Title of Invention: METHOD OF PREPARING AND

EXPANDING A POPULATION OF IMMUNE CELLS FOR CANCER THERAPY, POTENCY ASSAY FOR TUMOR RECOGNITION, BIOLOGICAL VACCINE PREPARATION AND EPITOPE TARGET FOR ANTIBODIES,

Technical Field The present invention relates to cancer immunotherapy and discloses a method of preparing and expanding a population of immune cells for anticancer directed therapy. The invention further discloses a potency assay for tumor recognition, a biological vaccine preparation to provide antitumor responses or antiviral responses and an epitope target for antibodies.

Background of the Invention Cancer Cancer is a major cause of death worldwide with an annual toll of almost 10 million. Last year, approximately 19.3 million people were diagnosed with cancer ¹ and pancreatic cancer was the 7 ^th leading cancer-related cause of death while also being the 12 ^th most incident cancer worldwide ² Standard cancer treatments, such as surgery, chemotherapy, and radiotherapy, have demonstrated limited efficacy for the treatment of patients with pancreatic cancer. Immunotherapy emerged as a path to bypass various immune evasion mechanisms and potentiate immune cells to improve their anti-tumor functions or - not mutually exclusive - to expand and activate immune cells that have not yet been effectively recruited in a patient to contribute to clinically relevant immune responses. Several different types of immunotherapies, from the least specific to the more tumor-directed, have been designed to overcome different tumor escape mechanisms. Pancreatic adenocarcinoma (PDAC) is one of the most lethal cancers, having a 5-year survival rate of around 5 - 10 % ³ . To this date, traditional chemotherapeutics have failed to produce significant improvements in pancreatic cancer survival and research efforts have been focused on the role of the immune system in the development and progression of this malignancy ^4-6 .The PDAC microenvironment tends to be pro-tumorigenic and immunosuppressive, moreover, it presents a desmoplastic phenotype that prevents T-cell infiltration, resulting in a more pronounced immune- suppressive effect, although more clinical research is needed to define immunological subtypes in PDACs, since some tumor lesions show during tumor evolution T-cell infiltration - that may be blunted by different immune-escape mechanisms ^4-9 Immune system and oncology interplay Tumors are complex ecosystems composed of neoplastic cells, extracellular matrix and accessory nonneoplastic cells which may include a broad array of inflammatory immune cells infiltrating into tumor tissue, sometimes more than 90% of a cancer lesion are non-cancer cells, consisting of connective tissue and non-transformed cells. Crosstalk between cancer cells and accessory cells contributes to tumor development. During tumor formation, the tissue architecture evolves into a specialized microenvironment that may either be acting in a pro-tumor or anti-tumor ⁴ directed fashion. The landscape of the tumor microenvironment (TME) represents a dynamic ecosystem controlled by the tumor to support its growth and survival via altered levels of metabolites, cytokines, nutrients, oxygen, and expression of immune checkpoints to promote immune evasion. Cancer cells use mechanisms to avoid immune detection and attack such as contact dependent factors (expression of immune system checkpoint ligands such as PD-L1 , CTLA-4, LAG-3, TIM-3, TIGIT), production of soluble immunosuppressive factors (such as IL-10 and TGF-[3) and downregulation of MHC class I on tumor cells. Through these mechanisms, cancer cells can achieve general immunosuppression and tumor progression ⁴ . Conventional a|3 T-cells orchestrate cellular immunity by recognizing short foreign peptides bound to specialized molecular ‘presentation proteins’ called MHC molecules complexed with the peptide on the surface of antigen-presenting cells (APCs) during the initiation of a cellular immune response, or on tumor cells during the immune effector phase. Under situations of infection and malignant transformation, the recognition of an MHC-peptide complex triggers elimination of the target by T-cells by effector mechanisms such as production of perforin and granzyme granules. One of the key factors in anti-tumor directed immune responses is IFN-y (IFN-gamma). A vast array of other cytokines plays a decisive role in anti-tumor immune responses, for instance, IL-17 production appears to be a double-edged sword; it may - for some tumor types - be helpful to fight off cancer cells early in the disease while facilitating disease progression in more advanced cancer disease, associated with the molecular phenotype signature. However, some IL-17 production is also needed for T-cells to enter the central nervous system (CNS) and may therefore be helpful if T-cell responses are needed in mediating anti-tumor directed immune responses (e.g., for metastatic disease) in the CNS. 0] The T-cell receptor (TCR) is comprised of an a-chain and a [3-chain, which are composed of complementary determining loops (CDR1 , CDR2 and CDR3) that are collectively unique to every individual TCR and amount to the enormous diversity of TCRs. The CDR3 region is the region where T-cell specificity resides and the CDR3 regions is (for a|3 T- cells) between 1 and 11 amino acids long. It provides exquisite specificity for MHC class I (for CD8 ⁺ T-cells) or MHC class II (for CD4 ⁺ ) restricted T- cells. The TCR engages with the 3D shape of the nominal target epitope that is buried and anchored in the MHC binding cleft within designated ‘pockets’ provided by the MHC class I molecules, while MHC class II presented peptides assume a different accommodation since MHC class I molecules are closed on both ends, while MHC class II molecules are open on one end and afford to accommodate larger peptide species. Similarities in the 3D recognition of nominal target epitopes have been described and are in part due to the similar shape, charge, size, and composition of amino acids recognized by ‘cross-reactive’ T-cells reacting to even non-related target epitopes between different human, bacterial or viral peptide antigens or non-related epitopes or different origins. ] Mucosal-associated invariant T-cells (MAIT) represent a different immune effector population and recognize different molecular targets, in part since they express a semi-invariant TCR; most of the MAIT family express the Va7.2 chain, while some other MAIT representatives express a different and more diverse set of TCRs. MAIT cells are a part of the unconventional T-cell family, like yb T-cells, that recognize metabolite antigens presented by MHC class l-like molecule (MR1) and CD1 , respectively ⁴ ’ ¹⁰ . MAIT cells recognize not only vitamin - metabolites, yet also drugs, intermediates of cellular metabolism and possibly shared and private tumor-associated antigens, which have not yet been defined up to now. ] Some subpopulations of yb T-cells, similarly as natural-killer T (NKT) cells, respond to phosphoantigens from infected cells and cancer cells within the context of butyrophilin molecules, yb T-cells may recognize their nominal target antigens in the context of CD1a-d, other classical or non-classical MHC molecules or - for some yb T-cell subsets - without classical or non-classical MHC molecules. Like other unconventional T- cell subsets, the MR1 -reactive T-cell family and the yb T-cell population is far more complex than initially believed and the same has been found to be true for their nominal target epitopes. Also, TCR yb T-cells have been found to recognize the ‘underside’ of the MR1 molecule underlying that MR1 recognition is very diverse; in part reflected by the fact that also TRAV1-2 and TRAV36 TCRs are also able to bind in a different fashion to MR1 with a different docking behavior. 3] Tumor infiltrating lymphocytes (TIL) therapy 4] Therapy of patients with tumor infiltrating lymphocytes (TIL) has been successful in patients with melanoma and achieved using expansion of TIL with interleukin-2 (IL-2) and pre-conditioning of the patient with chemotherapy, usually cyclophosphamide and fludarabine, with a response rate of patients with melanoma in the range of 50-70% and a long-term overall 5-year survival of 93% in patients who experienced a complete response. Other cancers, like cervical cancer, respond also to TIL therapy. 5] Patients with melanoma who previously had ipilimumab and IL-2 showed an overall response rate of 42%. Clinical response has been associated with neoepitope reactivity in TIL and certain T-cell phenotypes, for instance with a precursor or central memory T-cell subset (CD45RA ⁺ CCR7 ⁺ , CD45RA’CCR7 ⁺ ), or other phenotypes, e.g., the CD3 ⁺ CD8 ⁺ CD39’CD69’ phenotype. Although patients with melanoma profit from TIL therapy, progress in TIL therapy or using checkpoint inhibitors has been less successful in patients with cancers other than melanoma. Yet the use of TIL has been extended to patients with malignancies with different origins, i.e. , patients with HPV-positive cancer or patients with non-small cell lung cancer (NSCLC). There are individual patients with breast cancer, or patients with cholangiocarcinoma in whom TIL against an individual epitope are associated with clinical success, or a patient with colorectal cancer (CRC) whose TIL were directed against a mutant KRAS target epitope. 6] Immunological therapy of patients with PDAC has been proven in individual patients i.e., using Autologous Hematopoietic Stem Cell Transplantation (aHSCT). The TME in PDAC and immunological situation bears a unique profile as compared to other tumor types, i.e., the desmoplastic stroma and immuno-suppressing myeloid cells ¹¹ . Sparse T- cell infiltration has been quoted as one of the key factors for less responses for patients with PDAC, as well as a low frequency of mutations. ] TIL in patients with PDAC have been compared with TIL in patients with melanoma and, the landscape of TIL infiltrates in PDAC is different, i.e., sparse TIL within the tumor, TIL found at the rim between the tumor and non-malignant tissue. These paradigms have been changed, i.e., certain patients present with TIL detected in the tumor-associated stroma, TIL from individual patients with PDAC have been reported to recognize neoepitopes and autologous tumor cells. 8] TIL from multiple tumor regions provide more likely a more accurate coverage of anti-tumor reactive T-cells directed against tissue from which TIL were harvested, yet also directed against tumor cells at distant sites in a different tumor-organ microenvironment (e.g., with the primary tumor in the pancreas and metastatic lesions in the liver, lung, or other organs I tissues). Neo-epitope specific T-cells have also been attempted to be isolated from peripheral blood with limited success. 9] TIL from patients with PDAC have been reported to recognize autologous tumor cells and/or neoepitopes from patients with PDAC after expansion. A major challenge of expanding TIL from PDAC lesions is to obtain sufficient numbers of TIL and that these TIL are indeed tumor- reactive, which was estimated to be low in TIL from epithelial cancers, with bystander T-cells (i.e. , not tumor-directed T-cells) or viral-directed T- cells present in TIL preparations. Tumor antigens may be derived from frame-shift mutations, point-mutations, non-mutant immune cell target antigens that are expressed during embryonal development, antigens that are not exposed during thymic selection, antigens to which the cellular and humoral immune system has not been ‘tolerized’. Other factors, influencing immunogenicity have been less explored at this point, e.g. the different glycosylation status of proteins or other post-translational modifications. 0] The state of T-cell differentiation, as well as access of TIL into tissue are favorable factors. Not only the anatomical landscape of TIL, yet also the composition of TIL is differently associated with their tissue of origin, i.e., the presence of TCR yb T-cells or MAIT in patients with pancreatic cancer may shape the orchestrated cellular immune response of ‘classical’ MHC class I or MHC class II restricted responses, as well as the interplay with immune cells that are anti-tumor directed and restricted by ‘non-classical’ MHC molecules, e.g. CD1 d or MR1 .

