Artificial antigen presenting cells and uses thereof in the expansion of cells

Abstract

The present invention is targeted towards engineered (or artificial) feeder cell lines that are especially useful for expanding cells, such as lymphocytes and especially tumor infiltrating lymphocytes (TILs), and for use in adoptive cell transfer without the use of allogenic peripheral blood mononuclear cells (PBMCs). Instead of using PBMCs, a replenishable cell line expressing T cell stimulatory molecules is used in the rapid expansion protocol (REP) to stimulate the growth of TILs into therapeutically relevant numbers, and with a more potent phenotype than currently established expansion methods. An expansion with the engineered feeder cell line further enables a donor free expansion process by substituting the allogenic feeder cells used in the REP without the need for human serum as media substitute.

Description

[0001] Artificial antigen presenting cells and uses thereof in the expansion of cells

[0002] FIELD

[0003] The present invention is targeted towards engineered (or artificial) feeder cell lines that are especially useful for expanding cells, such as lymphocytes and especially tumor infiltrating lymphocytes (TILs), and for use in adoptive cell transfer without the use of allogenic peripheral blood mononuclear cells (PBMCs). Instead of using PBMCs, a replenishable cell line expressing T cell stimulatory molecules is used in the rapid expansion protocol (REP) to stimulate the growth of TILs into therapeutically relevant numbers, and with a more potent phenotype than currently established expansion methods. An expansion with the engineered feeder cell line further enables a donor free expansion process by substituting the allogenic feeder cells used in the REP without the need for human serum as media substitute.

[0004] BACKGROUND

[0005] Treatment of bulky, refractory cancers using adoptive autologous transfer of tumor infiltrating lymphocytes (TILs) represents a powerful approach to therapy for patients with poor prognoses. A large number of TILs are required for successful immunotherapy, and a robust and reliable process is needed for commercialization. This has been a challenge to achieve because of technical, logistical, and regulatory issues with cell expansion. IL-2-based TIL expansion followed by a “rapid expansion process” (REP) has become a preferred method for TIL expansion because of its speed and efficiency. However, although REP can result in a 1,000-fold expansion of TILs over a 14-day period, it requires a large excess (e.g., 200-fold) of irradiated allogeneic peripheral blood mononuclear cells (PBMCs), often from multiple donors, as feeder cells, as well as anti-CD3 antibody (OKT-3) and high doses of IL-2. Despite their high performance, PBMCs have multiple drawbacks, including the large numbers of allogeneic PBMCs required, the need to obtain PBMCs from multiple healthy donors, the resulting interdonor variability in PBMC viability after cryopreservation and variable TIL expansion results, the risk of undetected viral pathogens causing downstream patient infections, and the extensive and costly laboratory testing of each individual donor cell product to confirm sterility and quality (including viral contaminant testing) and to test expansion properties. Unfortunately, artificial antigen presenting cells (aAPCs) developed for use in the expansion of TILs have suffered from poor performance when compared to PBMCs, including alterations of the phenotypic properties of the input TILs, as well as poor expansion performance and / or high variability in expansion results. Because of the large number of potential cells that might be adapted for use as aAPCs and the unpredictability of identifying suitable candidates, the focus of aAPC development for polyclonal TILs to date has been on e.g., the well-established K562 cell line. Other engineeredK562 cells modified to express CD64, CD86, and 4-1 BBL were tested and achieved TIL expansion that was at best comparable to PBMCs, and most likely less than PBMCs, and also suffered from skewing of the polyclonal TIL phenotype to a less favorable CD8+ / CD4+ T cell ratio. Recently, K562 cells modified to express CD86, 4-1 BBL (CD137L), high affinity Fc receptor (CD64) and membranebound IL-15 have also been shown to propagate TIL (post-REP) at equivalent numbers compared to PBMC feeders, but with the additional complexity of membrane-bound IL-15. Overall, K562 modified aAPCs have not been shown to provide for consistent expansion of TILs with acceptable variability while also performing better than PBMCs in other measures including overall expansion cell counts. Alternative aAPCs besides K562 cells have been successful in other cell expansion methods, but have not achieved the same performance as PBMCs with the unique polyclonal subset of cells that make up TILs.

[0006] Recent TIL research has shown that a CD39-negative stem-like phenotype (CD39-CD69-) is associated with complete cancer regression and TIL persistence. Improved phenotypes are therefore advantageous in TIL therapy and ideally such phenotypes should be enriched in TIL expansion protocols that includes the use of aAPCs.

[0007] SUMMARY

[0008] In its broadest aspect, the present invention relates to an artificial feeder cell comprising: at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules selected from the group consisting of: CD64, CD32, CD86, CD80, OX40L, a membrane bound receptor with affinity toward OKT-3 and / or 4-1 BBL.

[0009] An aspect of the present invention related to a culture medium comprising one or more artificial feeder cell(s) according to the invention.

[0010] An aspect of the present invention relates to a method for expanding cells into a therapeutic population of cells comprising:

[0011] a) culturing autologous cells by obtaining a first population of cells from a tumor and / or tumor fragments resected from a mammal,

[0012] b) performing a first expansion by culturing the depleted population of cells in a cell culture medium, and optionally adding the artificial feeder cells according the present invention. c) performing a second expansion by supplementing the cell culture medium of the second population of cells with;

[0013] - additional IL-2 or a combination of IL-7, IL-15 and IL-21,

[0014] - anti-CD3 antibody, and

[0015] - the artificial feeder cell(s) according the present invention,to produce a third population of cells, wherein the third population of cells is a therapeutic population.

[0016] An aspect of the present invention relates to a process for expanding cells into a population of cells, that can be therapeutic, comprising the steps:

[0017] a. obtaining a population of cells from a tumor and / or tumor fragments resected from a mammal;

[0018] b. expanding the population of cells by culturing said population of cells in a cell culture medium, wherein feeder cells are added to the cell culture 0-4 times at predetermined timepoint(s) and / or predetermined number(s) of cells, to produce a second population of cells, optionally wherein the second population of cells is a therapeutic population of cells;

[0019] c. Optionally harvesting the second population of cells.

[0020] An aspect of the present invention relates to a population of cells expanded using the artificial feeder cell according to the invention, the culture medium according to the invention, the method according to the invention and / or the process according to the present disclosure.

[0021] An aspect of the present invention related to a population of cells and / or a composition according to the invention, wherein the cells are derived from a human in need for treatment for cancer.

[0022] DETAILED DESCRIPTION

[0023] The present invention provides the unexpected finding that engineered lineage cells, including myeloid like K562 cells, which have been modified (e.g., transduced) with additional costimulatory molecules, including CD86 (B7-2), 4-1 BBL (CD137L), and OX40L (CD134L), as well as CD64 to provide for superior and highly efficient expansions of TILs in large numbers with minimal variability, reduced cost, and no reliance on human blood samples as a source of PBMCs, with the benefit of using an artificial feeder cell, also referred to as an artificial antigen presenting cells (aAPC), which can be produced efficiently from a master cell bank. CD86 and 4-1 BBL, and OX40L are costimulatory molecules that provide costimulatory signals for T cell activation. CD64 is a high affinity Fc receptor allowing for coating of the cell with anti CD3, or other, antibodies to stimulate T cell activation. K562 cells transduced with additional costimulatory molecules are useful, for example, in the expansion of TILs for use in cancer immunotherapy and other therapies.

[0024] The present inventors have found that donor derived PBMCs used in the REP in the TIL expansion process can be replaced with an artificial feeder cell, engineered to express T cell stimulatory molecules. The stimulation with the artificial feeder cell leads to:Higher proportion / total cell number of favorable TILs (CD8+, T cells with an effector memory phenotype, CD8 T cells with a stem-like phenotype etc.),

[0025] Faster expansion

[0026] Donor free expansion process

[0027] A reduced number of steps during the culturing process

[0028] Higher reactivity of the cultured T cells (measured by “potency assay”),

[0029] Higher total number of reactive T cells,

[0030] Higher number of tumor specificities,

[0031] Higher frequency of tumor-specific T cells, and / or

[0032] Higher T-cell repertoire (based on TCR sequencing).

[0033] Number of specificities of the CD8+ T cells and their frequency in the TIL product can be defined by staining TILs with MHC multimer complexes displaying tumor peptides of interest that can be recognized by CD8+ T cells and / or by sequencing the T cell receptor repertoire. Thus, an aspect of the present invention relates to an artificial feeder cell comprising at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules selected from the group consisting of CD64, CD32, CD86, CD80, OX40L, a membrane bound receptor with affinity toward OKT-3 and / or 4-1 BBL.

[0034] Artificial feeder cells

[0035] In the present context, an artificial feeder cell is a cell that is used to induce the activation, proliferation and expression of one or more immune cells and / or cell cultures. For example, an immune cell / cell culture can be one or more of leukocytes, such as lymphocytes, such as tumour infiltrating lymphocytes (TILs). An artificial feeder cell may, for example, be a naturally occurring or synthetic cell which has been modified and / or engineered, and / or derived from cells that do not naturally occur in nature. The artificial feeder cell may be used interchangeably with artificial feeder cell, also referred to as artificial antigen presenting cells (aAPCs).

[0036] In its broadest aspect, the artificial feeder cell comprises at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules and optionally a promotor, such as a SFFV promotor, CMV promotor, mCMV promotor, EF-1 promotor or a UbiC promotor. In one or more embodiments, the artificial feeder cell comprises at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules as defined herein.In one or more embodiments, the artificial feeder cell comprises at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules as defined herein and a promotor, such as a SFFV promotor, CMV promotor, mCMV promotor, EF-1 promotor or a UbiC promotor.

[0037] In one or more embodiments, the artificial feeder cell comprises at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules as defined herein and a SFFV promotor. In one or more embodiments, the artificial feeder cell comprises at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules as defined herein and a CMV promotor. In one or more embodiments, the artificial feeder cell comprises at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules as defined herein and a mCMV promotor. In one or more embodiments, the artificial feeder cell comprises at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules as defined herein and a EF-1 promotor. In one or more embodiments, the artificial feeder cell comprises at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules as defined herein and a UbiC promotor.ln one or more embodiments, the one or more co-stimulatory molecules is CD64, CD32, CD86, CD80, OX40L, a membrane bound receptor with affinity toward OKT-3 and / or 4-1BBL.

[0038] In one or more embodiments, the one or more co-stimulatory molecules comprises CD64. In one or more embodiments, the one or more co-stimulatory molecules comprises CD32. In one or more embodiments, the one or more co-stimulatory molecules comprises CD86. In one or more embodiments, the one or more co-stimulatory molecules comprises CD80. In one or more embodiments, the one or more co-stimulatory molecules comprises OX40L. In one or more embodiments, the one or more co-stimulatory molecules comprises a membrane bound receptor with affinity toward OKT-3. In one or more embodiments, the one or more co-stimulatory molecules comprises 4-1 BBL.

[0039] In one or more embodiments, the one or more co-stimulatory molecules is CD64. In one or more embodiments, the one or more co-stimulatory molecules is CD32. In one or more embodiments, the co-stimulatory molecules is CD86. In one or more embodiments, the one or more co-stimulatory molecules is CD80. In one or more embodiments, the one or more co-stimulatory molecules is OX40L. In one or more embodiments, the one or more co-stimulatory molecules is a membrane bound receptor with affinity toward OKT-3. In one or more embodiments, the one or more co-stimulatory molecules is 4-1 BBL.

[0040] In one or more embodiments, the one or more co-stimulatory molecules is CD64 and CD86. In one or more embodiments, the one or more co-stimulatory molecules is CD64 and CD86 and CD32. In one or more embodiments, the one or more co-stimulatory molecules is CD64 and CD86 and CD80. In one or more embodiments, the one or more co-stimulatory molecules is CD64 and CD86and OX40L. In one or more embodiments, the one or more co-stimulatory molecules is CD64 and CD86 and a membrane bound receptor with affinity towards OKT-3. In one or more embodiments, the one or more co-stimulatory molecules is CD64 and CD86 and 4-1 BBL. In one or more embodiments, the one or more co-stimulatory molecules is CD64 and 4-1 BBL and OX40L and CD86.

[0041] In one or more embodiments, the one or more co-stimulatory molecules comprises CD64 and CD86. In one or more embodiments, the one or more co-stimulatory molecules comprises CD64 and CD86 and CD32. In one or more embodiments, the one or more co-stimulatory molecules comprises CD64 and CD86 and CD80. In one or more embodiments, the one or more co-stimulatory molecules comprises CD64 and CD86 and OX40L. In one or more embodiments, the one or more co-stimulatory molecules comprises CD64 and CD86 and a membrane bound receptor with affinity towards OKT-3. In one or more embodiments, the one or more co-stimulatory molecules comprises CD64 and CD86 and 4-1 BBL. In one or more embodiments, the one or more co-stimulatory molecules comprises CD64 and 4-1 BBL and OX40L and CD86.

[0042] In a further aspect, the artificial feeder cell comprises an expression system comprising the at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules, such as CD64, CD32, CD86, CD80, OX40L, a membrane bound receptor with affinity toward OKT-3 and / or 4-1BBL.

[0043] In one or more embodiments, the artificial feeder cell comprises an expression system comprising at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules. In one or more embodiments, the artificial feeder cell comprises an expression system comprising at least one or more nucleic acid molecules encoding CD64. In one or more embodiments, the artificial feeder cell comprises an expression system comprising at least one or more nucleic acid molecules encoding CD32. In one or more embodiments, the artificial feeder cell comprises an expression system comprising at least one or more nucleic acid molecules encoding CD86. In one or more embodiments, the artificial feeder cell comprises an expression system comprising at least one or more nucleic acid molecules encoding CD80. In one or more embodiments, the artificial feeder cell comprises an expression system comprising at least one or more nucleic acid molecules encoding OX40L. In one or more embodiments, the artificial feeder cell comprises an expression system comprising at least one or more nucleic acid molecules encoding a membrane bound receptor with affinity toward OKT-3. In one or more embodiments, the artificial feeder cell comprises an expression system comprising at least one or more nucleic acid molecules encoding 4-1BBL.In a further aspect, the artificial feeder cell comprises a myeloid cell, such as a K562 cell, EM-3 cell, MOLM-13 cell or MOLM-14 cell.

[0044] In one or more embodiments, the artificial feeder cell comprises a myeloid cell. In one or more embodiments, the artificial feeder cell comprises a K562 cell. In one or more embodiments, the artificial feeder cell comprises EM-3 cell. In one or more embodiments, the artificial feeder cell comprises a MOLM-13 cell. In one or more embodiments, the artificial feeder cell comprises a MOLM-14 cell.

[0045] One or more embodiments of the present invention relates to the artificial feeder cell of the present invention, wherein the at least one or more nucleic acid molecules encoding one or more costimulatory molecules are selected from: CD64 and / or CD86.

[0046] One or more embodiments of the present invention relates to the artificial feeder cell of the present invention, wherein the at least one or more nucleic acid molecules encoding one or more costimulatory molecules is CD64. One or more embodiments of the present invention relates to the artificial feeder cell of the present invention, wherein the at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules is CD64 and CD86. One or more embodiments of the present invention relates to the artificial feeder cell of the present invention, wherein the at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules is CD86.

[0047] One or more embodiments of the present invention relates to the artificial feeder cell of the present invention, wherein the at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is selected from the group consisting of:

[0048] a. A SFFV promotor or a homologue thereof with at least 80% sequence identity to SEQ ID NO: 9,

[0049] b. A CMV promotor or a homologue thereof with at least 80% sequence identity to SEQ ID NO: 10,

[0050] c. An mCMV promotor or a homologue thereof with at least 80% sequence identity to SEQ ID NO: 11.

[0051] d. A EF-1 promotor or a homologue thereof with at least 80% sequence identity to SEQ ID NOs: 12 or 13.

[0052] e. A UbiC promotor or a homologue thereof with at least 80% sequence identity to SEQ ID NO: 14.

[0053] One or more embodiments of the present invention relates to the artificial feeder cell of the present invention, wherein at least one or more nucleic acid molecules comprises at least one promotor,wherein the promotor is a SFFV promotor or a homologue thereof with at least 80% sequence identity to SEQ ID NO: 9. One or more embodiments of the present invention relates to the artificial feeder cell of the present invention, wherein at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a SFFV promotor or a homologue thereof with at least 85% sequence identity to SEQ ID NO: 9. One or more embodiments of the present invention relates to the artificial feeder cell of the present invention, wherein at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a SFFV promotor or a homologue thereof with at least 90% sequence identity to SEQ ID NO: 9. One or more embodiments of the present invention relates to the artificial feeder cell of the present invention, wherein at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a SFFV promotor or a homologue thereof with at least 95% sequence identity to SEQ ID NO: 9. One or more embodiments of the present invention relates to the artificial feeder cell of the present invention, wherein at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a SFFV promotor or a homologue thereof with at least 99% sequence identity to SEQ ID NO: 9.

[0054] One or more embodiments of the present invention relates to the artificial feeder cell of the present invention, wherein at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a CMV promotor or a homologue thereof with at least 80% sequence identity to SEQ ID NO: 10. One or more embodiments of the present invention relates to the artificial feeder cell of the present invention, wherein at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a CMV promotor or a homologue thereof with at least 85% sequence identity to SEQ ID NO: 10. One or more embodiments of the present invention relates to the artificial feeder cell of the present invention, wherein at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a CMV promotor or a homologue thereof with at least 90% sequence identity to SEQ ID NO: 10. One or more embodiments of the present invention relates to the artificial feeder cell of the present invention, wherein at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a CMV promotor or a homologue thereof with at least 95% sequence identity to SEQ ID NO: 10. One or more embodiments of the present invention relates to the artificial feeder cell of the present invention, wherein at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a CMV promotor or a homologue thereof with at least 99% sequence identity to SEQ ID NO: 10.

[0055] One or more embodiments of the present invention relates to the artificial feeder cell of the present invention, wherein at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is an mCMV promotor or a homologue thereof with at least 80% sequence identity to SEQ ID NO: 11. One or more embodiments of the present invention relates to the artificial feeder cell of the present invention, wherein at least one or more nucleic acid moleculescomprises at least one promotor, wherein the promotor is an mCMV promotor or a homologue thereof with at least 85% sequence identity to SEQ ID NO: 11. One or more embodiments of the present invention relates to the artificial feeder cell of the present invention, wherein at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is an mCMV promotor or a homologue thereof with at least 90% sequence identity to SEQ ID NO: 11. One or more embodiments of the present invention relates to the artificial feeder cell of the present invention, wherein at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is an mCMV promotor or a homologue thereof with at least 95% sequence identity to SEQ ID NO: 11. One or more embodiments of the present invention relates to the artificial feeder cell of the present invention, wherein at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is an mCMV promotor or a homologue thereof with at least 99% sequence identity to SEQ ID NO: 11.

[0056] One or more embodiments of the present invention relates to the artificial feeder cell of the present invention, wherein at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a EF-1 promotor or a homologue thereof with at least 80% sequence identity to SEQ ID NOs: 12 or 13.

[0057] One or more embodiments of the present invention relates to the artificial feeder cell of the present invention, wherein at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a EF-1 promotor or a homologue thereof with at least 80% sequence identity to SEQ ID NOs: 12. One or more embodiments of the present invention relates to the artificial feeder cell of the present invention, wherein at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a EF-1 promotor or a homologue thereof with at least 85% sequence identity to SEQ ID NOs: 12. One or more embodiments of the present invention relates to the artificial feeder cell of the present invention, wherein at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a EF-1 promotor or a homologue thereof with at least 90% sequence identity to SEQ ID NOs: 12. One or more embodiments of the present invention relates to the artificial feeder cell of the present invention, wherein at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a EF-1 promotor or a homologue thereof with at least 95% sequence identity to SEQ ID NOs: 12. One or more embodiments of the present invention relates to the artificial feeder cell of the present invention, wherein at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a EF-1 promotor or a homologue thereof with at least 99% sequence identity to SEQ ID NOs: 12.

[0058] One or more embodiments of the present invention relates to the artificial feeder cell of the present invention, wherein at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a EF-1 promotor or a homologue thereof with at least 80% sequenceidentity to SEQ ID NOs: 13. One or more embodiments of the present invention relates to the artificial feeder cell of the present invention, wherein at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a EF-1 promotor or a homologue thereof with at least 85% sequence identity to SEQ ID NOs: 13. One or more embodiments of the present invention relates to the artificial feeder cell of the present invention, wherein at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a EF-1 promotor or a homologue thereof with at least 90% sequence identity to SEQ ID NOs: 13. One or more embodiments of the present invention relates to the artificial feeder cell of the present invention, wherein at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a EF-1 promotor or a homologue thereof with at least 95% sequence identity to SEQ ID NOs: 13. One or more embodiments of the present invention relates to the artificial feeder cell of the present invention, wherein at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a EF-1 promotor or a homologue thereof with at least 99% sequence identity to SEQ ID NOs: 13.

[0059] One or more embodiments of the present invention relates to the artificial feeder cell of the present invention, wherein at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a UbiC promotor or a homologue thereof with at least 80% sequence identity to SEQ ID NO: 14. One or more embodiments of the present invention relates to the artificial feeder cell of the present invention, wherein at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a UbiC promotor or a homologue thereof with at least 85% sequence identity to SEQ ID NO: 14. One or more embodiments of the present invention relates to the artificial feeder cell of the present invention, wherein at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a UbiC promotor or a homologue thereof with at least 90% sequence identity to SEQ ID NO: 14. One or more embodiments of the present invention relates to the artificial feeder cell of the present invention, wherein at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a UbiC promotor or a homologue thereof with at least 95% sequence identity to SEQ ID NO: 14. One or more embodiments of the present invention relates to the artificial feeder cell of the present invention, wherein at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a UbiC promotor or a homologue thereof with at least 99% sequence identity to SEQ ID NO: 14.

[0060] One or more embodiments of the present invention relates to the artificial feeder cell of the present invention, wherein the at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a SFFV promotor or a homologue thereof with at least 80% sequence identity to SEQ ID NO: 9.One or more embodiments of the present invention relates to the artificial feeder cell of the present invention, wherein the at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is SEQ ID NO: 9.

[0061] One or more embodiments of the present invention relates to the artificial feeder cell of the present invention, wherein the artificial feeder cell is comprising an expression system comprising the at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules.

[0062] One or more embodiments of the present invention relates to the artificial feeder cell of the present invention, wherein the expression system is selected from the group consisting of one or more viral vectors, transposon-mediated gene transfer system(s) or genome-editing based expression system(s), such as CRISPR-based expression system(s).

[0063] One or more embodiments of the present invention relates to the artificial feeder cell of the present invention, wherein the one or more viral vectors are transduced.

[0064] One or more embodiments of the present invention relates to the artificial feeder cell of the present invention, wherein the one or more viral vectors are stably transduced.

[0065] One or more embodiments of the present invention relates to the artificial feeder cell of the present invention, wherein said artificial feeder cell is a myeloid cell.

[0066] One or more embodiments of the present invention relates to the artificial feeder cell of the present invention, wherein the myeloid cell is selected from the group consisting of: K562, EM-3, EM-2, MOLM-13, MOLM-14.

[0067] One or more embodiments of the present invention relates to the artificial feeder cell of the present invention, wherein the myeloid cell is a K562 cell.

[0068] In one or more embodiments, the artificial feeder cell(s) undergoes treatment that leads to cell death.

[0069] In one or more embodiments, the artificial feeder cell(s) are irradiated.

[0070] In one or more embodiments, the artificial feeder cell(s) have been cryopreserved.

[0071] In one or more embodiments, the artificial feeder cell(s) are irradiated before cryopreservation, while they are cryopreserved, or after they have been cryopreserved.

[0072] In one or more embodiments, the artificial feeder cell(s) are irradiated before cryopreservation. In one or more embodiments, the artificial feeder cell(s) are irradiated while they are

[0073] cryopreserved.In one or more embodiments, the artificial feeder cell(s) are irradiated after they have been cryopreserved.

[0074] In one or more embodiments, the artificial feeder cells are irradiated with at least 25 Gy. In one or more embodiments, the artificial feeder cells are irradiated with at least 50 Gy.

[0075] In one or more embodiments, the artificial feeder cells are irradiated using ionizing radiation such as x-rays and / or gamma rays. In one or more embodiments, the artificial feeder cells are irradiated using ionizing radiation. In one or more embodiments, the artificial feeder cells are irradiated using x-rays. In one or more embodiments, the artificial feeder cells are irradiated using gamma rays. In one or more embodiments, the artificial feeder cells are irradiated using x-rays and gamma rays. In one or more embodiments, the artificial feeder cells are irradiated using the artificial feeder cells are cryopreserved at a temperature of at least -70°C or colder. In one or more embodiments, the artificial feeder cells are irradiated using the artificial feeder cells are cryopreserved at a temperature of at least -75°C or colder. In one or more embodiments, the artificial feeder cells are irradiated using the artificial feeder cells are cryopreserved at a temperature of at least -80°C or colder.

[0076] Nucleic acid molecules

[0077] As used herein, the term “nucleic acid molecule”, refer to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form. Unless otherwise indicated, the term "nucleic acid molecule" refers a nucleic acid molecule in double stranded from, i.e. also including the complementary sequence of the nucleic acid molecule. It is understood that as a result of the degeneracy of the genetic code, a multitude of nucleic acid molecules encoding for the same amino acid sequence may be produced.

[0078] A nucleic acid molecule in the present disclosure, refers to either a naturally occurring nucleic acid molecule that has been isolated from its natural source, or an artificial nucleic acid molecule characterised by an artificially induced (i.e. produced in vitro using standard laboratory methods for making nucleic acid molecule) combination of two or more segments of DNA, which do not occur in that specific combination in nature. The components of a recombinant nucleic acid molecule (i.e. segments of DNA), however, need not be artificially induced and may be naturally occurring. Techniques for introducing exogenous nucleic acid molecules and / or inserting exogenous nucleic acid molecules (recombinant, heterologous) into a cell's hereditary information for inserting, deleting or altering the nucleotide sequence of a cell's genetic information are known to the skilled artisan.Encoding

[0079] In the present context, the term "encoding" as used herein is intended to mean that a nucleic acid molecule or set of nucleic acid molecules correspond to the sequence of amino acids in a protein following transcription and / or translation of the nucleic acid molecule(s). In the present context, such nucleic acid molecules encoding one or more co-stimulatory molecules would following transcription and / or translation of the nucleic acid molecule(s) result in production of a protein having the amino acid sequence of the specific co-stimulatory molecule(s), such as CD64 and / or a homologue thereof with at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, or 100% sequence identity with SEQ ID NO: 4, CD32 and / or a homologue thereof with at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, or 100% sequence identity with SEQ ID NO: 16, CD86 and / or a homologue thereof with at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, or 100% sequence identity with SEQ ID NO: 2, CD80 and / or a homologue thereof with at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, or 100% sequence identity with SEQ ID NO: 18, QX40L and / or a homologue thereof with at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, or 100% sequence identity with SEQ ID NO: 6, a membrane bound receptor with affinity toward OKT-3 and / or 4-1 BBL and / or a homologue thereof with at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 95%, or 100% sequence identity with SEQ ID NO: 8.

[0080] For example, a nucleic acid molecule encoding for the co-stimulatory molecule CD64 would following transcription and / or translation of the nucleic acid molecule(s) result in production of a protein having the amino acid sequence of CD64. In a further example, two nucleic acid molecules encoding for the co-stimulatory molecules CD64 and CD32 would following transcription and / or translation of the nucleic acid molecules result in production of the protein having the amino acid sequences of CD64 and CD32.

[0081] Genome integration

[0082] The terms ‘integration’ and / or ‘integration into a genome’ is meant to describe the integration of the one or more nucleic acid molecule(s) into the genome of the artificial feeder cell. Integration of the one or more nucleic acid molecule(s) may be transient, wherein the one or more nucleic acid molecule(s) is introduced and / or integrated into a target genome, wherein the introduced nucleic acid molecule(s) persists in cells for a limited period of time, for instance, several days. Integration of nucleic acid molecule(s) also encompasses inheritance of non-genomic DNA.

[0083] The term ‘stable integration’ and / or ‘stable integration into a genome’ is meant to describe stable integration of the nucleic acid molecule(s), wherein the nucleic acid molecule(s) is introduced and / or integrated into a target genome, and persists in cells long-term, supporting stable gene expression, and is often able to pass the said nucleic acid molecule(s) into the progeny of such cells.Thus, in one or more embodiments, at least one or more nucleic acid molecules encodes for one or more co-stimulatory molecules selected from the group consisting of: CD64, CD32, CD86, CD80, OX40L, a membrane bound receptor with affinity toward OKT-3 and / or 4-1 BBL.

[0084] In one or more embodiments, at least two or more nucleic acid molecules encodes for two or more co-stimulatory molecules selected from the group consisting of: CD64, CD32, CD86, CD80, OX40L, a membrane bound receptor with affinity toward OKT-3 and / or 4-1 BBL.

[0085] In one or more embodiments, at least three or more nucleic acid molecules encodes for three or more co-stimulatory molecules selected from the group consisting of: CD64, CD32, CD86, CD80, OX40L, a membrane bound receptor with affinity toward OKT-3 and / or 4-1 BBL.

[0086] In one or more embodiments, at least four or more nucleic acid molecules encodes for four or more co-stimulatory molecules selected from the group consisting of: CD64, CD32, CD86, CD80, OX40L, a membrane bound receptor with affinity toward OKT-3 and / or 4-1 BBL.

[0087] In one or more embodiments, at least five or more nucleic acid molecules encodes for five or more co-stimulatory molecules selected from the group consisting of: CD64, CD32, CD86, CD80, OX40L, a membrane bound receptor with affinity toward OKT-3 and / or 4-1 BBL.

[0088] In one or more embodiments, at least six or more nucleic acid molecules encodes for six or more co-stimulatory molecules selected from the group consisting of: CD64, CD32, CD86, CD80, OX40L, a membrane bound receptor with affinity toward OKT-3 and / or 4-1 BBL.

[0089] In one or more embodiments, seven nucleic acid molecules encodes for seven co-stimulatory molecules selected from the group consisting of: CD64, CD32, CD86, CD80, OX40L, a membrane bound receptor with affinity toward OKT-3 and / or 4-1 BBL.

[0090] In one or more embodiments, at least one or more nucleic acid molecules encodes for one or more co-stimulatory molecules selected from the group consisting of: CD64, CD32, CD86, CD80, OX40L, a membrane bound receptor with affinity toward OKT-3 and / or 4-1 BBL and are integrated into the genome of the artificial feeder cell.

[0091] In one or more embodiments, at least one or more nucleic acid molecules encodes for one or more co-stimulatory molecules selected from the group consisting of: CD64, CD32, CD86, CD80, OX40L, a membrane bound receptor with affinity toward OKT-3 and / or 4-1 BBL and are stably integrated into the genome of the artificial feeder cell.

[0092] In one or more embodiments, at least one or more nucleic acid molecules encodes for CD64. In one or more embodiments, at least one or more nucleic acid molecules encodes CD32. In one or more embodiments, at least one or more nucleic acid molecules encodes CD86. In one or more embodiments, at least one or more nucleic acid molecules encodes CD80. In one or more embodiments, at least one or more nucleic acid molecules encodes OX40L. In one or moreembodiments, at least one or more nucleic acid molecules encodes a membrane bound receptor with affinity toward OKT-3. In one or more embodiments, at least one or more nucleic acid molecules encodes for 4-1 BBL.

