL-2, IL-7, IL-15, IL-21, or any combination thereof) and optionally an anti-CD3 antibody. In some embodiments, non-adhesive PBMCs are contacted with an immune checkpoint inhibitor (e.g., an inhibitor of CTLA-4) before and / or during co-culture. In some implementations, the PBMC group is obtained from individuals who are receiving treatment.

[0220] In some embodiments, a method for treating cancer in an individual is provided, the method comprising: (a) contacting a population of PBMCs with a plurality of tumor antigen peptides to obtain an activated population of PBMCs; (b) administering an effective amount of the activated PBMCs to the individual; and (c) administering an effective amount of an inhibitor of CTLA-4 to the individual. In some embodiments, the inhibitor of CTLA-4 is an anti-CTLA-4 antibody, such as ipilimumab. In some embodiments, the activated PBMCs and the inhibitor of CTLA-4 are administered simultaneously, such as in the same composition. In some embodiments, the activated PBMCs and the inhibitor of CTLA-4 are administered sequentially. In some embodiments, the PBMCs are contacted with the plurality of tumor antigen peptides in the presence of a composition that favors the uptake of the plurality of tumor antigen peptides by antigen-presenting cells (such as dendritic cells) in the PBMCs. In some embodiments, the activated PBMCs are further contacted with IL-2. In some embodiments, the PBMCs are contacted with the plurality of tumor antigen peptides in the presence of an immune checkpoint inhibitor (such as an inhibitor of CTLA-4). In some embodiments, the activated PBMCs are administered at least three times. In some implementations, the interval between each administration of activated PBMCs is approximately 2 weeks to approximately 5 months (e.g., approximately 3 months). In some implementations, the activated PBMCs are administered intravenously. In some implementations, the PBMC group is obtained from the individual receiving treatment.

[0221] In some embodiments, activated T cells (or activated PBMCs) and immune checkpoint inhibitors are administered simultaneously. In some embodiments, activated T cells (or activated PBMCs) and immune checkpoint inhibitors are administered in a single composition. In some embodiments, the immune checkpoint inhibitor is present in a co-culture. In some embodiments, activated T cells (or activated PBMCs) and immune checkpoint inhibitors are mixed prior to administration (e.g., immediately before administration). In some embodiments, activated T cells (or activated PBMCs) and immune checkpoint inhibitors are administered simultaneously via a single composition.

[0222] In some embodiments, activated T cells (or activated PBMCs) and immune checkpoint inhibitors are administered sequentially. In some embodiments, the immune checkpoint inhibitor is administered before the administration of activated T cells (or activated PBMCs). In some embodiments, the immune checkpoint inhibitor is administered after the administration of activated T cells (or activated PBMCs).

[0223] Multiple tumor antigen peptides

[0224] All MASCT methods (including PBMC-based MASCT methods) and cell preparation methods described in this article use a variety of tumor antigen peptides (including neoantigen peptides) to prepare APCs (such as dendritic cells) and activated T cells or activated PBMCs that can elicit specific T cell responses in vitro and in vivo.

[0225] In some embodiments, each tumor antigen peptide in the MASCT method comprises any number of epitopes from about 1, 2, 3, 4, 5, or 10 epitopes from a single protein antigen (including neoantigens). In some embodiments, each of the multiple tumor antigen peptides comprises at least one epitope that can be recognized by a T-cell receptor. In some embodiments, the multiple tumor antigen peptides comprise at least one tumor antigen peptide containing at least two epitopes from a single protein antigen. The tumor antigen peptide may be a naturally derived peptide fragment from a protein antigen containing one or more epitopes, or an artificially designed peptide having one or more natural epitope sequences, wherein a linker peptide may optionally be positioned between adjacent epitope sequences. In some preferred embodiments, the epitopes contained in the same tumor antigen peptide are derived from the same protein antigen.

[0226] Tumor antigen peptides (including neoantigen peptides) may contain at least one MHC-I epitope, at least one MHC-II epitope, or both one or more MHC-I epitopes and one or more MHC-II epitopes. In some embodiments, the plurality of tumor antigen peptides includes at least one peptide containing an MHC-I epitope. In some embodiments, the plurality of tumor antigen peptides includes at least one peptide containing an MHC-II epitope. In some embodiments, at least one of the plurality of tumor antigen peptides contains both MHC-I and MHC-II epitopes.

[0227] Specialized design strategies can be applied to the sequences of tumor antigen peptides (including neoantigen peptides) to optimize the immune response to dendritic cells loaded with tumor antigen peptides. Typically, peptides longer than their precise epitope peptides can increase peptide uptake into antigen-presenting cells, such as dendritic cells. In some embodiments, based on the native sequence of the protein carrying the epitope, the MHC-I or MHC-II epitope sequence is extended at the N-terminus, C-terminus, or both ends to obtain an extended sequence suitable for presentation by class I and class II MHC molecules, and by different subtypes of MHC molecules in different individuals. In some embodiments, the epitope sequence is extended at one or both ends by any number of amino acid residues, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, or 20, to generate the extended epitope. In some embodiments, the peptide having the MHC-I or MHC-II epitope also includes additional amino acids flanking the epitope at the N-terminus, C-terminus, or both ends. In some embodiments, each of the plurality of tumor antigen peptides is about 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 amino acids in length. The different tumor antigen peptides in the plurality of tumor antigen peptides may have the same length or different lengths. In some embodiments, each of the plurality of tumor antigen peptides is about 20-40 amino acids in length.

[0228] In some embodiments, the amino acid sequences of one or more epitope peptides used to design the tumor antigen peptides of this application are based on sequences known in the art or available in public databases such as peptide databases (van der Bruggen P et al. (2013) "Peptide database: T cell-defined tumor antigens. Cancer Immunity"). URL: www.cancerimmunity.org / peptide / . In some embodiments, the amino acid sequences of said one or more epitope peptides are selected from SEQ ID NO:1-35.

[0229] In some embodiments, bioinformatics tools for T-cell epitope prediction are used to predict the amino acid sequence of one or more epitope peptides based on the sequence of the antigen protein (including neoantigens). Exemplary bioinformatics tools for T-cell epitope prediction are known in the art, for example, see Yang X. and Yu X. (2009) “An introduction to epitope prediction methods and software” Rev. Med. Virol. 19(2):77-96. In some embodiments, the sequence of the antigen protein is known in the art or available in public databases. In some embodiments, the sequence of the antigen protein (including neoantigens) is determined by sequencing a sample (such as a tumor sample) of an individual receiving treatment.

[0230] The present invention envisions tumor antigen peptides derived from any tumor antigens and epitopes (including neoantigens and neoepitaxes) known in the art, or specifically developed or predicted by the inventors using bioinformatics tools.

[0231] In some embodiments, the plurality of tumor antigen peptides includes a first core group of common tumor antigen peptides. In some embodiments, the plurality of tumor antigen peptides further includes a second group of cancer type-specific antigen peptides. In some embodiments, the plurality of tumor antigen peptides includes one or more neoantigen peptides. In some embodiments, the neoantigen peptide is a cancer type-specific antigen peptide. In some embodiments, the plurality of tumor antigen peptides consists of a first core group of common tumor antigen peptides. In some embodiments, the plurality of tumor antigen peptides consists of a first core group of common tumor antigen peptides and a second group of cancer type-specific antigen peptides. In some embodiments, the plurality of tumor antigen peptides consists only of neoantigen peptides. In some embodiments, the plurality of tumor antigen peptides includes a first core group of common tumor antigen peptides and one or more neoantigen peptides. In some embodiments, the plurality of tumor antigen peptides includes a first core group of common tumor antigen peptides, a second group of cancer type-specific antigen peptides, and one or more neoantigen peptides.

[0232] The first core group of common tumor antigen peptides are derived from tumor antigens that are typically expressed or overexpressed on the surface of various cancers of different types. Therefore, the first core group of common tumor antigen peptides can be used to prepare dendritic cells or activated T cells for use in any MASCT method (including PBMC-based MASCT methods) or other therapeutic or cell preparation methods described herein to treat individuals with different cancer types. For example, in some embodiments, the first core group of common tumor antigen peptides can be used in methods described herein for treating various cancers such as lung cancer, colon cancer, gastric cancer, prostate cancer, melanoma, lymphoma, pancreatic cancer, ovarian cancer, breast cancer, glioma, esophageal cancer, nasopharyngeal carcinoma, cervical cancer, renal cancer, or hepatocellular carcinoma. Exemplary tumor antigen peptides of the first core group include, but are not limited to, peptides derived from hTERT, p53, survivin, NY-ESO-1, CEA, CCND1, MET, RGS5, MMP7, VEGFR, and CDCA1. The first core group may comprise peptides derived from any number of tumor antigens selected from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 25, 30, 40, or 50. The first core group may comprise any number of common tumor antigen peptides selected from about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 25, 30, 40, or 50. In some embodiments, the first core group comprises more than one common tumor antigen peptide. In some embodiments, the first core group comprises about 10 to about 20 common tumor antigen peptides. In some embodiments, the first core group comprises common tumor antigen peptides having more than one epitope selected from SEQ ID NO: 1-24.

[0233] The second group of cancer type-specific antigenic peptides is derived from tumor antigens expressed or overexpressed only in one or a limited number of cancer types. Therefore, the second group of cancer type-specific antigenic peptides can be used to prepare dendritic cells, activated T cells, which are used in any MASCT method or other therapeutic or cell preparation methods described herein to treat individuals with specific cancer types. Exemplary cancer type-specific antigenic peptides for treating hepatocellular carcinoma (HCC) include, but are not limited to, peptides derived from AFP and GPC3. In some embodiments, one or more cancer-specific antigenic peptides are virus-specific antigenic peptides derived from a virus that can induce cancer or is associated with cancer development in an individual upon infection. In some embodiments, the virus-specific antigenic peptides are specific to the subtype of the virus infecting the individual. Exemplary virus-specific antigenic peptides for treating HCC patients co-infected with HBV include, but are not limited to, peptides derived from HBV core antigen and HBV DNA polymerase. In some embodiments, the virus-specific antigenic peptides contain at least one epitope selected from SEQ ID NO:31-35. In some embodiments, the second group comprises virus-specific antigenic peptides derived from HBV antigens, wherein the method is for treating hepatocellular carcinoma in an individual. In some embodiments, the second group comprises virus-specific antigenic peptides derived from HPV antigens, wherein the method is for treating cervical cancer in an individual. In some embodiments, the second group comprises virus-specific antigenic peptides derived from EBV antigens, wherein the method is for treating nasopharyngeal carcinoma in an individual. The second group of cancer type-specific antigenic peptides may include peptides derived from any number of cancer type-specific antigens selected from about 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 25, 30, 40, or 50. In some embodiments, the second group comprises more than one cancer type-specific antigenic peptide. In some embodiments, the second group comprises about 1 to about 10 cancer type-specific antigenic peptides. In some embodiments, the second group comprises a cancer type-specific antigenic peptide having at least one epitope selected from SEQ ID NO:25-35, wherein the cancer is hepatocellular carcinoma. In some embodiments, the type of cancer targeted by the cancer type-specific antigenic peptide is substantially selected from hepatocellular carcinoma, cervical cancer, nasopharyngeal carcinoma, breast cancer, and lymphoma.

[0234] In some embodiments, the plurality of tumor antigen peptides includes one or more neoantigen peptides (such as any combination of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more). The neoantigen peptide is derived from a neoantigen. A neoantigen is an antigen recently collected and expressed that is present in the tumor cells of an individual (such as an individual receiving cancer treatment). In some embodiments, the neoantigen is derived from a mutant protein antigen that is present only in cancer cells and not in normal cells. The neoantigen may be uniquely present in the tumor cells of an individual receiving cancer treatment (such as all tumor cells or a portion of tumor cells), or in an individual having a cancer type similar to that of the individual receiving treatment. In some embodiments, the neoantigen is a clonal neoantigen. In some embodiments, the neoantigen is a subclonal neoantigen. In some embodiments, the neoantigen is present in at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of the tumor cells in the individual. In some embodiments, the neoantigen peptide contains an MHC-I restricted neoepitope. In some embodiments, the neoantigen peptide contains a novel MHC-II restricted epitope. In some embodiments, the neoantigen peptide is designed (e.g., by extending the novel epitope at the N-terminus and C-terminus) to facilitate presentation of the novel epitope by both class I and class II MHC molecules. Exemplary neoantigen peptides include, but are not limited to, those derived from mutant KRAS (e.g., KRAS...). G12A ), PARP4 (e.g., PARP4) T1170I MLL3 (e.g., MLL3) C988F ) and MTHFR (e.g., MTHFR A222V The neoantigen peptide contains a novel epitope. In some embodiments, the neoantigen peptide includes an epitope with a point mutation in a sequence selected from SEQ ID No:41-45. In some embodiments, the neoantigen peptide includes an epitope selected from SEQ ID NO:36-40.

