Single vessel lymphocyte expansion

Abstract

The present invention relates to a population of lymphocytes comprising at least 90% CD3+ T cells and less than 5% B cells, wherein at least 70% of said T cell portion are viable, at least 20% are CD27 / CD28 double positive, and less than 10% are triple positive for CD45RA, CD57 and KLRG1, as well as to a method of expanding a population of lymphocytes specific for one or more antigens comprising a single culture phase.

Description

[Technical field]

[0001] 1. Background The present invention relates to lymphocytes for use in targeted tumor immunotherapy, such as adoptive T cell therapy, as well as production methods and kits comprising such cells. [Background technology]

[0002] The lymphocytes are preferably human lymphocytes, e.g., NK cells or T cells, including CD3+ T cells, CD8+ T cells, CD4+ T cells and γδ T cells. Most preferably, the cells of the invention are primary human T cells. The invention provides a population of suitable lymphocytes exhibiting a specific marker profile (i.e., high CD27 / CD28 expression and low CD45RA / CD57 / KLRG1 expression) and specificity for one or more defined antigens. Such antigens may be antigens characteristic of disease conditions, including infectious diseases (e.g., viral or bacterial infections) and cancer, and / or neoantigens selected from known neoantigens or identified in a sample taken from a subject, e.g., a patient to be treated. Pharmaceutical compositions comprising such lymphocytes are also provided, in particular pharmaceutical compositions for use in methods of treating diseases characterized by antigens or neoantigen expression.

[0003] The use of adoptive cell therapy (ACT), such as T cell therapy, has been demonstrated as an effective treatment for several diseases, including cancer.Adoptive cell therapy is a powerful treatment approach that uses naturally occurring antigen-specific lymphocytes, such as T cells, or uses lymphocytes that are made antigen-specific by genetic engineering, for example, to express recombinant T cell receptors or chimeric antigen receptors.However, a particular problem facing the wider development and use of such treatments has been the complexity and cost associated with the development and selection of cell therapy, i.e., the selection and expansion of cells with the desired specificity in the required quantity and quality.

[0004] A common drawback of adoptive cell therapy is the lack of sufficient cell numbers (approximately 10 9 To reach the target lymphocyte population (T cells), it is usually necessary to expand the cells ex vivo for several weeks and / or to use multiple culture phases, during which the cells are usually frozen. As a result, a large proportion of the cells may be lost due to the effects of freezing-thawing, and in addition, due to the long-term culture, T cells may become terminal effector cells, which may die immediately after infusion into the patient before reaching the target cells, tissues and / or organs. Therefore, there is a need in the art for a shorter expansion protocol that avoids the freeze-thaw cycles and results in a younger, more suitable lymphocyte population, i.e., a population of antigen-specific T cells that are not terminally differentiated and contain a low percentage of terminal effectors. Summary of the Invention [Problem to be solved by the invention]

[0005] 2. Overview The present invention relates to an improved method for the ex vivo expansion of lymphocytes, particularly antigen-specific lymphocytes. The method of the present invention allows for the ex vivo expansion of high cell numbers (e.g., at least approximately 10 cells) from a patient sample in a single, controlled culture vessel without the need to transfer the cell culture to a larger culture vessel during the process. 7 The method of the present invention has the advantage that it is possible to obtain a younger cell population, characterized by a small proportion of terminal effector cells and preferably high stemness. These characteristics allow the younger cells to proliferate efficiently after reinfusion and thus reach the target cell, tissue or organ before differentiating into terminal effector cells. The terminal effector cells are involved in the immediate attack of the cancer cells, whereas the younger cells are expected to respond in a sustained manner. [Means for solving the problem]

[0006] The present invention relates to the following items: A1. A population of lymphocytes comprising at least 90% CD3+ T cells and less than 5% B cells, wherein at least 70% of said T cell portion are viable, at least 50% are CD27 / CD28 double positive, and less than 10% are triple positive for CD45RA, CD57 and KLRG1. A2. A population of lymphocytes according to paragraph A1, wherein said T cells are specific for one or more markers and are not positive for KLRG1 as determined by flow cytometry. A3. A population of lymphocytes according to any one of items A1 or A2, wherein said T cell portion has an average telomere length of at least 5 kb, 6 kb, 7 kb, 8 kb, 9 kb. A4. A population of lymphocytes according to any one of items A1 to A3, wherein less than 10% of said T cell portion secretes at least one protein from the group consisting of IFN-γ, TNF-α, IL-4, IL-5, granzyme B and perforin. A5. At least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the T cells in the T cell portion are CD8 + A population of lymphocytes that are T cells, determined by any one of items A1 to A4. A6. A population of lymphocytes according to any one of items A1 to A5, wherein at least two T cells in said T cell portion are directed against different antigens. A7. A method for expanding a population of lymphocytes specific for one or more antigens, comprising a single culture phase, said single phase comprising: a) culturing a tissue or blood sample from a subject, the sample being known or suspected to contain lymphocytes, in the presence of said one or more antigens; or b) culturing T cells, the T cells being isolated from a tissue or blood sample from the subject, in the presence of said one or more antigens. Including, The culturing step comprises culturing the population of T cells to a concentration of at least 10×108 and continuing until the cells are cultured, said culturing step being at a temperature greater than 0° C. during said single culture phase. A8. The method according to paragraph A7(a) or A7(b), wherein the sample or the T cells are maintained at a temperature above 0° C. after isolation from the subject and prior to the culturing. A9. The method according to item A7 or A8, wherein the antigen is isolated or determined from the subject or the sample. A10. The method according to any one of items A7 to A9, wherein said one or more antigens are soluble peptides contained in the culture medium at a concentration of 0.1 to 10 μg / ml. A11. The method according to any one of items A7 to A10, wherein said culturing step comprises co-culturing with antigen-presenting cells (APCs) engineered to present said one or more antigens. A12. The method according to item A11, wherein the APCs comprise B cells. A13. The method according to item A12, wherein the B cells are recombinantly engineered to express the one or more antigens. A14. A population of T cells obtainable by the method according to any one of A7 to A13. A15. A population of lymphocytes according to any one of items A1 to A6 or a population of T cells according to item A14 for use as a medicament. B1. A method for expanding a population of lymphocytes specific for one or more antigens in a single controlled culture vessel, comprising: a) culturing a tissue or blood sample from a subject, the sample being known or suspected to contain lymphocytes, in the presence of said one or more antigens; or b) culturing lymphocytes, which are lymphocytes isolated from a tissue or blood sample from the subject, in the presence of said one or more antigens. wherein the lymphocytes are cultured in a conditioned culture medium. B2. The method according to item B1, wherein the conditioned culture medium is a culture medium in which at least one of the following parameters: pH, dissolved oxygen (DO) concentration, glucose concentration, lactate concentration and / or temperature is monitored and adjusted as necessary. B3. The method according to item B2, wherein the conditioned culture medium is a culture medium in which pH, ​​dissolved oxygen (DO) concentration, glucose concentration, lactate concentration and temperature are monitored and adjusted as necessary. B4. The method according to any one of items B1 to B3, comprising a step of adjusting the culture volume to the expansion rate of the lymphocytes. B5. The method according to item B4, wherein the culture volume is increased at least 2, 3, 4, 5 or 6 fold during lymphocyte expansion. B6. The method according to any one of items B1 to B5, comprising a step of dynamically culturing the lymphocyte culture using a conditioned culture medium. B7. The method according to any one of items B1 to B6, wherein the tissue sample is derived from a tumor, in particular the tissue sample is a tumor sample. B8. The method according to item B7, wherein the tumor comprises at least one neo-antigen. B9. The method according to any one of paragraphs B1 to B8, wherein the lymphocytes comprise tumor-infiltrating lymphocytes, in particular, the tumor-infiltrating lymphocytes are T cells. B10. The method according to any one of items B1 to B9, wherein the one or more antigens are added to the culture medium in the form of a peptide. B11. The method according to item B10, wherein the peptide is added to the culture medium at a concentration of 0.1 to 10 μg / ml. B12. The method according to any one of items B1 to B11, wherein the culturing step includes a step of co-culturing lymphocytes and antigen-presenting cells (APCs). B13. The method according to item B12, wherein the antigen presenting cells (APCs) are engineered to present one or more antigens. B14. The method according to item B12 or B13, wherein the antigen presenting cell (APC) comprises or is a B cell. B15. The method according to item B14, wherein the B cells are obtained by apheresis. B16. The method according to items B14 or B15, wherein the B cells are activated prior to addition to the lymphocytes. B17. The method according to item B16, wherein the B cells are activated with IL-21, IL-4 and / or CD40L. B18. The method according to any one of items B12-B17, wherein the antigen presenting cells (APCs) are genetically engineered to express one or more transgenes. B19. The method according to item B18, wherein the genetically engineered APCs are obtained by transfecting APCs with a nucleic acid encoding one or more transgenes. B20. The method according to item B18 or B19, wherein at least one of the one or more transgenes encodes an immunomodulatory factor. B21. The method according to item B20, wherein the immune modulator is selected from the group consisting of OX40L, 4-1BBL, CD80, CD86, CD83, CD70, CD40L, GITR-L, CD127L, CD30L (CD153), LIGHT, BTLA, ICOS-L (CD275), SLAM (CD150), CD662L, interleukin-12, interleukin-7, interleukin-15, interleukin-17, interleukin-21, interleukin-4, Bcl6, Bcl-XL, BCL-2, MCL1, STAT-5, and activators of one or more signal transduction pathways (e.g., the JAK / STAT pathway, the Akt / PKB signaling pathway, the BCR signaling pathway, and / or the BAFF / BAFFR signaling pathway). B22. The method according to items B20 or B21, wherein the immunomodulatory factor is one or more of OX40L, 4-1BBL and / or interleukin-12. B23. The method according to any one of items B7 to B22, wherein the presence of at least one of the one or more antigens has been confirmed in the tumor sample comprising lymphocytes prior to the culturing step. B24. The method according to any one of items B7 to B23, wherein at least one of the one or more antigens is a neoantigen, and the presence of said neoantigen has been confirmed in the tumor sample comprising lymphocytes prior to the culturing step. B25. The method according to item B23 or B24, wherein confirming the presence of at least one of the one or more antigens in the tumor sample comprises sequencing genomic DNA obtained from the tumor sample. B26. The method according to any one of items B1 to B25, comprising a step of activating lymphocytes during culture. B27. The method according to item B26, wherein the activation step comprises the addition of a CD3 agonist to the culture medium. B28. The method according to item B27, wherein the CD3 agonist is added to the culture medium 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 days after the initiation of the culture. B29. The method according to any one of items B21 to B28, wherein the culture medium is supplemented with human AB serum and / or IL-2. B30. The culturing step comprises culturing the population of T cells to at least 10 7 The method according to any one of items B1 to B29, continued until the cell is reached. B31. The method according to any one of items B1 to B30, wherein the culturing step is carried out at a temperature higher than 0° C. during the single culture phase. B32. The method according to any one of paragraphs B1 to B31, wherein the sample or the lymphocytes are maintained at a temperature above 0° C. after isolation from the subject and prior to the culturing. B33. A population of lymphocytes obtainable by the method according to any one of items B1 to B32. B34. A population of lymphocytes comprising at least 90% CD3+ T cells and less than 5% B cells, wherein at least 70% of the T cell portion are viable, at least 20% are CD27 / CD28 double positive, and less than 10% are triple positive for CD45RA, CD57 and KLRG1. B35. The population of lymphocytes according to item B34, wherein said T cells are specific for one or more antigens. B36. The population of lymphocytes according to any one of paragraphs B34 to B36, wherein less than 15% of said T cell portion secretes at least one protein from the group consisting of TNF-α, IL-4 and IL-5. B37. At least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the T cells in said T cell portion are CD8 + A population of lymphocytes according to any one of items B34 to B36 that are T cells. B38. A population of lymphocytes according to any one of paragraphs B34 to B37, wherein at least two T cells in said T cell portion are directed against different antigens. B39. The population of lymphocytes according to item B38, wherein at least one antigen is a neoantigen. B40. The T cell portion is at least 10 7 A population of lymphocytes determined by any one of items B34 to B39, containing T cells. B41. A pharmaceutical composition comprising a population of lymphocytes according to any one of items B33 to B40. B42. The pharmaceutical composition according to item B41, wherein the lymphocytes are suspended in a pharma- ceutically acceptable buffer. B43. The pharmaceutical composition according to item B42, wherein the pharma- ceutically acceptable buffer comprises about 0.9% NaCl, optionally containing up to 15% DMSO. B44. A population of lymphocytes according to any one of items B33 to B40 or a pharmaceutical composition according to any one of items B41 to B43 for use as a medicament. B45. A population of lymphocytes according to any one of items B33 to B40 or a pharmaceutical composition according to any one of items B41 to B43 for use in cancer treatment. B46. A population of lymphocytes or a pharmaceutical composition for use according to item B45, wherein the cancer treatment is adoptive cell therapy. B47. A population of lymphocytes or a pharmaceutical composition for use according to items B45 or B46, wherein the cancer treatment is an autologous cell therapy. B48. A method for treating cancer, comprising: a) providing a population of lymphocytes according to any one of items B33 to B40 or a pharmaceutical composition according to any one of items B41 to B43; and b) injecting the population of lymphocytes or the pharmaceutical composition into a subject suffering from cancer. A method comprising: B49. A method of treating cancer in a subject, comprising: a) surgically removing a tumor from a subject or taking a biopsy from a tumor in a subject; b) identifying at least one tumor antigen in the tumor sample obtained in step (a); c) expanding lymphocytes contained in the tumor sample obtained in step (a) by a method according to any one of items B1 to B33, in the presence of at least one tumor antigen identified in step (b) as being present in the tumor sample; d) injecting the expanded lymphocytes obtained in step (c) into the subject from whom the tumor sample was taken. The method includes: B50. The method according to item B49, wherein the tumor antigen is a tumor-associated antigen or a tumor-specific antigen. B51. The method according to any one of items B48 to B49, wherein the lymphocytes include tumor infiltrating lymphocytes (TILs). B52. The method according to item B51, wherein the TILs specifically recognize one or more tumor antigens. B53. The method according to item B52, wherein at least one tumor antigen is a neoantigen. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0007] Thus, in a particular embodiment, the present invention relates to a population of lymphocytes comprising at least 90% CD3+ T cells and less than 5% B cells, wherein at least 70% of said T cell portion are viable and at least 50% of said T cell portion are CD27 and / or CD28 positive.

[0008] Thus, in certain embodiments, the present invention relates to a population of lymphocytes comprising at least 90% CD3+ T cells. The term "CD3+ T cells" as used herein refers to a type of cell that expresses the CD3 marker. "CD3" as used herein refers to cluster of differentiation 3, a protein complex made up of four separate chains. In mammals, this complex contains the CD3γ chain, the CD3δ chain, and two CD3ε chains. These chains associate with a molecule known as the T cell receptor (TCR) and the ζ chain to generate an activation signal in T lymphocytes. The TCR, ζ chain, and CD3 molecule together constitute the TCR complex.

[0009] In certain embodiments, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the lymphocytes in the population of lymphocytes are CD3+ T cells.

[0010] Those skilled in the art are aware of methods for determining the percentage of CD3+ T cells in a population of cells. For example, the percentage of CD3+ T cells in a population of cells can be determined by flow cytometry using an antibody against CD3 and / or other suitable T cell specific surface markers.

[0011] In certain embodiments, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the lymphocytes in the population of lymphocytes are CD3+ and CD45+ T cells as determined by flow cytometry.

[0012] In certain embodiments, the population of lymphocytes may contain no more than 10% contaminants. In certain embodiments, the lymphocyte population is obtained by contacting a patient sample containing lymphocytes or isolated lymphocytes with B cells, particularly antigen-presenting B cells. Thus, in certain embodiments, the lymphocyte population can include a B cell population. In certain embodiments, the lymphocyte population includes less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% B cells.

[0013] Alternatively, the population of lymphocytes may comprise between 0.1% and 5% B cells, between 0.1% and 4% B cells, between 0.1% and 3% B cells, between 0.1% and 2% B cells, or between 0.1% and 1% B cells.

[0014] It should be understood that even if lymphocytes are initially cultured in the presence of B cells, the final population of lymphocytes may not contain B cells, since B cells cannot normally survive for extended periods in T cell specific medium. Thus, in certain embodiments, the population of lymphocytes by __ is substantially free of B cells, i.e., the number of B cells in the population may be below the limit of quantification by flow cytometry.

[0015] Those skilled in the art are aware of methods for determining the percentage of B cells in a population of cells. For example, B cells can be identified by flow cytometry using antibodies against B cell specific surface markers, such as CD19 or CD20.

[0016] The term "B cell" as used herein refers to a type of lymphocyte that plays a major role in humoral immune responses, as opposed to cell-mediated immune responses dominated by T cells. B cells are characterized by a B cell receptor (BCR) present on their outer surface, which allows the B cell to bind to its specific antigen. The main functions of B cells are (i) to produce antibodies against the specific antigen they recognize, (ii) to act as an antigen-presenting cell (APC), and (iii) to eventually become memory B cells after activation by interaction with their cognate antigen. B cells are essential components of the adaptive immune system. The term "B cell" includes long-lived plasma cells and memory B cells. The term "long-lived plasma B cell" as used herein refers to a subtype of B cells that resides primarily in the bone marrow and secretes antibodies continuously. The term "memory B cells," as used herein, refers to a subtype of B cells that are formed following initial infection and activation by interaction with their cognate antigen, reside primarily in peripheral lymphoid tissues, and differentiate into antibody-secreting cells (ASCs) upon re-encounter with the priming antigen, thus amplifying the antibody response. In certain embodiments, the B cells are memory B cells.

[0017] Other contaminants may be cells contained in the sample from which lymphocytes and / or B cells originate.For example, in certain embodiments, lymphocytes originate from tumor samples.In such embodiments, the preparation of lymphocytes may contain residual fractions of tumor cells.The abundance of tumor cells in the final population of lymphocytes can be determined by flow cytometry, for example by determining the abundance of CD45 negative cells in the population of lymphocytes.Alternatively or additionally, the residual tumor cells in the population of lymphocytes can be detected by qPCR, as known in the art.

[0018] In certain embodiments, the population of lymphocytes comprises less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, or less than 2% B cells (CD19+ or CD20+) and / or tumor cells (CD45-).

[0019] Alternatively, the population of lymphocytes may comprise between 0.1%-10%, between 0.1%-9%, between 0.1%-8%, between 0.1%-7%, between 0.1%-6%, between 0.1%-5%, between 0.1%-4% B cells, between 0.1%-3%, between 0.1%-2%, or between 0.1%-1% B cells (CD19+ or CD20+) and / or tumor cells (CD45-).

[0020] The population of lymphocytes according to the invention may further comprise NK cells (CD3-, CD56+) and / or NKT cells (CD3+, CD56+). Thus, in certain embodiments, the population of lymphocytes may comprise between 0.1% and 10%, between 0.1% and 9%, between 0.1% and 8%, between 0.1% and 7%, between 0.1% and 6%, between 0.1% and 5%, between 0.1% and 4% B cells, between 0.1% and 3%, between 0.1% and 2%, or between 0.1% and 1% B cells (CD19+ or CD20+) and / or tumor cells (CD45-) and / or NK cells (CD3-, CD56+) and / or NKT cells (CD3+, CD56+).

[0021] In the present invention, it is preferred that at least 70% of CD3+ T cells in a cell population are viable cells.Various methods for determining T cell viability are known in the art and are commercially available.Without being limited thereto, the viability of T cells in a lymphocyte population can be determined by proliferation assay or by live cell / dead cell differentiation staining.

[0022] In certain embodiments, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the CD3+ T cells in the population of lymphocytes are viable CD3+ T cells.

[0023] In certain embodiments, the viability of CD3+ T cells in a population of lymphocytes can be determined by flow cytometry using cell surface marker Annexin V (AnnV) and nucleic acid stain 7-amino-actinomycin D (7AAD) or propidium iodide (PI). Viable cells are double negative for AnnV and 7AAD / PI. Early apoptotic cells are positive for AnnV and negative for 7AAD / PI. Late apoptotic cells are positive for AnnV and positive for 7AAD / PI. Dead cells are negative for AnnV and positive for 7AAD / PI.

[0024] In certain embodiments, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of the CD3+ T cells in the population of lymphocytes are double negative for AnnV and 7AAD / PI.

[0025] Annexin V (or annexin V) is a cellular protein in the annexin family. In flow cytometry, annexin V is commonly used to detect apoptotic cells due to its ability to bind to phosphatidylserine, a marker of apoptosis, when it is on the outer leaflet of the plasma membrane.

[0026] 7-Aminoactinomycin D (7-AAD) is a fluorescent chemical with a strong affinity for DNA. It is used as a fluorescent marker for DNA in fluorescence microscopy and flow cytometry. It is taken up by cells upon loss of cell membrane integrity and intercalates into double-stranded DNA with high affinity for GC-rich regions, making it useful for chromosome banding studies.

[0027] Propidium iodide (or PI) is a fluorescent intercalating agent that can be used to stain cells and nucleic acids. PI binds to DNA by intercalating between bases with little or no sequence preference. Propidium iodide is used as a DNA stain in flow cytometry to assess cell viability or DNA content in cell cycle analysis, or in microscopy to visualize nuclei and other DNA-containing organelles. Because it is not membrane permeable, propidium iodide is useful for distinguishing between necrotic, apoptotic, and healthy cells based on membrane integrity.

[0028] Viability can further be determined by using a cell counter, such as, but not limited to, a NucleoCounter NC-202. That is, in certain embodiments, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of the CD3+ T cells in the population of lymphocytes are viable as determined by a cell counter, in particular a NucleoCounter NC-202.

[0029] Viability can further be determined by trypan blue cell counting as known in the art, i.e., in certain embodiments, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the CD3+ T cells in the population of lymphocytes are viable as determined by trypan blue cell counting.

[0030] It should be understood that the viability will vary depending on the method by which it is determined, in particular due to variations in the expression of cell markers such as AnnV. Thus, it is sufficient to obtain a viability of at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of CD3+ T cells in a population of lymphocytes by at least one suitable method known in the art, preferably one of the methods disclosed herein.

[0031] Furthermore, it is preferred that at least 50% of the CD3+ T cells in the lymphocyte population are CD27 and / or CD28 positive cells. In certain embodiments, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% or at least 95% of the T cell portion are CD27 and / or CD28 positive. It is preferred that at least 75% of the T cell portion are CD27 and / or CD28 positive.

[0032] Alternatively, at least 20% of the CD3+ T cells in the population of lymphocytes are CD27 and / or CD28 positive cells. In certain embodiments, at least 25%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50% of the T cell portion are CD27 and / or CD28 positive.

[0033] CD27 is a member of the tumor necrosis factor receptor superfamily. This receptor is required for the development and long-term maintenance of T cell immunity. It binds to the ligand CD70 and plays a key role in regulating B cell activation and immunoglobulin synthesis. CD27 is expressed primarily on naive, central memory (CM) and effector memory (EM) T cells, but not on terminal effector (TE) T cells.

[0034] CD28 is one of the proteins expressed on T cells that provides the costimulatory signal required for T cell activation and survival. T cell stimulation by CD28 in addition to the T cell receptor (TCR) can provide a strong signal for the production of various interleukins. Like CD27, CD28 is mainly expressed on naive, central memory (CM) and effector memory (EM) T cells, but not on terminal effector (TE) T cells.

[0035] As mentioned above, the T cells in the lymphocyte population preferably contain a small number of terminal effector T cells. Thus, in certain embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the CD3+ T cells in the lymphocyte population express the cell surface marker CD27. In other embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the CD3+ T cells in the lymphocyte population express the cell surface marker CD28. In other embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of the CD3+ T cells in the lymphocyte population express the cell surface markers CD27 and CD28.

[0036] Those skilled in the art are aware of the method for determining the percentage of CD27 and / or CD28 positive cells in a cell population.For example, the percentage of CD27 and / or CD28 positive cells in a cell population can be determined by flow cytometry.Antibodies against CD27 and CD28 are known in the art and are commercially available.

[0037] In a particular embodiment, the invention relates to a method according to the invention, wherein less than 10% of said T cell portion are positive for at least one of the markers from the group consisting of CD45RA, CD57 and KLRG1.

[0038] That is, CD3+ T cells within a population of lymphocytes may be further characterized by the absence of one or more senescence markers.

[0039] In certain embodiments, it is preferred that less than 10%, less than 9%, less than 8%, less than 7%, less than 6% or less than 5% of the CD3+ T cells in the population of lymphocytes are positive for the cell surface marker CD45RA.

[0040] The term "CD45RA" as used herein refers to the cluster of differentiation 45 isoform RA, or protein tyrosine phosphatase, receptor type, C (PTPRC). CD45RA, preferably in combination with CD57 and KLRG1, is widely accepted as a marker for terminal differentiation of CD8+ memory T cells. The percentage of CD45RA positive cells in a population of lymphocytes is preferably determined by flow cytometry using an antibody against CD45RA.

[0041] In certain embodiments, it is preferred that less than 10%, less than 9%, less than 8%, less than 7%, less than 6% or less than 5% of the CD3+ T cells within a population of lymphocytes are positive for the cell surface marker CD57.

[0042] The CD57 antigen (or HNK-1, LEU-7, or L2) is routinely used to identify terminally differentiated "senescent" cells that have reduced proliferative capacity and altered functional properties. The percentage of CD57 positive cells within a population of lymphocytes is preferably determined by flow cytometry using an antibody against CD57.

[0043] In certain embodiments, it is preferred that less than 10%, less than 9%, less than 8%, less than 7%, less than 6% or less than 5% of CD3+ T cells within a population of lymphocytes are positive for the cell surface marker KLRG1.

[0044] Killer cell lectin-like receptor subfamily G member 1 (KLRG1) is a protein that is encoded by the KLRG1 gene in humans. KLRG1 is expressed on NK cells and antigen-experienced T cells, and is hypothesized to be a marker of senescence. The percentage of KLRG1 positive cells in a population of lymphocytes is preferably determined by flow cytometry using an antibody against KLRG1.

[0045] In certain embodiments, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, or less than 5% of the CD3+ T cells in the population of lymphocytes are positive for at least one of the cell surface markers CD45RA, CD57, and / or KLRG1. In certain embodiments, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, or less than 5% of the CD3+ T cells in the population of lymphocytes are positive for two of the cell surface markers CD45RA, CD57, and / or KLRG1. In certain embodiments, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, or less than 5% of the CD3+ T cells in the population of lymphocytes are positive for all three of the cell surface markers CD45RA, CD57, and / or KLRG1.

[0046] In certain embodiments, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, or less than 5% of the CD3+ T cells in a population of lymphocytes are positive for at least one of the cell surface markers CD45RA and / or CD57. In certain embodiments, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, or less than 5% of the CD3+ T cells in a population of lymphocytes are double positive for CD45RA and CD57.

[0047] In certain embodiments, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, or less than 5% of the CD3+ T cells in a population of lymphocytes are positive for at least one of the cell surface markers KLRG1 and / or CD57. In certain embodiments, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, or less than 5% of the CD3+ T cells in a population of lymphocytes are double positive for KLRG1 and CD57.

[0048] In certain embodiments, more than 50%, more than 60%, more than 70%, more than 80%, more than 85%, more than 90%, or more than 95% of the CD3+ T cells in the population of lymphocytes are negative for at least one of the cell surface markers CD45RA, CD57, and / or KLRG1. In certain embodiments, more than 50%, more than 60%, more than 70%, more than 80%, more than 85%, more than 90%, or more than 95% of the CD3+ T cells in the population of lymphocytes are negative for two of the cell surface markers CD45RA, CD57, and / or KLRG1. In certain embodiments, more than 80%, more than 85%, more than 90%, or more than 95% of the CD3+ T cells in the population of lymphocytes are double negative for CD57 and KLRG1. In certain embodiments, greater than 80%, greater than 85%, greater than 90%, or greater than 95% of the CD3+ T cells in a population of lymphocytes are triple negative for CD45RA, CD57, and KLRG1.

[0049] In a particular embodiment, the present invention relates to a population of lymphocytes comprising at least 90% CD3+ T cells and less than 5% B cells, wherein at least 70% of said T cell portion are viable, at least 50% are CD27 / CD28 double positive, and at least 80% are double negative for CD57 and KLRG1.

[0050] In a particular embodiment, the present invention relates to a population of lymphocytes according to the present invention, wherein said T cell portion has an average telomere length of at least 5 kb, at least 6 kb, at least 7 kb, at least 8 kb, at least 9 kb. The skilled artisan is aware that the average telomere length depends on the starting material. For example, the average telomere length depends on the age of the patient from whom the starting material is taken.

[0051] Alternatively, or in addition to senescence markers CD45RA, CD57 and KLRG1, the lymphocyte population of the present invention can be characterized based on the average telomere length of the CD3+ T cells contained in the lymphocyte population.It is known in the art that the onset of replicative senescence is controlled by the length of telomeres, which are specialized structures at the ends of chromosomes that progressively shorten with each DNA replication cycle.Telomere shortening beyond a critical length induces p53-mediated growth arrest and senescence.

[0052] Methods for determining the average telomere length in cells of a cell population are known in the art and are described, for example, by Huang et al. (Scientific Reports Vol. 7, Article No.: 6785 (2017)).

[0053] In certain embodiments, CD3+ T cells within a population of lymphocytes can be characterized by an average telomere length of at least 5 kb, at least 6 kb, at least 7 kb, at least 8 kb, at least 9 kb, or at least 10 kb.

[0054] Instead of characterizing lymphocytes based on the average length of all telomeres of CD3+ T cells contained in the population of lymphocytes, the CD3+ T cells can also be characterized based on the average length of the shortest 20% of the telomeres. Thus, in a particular embodiment, the invention relates to a population of lymphocytes according to the invention, wherein the shortest 20% of the telomeres in said T cell portion have an average telomere length of at least 1 kb, at least 1.5 kb, at least 2 kb, at least 2.5 kb, at least 3 kb.

[0055] In a particular embodiment, the invention relates to a population of lymphocytes according to the invention, wherein less than 10% of said T cell portion secretes at least one protein from the group consisting of TNF-α, IL-4, IL-5, granzyme B and perforin.

[0056] In one particular embodiment, the invention relates to a population of lymphocytes according to the invention, wherein less than 15% of said T cell portion secretes at least one protein from the group consisting of TNF-α, IL-4 and IL-5.

[0057] In one particular embodiment, the invention relates to a population of lymphocytes according to the invention, wherein less than 10% of said T cell portion secretes at least one protein from the group consisting of TNF-α, IL-4 and IL-5.

[0058] In a particular embodiment, the present invention relates to a population of lymphocytes according to the present invention, wherein less than 15% of said T cell portion secrete TNF-α and less than 10% of said T cell portion secrete at least one protein from the group consisting of IL-4 and IL-5.

[0059] Alternatively, or in addition to the senescence markers CD45RA, CD57 and KLRG1, the lymphocyte population according to the present invention can be characterized based on the secretion profile of the CD3+ T cells contained in the lymphocyte population. It is known in the art that terminal effector T cells secrete different proteins than less differentiated T cells. Thus, the senescence of cells in a population of cells can be determined based on the proteins secreted by the cells in the population.

[0060] In certain embodiments, CD3+ T cells in a population of lymphocytes may be characterized in that less than 15% of these CD3+ T cells secrete TNF-α. In certain embodiments, CD3+ T cells in a population of lymphocytes may be characterized in that less than 1% of these CD3+ T cells secrete TNF-α. Tumor necrosis factor (TNF, cachexin, or cachectin; often called tumor necrosis factor alpha or TNF-α) is a cytokine - a small protein used by the immune system for cell signaling. TNF-α is primarily secreted by terminal effector T cells, but not by naive T cells and central memory T cells.

[0061] In certain embodiments, less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, or less than 5% of CD3+ T cells in a population of lymphocytes secrete detectable amounts of TNF-α.

[0062] In certain embodiments, CD3+ T cells in a population of lymphocytes may be characterized in that less than 15% of these CD3+ T cells secrete IL-4. In certain embodiments, CD3+ T cells in a population of lymphocytes may be characterized in that less than 10% of these CD3+ T cells secrete IL-4. Interleukin (IL-4) has many biological roles, including stimulating activated B and T cell proliferation and differentiation of B cells into plasma cells. IL-4 is primarily secreted by terminal effector T cells, but not by naive T cells and central memory T cells.

[0063] In certain embodiments, less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, or less than 5% of CD3+ T cells in a population of lymphocytes secrete detectable amounts of IL-4.

