Lymphocyte expansion

Abstract

The present invention relates to a method for expanding a population of lymphocytes 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; or (b) culturing lymphocytes, the lymphocytes being lymphocytes isolated from a tissue or blood sample from the subject; wherein the lymphocytes are expanded in a culture medium in which at least one of the following parameters is monitored and adjusted to a predefined value or range: pH, dissolved oxygen (DO) concentration, glucose concentration, lactate concentration, and / or temperature; and wherein the method comprises adjusting the culture volume to match the rate of lymphocyte expansion. Furthermore, 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 the T cell portion is viable and, optionally, less than 10% are triple positive for CD45RA, CD57, and KLRG1.

Description

[Technical Field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application is an international PCT application claiming priority to and benefit of European Patent Application No. 22205178.1, filed November 2, 2022, the entire contents of which are incorporated herein by reference.

[0002] 2. Incorporation by Reference to Sequence Listing This application contains a Sequence Listing that has been submitted via the Patent Center. The Sequence Listing, entitled AF1840 PCT BS.xml, created on November 1, 2023, and which is 180,550 bytes in size, is hereby incorporated by reference in its entirety. [Background technology]

[0003] 3.Background The present invention relates to lymphocytes for use in targeted tumor immunotherapy, such as adoptive T cell therapy, including CAR-T cell therapy, as well as production methods and kits containing such cells. The lymphocytes are preferably human lymphocytes, such as T cells, NK cells, or NKT cells, including CD3+ T cells, CD8+ T cells, CD4+ T cells, and γδ T cells. Most preferably, the cells of the present invention are primary human T cells. The present invention provides suitable lymphocyte populations that exhibit a specific marker profile (i.e., low CD45RA / CD57 / KLRG1 expression) and specificity for one or more defined antigens. Such antigens may be antigens specific to pathologies, including infectious diseases (e.g., viral or bacterial infections) and cancer, and / or neoantigens selected from known neoantigens or identified in samples collected from subjects, e.g., patients, to be treated. Pharmaceutical compositions containing such lymphocytes, particularly those for use in methods of treating diseases characterized by antigen or neoantigen expression, are also provided.

[0004] 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 lymphocytes that have been genetically engineered to be antigen-specific, 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 expense 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.

[0005] A common drawback of adoptive cell therapy is the lack of sufficient cell numbers (approximately 10 9 Achieving a high-throughput T cell population typically requires expanding the cells ex vivo for several weeks and / or using multiple culture phases, typically with the cells frozen between these phases. As a result, a large proportion of cells may be lost due to the effects of freeze-thawing. In addition, prolonged culture can result in T cells becoming terminal effector cells, which may die shortly after infusion into a patient before reaching the target cells, tissues, and / or organs. Thus, there is a need in the art for a more rapid expansion protocol that avoids 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 proportion of terminal effectors. Summary of the Invention [Problem to be solved by the invention]

[0006] 4. Overview The present invention relates to improved methods for the ex vivo expansion of lymphocytes, particularly antigen-specific lymphocytes. The methods of the present invention allow for the production 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. 9The method of the present invention has the advantage of being able to obtain younger cells (cells). Furthermore, the method of the present invention provides for a more rapid expansion of cells compared to available methods. As a result, a younger cell population can be obtained, characterized by a small proportion of terminal effector cells. These characteristics allow the younger cells to proliferate efficiently after reinfusion and thus reach the target cells, tissues, or organs before differentiating into terminal effector cells. Terminal effector cells are responsible for the immediate attack of cancer cells, while the younger cells are expected to provide a sustained response. [Means for solving the problem]

[0007] The present invention relates to the following items: 1. A method for expanding a population of lymphocytes 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; or b) culturing lymphocytes, which are lymphocytes isolated from a tissue or blood sample from the subject; Includes; The lymphocytes are expanded in 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 to a predefined value or range; The method comprises adjusting the culture volume to the rate of lymphocyte expansion.

[0008] 2. The method according to item 1, wherein the culture medium is a culture medium in which pH, ​​dissolved oxygen (DO) concentration, glucose concentration, lactate concentration and temperature are monitored and adjusted to predefined values ​​or ranges.

[0009] 3. A method according to item 1 or 2, wherein the culture volume is increased by at least 2, 3, 4, 5, or 6 fold during lymphocyte expansion.

[0010] 4. The method according to any one of items 1 to 3, comprising a step of dynamic culturing of lymphocytes.

[0011] 5. The method according to any one of items 1 to 4, wherein the tissue sample is a tumor sample.

[0012] 6. The method according to item 5, wherein the tumor sample contains at least one neoantigen.

[0013] 7. The method according to any one of items 1 to 6, wherein the population of lymphocytes comprises tumor-infiltrating lymphocytes, in particular, the tumor-infiltrating lymphocytes are T cells.

[0014] 8. The method according to any one of items 1 to 7, wherein the lymphocytes are expanded in the presence of one or more antigens.

[0015] 9. The method according to item 8, wherein the one or more antigens are contained in the tumor sample.

[0016] 10. The method according to item 9, wherein the tumor sample is the same tumor sample from which the lymphocytes were taken.

[0017] 11. The method according to any one of items 8 to 10, wherein the one or more antigens are added to the culture medium in the form of peptides.

[0018] 12. The method according to item 11, wherein the peptide is added to the culture medium at a concentration of 0.1 to 10 μg / ml.

[0019] 13. The method according to any one of items 1 to 12, wherein the culturing step comprises co-culturing lymphocytes with antigen-presenting cells (APCs) or artificial antigen-presenting cells (aAPCs).

[0020] 14. The method according to item 13, wherein the antigen-presenting cells (APCs) comprise or consist of B cells.

[0021] 15. The method according to item 14, wherein the B cells are obtained by apheresis.

[0022] 16. The method according to item 14 or 15, wherein the B cells are activated before addition to the lymphocytes.

[0023] 17. The method according to item 16, wherein the B cells are activated with IL-4 and / or CD40L.

[0024] 18. The method according to any one of items 13 to 17, wherein the antigen-presenting cells (APCs) are genetically engineered to express one or more transgenes.

[0025] 19. The method according to item 18, wherein the genetically engineered APC is obtained by introducing nucleic acids encoding one or more transgenes into the APC.

[0026] 20. The method according to item 18 or 19, wherein at least one of the one or more transgenes encodes an immunomodulatory factor.

[0027] 21. The method according to item 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), CD62L, 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 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).

[0028] 22. The method according to item 20 or 21, wherein the immunomodulatory factor is one or more of OX40L, 4-1BBL and / or interleukin-12.

[0029] 23. The method according to any one of items 8 to 22, wherein the presence of at least one of the one or more antigens has been confirmed in a tumor sample taken from the subject.