[0235] Neoantigen peptides can be selected based on the genetic profile of one or more tumor sites in an individual receiving treatment. In some embodiments, the genetic profile of the tumor sample includes whole-genome sequence information. In some embodiments, the genetic profile of the tumor sample includes exome sequence information. In some embodiments, the genetic profile of the tumor sample includes sequence information of cancer-related genes.

[0236] The neoantigen peptides suitable for use in this invention can be derived from any mutant protein in tumor cells, such as those encoded by mutant cancer-associated genes. In some embodiments, the neoantigen peptide comprises a single novel epitope derived from a cancer-associated gene. In some embodiments, the neoantigen peptide comprises more than one (e.g., two, three, or more) novel epitopes derived from cancer-associated genes. In some embodiments, the multiple tumor antigens comprise multiple neoantigen peptides derived from a single cancer-associated gene. In some embodiments, the multiple tumor antigens comprise multiple neoantigen peptides derived from more than one (e.g., any combination of two, three, four, five, or more) cancer-associated genes.

[0237] Cancer-associated genes are genes that are overexpressed or expressed only in cancer cells and not in normal cells. Exemplary cancer-related genes include, but are not limited to, ABL1, AKT1, AKT2, AKT3, ALK, ALOX12B, APC, AR, ARAF, ARID1A, ARID1B, ARID2, ASXL1, ATM, ATRX, AURKA, AURKB, AXL, B2M, BAP1, BCL2, BCL2L1, BCL2L12, BCL6, BCOR, BCORL1, BLM, BMPR1A, BRAF, BRCA1, BRCA2, BRD4, BRIP1, BUB1B, CADM2, CARD11, CBL, CBLB, CCND1, CCND2, CCND3, CCNE1, CD274, CD58, CD79B, CDC73, CDH1, CDK1, CDK2, CDK4, CDK5, CDK6, CDK9, CDKN1A, CDKN1B, CDKN1C. CDKN2A, CDKN2B, CDKN2C, CEBPA, CHEK2, CIITA, CREBBP, CRKL, CRLF2, CRTC1, CRTC2, CSF1R, CSF3R, CTNNB1, CUX1, CYLD, DDB2, DDR2, DEPDC5, DICER1, DIS3, DMD, DNMT3A, EED, EGFR, EP300, EPHA3, EPHA5, EPHA7, ERBB2, ERBB3, ERBB4, ERCC2, ERCC3, ERCC4, ERCC5, ESR1, ETV1, ETV4, ETV5, ETV6, EWSR1, EXT1, EXT2, EZH2, FAM46C, FANCA, FANCC, FANCD2, FANCE, FANCF, FANCG, FAS, FBXW7, FGFR1, FGFR2, FGFR3, FGFR4, FH, FKBP9, FLCN, FLT1, FLT3, FLT4, FUS, GATA3, GATA4, GATA6, GLI1, GLI2, GLI3, GNA11, GNAQ, GNAS, GNB2L1, GPC3, GSTM5, H3F3A, HNF1A, HRAS, ID3, IDH1, IDH2, IGF1R, IKZF1, IKZF3, INSIG1, JAK2, JAK3, KCNIP1, KDM5C, KDM6A, KDM6B, KDR, KEAP1, KIT, KRAS, LINC00894, LMO1, LMO2, LMO3, MAP2K1, MAP2K4, MAP3K1,MAPK1, MCL1, MDM2, MDM4, MECOM, MEF2B, MEN1, MET, MITF, MLH1, MLL (KMT2A), MLL2 (KTM2D), MPL, MSH2, MSH6, MTOR, MUTYH, MYB, MYBL1, MYC, MYCL1 (MYCL), MYCN, MYD88, NBN, NEGR1, NF1, NF2, NFE2L2, NFKBIA, NFKBIZ, NKX2-1, NOTCH1, NOTCH2, NPM1, NPRL2, NPRL3, NRAS, NTRK1, NTRK2, NTRK3, PALB2, PARK2, PAX5, PBRM1, PDCD1LG2, PDGFRA, PDGFRB, PHF6, PHOX2B, PIK3C2B, PIK3CA, PIK3R1, PIM1, PMS1, PMS2, PNRC1, PRAME, PRDM1, PRF1, PRKAR1A, PRKCI, PRKCZ, PRKDC, PRPF40B, PRPF8, PSMD13, PTCH1, PTEN, PTK2, PTPN11, PTPRD, QKI, RAD21, RAF1, RARA, RB1, RBL2, RECQL4, REL, RET, RFWD2, RHEB, RHPN2, ROS1, RPL26, RUNX1, SBDS, SDHA, SDHAF2, SDHB, SDHC, SDHD, SETBP1, SETD2, SF1, SF3B1, SH2B3, SLITRK6, SMAD2, SMAD4, SMARCA4, SMARCB1, SMC1A, SMC3, SMO, SOCS1, SOX2, SOX9, SQSTM1, SRC, SRSF2, STAG1, STAG2, STAT3, STAT6, STK11, SUFU, SUZ12, SYK, TCF3, TCF7L1, TCF7L2, TERC, TERT, TET2, TLR4, TNFAIP3, TP53, TSC1, TSC2, U2AF1, VHL, WRN, WT1, XPA, XPC, XPO1, ZNF217, ZNF708, and ZRSR2.

[0238] In some embodiments, the plurality of tumor antigen peptides comprises at least one (such as any combination of at least about 1, 5, 10, 15, 20, 25, 30, 35, or 40) epitopes selected from SEQ ID NO: 1-40. In some embodiments, the plurality of tumor antigen peptides comprises at least one (such as any combination of at least about 1, 5, 10, 15, 20, or 24) epitopes selected from SEQ ID NO: 1-24. In some embodiments, the plurality of tumor antigen peptides comprises at least one (such as any combination of about 1, 2, 3, 4, 5, or 6) epitopes selected from SEQ ID NO: 25-30. In some embodiments, the plurality of tumor antigen peptides comprises at least one (such as any combination of about 1, 2, 3, 4, or 5) epitopes selected from SEQ ID NO: 31-35. In some embodiments, the plurality of tumor antigen peptides comprises at least one (such as any combination of about 1, 2, 3, 4, or 5) epitopes selected from SEQ ID NO: 36-40. In some embodiments, the plurality of tumor antigen peptides include Figure 2B or Figure 2C At least one of common tumor antigen peptides (such as any combination of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10). In some embodiments, the multiple tumor antigen peptides include Figure 29A At least one of the following (such as any combination of at least about 1, 2, 3, 4, or 5) neoantigen peptides. In some embodiments, the multiple tumor antigen peptides include Figure 2B , Figure 2C or Figure 29A The plurality of tumor antigen peptides includes at least one (such as any combination of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more) tumor antigen peptides. In some embodiments, the plurality of tumor antigen peptides includes at least one (such as any combination of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more) tumor antigen peptide, each tumor antigen peptide comprising one or more epitopes encoded by oncology-associated genes selected from hTERT, p53, survivin, NY-ESO-1, CEA, CCND1, MET, RGS5, MMP7, VEGFR, AFP, GPC3, HBVc, HBVp, CDCA, KRAS, PARP4, MLL3 and MTHFR.

[0239] In some embodiments, the plurality of tumor antigen peptides includes at least 10 tumor antigen peptides. In some embodiments, each of the at least 10 tumor antigen peptides contains at least one epitope selected from SEQ ID NO:1-40. In some embodiments, each of the at least 10 tumor antigen peptides contains at least one epitope selected from SEQ ID NO:1-24. In some embodiments, the plurality of tumor antigen peptides includes... Figure 2B The tumor antigen peptides include at least 10 tumor antigen peptides. In some embodiments, the plurality of tumor antigen peptides includes those selected from... Figure 2C The tumor antigen peptides include at least 10 tumor antigen peptides. In some embodiments, the plurality of tumor antigen peptides includes Figure 29A At least one neoantigen peptide in it.

[0240] In some embodiments, a composition comprising at least 10 tumor antigen peptides is provided, wherein each of the at least 10 tumor antigen peptides comprises at least one epitope selected from SEQ ID NO:1-40. In some embodiments, a composition comprising at least 10 tumor antigen peptides is provided, wherein each of the at least 10 tumor antigen peptides comprises at least one epitope selected from SEQ ID NO:1-24. In some embodiments, a composition comprising... Figure 2B A composition comprising at least 10 tumor antigen peptides selected from [the tumor antigen peptides mentioned above]. In some embodiments, a composition comprising [the tumor antigen peptides mentioned above] is provided. Figure 2C Compositions of at least 10 tumor antigen peptides, including the tumor antigen peptides shown in Figure 29A. In some embodiments, compositions comprising at least 10 tumor antigen peptides are provided, each tumor antigen peptide containing an epitope encoded by an oncology-associated gene selected from hTERT, p53, survivin, NY-ESO-1, CEA, CCND1, MET, RGS5, MMP7, VEGFR, AFP, GPC3, HBVc, HBVp, CDCA, KRAS, PARP4, MLL3, and MTHFR.

[0241] In some embodiments, a separated dendritic cell population loaded with multiple tumor antigen peptides is provided, prepared by contacting the dendritic cell population with multiple tumor antigen peptides, wherein the multiple tumor antigen peptides include at least 10 tumor antigen peptides. In some embodiments, the multiple tumor antigen peptides include at least 10 tumor antigen peptides, which include a first core group of common tumor antigen peptides and optionally a second group of cancer type-specific antigen peptides. In some embodiments, the multiple tumor antigen peptides include one or more neoantigen peptides. In some embodiments, each of the at least 10 tumor antigen peptides contains at least one epitope selected from SEQ ID NO:1-40. In some embodiments, the multiple tumor antigen peptides include those selected from... Figure 2C and Figure 29A The tumor antigen peptides comprise at least 10 tumor antigen peptides. In some embodiments, the plurality of tumor antigen peptides includes at least 10 tumor antigen peptides, each tumor antigen peptide containing one or more epitopes encoded by cancer-associated genes, said epitopes being selected from hTERT, p53, survivin, NY-ESO-1, CEA, CCND1, MET, RGS5, MMP7, VEGFR, AFP, GPC3, HBVc, HBVp, CDCA, KRAS, PARP4, MLL3, and MTHFR.

[0242] In some embodiments, a method for preparing an activated T cell population is provided, the method comprising co-culturing the T cell population with a dendritic cell population loaded with a plurality of tumor antigen peptides, wherein the plurality of tumor antigen peptides comprises at least 10 tumor antigen peptides. In some embodiments, an isolated activated T cell population is provided by co-culturing a T cell population with a dendritic cell population loaded with a plurality of tumor antigen peptides, wherein the plurality of tumor antigen peptides comprises at least 10 tumor antigen peptides. In some embodiments, the plurality of tumor antigen peptides comprises at least 10 tumor antigen peptides, these tumor antigen peptides comprising a first core group of common tumor antigen peptides and optionally a second group of cancer type-specific antigen peptides. In some embodiments, the plurality of tumor antigen peptides comprises one or more neoantigen peptides. In some embodiments, each of the at least 10 tumor antigen peptides contains at least one epitope selected from SEQ ID NO:1-40. In some embodiments, the plurality of tumor antigen peptides comprises epitopes selected from... Figure 2C and Figure 29AThe tumor antigen peptides comprise at least 10 tumor antigen peptides. In some embodiments, the plurality of tumor antigen peptides includes at least 10 tumor antigen peptides, each containing one or more epitopes encoded by cancer-associated genes selected from hTERT, p53, survivin, NY-ESO-1, CEA, CCND1, MET, RGS5, MMP7, VEGFR, AFP, GPC3, HBVc, HBVp, CDCA, KRAS, PARP4, MLL3, and MTHFR.