[0064] In certain embodiments, CD3+ T cells in a population of lymphocytes may be characterized in that less than 15% of these CD3+ T cells secrete IL-5. In certain embodiments, CD3+ T cells in a population of lymphocytes may be characterized in that less than 10% of these CD3+ T cells secrete IL-5. By binding to the interleukin-5 receptor, interleukin-5 stimulates B cell growth and increases immunoglobulin-primarily IgA-secretion. It is also a critical mediator in eosinophil activation. IL-5 is primarily secreted by terminal effector T cells, but not by naive T cells and central memory T cells.

[0065] In certain embodiments, less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, or less than 5% of CD3+ T cells in a population of lymphocytes secrete detectable amounts of IL-5.

[0066] In certain embodiments, CD3+ T cells in a population of lymphocytes may be characterized in that less than 10% of these CD3+ T cells secrete granzyme B. Granzyme B (GrB) is a serine protease that is most commonly found in the granules of natural killer cells (NK cells) and cytotoxic T cells. It is secreted by these cells together with the pore-forming protein perforin to mediate apoptosis in target cells. Granzyme B is primarily secreted by terminal effector T cells and effector memory T cells, but not by naive T cells and central memory T cells.

[0067] In certain embodiments, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, or less than 5% of CD3+ T cells within a population of lymphocytes secrete detectable amounts of granzyme B.

[0068] In certain embodiments, CD3+ T cells in a population of lymphocytes may be characterized in that less than 10% of these CD3+ T cells secrete perforin. Perforin is a pore-forming cytotoxic protein found in the granules of cytotoxic T lymphocytes (CTLs) and natural killer cells (NK cells). Upon degradation, perforin binds to the plasma membrane of target cells and oligomerizes in a Ca2+-dependent manner to form pores in the target cells. The formed pores allow passive diffusion of a family of proapoptotic proteases known as granzymes into the target cells. Perforin is primarily secreted by terminal effector T cells, but not by naive and central memory T cells.

[0069] In certain embodiments, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, or less than 5% of CD3+ T cells in a population of lymphocytes secrete detectable amounts of perforin.

[0070] In certain embodiments, the majority of the cell population may express granzyme B and / or perforin. Thus, it is preferred herein that less than 15% or more preferably less than 10% of the T cell fraction contained in the population of cells secretes at least one protein, at least two proteins, or all proteins from the group consisting of TNF-α, IL-4, and IL-5.

[0071] In a particular embodiment, the invention relates to a population of lymphocytes according to the invention, in which less than 10% of the T cell portion secrete at least one protein from the group consisting of TNF-α, IL-4, IL-5, granzyme B and perforin. In a particular embodiment, the invention relates to a population of lymphocytes according to the invention, in which less than 10% of the T cell portion secrete two of the proteins from the group consisting of TNF-α, IL-4, IL-5, granzyme B and perforin. In a particular embodiment, the invention relates to a population of lymphocytes according to the invention, in which less than 10% of the T cell portion secrete three of the proteins from the group consisting of TNF-α, IL-4, IL-5, granzyme B and perforin. In a particular embodiment, the invention relates to a population of lymphocytes according to the invention, in which less than 10% of the T cell portion secrete four of the proteins from the group consisting of TNF-α, IL-4, IL-5, granzyme B and perforin. In one particular embodiment, the present invention relates to a population of lymphocytes according to the present invention, wherein less than 10% of said T cell portion secretes all of the proteins from the group consisting of TNF-α, IL-4, IL-5, granzyme B and perforin.

[0072] In certain embodiments, the invention relates to a population of lymphocytes according to the invention, wherein less than 10% of the T cell portion secrete at least one of the proteins from the group consisting of TNF-α, IL-4 and IL-5. In certain embodiments, the invention relates to a population of lymphocytes according to the invention, wherein less than 10% of the T cell portion secrete at least two of the proteins from the group consisting of TNF-α, IL-4 and IL-5. In certain embodiments, the invention relates to a population of lymphocytes according to the invention, wherein less than 10% of the T cell portion secrete all of the proteins from the group consisting of TNF-α, IL-4 and IL-5.

[0073] In the present invention, a cell is determined to secrete a specific protein if a detectable amount of said protein can be identified in an ELISpot assay. The enzyme immunospot (ELISpot) assay is a sensitive immunoassay that measures the frequency of cytokine-secreting cells at the single cell level. In this assay, cells are cultured on a surface coated with a specific capture antibody in the presence or absence of a stimulus. Proteins secreted by the cells, such as cytokines, will be captured by the specific antibody on the surface. After a suitable incubation time, the cells are removed and the secreted molecules are detected using a detection antibody in a procedure similar to that utilized by ELISA. The detection antibody is biotinylated and followed by a streptavidin-enzyme conjugate or the antibody is directly conjugated to the enzyme. By using a substrate with a product that precipitates rather than a soluble product, the final product becomes a visible spot on the surface. Each spot corresponds to an individual cytokine-secreting cell. The ELISpot assay captures the presence of cytokines immediately after secretion, which is very different from measurements that are distorted by receptor binding or protease degradation. This assay is considered to be one of the most sensitive cell assays available. The detection limit that is usually achieved can be 1 cell in 100,000 cells. Due to the high sensitivity of this assay, this assay is particularly useful for studying small populations of cells found in specific immune responses. ELISpot assays for determining the percentage of cells that secrete IFN-γ, TNF-α, IL-4, IL-5, granzyme B and perforin are known in the art.

[0074] Alternatively or additionally, the secretion of these proteins can be estimated by flow cytometry.For this, T cells must be fixed and permeabilized so that antibodies can be used to quantify the intracellular pool of each protein.Methods for quantifying the intracellular pool of TNF-α, IL-4, IL-5, granzyme B and / or perforin are known in the art.

[0075] In a particular embodiment, the invention relates to a population of lymphocytes according to the invention, wherein at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the T cells in said T cell portion are CD8+ T cells.

[0076] That is, the majority of T cells in a population of lymphocytes are preferably CD8+ T cells. As used herein, "CD8+ T cells" has its general meaning in the art and refers to a subset of T cells that express CD8 on their surface. They are MHC class I restricted and function as cytotoxic T cells. "CD8+ T cells" are also called cytotoxic T lymphocytes (CTLs), T killer cells, cytolytic T cells, or killer T cells. The CD8 antigen is a member of the immunoglobulin supergene family and is the associative recognition element in major histocompatibility complex class I restricted interactions. As used herein, the term "tumor-infiltrating CD8+ T cells" refers to a pool of CD8+ T cells of a patient that have migrated away from the bloodstream to the tumor.

[0077] Preferably, the second largest portion of T cells in the population of lymphocytes are CD4+ T cells. As used herein, the term "CD4+ T cells" refers to T cells that present the co-receptor CD4 on their surface. CD4 is a transmembrane glycoprotein that serves as a co-receptor for the T cell receptor (TCR), which can recognize specific antigens. In certain embodiments, the CD4+ T cells are T helper cells. T helper cells (TH cells) assist other white blood cells in immunological processes, including maturation of B cells into plasma cells and memory B cells, and activation of cytotoxic T cells and macrophages. Helper T cells are activated when they are presented with peptide antigens by MHC class II molecules expressed on the surface of antigen-presenting cells (APCs). Once activated, they divide rapidly and secrete small proteins called cytokines that control or support active immune responses. These cells can differentiate into one of several subtypes, including TH1, TH2, TH3, TH17, TH9 or TFH, which secrete different cytokines to promote different types of immune responses. Signaling from APC directs T cells to specific subtypes. In certain embodiments, CD4+ T cells are regulatory T cells. Regulatory T cells play an essential role in attenuating immune responses, preventing autoimmune diseases, and oral tolerance.

[0078] In certain embodiments, the invention relates to a population of lymphocytes according to the invention, wherein no more than 50%, no more than 40%, no more than 30%, no more than 20%, or no more than 10% of the T cells in said T cell portion are CD4+ T cells.

[0079] In certain embodiments, the invention relates to a population of lymphocytes according to the invention, wherein the ratio of CD8+ T cells to CD4+ T cells in said T cell portion is between 1:1 and 10:1. In certain embodiments, the invention relates to a population of lymphocytes according to the invention, wherein the ratio of CD8+ T cells to CD4+ T cells in said T cell portion is 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1 or greater than 10:1.

[0080] Those skilled in the art are aware of methods for determining the percentage of CD4+ and / or CD8+ T cells in a population of lymphocytes. For example, the percentage of CD4+ and / or CD8+ T cells in a population of lymphocytes can be determined by flow cytometry using antibodies against CD4 and / or CD8, respectively.

[0081] In a particular embodiment, the invention relates to a population of lymphocytes according to the invention, wherein at least two T cells in said T cell portion are directed against different antigens.

[0082] That is, the T cells contained in the population of lymphocytes preferably recognize more than one antigen. Obtaining the population of lymphocytes according to the present invention comprises a step of contacting these lymphocytes with a pool of different antigenic peptides. It is therefore envisaged to expand the T cells that recognize antigens from the pool of antigens. The pool of antigenic peptides may comprise more than 50, more than 100, more than 200, more than 300, more than 400, more than 500, or more than 1000 different antigenic peptides. Thus, in certain embodiments, the T cell portion contained in the population of lymphocytes may comprise at least 2, at least 5, at least 10, at least 25, at least 50, at least 75, at least 100, at least 150, at least 200 or at least 300 T cells, each T cell being directed against a different antigen. Non-limiting examples of antigens that may be recognized by the T cells contained in the population of lymphocytes are provided herein.

[0083] Preferably, the population of lymphocytes comprises a large number of cells suitable for use in adoptive cell transfer (ACT) therapy in humans. That is, the population of lymphocytes according to the present invention comprises at least 10 6 pieces, 10 7 pieces, 10 8 pieces, 10 9 pcs or 10 10 Preferably, the population of lymphocytes according to the present invention comprises 10 6 ~10 10 Between 10 and 10 CD3+ T cells, preferably 10 7 ~10 9 In certain embodiments, the population of lymphocytes according to the invention comprises between 10×10 T cells. 8 Contains T cells.

[0084] In a particular embodiment, the present invention relates to a population of lymphocytes for allogeneic cell transfer in humans, comprising at least 90% CD3+ T cells and less than 5% B cells, wherein at least 70% of said T cell portion are viable, at least 50% are CD27 / CD28 double positive, and less than 10% are triple positive for CD45RA, CD57 and KLRG1.

[0085] In a particular embodiment, the present invention relates to a population of lymphocytes for allogeneic cell transfer in humans, comprising at least 90% CD3+ T cells and less than 5% B cells, wherein at least 70% of said T cell portion are viable, at least 20% are CD27 / CD28 double positive, and less than 10% are triple positive for CD45RA, CD57 and KLRG1.

[0086] In a particular embodiment, the present invention relates to a population of lymphocytes for adoptive cell transfer therapy in humans, comprising at least 90% CD3+ T cells and less than 5% B cells, wherein at least 70% of said T cell portion are viable, at least 50% are CD27 / CD28 double positive, and less than 10% are positive for CD45RA and CD57.

[0087] In a particular embodiment, the present invention relates to a population of lymphocytes for adoptive cell transfer therapy in humans, comprising at least 90% CD3+ T cells and less than 5% B cells, wherein at least 70% of said T cell portion are viable, at least 20% are CD27 / CD28 double positive, and less than 10% are positive for CD45RA and CD57.

[0088] In a particular embodiment, the present invention relates to a population of lymphocytes for adoptive cell transfer therapy in humans, comprising at least 90% CD3+ T cells and less than 5% B cells, wherein at least 70% of said T cell portion are viable, at least 50% are CD27 / CD28 double positive, and less than 10% are positive for CD57 and KLRG1.

[0089] In a particular embodiment, the present invention relates to a population of lymphocytes for adoptive cell transfer therapy in humans, comprising at least 90% CD3+ T cells and less than 5% B cells, wherein at least 70% of said T cell portion are viable, at least 20% are CD27 / CD28 double positive, and less than 10% are positive for CD57 and KLRG1.

[0090] In a particular embodiment, the present invention relates to a population of lymphocytes for adoptive cell transfer therapy in humans, comprising at least 90% CD3+ T cells and less than 5% B cells, wherein at least 70% of said T cell portion are viable, at least 50% are CD27 / CD28 double positive, and more than 80% are negative for CD57 and KLRG1.

[0091] In a particular embodiment, the present invention relates to a population of lymphocytes for adoptive cell transfer therapy in humans, comprising at least 90% CD3+ T cells and less than 5% B cells, wherein at least 70% of said T cell portion are viable, at least 20% are CD27 / CD28 double positive, and more than 80% are negative for CD57 and KLRG1.

[0092] Preferably, the population of lymphocytes according to the invention is suitable for use in autologous cell therapy. Autologous cell therapy is a therapeutic intervention that uses cells of an individual that are cultured and expanded outside the body and reintroduced into the donor. Advantages of such an approach include minimizing the risks of systemic immunological reactions, bioincompatibility and disease transmission associated with non-autologous grafts or cells from the individual. Preferably, the cells contained in the population of lymphocytes according to the invention are obtained by ex vivo expansion of T cells of an individual and then infused back into the same individual.

[0093] Thus, in a particular embodiment, the present invention relates to a population of lymphocytes for autologous cell therapy in humans, comprising at least 90% CD3+ T cells and less than 5% B cells, wherein at least 70% of said T cell portion are viable, at least 50% are CD27 / CD28 double positive, and less than 10% are triple positive for CD45RA, CD57 and KLRG1.

[0094] Thus, in a particular embodiment, the present invention relates to a population of lymphocytes for autologous cell therapy in humans, comprising at least 90% CD3+ T cells and less than 5% B cells, wherein at least 70% of said T cell portion are viable, at least 20% are CD27 / CD28 double positive, and less than 10% are triple positive for CD45RA, CD57 and KLRG1.

[0095] In a particular embodiment, the present invention relates to a population of lymphocytes for autologous cell therapy in humans, comprising at least 90% CD3+ T cells and less than 5% B cells, wherein at least 70% of said T cell portion are viable, at least 50% are CD27 / CD28 double positive, and less than 10% are double positive for CD45RA and CD57.

[0096] In a particular embodiment, the present invention relates to a population of lymphocytes for autologous cell therapy in humans, comprising at least 90% CD3+ T cells and less than 5% B cells, wherein at least 70% of said T cell portion are viable, at least 20% are CD27 / CD28 double positive, and less than 10% are double positive for CD45RA and CD57.

[0097] In a particular embodiment, the present invention relates to a population of lymphocytes for autologous cell therapy in humans, comprising at least 90% CD3+ T cells and less than 5% B cells, wherein at least 70% of said T cell portion are viable, at least 50% are CD27 / CD28 double positive, and less than 10% are double positive for CD57 and KLRG1.

[0098] In a particular embodiment, the present invention relates to a population of lymphocytes for autologous cell therapy in humans, comprising at least 90% CD3+ T cells and less than 5% B cells, wherein at least 70% of said T cell portion are viable, at least 20% are CD27 / CD28 double positive, and less than 10% are double positive for CD57 and KLRG1.

[0099] In a particular embodiment, the present invention relates to a population of lymphocytes for autologous cell therapy in humans, comprising at least 90% CD3+ T cells and less than 5% B cells, wherein at least 70% of said T cell portion are viable, at least 50% are CD27 / CD28 double positive, and more than 80% are double negative for KLRG1 and CD57.

[0100] In a particular embodiment, the present invention relates to a population of lymphocytes for autologous cell therapy in humans, comprising at least 90% CD3+ T cells and less than 5% B cells, wherein at least 70% of said T cell portion are viable, at least 20% are CD27 / CD28 double positive, and more than 80% are double negative for KLRG1 and CD57.

[0101] The present invention also provides a method for producing a population of lymphocytes specific for one or more antigens as defined herein, comprising a single culture phase comprising: (a) culturing a tissue or blood sample from a subject, the sample being known or suspected to contain lymphocytes, in the presence of said one or more antigens; or (b) culturing lymphocytes, the lymphocytes being lymphocytes isolated from a tissue or blood sample from a subject, in the presence of said one or more antigens.

[0102] In certain embodiments, the culturing step comprises culturing at least 10×10 8This is continued until said T cell population of cells is obtained. At all times, the lymphocyte and / or T cell populations are maintained at a temperature above 0° C. during said single culture phase.

[0103] In certain embodiments, the culturing step comprises culturing at least 1×10 7 This is continued until said T cell population of cells is obtained. At all times, the lymphocyte and / or T cell populations are maintained at a temperature above 0° C. during said single culture phase.

[0104] In certain embodiments, the sample containing the population of lymphocytes and / or T cells is maintained at a temperature above 0° C. after isolation from the subject and prior to culture. However, it is understood that frozen samples can also be used in the methods of the present invention.

[0105] Previous expansion protocols for autologous tumor-infiltrating lymphocytes (TILs) consist of two phases. In the initial pre-REP phase, TILs are expanded for 3-5 weeks. In the subsequent REP phase, TILs obtained in the pre-REP phase are rapidly expanded for another 2 weeks. Between the pre-REP and REP phases, TILs are usually cryopreserved. The drawback of this long culture period, including an optional cryopreservation step, is that the majority of lymphocytes in the final product are terminal effector cells that die rapidly after infusion into the patient.

[0106] Therefore, an object of the present invention is to produce a high number of cells (10 10 20 30 40 50 60 65 80 70 85 90 95 100 150 160 170 180 190 200 250 260 270 280 300 350 400 450 500 550 600 7 The objective of the present invention is to establish a lymphocyte expansion protocol that can reach a total cell mass of 10 ...

[0107] The method of the invention is characterized in that the cells are cultured in a "conditioned culture medium". That is, certain parameters of the culture medium are monitored throughout the entire process and adjusted, if necessary, to predefined values. Suitable parameters of the culture medium to be monitored and / or adjusted during the method of the invention are disclosed elsewhere herein. With the aid of them, optimal growth conditions can be maintained throughout the entire process.

[0108] The method of the invention is further characterized in that it includes a step of "dynamic culturing". Dynamic culturing involves culturing the cells with a continuous flow of culture medium. Dynamic culturing includes both circulation, where conditioned culture medium is circulated within the growth chamber, and perfusion, where culture medium from the growth chamber is exchanged with fresh culture medium.

[0109] The method of expanding a desired T cell population from a sample, for example comprising lymphocytes and / or T cells, comprises presenting one or more antigens to T cells in the sample to be cultured. The antigens can be presented by any means known in the art and / or described herein suitable for inducing the expansion of T cells that specifically recognize one or more antigens. As an illustrative non-limiting example, one or more soluble antigens can be provided continuously in the culture medium (e.g., to maintain a steady-state concentration or a desired concentration range) or can be included in one or more specific periods of less than the entire culture phase. The soluble antigens can also be introduced at one or more discrete time points of the culture phase. Additionally or alternatively, the soluble antigens can be presented by antigen-presenting cells (APCs) to the lymphocyte sample and / or T cells during culture, as disclosed herein. The APCs are preferably B cells. The APCs can be engineered to present one or more desired antigens by any means known in the art or described herein. Alternatively or additionally, the APCs can be contacted with antigenic peptides by any means known in the art or described herein.

[0110] In certain embodiments, the one or more antigens added in the culturing step are contained in tumor sample.That is, tumor sample itself can simultaneously serve as lymphocyte source and antigen source.In such an embodiment, tumor sample can be co-cultured with APC in the absence of antigen peptide.

[0111] APCs can be recombinantly engineered to transiently or continuously express one or more antigens of interest. Recombinant engineering can be accomplished by any means known in the art or described herein, and is preferably accomplished by transduction using a viral vector or transfection using a plasmid or mRNA.

[0112] However, it is preferred that APCs, and in particular B cells, are contacted with chemically synthesized antigenic peptides, as explained in more detail below.

[0113] The antigen may be one or more known antigens that characterize disease or cancer, or may be determined by evaluating a patient to determine one or more neo-antigens.To this end, patient cells can be collected by biopsy and analyzed by mass spectrometry or scRNAseq to identify neo-antigens.The sequences obtained from these methods can then be analyzed using proprietary algorithms to identify and select relevant neo-antigens.

[0114] It is to be understood that the lymphocyte populations, isolated lymphocytes, and / or methods for their production and use are provided as tools for the treatment of disease (e.g., for use as a pharmaceutical or in pharmaceutical development and manufacturing), as well as have applicability as model systems for studying disease treatment. Thus, the lymphocytes of the invention disclosed herein are preferably human lymphocytes, more preferably primary human lymphocytes (e.g., including NK cells and T cells), most preferably primary human T cells (including CD3+ T cells, CD4+ T cells, CD8+ T cells, γδ T cells), although lymphocyte cell lines (whether of human or non-human origin), and lymphocytes that are primary cells of non-human origin, such as, but not limited to, primary lymphocytes, and lymphocytes from mice, rats, monkeys, apes, cats, and dogs, are also provided.

[0115] Of the more preferred primary human lymphocytes, the most preferred are primary human T cells. Thus, the present invention also provides a population of primary human T cells characterized by at least 90% CD3+ T cells and less than 5% B cells, wherein at least 70% of said T cell portion are viable, at least 50% are CD27 / CD28 double positive, and less than 10% are triple positive for CD45RA, CD57 and KLRG1.

[0116] Alternatively, the present invention provides a population of primary human T cells characterized by at least 90% CD3+ T cells and less than 5% B cells, wherein at least 70% of said T cell portion are viable, at least 20% are CD27 / CD28 double positive, and less than 10% are triple positive for CD45RA, CD57 and KLRG1.

[0117] Furthermore, the present invention also provides a population of primary human T cells characterized by at least 90% CD3+ T cells and less than 5% B cells, wherein at least 70% of said T cell portion are viable, at least 50% are CD27 / CD28 double positive, and greater than 80% are double positive for CD57 and KLRG1.

[0118] Furthermore, the present invention also provides a population of primary human T cells characterized by at least 90% CD3+ T cells and less than 5% B cells, wherein at least 70% of said T cell portion are viable, at least 20% are CD27 / CD28 double positive, and greater than 80% are double positive for CD57 and KLRG1.

[0119] The lymphocyte populations provided herein or produced according to the methods provided herein, whether human or not, and whether primary or not, may be comprised of any lymphocyte class or subclass known or believed to be useful in adoptive cell therapy and / or known or believed to be useful in in vitro or in vivo model systems, as known in the art or described herein. Non-limiting examples of lymphocyte classes encompassed by the present invention include lymphocyte populations including T cells (including CD3+ T cells, CD4+ T cells, CD8+ T cells, γδ T cells, invariant T cells), as well as B cells, macrophages and NK cells, and combinations thereof.

[0120] The population of cells (e.g., APC, preferably B cells) for use in the cell and / or their production method provided herein includes genetically engineered cells, which may be directly genetically engineered cells, i.e., cells directly subjected to genetic engineering, or may be cells derived from such engineered cells, e.g., daughter cells or descendants of directly genetically engineered cells. Any suitable genetic engineering method can be used, including but not limited to lipofection, CRISPR / CAS, calcium phosphate transfection, Sleeping Beauty transposon, PEG-mediated transfection, and transduction with viral vectors (e.g., lentiviral vectors). Exogenous nucleic acid molecules can be introduced into cells as linear molecules and / or as circular molecules (e.g., plasmids, mini-plasmids, or mRNA). In non-limiting embodiments, one or more of the lymphocytes within the lymphocyte populations of the invention can be engineered to express one or more immune modulators, such as OX40L, 4-1BBL, CD80, CD86, CD83, CD70, CD40L, GITR-L, CD127L, CD30L (CD153), LIGHT, BTLA, ICOS-L (CD275), SLAM (CD150), CD662L, interleukin-12, interleukin-7, interleukin-15, interleukin-17, interleukin-21, interleukin-4, Bcl6, Bcl-XL, BCL-2, MCL1, STAT-5, and / or activators of one or more signal transduction pathways (e.g., the JAK / STAT pathway, the Akt / PBK signaling pathway, the BCR signaling pathway, and / or the BAFF / BAFFR signaling pathway). Similarly, one or more APCs useful in the methods disclosed herein can be engineered to express one or more known antigens, or one or more neo-antigens determined from a patient sample.

[0121] APCs, particularly B cells, are preferably engineered to express one or more of the immune regulators OXO40L, 4-1BB and / or interleukin-12.

[0122] In certain embodiments, APCs, particularly B cells, are engineered to express OXO40L and 4-1BB.

[0123] In certain embodiments, APCs, particularly B cells, are engineered to express OXO40L and interleukin-12.

[0124] In certain embodiments, APCs, particularly B cells, are engineered to express 4-1BB and interleukin-12.

[0125] In certain embodiments, APCs, particularly B cells, are engineered to express OXO40L, 4-1BB and interleukin-12.

[0126] Nucleic acids encoding the above-mentioned immunomodulatory factors can be introduced into APCs, particularly B cells, by any method known in the art and / or disclosed herein. Preferably, mRNA encoding the above-mentioned immunomodulatory factors is introduced into APCs, particularly B cells, by transfection to transiently express the encoded protein.

[0127] The lymphocytes and lymphocyte populations of the present invention, preferably human lymphocytes, more preferably primary human lymphocytes, most preferably primary human T cells, are intended for use in therapy and can be autologous (i.e., the donor from which the cells are derived is the same subject as the recipient) or allogeneic (i.e., the donor from which the cells are derived is different from the recipient). If autologous, any suitable source known in the art or described herein can be used, including but not limited to the tumor environment, whether solid tumor cells (e.g., for tumor-infiltrating lymphocytes (TILs)) or circulating tumor cells; and peripheral blood (e.g., PBMCs). Preferably, the lymphocytes in the lymphocyte population are obtained by expanding TILs ex vivo.

[0128] If the cells are allogeneic, they can be further engineered or prepared so that they are not alloreactive. As understood in the art and as used herein, non-allo-reactive indicates that the cells have been engineered (e.g., engineered) to be incapable of being recognized as allogeneic cells (foreign cells) or to be capable of recognizing allogeneic cells (foreign cells). Similarly, the engineered lymphocytes of the present invention can additionally or alternatively be engineered to be not recognized by the recipient's immune system. As a non-limiting example in this embodiment, the lymphocytes of the present invention can have disruption or deletion of endogenous major histocompatibility complex (MHC). Such cells can have reduced or eliminated expression of endogenous MHC, thus preventing or reducing the activation of the recipient's immune system against self-cells.

[0129] As understood in the art, such non-allo-reactive cells are incapable of reacting with cells of a foreign host. Thus, non-allo-reactive cells derived from a third-party donor can be universal, i.e., recipient-independent. As explained above, non-allo-reactive cells can also include additional manipulations that render them incapable of eliciting an immune response and / or incapable of being recognized by the recipient's immune system, thus preventing them from being rejected. Such cells that are non-allo-reactive and / or incapable of eliciting an immune response or incapable of being recognized by the recipient's immune system can also be referred to as "off-the-shelf" cells, as known in the art. Lymphocytes can be made non-allo-reactive and / or incapable of eliciting or incapable of being recognized by the immune system by any means known in the art or described herein. In a non-limiting example, with respect to T cells, non-allo-reactive cells can have reduced or eliminated expression of endogenous T cell receptors (TCRs) compared to unmodified control cells. Such non-allo-reactive T cells may include modifications or deletions of genes involved in self-recognition, such as those encoding components of the TCR, including, but not limited to, the alpha and / or beta chains. Similarly, the genetically engineered lymphocytes disclosed herein may additionally or alternatively have reduced or eliminated expression of endogenous MHC compared to unmodified control cells. Such lymphocytes may include any modifications or genetic deletions known in the art or described herein to minimize or eliminate antigen presentation, in particular to avoid immunogenic surveillance and elimination in the recipient. As previously mentioned, non-allo-reactive cells that optionally avoid immune surveillance are broadly referred to in the art as "off-the-shelf" cells, and these terms are used interchangeably herein. Such non-allo-reactive / off-the-shelf leukocytes may be obtained from repositories.Genetic modifications known in the art or described herein to reduce or eliminate alloreactivity (i.e., to render the cells non-alloreactive) and / or to reduce or eliminate self-antigen presentation (i.e., to prevent them from eliciting an immune response or being recognized by the recipient's immune system) can be performed prior to, in parallel with, or after any other genetic manipulations in connection with the present invention.

[0130] The present invention also encompasses a population of lymphocytes, preferably human lymphocytes, obtainable by any of the methods disclosed herein.

[0131] The present invention provides a method of immunotherapy for treating disease, comprising the use of the cell or cell population disclosed herein.Therefore, a population of lymphocytes (preferably human lymphocytes, more preferentially primary human lymphocytes, most preferentially primary human T cells) as described herein is provided for use as a medicament.The present invention also provides a population of lymphocytes as disclosed herein in a pharmaceutically acceptable carrier in the form of a pharmaceutical composition.The medicaments and pharmaceutical compositions disclosed herein are particularly useful for adoptive cell therapy.

[0132] The lymphocyte populations, medicaments and / or pharmaceutical compositions of the present invention are useful for the treatment of cancer regardless of tumor type, as well as for the treatment of viral diseases, bacterial diseases such as tuberculosis (including antibiotic-resistant diseases), and parasitic diseases.

[0133] The lymphocyte populations, medicaments and / or pharmaceutical compositions of the invention may be administered in combination with an anti-neoplastic or immunomodulatory agent, such as, but not limited to, azacitidine, capecitabine, carmofur, cladribine, clofarabine, cytarabine, decitabine, floxuridine, fludarabine, fluorouracil, gemcitabine, mercaptopurine, nelarabine, pentostatin, tegafur, thioguanine, methotrexate, pemetrexed, raltitrexed, hydroxycarbamide, irinotecan, topotecan, daunorubicin, epirubicin, idarubicin, mitoxane, riboflavin ... introne, valrubicin, etoposide, teniposide, cabazitaxel, docetaxel, paclitaxel, vinblastine, vincristine, vindesine, vinflunine, vinorelbine, bendamustine, busulfan, carmustine, chlorambucil, chloromethine, cyclophosphamide, dacarbazine, fotemustine, ifosfamide, lomustine, melphalan, streptozotocin, temozolomide, carboplatin, cisplatin, nedaplatin, oxaliplatin, altretamine, bleomycin, bortezomib, dactinomycin, estram stin, ixabepilone, mitomycin, alemtuzumab, bevacizumab, cetuximab, denosumab, gemtuzumab, ozogamicin, ibritumomab tiuxetan, ipilimumab, nivolumab, ofatumumab, panitumumab, pembrolizumab, pertuzumab, rituximab, tositumomab, trastuzumab, afatinib, aflibercept, axitinib, bosutinib, crizotinib, dasatinib, erlotinib, gefitinib, imatinib, lapatinib, nilotinib, pazopanib, ponatinib, regorafenib, ruxolitinib, sorafenib , sunitinib, vandetanib, everolimus, temsirolimus, alitretinoin, bexarotene, isotretinoin, tamibarotene, tretinoin, lenalidomide, pomalidomide, thalidomide, panobinostat, romidepsin, valproic acid, vorinostat, anagrelide, arsenic trioxide, asparaginase, BCG vaccine, denileukin diftitox, vemurafenib, goserelin, toremifene, fulvestrant, bicalutamide, enzalutamide, apalutamide, darolutamide, anastrozole, letrozole, degarelix, abiraterone,Filgrastim, molgramostin, pegfilgrastim, lipecfilgrastim, valgrastim, levacetylmethadol, interferon gamma, interferon alpha-2b, interferon alpha-n1, interferon beta-1a, peginterferon alpha-2b, peginterferon beta-1a, lopeginterferon alpha-2v, tasonermin, histamine dihydrochloride, mifarmurtide, plerixafor, sipuleucel-T, daciprotim-T, muromonab-CD3, mycophenolate, sirolimus, leflunomide, efalizumab, natalizumab, abatacept, exon It may be used in combination with: lizumab, ofatumumab, fingolimod, eltrombopag, tofacitinib, teriflunomide, apremilast, vedolizumab, baricitinib, ozanimod, upadacitinib, filgotinib, etanercept, infliximab, adalimumab, certolizumab pegol, golimumab, valdecoxib, anakinra, rilonacept, ustekinumab, tocilizumab, canakinumab, secukinumab, lopinavir, ritonavir, brodalumab, ixekizumab, sarilumab, tacrolimus, voclosporin, thalidomide, methotrexate, lenalidomide, pirfenidone, pomalidomide, dimethyl fumarate, and dalvadostrocel. As used herein, combining with the lymphocyte populations, medicaments and / or pharmaceutical compositions of the present invention does not indicate that the lymphocyte therapy and one or more additional medicaments must be administered together, for example in the same infusion. Combining includes simultaneous administration and sequential administration in any order. Combining also includes administration schemes in which one or more agents are administered multiple times over a time frame, for example, of days, weeks or months, and the other agent(s) are administered only once or according to a different dosing scheme. Combining includes any scheme in which agents are intentionally administered such that there is at least some overlap in therapeutic effects.