[0030] 24. The method according to any one of items 8 to 23, wherein at least one of the one or more antigens is a neoantigen, and the presence of the neoantigen has been confirmed in a tumor sample taken from the subject.

[0031] 25. The method according to item 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.

[0032] 26. The method according to any one of items 1 to 4, wherein the lymphocytes are isolated from a blood sample.

[0033] 27. The method according to item 26, wherein the lymphocytes are genetically engineered to express a transgene.

[0034] 28. The method according to item 27, wherein the transgene encodes a chimeric antigen receptor.

[0035] 29. The method according to any one of items 1 to 28, wherein the lymphocytes are expanded in the presence of feeder cells.

[0036] 30. The method according to item 29, wherein the feeder cells are autologous or allogeneic cells.

[0037] 31. The method according to item 29 or 30, wherein the feeder cells are B cells, dendritic cells, T cells, macrophages and / or PBMCs.

[0038] 32. The method according to any one of items 29 to 31, wherein the feeder cells are irradiated cells.

[0039] 33. The method according to any one of items 1 to 32, comprising activating lymphocytes during culture.

[0040] 34. The method according to item 33, wherein the activation step comprises the addition of a CD3 agonist and / or a CD28 agonist to the culture medium.

[0041] 35. The method according to item 34, wherein the CD3 agonist is an agonist anti-CD3 antibody and / or the CD28 agonist is an agonist anti-CD28 antibody.

[0042] 36. The method according to item 35, wherein the anti-CD3 antibody and / or the anti-CD28 antibody is immobilized on solid particles.

[0043] 37. The method according to any one of items 34 to 36, wherein the CD3 agonist and / or CD28 agonist is added to the culture medium 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 days after the initiation of the culture.

[0044] 38. The method according to any one of items 1 to 37, wherein the culture medium is supplemented with human or synthetic AB serum, IL-2 and / or IL-15.

[0045] 39. The step of culturing comprises culturing the population of T cells to at least 10 9 39. The method according to any one of items 1 to 38, continued until a cell is reached.

[0046] 40. The method according to any one of items 1 to 39, wherein the culturing step is carried out at a temperature above 0°C.

[0047] 41. The method according to any one of items 1 to 40, wherein the sample or the lymphocytes are maintained at a temperature above 0°C after isolation from the subject and before culturing.

[0048] 42. The method according to any one of items 1 to 41, wherein the cells are collected from the culture vessel after expansion and, if desired, transferred to a second culture vessel for a second expansion.

[0049] 43. At least 1×10 9 43. The method according to item 42, wherein the cells are transferred from the first culture vessel to a second culture vessel.

[0050] 44. The method according to item 42 or 43, wherein the culture medium in the second culture vessel is supplemented with human or synthetic AB serum, IL-2, IL-15, nicotinamide and / or nicotinamide mononucleotide.

[0051] 45. The method according to any one of items 42 to 44, wherein the second expansion comprises a step of dynamic culture of lymphocytes, in particular a step of perfusion.

[0052] 46. ​​The method according to item 45, wherein the perfusion rate is in the range of 0.5 to 10 L / day, preferably 1 to 6 L / day.

[0053] 47. The cell concentration in the second culture vessel is at least 1 x 10 6 , preferably 2 x 10 6 47. The method according to any one of items 42 to 46, wherein the saturation level is 10 ...

[0054] 48. The method according to any one of items 42 to 47, wherein the second expansion is carried out for at least 2, 3, 4, 5, 6, or 7 days.

[0055] 49. A second expansion is performed to a second culture vessel containing at least 1 x 10 10 49. The method according to any one of items 42 to 48, which is stopped when the total cell number of cells is reached.

[0056] 50. A population of lymphocytes obtainable by the method of any one of items 1 to 49.

[0057] 51. 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 and less than 10% are triple positive for CD45RA, CD57, and KLRG1.

[0058] 52. A population of lymphocytes according to item 51, wherein the T cells are specific for one or more antigens.

[0059] 53. A population of lymphocytes according to item 51 or 52, wherein less than 15% of the T cell portion secretes IL-4 and / or IL-5 in response to an antigen.

[0060] 54. 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 according to any one of items 51-53 that are T cells.

[0061] 55. A population of lymphocytes according to any one of items 51 to 54, including CAR-T cells.

[0062] 56. A population of lymphocytes according to any one of items 51 to 54, wherein at least two T cells in the T cell portion are directed against different antigens.

[0063] 57. A population of lymphocytes according to item 56, in which at least one antigen is a neoantigen.

[0064] 58. The T cell portion comprises at least 10 9 A population of lymphocytes according to any one of items 51 to 57, containing T cells.

[0065] 59. A pharmaceutical composition comprising a population of lymphocytes according to any one of items 50 to 58.

[0066] 60. A pharmaceutical composition according to item 59, wherein the lymphocytes are suspended in a pharmacologically acceptable buffer solution.

[0067] 61. A pharmaceutical composition according to item 60, wherein the pharmaceutically acceptable buffer comprises about 0.9% NaCl and optionally not more than 15% DMSO.

[0068] 62. A population of lymphocytes according to any one of items 50 to 58 or a pharmaceutical composition according to any one of items 59 to 61 for use as a medicament.

[0069] 63. A population of lymphocytes according to any one of items 50 to 58 or a pharmaceutical composition according to any one of items 59 to 61 for use in cancer treatment.

[0070] 64. A population of lymphocytes or a pharmaceutical composition for use according to item 63, wherein the cancer treatment is adoptive cell therapy.

[0071] 65. A population of lymphocytes or a pharmaceutical composition for use according to item 63 or 64, wherein the cancer treatment is autologous cell therapy.

[0072] 66. A method of treating cancer, comprising: a) providing a population of lymphocytes according to any one of items 50 to 58 or a pharmaceutical composition according to any one of items 59 to 61; and b) injecting the population of lymphocytes or the pharmaceutical composition into a subject suffering from cancer. A method comprising:

[0073] 67. 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 1 to 49, 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; d) injecting the expanded lymphocytes obtained in step (c) into the subject from whom the tumor sample was taken. A method comprising:

[0074] 68. The method according to item 67, wherein the tumor antigen is a tumor-associated antigen or a tumor-specific antigen.

[0075] 69. The method according to any one of items 66 to 68, wherein the lymphocytes include tumor-infiltrating lymphocytes (TILs).

[0076] 70. The method according to item 69, wherein the TILs specifically recognize one or more tumor antigens.

[0077] 71. The method according to item 70, wherein at least one tumor antigen is a neoantigen. DETAILED DESCRIPTION OF THE INVENTION

[0078] 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 is viable.

[0079] 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 comprise the TCR complex.

[0080] 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.

[0081] 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 antibodies against CD3 and / or other suitable T cell-specific surface markers.

[0082] 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.

[0083] In certain embodiments, the population of lymphocytes may contain no more than 10% contaminants.