[0243] In some embodiments, a method for preparing an activated T cell population is provided, the method comprising: (a) inducing a monocyte population to differentiate into a dendritic cell population; (b) contacting the dendritic cell population with a plurality of tumor antigen peptides to obtain a dendritic cell population loaded with said plurality of tumor antigen peptides; and (c) co-culturing the dendritic cell population loaded with said plurality of tumor antigen peptides with a non-adhesive PBMC population to obtain an activated T cell population, wherein the monocyte population and the non-adhesive PBMC population are obtained from a PBMC population (such as from an individual), and wherein said plurality of tumor antigen peptides include at least 10 tumor antigen peptides. In some embodiments, an isolated activated T cell population is provided prepared by: (a) inducing a monocyte population to differentiate into a dendritic cell population; (b) contacting the dendritic cell population with a plurality of tumor antigen peptides to obtain a dendritic cell population loaded with the plurality of tumor antigen peptides; and (c) co-culturing the dendritic cell population loaded with the plurality of tumor antigen peptides with a non-adhesive PBMC population to obtain an activated T cell population, wherein the monocyte population and the non-adhesive PBMC population are obtained from a PBMC population (such as from an individual), and wherein the plurality of tumor antigen peptides comprises at least 10 tumor antigen peptides. In some embodiments, the plurality of tumor antigen peptides comprises at least 10 tumor antigen peptides, which include a first core group of common tumor antigen peptides and optionally a second group of cancer type-specific antigen peptides. In some embodiments, the plurality of tumor antigen peptides comprises one or more neoantigen peptides. In some embodiments, each of the at least 10 tumor antigen peptides contains at least one epitope selected from SEQ ID NO: 1-40. In some embodiments, the plurality of tumor antigen peptides comprises a subset of... Figure 2C and Figure 29A The tumor antigen peptides comprise at least 10 tumor antigen peptides. In some embodiments, the plurality of tumor antigen peptides includes at least 10 tumor antigen peptides, each containing one or more epitopes encoded by cancer-associated genes selected from hTERT, p53, survivin, NY-ESO-1, CEA, CCND1, MET, RGS5, MMP7, VEGFR, AFP, GPC3, HBVc, HBVp, CDCA, KRAS, PARP4, MLL3, and MTHFR.

[0244] In some embodiments, a method for treating cancer in an individual is provided, the method comprising contacting a population of PBMCs with a plurality of tumor antigen peptides to obtain an activated population of PBMCs, and administering an effective amount of the activated PBMCs to the individual, wherein the plurality of tumor antigen peptides comprises at least 10 tumor antigen peptides. In some embodiments, the population of PBMCs is contacted with the plurality of tumor antigen peptides in the presence of an immune checkpoint inhibitor such as an inhibitor of PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3. In some embodiments, the activated PBMCs are administered at least three times. In some embodiments, the interval between each administration of the activated PBMCs is from about 2 weeks to about 5 months (e.g., about 3 months). In some embodiments, the population of PBMCs is obtained from an individual receiving treatment. In some embodiments, the method further comprises administering an effective amount of an immune checkpoint inhibitor, such as an inhibitor of PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3, to the individual. In some embodiments, the plurality of tumor antigen peptides includes at least 10 tumor antigen peptides, comprising a first core group of common tumor antigen peptides and optionally a second group of cancer type-specific antigen peptides. In some embodiments, the plurality of tumor antigen peptides includes one or more neoantigen peptides. In some embodiments, each of the at least 10 tumor antigen peptides contains at least one epitope selected from SEQ ID NO:1-40. In some embodiments, the plurality of tumor antigen peptides includes... Figure 2C and Figure 29A The tumor antigen peptides comprise at least 10 tumor antigen peptides. In some embodiments, the plurality of tumor antigen peptides includes at least 10 tumor antigen peptides, each tumor antigen peptide containing one or more epitopes encoded by cancer-associated genes selected from hTERT, p53, survivin, NY-ESO-1, CEA, CCND1, MET, RGS5, MMP7, VEGFR, AFP, GPC3, HBVc, HBVp, CDCA, KRAS, PARP4, MLL3, and MTHFR.

[0245] In some embodiments, a method for treating cancer in an individual is provided, the method comprising optionally administering to the individual an effective amount of dendritic cells loaded with multiple tumor antigen peptides, and administering to the individual an effective amount of activated T cells, wherein the activated T cells are prepared by co-culturing a population of T cells with a population of dendritic cells loaded with said multiple tumor antigen peptides (e.g., in the presence of an immune checkpoint inhibitor), wherein said multiple tumor antigen peptides include at least 10 tumor antigen peptides. In some embodiments, the population of T cells is contacted with an immune checkpoint inhibitor (such as an inhibitor of PD-1, PD-L1, or CTLA-4) before and / or during co-culturing. In some embodiments, the population of dendritic cells loaded with said multiple tumor antigen peptides is prepared by contacting the population of dendritic cells with said multiple tumor antigen peptides. In some embodiments, the population of T cells and the population of dendritic cells are derived from the same individual. In some embodiments, the population of T cells, the population of dendritic cells, the population of PBMCs, or any combination thereof are derived from an individual receiving treatment. In some embodiments, the method further includes administering an effective amount of an immune checkpoint inhibitor to the individual, such as an inhibitor of PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3. In some embodiments, the plurality of tumor antigen peptides comprises at least 10 tumor antigen peptides, including a first core group of common tumor antigen peptides and optionally a second group of cancer type-specific antigen peptides. In some embodiments, the plurality of tumor antigen peptides comprises one or more neoantigen peptides. In some embodiments, each of the at least 10 tumor antigen peptides contains at least one epitope selected from SEQ ID NO:1-40. In some embodiments, the plurality of tumor antigen peptides comprises... Figure 2C and Figure 29A The tumor antigen peptides comprise at least 10 tumor antigen peptides. In some embodiments, the plurality of tumor antigen peptides includes at least 10 tumor antigen peptides, each tumor antigen peptide containing one or more epitopes encoded by cancer-associated genes selected from hTERT, p53, survivin, NY-ESO-1, CEA, CCND1, MET, RGS5, MMP7, VEGFR, AFP, GPC3, HBVc, HBVp, CDCA, KRAS, PARP4, MLL3, and MTHFR.

[0246] In some embodiments, a method for treating cancer in an individual is provided, the method comprising: (a) inducing a population of monocytes to differentiate into a population of dendritic cells; (b) contacting the dendritic cell population with a plurality of tumor antigen peptides to obtain a population of dendritic cells loaded with said plurality of tumor antigen peptides; (c) optionally administering an effective amount of the dendritic cells loaded with said plurality of tumor antigen peptides to the individual; (d) co-culturing the population of dendritic cells loaded with said plurality of tumor antigen peptides with a population of non-adhesive PBMCs to obtain a population of activated T cells; and (e) administering an effective amount of activated T cells to the individual, wherein the monocyte population and the non-adhesive PBMC population are obtained from a population of PBMCs (such as those derived from the individual), and wherein said plurality of tumor antigen peptides comprise at least 10 tumor antigen peptides. In some embodiments, the non-adhesive PBMC population is contacted with an immune checkpoint inhibitor (such as an inhibitor of PD-1, PD-L1, or CTLA-4) before and / or during co-culturing. In some embodiments, the method further includes administering an effective amount of an immune checkpoint inhibitor to the individual, such as an inhibitor of PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3. In some embodiments, the plurality of tumor antigen peptides comprises at least 10 tumor antigen peptides, including a first core group of common tumor antigen peptides and optionally a second group of cancer type-specific antigen peptides. In some embodiments, the plurality of tumor antigen peptides comprises one or more neoantigen peptides. In some embodiments, each of the at least 10 tumor antigen peptides contains at least one epitope selected from SEQ ID NO:1-40. In some embodiments, the plurality of tumor antigen peptides comprises... Figure 2C and Figure 29A The tumor antigen peptides comprise at least 10 tumor antigen peptides. In some embodiments, the plurality of tumor antigen peptides includes at least 10 tumor antigen peptides, each tumor antigen peptide containing one or more epitopes encoded by cancer-associated genes selected from hTERT, p53, survivin, NY-ESO-1, CEA, CCND1, MET, RGS5, MMP7, VEGFR, AFP, GPC3, HBVc, HBVp, CDCA, KRAS, PARP4, MLL3, and MTHFR.

[0247] Precise MASCT

[0248] This article also provides a precise MASCT approach tailored to the individual based on their genetics and treatment response. Any of the above MASCT methods can be customized to provide a precise MASCT approach.

[0249] In some implementations, the MASCT method described herein is particularly suitable for specific individual populations, such as individuals with low mutational loads in cancers (such as all or subpopulations of cancer cells) (such as in MHC genes) and / or individuals with one or more neoantigens.

[0250] Mutation load

[0251] In some embodiments, the MASCT method is particularly suitable for individuals with a low total mutational burden in their cancer. In some embodiments, the MASCT method is particularly suitable for individuals with a low mutational burden in cancer-related genes within their cancer. In some embodiments, the MASCT method is particularly suitable for individuals with a low mutational burden in immune genes related to T-cell responses within their cancer. In some embodiments, the MASCT method is particularly suitable for individuals with a low mutational burden in MHC genes within their cancer. Mutational burden can be the mutational burden in all cancer cells or cancer cell subsets (such as cells in primary or metastatic tumor sites, e.g., cells in a tumor biopsy sample).

[0252] Therefore, in some embodiments, a method for treating cancer in an individual is provided, the method comprising: (a) optionally administering an effective amount of dendritic cells loaded with a plurality of tumor antigen peptides; and (b) administering an effective amount of activated T cells to the individual, wherein the activated T cells are prepared by co-culturing a population of T cells with a population of dendritic cells loaded with said plurality of tumor antigen peptides, and wherein the individual has a low mutational burden in the cancer. In some embodiments, the interval between the administration of dendritic cells and the administration of activated T cells is about 7 days to about 21 days (e.g., about 7 days to about 14 days, about 14 days to about 21 days, about 10 days, or about 14 days). In some embodiments, the dendritic cells loaded with said plurality of tumor antigen peptides are administered subcutaneously. In some embodiments, the dendritic cells loaded with said plurality of tumor antigen peptides are administered at least three times. In some embodiments, the activated T cells are administered intravenously. In some embodiments, the activated T cells are administered at least three times. In some embodiments, the co-culture lasts about 7 days to about 21 days (e.g., about 7 days to about 14 days, or about 14 days to about 21 days). In some embodiments, the T cell population is contacted with an immune checkpoint inhibitor (such as an inhibitor of PD-1, PD-L1, or CTLA-4) before and / or during co-culture. In some embodiments, the dendritic cell population and the T cell population are derived from the same individual, such as an individual receiving treatment. In some embodiments, the multiple tumor antigen peptides include at least 10 tumor antigen peptides. In some embodiments, the multiple tumor antigen peptides include a first core group of common tumor antigen peptides and optionally a second group of cancer type-specific antigen peptides. In some embodiments, the multiple tumor antigen peptides include one or more neoantigen peptides. In some embodiments, the method further includes administering an effective amount of an immune checkpoint inhibitor, such as an inhibitor of PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3, to the individual. In some embodiments, the mutational burden of the cancer is determined by sequencing a tumor sample taken from the individual. In some implementations, an individual has a low mutation load (e.g., no more than about 10 mutations, no mutations in B2M and / or no mutations in functional regions) in one or more MHC genes (such as MHC-I genes) in cancer.