[0134] 3. Detailed Description 3.1 Lymphocytes for immunotherapy The present invention particularly relates to a population of lymphocytes (preferably human lymphocytes, more preferably primary human lymphocytes, most preferably primary human T cells) characterized by at least 90% CD3+ T cells and less than 5% B cells, wherein at least 70% of said T cell portion are viable, at least 50% are CD27 / CD28 double positive, and less than 10% are triple positive for CD45RA, CD57 and KLRG1. As used herein, with respect to cells or cell populations, the terms "primary" and similar terms correspond to their commonly understood meaning in the art, i.e., cells obtained directly from a living tissue (i.e., a biopsy such as a tumor sample or a blood sample) or cells from a subject that have not been passaged in culture or that have been passaged and maintained in culture but have not been immortalized. Preferably, the primary cells are primary human lymphocytes. Primary cells have undergone very few, if any, population doublings.

[0135] The population of lymphocytes according to the present invention may comprise any lymphocyte class, subclass or mixture thereof described herein as suitable for use, particularly for use in adoptive cell therapy, or known in the art as suitable.However, the method of the present invention may also be applicable for use other than therapy, for example in screening methods, and / or in model systems, such as model systems that serve in vitro assays or in vivo animal models.Non-limiting examples of lymphocytes (which may be primary lymphocytes or derived from cell lines) include NK cells, inflammatory T lymphocytes, cytotoxic T lymphocytes, helper T lymphocytes, CD4+ T lymphocytes, CD8+ T lymphocytes, γδ T lymphocytes, invariant T lymphocytes, NK lymphocytes, B lymphocytes and macrophages.

[0136] It is preferred herein that at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the CD3+ T cells contained in the population of lymphocytes are CD8+ T cells.

[0137] 3.2 Metabolic characterization A population of lymphocytes (preferably human lymphocytes, more preferably primary human lymphocytes, most preferably primary human T cells) can be analyzed for expression of one or more phenotypic markers after expansion. In some embodiments, the markers are selected from one or more of TCRab (i.e., TCR alpha / beta), CD57, CD28, CD4, CD27, CD56, CD8a, CD45RA, CD8a, CCR7, CD4, CD3, CD38, CD45RA, and HLA-DR. In some embodiments, expression of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 markers is examined.

[0138] A population of lymphocytes (preferably human lymphocytes, more preferably primary human lymphocytes, most preferably primary human T cells) can be analyzed for expression of one or more regulatory markers. In some embodiments, the regulatory markers are selected from one or more of CD137, CD8a, Lag3, CD4, CD3, PD-1, TIM-3, CD69, CD8a, TIGIT, CD4, CD3, KLRG1 and CD154.

[0139] It is preferred to analyze a population of lymphocytes (preferably human lymphocytes, more preferably primary human lymphocytes, most preferably primary human T cells) for the expression of both one or more phenotypic markers and one or more regulatory markers.Thus, a population of lymphocytes (preferably human lymphocytes, more preferably primary human lymphocytes, most preferably primary human T cells) can be analyzed for the expression of one or more of TCRab (i.e. TCR alpha / beta), CD57, CD28, CD4, CD27, CD56, CD8a, CD45RA, CD8a, CCR7, CD4, CD3, CD38, CD45RA, HLA-DR, CD137, CD8a, Lag3, CD4, CD3, PD-1, TIM-3, CD69, CD8a, TIGIT, CD4, CD3, KLRG1 and CD154. Preferably, at least 50% of the CD3+ T cells in the lymphocyte population are CD27 / CD28 double positive, and less than 10% of the CD3+ T cells in the lymphocyte population are triple positive for CD45RA, CD57 and KLRG1.

[0140] Alternatively, it is preferred that at least 50% of the CD3+ T cells contained in the lymphocyte population are CD27 / CD28 double positive and greater than 80% of the CD3+ T cells contained in the lymphocyte population are double negative for CD57 and KLRG1.

[0141] Preferably, the presence of the above-mentioned markers on the cell surface of CD3+ T cells contained within the population of lymphocytes is determined by flow cytometry.

[0142] As used herein, the term "flow cytometry" refers to an assay in which the proportion of material in a sample (e.g., lymphocytes containing a particular marker) is determined by labeling the material (e.g., by binding a labeled antibody to the material), passing a fluid stream containing the material through a beam of light, separating the light emitted from the sample into component wavelengths by a series of filters and mirrors, and detecting the light.

[0143] A number of flow cytometers are commercially available, including, for example, those from Becton Dickinson FACScan and FACScaliber (BD Biosciences, Mountain View, Calif.). Antibodies that can be used for FACS analysis are widely available commercially.

[0144] In some embodiments, the viability of the population of lymphocytes (preferably human lymphocytes, more preferably primary human lymphocytes, most preferably primary human T cells) is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 98%. The viability of lymphocytes can be determined by any one of the methods known in the art, such as those disclosed herein above.

[0145] A population of lymphocytes (preferably human lymphocytes, more preferably primary human lymphocytes, most preferably primary human T cells) can be assessed for interferon-γ (IFN-γ) secretion in response to stimulation with either anti-CD3 antibodies (e.g., OKT3) or co-culture with autologous tumor digests or stimulation with antigens and / or neo-antigenic peptides. The skilled artisan is aware that antigens and / or neo-antigenic peptides should be presented in an MHC-dependent manner.

[0146] In some embodiments, the health of TILs is measured by IFN-gamma (IFN-γ) secretion. In some embodiments, IFN-γ secretion indicates active T cells in the expanded population. In some embodiments, a potency assay for IFN-γ production is utilized. IFN-γ production is another measure of cytotoxicity. IFN-γ production can be measured by determining the level of the cytokine IFN-γ in the medium of a population of lymphocytes (preferably human lymphocytes, more preferably primary human lymphocytes, most preferably primary human T cells) provided and produced according to the methods herein, which can be analyzed after stimulation with antibodies against CD3, CD28, and / or CD137 / 4-1BB. IFN-γ levels in the medium from these stimulated populations of lymphocytes (preferably human lymphocytes, more preferably primary human lymphocytes, most preferably primary human T cells) can be determined by using / measuring IFN-γ release. In some embodiments, IFN-γ secretion is increased 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold or more relative to corresponding cells in the sample before expansion.

[0147] In some embodiments, telomere length can be used as a measure of cell viability and / or cell function. In some embodiments, telomeres are surprisingly the same length in lymphocyte populations produced by the present invention compared to lymphocyte populations prepared using methods other than those provided in the present invention. A variety of methods have been used to measure telomere length in genomic DNA and cell preparations. Telomere restriction fragment (TRF) analysis is the gold standard for measuring telomere length. However, a major limitation of TRF is that it requires large amounts of DNA. Two techniques that are widely used to measure telomere length can be utilized with the present invention: fluorescent in situ hybridization (e.g., FISH; Agilent Technologies, Santa Clara, Calif.) and quantitative PCR. In some embodiments, there is no change in telomere length between the lymphocytes (or any subpopulations thereof, e.g., T cells) initially collected from the sample and the lymphocyte and / or T cell populations after expansion.

[0148] 3.3 Source of lymphocytes The primary lymphocytes described herein can be isolated and / or obtained by any method known in the art or described herein from a number of tissue sources, including, but not limited to, peripheral blood mononuclear cells isolated from blood samples, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, splenic tissue, and / or tumor. In the methods of the present invention, the isolated cells and / or samples used, for example, to generate a population of lymphocytes (preferably human lymphocytes, more preferably primary human lymphocytes, most preferably primary human T cells), are preferably obtained and / or isolated from a population derived from a tumor sample, whether solid or circulating (e.g., for the isolation of TILs) or from a population derived from an infected tissue (e.g., tissue infected with a virus, bacteria or parasite). Methods for isolating / obtaining a specific population of lymphocytes from a patient or donor are well known in the art and include, as a first step, for example, isolating / obtaining a donor or patient sample known or expected to contain such cells.

[0149] For example, lymphocytes (preferably human lymphocytes, more preferably primary human lymphocytes, most preferably primary human T cells (including TILs)) can be obtained from a patient tumor sample and then expanded into a larger population. Such expanded cells and / or populations can be cryopreserved as needed after expansion for storage and handling prior to administration.

[0150] Patient tumor samples can be obtained using methods known in the art, typically by surgical removal, needle biopsy, or other means to obtain a sample containing a mixture of tumor and lymphocytes. Generally, tumor samples can be from any solid tumor, including primary tumors, invasive tumors, or metastatic tumors. Tumor samples can also be liquid tumors, such as tumors obtained from hematological malignancies. Solid tumors can be from any cancer type, including, but not limited to, squamous cell carcinoma, basal cell carcinoma, and melanoma, including, but not limited to, breast, pancreatic, prostate, colorectal, lung, brain, kidney, stomach, and skin. Most preferably, the sample is one known or suspected to contain T cells, particularly TILs. In some embodiments, useful TILs are obtained from malignant melanoma tumors, as these have been reported to have particularly high levels of lymphocytes, particularly TILs.

[0151] The term "solid tumor" refers to an abnormal mass of tissue that does not usually contain cysts or liquid areas. Solid tumors can be benign or malignant. The term "solid tumor cancer" refers to a malignant, neoplastic, or cancerous solid tumor. Solid tumor cancers include, but are not limited to, sarcomas, carcinomas, and lymphomas, such as lung cancer, breast cancer, triple-negative breast cancer, prostate cancer, colon cancer, rectal cancer, and bladder cancer. In some embodiments, the cancer is selected from cervical cancer, head and neck cancer (including, for example, head and neck squamous cell carcinoma (HNSCC)), glioblastoma, ovarian cancer, sarcoma, pancreatic cancer, bladder cancer, breast cancer, triple-negative breast cancer, and small cell lung cancer. The tissue structure of a solid tumor includes interdependent tissue compartments, including parenchymal cells (cancer cells) and supporting stromal cells, into which the cancer cells are distributed and which may provide a supportive microenvironment.

[0152] After isolating / obtaining the sample, the desired cells, for example, human lymphocytes and / or T cells (e.g., TILs), can be cultured under conditions that allow preferential growth and expansion of cells of the desired cell class, subclass, or with the desired specificity. In particular, this method allows the isolation / obtainment of populations that maintain stemness and exhibit a low percentage of terminal effector cells, which are known in the art to have increased replicative capacity and / or high cell killing activity capacity. Such cells are characterized by high expression of CD27 and CD28, low expression of CD45RA, CD57, and KLRG1, and low secretion of TNF-α, IL-4, IL-5, and optionally granzyme B and perforin, as disclosed elsewhere herein.

[0153] 3.4 Antigen specificity The present invention provides a method for generating lymphocytes (preferably human lymphocytes, more preferably primary human lymphocytes, and most preferably primary human T cells (such as TILs)) with defined specificity, i.e. with targeted killing activity directed to cells expressing a particular antigen. As is known in the art, lymphocyte responses, particularly T cell responses, depend on the recognition of peptides by the T cell receptor, particularly in the context of the MHC complex. Thus, the present invention provides for the culture of a lymphocyte population in the presence of a peptide against which a desired response is to be directed. For example, the peptide may be a known antigen associated with a disease and / or an antigen determined in the subject to be treated, e.g. a neo-antigen as determined from the analysis of a tumor sample or a sample of infected tissue. A sample comprising lymphocytes and / or lymphocyte cultures may be exposed to between 2 and 300 peptides (whether as soluble peptides or presented by antigen presenting cells (APCs) as described herein).

[0154] The peptides to be included in the culture with lymphocytes (or in the sample containing lymphocytes) may be in soluble form. When soluble peptides are used, they may be cultured with lymphocytes at a concentration of 0.1-10 micromolar, 0.5-5 micromolar, or 1-2 micromolar. Alternatively or additionally, the peptides in the culture may be presented by APCs as known in the art.

[0155] Antigenic peptides are preferably added to the culture such that they can be presented to lymphocytes by B cells in an MHC-dependent manner. Preferably, the peptides added to the lymphocytes have a length between 9 and 35 amino acids, between 9 and 30 amino acids, between 9 and 25 amino acids. In certain embodiments, the antigenic peptides added to the lymphocytes are peptides presented by MHC class I molecules. Such peptides typically have a length of 9 to 12 amino acids. In certain embodiments, the antigenic peptides added to the lymphocytes are peptides presented by MHC class II molecules. Such peptides typically have a length of 13 to 25 amino acids. In certain embodiments, the antigenic peptides added to the lymphocytes can be a mix of peptides presented by MHC class I or MHC class II molecules. Such peptides can have a length of 9 to 25 amino acids. However, the peptides added to the culture can also be longer peptides that can be taken up and processed by APCs into shorter peptides that can be presented in an MHC-dependent manner.

[0156] Non-limiting examples of APCs useful in the methods herein include B cells. It is known that B cells stimulate a certain population of lymphocytes, particularly T cells (including TILs), that are responsive to antigens presented. APCs, for example, B cells, can be either from allogeneic sources (one or more apheresis products from one or more donors) or autologous, as described herein. APCs can be collected from frozen or fresh apheresis products according to methods known in the art. For B cells, they can be selected using LOVO (Fresenius Kabi), Prodigy (Miltenyi biotec), EKKO (Millipore, Sigma) instruments or other cell separation techniques. APCs, particularly B cells, can be activated, for example, using antibody CD40-coated beads (Miltenyi Biotec and / or Adipogen). Autologous or allogeneic APCs can be treated with mRNA to express the antigens disclosed herein. In addition, APCs can be cultured in the presence of nucleotide sequences containing the sequences of the recovered peptides, and the same transduction could be performed on TILs or T cells in culture.

[0157] APCs, e.g., B cells, can be engineered to present the desired antigen by any means known in the art or described herein, e.g., coated with peptides for presentation in the context of MHC on the cell surface, or recombinantly engineered to express and process the antigen. In a non-limiting example, APCs can be either incubated and expanded for 0-4 days in static culture or in a bioreactor, or immediately transfected and / or expanded for up to 4 days, prior to culture with a sample known or believed to contain lymphocytes. Bioreactors for the culture of APCs include, but are not limited to, ADVA (from ADVA Biotech); WAVE Bioreactor (Cytiva), GRex (Wilson Wolff), Ori Bioreactor (Ori), and Cocoon (Lonza). Alternatively, APCs can be cultured in gas-permeable culture bags. For B cells, quality can be assessed by testing for CD20+ cells. In certain embodiments, 85% or more of the cells in the B cell culture are CD20+.

[0158] In certain embodiments, B cells are prepared before they are added to lymphocytes. First, B cells can be obtained from PBMCs by cell selection. PBMCs are preferably obtained by apheresis. If B cells (or any other type of APC) are used to prepare a population of lymphocytes for autologous cell therapy, B cells need to be taken from the same patient as lymphocytes.

[0159] Kits for isolating B cells from PBMCs are known in the art and commercially available. The isolated B cells are preferably activated before adding them to the lymphocytes. Preferably, the B cells are activated for 0-20 days, 0-15 days, 0-12 days, 0-10 days, 0-7 days, 0-5 days or 0-2 days. In certain embodiments, the B cells may be activated for 1-48 hours, 8-48 hours or 12-36 hours. For example, activation of the B cells may be achieved by contacting the B cells with IL-4 and / or CD40L. Additionally, the B cells may be activated in the presence of IL-21.

[0160] When APC is transfected to express an antigen of interest, it can be carried out by any means known in the art, including but not limited to electroporation, PEG, lipofection or Crispr Cas.Alternatively or in addition, APC can be transfected to express an immune regulator, such as, for example, OX40L, 4-1BBL, CD80, CD86, CD83, CD70, CD40L, GITR-L, CD127L, CD30L (CD153), LIGHT, BTLA, ICOS-L (CD275), SLAM (CD150), CD662L, interleukin-12, interleukin-7, interleukin-15, interleukin-17, interleukin-21, interleukin-4, Bcl6, Bcl-XL, BCL-2, MCL1 or STAT-5. Alternatively or in addition, the APCs can be transfected with one or more activators of at least one signaling pathway, e.g., the JAK / STAT pathway, the Akt / PBK AKT signaling pathway, the BCR signaling pathway, or the BAFF / BAFFR signaling pathway.

[0161] In a non-limiting example, the APC has the sequence of SEQ ID NO:1: MVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVL; or SEQ ID NO: 2: ATGGTATCACATCGGTATCCTCGAATTCAAAGTATCAAAGTACAATTTACCGAATATAAGAAGGAGAAAGGTTTCATCCTCACTTCCCAAAAGGAGGATGAAATCATGAAGGTGCAGAACAACTCAGTCATCATCAACTGTGATGGGTTTTATCTCATCTCCCTGAAGGGCTACTTCTCCCAGGAAGTCAACATTAGCCTT CATTACCAGAAGGATGAGGAGCCCCTCTTCCAACTGAAGAAGGTCAGGTCTGTCAACTCCTTGATGGTGGCCTCTCTGACTTACAAAGACAAAGTCTACTTGAATGTGACCACTGACAATACCTCCCTGGATGACTTCCATGTGAATGGCGGAGAACTGATTCTTATCCATCAAAATCCTGGTGAATTCTGTGTCCTTTGA The present invention can express human OX40L encoded by the DNA sequence shown below.

[0162] In another non-limiting example, the APC comprises SEQ ID NO:3: MEGEGVQPLDENLENGSRPRFKWKKTLRLVVSGIKGAGMLLCFIYVCLQLSSSPAKDPPIQRLRGAVTRCEDGQLFISSYKNEYQTMEVQNNSVVIKCDGLYIIYLKGSFFQEVKIDHFREDHNPISIPMLNDGRRIVFTVVASLAFKDKVYLTVNAPDTLCEHLQINDGELIVVQLTPGYCAPEGSYHSTVNQVPL; or SEQ ID NO: 4: ATGGAAGGGGAAGGGGTTCAACCCCTGGATGAGAATCTGGAAAACGGATCAAGGCCAAGATTCAAGTGGAAGAAGACGCTAAGGCTGGTGGTCTCTGGGATCAAGGGAGCAGGGATGCTTCTGTGCTTCATCTATGTCTGCCTGCAACT CTCTTCCTCTCCGGCAAAGGACCCTCCAATCCAAAGACTCAGAGGAGCAGTTACCAGATGTGAGGATGGCAACTATTCATCAGCTCATACAAGAATGAGTATCAAACTATGGAGGTGCAGAACAATTCGGTTGTCATCAAGTGCGATG GGCTTTATATCATCTACCTGAAGGGCTCCTTTTTCCAGGAGGTCAAGATTGACCTTCATTTCCGGGAGGATCATAATCCCATCTCTATTCCAATGCTGAACGATGGTCGAAGGATTGTCTTCACTGTGGTGGCCTCTTTGGCTTTCAAA GATAAAGTTTACCTGACTGTAAATGCTCCTGATACTCTCTGCGAACACCTCCAGATAAATGATGGGGAGCTGATTGTTGTCCAGCTAACGCCTGGATACTGTGCTCCTGAAGGATCTTACCACAGCACTGTGAACCAAGTACCACTGTGA The mouse OX40L encoded by the DNA sequence shown in is capable of being expressed.

[0163] In another non-limiting example, the APC comprises SEQ ID NO:5: MEYASDASLDPEAPWPPAPRARACRVLPWALVAGLLLLLLLAAACAVFLACPWAVSGARASPGSAASPRLREGPELSPDDPAGLLDLRQGMFAQLVAQNVLLIDGPLSWYSDPGLAGVSLTGGLSYK EDTKELVVAKAGVYYVFFQLELRRVVAGEGSGSVSLALHLQPLRSAAGAAALALTVDLPPASSEARNSAFGFQGRLLHLSAGQRLGVHLHTEARARHAWQLTQGATVLGLFRVTPEIPAGLPSPRSE; or human 4-1BBL, as represented by SEQ ID NO: 6: ATGGAATACGCCTCTGACGCTTCACTGGACCCCGAAGCCCCGTGGCCTCCCGCGCCCCGCTCGCGCCTGCCGCGTACTGCCTTGGGCCCTGGTCGCGGGGCTGCTGCTGCTGCTGCTGCTCGCTGCCGCCTGCGCCGTCTTCCTCGCCTGCCCCTGGGCCGTGTCCGGGGCTCGCGCCTCGCCCGGCTC CGCGGCCAGCCCGAGACTCCGCGAGGGTCCCGAGCTTTCGCCCGACGATCCCGCCGGCCTCTTGGACCTGCGGCAGGGCATGTTTGCGCAGCTGGTGGCCCAAAATGTTCTGCTGATCGATGGGCCCCTGAGCTGGTACAGTGACCCAGGCCTGGCAGGCGTGTCCCTGACGGGGGGCCTGAGCTACAAAG AGGACACGAAGGAGCTGGTGGTGGCCAAGGCTGGAGTCTACTATGTCTTCTTTCAACTAGAGCTGCGGCGCGTGGTGGCCGGCGAGGGCTCAGGCTCCGTTTCACTTGCGCTGCACCTGCAGCCACTGCGCTCTGCTGCTGGGGCCGCCGCCCTGGCTTTGACCGTGGACCTGCCACCCGCCTCCTCCGAG GCTCGGAACTCGGCCTTCGGTTTCCAGGGCCGCTTGCTGCACCTGAGTGCCGGCCAGCGCCTGGGCGTCCATCTTCACACTGAGGCCAGGGCACGCCATGCCTGGCAGCTTACCCAGGGCCCACAGTCTTGGGACTCTTCCGGGTGACCCCCGAAATCCCAGCCGGACTCCCTTCACCGAGGTCGGAATAA The human 4-1BBL encoded by the DNA sequence shown in is capable of being expressed.

[0164] In another non-limiting example, the APC comprises SEQ ID NO:7: MDQHTLDVEDTADARHPAGTSCPSDAALLRDTGLLADAALLSDTVRPTNAALPTDAAYPAVNVRDREAAWPPALNFCSRHPKLYGLVALVLLLLIAACVPIFTRTEPRPALTITTSPNLGTRENNADQVTPVSHIGCPNTTQQGSPVFAKLLAKN QASLCNTTLNWHSQDGAGSSYLSQGLRYEEDKKELVVDSPGLYYVFLELKLSPTFTNTGHKVQGWVSLVLQAKPQVDDFDNLALTVELFPCSMENKLVDRSWSQLLLLKAGHRLSVGLRAYLHGAQDAYRDWELSYPNTTSFGLFLVKPDNPWE; or SEQ ID NO: 8: ATGGACCAGCACACACTTGATGTGGAGGATACCGCGGATGCCAGACATCCAGCAGGTACTTCGTGCCCCTCGGATGCGGCGCTCCTCAGAGATACCGGGCTCCTCGCGGACGCTGCGCTCCTCTCAGATACTGTGCGCCCCACAAATGCCGCGCTCCCCACGGATGCTGCCTACCCTGCGGTTAATGTTCGGGATCGCGAGGCCGCGTGGCCGCCTGCACTGAACTTCTGTTCCCGCCACCCAAAGCTCTATGGCCTAGTCGCTTTGGTTTTGCTGCTTCTGATCGCCGCCTGTGTTCCTATCTTCACCCGCACCGAGCCTCGGCCAGCGCTCACAATCACCACCTCGCCCAACCTGGGTACCCGAGAGAATAATGCAGACCAGGTCACCCCTGTTTCCCACATTGGCTGCCCCAACACTACACAACAGGGCTCTCCTGTGTTCGCCAAGCTACTGGCTAAAAACCAAGCATCGTTGTGCAATACAACTCTGAACTGGCACAGCCAAGATGGAGCTGGGAGCTCATACCTATCTCAAGGTCTGAGGTACGAAGAAGACAAAAAGGAGTTGGTGGTAGACAGTCCCGGGCTCTACTACGTATTTTTGGAACTGAAGCTCAGTCCAACATTCACAAACACAGGCCACAAGGTGCAGGGCTGGGTCTCTCTTGTTTTGCAAGCAAAGCCTCAGGTAGATGACTTTGACAACTTGGCCCTGACAGTGGAACTGTTCCCTTGCTCCATGGAGAACAAGTTAGTGGACCGTTCCTGGAGTCAACTGTTGCTCCTGAAGGCTGGCCACCGCCTCAGTGTGGGTCTGAGGGCTTATCTGCATGGAGCCCAGGATGCATACAGAGACTGGGAGCTGTCTTATCCCAACACCACCAGCTTTGGACTCTTTCTTGTGAAACCCGACAACCCATGGGAATGA It can express mouse 4-1BBL encoded by the DNA sequence shown in .

[0165] In another non-limiting example, the APC comprises SEQ ID NO:9: MEVPPPAPRSFLCRALCLFPRVFAAEAVTADSEVLEERQKRLPYVPEPYYPESGWDRLRELFGKDEQQRISKDLANICKTAATAGIIGWVYGGIPAFIHAKQQYIEQSQAEIYHNRFDAVQSAHRAATRGFIRYGWRWGWRTA VFVTIFNTVNTSLNVYRNKDALSHFVIAGAVTGSLFRINVGLRGLVAGGIIGALLGTPVGGLLMAFQKYSGETVQERKQKDRKALHELKLEEWKGRLQVTEHLPEKIESSLQEDEPENDAKKIEALLNLPRNPSVIDKQDKD; or SEQ ID NO: 10: ATGGAGGTGCCGCCACCGGCACCGCGGAGCTTTCTCTGTAGAGCATTGTGCCTATTTCCCCGAGTCTTTGCTGCCGAAGCTGTGACTGCCGATTCGGAAGTCCTTGAGGAGCGTCAGAAGCGGCTTCCCTACGTCCCAGAGCCCTATTACCCGGAATCTGGATGGGACCGCCTCCGGGAGCTGTTTGGCAAAGATGAACAGCAGAGAATTTCAA AGGACCTTGCTAATATCTGTAAGACGGCAGCTACAGCAGGCATCATTGGCTGGGTGTATGGGGGAATACCAGCTTTTATTCATGCTAAACAACAATACATTGAGCAGAGCCAGGCAGAAATTTATCATAACCGGTTTGATGCTGTGCAATCTGCACATCGTGCTGCCACACGAGGCTTCATTCGTTATGGCTGGCGCTGGGGTTGGAGAACTGCA GTGTTTGTGACTATATTCAACACAGTGAACACTAGTCTGAATGTATACCGAAATAAAGATGCCTTAAGCCATTTTGTAATTGCAGGAGCTGTCACGGGAAGTCTTTTTAGGATAAACGTAGGCCTGCGTGGCCTGGTGGCTGGTGGCATAATTGGAGCCTTGCTGGGCACTCCTGTAGGAGGCCTGCTGATGGCATTTCAGAAGTACTCTGGTG AGACTGTTCAGGAAAGAAAACAGAAGGATCGAAAGGCACTCCATGAGCTAAAACTGGAAGAGTGGAAAGGCAGACTACAAGTTACTGAGCACCTCCCTGAGAAAATTGAAAGTAGTTTACAGGAAGATGAACCTGAGAATGATGCTAAGAAAATTGAAGCACTGCTAAACCTTCCTAGAAACCCTTCAGTAATAGATAAACAAGACAAGGACTGA The present invention can express human CD80 encoded by the DNA sequence shown in

[0166] In another non-limiting example, the APC comprises SEQ ID NO:11: MACNCQLMQDTPLLKFPCPRLILLFVLLIRLSQVSSDVDEQLSKSVKDKVLLPCRYNSPHEDESEDRIYWQKHDKVVLSVIAGKLKVWPEYKNRTLYDNTTYSLIILGLVLSDRGTYSCVVQKKERGTYEVKHLALVKLSIKADFSTPNITES GNPSADTKRITCFASGGFPKPRFSWLENGRELPGINTTISQDPESELYTISSQLDFNTTRNHTIKCLIKYGDAHVSEDFTWEKPPEDPPDSKNTLVLFGAGFGAVITVVVIVVIIKCFCKHRSCFRRNEASRETNNSLTFGPEEALAEQTVFL; or SEQ ID NO: 12: ATGGCTTGCAATTGTCAGTTGATGCAGGATACACCACTCCTCAAGTTTCCATGTCCAAGGCTCATTCTTCTCTTTGTGCTGCTGATTCGTCTTTCACAAGTGTCTTCAGATGTTGATGAACAACTGTCCAAGTCAGTGAAAGATAAGGTATTGCTGCCTTGCCGTTACAACTCTCCTCATGAAGATGAGTCTGAAGACCGAATCTACTGGCAAAAACATGACAAAGTGGTGCTGTCTGTCATTGCTGGGAAACTAAAAGTGTGGCCCGAGTATAAGAACCGGACTTTATATGACAACACTACCTACTCTCTTATCATCCTGGGCCTGGTCCTTTCAGACCGGGGCACATACAGCTGTGTCGTTCAAAAGAAGGAAAGAGGAACGTATGAAGTTAAACACTTGGCTTTAGTAAAGTTGTCCATCAAAGCTGACTTCTCTACCCCCAACATAACTGAGTCTGGAAACCCATCTGCAGACACTAAAAGGATTACCTGCTTTGCTTCCGGGGGTTTCCCAAAGCCTCGCTTCTCTTGGTTGGAAAATGGAAGAGAATTACCTGGCATCAATACGACAATTTCCCAGGATCCTGAATCTGAATTGTACACCATTAGTAGCCAACTAGATTTCAATACGACTCGCAACCACACCATTAAGTGTCTCATTAAATATGGAGATGCTCACGTGTCAGAGGACTTCACCTGGGAAAAACCCCCAGAAGACCCTCCTGATAGCAAGAACACACTTGTGCTCTTTGGGGCAGGATTCGGCGCAGTAATAACAGTCGTCGTCATCGTTGTCATCATCAAATGCTTCTGTAAGCACAGAAGCTGTTTCAGAAGAAATGAGGCAAGCAGAGAAACAAACAACAGCCTTACCTTCGGGCCTGAAGAAGCATTAGCTGAACAGACCGTCTTCCTT It can express mouse CD80 encoded by the DNA sequence shown in

[0167] In another non-limiting example, the APC is SEQ ID NO: 13: MGRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVLANFSQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGVMQKSQDNVTELYDVSISLSVS FPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIPWITAVLPTVIICVMVFCLILWKWKKKKRPRNSYKCGTNTMEREESEQTKKREKIHIPERSDEAQRVFKSSKTSSCDKSDTCF; or SEQ ID NO: 14: ATGGGCCGCACAAGTTTTGATTCGGACAGTTGGACCCTGAGACTTCACAATCTTCAGATCAAGGACAAGGGCTTGTATCAATGTATCATCCATCACAAAAAGCCCACAGGAATGATTCGCATCCACCAGATGAATTCTGAACTGTCAGTGCTTGCTAACTTCAGTCAACCTGAAATAGTACCAATT TCTAATATAACAGAAAATGTGTACATAAATTTGACCTGCTCATCTATACACGGTTACCCAGAACCTAAGAAGATGAGTGTTTTGCTAAGAACCAAGAATTCAACTATCGAGTATGATGGTGTTATGCAGAAATCTCAAGATAATGTCACAGAACTGTACGACGTTTCCATCAGCTTGTCTGTTTCA TTCCCTGATGTTACGAGCAATATGACCATCTTCTGTATTCTGGAAACTGACAAGACGCGGCTTTTATCTTCACCTTTCTCTATAGAGCTTGAGGACCCTCAGCTCCCCCAGACCACATTCCTTGGATTACAGCTGTACTTCCAACAGTTATTATATGTGTGATGGTTTTCTGTCTAATTCTATGG AAATGGAAGAAGAAGAAGCGGCCTCGCAACTCTTATAAATGTGGAACCAACACAATGGAGAGGGAAGAGAGTGAACAGACCAAGAAAAGAGAAAAAATCCATATACCTGAAAGATCTGATGAAGCCCAGCGTGTTTTAAAGTTCGAAGACATCTTCATGCGACAAAAGTGATACATGTTTTTAA The human CD86 encoded by the DNA sequence shown in is capable of being expressed.