[0084] 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 population of B cells. 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.

[0085] Alternatively, the lymphocyte population 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.

[0086] 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 typically cannot survive for extended periods in T cell-specific medium. Thus, in certain embodiments, the population of lymphocytes according to the present invention is substantially free of B cells; that is, the number of B cells in the population may be below the limit of quantification by flow cytometry.

[0087] 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.

[0088] 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 enables the B cell to bind to its specific antigen. The primary 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 cell that resides primarily in the bone marrow and continuously secretes antibodies. The term "memory B cells," as used herein, refers to a subtype of B cells that are formed after primary 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.

[0089] 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, lymphocyte preparations may contain residual fractions of tumor cells and other cell types contained in tumor samples.The abundance of tumor cells in the final lymphocyte population can be determined by flow cytometry, for example, by determining the abundance of CD45-negative cells in the lymphocyte population.Alternatively or additionally, residual tumor cells in the lymphocyte population can be detected by qPCR or digital PCR, which are known in the art.

[0090] In certain embodiments, the lymphocyte population (CD45+ of live cells) 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-).

[0091] Alternatively, the lymphocyte population 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-).

[0092] In certain embodiments, the population of lymphocytes (CD45+ of live cells) 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% of cells that are CD19-, CD14-, CD3-, CD16 / 56-.

[0093] Alternatively, the lymphocyte (CD45+ of live cells) population may contain 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% of cells that are CD19-, CD14-, CD3-, and CD16 / 56-.

[0094] The lymphocyte population according to the present invention may further comprise NK cells (CD3-, CD56+) and / or NKT cells (CD3+, CD56+). Thus, in certain embodiments, the lymphocyte population 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%, 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+).

[0095] In the present invention, it is preferred that at least 70% of CD3+ T cells in a population of cells are viable cells. Various methods for determining T cell viability are known in the art and commercially available. Without limitation, the viability of T cells in a population of lymphocytes can be determined by live / dead cell differentiation staining.

[0096] 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.

[0097] Viability can 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 lymphocyte population are viable as determined by a cell counter, particularly a NucleoCounter NC-202.

[0098] 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.

[0099] It is understood that viability will vary depending on the method by which it is determined. 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.

[0100] Furthermore, it is preferred that at least 2, 5, 10, 15, 20, 25, or 30% of the CD3+ T cells in the lymphocyte population are CD27 and / or CD28 positive cells, hi certain embodiments, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, or at least 80% of the T cell portion are CD27 and / or CD28 positive.

[0101] Thus, in certain embodiments, the present invention relates to a population of lymphocytes for adoptive cell transfer in humans, 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 30% are CD27 and / or CD28 positive, and less than 10% are triple positive for CD45RA, CD57, and KLRG1.

[0102] 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 an important role in regulating B cell activation and immunoglobulin synthesis. CD27 is primarily expressed on naive, central memory (CM), and effector memory (EM) T cells, but not on terminal effector (TE) T cells.

[0103] CD28 is one of the proteins expressed on T cells that provides costimulatory signals necessary for T cell activation and survival. T cell stimulation by CD28 in addition to the T cell receptor (TCR) can provide a powerful signal for the production of various interleukins. Like CD27, CD28 is primarily expressed on naive, central memory (CM), and effector memory (EM) T cells, but not on terminal effector (TE) T cells.

[0104] As noted above, the T cells in the lymphocyte population preferably include a small number of terminal effector T cells. Thus, in certain embodiments, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or at least 60% of the CD3+ T cells in the lymphocyte population express the cell surface marker CD27. In other embodiments, at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, or at least 60% of the CD3+ T cells in the lymphocyte population express the cell surface marker CD28.

[0105] Those skilled in the art know how to determine 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.The antibodies against CD27 and CD28 are known in the art and are commercially available.

[0106] In a particular embodiment, the present invention relates to a method according to the invention, wherein less than 10% of said T cell fraction are positive for at least one, preferably two, more preferably all, of the markers from the group consisting of CD45RA, CD57 and KLRG1.

[0107] That is, CD3+ T cells within a population of lymphocytes may be further characterized by the absence of one or more senescence markers.

[0108] 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 the cell surface marker CD45RA.

[0109] 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 of terminal differentiation of CD8+ memory T cells. The percentage of CD45RA-positive cells within a population of lymphocytes is preferably determined by flow cytometry using an antibody against CD45RA.

[0110] Terminally differentiated effector memory (Temra) cells typically express CD45RA but not CCR7 (CD45RA+CCR7-). In certain embodiments, the lymphocyte population contains less than 30%, less than 25%, less than 20%, less than 15%, or less than 10% cells that are CD45RA positive and CCR7 negative (CD45RA+CCR7-).

[0111] Naive T cells, on the other hand, express both CD45RA and CCR7. Thus, in certain embodiments, a population of lymphocytes contains less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, or less than 5% cells that are positive for CD45RA and CCR7 (CD45RA+CCR7+).

[0112] In certain embodiments, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, 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 CD57.

[0113] 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.

[0114] In certain embodiments, less than 85%, less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, 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 KLRG1.

[0115] Killer cell lectin-like receptor subfamily G member 1 (KLRG1) is a protein 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. However, KLRG1 expression is reversible. The percentage of KLRG1-positive cells in a lymphocyte population is preferably determined by flow cytometry using an antibody against KLRG1.

[0116] In certain embodiments, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, 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, CD57, and / or KLRG1. In preferred embodiments, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, 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 two of the cell surface markers CD45RA, CD57, and / or KLRG1. In more preferred embodiments, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, 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 lymphocyte population are positive for all three of the cell surface markers CD45RA, CD57, and KLRG1.

[0117] In certain embodiments, less than 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, or 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 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, or 5% of the CD3+ T cells in a population of lymphocytes are double positive for CD45RA and CD57.

[0118] In certain embodiments, less than 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, or 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 60%, 50%, 40%, 30%, 20%, 10%, 9%, 8%, 7%, 6%, or 5% of the CD3+ T cells in a population of lymphocytes are double positive for KLRG1 and CD57.

[0119] In certain embodiments, greater than 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, or 95% of the CD3+ T cells in a population of lymphocytes are negative for at least one of the cell surface markers CD45RA, CD57, and / or KLRG1. In certain embodiments, greater than 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, or 95% of the CD3+ T cells in a population of lymphocytes are negative for two of the cell surface markers CD45RA, CD57, and / or KLRG1. In certain embodiments, greater than 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, or 95% of the CD3+ T cells in a population of lymphocytes are double negative for CD57 and KLRG1. In certain embodiments, greater than 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, or 95% of the CD3+ T cells in a population of lymphocytes are triple negative for CD45RA, CD57, and KLRG1.

[0120] In a specific 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 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% are double negative for CD57 and KLRG1.

[0121] 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 IL-4 and / or IL-5.