[0253] In some embodiments, a method for treating cancer in an individual is provided, the method comprising: (a) selecting the individual for the method based on the mutational burden in the cancer; (b) optionally administering an effective amount of dendritic cells loaded with a plurality of tumor antigen peptides; and (c) administering an effective amount of activated T cells to the individual, wherein the activated T cells are prepared by co-culturing a population of T cells with a population of dendritic cells loaded with the plurality of tumor antigen peptides. In some embodiments, the interval between the administration of dendritic cells and the administration of activated T cells is about 7 days to about 21 days (e.g., about 7 days to about 14 days, about 14 days to about 21 days, about 10 days, or about 14 days). In some embodiments, the dendritic cells loaded with the plurality of tumor antigen peptides are administered subcutaneously. In some embodiments, the dendritic cells loaded with the plurality of tumor antigen peptides are administered at least three times. In some embodiments, the activated T cells are administered intravenously. In some embodiments, the activated T cells are administered at least three times. In some embodiments, the co-culture lasts about 7 days to about 21 days (e.g., about 7 days to about 14 days, or about 14 days to about 21 days). In some embodiments, the T cell population is contacted with an immune checkpoint inhibitor (such as an inhibitor of PD-1, PD-L1, or CTLA-4) before and / or during co-culture. In some embodiments, the dendritic cell population and the T cell population are derived from the same individual, such as an individual receiving treatment. In some embodiments, the multiple tumor antigen peptides include at least 10 tumor antigen peptides. In some embodiments, the multiple tumor antigen peptides include a first core group of common tumor antigen peptides and optionally a second group of cancer type-specific antigen peptides. In some embodiments, the multiple tumor antigen peptides include one or more neoantigen peptides. In some embodiments, the method further includes administering an effective amount of an immune checkpoint inhibitor, such as an inhibitor of PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3, to the individual. In some embodiments, the mutational burden of the cancer is determined by sequencing a tumor sample taken from the individual. In some implementations, an individual has a low mutation load (e.g., no more than about 10 mutations, no mutations in B2M and / or no mutations in functional regions) in one or more MHC genes (such as MHC-I genes) in cancer.

[0254] In some embodiments, a method for treating cancer in an individual is provided, the method comprising: (a) optionally administering an effective amount of dendritic cells loaded with a plurality of tumor antigen peptides; and (b) administering an effective amount of activated T cells to the individual, wherein the activated T cells are prepared by co-culturing a population of T cells with a population of dendritic cells loaded with the plurality of tumor antigen peptides, and wherein the individual is selected for treatment based on having a low mutational burden in the cancer. In some embodiments, the interval between the administration of dendritic cells and the administration of activated T cells is from about 7 days to about 21 days (e.g., from about 7 days to about 14 days, from about 14 days to about 21 days, from about 10 days, or from about 14 days). In some embodiments, the dendritic cells loaded with the plurality of tumor antigen peptides are administered subcutaneously. In some embodiments, the dendritic cells loaded with the plurality of tumor antigen peptides are administered at least three times. In some embodiments, the activated T cells are administered intravenously. In some embodiments, the activated T cells are administered at least three times. In some embodiments, the co-culture lasts from about 7 days to about 21 days (e.g., from about 7 days to about 14 days, or from about 14 days to about 21 days). In some embodiments, the T cell population is contacted with an immune checkpoint inhibitor (such as an inhibitor of PD-1, PD-L1, or CTLA-4) before and / or during co-culture. In some embodiments, the dendritic cell population and the T cell population are derived from the same individual, such as an individual receiving treatment. In some embodiments, the multiple tumor antigen peptides include at least 10 tumor antigen peptides. In some embodiments, the multiple tumor antigen peptides include a first core group of common tumor antigen peptides and optionally a second group of cancer type-specific antigen peptides. In some embodiments, the multiple tumor antigen peptides include one or more neoantigen peptides. In some embodiments, the method further includes administering an effective amount of an immune checkpoint inhibitor, such as an inhibitor of PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3, to the individual. In some embodiments, the mutational burden of the cancer is determined by sequencing a tumor sample taken from the individual. In some implementations, an individual has a low mutation load (e.g., no more than about 10 mutations, no mutations in B2M and / or no mutations in functional regions) in one or more MHC genes (such as MHC-I genes) in cancer.

[0255] In some embodiments, a method for treating cancer in an individual is provided, the method comprising: (a) inducing a population of monocytes to differentiate into a population of dendritic cells (e.g., in the presence of GM-CSF and IL-4); (b) contacting the population of dendritic cells with a plurality of tumor antigen peptides (e.g., in the presence of a plurality of Toll-like receptor (TLR) agonists) to obtain a population of dendritic cells loaded with the plurality of tumor antigen peptides; (c) optionally administering an effective amount of the dendritic cells loaded with the plurality of tumor antigen peptides to the individual; (d) co-culturing the population of dendritic cells loaded with the plurality of tumor antigen peptides with a population of non-adhesive PBMCs (e.g., in the presence of a plurality of cytokines and optionally an anti-CD3 antibody) to obtain a population of activated T cells; and (e) administering an effective amount of activated T cells to the individual, wherein the population of monocytes and the non-adhesive PBMCs are obtained from a population of PBMCs (e.g., from the individual), and wherein the individual has a low mutational burden in the cancer. In some embodiments, the interval between the administration of dendritic cells and the administration of activated T cells is approximately 7 to approximately 21 days (e.g., approximately 7 to approximately 14 days, approximately 14 to approximately 21 days, approximately 10 days, or approximately 14 days). In some embodiments, dendritic cells loaded with the multiple tumor antigen peptides are administered subcutaneously. In some embodiments, dendritic cells loaded with the multiple tumor antigen peptides are administered at least three times. In some embodiments, activated T cells are administered intravenously. In some embodiments, activated T cells are administered at least three times. In some embodiments, co-culture lasts approximately 7 to approximately 21 days (e.g., approximately 7 to approximately 14 days, or approximately 14 to approximately 21 days). In some embodiments, non-adhesive PBMCs are contacted with immune checkpoint inhibitors (such as inhibitors of PD-1, PD-L1, or CTLA-4) before and / or during co-culture. In some embodiments, the multiple tumor antigen peptides include at least 10 tumor antigen peptides. In some embodiments, the multiple tumor antigen peptides include a first core group of common tumor antigen peptides and optionally a second group of cancer type-specific antigen peptides. In some embodiments, the plurality of tumor antigen peptides includes one or more neoantigen peptides. In some embodiments, the method further includes administering an effective amount of an immune checkpoint inhibitor to the individual, such as an inhibitor of PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3. In some embodiments, the mutational burden of the cancer is determined by sequencing a tumor sample taken from the individual. In some embodiments, the individual has a low mutational burden (e.g., no more than about 10 mutations, no mutations in B2M, and / or no mutations in functional regions) in one or more MHC genes (such as MHC-I genes) in the cancer.

[0256] In some embodiments, a method for treating cancer in an individual is provided, the method comprising: (a) selecting an individual for the method based on the mutational burden in the cancer; (b) (e.g., in the presence of GM-CSF and IL-4) inducing a population of monocytes to differentiate into a population of dendritic cells; (c) (e.g., in the presence of multiple Toll-like receptor (TLR) agonists) contacting the population of dendritic cells with multiple tumor antigen peptides to obtain a population of dendritic cells loaded with said multiple tumor antigen peptides; (d) optionally administering an effective amount of the dendritic cells loaded with said multiple tumor antigen peptides to the individual; (e) (e.g., in the presence of multiple cytokines and optionally an anti-CD3 antibody) co-culturing the population of dendritic cells loaded with said multiple tumor antigen peptides with a population of non-adhesive PBMCs to obtain a population of activated T cells; and (f) administering an effective amount of activated T cells to the individual, wherein the monocyte population and the non-adhesive PBMC population are obtained from a population of PBMCs (e.g., from the individual). In some embodiments, the interval between the administration of dendritic cells and the administration of activated T cells is approximately 7 to approximately 21 days (e.g., approximately 7 to approximately 14 days, approximately 14 to approximately 21 days, approximately 10 days, or approximately 14 days). In some embodiments, dendritic cells loaded with the multiple tumor antigen peptides are administered subcutaneously. In some embodiments, dendritic cells loaded with the multiple tumor antigen peptides are administered at least three times. In some embodiments, activated T cells are administered intravenously. In some embodiments, activated T cells are administered at least three times. In some embodiments, co-culture lasts approximately 7 to approximately 21 days (e.g., approximately 7 to approximately 14 days, or approximately 14 to approximately 21 days). In some embodiments, non-adhesive PBMCs are contacted with immune checkpoint inhibitors (such as inhibitors of PD-1, PD-L1, or CTLA-4) before and / or during co-culture. In some embodiments, the multiple tumor antigen peptides include at least 10 tumor antigen peptides. In some embodiments, the multiple tumor antigen peptides include a first core group of common tumor antigen peptides and optionally a second group of cancer type-specific antigen peptides. In some embodiments, the multiple tumor antigen peptides include one or more neoantigen peptides. In some embodiments, the method further includes administering an effective amount of an immune checkpoint inhibitor to the individual, such as an inhibitor of PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3. In some embodiments, the mutational burden of the cancer is determined by sequencing a tumor sample taken from the individual. In some embodiments, the individual has a low mutational burden (e.g., no more than about 10 mutations, no mutations in B2M, and / or no mutations in functional regions) in one or more MHC genes (such as MHC-I genes) in the cancer.

[0257] In some embodiments, a method for treating cancer in an individual is provided, the method comprising: (a) inducing a population of monocytes to differentiate into a population of dendritic cells (e.g., in the presence of GM-CSF and IL-4); (b) contacting the dendritic cell population with a plurality of tumor antigen peptides (e.g., in the presence of a plurality of Toll-like receptor (TLR) agonists) to obtain a population of dendritic cells loaded with said plurality of tumor antigen peptides; (c) optionally administering an effective amount of the dendritic cells loaded with said plurality of tumor antigen peptides to the individual; (d) co-culturing the population of dendritic cells loaded with said plurality of tumor antigen peptides with a population of non-adhesive PBMCs (e.g., in the presence of a plurality of cytokines and optionally an anti-CD3 antibody) to obtain a population of activated T cells; and (e) administering an effective amount of activated T cells to the individual, wherein the monocyte population and the non-adhesive PBMC population are obtained from a population of PBMCs (e.g., from the individual), and wherein the individual is selected for treatment based on having a low mutational load in the cancer. In some embodiments, the interval between the administration of dendritic cells and the administration of activated T cells is approximately 7 to approximately 21 days (e.g., approximately 7 to approximately 14 days, approximately 14 to approximately 21 days, approximately 10 days, or approximately 14 days). In some embodiments, co-culture lasts approximately 7 to approximately 21 days (e.g., approximately 7 to approximately 14 days, or approximately 14 to approximately 21 days). In some embodiments, non-adhesive PBMCs are contacted with an immune checkpoint inhibitor (such as an inhibitor of PD-1, PD-L1, or CTLA-4) before and / or during co-culture. In some embodiments, activated T cells are administered intravenously. In some embodiments, activated T cells are administered at least three times. In some embodiments, dendritic cells loaded with the multiple tumor antigen peptides are administered subcutaneously. In some embodiments, dendritic cells loaded with the multiple tumor antigen peptides are administered at least three times. In some embodiments, the multiple tumor antigen peptides include at least 10 tumor antigen peptides. In some embodiments, the multiple tumor antigen peptides include a first core group of common tumor antigen peptides and optionally a second group of cancer type-specific antigen peptides. In some embodiments, the plurality of tumor antigen peptides includes one or more neoantigen peptides. In some embodiments, the method further includes administering an effective amount of an immune checkpoint inhibitor to the individual, such as an inhibitor of PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3. In some embodiments, the mutational burden of the cancer is determined by sequencing a tumor sample taken from the individual. In some embodiments, the individual has a low mutational burden (e.g., no more than about 10 mutations, no mutations in B2M, and / or no mutations in functional regions) in one or more MHC genes (such as MHC-I genes) in the cancer.

[0258] In some embodiments, a method for treating cancer in an individual is provided, the method comprising: (a) (e.g., in the presence of an immune checkpoint inhibitor) contacting a population of PBMCs with a plurality of tumor antigen peptides to obtain an activated population of PBMCs; and (b) administering an effective amount of activated PBMCs to the individual, wherein the individual has a low mutational burden in the cancer. In some embodiments, the activated PBMCs are administered at least three times. In some embodiments, the interval between each administration of the activated PBMCs is from about 2 weeks to about 5 months (e.g., about 3 months). In some embodiments, the activated PBMCs are administered intravenously. In some embodiments, the population of PBMCs is obtained from an individual receiving treatment. In some embodiments, the plurality of tumor antigen peptides comprises at least 10 tumor antigen peptides. In some embodiments, the plurality of tumor antigen peptides comprises a first core group of common tumor antigen peptides and optionally a second group of cancer type-specific antigen peptides. In some embodiments, the plurality of tumor antigen peptides comprises one or more neoantigen peptides. In some embodiments, the method further comprises administering an effective amount of an immune checkpoint inhibitor to the individual, such as an inhibitor of PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3. In some implementations, the mutational burden of the cancer is determined by sequencing a tumor sample taken from the individual. In some implementations, the individual has a low mutational burden (e.g., no more than about 10 mutations, no mutations in B2M, and / or no mutations in functional regions) in one or more MHC genes (such as MHC-I genes) in the cancer.