[0168] In another non-limiting example, the APC has the sequence of SEQ ID NO: 15: MDPRCTMGLAILIFVTVLLISDAVSVETQAYFNGTAYLPCPFTKAQNISLSELVVFWQDQQKLVLYEHYLGTEKLDSVNAKYLGRTSFDRNNWTLRLHNVQIKDMGSYDCFIQKKPPTGSIILQQTLTELSVIANFSEPEIKLAQNVTGNSGINL TCTSKQGHPKPKKMYFLITNSTNEYGDNMQISQDNVTELFSISNSLSLSFPDGVWHMTVVCVLETESMKISSKPLNFTQEFPSPQTYWKEITASVTVALLLVMLLIIVCHKKPNQPSRPSNTASKLERDSNADRETINLKELEPQIASAKPNAE; or SEQ ID NO: 16: ATGGACCCCAGATGCACCATGGGCTTGGCAATCCTTATCTTTGTGACAGTCTTGCTGATCTCAGATGCTGTTTCCGTGGAGACGCAAGCTTATTTCAATGGGACTGCATATCTGCCGTGCCCATTTACAAAGGCTCAAAACATAAGCCTGAGTGAGCTGGTAGTATTTTGGCAGGACCAGCAAAAGTTGGTTCTGTACGAGCACTATTTGGGCACAGAGAAACTTGATAGTGTGAATGCCAAGTACCTGGGCCGCACGAGCTTTGACAGGAACAACTGGACTCTACGACTTCACAATGTTCAGATCAAGGACATGGGCTCGTATGATTGTTTTATACAAAAAAAGCCACCCACAGGATCAATTATCCTCCAACAGACATTAACAGAACTGTCAGTGATCGCCAACTTCAGTGAACCTGAAATAAAACTGGCTCAGAATGTAACAGGAAATTCTGGCATAAATTTGACCTGCACGTCTAAGCAAGGTCACCCGAAACCTAAGAAGATGTATTTTCTGATAACTAATTCAACTAATGAGTATGGTGATAACATGCAGATATCACAAGATAATGTCACAGAACTGTTCAGTATCTCCAACAGCCTCTCTCTTTCATTCCCGGATGGTGTGTGGCATATGACCGTTGTGTGTGTTCTGGAAACGGAGTCAATGAAGATTTCCTCCAAACCTCTCAATTTCACTCAAGAGTTTCCATCTCCTCAAACGTATTGGAAGGAGATTACAGCTTCAGTTACTGTGGCCCTCCTCCTTGTGATGCTGCTCATCATTGTATGTCACAAGAAGCCGAATCAGCCTAGCAGGCCCAGCAACACAGCCTCTAAGTTAGAGCGGGATAGTAACGCTGACAGAGAGACTATCAACCTGAAGGAACTTGAACCCCAAATTGCTTCAGCAAAACCAAATGCAGAGTGA It can express mouse CD86 encoded by the DNA sequence shown in

[0169] In another non-limiting example, the APC is SEQ ID NO:17: METPQEDHLRGQHYHQKGQNGSFDAPNERPYSLKIRNTTSCNSGTYRCTLQDPDGQRNLSGKVILRVTGCPAQRKEETFKKYRAEIVLLLALVIFYLTLIIFTCKFARLQSIFPDFSKAGMERAFLPVTSPNKHLGLVTPHKTELV; or SEQ ID NO: 18: ATGGAGACCCCCAGGAAGACCACCTCAGGGACAGCACTATCATCAGAAGGGGCAAAATGGTTCTTTCGACGCCCCCAATGAAAGGCCCTATTCCCTGAAGATCCGAAACACTACCAGCTGCAACTCGGGGACATACAGGTGCACTCTGCAGGACCCGGATGGGCAGAGAAACCTAAGTGGCAAGGTGATCTTGAGAGTGACAGGATGCCCTGCACAGC GTAAAGAAGAGACTTTTAAGAAATACAGAGCGGAGATTGTCCTGCTGCTGGCTCTGGTTATTTTCTACTTAACACTCATCATTTTCACTTGTAAGTTTGCACGGCTACAGAGTATCTTCCCAGATTTTTCTAAAGCTGGCATGGAACGAGCTTTTCTCCCAGTTACCTCCCCAAATAAGCATTTAGGGCTAGTGACTCCTCACAAGACAGAACTGGTATGA The present invention can express human CD83 encoded by the DNA sequence shown in

[0170] In another non-limiting example, the APC has the sequence of SEQ ID NO:19: MSQGLQLLFLGCACSLAPAMAMREVTVACSETADLPCTAPWDPQLSYAVSWAKVSESGTESVELPESKQNSSFEAPRRRAYSLTIQNTTICSSGTYRCALQELGGQRNLSGTVVLKVTGCPKEATESTFRKYRAEAVLLFSLVVFYLTLIIFTCKFARLQSIFPDISKPGTEQAFLPVTSPSKHLGPVTLPKTETV; or SEQ ID NO: 20: ATGTCGCAAGGCCTCCAGCTCCTGTTTTCTAGGCTGCGCCTGCAGCCTGGCACCCGCGATGGCGATGCGGGAGGTGACGGTGGCTTGCTCCGAGACCGCCGACTTGCCTTGCACAGCGCCCTGGGACCCGCAGCTCTCCTATGCAGTG TCCTGGGCCAAGGTCTCCGAGAGTGGCACTGAGAGTGTGGAGCTCCCGGAGAGCAAGCAAAACAGCTCCTTCGAGGCCCCCAGGAGAAGGGCCTATTCCCTGACGATCCAAAACACTACCATCTGCAGCTCGGGCACCTACAGGTGTG CCCTGCAGGAGCTCGGAGGGCAGCGCAACTTGAGCGGCACCGTGGTTCTGAAGGTGACAGGATGCCCCAAGGAAGCTACAGAGTCAACTTTCAGGAAGTACAGGGCAGAAGCTGTGTTGCTCTTCTCTCTGGTTGTTTTCTACCTGAC ACTCATCATTTTCACCTGCAAATTTGCACGACTACAAAGCATTTTCCCAGATATTTCTAAACCTGGTACGGAACAAGCTTTTCTTCCAGTCACCTCCCCAAGCAAACATTTGGGGCCAGTGACCCTTCTAAGACAGAAACGGTATGA The mouse CD83 encoded by the DNA sequence shown in is capable of being expressed.

[0171] In another non-limiting example, the APC comprises SEQ ID NO:21: MPEEGSGCSVRRRPYGCVLRAALVPLVAGLVICLVVCIQRFAQAQQQLPLESLGWDVAELQLNHTGPQQDPRLYWQGGPALGRSFLHGPELDKGQLRIHRDGIY MVHIQVTLAICSSTTASRHHPTTLAVGICSPASRSISLLRLSFHQGLFGFWNWGLKVKCFLRHLIWTAHCFIPLTQLVFMQALQSWRNHHCSHFTDEENRGVNR; or SEQ ID NO: 22: ATGCCGGAGGAGGGTTCGGGCTGCTCGGTGCGGCGCAGGCCCTATGGGTGCGTCCTGCGGGCTGCTTTGGTCCCATTGGTCGCGGGCTTGGTGATCTGCCTCGTGGTGTGCATCCAGCGCTTCGCACAGGCTCAGCAGCAGCTGCCGCTCGAGTCA CTTGGGTGGGACGTAGCTGAGCTGCAGCTGAATCACACAGGACCTCAGCAGGACCCCAGGCTATACTGGCAGGGGGGCCCAGCACTGGGCCGCTCCTTCCTGCATGGACCAGAGCTGGACAAGGGGCAGCTACGTATCCATCGTGATGGCATCTACA TGGTACACATCCAGGTGACGCTGGCCATCTGCTCCTCCACGACGGCCTCCAGGCACCACCCCACCACCCTGGCCGTGGGAATCTGCTCTCCCGCCTCCCGTAGCATCAGCCTGCTGCGTCTCAGCTTCCACCAAGGGCTTTTTGGATTTTGGAACTG GGGACTCAAAGTCAAGTGCTTCTTACGGCATTTAATATGGACTGCACACTGTTTTATCCCATTAACTCAGCTCGTGTTCATGCAAGCCCTACAAAGCTGGAGGAATCATCATTGTTCCCATTTCACAGATGAGGAAAACAGAGGCGTAAACCGTTGA The human CD70 encoded by the DNA sequence shown in is capable of being expressed.

[0172] In another non-limiting example, the APC has the sequence of SEQ ID NO:23: MPEEGRPCPWVRWSGTAFQRQWPWLLLVVFITVFCCWFHCSGLLSKQQQRLLEHPEPHTAELQLNLTVPRKDPTLRWGAGPALGRSFTHGPELEEGHLRIHQDGLYRLHIQVTLANCSSPGSTLQHRATLAVGICSPAAHGISLLRGRFGQDCTVALQRLTYLVHGDVLCTNLTLPLLPSRNADETFFGVQWICP; or SEQ ID NO: 24: ATGCCGGAGGAAGGTCGCCCTTGCCCCTGGGTTCGCTGGAGCGGGACCGCGTTCCAGCGCCAATGGCCATGGCTGCTGCTGGTGGTGTTTATTACTGTGTTTTGCTGTTGGTTTCATTGTAGCGGACTACTCAGTAAGCAGCAACAG AGGCTGCTGGAGCACCCTGAGCCGCACACAGCTGAGTTACAGCTGAATCTCACAGTTCCTCGGAAGGACCCCACACTGCGCTGGGGAGCAGGCCCAGCCTTGGGAAGGTCCTTCACACACGGACCAGAGCTGGAGGAGGGCCATCTG CGTATCCATCAAGATGGCCTCTACAGGCTGCATATCCAGGTGACACTGGCCAACTGCTCTTCCCCAGGCAGCACCCTGCAGCACAGGGCCACCCTGGCTGTGGGCATCTGCTCCCCGCTGCGCACGGCATCAGCTTGCTGCGTGGG CGCTTTGGACAGGACTGTACAGTGGCATTACAGCGCCTGACATACCTGGTCCACGGAGATGTCCTCTGTACCAACCTCACCCTGCCTCTGCTGCCGTCCCGCAACGCTGATGAGACCTTCTTTGGAGTTCAGTGGATATGCCCTTGA The mouse CD70 encoded by the DNA sequence shown in is capable of being expressed.

[0173] In another non-limiting example, the APC comprises SEQ ID NO:25: MTILGTTFGMVFSLLQVVSGESGYAQNGDLEDAELDDYSFSCYSQLEVNGSQHSLTCAFEDPDVNITNLEFEICGALVEVKCLNFRKLQEIYFIETKKFLLIGKSNICVKVGEKSLTCKKIDLTTIVKPEA PFDLSVVYREGANDFVVTFNTSHLQKKYVKVLMHDVAYRQEKDENKWTHVNLSSTKLTLLQRKLQPAAMYEIKVRSIPDHYFKGFWSEWSPSYYFRTPEINNSSGLSLSYGPVSPIIRRLWNIFVRNQEK; or SEQ ID NO: 26: ATGACAATTCTAGGTACAACTTTTGGCATGGTTTTTTCTTTACTTCAAGTCGTTTCTGGAGAAAGTGGCTATGCTCAAAATGGAGACTTGGAAGATGCAGAACTGGATGACTACTCATTCTCATGCTATAGCCAGTTGGAAGTGAATGGATCGCAGCACTCACTGACCTGTGCTTTTGAGGACCCAGATGTCAACA TCACCAATCTGGAATTTGAAATATGTGGGGCCCTCGTGGAGGTAAAGTGCCTGAATTTCAGGAAACTACAAGAGATATATTTCATCGAGACAAAGAAATTCTTACTGATTGGAAAGAGCAATATATGTGTGAAGGTTGGAGAAAAGAGTCTAACCTGCAAAAAAATAGACCTAACCACTATAGTTAAACCTGAGGCT CCTTTTGACCTGAGTGTCGTCTATCGGGAAGGAGCCAATGACTTTGTGGTGACATTTAATACATCACACTTGCAAAAGAAGTATGTAAAAGTTTTAATGCACGATGTAGCTTACCGCCAGGAAAAGGATGAAAACAAATGGACGCATGTGAATTTATCCAGCACAAAGCTGACACTCCTGCAGAGAAAGCTCCAAC CGGCAGCAATGTATGAGATTAAAGTTCGATCCATCCCTGATCACTATTTTAAAGGCTTCTGGAGTGAATGGAGTCCAAGTTATTACTTCAGAACTCCAGAGATCAATAATAGCTCAGGATTAAGCCTATCGTATGGCCCAGTCTCCCCGATCATAAGAAGACTCTGGAACATCTTTGTAAGAAACCAAGAAAAGTGA The present invention can express human IL7 / CD127 encoded by the DNA sequence shown in

[0174] In another non-limiting example, the APC has the sequence of SEQ ID NO:27: MMALGRAFAIVFCLIQAVSGESGNAQDGDLEDADADDHSFWCHSQLEVDGSQHLLTCAFNDSDINTANLEFQICGALLRVKCLTLNKLQDIYFIKTSEFLLIGSSNICVKLGQKNLTCKNMAINTIVKAEAPSDLKVVYRKEA NDFLVTFNAPHLKKYLKKVKHDVAYRPARGESNWTHVSLFHTRTTIPQRKLRPKAMYEIKVRSIPHNDYFKGFWSEWSPSSTFETPEPKNQGGWDPVLPSVTILSLFSVFLLVILAHVLWKKRIKPVVWPSLPDHKKTLEQL; or SEQ ID NO: 28: ATGATGGCTCTGGGTAGAGCTTTCGCTATAGTTTTCTGCTTAATTCAAGCTGTTTCTGGAGAAAGTGGAAATGCCCAGGATGGAGACCTAGAAGATGCAGACGCGGACGATCACTCCTTCTGGTGCCACAGCCAGTTGGAAGTGGATGGAAGTCAACATTTATTGACTTGTGCTTTTAATGACTCAGACATCAACACAGCTAATCTGGAATTTCA AATATGTGGGGCTCTTTTACGAGTGAAATGCCTAACTCTTAACAAGCTGCAAGATATATATTTTATAAAGACATCAGAATTCTTACTGATTGGTAGCAGCAATATATGTGTGAAGCTTGGACAAAAGAATTTAACTTGCAAAAATATGGCTATAAACACAATAGTTAAAGCCGAGGCTCCCTCTGACCTGAAAGTCGTTTATCGCAAAGAAGCAA ATGATTTTTTGGTGACATTTAATGCACCTCACTTGAAAAAGAAATATTTAAAAAAAGTAAAGCATGATGTGGCCTACCGCCCAGCAAGGGGTGAAAGCAACTGGACGCATGTATCTTTATTCCACACAAGAACAACAATCCCACAGAGAAAACTACGACCAAAAGCAATGTATGAAATCAAAGTCCGATCCATTCCCCATAACGATTACTTCAAA GGCTTCTGGAGCGAGTGGAGTCCAAGTTCTACCTTCGAAACTCCAGAACCCAAGAATCAAGGAGGATGGGATCCTGTCTTGCCAAGTGTCACCATTCTGAGTTTGTTCTCTGTGTTTTTGTTGGTCATCTTAGCCCATGTGCTATGGAAAAAAAGGATTAAACCTGTCGTATGGCCTAGTCTCCCCGATCATAAGAAAACTCTGGAACAACTATAG The mouse IL7 / CD127 encoded by the DNA sequence shown in is capable of expressing.

[0175] In another non-limiting example, the APC has the sequence of SEQ ID NO:29: MDPGLQQALNGMAPPGDTAMHVPAGSVASHLGTTSRSYFYLTTATLALCLVFTVATIMVLVVQRTDSIPNSPDNVPLKGGNCSEDLLCILKRAPFKKSWAYLQVAKHLNKTKLSWNKDGILHGVRYQDGNLVIQFPDYCGMILHHSHSTLDSGKGHCCLETLQP; or SEQ ID NO: 30: ATGGACCCAGGGCTGCAGCAAGCACTCAACGGAATGGCCCCTCCTGGAGACACAGCCATGCATGTGCCGGCGGGCTCCGTGGCCAGCCACCTGGGGACCACGAGCCGCAGCTATTTCTATTTG ACCACAGCCACTCTGGCTCTGTGCCTTGTCTTCACGGTGGCCACTATTATGGTGTTGGTCGTTCAGAGGACGGACTCCATTCCCAACTCACCTGACAACGTCCCCCTCAAAGGAGGAAATTGCT CAGAAGACCTCTTATGTATCCTGAAAAGGGCTCCATTCAAGAAGTCATGGGCCTACCTCCAAGTGGCAAAGCATCTAAACAAAACCAAGTTGTCTTGGAACAAAGATGGCATTCTCCATGGAGTCAGATATCAGGATGGGAATCTGGTGATCCAATTCCCTGATTACTGTGGCATGATCCTCCACCATTCACACTCTACCCTGGACTCTGGGAAGGGACACTGCTGCCTTGAAACTCTACAACCCTGA The present invention can express human CD30L encoded by the DNA sequence shown below.

[0176] In another non-limiting example, the APC has the sequence of SEQ ID NO:31: MEPGLQQAGSCGAPSDPAMQVQPGSVASPWRSTRPWRSTSRSYFYLSTTALVCLVVAVAIILVLVVQKKDSTPNTTEKAPLKGGNCSEDLFCTLKSTPSKKSWAYLQVSKHLNNTKLSWNEDGTIHGLIYQDGNLIVQFPGLYFIVCQLQFLVQCSNHSVDLTLQLLINSKIKKQTLVTVCESGVQSKNIYQNLSQFLLHYLQVNSTISVRVDNFQYVD; でしまするなゃるCD30L、さんなれますまする32: ATGGAGCCAGGGCTGCAACAAGCAGGCAGCTGTGGGGCTCCTTCCCCTGACCCAGCCATGCAGGTGCAGCCCGGCTCGGTAGCCAGCCCCTGGAGAAGCACGAGGCCCTGGAGAAGCACAAGTCGCAGCTACTTCTACCTCAGCACCACCGACTGGTGTCCTTGTTGTGGCAGTGGCG ATCATTCTGGTACTGGTAGTCCAGAAAAAGGACTCCACTCCAAATACAACTGAGAAGGCCCCCCTTAAAGGAGGAAATTGCTCAGAGGATCTCTTCTGTACCCTGAAAGTACTCCATCCAAGAAGTCATGGGCCATCTCCAAGTGTCAAAGCATCTCAAACAATACCAAACTGTCATGG AACGAAGATGGCACCATCCACGGACTCATATACCAGGACGGGAACCTGATAGTCCAATTCCCTGGCTTGTACTTCATCGTTTGCCAACTGCAGTTCCTCGTGCAGTGCTCAAATCATTCTGTGGACCTGACATTGCAGCTCCTCATCAATTCCAAGATCAAAAAGCAGACGTTGGTAACAA GTGTGTGAGTCTGGAGTTCAGAGTAAGAACATCTACCAGAATCTCTTCACGTTTTGCTGCATTACTTACAGGTCAACTCTACCATATCAGTCAGGGTGGATAATTTCCAGTATGTGGATACAAACACTTTCCCTCTTGATAATGTGCTATCCGTCTTCTTTATATAGTAGCTCAGACTGA The mouse CD30L encoded by the DNA sequence shown in is capable of being expressed.

[0177] In another non-limiting example, the APC has the sequence of SEQ ID NO: 33: MEPPGDWGPPPWRSTPKTDVLRLVLYLTFLGAPCYAPALPSCKEDEYPVGSECCPKCSPGYRVKEACGELTGTVCEPCPPGTYIAHLNGLSKCLQCQMCDPAMGLRASRNCSRTEN AVCGCSPGHFCIVQDGDHCAACRAYATSSPGQRVQKGGTESQDTLCQNCPPGTFSPNGTLEECQHQTKCSWLVTKAGAGTSSSHWVWWFLSGSLVIVIVCSTVGLIICVKRRKPR; or SEQ ID NO: 34: ATGGAGCCTCCTGGAGACTGGGGGCCTCCTCCCTGGAGATCCACCCCCAAAACCGACGTCTTGAGGCTGGTGCTGTATCTCACCTTCCTGGGAGCCCCCTGCTACGCCCCAGCTCTGCCGTCCTGCAAGGAGGACGAGTACCCAGTGGGCTCCGAGTGCTGCCCCAAGTGCAG TCCAGGTTATCGTGTGAAGGAGGCCTGCGGGGAGCTGACGGGCACAGTGTGTGAACCCTGCCCTCCAGGCACCTACATTGCCCACCTCAATGGCCTAAGCAAGTGTCTGCAGTGCCAAATGTGTGACCCAGCCATGGGCCTGCGCGCGAGCCGGAACTGCTCCAGGACAGAGAA CGCCGTGTGGCTGCAGCCCAGGCCACTTCTGCATCGTCCAGGACGGGGACCACTGCGCCGCGTGCCGCGCTTACGCCACCTCCAGCCCGGGCCAGAGGGTGCAGAAGGGAGGCACCGAGAGTCAGGACACCCTGTGTCAGAACTGCCCCCCGGGGACCTTCTCTCCCAATG GGACCCTGGAGGAATGTCAGCACCAGACCAAGTGCAGCTGGCTGGTGACGAAGGCCGGAGCTGGGACCAGCAGCTCCCACTGGGTATGGTGGTTTCTCTCAGGGAGCCTCGTCATCGTCATTGTTTGCTCCACAGTTGGCCTAATCATATGTGTGAAAAGAAGAAAGCCAAGGG The human LIGHT encoded by the DNA sequence shown in is capable of being expressed.

[0178] In another non-limiting example, the APC has the sequence of SEQ ID NO:35: MESVVQPSVFVVDGQTDIPFRRLEQNHRRRRCGTVQVSLALVLLLGAGLATQGWFLLRLHQRLGDIVAHLPDGGKGSWEKLIQDQRSHQANPAAHLTGANASLIGGPLLWETRLGLAF LRGLTYHDGALVTMEPGYYYVYSKVQLSGVGCPQGLANGLPITHGLYKRTSRYPKELELLVSRRSPCGRANSSRVWWDSSFLGGVVHLEAGEEVVVRVPGNRLVRPRDGTRSYFGAFMV; or mouse LIGHT, as represented by SEQ ID NO: 36: ATGGAGAGTGTGGTACAGCCTTCAGTGTTTGTGGTGGATGGACAGACGGACATCCCATTCAGGCGGCTGGAACAGAACCACCGGAGACGGCGCTGTGGCACTGTCCAGGTCAGCCTGGCCCTGGTGCTGCTGCTAGGTGCTGGGCTGGCCACTCAGGGCTGGTTTCTCCTGAGACTGCAT CAACGTCTTGGAGACATAGTAGCTCATCTGCCAGATGGAGGCAAAGGCTCCTGGGAGAAGCTGATACAAGATCAACGATCTCACCAGGCCAACCCAGCAGCACATCTTACAGGAGCCAACGCCAGCTTGATAGGTATTGGTGGACCTCTGTTATGGGAGACACGACTTGGCCTGGCCTTC TTGAGGGGCTTGACGTATCATGATGGGCCCTGGTGACCATGGAGCCCGGTTACTACTATGTGTACTCCAAAGTGCAGCTGAGCGGCGTGGGCTGCCCCCAGGGGCTGGCCAATGCCTCCCCATCACCCATGGACTATACAAGCGCACATCCCGCTACCCGAAGGAGTTAGAACTGCTG GTCAGTCGGCGGTCACCCTGTGGCCGGGCCAACAGCTCCCGAGTCTGGTGGGACAGCAGCTTCCTGGGCGGCGTGGTACATCTGGAGGCTGGGGAAGAGGTGGTGGTCCGCGTGCCTGGAAACCGCCTGGTCAGACCACGTGACGGCACCAGGTCCTATTTCGGAGCTTTCATGGTCTGA The mouse LIGHT encoded by the DNA sequence shown in is capable of being expressed.

[0179] In another non-limiting example, the APC has the sequence of SEQ ID NO:37: MKTLPAMLGTGKLFWVFFLIPYLDIWNIHGKESCDVQLYIKRQSEHSILAGDPFELECPVKYCANRPHVTWCKLNGTTCVKLEDRQTSWKEEKNISFFILHFEPVLPNDNGSYRCSANFQSNLIESHSTTLYVTDVKSASERPSK DEMASRPWLLYRLLPLGGLPLLITTCFCLFCCLRRHQGKQNELSDTAGREINLVDAHLKSEQTEASTRQNSQVLLSETGIYDNDPDLCFRMQEGSEVYSNPCLEENKPGIVYASLNHSVIGPNSRLARNVKEAPTEYASICVRS; or SEQ ID NO: 38: ATGAAGACATTGCCTGCCATGCTTGGAACTGGGAAATTTTGGGTCTTCTTCTTAATCCCATATCTGGACATCTGGAACATCCATGGGAAAGAATCATGTGATGTACAGCTTTATATAAAGAGACAATCTGAACACTCCATCTTAGCAGGAGATCCCTTTGAACTAGAATGCCCTGTGAAATACTGTGCTAACAGGCCTCATGTGACTTGGTGCA AGCTCAATGGAACAACATGTGTAAAACTTGAAGATAGACAAACAAGTTGGAAGGAAGAGAAGAACATTTCATTTTTCATTCTACATTTTGAACCAGTGCTTCCTAATGACAATGGGTCATACCGCTGTTCTGCAAATTTTCAGTCTAATCTCATTGAAAGCCACTCAACAACTCTTTATGTGACAGATGTAAAAAGTGCCTCAGAACGACCCTCCAAG GACGAAATGGCAAGCAGACCCTGGCTCCTGTATCGTTTACTTCCTTTGGGGGGATTGCCTCTACTCATCACTACCTGTTTCTGCCTGTTCTGCTGCCTGAGAAGGCACCAAGGAAAGCAAAATGAACTCTGACACAGCAGGAAGGGAAATTAACCTGGTTGATGCTCACCTTAAGAGTGAGCAAACAGAAGCAAGCACCAGGCAAAATTCCCAAG TACTGCTATCAGAAACTGGAATTTATGATAATGACCCTGACCTTTGTTTCAGGATGCAGGAAGGGTCTGAAGTTTATTCTAATCCATGCCTGGAAGAAAACAAACCAGGCATTGTTTATGCTTCCCTGAACCATTCTGTCATTGGACCGAACTCAAGACTGGCAAGAAATGTAAAAGAAGCACCAACAGAATATGCATCCATATGTGTGAGGAGTTAA The present invention can express human BTLA encoded by the DNA sequence shown in

[0180] In another non-limiting example, the APC has the sequence of SEQ ID NO:39: MKTVPAMLGTPRLFREFFILHLGLWSILCEKATKRNDEECPVQLTITRNSKQSARTGELFKIQCPVKYCVHRPNVTWCKHNGTICVPLEVSPQLYTSWEENQSVPVFVLHFKPIHLSDNGSYSCSTNFNSQVINSHSVTIHVTERTQNSSEHP LIISDIPDATNASGPSTMEERPGRTWLLYTLLPLGALLLLLACVCLLCFLKRIQGKEKKPSDLAGRDTNLVDIPASSRTNHQALPSGTGIYDNDPWSSMQDESELTISLQSERNNQGIVYASLNHCVIGRNPRQENNMQEAPTEYASICVRS; or SEQ ID NO: 40: ATGAAGACAGTGCCTGCCATGCTTGGGACTCCTCGGTTATTTAGGGAATTCTTCATCCTCCATCTGGGCCTCTGGAGCATCCTTTGTGAGAAAGCTACTAAGAGGAATGATGAAGAGTGTCCAGTGCAACTTACTATTACGAGGAATTCCAAACAGTCTGCCAGGACAGGAGAGTTATTTAAAATTCAATGTCCTGTGAAATACTGTGTTCATAGACCTAATGTGACTTGGTGTAAGCACAATGGAACAATCTGTGTACCCCTTGAGGTTAGCCCTCAGCTATACACTAGTTGGGAAGAAAATCAATCAGTTCCGGTTTTTGTTCTCCACTTTAAACCAATACATCTCAGTGATAATGGGTCGTATAGCTGTTCTACAAACTTCAATTCTCAAGTTATTAATAGCCATTCAGTAACCATCCATGTGACAGAAAGGACTCAAAACTCTTCAGAACACCCACTAATAATATCTGACATCCCAGATGCCACCAATGCCTCAGGACCATCCACCATGGAAGAGAGGCCAGGCAGGACTTGGCTGCTTTACACCTTGCTTCCTTTGGGGGCATTGCTTCTGCTCCTTGCCTGTGTCTGCCTGCTCTGCTTTCTGAAAAGGATCCAAGGGAAAGAAAAGAAGCCTTCTGACTTGGCAGGAAGGGACACTAACCTGGTTGATATTCCAGCCAGTTCCAGGACAAATCACCAAGCACTGCCATCAGGAACTGGAATTTATGATAATGATCCCTGGTCTAGCATGCAGGATGAATCTGAATTGACAATTAGCTTGCAATCAGAGAGAAACAACCAGGGCATTGTTTATGCTTCTTTGAACCATTGTGTTATTGGAAGGAATCCAAGACAGGAAAACAACATGCAGGAGGCACCCACAGAATATGCATCCATTTGTGTGAGAAGTTAA It can express mouse BTLA encoded by the DNA sequence shown in .

[0181] In another non-limiting example, the APC has the sequence of SEQ ID NO:41: MRLGSPGLLFLLFSSLRADTQEKEVRAMVGSDVELSCACPEGSRFDLNDVYVYWQTSESKTVVTYHIPQNSSLENVDSRYRNRALMSPAGMLRGDFSLRLFNVTPQDEQKFHCLVLSQSLGFQEVLSVEVTLHVAANFSVPVVSAPHSPSQDELT FTCTSINGYPRPNVYWINKTDNSLLDQALQNDTVFLNMRGLYDVVSVLRIARTPSVNIGCCIENVLLQQNLTVGSQTGNDIGERDKITENPVSTGEKNAATWSILAVLCLLVVVAVAIGWVCRDRCLQHSYAGAWAVSPETELTESWNLLLLLS; or human ICOS-L as shown in SEQ ID NO: 42: ATGCGGCTGGGCAGTCCTGGACTGCTCTTCCTGCTCTTCAGCAGCCTTCGAGCTGATACTCAGGAGAAGGAAGTCAGAGCGATGGTAGGCAGCGACGTGGAGCTCAGCTGCGCTTGCCCTGAAGGAAGCCGTTTTGATTTAAATGATGTTTACGTATATTGGCAAACCAGTGAGTCGAAAACCGTGGTGACCTACCACATCCCACAGAACAGCTCCTTGGAAAACGTGGACAGCCGCTACCGGAACCGAGCCCTGATGTCACCGGCCGGCATGCTGCGGGGCGACTTCTCCCTGCGCTTGTTCAACGTCACCCCCCAGGACGAGCAGAAGTTTCACTGCCTGGTGTTGAGCCAATCCCTGGGATTCCAGGAGGTTTTGAGCGTTGAGGTTACACTGCATGTGGCAGCAAACTTCAGCGTGCCCGTCGTCAGCGCCCCCCACAGCCCCTCCCAGGATGAGCTCACCTTCACGTGTACATCCATAAACGGCTACCCCAGGCCCAACGTGTACTGGATCAATAAGACGGACAACAGCCTGCTGGACCAGGCTCTGCAGAATGACACCGTCTTCTTGAACATGCGGGGCTTGTATGACGTGGTCAGCGTGCTGAGGATCGCACGGACCCCCAGCGTGAACATTGGCTGCTGCATAGAGAACGTGCTTCTGCAGCAGAACCTGACTGTCGGCAGCCAGACAGGAAATGACATCGGAGAGAGAGACAAGATCACAGAGAATCCAGTCAGTACCGGCGAGAAAAACGCGGCCACGTGGAGCATCCTGGCTGTCCTGTGCCTGCTTGTGGTCGTGGCGGTGGCCATAGGCTGGGTGTGCAGGGACCGATGCCTCCAACACAGCTATGCAGGTGCCTGGGCTGTGAGTCCGGAGACAGAGCTCACTGAATCCTGGAACCTGCTCCTTCTGCTCTCGTGA It can express human ICOS-L that can be encoded by the DNA sequence shown in

[0182] In another non-limiting example, the APC has the sequence of SEQ ID NO: 43: CPCFVSLGTRQPVWKKLHVSSGFFSGLGLFLLLLSSLCAASAETEVGAMVGSNVVLSCIDPHRRHFNLSGLYVYWQIENPEVSVTYYLPYKSPGINVDSSYKNRGHLSLDSMKQGNFSLYLKNVTPQDTQEFTCRVFMNTATELVKILEEVVRLRVAANFSTPVISTSDSSN PGQERTYTCMSKNGYPEPNLYWINTTDNSLIDTALQNNTVYLNKLGLYDVISTLRLPWTSRGDVLCCVENVALHQNITSISQAESFTGNNTKNPQETHNNELKVLVPVLAVLAAAAFVSFIIYRRTRPHRSYTGPKTVQLELTDTWAPVPYQDYLIPRYLMSPCLKTRGLP; or SEQ ID NO: 44: The mouse ICOS-L encoded by the DNA sequence shown in is capable of being expressed.