[21] Not only aβ TIL, yet yb TIL react to epithelial cells. Anti-CD3 stimulation and allogeneic feeder cells, or, in some protocols, co-stimulation of matrix-bound CD3-CD28 have also been used to expand anti-tumor directed immune cells. Additional stimuli have been tested to obtain ‘better’ TIL i.e. , increased potassium and acetate, inducing increased ‘T- cell sternness’, or reducing T-cell senescence ¹² . ] Furthermore, bacterial, and fungal species have been described to be associated with pancreatic cancer progression ¹³ , and - vice versa - with better prognosis: long-term survivors with pancreatic cancer exhibit a different microbiome ¹⁴ , which suggests the possible role of MAIT in pancreatic cancer outcome. Riquelme et al. (2019) ¹⁵ demonstrated an intra-tumoral microbiome signature involving Pseudoxanthomas, Streptomyces, Saccharopolyspora and Bacillus that was predictive of survival in patients with pancreatic ductal adenocarcinoma. Some of these bacterial species or related families have been implicated in inflammatory diseases associated with IFN-y production. This could imply there is cross-reactivity between T-cells that recognize tumor antigens and microbial antigens present in the tumor tissue, or tumor-associated tissue components. 3] Due to the above, the present application suggests that target antigens could be recognized by clinically relevant immune cells and, therefore, boost the immune response in cancer therapy when administered to a patient in need thereof as a biological vaccine preparation. 4] The present application provides a method of preparing and expanding a population of clinically relevant immune cells using interleukins, cardiolipin and feeder cells, wherein the expansion is facilitated by adding an antibody directed against CD3, a component of the T-cell receptor, in order to increase tumor-reactive T-cells in an immune cell product. documents 5] Document WO 2020/172202 A1 discloses methods for manufacturing T-cells which express a novel group of cell surface receptors that recognize peptides on the surface of a target cell, as well as populations of T cells produced by the methods and pharmaceutical compositions thereof. 6] More specifically, the method comprises the steps of processing a biological sample containing a population of T lymphocyte cells obtained from a donor subject that has a tumor to produce a population of T lymphocyte cells and then stimulating such population with one or more T-cell stimulating agents under conditions for expansion. ] In KR 102182555 B1 , a method for screening a common cancer antigen or neoantigen commonly expressed in cancer tissues is disclosed. The screening method efficiently screens a peptide capable of specifically binding to a human leukocyte antigen expressed in cancer tissues. The peptide has high immunogenicity, so it can be usefully used in cancer treatment vaccines. 8] In document WO 2020/180648 A1 , it is disclosed a method for separately isolating antigen-binding T-cells and antigen-activated T-cells derived from an initial population of peripheral blood mononuclear cells, and for identifying clonotypes of the blood receptor overlapping T cells. In this specific document, antigens include personal and shared neoantigens as well as testicular cancer antigens. T cell receptor clonotypes identified by screening anti-cancer - associated antigens, can further be used to develop cancer treatment therapies, e.g. by cloning the nominal tumor-target specific T-cell receptor which could be used with an appropriate vector system - to transfer antigen specificity restricted by ‘classical’ (e.g. MHC class I or MHC class II) or non-classical (e.g. MR1 or CD1 ) restricting molecules - into recipient immune cells. Summary of the Invention 9] The present invention is based on the fact that private or commonly shared tumor-associated antigens could be recognized by clinically relevant immune cells. Such target antigens could be used to prepare a biological vaccine preparation to provide anti-tumor response or antiviral response by expanding a certain set of T-cells or B-cells and boosting the immune response in anti-cancer directed therapy.

[30] Therefore, the present invention relates, in a first aspect, to a method of preparing and expanding a population of immune cells, wherein a body sample is first obtained from a mammal and then cultured for three expansion steps to obtain anti-tumor directed immune cells selected from tumor-infiltrating lymphocytes, peripheral blood mononuclear cells, or immune cells harvested from non-cancer tissue from a distant anatomical site, for instance skin (devoid of cancer cells) yet provides an environment that enriches for tumor antigen specific T-cells. ] In a second aspect, the invention relates to a potency assay for tumor recognition, wherein the clinically relevant immune cells previously obtained are challenged with at least one target antigen to detect a change in a cytokine or immune effector molecule production. ] The present invention further relates, in a third aspect, to a biological vaccine preparation to provide anti-tumor response or antiviral response, comprising target antigens that lead to the expansion of a certain set of T- cells or B-cells. Also, the target antigen serves as a target for antibody molecules, or protein that binds specifically to it, whose responses are directed against cancer cells, which could be used for antibody therapy or construction of a chimeric antigen receptor construct. 3] In a fourth aspect, the present invention discloses an epitope target for antibodies that serves as a viable target that can be used to construct chimeric antigen receptors. scription of Drawings 4] In order to promote an understanding of the principles according to the modalities of the present invention, reference will be made to the embodiments illustrated in the figures and the language used to describe them. 5] It should also be understood that there is no intention to limit the scope of the invention to the content of the figures and that modifications to the inventive features illustrated herein, as well as additional applications of the principles and embodiments illustrated, which would normally occur to a person skilled in the art having the possession of this description, are considered within the scope of the claimed invention. 6] [Fig.1 ] shows an example of a tumor resection, the first steps of TIL propagation and examples of selection of tumor areas for TIL expansion guided by anatomical structures, wherein (a) is a gross specimen after resection, (b) is the anterior view of the specimen, (c) is the posterior view of the specimen, (d) is the opened tumor specimen for TIL propagation, the entire middle section is used for TIL expansion. Parallel sections to the left and right are prepared for standard histology, immunohistology and gene expression analysis, including spatial transcriptom ics and (e) is the tissue piece where TIL mobilized by the method described in the current application. Parallel sections are prepared for histopathology to guide the selection of tissues for expansion and to understand better the biology of anti-tumor responses, as well as to select the most promising T-cell infiltrating area of the tumor, while avoiding areas that may contain immune cells that may facilitate tumor progression or mediate immune- suppression. ] [Fig.2] shows examples of selection of tumor areas for TIL expansion, wherein (a) is the mobilized tumor piece for TIL propagation, (b) is the dissected tumor pieces and minced into 1 - 3 mm ³ pieces to be placed into culture vessels, ice-cold PBS aids to identify distinct anatomical areas and the selection is guided by the tumor piece anatomy and if immediately available histopathology, marker analysis, e.g. by immunohistochemistry, or by gene expression analysis, (c) is the selection of distinct tumor areas along anatomical structures, cutting of tumor areas into distinct 1 mm ³ pieces that are individually placed into 24 well plates and (d) is the HE staining parallel section of the tumor piece used for TIL propagation. 8] [Fig.3] shows examples of the HE staining parallel section of the tumor piece used for TIL propagation, histology aids to define regions with tumor cells and TIL infiltrates as well as tertiary lymphoid structures (TLS) to link with TIL propagation, wherein (a) corresponds to histopathology in 5 mm scale, (b) corresponds to histopathology in 1 mm scale, (c) corresponds to histopathology in 100 pm scale, (d) corresponds to histopathology in 50 pm scale and (e) corresponds to histopathology in 200 pm scale. 9] [Fig.4] is an immunohisto-fluorescence analysis showing the association of strong IL-17 expression in distinct regions of the tumor lesion surrounded by CD3 ⁺ T-lymphocytes - imprinting on T-cell biology and subsequent TIL expansion. 0] [Fig.5] is an immunohisto-fluorescence analysis showing the association of IL-7 expression in distinct regions of the tumor lesion surrounded by CD3 ⁺ T-lymphocytes - imprinting on T-cell biology and subsequent TIL expansion, wherein IL-7 binding to its nominal receptor CD127 represents a strong T-cell survival factor as well as a strong anti- TGF-beta antagonist, enriching tumor-reactive TIL. ] [Fig.6] shows the production of IFN-y and IL-17A in peripheral blood mononuclear cells (PBMCs) from buffy coats, PBMCs from healthy donors and unprocessed blood from healthy donors incubated with different concentrations of cardiolipin and in the presence of the nominal target antigens and controls, wherein (a) corresponds to production of IFN-y with titration at 0 nM at day 3 in PBMCs from buffy coats, (b) corresponds to production of IFN-y with titration at 0 nM at day 7 in PBMCs from buffy coats, (c) corresponds to production of IFN-y with titration at 50 nM at day 3 in PBMCs from buffy coats, (d) corresponds to production of IFN-y with titration at 50 nM at day 7 in PBMCs from buffy coats, (e) corresponds to production of IFN-y with titration at 150 nM at day 3 in PBMCs from buffy coats, (f) corresponds to production of IFN-y with titration at 150 nM at day 7 in PBMCs from buffy coats, (g) corresponds to production of IFN-y with titration at 250 nM at day 3 in PBMCs from buffy coats, (h) corresponds to production of IFN-y with titration at 250 nM at day 7 in PBMCs from buffy coats, (i) corresponds to production of IFN-y with titration at 350 nM at day 3 in PBMCs from buffy coats, (j) corresponds to production of IFN-y with titration at 350 nM at day 7 in PBMCs from buffy coats, (k) corresponds to production of IFN-y with titration at 450 nM at day 3 in PBMCs from buffy coats, (I) corresponds to production of IFN-y with titration at 450 nM at day 7 in PBMCs from buffy coats, (m) corresponds to production of IL-17A with titration at 0 nM at day 3 in PBMCs from buffy coats, (n) corresponds to production of IL-17A with titration at 0 nM at day 7 in PBMCs from buffy coats in PBMCs from buffy coats, (o) corresponds to production of IL-17A with titration at 50 nM at day 3 in PBMCs from buffy coats, (p) corresponds to production of IL-17A with titration at 50 nM at day 7 in PBMCs from buffy coats, (q) corresponds to production of IL-17A with titration at 150 nM at day 3 in PBMCs from buffy coats, (r) corresponds to production of IL-17A with titration at 150 nM at day 7 in PBMCs from buffy coats, (s) corresponds to production of IL-17A with titration at 250 nM at day 3 in PBMCs from buffy coats, (t) corresponds to production of IL-17A with titration at 250 nM at day 7 in PBMCs from buffy coats, (u) corresponds to production of IL-17A with titration at 350 nM at day 3 in PBMCs from buffy coats, (v) corresponds to production of IL-17A with titration at 350 nM at day 3 in PBMCs from buffy coats, (w) corresponds to production of IL-17A with titration at 450 nM at day 3 in PBMCs from buffy coats, (x) corresponds to production of IL-17A with titration at 450 nM at day 7 in PBMCs from buffy coats, (y) corresponds to production of IFN-y with titration at 0 nM at day 3 in PBMCs from healthy donors, (z) corresponds to production of IFN-y with titration at 0 nM at day 7 in PBMCs from healthy donors, (aa) corresponds to production of IFN-y with titration at 50 nM at day 3 in PBMCs from healthy donors, (bb) corresponds to production of IFN-y with titration at 50 nM at day 7 in PBMCs from healthy donors, (cc) corresponds to production of IFN-y with titration at 150 nM at day 3 in PBMCs from healthy donors, (dd) corresponds to production of IFN-y with titration at 150 nM at day 7 in PBMCs from healthy donors, (ee) corresponds to production of IFN-y with titration at 250 nM at day 3 in PBMCs from healthy donors, (ff) corresponds to production of IFN-y with titration at 250 nM at day 7 in PBMCs from healthy donors, (gg) corresponds to production of IFN-y with titration at 350 nM at day 3 in PBMCs from healthy donors, (hh) corresponds to production of IFN-y with titration at 350 nM at day 7 in PBMCs from healthy donors, (ii) corresponds to production of IFN-y with titration at 450 nM at day 3 in PBMCs from healthy donors, (jj) corresponds to production of IFN-y with titration at 450 nM at day 7 in PBMCs from healthy donors, (kk) corresponds to production of IL-17A with titration at 0 nM at day 3 in PBMCs from healthy donors, (II) corresponds to production of IL-17A with titration at 0 nM at day 7 in PBMCs from healthy donors, (mm) corresponds to production of IL-17A with titration at 50 nM at day 3 in PBMCs from healthy donors, (nn) corresponds to production of IL-17A with titration at 50 nM at day 7 in PBMCs from healthy donors, (oo) corresponds to production of IL-17A with titration at 150 nM at day 3 in PBMCs from healthy donors, (pp) corresponds to production of IL-17A with titration at 150 nM at day 7 in PBMCs from healthy donors, (qq) corresponds to production of IL-17A with titration at 250 nM at day 3 in PBMCs from healthy donors, (rr) corresponds to production of IL-17A with titration at 250 nM at day 7 in PBMCs from healthy donors, (ss) corresponds to production of IL-17A with titration at 350 nM at day 3 in PBMCs from healthy donors, (tt) corresponds to production of IL-17A with titration at 350 nM at day 7 in PBMCs from healthy donors, (uu) corresponds to production of IL-17A with titration at 450 nM at day 3 in PBMCs from healthy donors, (vv) corresponds to production of IL-17A with titration at 450 nM at day 7 in PBMCs from healthy donors, (ww) corresponds to production of IFN-y with titration at 0 nM at day 3 in whole blood from healthy donors, (xx) corresponds to production of IFN-y with titration at 0 nM at day 7 in whole blood from healthy donors, (yy) corresponds to production of IFN-y with titration at 50 nM at day 3 in whole blood from healthy donors, (zz) corresponds to production of IFN-y with titration at 50 nM at day 7 in whole blood from healthy donors, (aaa) corresponds to production of IFN- y with production of IFN-y with titration at 150 nM at day 3 in whole blood from healthy donors, (bbb) corresponds to production of IFN-y with titration at 150 nM at day 7 in whole blood from healthy donors, (ccc) corresponds to production of IFN-y with titration at 250 nM at day 3 in whole blood from healthy donors, (ddd) corresponds to production of IFN- y with titration at 250 nM at day 7 in whole blood from healthy donors, (eee) corresponds to production of IFN-y with titration at 350 nM at day 3 in whole blood from healthy donors, (fff) corresponds to production of IFN-y with titration at 350 nM at day 7 in whole blood from healthy donors, (ggg) corresponds to production of IFN-y with titration at 450 nM at day 3 in whole blood from healthy donors, (hhh) corresponds to production of IFN-y with titration at 450 nM at day 7 in whole blood from healthy donors, (iii) corresponds to production of IL-17A with titration at 0 nM at day 3 in whole blood from healthy donors, (jjj) corresponds to production of IL-17A with titration at 0 nM at day 7 in whole blood from healthy donors, (kkk) corresponds to production of IL-17A with titration at 50 nM at day 3 in whole blood from healthy donors, (III) corresponds to production of IL-17A with titration at 50 nM at day 7 in whole blood from healthy donors, (mmm) corresponds to production of IL-17A with titration at 150 nM at day 3 in whole blood from healthy donors, (nnn) corresponds to production of IL-17A with titration at 150 nM at day 7 in whole blood from healthy donors, (ooo) corresponds to production of IL-17A with titration at 250 nM at day 3 in whole blood from healthy donors, (ppp) corresponds to production of IL-17A with titration at 250 nM at day 7 in whole blood from healthy donors, (qqq) corresponds to titration at 350 nM at day 3 in whole blood from healthy donors, (rrr) corresponds to production of IL-17A with titration at 350 nM at day 7 in whole blood from healthy donors, (sss) corresponds to production of IL-17A with titration at 450 nM at day 3 in whole blood from healthy donors, (ttt) corresponds to production of IL-17A with titration at 450 nM at day 7 in whole blood from healthy donors. The medium (as well as all positive and antigen responses) contained IL-7, IL- 15, and IL-21 , which represents the initial step of the TIL expansion process. Negative control: medium alone. Positive control: OKT3 - T-cell receptor cross linking via the anti-CD3 directed antibody; PHA - phytohemagglutinin. Antigens: CMV - overlapping peptides covering CMV pp65; EBNA - overlapping peptides covering EBNA3; M1 - peptides covering the M1 protein from the Flu virus, a target that is conserved among different strains; ESAT-6 - peptides covering the immunodominant M. tuberculosis ESAT-6 target (LEEKKGNYWTDH); HG - immunodominant hemagglutinin target (VEPGDKITFEATGNL). ] [Fig.7] is a Western Blot assay to determine mitochondrial isolation from PBMC, PBMCs pre-treated with Doxirubicin or and the PANC-1 (pancreatic cancer) tumor cell line. 3] [Fig.8] are histopathology photographs showing the presence of yσ ⁺ T- cells that can be expanded for TIL propagation. 4] [Fig.9] graphically shows that cardiolipin leads to increased frequency of yσ ⁺ TIL, wherein (a) is the percentage of yσ ⁺ TIL expanded and (b) is the percentage of V51 ⁺ TIL with or without cardiolipin, (c) shows that the presence of feeder cells does not affect cardiolipin expanded yσ ⁺ TIL and (d) V51 ⁺ TIL. 5] [Fig.10] graphically shows no difference between cells expanded with and without cardiolipin wherein (a) is CD107a induction assay, (b) is the percentage of Tregs, (c) is the production of IFN-y by 10 ⁵ cells in 24 hours and (d) is the production of IL-17A by 10 ⁵ cells in 24 hours. 6] [Fig.11 ] graphically shows similar frequency of activation and exhaustion markers, measured by flow cytometry, in different TIL subpopulations independent of cardiolipin, wherein (a) are CD4 ⁺ T-cells and (b) are CD8 ⁺ T-cells. ] [Fig.12] graphically shows the differentiation and maturation status of peripheral blood mononuclear cells expanded with the triple TIL expansion protocol, i.e., first with IL-7/1 IL-15/IL-21 , followed by IL-2/IL- 7/IL-15 in the presence of Cardiolipin and cross-linking of the T-cell receptor using anti-CD3-lgG1 . Note that no feeder cells were used. The data show that the frequency of precursor (CD45RA ⁺ CCR7 ⁺ ), central memory (CD45RA’CCR7 ⁺ ), effector memory (CD45RA’CCR7’) and terminally differentiated effector cells (CD45RA ⁺ CCR7’) in CD8 ⁺ PBMCs. Each dot represents PBMCs from an individual patient with cancer. The protocol does not drive peripheral T-cells into terminal differentiation, yet rather into a memory T-cell subset which is advantageous as recipient cells for transgenic immune recognition receptors, i.e., antibodies or T- cells for CAR-T-cell development. Note a decrease in frequency of terminally differentiated effector cells and enrichment in cellular memory cells which has been associated with increased clinical responsiveness in active cellular therapy. 8] [Fig.13] graphically shows the increased recognition of tumor associated antigens in the presence of cardiolipin in expanded TIL, wherein (a) is control, MART-1 , and infectious diseases antigens, (b) is Mucin 4 peptides, (c) Mesothelin-derived peptides and (d) KRAS peptides from wildtype or mutant KRAS molecules. 9] [Fig.14] graphically shows that the MCC950 inhibitor reduces the tumor associated antigen recognition in TIL underlining that Cardiolipin uses the NRLP3 pathway. 0] [Fig.15] shows the genes differentially expressed in TIL grown with and without cardiolipin. ] [Fig.16] shows the RNA expression profile of TIL with or without cardiolipin, wherein (a) genes associated with Th1 profile, (b) genes associated with Th2 profile, (c) genes associated with Th17 profile, (d) genes associated with Tfh profile, (e) genes associated with Treg profile, (f) genes associated with T-cell activation, (g) genes associated with immune response, (h) genes associated with memory phenotype, (i) genes associated with effector differentiation phenotype, (j) genes associated with exhaustion phenotype. ] [Fig.17] graphically shows seven a[3 ⁺ TIL lines which were tested against autologous tumor. Strong and specific recognition shown be either blocking with an anti-MHC class I or -class II (HLA-DR) specific mAb. 3] [Fig.18] graphically shows five yσ ⁺ TIL lines which were tested against autologous cancer cells. Recognition of tumor cells is CD1d restricted using a blocking anti-CD1d antibody. 4] [Fig.19] is an example of T-cell repertoire in the tumor tissue, where TCR VA/B or VG/D family is visualized in a different color and the size of the CDR3 region is associated with the relative frequency of RNA TCR reads, wherein (a) is the TCR a chain, (b) is the TCR β chain, (c) is the TCR y chain and (d) is the TCR σ chain.