[0259] In some embodiments, a method for treating cancer in an individual is provided, the method comprising: (a) selecting the individual for the method based on the mutational burden in the cancer; (b) (e.g., in the presence of an immune checkpoint inhibitor) contacting a population of PBMCs with a plurality of tumor antigen peptides to obtain an activated population of PBMCs; and (c) administering an effective amount of activated PBMCs to the individual. In some embodiments, the activated PBMCs are administered at least three times. In some embodiments, the interval between each administration of the activated PBMCs is from about 2 weeks to about 5 months (e.g., about 3 months). In some embodiments, the activated PBMCs are administered intravenously. In some embodiments, the population of PBMCs is obtained from the individual receiving treatment. In some embodiments, the plurality of tumor antigen peptides comprises at least 10 tumor antigen peptides. In some embodiments, the plurality of tumor antigen peptides comprises a first core group of common tumor antigen peptides and optionally a second group of cancer type-specific antigen peptides. In some embodiments, the plurality of tumor antigen peptides comprises one or more neoantigen peptides. In some embodiments, the method further includes administering an effective amount of an immune checkpoint inhibitor to the individual, such as an inhibitor of PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3. In some embodiments, the mutational burden of the cancer is determined by sequencing a tumor sample taken from the individual. In some embodiments, the individual has a low mutational burden (e.g., no more than about 10 mutations, no mutations in B2M, and / or no mutations in functional regions) in one or more MHC genes (such as MHC-I genes) in the cancer.

[0260] In some embodiments, a method for treating cancer in an individual is provided, the method comprising: (a) (e.g., in the presence of an immune checkpoint inhibitor) contacting a population of PBMCs with a plurality of tumor antigen peptides to obtain an activated population of PBMCs; and (b) administering an effective amount of activated PBMCs to the individual, wherein the individual is selected for treatment based on having a low mutational load in the cancer. In some embodiments, the activated PBMCs are administered at least three times. In some embodiments, the interval between each administration of the activated PBMCs is from about 2 weeks to about 5 months (e.g., about 3 months). In some embodiments, the activated PBMCs are administered intravenously. In some embodiments, the population of PBMCs is obtained from an individual receiving treatment. In some embodiments, the plurality of tumor antigen peptides comprises at least 10 tumor antigen peptides. In some embodiments, the plurality of tumor antigen peptides comprises a first core group of common tumor antigen peptides and optionally a second group of cancer type-specific antigen peptides. In some embodiments, the plurality of tumor antigen peptides comprises one or more neoantigen peptides. In some embodiments, the method further includes administering an effective amount of an immune checkpoint inhibitor to the individual, such as an inhibitor of PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3. In some embodiments, the mutational burden of the cancer is determined by sequencing a tumor sample taken from the individual. In some embodiments, the individual has a low mutational burden (e.g., no more than about 10 mutations, no mutations in B2M, and / or no mutations in functional regions) in one or more MHC genes (such as MHC-I genes) in the cancer.

[0261] In some embodiments, a low mutational load of one or more genes is a low number of accumulated mutations in said one or more genes. In some embodiments, a low mutational load is indicated by any number of mutations not exceeding about 500, 400, 300, 200, 100, 50, 40, 30, 20, 10, 5, or fewer. In some embodiments, a low mutational load of one or more MHC genes is indicated by any number of mutations not exceeding about 50, 40, 30, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1. In some embodiments, a low mutational load of one or more genes is a low ratio between the number of accumulated mutations in said one or more genes (such as MHC genes) and the total number of mutations in a selected group of genes (such as oncogenes) or the entire genome. In some embodiments, a ratio of the number of mutations in one or more MHC genes to the total number of the 333 cancer-associated genes described in Example 5 that is less than any of the ratios of 1:10, 1:15, 1:20, 1:25, 1:30, 1:40, 1:50, 1:60, 1:70, 1:80, 1:90, 1:100, 1:200 or less indicates a low mutational load in one or more MHC genes.

[0262] In some embodiments, the one or more MHC genes include MHC class I genes (or loci). In some embodiments, the one or more MHC genes include MHC class II genes (or loci). In some embodiments, the individual is a human individual, and the one or more MHC genes are selected from HLA-A, HLA-B, HLA-C, and B2M.

[0263] Exemplary mutations include, but are not limited to, deletions, frameshifts, insertions, insertion-deletion, missense mutations, nonsense mutations, point mutations, copy number variations, single nucleotide variations (SNVs), silencing mutations, splice site mutations, splice variants, gene fusions, and translocations. In some embodiments, copy number variations in MHC genes are caused by structural rearrangements of the genome, including deletions, duplications, inversions, and translocations of chromosomes or segments thereof. In some embodiments, the mutations in one or more MHC genes are selected from point mutations, frameshift mutations, gene fusions, and copy number variations. In some embodiments, the mutation is located in a protein-coding region of an MHC gene. In some embodiments, the mutation is a non-synonymous mutation. In some embodiments, the mutation is not a polymorphism. In some embodiments, the mutation is present in normal cells of an individual. In some embodiments, the mutation is not present in normal cells of an individual. In some embodiments, the mutation affects the physiological, chemical, or functional properties of the MHC molecule encoded by the affected gene, such as stability or binding affinity. In some embodiments, the mutation results in an irreversible defect in the MHC molecule. In some embodiments, the mutation reduces the binding affinity of the MHC molecule to T cell epitopes and / or T cell receptors. In some embodiments, the mutation is a loss-of-function mutation. In some embodiments, the mutation results in a reversible defect in the MHC molecule. In some embodiments, the mutation does not affect the binding affinity of the MHC molecule to T cell epitopes and / or T cell receptors. In some embodiments, the mutation is a somatic mutation. In some embodiments, the mutation is a germline mutation.

[0264] The mutation included in the mutational burden may be present in all cancer cells or in a subset of cancer cells. In some embodiments, the mutation is present in all cancer cells of an individual. In some embodiments, the mutation is present in all cancer cells at the tumor site. In some embodiments, the mutation is clonal. In some embodiments, the mutation is subclonal. In some embodiments, the mutation is present in at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of the cancer cells of an individual.

[0265] Mutations in certain MHC genes and / or certain domains or locations of one or more of the aforementioned MHC genes can have a more profound impact on an individual's clinical response to the MASCT method described herein. For example, loss-of-function mutations can occur in the leader peptide sequence, α3 domain (which binds to the CD8 helper receptor on T cells), α1 peptide-binding domain, or α2 peptide-binding domain of HLA molecules; see, for example, Shukla S. et al. Nature Biotechnology 33, 1152-1158 (2015), which is incorporated herein by reference. Mutations in the B2M (β2-macroglobulin) gene can also promote a tumor escape phenotype. See, for example, Monica B et al. Cancer Immunol. Immu., (2012) 61: 1359-1371 (Monica B et al., Cancer Immunology and Immunotherapy, 2012, Vol. 61, pp. 1359-1371). In some embodiments, the presence of any number (such as 1, 2, 3, 4, 5 or more) of mutations in functional regions of the one or more MHC genes, such as leader peptide sequences, α1 domains, α2 domains, or α3 domains, indicates a high mutational burden. In some embodiments, the presence of any number (such as 1, 2, 3, 4, 5 or more) of loss-of-function mutations in the one or more MHC genes (such as HLA-A, HLA-B, or HLA-C genes in human individuals) indicates a high mutational burden. In some embodiments, a low mutational load in the one or more MHC genes does not include mutations in functional regions of the one or more MHC genes (such as HLA-A, HLA-B, or HLA-C genes), said functional regions including leader peptide sequences, α1 domains (e.g., residues directly contacting the CD8 helper receptor), α2 domains, and α3 domains (e.g., residues directly contacting epitopes). In some embodiments, the presence of any number of mutations (such as loss-of-function mutations) in the B2M gene indicates a high mutational load. In some embodiments, a low mutational load in the one or more MHC genes does not include mutations in the B2M gene.

[0266] The mutational load of one or more genes (such as MHC genes) can be determined by any method known in the art, including but not limited to genomic DNA sequencing, exome sequencing or other DNA sequencing-based methods using Sanger sequencing or next-generation sequencing platforms; polymerase chain reaction assays; in situ hybridization assays; and DNA microarrays.

[0267] In some embodiments, the mutational load of one or more MHC genes is determined by sequencing a tumor sample taken from an individual. In some embodiments, the sequencing is next-generation sequencing. In some embodiments, the sequencing is whole-genome sequencing. In some embodiments, the sequencing is exome sequencing. In some embodiments, the sequencing is targeted sequencing of candidate genes such as cancer-associated genes plus HLA genes. For example, ONCOGXONE TM Plus (Admera Health) can be used to sequence cancer-related genes and HLA loci at high sequencing depth. In some implementations, the same sequencing data can be used to determine the mutational load of one or more MHC genes and identify neoantigens in an individual.

[0268] In some embodiments, the tumor sample is a tissue sample. In some embodiments, the tumor sample is a tumor biopsy sample, such as tumor cells obtained through fine-needle aspiration or laparoscopy (e.g., including tumor stroma). In some embodiments, the tumor sample is freshly obtained. In some embodiments, the tumor sample is frozen. In some embodiments, the tumor sample is a formaldehyde-fixed paraffin-embedded (FFPE) sample. In some embodiments, the tumor sample is a cell sample. In some embodiments, the tumor sample contains circulating metastatic cancer cells. In some embodiments, the tumor sample is obtained by sorting circulating tumor cells (CTCs) from blood. In some embodiments, nucleic acids (such as DNA and / or RNA) are extracted from the tumor sample for sequencing analysis. In some embodiments, the sequencing data of the tumor sample is compared with the sequencing data of a reference sample (such as a sample from healthy tissue taken from the same individual or a sample from a healthy individual) to identify mutations in the tumor cells and determine the mutation load. In some embodiments, the sequencing data of the tumor sample is compared with a reference sequence from a genome database to identify mutations in the tumor cells and determine the mutation load.

[0269] Neoantigen peptide

[0270] In some implementations, the MASCT method described herein is particularly suitable for treating individuals with one or more neoantigens. Any MASCT method described herein using one or more neoantigen peptides from the plurality of tumor antigen peptides may further include the step of selecting an individual for this treatment based on the presence of one or more (such as at least five) neoantigens in the individual, and / or the steps of: (i) identifying the individual's neoantigens; and (ii) incorporating a neoantigen-derived neoantigen peptide into the plurality of tumor antigen peptides for use in the MASCT method.

[0271] Therefore, in some embodiments, a method for treating cancer in an individual is provided, the method comprising: (a) identifying neoantigens in the individual; (b) incorporating a neoantigen peptide into a plurality of tumor antigen peptides, wherein the neoantigen peptides contain a novel epitope of the neoantigen; (c) preparing a population of dendritic cells loaded with the plurality of tumor antigen peptides; (d) optionally administering an effective amount of dendritic cells loaded with the plurality of tumor antigen peptides; (e) co-culturing a population of T cells with the population of dendritic cells loaded with the plurality of tumor antigen peptides; and (f) administering an effective amount of activated T cells to the individual, wherein the individual has one or more neoantigens. In some embodiments, the interval between the administration of dendritic cells and the administration of activated T cells is about 7 days to about 21 days (e.g., about 7 days to about 14 days, about 14 days to about 21 days, about 10 days, or about 14 days). In some embodiments, the co-culture lasts about 7 days to about 21 days (e.g., about 7 days to about 14 days, or about 14 days to about 21 days). In some embodiments, the T cell population is contacted with an immune checkpoint inhibitor (such as an inhibitor of PD-1, PD-L1, or CTLA-4) before and / or during co-culture. In some embodiments, dendritic cells loaded with the multiple tumor antigen peptides are administered subcutaneously. In some embodiments, dendritic cells loaded with the multiple tumor antigen peptides are administered at least three times. In some embodiments, activated T cells are administered intravenously. In some embodiments, activated T cells are administered at least three times. In some embodiments, the dendritic cell population and the T cell population are derived from the same individual, such as an individual receiving treatment. In some embodiments, the multiple tumor antigen peptides include at least 10 tumor antigen peptides. In some embodiments, the multiple tumor antigen peptides include a first core group of common tumor antigen peptides and optionally a second group of cancer type-specific antigen peptides. In some embodiments, the multiple tumor antigen peptides include multiple neoantigen peptides. In some embodiments, the method further includes administering an effective amount of an immune checkpoint inhibitor, such as an inhibitor of PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3, to the individual. In some implementations, individuals are selected for the treatment based on having a low mutational load in cancer (such as in one or more MHC genes).