[0183] In another non-limiting example, the APC has the sequence of SEQ ID NO: 45: MDPKGLLSLTFVLFLSLAFGASYGTGGRMMNCPKILRQLGSKVLLPLTYERINKSMNKSIHIVVTMAKSLENSVENKIVSLDPSEAGPPRYLGDRYKFYLENLTLGIRESRKEDEGWYLMTLEKNVSVQRFCLQLRLYEQVSTPEIKVLNKTQENGTCTLILGCTVEK GDHVAYSWSEKAGTHPLNPANSSHLLSLTLGPQHADNIYICTVSNPISNNSQTFSPWPGCRTDPSETKPWAVYAGLLGGVIMILIMVVILQLRRRGKTNHYQTTVEKKSLTIYAQVQKPGPLQKKLDSFPAQDPCTTIYVAATEPVPESVQETNSITVYASVTLPES; or SEQ ID NO: 46: The present invention can express human CD150 encoded by the DNA sequence shown in

[0184] In another non-limiting example, the APC has the sequence of SEQ ID NO:47: MDPKGSLSWRILLFLSLAFELSYGTGGGVMDCPVILQKLGQDTWLPLTNEHQINKSVNKSVRILVTMATSPGSKSNKKIVSFDLSKGSYPDHLEDGYHFQSKNLSLKILGNRRESEGWYLVSVEENVSVQQFCKQLKLYEQVSPPEIKVLNKTQENENGTCSLLLACTVKKG DHVTYSWSDEAGTHLLSRANRSHLLHITLSNQHQDSIYNCTASNPVSSISRTFNLSSQACKQESSSESSPWMQYTLVPLGVVIIFILVFTAIIMMKRQGKSNHCQPPVEEKSLTIYAQVQKSGPQEKKLHDALTDQDPCTTIYVAATEPAPESVQEPNPTTVYASVTLPES; or SEQ ID NO: 48: The mouse CD150 encoded by the DNA sequence shown in is capable of being expressed.

[0185] In another non-limiting example, the APC has the sequence of SEQ ID NO:49: MWPPGSASQPPPSPAAATGLHPAARPVSLQCRLSMCPARSLLLVATLVLLDHLSLARNLPVATPDPGMFPCLHHSQNLLRAVSNMLQKNESCLNSRETSFITNGSCLASRKTSFMMALCLSSIYEDLKMYQVEFKTMNAKLLMDPKRQIFLDQNMLAVIDELMQALNFNSETVPQKSSLEEPDFYKTKIKLCILLHAFRIRAVTIDRVMSYLNAS; or SEQ ID NO: 50: ATGTGGCCCCCTGGGTCAGCCTCCCAGCCACCGCCCTCACCTGCCGCGGCCACAGGTCTGCATCCAGCGGCTCGCCCTGTGTCCCTGCAGTGCCGGCTCAGCATGTGTCCAGCGCAGCCTCCTCCTTGTGGCTACCCTGGTCCTCCTGGACCACCTCAGT TTGGCCAGAAACCTCCCCGTGGCCACTCCAGACCCAGGAATGTTCCCATGCCTTCACCACTCCCAAAACCTGCTGAGGGCCGTCAGCAACATGCTCCAGAAGAATGAGAGTTGCCTAAATTCCAGAGAGACCTCTTTCATAACTAATGGGAGTTGCCTGGCC TCCAGAAAGACCTCTTTTATGATGGCCCTGTGCCTTAGTAGTATTTATGAAGACTTGAAGATGTACCAGGTGGAGTTCAAGACCATGAATGCAAAGCTTCTGATGGATCCTAAGAGGCAGATCTTTCTAGATCAAAACATGCTGGCAGTTATTGATGAGCTG ATGCAGGCCCTGAATTTCAACAGTGAGACTGTGCCACAAAAATCCTCCCTTGAAGAACCGGATTTTTATAAAAACTAAAATCAAGCTCTGCATACTTCTTCATGCTTTCAGAATTCGGGCAGTGACTATTGATAGAGTGATGAGCTATCTGAATGCTTCCTAA The human IL-12 encoded by the DNA sequence shown in is capable of being expressed.

[0186] In another non-limiting example, the APC has the sequence of SEQ ID NO:51: MCPQKLTISWFAIVLLVSPLMAMWELEKDVYVVEVDWTPDAPGETVNLTCDTPEEDDITWTSDQRHGVIGSGKTLTITVKEFLDAGQYTCHKGGETLSHSHLLLHKKENGIWSTEILKNFKNKTFLKCEAPNYSGRFTCSWLVQRNMDLKFNIKSSSSSPDSRAVTCG MASLSAEKVTLDQRDYEKYSVSCQEDVTCPTAEETLPIELALEARQQNKYENYSTSFFIRDIIKPDPPKNLQMKPLKNSQVEVSWEYPDSWSTPHSYFSLKFFVRIQRKKEKMKETEEGCNQKGAFLVEKTSTEVQCKGGNVCVQAQDRYYNSSCSKWACVPCRVRS; or SEQ ID NO: 52: The mouse IL-12 encoded by the DNA sequence shown in is capable of expressing mouse IL-12.

[0187] In another non-limiting example, the APC has the sequence of SEQ ID NO:53: MFHVSFRYIFGLPPLILVLLPVASSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH; or SEQ ID NO: 54: ATGTTCCATGTTTCTTTTAGGTATATCTTTGGACTTCCTCCCCTGATCCTTGTTCTGTTGCCAGTAGCATCATCTGATTGTGATATTGAAGGTAAAGATGGCAAACAATATGAGAGTGTTCTAATGGTCAGCATCGATCAATTATTGGACAGCATGAAAGAAATTGGTAGCAATTGCCTGAATAATGAATTTAACTTTTT TAAAAGACATATCTGTGATGCTAATAAGGTTAAAGGAAGAAAACCAGCTGCCCTGGGTGAAGCCCAACCAACAAAGAGTTTGGAAGAAAATAAATCTTTAAAGGAACAGAAAAAACTGAATGACTTGTGTTTCCTAAAGAGACTATTACAAGAGATAAAAACTTGTTGGAATAAAATTTTGATGGGCACTAAAGAACACTG The human IL-7 encoded by the DNA sequence shown in is capable of being expressed.

[0188] In another non-limiting example, the APC has the sequence of SEQ ID NO:55: MFHVSFRYIFGIPPLILVLLPVTSSECHIKDKEGKAYESVLMISIDELDKMTGTDSNCPNNEPNFFRKHVCDDTKEAAFLNRAARKLKQFLKMNISEEFNVHLLTVSQGTQTLVNCTSKEEKNVKEQKKNDACFLKRLLREIKTCWNKILKGSI; or SEQ ID NO: 56: ATGTTCCATGTTTCTTTTAGATATATCTTTGGAATTCCTCCACTGATCCTTGTTCTGCTGCCTGTCACATCATCTGAGTGCCACATTAAAGACAAAGAAGGTAAAGCATATGAGAGTGTACTGATGATCAGCATCGATGAATTGGACAAAATGACAGGAACTGATAGTAATTGCCCGAATAATGAACCAAACTTTTTTAGAAAACATGTATGTGATGATACAAAGGAAGCTG CTTTTCTAAATCGTGCTGCTCGCAAGTTGAAGCAATTTCTTAAAATGAATATCAGTGAAGAATTCAATGTCCACTTACTAACAGTATCACAAGGCACACAAACACTGGTGAACTGCACAAGTAAGGAAGAAAAAAACGTAAAGGAACAGAAAAAGAATGATGCATGTTTCCTAAAGAGACTACTGAGAGAAATAAAAACTTGTTGGAATAAAATTTTGAAGGGCAGTATATAA The mouse IL-7 encoded by the DNA sequence shown in is capable of expressing mouse IL-7.

[0189] In another non-limiting example, the APC has the sequence of SEQ ID NO:57: MRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS; or SEQ ID NO: 58: ATGAGAATTTCGAAACCACATTTGAGAAGTATTTCCATCCAGTGCTACTTGGTTTACTTCTAAACAGTCATTTTCTAACTGAAGCTGGCATTCATGTCTTCATTTTGGGCTGTTTCAGTGCAGGGCTTCCTAAAACAGAAGCCAACTGGGTGAATGTAATAAGTGATTTGAAAAAAATTGAAGATCTTATTCAATCTATGCATATTGATGCTACTTTATATACGGAAAGTGATGTTCACCCCA GTTGCAAAGTAACAGCAATGAAGTGCTTTCTTGGAGTTACAAGTTATTTCACTTGAGTCCGGAGATGCAAGTATTCATGATACAGTAGAAAATCTGATCATCCTAGCAAACAGTTTGTCTTCTAATGGGAATGTAACAGAATCTGGATGCAAAGAATGTGAGGAACTGGAGGAAAAAAATATTAAAGAATTTTTGCAGAGTTTTGTACATATTGTCCAAATGTTCATCAACACTTCTTGA The human IL-15 encoded by the DNA sequence shown in is capable of being expressed.

[0190] In another non-limiting example, the APC has the sequence of SEQ ID NO:59: MDWPHNLLFLLTISIFLGLGQPRSPKSKRKGQGRPGPLAPGPHQVPLDLVSRMKPYARMEEYERNIEEMVAQLRNSSELAQRKCEVNLQLWMSNKRSLSSPWGYSINHDPSRIPVDLPEARCLCLGCVNPFTMQEDRSMVSVPVFSQVPVRRRLCPPPPRTGPCRQRAVMETIAVGCTCIF; or SEQ ID NO: 60: ATGGACTGGCCTCACAACCTGCTGTTTCTTCTTACCATTTCCATCTTCCTGGGGCTGGGCCAGCCCAGGAGCCCAAAAGCAAGAGGAAGGGGCAAGGGCGGCCTGGGCCCCTGGCCCCTGGCCCTCACCAGGTG CCACTGGACCTGGTGTCACGGATGAAACCGTATGCCCGCATGGAGGAGTATGAGAGGAACATCGAGGAGATGGTGGCCCAGCTGAGGAACAGCTCAGAGCTGGCCCAGAGAAAGTGTGAGGTCAACTTGCAGCTGT GGATGTCCAACAAGAGGAGCCTGTCTCCCTGGGGCTACAGCATCAACCACGACCCCAGCCGTATCCCCGTGGACCTGCCGGAGGCACGGTGCCTGTGTCTGGGCTGTGTGAACCCCTTCACCATGCAGGAGGACCG CAGCATGGTGAGCGTGCCGGTGTTCAGCCAGGTTCCTGTGCGCCGCCGCCTCTGCCCGCCACCGCCCCGCACAGGGCCTTGCCGCCAGCGCGTCATGGAGACCATCGCTGTGGGCTGCACCTGCATCTTCTGA The human IL-17 encoded by the DNA sequence shown in is capable of being expressed.

[0191] In another non-limiting example, the APC has the sequence of SEQ ID NO:61: MSPGRASSVSLMLLLLLSLAATVKAAAIIPQSSACPNTEAKDFLQNVKVNLKVFNSLGAKVSSRRPSDYLNRSTSPWTLHRNEDPDRYPSVIWEAQCRHQRCVNAEGKLDHHMNSVLIQQEILVLKREPESCPFTFRVEKMLVGVGCTCVASIVRQAA; or SEQ ID NO: 62: ATGAGTCCAGGGAGAGCTTCATCTGTGTCTCTGATGCTGTTGCTGCTGCTGAGCCTGGCGGCTACAGTGAAGGCAGCAGCGATCATCCCTCAAAGCTCAGCGTGTCCAAACACTGAGGCCAAGGACTTCCTCCAGAATGTGAAGGTCAACCTCAAAGTCTTTAACTCCCTTGGCGCAAGTGAGCTCCAGAAGGCCCTCAGACTACCTCAACCGTTCCACGTCACCCTGGACTCTCC ACCGCAATGAAGACCCTGATAGATATCCCTCTGTGATCTGGGAAGCTCAGTGCCGCCACCAGCGCTGTGTCAATGCGGAGGGAAAGCTGGACCACCACATGAATTCTGTTCTCATCCAGCAAGAGATCCTGGTCCTGAAGAGGGAGCCTGAGAGCTGCCCCTTCACTTTCAGGGTCGAGAAGATGCTGGTGGGTGTGGGCTGCACCTGCGTGGCCTCGATTGTCCGCCAGGCAGCCTAA The mouse IL-17 encoded by the DNA sequence shown in is capable of expressing mouse IL-17.

[0192] In another non-limiting example, the APC has the sequence of SEQ ID NO: 63: MRSSPGNMERIVICLMVIFLGTLVHKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS; or SEQ ID NO: 64: ATGAGATCCAGTCCTGGCAACATGGAGAGGATTGTCATCTGTCTGATGGTCATCTTCTTGGGGACACTGGTCCACAAATCAAGCTCCCAAGGTCAAGATCGCCACATGATTAGAATGCGTCA ACTTATAGATATTGTTGATCAGCTGAAAAATTATGTGAATGACTTGGTCCCTGAATTTCTGCCAGCTCCAGAAGATGTAGAGACAAACTGTGAGTGGTCAGCTTTTTCCTGCTTTCAGAAGG CCCAACTAAAGTCAGCAAATACAGGAAACAATGAAAGGATAATCAATTATCAATTAAAAAAGCTGAAGAGGAAACCACCTTCCACAAATGCAGGGAGAAGACAGAAACACAGACTAACATGCCCTTCATGTGATTCTTATGAGAAAAAACCACCCAAAGAATTCCTAGAAAGATTCAAATCACTTCTCCAAAAGATGATTCATCAGCATCTGTCCTCTAGAACACACGGAAGTGAAGATTCCTGA The human IL-21 encoded by the DNA sequence shown in is capable of being expressed.

[0193] In another non-limiting example, the APC has the sequence of SEQ ID NO: 65: MERTLVCLVVIFLGTVAHKSSPQGPDRLLIRLRHLIDIVEQLKIYENDLDPELLSAPQDVKGHCEHAAFACFQKAKLKPSNPGNNKTFIIDLVAQLRRRLPARRGGKKQKHIAKCPSCDSYEKRTPKEFLERLKWLLQKMIHQHLS; or SEQ ID NO: 66: ATGGAGAGGACCCTTGTCTGTCTGGTAGTCATCTTCTTGGGGACAGTGGCCCATAAATCAAGCCCCCAAGGGCCAGATCGCCTCCTGATTAGACTTCGTCACCTTATTGACATTGTTGAACAGCTGAAAATCTATGAAAATGACTTGGATCCTGAACTTCTATCAGCTCCACAAGATGTAAAGGGGCACTGTGAGCATGCAGCTTTTGCCTGTTTTCAGA AGGCCAAACTCAAGCCATCAAACCCTGGAAACAAATAAGACATTCATCATTGACCTCGTGGCCCAGCTCAGGAGGAGGCTGCCTGCCAGGAGGGGAGGAAAGAAACAGAAGCACATAGCTAAATGCCCTTCCTGTGATTCGTATGAGAAAAGGACACCCAAAGAATTCCTAGAAAGACTAAAATGGCTCCTTCAAAAGATGATTCATCAGCATCTCTCCTAG The mouse IL-21 encoded by the DNA sequence shown in is capable of being expressed.

[0194] In another non-limiting example, the APC has the sequence of SEQ ID NO:67: MKVLLRLICFIALLISSLEADKCKEREEKIILVSSANEIDVRPCPLNPNEHKGTITWYKDDSKTPVSTEQASRIHQHKEKLWFVPAKVEDSGHYYCVVRNSSYCLRIKISAK FVENEPNLCYNAQAIFKQKLPVAGDGGLVCPYMEFFKNENNELPKLQWYKDCKPLLLDNIHFSGVKDRLIVMNVAEKHRGNYTCHASYTYLGKQYPITRVIEFITLEENKPT RPVIVSPANETMEVDLGSQIQLICNVTGQLSDIAYWKWNGSVIDEDDPVLGEDYYSVENPANKRRSTLITVLNISEIESRFYKHPFTCFAKNTHGIDAAYIQLIYPVTNFQK HMIGICVTLTVIIVCSVFIYKIFKIDIVLWYRDSCYDFLPIKASDGKTYDAYILYPKTVGEGSTSDCDIFVFKVLPEVLEKQCGYKLFIYGRDDYVGEGMCVMEQSKGLLL; or SEQ ID NO: 68: The human IL-1 encoded by the DNA sequence shown in is capable of being expressed.

[0195] In another non-limiting example, the APC has the sequence of SEQ ID NO:69: ; or SEQ ID NO: 70: ATGGAGAATATGAAAGTGCTACTGGGGCTCATTTGTCTCAT G The mouse IL-1 encoded by the DNA sequence shown in is capable of being expressed.

[0196] In another non-limiting example, the APC has the sequence of SEQ ID NO:71: MGSPAAPEGALGYVREFTRHSSDVLGNLNELRLRGILTDVTLLVGGQPLRAHKAVLIACSGFFYSIFRGRAGVGVDVLSLPGGPEARGFAPLLDFMYTSRLRLSPATAPAVLAAAATYLQM EHVVQACHRFIQASYEPLGISLRPLEAEPPTPPTAPPPGSPRRSEGHPPDPPTESRSCSQGPPSSPASPDPKACNWKKYKYIVLNSQASQAGSLVGERSSGQPCPQARLPSGDEASSSSSSS SSSSEEGPIPGPQSRLSPTAATVQFKCGAPASTPYLLTSQAQDTSGSPSERARPLPGSEFSCQNCEAVAGCSSGLDSLVPGDEDKPYKCQLCRSSFRYKGNLASHRTVHTGEKPYHCSI CGARFNRPANLKTHSRIHSGEKPYKCETCGSRFVQVAHLRAHVLIHTGEKPYPCPTCGTRFRHLQTLKSHVRIHTGEKPYHCDPCGLHFRHKSQLRLHLRQKHGAATNTKVHYHILGGP; or SEQ ID NO: 72: The human BCL-6 encoded by the DNA sequence shown in is capable of being expressed.

[0197] In another non-limiting example, the APC has the sequence of SEQ ID NO: 73: ; or SEQ ID NO: 74: The mouse BCL-6 encoded by the DNA sequence shown in is capable of being expressed.

[0198] In another non-limiting example, the APC has the sequence of SEQ ID NO: 75: MSQSNRELVVDFLSYKLSQKGYSWSQFSDVEENRTEAPEGTESEMETPSAINGNPSWHLADSPAVNGATGHSSSLDAREVIPMAAVKQALREAGDEFELRYRRAFSDLTSQLHITPG TAYQSFEQVVNELFRDGVNWGRIVAFFSFGGALCVESVDKEMQVLVSRIAAWMATYLNDHLEPWIQENGGWDTFVELYGNNAAAESRKGQERFNRWFLTGMTVAGVVLLGSLFSRK; or SEQ ID NO: 76: ATGTCTCAGAGCAACCGGGAGCTGGTGGTTGACTTTCTCTCCTACAAGCTTTCCCAGAAAGGATACAGCTGGAGTCAGTTTAGTGATGTGGAAGAGAACAGGACTGAGGCCCAGAAGGGACTGAATCGGAGATGGAGACCCCCAGTGCCATCAATGGCAACCCATCCTGGCACC TGGCAGACAGCCCCGCGGTGAATGGAGCCACTGGCCACAGCAGCAGTTTGGATGCCCGGGAGGTGATCCCCATGGCAGCAGTAAAGCAAGCGCTGAGGGAGGCAGGCGACGAGTTTGAACTGCGGTACCGGCGGGCATTCAGTGACCTGACATCCCAGCTCCACATCACCCCAGGG ACAGCATATCAGAGCTTTGAACAGGTAGTGAATGAACTCTTCCGGGATGGGGTAAACTGGGGTCGCATTGTGGCCTTTTTCTCCTTCGGCGGGGCACTGTGCGTGGAAAGCGTAGACAAGGAGATGCAGGTATTGGTGAGTCGGATCGCAGCTTGGATGGCCACTTACCTGAATG ACCACCTAGAGCCTTGGATCCAGGAGAACGGCGGCTGGGATACTTTTGTGGAACTCTATGGGAACAATGCAGCAGCCGAGAGCCGAAAGGGCCAGGAACGCTTCAACCGCTGGTTCCTGACGGGCATGACTGTGGCCGGCGTGGTTCTGCTGGGCTCACTCTTCAGTCGGAAATGA The present invention can express human BCLXL encoded by the DNA sequence shown below.

[0199] In another non-limiting example, the APC has the sequence of SEQ ID NO:77: MSQSNRELVVDFLSYKLSQKGYSWSQFSDVEENRTEAPEETEAERETPSAINGNPSWHLADSPAVNGATGHSSSLDAREVIPMAAVKQALREAGDEFELRYRRAFSDLTSQLHITPGTAYQSFEQVVNELFRDGVNWGRIVAFFSFGGALCVESVDKEMQVLVSRIASWMATYLNDHLEPWIQENGGWDTFVDLYGNNAAAESRKGQERFNRWFLTGMTVAGVVLLGSLFSRK; でしまするまするBCLXL、さるんますまする78: ATGTCTCAGAGCAACCGGGAGCTGGTGGTCGACTTTCTCTCCTACAAGCTTTCCCAGAAAGGATACAGCTGGAGTCAGTTTAGTGATGTCGAAGAGAATAGGACTGAGGCCCCAGAAAAACTGAAGCAGAGAGGGAGACCCCCAGTGCCATCAATGGCAACCCATCCTGGCACCC TGGCGGATAGCCCGGCCGTGAATGGAGCCACTGGCCACAGCAGCAGTTTGGATGCGCGGGAGGTGATTCCCATGGCAGCAGTGAAGCAAGCGCTGAGAGAGGCAGGCGATGAGTTTGACTGCGGTACCGGAGAGCGTTCAGTGATCTAACATCCCAGCTTCACATAACCCCAGG ACCGCGTATCAGAGCTTTGAGCAGGTAGTGGAATGAACTCTTTCGGGATGGAGTAAACTGGGGTCGCATCGTGGCCTTTTCTCCTTTGGCGGGGCACTGTGCGTGGAAAGCGTAGACAAGGAGATGCAGGTATTGGTGAGTCGGATTGCAAGTTGGATGGCCACCTATCTGAATG ACCACCTAGAGCCTTGGATCCAGGAGAACGGCGGCTGGGACACTTTTGTGGATCTCTCAGGGAACAATGCAGCAGCCGAGAGCCGAAAGGCCAGGAGCGCTTCAACCGCTGGTTCCTGACGGGCATGACTGTGGCTGGTGTGGTTCTGCTGGGCTCACTCTTCAGTCGGAAGTGA The mouse BCLXL encoded by the DNA sequence shown in is capable of being expressed.

[0200] In another non-limiting example, the APC has the sequence of SEQ ID NO:79: MAHAGRTGYDNREIVMKYIHYKLSQRGYEWDAGDVGAAPPGAAPAPGIFSSQPGHTPHPAASRDPVARTSPLQTPAAPGAAAGPALSPVPPVVHLTLRQAGDDFSRRYRRDF AEMSSQLHLTPFTARGRFATVVEELFRDGVNWGRIVAFFEFGGVMCVESVNREMSPLVDNIALWMTEYLNRHLHTWIQDNGGWDAFVELYGPSMRPLFDFSWLSLKTLLSLA; or SEQ ID NO: 80: ATGGCGCACGCTGGGAGAACAGGGTACGATAACCGGGAGATAGTGATGAAGTACATCCATTATAAGCTGTCGCAGAGGGGCTACGAGTGGGATGCGGGAGATGTGGGCGCCGCCCCCGGGGGCCGCCCCCGCACCGGGCATCTTCTCCTCCAGCCCGGGCACACGCCCCATCCAGCC GCATCCCGGGACCCGGTCGCCAGGACCTCGCCGCTGCAGACCCCGGCTGCCCCCGGCGCCGCCGCGGGGCCTGCGCTCAGCCCGGTGCCACCTGTGGTCCACCTGACCCTCCGCCAGGCCGGCGACGACTTCTCCCGCCGCTACCGCCGCGACTTCGCCGAGATGTCCAGCCAGCTGCAC CTGACGCCCTTCACCGCGCGGGGACGCTTTGCCACGGTGGTGGAGGAGCTCTTCAGGGACGGGGTGAACTGGGGGAGGATTGTGGCCTTCTTTGAGTTCGGTGGGGTCATGTGTTGGAGAGCGTCAACCGGGATGTCGCCCCTGGTGGACAACATCGCCCTGTGGATGACTGAGTAC CTGAACCGGCACCTGCACACCTGGATCCAGGATAACGGAGGCTGGGATGCCTTTGTGGAACTGTACGGCCCCAGCATGCGGCCTCTGTTTGATTTCTCCTGGCTGTCTCTGAAGACTCTGCTCAGTTTGGCCCTGGTGGGAGCTTGCATCACCCTGGGTGCCTATCTGGGCCACAAGTGA The present invention can express human BCL 2 encoded by the DNA sequence shown in

[0201] In another non-limiting example, the APC has the sequence of SEQ ID NO:81: MAQAGRTGYDNREIVMKYIHYKLSQRGYEWDAGDADAAPLGAAPTPGIFSFQPESNPMPAVHRDMAARTSPLRPLVATAGPALSPVPPVVHLTLRRAGDDFSRRYRRDFAEMSSQLHLTPFTARGRFATVVEELFRDGVNWGRIVAFFEFGGVMCVESVNREMSPLVDNIALWMTEYLNRHLHTWIQDNGGWDAFVELYGPSMRPLFDFSWLSLKTLL; mouse BCL2 shown in, or SEQ ID NO: 82: ATGGCGCAAGCCGGGAGAACAGGGTATGATAACCGGGAGATCGTGATGAAGTACATACATTATAAGCTGTCACAGAGGGGCTACGAGTGGGATGCTGGAGATGCGGACGCGGCGCCCCTGGGGGCTGCCCCCACCCCTGGCATCTTCTCCTTCCAGCCTGAGAGCAACCCAATGCCCGCTGTGCACCGGGACATGGCTGCCAGGACGTCTCCTCTCAGGCCCCTCGTTGCCACCGCTGGGCCTGCGCTCAGCCCTGTGCCACCTGTGGTCCATCTGACCCTCCGCCGGGCTGGGGATGACTTCTCTCGTCGCTACCGTCGTGACTTCGCAGAGATGTCCAGTCAGCTGCACCTGACGCCCTTCACCGCGAGGGGACGCTTTGCCACGGTGGTGGAGGAACTCTTCAGGGATGGGGTGAACTGGGGGAGGATTGTGGCCTTCTTTGAGTTCGGTGGGGTCATGTGTGTGGAGAGCGTCAACAGGGAGATGTCACCCCTGGTGGACAACATCGCCCTGTGGATGACTGAGTACCTGAACCGGCATCTGCACACCTGGATCCAGGATAACGGAGGCTGGGATGCCTTTGTGGAACTATATGGCCCCAGCATGCGACCTCTGTTTGATTTCTCCTGGCTGTCTCTGAAGACCCTGCTCAGCCTGGCCCTGGTCGGGGCCTGCATCACTCTGGGTGCATACCTGGGCCACAAGTGA The mouse BCL 2 encoded by the DNA sequence shown in

[0202] In another non-limiting example, the APC has the sequence of SEQ ID NO:83: MFGLKRNAVIGLNLYCGGAGLGAGSGGATRPGGRLLATEKEASARREIGGGEAGAVIGGSAGASPPSTLTPDSRRVARPPPIGAEVPDVTATPARLLFFAPTRRAAPLEEMEAPAADAIMSPEEELDGYEPEPLGKRPAVLPLLELVGESGNNTSTDGSLPSTPPPAEEEEDELY RQSLEIISRYLREQATGAKDTKPMGRSGATSRKALETLRRVGDGVQRNHETAFQGMLRKLDIKNEDDVKSLSRVMIHVFSDGVTNWGRIVTLISFGAFVAKHLKTINQESCIEPLAESITDVLVRTKRDWLVKQRGWDGFVEFFHVEDLEGGIRNVLLAFAGVAGVGAGLAYLIR; or SEQ ID NO: 84: The present invention can express human MCL 1 encoded by the DNA sequence shown in

[0203] In another non-limiting example, the APC has the sequence of SEQ ID NO:85: MFGLRRNAVIGLNLYCGGASLGAGGGSPAGARLVAEEAKARREGGGEAALLPGARVVARPPPVGAEDPDVTASAERRLHKSPGLLAVPPEEMAASAAAAIVSPEEELDGCEPEAIGKRPAVLPLLERVSEAAKSSGADGSLPSTPPPPEEEEDDLYRQSLEIISRY LREQATGSKDSKPLGEAGAAGRRALETLRRVGDGVQRNHETAFQGMLRKLDIKNEGDVKSFSRVMVHVFKDGVTNWGRIVTLISFGAFVAKHLKSVNQESFIEPLAETITDVLVRTKRDWLVKQRGWDGFVEFFHVQDLEGGIRNVLLAFAGVAGVGAGLAYLIR; or SEQ ID NO: 86: ATGTTTGGCCTGCGGAGAAACGCGGTCATCGGCTTGAACCTGTACTGCGGCGGCGCCAGCCTCGGCGCGGGCGGCGGTTCTCCGGCAGGGGCGCGCCTGGTGGCCGAGGAGGCCAAGGCGCGGCGCGAGGGGGGAGGGGAGGCCGCCCTGCTGCCCGGCGCGCGGGTGGTCGCCCGGCCGCCGCCCGTGGGCGCCGAGGACCCCGACGTCACCGCGTCGGCCGAAAGGCGGCTGCATAAGTCGCCCGGCCTCCTCGCCGTGCCGCCCGAGGAGATGGCCGCGTCGGCCGCCGCCGCCATCGTGTCTCCGGAGGAGGAACTGGACGGCTGCGAGCCGGAGGCCATCGGCAAGCGCCCGGCCGTGCTGCCCCTCCTGGAGCGCGTGAGCGAGGCGGCCAAGAGCTCCGGGGCCGACGGCTCTCTGCCCTCCACGCCGCCGCCGCCCGAGGAGGAAGAGGACGACCTATACCGCCAGTCGCTGGAGATCATCTCGCGCTACTTGCGGGAGCAGGCGACCGGCTCCAAGGACTCGAAGCCTCTGGGCGAGGCGGGCGCGGCGGGCCGGAGAGCGCTGGAGACCCTGCGGCGCGTGGGCGACGGCGTGCAGCGCAACCACGAGACGGCCTTCCAGGGCATGCTCCGGAAACTGGACATTAAAAACGAAGGCGATGTTAAATCTTTTTCTCGAGTAATGGTCCATGTTTTCAAAGATGGCGTAACAAACTGGGGCAGGATTGTGACTCTTATTTCTTTCGGTGCCTTTGTGGCCAAACACTTAAAGAGCGTAAACCAAGAAAGCTTCATCGAACCATTAGCAGAAACTATCACAGATGTTCTTGTAAGGACGAAACGGGACTGGCTTGTCAAACAAAGAGGCTGGGATGGGTTTGTGGAGTTCTTCCACGTACAGGACCTAGAAGGCGGCATCAGAAATGTGCTGCTGGCTTTTGCGGGTGTTGCTGGAGTAGGGGCTGGTCTGGCATATCTAATAAGATAG The mouse MCL 1 encoded by the DNA sequence shown in

[0204] In another non-limiting example, the APC has the sequence of SEQ ID NO:87: MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT; or SEQ ID NO: 88: ATGTACAGGATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACAAACAGTGCACCTACTTCAAGTTCTACAAAGAAAACACAGCTACAACTGGAGCATTTACTGCTGGATTTACAGATGATTTTGAATGGAATTAATAATTACAAGAATCCCAAACTCACCAGGATGCTCACATTTAAGTTTTACATGCCCAAGAAGGCCACAGAACTGAAACATCTTCAG TGTCTAGAAGAAGAACTCAAACCTCTGGAGGAAGTGCTAAATTTAGCTCAAAGCAAAAACTTTCACTTAAGACCCAGGGACTTAATCAGCAATATCAACGTAATAGTTCTGGAACTAAAGGGATCTGAAACAACATTCATGTGTGAATATGCTGATGAGACAGCAACCATTGTAGAATTTCTGAACAGATGGATTACCTTTTGTCAAAGCATCATCTCAACACTGACTTGA The human IL-2 encoded by the DNA sequence shown in is capable of being expressed.