[55] [Fig.20] is an example of T-cell repertoire in expanded TIL, where TCR VA/B or VG/D family is visualized in a different color and the size of the CDR3 region is associated with the relative frequency of RNA TCR reads, wherein (a) is the TCR a chain, (b) is the TCR β chain, (c) is the TCR y chain, (d) is the TCR σ chain and (e) is a Tumor Autologous Recognition assay showing IFN-y production in a HLA-DR restricted fashion. 6] [Fig.21 ] is an example of the negatively sorted TCR γ5 repertoire in expanded TIL, wherein each VG/D family is visualized in a different color and the size of the CDR3 region is associated with the relative frequency of RNA TCR reads, wherein (a) is the TCR y chain, (b) is the TCR 5 chain, (c) represents an tumor autologous recognition assay showing IFN-y production in a CD1 d restricted fashion and (d) is a histopathology photograph showing the presence of yσ ⁺ T-cells from which this TCR γ5 T-cell line was originally derived. ] [Fig.22] graphically shows that four yσ ⁺ TIL lines were tested for recognition of autologous IL-4/GM-CSF generated autologous APCs. That were fed with mitochondria isolated from PBMCs. Mitochondria from non-manipulated PBMCs did not lead to IFN-y production. Doxirubicin pretreated of PBMCs, followed by the isolation of mitochondria resulted in CD1 d restricted recognition in 2/4 GD ⁺ TIL. The same was found to be true for autologous APCs pulsed with mitochondria from PANC-1 , a pancreatic cancer tumor cell line. 8] [Fig.23] graphically shows seven yσ ⁺ TIL lines tested for recognition of PANC-1 cells. 7/9 yσ ⁺ TIL recognized PANC1 in a CD1d restricted fashion. This could not be changed by incubating PANC1 with doxirubicin, leflunamide or rapamycin. Values are pg IFN-y/7 days. 9] [Fig.24] shows the results of potency assay in forty different TIL lines. 0] [Fig.25] shows the results of potency assay in 12 different PBMCs samples. ] [Fig.26] shows the results of potency assay in forty different TIL lines wherein (a) are the controls and (b) are the MUC4 antigens. ] [Fig. 27] shows the results of potency assay in 12 different PBMCs samples wherein (a) are the controls and (b) are the MUC4 antigens. 3] [Fig.28] graphically shows the frequency of TCRVa7.2 ⁺ in CD3 ⁺ cells in PBMCs and TIL expanded from pancreatic malignancies. 4] [Fig.29] graphically shows the frequency of MR1 -tetramer binding events, wherein (a) is the frequency of MR1 -6FP tetramer-binding events in PBMCs and TIL of patients with pancreatic malignancies, (b) is the frequency of TCRVa7.2 ⁺ events in MR1 -6FP tetramer positive cells, both in PBMCs and TIL, (c) is the frequency of MR1 -5-0P-RU tetramer-binding events in PBMCs and TIL of patients with pancreatic malignancies and (d) is the frequency of TCRVa7.2 ⁺ and CD161 ⁺ events in MR1 -5-OP-RU tetramer positive cells, both in PBMCs and TIL. 5] [Fig.30] graphically shows the immunophenotypic analysis of MAIT, wherein (A) is the frequency of CD161 in TCRVa7.2 ⁺ cells in PBMCs and TIL from patients with pancreatic malignancies, (B) is the frequency of CD26 in TCRVa7.2 ⁺ cells in PBMCs and TIL from patients with pancreatic malignancies, (C) is the frequency of CD95 in TCRVa7.2 ⁺ cells in PBMCs and TIL from patients with pancreatic malignancies, (D) is the frequency of CD103 ⁺ in TCRVa7.2 ⁺ cells in PBMCs and TIL from patients with pancreatic malignancies and (E) is the frequency of CD69 in TCRVa7.2 ⁺ cells in PBMCs and TIL from patients with pancreatic malignancies. 6] [Fig.31 ] graphically shows the production of IFN-y (pg I mL) produced from VA7.2 T-cells reacting against autologous tumor cells from five patients. Blocking T-cell reactivity with an anti-MR1 antibody shows that VA7-2 T-cells recognize the tumor target in an MR1 -restricted fashion. ] [Fig.32] graphically shows the production of IFN-y (pg I mL) of TCRVa7.2 ⁺ cells challenged with PANC-1 after downregulation of MR1 with siRNA in VA7-2 sorted TIL from five different patients with cancer. 8] [Fig. 33] graphically shows the frequencies of IFN-y, IL-17A, CD107a and Perforin in the TCRVa7.2 ⁺ cells (MAIT), CD8 ⁺ , CD4 ⁺ , DP (double- positive, i.e. CD4-CD8’) and DN (double-negative) gated in CD3 ⁺ T-cells of patients with a pancreatic malignancy stimulated for 8h with (a) bacteria and (b) bacterial supernatants (bacterial products).