[0093] In one or more embodiments, at least one or more nucleic acid molecules encodes for CD64 and CD86. In one or more embodiments, at least one or more nucleic acid molecules encodes for CD64 and CD86 and CD32.

[0094] In one or more embodiments, at least one or more nucleic acid molecules encodes for CD64 and CD86 and CD80.

[0095] In one or more embodiments, at least one or more nucleic acid molecules encodes for CD64 and CD86 and OX40L.

[0096] In one or more embodiments, at least one or more nucleic acid molecules encodes for CD64 and CD86 and a membrane bound receptor with affinity towards OKT-3.

[0097] In one or more embodiments, at least one or more nucleic acid molecules encodes for CD64 and CD86 and 4-1 BBL.

[0098] In one or more embodiments, at least one or more nucleic acid molecules encodes for CD64 and 4-1 BBL and OX40L and CD86.

[0099] Co-stimulatory molecules

[0100] In the present context, co-stimulatory molecules are a heterogenous group of cell surface molecules that act to amplify or counteract the initial activating signals provided to T cells from the T cell receptor (TOR) following its interaction with an antigen / major histocompatibility complex (MHC), thereby influencing T cell differentiation and fate. Activation of adaptive immune responses requires signals through antigen-specific T- and B-cell receptors. However, this antigen-specific signal alone is not sufficient to drive the activation of naive T cells and requires a second antigenindependent nonspecific signal known as costimulatory signal. The primary source of this costimulatory signal are interactions between the T-cell integral membrane proteins and their corresponding ligands on the ARC. Examples of co-stimulatory molecules include CD64, CD32, CD86, CD80, OX40L, a membrane bound receptor with affinity towards OKT-3 and 4-1 BBL.

[0101] In one aspect, K562 cells transduced with additional costimulatory molecules are useful, for example, in the expansion of TILs for use in cancer immunotherapy and other therapies. These aspects are described herein.

[0102] CD64

[0103] CD64 (Gene ID: 2209) is defined as a type of integral membrane glycoprotein known as an Fc receptor that binds monomeric IgG-type antibodies with high affinity. It is more commonly known as Fc-gamma receptor 1 (FcyRI). It is a transmembrane glycoprotein of 72-kDa.One or more aspects of the present invention relates to a CD64 protein encoded by the nucleic acid sequence according to SEQ ID NO. 3 or a homologue thereof with at least 80 % sequence identity to SEQ ID NO. 3. This homologue can be with at least 80 % sequence identity to SEQ ID NO. 3. This homologue can be with at least 90 % sequence identity to SEQ ID NO. 3. This homologue can be with at least 90 % sequence identity to SEQ ID NO. 3.

[0104] In one or more embodiments, the CD64 protein is encoded by a nucleic acid sequence with at least 80% sequence identity according to SEQ ID NO. 3 or a homologue thereof with at least 80 % sequence identity to SEQ ID NO. 3. In one or more embodiments, the CD64 protein is encoded by a nucleic acid sequence with at least 85% sequence identity according to SEQ ID NO. 3 or a homologue thereof with at least 85 % sequence identity to SEQ ID NO. 3. In one or more embodiments, the CD64 protein is encoded by a nucleic acid sequence with at least 90% sequence identity according to SEQ ID NO. 3 or a homologue thereof with at least 90 % sequence identity to SEQ ID NO. 3. In one or more embodiments, the CD64 protein is encoded by a nucleic acid sequence with at least 95% sequence identity according to SEQ ID NO. 3 or a homologue thereof with at least 95 % sequence identity to SEQ ID NO. 3. In one or more embodiments, the CD64 protein is encoded by a nucleic acid sequence with at least 99% sequence identity according to SEQ ID NO. 3 or a homologue thereof with at least 99 % sequence identity to SEQ ID NO. 3. In one or more embodiments, the CD64 protein is encoded by the nucleic acid sequence according to SEQ ID NO. 3.

[0105] One or more aspects of the present invention relates to a CD64 protein according to SEQ ID NO. 4 or a homologue thereof with at least 80 % sequence identity to SEQ ID NO. 4. This homologue can be with at least 80 % sequence identity to SEQ ID NO. 4. This homologue can be with at least 90 % sequence identity to SEQ ID NO. 4. This homologue can be with at least 90 % sequence identity to SEQ ID NO. 4.

[0106] In one or more embodiments, the CD64 protein has at least 80% sequence identity according to SEQ ID NO. 4 or a homologue thereof with at least 80 % sequence identity to SEQ ID NO. 4. In one or more embodiments, the CD64 protein has at least 85% sequence identity according to SEQ ID NO. 4 or a homologue thereof with at least 85 % sequence identity to SEQ ID NO. 4. In one or more embodiments, the CD64 protein has at least 90% sequence identity according to SEQ ID NO. 4 or a homologue thereof with at least 90 % sequence identity to SEQ ID NO. 4. In one or more embodiments, the CD64 protein has at least 95% sequence identity according to SEQ ID NO. 4 or a homologue thereof with at least 95 % sequence identity to SEQ ID NO. 4. In one or more embodiments, the CD64 protein has at least 99% sequence identity according to SEQ ID NO. 4 or a homologue thereof with at least 99 % sequence identity to SEQ ID NO. 4. In one or more embodiments, the CD64 protein is SEQ ID NO. 4.CD32

[0107] CD32 (Gene ID: 2212), also known as FcyRII or FCGR2, is a surface receptor glycoprotein. CD32 can be found on the surface of a variety of immune cells. CD32 has two major functions - cellular response regulation, and the uptake of immune complexes. In humans, there are three major CD32 subtypes: CD32A, CD32B, and CD32C. It can be found on Langerhans cells, mast cells, basophils, eosinophils, monocytes, megakaryocytes, and a subpopulation of activated CD4+ T cells.

[0108] One or more aspects of the present invention relates to a CD32 protein encoded by the nucleic acid sequence according to SEQ ID NO. 15 or a homologue thereof with at least 80 % sequence identity to SEQ ID NO. 15. This homologue can be with at least 80 % sequence identity to SEQ ID NO. 15. This homologue can be with at least 90 % sequence identity to SEQ ID NO. 15. This homologue can be with at least 90 % sequence identity to SEQ ID NO. 15.

[0109] In one or more embodiments, the CD32 protein is encoded by a nucleic acid sequence with at least 80% sequence identity according to SEQ ID NO. 15 or a homologue thereof with at least 80 % sequence identity to SEQ ID NO. 15. In one or more embodiments, the CD32 protein is encoded by a nucleic acid sequence with at least 85% sequence identity according to SEQ ID NO. 15 or a homologue thereof with at least 85 % sequence identity to SEQ ID NO. 15. In one or more embodiments, the CD32 protein is encoded by a nucleic acid sequence with at least 90% sequence identity according to SEQ ID NO. 15 or a homologue thereof with at least 90 % sequence identity to SEQ ID NO. 15. In one or more embodiments, the CD32 protein is encoded by a nucleic acid sequence with at least 95% sequence identity according to SEQ ID NO. 15 or a homologue thereof with at least 95 % sequence identity to SEQ ID NO. 15. In one or more embodiments, the CD32 protein is encoded by a nucleic acid sequence with at least 99% sequence identity according to SEQ ID NO. 15 or a homologue thereof with at least 99 % sequence identity to SEQ ID NO. 15. In one or more embodiments, the CD32 protein is encoded by the nucleic acid sequence according to SEQ ID NO. 15.

[0110] One or more aspects of the present invention relates to a CD32 protein according to SEQ ID NO. 2 or a homologue thereof with at least 80 % sequence identity to SEQ ID NO. 16. One or more aspects of the present invention relates to a CD32 protein according to SEQ ID NO. 16 or a homologue thereof with at least 80 % sequence identity to SEQ ID NO. 16. This homologue can be with at least 80 % sequence identity to SEQ ID NO. 16. This homologue can be with at least 90 % sequence identity to SEQ ID NO. 16. This homologue can be with at least 90 % sequence identity to SEQ ID NO. 16.In one or more embodiments, the CD32 protein has at least 80% sequence identity according to SEQ ID NO. 16 or a homologue thereof with at least 80 % sequence identity to SEQ ID NO. 16. In one or more embodiments, the CD32 protein has at least 85% sequence identity according to SEQ ID NO. 16 or a homologue thereof with at least 85 % sequence identity to SEQ ID NO. 16. In one or more embodiments, the CD32 protein has at least 90% sequence identity according to SEQ ID NO. 16 or a homologue thereof with at least 90 % sequence identity to SEQ ID NO. 16. In one or more embodiments, the CD32 protein has at least 95% sequence identity according to SEQ ID NO. 16 or a homologue thereof with at least 95 % sequence identity to SEQ ID NO. 16. In one or more embodiments, the CD32 protein has at least 99% sequence identity according to SEQ ID NO. 16 or a homologue thereof with at least 99 % sequence identity to SEQ ID NO. 16. In one or more embodiments, the CD32 protein is SEQ ID NO. 16.

[0111] CD86

[0112] CD86 (Gene ID: 942) is a 70-kDa glycoprotein made up of 329 amino acids, a transmembrane region, and a cytoplasmic domain. CD86 is constitutively expressed on interdigitating dendritic cells (DCs), Langerhans cells, peripheral blood DCs, memory B cells and germinal center B cells, and macrophages. Along with CD80, CD86 provides costimulatory signals necessary for T cell activation and survival.

[0113] One or more aspects of the present invention relates to a CD86 protein encoded by the nucleic acid sequence according to SEQ ID NO. 1 or a homologue thereof with at least 80 % sequence identity to SEQ ID NO. 1. This homologue can be with at least 80 % sequence identity to SEQ ID NO. 1. This homologue can be with at least 90 % sequence identity to SEQ ID NO. 1. This homologue can be with at least 90 % sequence identity to SEQ ID NO. 1.

[0114] In one or more embodiments, the CD86 protein is encoded by a nucleic acid sequence with at least 80% sequence identity according to SEQ ID NO. 1 or a homologue thereof with at least 80 % sequence identity to SEQ ID NO. 1. In one or more embodiments, the CD86 protein is encoded by a nucleic acid sequence with at least 85% sequence identity according to SEQ ID NO. 1 or a homologue thereof with at least 85 % sequence identity to SEQ ID NO. 1. In one or more embodiments, the CD86 protein is encoded by a nucleic acid sequence with at least 90% sequence identity according to SEQ ID NO. 1 or a homologue thereof with at least 90 % sequence identity to SEQ ID NO. 1. In one or more embodiments, the CD86 protein is encoded by a nucleic acid sequence with at least 95% sequence identity according to SEQ ID NO. 1 or a homologue thereof with at least 95 % sequence identity to SEQ ID NO. 1. In one or more embodiments, the CD86 protein is encoded by a nucleic acid sequence with at least 99% sequence identity accordingto SEQ ID NO. 1 or a homologue thereof with at least 99 % sequence identity to SEQ ID NO. 1. In one or more embodiments, the CD86 protein is encoded by a nucleic acid sequence according to SEQ ID NO. 1.

[0115] One or more aspects of the present invention relates to a CD86 protein according to SEQ ID NO. 2 or a homologue thereof with at least 80 % sequence identity to SEQ ID NO. 2. This homologue can be with at least 80 % sequence identity to SEQ ID NO. 2. This homologue can be with at least 90 % sequence identity to SEQ ID NO. 2. This homologue can be with at least 90 % sequence identity to SEQ ID NO. 2.

[0116] In one or more embodiments, the CD86 protein has at least 80% sequence identity according to SEQ ID NO. 2 or a homologue thereof with at least 80 % sequence identity to SEQ ID NO. 2. In one or more embodiments, the CD86 protein has at least 85% sequence identity according to SEQ ID NO. 2 or a homologue thereof with at least 85 % sequence identity to SEQ ID NO. 2. In one or more embodiments, the CD86 protein has at least 90% sequence identity according to SEQ ID NO. 2 or a homologue thereof with at least 90 % sequence identity to SEQ ID NO. 2. In one or more embodiments, the CD86 protein has at least 95% sequence identity according to SEQ ID NO. 2 or a homologue thereof with at least 95 % sequence identity to SEQ ID NO. 2. In one or more embodiments, the CD86 protein has at least 99% sequence identity according to SEQ ID NO. 2 or a homologue thereof with at least 99 % sequence identity to SEQ ID NO. 2. In one or more embodiments, the CD86 protein is SEQ ID NO. 2.

[0117] CD80

[0118] CD80 (Gene ID: 941) is a transmembrane glycoprotein and a member of the Ig superfamily. It is composed of 288 amino acids, and its mass is 33 kDa. CD80 (also known as B7-1) is an inducible co-stimulatory molecule on antigen-presenting cells. CD80 and CD86 (B7-2), provide a signal to the T cell CD28 receptor which amplifies T-cell antigen receptor signaling. CD80 is present specifically on DC, activated B-cells, and macrophages, but also T-cells.

[0119] One or more aspects of the present invention relates to a CD80 protein encoded by the nucleic acid sequence according to SEQ ID NO. 17 or a homologue thereof with at least 80 % sequence identity to SEQ ID NO. 17. This homologue can be with at least 80 % sequence identity to SEQ ID NO. 17. This homologue can be with at least 90 % sequence identity to SEQ ID NO. 17. This homologue can be with at least 90 % sequence identity to SEQ ID NO. 17.

[0120] In one or more embodiments, the CD80 protein is encoded by a nucleic acid sequence with at least 80 % sequence identity according to SEQ ID NO. 17 or a homologue thereof with at least 80% sequence identity to SEQ ID NO. 17. In one or more embodiments, the CD80 protein is encoded by a nucleic acid sequence with at least 85% sequence identity according to SEQ ID NO. 17 or a homologue thereof with at least 85 % sequence identity to SEQ ID NO. 17. In one or more embodiments, the CD80 protein is encoded by a nucleic acid sequence with at least 90 % sequence identity according to SEQ ID NO. 17 or a homologue thereof with at least 90 % sequence identity to SEQ ID NO. 17. In one or more embodiments, the CD80 protein is encoded by a nucleic acid sequence with at least 95 % sequence identity according to SEQ ID NO. 17 or a homologue thereof with at least 95 % sequence identity to SEQ ID NO. 17. In one or more embodiments, the CD80 protein is encoded by a nucleic acid sequence with at least 99% sequence identity according to SEQ ID NO. 17 or a homologue thereof with at least 99 % sequence identity to SEQ ID NO. 17. In one or more embodiments, the CD80 protein is encoded by a nucleic acid sequence according to SEQ ID NO. 17.

[0121] One or more aspects of the present invention relates to a CD80 protein according to SEQ ID NO.

[0122] 18 or a homologue thereof with at least 80 % sequence identity to SEQ ID NO. 18. This homologue can be with at least 80 % sequence identity to SEQ ID NO. 18. This homologue can be with at least 90 % sequence identity to SEQ ID NO. 18. This homologue can be with at least 90 % sequence identity to SEQ ID NO. 18.

[0123] In one or more embodiments, the CD80 protein has at least 80% sequence identity according to SEQ ID NO. 18 or a homologue thereof with at least 80 % sequence identity to SEQ ID NO. 18. In one or more embodiments, the CD80 protein has at least 85% sequence identity according to SEQ ID NO. 18 or a homologue thereof with at least 85 % sequence identity to SEQ ID NO. 18. In one or more embodiments, the CD80 protein has at least 90% sequence identity according to SEQ ID NO. 18 or a homologue thereof with at least 90 % sequence identity to SEQ ID NO. 18. In one or more embodiments, the CD80 protein has at least 95% sequence identity according to SEQ ID NO. 18 or a homologue thereof with at least 95 % sequence identity to SEQ ID NO. 18. In one or more embodiments, the CD80 protein has at least 99% sequence identity according to SEQ ID NO. 18 or a homologue thereof with at least 99 % sequence identity to SEQ ID NO. 18. In one or more embodiments, the CD80 protein is SEQ ID NO. 18.

[0124] OX40L

[0125] OX40L (Gene ID: 7292), also known as CD252, is the ligand for 0X40 and is stably expressed on many antigen-presenting cells such as DC2s (a subtype of dendritic cells), macrophages and activated B lymphocytes. The 0X40 molecule, conversely, is present on the surface of activated T lymphocytes (mainly CD4+ T cells). The ligation of OX40-OX40L is a source of survival signal for Tcells and enables the development of memory T cells. 0X40 functions as a late costimulatory receptor and artificial engagement by OX40L stimulates proliferation, cytokine secretion, and survival of T cells.

[0126] One or more aspects of the present invention relates to a OX40L protein encoded by the nucleic acid sequence according to SEQ ID NO. 5 or a homologue thereof with at least 80 % sequence identity to SEQ ID NO. 5. This homologue can be with at least 80 % sequence identity to SEQ ID NO. 5. This homologue can be with at least 90 % sequence identity to SEQ ID NO. 5. This homologue can be with at least 90 % sequence identity to SEQ ID NO. 5.

[0127] In one or more embodiments, the OX40L protein is encoded by a nucleic acid sequence with at least 80 % sequence identity according to SEQ ID NO. 5 or a homologue thereof with at least 80 % sequence identity to SEQ ID NO. 5. In one or more embodiments, the OX40L protein is encoded by a nucleic acid sequence with at least 85 % sequence identity according to SEQ ID NO. 5 or a homologue thereof with at least 85 % sequence identity to SEQ ID NO. 5. In one or more embodiments, the OX40L protein is encoded by a nucleic acid sequence with at least 90 % sequence identity according to SEQ ID NO. 5 or a homologue thereof with at least 90 % sequence identity to SEQ ID NO. 5. In one or more embodiments, the OX40L protein is encoded by a nucleic acid sequence with at least 95 % sequence identity according to SEQ ID NO. 5 or a homologue thereof with at least 95 % sequence identity to SEQ ID NO. 5. In one or more embodiments, the OX40L protein is encoded by a nucleic acid sequence with at least 99 % sequence identity according to SEQ ID NO. 5 or a homologue thereof with at least 99 % sequence identity to SEQ ID NO. 5. In one or more embodiments, the OX40L protein is encoded by a nucleic acid sequence according to SEQ ID NO. 5.

[0128] One or more aspects of the present invention relates to a OX40L protein according to SEQ ID NO.

[0129] 6 or a homologue thereof with at least 80 % sequence identity to SEQ ID NO. 6. This homologue can be with at least 80 % sequence identity to SEQ ID NO. 6. This homologue can be with at least 90 % sequence identity to SEQ ID NO. 6. This homologue can be with at least 90 % sequence identity to SEQ ID NO. 6.

[0130] In one or more embodiments, the OX40L protein has at least 80% sequence identity according to SEQ ID NO. 6 or a homologue thereof with at least 80 % sequence identity to SEQ ID NO. 6. In one or more embodiments, the OX40L protein has at least 85% sequence identity according to SEQ ID NO. 6 or a homologue thereof with at least 85 % sequence identity to SEQ ID NO. 6. In one or more embodiments, the OX40L protein has at least 90% sequence identity according to SEQ ID NO. 6 or a homologue thereof with at least 90 % sequence identity to SEQ ID NO. 6. In one or more embodiments, the OX40L protein has at least 95% sequence identity according to SEQ ID NO. 6 or a homologue thereof with at least 95 % sequence identity to SEQ ID NO. 6. Inone or more embodiments, the OX40L protein has at least 99% sequence identity according to SEQ ID NO. 6 or a homologue thereof with at least 99 % sequence identity to SEQ ID NO. 6. In one or more embodiments, the OX40L protein is SEQ ID NO. 6.

[0131] 4-1BBL

[0132] 4-1 BBL (Gene ID: 8744), also known as CD137L is a type 2 transmembrane glycoprotein receptor that is found on APCs and binds to 4-1 BB (also known as CD137). The 4-1BB / 4-1BBL complex belongs to the TNFR: TNF superfamily, which is expressed on activated T Lymphocytes. The 4-1BB / 4-1BBL complex together with a signal provided by a T-cell receptor can provide costimulatory signals to CD4+ and CD8+ T cells in mice, leading to the activation of CD4+ and CD8+ T cells. One or more aspects of the present invention relates to a 4-1 BBL protein encoded by the nucleic acid sequence according to SEQ ID NO. 7 or a homologue thereof with at least 80 % sequence identity to SEQ ID NO. 7. This homologue can be with at least 80 % sequence identity to SEQ ID NO. 7. This homologue can be with at least 90 % sequence identity to SEQ ID NO. 7. This homologue can be with at least 90 % sequence identity to SEQ ID NO. 7.

[0133] In one or more embodiments, the 4-1 BBL protein is encoded by a nucleic acid sequence with at least 80 % sequence identity according to SEQ ID NO. 7 or a homologue thereof with at least 80 % sequence identity to SEQ ID NO. 7. In one or more embodiments, the 4-1 BBL protein is encoded by a nucleic acid sequence with at least 85 % sequence identity according to SEQ ID NO. 7 or a homologue thereof with at least 85 % sequence identity to SEQ ID NO. 7. In one or more embodiments, the 4-1 BBL protein is encoded by a nucleic acid sequence with at least 90 % sequence identity according to SEQ ID NO. 7 or a homologue thereof with at least 90 % sequence identity to SEQ ID NO. 7. In one or more embodiments, the 4-1 BBL protein is encoded by a nucleic acid sequence with at least 95 % sequence identity according to SEQ ID NO. 7 or a homologue thereof with at least 95 % sequence identity to SEQ ID NO. 7. In one or more embodiments, the 4-1BBL protein is encoded by a nucleic acid sequence with at least 99 % sequence identity according to SEQ ID NO. 7 or a homologue thereof with at least 99 % sequence identity to SEQ ID NO. 7. In one or more embodiments, the 4-1 BBL protein is encoded by a nucleic acid sequence according to SEQ ID NO. 7.

[0134] One or more aspects of the present invention relates to a 4-1 BBL protein according to SEQ ID NO.

[0135] 8 or a homologue thereof with at least 80 % sequence identity to SEQ ID NO. 8. This homologue can be with at least 80 % sequence identity to SEQ ID NO. 8. This homologue can be with at least 90 % sequence identity to SEQ ID NO. 8. This homologue can be with at least 90 % sequence identity to SEQ ID NO. 8.In one or more embodiments, the 4-1 BBL protein has at least 80% sequence identity according to SEQ ID NO. 8 or a homologue thereof with at least 80 % sequence identity to SEQ ID NO. 8. In one or more embodiments, the 4-1 BBL protein has at least 85% sequence identity according to SEQ ID NO. 8 or a homologue thereof with at least 85 % sequence identity to SEQ ID NO. 8. In one or more embodiments, the 4-1 BBL protein has at least 90% sequence identity according to SEQ ID NO. 8 or a homologue thereof with at least 90 % sequence identity to SEQ ID NO. 8. In one or more embodiments, the 4-1 BBL protein has at least 95% sequence identity according to SEQ ID NO. 8 or a homologue thereof with at least 95 % sequence identity to SEQ ID NO. 8. In one or more embodiments, the 4-1 BBL protein has at least 99% sequence identity according to SEQ ID NO. 8 or a homologue thereof with at least 99 % sequence identity to SEQ ID NO. 8. In one or more embodiments, the 4-1 BBL protein is SEQ ID NO. 8.

[0136] A membrane bound receptor with affinity toward OKT-3

[0137] Membrane bound receptors are inserted into the plasma membrane where they serve as receptors for antigen, examples of which include surface-bound IgD or IgM antibodies. OKT-3 is a murine monoclonal antibody of the immunoglobulin lgG2a isotype. The target of OKT-3, CD3, is a 17-20 kilodalton (kD) molecule that is part of a multimolecular complex found only on mature T cells and medullary thymocytes. This complex is uniquely situated next to the T-cell receptor for antigen. OKT-3 activates the T cell receptor and stimulates expansion. In the context of the present disclosure, the terms ‘OKT3’ and ‘OKT-3’ may be used interchangeably.

[0138] A membrane bound receptor, potentially an antibody, with affinity for other antibodies directed against CD3. The antibodies that bind CD3, a part of the TOR where binding causes activation of the TOR, can be OKT-3, or a humanized version of OKT-3, or UCHT1, 12F6, otelixizumab, foralumab.

[0139] A membrane bound receptor with affinity towards CD3. Potentially an antibody. The receptor serves to bind to CD3, that is a co-receptor of the TOR. Binding of CD3 by the receptor results in activation of the TOR that stimulates expansion of T cells. Could be a membrane bound version of OKT-3, UCHT1, 12F6, otelixizumab, foralumab.

[0140] In one aspect, the artificial feeder cell comprises at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules selected from a group consisting of CD64, CD32, CD86, CD80, OX40L, a membrane bound receptor with affinity towards OKT-3 and / or 4-1 BBL. In a further aspect, the artificial feeder cell comprises at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules selected from a group consisting of CD64 and / or CD86.In one or more embodiments, the artificial feeder cell comprises at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules selected from a group consisting of CD64 and / or CD86 and / or CD32.

[0141] In one or more embodiments, the artificial feeder cell comprises at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules selected from a group consisting of CD64 and / or CD86 and / or CD80.

[0142] In one or more embodiments, the artificial feeder cell comprises at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules selected from a group consisting of CD64 and / or CD86 and / or OX40L.

[0143] In one or more embodiments, the artificial feeder cell comprises at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules selected from a group consisting of CD64 and / or CD86 and / or a membrane bound receptor with affinity towards OKT-3.

[0144] In one or more embodiments, the artificial feeder cell comprises at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules selected from a group consisting of CD64 and / or CD86 and / or 4-1 BBL.

[0145] In one or more embodiments, the artificial feeder cell comprises at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules selected from a group consisting of CD64 and / or 4-1 BBL and / or OX40L and / or CD86. This can e.g., be seen in example 4.

[0146] In one or more embodiments, the artificial feeder cell is a myeloid cell comprising at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules selected from a group consisting of CD64, CD32, CD86, CD80, OX40L, a membrane bound receptor with affinity towards OKT-3 and / or 4-1 BBL.

[0147] In one or more embodiments, the artificial feeder cell is a myeloid cell comprising at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules selected from a group consisting of CD64 and / or CD86.

[0148] In one or more embodiments, the artificial feeder cell is a myeloid cell comprising at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules selected from a group consisting of CD64 and / or CD86 and / or CD32.

[0149] In one or more embodiments, the artificial feeder cell is a myeloid cell comprising at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules selected from a group consisting of CD64 and / or CD86 and / or CD80.In one or more embodiments, the artificial feeder cell is a myeloid cell comprising at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules selected from a group consisting of CD64 and / or CD86 and / or OX40L.

[0150] In one or more embodiments, the artificial feeder cell is a myeloid cell comprising at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules selected from a group consisting of CD64 and / or CD86 and / or a membrane bound receptor with affinity towards OKT-3. In one or more embodiments, the artificial feeder cell is a myeloid cell comprising at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules selected from a group consisting of CD64 and / or CD86 and / or 4-1 BBL.

[0151] In one or more embodiments, the artificial feeder cell is a myeloid cell comprising at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules selected from a group consisting of CD64 and / or 4-1 BBL and / or OX40L and / or CD86.

[0152] In one or more embodiments, the artificial feeder cell is a myeloid cell selected from a group consisting of K562, EM-2, EM-3, MOLM-13, MOLM-14, comprising at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules selected from a group consisting of CD64, CD32, CD86, CD80, OX40L, a membrane bound receptor with affinity towards OKT-3 and / or 4-1BBL.

[0153] In one or more embodiments, the artificial feeder cell is a myeloid cell selected from a group consisting of K562, EM-2, EM-3, MOLM-13, MOLM-14, comprising at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules selected from a group consisting of CD64 and / or CD86.

[0154] In one or more embodiments, the artificial feeder cell is a myeloid cell selected from a group consisting of K562, EM-2, EM-3, MOLM-13, MOLM-14, comprising at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules selected from a group consisting of CD64 and / or CD86 and / or CD32.

[0155] In one or more embodiments, the artificial feeder cell is a myeloid cell selected from a group consisting of K562, EM-2, EM-3, MOLM-13, MOLM-14, comprising at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules selected from a group consisting of CD64 and / or CD86 and / or CD80.

[0156] In one or more embodiments, the artificial feeder cell is a myeloid cell selected from a group consisting of K562, EM-2, EM-3, MOLM-13, MOLM-14, comprising at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules selected from a group consisting of CD64 and / or CD86 and / or OX40L.In one or more embodiments, the artificial feeder cell is a myeloid cell selected from a group consisting of K562, EM-2, EM-3, MOLM-13, MOLM-14, comprising at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules selected from a group consisting of CD64 and / or CD86 and / or a membrane bound receptor with affinity towards OKT-3.

[0157] In one or more embodiments, the artificial feeder cell is a myeloid cell selected from a group consisting of K562, EM-2, EM-3, MOLM-13, MOLM-14, comprising at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules selected from a group consisting of CD64 and / or CD86 and / or 4-1 BBL.

[0158] In one or more embodiments, the artificial feeder cell is a myeloid cell selected from a group consisting of K562, EM-2, EM-3, MOLM-13, MOLM-14 comprising at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules selected from a group consisting of CD64 and / or 4-1 BBL and / or OX40L and / or CD86.

[0159] In one or more embodiments, the artificial feeder cell is a K562 cell comprising at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules selected from a group consisting of CD64, CD32, CD86, CD80, OX40L, a membrane bound receptor with affinity towards OKT-3 and / or 4-1 BBL.

[0160] In one or more embodiments, the artificial feeder cell is a K562 cell comprising at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules selected from a group consisting of CD64 and / or CD86.

[0161] In one or more embodiments, the artificial feeder cell is a K562 cell comprising at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules selected from a group consisting of CD64 and / or CD86 and / or CD32.

[0162] In one or more embodiments, the artificial feeder cell is a K562 cell comprising at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules selected from a group consisting of CD64 and / or CD86 and / or CD80.

[0163] In one or more embodiments, the artificial feeder cell is a K562 cell comprising at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules selected from a group consisting of CD64 and / or CD86 and / or OX40L.

[0164] In one or more embodiments, the artificial feeder cell is a K562 cell comprising at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules selected from a group consisting of CD64 and / or CD86 and / or a membrane bound receptor with affinity towards OKT-3. In one or more embodiments, the artificial feeder cell is a K562 cell comprising at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules selected from a group consisting of CD64 and / or CD86 and / or 4-1 BBL.In one or more embodiments, the artificial feeder cell is a cell selected from a group consisting of K562, EM-2, EM-3, MOLM-13, MOLM-14 comprising at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules selected from a group consisting of CD64 and / or 4-1BBL and / or OX40L and / or CD86.

[0165] In one or more embodiments, the artificial feeder cell is a cell selected from a group consisting of K562, EM-2, EM-3, MOLM-13, MOLM-14 comprising at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules selected from a group consisting of CD64 and 4-1BBL and OX40L and CD86.

[0166] Promotors

[0167] In an aspect of the invention, the artificial feeder cell comprises at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules selected from the group consisting of: CD64, CD32, CD86, CD80, OX40L, a membrane bound receptor with affinity toward OKT-3 and / or 4-1 BBL, wherein the at least one or more nucleic acid molecules comprises at least one promotor. The term promotor designates DNA sequences which provide a site for initiation of the transcription into mRNA. Promotors usually lie upstream of (i.e. preceding) a gene in a DNA sequence. In the present context, promotors may be naturally occurring sequences derived from different species, naturally occurring or synthetic DNA sequences which have been modified and / or genetically engineered, and / or derived from DNA sequences that do not naturally occur in nature. Examples of promotors include SFFV promotors, CMV promotors, mCMV promotors, EF-1 promotors and UbiC promotors. The terms ‘promotor’ and ‘promoter’ may be used interchangeably.