[0205] In another non-limiting example, the APC has the sequence of SEQ ID NO:89: MYSMQLASCVTLTLVLLVNSAPTSSSTSSSTAEAQQQQQQQQQQQHLEQLLMDLQELLSRMENYRNLKLPRMLTFKFYLPKQATELKDLQCLEDELGPLRHVLDLTQSKSFQLEDAENFISNIRVTVVKLKGSDNTFECQFDDES; or SEQ ID NO: 90: ATGTACAGCATGCAGCTCGCATCCTGTGTCACATTGACACTTGTGCTCCTTGTCAACAGCGCACCCACTTCAAGCTCCACTTCAAGCTCTACAGCGGAAGCACAGCAGCAGCAGCAGCAGCAGCAGC AGCAGCAGCAGCACCTGGAGCAGCTGTTGATGGACCTACAGGAGCTCCTGAGCAGGATGGAGAATTACAGGAACCTGAAACTCCCCAGGATGCTCACCTTCAAATTTTACTTGCCCAAGCAGGCCACA GAATTGAAAGATCTTCAGTGCCTAGAAGATGAACTTGGACCTCTGCGGCATGTTCTGGATTTGACTCAAAGCAAAAGCTTTCAATTGGAAGATGCTGAGAATTTCATCAGCAATATCAGAGTAACTG TTGTAAAACTAAAGGGCTCTGACAACACATTTGAGTGCCAATTCGATGATGAGTCAGCAACTGTGGTGGACTTTCTGAGGAGATGGATAGCCTTCTGTCAAAGCATCATCTCAACAAGCCCTCAATAA The mouse IL-2 encoded by the DNA sequence shown in is capable of being expressed.

[0206] In another non-limiting example, the APC has the sequence of SEQ ID NO:91: MIETYNQTSPRSAATGLPISMKIFMYLLTVFLITQMIGSALFAVYLHRRLDKIEDERNLHEDFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEETKKENSFEMQKGDQNPQIAAHVISEAS SKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL; or SEQ ID NO: 92: ATGATCGAAACATACAACCAAACTTCTCCCCGATCTGCGGCCACTGGACTGCCCATCAGCATGAAAATTTTTATGTATTTACTTACTGTTTTTCTTATCACCCAGATGATTGGGTCAGCACTTTTTGCTGTGTATCTTCATAGAAGGTTGGACAAGATAGAAGATGAAAGGAATCTTCATGAAGATTTTGTATTCA TGAAAACGATACAGAGATGCAACACAGGAGAAAGATCCTTATCCTTACTGAACTGTGAGGAGATTAAAAGCCAGTTTGAAGGCTTTGTGAAGGATATAATGTTAAACAAAGAGGAGACGAAGAAAGAAAACAGCTTTGAAATGCAAAAAGGTGATCAGAATCCTCAAATTGCGGCACATGTCATAAGTGAGGCCAGC AGTAAAACAACATCTGTGTTACAGTGGGCTGAAAAAAGGATACTACACCATGAGCAACAACTTGGTAACCCTGGAAAATGGGAAACAGCTGACCGTTAAAAGACAAGGACTCTATTATATCTATGCCCAAGTCACCTTCTGTTCCAATCGGGAAGCTTCGAGTCAAGCTCCATTTATAGCCAGCCTCTGCCTAAAGT CCCCCGGTAGATTCGAGAGAATCTTACTCAGAGCTGCAAATACCCACAGTTCCGCCAAACCTTGCGGGCAACAATCCATTCACTTGGGAGGAGTATTTGAATTGCAACCAGGTGCTTCGGTGTTTGTCAATGTGACTGATCCAAGCCAAGTGAGCCATGGCACTGGCTTCACGTCCTTTGGCTTACTCAAACTCTGA The present invention can express human CD40L encoded by the DNA sequence shown below.

[0207] In another non-limiting example, the APC has the sequence of SEQ ID NO:93: MIETYSQPSPRSVATGLPASMKIFMYLLTVFLITQMIGSVLFAVYLHRRLDKVEEEVNLHEDFVFIKKLKRCNKGEGSLSLLNCEEMRRQFEDLVKDITLNKEEKKENSFEMQRGDEDPQIAAHVVSEAN SNAASVLQWAKKGYYTMKSNLVMLENGKQLTVKREGLYYVYTQVTFCSNREPSSQRPFIVGLWLKPSSGSERILLKAANTHSSSQLCEQQSVHLGGVFELQAGASVFVNVTEASQVIHRVGFSSFGLLKL; or SEQ ID NO: 94: ATGATAGAAACATACAGCCAACCTTCCCCCAGATCCGTGGCAACTGGACTTCCAGCGAGCATGAAGATTTTTATGTATTTACTTACTGTTTTCCTTATCACCCAAATGATTGGATCTGTGCTTTTTGCTGTGTATCTTCATAGAAGATTGGATAAGGTCGAAGAGGAAGTAAACCTTCATGAAGATTTTGTATTC ATAAAAAAGCTAAAGAGATGCAACAAAGGAGAAGGATCTTTATCCTTGCTGAACTGTGAGGAGATGAGAAGGCAATTTGAAGACCTTGTCAAGGATATAACGTTAAACAAAGAAGAGAAAAAAGAAAACAGCTTTGAAATGCAAAGGTGATGAGGATCCTCAAATTGCAGCACACGTTGTAAGCGAAGCCAACA GTAATGCAGCATCCGTTCTACAGTGGGCCAAGAAAGGATATTATACCATGAAAAGCAACTTGGTAATGCTTGAAAATGGGAAACAGCTGACGGTTAAAAGAGAAGGACTCTATTATGTCTACACTCAAGTCACCTTCTGCTCTAATCGGGAGCCTTCGAGTCAACGCCCATTCATCGTCGGCCTCTGGCTGAAGCC CAGCAGTGGATCTGAGAGAATCTTACTCAAGGCGGCAAATACCCACAGTTCCTCCCAGCTTTGCGAGCAGCAGTCTGTTCACTTGGGCGGAGTGTTTGAATTACAAGCTGGTGCTTCTGTTTGTCAACGTGACTGAAGCAAGCCAAGTGATCCACAGAGTTGGCTTCTCATCTTTTGGCTTACTCAAACTCTGA The mouse CD40L encoded by the DNA sequence shown in is capable of being expressed.

[0208] In another non-limiting example, the APC has the sequence of SEQ ID NO:95: MCLSHLENMPLSHSRTQGAQRSSWKLWLFCSIVMLLFLCSFSWLIFIFLQLETAKEPCMAKFGPLPSKWQMASSEPPCVNKVSDWKLEILQNGLYLIYGQVAPNANYNDVAPFEVRLYKNKDMIQTLTNKSKIQNVGGTYELHVGDTIDLI; or SEQ ID NO: 96: ATGTGTTTGAGCCACTTGGAAAATATGCCTTTAAGCCATTCAAGAACTCAAGGAGCTCAGAGATCATCCTGGAAGCTGTGGCTCTTTGCTCAATAGTTATGTTGCTATTTCTTTGCTCCTTCAGTTGGCTAA TCTTTATTTTTCTCCAATTAGAGACTGCTAAGGAGCCCTGTATGGCTAAGTTTGGACCATTACCCTCAAAATGGCAAATGGCATCTTCTGAACCTCCTTGGCGTGAATAAGGTGTCTGACTGGAAGCTGGAGATA CTTCAGAATGGCTTATATTTAATTTATGGCCAAGTGGCTCCCAATGCAAACTACAATGATGTAGCTCCTTTTGAGGTGCGGCTGTATAAAAACAAAGACATGATACAAACTCTAACAAACAAATCTAAAATCC AAAATGTAGGAGGGACTTATGAATTGCATGTTGGGGACACCATAGACTTGATATTCAACTCTGAGCATCAGGTTCTAAAAAATAATACATACTGGGGTATCATTTTACTAGCAAATCCCAATTCATCTCCTAG The human GITR-L encoded by the DNA sequence shown in is capable of being expressed.

[0209] In another non-limiting example, the APC is SEQ ID NO: 97: MEEMPLRESSPQRAERCKKSWLLCIVALLLMLLCSLGTLIYTSLKPTAIESCMVKFELSSSKWHMTSPKPHCVNTTSDGKLKILQSGTYLIYGQVIPVDKKYIKDNAPFVVQIYKKNDVLQTLMNDFQILPIGGVYELHAGDNIYLKFNSKDHIQKTNTYWGII; or SEQ ID NO: 98: ATGGAGGAAATGCCTTTTGAGAGAATCAAGTCCTCAAAGGGCAGAGAGGTGCAAGAAGTCATGGCTCTTGTGCATAGTGGCTCTGTTACTGATGTTGCTCTGTTCTTTGGGTACACTGATCTATACTTCAC TCAAGCCAACTGCCATCGAGTCCTGCATGGTTAAGTTTGAACTATCATCCTCAAAATGGCACATGACATCTCCCAAACCTCACTGTGTGAATACGACATCTGATGGGAAGCTGAAGATACTGCAGAGTGGC ACATATTTAATCTACGGCCAAGTGATTCCTGTGGATAAGAAATACATAAAAGACAATGCCCCCTTCGTAGTACAGATATATAAAAAGAATGATGTCCTACAAACTCTAATGAATGATTTTCAAATCTTGC CTATAGGAGGGGTTTATGAACTGCATGCTGGAGATAACATATATCTGAAGTTCAACTCTAAAGACCATATTCAGAAAACTAACACATACTGGGGGATCATCTTAATGCCTGATCTACCATTCATCTCTTAG The mouse GITR-L encoded by the DNA sequence shown in is capable of being expressed.

[0210] In another non-limiting example, the APC is SEQ ID NO: 99: MGHLSAPLHRVRVPWQGLLLTASLLTFWNPPTTAQLTTESMPFNVAEGKEVLLLVHNLPQQLFGYSWYKGERVDGNRQIVGYAIGTQQATPGPANSGRETIYPNASLLIQNVTQND TGFYTLQVIKSDLVNEEATGQFHVYPELPKPSISSNNSNPVEDKDAVAFTCEPETQDTTYLWWINNQSLPVSPRLQLSNGNRTLTLLSVTRNDTGPYECEIQNPVSANRSDPVTLN VTYGPDTPTISPSDTYYRPGANLSLSCYAASNPPAQYSWLINGTFQQSTQELFIPNITVNNSGSYTCHANNSVTGCNRTTVKTIIVTELSPVVAKPQIKASKTTVTGDKDSVNLTC STNDTGISIRWFFKNQSLPSSERMKLSQGNTTLSINPVKREDAGTYWCEVFNPISKNQSDPIMLNVNYNALPQENGLSPGAIAGIVIGVVALVALIAVALACFLHFGKTGSSGPLQ; or SEQ ID NO: 100: The present invention can express human CD66a encoded by the DNA sequence shown in

[0211] In another non-limiting example, the APC comprises the sequence of SEQ ID NO: 101: MELASAHLHKGQVPWGGLLLTASLLASWSPATTAEVTIEAVPPQVAEDNNVLLLVHNLPLALGAFAWYKGNTTAIDKEIARFVPNSNNMNFTGQAYSGREIIYSNGSLLFQMITM KDMGVYTLDMTDENYRRTQATVRFHVHPILLKPNITSNNSNPVEGDDSVSLTCDSYTDPDNINYLWSRNGESLSEGDRLKLSEGNRTLTLLNVTRNDTGPYVCETRNPVSVNRSD PFSLNIIYGPDTPIISPSDIYLHPGSNLNLSCHAASNPPAQYFWLINEKPHASSQELFIPNITTNNSGTYTCFVNNSVTGLSRTTVKNITVLEPVTQPFLQVTNTTVKELDSVTL TCLSNDIGANIQWLFNSQSLQLTERMTLSQNNSILRIDPIKREDAGEYQCEISNPVSVRRSNSIKLDIIFDPTQGGLSDGAIAGIVIGVVAGVALIAGLAYFLYSRKSGGSGSF; or SEQ ID NO: 102: The mouse CD66a encoded by the DNA sequence shown in is capable of being expressed.

[0212] In certain embodiments, the APCs, particularly B cells, are engineered to express nucleic acids encoding OX40L (SEQ ID NO: 1), 4-1BB (SEQ ID NO: 5) and / or IL-12 (SEQ ID NO: 49). In certain embodiments, the APCs, particularly B cells, are engineered to express nucleic acids encoding at least two of OX40L (SEQ ID NO: 1), 4-1BB (SEQ ID NO: 5) and / or IL-12 (SEQ ID NO: 49). In certain embodiments, the APCs, particularly B cells, are engineered to express nucleic acids encoding OX40L (SEQ ID NO: 1), 4-1BB (SEQ ID NO: 5) and IL-12 (SEQ ID NO: 49). In certain embodiments, the nucleic acids encoding OX40L (SEQ ID NO: 1), 4-1BB (SEQ ID NO: 5) and / or IL-12 (SEQ ID NO: 49) are mRNA transfected into the expanded B cells prior to contact with lymphocytes.

[0213] In certain embodiments, APC cultures should be at least 50% B cells with detectable cytokine secretion either in the B cell culture itself or in co-culture with leukocytes, such as T cells.

[0214] 3.5 Expansion and culture media Lymphocyte culture is an expansion culture, i.e., selectively expanding a desired class or subclass of lymphocytes (preferably human lymphocytes, more preferably primary human lymphocytes, and most preferably primary human T cells (including TILs)) specific for a desired antigen (e.g., expressed by a subject sample of tumor or infected tissue). Expansion can be carried out in any suitable bioreactor known in the art or described herein, including, but not limited to, GREX (Wilson Wolff), Cytiva Wave bioreactor, Ori (Ori Biotech), Cocoon (Lonza), and ADVA (ADVA Biotech). Equipment, such as ADVA (ADVA Biotech), LOVO (Fresenius Kabi), EKKO Millipore Sigma), Sepia (Cytiva), Elite, Miltenyi Prodigy, or similar cell selection equipment, can also be used to select and collect cells.

[0215] It is preferred herein that the method of the invention is performed in a "single controlled culture vessel", i.e. the entire expansion protocol from a patient-derived sample to a final cell population is preferably performed in a single culture vessel, without the need to transfer the culture to a larger vessel once the cell culture volume has increased.

[0216] Within the present invention, the single culture vessel is preferably the growth chamber of a bioreactor. The growth chamber may have a shape that allows the volume of the cell culture to be adjusted throughout the process. In certain embodiments, the growth chamber has an inverted cone shape, or any other shape that tapers toward the bottom of the growth chamber. A growth chamber with such a shape allows the initial culture in a relatively small volume. At the same time, such a growth chamber offers the possibility of increasing the initial culture volume many times, thus allowing the initial cell population to be expanded on a large scale without the need to switch to a larger vessel.

[0217] It is preferred herein that a single culture vessel is "controlled". A culture vessel is controlled if at least one parameter of the culture medium in the culture vessel can be monitored and adjusted if necessary. Preferably, one or more of the parameters of the culture medium disclosed herein can be monitored and adjusted in a single controlled culture vessel according to the present invention.

[0218] Any suitable cell culture medium known in the art or described herein can be used for expansion. Non-limiting examples include commercially available media such as PRIME-XV (Irvine Scientific), X-Vivo (Lonza), Excellerate (R&D Systems), CTS Optimizer (Thermo Fisher), LymphoOne T Cell Medium (Takara), Stemline, ATCC Media (LGC Standards), and ImmunoCult™-XF T Cell Expansion Medium. The expansion medium may contain IL-2 or variant IL 2, where the variant versions, in non-limiting embodiments, include any of the following mutations, alone or in combination: M1 (Q22V, Q126A, I129D, S130G), M2 (L18N, Q126Y, S136R, M3 Q13Y, Q126Y, I129D, S1230R), and / or M4 (L18N, Q22V, T123A, S130R). In addition, the IL-2 variant may be any of the IL-2 variants disclosed in WO2011 / 063770 or US Pat. No. 8,759,486, which are incorporated herein by reference in their entirety.

[0219] The medium may further contain 0.5 g / l to 20 g / l glucose; additional vitamins, including MEM vitamin mix, glutamine; Pluronic; and one or more mitogens, including phytohemagglutinin (PHA), concanavalin A (ConA), pokeweed mitogen (PWM), mezerein (Mzn), and / or tetradecanoylphorbol acetate (TPA).

[0220] Preferably, the lymphocytes are cultured in an ADVA bioreactor, in particular an ADVA X3 bioreactor.

[0221] The culture medium may contain IL-2 or a variant thereof under conditions that favor the growth of lymphocytes (preferably human lymphocytes, more preferably primary human lymphocytes, most preferably including primary human T cells (TILs)) over tumor and other cells. In some embodiments, the IL is recombinant human IL-2 (rhIL-2). The culture medium may contain about 5,000 IU / mL to about 9,000 IU / mL of IL-2, about 6,000 IU / mL to about 8,000 IU / mL of IL-2, or about 6,000 IU / mL to about 7,000 IU / mL of IL-2. The culture medium may contain about 10,000 IU / mL IL-2, about 9,000 IU / mL IL-2, about 8,000 IU / mL IL-2, about 7,000 IU / mL IL-2, about 6000 IU / mL IL-2, about 5,000 IU / mL IL-2, about 4,000 IU / mL, about 3,000 IU / mL IL-2, or about 1,000 IU / mL IL-2. Preferably, the medium is supplemented with IL-2 or an active variant thereof throughout the culture process. Preferably, IL-2 or an active variant thereof is added to the culture medium to a final concentration of about 3000 IU / mL.

[0222] Additionally or alternatively, the culture medium may contain human AB serum (hAB). The culture medium may contain about 1% to about 20% hAB, about 4% to about 18% hAB, about 6% to about 15% hAB, or about 8% to about 12% hAB. The culture medium may contain about 2.5% hAB, about 5% hAB, about 7.5% hAB, about 10% hAB, about 12.5% ​​hAB, about 15% hAB, about 17.5 hAB, or about 20% hAB. Instead of hAB, a substitute for hAB may be used, such as human serum (huS) or platelet lysate (hPL), or any synthetic hAB variant known in the art.

[0223] Additionally or alternatively, the culture medium may contain IL-15. The culture medium may contain about 100 IU / mL to about 500 IU / mL of IL-15, about 100 IU / mL to about 400 IU / mL of IL-15, about 100 IU / mL to about 300 IU / mL of IL-15, or about 100 IU / mL to about 200 IU / mL of IL-15. The culture medium may contain about 500 IU / mL IL-15, about 400 IU / mL IL-15, about 300 IU / mL IL-15, about 200 IU / mL IL-15, about 180 IU / mL IL-15, about 160 IU / mL IL-15, about 140 IU / mL IL-15, about 120 IU / mL IL-15, or about 100 IU / mL IL-15.

[0224] Additionally or alternatively, the culture medium may contain IL-21. The culture medium may contain about 0.5 IU / mL to about 20 IU / mL of IL-21, about 0.5 IU / mL to about 15 IU / mL of IL-21, about 0.5 IU / mL to about 12 IU / mL of IL-21, about 0.5 IU / mL to about 10 IU / mL of IL-21, about 0.5 IU / mL to about 5 IU / mL of IL-21, or about 0.5 IU / mL to about 1 IU / mL of IL-21. The culture medium may contain about 20 IU / mL, about 15 IU / mL, about 12 IU / mL, about 10 IU / mL, about 5 IU / mL, about 4 IU / mL, about 3 IU / mL, about 2 IU / mL, about 1 IU / mL, or about 0.5 IU / mL of IL-21.

[0225] It is preferred herein that the APC in the medium is genetically engineered to produce IL-12. However, rather than using genetically engineered APC, IL-12 can also be added to the culture medium as a supplement at any suitable concentration to support lymphocyte expansion.

[0226] The cell culture medium may also include one or more TNFRSF agonists. In some embodiments, the TNFRSF agonist includes a 4-1BB agonist, which may be, in non-limiting examples, urelumab, utomirumab, EU-101, or a fusion protein, fragment, derivative, variant, or biosimilar thereof, and the TNSFR agonist may also include a combination of agonists listed herein and / or known in the art. The TNFRSF agonist may be added at a concentration sufficient to achieve a concentration of between 0.1 μg / mL and 100 μg / mL, or between 20 μg / mL and 40 μg / mL in the cell culture medium.

[0227] The method of the present invention preferably comprises the following modes: a) Batch mode: during this step, the tumor sample is co-cultured with APCs in batch mode. During this static expansion step, no or very limited lymphocyte expansion occurs. Preferably, during the initiation expansion step, pH and dissolved oxygen (DO) concentration are monitored and controlled and adjusted as necessary.

[0228] b) Fed-batch mode: When lymphocytes are expanded in batch culture, changes in the composition of the culture medium will be observed. In particular, the concentration of glucose in the culture medium will drop and lactate will accumulate. In order to maintain the glucose and lactate concentrations within a defined range, fresh medium (containing glucose and no lactate) is fed to the growth chamber to increase the glucose concentration and decrease the lactate concentration in the culture medium. During the fed-batch mode, the pH, DO concentration, glucose concentration and lactate concentration of the culture medium are preferably monitored and adjusted if necessary. Due to the addition of culture medium during the fed-batch mode, the culture volume will increase. The fed-batch mode is preferably continued until the bioreactor reaches a defined volume.

[0229] c) Circulation mode: Once a defined volume of the bioreactor is reached, the culture medium is circulated in / out of the growth chamber. That is, the culture medium may be removed from the growth chamber and then circulated back to the growth chamber. During circulation mode, the pH, DO concentration, glucose concentration and lactate concentration of the culture medium are preferably monitored. The pH and DO concentration can be adjusted to meet defined values, if necessary. The circulation mode is preferably performed until the glucose and / or lactate concentrations are outside of a predefined tolerance range.

[0230] d) Perfusion mode: When the glucose and / or lactate concentrations are no longer within predefined tolerances, the bioreactor will switch to perfusion mode. That is, the growth medium is constantly or stepwise removed from the growth chamber to waste and fresh culture medium is added at the same time. During perfusion mode, the pH, DO concentration, glucose concentration and lactate concentration of the culture medium are preferably monitored. The pH and DO concentration can be adjusted to meet defined values, if necessary. By adjusting the perfusion rate, the glucose and lactate concentrations can be fine-tuned.

[0231] In the first step, the tumor sample is cultured in the growth chamber of the bioreactor during batch mode for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 days. During this time, the TILs contained in the tumor sample will migrate out of the tumor sample. However, it should be understood that lymphocytes may also be expanded during batch mode, at least to some extent, for example by activation with APCs. It is preferred herein that the batch mode is performed immediately before the subsequent expansion step in the same bioreactor. However, the batch mode may also be omitted or shortened when the tumor sample is processed / before it is added to the bioreactor. For example, the tumor fragments can be enzymatically digested and then the resulting TILs can be transferred to the bioreactor for the expansion step.

[0232] The batch mode is preferably carried out in batch mode, i.e. no fresh culture medium is added during this step, however, the pH and dissolved oxygen levels are preferably adjusted, monitored and maintained within predefined ranges during the batch mode, if necessary.

[0233] It is preferred that the APCs and / or at least one antigen are added to the growth chamber along with the tumor sample during batch mode, however, the APCs and / or antigens can also be added to the TILs at a later time point.

[0234] Preferably, the APCs and antigen are added to the tumor sample in the growth chamber prior to the addition of the activating anti-CD3 antibody. Preferably, the APCs and optionally the antigen are added to the tumor sample at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days prior to the activating anti-CD3 antibody.

[0235] In a preferred embodiment, lymphocytes are co-cultured with antigen presenting cells (APCs), in particular with B cells. The lymphocytes and APCs can be mixed in a ratio that allows sufficient availability of MHC-presented antigen peptides by the lymphocytes.

[0236] Furthermore, it is known that APCs, and especially B cells, secrete cytokines that can activate T cells and thus induce T cell expansion. Thus, lymphocytes and APCs can be mixed in a ratio that allows sufficient availability of APC-secreted cytokines by lymphocytes and costimulation of lymphocytes.

[0237] In certain embodiments, B cells are cultured with tumor fragments known or suspected to contain lymphocytes, particularly TILs. 3 One tumor fragment had a size of approximately 1 × 10 4 , 5×10 4 , 10×10 4 , 25×10 4 , 50×10 4 , 75×10 4 , 100×10 4 , 250×10 4 , 500×10 4 , 750×10 4 or 1000 x 10 4 , 2500×10 4 , 5000×10 4 , 7500×10 4 , 10000×10 4 In a particularly preferred embodiment, the B cells are contacted with 1 to 3 mm 3 One tumor fragment with a size of approximately 10 5 ~10 7 B cells, and more preferably about 10 6 It is particularly preferred to contact the individual B cells.

[0238] In certain embodiments, 1 to 3 mm 3 Between 10 and 1000 tumor fragments having a size of 1 to 3 mm are added to the culture. 3 Between 25 and 500, preferably between 50 and 250, more preferably between 50 and 150, and most preferably between 50 and 100 tumor fragments having a size of 0.1-0.2 mm are added to the culture.

[0239] Alternatively, B cells can be cultured with isolated lymphocytes, particularly isolated T cells.In certain embodiments, T cells can be isolated from blood by any method known in the art.In certain embodiments, T cells can be tumor-infiltrating lymphocytes isolated from tumor samples, for example, by enzymatic digestion of tumor samples. In certain embodiments, the initial ratio of T cells to B cells in the culture is about 1:10000, 1:9000, 1:8000, 1:7000, 1:6000, 1:5000, 1:4000, 1:3000, 1:2000 1:1000, 1:900, 1:800, 1:700, 1:600, 1:500, 1:400, 1:300, 1:200, 1:100, 1:90, 1:80, 1:70, 1:60, 1:50, 1:40, 1:30, 1:20, 1:10, 1:9, 1:8, 1:7, 1:6, 1:5, 1:4, 1:3, 1:2 or 1:1. Preferably, the initial ratio of T cells to B cells is between 1:10000 and 1:100, more preferably between 1:3000 and 1:300.

[0240] After an initial lag period, lymphocytes in the growth chamber begin to expand in the presence of antigen-presenting cells that present a suitable antigen. Once lymphocytes begin to expand, it is preferred herein that the composition and / or volume of the growth medium is adjusted based on the rate of lymphocyte expansion (transition from batch to fed-batch mode). To this end, certain parameters of the culture medium must be continuously monitored.

[0241] The batch mode is followed by a fed-batch mode during which fresh culture medium is added to the growth chamber in order to adjust and / or maintain the composition of the culture medium in the growth chamber. To this end, one or more parameters of the culture medium in the growth chamber must be monitored and, if necessary, adjusted to a predefined range or value. The parameters include, but are not limited to, pH, dissolved oxygen (DO) concentration, glucose concentration, lactate concentration, glutamine concentration, glutamate concentration, and temperature. The concentrations of glucose and lactate, and optionally glutamate and / or glutamine, are preferably adjusted by adding fresh culture medium to the growth chamber. The pH and / or FO can be adjusted by adjusting the oxygen and / or carbon dioxide in the head space of the growth chamber. The temperature of the culture medium can be adjusted with a heating element.

[0242] When fresh culture medium is added to the growth chamber during fed-batch mode, the fresh culture medium is preferably added near the bottom of the growth chamber such that the fresh medium entering the growth chamber is in direct contact with the lymphocytes, which are preferably separated from the inlet near the bottom of the growth chamber by a membrane or perforated barrier.

[0243] The fed-batch mode ultimately results in an increased culture volume. Since the rate at which fresh culture medium is added to the growth chamber depends on the composition of nutrients (i.e., glucose) and / or the production of metabolites (i.e., lactate), the volume of the cell culture during fed-batch mode correlates with the expansion rate of lymphocytes. Thus, in certain embodiments, the method according to the present invention includes a step of adjusting the volume of the culture medium according to the expansion rate of lymphocytes in the growth chamber.

[0244] In certain embodiments, the culture volume will be increased at least 2, 3, 4, 5 or 6 fold during fed-batch mode. Preferably, the fed-batch mode is performed until the maximum or defined volume of the growth chamber is reached.

[0245] When a defined cell culture volume is reached in the growth chamber, e.g., the maximum volume of the growth chamber, the bioreactor can be set in a circulation mode, i.e., the culture medium can be removed from the growth chamber and then added back to the growth chamber. Preferably, the culture medium is removed near the surface of the culture medium in the growth chamber and added back to the bottom of the growth chamber, thus creating a flow of culture medium along the lymphocytes in the growth chamber.

[0246] During recirculation mode, the same parameters as during fed-batch mode are preferably monitored. No nutrients can be added in the form of fresh medium after the culture has reached its final volume. However, during recirculation mode, pH (by CO2), DO (by O2) and temperature (by a heating element) can be adjusted.

[0247] It should be noted that circulation is primarily performed to reduce the consumption of fresh medium, but the circulation mode may be omitted and instead the fed-batch mode may be followed immediately by the perfusion mode.

[0248] Finally, during the perfusion mode, medium is constantly or gradually removed from the growth chamber and replaced with fresh medium. As for the circulation mode, the used medium is preferably removed near the surface of the culture medium in the growth chamber and fresh medium is added to the bottom of the growth chamber so that it is in contact with the lymphocytes in the growth chamber.

[0249] During perfusion mode, the same parameters are preferably monitored as discussed above for fed-batch and circulation modes. The perfusion rate can be adjusted according to the composition of nutrients (i.e., glucose) or the formation of metabolites (i.e., lactate).

[0250] The bioreactor comprises a conditioning chamber connected to the growth chamber via at least one outlet. That is, the culture medium can be added to the growth chamber from the conditioning chamber. Preferably, the conditioning chamber further comprises at least one inlet that can pump the medium from the growth chamber to the conditioning chamber. The conditioning chamber connected to the growth chamber via at least one inlet and at least one outlet can be used to circulate the culture medium in the growth chamber.

[0251] The conditioning chamber can be used to adjust the temperature of the culture medium before it is added to the growth chamber during fed-batch, circulation and / or perfusion modes. Additionally, one or more parameters used in the culture medium can be adjusted in the conditioning chamber before the conditioned medium is added to the growth chamber.

[0252] The conditioning chamber and / or the growth chamber preferably comprises one or more sensors that allow monitoring of one or more parameters of the culture medium. That is, the conditioning chamber may comprise a sensor for monitoring at least one parameter of the culture medium selected from pH, dissolved oxygen (DO) concentration, glucose concentration, lactate concentration, glutamine concentration, glutamic acid concentration and temperature. However, the bioreactor may also comprise an analysis unit, in which one or more parameters of the culture medium are determined. The analysis unit may be connected to the growth chamber, so that the culture medium can be transferred from the growth chamber to the analysis unit either permanently or at defined intervals. In certain embodiments, the glucose and lactate concentrations, and optionally the glutamic acid / glutamine concentrations, are measured in the analysis unit by any suitable method known in the art.

[0253] For each parameter of the culture medium, a tolerance range can be defined. Then, for each individual parameter, the culture medium in the growth chamber is monitored to see if it is within the predefined tolerance range for said parameter. Certain parameters, such as pH, dO or temperature, can be constantly monitored. However, the determination of other parameters, such as glucose or lactate concentration, can take more time and therefore can be performed at certain intervals. For example, and without limitation, certain parameters can be determined every minute, every 5 minutes, every 10 minutes, every 15 minutes, every 30 minutes, or every 60 minutes.

[0254] The expansion of lymphocytes leads to the consumption of medium components (e.g., glucose, glutamic acid or glutamine) and the accumulation of metabolites (e.g., lactate or ammonium) in the culture medium. These changes in the composition of the culture medium may result in one or more parameters no longer falling within a predefined acceptable range or exceeding a predefined threshold. In this case, culture medium is replenished in the growth chamber so that each parameter is again within the acceptable range.

[0255] It will be appreciated that the bioreactor for the above process is equipped at least with a growth chamber connected to a supply of fresh medium and a waste container, and further includes the pumps necessary for adding fresh medium to the growth chamber and removing used medium from the growth chamber.

[0256] However, it is preferred herein that the bioreactor for the above process further comprises a conditioning chamber and a pump required to circulate the culture medium between the growth chamber and the conditioning chamber. Additional pumps will be required to connect the growth chamber and / or the conditioning chamber to a supply of fresh culture medium and / or to a waste container. Furthermore, the growth chamber and / or the conditioning chamber may be equipped with suitable sensors to monitor the parameters of the culture medium throughout the process. Suitable devices for the single-step expansion of lymphocytes as described above are known in the art and include, but are not limited to, the ADVA X3 bioreactor. Furthermore, bioreactors such as those disclosed in WO2021 / 148878 can be used in the method according to the present invention. WO2021 / 148878 is fully incorporated herein by reference.