[69] [Fig.34] graphically shows the frequencies of IFN-y, IL-17A, CD107a and Perforin in the TCRVa7.2 ⁺ cells (MAIT), CD8 ⁺ , CD4 ⁺ , DP and DN gated in CD3 ⁺ T-cells of patients with pancreatic malignancies stimulated for 8h with (a) bacteria and (b) bacterial supernatants (bacterial products). 0] [Fig.35] is an example of T-cell repertoire in the tumor tissue, where TCR VA/B or VG/D family is visualized in a different color and the size of the CDR3 region is associated with the relative frequency of RNA TCR reads, wherein (a) is the TCR a chain, (b) is the TCR β chain, (c) is the TCR y chain and (d) is the TCR σ chain. ] [Fig.36] is an example of T-cell repertoire in expanded TIL, where TCR VA/B or VG/D family is visualized in a different color and the size of the CDR3 region is associated with the relative frequency of RNA TCR reads, wherein (a) is the TCR a chain, (b) is the TCR β chain, (c) is the TCR y chain and (d) is the TCR σ chain. ] [Fig.37] is an example of sorted TCRVa7.2 repertoire in expanded TIL, where TCR VA/B or VG/D family is visualized in a different color and the size of the CDR3 region is associated with the relative frequency of RNA TCR reads, wherein (a) is the TCR a chain and (b) is the TCR β chain. 3] [Fig.38] is an example of T-cell repertoire in the tumor tissue, where TCR VA/B or VG/D family is visualized in a different color and the size of the CDR3 region is associated with the relative frequency of RNA TCR reads, wherein (a) is the TCR a chain, (b) is the TCR β chain, (c) is the TCR Y chain and (d) is the TCR σ chain. 4] [Fig.39] is an example of T-cell repertoire in expanded TIL, where TCR VA/B or VG/D family is visualized in a different color and the size of the CDR3 region is associated with the relative frequency of RNA TCR reads, wherein (a) is the TCR a chain, (b) is the TCR β chain, (c) is the TCR y chain and (d) is the TCR σ chain. 5] [Fig.40] is an example of sorted TCRVa7.2 repertoire in expanded TIL, where TCR VA/B or VG/D family is visualized in a different color and the size of the CDR3 region is associated with the relative frequency of RNA TCR reads, wherein (a) is the TCR a chain and (b) is the TCR β chain.

[76] [Fig.41 ] is an example of sorted MR1 -6FP tetramer-binding repertoire in expanded TIL, where TCR VA/B or VG/D family is visualized in a different color and the size of the CDR3 region is associated with the relative frequency of RNA TCR reads, wherein (a) is the TCR a chain and (b) is the TCR β chain and (c) graphically shows the production of IFN-y (pg I mL) in TIL derived from a patient with cancer in a-MR1 restricted fashion. ] [Fig.42] graphically shows the percentage of yσ ⁺ and TCRVa7.2 ⁺ cells in MR1-6FP tetramer-binding cells. 8] [Fig.43] is an example of the is an example of T-cell repertoire in expanded TIL, where TCR VA/B or VG/D family is visualized in a different color and the size of the CDR3 region is associated with the relative frequency of RNA TCR reads, wherein (a) is the TCR a chain, (b) is the TCR p chain, (c) is the TCR y chain and (d) is the TCR σ chain. 9] [Fig.44] is an example of sorted MR1 -6FP tetramer-binding repertoire in expanded TIL, where TCR VA/B or VG/D family is visualized in a different color and the size of the CDR3 region is associated with the relative frequency of RNA TCR reads, wherein (a) is the TCR a chain, (b) is the TCR p chain, (c) is the TCR y chain and (d) is the TCR σ chain. Description of the Invention 0] Hereinafter, the best mode for carrying out the present invention is described in detail. ] The present invention discloses, in a first aspect, a method of preparing and expanding a population of immune cells directed against tumor cells, tumor-precursor cells, non-tumor cells facilitating tumor transformation or cells facilitating tumor progression for cancer therapy, comprising the steps of:

(i) providing a body sample obtained from a mammal, in particular a tissue sample or a body liquid sample, comprising tumor cells, areas of non- tumor cells and immune cells or areas of tumor cells and immune cells, immune cells in close proximity to the tumor cells or distant to tumor cells or immune cells capable of killing tumor cells or controlling tumor cell proliferation, T-cells favoring or stopping cells that promote tumor transformation or tumor progression, tumor cell activity or tumor cell movement within tissues or in the organism over a longer period of time; and

(ii) culturing the body sample to expand the immune cell populations ex vivo. ] In a preferred embodiment of the present method, when the body sample is a tissue sample, such tissue is selected from tissue containing tumor cells or tissue close to cancer lesions that does not contain tumor cells, as well as healthy tissue, for instance T-cell infiltrated skin. When the body sample is a body liquid sample, such body liquid sample is selected from cerebrospinal fluid, blood or synovial, pleural effusion, bone marrow or material from the peritoneum. 3] The tissue samples can be obtained from patients who did not undergo any prior therapy or patients who underwent radiotherapy, chemotherapy, small-molecule drugs, or therapy with checkpoint inhibitors, or any combination thereof. 4] Preferably, the tissue sample is a 1-3 mm ³ piece collected from a 2-3 mm distance from the artery or vein or lymph vessel of the tumor based on surgically and clinically relevant locations where recurrences often take place or areas where cancer stem cells and immune cells are located. The tissue sample is further dissected based on its anatomical or microscopical architecture and I or based on an anti-tumorigenic or pro- tumorigenic protein or gene expression profile, including epigenetic differences and I or differences in microRNA. 5] The culturing of the body sample to expand the population of immune cells comprises three steps, namely:

- a first expansion step comprising an incubation in culture medium supplemented with the interleukins selected from IL-7, IL-15, and IL-21 and cardiolipin and optionally human serum;

- a second expansion step at day 2 following the day of tumor tissue harvest with an anti-CD3 antibody, crosslinking the T-cell receptor with or without cytokine-activated irradiated feeder cells cultured (prior to adding to the TIL culture) for 4 to 168 hours in the presence of interleukins; and

- a third expansion step at day 4 or 5 comprising an incubation in culture medium changed to IL-2, IL-7 and IL-15 supplemented with cardiolipin over the entire culture period with or without repetitively adding an anti- CD3 antibody, crosslinking the T-cell receptor with or without cytokine- activated irradiated feeder cells cultured for 4 to 168 hours in the presence of interleukins. 6] The expansion steps are performed by adding solely culture medium, or by adding no culture medium or a limited amount of culture medium depending on the concentration of the starting solution plus

- adding additional amino acids selected from essential amino acids plus acetate, wherein the amino acids are in the range of 0.001 mg/L to 1 mg/L and the acetate is in the range of 0.001 mg/L to 1 mg/L;

- adjusting the pH with sodium bicarbonate to the range of 7.3 to 7.5 and the glucose concentration between 1 .6 and 7 g/L by adding extra glucose to the medium; and

- allowing increasing lactate in the range of 1 mmol/L to 60 mmol/L. ] In a preferred embodiment of the present method, the culture medium in the first expansion step further comprises interleukins selected from IL- 7 ranging from 10 lU/mL to 6000 lU/mL, IL-15 ranging from 5 lU/mL to 1000 lU/mL, IL-21 ranging from 0.001 lU/mL to 100 lU/mL, cardiolipin ranging from 10 to 10000 nM, and human serum from 0.1 up to 10 %.8] In a preferred embodiment of the present method, the culture medium in the second expansion step further comprises an anti-CD3 antibody selected from the anti-CD3 complex, which crosslinks the T-cell receptor with or without cytokine-activated irradiated feeder cells cultured for 4 to 168 hours in the presence of interleukins. The anti-CD3 antibody ranges from 10 to 3000 ng I mL and the cytokine-activated irradiated feeder cells, when present, are ranging from 0.1 to 5 million feeder cells I well, preferably 1 million cells / well. 9] In the third expansion step, the culture medium is changed to IL-2, IL-7 and IL-15 and the interleukins are selected from IL-2 ranging from 300 lU/mL to 6000 lU/mL, IL-7 ranging from 10 lU/mL to 6000 lU/mL and IL- 15 ranging from 10 lU/mL to 1000 lU/mL and cardiolipin ranging from 10 to 10000 nM. 0] When present, the anti-CD3 antibody is selected from the anti-CD3 complex and ranges from 10 to 3000 ng I mL and the cytokine-activated irradiated feeder cells ranges from 0.1 to 5 million feeder cells I well, preferably 1 million cells I well. ] The feeder cells I well are added every 7-14 days, preferably 7-10 days, in a ratio of feeder cells to the immune cells is in the range from 1 :1 up to 400:1 , preferably in a range of 10:1 (feeder cell: T-cell) along with a crosslinking anti-CD3 directed antibody in the range of 10 to 3000 ng /mL, preferably at 30 ng/mL. ] The expanded population of immune cells directed against tumor cells are selected from the group consisting of tumor-infiltrating lymphocytes or peripheral blood mononuclear cells, preferably selected from the group consisting of double-positive (CD4 ⁺ CD8 ⁺ ) T-cells, double-negative (CD4- CD8-) T-cells, CD4 ⁺ CD8- or CD4-CD8 ⁺ T-cells, γ5 T-cells, MAIT cells, MR1 reactive T-cells, T-cells producing Th1 cytokines, T-cells expressing cytotoxic molecules or producing IL-9, a|3 T-cells, including mixed populations of tissue resident immune-cells comprising at least one of the listed immune cell subsets. 3] Preferably, the immune cells directed against tumor cells are tumor- infiltrating lymphocytes or T-cells isolated from PBMCs or skin, antibody- sorted or a recombinant classical or non-classical MHC molecule loaded with the appropriate target antigen guiding antigen-specific selection comprising, but not limited to, the CDR3 region as set for in any of SEQ ID NO 1 to SEQ ID NO 410. 4] As regards the sequence listing, TCRs sequences are shown as follows:

[95] SEQ ID NOs 1 -20, 41 -80, 111 -130, 253-272, 315-334, 355-394 list the most frequent TCRs in the tumor section from which TIL were expanded;6] SEQ ID NOs 21 -40, 131 -152, 273-292, 335-354 exhibit the most frequent TCRs recognizing autologous pancreatic cancer cells in an MHC class I (or class II) restricted fashion; ] SEQ ID NOs 81 -110 show the most frequent TCRs CD1 d restricted and tumor-reactive T-cells; 8] SEQ ID NOs 153-172 and 295-316 show the most frequent TCRs Va 7.2 enriched T-cells which recognize autologous tumor cells in an MR1 restricted fashion; 9] SEQ ID NOs 173-252 show the most frequent TCRs MR1 -6FP binding T-cells; 00] SEQ ID NOs 395-400 show the most frequent antigen specific TCRs against LRP1 B mutant (VSKRLKFSRDLSLDP); 01 ] SEQ ID NOs 401 -404 show the most frequent antigen specific TCRs against Mesothelin (HRLSEPPEDLDALPL); and 02] SEQ ID NOs 405-410 most frequent antigen specific TCRs against UQCRFS1 mutant (FVSSMSASAWLALAKIEIKLSD) restricted by DPB1 *04:02. 03] The present invention further discloses, in a second aspect, a potency assay for tumor recognition comprising the steps of:

(i) challenging the clinically relevant immune cells as set for in SEQ ID NO 517 to SEQ ID NO 611 and for SEQ ID NO 796 to SEQ ID NO 832 with at least one target antigen of human, bacterial, viral, helminth, protozoan or bacteriophage origin or their related mimicry analogues, provided that the analogue presents 50 - 100 % of similarity in amino acids or the analogue presents different amino acids, but with similarity based on size, structure, or charge and