[0122] In one particular embodiment, the invention relates to a population of lymphocytes according to the invention, wherein less than 5% of said T cell portion secretes IL-4 and / or IL-5.

[0123] In one particular embodiment, the invention relates to a population of lymphocytes according to the invention, wherein less than 1% of said T cell fraction secretes aIL-4 and / or IL-5.

[0124] In one particular embodiment, the invention relates to a population of lymphocytes according to the invention, wherein less than 1% of said T cell fraction secretes IL-4 and IL-5.

[0125] In certain embodiments, CD3+ T cells within a population of lymphocytes may be characterized by less than 15% of the CD3+ T cells secreting IL-4. In certain embodiments, CD3+ T cells within a population of lymphocytes may be characterized by less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the CD3+ T cells secreting IL-4. Interleukin 4 (IL-4) has many biological roles, including stimulating activated B cell and T cell proliferation and differentiation of B cells into plasma cells.

[0126] In certain embodiments, less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the CD3+ T cells in a population of lymphocytes secrete detectable amounts of IL-4.

[0127] In certain embodiments, CD3+ T cells in a population of lymphocytes may be characterized by less than 15% of these CD3+ T cells secreting IL-5. In certain embodiments, CD3+ T cells in a population of lymphocytes may be characterized by less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of these CD3+ T cells secreting 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.

[0128] In certain embodiments, less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of the CD3+ T cells in a population of lymphocytes secrete detectable amounts of IL-5.

[0129] In the present invention, cells are determined to secrete a specific protein if a detectable amount of the protein can be identified in an ELISpot assay. The enzyme-linked immunospot (ELISpot) assay is a highly 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, are captured by the specific antibody on the surface. After an appropriate incubation period, the cells are removed, and the secreted molecules are detected using a detection antibody in a procedure similar to that used 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 precipitating product 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 significantly different from measurements distorted by receptor binding or protease degradation. This assay is considered one of the most sensitive cellular assays available. The detection limit typically achieved can be 1 cell in 100,000 cells. Due to the high sensitivity of this assay, it is particularly useful for studying small populations of cells found in specific immune responses. ELISpot assays for determining the percentage of cells secreting IL-4 and IL-5 are known in the art.

[0130] Alternatively or additionally, the secretion of these proteins can be estimated by flow cytometry. For this purpose, T cells must be fixed and permeabilized so that antibodies can be used to quantify the intracellular pools of each protein. Methods for quantifying the intracellular pools of IL-4 and IL-5 are known in the art.

[0131] In a specific 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.

[0132] That is, it is preferred that the majority of T cells in a lymphocyte population are 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 a patient's CD8+ T cells that have migrated from the bloodstream to the tumor.

[0133] Preferably, the second largest portion of T cells in the lymphocyte population are CD4+ T cells. As used herein, the term "CD4+ T cells" refers to T cells that display the co-receptor CD4 on their surface. CD4 is a transmembrane glycoprotein that functions as a co-receptor for the T cell receptor (TCR), which can recognize specific antigens. In certain embodiments, CD4+ T cells are T helper cells. T helper cells (TH cells) assist other white blood cells in immunological processes, including the maturation of B cells into plasma cells and memory B cells, and the 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 into 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.

[0134] 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.

[0135] 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 the T cell portion is between 1:1 and 20:1. 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 the 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.

[0136] 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.

[0137] 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.

[0138] That is, T cells contained in the lymphocyte population preferably recognize more than one antigen. Obtaining a lymphocyte population according to the present invention may include contacting these lymphocytes with a pool of different antigenic peptides. Therefore, it is envisioned that T cells that recognize antigens from a pool of antigens are primarily expanded. The pool of antigenic peptides may contain 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 lymphocyte population may include 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 directed against a different antigen. Non-limiting examples of antigens that can be recognized by T cells contained in the lymphocyte population are provided herein.

[0139] 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 9 Preferably, the population of lymphocytes according to the present invention comprises 10 CD3+ T cells. 6 ~10 10 Between 10 and 10 CD3+ T cells, preferably 10 9 ~10 11 Between 10 and 10 T cells, more preferably 10 9 ~10 10 Contains T cells between individuals.

[0140] In a specific embodiment, the present invention relates to a population of lymphocytes for adoptive cell transfer in humans, comprising at least 90% CD3+ T cells and less than 5% B cells, wherein at least 70% of the T cell portion are viable and less than 10% are triple positive for CD45RA, CD57, and KLRG1.

[0141] In a specific 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 the T cell portion are viable and less than 10% are positive for CD45RA and CD57.

[0142] In certain embodiments, 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 the T cell portion are viable and less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, or less than 10% are positive for CD57 and KLRG1.

[0143] In certain embodiments, 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 and more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, more than 80% are negative for CD57 and KLRG1.

[0144] In a specific 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 the T cell portion is viable.

[0145] It should be noted that the expression of cell surface markers may vary depending on the starting material from which lymphocytes are expanded. Thus, the expression of one or more of the surface markers and / or cytokines listed herein above may fall outside the limits and / or ranges defined herein above.

[0146] Preferably, the lymphocyte populations of the present invention are suitable for use in autologous cell therapy. Autologous cell therapy is a therapeutic intervention that uses an individual's cells, which are cultured and expanded ex vivo and then 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 lymphocyte populations of the present invention are obtained by ex vivo expansion of the individual's T cells and then infused back into the same individual.

[0147] Thus, in a particularly preferred 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 and, optionally, less than 10% are triple positive for CD45RA, CD57 and KLRG1.

[0148] In a specific 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 the T cell portion are viable and, optionally, less than 10% are double positive for CD45RA and CD57.

[0149] In a specific 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 the T cell portion are viable and, optionally, less than 10% are double positive for CD57 and KLRG1.

[0150] In a specific 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 the T cell portion are viable and, optionally, greater than 80% are double negative for KLRG1 and CD57.

[0151] The present invention also provides methods for producing a population of lymphocytes specific for one or more antigens as defined herein, comprising a single culture phase, the single phase comprising: (a) culturing a tissue or blood sample from a subject, the sample being known or suspected to contain lymphocytes; or (b) culturing lymphocytes, the lymphocytes being lymphocytes isolated from a tissue or blood sample from a subject. In certain embodiments, the tissue, blood sample, and / or lymphocytes are cultured in the presence of one or more antigens.

[0152] In certain embodiments, the culturing step comprises culturing for at least 10 9 This is continued until the 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 the single culture phase.

[0153] 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 should be understood that frozen samples can also be used in the methods of the present invention.

[0154] 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 transferred to a larger bioreactor and rapidly expanded for an additional 2 weeks. Between the pre-REP and REP phases, TILs are typically cryopreserved. The drawback of this long culture period, including the optional cryopreservation step, is that the majority of lymphocytes in the final product are terminal effector cells that die rapidly after infusion into patients.