[0168] In the context of the present invention, the SFFV and CMV promotors are constitutively active and provide high-level transcriptional activity, resulting in robust and rapid expression of the encoded gene. Such promoters are particularly advantageous where maximal protein production is desired. Promoters such as EF-1 and UbiC provide stable and uniform expression across diverse cell types, including dividing and non-dividing cells. The mCMV promoter provide moderate constitutive expression with reduced promoter strength compared to full-length CMV, allowing improved balance between expression level and cellular viability, and reducing cellular stress or potential toxicity associated with excessive expression. An SFFV promotor is a promotor derived from the spleen focus forming virus, and / or a homologue thereof with at least 50%, such as 60%, such as 70%, such as 75%, such as 80%, such as 85%, such as 90%, such as 95%, such as 99%, such as 99.9% or such as 100% identity to SEQ ID NO: 9.

[0169] A CMV promotor is a promotor derived from cytomegalovirus, such as a human cytomegalovirus, and / or a homologue thereof with at least 50%, such as 60%, such as 70%, such as 75%, such as 80%, such as 85%, such as 90%, such as 95%, such as 99%, such as 99.9% or such as 100% identity to SEQ ID NO: 10.An mCMV promotor is a promotor derived from a murine cytomegalovirus, and / or a homologue thereof with at least 50%, such as 60%, such as 70%, such as 75%, such as 80%, such as 85%, such as 90%, such as 95%, such as 99%, such as 99.9% or such as 100% identity to SEQ ID NO: 11.

[0170] An EF-1 promotor is the promotor of human elongation factor (EF-1) , and / or a homologue thereof with at least 50%, such as 60%, such as 70%, such as 75%, such as 80%, such as 85%, such as 90%, such as 95%, such as 99%, such as 99.9% or such as 100% identity to SEQ ID NOs: 12 or 13. Examples of EF-1 promotors include an EF1a promotor, such as an EF1a-1 and / or EF1a-2 promotor. In the present context, further examples of an EF-1 promotor include an EF-1 core promotor, and / or a homologue thereof with at least 50%, such as 60%, such as 70%, such as 75%, such as 80%, such as 85%, such as 90%, such as 95%, such as 99%, such as 99.9% or such as 100% identity to SEQ ID NO: 12 and / or an EF-1 hybrid promotor, and / or a homologue thereof with at least 50%, such as 60%, such as 70%, such as 75%, such as 80%, such as 85%, such as 90%, such as 95%, such as 99%, such as 99.9% or such as 100% identity to SEQ ID NO: 13.

[0171] A UbiC promotor is the promotor of Ubiquitin C, such as human UbiC promotor, and / or a homologue thereof with at least 50%, such as 60%, such as 70%, such as 75%, such as 80%, such as 85%, such as 90%, such as 95%, such as 99%, such as 99.9% or such as 100% identity to SEQ ID NO: 14.

[0172] Sequence identity

[0173] The term "sequence identity" as used herein describes the relatedness between two amino acid sequences or between two nucleotide sequences, i.e., a candidate sequence (e.g., a nucleic acid sequence encoding an alkaline phosphatase) and a reference sequence (such as a prior art sequence) based on their pairwise alignment. For purposes disclosed herein, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mo / . Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277,), preferably version 5.0.0 or later (available at https: / / www.ebi.ac.uk / Tools / psa / emboss needle / ). The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of 30 BLOSUM62) substitution matrix. The output of Needle labeled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: (Identical Residues x 100) / (Length of Alignment - Total Number of Gaps in Alignment).

[0174] For purposes disclosed herein, the sequence identity between two nucleotide sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1 970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European MolecularBiology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), 10 preferably version 5.0.0 or later. The parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the DNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The output of Needle labelled "longest identity" (obtained using the -nobrief option) is used as the percent identity and is calculated as follows: (Identical Deoxyribonucleotides x 100) / (Length of Alignment — Total Number of Gaps in Alignment).

[0175] Homologue

[0176] A homologue of a nucleic acid sequence as described herein is a nucleic acid molecule with alterations in the genetic code, which retain its original functionality. A homologue may be obtained by mutagenesis or may be natural occurring variants from the same or other species. The homologue should have a remaining functionality of at least 50%, at least 60%, at least 70%, at least 80 %, at least 90% or 100% compared to the functionality of the nucleic acid molecule.

[0177] A homologue of any one of the nucleic acid sequences can also have a higher functionality. In the case of promotors, a homologue of any one of the nucleic acid sequences, as disclosed herein, should ideally be able to exercise expression of the one or more co-stimulatory molecules.

[0178] In an aspect of the invention, the artificial feeder cell comprises at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules selected from the group consisting of: CD64, CD32, CD86, CD80, OX40L, a membrane bound receptor with affinity toward OKT-3 and / or 4-1 BBL, wherein the at least one or more nucleic acid molecules comprises at least one promotor.

[0179] In a further aspect of the invention, the artificial feeder cell comprises at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules selected from the group consisting of: CD64 and / or CD86, wherein the at least one or more nucleic acid molecules comprises at least one promotor.

[0180] In a further aspect of the invention, the artificial feeder cell comprises at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules selected from the group consisting of: CD64 and / or CD86, wherein the at least one or more nucleic acid molecules comprises at least one promotor.

[0181] In a further aspect of the invention, the artificial feeder cell comprises at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules selected from the group consisting of: CD64 and / or CD86 and / or 4-1 BBL and / or OX40L, wherein the at least one or more nucleic acid molecules comprises at least one promotor.Thus, in one or more embodiments, the at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a SFFV promotor or a homologue thereof with at least 70% sequence identity to SEQ ID NO: 9.

[0182] In one or more embodiments, the at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a SFFV promotor or a homologue thereof with at least 75% sequence identity to SEQ ID NO: 9.

[0183] In one or more embodiments, the at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a SFFV promotor or a homologue thereof with at least 80% sequence identity to SEQ ID NO: 9.

[0184] In one or more embodiments, the at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a SFFV promotor or a homologue thereof with at least 85% sequence identity to SEQ ID NO: 9.

[0185] In one or more embodiments, the at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a SFFV promotor or a homologue thereof with at least 90% sequence identity to SEQ ID NO: 9.

[0186] In one or more embodiments, the at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a SFFV promotor or a homologue thereof with at least 95% sequence identity to SEQ ID NO: 9.

[0187] In one or more embodiments, the at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a SFFV promotor or a homologue thereof with at least 99% sequence identity to SEQ ID NO: 9.

[0188] In one or more embodiments, the at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is SEQ ID NO: 9.

[0189] In one or more embodiments, the at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a CMV promotor or a homologue thereof with at least 70% sequence identity to SEQ ID NO: 10.

[0190] In one or more embodiments, the at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a CMV promotor or a homologue thereof with at least 75% sequence identity to SEQ ID NO: 10.In one or more embodiments, the at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a CMV promotor or a homologue thereof with at least 80% sequence identity to SEQ ID NO: 10.

[0191] In one or more embodiments, the at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a CMV promotor or a homologue thereof with at least 85% sequence identity to SEQ ID NO: 10.

[0192] In one or more embodiments, the at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a CMV promotor or a homologue thereof with at least 90% sequence identity to SEQ ID NO: 10.

[0193] In one or more embodiments, the at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a CMV promotor or a homologue thereof with at least 95% sequence identity to SEQ ID NO: 10.

[0194] In one or more embodiments, the at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a CMV promotor or a homologue thereof with at least 99% sequence identity to SEQ ID NO: 10.

[0195] In one or more embodiments, the at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is SEQ ID NO: 10.

[0196] In one or more embodiments, the at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a mCMV promotor or a homologue thereof with at least 70% sequence identity to SEQ ID NO: 11.

[0197] In one or more embodiments, the at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a mCMV promotor or a homologue thereof with at least 75% sequence identity to SEQ ID NO: 11.

[0198] In one or more embodiments, the at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a mCMV promotor or a homologue thereof with at least 80% sequence identity to SEQ ID NO: 11.

[0199] In one or more embodiments, the at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a mCMV promotor or a homologue thereof with at least 85% sequence identity to SEQ ID NO: 11.In one or more embodiments, the at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a mCMV promotor or a homologue thereof with at least 90% sequence identity to SEQ ID NO: 11.

[0200] In one or more embodiments, the at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a mCMV promotor or a homologue thereof with at least 95% sequence identity to SEQ ID NO: 11.

[0201] In one or more embodiments, the at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a mCMV promotor or a homologue thereof with at least 99% sequence identity to SEQ ID NO: 11.

[0202] In one or more embodiments, the at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is SEQ ID NO: 11.

[0203] In one or more embodiments, the at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a EF-1 promotor or a homologue thereof with at least 70% sequence identity to SEQ ID NOs: 12 or 13.

[0204] In one or more embodiments, the at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a EF-1 promotor or a homologue thereof with at least 75% sequence identity to SEQ ID NOs: 12 or 13.

[0205] In one or more embodiments, the at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a EF-1 promotor or a homologue thereof with at least 80% sequence identity to SEQ ID NOs: 12 or 13.

[0206] In one or more embodiments, the at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a EF-1 promotor or a homologue thereof with at least 85% sequence identity to SEQ ID NOs: 12 or 13.

[0207] In one or more embodiments, the at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a EF-1 promotor or a homologue thereof with at least 90% sequence identity to SEQ ID NOs: 12 or 13.

[0208] In one or more embodiments, the at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a EF-1 promotor or a homologue thereof with at least 95% sequence identity to SEQ ID NOs: 12 or 13.In one or more embodiments, the at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a EF-1 promotor or a homologue thereof with at least 99% sequence identity to SEQ ID NOs: 12 or 13.

[0209] In one or more embodiments, the at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is one of SEQ ID NOs: 12 or 13.

[0210] In one or more embodiments, the at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a UbiC promotor or a homologue thereof with at least 70% sequence identity to SEQ ID NO: 14.

[0211] In one or more embodiments, the at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a UbiC promotor or a homologue thereof with at least 75% sequence identity to SEQ ID NO: 14.

[0212] In one or more embodiments, the at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a UbiC promotor or a homologue thereof with at least 80% sequence identity to SEQ ID NO: 14.

[0213] In one or more embodiments, the at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a UbiC promotor or a homologue thereof with at least 85% sequence identity to SEQ ID NO: 14.

[0214] In one or more embodiments, the at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a UbiC promotor or a homologue thereof with at least 90% sequence identity to SEQ ID NO: 14.

[0215] In one or more embodiments, the at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a UbiC promotor or a homologue thereof with at least 95% sequence identity to SEQ ID NO: 14.

[0216] In one or more embodiments, the at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is a UbiC promotor or a homologue thereof with at least 99% sequence identity to SEQ ID NO: 14.

[0217] In one or more embodiments, the at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is SEQ ID NO: 14.

[0218] Expression systemsThe term expression system as used herein refers to a system designed for the production and / or expression of proteins within a cell and / or on its surface, such as cell surface markers, such as costimulatory molecules, including CD64, CD32, CD86, CD80, OX40L, a membrane bound receptor with affinity toward OKT-3 and / or 4-1 BBL. Expression systems can be used to genetically modify cells, such as the artificial feeder cell of the present invention. Examples of expression systems include viral vectors and / or viral-free systems such as transposon-mediated gene transfer systems and the use of genome editing (e.g. CRISPR).

[0219] In one or more embodiments, the expression system is selected from the group consisting of one or more viral vectors, transposon-mediated gene transfer system(s) or genome-editing based expression system(s), such as CRISPR-based expression system(s).

[0220] In one or more embodiments, the expression system is one or more viral vectors. In one or more embodiments, the expression system is transposon-mediated gene transfer system(s). In one or more embodiments, the expression system is genome-editing based expression system(s). In one or more embodiments, the expression system is CRISPR-based expression system(s).

[0221] Viral vectors

[0222] Viral vectors, as used herein, refer to modified viruses designed to deliver genetic material into cells. This process can be performed inside an organism or in cell culture. Viral vectors may be transduced, the process by which foreign nucleic acid sequence(s) is / are introduced into a cell by a virus or viral vector. Such transduction may or may not be stable. Stable transduction as defined herein refers to the introduction of foreign nucleic acid sequences into a cell by a virus or viral vector, wherein the foreign nucleic acid sequences is integrated into the genome of said cell, and is incorporated in the genome of at least one future generation / passage of said cell. Examples of foreign nucleic acid sequences include nucleic acid sequence(s) encoding one or more co-stimulatory molecules of the present invention. Viral vector and / or transduction systems are known in the art and are described, e.g., in Levine, et al., Proc. Nat'l Acad. Sci. 2006, 103, 17372-77; Zufferey, et al., Nat. Biotechnol. 1997, 15, 871-75; Dull, et al., J. Virology 1998, 72, 8463-71, and include well-known systems such as lentiviruses and / or a lentiviral transduction systems, and / or gamma-retroviral transduction systems (e.g., Cepko and Pear, Cur. Prot. Mol. Biol. 1996, 9.9.1-9.9.16). Examples of a viral vector in the present invention include the use of a lentiviral transduction system encoding the one or more co-stimulatory molecules described herein.

[0223] Transposon-mediated gene transfer systems

[0224] Transposon-mediated gene transfer systems are known in the art and include systems wherein the transposase is provided as DNA expression vector or as an expressible RNA or a protein such that long-term expression of the transposase does not occur in the transgenic cells, for example, atransposase provided as an mRNA (e.g., an mRNA comprising a cap and poly-A tail). Suitable transposon-mediated gene transfer systems, including the salmonid-type Tel-like transposase (SB or Sleeping Beauty transposase), such as SB10, SB11, and SB100*, PiggyBac transposase, and engineered enzymes with increased enzymatic activity, are described in, e.g., Hackett, et al., Mol. Therapy 2010, 18, 674-83.

[0225] Genome editing-based expression systems

[0226] Genome editing-based expression systems are well known in the art, wherein gene editing is utilized to integrate a foreign nucleic acid sequence(s) into a target genome. For example, integration of a foreign nucleic acid sequence(s) into a target genome in the present invention, may the integration of nucleic acid sequences encoding one or more co-stimulatory molecules into the genome of the artificial feeder cell of the present invention. Examples of genome editing-based expression systems include the use of CRISPR, zinc-finger nucleases, non-homologous end joining (NHEJ) pathways, homology-directed repair (HDR) pathways such as homologous recombination. As used herein, the term “CRISPR-based expression system” refers to a genome editing-based expression system wherein CRISPR is used.

[0227] Thus, in an aspect of the present invention, the artificial feeder cell comprises an expression system comprising the at least one or more nucleic acid molecules encoding one or more costimulatory molecules, such as CD64, CD32, CD86, CD80, OX40L, a membrane bound receptor with affinity toward OKT-3 and / or 4-1 BBL.

[0228] In one or more embodiments, the expression system comprises one or more viral vectors, such as one, two, three, four, five, six or seven viral vectors. In one or more embodiments, the expression system comprises one viral vector. In one or more embodiments, the expression system comprises two viral vectors. In one or more embodiments, the expression system comprises three viral vectors. In one or more embodiments, the expression system comprises one viral vector. In one or more embodiments, the expression system comprises four viral vectors. In one or more embodiments, the expression system comprises one viral vector. In one or more embodiments, the expression system comprises five viral vectors. In one or more embodiments, the expression system comprises one viral vector. In one or more embodiments, the expression system comprises six viral vectors. In one or more embodiments, the expression system comprises one viral vector. In one or more embodiments, the expression system comprises seven viral vectors.

[0229] In one or more embodiments, the one or more viral vectors is / are transduced.

[0230] In one or more embodiments, the one or more viral vectors is / are stably transduced.

[0231] In one or more embodiments, the one or more viral vectors comprises one or more lentivirus(es) and / or lentiviral transduction system(s). In one or more embodiments, the one or more viral vectorscomprises one or more lentivirus(es). In one or more embodiments, the one or more viral vectors comprises one or more lentiviral transduction system(s). In one or more embodiments, the one or more viral vectors comprises one or more lentivirus(es) and lentiviral transduction system(s). In one or more embodiments, the one or more lentivirus(es) and / or lentiviral transduction system(s) is / are transduced. In one or more embodiments, the one or more lentivirus(es) is / are transduced. In one or more embodiments, the one or more lentiviral transduction system(s) is / are transduced. In one or more embodiments, the one or more lentivirus(es) and lentiviral transduction system(s) is / are transduced.

[0232] In one or more embodiments, the one or more lentivirus(es) and / or lentiviral transduction system(s) is / are stably transduced. In one or more embodiments, the one or more lentivirus(es) is / are stably transduced. In one or more embodiments, the one or more lentiviral transduction system(s) is / are stably transduced. In one or more embodiments, the one or more lentivirus(es) and lentiviral transduction system(s) is / are stably transduced.

[0233] In one or more embodiments, the one or more viral vectors comprises one or more gamma-retroviral transduction system(s).

[0234] In one or more embodiments, the one or more gamma-retroviral transduction system(s) is / are transduced.

[0235] In one or more embodiments, the one or more gamma-retroviral transduction system(s) is / are stably transduced.

[0236] In one or more embodiments, the expression system comprises one or more transposon-mediated gene transfer system(s).

[0237] In one or more embodiments, the expression system comprises one or more transposon-mediated gene transfer system(s) and the at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules are integrated into the genome of the artificial feeder cell.

[0238] In one or more embodiments, the expression system comprises one or more transposon-mediated gene transfer system(s) and the at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules are stably integrated into the genome of the artificial feeder cell.

[0239] In one or more embodiments, the expression system comprising the one or more transposon-mediated gene transfer system(s) comprises Sleeping Beauty transposase(s), such as SB10, SB11, and SB100*.

[0240] In one or more embodiments, the Sleeping Beauty transposase(s) is SB10. In one or more embodiments, the Sleeping Beauty transposase(s) is SB11. In one or more embodiments, the Sleeping Beauty transposase(s) is SB100*. In one or more embodiments, the Sleeping Beautytransposase(s) comprises SB10. In one or more embodiments, the Sleeping Beauty transposase(s) comprises SB11. In one or more embodiments, the Sleeping Beauty transposase(s) comprises SB100*. In one or more embodiments, the Sleeping Beauty transposase(s) comprises SB10, SB11, and SB100*. In one or more embodiments, the Sleeping Beauty transposase(s) comprises SB10 and SB11. In one or more embodiments, the Sleeping Beauty transposase(s) comprises SB10, and SB100*.

[0241] In one or more embodiments, the expression system comprising the one or more transposon-mediated gene transfer system(s) comprises Sleeping Beauty transposase(s), such as SB10, SB11, and SB100* and the at least one or more nucleic acid molecules encoding one or more costimulatory molecules are integrated into the genome of the artificial feeder cell.

[0242] In one or more embodiments, the expression system comprising the one or more transposon-mediated gene transfer system(s) comprises Sleeping Beauty transposase(s), such as SB10, SB11, and SB100* and the at least one or more nucleic acid molecules encoding one or more costimulatory molecules are stably integrated into the genome of the artificial feeder cell.

[0243] In one or more embodiments, the expression system comprising the one or more transposon-mediated gene transfer system(s) comprises PiggyBac transposase(s) and the at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules are integrated into the genome of the artificial feeder cell.

[0244] In one or more embodiments, the one or more transposon-mediated gene transfer system(s) comprises PiggyBac transposase(s).

[0245] In one or more embodiments, the expression system comprising the one or more transposon-mediated gene transfer system(s) comprises PiggyBac transposase(s) and the at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules are stably integrated into the genome of the artificial feeder cell.

[0246] In one or more embodiments, the expression system comprises one or more genome editingbased expression systems.

[0247] In one or more embodiments, the expression system comprises one or more genome editingbased expression systems and the at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules are integrated into the genome of the artificial feeder cell.

[0248] In one or more embodiments, the expression system comprises one or more genome editingbased expression systems and the at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules are stably integrated into the genome of the artificial feeder cell. In one or more embodiments, the one or more genome editing-based expression system(s) comprises one or more CRISPR-based expression system(s) and the at least one or more nucleicacid molecules encoding one or more co-stimulatory molecules are integrated into the genome of the artificial feeder cell.

[0249] In one or more embodiments, the one or more genome editing-based expression system(s) comprises one or more CRISPR-based expression system(s) and the at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules are stably integrated into the genome of the artificial feeder cell.

[0250] Myeloid cell

[0251] The term "myeloid cell" as used herein refers to cells of the myeloid lineage or derived therefrom. The myeloid lineage includes a number of morphologically, phenotypically, and functionally distinct cell types including different subsets of granulocytes (neutrophils, eosinophils, and basophils), monocytes, macrophages, erythrocytes, megakaryocytes, and mast cells. Examples of myeloid cells in the present invention include K562 cells, EM-3 cells, EM-2 cells, MOLM-14 cells and / or MOLM-13 cells.

[0252] K562 cells

[0253] “K562” refers to a human leukemia cell line which was established from the bone marrow of a chronic myelogenous leukemia patient, and initial phenotypic characterization indicated the presence of at least the following markers: CD7, CD58. The K562 cell line is deposited at DSMZ under Accession No. ACC10. As used herein the term "K562 cell" refers to a K562 cell and / or a cell derived from the deposited K562 parental cell line.

[0254] EM-3 and EM-2 cells

[0255] "EM-3" refers to a human cell line was established from the bone marrow of a patient with Philadelphia chromosome-positive CIVIL. Konopka, et al., Proc. Nat'l Acad. Sci. USA 1985, 82, 1810-4. Phenotypic characterization for EM-3 cells indicates the presence of at least the following markers: CD13, CD15, and CD33. The EM-3 cell line is deposited at DSMZ under Accession No. ACC134. As used herein the term “EM-3 cell” refers to an EM-3 cell and / or cell derived from the deposited EM-3 parental cell line. The closely related EM-2 cell line is deposited at DSMZ under Accession No. ACC135. As used herein the term “EM-2 cell” refers to a EM-2 cell and / or a cell derived from the deposited EM-2 parental cell line.

[0256] MOLM-14 and MOLM-13 cells

[0257] "MOLM-14" refers to a human leukemia cell line which was established from the peripheral blood of a patient with relapsed acute monocytic leukemia, and initial phenotypic characterization indicated the presence of at least the following markers: CD4, CD9, CD1la, CD13, CD14, CD15, CD32, CD33, CD64, CD65, CD87, CD92, CD93, CD116, CD118, and CD155. Matsuo, et al., Leukemia 1997, 11 1469-77. Additional phenotypic characterization of MOLM-14 found higher levels of HLA-A / B / C, CD64, CD80, ICOS-L, CD58, and lower levels of CD86. The MOLM-14 cell line is deposited at DSMZunder Accession No. ACC777. The closely related MOLM-13 cell line is deposited at DSMZ under Accession No. ACC554. As used herein the term "MOLM-14 cell" refers to a MOLM-14 cell and / or a cell derived from the deposited MOLM-14 parental cell line. As used herein the term “MOLM-13 cell” refers to a MOLM-13 cell and / or cell derived from the deposited MOLM-13 parental cell line.

[0258] Thus, in one or more embodiments, the artificial feeder cell is a myeloid cell.

[0259] In one or more embodiments, the myeloid cell is selected from the group consisting of: K562, EM-3, EM-2, MOLM-13, MOLM-14.

[0260] In one or more embodiments, the myeloid cell is a K562 cell.

[0261] In one or more embodiments, the myeloid cell is a EM-3 cell.

[0262] In one or more embodiments, the myeloid cell is a EM-2 cell.

[0263] In one or more embodiments, the myeloid cell is a MOLM-13 cell.

[0264] In one or more embodiments, the myeloid cell is a MOLM-14 cell.

[0265] Culture medium

[0266] The term ‘culture medium’ is meant to describe a solid, liquid, or semi-solid designed to support the growth of a population of microorganisms or cells via the process of cell proliferation. Cell culture mediums often include additional compounds to facilitate cell growth and proliferation. For example, such compounds include hormones, cytokines, sera and / or growth factors. Cell culture mediums may also include reduced serum or may be serum-free. Examples of such compounds include IL-2, IL-21, IL-7, IL-12, IL-15 and / or any combinations thereof, anti-CD3 antibody, TME stimulators from the group of “Inhibitors” as defined herein. For example, the culture medium according to the present invention may also comprise one or more artificial feeder cell(s) according to the present invention. The terms ‘culture medium’ and ‘cell culture medium’ can be used interchangeably.

[0267] In an aspect of the present invention, a culture medium comprises the one or more artificial feeder cell(s) according to the present invention.

[0268] Addition of additional compounds such as those defined herein, may facilitate the growth and rate of expansion of the population of cells. These are described in the text and examples herein.In a further aspect of the present invention, a culture medium comprises the one or more artificial feeder cell(s) according to the present invention, wherein the culture medium comprises IL-2, IL-21, IL-7, IL-12, IL-15 and / or any combinations thereof.

[0269] In one or more embodiments, the culture medium comprises the one or more artificial feeder cell(s) according to the present invention.

[0270] In one or more embodiments, the culture medium comprises IL-2 and IL-21.

[0271] In one or more embodiments, the culture medium comprises IL-2 and IL-7.

[0272] In one or more embodiments, the culture medium comprises IL-2 and IL-12.

[0273] In one or more embodiments, the culture medium comprises IL-2 and IL-15.

[0274] In one or more embodiments, the culture medium comprises IL-2 and IL-21 and IL-7.

[0275] In one or more embodiments, the culture medium comprises IL-2 and IL-21 and IL-12.

[0276] In one or more embodiments, the culture medium comprises IL-2 and IL-21 and IL-15.

[0277] In one or more embodiments, the culture medium comprises IL-7 and IL-15 and IL-21.

[0278] In one or more embodiments, the culture medium is a serum-free medium.

[0279] In one or more embodiments, the culture medium comprises IL-2, and serum-free medium.

[0280] In one or more embodiments, the culture medium comprises IL-2, IL-21 and serum-free medium.

[0281] In one or more embodiments, the culture medium comprises IL-2, IL-21, IL-7, and serum-free medium.

[0282] In one or more embodiments, the culture medium is cryopreserved at a temperature of at least -70°C or colder. In one or more embodiments, the culture medium is cryopreserved at a temperature of at least -75°C or colder. In one or more embodiments, the culture medium is cryopreserved at a temperature of at least -80°C or colder. In one or more embodiments, the culture medium is cryopreserved at a temperature of at least -85°C or colder.In one or more embodiments, the artificial feeder cell comprises at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules selected from a group consisting of CD64, CD32, CD86, CD80, OX40L, a membrane bound receptor with affinity towards OKT-3 and / or 4-1 BBL and is cryopreserved. The CD64 mentioned herein is CD64 or a homologue thereof with at least 80% sequence identity to SEQ ID NO: 3. The CD32 mentioned herein is CD32 or a homologue thereof with at least 80% sequence identity to SEQ ID NO: 15 . The CD86 mentioned herein is CD86 or a homologue thereof with at least 80% sequence identity to SEQ ID NO: 1. The CD80 mentioned herein is CD80 or a homologue thereof with at least 80% sequence identity to SEQ ID NO: 17. The QX40L mentioned herein is QX40L or a homologue thereof with at least 80% sequence identity to SEQ ID NO: 5. The 4-1 BBL mentioned herein is 4-1 BBL or a homologue thereof with at least 80% sequence identity to SEQ ID NO: 7.

[0283] In one or more embodiments, the artificial feeder cell is cryopreserved and comprises at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules selected from a group consisting of CD64 and CD86. The CD64 mentioned herein is CD64 or a homologue thereof with at least 80% sequence identity to SEQ ID NO: 3. The CD86 mentioned herein is CD86 or a homologue thereof with at least 80% sequence identity to SEQ ID NO: 1.

[0284] In one or more embodiments, the artificial feeder cell is cryopreserved and comprises at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules selected from a group consisting of CD64 and / or 4-1 BBL and / or QX40L and / or CD86. This can e.g., be seen in example 4. The CD64 mentioned herein is CD64 or a homologue thereof with at least 80% sequence identity to SEQ ID NO: 3. The CD86 mentioned herein is CD86 or a homologue thereof with at least 80% sequence identity to SEQ ID NO: 1. The QX40L mentioned herein is QX40L or a homologue thereof with at least 80% sequence identity to SEQ ID NO: 5. The 4-1 BBL mentioned herein is 4-1 BBL or a homologue thereof with at least 80% sequence identity to SEQ ID NO: 7.

[0285] In one or more embodiments, the artificial feeder cell is a cryopreserved myeloid cell comprising at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules selected from a group consisting of CD64, CD32, CD86, CD80, QX40L, a membrane bound receptor with affinity towards OKT-3 and / or 4-1 BBL and is cryopreserved. The CD64 mentioned herein is CD64 or a homologue thereof with at least 80% sequence identity to SEQ ID NO: 3. The CD32 mentioned herein is CD32 or a homologue thereof with at least 80% sequence identity to SEQ ID NO: 15. The CD86 mentioned herein is CD86 or a homologue thereof with at least 80% sequence identity to SEQ ID NO: 1. The CD80 mentioned herein is CD80 or a homologue thereof with at least 80% sequence identity to SEQ ID NO: 17. The QX40L mentioned herein is QX40L or a homologue thereof with at least 80% sequence identity to SEQ ID NO: 5. The 4-1 BBL mentioned herein is 4-1 BBL or a homologue thereof with at least 80% sequence identity to SEQ ID NO: 7.In one or more embodiments, the artificial feeder cell is a cryopreserved myeloid cell comprising at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules selected from a group consisting of CD64 and CD86. The CD64 mentioned herein is CD64 or a homologue thereof with at least 80% sequence identity to SEQ ID NO: 3. The CD86 mentioned herein is CD86 or a homologue thereof with at least 80% sequence identity to SEQ ID NO: 1.

[0286] In one or more embodiments, the artificial feeder cell is a cryopreserved myeloid cell comprising at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules selected from a group consisting of CD64 and / or 4-1 BBL and / or OX40L and / or CD86. This can e.g., be seen in example 4. The CD64 mentioned herein is CD64 or a homologue thereof with at least 80% sequence identity to SEQ ID NO: 3. The CD86 mentioned herein is CD86 or a homologue thereof with at least 80% sequence identity to SEQ ID NO: 1. The QX40L mentioned herein is QX40L or a homologue thereof with at least 80% sequence identity to SEQ ID NO: 5. The 4-1 BBL mentioned herein is 4-1 BBL or a homologue thereof with at least 80% sequence identity to SEQ ID NO: 7.

[0287] Expansion of tumor infiltrating lymphocytes (TILs)

[0288] Method

[0289] An aspect of the present invention relates to a method for expanding cells into population of cells, that can be therapeutic, comprising: culturing autologous cells by obtaining a first population of cells from a tumor and / or tumor fragments resected from a mammal, performing a first expansion by culturing the depleted population of cells in a cell culture medium, and optionally adding the artificial feeder cells according to the present invention, and performing a second expansion by supplementing the cell culture medium of the second population of cells with;

[0290] - additional IL-2 or a combination of IL-7, IL-15 and IL-21,

[0291] - anti-CD3 antibody, and

[0292] - the artificial feeder cell(s) according to the present invention,

[0293] to produce a third population of cells, wherein the third population of cells, which can be a therapeutic population.