(ii) detecting a change in a cytokine or immune effector molecule production selected from cytokines or chemokines, preferably in particular IFN-y production, cell proliferation, cytotoxicity, immune signaling and I or intracellular phosphorylation of signaling molecules associated with an anti-tumor immune response. 04] Preferably, the target antigen is a wild-type, a mutated private or commonly shared tumor-associated antigen, an antigen that is preferentially expressed during embryonal or fetal development and/or specifically presented by tumor cells or non-tumor cells supporting tumor- cells or driving tumorigenesis. The mutation is selected from point mutations, frameshift mutations, or antigens preferentially expressed during fetal I embryonal development; the point mutation residing preferably in the middle of the antigen. 05] Preferably, the target antigen comprises a length of 7 to 25 amino acids. More preferably, the target antigen is a 15-mer peptide or binding to MHC class II, or between 8-11 amino acids, preferably 9 amino acids, preferably binding to MHC class I target antigens or antigens presented by CD1 molecules or MR1 molecules. The antigen may undergo pre- or post-translational modification concerning sugar or lipid moieties. 06] In a preferred embodiment of the present invention, the private tumor- associated antigen further comprises a sequence as set for in any of SEQ ID NO 556 to SEQ ID NO 611 and the commonly shared tumor- associated antigen further comprises a sequence as set for in any of SEQ ID NO 517 to SEQ ID NO 555 and SEQ ID NO 796 to SEQ ID NO 832. 07] In a particular embodiment of the present invention, the private tumor- associated targets are obtained from

- providing a body sample from a patient, in particular a tumor sample;

- performing whole tumor exome sequencing as compared to a non-tumor sample from the same patient;

- performing RNA or DNA sequencing of the tumor sample;

- preparing a construct with the mutation in the middle of the peptide sequence flanked by 7 amino acids to the left and to the right of the mutant amino acid residue;

- constructing a synthetic peptide containing at least 12 to 17 amino acids of the identified frameshift mutation; - tailoring peptides to best fit according to the patient’s individual MHC class I and MHC class II genetic background;

- blocking classical MHC molecules, selected from MHC class I and MHC class II proteins, or non — classical MHC molecules, selected from MR1 or CD1 a-d, preferably CD1 d using appropriate blocking antibodies or using siRNA;

- analyzing the tumor sample to identify a specific T-cell receptor reaction with a wild-type or mutant target from a tumor-associated antigen provided from a cellular protein or a mitochondrial target;

- identifying a mutated target antigen or a series of mutated target antigens in the tumor sample, provided that the mutated target antigen(s) does not elicit pro-tumorigenic or immune-suppressive functions;

- producing synthetic peptides with similar amino acid composition and I or a similar 3D structure with the mutated target antigen in lieu of the tumor cells or other target antigen presenting cells; wherein the specific T-cell receptor is T-cell receptor a|3, T-cell receptor yb or T-cell receptors expressed by MAIT-cells or MR1 reactive T-cells. 08] Preferably, the specific T-cell receptor is a T-cell receptor as set for in any of SEQ ID NO 1 to SEQ ID NO 410 and the reaction between the specific T-cell receptor and the target from the tumor-associated antigen give rise to immune effector functions in responding T-cells that are anti- tumor directed or - more specifically, directed to tumor-stem cells, based on cytotoxicity, proliferation, apoptosis-inducing molecules, or the quality and quantity of cytokine-mediated tumor cell death, inhibition of tumor cell proliferation, induction of differentiation or aging of the tumor cells. 09] In a third aspect, the present invention further discloses a biological vaccine preparation to provide anti-tumor response or antiviral response, wherein it comprises target antigens that lead to the expansion of a certain set of T-cells or B-cells. 0] Additionally, the target antigens that show cross-reactivity to human self-proteins and antigenic structures that induce factors that are pro- tumorigenic and I or induce autoimmune responses are removed from the preparation, based on antigen-specific immune reactivity, e.g., cytokine production, using IFN-y, IL-17 or Th2- based cytokines as the signature immune readouts or their related RNA signatures associated with Th1 I Th2 I Th17 or Th9 responses. 1] The target antigens are private or commonly shared tumor-associated targets specifically presented by tumor cells or non-tumor cells supporting tumor-cells or driving tumorigenesis by non-transformed cells that support malignant transformation, or support transformed cells. 2] Preferably, the private or commonly shared tumor-associated target is selected from wild-type or mutant, not excluding fusion proteins or frameshift mutations, 15-mer peptides or binding to or binding to MHC class II, or between 8-11 amino acids, preferably 9 amino acids, preferably binding to MHC class I target antigens or antigens presented by CD1 molecules or MR1 molecules. The private or commonly shared tumor-associated target can also be selected from wild-type or dysfunctional or damaged mitochondrial-associated molecules that act as tumor-associated targets in humans. The antigens may be glycosylated, phosphorylated or lactylated. The tumor-associated antigens may be differentially expressed during fetal or embryonal development or not readily accessible post-partum due to limited access of immune cells to the nominal target antigen. 3] Additionally, the private tumor-associated target is as set for in any of SEQ ID NO 556 to SEQ ID NO 611 and the commonly shared tumor- associated target is as set for in any of SEQ ID NO 517 to SEQ ID NO 555 and SEQ ID NO 796 to SEQ ID NO 832. For both cases, the tumor- associated target may also be an amino acid sequence presenting up to 70% or more in amino acid exchanges, provided that the individual amino acids comprise a similar chemically 3D structure or an amino acid sequence presenting different amino acids, but with similarity based on size, structure or charge, or an amino acid sequence which is part of a chimeric antigen receptor construct. 4] Preferably, the specific T-cell receptor is a T-cell receptor as set for in any of SEQ ID NO 411 to SEQ ID NO 516 to the target antigens as set for in any of SEQ ID NO 612 to SEQ ID NO 795, wherein: 5] SEQ ID NOs 411 -442 show the most frequent antigen specific TCRs against LP(V)RDLPQGF from SARS-CoV-2 spike (positions 212-220 QHD43416.1 ) tailored for HLA-B3501 6] SEQ ID NOs 443-474 show the most frequent antigen specific TCRs against LP(V)RDLVTGF from Human U11/U12 snRNP 35 kDa protein (positions 81 -90 Q16560.1 ) tailored for HLA-B3501 ; 7] SEQ ID NOs 475-478 show the most frequent antigen specific TCRs against LP(D)SKVGGNY from SARS-CoV-2 spike (positions 440-449 QHD43416.1 ) tailored for HLA-B3501 ; 8] SEQ ID NOs 479-484 show the most frequent antigen specific TCRs against LR(D)SKVGGNY from SARS-CoV-2 spike (positions 440-449 QHD43416.1 ) tailored for HLA-C0701 ; 9] SEQ ID NOs 485-496 show the most frequent antigen specific TCRs against LR(K)SKHGGNY from Human Tensin-1 (positions 47-55 Q9HBL0.2) tailored for HLA-C0701 ; 0] SEQ ID NOs 497-504 show the most frequent antigen specific TCRs against LR(V)RDLPQGF from SARS-CoV-2 spike (positions 212-220 QHD43416.1 ) tailored for HLA-C0701 ; 1] SEQ ID NOs 505-512 show the most frequent antigen specific TCRs against LVQDLAQGF from Tryptophan--tRNA ligase, mitochondrial (positions 188-196 Q9UGM6.1 ) tailored for HLA-C0701 ; and 2] SEQ ID NOs 513-516 show the most frequent antigen specific TCRs against LR(V)RDLVTGF from Human U11/1112 snRNP 35 kDa protein (positions 81 -90 Q16560.1 ) tailored for HLA-C0701. 3] In a fourth aspect, the present invention discloses epitope target for antibodies that serves as a viable target that can be used to construct chimeric antigen receptors.

[124] More specifically, the epitope targets for antibodies are directed against PVTSLSSVSTGDTTP from MUC4 or parts of said epitope, preferably 3-5 or 3-6, 3-7, or 3-8 amino acids or amino acids of a similar size and charge, resulting in a similar 3D structure binding to the epitope from MUC4, which can be used to construct chimeric antigen receptors targeting MUC4.

[125] The MUC4 peptide is targeted by human IgG in serum from patients with cancer. This epitope is present in different areas of MUC4 and exhibits therefore multiple docking sites for an anti-MUC4 directed antibody response or as a target for chimeric antigen receptors, since it is targeted by naturally occurring antibody responses in patients with cancer.

Examples

[126] Hereinafter, reference is made to the examples of the present invention, which aim to describe it in more details as regards tests that have been conducted and experimental results that have been achieved.

[127] Unless otherwise indicated, all technical and scientific terms used in this document have the same meaning as commonly understood by someone skilled in the art to which this invention belongs.

[128] Methods and materials are described in this document for use in the present invention; other suitable methods and materials known in the art can also be used. The materials, methods and examples are illustrative only and are not intended to be limiting.

[129] Body samples: tissue

[130] Tumor specimens were removed from primary or metastatic tumor lesions (see Figure 8) listed in the Table 1 below. The tumor specimen was removed and a wedge from the tumor and parallel sections were performed by the pathologist.

[131] Table 1 based on anatomical areas and tissue ‘stiffness’, defined by desmoplastic region, i.e. , at least 2-3 mm from arterial vessels or from infiltration that is more visible upon bathing the piece in cold PBS (Figures 1 to 3), and /or based on differential gene expression and consequent cytokine expression in the tumor and adjacent tissue, for example IL-7 or IL-17. (Figures 4 and 5). 33] Each tumor area was dissected into small tumor pieces and placed in individual 24 well plates with 3-5 pieces I well of 1 mm ³ in 1 mL of CellGenix GMP DC media, or alternatively Lonza X VIVO 15 media supplemented with 10% Human Serum and 1000 lU/mL IL-7, 150 lU/mL IL-15 and 1 lU/mL IL21. Serum free medium can also be used or a range of serum between 1 - 10%. 34] Expansion of TIL 35] Outgrowth of TIL was differently associated with the different TIL infiltrates that had great impact on T-cell activation, exhaustion, composition, and maturation markers, as can be seen on Tables 2 and 3 below. ble 2 - Different TIL populations from different tumor regions (D3290).