[0155] Therefore, it is an object of the present invention to produce lymphocytes at high cell numbers (10 - 16) in 2-8 weeks, preferably 2-6 weeks, more preferably 2-4 weeks, without the need for a cryopreservation step and / or the need to transfer lymphocytes from one bioreactor to another. 9The goal is to establish a lymphocyte expansion protocol that can reach a maximum cell mass of 10 ...

[0156] In certain embodiments, the present invention relates to a method for expanding a population of lymphocytes in a single controlled culture vessel, comprising the steps of: (a) culturing a tissue or blood sample from a subject, the sample being known or suspected to contain lymphocytes, or (b) culturing lymphocytes, the lymphocytes being lymphocytes isolated from a tissue or blood sample from a subject; wherein the lymphocytes are expanded in 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 to a predefined value or range; and wherein the method comprises adjusting the culture volume to match the rate of expansion of the lymphocytes.

[0157] The method of the present 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 that are monitored and / or adjusted during the method of the present invention are disclosed elsewhere herein. Optimal growth conditions can be maintained throughout the entire process.

[0158] The method of the present invention is further characterized by including a step of "dynamic culturing." Dynamic culturing involves culturing cells with a continuous flow of culture medium. Dynamic culturing includes both circulation, in which conditioned culture medium is circulated within the growth chamber, and perfusion, in which culture medium from the growth chamber is exchanged with fresh culture medium.

[0159] Finally, lymphocytes are expanded in a culture vessel that allows for adaptation of the culture volume to the lymphocyte expansion rate. Thus, the method of the present invention allows for the cultivation of large numbers of cells (at least 109 Cells) can be obtained directly from a patient sample.

[0160] In certain embodiments, the method according to the present invention is used to expand autologous T cells, preferably autologous TILs. To this end, the cells are preferably expanded in the presence of an antigen to selectively activate T cells in a TCR-dependent manner. For the expansion of autologous TILs, the cells are preferably cultured in the presence of at least one antigen and at least one type of antigen-presenting cell. In certain embodiments, autologous TILs can be co-cultured with antigen-presenting cells, particularly B cells or artificial B cells, in the presence of a tumor sample, preferably the same tumor sample from which the TILs were collected. In certain embodiments, the B cells are genetically engineered before contacting with the TILs.

[0161] Thus, methods for expanding a desired T cell population from a sample containing, for example, lymphocytes and / or T cells, include presenting one or more antigens to T cells within 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.

[0162] As an illustrative, non-limiting example, one or more soluble antigens can be continuously provided to the culture medium (e.g., to maintain a steady-state concentration or a desired concentration range), or can be included for one or more specific periods less than the entire culture phase. The soluble antigen can also be introduced at one or more discrete time points during the culture phase. Additionally or alternatively, the soluble antigen can be presented to the lymphocyte sample and / or T cells by antigen-presenting cells (APCs) 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.

[0163] 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 embodiments, tumor sample can be co-cultured with APC in the absence of antigen peptide.

[0164] APC can be engineered by recombinant technology to transiently or continuously express one or more antigens of interest.For example, the nucleic acid molecule encoding either antigen peptide or larger polypeptide containing one or more antigen peptides can be introduced into APC by genetic engineering method to promote the presentation of one or more antigens by APC.Recombinant engineering can be achieved by any means known in the art or described herein, and preferably by transduction using viral vector or transfection using plasmid or mRNA.

[0165] Alternatively or additionally, APCs, particularly B cells, can be contacted with chemically synthesized antigenic peptides, as described in more detail below.

[0166] The antigen can be one or more known antigens that characterize disease or cancer, or can be determined by evaluating a patient to determine one or more neoantigens.To this end, patient cells can be collected by biopsy and analyzed by mass spectrometry or scRNAseq to identify neoantigens.The sequences obtained from these methods can then be analyzed using proprietary algorithms to identify and select relevant neoantigens.

[0167] It will be appreciated that the lymphocyte populations, isolated lymphocytes, and / or methods for their production and use not only serve as tools for the treatment of disease (e.g., for use as a pharmaceutical or in the development and manufacture of pharmaceuticals), but also have applicability as model systems for studying disease therapy. Accordingly, the lymphocytes of the invention disclosed herein are preferably human lymphocytes, more preferably primary human lymphocytes (including, e.g., NK cells and T cells), and 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, including, but not limited to, primary lymphocytes, and lymphocytes from mice, rats, monkeys, apes, cats, and dogs, are also provided.

[0168] Of the more preferred primary human lymphocytes, primary human T cells are most preferred. 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 the T cell portion is viable, and optionally less than 60%, 50%, 40%, 30%, 20%, or 10% are triple positive for CD45RA, CD57, and KLRG1.

[0169] 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 the T cell portion are viable and, optionally, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, or greater than 80% are double negative for CD57 and KLRG1.

[0170] The lymphocyte populations provided herein or produced according to the methods provided herein, whether human or not, and whether primary or not, can be composed of any lymphocyte class or subclass known in the art or described herein that is 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. Non-limiting examples of lymphocyte classes encompassed by the present invention include lymphocyte populations that include 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.

[0171] The population of cells (e.g., APCs, preferably B cells) for use in the cells and / or their production methods provided herein includes genetically engineered cells, and these cells can be directly genetically engineered cells, i.e., cells directly subjected to genetic engineering, or cells derived from such engineered cells, for example, 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).

[0172] In non-limiting embodiments, one or more of the APCs to be cultured with the lymphocyte populations of the present invention can be engineered to express one or more immunomodulatory factors, e.g., 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 patient samples.

[0173] APCs, particularly B cells, are preferably engineered to express one or more of the immunoregulatory factors OX40L, 4-1BBL and / or interleukin-12.

[0174] In certain embodiments, APCs, particularly B cells, are engineered to express OX40L and 4-1BBL.

[0175] In certain embodiments, APCs, particularly B cells, are engineered to express OX40L and interleukin-12.

[0176] In certain embodiments, APCs, particularly B cells, are engineered to express 4-1BBL and interleukin-12.

[0177] In certain embodiments, APCs, particularly B cells, are engineered to express OX40L, 4-1BBL and interleukin-12.

[0178] 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.

[0179] The lymphocytes and lymphocyte populations of the present invention, preferably human lymphocytes, more preferably primary human lymphocytes, and 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, solid tumor cells (e.g., for tumor-infiltrating lymphocytes (TILs)) or circulating tumor cells from the tumor environment; and peripheral blood (e.g., PBMCs). Preferably, the lymphocytes in the lymphocyte population are obtained by ex vivo expansion of TILs.