[0257] The growth chamber is a chamber suitable for culturing lymphocytes, in particular T cells. It is preferred herein that the growth chamber is suitable for culturing lymphocytes by circulation and / or perfusion mode, i.e., the growth chamber comprises at least one inlet for adding fresh or conditioned culture medium to the growth chamber, and at least one outlet for removing the culture medium from the growth chamber (either to a waste container or to a conditioning chamber).

[0258] Preferably, the inlet for adding fresh or conditioned medium to the growth chamber is located near the bottom of the growth chamber and the outlet is located at the top portion of the growth chamber, so that culture medium can be removed from near the surface of the culture medium within the growth chamber. By adding culture medium at the bottom of the growth chamber and removing it from the top of the growth chamber, a flow of culture medium will be created along the lymphocytes to adequately provide nutrients to the lymphocytes.

[0259] In certain embodiments, the growth chamber may include multiple outlets at a top portion of the growth chamber that are arranged at different heights. Having multiple outlets at different heights allows the growth chamber to be filled with different volumes of culture medium while still being able to remove the culture medium near the surface of the culture medium within the growth chamber.

[0260] Preferably, the cells are separated from the inlet at the bottom of the growth chamber by a perforated barrier. A growth chamber that can be used in the method of the invention for culturing lymphocytes is disclosed in WO2018037402, which is incorporated herein by reference in its entirety.

[0261] When the lymphocytes are provided in a recirculated circulating culture medium, the bioreactor preferably includes a conditioning chamber that can adjust the composition of the culture medium according to predefined parameters. The conditioning chamber preferably includes one or more inlets that can replenish the culture medium to the conditioning chamber. In addition, the conditioning chamber can include one or more sensors for monitoring parameters of the culture medium in the conditioning chamber. In addition, the conditioning chamber can include an agitator to facilitate mixing of the culture medium and supplements in the conditioning chamber. The conditioning chamber can further include a heating element to maintain the culture medium at a predefined temperature.

[0262] As mentioned above, the bioreactor may include a number of sensors for monitoring parameters in the culture medium. The sensors are preferably located in the growth chamber and / or the conditioning chamber. Alternatively or in addition, one or more sensors may be located in the connection between the growth chamber and the conditioning chamber and / or in an analysis unit connected to the growth chamber and / or the conditioning chamber.

[0263] The conditioned culture medium can be based on any culture medium suitable for culturing lymphocytes.In particular, the conditioned growth medium can be based on any culture medium suitable for culturing T cells.In particular, the conditioned growth medium can be based on any T cell culture medium disclosed herein.

[0264] In certain embodiments, the conditioned culture medium is maintained at a defined pH range. Sensors for measuring the pH of fluids are well known in the art and are commonly used in bioreactors. The conditioned growth medium according to the present invention is preferably maintained at a pH range of 6-8, preferably 6.5-7.5, more preferably 7.0-7.4. Maintenance of pH in the culture medium can be achieved by titrating the culture medium with an acid or base, or more preferably by adjusting the CO2 concentration in the growth and / or conditioning chamber.

[0265] In certain embodiments, a defined dissolved oxygen (DO) concentration is maintained in the conditioned growth medium. Sensors or probes for measuring dissolved oxygen concentration in fluids are well known in the art and are commonly used in bioreactors. The conditioned growth medium according to the present invention is preferably maintained at a DO concentration in the range of 10% to 100% DO, preferably 20% to 90% DO, more preferably 30% to 80% DO. Maintenance of the DO concentration in the culture medium can be achieved by sparging the culture medium with air or oxygen.

[0266] In certain embodiments, a defined glucose concentration is maintained in the conditioned growth medium. Sensors or methods for continuously measuring glucose concentration in a fluid are known in the art and are commonly used in bioreactors. The conditioned growth medium according to the present invention is preferably maintained at a glucose concentration in the range of 0.5-10 g / L glucose, preferably 1-8 g / L glucose, more preferably 2-6 g / L glucose. Maintenance of the glucose concentration in the culture medium can be achieved by adding a concentrated glucose solution to the culture medium. However, within the present invention, it is preferred that the glucose concentration in the culture medium is maintained by supplementing the culture medium with fresh culture medium.

[0267] In certain embodiments, a defined glutamate concentration is maintained in the conditioned growth medium. Sensors or methods for continuously measuring glutamate concentration in a fluid are known in the art and are commonly used in bioreactors. Maintenance of glutamate concentration in the culture medium can be achieved by adding a concentrated glutamate solution to the culture medium. However, within the present invention, it is preferred that the glutamate concentration in the culture medium is maintained by supplementing the culture medium with fresh culture medium.

[0268] In certain embodiments, a defined glutamine concentration is maintained in the conditioned growth medium. Sensors or methods for continuously measuring glutamine concentration in fluids are known in the art and are commonly used in bioreactors. The maintenance of glutamine concentration in the culture medium can be achieved by adding a concentrated glutamine solution to the culture medium. However, within the present invention, it is preferred that the glutamine concentration in the culture medium is maintained by supplementing the culture medium with fresh culture medium.

[0269] In certain embodiments, a defined lactate concentration is maintained in the conditioned growth medium. Sensors or methods for continuously measuring lactate concentration in a fluid are known in the art and are commonly used in bioreactors. The culture medium according to the present invention is preferably conditioned such that the lactate concentration is maintained at less than 15 mM g / L lactate, preferably 10 mM g / L lactate, more preferably 5 mM g / L lactate. Maintaining the lactate concentration in the culture medium below a defined threshold can be achieved by diluting the culture medium with fresh culture medium.

[0270] In certain embodiments, the conditioned growth medium is maintained at a defined temperature. Sensors for continuously measuring the temperature in a fluid are known in the art and are commonly used in bioreactors. The culture medium according to the invention is preferably maintained at a temperature in the range of 35-39°C, preferably 36-38°C, more preferably 36.5-37.5°C. Maintaining the temperature of the culture medium within the defined range can be achieved by heating means contained within the bioreactor.

[0271] While it is possible to replenish the growth medium in the growth chamber, it is preferred to replenish the growth medium in the conditioning chamber to prevent direct contact of the lymphocytes with high concentrations of supplements, however DO and pH are preferably regulated directly in the growth chamber by adjusting the composition of CO2 and O2 in the head space of the growth chamber.

[0272] In certain embodiments, a conditioned culture medium is a medium in which at least one of the parameters pH, DO, glucose concentration, lactate concentration, glutamate concentration, glutamine concentration and / or temperature is maintained within a defined range as disclosed herein.

[0273] In certain embodiments, a conditioned culture medium is a medium in which at least two of the parameters pH, DO, glucose concentration, lactate concentration, glutamate concentration, glutamine concentration and / or temperature are maintained within defined ranges as disclosed herein.

[0274] In certain embodiments, a conditioned culture medium is a medium in which at least three of the parameters pH, DO, glucose concentration, lactate concentration, glutamate concentration, glutamine concentration and / or temperature are maintained within defined ranges as disclosed herein.

[0275] In certain embodiments, a conditioned culture medium is a medium in which at least four of the parameters pH, DO, glucose concentration, lactate concentration, glutamate concentration, glutamine concentration and / or temperature are maintained within defined ranges as disclosed herein.

[0276] In certain embodiments, a conditioned culture medium is a medium in which at least five of the parameters pH, DO, glucose concentration, lactate concentration, glutamate concentration, glutamine concentration and / or temperature are maintained within defined ranges as disclosed herein.

[0277] In certain embodiments, a conditioned culture medium is a medium in which at least six of the parameters pH, DO, glucose concentration, lactate concentration, glutamate concentration, glutamine concentration and / or temperature are maintained within defined ranges as disclosed herein.

[0278] In certain embodiments, a conditioned culture medium is a medium in which the parameters pH, DO, glucose concentration, lactate concentration, glutamate concentration, glutamine concentration and temperature are all maintained within the defined ranges disclosed herein.

[0279] In certain embodiments, a conditioned culture medium is a medium in which the parameters pH, DO, glucose concentration, lactate concentration and temperature are all maintained within the defined ranges disclosed herein.

[0280] It should be noted that additional parameters in the conditioned growth medium can be controlled. Additional parameters and suitable probes / methods for determining the above-mentioned parameters are summarized in Reyes et al., Processes 2022, Vol. 10, No. 189, https: / / doi.org / 10.3390 / pr10020189, which is fully incorporated herein by reference.

[0281] It should be noted that during operation of the bioreactors and bioreactor systems of the present application, liquids, e.g., growth medium, can be supplied by perfusion (constant exchange of medium in and waste out), by circulation (constant exchange of medium by circulation), or by fed-batch (addition of certain nutrients to the growth medium).

[0282] It is preferred herein to perfuse the lymphocytes with conditioned culture medium during the expansion phase. That is, during the expansion phase, conditioned culture medium is supplied to the lymphocytes while the growth medium is removed from the bioreactor at the same time. Preferably, the perfusion of lymphocytes is performed as disclosed in WO2018 / 037402, which is fully incorporated herein by reference.

[0283] The expansion of lymphocytes requires the presence of activation signals. In the method of the present invention, lymphocytes are preferably first activated by a group of antigen-presenting cells (APCs) that are co-cultured with lymphocytes. It is preferred herein that lymphocytes are co-cultured with APCs for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 days. APCs are preferably activated B cells as disclosed herein.

[0284] It should be understood that most APCs survive only a limited number of days in T cell culture, therefore, it is preferred that additional activating factors are added to the lymphocytes during the process.

[0285] In certain embodiments, the activator is an anti-CD3 antibody. Any anti-CD3 antibody that can activate lymphocytes, particularly T cells, can be used in the method of the present invention. Preferably, the anti-CD3 antibody OKT-3 is used to activate lymphocytes in culture.

[0286] The cell culture medium may be supplemented with an OKT-3 antibody component alone or in combination with one or more of the cytokines disclosed herein. The culture medium may contain a final concentration of 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 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, or about 1 μg / mL of OKT-3 antibody. The cell culture medium may contain OKT-3 antibody at a concentration 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, or between 50 ng / mL and 100 ng / mL. In some embodiments, the cell culture medium does not contain OKT-3 antibody. In a preferred embodiment, the OKT-3 antibody is added to the culture medium to obtain a final concentration of about 100 ng / mL.

[0287] It is preferred herein that the anti-CD3 antibody, in particular the OKT-3 antibody, is added to the cell culture after the addition of APC. Preferably, the anti-CD3 antibody, in particular the OKT-3 antibody, is added to the culture after the lymphocytes have been cultured in the presence of APC for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 days. In a particularly preferred embodiment, the anti-CD3 antibody, in particular the OKT-3 antibody, is added to the culture after the lymphocytes have been cultured in the presence of APC for 8 to 12 days, even more preferably for 9 to 11 days, most preferably for 10 days.

[0288] In certain embodiments, lymphocytes are first cultured with the B cells and the pool of peptides for 8 to 12 days, even more preferably 9 to 11 days, and most preferably 10 days, before anti-CD3 antibodies, in particular OKT-3 antibodies, are added to the culture.

[0289] In certain embodiments, activator, such as anti-CD3 antibody, can be added to lymphocytes more than once.That is, in certain embodiments, anti-CD3 antibody, such as OKT-3, can be added to lymphocytes twice, and the second dose of antibody is given 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days after the first day.In certain embodiments, anti-CD3 antibody, such as OKT-3, can be added to lymphocytes multiple times, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days apart.

[0290] The expansion phase may last from 5 to 35 days. The expansion phase may be from 5 to 30 days, 5 to 25 days, 5 to 20 days, or 5 to 15 days. In certain embodiments, the expansion phase is only 15 days. In certain embodiments, the expansion phase may be from 25 to 50 days, 25 to 45 days, 25 to 40 days, or 25 to 35 days. It is further preferred that the sample containing lymphocytes and / or T cells is maintained above 0° C. prior to expansion, and is maintained above 0° C. throughout the expansion.

[0291] Briefly, once obtained from the source, the sample of cells and / or T cells to be subjected to expansion should be cultured to a desired yield, preferably at least 1×10 7 It is preferred that the cells are not frozen at any point until they reach a population of at least 1×10 cells. 7 , 5×10 7 , 10×10 7 , 15×10 7 , 20×10 7 , 25×10 7 , 30×10 7 , 35×10 7 , 40×10 7, 45×10 7 , 50×10 7 , 55×10 7 , 60×10 7 , 65×10 7 , 70×10 7 , 75×10 7 , 80×10 7 , 85×10 7 , 90×10 7 , 95×10 7 , or at least 100×10 7 Preferably, expansion is continued under conditions as described herein until at least 10×10 T cells are obtained. 8 This is continued until T cells are obtained.

[0292] As described herein, the culture may also include feeder cells known in the art, e.g., B cells, dendritic cells, T cells, macrophages and / or PBMCs, which may be autologous or allogeneic cells. It is also possible to use cytokines in the medium instead of feeder cells. Feeder cells can be added before the start of the culture or on any day of the expansion culture. The final yield of expansion is preferably 1×10 target cells (e.g., T cells). 7 ~1000×10 7 Between 10 and 10, more preferably 10×10 7 ~1000×10 7 In a preferred embodiment, the expanded population is at least 90% CD3+, contains at least 15% cells that react with the desired antigen, e.g., neo-antigen recovered from / identified in the patient, contains a majority of CD8+ cells, and has a viability of at least 70%. It is further preferred that at least half of the T cells that respond to stimulation with the neo-antigen peptide generate a sustained response in the patient. To that end, peripheral lymphocytes can be recovered from the patient and tested for the presence of the neo-antigen in an ELISpot assay.

[0293] Specific population of lymphocytes can be separated from other components of sample and / or culture. Methods for separating specific population of desired cells from sample are known, including, but not limited to, leukapheresis for obtaining T cells from peripheral blood sample from patient or donor; isolating / obtaining specific population from sample using FACSort device; and selecting specific population manually or using micromanipulator from fresh biopsy specimen containing live leukocytes (see, for example, Dudley, Immunother. 26 (2003), 332-342; Robbins, Clin. Oncol. 29 (20011), 917-924; Leisegang, J. Mol. Med. 86 (2008), 573-58). The term "fresh biopsy specimen" refers to tissue sample (e.g., tumor tissue, infected tissue, or blood sample) that is or will be removed and / or isolated from subject by surgery or any other known means.

[0294] As is well known in the art, it is also possible to isolate / obtain and culture / select one or more specific subpopulations of white blood cells, such as, for example, the most preferred T cells. Such methods include, but are not limited to, the isolation and culture of lymphocyte subpopulations, such as, for example, CD3+, CD28+, CD4+, CD8+, and γδ subclasses, as well as the isolation and culture of other primary lymphocyte populations, such as, for example, NK T cells, B cells, or macrophages. Such selection methods may include, for example, positive and / or negative selection techniques, in which samples are incubated with specific combinations of antibodies and / or cytokines to select the desired subpopulation. Those skilled in the art can easily adjust the components of the selection medium and / or the method and length of selection using methods well known in the art. Longer incubation times may be used to isolate the desired population in any situation where the desired cells are expected to be fewer compared to other cell types, such as, for example, when isolating tumor infiltrating lymphocytes (TILs) from tumor tissue or from immunocompromised individuals. Those skilled in the art will also recognize that multiple rounds of selection can be used in the methods of the present disclosure.

[0295] Enrichment of the desired population can also be achieved by negative selection, for example, by using a combination of antibodies against surface markers specific to the cells to be negatively selected. In a non-limiting example, cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry can be used, using a cocktail of monoclonal antibodies against cell surface markers present on the cells to be negatively selected. For example, to enrich CD4+ cells by negative selection, a monoclonal antibody cocktail can be used, typically including antibodies specific for, for example, CD14, CD20, CD11b, CD16, HLA-DR, and CD8. The methods disclosed herein also include removing regulatory immune cells, for example, CD25+ T cells, from the population to be expanded or otherwise contained in the culture. Such methods include the use of anti-CD25 antibodies or fragments thereof, or CD25-binding ligands, for example, IL-2.

[0296] The donor and / or recipient of the leukocytes and / or leukocyte populations disclosed herein, including the subject to be treated with allogeneic or autologous leukocytes, can be any living organism (e.g., a mammal) capable of eliciting an immune response. Examples of donors and / or recipients as used herein include humans, dogs, cats, mice, rats, monkeys and apes, and transgenic species thereof, preferably humans.

[0297] 3.6 Antigens and neoantigens In the present invention, it is preferred that the T cells contained in the population of lymphocytes specifically recognize one or more predetermined antigens, which can be achieved by exposing the lymphocytes to the predetermined antigens during the culture process, which will promote the expansion of T cells that specifically recognize these antigens.

[0298] As disclosed in more detail above, antigen is preferably presented to lymphocytes by antigen presenting cells, particularly B cells.Methods for achieving the presentation of specific antigens by APC are disclosed herein and include genetic engineering of APC or adding synthetic peptides to APC.Alternatively, homogenized tumor samples can be added to APC.

[0299] Neoantigens arise as a result of somatic mutations in tumor cells, and are therefore expressed only in tumor cells, not in normal cells. Because normal cells do not express neoantigens, they are considered non-self by the immune system. As a result, targeted neoantigens do not easily induce autoimmunity. Therefore, neoantigens are ideal targets for therapeutic cancer vaccines and T cell-based cancer immunotherapy. By exploiting the immune activity of neoantigens, synthetic neoantigen drugs can be designed according to the tumor cell mutation context to achieve therapeutic effects.

[0300] In certain embodiments, the presented antigen is retrieved by sequencing the tumor or peripheral blood cells or other possible antigen sources (e.g., tumor samples, or samples of infected tissues) of the patient to be treated, and identified by suitable algorithms. Such algorithms are well known in the art, and include, for example, Neon (Neon Therapeutics) and Achilles (Achilles Therapeutics). The identification of neo-antigens in tumor samples is disclosed in, but not limited to, WO2017 / 106638, WO2011 / 143656, WO2017 / 011660, WO2018 / 213803 or WO2021 / 116714, and these reference patent documents are fully incorporated herein by reference.

[0301] Neo-antigen peptides that may be used in the methods according to the present invention are disclosed in WO2016 / 187508, which is hereby incorporated by reference in its entirety.

[0302] Within the method according to the invention, it is preferred to contact lymphocytes, preferably APCs, with a pool of chemically synthesised peptides.

[0303] The pool of chemically synthesized peptides can be specifically designed for the subject to be treated with the population of lymphocytes. For example, the pool of peptides can include multiple antigen and / or neo-antigen peptides known to be associated with the particular type of cancer the subject is suffering from.

[0304] Alternatively, the pool of peptides may be personalized for a subject suffering from cancer, i.e., the pool of peptides may include antigen and / or neo-antigenic peptides identified as present in the subject's tumor.

[0305] The pool of peptides may also comprise a mixture of "known" and "personalized" antigenic and / or neo-antigenic peptides.

[0306] Preferably, the pool of chemically synthesized peptides consists of or comprises neo-antigenic peptides. More preferably, the neo-antigenic peptides contained in the pool of chemically synthesized peptides are those identified in a tumor sample of the same subject from which the lymphocytes for the culture process were taken.

[0307] The identified neo-antigens can range in length from between 6-20 amino acids or between 9-25 amino acids. Alternatively, complete MHC complexes (maximum size 45 KDa) carrying the neo-antigen peptide can be contacted with a population of cells. In certain embodiments, the invention also encompasses the use of antigens described herein (whether known or identified according to the methods of the invention) to attract and collect peripheral immune cells (including T cells, B cells, NK cells or macrophages).

[0308] In certain embodiments, neoantigens are not individually identified, but rather are presented by adding samples of tumor or infected tissue, particularly encapsulated samples, to lymphocyte cultures.

[0309] 3.7 Genetic manipulation One or more cells useful in the methods disclosed herein, such as lymphocytes (preferably human lymphocytes, more preferably primary human lymphocytes, most preferably including primary human T cells (TILs)), feed cells and / or APCs (e.g. B cells), can be genetically engineered to present a desired antigen suitable for stimulating and / or activating T cells specific for that antigen. The genetically engineered lymphocytes may transiently or stably express the encoded polypeptide. Expression may be constitutive or constitutional depending on the system used, as known in the art. The encoding nucleic acid may or may not be stably integrated into the genome of the engineered cell.

[0310] Methods for genetically engineering cells (e.g., feeder cells and / or one or more APCs, e.g., B cells) to express a polypeptide of interest are known in the art and can generally be divided into physical, chemical and biological methods. The method appropriate for a given cell type and intended use can be easily determined by the skilled artisan using prevailing general knowledge. Such methods for genetically engineering cells by introduction of a nucleic acid molecule / sequence (e.g., in an expression vector) encoding a polypeptide of interest include, but are not limited to, chemical and electroporation methods, calcium phosphate methods, cationic lipid methods, and liposome methods. The nucleic acid molecule / sequence to be transduced can be conventionally and highly efficiently transduced by using commercially available transfection reagents and / or by any suitable method known in the art or described herein. In addition to the method of genetically engineering cells with a nucleic acid molecule that comprises or consists of a DNA sequence, the method disclosed herein can also be carried out using mRNA transfection. "MRNA transfection" refers to a method well known to those skilled in the art for transiently expressing a protein of interest.

[0311] Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like; see, e.g., Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, vols. 1-4, Cold Spring Harbor Press, NY.

[0312] Biological methods for introducing polynucleotide of interest into host cells include the use of DNA and RNA vectors.Viral vectors, especially retroviral vectors, have become the most widely used method for inserting genes into mammalian cells.Therefore, retroviral vectors are preferred for use in the methods and cells disclosed herein.Viral vectors can be derived from a variety of different viruses, including but not limited to lentivirus, poxvirus, herpes simplex virus type I, adenovirus and adeno-associated virus; see, for example, U.S. Patent No. 5,350,674 and U.S. Patent No. 5,585,362. Non-limiting examples of retroviral vectors suitable for transducing T cells include SAMEN CMV / SRa (Clay et al., J. Immunol. 163 (1999), 507-513), LZRS-id3-IHRES (Heemskerk et al., J. Exp. Med. 186 (1997), 1597-1602), FeLV (Neil et al., Nature 308 (1984), 814-820), SAX (Kantoff et al., Proc. Natl. Acad. Sci. USA 83 (1986), 6563-6567), pDOL (Desiderio, J. Exp. Med. 167 (1988), 372-388), N2 (Kasid et al., Proc. Natl. Acad. Sci. USA 27 (2001), 111-113), and the like. 87 (1990), 473-477), LNL6 (Tiberghien et al., Blood 84 (1994), 1333-1341), pZipNEO (Chen et al., J. Immunol. 153 (1994), 3630-3638), LASN (Mullen et al., Hum. Gene Ther. 7 (1996), 1123-1129), pG1XsNa (Taylor et al., J. Exp. Med. 184 (1996), 2031-2036), LCNX (Sun et al., Hum. Gene Ther. 8 (1997), 1041-1048), SFG (Gallardo et al., Blood 90 (1997), LXSN (Sun et al., Hum. Gene Ther.8 (1997), 1041-1048), SFG (Gallardo et al., Blood 90 (1997), 952-957), HMB-Hb-Hu (Vieillard et al., Proc. Natl. Acad. Sci. USA 94 (1997), 11595-11600), pMV7 (Cochlovius et al., Cancer Immunol. Immunother. 46 (1998), 61-66), pSTITCH (Weitjens et al., Gene Ther 5 (1998), 1195-1203), pLZR (Yang et al., Hum. Gene Ther. 10 (1999), 123-132), pBAG (Wu et al., Hum. Gene Ther. Ther. 10 (1999), 977-982), rKat.43.267bn (Gilham et al., J. Immunother. 25 (2002), 139-151), pLGSN (Engels et al., Hum. Gene Ther. 14 (2003), 1155-1168), pMP71 (Engels et al., Hum. Gene Ther. 14 (2003), 1155-1168), pGCSAM (Morgan et al., J. Immunol. 171 (2003), 3287-3295), pMSGV (Zhao et al., J. Immunol. 174 (2005), 4415-4423), or pMX (de Witte et al., J. Immunol. 181 (2008), 5128-5136). Most preferred are lentiviral vectors. Non-limiting examples of suitable lentiviral vectors for transducing T cells include, for example, PL-SIN lentiviral vectors (Hotta et al., Nat Methods. 6 (2009), 370-376), p156RRL-sinPPT-CMV-GFP-PRE / NheI (Campeau et al., PLoS One vol. 4 (2009), e6529), pCMVR8.74 (Addgene Catalog No. 22036), FUGW (Lois et al., Science 295 (2002), 868-872, pLVX-EF1 (Addgene Catalog No. 64368), pLVE (Brunger et al., Proc Natl Acad Sci USA 111 (2014), E798-806), pCDH1-MCS1-EF1 (Hu et al., Mol Cancer Res.7 (2009), 1756-1770), pSLIK (Wang et al., Nat Cell Biol. 16 (2014), 345-356), pLIM1 (Solomon et al., Nat Genet. 45 (2013), 1428-30), pLX302 (Kang et al., Sci Signal. 6 (2013), rs13), pHR-IG (Xie et al., J Cereb Blood Flow Metab. 33 (2013), 1875-85), pRRLSIN (Addgene catalog no. 62053), pLS (Miyoshi et al., J Virol. 72 (1998), 8150-8157), pLL3.7 (Lazebnik et al., J Biol. Chem. 283 (2008), 11078-82), FRIG (Raissi et al., Mol Cell Neurosci. 57 (2013), 23-32), pWPT (Ritz-Laser et al., Diabetologia. 46 (2003), 810-821), pBOB (Marr et al., J Mol Neurosci. 22 (2004), 5-11), and pLEX (Addgene catalog number 27976).

[0313] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes.Exemplary colloidal systems for use as delivery vehicles in vitro and in vivo are liposomes (e.g., artificial membrane vesicles).Other state-of-the-art targeted delivery methods of nucleic acids are available, such as delivery of polynucleotides in targeted nanoparticles or other suitable submicron-sized delivery systems.

[0314] Regardless of the method used to introduce exogenous nucleic acid into host cells (e.g., lymphocytes (preferably human lymphocytes, more preferably primary human lymphocytes, most preferably including primary human T cells (TILs)), feeder cells and / or APCs (e.g., B cells)), a variety of assays can be performed to confirm the presence of recombinant DNA sequences in the target cells (i.e., to confirm that the cells have been genetically engineered according to the methods disclosed herein). Such assays include, for example, "molecular biological" assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; "biochemical" assays, such as , detection of the presence or absence of a particular polypeptide, for example, by immunological means (ELISA and / or Western blot), or by assays described herein to identify whether cells exhibit properties or activities associated with an engineered polypeptide, i.e., assays to evaluate whether lymphocytes (more preferably human primary lymphocytes, e.g., NK cells or T cells) exhibit CCR8 activity. It is recognized that such assays are also applicable to testing expression and / or endogenous activity of endogenously expressed proteins, for example, to evaluate and / or select populations based on endogenous function.

[0315] The cells of the invention can be engineered with nucleic acid molecules to express other polypeptides believed or known to be useful in adoptive lymphocyte therapy, for example, with nucleic acid sequences encoding exogenous T cell receptors, chimeric antigen receptors (CARs) specific for the tumor of interest, exogenous cytokine receptors (whose sequences may or may not be modified compared to the endogenous / wild-type sequence), and / or endogenous cytokine receptors having modified sequences compared to the wild-type sequence (i.e., modified endogenous cytokine receptors). Alternatively or in addition, one or more of the T cells in the population of the invention can be further genetically modified to prevent expression of an endogenous T cell receptor, such that the receptor is not expressed or is expressed at a reduced level compared to T cells without such modification.

[0316] As used herein, "foreign T cell receptor" or "foreign TCR" refers to a TCR whose sequence is introduced into the genome of a lymphocyte (preferably a human lymphocyte, more preferably a primary human lymphocyte, most preferably including a primary human T cell ((TIL)) that may or may not endogenously express that TCR. Expression of a foreign TCR on an immune effector cell confers specificity for a particular epitope or antigen (e.g., an epitope or antigen that is preferentially present on the surface of cancer cells or other disease-causing cells). Such foreign T cell receptors can comprise alpha and beta chains, or alternatively may comprise gamma and delta chains. Foreign TCRs useful in the present invention can have specificity for any antigen or epitope of interest.

[0317] The lymphocyte (preferably comprising human lymphocytes, more preferably primary human lymphocytes, and most preferably primary human T cells ((TILs)) populations of the present invention can be further modified to express chimeric antigen receptors (also referred to as "CARs") as known in the art. Chimeric antigen receptors (CARs) are well known in the art and refer to engineered receptors that confer or graft specificity for an antigen onto lymphocytes (most preferably primary human T cells). CARs typically comprise an extracellular ligand-binding domain or portion and an intracellular domain that comprises one or more stimulatory domains that transduce signals necessary for lymphocyte (e.g., T cell) activation. In some embodiments, the extracellular ligand-binding domain or portion binds to a particular epitope or antigen (e.g., associated with cancer, e.g., The chimeric antigen receptor may be in the form of a single chain variable fragment (scFv) derived from a monoclonal antibody that provides specificity for an antigen or epitope of interest (epitope or antigen that is preferentially expressed on the surface of cancer cells or other disease-causing cells). The extracellular ligand-binding domain may be specific for an antigen or epitope of interest. The intracellular stimulatory domain typically comprises the intracellular domain signaling domain of a non-TCR T cell stimulatory / agonist receptor. Such cytoplasmic signaling domains may include, for example, but are not limited to, the intracellular signaling domains of CD3zeta, CD28, 4-1BB, OX40, or combinations thereof. The chimeric antigen receptor may further comprise additional structural elements, including a transmembrane domain that is connected to the extracellular ligand-binding domain via a hinge or spacer sequence.

[0318] One or more lymphocytes in the population of lymphocytes of the invention (preferably including human lymphocytes, more preferably primary human lymphocytes, and most preferably primary human T cells ((TILs)) can be genetically modified to express one or more additional exogenous cytokine receptors (which may have a wild-type sequence or may have an amino acid sequence that is modified compared to that of the endogenous / wild-type sequence) and / or one or more endogenous cytokine receptors having a sequence that is modified from that of the endogenous sequence. As used herein, "exogenous cytokine receptor" refers to a cytokine receptor whose sequence is introduced into the genome of a lymphocyte (preferably including human lymphocytes, more preferably primary human lymphocytes, and most preferably primary human T cells ((TILs)) that does not endogenously express that receptor. Similarly, "endogenous cytokine receptor" refers to a receptor whose sequence is introduced into the genome of such lymphocytes that endogenously express that receptor. The introduced exogenous or endogenous cytokine receptor can be modified to alter the function of the receptor normally exhibited in its endogenous environment. For example, dominant-negative mutants of a receptor are known to bind a ligand, but the ligand-receptor interaction does not elicit the endogenous activity normally associated with such an interaction. Expression of an exogenous cytokine receptor (modified or not) and / or a modified endogenous receptor can confer a ligand-specific activity not normally exhibited by the lymphocyte, or, in the case of a dominant-negative modification, can act as a ligand sink to bind the cytokine and prevent and / or reduce the ligand-specific activity.

[0319] 3.8 Non-alloreactive T cells The population of lymphocytes (preferably human lymphocytes, more preferably primary human lymphocytes, most preferably primary human T cells (e.g., TILs)) obtainable by the methods described herein are useful as medicines, for example in the treatment of cancer. They, and the treatments based on their use, can be either part of an autoimmunotherapy or part of an allogeneic immunotherapy treatment. As understood in the art, "autologous" in the context of immunotherapy methods refers to the situation where the origin of the population used in the treatment is from the patient to be treated, and the donor of the lymphocytes and the recipient of the immunotherapy (i.e., cell transfer) are the same. "Allogeneic" in the context of immunotherapy methods refers to the situation where the origin of the lymphocytes or the population of lymphocytes used in the immunotherapy is from a donor that is genetically distinct from the patient.