36] As can be seen in Table 2, TIL from tumor D3290 were expanded using identical conditions and flow cytometric analysis has been performed. Different phenotypes in TIL were observed, when using different tumor regions in the three-expansion step herein proposed.37] CD4 ⁺ TIL and CD8 ⁺ TIL showed increased frequency in Zone 1 . A CD39’CD69’phenotype in the TIL product has been identified with increased responsiveness to therapy. Central memory T-cells (TCM) defined by the markers CD45RA CCR7 ⁺ are also associated with increased responsiveness in the TIL product. 38] It is also of note that Zone 2 has - independent of the CD4 ⁺ or CD8 ⁺ components - an increased frequency of this T-cell subpopulation. In contrast, the frequency of TEM (effector memory T-cells) is increased in TIL from Zone 1 . T-cell activation marker 4-1 BB has been associated with T-cell activation as well as with a T-cell population enriched in antigen- specific T-cells, which is higher in TIL from Zone 2 independent of the CD4 ⁺ or CD8 ⁺ T-cell phenotype. 39] It is also of note that CD4 ⁺ CD8 ⁺ (double-positive) CD3 ⁺ TIL represent highly activated CD4 ⁺ T-cells that express the CD8a chain; CD4 CD8’ (double-negative T-cells) represent here highly activated T-cells that have downregulated the CD4 ⁺ or CD8 ⁺ co-receptor. Rarely they represent thymic emigrants that are independent of CD4 ⁺ or CD8 ⁺ co-receptor help. 40] Phenotypic analysis of TIL from different regions represents biologically relevant differences in T-cell function, activation, tissue access, maturation, and differentiation - as well as markers that have been linked of the TIL phenotype to clinical responsiveness. 41] Table 3 - Differential cytokine production in TIL associated with distinct tumor areas

42] In table 3, TIL were seated in at 10e ⁵ cells I well in triplicates in medium containing IL-2 I IL-7 I IL-15 + cardiolipin (medium) or in IL-21 IL-7 / IL-15 + cardiolipin plus anti-CD3-lgG1 at 30 ng/mL to crosslink the T-cell receptor. 43] Supernatants were harvested after 24 hours in order to test for maximal T-cell activation defined in 24 hrs. 1 10e ⁵ cells for comparison. The delta between maximal stimulation (b) and constitutive cytokine production (A) is summarized in row “delta (B-A)”. 44] To compare the effect of cardiolipin in regard to cytokine production, the culture wells were supplemented with cardiolipin at different concentrations (0, 50, 150, 250, 350 and 450 nM) in culture wells with medium alone, positive controls, as well as commonly recognized target antigens, e.g., CMV, EBNA3, Flu-M1 , ESAT-6, hemagglutinin (Figure 6). 45] T-cell expansion medium was supplemented with 250 nM of cardiolipin, a concentration that showed favorable production of IFN-y in PBMCs from healthy individuals stimulated with OKT3 (30 ng/mL) and cardiolipin. 46] PBMCs of healthy individuals expanded in the presence of cardiolipin without interleukins increased the frequency of γ5 T-cells (Table 4). 47] Table 4 - Percentage of γ5 T-cells in healthy individuals PBMCs in the presence or absence of cardiolipin. 48] TIL cultures were initiated by placing tumor pieces in individual 24 well culture vessels and fed as needed. The IL-71 IL-15 / IL-21 cytokine mix was used to test whether cardiolipin would be able to (i) increase the frequency of TCR γ5 T-cells, (ii) induce changes in T-cell phenotype or function and (iii) increase tumor antigen recognition defined by IFN-y production. 49] Except for testing the effect of cardiolipin, TIL were expanded using the following protocol: tumor pieces were cultured for 4 days with IL-7 (1000 lU/mL), 15 (150 lU/mL) and IL-21 (1 lU/mL), supplemented with Cardiolipin (step 1 ), followed by adding anti-CD3 (30ng/mL) and irradiated (with 40 Gy) allogeneic feeder cells at 10e6 cells/well (in a 24 well culture dish) from 3 donors at day 2 (step 2) and the medium was changed after 3-5 days to IL-2 (1000 lU/mL), IL-7 (1000 lU/mL) and IL-15 (150 IU /mL), also supplemented with Cardiolipin (step 3). Feeder cells were added for expansion every week starting tumor pieces were carefully removed around day 5-10 days after culture initiation. Cells were split as needed and expanded for individual patient TIL up to 1x 10e ⁹ TIL for testing. Cardiolipin was present during the entire culture period at 250 nM. 50] Peptide recognition assays. 51] Peptides from tumor-associated antigens (TAAs), listed in Table 5 were placed at 1 ug/well in duplicates using anti-CD3 at 30ng/mL as well as PHA at 5 pg/mL as positive controls. 52] Table 5

53] TIL were seeded at 10 ⁴ / well in medium with 10% human serum added 24h later and reduced cytokine concentrations (IL-2 at 100 lU/mL, IL-15 at 100 lU/mL). Supernatants were tested for IFN-y production with the Human IFN-y ELISA basic kit (HRP) (Mabtech, 3420-1 H-6) according to the manufacturer’s instructions. The assay can also be performed using IL-7 at 100 lU /mL and 100 IU IL-15 /mL). 54] Testing of the NRLP3 pathway 55] Cardiolipin has been shown to mediate its effects via the NRLP3 pathway and in order to test this hypothesis, TIL were expanded in the presence of IL-7, IL-15, and IL-21 (1000 lU/mL, 150 lU/mL, 1 lU/mL) in CellGenix GMP DC media supplemented with 10% serum in the presence or absence of the NRLP3 pathway inhibitor MCC950 (10uM/mL) throughout the culture. 56] TIL were expanded as outlined above and tested for recognition of a panel of 26 KRAS peptides (listed in Table 6 below). The number of peptide targets recognized as well as the amount of IFN-y in pg/10e ⁴ TIL/target/7 days was analyzed and compared in the cardiolipin-positive versus negative group testing with Human IFN-y ELISA basic kit (HRP). 57] Table 6 58] Blocking antibodies 59] MHC class I responses were blocked using mAb clone w6/32 (mouse lgG2a) at 10 pg/mL, anti-HLA-DR clone L243 (mouse lgG2a) at 10 pg/mL, anti-HLA-DP clone B7/21 (mouse lgG3) at 10 ug/mL, anti-CD1d clone CD1d42 (mouse lgG1 ) at 10 pg/mL. Isotype control antibodies included mouse lgG2a and mouse lgG1 at 10 pg/mL. MR1 responses were blocked using the mAb clone 26.5 (mouse lgG2a) at 10 pg/mL. All antibodies are commercially available. 60] siRNA 61] siRNA against MR1 was used to silence MR1 expression using Silencer Select Pre-designed siRNA (Ambion, Thermo Fisher)). A scramble siRNA was used as negative control (Ambion, Thermo Fisher). Briefly, target cells were seeded 24h before transfection, both siRNAs were used at 10 pM with lipofectamine RNA iMAX diluted in OptiMEM, incubated for 5 minutes, and added on top of the cells. Cells were incubated for 48h before being used on the respective assays. 62] yb T-cell isolation 63] yb T-cells were negatively sorted using a commercial kit from Stem Cell. Purity of negatively sorted cells was tested by flow cytometry using the DURAclone panel IM TCRs. 64] Cellular recognition assay 65] Autologous tumor, confirmed by standard histology, was viably snap frozen and then thawed to be used as targets in recognition assays once TIL had been expanded. Small autologous tumor pieces (1 mm ³ ) from tumor areas that contained tumor cells were tested in duplicates. TIL were incubated with tumor pieces or allogeneic tumor cells in CellGenix GMP DC media supplemented with 10% serum in the presence of IL-2 (50 lU/mL) and IL-15 (10 lU/mL) for testing MAIT -cells. TCR ap T-cells or TCR yb cells were tested for autologous or allogeneic tumor recognition using 100 IU IL-2 /mL and 100 IU IL-15 /mL; or using 100 IU IL-7/mL combined with 100 IU IL-15 /mL. The allogeneic tumor cell line PANC-1 obtained from ATCC (CRL-1469) derived from pancreatic duct epithelioid carcinoma, was maintained in DMEM high glucose, GlutaMAX Supplement, and served as a target for sorted yb T-cells or Va7.2 sorted (MAIT) T-cells. 66] Mitochondrial isolation 67] Mitochondria were isolated from PBMCs from healthy blood donors provided by the National Blood Bank Lisbon, Portugal, and were isolated using the commercial isolation kit Mitochondria Isolation Kit for culture cells (Thermo Scientific). Mitochondrial isolation was confirmed by Western Blot using the commercial antibody TOM20 (clone EPR15581 - 54) (Figure 7) detecting an antigen-specific band at 16 kDa.

[168] Mitochondria were isolated from PBMCs from healthy donors cultured for 48h at 2x10 ⁶ /mL without cytokines or cultured with doxorubicin 1 uM for 12h, which induces mitochondrial abnormalities. PANC-1 cells were used as a source for mitochondria using the commercial isolation kit Mitochondria Isolation Kit for culture cells (Thermo Scientific).

Mitochondria were also isolated from PANC1 cells treated with or without leflunomide (that induces mitochondrial fusion) at 50 pM for 48h at ng I mL or rapamycin (10 nM for 12h). 69] Mitochondrial presentation assays. 0] For APC differentiation in vitro, PBMCs (from the patients whose isolated TCR yσ ⁺ T-cells were tested) were obtained from heparinized blood and plated onto flat bottom 96-well culture plates in the presence of serum-free RPMI medium for 2h at 37°C and 5% CO2. Then, non- adherent cells were washed with PBS and adherent cells were cultured in RPMI with 5% Human serum and cytokines GM-CSF (800 ILI/mL) and IL- 4 (800 ILI/mL) for an additional 5 days. 1] APCs (antigen presenting cells) were exposed to different mitochondria solutions (100 pg/mL) overnight and the next day, yσ ⁺ T-cells were seeded on APCs exposed to mitochondria at 10 ⁴ cells/well, blocking antibodies were added (e.g., anti-CD1 d or isotype controls) and supernatants were tested for IFN-y production. 2] Whole Exome Sequencing pipeline 3] Exome sequencing data was aligned using the Burrows-Wheeler Aligner to human genome build hg19. Duplicates were marked using Picard’s MarkDuplicates tool and base recalibration was performed using GATK. Once the data was recalibrated, samtools was used to create the tumor tissue and, for comparison, the non-transformed tissue pileup files. 4] Four different tools were used to call mutations: Mutect2, Varscan2, Strelka2 and Lancet. A consensus VCF file containing mutations called from at least two tools is then annotated using the Ensembl Variant Effect Predictor. Candidate neoantigens were identified using pVACtools, wherein two types of peptides are predicted: peptides with 15 residues where the alteration is centered or the full downstream protein sequence in case of a frameshift, that are used for immunoassays, and peptides of different lengths tailored for the MHC typing of the patient that are candidates for PCV. 5] The entire set of tumor mutations for the TIL from patients D1309 and D1 313 are listed in the Table 7 below. Peptides selected for TIL recognition were selected based on (i) driver mutations, (ii) frequency of reads I million reads, (iii) frameshift mutations, (iv) binding to MHC class I - or class II molecules and (v) the possibility to synthetically produce the peptides. 6] Table 7 7] RNA extraction and sequencing 8] RNA from TIL was isolated using the commercially available RNeasy Mini Kit (Qiagen) and sequencing was done using the Illumina NovaSeq 6000 system with 100 bp paired end read lengths. Adapters were removed. 9] Transcriptome sequencing data alignment was performed using STAR two-pass method to human genome build hg19. Duplicates were marked using the Picard’s MarkDuplicates tool and reads were split using the GATK SplitNCigarReads tool and variants were called using Varscan2. STAR-Fusion was used to identify candidate fusion transcripts and its output was annotated using AGFusion. 80] TCR library preparation and sequencing 81] The NGS libraries covering human TCRa, TCR[3, TCRy and TCR5 CDR3 regions were prepared using the commercially available iR- RepSeq-plus 7-Chain Cassette. Briefly, 1 pg of total RNA was loaded into the disposable cassette which contains all necessary reagents for library amplification, purification and processed by iR-Processor (iRepertoire).82] All four chains were amplified in a single assay using a strategy which allows the incorporation of unique molecular identifiers (UMIs) during the reverse transcription (RT) step. The final constructed library includes Illumina dual index sequencing adapters, a 10-nucleotide UMIs, and an 8- nucleotide internal barcode associated with the C-gene primer. 83] After quality control, amplified libraries were multiplexed and pooled for sequencing on the Illumina NextSeq platform with a 300-cycle kit (300 single-end reads). The samples were analyzed using iRepertoire’s proprietary YPL5-UMI bioinformatics pipeline which condenses the sequences by UMI, removes sequencing artifacts, and identifies CDR3 regions by mapping the reads to reference sequences. 84] Flow cytometric analysis 85] TIL and PBMCs were analyzed using several DuraClone (Beckman Coulter) Panels, always with addition of the LIVE/DEAD™ Fixable Yellow Dead Cell Stain Kit: DuraClone IM T-cell Subsets tube adding LAG-3 BV650 and CD95 BV785 and Duraclone IM TCRs tube. Besides, another panel with commercially available antibodies were used, which included CD4 Pacific Blue, CD8 KromeOrange, CXCR3 BV650, CD95 BV785, CD103 FITC, CCR7 PE, CCR4 PE/Dazzle, TCR pan-y/5 PC5.5, CCR6 PC-7, CCR9 Alexa Fluor 647, CD45RA AF700, CD3 APC-A750 and LIVE/DEAD™ Fixable Yellow Dead Cell Stain Kit. After 15 minutes incubation, T-cells and PBMCs were washed in PBS-2% FBS and acquired using a CytoFlex LX flow cytometer from Beckman Coulter. Analysis was performed using FlowJo software. 86] Tree detection assay 87] Briefly, cells were stained extracellularly with CD3 PE (clone UCHT-1 , BD 555749), CD4 V450 (clone RPA-T4, BD 561838), CD8 APC-Cy7 (clone SK1 , BD 557834), CD25 PE-Cy7 (clone 2A3, BD 335824), CD127 APC (clone R34.34, Beckman Coulter B42026) for 15 minutes in ice and then washed and permeabilized using Biolegend’s True-Nuclear Transcription Factor Buffer Set according to manufacturer’s instructions. 88] Cells were then stained intracellularly with F0XP3 Alexa488 antibody (clone 259D/C7, BD 560047) or an lgG1 isotype control Alexa 488 (BD 557702), and CD3 PE (clone UCHT-1 , BD 555749). Tregs cells were identified by flow cytometry as CD3 ⁺ CD4 ⁺ CD25 ^high CD127’FoxP3 ⁺ ). 89] CD107a assay 90] Briefly, positive control cells were stimulated with 1 pg/mL of PMA; non-stimulated cells served as the control and data were performed in triplicates. Both stimulated and non-stimulated cells were incubated with a protein transport inhibitor containing monensin (BD, 51 -2092kz) and CD107a PE antibody (clone H4A3, BD 555801 ) for 2 hours. Then an extracellular staining was performed using CD3 PE-Cy7 (clone UCHT1 , BD 563423), CD4 V450 (clone RPA-T4, BD 561838) and CD8 APC-Cy7 (clone SK1 , BD 557834). Control cells are PBMCs that had been cultured for 48-72 hours in the presence of 300 ILI/mL IL-2 and 50 ILI/mL IL-15. The assay is considered as positive if the positive control cells (PMA stimulated) have at least 2x the SD of the triplicates of the negative control. 91] IFN-y induction assay 92] This assay is used to measure the IFN-y production after anti-CD3 induced activation during 24h in 10 ⁵ cells. Briefly, TIL are seeded in triplicates (10 ⁵ cells/wells) in TIL medium supplemented with 10% Human Serum and 1000 lU/mL IL-2, IL-7 1000 lU/mL and 150 lU/mL IL-15 (i.e. the 3 ^rd step of the TIL expansion protocol) in the presence of an anti-CD3 crosslinking antibody 30ng/mL (for the positive control wells. There are also 3 wells seeded with medium and no cells as an additional control for IFN-y that may be present in the human serum as a part of the cell culture medium. After 24h incubation at 37°C and 5% CO2, supernatants are harvested and an IFN-y ELISA (Human IFN-y ELISA basic kit (HRP) (Mabtech, 3420-1 H-6)) is run according to manufacturer’s instructions.