[0180] If the cells are allogeneic, they can be further genetically engineered or prepared so that they are not alloreactive. As understood in the art and as used herein, non-alloreactive indicates that the cells have been engineered (e.g., genetically engineered) to render them incapable of being recognized as allogeneic (foreign) cells or to render them capable of recognizing allogeneic (foreign) cells. Similarly, the genetically engineered lymphocytes of the present invention can additionally or alternatively be engineered to render them unrecognizable by the recipient's immune system. As a non-limiting example of this embodiment, the lymphocytes of the present invention can have disrupted or deleted endogenous major histocompatibility complex (MHC). Such cells can have reduced or absent expression of endogenous MHC, thus preventing or reducing activation of the recipient's immune system against autologous cells.

[0181] As understood in the art, such non-alloreactive cells are incapable of reacting with cells of a foreign host. Thus, non-alloreactive cells derived from a third-party donor can be universal, i.e., recipient-independent. As explained above, non-alloreactive cells may 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 their rejection. Such cells that are non-alloreactive and / or incapable of eliciting an immune response or being recognized by the recipient's immune system may also be referred to as "off-the-shelf" cells, as known in the art. Lymphocytes can be made non-alloreactive and / or incapable of eliciting or 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-alloreactive cells may have reduced or eliminated expression of endogenous T cell receptors (TCRs) compared to unmodified control cells. Such non-alloreactive T cells may contain 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 contain any modifications or gene deletions known in the art or described herein to minimize or eliminate antigen presentation, particularly to avoid immunogenic surveillance and elimination in the recipient. As previously mentioned, non-alloreactive 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-alloreactive / 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 autoantigen presentation (i.e., to prevent them from eliciting an immune response or being recognized by the recipient's immune system) can be performed before, in parallel with, or after any other genetic manipulations in connection with the present invention.

[0182] The present invention also encompasses populations of lymphocytes, preferably human lymphocytes, obtainable by any of the methods disclosed herein.

[0183] The present invention provides a method of immunotherapy for treating disease, comprising the use of the cells or cell populations disclosed herein.Therefore, a population of lymphocytes (preferably human lymphocytes, more preferably primary human lymphocytes, most preferably primary human T cells) described herein is provided for use as a medicament.The present invention also provides a population of lymphocytes 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.

[0184] 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.

[0185] The lymphocyte populations, medicaments and / or pharmaceutical compositions of the present invention can 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, mitoxantrone, 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 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, lipegfilgrastim, valgrastim, levacetylmethadol, interferon, Gamma, interferon alfa-2b, interferon alfa-n1, interferon beta-1a, peginterferon alfa-2b, peginterferon beta-1a, lopeginterferon alfa-2v, tasonermin, histamine dihydrochloride, mifamurtide, plerixafor, sipuleucel-T, dasiprotim-T, muromonab-CD3, mycophenolate, sirolimus, leflunomide, efalizumab, natalizumab, abatacept, exlizumab, ofatumumab, fingolimod, eltrombopag, tofacitinib, teriflunomide It can be used in combination with: ibuprofen, 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, over several days, weeks, or months, and other agent(s) are administered only once or according to a different dosing scheme. Combining includes any scheme in which agents are intentionally administered so that their therapeutic effects overlap to some extent.

[0186] 5. Detailed Description 5.1 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 the T cell portion is viable, and optionally 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 living tissue (i.e., a biopsy such as a tumor or blood sample) or cells from a subject that have not been passaged in culture or that have been passaged and maintained in culture but not immortalized. Preferably, the primary cells are primary human lymphocytes. Primary cells have undergone few, if any, population doublings.

[0187] The lymphocyte populations of the present invention can include any lymphocyte class, subclass, or mixture thereof described herein or known in the art to be suitable for use, particularly in adoptive cell therapy. However, the methods of the present invention could also be applicable to non-therapeutic uses, such as in screening methods and / or in model systems, such as model systems useful for 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.

[0188] 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 in the lymphocyte population are CD8+ T cells.

[0189] 5.2 Metabolic characterization After expansion, a population of lymphocytes (preferably human lymphocytes, more preferably primary human lymphocytes, and most preferably primary human T cells) can be analyzed for expression of one or more phenotypic markers. 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.

[0190] A population of lymphocytes (preferably human lymphocytes, more preferably primary human lymphocytes, and 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.

[0191] Preferably, a population of lymphocytes (preferably human lymphocytes, more preferably primary human lymphocytes, and most preferably primary human T cells) is analyzed for 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, and most preferably primary human T cells) can be analyzed for 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, less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10% of the CD3+ T cells in the lymphocyte population are triple positive for CD45RA, CD57 and KLRG1.

[0192] Alternatively, it is preferred that more than 20%, more than 30%, more than 40%, more than 50%, more than 60%, more than 70%, or more than 80% of the CD3+ T cells contained in the lymphocyte population are double negative for CD57 and KLRG1.

[0193] Preferably, the presence of the above-mentioned markers on the cell surface of CD3+ T cells contained in the lymphocyte population is determined by flow cytometry.

[0194] 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 with a series of filters and mirrors, and detecting the light.

[0195] Numerous 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.

[0196] In some embodiments, the viability of the population of lymphocytes (preferably human lymphocytes, more preferably primary human lymphocytes, and 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%. Lymphocyte viability can be determined by methods known in the art, such as any one of the methods disclosed herein above.

[0197] A population of lymphocytes (preferably human lymphocytes, more preferably primary human lymphocytes, and most preferably primary human T cells) can be assessed for interferon-γ (IFN-γ) secretion in response to stimulation with either an anti-CD3 antibody (e.g., OKT3) or co-culture with an autologous tumor sample or tumor digest, or stimulation with antigens and / or neoantigenic peptides. Those skilled in the art will recognize that antigens and / or neoantigenic peptides should be presented in an MHC-dependent manner. In certain embodiments, greater than 6%, greater than 7%, greater than 8%, or greater than 9% of the CD3+ cells contained in the population of lymphocytes secrete IFN-γ in response to an autologous tumor sample.

[0198] In some embodiments, the health of TILs is measured by IFN-gamma (IFN-γ) secretion. In some embodiments, IFN-γ secretion indicates activated T cells within 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 culture 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 culture 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 measuring IFN-γ release. In some embodiments, IFN-γ secretion is increased by 1-fold, 2-fold, 3-fold, 4-fold, or 5-fold or more compared to corresponding cells in the sample before expansion and / or activation.

[0199] 5.3 Lymphocyte Sources 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 a blood sample, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, splenic tissue, and / or tumor.

[0200] In a preferred embodiment, the isolated cells and / or samples used in the methods of the invention, e.g., to generate a population of lymphocytes (preferably human lymphocytes, more preferably primary human lymphocytes, most preferably primary human T cells), are 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 infected tissue (e.g., tissue infected with a virus, bacteria, or parasite). Methods for isolating / obtaining specific populations of lymphocytes from patients or donors are well known in the art and involve, as a first step, for example, isolating / obtaining a donor or patient sample known or suspected to contain such cells.