[0294] An aspect of the present invention relates to a method for expanding cells into a population of cells, that can be therapeutic, comprising:

[0295] a) culturing autologous cells by obtaining a first population of cells from a tumor and / or tumor fragments resected from a mammal,

[0296] b) performing a first expansion by culturing the first population of cells in a cell culture medium, and optionally adding the artificial feeder cells according to the present invention to produce a second population of cells,

[0297] c) performing a second expansion by supplementing the cell culture medium of the second population of cells comprising;- additional IL-2, a combination of IL-7, IL-15 and IL-21, or a combination of IL-2, IL-7 and IL-21,

[0298] - anti-CD3 antibody, and

[0299] - the artificial feeder cell(s) according to the present invention,

[0300] to produce a third population of cells, optionally wherein the third population of cells is a therapeutic population.

[0301] One or more embodiments of the present invention relates to the method of the present invention, wherein the culture medium of step b) comprises the culture medium of the present invention.

[0302] In one or more embodiments, the second expansion by supplementing the cell culture medium of the second population of cells comprises additional IL-2. In one or more embodiments, the second expansion by supplementing the cell culture medium of the second population of cells comprises a combination of IL-7, IL-15 and IL-21. In one or more embodiments, the second expansion by supplementing the cell culture medium of the second population of cells comprises a combination of IL-2, IL-7 and IL-21.

[0303] In one or more embodiments, the second expansion by supplementing the cell culture medium of the second population of cells comprises anti-CD3 antibody.

[0304] In one or more embodiments, the second expansion by supplementing the cell culture medium of the second population of cells comprises the artificial feeder cell(s) according to the present invention.

[0305] In one or more embodiments, the third population of cells is a therapeutic population.

[0306] One or more embodiments of the present invention relates to the method of the present invention, wherein IL-2 is added together with IL-21 which has shown to have positive effects in the examples of this disclosure.

[0307] One or more embodiments of the present invention relates to the method of the present invention, comprising the step of performing a depletion of suppressive cells, including regulatory T cells, and / or blocking negative signals to obtain a depleted population of cells, optionally by the addition of one or more TME stimulators from the group of “Inhibitors”, wherein the step is performed between step a) and b).

[0308] One or more embodiments of the present invention relates to the method of the present invention, comprising the step of performing a depletion of suppressive cells, including regulatory T cells, and / or blocking negative signals to obtain a depleted population of cells, by the addition of one ormore TME stimulators from the group of “Inhibitors”, wherein the step is performed between step a) and b).

[0309] One or more embodiments of the present invention relates to the method of the present invention, comprising the step of performing a depletion of suppressive cells, including regulatory T cells, and blocking negative signals to obtain a depleted population of cells, by the addition of one or more TME stimulators from the group of “Inhibitors”, wherein the step is performed between step a) and b).

[0310] One or more embodiments of the present invention relates to the method of the present invention, comprising the step of blocking negative signals to obtain a depleted population of cells, by the addition of one or more TME stimulators from the group of “Inhibitors”, wherein the step is performed between step a) and b).

[0311] One or more embodiments of the present invention relates to the method of the present invention, comprising the step of performing a depletion of suppressive cells, including regulatory T cells, and / or blocking negative signals to obtain a depleted population of cells, optionally by the addition of one or more TME stimulators from the group of “Inhibitors”, wherein the step is performed between step a) and b), and wherein the first population of cells in step b) is the depleted population of cells.

[0312] One or more embodiments of the present invention relates to the method of the present invention, comprising the step of performing a depletion of suppressive cells, including regulatory T cells, and / or blocking negative signals to obtain a depleted population of cells, and wherein the first population of cells in step b) is the depleted population of cells.

[0313] One or more embodiments of the present invention relates to the method of the present invention, comprising the step of performing a depletion of suppressive cells, including regulatory T cells, and / or blocking negative signals to obtain a depleted population of cells, by the addition of one or more TME stimulators from the group of “Inhibitors”, wherein the step is performed between step a) and b), and wherein the first population of cells in step b) is the depleted population of cells.

[0314] One or more embodiments of the present invention relates to the method of the present invention, comprising the step of performing a depletion of suppressive cells, including regulatory T cells, and blocking negative signals to obtain a depleted population of cells, optionally by the addition of one or more TME stimulators from the group of “Inhibitors”, wherein the step is performed between step a) and b), and wherein the first population of cells in step b) is the depleted population of cells. One or more embodiments of the present invention relates to the method of the present invention, comprising the step of performing a depletion of suppressive cells, including regulatory T cells, and wherein the first population of cells in step b) is the depleted population of cells.One or more embodiments of the present invention relates to the method of the present invention, comprising the step of blocking negative signals to obtain a depleted population of cells, by the addition of one or more TME stimulators from the group of “Inhibitors”, wherein the step is performed between step a) and b), and wherein the first population of cells in step b) is the depleted population of cells.

[0315] One or more embodiments of the present invention related to a method according to the present disclosure, wherein the cells are selected from the group consisting of tumor infiltrating lymphocytes (TILs), NK cells, TCR-T cells, and CAR-T cells.

[0316] In one or more embodiments, the cells are tumor infiltrating lymphocytes (TILs) In one or more embodiments, the cells are NK cells. In one or more embodiments, the cells are TCR-T cells. In one or more embodiments, the cells are CAR-T cells.

[0317] One or more embodiments of the present invention related to a method according to the present disclosure, wherein the cells are tumor infiltrating lymphocytes (TILs).

[0318] One or more embodiments of the present invention related to the cells and methods according to the present disclosure, wherein the cells are NK cells. One or more embodiments of the present invention related to the cells and methods according to the present disclosure, wherein the cells are TCR-T cells. One or more embodiments of the present invention related to the cells and methods according to the present disclosure, wherein the cells are CAR-T cells.

[0319] One or more embodiments of the present invention related to a method according to the present disclosure, wherein the cells in step b) are T cells or NK cells.

[0320] One or more embodiments of the present invention related to a method according to the present disclosure, wherein the artificial feeder cell(s) undergoes treatment that leads to cell death.

[0321] One or more embodiments of the present invention related to a method according to the present disclosure, wherein artificial feeder cell(s) are irradiated.

[0322] One or more embodiments of the present invention related to a method according to the present disclosure, wherein the artificial feeder cell(s) have been cryopreserved before use in the method.

[0323] One or more embodiments of the present invention related to a method according to the present disclosure, wherein artificial feeder cell(s) are irradiated before cryopreservation, while they are cryopreserved, or after they have been cryopreserved.One or more embodiments of the present invention related to a method according to the present disclosure, wherein the one or more tumor(s) and / or tumor fragment(s) in step a) are resected from a mammal diagnosed with cancer and / or undergoing treatment for cancer.

[0324] One or more embodiments of the present invention related to a method according to the present disclosure, wherein the one or more tumor(s) and / or tumor fragment(s) in step a) are resected from a human diagnosed with cancer and / or undergoing treatment for cancer.

[0325] One or more embodiments of the present invention related to a method according to the present disclosure, wherein the TILs and artificial feeder cells in step c) are cultured in IL-2 and IL-21 and / or the culture medium according to the present invention.

[0326] One or more embodiments of the present invention related to a method according to the present disclosure, wherein the TILs and artificial feeder cells in step c) are cultured in a ratio between 1 TIL:5 artificial feeder cells and 1 TIL: 150 artificial feeder cells.

[0327] One or more embodiments of the present invention related to a method according to the present disclosure, wherein the TILs and artificial feeder cells in step c) are cultured in a ratio between 1 TIL: 10 artificial feeder cells and 1 TIL:200 artificial feeder cells.

[0328] One or more embodiments of the present invention related to a method according to the present disclosure, wherein the TILs and artificial feeder cells in step c) are cultured in a ratio between 1 TIL:20 artificial feeder cells and 1 TIL: 150 artificial feeder cells.

[0329] One or more embodiments of the present invention related to a method according to the present disclosure, wherein the TILs and artificial feeder cells in step c) are cultured in a ratio between 1 TIL:20 artificial feeder cells and 1 TIL: 110 artificial feeder cells.

[0330] One or more embodiments of the present invention related to a method according to the present disclosure, wherein the TILs and artificial feeder cells in step c) are cultured in a ratio between 1 TIL:30 artificial feeder cells and 1 TIL:60 artificial feeder cells.

[0331] One or more embodiments of the present invention related to a method according to the present disclosure, wherein the population of artificial feeder cells are irradiated with at least 25 Gy before use in the method.

[0332] One or more embodiments of the present invention related to a method according to the present disclosure, wherein the population of artificial feeder cells are irradiated with at least 50 Gy before use in the method.One or more embodiments of the present invention related to a method according to the present disclosure, wherein the population of artificial feeder cells are irradiated using ionizing radiation such as x-rays and / or gamma rays.

[0333] One or more embodiments of the present invention related to a method according to the present disclosure, wherein the population of artificial feeder cells in step a) and b) are cryopreserved at a temperature of at least -70°C or colder before use in the method. In one or more embodiments, the population of artificial feeder cells in step a) and b) are cryopreserved at a temperature of at least -75°C or colder before use in the method. In one or more embodiments, the population of artificial feeder cells in step a) and b) are cryopreserved at a temperature of at least -80°C or colder before use in the method. In one or more embodiments, the population of artificial feeder cells in step a) and b) are cryopreserved at a temperature of at least -85°C or colder before use in the method.

[0334] An aspect of the present invention relates to a method for expanding tumour infiltrating lymphocytes (TILs) into a therapeutic population of TILs comprising culturing autologous T cells by obtaining a first population of TILs from a tumour and / or tumour fragments resected from a mammal; performing a first expansion by culturing the first population of TILs in a cell culture medium; performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, anti-CD3 antibody, and the artificial feeder cell(s) (aAPCs) according to the present invention, to produce a third population of TILs, wherein the third population of TILs is a therapeutic population.

[0335] Therapeutic population of cells

[0336] In the present context, a therapeutic population of cells is taken to mean a population of cells, which are in sufficient in number for use in therapy. For example, in certain circumstances, for TILs and TCR-T cells, this may be >1 billion cells. In further examples describing CAR-Ts the number of cells may be in the range of millions to hundreds of millions.

[0337] A further aspect of the present invention relates to expanded tumor infiltrating lymphocytes (TILs) for use in promoting regression of a cancer in a subject with cancer, the regression comprising the steps of culturing autologous T cells by obtaining a first population of TILs from a tumour and / or tumour fragments resected from a mammal; performing a first expansion by culturing the first population of TILs in a cell culture medium; performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2, anti-CD3 antibody, and the artificial feeder cell(s) (aAPCs) according to the present invention, to produce a third population of TILs, wherein the third population of TILs is a therapeutic population, and after administeringnonmyeloablative lymphodepleting chemotherapy, administering to the mammal the therapeutic population of T cells, wherein the T cells administered to the mammal with or without IL-2 treatment, whereupon the regression of the cancer in the mammal is promoted. The promotion of regression of cancer may be a treatment of the cancer.

[0338] These methods can include the step of performing a depletion of suppressive cells, including regulatory T cells, and / or blocking negative signals to obtain a depleted population of TILs, optionally by the addition of one or more TME stimulators from the group of “Inhibitors”. This step can be performed on the first population of TILs from a tumour and / or tumour fragments resected from a mammal.

[0339] Such step of performing a depletion of suppressive cells, including regulatory T cells, and / or blocking negative signals to obtain a depleted population of TILs, optionally by the addition of one or more TME stimulators from the group of “Inhibitors” can comprise addition of the artificial antigen presenting cells (aAPCs) of the present invention.

[0340] In one or more embodiments of the present invention, the method further comprises a step e: e) performing a further expansion on the third population of lymphocytes, the further expansion comprising viii. Addition of feeder cells.

[0341] In one or more embodiments of the present invention, the method further comprises the performance of step c), d) or e) generates a population of lymphocytes, which can be harvested in a step f): ix: Harvesting of lymphocytes.

[0342] An aspect of the present invention related to a composition according to the invention, wherein the cells are derived from a human in need for treatment for cancer.

[0343] An aspect of the present invention related to a composition, wherein the TILs are derived from a human in need for treatment for cervical, ovarian and / or lung cancer.

[0344] An aspect of the present invention related to a population of TILs according to the present invention or the composition according to the present invention for use as a medicament.

[0345] A population of TILs according to the present invention or the composition according to the present invention for use in the treatment of cancer.

[0346] A population of TILs according to the present invention or the composition according to the present invention for use in the treatment of a cancer selected from the group consisting of cervical, ovarian, head and neck (including oral cancer, oropharynx cancer), HPV induced cancer, and thyroid cancer (including anaplastic thyroid carcinoma) and / or pulmonary cancer.An aspect of the present invention related to a method for manufacturing the artificial feeder cell according to the present invention, said method comprising culturing one or more artificial feeder cells in a flask or a bioreactor.

[0347] An aspect of the present invention related to a method according to the present invention, wherein step c) is performed on 20x106cells. An aspect of the present invention related to a method according to the present invention, wherein step c) is performed on 20x106cells. An aspect of the present invention related to a method according to the present invention, wherein step c) is performed on 20x107cells. An aspect of the present invention related to a method according to the present invention, wherein step c) is performed on 20x108cells.

[0348] An aspect of the present invention wherein step c is performed on all cells from step b.

[0349] An aspect of the present invention wherein step c is performed on the majority of the cells from step b. One or more embodiments of the present disclosure describes the method of the present invention, wherein step c is performed on all cells from step b.

[0350] One or more embodiments of the present disclosure describes the method of the present invention, wherein step c is performed on the majority of the cells from step b.

[0351] An aspect of the present invention related to a method according to the present invention, wherein step c) is performed on 20x106cells that have been cryopreserved before step c). An aspect of the present invention related to a method according to the present invention, wherein step c) is performed on 20x107cells that have been cryopreserved before step c). An aspect of the present invention related to a method according to the present invention, wherein step c) is performed on 20x108cells that have been cryopreserved before step c).

[0352] TME stimulators

[0353] The term “TME stimulators” relates to substances (or agents) that have the ability to create a favorable microenvironment within the tumor where lymphocytes, such as exhausted T cells can be reinvigorated in order to expand manyfold and restore their anti-tumor functionality. Thus, in one or more embodiments, the one or more TME stimulators are selected from the groups consisting of: (x) one or more substances that are capable of antagonizing and / or inhibiting receptors expressed on T cells (or their ligands) known to cause T-cell downregulation, deactivation and / or exhaustion, (y) one or more substances that are capable of agonizing and / or stimulating receptors expressed on lymphocytes, such as T cells known to cause T-cell upregulation, activation, and / or reinvigoration, (z) one or more substances that are capable of antagonizing and / or inhibiting soluble molecules and cytokines and their receptors known to cause T-cell downregulation, deactivation, and / or exhaustion, and (v) one or more substances that are capable ofdownregulating and / or depleting suppressive cells, including regulatory T cells, thereby favoring ex-vivo effector T-cell expansion.

[0354] In one or more embodiments, the substances that are capable of antagonizing and / or inhibiting receptors expressed on T cells (or their ligands) known to cause T-cell downregulation, deactivation and / or exhaustion are selected from the groups consisting of: A: substances that act through the PD-1 receptor on T cells, B: substances that act through the CTLA-4 receptor on T cells, C: substances that act through the LAG-3 receptor on T cells, D: substances that act through the TIGIT / CD226 receptor on T cells, E: substances that act through the KIR receptor on T cells, F: substances that act through the TIM-3 receptor on T cells, G: substances that act through the BTLA receptor on T cells, and H: substances that act through the A2aR receptor on T cells. It is to be understood that the definition of substances that act through a given receptor also can cover the same receptors ligand. This means e.g. that for the PD-1 receptor, substances that target the PD-L1 or PD-L2 can also be covered. Group A can therefore cover substances that act through the PD-1 receptor on T cells as well as its ligand(s).

[0355] The substances of the present invention can be an antibody. The substances of the present invention can be a peptide. The substances of the present invention can be a small molecule. The term “antibody” refers to an antibody or variant thereof e.g., a monoclonal antibody including human, humanized, chimeric, or murine antibodies, or F(ab')2 or Fab fragment, biosimilar, or Nanobody.

[0356] Thus, an aspect of the present invention relates to a method for expanding lymphocytes into an expanded population of lymphocytes, the method comprising, a) culturing lymphocytes by obtaining a first population of lymphocytes from a sample resected from a mammal, and b) performing a depletion of suppressive cells, including regulatory T cells, and / or blocking negative signals by the addition of one or more TME stimulators from the group of “Inhibitors” to obtain a depleted population of lymphocytes. The method can further comprise a step c) performing a first expansion by culturing the depleted population of lymphocytes to produce a first population of expanded lymphocytes, the expansion comprising one or more of the steps i-iv: i. Addition of feeder cells, ii. Addition of an anti-CD3 antibody, Hi. Addition of one or more TME stimulators, which are cytokines selected from the group consisting of IL-2, IL-7, IL-12, IL-15, and IL-21, and iv.

[0357] Addition of TME stimulators from the “Stimulator” group. The aAPCs of the present invention can be added to step b), to step c), or to both step b) and step c)

[0358] The one or more TME stimulators from the group of “Inhibitors” to obtain a depleted population of TILs with or without the addition of “cytokines”.

[0359] In one or more embodiments, the substance of group A is an antibody selected from one or more from the group consisting of pembrolizumab, nivolumab, cemiplimab, sym021, atezolizumab, avelumab, durvalumab, Toripalimab, Sintilimab, Camrelizumab, Tislelizumab, Sasanlimab, andDostarlimab. In one or more embodiments, the substance of group A is a small molecule selected from one or more from the group consisting of MAX-10181, YPD-29B, IMMH-010, INCB086550, GS-4224, DPPA-1, TPP-1 , BMS-202, CA-170, JQ1, eFT508, Osimertinib, PlatycodinD, PD-LYLSO, Curcumin, and Metformin. In one or more embodiments, the substance of group A is selected from one or more from the group consisting of pembrolizumab, nivolumab, cemiplimab, sym021, atezolizumab, avelumab, durvalumab, Toripalimab, Sintilimab, Camrelizumab, Tislelizumab, Sasanlimab, Dostarlimab, MAX-10181, YPD-29B, IMMH-010, INCB086550, GS-4224, DPPA-1, TPP-1 , BMS-202, CA-170, JQ1, eFT508, Osimertinib, PlatycodinD, PD-LYLSO, Curcumin, and Metformin. In one or more embodiments, the substance of group A is pembrolizumab. In one or more embodiments, the substance of group A is nivolumab. In one or more embodiments, the substance of group A is cemiplimab. In one or more embodiments, the substance of group A is sym021. In one or more embodiments, the substance of group A is atezolizumab. In one or more embodiments, the substance of group A is avelumab. In one or more embodiments, the substance of group A is durvalumab. In one or more embodiments, the substance of group A is Toripalimab. In one or more embodiments, the substance of group A is Sintilimab. In one or more embodiments, the substance of group A is Camrelizumab. In one or more embodiments, the substance of group A is Tislelizumab. In one or more embodiments, the substance of group A is Sasanlimab. In one or more embodiments, the substance of group A is Dostarlimab. In one or more embodiments, the substance of group A is MAX-10181. In one or more embodiments, the substance of group A is YPD-29B. In one or more embodiments, the substance of group A is IMMH-010. In one or more embodiments, the substance of group A is INCB086550. In one or more embodiments, the substance of group A is GS-4224. In one or more embodiments, the substance of group A is DPPA-1. In one or more embodiments, the substance of group A is TPP-1. In one or more embodiments, the substance of group A is BMS-202. In one or more embodiments, the substance of group A is CA-170. In one or more embodiments, the substance of group A is JQ1. In one or more embodiments, the substance of group A is eFT508. In one or more embodiments, the substance of group A is Osimertinib. In one or more embodiments, the substance of group A is PlatycodinD. In one or more embodiments, the substance of group A is PD-LYLSO. In one or more embodiments, the substance of group A is Curcumin. In one or more embodiments, the substance of group A is Metformin.

[0360] In one embodiment group A is a substance that acts through the PD-1 receptor by blocking the interaction with its ligand including PD-L1 / PD-L2. In one embodiment group A is a substance that acts through the PD-L1 / L2 by blocking the interaction with its receptor including PD-1. In one embodiment group A is a substance that blocks the interaction between PD-1 and PD-L1 / PD-L2. In one embodiment group A is a substance that blocks the signaling of the PD-1 receptor and / or its downstream signaling pathways. In one embodiment group A is a substance that downregulatesthe expression of PD-L1 / PD-L2. In one embodiment group A is a substance that promotes degradation of PD-L1 / PD-L2.

[0361] In one or more embodiments, the substance of group B is selected from one or more antibodies from the group consisting of ipilimumab and tremelimumab. In one or more embodiments, the substance of group B is ipilimumab. In one or more embodiments, the substance of group B is tremelimumab.

[0362] In one embodiment group B is a substance that acts through the CTLA4 receptor by blocking the interaction with its ligand including B7-1 or B7-2. In one embodiment group B is a substance that acts through the B7-1 or B7-2 by blocking the interaction with its receptor including CTLA4. In one embodiment group B is a substance that blocks the interaction between CTLA4 and B7-1 or B7-2. In one embodiment group B is a substance that blocks the signaling of the B7-1 or B7-2 receptor and / or its downstream signaling pathways. In one embodiment group B is a substance that blocks the signaling of the CTLA4 receptor and / or its downstream signaling pathways. In one embodiment group B is a substance that induces cell death upon binding the CTLA4 receptor. In one embodiment group B is an antibody with antibody-dependent cellular cytotoxicity (ADCC). In one embodiment group B is a substance that depletes CTLA4 expressing cells. In one embodiment group B is a substance that depletes regulatory T cells through binding CTLA4 and killing the cell. In one embodiment group B is a substance capable of blocking the interaction of CTLA4 and its ligand, and mediating cell specific cytotoxicity through binding to CTLA4.

[0363] In one or more embodiments, the substance of group C is selected from one or more from the group consisting of relatlimab, eftilagimo alpha, sym022, BMS-986016, and GSK28-31781.

[0364] In one embodiment group C is a substance that acts through the LAG3 receptor by blocking the interaction with its receptor. In one embodiment group C is a substance that blocks the signaling of the LAG3 receptor and / or its downstream signaling pathways.

[0365] In one or more embodiments, the substance of group D is tiragolumab or Liothyronine. In one or more embodiments, the substance of group D is tiragolumab. In one or more embodiments, the substance of group D is Liothyronine.

[0366] In one embodiment group D is a substance that acts through the TIGIT receptor by blocking the interaction with its receptor. In one embodiment group D is a substance that blocks the signaling of the TIGIT receptor and / or its downstream signaling pathways. In one embodiment group D is a substance that induces cell death upon binding the TIGIT receptor. In one embodiment group D is an antibody with antibody-dependent cellular cytotoxicity (ADCC). In one embodiment group D is a substance capable of blocking the interaction of TIGIT and its ligand, and mediating cell specific cytotoxicity through binding to TIGIT.In one or more embodiments, the substance of group E is lirilumab. In one or more embodiments, the substance of group F is sym023. In one or more embodiments, the substance of group G is 40E4 and PJ196.

[0367] In one or more embodiments, the substances that are capable of agonizing and / or stimulating receptors expressed on T cells known to cause T-cell upregulation, activation, and / or reinvigoration are selected from the groups consisting of: I: substances that act through the OX40 / CD134 receptor on T cells, J: substances that act through the 4-1BB / CD137 receptor on T cells, K: substances that act through the CD28 receptor on T cells, L: substances that act through the ICOS receptor on T cells, M: substances that act through the GITR receptor on T cells, N: substances that act through the CD40L receptor on T cells, and O: substances that act through the CD27 receptor on T cells.

[0368] In one or more embodiments, the substance of group J is selected from one or more antibodies from the group consisting of urelumab and utomilumab. In one or more embodiments, the substance of group J is selected from one or more peptides from the group consisting of BCY7835, and BCY7838 from Bicycle Therapeutics. In one or more embodiments, the substance of group J is selected from one or more from the group consisting of BCY7835, BCY7838, urelumab and utomilumab. In one or more embodiments, the substance of group J is urelumab. In one or more embodiments, the substance of group J is utomilumab. In one or more embodiments, the substance of group J is BCY7835. In one or more embodiments, the substance of group J is BCY7838.

[0369] In one embodiment group J is a substance that act through the 4-1BB / CD137 receptor. In one embodiment group J is a substance that act through the 4-1BB / CD137 receptor and stimulates the growth of T cells. In one embodiment group J is a substance that act through the 4-1BB / CD137 receptor and stimulates antigen presenting cells (APC).

[0370] The group J substances can be used in combination with an anti-CD3 substance such as OKT-3. One combination can therefore be urelumab and OKT-3 (urelumab / OKT-3). Another combination can be utomilumab and OKT-3 (utomilumab / OKT-3). An anti-CD3 substances, such as OKT-3, belongs to group W as defined herein. In one embodiment group W is a substance that binds and activates CD3 on T cells. In one or more embodiments, the substance of group K is theralizumab. In one or more embodiments, the substance of group O is valilumab.

[0371] In one or more embodiments, one or more of the substances of group A can be combined with one or more of the substances of group B. In one or more embodiments, one or more of the substances of group A can be combined with one or more of the substances of group B, and with one or more of the substances of group J. These combinations are shown to be effective in the examples of the present disclosure. This means that one or more substances of group A selectedfrom one or more from the group consisting of pembrolizumab, nivolumab, cemiplimab, sym021, atezolizumab, avelumab can be combined with one or more of the substances of group B which is selected from one or more from the group consisting of ipilimumab and tremelimumab. These can then be combined with one or more substances of group J which is selected from one or more from the group consisting of urelumab and utomilumab. The group J substances can be used in combination with an anti-CD3 substance such as OKT-3. One combination can therefore be one or more substances of group A selected from one or more from the group consisting of pembrolizumab, nivolumab, cemiplimab, sym021, atezolizumab, avelumab combined with ipilimumab from group B and urelumab from group J. A specific selection can be pembrolizumab combined with ipilimumab from group B and urelumab from group J, with or without an anti-CD3 substance such as OKT-3.

[0372] In one or more embodiments, the substances that are capable of antagonizing and / or inhibiting soluble molecules and cytokines and their receptors known to cause T-cell downregulation, deactivation, and / or exhaustion are selected from the groups consisting of: P: substances that act through the IDO1 / 2 receptor on T cells, Q: substances that act through the TGFp receptor on T cells, R: substances that act through the IL-10 receptor on T cells, and S: substances that act through the IL-35 receptor on T cells.

[0373] In one or more embodiments, the substance of group P is epacedostat. In one or more embodiments, the substance of group Q is linrodostat. In one or more embodiments, the substance of group R is galunisertib.

[0374] In one or more embodiments, the substances that are capable of downregulating and / or depleting suppressive cells, including regulatory T cells, thereby favoring ex-vivo effector T-cell expansion are selected from the groups consisting of: T: cyclophosphamides, U: TKIs, V: substances that act through aCD25, and X: IL2 / Diphteria toxin fusions.

[0375] In one or more embodiments, the substance of group U is sunitinib. In one or more embodiments, the substance of group V is selected from one or more from the group consisting of sorafenib, imatinib and daclizumab. In one or more embodiments, the substance of group X is dinileukin diftitox.

[0376] Using the approaches presented herein allows for dose levels that are much higher than would be tolerated in vivo. The methods of the present invention are therefore ex vivo. The concentrations can therefore be at least twice as high as the maximum allowed dose tolerated in vivo. The concentration can be even higher such as 5-10 times as high as the maximum allowed dose tolerated in vivo. Thus, in one or more embodiments, the concentration of substance in is 0.1 pg / mL to 300 pg / mL. The concentration can also be 1 pg / mL to 100 pg / mL. The concentration can also be 10 pg / mL to 100 pg / mL. The concentration can also be 1 pg / mL to 10 pg / mL.In one or more embodiments, the therapeutic population of lymphocytes, such as T cells are used to treat a cancer type selected from the groups consisting of: 1: solid tumors, 2: ICI naive tumors, 3: MSI-H tumors, 4: Hematological tumors, 5: Hyper-mutated tumors (such as POL-E and POL-D mutated tumors), and 6: virus-induced tumors.

[0377] In one or more embodiments of the present invention, step b) comprises all of steps i-iv.

[0378] In one or more embodiments of the present invention, step a) and step b) are performed at least one day apart, such as at least two days apart, at least three days apart, or at least four days apart, two days apart, at three days apart, or at four days apart.

[0379] In one or more embodiments of the present invention, step b optionally further comprises: Addition of feeder cells. In one or more embodiments of the present invention, step b optionally further comprises: Addition of an anti-CD3 antibody. In one or more embodiments of the present invention, step b optionally further comprises: Addition of one or more cytokines selected from the group consisting of IL-2, IL-7, IL-12, IL-15, and IL-21.

[0380] The one or more TME stimulators from the group of “cytokines” can be added in step b). IL-2, IL-4, IL-7, IL-12, IL-15, and / or IL-21, as defined above are “cytokines”. One specific cytokine is IL-2, which can be used in any of the steps of the present invention.

[0381] An embodiment of the present invention relates to the uses and methods of the present invention, wherein the group of “Inhibitors” that function by antagonizing / inhibiting receptors expressed on T cells (or their ligands) known to cause T-cell downregulation / deactivation / exhaustion. The group of “Inhibitors” can be selected from the group consisting one or more of: A) substances that act through the PD-1 receptor (or their ligands) on T cells or its ligands PD-L1 or PD-L2, B) substances that act through the CTLA-4 receptor (or their ligands) on T cells, C) substances that act through the LAG-3 receptor (or their ligands) on T cells, D) substances that act through the TIGIT / CD226 receptor on T cells, E) substances that act through the KIR receptor (or their ligands) on T-cells, F) substances that act through the TIM-3 receptor (or their ligands) on T cells, G) substances that act through the BTLA receptor (or their ligands) on T cells, and H) substances that act through the A2aR receptor (or their ligands) on T-cells.

[0382] An embodiment of the present invention relates to the uses and methods of the present invention, wherein the group of “Inhibitors” are selected from the group consisting one or more of: A) substances that act through the PD-1 receptor (or their ligands) on T cells, B) substances that act through the CTLA-4 receptor (or their ligands) on T cells, C) substances that act through the LAG-3 receptor (or their ligands) on T cells, and D) substances that act through the TIGIT / CD226 receptor (or their ligands) on T cells.An embodiment of the present invention relates to the uses and methods of the present invention, wherein group of “Inhibitors” are: A: substances that act through the PD-1 receptor on T cells (or their ligands), and B: substances that act through the CTLA-4 receptor (or their ligands) on T cells. An embodiment of the present invention relates to the uses and methods of the present invention, wherein the group of “Inhibitors” are: A: substances that act through the PD-1 receptor (or their ligands) on T cells, B: substances that act through the CTLA-4 receptor (or their ligands) on T cells, and C) substances that act through the LAG-3 receptor on T cells.

[0383] An embodiment of the present invention relates to the uses and methods of the present invention, wherein the group of “Inhibitors” are: A: substances that act through the PD-1 receptor (or their ligands) on T cells (or their ligands), B: substances that act through the CTLA-4 receptor (or their ligands) on T cells, and D) substances that act through the TIGIT / CD226 receptor on T cells.

[0384] An embodiment of the present invention relates to the uses and methods of the present invention, wherein the group of “Inhibitors” are: A) substances that act through the PD-1 receptor on T cells (or their ligands), B) substances that act through the CTLA-4 receptor (or their ligands) on T cells, C) substances that act through the LAG-3 receptor (or their ligands) on T cells, and D) substances that act through the TIGIT / CD226 receptor (or their ligands) on T cells.