[ 93] Western-Blot assay

[194] A Western-Blot assay was performed to assess the quality of the mitochondria extraction from PANC-1 and healthy PBMCs using a monoclonal antibody against the mitochondrial specific protein TOMM20. Briefly, mitochondrial protein extracts were first measured to assess their concentration by a Bradford assay and equal amounts of protein for each condition were lysed using RIPA buffer. The protein extracts were added to an SDS-PAGE gel and run at a constant voltage of 100V for 1 hour. Then the protein extracts were transferred from the gel to a membrane and blocked in PBS-5% non-fat milk. Then the membrane was incubated with primary antibody rabbit anti-human TOMM-20 (clone EPR15581 -54, ab186735, abeam) diluted in PBS-5% non-fat milk overnight at 4°C, washed three times in TBS-Tween and incubated for 1 hour in secondary antibody anti-rabbit at room temperature. Finally, the membrane was washed three times and developed using the kit Pierce™ ECL Plus Western Blotting Substrate. 95] Statistical analysis 96] The statistical analysis was performed for antigen recognition only and used a t-student test with software Prism 7. Statistical significance was considered with p value minor than 0.05 to detect differences in tumor- associated target recognition by TIL. 97] Va7.2 ⁺ or MR1 -reactive T-cell analysis in TIL 98] TIL were washed with PBS (Coming Life Sciences, New York, USA) and incubated for 15 min with antibodies at room temperature (except the MR1 -tetramers that required 40 minutes of incubation, then the cells were washed, and the second layer of antibodies was added for incubation). The MR1 tetramer technology was developed jointly by Dr. James McCluskey, Dr. Jamie Rossjohn, and Dr. David Fairlie, and the material was produced by the NIH Tetramer Core Facility as permitted to be distributed by the University of Melbourne. 99] After the staining, cells were washed with PBS (Coming Life Sciences, New York, USA), resuspended in 200 pL of PBS (Coming Life Sciences, New York, USA) and analyzed by flow cytometry (CytoFLEX, Beckman Coulter, Brea, CA, USA). Data analysis was performed using FlowJo software version 10.7.1. In detail, MR1 -6FP and MR1 -5-OP-RU tetramers were kindly provided by the NIH facility. 00] a-TCRVa7.2 (clone 3C10), a-CCR7 (clone G043H7), a-CD69 (clone FN50), a-CD161 (clone HP-3G10), a-CD3 (clone UCHT1 ), a-CD26 (clone BA5b), and a-CD95 (clone DX2) are from Biolegend, CA, USA; a- CD45RA (clone 2H4) is from BD Biosciences, CA, USA; a-CD161 (clone HP-3G10) is also from ExBIO, Vestec, Czech Republic; and a-CD4 (clone 13B8.2), a-CD8 (clone B9.11 ), a-CD103 (clone 2G5) are from Beckman Coulter, Brea, Ca, USA.MAIT were isolated with the immunomagnetic EasySep™ Release Human PE Positive Selection Kit (STEMCELL™ Tecnologies), using a-TCRVa7.2 PE-conjugated antibody (clone 3C10, Biolegend, CA, USA). The sorting comprises FcR blocking to prevent unwanted binding of antibodies and staining with the PE antibody. MAITs are labelled with a-TCRVa7.2 antibody followed by incubation with PE- binding magnetic beads. The desired cells are then magnetically separated and once removed from the magnet, are able to be collected from the tube. A release buffer is used to then remove the PE-binding beads. 01] After calculating cell concentration, MAIT underwent functional assays, i.e. , recognition of autologous tumor cells and blocking with anti-MR1 specific monoclonal antibodies as well as appropriate control antibodies. The allogeneic tumor cell line PANC-1 was also tested a s target and MR1 restriction was shown using MR1 -specific siRNA as well as scrambled RNA. 02] Bacterial recognition by MAIT 03] Five strains of bacteria were grown- Saccharopolyspora rectivirgula (strain 683 [DSM 43747 INMI 683, VKM-A-810] ATCC), Bacillus clausii (strain DSM 8716 [C360, NCIB 1039, PN-23] ATCC), Pseudoxanthomonas mexicana (strain AMX 26B ATCC), Streptomyces mycarofaciens (strain SF-837 ATCC) and Enterococcus faecalis (strain AGR 329 ATCC) - in 50 mL tubes Bacteria concentration was evaluated using a Bacteria Counting Kit (Invitrogen, Thermo Fisher Scientifics, MA, USA), which enumerates bacteria by flow cytometry using a calibrated suspension of microspheres beads and a nucleic acid that penetrates bacteria (both Gram-negative and positive). 04] The staining of beads plus bacteria are read in the green fluorescence channel and distinguished on a plot of forward scatter versus FITC. The density of the bacteria in the sample was calculated from the ratio of bacterial signals to microsphere signals. TIL were exposed to the different bacterial species which were reported to be associated with increased survival in patients with pancreatic cancer. Either the (washed) bacterial directly or the bacterial supernatants were used to stimulate TIL, supernatants were then harvested and tested for cytokine production by ELISA. 05] Results 06] As previously disclosed, TIL from sixteen patients (listed in Table 1 ) with various amounts of yσ ⁺ TIL infiltrations (see Figure 8) were expanded in the presence of IL-7, IL-15, and IL-21 and in the presence or absence of cardiolipin. 07] Differences were found in the frequency of γ5 TIL (Figure 9) which showed to be increased in TIL expanded in cardiolipin. However, no differences regarding IFN-y production, IL-17 production, nor CD107 induction or regulatory T-cells (Treg) in TIL, without or without cardiolipin, were observed (Figure 10). Differences regarding T-cell maturation, activation, or exhaustion markers in CD4 ⁺ , CD8 ⁺ , double-positive (CD4 ⁺ CD8 ⁺ ), double-negative (CD4’CD8’) or yσ ⁺ TIL (Figure 11 ) were not observed. 08] Using the identical 3 step expansion process (using cardiolipin, IL-7 I IL-15 / IL-21 followed by IL-2 I IL-7 I IL-15 and anti-CD3 stimulation) in peripheral blood mononuclear cells (PBMCs) exhibited that the method does not drive T-cell into terminal differentiation. 09] It can be observed an decrease in precursor cells, yet also an increase in central memory and effector memory T-cells and a reduction in frequency of terminally differentiated T-cells (Figure 12), which makes the expansion method suitable to activate and expand peripheral T-cells as recipients for transgenic immune recognition receptors, e.g. T-cell receptor or antibodies (chimeric antigen receptor technology) using different genetic transfer methods, for instance mRNA, CRISPR- platforms, retroviral or lentiviral vectors, not excluding alternative genetic delivery platforms. 0] Mutant tumor antigen-specific T-cells could also be expanded using the 3-step expansion method from (tumor cells) free healthy skin that exhibited T-cell infiltration microscopically and clinically as erythema I papule-like lesions. In contrast, PBMCs harvested at the same day did not yield tumor antigen specific T-cells demonstrating that i) the anatomical location as well as ii) the expansion method allowed for tumor - antigen specific T-cells that could give as a source for cell-therapy or antigen-specific -T-cell receptors, as can be seen in Table 8 below. 1] Table 8 - Mutant tumor antigen specific T-cells expanded from skin lesions from patients with solid cancer - 2] TIL expanded in the presence or absence of cardiolipin were tested for recognition of commonly shared tumor-associated antigens (e.g., mesothelin, KRAS listed in the Table 5), for the amount of IFN-y production directed against each individual target. TIL expanded in cardiolipin recognized a high number of KRAS as well as mesothelin targets, which suggests that cardiolipin facilitates expansion of antigen- specific T-cells (Figure 13). 3] Since cardiolipin has been reported to activate the NRLP3 inflammasome, aliquots of these same TIL preparation were expanded and the absence or presence of the NRLP3 inhibitor (MCC950) was investigated. The preparation was tested against a panel of wild-type and mutant KRAS epitopes (listed in the Table 6) to gauge different peptide- specific T-cell responses (Figure 14) that showed an increased number of epitopes recognized and stronger IFN-y production, suggesting the inhibition of the NRLP3 pathway statistically reduces the function of cardiolipin. 4] RNA sequencing was performed in seven of TIL expansions (i.e. from seven individual patients) with or without cardiolipin and tested for differential gene expression (Figure 15). Genes with extremely low counts across samples were removed because they provide little evidence for differential expression and may interfere with some of the statistical approximations used. 5] DESeq2 was used with a False Discovery Rate <0.10 as a cut-off value to estimate differential expression between the TIL grown with and without cardiolipin, two genes were found to be over-expressed in TIL expanded with cardiolipin, CXCL9 and CXCL10, and the gene expression of KBTBD11 (a gene that influences the nuclear factor of activated T cell cytoplasmic-1 (NFATcl ) pathway) was statistically reduced in TIL expanded with cardiolipin. 6] Both CXCL9 and CXCL10 are cytokines that belong to the intercrine alpha family and bind to CXCR3. They are a chemotactic factor for activated T-cells that affects the growth, movement, or activation state of cells that participate in immune and inflammatory response. CXCL9 and CSCL10 induce chemotaxis and facilitate extravasation of immune cells thereby enabling T-cells to enter tissues, a positive feature in the biology of tumor - infiltrating lymphocytes). The raw counts for individual RNA transcript were also normalized using transcripts per million (TPM) and heatmaps focusing on gene signatures and markers strongly associated with immune responses were built (Figure 16). 7] Next, TIL expansion from patients with various amounts and spatial presence of γ5 T-cells were tested in the presence of cardiolipin for recognition of autologous tumor cells by TCR aβ and γ5 TIL. TIL were expanded, for the first 3-5 days, with IL-7, IL-15, and IL-21 , followed by a switch to IL2, IL-7 and IL-15. γ5 TIL were then negatively sorted and γ5 TIL purity was shown to be in the range of 98% (Figure 16). Unsorted TIL or TCR aβ TIL, depleted of γ5 TIL, recognized the autologous tumors (Figure 17). 8] 9 TIL were tested for recognition of autologous tumor cells, wherein 4/9 were HLA-DR restricted, 3/9 were HLA-A/B/C restricted, 1/9 was both HLA-DR and HLA-A/B/C restricted and 1/9 showed CD1d restriction (a teratoma) shown by blocking with specific antibodies. Pure sorted γ5 TIL (5/5) recognized the autologous tumor and were shown to be blocked with anti-CD1 d, yet not with control antibodies. TCR analysis was performed to gauge the presence of TCR a|3 in MHC class I or MHC-class II, as well as in sorted TCR GD-reactive CD1 d T-cells (Figure 18, 19, 20 and 21 ). 9] To identify the yd and aβ TIL recognition, further experiments were conducted. 0] Autologous IL-4/GM-CSF antigen presenting cells were differentiated from monocytes prepared from PBMCs in 4/4 patients and were pulsed with different mitochondrial preparations (Figure 22). Mitochondria isolated from healthy, non-manipulated PBMCs did not yield APC recognition defined by IFN-y production. In contrast, 2/4 TIL recognized mitochondria prepared from PBMCs exposed to doxorubicin (which damages mitochondria) pretreated PBMCs, the same was also found to be true for mitochondria prepared from the pancreatic cancer cell line PANC-1 , showing that cardiolipin expanded TCR GD ⁺ TIL recognized both damaged mitochondria and mitochondria obtained from tumor cells but not mitochondria from healthy cells. 1] Next, 7 TCR yb TIL lines were examined and 5/7 yb ⁺ TIL recognized PANC-1 cells in a CD1 d restricted fashion shown by IFN-y production and blocking with specific antibodies, yet not with the appropriate controls. PANC-1 recognition was not changed by pretreatment of PANC-1 cells with doxorubicin (which damages further mitochondria), nor with Leflunomide (that leads to fusion of mitochondria) or rapamycin (that interferes with the antigen processing and presentation pathway) (Figure 23). 2] In order to examine more specificity in TIL from patient D1309 and D1313, synthetic peptides were tested for TIL recognition defined by IFN- y production. Commonly shared tumor-associated antigens (listed in Table 5) and private tumor mutations (Table 7) were tested for TIL recognition from patient 1309 and D1313 that recognized several commonly shared epitopes and neoepitopes. TIL from patient D1313 recognized a mutant epitope from a mitochondrial protein which was found to be HLA-DP restricted. TIL from patient D1309 recognized a mutant driver gene involved in PDAC oncogenesis. 3] In summary, cardiolipin activated TIL resulted in increased frequencies of tumor-reactive T-cells in general, resulted in expansion of yb TIL that were tumor-specific and CD1 d restricted. In order to define in greater detail, the nature of the yb TIL reactivity, mitochondria from healthy donor PBMCs, from doxorubicin-treated PBMCs or with mitochondria from the allogeneic tumor cell line PANC-1 were isolated, yb TIL recognized the autologous tumor in a CD1 d restricted fashion and the same yb TIL recognized the pancreatic cancer cell line PANC1 , also CD1 d restricted. 4] One of the candidate targets are damaged mitochondria, either induced by doxorubicin from healthy cells or mitochondria isolated from cancer cells. Cardiolipin also facilitates the expansion of tumor-reactive a|3 TIL, they recognized the autologous tumor cells in an MHC class I or class II - restricted fashion and analysis of 2 TIL in greater detail showed that commonly shared, as well as private mutant neoepitopes, are being recognized, e.g., from a mutant PDAC driver gene or a gene from a mitochondrion suggesting that mitochondrial targets are viable sources for TIL target recognition. Cardiolipin induced up-regulation of CXCL9 and CXCL10 expression in TIL, factors that facilitate the invasion of TIL into solid cancers. This makes this protocol unique, combining a|3 and yb T- cell expansion and taking into consideration the starting material in the process since PDAC lesions, or lesions from other solid tumors, are heterogenous in tumor cells, yet also in T-cell infiltrates. TIL can be expanded up to 1x10 ⁹ cells as shown in these experiments. 5] A tumor specific cellular immune response can be seen a very specific autoimmune response directed against mutant or non-mutant self- proteins that are preferentially or exclusively expressed in transformed tissue. The 3-step expansion method allowed to gauge for cytokine production in response to viral I cross-reactive epitopes recognizing human self-proteins, as can be seen on Table 9 below.