[0201] 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, if desired, after expansion for storage and handling prior to administration.

[0202] Patient tumor samples can be obtained using methods known in the art, typically by surgical resection, needle biopsy, or other means for obtaining a sample containing a mixture of tumor and lymphocytes. Generally, tumor samples can be from any solid tumor, including primary, invasive, or metastatic tumors. Tumor samples can also be liquid tumors, such as those 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, those of the breast, pancreas, prostate, colorectum, 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.

[0203] The term "solid tumor" refers to an abnormal mass of tissue that usually does not 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 histology of a solid tumor comprises interdependent tissue compartments, including parenchymal cells (cancer cells) and supporting stromal cells, into which cancer cells are dispersed and which may provide a supportive microenvironment.

[0204] In certain embodiments, the tumor sample is obtained from a lung cancer patient, for example, but not limited to, a patient suffering from lung adenocarcinoma or small cell lung cancer.

[0205] After isolating / obtaining the sample, desired cells, e.g., human lymphocytes and / or T cells (e.g., TILs), can be cultured under conditions that allow for the preferential growth and expansion of cells of the desired cell class, subclass, or with the desired specificity. In particular, this method allows for 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. Such cells may be characterized by low expression of CD45RA, CD57, and KLRG1, and low secretion of IL-4 and IL-5, as disclosed elsewhere herein.

[0206] In another preferred embodiment, the method of the present invention is used to expand genetically modified lymphocytes, particularly CAR-T cells. To this end, lymphocytes are preferably collected from a subject's blood sample. In a particularly preferred embodiment, a peripheral blood mononuclear cell (PBMC) sample collected from the subject's blood sample is used to produce CAR-T cells in the method of the present invention.

[0207] When the method of the present invention is used to produce CAR-T cells, T cells must be genetically engineered with a polynucleotide encoding chimeric antigen receptor (CAR) during or before the expansion step.The nucleic acid constructs encoding CARs directed to various targets and the methods of introducing these constructs into T cells are well known in the art.For example, the nucleic acid constructs encoding CARs can be introduced into T cells by utilizing viral vectors, transposons, gene editing methods such as CRISPR-Cas9, or a combination thereof.

[0208] The term "chimeric antigen receptor," as used herein, is defined as a cell surface receptor that contains an extracellular ligand-binding or antigen-binding domain, a transmembrane domain, and a cytoplasmic costimulatory signaling domain in a combination not naturally found together on a single protein. This specifically includes receptors in which the extracellular and cytoplasmic domains are not naturally found together on a single receptor protein. Furthermore, chimeric antigen receptors differ from TCRs expressed on native T cell lymphocytes. As described in U.S. Patent Nos. 5,359,046, 5,686,281, and 6,103,521 (the contents of which are incorporated herein by reference in their entireties), the extracellular domain can be derived from any of a wide variety of extracellular domains associated with ligand binding and / or signal transduction or secreted proteins. The extracellular domain can be part of a protein that is monomeric, homodimeric, heterodimeric, or associated with multiple proteins in a noncovalent complex.

[0209] In particular, in preferred embodiments, the extracellular domain may comprise an Ig heavy chain, which may be covalently associated with an Ig light chain due to the presence of the CH1 and hinge regions, or with another Ig heavy / light chain complex due to the presence of the hinge, CH2, and CH3 domains. In the latter case, the heavy / light chain complex bound to the chimeric construct may constitute an antibody with a specificity distinct from that of the chimeric construct. Depending on the antibody function, desired structure, and signaling, the entire chain may be used, or a truncated chain may be used in which all or part of the CH1, CH2, or CH3 domains have been removed, or all or part of the hinge region has been removed.

[0210] As described herein, in some embodiments, the extracellular domain of the CAR is derived from an immunoglobulin. The term "antibody," as used herein, refers to a peptide or polypeptide derived from, mimicking, or substantially encoded by an immunoglobulin gene(s) or fragment(s) thereof, which is capable of specifically binding to an antigen or epitope. See, e.g., Fundamental Immunology, 3rd ed., W.E. Paul (ed.), Raven Press, NY (1993); Wilson (1994; J. Immunol. Methods 175:267-273; Yarmush (1992) J. Biochem. Biophys. Methods 25:85-97. The term antibody includes (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CH1 domains; (ii) a F(ab')2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; and (v) a dAb fragment (Ward et al. (1989) Nature 1999, 11:147-151), which consists of the VH domain. 341:544-546); and (vi) isolated complementarity determining regions (CDRs), comprising an antigen-binding portion, or "antigen-binding site," (e.g., fragments, subsequences, complementarity determining regions (CDRs)) that retain the ability to bind to the antigen. Single chain antibodies are also included by reference in the term "antibody."

[0211] When anti-tumor chimeric antigen receptors are used, the tumor may be of any type, as long as it has a cell surface antigen that can be recognized by the chimeric receptor. In some embodiments of the aspects described herein, the chimeric antigen receptor may be directed against any cancer for which a specific monoclonal antibody exists or can be produced. For example, cancers such as neuroblastoma, small cell lung cancer, melanoma, ovarian cancer, renal cell carcinoma, colon cancer, Hodgkin's lymphoma, and acute lymphoblastic leukemia (e.g., childhood acute lymphoblastic leukemia) have antigens known to be targetable by chimeric antigen receptors. The systems and methods described herein can be used in immunotherapy for cancer treatment, such as the treatment of lung cancer, melanoma, breast cancer, prostate cancer, colon cancer, renal cell carcinoma, ovarian cancer, neuroblastoma, rhabdomyosarcoma, leukemia, and lymphoma. The systems and methods described herein can be used in combination with other types of cancer therapy, such as chemotherapy, surgery, radiation, gene therapy, etc., as described below.

[0212] The extracellular domain of the CAR-T cells according to the present invention is not limited to any particular antigen. In certain embodiments, the extracellular domain of the CAR-T cells according to the present invention is directed to an antigen associated with a solid or liquid tumor, such as, but not limited to, TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-1Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, prostase, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-ab l, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, legumain, HPV E6, E7, MAGE A1, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostein, survivin and telomerase, PCTA-1 / galectin 8, MelanA / MARTl, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mutspecifically targeting hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, and IGLL1; in certain embodiments, the antigen is CD19. In certain embodiments, the antigen is BCMA.