[0385] An embodiment of the present invention relates to the uses and methods of the present invention, wherein the group of “Inhibitors” are selected from the group consisting one or more of: P) substance that act through the molecule IDO, Q) substances that act through the TGFp molecule TGFp receptor (or their ligands) on T cells, R) substances that act through the IL-10 molecule or IL-10 receptor (or their ligands) on T cells, and S) substances that act through the IL-35 molecule or IL-35 receptor (or their ligands) on T-cells. This group work by “Soluble inhibition” by antagonizing / inhibiting soluble molecules and cytokines and their receptors known to cause T-cell downregulation / deactivation / exhaustion.

[0386] An embodiment of the present invention relates to the uses and methods of the present invention, wherein the group of “Inhibitors” are selected from the group consisting of one or more of: T) cyclophosphamides, U) TKIs, V) substances that act through aCD25, and X) IL2 / Diphteria toxin fusions. This group works by adding factors known to downregulate and / or deplete regulatory T cells thereby favoring ex-vivo effector T-cell expansion.

[0387] An embodiment of the present invention relates to the uses and methods of the present invention, wherein the group of “Stimulator” are selected from the group consisting one or more of: I) substances that act through the OX40 / CD134 receptor (or their ligands) on T cells, J) substances that act through the 4-1BB / CD137 receptor (or their ligands) on T cells, K) substances that act through the CD28 receptor (or their ligands) on T cells, L) substances that act through the ICOS receptor (or their ligands) on T cells, M) substances that act through the GITR receptor (or theirligands) on T cells, N) substances that act through the CD40L receptor (or their ligands) on T cells, O) substances that act through the CD27 receptor (or their ligands) on T cells, and W) substances that act through CD-3. These are ’’Stimulators” that work by agonizing / stimulating receptors expressed on T cells known to cause T-cell upregulation / activation / reinvigoration.

[0388] An embodiment of the present invention relates to the uses and methods of the present invention, wherein the group of “Stimulator” is: J) substances that act through the 4-1BB / CD137 receptor (or their ligands) on T cells.

[0389] An embodiment of the present invention relates to the uses and methods of the present invention, wherein: the group of “Inhibitors” in step b) are: A: substances that act through the PD-1 receptor on T cells (or their ligands), and B: substances that act through the CTLA-4 receptor (or their ligands) on T cells, and wherein the group of “Stimulator” in step c) is: J) substances that act through the 4-1BB / CD137 receptor (or their ligands) on T cells. One or more cytokines can be added to steps b) and / or c).

[0390] In one or more embodiments of the present invention, the TME stimulators from the group of “Inhibitors” added in step b) comprises substances that act through the PD-1 receptor on T-cells (group A), and / or substances that act through the CTLA-4 receptor (or their ligands) on T-cells (group B).

[0391] In one or more embodiments of the present invention, the TME stimulators added in step b) further comprises substances that act through the LAG-3 receptor (or their ligands) on T-cells (Group C), and / or substances that act through the TIGIT / CD226 receptor (or their ligands) on T-cells (Group D).

[0392] In one or more embodiments of the present invention, the TME stimulators from the “Stimulator” group added in step c) comprises substances that act through the 4-1BB / CD137 receptor (or their ligands) on T-cells (group J).

[0393] In one or more embodiments, the substance of group A is an anti-PD-1 antibody. In one or more embodiments, the substance of group B is an anti-CTLA-4 antibody. In one or more embodiments, the substance of group C is an anti-LAG-3 antibody. In one or more embodiments, the substance of group D is an anti-TIGIT antibody. In one or more embodiments, the substance of group J is an anti-4-1 BB antibody.

[0394] In one or more embodiments, the substance of group A comprises an anti-PD-1 antibody. In one or more embodiments, the substance of group B comprises an anti-CTLA-4 antibody. In one or more embodiments, the substance of group C comprises an anti-LAG-3 antibody. In one or more embodiments, the substance of group D comprises an anti-TIGIT antibody. In one or more embodiments, the substance of group J comprises an anti-4-1 BB antibody.In one or more embodiments, the substance of group A consists of one or more anti-PD-1 antibodies. In one or more embodiments, the substance of group B consists of one or more anti-CTLA-4 antibodies. In one or more embodiments, the substance of group C consists of one or more anti-LAG-3 antibodies. In one or more embodiments, the substance of group D consists of one or more anti-TIGIT antibodies. In one or more embodiments, the substance of group J consists of one or more anti-4-1 BB antibodies.

[0395] In one or more embodiments, the one or more substances of group A is an anti-PD-1 antibody. In one or more embodiments, the one or more substances of group B is an anti-CTLA-4 antibody. In one or more embodiments, the one or more substances of group C is an anti-LAG-3 antibody. In one or more embodiments, the one or more substances of group D is an anti-TIGIT antibody. In one or more embodiments, the one or more substances of group J is an anti-4-1 BB antibody.

[0396] In one or more embodiments, the one or more substances of group A comprises an anti-PD-1 antibody. In one or more embodiments, the one or more substances of group B comprises an anti-CTLA-4 antibody. In one or more embodiments, the one or more substances of group C comprises an anti-LAG-3 antibody. In one or more embodiments, the one or more substances of group D comprises an anti-TIGIT antibody. In one or more embodiments, the one or more substances of group J comprises an anti-4-1 BB antibody.

[0397] In one or more embodiments, the one or more substances of group A consists of one or more anti-PD-1 antibodies. In one or more embodiments, the one or more substances of group B consists of one or more anti-CTLA-4 antibodies. In one or more embodiments, the one or more substances of group C consists of one or more anti-LAG-3 antibodies. In one or more embodiments, the one or more substances of group D consists of one or more anti-TIGIT antibodies. In one or more embodiments, the one or more substances of group J consists of one or more anti-4-1 BB antibodies.

[0398] In one or more embodiments, the one or more substances of group A consists of one or more anti-PD-1 antibodies. In one or more embodiments, the one or more substances of group B consists of one or more anti-CTLA-4 antibodies. In one or more embodiments, the one or more substances of group C consists of one or more anti-LAG-3 antibodies. In one or more embodiments, the one or more substances of group D consists of one or more anti-TIGIT antibodies. In one or moreembodiments, the one or more substances of group J consists of one or more anti-4-1 BB antibodies.

[0399] In one or more embodiments, the one or more anti-PD-1 antibodies is selected from one or more antibodies from the group consisting of pembrolizumab, nivolumab, cemiplimab, sym021 , atezolizumab, avelumab, durvalumab, Toripalimab, Sintilimab, Camrelizumab, Tislelizumab, Sasanlimab, and Dostarlimab. In one or more embodiments, the one or more anti-PD-1 antibodies is pembrolizumab. In one or more embodiments, the one or more anti-PD-1 antibodies is nivolumab. In one or more embodiments, the one or more anti-PD-1 antibodies is cemiplimab. In one or more embodiments, the one or more anti-PD-1 antibodies is sym021. In one or more embodiments, the one or more anti-PD-1 antibodies is atezolizumab. In one or more embodiments, the one or more anti-PD-1 antibodies is avelumab. In one or more embodiments, the one or more anti-PD-1 antibodies is durvalumab. In one or more embodiments, the one or more anti-PD-1 antibodies is Toripalimab. In one or more embodiments, the one or more anti-PD-1 antibodies is Sintilimab. In one or more embodiments, the one or more anti-PD-1 antibodies is Camrelizumab. In one or more embodiments, the one or more anti-PD-1 antibodies is Tislelizumab. In one or more embodiments, the one or more anti-PD-1 antibodies is Sasanlimab. In one or more embodiments, the one or more anti-PD-1 antibodies is Dostarlimab.

[0400] In one or more embodiments, the anti-PD-1 antibody is selected from one or more antibodies from the group consisting of pembrolizumab, nivolumab, cemiplimab, sym021, atezolizumab, avelumab, durvalumab, Toripalimab, Sintilimab, Camrelizumab, Tislelizumab, Sasanlimab, and Dostarlimab. In one or more embodiments, the anti-PD-1 antibody is pembrolizumab. In one or more embodiments, the anti-PD-1 antibody is nivolumab. In one or more embodiments, the anti-PD-1 antibody is cemiplimab. In one or more embodiments, the anti-PD-1 antibody is sym021. In one or more embodiments, the anti-PD-1 antibody is atezolizumab. In one or more embodiments, the anti-PD-1 antibody is avelumab. In one or more embodiments, the anti-PD-1 antibody is durvalumab. In one or more embodiments, the anti-PD-1 antibody is Toripalimab. In one or more embodiments, the anti-PD-1 antibody is Sintilimab. In one or more embodiments, the anti-PD-1 antibody is Camrelizumab. In one or more embodiments, the anti-PD-1 antibody is Tislelizumab. In one or more embodiments, the anti-PD-1 antibody is Sasanlimab. In one or more embodiments, the anti-PD-1 antibody is Dostarlimab.

[0401] In one or more embodiments, the one or more anti-CTLA-4 antibodies is selected from one or more antibodies from the group consisting of ipilimumab and tremelimumab. In one or moreembodiments, the one or more anti-CTLA-4 antibodies is ipilimumab. In one or more embodiments, the one or more anti-CTLA-4 antibodies is tremelimumab.

[0402] In one or more embodiments, the anti-CTLA-4 antibody is selected from one or more antibodies from the group consisting of ipilimumab and tremelimumab. In one or more embodiments, the the anti-CTLA-4 antibody is ipilimumab. In one or more embodiments, the the anti-CTLA-4 antibody is tremelimumab.

[0403] In one or more embodiments, the anti-LAG-3 antibody is selected from one or more from the group consisting of relatlimab, eftilagimo alpha, sym022, BMS-986016, and GSK28-31781. In one or more embodiments, the anti-LAG-3 antibody is relatlimab. In one or more embodiments, the anti-LAG-3 antibody is eftilagimo alpha. In one or more embodiments, the anti-LAG-3 antibody is sym022. In one or more embodiments, the anti-LAG-3 antibody is BMS-986016. In one or more embodiments, the anti-LAG-3 antibody is GSK28-31781.

[0404] In one or more embodiments, the one or more anti-LAG-3 antibodies is selected from one or more from the group consisting of relatlimab, eftilagimo alpha, sym022, BMS-986016, and GSK28-31781. In one or more embodiments, the one or more anti-LAG-3 antibodies is relatlimab. In one or more embodiments, the one or more anti-LAG-3 antibodies is eftilagimo alpha. In one or more embodiments, the one or more anti-LAG-3 antibodies is sym022. In one or more embodiments, the one or more anti-LAG-3 antibodies is BMS-986016. In one or more embodiments, the one or more anti-LAG-3 antibodies is GSK28-31781.

[0405] In one or more embodiments, the anti-TIG IT antibody is tiragolumab or Liothyronine. In one or more embodiments, the anti-TIG IT antibody is tiragolumab. In one or more embodiments, the anti-TIGIT antibody is Liothyronine.

[0406] In one or more embodiments, the one or more anti-TIGIT antibodies is tiragolumab or Liothyronine. In one or more embodiments, the one or more anti-TIGIT antibodies is tiragolumab. In one or more embodiments, the one or more anti-TIGIT antibodies is Liothyronine.

[0407] In one or more embodiments, the anti-4-1 BB antibody is selected from one or more antibodies from the group consisting of urelumab and utomilumab. In one or more embodiments the anti-4-1 BB antibody is urelumab. In one or more embodiments, the anti-4-1 BB antibody is utomilumab. In one or more embodiments, the one or more anti-4-1 BB antibodies is selected from one or more antibodies from the group consisting of urelumab and utomilumab. In one or more embodiments,the one or more anti-4-1 BB antibodies is urelumab. In one or more embodiments, the one or more anti-4-1 BB antibodies is utomilumab.

[0408] In one or more embodiments of the present invention, the method of the present invention comprises the step of: Performing a second expansion on the second population of lymphocytes obtained to produce a third population of lymphocytes, the second expansion comprising one or more of the v-vi steps, v. Addition of feeder cells, vi. Addition of an anti-CD3 antibody, vii. Addition of one or more cytokines selected from the group consisting of IL-2, IL-7, IL-12, IL-15, and IL-21. The cytokine can be IL-2. The term “IL-2” (also referred to herein as “IL2”) refers to the T-cell growth factor known as interleukin-2, and includes all forms of IL-2 including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof.

[0409] After preparation of the tumor fragments, the resulting cells (i.e., fragments) are cultured in media containing IL-2 under conditions that favor the growth of TILs over tumor and other cells. In some embodiments, the tumor digests are incubated in e.g. 2 mL wells in media comprising inactivated human AB serum (or, in some cases, as outlined herein, in the presence of feeder cells, such as a aAPC cell population) with 6000 lU / mL of IL-2. This primary cell population is cultured for a period of days, generally from 6 to 14 days, resulting in a bulk TIL population, generally about 1 x 106to 1 x 108bulk TIL cells. In some embodiments, the growth media during the first expansion comprises IL-2 or a variant thereof. In some embodiments, the IL-2 is recombinant human IL-2 (rhlL-2). In some embodiments the IL-2 stock solution has a specific activity of 20-30 x 106lU / mg for a 1 mg vial. In some embodiments the IL-2 stock solution has a specific activity of 20 x 106lU / mg for a 1 mg vial. In some embodiments the IL-2 stock solution has a specific activity of 25 x 106lU / mg for a 1 mg vial. In some embodiments the IL-2 stock solution has a specific activity of 30x 106lU / mg for a 1 mg vial. In some embodiments, the IL- 2 stock solution has a final concentration of 4-8 x 106lU / mg of IL-2. In some embodiments, the IL- 2 stock solution has a final concentration of 5-7x 106lU / mg of IL-2. In some embodiments, the IL- 2 stock solution has a final concentration of 6 x 106lU / mg of IL-2. In some embodiments, the IL-2 stock solution is prepare as described in the examples. In some embodiments, the first expansion culture media comprises about 10,000 lU / mL of IL-2, about 9,000 lU / mL of IL-2, about 8,000 lU / mL of IL-2, about 7,000 lU / mL of IL-2, about 6000 lU / mL of IL-2 or about 5,000 lU / mL of IL-2. In some embodiments, the first expansion culture media comprises about 9,000 lU / mL of IL-2 to about 5,000 lU / mL of IL-2. In some embodiments, the first expansion culture media comprises about 8,000 lU / mL of IL-2 to about 6,000 lU / mL of IL-2. In some embodiments, the first expansion culture media comprises about 7,000 lU / mL of IL-2 to about 6,000 lU / mL of IL-2. In some embodiments, the first expansion culture media comprises about 6,000 lU / mL of IL-2. In an embodiment, the cell culture medium further comprises IL-2. Insome embodiments, the cell culture medium comprises about 3000 lU / mL of IL-2. In an embodiment, the cell culture medium further comprises IL-2. In a preferred embodiment, the cell culture medium comprises about 3000 lU / mL of IL-2. In an embodiment, the cell culture medium comprises about 1000 lU / mL, about 1500 lU / mL, about 2000 lU / mL, about 2500 lU / mL, about 3000 lU / mL, about 3500 lU / mL, about 4000 lU / mL, about 4500 lU / mL, about 5000 lU / mL, about 5500 lU / mL, about 6000 lU / mL, about 6500 lU / mL, about 7000 lU / mL, about 7500 lU / mL, or about 8000 lU / mL of IL-2. In an embodiment, the cell culture medium comprises between 1000 and 2000 lU / mL, between 2000 and 3000 lU / mL, between 3000 and 4000 lU / mL, between 4000 and 5000 lU / mL, between 5000 and 6000 lU / mL, between 6000 and 7000 lU / mL, between 7000 and 8000 lU / mL, or about 8000 lU / mL of IL-2.

[0410] IL-2, IL-4, IL-7, IL-12, IL-15, and / or IL-21 can be added to step (b) and / or (c) of the present methods. Sometimes this is also done in step d) or step (e). A preferred embodiment relates to IL-2 to be added to step (b) and / or (c) of the present methods. These are part of the definition “cytokines” and part of the group of “cytokines” mentioned herein. The term “IL-4” (also referred to herein as “IL4”) refers to the cytokine known as interleukin 4, which is produced by Th2 T cells and by eosinophils, basophils, and mast cells. IL-4 regulates the differentiation of naive helper T cells (ThO cells) to Th2 T cells. The term “IL-7” (also referred to herein as “IL7”) refers to a glycosylated tissue-derived cytokine known as interleukin 7, which may be obtained from stromal and epithelial cells, as well as from dendritic cells. The term “IL-15” (also referred to herein as “IL15”) refers to the T cell growth factor known as interleukin-15, and includes all forms of IL-15 including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. The term “IL-21” (also referred to herein as “IL21 ”) refers to the pleiotropic cytokine protein known as interleukin-21, and includes all forms of IL-21 including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof. Interleukin 12 (IL-12) is an interleukin that is naturally produced by dendritic cells, macrophages, neutrophils, and human B-lymphoblastoid cells (NC-37) in response to antigenic stimulation. The term “IL-12” (also referred to herein as “IL12”) refers to the pleiotropic cytokine protein known as interleukin-12, and includes all forms of IL-12 including human and mammalian forms, conservative amino acid substitutions, glycoforms, biosimilars, and variants thereof.

[0411] In one or more embodiments of the present invention, the method further comprises a step e: e) performing a further expansion on the third population of lymphocytes, the further expansion comprising viii. Addition of feeder cells.

[0412] In one or more embodiments of the present invention, the method further comprises the performance of step c), d) or e) generates a population of lymphocytes, which can be harvested in a step f): ix: Harvesting of lymphocytes. The lymphocytes can be harvested by storing them in a suitable container or bag. The lymphocytes from generated in step e) will be in an amount that isclinically, or therapeutically, relevant. In the present context is clinically and therapeutically relevant used interchangeably.

[0413] Thus, step f) can further comprise; step x: after administering nonmyeloablative lymphodepleting chemotherapy, administering to the mammal the therapeutic population of T cells, wherein the T cells administered to the mammal, optionally followed by IL-2 infusion, whereupon the regression of the cancer in the mammal is promoted.

[0414] In an embodiment, the ratio of lymphocytes, such as TILs to aAPCs in the second expansion is about 1 to 25, about 1 to 50, about 1 to 100, about 1 to 125, about 1 to 150, about 1 to 175, about 1 to 200, about 1 to 225, about 1 to 250, about 1 to 275, about 1 to 300, about 1 to 325, about 1 to 350, about 1 to 375, about 1 to 400, or about 1 to 500. In an embodiment, the ratio of lymphocytes, such as TILs to aAPCs in the second expansion is between 1 to 50 and 1 to 300. In an embodiment, the ratio of lymphocytes, such as TILs to aAPCs in the second expansion is between 1 to 100 and 1 to 200.

[0415] Thus, in one or more embodiments of the present invention, the method further comprises the aAPCs added are in the the ratio of lymphocytes, such as TILs to aAPCs in the second expansion is between 1 to 50 and 1 to 300. Feeder cells and aAPCs are in the present context used interchangeably.

[0416] In an embodiment all of the lymphocytes, such as TILs obtained in step c are transferred to step d and co-cultured with a fixed number of aAPCs. For example if 10-100 or 1-200 million cells are obtained in step c, all of them are transferred to step d and cultured with 4 billion aAPC. The ratio or cells to aAPCs, such as aAPCs can be 1:200. It could also be in increments, 10-50 million TILs are co-cultured with 4 billion aAPCs. 50-100 million TILs are co-cultured with 10 billion aAPCs. In an embodiment the lymphocytes, such as TILs obtained in step c, are cultures with aAPCs in a concentration of about 0,1-1 million cells / cm2, of about 1 to 2 million cells / cm2of about 1 to 3 million cells / cm2of about 1 to 5 million cells / cm2, of about 1 to 10 million cells / cm2, of about 1 to 20 million cells / cm2, of about 1 to 50 million cells / cm2. The aAPCs can also be in a concentration of 1, 2, 3, 4 or 5 million cells / cm2.

[0417] In an embodiment, lymphocytes, such as TILs, expanded using aAPCs of the present disclosure are administered to a patient as a pharmaceutical composition. In an embodiment, the pharmaceutical composition is a suspension of lymphocytes, such as TILs, in a sterile buffer. Lymphocytes, such as TILs, expanded using aAPCs of the present disclosure may be administered by any suitable route as known in the art. In some embodiments, the T cells are administered as a single intra-arterial or intravenous infusion, which preferably lasts approximately 30 to 60 minutes. Other suitable routes of administration include intraperitoneal, intrathecal, intra-tumoral, and intralymphatic. In one or more embodiments, the therapeutic population of lymphocytes, such as TILs, are infused into a patient.

[0418] In one or more embodiments, the cells are removed from the cell culture and cryopreserved in a storage medium prior to performing any of the steps of the present invention, i.e. step b), step c) d), step e) or step f), and also after any of the individual substeps from i to x.

[0419] In one or more embodiments, the method further comprises the step of transducing the first population of TILs with an expression vector comprising a nucleic acid encoding a chimeric antigen receptor (CAR) comprising a single chain variable fragment antibody fused with at least one endodomain of a T-cell signaling molecule.

[0420] In one or more embodiments, step (c) further comprises a step of removing the cells from the cell culture medium.

[0421] In one or more embodiments, step (a) further comprises processing of the resected tumor into multiple tumor fragments, such as 4 to 50 fragments, such as 20 to 30 fragments. In one or more embodiments, the fragments have a size of about 1 to 50 mm3. In one or more embodiments, the fragments have a size of about 5 to 50 mm3. In one or more embodiments, the fragments have a size of about 0.1 to 10 mm3. In one or more embodiments, the fragments have a size of about 0.1 to 1 mm3. In one or more embodiments, the fragments have a size of about 0.5 to 5 mm3. In one or more embodiments, the fragments have a size of about 1 to 10 mm3. In one or more embodiments, the fragments have a size of about 1 to 3 mm3. The terms “fragmenting”, “fragment,” and “fragmented”, as used herein to describe processes for disrupting a tumor, includes mechanical fragmentation methods such as crushing, slicing, dividing, and morcellating tumor tissue as well as any other method for disrupting the physical structure of tumor tissue.

[0422] In one or more embodiments, the mammal is a human. In some embodiments, the TILs are obtained from tumor fragments. In some embodiments, the tumor fragment is obtained by sharp dissection. In some embodiments, the tumor fragment is between about 0.1 mm3and 10 mm3. In some embodiments, the tumor fragment is between about 1 mm3and 10 mm3. In some embodiments, the tumor fragment is between about 1 mm3and 8 mm3. In some embodiments, the tumor fragment is about 1 mm3. In some embodiments, the tumor fragment is about 2 mm3. In some embodiments, the tumor fragment is about 3 mm3. In some embodiments, the tumor fragment is about 4 mm3. In some embodiments, the tumor fragment is about 5 mm3. In some embodiments, the tumor fragment is about 6 mm3. In some embodiments, the tumor fragment is about 7 mm3. In some embodiments, the tumor fragment is about 8 mm3. In some embodiments, the tumor fragment is about 9 mm3. In some embodiments, the tumor fragment is about 10 mm3. In some embodiments, the tumors are 1-4 mm x 1-4 mm x 1-4 mm. In some embodiments, the tumors are 1 mm x 1 mm x 1 mm. In some embodiments, the tumors are 2 mm x 2 mm x 2 mm. Insome embodiments, the tumors are 3 mm x 3 mm x 3 mm. In some embodiments, the tumors are 4 mm x 4 mm x 4 mm. Currently fairly large fragment sizes are needed (more than 5 mm3). The present invention allows for the use of smaller fragments because the cells grow in a more optimized way reaching the cell count needed for treatment faster. The use of smaller fragments means that patients that until now have not been treatable because e.g. because their tumor has been too small or because it only has been possible to obtain a small tumor sample, now can be treated. The size of the fragments used in the methods of the present invention can therefore be important.

[0423] In some embodiments, the tumor fragmentation is performed in order to maintain the tumor internal structure. In some embodiments, the tumor fragmentation is performed without preforming a sawing motion with a scalpel. In some embodiments, the TILs are obtained from tumor digests. In some embodiments, tumor digests were generated by incubation in enzyme media, for example but not limited to RPMI 1640, 2 mM GlutaMAX,10 mg / mL gentamicin, 30 U / mL DNase, and 1.0 mg / mL collagenase, followed by mechanical dissociation (GentleMACS, Miltenyi Biotec, Auburn, CA). After placing the tumor in enzyme media, the tumor can be mechanically dissociated for approximately 1 minute. The solution can then be incubated for 30 minutes at 37 °C in 5% CO2 and it then mechanically disrupted again for approximately 1 minute. After being incubated again for 30 minutes at 37 °C in 5% CO2, the tumor can be mechanically disrupted a third time for approximately 1 minute. In some embodiments, after the third mechanical disruption if large pieces of tissue were present, 1 or 2 additional mechanical dissociations were applied to the sample, with or without 30 additional minutes of incubation at 37 °C in 5% CO2. In some embodiments, at the end of the final incubation if the cell suspension contained a large number of red blood cells or dead cells, a density gradient separation using Ficoll can be performed to remove these cells. In one or more embodiments, the cell culture medium is provided in a container selected from the group consisting of a G-Rex container and a Xuri cellbag.

[0424] In one or more embodiments of the present invention, the method further comprises the lymphocytes are tumor infiltrating lymphocytes (TILs) and the therapeutic lymphocyte population is a therapeutic tumor infiltrating lymphocyte (TIL) population.

[0425] By “tumor infiltrating lymphocytes” or “TILs” herein is meant a population of cells originally obtained as lymphocytes that have left the bloodstream of a subject and migrated into a tumor. TILs include, but are not limited to, CD8+ cytotoxic T cells (lymphocytes), Th1 and Th17 CD4+ T cells (CD4+ helper cells), natural killer cells, dendritic cells and Ml macrophages. TILs include both primary and secondary TILs. “Primary TILs” are those that are obtained from patient tissue samples as outlined herein (sometimes referred to as “freshly harvested”), and “secondary TILs” are any TIL cell populations that have been expanded or proliferated as discussed herein. TILs can generally bedefined either biochemically, using cell surface markers, or functionally, by their ability to infiltrate tumors and induce tumor cell killing. TILs can be generally categorized by expressing one or more of the following biomarkers: CD4, CD8, TCR ab, CD27, CD28, CD56, CCR7, CD45Ra, CD95, PD-1, LAG-3, TIM-3, CD69, CD103, CD107a, TNFa, IFNg, CD3, and CD25. Additionally, and alternatively, TILs can be functionally defined by their ability to infiltrate solid tumors upon reintroduction into a patient. TILs may further be characterized by potency - for example, TILs may be considered potent if, for example, interferon (IFN) release is greater than about 50 pg / mL, greater than about 100 pg / mL, greater than about 150 pg / mL, or greater than about 200 pg / mL or interferon (IFN), tumor-necrosis-factor (TNF) production can be detected intracellularly and CD107a on the cell surface upon stimulation with coated beads or tumor cells (tumor cell lines or tumor digest). Functionality of these stimulated TILs can for example be further characterized by classification into single-, double-, or triple positivity for TNF and / or IFN and / or CD107a, whereby triple positive TILs are considered the most functional. Potency of the TIL product can be further characterized by analyzing direct tumor cell killing by detection of apoptosis and / or proliferation of tumor cells.

[0426] In one or more embodiments of the present invention, wherein the lymphocytes are cryopreserved after any one or more of the step(s). Cryopreservation ensures that the cells at any stage (after any step) can be frozen and stored and / or transported. The subsequent steps can therefore be performed at separate locations. Number of specificities of the CD8+ T cells and their frequency in the TIL product can be defined by staining TILs with MHC multimer complexes displaying tumor peptides of interest that can be recognized by CD8+ T cells and / or by sequencing the T cell receptor repertoire.

[0427] An aspect of the present invention relates to a population of lymphocytes that comprises one or TME stimulators as disclosed herein. The population can also comprise feeder cells. These populations can have a favourable makeup which can be beneficial for several purposes, including for TIL therapy.

[0428] One or more embodiments of the present invention related to the the method of the present invention wherein the lymphocytes are NK cells B cells, or T-cells, like T cells trained and / or expanded with their specific antigen-peptide(s) presented on an MHC molecule or presented by APC; TCR engineered T cells or CAR T cells.

[0429] One or more embodiments of the present invention relates to the method of the present invention wherein lymphocytes are T cells. The T cells can be tumor infiltrating lymphocytes (TILs).

[0430] An aspect of the present invention relates to including a time lapse between steps b) and c), i.e. where the two steps are performed with a certain time period apart.In one or more embodiments of the present invention, the method further comprises step b) and step c) are performed in time lapse, such as one day apart, or such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days apart.

[0431] An embodiment of the present invention relates to the uses and methods of the present invention, wherein the step b) and step c) are performed 1-8 days apart. An embodiment of the present invention relates to the uses and methods of the present invention, wherein the step b) and step c) are performed 2-8 days apart. An embodiment of the present invention relates to the uses and methods of the present invention, wherein the step b) and step c) are performed 3-8 days apart. An embodiment of the present invention relates to the uses and methods of the present invention, wherein the step b) and step c) are performed 4-8 days apart. An embodiment of the present invention relates to the uses and methods of the present invention, wherein the step b) and step c) are performed 5-8 days apart. An embodiment of the present invention relates to the uses and methods of the present invention, wherein the step b) and step c) are performed 6-8 days apart. An embodiment of the present invention relates to the uses and methods of the present invention, wherein the step b) and step c) are performed 7-8 days apart. An embodiment of the present invention relates to the uses and methods of the present invention, wherein the step b) and step c) are performed 2-7 days apart. An embodiment of the present invention relates to the uses and methods of the present invention, wherein the step b) and step c) are performed 3-7 days apart. An embodiment of the present invention relates to the uses and methods of the present invention, wherein the step b) and step c) are performed 4-7 days apart. An embodiment of the present invention relates to the uses and methods of the present invention, wherein the step b) and step c) are performed 5-7 days apart. An embodiment of the present invention relates to the uses and methods of the present invention, wherein the step b) and step c) are performed 6-7 days apart. An embodiment of the present invention relates to the uses and methods of the present invention, wherein the step b) and step c) are performed 2-6 days apart. An embodiment of the present invention relates to the uses and methods of the present invention, wherein the step b) and step c) are performed 3-6 days apart. An embodiment of the present invention relates to the uses and methods of the present invention, wherein the step b) and step c) are performed 4-6 days apart. An embodiment of the present invention relates to the uses and methods of the present invention, wherein the step b) and step c) are performed 5-6 days apart. An embodiment of the present invention relates to the uses and methods of the present invention, wherein the step b) and step c) are performed 2-5 days apart. An embodiment of the present invention relates to the uses and methods of the present invention, wherein the step b) and step c) are performed 3-5 days apart. An embodiment of the present invention relates to the uses and methods of the present invention, wherein the step b) and step c) are performed 4-5 days apart. An embodiment of the present invention relates to the uses and methods of the present invention, wherein the step b) and step c)are performed 2-4 days apart. An embodiment of the present invention relates to the uses and methods of the present invention, wherein the step b) and step c) are performed 3-4 days apart. An embodiment of the present invention relates to the uses and methods of the present invention, wherein the step b) and step c) are performed 4-16 days apart. An embodiment of the present invention relates to the uses and methods of the present invention, wherein the step b) and step c) are performed 8-14 days apart. An embodiment of the present invention relates to the uses and methods of the present invention, wherein the step b) and step c) are performed 6-12 days apart. An embodiment of the present invention relates to the uses and methods of the present invention, wherein the step b) and step c) are performed 6-14 days apart. An embodiment of the present invention relates to the uses and methods of the present invention, wherein the step b) and step c) are performed 10-15 days apart. An embodiment of the present invention relates to the uses and methods of the present invention, wherein the step b) and step c) are performed on two consecutive days. This means that step b) for example can be performed during a given day (for example a workday), and then step c) is performed the next day (for example the next workday). This means that the time delay (TD) or time lapse can be less than 1 day, for example 20 hours, 18-24 hours, or 14-20 hours.