[0272] In some embodiments, a method for treating cancer in an individual is provided, the method comprising: (a) selecting the individual for the treatment based on the presence of one or more (such as at least five) neoantigens in the individual; (b) identifying the neoantigens in the individual; (c) incorporating a neoantigen peptide into a plurality of tumor antigen peptides, wherein the neoantigen peptides contain novel epitopes of the neoantigens; (d) preparing a population of dendritic cells loaded with the plurality of tumor antigen peptides; (e) optionally administering an effective amount of dendritic cells loaded with the plurality of tumor antigen peptides; (f) co-culturing a population of T cells with the population of dendritic cells loaded with the plurality of tumor antigen peptides; and (g) administering an effective amount of activated T cells to the individual. In some embodiments, the interval between the administration of dendritic cells and the administration of activated T cells is from about 7 days to about 21 days (such as from about 7 days to about 14 days, from about 14 days to about 21 days, from about 10 days, or from about 14 days). In some embodiments, the co-culture lasts from about 7 days to about 21 days (such as from about 7 days to about 14 days, or from about 14 days to about 21 days). In some embodiments, the T cell population is contacted with an immune checkpoint inhibitor (such as an inhibitor of PD-1, PD-L1, or CTLA-4) before and / or during co-culture. In some embodiments, dendritic cells loaded with the multiple tumor antigen peptides are administered subcutaneously. In some embodiments, dendritic cells loaded with the multiple tumor antigen peptides are administered at least three times. In some embodiments, activated T cells are administered intravenously. In some embodiments, activated T cells are administered at least three times. In some embodiments, the dendritic cell population and the T cell population are derived from the same individual, such as an individual receiving treatment. In some embodiments, the multiple tumor antigen peptides include at least 10 tumor antigen peptides. In some embodiments, the multiple tumor antigen peptides include a first core group of common tumor antigen peptides and optionally a second group of cancer type-specific antigen peptides. In some embodiments, the multiple tumor antigen peptides include multiple neoantigen peptides. In some embodiments, the method further includes administering an effective amount of an immune checkpoint inhibitor, such as an inhibitor of PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3, to the individual. In some implementations, individuals are selected for the treatment based on having a low mutational load in cancer (such as in one or more MHC genes).

[0273] In some embodiments, a method for treating cancer in an individual is provided, the method comprising: (a) identifying neoantigens in the individual; (b) incorporating a neoantigen peptide into a plurality of tumor antigen peptides, wherein the neoantigen peptides contain a novel epitope of the neoantigen; (c) preparing a population of dendritic cells loaded with the plurality of tumor antigen peptides; (d) optionally administering an effective amount of dendritic cells loaded with the plurality of tumor antigen peptides; (e) co-culturing a population of T cells with the population of dendritic cells loaded with the plurality of tumor antigen peptides; and (f) administering an effective amount of activated T cells to the individual, wherein the individual is selected for the treatment based on having one or more (such as at least five) neoantigens. In some embodiments, the interval between the administration of dendritic cells and the administration of activated T cells is from about 7 days to about 21 days (such as from about 7 days to about 14 days, from about 14 days to about 21 days, from about 10 days, or from about 14 days). In some embodiments, the co-culture lasts from about 7 days to about 21 days (such as from about 7 days to about 14 days, or from about 14 days to about 21 days). In some embodiments, the T cell population is contacted with an immune checkpoint inhibitor (such as an inhibitor of PD-1, PD-L1, or CTLA-4) before and / or during co-culture. In some embodiments, dendritic cells loaded with the multiple tumor antigen peptides are administered subcutaneously. In some embodiments, dendritic cells loaded with the multiple tumor antigen peptides are administered at least three times. In some embodiments, activated T cells are administered intravenously. In some embodiments, activated T cells are administered at least three times. In some embodiments, the dendritic cell population and the T cell population are derived from the same individual, such as an individual receiving treatment. In some embodiments, the multiple tumor antigen peptides include at least 10 tumor antigen peptides. In some embodiments, the multiple tumor antigen peptides include a first core group of common tumor antigen peptides and optionally a second group of cancer type-specific antigen peptides. In some embodiments, the multiple tumor antigen peptides include multiple neoantigen peptides. In some embodiments, the method further includes administering an effective amount of an immune checkpoint inhibitor, such as an inhibitor of PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3, to the individual. In some implementations, individuals are selected for the treatment based on having a low mutational load in cancer (such as in one or more MHC genes).

[0274] In some embodiments, a method for treating cancer in an individual is provided, the method comprising: (a) identifying neoantigens in the individual; (b) incorporating a neoantigen peptide into a plurality of tumor antigen peptides, wherein the neoantigen peptide contains a novel epitope in the neoantigen; (c) (e.g., in the presence of GM-CSF and IL-4) inducing a population of monocytes to differentiate into a population of dendritic cells; (d) (e.g., in the presence of a plurality of Toll-like receptor (TLR) agonists) contacting the population of dendritic cells with a plurality of tumor antigen peptides to obtain a population of dendritic cells loaded with said plurality of tumor antigen peptides; (e) optionally administering an effective amount of the dendritic cells loaded with said plurality of tumor antigen peptides to the individual; (f) (e.g., in the presence of a plurality of cytokines and optionally an anti-CD3 antibody) co-culturing the population of dendritic cells loaded with said plurality of tumor antigen peptides with a population of non-adhesive PBMCs to obtain a population of activated T cells; and (g) administering an effective amount of activated T cells to the individual, wherein the monocyte population and the non-adhesive PBMC population are obtained from a population of PBMCs (e.g., from the individual), and wherein the individual possesses one or more neoantigens. In some embodiments, the interval between the administration of dendritic cells and the administration of activated T cells is approximately 7 to approximately 21 days (e.g., approximately 7 to approximately 14 days, approximately 14 to approximately 21 days, approximately 10 days, or approximately 14 days). In some embodiments, co-culture lasts approximately 7 to approximately 21 days (e.g., approximately 7 to approximately 14 days, or approximately 14 to approximately 21 days). In some embodiments, non-adhesive PBMCs are contacted with an immune checkpoint inhibitor (such as an inhibitor of PD-1, PD-L1, or CTLA-4) before and / or during co-culture. In some embodiments, activated T cells are administered intravenously. In some embodiments, activated T cells are administered at least three times. In some embodiments, dendritic cells loaded with the multiple tumor antigen peptides are administered subcutaneously. In some embodiments, dendritic cells loaded with the multiple tumor antigen peptides are administered at least three times. In some embodiments, the multiple tumor antigen peptides include at least 10 tumor antigen peptides. In some embodiments, the multiple tumor antigen peptides include a first core group of common tumor antigen peptides and optionally a second group of cancer type-specific antigen peptides. In some embodiments, the multiple tumor antigen peptides include multiple neoantigen peptides of the individual. In some embodiments, the method further includes administering an effective amount of an immune checkpoint inhibitor to the individual, such as an inhibitor of PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3. In some embodiments, individuals are selected for this treatment based on having a low mutational burden in the cancer (such as in one or more MHC genes).

[0275] In some embodiments, a method for treating cancer in an individual is provided, the method comprising: (a) selecting the individual for the treatment based on the presence of one or more (e.g., at least five) neoantigens in the individual; (b) identifying the neoantigens in the individual; (c) incorporating a neoantigen peptide into a plurality of tumor antigen peptides, wherein the neoantigen peptides contain novel epitopes of the neoantigens; (d) (e.g., in the presence of GM-CSF and IL-4) inducing a population of monocytes to differentiate into a population of dendritic cells; (e.g., in the presence of a plurality of Toll-like receptor (TLR) agonists) contacting the population of dendritic cells with a plurality of tumor antigen peptides to obtain a population of dendritic cells loaded with said plurality of tumor antigen peptides; (f) optionally administering an effective amount of the dendritic cells loaded with said plurality of tumor antigen peptides to the individual; (g) (e.g., in the presence of a plurality of cytokines and optionally an anti-CD3 antibody) co-culturing the population of dendritic cells loaded with said plurality of tumor antigen peptides with a population of non-adhesive PBMCs to obtain a population of activated T cells; and (h) administering an effective amount of activated T cells to the individual, wherein the monocyte population and the non-adhesive PBMC population are derived from (e.g., from the individual) PBMC populations are obtained. In some embodiments, the interval between the administration of dendritic cells and the administration of activated T cells is approximately 7 to approximately 21 days (e.g., approximately 7 to approximately 14 days, approximately 14 to approximately 21 days, approximately 10 days, or approximately 14 days). In some embodiments, co-culture lasts approximately 7 to approximately 21 days (e.g., approximately 7 to approximately 14 days, or approximately 14 to approximately 21 days). In some embodiments, non-adhesive PBMC populations are contacted with immune checkpoint inhibitors (such as inhibitors of PD-1, PD-L1, or CTLA-4) before and / or during co-culture. In some embodiments, activated T cells are administered intravenously. In some embodiments, activated T cells are administered at least three times. In some embodiments, dendritic cells loaded with the multiple tumor antigen peptides are administered subcutaneously. In some embodiments, dendritic cells loaded with the multiple tumor antigen peptides are administered at least three times. In some embodiments, the multiple tumor antigen peptides include at least 10 tumor antigen peptides. In some embodiments, the multiple tumor antigen peptides include a first core group of common tumor antigen peptides and optionally a second group of cancer type-specific antigen peptides. In some embodiments, the multiple tumor antigen peptides include multiple neoantigen peptides of the individual. In some embodiments, the method further includes administering an effective amount of an immune checkpoint inhibitor to the individual, such as an inhibitor of PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3. In some embodiments, individuals are selected for this treatment based on having a low mutational burden in the cancer (such as in one or more MHC genes).

[0276] In some embodiments, a method for treating cancer in an individual is provided, the method comprising: (a) identifying neoantigens in the individual; (b) incorporating a neoantigen peptide into a plurality of tumor antigen peptides, wherein the neoantigen peptides contain a novel epitope of the neoantigen; (c) (e.g., in the presence of GM-CSF and IL-4) inducing a population of monocytes to differentiate into a population of dendritic cells; (d) (e.g., in the presence of a plurality of Toll-like receptor (TLR) agonists) contacting the population of dendritic cells with a plurality of tumor antigen peptides to obtain a population of dendritic cells loaded with said plurality of tumor antigen peptides; (e) optionally administering an effective amount of the dendritic cells loaded with said plurality of tumor antigen peptides to the individual; (f) (e.g., in the presence of a plurality of cytokines and optionally an anti-CD3 antibody) co-culturing the population of dendritic cells loaded with said plurality of tumor antigen peptides with a population of non-adhesive PBMCs to obtain a population of activated T cells; and (g) administering an effective amount of activated T cells to the individual, wherein the monocyte population and the non-adhesive PBMC population are derived from PBMCs (e.g., from the individual). The population is obtained, and individuals are selected for this treatment based on having one or more (such as at least five) neoantigens. In some embodiments, the interval between the administration of dendritic cells and the administration of activated T cells is about 7 days to about 21 days (such as about 7 days to about 14 days, about 14 days to about 21 days, about 10 days, or about 14 days). In some embodiments, co-culture lasts about 7 days to about 21 days (such as about 7 days to about 14 days, or about 14 days to about 21 days). In some embodiments, non-adhesive PBMC populations are contacted with immune checkpoint inhibitors (such as inhibitors of PD-1, PD-L1, or CTLA-4) before and / or during co-culture. In some embodiments, activated T cells are administered intravenously. In some embodiments, activated T cells are administered at least three times. In some embodiments, dendritic cells loaded with the multiple tumor antigen peptides are administered subcutaneously. In some embodiments, dendritic cells loaded with the multiple tumor antigen peptides are administered at least three times. In some embodiments, the multiple tumor antigen peptides include at least 10 tumor antigen peptides. In some embodiments, the multiple tumor antigen peptides include a first core group of common tumor antigen peptides and optionally a second group of cancer type-specific antigen peptides. In some embodiments, the multiple tumor antigen peptides include multiple neoantigen peptides specific to the individual. In some embodiments, the method further includes administering an effective amount of an immune checkpoint inhibitor to the individual, such as an inhibitor of PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3. In some embodiments, individuals are selected for this treatment based on a low mutational burden in the cancer (such as in one or more MHC genes).