[226] Table 9 7] Testing of commonly shared antigens or private antigens for immune effector functions in TIL versus PBMCs harvested at the same time point aid to show that the 3 step-expanded TIL contain cancer target specific T- cells that are not present in PBMCs, it will also aid to select for different TIL products. The simultaneous measurement of IFN-y versus IL-17 aids also to select for T-cells that i) exclusively or preferentially produce IFN-y, but not IL-17 in response to cancer target antigens, it also aids to define target antigens that elicit in the T-cell populations an antigen-specific IL- 17 response but not an IFN-y response. Such T-cell products may either not be considered for therapy since IL-17 may facilitate tumor - progression; alternatively, IL-17 antigens, including autoimmune, cancer or viral targets that elicit IL-17 responses may be removed from vaccine formulations. 8] The differential recognition cytokine production against different molecularly defined target antigens is shown in Table 10 below. Strong IFN-y production in the positive controls (PHA or anti-CD3 cross-linking), only limited amount of IL-17 production in response to the positive control PHA. Yet distinct IL-17 production in response to defined peptide targets (e.g., KRAS or mesothelin) which may reflect antigen-driven specific T- cell proliferation leading to IL-17 production. 9] Table 10

reactivity. 10e ⁴ TIL or 10e ⁴ PBMC / well were co-cultured with the peptides listed in Table 1 1 below for 7 days and the supernatants were tested for IFN-y production. Differential recognition in TIL, but in PBMCs of KRAS peptide targets suggests that the expanded TIL product is enriched for mutant target specific T-cells and suitable for the active cellular therapy of patients with cancer. 31] Table 11

32] TIL lines (some from different areas) as well as PBMCs were evaluated for cytokine production to a broad panel commonly shared target antigens as described in this application that allowed to obtain a functional and molecular fingerprint of expanded TIL (Figure 24 and 25). This could also be done for a more specific questions, e.g., for MUC4 wild-type or mutant specific T-cell responses (Figure 26 and 27), or the detection of biologically relevant B-cell targets contained in MUC-4 repetitive epitopes. 33] In order to further characterize the immune cells expanded from a solid tissue or a liquid tissue sample, we analyzed the presence of MAIT cells in TIL and PBMCs (Figure 28), since they may also contribute to anti- tumor directed immune responses either by direct recognition of MR-1 presented targets, or - not mutually exclusive - by MAIT cells that recognized bacterial species, or their related products. This is biologically relevant since the presence of distinct bacterial species has been reported to be associated with increased survival in patients with pancreatic cancer. 34] MAIT can discriminate between foreign and commensal bacteria by recognizing riboflavin precursors found in most bacteria and yeasts allowing the use of MR1 tetramers loaded with these molecules to selectively activate MAIT. 6-FP (6 Formyl-Pterin, a folic acid derivate) is recognized by MAIT but does not activate them, in contrast to 5-OP-RU which is a potent activator. Some TCR yσ ⁺ T-cells have also been described to recognize the ‘underside’ of the MR1 molecule, most likely independent of the nominal ligand. Yet it could be possible that individual, as yet ill-defined MR1 ligands, shape the ‘underside’ of MR1 in such a way that TCR yσ ⁺ T-cells preferentially bind to certain MR1 molecules loaded with targets that facilitate the interaction of the yσ ⁺ T-cell receptor with the MR1 molecules. 35] TIL expanded with the method described in the present invention also contain MAIT cells, either defined by the presence of the TCR VA7.2 ⁺ T- cells, or MAIT. 6-FP (6 Formyl-Pterin, a folic acid derivate) or MR1 presented 5-OP-RU antigens (Figure 29 and 30). VA7.2- sorted MAIT cells recognize the autologous tumor cells in an MR1 restricted fashion, defined by IFN-y production and they also recognize allogeneic tumor cell lines in an MR1 restricted fashion (Figure 31 ). 36] Specificity is shown by reducing MR1 expression using siRNA specifically targeting MR1 , yet not scrambled control siRNA (Figure 32). Bacterial recognition is detected in VA7.2 TIL against individual bacterial species associated with improved survival, as defined by IFN-y ⁺ frequency, or perforin/CD107a ⁺ frequency, yet not IL17 ⁺ frequency, some of these bacterial responses are pronounced in the double-positive CD4 ⁺ CD8 ⁺ T-cell TIL population (Figure 33 and 34). 37] T-cell receptor analysis was performed in the tumor specimens (Figure 35 and 38), in the final expanded TIL product (Figure 36, 39 43) as well as in the TCRVa7.2 sorted T-cells (Figure 37 and 40). MR1 -6FP sorted cells show MR1 restricted tumor recognition (Figure 41 and 44). Flow cytometric analysis clearly showed that the MR-1 6-formylpterin reactive T-cells have a higher percentage of yσ ⁺ T-cells than TCRVa7.2 cells (Figure 42) suggesting that MR1 -6FP + targets could be preferentially recognized by TCR yσ ⁺ T-cells.

40] Table 13 OD readout - corresponding to table 12

41] Sera from different patients with PDAC (labeled as S1 -S15) were incubated with the target epitope at different dilutions in phosphate- buffered saline (PBS) (1 :2 and 1 :10) and reactivity was tested based on the OD reading. Differential responses with strong IgG binding reactivity to the nominal MUC4 epitope for instance in sera from patient S1 and S15 (see layout in Tables 12 and 13). 42] The subject matter described above is provided as an illustration of the present invention and, therefore, should not be construed to limit it. The terminology employed for the purpose of describing preferred embodiments of the present invention should not be restricted to them. 43] As used in the description, defined and indefinite articles, in their singular form, are intended for interpretation to also include plural forms, unless the context of the description explicitly indicates otherwise. 44] Undefined articles "one" should generally be interpreted as "one or more", unless the meaning of a singular modality is clearly defined in a specific situation. 45] It will be understood that the terms "understand" and "include", when used in this description, specify the presence of characteristics, elements, components, steps, and related operations, but do not exclude the possibility of other characteristics, elements, components, steps, and operations as well contemplated. 46] As used throughout this patent application, the term "or" is used in an inclusive sense rather than an exclusive sense, unless the exclusive meaning is clearly defined in a specific situation. In this context, a phrase of the type "X uses A or B" should be interpreted as including all relevant inclusive combinations, for example "X uses A", "X uses B" and "X uses A and B". 47] In addition, the articles “a” and “an” as used in this application and the appended claims should generally be construed to mean “one or more” unless specified otherwise or clear from the context to be directed to a singular form.

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