[0432] In one or more embodiments of the present invention, step b) and step c) are performed 1-2 days apart. In one or more embodiments of the present invention, step b) and step c) are performed 1-3 days apart. In one or more embodiments of the present invention, step b) and step c) are performed 1-4 days apart. In one or more embodiments of the present invention, step b) and step c) are performed 1-5 days apart. In one or more embodiments of the present invention, step b) and step c) are performed 1-6 days apart. In one or more embodiments of the present invention, step b) and step c) are performed 1-7 days apart. In one or more embodiments of the present invention, step b) and step c) are performed 2-4 days apart. In one or more embodiments of the present invention, step b) and step c) are performed 4-8 days apart.

[0433] The methods of the present invention, one or more steps from step (a) to step (e), can be performed in a closed system. The term “closed system” refers to a system that is closed to the outside environment. Any closed system appropriate for cell culture methods can be employed with the methods of the present invention. Closed systems include, for example, but are not limited to closed G-Rex containers.

[0434] In one or more embodiments of the present invention, the concentration of the TME stimulators, substances, is 0.1 pg / mL to 300 pg / mL, such as 1 pg / mL to 100 pg / mL, such as 10 pg / mL to 100 pg / mL, such as 1 pg / mL to 10 pg / mL, such as 2-20 pg / mL.

[0435] In one or more embodiments of the present invention, step c) and step d) are performed in time lapse, such as one day apart, or such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 days apart.In one or more embodiments of the present invention, step c) and step d) are performed 1-2 days apart. In one or more embodiments of the present invention, step c) and step d) are performed 1-3 days apart. In one or more embodiments of the present invention, step c) and step d) are performed 1-4 days apart. In one or more embodiments of the present invention, step c) and step d) are performed 1-5 days apart. In one or more embodiments of the present invention, step c) and step d) are performed 1-6 days apart. In one or more embodiments of the present invention, step c) and step d) are performed 1-7 days apart. In one or more embodiments of the present invention, the step c) and step d) are performed 2-4 days apart. In one or more embodiments of the present invention, step c) and step d) are performed 4-8 days apart. An embodiment of the present invention relates to the uses and methods of the present invention, wherein the step c) and step d) are performed 4-16 days apart. An embodiment of the present invention relates to the uses and methods of the present invention, wherein the step c) and step d) are performed 8-14 days apart. An embodiment of the present invention relates to the uses and methods of the present invention, wherein the step c) and step d) are performed 6-12 days apart. An embodiment of the present invention relates to the uses and methods of the present invention, wherein the step c) and step d) are performed 6-14 days apart. An embodiment of the present invention relates to the uses and methods of the present invention, wherein the step c) and step d) are performed 10-15 days apart. In one or more embodiments of the present invention, steps (a) through (e) are performed within a period of about 15 days to about 25 days, such as 15 days, such as 16 days, such as 17 days, such as 18 days, such as 19 days, such as 19 days, such as 20 days, such as 21 days, such as 22 days, such as 23 days, such as 24 days, such as 25 days.

[0436] In one or more embodiments of the present invention, steps (a) through (f) are performed within a period of about 15 days to about 25 days, such as 15 days, such as 16 days, such as 17 days, such as 18 days, such as 19 days, such as 19 days, such as 20 days, such as 21 days, such as 22 days, such as 23 days, such as 24 days, such as 25 days.

[0437] In one or more embodiments of the present invention, the therapeutic population of T cells is used to treat a cancer type selected from the groups consisting of breast cancer, renal cell cancer, bladder cancer, melanoma, cervical cancer, gastric cancer, colorectal cancer, lung cancer, head and neck cancer, ovarian cancer, Hodgkin lymphoma, pancreatic cancer, liver cancer, and sarcomas.

[0438] In one or more embodiments, the therapeutic population of T cells is used to treat a breast cancer. In one or more embodiments, the therapeutic population of T cells is used to treat renal cell cancer. In one or more embodiments, the therapeutic population of T cells is used to treat bladder cancer. In one or more embodiments, the therapeutic population of T cells is used to treat melanoma. In one or more embodiments, the therapeutic population of T cells is used to treat cervical cancer. Inone or more embodiments, the therapeutic population of T cells is used to treat gastric cancer. In one or more embodiments, the therapeutic population of T cells is used to treat colorectal cancer. In one or more embodiments, the therapeutic population of T cells is used to treat lung cancer. In one or more embodiments, the therapeutic population of T cells is used to treat head and neck cancer. In one or more embodiments, the therapeutic population of T cells is used to treat ovarian cancer. In one or more embodiments, the therapeutic population of T cells is used to treat Hodgkin lymphoma. In one or more embodiments, the therapeutic population of T cells is used to treat pancreatic cancer. In one or more embodiments, the therapeutic population of T cells is used to treat liver cancer. In one or more embodiments, the therapeutic population of T cells is used to treat sarcomas.

[0439] In one or more embodiments, the therapeutic population of cells is used to treat a breast cancer. In one or more embodiments, the therapeutic population of cells is used to treat renal cell cancer. In one or more embodiments, the therapeutic population of cells is used to treat bladder cancer. In one or more embodiments, the therapeutic population of cells is used to treat melanoma. In one or more embodiments, the therapeutic population of cells is used to treat cervical cancer. In one or more embodiments, the therapeutic population of cells is used to treat gastric cancer. In one or more embodiments, the therapeutic population of cells is used to treat colorectal cancer. In one or more embodiments, the therapeutic population of cells is used to treat lung cancer. In one or more embodiments, the therapeutic population of cells is used to treat head and neck cancer. In one or more embodiments, the therapeutic population of cells is used to treat ovarian cancer. In one or more embodiments, the therapeutic population of cells is used to treat Hodgkin lymphoma. In one or more embodiments, the therapeutic population of cells is used to treat pancreatic cancer. In one or more embodiments, the therapeutic population of cells is used to treat liver cancer. In one or more embodiments, the therapeutic population of cells is used to treat sarcomas.

[0440] In one or more embodiments, the therapeutic population of TILs is used to treat a breast cancer. In one or more embodiments, the therapeutic population of TILs is used to treat renal cell cancer. In one or more embodiments, the therapeutic population of TILs is used to treat bladder cancer. In one or more embodiments, the therapeutic population of TILs is used to treat melanoma. In one or more embodiments, the therapeutic population of TILs is used to treat cervical cancer. In one or more embodiments, the therapeutic population of TILs is used to treat gastric cancer. In one or more embodiments, the therapeutic population of TILs is used to treat colorectal cancer. In one or more embodiments, the therapeutic population of TILs is used to treat lung cancer. In one or more embodiments, the therapeutic population of TILs is used to treat head and neck cancer. In one or more embodiments, the therapeutic population of TILs is used to treat ovarian cancer. In one or more embodiments, the therapeutic population of TILs is used to treat Hodgkin lymphoma. In oneor more embodiments, the therapeutic population of TILs is used to treat pancreatic cancer. In one or more embodiments, the therapeutic population of TILs is used to treat liver cancer. In one or more embodiments, the therapeutic population of TILs is used to treat sarcomas.

[0441] Thus, a further aspect of the present invention relates to expanded tumor infiltrating lymphocytes (TILs) for use in promoting regression of a cancer in a subject with cancer, the regression comprising the steps of: a) culturing autologous T cells by obtaining a first population of TILs from a tumor resected from a mammal, b) performing a depletion of suppressive cells, including regulatory T cells, and / or blocking negative signals by the addition of one or more TME stimulators from the group of “Inhibitors” to obtain a depleted population of TILs with or without the addition of “cytokines”, c) performing a first expansion by culturing the depleted population of TILs in a cell culture medium comprising: one or more TME stimulators from the group of “cytokines”, and / or one or more of the TME stimulators from the “Stimulator” group to produce a second population of TILs, d) performing a second expansion by supplementing the cell culture medium of the second population of TILs with additional IL-2 and / or other cytokines from the “cytokine” group, anti-CD3 antibody, and antigen presenting cells (APCs), to produce a third population of TILs, wherein the third population of TILs is a therapeutic population; and e) after administering nonmyeloablative lymphodepleting chemotherapy, administering to the mammal the therapeutic population of T cells, wherein the T cells administered to the mammal with or without IL-2 treatment, whereupon the regression of the cancer in the mammal is promoted.

[0442] One of the key findings has been that more TILs can be generated faster. This has high value because there is a certain amount of cells that are needed in order to be relevant for medical treatment. More cells faster will drive down the costs for production and also provide treatment to the patient faster. In one or more embodiments, step b) or step (c) results in 1 x 106to 1x 107cells, such as 2 x 106to 5x 106cells. In one or more embodiments, step b) or step (c results in 5 x 106to 1x 107cells. In one or more embodiments, step b) or step (c results in 1 x 106to 5x 107cells. In one or more embodiments, step b) or step (c results in 1 x 107to 5x 107cells. In one or more embodiments, step b) or step (c results in 1 x 107to 1x 1012cells, such as 1 x 108to 5x 109cells, such as 1 x 109to 5x 109cells, such as 1 x 108to 5x 1O10cells, such as 1 x 109to 5x 1011cells. In one or more embodiments, step b) or step (c results in an at least 104fold increase as compared to the number of cells after the expansion in step b) or step (c), such as at least 103fold increase, such as at least 102fold increase, such as at least 10 fold increase. In one or more embodiments, step step b) or step (c results in 1 x 107to 1x 101° cells. In one or more embodiments, step b) or step (c results in 1 x 107to 1x 109cells. In one or more embodiments, step b) or step (c results in 1 x 107to 1x 108cells. In one or more embodiments, step b) or step (c results in 1 x 101°to 1x 1011cells. In one or more embodiments, step b) or step (c results in 1 x 1011to 2x 1011cells. In one ormore embodiments, step b) or step (c results in at least 1x 1011cells. In one or more embodiments, step b) or step (c results in at least 2x 1011cells.

[0443] In one or more embodiments of the present invention, step b) or step (c results in 1 x 107to 1x 1012cells, such as 1 x 108to 5x 109cells, such as 1 x 109to 5x 109cells, such as 1 x 108to 5x 1010 cells, such as 1 x 109to 5x 1011cells.

[0444] In one or more embodiments of the present invention, the anti-CD3 antibody is OKT3.

[0445] The term “anti-CD3 antibody” refers to an antibody or variant thereof e.g., a monoclonal antibody and including human, humanized, chimeric or murine antibodies, or biosimilars which are directed against the CD3 receptor in the T-cell antigen receptor of mature T cells. Anti-CD3 antibodies include OKT3, also known as muromonab. Anti-CD3 antibodies also include the UHCT1 clone, also known as T3 and CD3e. Other anti-CD3 antibodies include, for example, otelixizumab, teplizumab, and visilizumab. In an embodiment, the cell culture medium comprises OKT3 antibody. In some embodiments, the cell culture medium comprises about 30 ng / mL of OKT3 antibody. In an embodiment, the cell culture medium comprises about 0.1 ng / mL, about 0.5 ng / mL, about 1 ng / mL, about 2.5 ng / mL, about 5 ng / mL, about 7.5 ng / mL, about 10 ng / mL, about 12 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 200 ng / mL, about 500 ng / mL, and about 1 pg / mL of OKT3 antibody. In an embodiment, the cell culture medium comprises between 0.1 ng / mL and 1 ng / mL, between 1 ng / mL and 5 ng / mL, between 5 ng / mL and 10 ng / mL, between 10 ng / mL and 20 ng / mL, between 20 ng / mL and 30 ng / mL, between 30 ng / mL and 40 ng / mL, between 40 ng / mL and 50 ng / mL, and between 50 ng / mL and 100 ng / mL of OKT3 antibody. In some embodiments, the cell culture medium does not comprise OKT3 antibody. Cytokines can be added in 0,1 ng / mL-10 ng / mL, 1 ng / mL-100 ng / mL, or in 1-100 ng / mL.

[0446] In one or more embodiments of the present invention, the mammal is a human individual.

[0447] In one or more embodiments of the present invention, the anti-CD3 antibody is selected from the group consisting of a monoclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, a murine antibody, a F(ab')2 or Fab fragment, a biosimilar, and a Nanobody.

[0448] In one or more embodiments of the present invention, the substance of any one or more of selected from groups A, B, J, C and / D is an antibody.

[0449] In one or more embodiments of the present invention, the antibody is selected from the group consisting of a monoclonal antibody, a human antibody, a humanized antibody, a chimeric antibody, a murine antibody, a F(ab')2 or Fab fragment, a biosimilar, and a Nanobody.In one or more embodiments of the present invention, group A is selected from one or more from the group consisting of pembrolizumab, nivolumab, cemiplimab, sym021, atezolizumab, avelumab, durvalumab, Toripalimab, Sintilimab, Camrelizumab, Tislelizumab, Sasanlimab, Dostarlimab, MAX-10181, YPD-29B, IMMH-010, INCB086550, GS-4224, DPPA-1 , TPP-1 , BMS-202, CA-170, JQ1, eFT508, Osimertinib, PlatycodinD, PD-LYLSO, Curcumin, and Metformin.

[0450] In one or more embodiments of the present invention, group B is selected from one or more antibodies from the group consisting of ipilimumab and tremelimumab.

[0451] In one or more embodiments of the present invention, the substance of group J is selected from one or more from the group consisting of urelumab, utomilumab, BCY7835, and BCY7838.

[0452] Activators of Nrf2 have been shown to reduce and / or preventing the suppression of lymphocytes, such as a T cell, such as TIL, activity caused by one or more stress; and / or inducing and / or maintaining and / or increasing lymphocyte, such as T cell activity in the presence of one or more stress; and / or increasing lymphocyte, such as T cell survival in response to one or more stress. Stress in this context can e.g., be caused by reactive oxygen species (ROS) that are chemically reactive oxygen derivates, such as superoxide radicals (O2-), hydroxyl radicals (OH) or hydrogen peroxide (H2O2) produced by cancer cells and / or immune cells such as Myeloid Derived Suppressor Cells (MDSCs), that can cause deactivation of immune effector cells such as T-cell and NK cells and thereby cause the therapeutic cell product to lose its anti-cancer effect. Nrf2 activation can revert this problem. Thus, in one or more embodiments of the present invention, the method further comprises the step of contacting one or more lymphocytes with an activator of Nrf2 in any of the steps of the present inventions, such as step b), step c), step d), step e) and / or step f). This can also be done during any of steps i-x, and especially before administration to a patient, i.e. after step ix and before step x. The activator of Nrf2 can be auranofin.

[0453] An aspect of the present invention relates to a population of lymphocytes obtainable by a method of the present invention. These lymphocytes can be used in the treatment of cancer.

[0454] In an embodiment, the invention includes a method of treating a cancer with a population of TILs, or use of the TILs to treat cancer, wherein a patient is pre-treated with non-myeloablative chemotherapy prior to an infusion of TILs according to the present disclosure. In an embodiment, the non-myeloablative chemotherapy is cyclophosphamide 60 mg / kg / d for 2 days (days 7 and 2 prior to TIL infusion) and fludarabine 25 mg / m2 / d for 5 days (days 5 to 1 prior to TIL infusion). In an embodiment, after non-myeloablative chemotherapy and TIL infusion (at day 0) according to the present disclosure, the patient receives an intravenous infusion of IL-2 intravenously at 100,000 to 1 ,000,000 such as 720,000 ID / kg every 8 hours to physiologic tolerance, or such as such as 600,000 ID / kg every 8 hours to physiologic tolerance.In an embodiment, the non-myeloablative chemotherapy is cyclophosphamide 500 mg / m2 / day i.v. for 3 days on day -4, -3, -2 and fludarabine 30 mg / m2 / day i.v. for 2 days on day -4, -3 followed by TIL infusion on day 0. In an embodiment, after non-myeloablative chemotherapy and TIL infusion (at day 0) according to the present disclosure, the patient receives an intravenous infusion of IL-2 intravenously at 720,000 lU / kg every 8 hours to physiologic tolerance.

[0455] An aspect of the present invention relates to a population of tumor infiltrating lymphocytes (TILs) according to the present invention, wherein the population of clinically (therapeutically) relevant TILs, has at least 5 % percentage of CD39- / CD69- CD8 T cells based on an average of individual 10 patient batches. This can also be on an average of individual 5 patient batches or 20 patient batches.

[0456] In one or more embodiments of the present invention, the population of clinically (therapeutically) relevant TILs, has at least 8 % percentage of CD39- / CD69- CD8 T cells based on an average of individual 10 patient batches.

[0457] In one or more embodiments of the present invention, the population of clinically (therapeutically) relevant TILs, has at least 10 % percentage of CD39- / CD69- CD8 T cells based on an average of individual 10 patient batches.

[0458] In one or more embodiments of the present invention, the population of clinically (therapeutically) relevant TILs, has at least 12 % percentage of CD39- / CD69- CD8 T cells based on an average of individual 10 patient batches.

[0459] In one or more embodiments of the present invention, the population of clinically (therapeutically) relevant TILs, has at least 15 % percentage of CD39- / CD69- CD8 T cells based on an average of individual 10 patient batches.

[0460] In one or more embodiments of the present invention, the population of clinically (therapeutically) relevant TILs, has at least 20 % percentage of CD39- / CD69- CD8 T cells based on an average of individual 10 patient batches.

[0461] In one or more embodiments of the present invention, the population of clinically (therapeutically) relevant TILs, has at least 30 % percentage of CD39- / CD69- CD8 T cells based on an average of individual 10 patient batches.

[0462] In one or more embodiments of the present invention, the population of clinically (therapeutically) relevant TILs, has at least 40 % percentage of CD39- / CD69- CD8 T cells based on an average of individual 10 patient batches.

[0463] In one or more embodiments of the present invention, the population of clinically (therapeutically) relevant TILs, has at least 50 % percentage of CD39- / CD69- CD8 T cells based on an average of individual 10 patient batches.In one or more embodiments of the present invention, the population of clinically (therapeutically) relevant TILs, has at least 60 % percentage of CD39- / CD69- CD8 T cells based on an average of individual 10 patient batches.

[0464] In one or more embodiments of the present invention, the population of clinically relevant TILs comprises trace amounts of TME stimulators, such as a substance selected from the group consisting of group J, group A, and group B. With TME stimulators, especially in time delay (TD) conditions the present inventors are able to increase the stem-like CD8 T cells and decrease the CD39+ CD69+ cells. Increased % of stem-like CD39- CD69- CD8 T cells with TME stimulators was also observed. Increased number of these stem-like CD8 T cells particularly in JAB TD condition (It was shown that responders to ACT had higher numbers of stem-like CD39- CD69- CD8 T cells in the infusion products). Thus, these effects are seen when step b) and step c) are performed in time lapse. A and B can be added in step b) while J is added in step c) to give the effect. Thus, one or more embodiments of the present invention relates to a method disclosed herein where in substances from group A and B are added in step b) for the first expansion followed by a time delay before a substance from group J is added in step c) for the second expansion.

[0465] In one or more embodiments of the present invention, the population of clinically relevant TILs comprises one or more aAPCs according to the present invention. The person skilled in the art will be able to identify a population of clinically relevant TILs comprises one or more aAPCs according to the present invention by using methods known in the art, such as PCR to identify the specific nucleic acid sequences that have been introduced into the aAPCs of the present invention.

[0466] The use aAPCs of the present invention for the expansion of TILs, and optionally combined with the time delay dscribed herein, will as described in the examples of the present disclosure show favorable results with more T and CD8 T cells, such as cells with increased %CD28+ CD8 T cells, less % CD39+ CD69+ and more % CD39- CD69- CD8 T cells. The person skilled in the art will be able to identify a population of clinically relevant TILs that comprises such subpopulations, e.g. using FACS or similar methods in the art. The examples herein clearly describe that it is possible to identify such subpopulations.

[0467] Process

[0468] An aspect of the present invention describes a process for expanding cells into a population of cells, that can be therapeutic, comprising the steps:

[0469] a. obtaining a population of cells from a tumor and / or tumor fragments resected from a mammal;

[0470] b. expanding the population of cells by culturing said population of cells in a cell culture medium, wherein feeder cells are added to the cell culture 0-4 times atpredetermined timepoint(s) and / or predetermined number(s) of cells, to produce a second population of cells, optionally wherein the second population of cells is a therapeutic population of cells.

[0471] c. Optionally harvesting the second population of cells.

[0472] An aspect of the present invention relates to a process for expanding cells into a population of cells, that can be therapeutic, comprising the steps:

[0473] a. obtaining a population of cells from a tumor and / or tumor fragments resected from a mammal;

[0474] b. expanding the population of cells by culturing said population of cells in a cell culture medium, optionally wherein feeder cells are added to the cell culture: i. at one or more predetermined timepoint(s) and / or, ii. when said population of cells have expanded to one or more predetermined number(s) of cells;

[0475] to produce a second population of cells, optionally wherein the second population of cells is a therapeutic population of cells;

[0476] c. Optionally harvesting the second population of cells.

[0477] An aspect of the present invention describes a process for expanding cells into a population of cells, that can be therapeutic, comprising the steps:

[0478] a. obtaining a population of cells from a tumor and / or tumor fragments resected from a mammal;

[0479] b. expanding the population of cells by culturing said population of cells in a cell culture medium, wherein feeder cells are added to the cell culture at least twice at predetermined timepoint(s), to produce a second population of cells. c. Optionally harvesting the second population of cells.

[0480] An aspect of the present invention describes a process for expanding cells into a population of cells, that can be therapeutic, comprising the steps:

[0481] a. obtaining a population of cells from a tumor and / or tumor fragments resected from a mammal;

[0482] b. expanding the population of cells by culturing said population of cells in a cell culture medium, optionally wherein feeder cells are added to the cell culture at least once at predetermined timepoint(s) or at predetermined number(s) of the population of cells, to produce a second population of cells.

[0483] c. Optionally harvesting the second population of cells.In the least, the process of the present invention results in clinically relevant TILs which can be generated through a continuous process utilizing feeder cells and TIL stimulators early in the process and scaling the culture vessels appropriately, compared to the two-step method currently used in the expansions of TILs.

[0484] The inventors have also identified specific timepoints which are advantageous for the growth and expansion of a population cells such as T cells and TILs.

[0485] Furthermore, the process of the present invention has been shown to result in a faster expansion of T cells in a shorter amount of time. In addition, said process uses less feeder cells compared to the traditional process. For example, the process of the present invention has been shown to result in a reduction of irradiated feeder cells of 84-96% when compared to a traditional REP process to expand a similar number of cells.

[0486] Amongst other advantages, the process of the present invention can be performed with irradiated PBMCs or irradiated artificial feeder cells and that the whole culture can be performed in serum-free media (SFM) resulting in a donor-free end-to-end process.

[0487] In one or more embodiments, adding feeder cells to the cell culture produces a second population of cells. In one or more embodiments, adding feeder cells to the cell culture at predetermined timepoint(s) and / or predetermined number(s) of cells produces a second population of cells.

[0488] In one or more embodiments, the feeder cells comprise or consist of the artificial feeder cells according to the present invention. In one or more embodiments, the feeder cells comprise the artificial feeder cells according to the present invention. In one or more embodiments, the feeder cells consist of the artificial feeder cells according to the present invention.

[0489] In one or more embodiments, the feeder cells comprise PBMCs. In one or more embodiments, the feeder cells consist of PBMCs.

[0490] In one or more embodiments, the feeder cells comprise PBMCs and the artificial feeder cells of the present invention. In one or more embodiments, the feeder cells consist of PBMCs and the artificial feeder cells of the present invention.

[0491] In one or more embodiments, the feeder cells are added to the cell culture once. In one or more embodiments, the feeder cells are added to the cell culture twice. In one or more embodiments, the feeder cells are added to the cell culture three times. In one or more embodiments, the feeder cells are added to the cell culture four times.In one or more embodiments, feeder cells are added to the cell culture at least once in step b). In one or more embodiments, feeder cells are added to the cell culture at least two times in step b). In one or more embodiments, feeder cells are added to the cell culture at least three times in step b). In one or more embodiments, feeder cells are added to the cell culture at least four times in step b).

[0492] In one or more embodiments, feeder cells are added to the cell culture once in step b). In one or more embodiments, feeder cells are added to the cell culture two times in step b). In one or more embodiments, feeder cells are added to the cell culture three times in step b). In one or more embodiments, feeder cells are added to the cell culture four times in step b).

[0493] In one or more embodiments of the process of the present invention, step c) describes harvesting the second population of cells.

[0494] In one or more embodiments, feeder cells are added to the cell culture: i. at one or more predetermined timepoint(s) and / or, ii. when said population of cells have expanded to one or more predetermined number(s) of cells. In one or more embodiments, feeder cells are added to the cell culture: i. at one or more predetermined timepoint(s) or, ii. when said population of cells have expanded to one or more predetermined number(s) of cells. In one or more embodiments, feeder cells are added to the cell culture: i. at one or more predetermined timepoint(s). In one or more embodiments, feeder cells are added to the cell culture: when said population of cells have expanded to one or more predetermined number(s) of cells. In one or more embodiments, feeder cells are added to the cell culture: i. at one or more predetermined timepoint(s) and, ii. when said population of cells have expanded to one or more predetermined number(s) of cells.

[0495] In one or more embodiments, the feeder cells are added to the cell culture one or more times. In one or more embodiments, the feeder cells are added to the cell culture two or more times. In one or more embodiments, the feeder cells are added to the cell culture three or more times. In one or more embodiments, the feeder cells are added to the cell culture four or more times.

[0496] In one or more embodiments, the feeder cells are added to the cell culture one or more times at predetermined timepoint(s) and / or predetermined number(s) of cells. In one or more embodiments, the feeder cells are added to the cell culture two or more times at predetermined timepoint(s) and / or predetermined number(s) of cells. In one or more embodiments, the feeder cells are added to the cell culture three or more times at predetermined timepoint(s) and / or predetermined number(s) of cells. In one or more embodiments, the feeder cells are added to the cell culture four or more times at predetermined timepoint(s) and / or predetermined number(s) of cells.

[0497] In one or more embodiments, the feeder cells are added to the cell culture 1-4 times at predetermined timepoint(s) and / or predetermined number(s) of cells. In one or more embodiments,the feeder cells are added to the cell culture 1-3 times at predetermined timepoint(s) and / or predetermined number(s) of cells. In one or more embodiments, the feeder cells are added to the cell culture 1-2 times at predetermined timepoint(s) and / or predetermined number(s) of cells. In one or more embodiments, the feeder cells are added to the cell culture 2-4 times at predetermined timepoint(s) and / or predetermined number(s) of cells. In one or more embodiments, the feeder cells are added to the cell culture 2-3 times at predetermined timepoint(s) and / or predetermined number(s) of cells. In one or more embodiments, the feeder cells are added to the cell culture 2-5 times at predetermined timepoint(s) and / or predetermined number(s) of cells. In one or more embodiments, the feeder cells are added to the cell culture one time at predetermined timepoint(s) and / or predetermined number(s) of cells. In one or more embodiments, the feeder cells are added to the cell culture two times at predetermined timepoint(s) and / or predetermined number(s) of cells. In one or more embodiments, the feeder cells are added to the cell culture three times at predetermined timepoint(s) and / or predetermined number(s) of cells. In one or more embodiments, the feeder cells are added to the cell culture four times at predetermined timepoint(s) and / or predetermined number(s) of cells.

[0498] In one or more embodiments, the feeder cells are added to the cell culture 1-4 times at predetermined timepoint(s). In one or more embodiments, the feeder cells are added to the cell culture 1-3 times at predetermined timepoint(s). In one or more embodiments, the feeder cells are added to the cell culture 1-2 times at predetermined timepoint(s). In one or more embodiments, the feeder cells are added to the cell culture 2-4 times at predetermined timepoint(s). In one or more embodiments, the feeder cells are added to the cell culture 2-3 times at predetermined timepoint(s). In one or more embodiments, the feeder cells are added to the cell culture 2-5 times at predetermined timepoint(s). In one or more embodiments, the feeder cells are added to the cell culture one time at a predetermined timepoint. In one or more embodiments, the feeder cells are added to the cell culture two times at predetermined timepoint(s). In one or more embodiments, the feeder cells are added to the cell culture three times at predetermined timepoint(s). In one or more embodiments, the feeder cells are added to the cell culture four times at predetermined timepoint(s).

[0499] In one or more embodiments, the feeder cells are added to the cell culture 1-4 times at predetermined number(s) of cells. In one or more embodiments, the feeder cells are added to the cell culture 1-3 times at predetermined number(s) of cells. In one or more embodiments, the feeder cells are added to the cell culture 1-2 times at predetermined number(s) of cells. In one or more embodiments, the feeder cells are added to the cell culture 2-4 times at predetermined number(s) of cells. In one or more embodiments, the feeder cells are added to the cell culture 2-3 times at predetermined number(s) of cells. In one or more embodiments, the feeder cells are added to the cell culture 2-5 times at predetermined number(s) of cells. In one or more embodiments, the feeder cells are added to the cell culture one time at predetermined number(s) of cells. In one or moreembodiments, the feeder cells are added to the cell culture two times at predetermined number(s) of cells. In one or more embodiments, the feeder cells are added to the cell culture three times at predetermined number(s) of cells. In one or more embodiments, the feeder cells are added to the cell culture four times at predetermined number(s) of cells.

[0500] In one or more embodiments, the feeder cells are added to the cell culture 1-4 times at predetermined timepoint(s) and predetermined number(s) of cells. In one or more embodiments, the feeder cells are added to the cell culture 1-3 times at predetermined timepoint(s) and predetermined number(s) of cells. In one or more embodiments, the feeder cells are added to the cell culture 1-2 times at predetermined timepoint(s) and predetermined number(s) of cells. In one or more embodiments, the feeder cells are added to the cell culture 2-4 times at predetermined timepoint(s) and / or predetermined number(s) of cells. In one or more embodiments, the feeder cells are added to the cell culture 2-3 times at predetermined timepoint(s) and predetermined number(s) of cells. In one or more embodiments, the feeder cells are added to the cell culture 2-5 times at predetermined timepoint(s) and predetermined number(s) of cells. In one or more embodiments, the feeder cells are added to the cell culture one time at predetermined timepoint(s) and predetermined number(s) of cells. In one or more embodiments, the feeder cells are added to the cell culture two times at predetermined timepoint(s) and predetermined number(s) of cells. In one or more embodiments, the feeder cells are added to the cell culture three times at predetermined timepoint(s) and / or predetermined number(s) of cells. In one or more embodiments, the feeder cells are added to the cell culture four times at predetermined timepoint(s) and predetermined number(s) of cells.