[0277] In some embodiments, a method for treating cancer in an individual is provided, the method comprising: (a) identifying neoantigens in the individual; (b) incorporating a neoantigen peptide into a plurality of tumor antigen peptides, wherein the neoantigen peptide contains a novel epitope of the neoantigen; (c) (e.g., in the presence of an immune checkpoint inhibitor) contacting a group of PBMCs with the plurality of tumor antigen peptides to obtain an activated group of PBMCs; and (d) administering an effective amount of activated PBMCs to the individual, wherein the individual has one or more neoantigens. In some embodiments, the activated PBMCs are administered at least three times. In some embodiments, the interval between each administration of the activated PBMCs is from about 2 weeks to about 5 months (e.g., about 3 months). In some embodiments, the activated PBMCs are administered intravenously. In some embodiments, the group of PBMCs is obtained from an individual receiving treatment. In some embodiments, the plurality of tumor antigen peptides comprises at least 10 tumor antigen peptides. In some embodiments, the plurality of tumor antigen peptides comprises a first core group of common tumor antigen peptides and optionally a second group of cancer type-specific antigen peptides. In some embodiments, the plurality of tumor antigen peptides comprises a plurality of neoantigen peptides. In some embodiments, the method further includes administering an effective amount of an immune checkpoint inhibitor to the individual, such as an inhibitor of PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3. In some embodiments, individuals are selected for this treatment based on having a low mutational burden in the cancer (such as in one or more MHC genes).

[0278] In some embodiments, a method for treating cancer in an individual is provided, the method comprising: (a) selecting the individual for the treatment based on the presence of one or more (e.g., at least five) neoantigens in the individual; (b) identifying the individual's neoantigens; (c) incorporating a neoantigen peptide into a plurality of tumor antigen peptides, wherein the neoantigen peptide contains a novel epitope of the neoantigen; (d) (e.g., in the presence of an immune checkpoint inhibitor) contacting a PBMC population with the plurality of tumor antigen peptides to obtain an activated PBMC population; and (e) administering an effective amount of activated PBMCs to the individual. In some embodiments, the activated PBMCs are administered at least three times. In some embodiments, the interval between each administration of the activated PBMCs is from about 2 weeks to about 5 months (e.g., about 3 months). In some embodiments, the activated PBMCs are administered intravenously. In some embodiments, the PBMC population is obtained from the individual receiving treatment. In some embodiments, the plurality of tumor antigen peptides comprises at least 10 tumor antigen peptides. In some embodiments, the plurality of tumor antigen peptides comprises a first core group of common tumor antigen peptides and optionally a second group of cancer type-specific antigen peptides. In some embodiments, the plurality of tumor antigen peptides comprises a plurality of neoantigen peptides. In some embodiments, the method further includes administering an effective amount of an immune checkpoint inhibitor to the individual, such as an inhibitor of PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3. In some embodiments, individuals are selected for this treatment based on having a low mutational burden in the cancer (such as in one or more MHC genes).

[0279] In some embodiments, a method for treating cancer in an individual is provided, the method comprising: (a) identifying neoantigens in the individual; (b) incorporating a neoantigen peptide into a plurality of tumor antigen peptides, wherein the neoantigen peptide contains a novel epitope of the neoantigen; (c) (e.g., in the presence of an immune checkpoint inhibitor) contacting a group of PBMCs with the plurality of tumor antigen peptides to obtain an activated group of PBMCs; and (d) administering an effective amount of the activated PBMCs to the individual, wherein the individual is selected for the treatment based on having one or more (e.g., at least five) neoantigens. In some embodiments, the activated PBMCs are administered at least three times. In some embodiments, the interval between each administration of the activated PBMCs is from about 2 weeks to about 5 months (e.g., about 3 months). In some embodiments, the activated PBMCs are administered intravenously. In some embodiments, the group of PBMCs is obtained from the individual receiving treatment. In some embodiments, the plurality of tumor antigen peptides comprises at least 10 tumor antigen peptides. In some embodiments, the plurality of tumor antigen peptides comprises a first core group of common tumor antigen peptides and optionally a second group of cancer type-specific antigen peptides. In some embodiments, the plurality of tumor antigen peptides comprises a plurality of neoantigen peptides. In some embodiments, the method further includes administering an effective amount of an immune checkpoint inhibitor to the individual, such as an inhibitor of PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, or LAG-3. In some embodiments, individuals are selected for this treatment based on having a low mutational burden in the cancer (such as in one or more MHC genes).

[0280] Individuals may possess any number (e.g., at least 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 50, 100 or more) of neoantigens to benefit from the MASCT method, which includes multiple tumor antigen peptides comprising neoantigen peptides. In some embodiments, the MASCT method is particularly suitable for individuals possessing at least about 4, 5, 6, 7, 8, 10, 15, 20, 50, 100 or more of neoantigens. In some embodiments, the neoantigen comprises one or more novel epitopes. In some embodiments, the MASCT method is particularly suitable for individuals possessing at least about 4, 5, 6, 7, 8, 10, 15, 20, 50, 100 or more of novel epitopes. In some embodiments, the T-cell epitope is an MHC-I restricted epitope. In some embodiments, the novel epitope has a higher affinity for the individual's MHC molecules compared to the corresponding wild-type T-cell epitope. In some embodiments, the neoantigen (or neoepitope) has a higher affinity for the model T cell receptor compared to the corresponding wild-type T cell epitope. In some embodiments, the neoantigen (or neoepitope) is a clonal neoantigen. In some embodiments, the neoantigen (or neoepitope) is a subclonal neoantigen. In some embodiments, the neoantigen (or neoepitope) is present in at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of the tumor cells in an individual.

[0281] The number of neoantigens can be combined with other biomarkers or selection criteria to select individuals for any of the MASCT methods described herein. In some embodiments, the MASCT method is particularly suitable for individuals with a low mutational burden in cancer cells (such as in one or more MHC genes) and any number of neoantigens from at least about 4, 5, 6, 7, 8, 10 or more (such as neoantigens with high-affinity MHC-I restricted epitopes).

[0282] In some implementations, methods are provided to provide prognostic information for individuals based on mutational burden in their cancer and / or the number of neoantigens in the individual, wherein this prognosis predicts the individual's clinical response to any of the MASCT methods described herein. In some implementations, individuals are categorized based on this prognosis into one of three groups: (1) those who benefit from MHC-restricted interventions (such as MASCT therapy); (2) those who potentially benefit from MHC-restricted interventions (such as MASCT therapy); and (3) those who do not benefit from MHC-restricted interventions (such as MASCT therapy). In some implementations, an individual is predicted to benefit from MHC-restricted interventions (such as MASCT therapy) if they do not have mutations in the B2M gene, do not have mutations in functional regions of MHC genes (such as leader peptide sequences, α1 domains, α2 domains, or α3 domains), and have no more than two mutations and / or more than five mutations in MHC-I genes (such as HLA-I A, B, and / or C genes). In some implementations, an individual is predicted to potentially benefit from an MHC-restricted intervention (such as MASCT) if they do not have mutations in the B2M gene, do not have mutations in functional regions of MHC genes (such as leader peptide sequences, α1 domains, α2 domains, or α3 domains), and have no more than about 10 mutations and / or no more than 5 mutations in MHC-I genes (such as HLA-IA, B, and / or C genes). In some implementations, an individual is predicted not to benefit from an MHC-restricted intervention (such as MASCT) if they have a mutation in the B2M gene or a high mutational load (such as at least 10 mutations) in an MHC gene (such as an MHC-I gene). In some implementations, an individual is selected for the MASCT approach if they are predicted to benefit from or potentially benefit from an MHC-restricted intervention (such as MASCT).

[0283] Any number (such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) of neoantigen peptides can be designed based on an individual's neoantigens and incorporated into said plurality of tumor antigen peptides for use in any MASCT method described herein. In some embodiments, the plurality of tumor antigen peptides comprises a single neoantigen peptide. In some embodiments, the plurality of tumor antigen peptides comprises a plurality of neoantigen peptides. Each neoantigen peptide may contain one or more novel epitopes derived from an individual's neoantigen. In some embodiments, the novel epitopes are T-cell epitopes. Methods for designing neoantigen peptides based on neoantigens are described in the section “Multiple Tumor Antigen Peptides”.

[0284] Neoantigens in an individual can be identified using any method known in the art. In some embodiments, neoantigens are identified based on a genetic map of a tumor sample taken from the individual. Each neoantigen contains one or more novel epitopes. In some embodiments, the one or more novel epitopes in the neoantigen are identified based on a genetic map of the tumor sample. A genetic map of a tumor sample can be provided using any known genetic mapping analysis method such as next-generation sequencing (NGS), microarrays, or proteomics.

[0285] In some implementations, neoantigens are identified by sequencing tumor samples taken from an individual. In some implementations, sequencing is next-generation sequencing. In some implementations, sequencing is whole-genome sequencing. In some implementations, sequencing is exome sequencing. In some implementations, sequencing is targeted sequencing of candidate genes such as cancer-associated genes. Many commercial NGS cancer testing packages, such as ONCOGXONE, are available. TM Plus (Admera Health) can be used to sequence cancer-related genes at high sequencing depth.

[0286] In some embodiments, the tumor sample is a tissue sample. In some embodiments, the tumor sample is a tumor biopsy sample, such as tumor cells obtained through fine-needle aspiration or laparoscopy (e.g., including tumor stroma). In some embodiments, the tumor sample is freshly obtained. In some embodiments, the tumor sample is frozen. In some embodiments, the tumor sample is a formaldehyde-fixed paraffin-embedded (FFPE) sample. In some embodiments, the tumor sample is a cell sample. In some embodiments, the tumor sample contains circulating metastatic cancer cells. In some embodiments, the tumor sample is obtained by sorting circulating tumor cells (CTCs) from blood. In some embodiments, nucleic acids (such as DNA and / or RNA) are extracted from the tumor sample for sequencing analysis. In some embodiments, proteins are extracted from the tumor sample for sequencing analysis.

[0287] In some embodiments, the genetic map of a tumor sample is compared with the genetic map of a reference sample (such as a sample from healthy tissue taken from the same individual or a sample from a healthy individual) to identify candidate mutant genes in tumor cells. In some embodiments, the genetic map of a tumor sample is compared with a reference sequence from a genomic database to identify candidate mutant genes in tumor cells. In some embodiments, the candidate mutant genes are cancer-related genes. In some embodiments, each candidate mutant gene contains one or more mutations, such as non-synonymous substitutions, insertions or deletions (insertions or deletions), or gene fusions, which can generate neoantigens. Common single nucleotide polymorphisms (SNPs) are excluded from the candidate mutations.

[0288] In some embodiments, novel epitopes in neoantigens are identified from candidate mutant proteins. In some embodiments, novel epitopes are predicted by computer (in silico). Exemplary bioinformatics tools for T-cell epitope prediction are known in the art, for example, see Yang X. and Yu X. (2009) “An introduction to epitopeeprediction methods and software” Rev. Med. Virol. 19(2):77-96. Factors considered in T-cell epitope prediction algorithms include, but are...

Claims

1. Use of activated T cells in a method of preparing a treatment for cancer in an individual, wherein the method includes administering an effective amount of activated T cells to the individual, wherein the activated T cells are prepared by co-culturing a population of T cells with a population of dendritic cells loaded with a plurality of tumor antigen peptides, wherein the plurality of tumor antigen peptides comprises at least 10 tumor antigen peptides, wherein each of the at least 10 tumor antigen peptides comprises at least one epitope selected from SEQ ID NO: 1-40, wherein the cancer is selected from hepatocellular carcinoma, cervical cancer, and colorectal cancer.