[0320] The population of lymphocytes that can be obtained by the method of the present invention and / or disclosed herein can be genetically modified before, during or after expansion so that they can be used in allogeneic treatment. As known in the art, this is not only an effort to promote proper engraftment, but also an effort to minimize undesirable graft-versus-host immune reactions. In the context of the present invention, such non-allo-reactive manipulation can be actively performed in combination with other genetic engineering methods herein, for example, such non-allo-reactive manipulation is performed before, in parallel with, or after the genetic engineering method (e.g., for the expression of foreign T cell receptors and / or CARs), and / or at any time during or after expansion. Thus, the method of the present invention can include the steps of obtaining a sample that is known or believed to contain lymphocytes (e.g., T cells (preferably TILs)) from a donor, and inactivating those genes involved in MHC recognition, as is known in the art. Such methods generally rely on the destruction of endogenous TCR. TCR contains two peptide chains, alpha and beta, which assemble to form a heterodimer that further associates with the CD3 transducing subunit to form the T cell receptor complex present on the cell surface. Each alpha and beta chain of the TCR consists of an immunoglobulin-like N-terminal variable (V) and constant (C) region, a hydrophobic transmembrane domain, and a short cytoplasmic region. For immunoglobulin molecules, the variable regions of the alpha and beta chains are generated by V(D)J recombination, which generates a large diversity of antigen specificity within a population of T cells. However, in stark contrast to immunoglobulins that recognize intact antigens, T cells are activated by processed peptide fragments associated with MHC molecules, thereby introducing an additional dimension to antigen recognition by T cells, known as MHC restriction. Differences in the recognition of donor and recipient MHC by the T cell receptor are responsible for T cell proliferation and the potential development of graft-versus-host immune responses that can manifest as graft-versus-host disease (GVHD) in severe cases.It is known that normal surface expression of the TCR depends on the coordinate synthesis and assembly of all seven components of the complex. Inactivation of the TCR alpha or TCR beta genes (and thus the expressed peptide) can result in the loss of the TCR from the surface of the T cell, which can prevent recognition of alloantigens (and thus GVHD), thereby rendering the cells non-allogeneic.

[0321] Alternatively, the non-allo-reactive engineering method may have been performed separately, such as to establish a universal, patient-independent source or cells that are available for purchase from a depository of prepared cells and can then be expanded according to the methods disclosed herein. Thus, the present invention also encompasses the use of lymphocytes (i.e., off-the-shelf lymphocytes), preferably primary lymphocytes, that have been purchased from a depository and / or that have already been engineered for expression of one or more desired peptides disclosed herein, e.g., engineered to express an exogenous TCR or CAR. Alternatively, the methods disclosed herein are applicable to non-allogeneic primary lymphocytes, preferably human lymphocytes, more preferably primary human lymphocytes, and most preferably primary human T cells ((TILs)), i.e., "off-the-shelf" primary human lymphocytes.

[0322] Similarly, the population of lymphocytes of the present invention or obtainable by the methods disclosed herein can additionally or alternatively be further engineered prior to, in parallel with, or after expansion to eliminate or reduce their ability to elicit an immune response and / or eliminate or reduce their recognition by the host immune system. This is an effort to minimize or eliminate host-versus-graft immune reactions. As with non-allo-reactive engineering, engineering of cells to reduce or eliminate their susceptibility to the host immune system (and / or their ability to elicit a host immune response) can be performed prior to, in parallel with, or after any other engineering method disclosed herein. As a non-limiting exemplary embodiment, engineering of cells to reduce or eliminate their susceptibility to the host immune system (and / or their ability to elicit a host immune response) can be performed by reducing or eliminating the expression of endogenous major histocompatibility complexes.

[0323] 3.9 Pharmaceutical Compositions In a particular embodiment, the present invention relates to a pharmaceutical composition comprising a population of lymphocytes according to the present invention.

[0324] The lymphocyte population of the present invention is intended for use in adoptive cell transfer (ACT) therapy in humans. That is, the cells contained in the lymphocyte population are preferably suspended in a liquid that is suitable for injection into the human body. Suitable liquids for suspending the cells contained in the lymphocyte population include, but are not limited to, pharma- ceutically acceptable buffers.

[0325] In certain embodiments, the pharma- ceutically acceptable buffer may be a sodium chloride buffer. In certain embodiments, the pharma-ceutically acceptable buffer may be a 0.9% NaCl buffer. In certain embodiments, the pharma-ceutically acceptable buffer may be supplemented with at least 5%, 10%, 15% or 20% DMSO to allow freezing of the lymphocyte population. In certain embodiments, the pharma-ceutically acceptable buffer may contain between 0 and 15% DMSO. That is, the pharma-ceutically acceptable buffer may contain 0.9% NaCl and 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15% DMSO.

[0326] Preferably, the pharmaceutical composition is substantially free of bacterial contaminants, especially mycoplasma. Absence of bacteria / mycoplasma can be tested by devices or kits known in the art, such as, but not limited to, BacTec device and / or MycoSeq kit. In addition, the pharmaceutical composition is preferably substantially free of endotoxin.

[0327] The term "medicament" is used interchangeably with the term "pharmaceutical composition" and relates to a composition suitable for administration to a patient, preferably a human patient. Thus, the present invention provides a population of lymphocytes (preferably human lymphocytes, more preferably primary human lymphocytes, most preferably including primary human T cells ((TILs) - which may or may not be further genetically engineered to express one or more desired peptides or receptors) for use as a medicament, and a method for producing such a population of lymphocytes for such use. The medicament / pharmaceutical composition may be administered to an allogeneic recipient, i.e. to a recipient that is a different individual that donates the T cells, or may be administered to an autologous recipient, i.e. in this case the recipient patient also donates the T cells. Alternatively, the medicament / pharmaceutical composition may comprise non-allogeneic lymphocytes ("off-the-shelf" lymphocytes as known in the art). Regardless of the patient's species, the donor and recipient (patient) are of the same species. Preferably, the patient / recipient is human.

[0328] In preparing a pharmaceutical formulation according to the invention, the expanded population of lymphocytes (preferably human lymphocytes, more preferably primary human lymphocytes, most preferably including primary human T cells ((TILs)) is typically mixed with a pharma- ceutically acceptable carrier, excipient and / or diluent, and the resulting composition is administered to a subject. The carrier must, of course, be acceptable in the sense of being compatible with the other components in the formulation and not deleterious to the subject or the engineered cells. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline, water, emulsions, such as oil / water emulsions, various types of wetting agents, sterile solutions, and the like. The carrier may be a solution that is isotonic with the blood of the recipient. Compositions containing such carriers may be formulated according to well-known conventional methods. The pharmaceutical compositions of the invention may further comprise one or more additional agents useful for the treatment of a disease in a subject. The pharmaceutical compositions of the invention may be administered in It may further include biomolecules known to be beneficial to lymphocyte function or activity, including, but not limited to, cytokines that promote cell proliferation and engraftment in vivo (e.g., IL-2, IL-7, IL-15, and / or IL-21). The lymphocyte populations of the present invention may be administered in the same composition as one or more additional agents or biomolecules, or alternatively, may be co-administered in separate compositions.

[0329] The pharmaceutical compositions described herein can be used in combination with chemotherapeutic agents. Exemplary chemotherapeutic agents include anthracyclines (e.g., doxorubicin (e.g., liposomal doxorubicin)), vinca alkaloids (e.g., vinblastine, vincristine, vindesine, vinorelbine), alkylating agents (e.g., cyclophosphamide, dacarbazine, melphalan, ifosfamide, temozolomide), immune cell antibodies (e.g., alemtuzumab, gemtuzumab, rituximab, ofatumumab, tositumomab, brentuximab, rit ... ximab), antimetabolites (including, for example, folate antagonists, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors (e.g., fludarabine)), mTOR inhibitors, TNFR glucocorticoid-inducible TNFR-related protein (GITR) agonists, proteasome inhibitors (e.g., aclacinomycin A, gliotoxin, or bortezomib), immunomodulators, such as thalidomide or thalidomide derivatives (e.g., lenalidomide).

[0330] Common chemotherapy agents that may be considered for use in combination therapy include anastrozole, bicalutamide, bleomycin sulfate, busulfan, capecitabine, N4-pentoxycarbonyl-5-deoxy-5-fluorocytidine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, cyclophosphamide, cytarabine, cytosine arabinoside, cytarabine liposome injection, dacarbazine, dactinomycin, daunorubicin hydrochloride, daunorubicin citrate liposome injection, dexamethasone, docetaxel, doxorubicin hydrochloride, etoposide, and fludarabine. These include ribacillin phosphate, 5-fluorouracil, flutamide, tezacitabine, gemcitabine, hydroxyurea (Hydrea®), idarubicin, ifosfamide, irinotecan, L-asparaginase, leucovorin calcium, melphalan, 6-mercaptopurine, methotrexate, mitoxantrone, mylotarg, paclitaxel, yttrium 90 / MX-DTPA, pentostatin, tamoxifen citrate, teniposide, 6-thioguanine, thiotepa, tirapazamine, topotecan hydrochloride, vinblastine, vincristine, and vinorelbine.

[0331] Anti-cancer drugs for use in combination with the lymphocyte populations of the present invention include, but are not limited to, anthracyclines; alkylating agents; metabolic antagonists; drugs that inhibit the calcium-dependent phosphatase calcineurin or the p70S6 kinase FK506 or that inhibit p70S6 kinase; mTOR inhibitors; immunomodulatory agents; anthracyclines; vinca alkaloids; proteosome inhibitors; GITR agonists; protein tyrosine phosphatase inhibitors; CDK4 kinase inhibitors; BTK inhibitors; MKN kinase inhibitors; DGK kinase inhibitors; or oncolytic viruses.

[0332] Exemplary antimetabolites include, but are not limited to, pyrimidine analogs, purine analogs, and adenosine deaminase inhibitors: methotrexate, 5-fluorouracil, floxuridine, cytarabine, 6-mercaptopurine, 6-thioguanine, fludarabine phosphate, pentostatin, pemetrexed, raltitrexed, cladribine, clofarabine, azacitidine, decitabine, and gemcitabine.

[0333] Exemplary alkylating agents include, but are not limited to, nitrogen mustards, uracil mustard, ethylenimine derivatives, alkylsulfonate esters, nitrosoureas, triazenes, chlormethine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, temozolomide, thiotepa, busulfan, carmustine, lomustine, streptozocin, dacarbazine, olanzapine ... These include xaliplatin, temozolomide, dactinomycin, melphalan, altretamine, carmustine, bendamustine, busulfan, carboplatin, lomustine, cisplatin, chlorambucil, cyclophosphamide, dacarbazine, altretamine, ifosfamide, prednumustine, procarbazine, mechlorethamine, streptozocin, thiotepa, cyclophosphamide, and bendamustine HCl.

[0334] 3.10 Therapeutic Applications The population of lymphocytes (preferably comprising human lymphocytes, more preferably primary human lymphocytes, and most preferably primary human T cells ((TILs)) of the present invention or obtainable by the methods disclosed herein are contemplated for use as a medicament in the treatment of diseases, including but not limited to cancer or precancerous conditions. The term "cancer" or "proliferative disease" as used herein means any disease, condition, trait, genotype or phenotype characterized by unregulated cell growth or replication as known in the art. Since the unique characteristics of a cancer / proliferative disease or precancerous condition are not relevant to the methods disclosed herein, i.e., since the population of lymphocytes is specifically expanded to be selective for a desired antigen, e.g., neo-antigen of a particular cancer, the cancer / proliferative disease that may be treated according to the methods disclosed herein and with the population of lymphocytes includes all types of tumors, lymphomas and carcinomas.

[0335] Non-limiting examples of such cancers include colorectal cancer, brain cancer, ovarian cancer, prostate cancer, pancreatic cancer, breast cancer, renal cancer, nasopharyngeal cancer, hepatocellular carcinoma, melanoma, skin cancer, oral cancer, head and neck cancer, esophageal cancer, gastric cancer, cervical cancer, bladder cancer, lymphoma, chronic or acute leukemia (e.g., of B, T and myeloid origin), sarcoma, lung cancer and multidrug resistant cancer.

[0336] The terms "treatment", "treating" and the like are used herein to generally mean obtaining a desired pharmacological and / or physiological effect. This effect may be prophylactic in that it completely or partially prevents a disease or its symptoms, and / or it may be therapeutic in that it partially or completely cures a disease or condition and / or adverse effects caused by a disease or condition. The term "treatment" as used herein encompasses any treatment of a disease or condition in a subject, including (a) preventing and / or ameliorating a proliferative disease (preferably cancer) in a subject who may be predisposed to the disease; (b) suppressing the disease, i.e., arresting its progression, e.g., suppressing the progression of cancer; (c) alleviating the disease, i.e., causing regression of the disease, e.g., suppression of cancer; and / or (d) preventing, suppressing, or alleviating any symptoms or adverse effects associated with the disease or condition. Preferably, the term "treatment" as used herein relates to medical intervention in an already manifest disorder, e.g., treatment of a diagnosed cancer.

[0337] The treatment or therapy (i.e., including the use of a medicament / pharmaceutical composition comprising a population of lymphocytes disclosed herein or obtainable by a method disclosed herein) can be administered alone or in combination with a suitable treatment protocol for a particular disease or condition known in the art. Non-limiting examples of such protocols include, but are not limited to, administration of analgesics, administration of chemotherapeutic agents, radiation therapy, and surgical management of a disease, condition, or a symptom thereof. Thus, the treatment regimen disclosed herein includes administration of a population of lymphocytes as disclosed herein or obtainable by a method disclosed herein, together with one such treatment protocol, or together with one or more such treatment protocols, without any of the treatment protocols described herein or known in the art suitable for the treatment or prevention of either disease, condition, or a symptom thereof. Administration "in combination" or use "with" other known therapies includes administration of the medicament / pharmaceutical composition of the present invention before, during, after, or in parallel with any of the combination therapies described herein or known in the art. The pharmaceutical compositions / medicaments disclosed herein may be administered during active disease or during periods of remission or low disease activity, alone or in combination with other therapies or treatments.

[0338] When administered in combination, the population of lymphocytes of the present invention or obtainable by the method of the present invention may be administered in an amount or dosage that is greater than, less than, or the same as the amount or dosage that each treatment or agent would be used individually, e.g., as a monotherapy. In certain embodiments, the amount or dosage of the lymphocyte therapy and / or at least one additional agent or treatment is less (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dosage of the corresponding treatment or agent used individually.

[0339] The population of lymphocytes of the present invention and / or obtainable by the methods disclosed herein can further be rendered resistant to chemotherapeutic agents used as standard treatments as described herein or known in the art. Engineering the population of lymphocytes of the present invention to be such resistant is expected to aid in the selection and expansion of such engineered lymphocytes in vivo in patients undergoing chemotherapy or immunosuppression.

[0340] The population of lymphocytes obtainable by the methods of the present invention and / or disclosed herein can undergo robust in vivo T cell expansion upon administration to a patient and can remain / persist in body fluids for an extended period of time, preferably one week, more preferably two weeks, and even more preferably at least one month. The population of lymphocytes obtainable by the methods of the present invention and / or disclosed herein can also be further manipulated with a safety switch allowing for potential administration of cell therapy. Such safety switches potentially useful for cell therapy are known in the art and include (but are not limited to) engineering cells to express targets that allow for antibody removal (e.g., truncated EGFR; Paszkiewicz et al., J Clin Invest 126 (2016), 4262-4272), introduction of artificial targets for small molecule inhibitors (e.g., HSV-TK; Liang et al., Nature 563 (2018), 701-704), and introduction of inducible cell death genes (e.g., icaspase; Minagawa et al., Methods Mol Biol 1895 (2019), 57-73).

[0341] Administration of the population of lymphocytes according to the invention can be by any conventional method, including by aerosol inhalation, injection, oral ingestion, infusion, implantation or transplantation. The medicaments and compositions described herein can be administered subcutaneously, intradermally, intratumorally, intranodal, intramedullary, intramuscularly, intravenously or intralymphatic injection, or intraperitoneally. The lymphocytes, medicaments and / or compositions of the invention are preferably administered by intravenous injection.

[0342] The dosage regimen will be determined by the attending physician and clinical factors. As is well known in the medical arts, the dosage for any one patient depends on many factors, including the patient's size, body surface area, age, the specific compound to be administered, sex, frequency and route of administration, overall health, and other drugs administered in parallel. For example, the lymphocyte population of the present invention and / or obtainable by the method disclosed herein may be administered to a subject in the form of 10 4 ~10 10 T cells / kg body weight, preferably 10 5 ~10 6 In the context of the present invention, lymphocytes may be administered at a dose of about 10 T cells / kg body weight. 5 ~10 6 Starting at a target dose of T cells / kg body weight, followed by 10 10 The cells, or population of cells, may be administered in one dose or multiple doses, such as by increasing the dose of T cells / kg body weight.

[0343] In certain embodiments, the present invention provides a method for treating cancer, comprising: a) providing a population of lymphocytes according to the invention or a pharmaceutical composition according to the invention; and b) injecting the population of lymphocytes or the pharmaceutical composition into a subject suffering from cancer. The present invention relates to a method comprising the steps of:

[0344] It is preferred herein that the population of lymphocytes or the pharmaceutical composition according to the invention is used in autologous cell therapy, in particular for the treatment of cancer. That is, it is preferred herein that the lymphocytes contained in the population of lymphocytes or the pharmaceutical composition according to the invention are obtained by expanding a sample of lymphocytes taken from a subject suffering from cancer. The population of lymphocytes, preferably in the form of a pharmaceutical composition, can then be infused back into the same subject.

[0345] When used in autologous cell therapy, the lymphocytes in the composition of lymphocytes preferably specifically attack the tumor of the subject.To do so, at least a portion of the lymphocytes in the population of lymphocytes must recognize an antigen present in the tumor of the subject.To ensure that at least a portion of the lymphocytes in the population of lymphocytes recognizes an antigen present in the tumor of the subject, the lymphocytes are preferably expanded in the presence of an antigen peptide previously identified as present in the tumor of the subject.

[0346] Thus, in certain embodiments, the present invention provides a method for treating cancer in a subject, comprising: a) surgically removing a tumor from a subject or taking a biopsy from a tumor in a subject; b) identifying at least one tumor antigen in the tumor sample obtained in step (a); c) expanding lymphocytes in the tumor sample obtained in step (a) by the method according to the invention, in the presence of at least the antigen identified in step (b) as being present in the tumor sample; d) injecting the expanded lymphocytes into the subject from whom the tumor sample was taken. The present invention relates to a method comprising the steps of:

[0347] The term "tumor antigen" as used throughout the present specification refers to an antigen that is uniquely or differentially expressed by tumor cells, whether intracellularly or on the tumor cell surface (preferably on the tumor cell surface), compared to normal or non-neoplastic cells. For example, tumor antigens may be present in or on tumor cells, and may not normally be present in or on normal cells or non-neoplastic cells (e.g., may only be expressed by a limited number of normal tissues, such as testis and / or placenta), or tumor antigens may be present in or on tumor cells in greater amounts than in or on normal or non-neoplastic cells, or tumor antigens may be present in or on tumor cells in a form different from that found in or on normal or non-neoplastic cells. Thus, the term includes tumor-specific antigens (TSAs), including tumor-specific membrane antigens, tumor-associated antigens (TAAs), including tumor-associated membrane antigens, tumor-embryo antigens, growth factor receptors, growth factor ligands, and the like. The term further includes cancer / testis (CT) antigens.

[0348] Examples of tumor antigens include, but are not limited to, β-human chorionic gonadotropin (βHCG), glycoprotein 100 (gp100 / Pmel17), carcinoembryonic antigen (CEA), tyrosinase, tyrosinase-related protein 1 (gp75 / TRP-1), tyrosinase-related protein 2 (TRP-2), NY-BR-1, NY-CO-58, NY-ESO-1, MN / gp250, idiotype, telomerase, synovial sarcoma X breakpoint 2 (SSX2), murine sarcoma idiotype 2 (MSX2), and cytoplasmic sarcoma idiotype 2 (CYP2A1). Tumor antigens include melanoma antigen 1 (MUC1), melanoma-associated antigen (MAGE) family of antigens, high molecular weight melanoma-associated antigen (HMW-MAA), melanoma antigen 1 recognized by T cells (MART1), Wilms tumor gene 1 (WT1), HER2 / neu, mesothelin (MSLN), alpha fetoprotein (AFP), cancer antigen 125 (CA-125), and aberrant forms of ras or p53 (see also WO2016187508A2). Tumor antigens can also be subject-specific (e.g., subject-specific neoantigens; see, e.g., U.S. Patent No. 9,115,402, and International Patent Application Publication Nos. WO2016 / 100977, WO2014 / 168874, WO2015 / 085233, and WO2015 / 095811).

[0349] In a preferred embodiment, the population of lymphocytes for use in the treatment of cancer comprises neo-TILs. Neo-TILs are tumor-infiltrating lymphocytes, preferably T cells, that specifically recognize neo-antigens. Neo-TILs can be specifically expanded by contacting tumor samples, or T cells obtained from tumor samples, with neo-antigen peptides as described in more detail herein. Preferably, the presence of neo-antigens has been confirmed in patients receiving the population of lymphocytes that comprises neo-TILs.

[0350] In the above detailed description of the invention, numerous individual elements, characterizing features, techniques and / or steps are disclosed. It is readily apparent that each of these has merit not only when considered or used individually and alone, but also when considered or used in combination with one another. Thus, to avoid excessively repetitive and lengthy text, the present description refrains from reiterating all possible combinations and permutations. However, whether expressly stated or not, such combinations are fully within the scope of the presently disclosed subject matter.

[0351] Technical and scientific terms used herein are intended to have the same meaning as commonly understood by those skilled in the art, unless otherwise defined. References to technology used herein are intended to refer to technology as commonly understood in the art, including variations of the technology or equivalent technology substitutions that would be apparent to one of ordinary skill in the art.

[0352] 4. Working Example 4.1 Preparation of B cells B cells are obtained from frozen apheresis samples. After thawing, the apheresis samples are washed and B cells are isolated using a commercially available B cell isolation kit. The isolated B cells are then activated by adding IL-4 (final concentration: 200 IU / ml) and CD40L (final concentration: 1 μg / ml).

[0353] After the activation step and before contacting with T lymphocytes, the B cells are transfected with mRNAs encoding 4-1BB, OX40L and IL-12 by mixing the B cells with the mRNAs and transfecting the cells using an electroporation device and a suitable electroporation buffer.

[0354] The electroporated B cells are resuspended in medium supplemented with 200 μg / mL Pen-Strep and 10% human AB serum (hAB). The resuspended B cells are stored or used directly for the expansion of T lymphocytes as antigen presenting cells (APCs).

[0355] The goal is 100 x 10 6 The aim is to prepare 100 B cells in a volume of 40 mL.

[0356] 4.2 Tumor sample preparation Tumor specimens (fresh or cryopreserved) were cut into small pieces (1–3 mm 3 The aim is to prepare 60 tumor fragments in 50 mL of supplemented medium.

[0357] Alternatively, tumor samples are dissociated using commercially available kits (which include a step of enzymatic digestion of the tumor sample) and the resulting lymphocytes are prepared in supplemented medium.

[0358] 4.3 Preparation of peptide solutions Prepare stock solutions of chemically synthesized peptides (a peptide library containing 2-100 different peptides with lengths of 9-25 amino acids). The desired peptide stock concentration is 100 µg / mL dissolved in 20% DMSO.

[0359] 4.4 Expansion of T lymphocytes 60 tumor fragments or equivalent and electroporated B cells are seeded in appropriate medium into an ADVA bioreactor (ADVA biotechnology).

[0360] - 100 × 10 in 40 mL of medium supplemented with 200 μg / mL Pen-Strep and 10% human AB serum (hABS). 6 B cells (see section 4.1).

[0361] - 60 tumor fragments (1-2 mm) in 50 mL of medium supplemented with 200 μg / mL Pen-Strep, 10% hABS and 6000 iU / mL IL-2. 3 ) (see Section 4.2).

[0362] B cells and tumor fragments are cultured in ADVA X3 bioreactors in batch mode for 1 day. (pH and dO are monitored and CO2 / O2 in the headspace of the growth chamber is adjusted as needed. After 24 hours, peptides are added to the ADVA X3 bioreactors.

[0363] Continue in batch mode while monitoring pH, dO, glucose and lactate concentrations. Increase culture volume by adding fresh medium to keep all four parameters within range.

[0364] Day 10: Activate lymphocytes (+ / - 5 days) - 15 mL of activation medium containing the anti-CD3 antibody OKT3 is added to obtain a final OKT3 concentration of 100 ng / mL in the culture.

[0365] Thereafter, IL-2 is added every 3 days to keep IL-2 concentrations high. Continue enriching the culture medium based on pH, DO, glucose and lactate concentrations. Switch from fed-batch mode to recirculation mode and finally to perfusion mode based on process parameters. Harvest cells with ADVA X3, change media and prepare cells for final formulation. Dispense / aliquot formulated cells and freeze for storage until analysis.

Claims

1. A method for expanding a population of lymphocytes specific for one or more antigens in a single, controlled culture vessel, comprising: a) culturing a tissue or blood sample from a subject, known or suspected to contain lymphocytes, in the presence of said one or more antigens; or b) culturing lymphocytes isolated from a tissue or blood sample from a subject in the presence of said one or more antigens wherein the lymphocytes are cultured in a conditioned culture medium.

2. The method according to claim 1, wherein the conditioned culture medium is a culture medium in which at least one of the following parameters: pH, dissolved oxygen (DO) concentration, glucose concentration, lactate concentration and / or temperature is monitored and adjusted as necessary.

3. The method according to claim 2, wherein the conditioned culture medium is a culture medium in which pH, dissolved oxygen (DO) concentration, glucose concentration, lactate concentration and temperature are monitored and adjusted as necessary.

4. The method according to claim 1, further comprising adjusting the culture volume according to the expansion rate of the lymphocytes.

5. The method according to claim 4, wherein the culture volume increases by at least 2, 3, 4, 5 or 6 times during the expansion of the lymphocytes.

6. The method according to claim 1, further comprising dynamically culturing the lymphocytes using the conditioned culture medium.

7. The method according to claim 1, wherein the tissue sample is derived from a tumor, in particular, the tissue sample is a tumor sample.

8. The method according to claim 7, wherein the tumor contains at least one neoantigen.

9. The method according to claim 1, wherein the lymphocytes include tumor-infiltrating lymphocytes, in particular, the tumor-infiltrating lymphocytes are T cells.

10. The method according to claim 1, wherein one or more antigens are added to the culture medium in the form of peptides.

11. The method according to claim 10, wherein the peptide is added to the culture medium at a concentration of 0.1 to 10 μg / ml.

12. The method according to claim 1, wherein the culturing step comprises co-culturing the lymphocytes and antigen-presenting cells (APCs).

13. The method according to claim 12, wherein the antigen-presenting cells (APCs) are engineered to present one or more antigens.

14. The method according to claim 12, wherein the antigen-presenting cells (APCs) comprise B cells or are B cells.

15. The method according to claim 14, wherein the B cells are obtained by apheresis.

16. The method according to claim 14, wherein the B cells are activated prior to addition to the lymphocytes.

17. The method according to claim 16, wherein the B cells are activated with IL-4 and / or CD40L.

18. The method according to claim 12, wherein the antigen-presenting cells (APCs) are genetically engineered to express one or more transgenes.

19. The method according to claim 18, wherein the genetically engineered APCs are obtained by transfecting the APCs with a nucleic acid encoding the one or more transgenes.

20. The method according to claim 18, wherein at least one of the one or more transgenes encodes an immunomodulatory factor. **Claim 21** The method according to claim 20, wherein the immunomodulatory factor is selected from the group consisting of OX40L, 4-1BBL, CD80, CD86, CD83, CD70, CD40L, GITR-L, CD127L, CD30L (CD153), LIGHT, BTLA, ICOS-L (CD275), SLAM (CD150), CD662L, interleukin-12, interleukin-7, interleukin-15, interleukin-17, interleukin-21, interleukin-4, Bcl6, BCLXL, BCL-2, MCL1, STAT-5, and activators of one or more signaling pathways (e.g., JAK / STAT pathway, Akt / PKB signaling pathway, BCR signaling pathway, and / or BAFF / BAFFR signaling pathway). **Claim 22** The method according to claim 20, wherein the immunomodulatory factor is one or more of OX40L, 4-1BB, and / or interleukin 12. **Claim 23** The method according to claim 7, wherein the presence of at least one of the one or more antigens has been confirmed in the tumor sample containing the lymphocytes prior to the culturing step. **Claim 24** The method according to claim 7, wherein at least one of the one or more antigens is a neoantigen, and the presence of the neoantigen has been confirmed in the tumor sample containing the lymphocytes prior to the culturing step. **Claim 25** The method according to claim 23 or 24, wherein confirming the presence of at least one of the one or more antigens in the tumor sample comprises sequencing genomic DNA obtained from the tumor sample. **Claim 26** The method according to any one of claims 1 to 24, comprising the step of activating the lymphocytes during culturing. **Claim 27** The method according to claim 26, wherein the activating step comprises adding a CD3 agonist to the culture medium. **Claim 28**: The method according to claim 27, wherein the CD3 agonist is added to the culture medium 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 days later. **Claim 29**: The method according to claim 1, wherein the culture medium is supplemented with human AB serum and / or IL-2. **Claim 30**: The method according to claim 1, wherein the culturing step is continued until the T cell population reaches at least 10^7 cells. **Claim 31**: The method according to claim 1, wherein the culturing step is performed at a temperature higher than 0°C. **Claim 32**: The method according to claim 1, wherein the sample or the lymphocytes are maintained at a temperature higher than 0°C after isolation from the subject and before culturing. **Claim 33**: A population of lymphocytes obtainable by the method according to claim 1. **Claim 34**: A population of lymphocytes comprising at least 90% CD3+ T cells and less than 5% B cells, wherein at least 70% of the T cell portion is viable, at least 20% is CD27 / CD28 double positive, and less than 10% is triple positive for CD45RA, CD57 and KLRG1. **Claim 35**: The population of lymphocytes according to claim 34, wherein the T cells are specific for one or more antigens. **Claim 36**: The population of lymphocytes according to claim 34, wherein less than 15% of the T cell portion secretes at least one protein from the group consisting of TNF-α, IL-4 and IL-5. **Claim 37**: The population of lymphocytes according to claim 34, wherein at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the T cells in the T cell portion are CD8+ T cells. **Claim 38**: The population of lymphocytes according to claim 34, wherein at least two T cells in the T cell portion are directed against different antigens. **Claim 39**: The population of lymphocytes according to claim 38, wherein at least one antigen is a neoantigen.

40. The population of lymphocytes according to claim 34, wherein the T cell portion comprises at least 10^7 T cells.

41. A pharmaceutical composition comprising the population of lymphocytes according to claim 33.

42. A pharmaceutical composition comprising the population of lymphocytes according to claim 34.

43. The pharmaceutical composition according to claim 41 or 42, wherein the lymphocytes are suspended in a pharmaceutically acceptable buffer.

44. The pharmaceutical composition according to claim 43, wherein the pharmaceutically acceptable buffer comprises about 0.9% NaCl and, optionally, up to 15% DMSO.

45. The population of lymphocytes according to claim 33 or 34 or the pharmaceutical composition according to claim 41 or 42 for use as a medicament.

46. The population of lymphocytes according to claim 33 or 34 or the pharmaceutical composition according to claim 41 or 42 for use in the treatment of cancer.

47. The population of lymphocytes or the pharmaceutical composition for use according to claim 46, wherein the cancer treatment is adoptive cell therapy.

48. The population of lymphocytes or the pharmaceutical composition for use according to claim 46, wherein the cancer treatment is autologous cell therapy.

49. For injecting into a subject suffering from cancer for treating the cancer of the subject, the population of lymphocytes according to claim 33 or 34 or the pharmaceutical composition according to claim 41 or 42.

50. A method for producing lymphocytes for injecting into a subject suffering from cancer for treating the cancer of the subject, comprising: a) surgically removing a tumor from the subject or taking a biopsy from the subject's tumor; b) identifying at least one tumor antigen in the tumor sample obtained in step (a); c) expanding the lymphocytes contained in the tumor sample obtained in step (a) by the method according to claim 1, wherein the lymphocytes are expanded in the presence of at least one tumor antigen identified in step (b) as being present in the tumor sample; A method comprising the steps of: **Claim 51**: The method according to claim 50, wherein the tumor antigen is a tumor-associated antigen or a tumor-specific antigen. **Claim 52**: The method according to claim 50, wherein the lymphocytes include tumor-infiltrating lymphocytes (TIL). **Claim 53**: The method according to claim 52, wherein the TIL specifically recognizes one or more tumor antigens. **Claim 54**: The method according to claim 53, wherein at least one tumor antigen is a neoantigen.