[0501] Predetermined ti me point (s)

[0502] In the context of the present process of the invention, the term ‘predetermined timepoint(s)’ describes the addition of the feeder cells at select points in time, wherein said addition of said feeder cells can facilitate the growth of the culture. These may be, for example, between 1-3 days, such as 2 days after culture initiation, 6-8 days, such as 7 days after culture initiation and / or optionally 12-15 days, such as 14 days after culture initiation. The addition of feeder cells may be performed one or more times during the process of the present invention at said predetermined timepoint(s). Adding feeder cells at predetermined timepoint(s) is based on the assumption that they will die since they have been irradiated or otherwise treated to make them incapable of cell division and thus will disappear from the culture shortly after. Adding feeder cells on multiple occasions extends the stimulation provided. They are likewise added when the culture is scaled up to alleviate the reduction in cell density, until the culture expands further. The term ‘predetermined timepoint’ may be used interchangeably with the term ‘predetermined period of time’. The term ‘one or more predetermined timepoint(s)’ may be used interchangeably with ‘predeterminedtimepoint(s)’. The term ‘predetermined timepoint(s) may be used interchangeably with predetermined timepoint(s)’.

[0503] In one or more embodiments, feeder cells are added to the cell culture at one or more predetermined timepoint(s). In one or more embodiments, feeder cells are added to the cell culture at two or more predetermined timepoint(s). In one or more embodiments, feeder cells are added to the cell culture at three or more predetermined timepoint(s). In one or more embodiments, feeder cells are added to the cell culture at four or more predetermined timepoint(s). In one or more embodiments, feeder cells are added to the cell culture at one predetermined timepoint. In one or more embodiments, feeder cells are added to the cell culture at two predetermined timepoint(s). In one or more embodiments, feeder cells are added to the cell culture at three predetermined timepoint(s). In one or more embodiments, feeder cells are added to the cell culture at four predetermined timepoint(s). In one or more embodiments, feeder cells are added to the cell culture at five predetermined timepoint(s). In one or more embodiments, feeder cells are added to the cell culture at 1-4 predetermined timepoint(s). In one or more embodiments, feeder cells are added to the cell culture at 1-5 predetermined timepoint(s). In one or more embodiments, feeder cells are added to the cell culture at 1-3 predetermined timepoint(s). In one or more embodiments, feeder cells are added to the cell culture at 1-2 predetermined timepoint(s). In one or more embodiments, feeder cells are added to the cell culture at 2-4 predetermined timepoint(s). In one or more embodiments, feeder cells are added to the cell culture at 2-3 predetermined timepoint(s). In one or more embodiments, feeder cells are added to the cell culture at 2-5 predetermined timepoint(s). In one or more embodiments, feeder cells are added to the cell culture at 3-4 predetermined timepoint(s).

[0504] The term ‘X-Y predetermined timepoint(s)’ describes the step of adding feeder cells at integers between X and Y. For example, using X as 1 and Y as 4, 1-4 predetermined timepoint(s) describes the addition of feeder cells either once, twice, three times or four times in the process.

[0505] In one or more embodiments, the one or more predetermined timepoint(s) is 1-3 days after culture initiation, 6-8 days after culture initiation and / or 12-15 days after culture initiation. In one or more embodiments, the one or more predetermined timepoint(s) is 1-3 days after culture initiation. In one or more embodiments, the one or more predetermined timepoint(s) is 6-8 days after culture initiation. In one or more embodiments, the one or more predetermined timepoint(s) is 12-15 days after culture initiation. In one or more embodiments, the one or more predetermined timepoint(s) is 1-3 days after culture initiation and 6-8 days after culture initiation. In one or more embodiments, the one or more predetermined timepoint(s) is 1-3 days after culture initiation, and 12-15 days after culture initiation. In one or more embodiments, the one or more predetermined timepoint(s) is 1-3 days after culture initiation, 6-8 days after culture initiation and 12-15 days after culture initiation. In one or more embodiments, the one or more predetermined timepoint(s) is 2 days after cultureinitiation, 7 days after culture initiation and / or 14 days after culture initiation. In one or more embodiments, the one or more predetermined timepoint(s) is 2 days after culture initiation. In one or more embodiments, the one or more predetermined timepoint(s) is 7 days after culture initiation. In one or more embodiments, the one or more predetermined timepoint(s) is 14 days after culture initiation. In one or more embodiments, the one or more predetermined timepoint(s) is 2 days after culture initiation and 7 days after culture initiation. In one or more embodiments, the one or more predetermined timepoint(s) is 2 days after culture initiation, and 14 days after culture initiation. In one or more embodiments, the one or more predetermined timepoint(s) is 2 days after culture initiation, 7 days after culture initiation and 14 days after culture initiation.

[0506] In one or more embodiments, the one or more predetermined timepoint(s) is 1 day after culture initiation. In one or more embodiments, the one or more predetermined timepoint(s) is 2 days after culture initiation. In one or more embodiments, the one or more predetermined timepoint(s) is 3 days after culture initiation. In one or more embodiments, the one or more predetermined timepoint(s) is 4 days after culture initiation. In one or more embodiments, the one or more predetermined timepoint(s) is 5 days after culture initiation. In one or more embodiments, the one or more predetermined timepoint(s) is 6 days after culture initiation. In one or more embodiments, the one or more predetermined timepoint(s) is 7 days after culture initiation. In one or more embodiments, the one or more predetermined timepoint(s) is 8 days after culture initiation. In one or more embodiments, the one or more predetermined timepoint(s) is 9 days after culture initiation. In one or more embodiments, the one or more predetermined timepoint(s) is 10 days after culture initiation. In one or more embodiments, the one or more predetermined timepoint(s) is 11 days after culture initiation. In one or more embodiments, the one or more predetermined timepoint(s) is 12 days after culture initiation. In one or more embodiments, the one or more predetermined timepoint(s) is 13 days after culture initiation. In one or more embodiments, the one or more predetermined timepoint(s) is 14 days after culture initiation. In one or more embodiments, the one or more predetermined timepoint(s) is 15 days after culture initiation.

[0507] In one or more embodiments, the one or more predetermined timepoint(s) comprises 1 day after culture initiation. In one or more embodiments, the one or more predetermined timepoint(s) comprises 2 days after culture initiation. In one or more embodiments, the one or more predetermined timepoint(s) comprises 3 days after culture initiation. In one or more embodiments, the one or more predetermined timepoint(s) comprises 4 days after culture initiation. In one or more embodiments, the one or more predetermined timepoint(s) comprises 5 days after culture initiation. In one or more embodiments, the one or more predetermined timepoint(s) comprises 6 days after culture initiation. In one or more embodiments, the one or more predetermined timepoint(s) comprises 7 days after culture initiation. In one or more embodiments, the one or more predetermined timepoint(s) comprises 8 days after culture initiation. In one or more embodiments, the one or more predetermined timepoint(s) comprises 9 days after culture initiation. In one ormore embodiments, the one or more predetermined timepoint(s) comprises 10 days after culture initiation. In one or more embodiments, the one or more predetermined timepoint(s) comprises 11 days after culture initiation. In one or more embodiments, the one or more predetermined timepoint(s) comprises 12 days after culture initiation. In one or more embodiments, the one or more predetermined timepoint(s) comprises 13 days after culture initiation. In one or more embodiments, the one or more predetermined timepoint(s) comprises 14 days after culture initiation. In one or more embodiments, the one or more predetermined timepoint(s) comprises 15 days after culture initiation.

[0508] In one or more embodiments, the feeder cells are added to the cell culture at least once at predetermined timepoint(s). In one or more embodiments, the feeder cells are added to the cell culture at least two times at predetermined timepoint(s). In one or more embodiments, the feeder cells are added to the cell culture at least three times at predetermined timepoint(s). In one or more embodiments, the feeder cells are added to the cell culture at least four times at predetermined timepoint(s).

[0509] In one or more embodiments, the feeder cells are added to the cell culture at least once at predetermined timepoint(s) in step b.. In one or more embodiments, the feeder cells are added to the cell culture at least two times at predetermined timepoint(s) in step b.. In one or more embodiments, the feeder cells are added to the cell culture at least three times at predetermined timepoint(s) in step b.. In one or more embodiments, the feeder cells are added to the cell culture at least four times at predetermined timepoint(s) in step b..

[0510] In one or more embodiments, the feeder cells are added to the cell culture once at a predetermined timepoint in step b.. In one or more embodiments, the feeder cells are added to the cell culture two times at predetermined timepoint(s) in step b.. In one or more embodiments, the feeder cells are added to the cell culture three times at a predetermined timepoint(s) in step b.. In one or more embodiments, the feeder cells are added to the cell culture four times at a predetermined timepoint(s) in step b..

[0511] In one or more embodiments, the feeder cells are added to the cell culture once at a predetermined timepoint. In one or more embodiments, the feeder cells are added to the cell culture two times at predetermined timepoint(s). In one or more embodiments, the feeder cells are added to the cell culture three times at predetermined timepoint(s). In one or more embodiments, the feeder cells are added to the cell culture four times at predetermined timepoint(s).

[0512] In one or more embodiments, the predetermined timepoint(s) is 1-3 days after culture initiation, 6-8 days after culture initiation and / or 12-15 days after culture initiation. In one or more embodiments, the predetermined timepoint(s) is 1-3 days after culture initiation and 6-8 days after cultureinitiation. In one or more embodiments, the predetermined timepoint(s) is 1-3 days after culture initiation and 12-15 days after culture initiation. In one or more embodiments, the predetermined timepoint(s) is 1-3 days after culture initiation, 6-8 days after culture initiation and 12-15 days after culture initiation.

[0513] In one or more embodiments, the predetermined timepoint(s) comprises 1-3 days after culture initiation, 6-8 days after culture initiation and / or 12-15 days after culture initiation. In one or more embodiments, the predetermined timepoint(s) comprises 1-3 days after culture initiation and 6-8 days after culture initiation. In one or more embodiments, the predetermined timepoint(s) comprises 1-3 days after culture initiation and 12-15 days after culture initiation. In one or more embodiments, the predetermined timepoint(s) comprises 1-3 days after culture initiation, 6-8 days after culture initiation and 12-15 days after culture initiation.

[0514] In one or more embodiments, the predetermined timepoint(s) is 1-3 days after culture initiation. In one or more embodiments, the predetermined timepoint(s) is 6-8 days after culture initiation. In one or more embodiments, the predetermined timepoint(s) is 12-15 days after culture initiation. In one or more embodiments, the predetermined timepoint(s) comprises 1-3 days after culture initiation. In one or more embodiments, the predetermined timepoint(s) comprises 6-8 days after culture initiation. In one or more embodiments, the predetermined timepoint(s) comprises 12-15 days after culture initiation.

[0515] In one or more embodiments, the predetermined timepoint(s) is 1 day after culture initiation. In one or more embodiments, the predetermined timepoint(s) is 2 days after culture initiation. In one or more embodiments, the predetermined timepoint(s) is 3 days after culture initiation. In one or more embodiments, the predetermined timepoint(s) is 4 days after culture initiation. In one or more embodiments, the predetermined timepoint(s) is 5 days after culture initiation. In one or more embodiments, the predetermined timepoint(s) is 6 days after culture initiation. In one or more embodiments, the predetermined timepoint(s) is 7 days after culture initiation. In one or more embodiments, the predetermined timepoint(s) is 8 days after culture initiation. In one or more embodiments, the predetermined timepoint(s) is 9 days after culture initiation. In one or more embodiments, the predetermined timepoint(s) is 10 days after culture initiation. In one or more embodiments, the predetermined timepoint(s) is 11 days after culture initiation. In one or more embodiments, the predetermined timepoint(s) is 12 days after culture initiation. In one or more embodiments, the predetermined timepoint(s) is 13 days after culture initiation. In one or more embodiments, the predetermined timepoint(s) is 14 days after culture initiation. In one or more embodiments, the predetermined timepoint(s) is 15 days after culture initiation.

[0516] In one or more embodiments, the predetermined timepoint(s) comprises 1 day after culture initiation. In one or more embodiments, the predetermined timepoint(s) comprises 2 days afterculture initiation. In one or more embodiments, the predetermined timepoint(s) comprises 3 days after culture initiation. In one or more embodiments, the predetermined timepoint(s) comprises 4 days after culture initiation. In one or more embodiments, the predetermined timepoint(s) comprises 5 days after culture initiation. In one or more embodiments, the predetermined timepoint(s) comprises 6 days after culture initiation. In one or more embodiments, the predetermined timepoint(s) comprises 7 days after culture initiation. In one or more embodiments, the predetermined timepoint(s) comprises 8 days after culture initiation. In one or more embodiments, the predetermined timepoint(s) comprises 9 days after culture initiation. In one or more embodiments, the predetermined timepoint(s) comprises 10 days after culture initiation. In one or more embodiments, the predetermined timepoint(s) comprises 11 days after culture initiation. In one or more embodiments, the predetermined timepoint(s) comprises 12 days after culture initiation. In one or more embodiments, the predetermined timepoint(s) comprises 13 days after culture initiation. In one or more embodiments, the predetermined timepoint(s) comprises 14 days after culture initiation. In one or more embodiments, the predetermined timepoint(s) comprises 15 days after culture initiation.

[0517] Predetermined number(s) of cells

[0518] In one or more embodiments, feeder cells are added to the cell culture when the population of cells have expanded to one or more predetermined number(s) of cells.

[0519] In the context of the present process of the invention, the term ‘predetermined number of cells' describes the number of cells required in the population of cells expanded in step b), so that the cells are not too dense in the culture and that the addition of feeder cells facilitates the growth of the culture. Thus, the term ‘predetermined number(s) of cells’ or ‘one or more predetermined number(s) of cells’ is taken to mean one or more predetermined values which describe the number of cells required in the population of cells expanded in step b), so that the cells are not too dense in the culture and that the addition of feeder cells facilitates the growth of the culture. For example, the predetermined number(s) of cells, assuming two predetermined numbers of cells, may be e.g., 1x106cells / cm2as the first predetermined number of cells, and 1x107cells / cm2as the second predetermined number of cells. Thus, in this example, feeder cells are added to the culture when the population of cells in step b. has expanded to 1x106cells / cm2and 1x107cells / cm2, respectively. Said predetermined number(s) of cells’ may also be a singular value e.g. 1x106cells / cm2.

[0520] Said predetermined number(s) of cells may correlate with the predetermined timepoint(s) of the present disclosure, such as wherein the population of cells have expanded to 1x106cells / cm2, 2 days after culture initiation. In this example, 1x106cells / cm2is a predetermined number of cells and 2 days is the predetermined timepoint.Methods to count said predetermined number of cells are known to a skilled person e.g., methods for cell counting, analysing lactate readings, metabolites or similar.

[0521] In one or more embodiments, a predetermined number of cells is between 1x105cells / cm2to 1x107cells / cm2. In one or more embodiments a predetermined number of cells is between 100x105cells / cm2to 100x106cells / cm2. In one or more embodiments, a predetermined number of cells is between 1x106cells / cm2to 100x106cells / cm2. In one or more embodiments, a predetermined number of cells is between 5x106cells / cm2to 20 x106cells / cm2. In one or more embodiments a predetermined number of cells is 10x106cells / cm2.

[0522] In one or more embodiments, the predetermined number(s) of cells is between 1x105cells / cm2to 1x107cells / cm2. In one or more embodiments, the predetermined number(s) of cells is between 100x105cells / cm2to 100x106cells / cm2. In one or more embodiments, the predetermined number(s) of cells is between 1x106cells / cm2to 100x106cells / cm2. In one or more embodiments, the predetermined number(s) of cells is between 5x106cells / cm2to 20 x106cells / cm2. In one or more embodiments the predetermined number(s) of cells is 10x106cells / cm2.

[0523] In one or more embodiments, the predetermined number(s) of cells comprises a predetermined number between 1x105cells / cm2to 1x107cells / cm2. In one or more embodiments the predetermined number(s) of cells comprises a predetermined number between 100x105cells / cm2to 100x106cells / cm2. In one or more embodiments, the predetermined number(s) of cells comprises a predetermined number between 1x106cells / cm2to 100x106cells / cm2. In one or more embodiments, the predetermined number(s) of cells comprises a predetermined number between 5x106cells / cm2to 20 x106cells / cm2. In one or more embodiments the predetermined number(s) of cells comprises a predetermined number between 10x106cells / cm2.

[0524] In one or more embodiments, the predetermined number(s) of cells is a predetermined number between 1x105cells / cm2to 1x107cells / cm2. In one or more embodiments the predetermined number(s) of cells is a predetermined number between 100x105cells / cm2to 100x106cells / cm2. In one or more embodiments, the predetermined number(s) of cells is a predetermined number between 1x106cells / cm2to 100x106cells / cm2. In one or more embodiments, the predetermined number(s) of cells is a predetermined number between 5x106cells / cm2to 20 x106cells / cm2. In one or more embodiments the predetermined number(s) of cells is a predetermined number between 10x106cells / cm2.

[0525] In one or more embodiments, the one or more predetermined numbers of cells is between 1x105cells / cm2to 1x107cells / cm2. In one or more embodiments the one or more predeterminednumbers of cells is between 100x105cells / cm2to 100x106cells / cm2. In one or more embodiments, the one or more predetermined numbers of cells is between 1x106cells / cm2to 100x106cells / cm2. In one or more embodiments, the one or more predetermined numbers of cells is between 5x106cells / cm2to 20 x106cells / cm2. In one or more embodiments the one or more predetermined numbers of cells is 10x106cells / cm2.

[0526] In one or more embodiments, the one or more predetermined numbers of cells comprises a predetermined number between 1x105cells / cm2to 1x107cells / cm2. In one or more embodiments the one or more predetermined numbers of cells comprises a predetermined number between 100x105cells / cm2to 100x106cells / cm2. In one or more embodiments, the one or more predetermined numbers of cells comprises a predetermined number between 1x106cells / cm2to 100x106cells / cm2. In one or more embodiments, the one or more predetermined numbers of cells comprises a predetermined number between 5x106cells / cm2to 20 x106cells / cm2. In one or more embodiments the one or more predetermined numbers of cells comprises a predetermined number between 10x106cells / cm2.

[0527] In one or more embodiments, the one or more predetermined numbers of cells is a predetermined number between 1x105cells / cm2to 1x107cells / cm2. In one or more embodiments the one or more predetermined numbers of cells is a predetermined number between 100x105cells / cm2to 100x106cells / cm2. In one or more embodiments, the one or more predetermined numbers of cells is a predetermined number between 1x106cells / cm2to 100x106cells / cm2. In one or more embodiments, the one or more predetermined numbers of cells is a predetermined number between 5x106cells / cm2to 20 x106cells / cm2. In one or more embodiments the one or more predetermined numbers of cells is a predetermined number between 10x106cells / cm2.

[0528] Culture initiation

[0529] The term culture initiation describes the start of the population of cells from a tumor and / or tumor fragments resected from a mammal, such as described in step a) of the process of the present invention. Said culture initiation is taken as day 0 in the process and / or method of the present disclosure.

[0530] One or more embodiments of the present disclosure describes the process of the present invention comprising a step of adding the feeder cells 1-3 days after culture initiation in step b).

[0531] One or more embodiments of the present disclosure describes the process of the present invention comprising a step of adding the feeder cells 6-8 days after culture initiation in step b).One or more embodiments of the present disclosure describes the process of the present invention comprising a step of adding the feeder cells 1-3 days after culture initiation in step b) and 6-8 days after culture initiation in step b).

[0532] One or more embodiments of the present disclosure describes the process of the present invention comprising a step of adding the feeder cells 12-15 days after culture initiation in step b).

[0533] One or more embodiments of the present disclosure describes the process of the present invention comprising a step of adding the feeder cells 1-3 days after culture initiation in step b) and 6-8 days after culture initiation in step b), and 12-15 days after culture initiation.

[0534] One or more embodiments of the present disclosure describes the process of the present invention comprising a step of adding the feeder cells 1-3 days after culture initiation in step b) and 12-15 days after culture initiation.

[0535] One or more embodiments of the present disclosure describes the process of the present invention comprising a step of adding the feeder cells 2 days after culture initiation in step b).

[0536] One or more embodiments of the present disclosure describes the process of the present invention comprising a step of adding the feeder cells 7 days after culture initiation in step b).

[0537] One or more embodiments of the present disclosure describes the process of the present invention comprising a step of adding the feeder cells 14 days after culture initiation in step b).

[0538] TME stimulators and anti-CD3 antibody

[0539] In one or more embodiments, one or more TME stimulators are added. In one or more embodiments, one or more TME stimulators are added in step b).

[0540] In one or more embodiments, the one or more TME stimulators are added in step b) at culture initiation. In one or more embodiments, the one or more TME stimulators are added at culture initiation.

[0541] In one or more embodiments, the one or more TME stimulators are added in step b) simultaneously with the feeder cells. In one or more embodiments, the one or more TME stimulators are added simultaneously with the feeder cells.In one or more embodiments, the one or more TME stimulators are added in step b) simultaneously with the feeder cells. In one or more embodiments, the one or more TME stimulators are added simultaneously with the feeder cells.

[0542] In one or more embodiments, the one or more TME stimulators are added in step b) simultaneously with the feeder cells at least once. In one or more embodiments, the one or more TME stimulators are added in step b) simultaneously with the feeder cells at least twice. In one or more embodiments, the one or more TME stimulators are added in step b) simultaneously with the feeder cells at least three times. In one or more embodiments, the one or more TME stimulators are added in step b) simultaneously with the feeder cells at least four times. In one or more embodiments, the one or more TME stimulators are added in step b) simultaneously with the feeder cells once. In one or more embodiments, the one or more TME stimulators are added in step b) simultaneously with the feeder cells twice. In one or more embodiments, the one or more TME stimulators are added in step b) simultaneously with the feeder cells three times. In one or more embodiments, the one or more TME stimulators are added in step b) simultaneously with the feeder cells four times.

[0543] In one or more embodiments, feeder cells are added to the cell culture at least two times or at least three times in step b) and no more than five times. In one or more embodiments, feeder cells are added to the cell culture no more than five times in step b). In one or more embodiments, feeder cells are added to the cell culture no more than four times in step b). In one or more embodiments, feeder cells are added to the cell culture 0-5 times in step b). In one or more embodiments, feeder cells are added to the cell culture 1-4 times in step b). In one or more embodiments, feeder cells are added to the cell culture 1-5 times. In one or more embodiments, feeder cells are added to the cell culture 0-4 times in step b). In one or more embodiments, feeder cells are added to the cell culture 1-3 times in step b). In one or more embodiments, feeder cells are added to the cell culture 0-3 times in step b). In one or more embodiments, feeder cells are added to the cell culture 2-3 times in step b). In one or more embodiments, feeder cells are added to the cell culture two times or three times.

[0544] In one or more embodiments, feeder cells are added to the cell culture no more than five times. In one or more embodiments, feeder cells are added to the cell culture no more than four times. In one or more embodiments, feeder cells are added to the cell culture 0-5 times. In one or more embodiments, feeder cells are added to the cell culture 1-4 times. In one or more embodiments, feeder cells are added to the cell culture 1-5 times. In one or more embodiments, feeder cells are added to the cell culture 0-4 times. In one or more embodiments, feeder cells are added to the cell culture 1-3 times. In one or more embodiments, feeder cells are added to the cell culture 0-3 times.In one or more embodiments, feeder cells are added to the cell culture 2-3 times. In one or more embodiments, feeder cells are added to the cell culture two times or three times.

[0545] In one or more embodiments, the one or more TME stimulators are added simultaneously with the feeder cells at least once. In one or more embodiments, the one or more TME stimulators are added simultaneously with the feeder cells at least twice. In one or more embodiments, the one or more TME stimulators are added simultaneously with the feeder cells at least three times. In one or more embodiments, the one or more TME stimulators are added simultaneously with the feeder cells at least four times. In one or more embodiments, the one or more TME stimulators are added in simultaneously with the feeder cells once. In one or more embodiments, the one or more TME stimulators are added in simultaneously with the feeder cells twice. In one or more embodiments, the one or more TME stimulators are added simultaneously with the feeder cells three times. In one or more embodiments, the one or more TME stimulators are added simultaneously with the feeder cells four times.

[0546] In one or more embodiments, the one or more TME stimulators are added in step b) simultaneously with the feeder cells at each step of adding said feeder cells. In one or more embodiments, the one or more TME stimulators are added simultaneously with the feeder cells at each step of adding said feeder cells.

[0547] In one or more embodiments, the one or more TME stimulators and / or an anti-CD3 antibody is added 0-3 days after culture initiation. In one or more embodiments, the one or more TME stimulators and / or an anti-CD3 antibody is added 1-3 days after culture initiation. In one or more embodiments, the one or more TME stimulators and / or an anti-CD3 antibody is added 6-8 days after culture initiation. In one or more embodiments, the one or more TME stimulators and / or an anti-CD3 antibody is added 12-15 days after culture initiation.

[0548] In one or more embodiments, the one or more TME stimulators is added 1-3 days after culture initiation. In one or more embodiments, the one or more TME stimulators is added 6-8 days after culture initiation. In one or more embodiments, the one or more TME stimulators is added 12-15 days after culture initiation.

[0549] In one or more embodiments, an anti-CD3 antibody is added 1-3 days after culture initiation. In one or more embodiments an anti-CD3 antibody is added 6-8 days after culture initiation. In one or more embodiments, an anti-CD3 antibody is added 12-15 days after culture initiation.

[0550] In one or more embodiments, the one or more TME stimulators and an anti-CD3 antibody is added 1-3 days after culture initiation. In one or more embodiments, the one or more TME stimulatorsand an anti-CD3 antibody is added 6-8 days after culture initiation. In one or more embodiments, the one or more TME stimulators and an anti-CD3 antibody is added 12-15 days after culture initiation.

[0551] In one or more embodiments, one or more TME stimulators is added at culture initiation. In one or more embodiments, one or more TME stimulators is added 1 day after culture initiation. In one or more embodiments, one or more TME stimulators is added 2 days after culture initiation. In one or more embodiments, one or more TME stimulators is added 3 days after culture initiation. In one or more embodiments, one or more TME stimulators is added 4 days after culture initiation. In one or more embodiments, one or more TME stimulators is added 5 days after culture initiation. In one or more embodiments, one or more TME stimulators is added 6 days after culture initiation. In one or more embodiments, one or more TME stimulators is added 7 days after culture initiation. In one or more embodiments, one or more TME stimulators is added 8 days after culture initiation. In one or more embodiments, one or more TME stimulators is added 9 days after culture initiation. In one or more embodiments, one or more TME stimulators is added 10 days after culture initiation. In one or more embodiments, one or more TME stimulators is added 11 days after culture initiation. In one or more embodiments, one or more TME stimulators is added 12 days after culture initiation. In one or more embodiments, one or more TME stimulators is added 13 days after culture initiation. In one or more embodiments, one or more TME stimulators is added 14 days after culture initiation. In one or more embodiments, one or more TME stimulators is added 15 days after culture initiation.

[0552] In one or more embodiments, an anti-CD3 antibody is added at culture initiation. In one or more embodiments, an anti-CD3 antibody is added 1 day after culture initiation. In one or more embodiments, an anti-CD3 antibody is added 2 days after culture initiation. In one or more embodiments, an anti-CD3 antibody is added 3 days after culture initiation. In one or more embodiments, an anti-CD3 antibody is added 4 days after culture initiation. In one or more embodiments, an anti-CD3 antibody is added 5 days after culture initiation. In one or more embodiments, an anti-CD3 antibody is added 6 days after culture initiation. In one or more embodiments, an anti-CD3 antibody is added 7 days after culture initiation. In one or more embodiments, an anti-CD3 antibody is added 8 days after culture initiation. In one or more embodiments, an anti-CD3 antibody is added 9 days after culture initiation. In one or more embodiments, an anti-CD3 antibody is added 10 days after culture initiation. In one or more embodiments, an anti-CD3 antibody is added 11 days after culture initiation. In one or more embodiments, an anti-CD3 antibody is added 12 days after culture initiation. In one or more embodiments, an anti-CD3 antibody is added 13 days after culture initiation. In one or more embodiments, an anti-CD3 antibody is added 14 days after culture initiation. In one or more embodiments, an anti-CD3 antibody is added 15 days after culture initiation.In one or more embodiments, one or more TME stimulators and an anti-CD3 antibody is added at culture initiation. In one or more embodiments, one or more TME stimulators and an anti-CD3 antibody is added 1 day after culture initiation. In one or more embodiments, one or more TME stimulators and an anti-CD3 antibody is added 2 days after culture initiation. In one or more embodiments, one or more TME stimulators and an anti-CD3 antibody is added 3 days after culture initiation. In one or more embodiments, one or more TME stimulators and an anti-CD3 antibody is added 4 days after culture initiation. In one or more embodiments, one or more TME stimulators and an anti-CD3 antibody is added 5 days after culture initiation. In one or more embodiments, one or more TME stimulators and an anti-CD3 antibody is added 6 days after culture initiation. In one or more embodiments, one or more TME stimulators and an anti-CD3 antibody is added 7 days after culture initiation. In one or more embodiments, an anti-CD3 antibody is added 8 days after culture initiation. In one or more embodiments, one or more TME stimulators and an anti-CD3 antibody is added 9 days after culture initiation. In one or more embodiments, one or more TME stimulators and an anti-CD3 antibody is added 10 days after culture initiation. In one or more embodiments, one or more TME stimulators and an anti-CD3 antibody is added 11 days after culture initiation. In one or more embodiments, one or more TME stimulators and an anti-CD3 antibody is added 12 days after culture initiation. In one or more embodiments, one or more TME stimulators and an anti-CD3 antibody is added 13 days after culture initiation. In one or more embodiments, one or more TME stimulators and an anti-CD3 antibody is added 14 days after culture initiation. In one or more embodiments, one or more TME stimulators and an anti-CD3 antibody is added 15 days after culture initiation.

[0553] In one or more embodiments, the one or more TME stimulators are selected from the group consisting of: a substance of group A, a substance of group B, a substance of group C, a substance of group D, a substance of group J.

[0554] In one or more embodiments, the one or more TME stimulators comprises a substance of group A, a substance of group B, a substance of group C, a substance of group D, a substance of group J or any combination thereof.

[0555] In one or more embodiments, the one or more TME stimulators comprises a substance of group A. In one or more embodiments, the one or more TME stimulators comprises a substance of group B. In one or more embodiments, the one or more TME stimulators comprises a substance of group C. In one or more embodiments, the one or more TME stimulators comprises a substance of group D. In one or more embodiments, the one or more TME stimulators comprises a substance of group J.

[0556] In one or more embodiments, the one or more TME stimulators comprises a substance of group A and a substance of group B. In one or more embodiments, the one or more TME stimulatorscomprises a substance of group A and a substance of group J. In one or more embodiments, the one or more TME stimulators comprises a substance of group B and a substance of group J. In one or more embodiments, the one or more TME stimulators comprises a substance of group J and a substance of group A and a substance of group B.

[0557] In one or more embodiments, the substance of group A is added at culture initiation. In one or more embodiments, the substance of group A is added 1-3 days after culture initiation. In one or more embodiments, the substance of group A is added 1 day after culture initiation. In one or more embodiments, the substance of group A is added 2 days after culture initiation. In one or more embodiments, the substance of group A is added 3 days after culture initiation.

[0558] In one or more embodiments, the substance of group B is added at culture initiation. In one or more embodiments, the substance of group B is added 1-3 days after culture initiation. In one or more embodiments, the substance of group B is added 1 day after culture initiation. In one or more embodiments, the substance of group B is added 2 days after culture initiation. In one or more embodiments, the substance of group B is added 3 days after culture initiation.

[0559] In one or more embodiments, the substance of group J is added 1-3 days after culture initiation. In one or more embodiments, the substance of group J is added 1 day after culture initiation. In one or more embodiments, the substance of group J is added 2 days after culture initiation. In one or more embodiments, the substance of group J is added 3 days after culture initiation.