2. The use according to claim 1, wherein the individual has previously been administered an effective amount of dendritic cells loaded with the plurality of tumor antigen peptides.

3. The use according to claim 1, wherein the method further comprises administering to the individual an effective amount of the dendritic cells loaded with the plurality of tumor antigen peptides.

4. The use according to claim 3, wherein the dendritic cells are administered prior to the administration of the activated T cells.

5. The use according to claim 4, wherein the dendritic cells are administered 7 to 21 days prior to the administration of the activated T cells.

6. The use according to any one of claims 1 to 5, wherein the use further comprises preparing the activated T cells by co-culturing the T cell population with the dendritic cell population loaded with the plurality of tumor antigen peptides.

7. The use according to claim 6, wherein the T cell population is co-cultured with the dendritic cell population loaded with the plurality of tumor antigen peptides for 7 to 21 days.

8. The use according to any one of claims 1-5, wherein the T cell population is contacted with an immune checkpoint inhibitor prior to the co-culture.

9. The use according to any one of claims 1-5, wherein, in the presence of an immune checkpoint inhibitor, the T cell population is co-cultured with the dendritic cell population loaded with the plurality of tumor antigen peptides.

10. The use according to claim 8, wherein the immune checkpoint inhibitor is an inhibitor of an immune checkpoint molecule selected from PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, and LAG-3.

11. The use according to any one of claims 1-5, wherein the use further comprises preparing the dendritic cell population loaded with the plurality of tumor antigen peptides.

12. The use according to claim 11, wherein the dendritic cell population loaded with the plurality of tumor antigen peptides is prepared by contacting the dendritic cell population with the plurality of tumor antigen peptides.

13. The use according to claim 12, wherein the dendritic cell population loaded with the plurality of tumor antigen peptides is prepared by contacting the dendritic cell population with the plurality of tumor antigen peptides in the presence of a composition that facilitates the uptake of the plurality of tumor antigen peptides by the dendritic cells.

14. The use according to any one of claims 1-5, wherein the T cell population and the dendritic cell population originate from the same individual.

15. The use according to claim 14, wherein the T cell population and the dendritic cell population are derived from the individual receiving treatment.

16. The use according to any one of claims 1-5, wherein the activated T cells are administered to the individual at least three times.

17. The use according to claim 16, wherein the interval between each application of the activated T cells is 0.5 months to 5 months.

18. The use according to any one of claims 1-5, wherein the activated T cells are administered intravenously.

19. The use according to any one of claims 1-5, wherein the activated T cells are at least 3 × 10 9 Dosage administration per cell / individual.

20. The use according to any one of claims 1-5, wherein the activated T cells are at a concentration of 1 × 10⁻⁶. 9 Up to 1×10 10 Apply per cell / individual.

21. The use according to any one of claims 1-5, wherein the dendritic cells loaded with the plurality of tumor antigen peptides are administered at least three times.

22. The use according to claim 21, wherein the interval between each application of the dendritic cells is 0.5 months to 5 months.

23. The use according to any one of claims 1-5, wherein the dendritic cells loaded with the plurality of tumor antigen peptides are administered subcutaneously.

24. The use according to any one of claims 1-5, wherein the dendritic cells are in a concentration of 1 × 10⁻⁶. 6 Up to 5×10 6 Dosage administration per cell / individual.

25. The use according to any one of claims 1-5, wherein each of the plurality of tumor antigen peptides is 20 to 40 amino acids in length.

26. The use according to any one of claims 1-5, wherein the plurality of tumor antigen peptides comprises naturally occurring tumor antigen peptides or synthetic tumor antigen peptides.

27. The use according to any one of claims 1-5, wherein the plurality of tumor antigen peptides comprises at least one peptide containing an MHC-I epitope.

28. The use according to any one of claims 1-5, wherein the plurality of tumor antigen peptides comprises at least one peptide containing an MHC-II epitope.

29. The use according to claim 27, wherein the at least one peptide containing an MHC-I epitope further comprises, at an N-terminus, a C-terminus, or both of these terms, an additional amino acid located adjacent to the epitope.

30. The use according to any one of claims 1-5, wherein the plurality of tumor antigen peptides comprises a first core group of common tumor antigen peptides.

31. The use according to any one of claims 1-5, wherein the plurality of tumor antigen peptides further comprises a second group of cancer type-specific antigen peptides.

32. The use according to claim 30, wherein the first core group of common tumor antigen peptides comprises 10 to 20 common tumor antigen peptides.

33. The use according to claim 31, wherein the second group of cancer type-specific antigenic peptides comprises 1 to 10 cancer type-specific antigenic peptides.

34. The use according to any one of claims 1-5, wherein the plurality of tumor antigen peptides includes neoantigen peptides.

35. The use according to claim 34, wherein the neoantigen peptide is selected based on a genetic profile of a tumor sample from the individual.

36. The use according to any one of claims 1-5, wherein the use further comprises administering an effective amount of an immune checkpoint inhibitor to the individual.

37. The use according to claim 36, wherein the immune checkpoint inhibitor is an inhibitor of an immune checkpoint molecule selected from PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, and LAG-3.

38. The use according to any one of claims 1-5, wherein the individual is selected for the treatment method based on the mutational burden in the cancer.

39. The use according to any one of claims 1-5, wherein the individual has a low mutational burden in the cancer.

40. The use according to claim 38, wherein the individual has a low mutational load in one or more MHC genes.

41. The use according to claim 38, wherein the individual has no more than 10 mutations in one or more of the MHC genes.

42. The use according to claim 38, wherein the individual does not have mutations in B2M.

43. The use according to claim 38, wherein the individual does not have mutations in the functional regions of the one or more MHC genes.

44. The use according to claim 38, wherein the mutational burden of the cancer is determined by sequencing a tumor sample from the individual.

45. The use according to any one of claims 1-5, wherein the individual is selected for the treatment based on having one or more neoantigens of the cancer.

46. ​​The use according to any one of claims 1-5, wherein the individual has at least 5 neoantigens.

47. The use according to claim 45, wherein the use further comprises identifying a neoantigen of the cancer, and incorporating a neoantigen peptide into the plurality of tumor antigen peptides, wherein the neoantigen peptide comprises a novel epitope of the neoantigen.

48. The use according to claim 45, wherein the neoantigen is identified by sequencing a tumor sample from said individual.

49. The use according to claim 44, wherein the sequencing is targeted sequencing of cancer-related genes.

50. The use according to claim 47, wherein the use further comprises determining the affinity of the new epitope for an MHC molecule.

51. The use according to claim 47, wherein the use further comprises determining the affinity of the complex comprising the novel epitope and the MHC molecule for a T-cell receptor.

52. The use according to claim 50, wherein the MHC molecule originates from the individual.

53. The use according to claim 51, wherein the MHC molecule originates from the individual.

54. The use according to any one of claims 1-5, wherein the use further comprises monitoring the individual after the administration of the activated T cells.

55. The use according to claim 54, wherein the monitoring includes determining the number of circulating tumor cells (CTCs) in the individual.

56. The use according to claim 54, wherein the monitoring includes detecting a specific immune response in the individual against the plurality of tumor antigen peptides.

57. The use according to claim 56, wherein the plurality of tumor antigen peptides are modulated based on the specific immune response to provide a plurality of customized tumor antigen peptides.

58. The use according to claim 57, wherein the treatment method is repeated using the plurality of customized tumor antigen peptides.

59. The use according to any one of claims 1-5, wherein the individual is a human individual.

60. A method for preparing an activated T cell population, the method comprising: a) Inducing the differentiation of monocyte populations into dendritic cell populations; b) Contacting the dendritic cell population with a plurality of tumor antigen peptides to obtain a dendritic cell population loaded with the plurality of tumor antigen peptides, wherein the plurality of tumor antigen peptides comprises at least 10 tumor antigen peptides, wherein each of the at least 10 tumor antigen peptides comprises at least one epitope selected from SEQ ID NO: 1-40; and c) The dendritic cell population loaded with the various tumor antigen peptides is co-cultured with a non-adhesive PBMC population to obtain the activated T cell population; The mononuclear cell population and the non-adhesive PBMC population are obtained from the PBMC population of an individual.

61. The method of claim 60, wherein step b) comprises contacting the dendritic cell population with the plurality of tumor antigen peptides in the presence of a composition that facilitates the uptake of the plurality of tumor antigen peptides by the dendritic cells.

62. The method of claim 61, wherein step b) further comprises contacting the dendritic cell population loaded with the plurality of tumor antigen peptides with a plurality of Toll-like receptor (TLR) agonists to induce maturation of the dendritic cell population loaded with the plurality of tumor antigen peptides.

63. The method of claim 61, wherein step c) further comprises contacting the activated T cell population with a variety of cytokines to induce the proliferation and differentiation of the activated T cell population.

64. The method according to any one of claims 60-63, wherein the plurality of cytokines includes IL-2, IL-7, IL-15 or IL-21.

65. The method according to any one of claims 60-63, wherein the non-adhesive PBMC population is contacted with an immune checkpoint inhibitor prior to the co-culture.

66. The method according to any one of claims 60-63, wherein step c) comprises co-culturing the dendritic cell population loaded with the plurality of tumor antigen peptides with the non-adhesive PBMC population in the presence of an immune checkpoint inhibitor.

67. The method of claim 65, wherein the immune checkpoint inhibitor is an inhibitor of an immune checkpoint molecule selected from PD-1, PD-L1, CTLA-4, IDO, TIM-3, BTLA, VISTA, and LAG-3.

68. The method according to any one of claims 60-63, wherein each of the plurality of tumor antigen peptides is 20 to 40 amino acids in length.

69. The method according to any one of claims 60-63, wherein the plurality of tumor antigen peptides comprises at least one peptide containing an MHC-I epitope.

70. The method according to any one of claims 60-63, wherein the plurality of tumor antigen peptides comprises at least one peptide containing an MHC-II epitope.

71. The method of claim 69, wherein the at least one peptide containing an MHC-I epitope further comprises, at an N-terminus, a C-terminus, or both of these terms, an additional amino acid located adjacent to the epitope.

72. The method of claim 70, wherein the at least one peptide containing an MHC-II epitope further comprises, at an N-terminus, a C-terminus, or both of these terms, an additional amino acid located adjacent to the epitope.

73. The method according to any one of claims 60-63, wherein the plurality of tumor antigen peptides comprises a first core group of common tumor antigen peptides.

74. The method according to any one of claims 60-63, wherein the plurality of tumor antigen peptides further comprises a second group of cancer type-specific antigen peptides.

75. The method of claim 73, wherein the first core group comprises 10 to 20 common tumor antigen peptides.

76. The method of claim 74, wherein the second group comprises 1 to 10 cancer type-specific antigenic peptides.

77. The method according to any one of claims 60-62, wherein the plurality of tumor antigen peptides includes neoantigen peptides.

78. The method of claim 77, wherein the neoantigen peptide is selected based on a genetic profile of a tumor sample from the individual.

79. A method for cloning tumor-specific T-cell receptors, the method comprising: (a) Treating an individual using a method for treating cancer as defined in any one of claims 1 to 4; (b) Isolating T cells from the individual, wherein the T cells specifically recognize tumor antigen peptides among the plurality of tumor antigen peptides; and (c) Cloning a T-cell receptor from the T-cell to provide the tumor-specific T-cell receptor.

80. The method of claim 79, wherein the individual has a strong specific immune response to the tumor antigen peptide.

81. The method of claim 79, wherein the T cells are isolated from a PBMC sample of the individual.

82. The method according to any one of claims 79-81, wherein the tumor antigen peptide is a neoantigen peptide.

83. A tumor-specific T-cell receptor cloned using the method of any one of claims 79-81.

84. An isolated T cell comprising the tumor-specific T cell receptor of claim 83.

85. Use of the isolated T cells according to claim 84 in a medicament administered in a method for treating cancer in an individual, wherein the method comprises administering an effective amount of the isolated T cells to the individual, wherein the cancer is selected from hepatocellular carcinoma, cervical cancer, and colorectal cancer.

86. A population of isolated cells prepared by the method of any one of claims 79-81.

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