[0560] In one or more embodiments, an anti-PD-1 antibody is added at culture initiation. In one or more embodiments, an anti-PD-1 antibody is added 1-3 days after culture initiation. In one or more embodiments an anti-PD-1 antibody is added 1 day after culture initiation. In one or more embodiments, an anti-PD-1 antibody is added 2 days after culture initiation. In one or more embodiments, an anti-PD-1 antibody is added 3 days after culture initiation.

[0561] In one or more embodiments, an anti-CTLA-4 antibody is added at culture initiation. In one or more embodiments, an anti-CTLA-4 antibody is added 1-3 days after culture initiation. In one or more embodiments, an anti-CTLA-4 antibody is added 1 day after culture initiation. In one or more embodiments, an anti-CTLA-4 antibody is added 2 days after culture initiation. In one or more embodiments, an anti-CTLA-4 antibody is added 3 days after culture initiation.

[0562] In one or more embodiments, an anti-4-1 BB antibody is added 1-3 days after culture initiation. In one or more embodiments, an anti-4-1 BB antibody is added 1 day after culture initiation. In one or more embodiments, an anti-4-1 BB antibody is added 2 days after culture initiation. In one or more embodiments, an anti-4-1 BB antibody is added 3 days after culture initiation.In one or more embodiments, an anti-CD3 antibody is added in step b).

[0563] In one or more embodiments, an anti-CD3 antibody is added in step b) simultaneously with the feeder cells, with the feeder cells at least once or at each step of adding said feeder cells. In one or more embodiments, an anti-CD3 antibody is added in step b) simultaneously with the feeder cells. In one or more embodiments, an anti-CD3 antibody is added in step b) with the feeder cells at least once. In one or more embodiments, an anti-CD3 antibody is added in step b) at each step of adding said feeder cells.

[0564] Concentration

[0565] The term ‘concentration of X feeder cells / cm2’ describes the number of feeder cells (X) per square centimeter of cell culture bottom surface area. Unless explicitly stated otherwise, said feeder cells may be any type of feeder cell as described herein (e.g. the artificial feeder cell of the present invention).

[0566] One or more embodiments of the present disclosure describes the process of the present invention, wherein the feeder cells in step b) are added at a concentration of 0.05-0.3 x106feeder cells / cm2when performing step b) and / or at a concentration of 0.1 -1.2 x106feeder cells / cm2at least once when performing step b).

[0567] One or more embodiments of the present disclosure describes the process of the present invention, wherein the feeder cells in step b) are added at a concentration of 0.05-0.3 x106feeder cells / cm2when performing step b) and at a concentration of 0.1 -1.2 x106feeder cells / cm2at least once when performing step b).

[0568] One or more embodiments of the present disclosure describes the process of the present invention, wherein the feeder cells in step b) are added at a concentration of 0.05-0.3 x106feeder cells / cm2when performing step b) or at a concentration of 0.1-1.2 x106feeder cells / cm2at least once when performing step b).

[0569] One or more embodiments of the present disclosure describes the process of the present invention, wherein the feeder cells in step b) are added at a concentration of 0.05-0.3 x106feeder cells / cm2when performing step b).

[0570] One or more embodiments of the present disclosure describes the process of the present invention, wherein the feeder cells in step b) are added at a concentration of 0.1 -1.2 x106feeder cells / cm2at least once when performing step b).One or more embodiments of the present disclosure describes the process of the present invention, wherein the feeder cells in step b) are added at a concentration of 0.1 -1.2 x106feeder cells / cm2once when performing step b).

[0571] One or more embodiments of the present disclosure describes the process of the present invention, wherein the feeder cells in step b) are added at a concentration of 0.1 -1.2 x106feeder cells / cm2twice when performing step b).

[0572] One or more embodiments of the present disclosure describes the process of the present invention, wherein the feeder cells in step b) are added at a concentration of 0.1 -1.2 x106feeder cells / cm2three times when performing step b).

[0573] One or more embodiments of the present disclosure describes the process of the present invention wherein the feeder cells in step b) are added at a concentration of 0.05-0.075 x106artificial feeder cells / cm2when performing step b) and / or at a concentration of 0.1 -0.3 x106artificial feeder cells / cm2at least once when performing step b).

[0574] One or more embodiments of the present disclosure describes the process of the present invention wherein the feeder cells in step b) are added at a concentration of 0.05-0.075 x106artificial feeder cells / cm2when performing step b) and at a concentration of 0.1 -0.3 x106artificial feeder cells / cm2at least once when performing step b).

[0575] One or more embodiments of the present disclosure describes the process of the present invention wherein the feeder cells in step b) are added at a concentration of 0.05-0.075 x106artificial feeder cells / cm2when performing step b) or at a concentration of 0.1-0.3 x106artificial feeder cells / cm2at least once when performing step b).

[0576] One or more embodiments of the present disclosure describes the process of the present invention wherein the feeder cells in step b) are added at a concentration of 0.05-0.075 x106artificial feeder cells / cm2when performing step b).

[0577] One or more embodiments of the present disclosure describes the process of the present invention wherein the feeder cells in step b) are added at a concentration of 0.1 -0.3 x106artificial feeder cells / cm2at least once when performing step b).

[0578] One or more embodiments of the present disclosure describes the process of the present invention wherein the feeder cells in step b) are added at a concentration of 0.1-0.3 x106artificial feeder cells / cm2once when performing step b).One or more embodiments of the present disclosure describes the process of the present invention wherein the feeder cells in step b) are added at a concentration of 0.1 -0.3 x106artificial feeder cells / cm2twice when performing step b).

[0579] One or more embodiments of the present disclosure describes the process of the present invention wherein the feeder cells in step b) are added at a concentration of 0.1 -0.3 x106artificial feeder cells / cm2three times when performing step b).

[0580] One or more embodiments of the present disclosure describes the process of the present invention wherein the feeder cells in step b) are added at a concentration of 0.1 -0.3 x106PBMCs / cm2when performing step b) and / or at a concentration of 0.8-1.2 x106PBMCs / cm2at least once when performing step b).

[0581] One or more embodiments of the present disclosure describes the process of the present invention wherein the feeder cells in step b) are added at a concentration of 0.1 -0.3 x106PBMCs / cm2when performing step b) and at a concentration of 0.8-1.2 x106PBMCs / cm2at least once when performing step b).

[0582] One or more embodiments of the present disclosure describes the process of the present invention wherein the feeder cells in step b) are added at a concentration of 0.1 -0.3 x106PBMCs / cm2when performing step b) or at a concentration of 0.8-1.2 x106PBMCs / cm2at least once when performing step b).

[0583] One or more embodiments of the present disclosure describes the process of the present invention wherein the feeder cells in step b) are added at a concentration of 0.1 -0.3 x106PBMCs / cm2when performing step b).

[0584] One or more embodiments of the present disclosure describes the process of the present invention wherein the feeder cells in step b) are added at a concentration of 0.8-1.2 x106PBMCs / cm2at least once when performing step b).

[0585] One or more embodiments of the present disclosure describes the process of the present invention wherein the feeder cells in step b) are added at a concentration of 0.8-1.2 x106PBMCs / cm2once when performing step b).

[0586] One or more embodiments of the present disclosure describes the process of the present invention wherein the feeder cells in step b) are added at a concentration of 0.8-1.2 x106PBMCs / cm2twice when performing step b).One or more embodiments of the present disclosure describes the process of the present invention wherein the feeder cells in step b) are added at a concentration of 0.8-1.2 x106PBMCs / cm2three times when performing step b).

[0587] One or more embodiments of the present disclosure describes the process of the present invention comprising a step of adding the feeder cells at a concentration of 0.05-0.3 x106feeder cells / cm21-3 days after culture initiation in step b) and adding the feeder cells at a concentration of 0.1-1.2 x106feeder cells / cm26-8 days after culture initiation in step b), and optionally adding the feeder cells at a concentration of 0.1-1.2 x106feeder cells / cm212-15 days after culture initiation.

[0588] One or more embodiments of the present disclosure describes the process of the present invention comprising a step of adding the feeder cells at a concentration of 0.05-0.3 x106feeder cells / cm21-3 days after culture initiation in step b) and adding the feeder cells at a concentration of 0.1-1.2 x106feeder cells / cm26-8 days after culture initiation in step b), and adding the feeder cells at a concentration of 0.1-1.2 x106feeder cells / cm212-15 days after culture initiation.

[0589] One or more embodiments of the present disclosure describes the process of the present invention comprising a step of adding the feeder cells at a concentration of 0.05-0.075 x106artificial feeder cells / cm21-3 days after culture initiation in step b) and adding the feeder cells at a concentration of 0.1 -0.3 x106artificial feeder cells / cm26-8 days after culture initiation in step b), and optionally adding the feeder cells at a concentration of 0.1-0.3 x106artificial feeder cells / cm212-15 days after culture initiation.

[0590] One or more embodiments of the present disclosure describes the process of the present invention comprising a step of adding the feeder cells at a concentration of 0.05-0.075 x106artificial feeder cells / cm21-3 days after culture initiation in step b) and adding the feeder cells at a concentration of 0.1 -0.3 x106artificial feeder cells / cm26-8 days after culture initiation in step b), and adding the feeder cells at a concentration of 0.1-0.3 x106artificial feeder cells / cm212-15 days after culture initiation.

[0591] One or more embodiments of the present disclosure describes the process of the present invention comprising a step of adding the feeder cells at a concentration of 0.1-0.3 x106PBMCs / cm21-3 days after culture initiation in step b) and adding the feeder cells at a concentration of 0.8-1.2 x106PBMCs / cm26-8 days after culture initiation in step b), and optionally adding the feeder cells at a concentration of 0.8-1.2 x106PBMCs / cm212-15 days after culture initiation.

[0592] One or more embodiments of the present disclosure describes the process of the present invention comprising a step of adding the feeder cells at a concentration of 0.1-0.3 x106PBMCs / cm21-3 days after culture initiation in step b) and adding the feeder cells at a concentration of 0.8-1.2 x106PBMCs / cm26-8 days after culture initiation in step b), and adding the feeder cells at a concentration of 0.8-1.2 x106PBMCs / cm212-15 days after culture initiation.

[0593] One or more embodiments of the present disclosure describes the process of the present invention comprising adding the feeder cells 1-3 days after culture initiation in step b) and 6-8 days after culture initiation in step b), and optionally 12-15 days after culture initiation. One or more embodiments of the present disclosure describes the process of the present invention comprising adding the feeder cells 1-3 days after culture initiation in step b) and 6-8 days after culture initiation in step b). One or more embodiments of the present disclosure describes the process of the present invention comprising adding the feeder cells 1-3 days after culture initiation in step b) and 6-8 days after culture initiation in step b) and 12-15 days after culture initiation. One or more embodiments of the present disclosure describes the process of the present invention comprising adding the feeder cells 1-3 days after culture initiation in step b). One or more embodiments of the present disclosure describes the process of the present invention comprising adding the feeder cells 6-8 days after culture initiation in step b). One or more embodiments of the present disclosure describes the process of the present invention comprising adding the feeder cells 12-15 days after culture initiation in step b).

[0594] One or more embodiments of the present disclosure describes the process of the present invention wherein a total of less than 700x106feeder cells are used during the culture. One or more embodiments of the present disclosure describes the process of the present invention wherein a total of less than 650x106feeder cells are used during the culture. One or more embodiments of the present disclosure describes the process of the present invention wherein a total of less than 630x106feeder cells are used during the culture. One or more embodiments of the present disclosure describes the process of the present invention wherein a total of less than 150x106irradiated feeder cells are used during the culture. One or more embodiments of the present disclosure describes the process of the present invention wherein a total of less than 130x106irradiated feeder cells are used during the culture.

[0595] One or more embodiments of the present disclosure describes the process of the present invention wherein a total of less than 700x106feeder cells are used during the culture before harvest. One or more embodiments of the present disclosure describes the process of the present invention wherein a total of less than 650x106feeder cells are used during the culture before harvest. One or more embodiments of the present disclosure describes the process of the present invention wherein a total of less than 630x106feeder cells are used during the culture before harvest. One or more embodiments of the present disclosure describes the process of the present invention wherein a total of less than 150x106irradiated feeder cells are used during the culture before harvest. One or more embodiments of the present disclosure describes the process of the presentinvention wherein a total of less than 130x106irradiated feeder cells are used during the culture before harvest.

[0596] One or more embodiments of the present disclosure describes the process of the present invention wherein the second population of cells in step c) is harvested between 20-30 days after culture initiation, preferably 22-27 days after culture initiation, preferably 24-26 days after culture initiation, preferably 24 days after culture initiation. One or more embodiments of the present disclosure describes the process of the present invention wherein the second population of cells in step c) is harvested between 20-30 days after culture initiation. One or more embodiments of the present disclosure describes the process of the present invention wherein the second population of cells in step c) is harvested between 22-27 days after culture initiation. One or more embodiments of the present disclosure describes the process of the present invention wherein the second population of cells in step c) is harvested between 23-25 days after culture initiation. One or more embodiments of the present disclosure describes the process of the present invention wherein the second population of cells in step c) is harvested 24-26 days after culture initiation. One or more embodiments of the present disclosure describes the process of the present invention wherein the second population of cells in step c) is harvested 20 days after culture initiation. One or more embodiments of the present disclosure describes the process of the present invention wherein the second population of cells in step c) is harvested 21 days after culture initiation. One or more embodiments of the present disclosure describes the process of the present invention wherein the second population of cells in step c) is harvested 22 days after culture initiation. One or more embodiments of the present disclosure describes the process of the present invention wherein the second population of cells in step c) is harvested 23 days after culture initiation. One or more embodiments of the present disclosure describes the process of the present invention wherein the second population of cells in step c) is harvested 24 days after culture initiation. One or more embodiments of the present disclosure describes the process of the present invention wherein the second population of cells in step c) is harvested 25 days after culture initiation. One or more embodiments of the present disclosure describes the process of the present invention wherein the second population of cells in step c) is harvested 26 days after culture initiation. One or more embodiments of the present disclosure describes the process of the present invention wherein the second population of cells in step c) is harvested 27 days after culture initiation.

[0597] One or more embodiments of the present disclosure describes the process of the present invention wherein the second population of cells after performing steps b) or c) is a therapeutic population of cells. One or more embodiments of the present disclosure describes the process of the present invention wherein the second population of cells after performing step b) is a therapeutic population of cells. One or more embodiments of the present disclosure describes the process of the present invention wherein the second population of cells after performing step c) is a therapeutic population of cells.One or more embodiments of the present disclosure describes the process of the present invention wherein the second population of cells in step c) is harvested between 20-30 days after culture initiation and wherein the population of cells after performing steps b) or c) is a therapeutic population of cells. One or more embodiments of the present disclosure describes the process of the present invention wherein the second population of cells in step c) is harvested between 20-30 days after culture initiation and wherein the population of cells after performing step b) is a therapeutic population of cells. One or more embodiments of the present disclosure describes the process of the present invention wherein the second population of cells in step c) is harvested between 20-30 days after culture initiation and wherein the population of cells after performing step c) is a therapeutic population of cells. One or more embodiments of the present disclosure describes the process of the present invention wherein the second population of cells in step c) is harvested between 22-27 days after culture initiation and wherein the population of cells after performing step b) is a therapeutic population of cells. One or more embodiments of the present disclosure describes the process of the present invention wherein the second population of cells in step c) is harvested between 22-27 days after culture initiation and wherein the population of cells after performing step c) is a therapeutic population of cells. One or more embodiments of the present disclosure describes the process of the present invention wherein the second population of cells in step c) is harvested between 21-25 days after culture initiation and wherein the population of cells after performing step b) is a therapeutic population of cells. One or more embodiments of the present disclosure describes the process of the present invention wherein the second population of cells in step c) is harvested between 21-25 days after culture initiation and wherein the population of cells after performing step c) is a therapeutic population of cells. One or more embodiments of the present disclosure describes the process of the present invention wherein the second population of cells in step c) is harvested 24 days after culture initiation and wherein the population of cells after performing step b) is a therapeutic population of cells. One or more embodiments of the present disclosure describes the process of the present invention wherein the second population of cells in step c) is harvested 24 days after culture initiation and wherein the population of cells after performing step c) is a therapeutic population of cells.

[0598] One or more embodiments of the present disclosure describes the process of the present invention wherein the second population of cells after performing steps b) or c) is at least 1 x 107to 1x 1012cells, such as 1 x 108to 5x 109cells, such as 1 x 109to 5x 109cells, such as 1 x 108to 5x 101° cells, such as 1 x 109to 5x 1011cells.

[0599] One or more embodiments of the present disclosure describes the process of the present invention wherein the second population of cells after performing steps b) or c) is at least 1 x 107to 1x 1012cells, such as 1 x 108to 5x 109cells, such as 1 x 109to 5x 109cells, such as 1 x 108to 5x 101° cells, such as 1 x 109to 5x 1011cells. One or more embodiments of the present disclosuredescribes the process of the present invention wherein the second population of cells after performing steps b) or c) is at least 1 x 107to 1x 1012cells. One or more embodiments of the present disclosure describes the process of the present invention wherein the second population of cells after performing steps b) or c) is at least 1 x 108to 5x 109cells. One or more embodiments of the present disclosure describes the process of the present invention wherein the second population of cells after performing steps b) or c) is at least 1 x 109to 5x 109cells. One or more embodiments of the present disclosure describes the process of the present invention wherein the second population of cells after performing steps b) or c) is at least 1 x 108to 5x 101° cells.

[0600] One or more embodiments of the present disclosure describes the process of the present invention wherein the second population of cells after performing step b) is at least 1 x 107to 1x 1012cells, such as 1 x 108to 5x 109cells, such as 1 x 109to 5x 109cells, such as 1 x 108to 5x 101° cells, such as 1 x 109to 5x 1011cells. One or more embodiments of the present disclosure describes the process of the present invention wherein the second population of cells after performing step b) is at least 1 x 107to 1x 1012cells. One or more embodiments of the present disclosure describes the process of the present invention wherein the second population of cells after performing step b) is at least 1 x 108to 5x 109cells. One or more embodiments of the present disclosure describes the process of the present invention wherein the second population of cells after performing step b) is at least 1 x 109to 5x 109cells. One or more embodiments of the present disclosure describes the process of the present invention wherein the second population of cells after performing step b) is at least 1 x 108to 5x 101° cells.

[0601] One or more embodiments of the present disclosure describes the process of the present invention wherein the second population of cells after performing step c) is at least 1 x 107to 1x 1012cells, such as 1 x 108to 5x 109cells, such as 1 x 109to 5x 109cells, such as 1 x 108to 5x 101° cells, such as 1 x 109to 5x 1011cells. One or more embodiments of the present disclosure describes the process of the present invention wherein the second population of cells after performing step c) is at least 1 x 107to 1x 1012cells. One or more embodiments of the present disclosure describes the process of the present invention wherein the second population of cells after performing step c) is at least 1 x 108to 5x 109cells. One or more embodiments of the present disclosure describes the process of the present invention wherein the second population of cells after performing step c) is at least 1 x 109to 5x 109cells. One or more embodiments of the present disclosure describes the process of the present invention wherein the second population of cells after performing step c) is at least 1 x 108to 5x 101° cells. One or more embodiments of the present disclosure describes the process of the present invention wherein the second population of cells after performing step c) is at least 1 x 107to 1x 1012cells, such as 1 x 108to 5x 109cells, such as 1 x 109to 5x 109cells, such as 1 x 108to 5x 101° cells, such as 1 x 109to 5x 1011cells.Cell divisions

[0602] Methods to calculate the number of cell divisions are known to a skilled person. The lower number of cell divisions results in a less expanded population of TILs that reaches the final dose in shorter time. Furthermore, there is a lower risk of losing tumor-reactive cells due to dilution when the TIL population is reduced from the young TIL to the start of the REP culture.

[0603] An example method to calculate the number of cell divisions is given below:

[0604] Starting amount: 1 million = 1 x 106cells

[0605] Final amount : 55 billion = 5.5 x 1O10cells

[0606] Fold-increase

[0607] c c i nio

[0608] - - — = 5.5 X 104= 55,000

[0609] 1 x 106

[0610] Number of doublings n

[0611] 2" = 55,000

[0612] n = log2(55,000)

[0613] Using base-10 logs:

[0614] loglo(55,000)

[0615] i = - log 10 (2)

[0616] loglo(55,000) ® 4.740

[0617] log10(2) = 0.301

[0618]

[0619] One or more embodiments of the present disclosure describes the process of the present invention wherein step c) is performed after less than 30 cell divisions in the cell culture. One or more embodiments of the present disclosure describes the process of the present invention wherein step c) is performed after less than 25 cell divisions in the cell culture. One or more embodiments of the present disclosure describes the process of the present invention wherein step c) is performed after less than 22 cell divisions in the cell culture. One or more embodiments of the present disclosure describes the process of the present invention wherein step c) is performed after less than 20 cell divisions in the cell culture. One or more embodiments of the present disclosure describes the process of the present invention wherein step c) is performed after less than 18 cell divisions in the cell culture. One or more embodiments of the present disclosure describes the process of the present invention wherein step c) is performed after less than 17 cell divisions in the cell culture. One or more embodiments of the present disclosure describes the process of the present invention wherein step c) is performed after less than 16 cell divisions in the cell culture.One or more embodiments of the present disclosure describes the process of the present invention, wherein the culture medium is a serum-free medium.

[0620] One or more embodiments of the present disclosure describes the process of the present invention wherein the culture medium in step b) comprises the culture medium of the present invention.

[0621] One or more embodiments of the present disclosure describes the process of the present invention comprising the step of performing a depletion of suppressive cells, including regulatory T cells, and / or blocking negative signals to obtain a depleted population of cells, optionally by the addition of one or more TME stimulators from the group of “Inhibitors”, wherein the step is performed between step a) and b).

[0622] One or more embodiments of the present disclosure describes the process of the present invention comprising the step of performing a depletion of suppressive cells, including regulatory T cells, and blocking negative signals to obtain a depleted population of cells, optionally by the addition of one or more TME stimulators from the group of “Inhibitors”, wherein the step is performed between step a) and b).

[0623] One or more embodiments of the present disclosure describes the process of the present invention comprising the step of performing a depletion of suppressive cells, including regulatory T cells, or blocking negative signals to obtain a depleted population of cells, optionally by the addition of one or more TME stimulators from the group of “Inhibitors”, wherein the step is performed between step a) and b).

[0624] One or more embodiments of the present disclosure describes the process of the present invention comprising the step of performing a depletion of suppressive cells, including regulatory T cells, and / or blocking negative signals to obtain a depleted population of cells, by the addition of one or more TME stimulators from the group of “Inhibitors”, wherein the step is performed between step a) and b).

[0625] One or more embodiments of the present disclosure describes the process of the present invention comprising the step of performing a depletion of suppressive cells, including regulatory T cells, or blocking negative signals to obtain a depleted population of cells, by the addition of one or more TME stimulators from the group of “Inhibitors”, wherein the step is performed between step a) and b).

[0626] One or more embodiments of the present disclosure describes the process of the present invention comprising the step of performing a depletion of suppressive cells, including regulatory T cells, andblocking negative signals to obtain a depleted population of cells, by the addition of one or more TME stimulators from the group of “Inhibitors”, wherein the step is performed between step a) and b).

[0627] One or more embodiments of the present disclosure describes the process of the present invention, wherein the cells are selected from the group consisting of tumor infiltrating lymphocytes (TILs), NK cells, TCR-T cells, and CAR-T cells.

[0628] One or more embodiments of the present disclosure describes the process of the present invention, wherein the cells are tumor infiltrating lymphocytes (TILs). One or more embodiments of the present disclosure describes the process of the present invention, wherein the cells are NK cells. One or more embodiments of the present disclosure describes the process of the present invention, wherein the cells are TCR-T cells. One or more embodiments of the present disclosure describes the process of the present invention, wherein the cells are CAR-T cells.

[0629] One or more embodiments of the present disclosure describes the process of the present invention, wherein the cells are wherein the cells are T cells or NK cells.

[0630] One or more embodiments of the present disclosure describes the process of the present invention, wherein the artificial feeder cell(s) undergoes treatment that leads to cell death.

[0631] One or more embodiments of the present disclosure describes the process of the present invention, wherein the artificial feeder cell(s) are irradiated.

[0632] One or more embodiments of the present disclosure describes the process of the present invention wherein the artificial feeder cell(s) have been cryopreserved before use in the process.

[0633] One or more embodiments of the present disclosure describes the process of the present invention wherein artificial feeder cell(s) are irradiated before cryopreservation, while they are cryopreserved, or after they have been cryopreserved.

[0634] One or more embodiments of the present disclosure describes the process of the present invention wherein artificial feeder cell(s) are irradiated before cryopreservation.

[0635] One or more embodiments of the present disclosure describes the process of the present invention wherein artificial feeder cell(s) are irradiated while they are cryopreserved.

[0636] One or more embodiments of the present disclosure describes the process of the present invention wherein artificial feeder cell(s) are irradiated after they have been cryopreserved.One or more embodiments of the present disclosure describes the process of the present invention wherein the one or more tumor(s) and / or tumor fragment(s) in step a) are resected from a mammal diagnosed with cancer and / or undergoing treatment for cancer.

[0637] One or more embodiments of the present disclosure describes the process of the present invention wherein the one or more tumor(s) and / or tumor fragment(s) in step a) are resected from a mammal diagnosed with cancer.

[0638] One or more embodiments of the present disclosure describes the process of the present invention wherein the one or more tumor(s) and / or tumor fragment(s) in step a) are resected from a mammal undergoing treatment for cancer.

[0639] One or more embodiments of the present disclosure describes the process of the present invention wherein the one or more tumor(s) or tumor fragment(s) in step a) are resected from a mammal diagnosed with cancer.

[0640] One or more embodiments of the present disclosure describes the process of the present invention wherein the one or more tumor(s) or tumor fragment(s) in step a) are resected from a mammal undergoing treatment for cancer.

[0641] One or more embodiments of the present disclosure describes the process of the present invention wherein the one or more tumor(s) and tumor fragment(s) in step a) are resected from a mammal diagnosed with cancer.

[0642] One or more embodiments of the present disclosure describes the process of the present invention wherein the one or more tumor(s) and tumor fragment(s) in step a) are resected from a mammal undergoing treatment for cancer.

[0643] One or more embodiments of the present disclosure describes the process of the present invention wherein the one or more tumor(s) and / or tumor fragment(s) in step a) are resected from a human diagnosed with cancer and / or undergoing treatment for cancer.

[0644] One or more embodiments of the present disclosure describes the process of the present invention wherein the one or more tumor(s) and / or tumor fragment(s) in step a) are resected from a human diagnosed with cancer.

[0645] One or more embodiments of the present disclosure describes the process of the present invention wherein the one or more tumor(s) and / or tumor fragment(s) in step a) are resected from a human undergoing treatment for cancer.One or more embodiments of the present disclosure describes the process of the present invention wherein the one or more tumor(s) or tumor fragment(s) in step a) are resected from a human diagnosed with cancer.

[0646] One or more embodiments of the present disclosure describes the process of the present invention wherein the one or more tumor(s) or tumor fragment(s) in step a) are resected from a human undergoing treatment for cancer.

[0647] One or more embodiments of the present disclosure describes the process of the present invention wherein the one or more tumor(s) and tumor fragment(s) in step a) are resected from a human diagnosed with cancer.

[0648] One or more embodiments of the present disclosure describes the ...

Claims

CLAIMS1. An artificial feeder cell comprising:a. at least one or more nucleic acid molecules encoding one or more co-stimulatory molecules selected from the group consisting of: CD64, CD32, CD86, CD80, OX40L, a membrane bound receptor with affinity toward OKT-3 and / or 4-1 BBL.

2. The artificial feeder cell according to claim 1 , wherein the at least one or more nucleic acid molecules comprises at least one promotor, wherein the promotor is selected from the group consisting of:a. A SFFV promotor or a homologue thereof with at least 80% sequence identity to SEQ ID NO: 9,b. A CMV promotor or a homologue thereof with at least 80% sequence identity to SEQ ID NO: 10,c. An mCMV promotor or a homologue thereof with at least 80% sequence identity to SEQ ID NO: 11.d. A EF-1 promotor or a homologue thereof with at least 80% sequence identity to SEQ ID NOs: 12 or 13.e. A UbiC promotor or a homologue thereof with at least 80% sequence identity to SEQ ID NO: 14.

3. The artificial feeder cell according to any of the previous items, wherein said artificial feeder cell is a myeloid cell selected from the group consisting of: K562, EM-3, EM-2, MOLM-13, MOLM-14.

4. A culture medium comprising one or more artificial feeder cell(s) according to any of the previous claims.

5. The culture medium comprising one or more artificial feeder cell according to claim 4, wherein the culture medium comprises IL-2 and IL-21.

6. The culture medium according to any one of claims 4-5, wherein the culture medium comprises IL-2, IL-7, and IL-21.

7. The culture medium according to any one of claims 4-6, wherein the culture medium comprises IL-7, IL-15 and IL-21.

8. The artificial feeder cell according to claims 1-3, or the culture medium according to claims 4-7, which is / are cryopreserved at a temperature of at least -70°C or colder.

9. A method for expanding cells into a population of cells, that can be therapeutic, comprising: a) culturing autologous cells by obtaining a first population of cells from a tumor and / or tumor fragments resected from a mammal,b) performing a first expansion by culturing the first population of cells in a cell culture medium, and optionally adding the artificial feeder cells according to claims 1-3 or 8 to produce a second population of cells,c) performing a second expansion by supplementing the cell culture medium of the second population of cells comprising;- additional IL-2, a combination of IL-7, IL-15 and IL-21, or a combination of IL-2, IL-7 and IL-21,- anti-CD3 antibody, and- the artificial feeder cell(s) according to claims 1-3 or 8,to produce a third population of cells, optionally wherein the third population of cells is a therapeutic population.

10. The method according to claim 9, comprising the step of performing a depletion of suppressive cells, including regulatory T cells, and / or blocking negative signals to obtain a depleted population of cells, optionally by the addition of one or more TME stimulators from the group of “Inhibitors”, wherein the step is performed between step a) and b), and wherein the first population of cells in step b) is the depleted population of cells and wherein the cells are tumor infiltrating lymphocytes (TILs).

11. A process for expanding cells into a population of cells, that can be therapeutic, comprising the steps:a. obtaining a population of cells from a tumor and / or tumor fragments resected from a mammal;b. expanding the population of cells by culturing said population of cells in a cell culture medium, wherein feeder cells are added to the cell culture 1-4 times at predetermined timepoint(s) and / or predetermined number(s) of cells, to produce a second population of cells, optionally wherein the second population of cells is a therapeutic population of cells;c. Optionally harvesting the second population of cells.

12. The process according to claim 11 , wherein the feeder cells comprise or consists of the artificial feeder cells according to any one of claims 1-3 or 8.

13. The process according to any one of claims 11-12, wherein the predetermined timepoint(s) is 1-3 days after culture initiation, 6-8 days after culture initiation and / or 12-15 days after culture initiation.

14. The process according to any one of claims 11-13, wherein the predetermined number(s) of cells is a predetermined number between 1x105cells / cm2to 1x107cells / cm2.

15. A population of cells expanded using the artificial feeder cell according to claims 1-3 or 8, the culture medium according to claims 4-7, the method according to claims 9-10, and / or the process according to claims 11-14.

16. A composition comprising the artificial feeder cell according to claims 1-3 or 8 and / or the culture medium according to claims 4-7.

17. The population of cells according to claim 15 or the composition according to claim 16 for use as a medicament.

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