[0213] The transmembrane domain of the CAR may be contributed by a protein that contributes to the multispecific extracellular inducer clustering domain, a protein that contributes to the effector function signaling domain, a protein that contributes to the growth signaling portion, or a completely different protein. In most cases, it will be convenient for the transmembrane domain to be naturally associated with one of the domains. In some cases, it may be desirable to use a transmembrane domain of the ζ, η, or FcεRlγ chain that contains a cysteine ​​residue capable of disulfide bonding, so that the resulting chimeric protein can form a disulfide-linked dimer with itself or with an unmodified version of the ζ, η, or FcεRlγ chain or a related protein. In some embodiments, the transmembrane domain is selected or modified by amino acid substitution to avoid binding to the transmembrane domain of the same or different surface membrane protein, in order to minimize interaction with other members of the receptor complex. In other embodiments, the transmembrane domains of ζ, η, or FcεRlγ chain and β, MB1 (Igα), B29, or CD3γ, ζ, or ε are utilized to maintain physical association with other members of the receptor complex. Examples of suitable transmembrane domains for use in agents for generating CAR T cells used in the methods described herein include the constant (Fc) region of immunoglobulin, human CD8a, and artificial linkers that help distance targeting moieties from the cell surface to improve access and binding to target cells. However, any transmembrane domain that is sufficient to anchor CAR within the membrane can be used. Those skilled in the art are aware of the numerous transmembrane domains and structural elements (such as lipophilic amino acid regions) that form transmembrane domains in numerous membrane proteins, and can therefore select sequences as needed without undue experimentation.

[0214] The cytoplasmic domain of a chimeric antigen receptor for use in the methods described herein can include a signaling domain (e.g., a costimulatory signaling domain) alone or in combination with any other desired cytoplasmic domain useful in this chimeric antigen receptor type, such as the 4-1BB signaling domain, the CD3ζ signaling domain, and / or the CD28 signaling domain. The 4-1BB, CD3ζ, and CD28 signaling domains have been well characterized, including, for example, their use in chimeric receptors. In some embodiments, the cytoplasmic domain of a chimeric receptor can include the 4-1BB signaling domain alone or in combination with any other desired cytoplasmic domain useful in this chimeric receptor type. In some embodiments, the extracellular domain includes a single-chain variable domain of a monoclonal antibody, the transmembrane domain includes the hinge and transmembrane domains of CD8a, and the cytoplasmic domain includes the signaling domain of CD3ζ and the signaling domain of 4-1BB. The CD8a hinge and transmembrane domain contains 69 amino acids translated from 207 nucleotides at positions 815 to 1021 of GenBank accession number NM_001768. The CD3ζ signaling domain contains 112 amino acids translated from 339 nucleotides at positions 1022 to 1360 of GenBank accession number NM_000734.

[0215] Specific populations of lymphocytes can be separated from other components of sample and / or culture.Methods for separating specific populations of desired cells from sample are known, and include but are not limited to, for example, leukapheresis to obtain T cells from peripheral blood samples from patients or donors; isolation / obtaining specific populations from samples using FACSort devices; and manual or micromanipulator selection of specific populations from fresh biopsy specimens 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 the tissue sample (for example, tumor tissue, infected tissue, or blood sample) that has been or will be removed and / or isolated from a subject by surgery or any other known means.

[0216] As is well known in the art, it is also possible to isolate / obtain and culture / select one or more specific subpopulations of leukocytes, such as the most preferred T cells. Such methods include, but are not limited to, the isolation and culture of lymphocyte subpopulations, such as CD3+, CD28+, CD4+, CD8+, and γδ subclasses, as well as the isolation and culture of other primary lymphocyte populations, such as NK T cells, B cells, or macrophages. Such selection methods may include, for example, positive and / or negative selection techniques in which a sample is incubated with a specific combination of antibodies and / or cytokines to select for 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 can be used to isolate the desired population in any situation where the desired cells are expected to be less abundant than 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.

[0217] Enrichment of a desired population can also be achieved by negative selection, for example, using a combination of antibodies against surface markers specific to the negatively selected cells. 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 negatively selected cells. For example, to enrich CD4+ cells by negative selection, a monoclonal antibody cocktail typically containing antibodies specific for CD14, CD20, CD11b, CD16, HLA-DR, and CD8 can be used. The methods disclosed herein also include removing regulatory immune cells, such as 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, such as IL-2.

[0218] Donors and / or recipients of the leukocytes and / or populations of leukocytes disclosed herein, including subjects 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.

[0219] 5.4 Antigen specificity The present invention provides methods 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., targeted killing activity directed against cells expressing a particular antigen. As is known in the art, lymphocyte responses, particularly T cell responses, depend on the recognition of peptides by T cell receptors, particularly in the context of MHC complexes. Thus, the present invention provides for the cultivation of a lymphocyte population in the presence of an antigen against which a desired response is to be directed.

[0220] In certain embodiments, the antigen may be in the form of a peptide that is added to the culture. For example, the peptide may be a known antigen associated with a disease and / or may be an antigen determined in the subject to be treated, e.g., a neoantigen as determined from analysis of a tumor sample or a sample of infected tissue. A sample containing 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).

[0221] Peptides to be included in the culture with lymphocytes (or in the sample containing lymphocytes) can be in soluble form. When soluble peptides are used, they can be cultured with lymphocytes at a concentration of 0.1 to 10 micromolar, 0.5 to 5 micromolar, or 1 to 2 micromolar. Alternatively or additionally, the peptides in the culture can be presented by APCs, as known in the art.

[0222] Antigenic peptides are preferably added to the culture so that they can be presented to lymphocytes by B cells in an MHC-dependent manner. Preferably, the peptides added to lymphocytes are between 9 and 35 amino acids, between 9 and 30 amino acids, or between 9 and 25 amino acids in length. In certain embodiments, the antigenic peptides added to 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 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 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, peptides added to the culture can also be longer peptides, which can be taken up and processed by APCs to form shorter peptides that can be presented in an MHC-dependent manner.

[0223] In some embodiments, the method of the present invention is used to expand TIL.In such embodiments, antigen may be contained in tumor sample.That is, tumor sample can serve as lymphocyte source and antigen source at the same time.Expanding lymphocytes in the presence of tumor sample has the advantage that tumor-specific lymphocytes can be selectively expanded.However, in certain embodiments, lymphocytes can be expanded in the presence of antigen peptide and tumor sample.

[0224] Regardless of whether lymphocytes are expanded in the presence of peptides or tumor samples, it is preferred herein that lymphocytes be co-cultured with APCs. Non-limiting examples of APCs useful in the methods herein include B cells. It is known that B cells stimulate specific populations of lymphocytes, particularly T cells (including TILs), that are responsive to presented antigens. APCs, e.g., B cells, can be either from an allogeneic source (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. Regarding B cells, they can be selected using a Prodigy (Miltenyi Biotec) instrument 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 engineered with mRNA to express the antigens disclosed herein or to express transgenes, particularly transgenes encoding immunomodulatory factors. 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.

[0225] APCs, e.g., B cells, can be modified to present a 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 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, before culturing with a sample known or suspected to contain leukocytes. Bioreactors for culturing 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. B cells can be assessed for quality by testing for CD19+ and / or CD20+ cells. In certain embodiments, 85% or more of the cells in the B cell culture are CD19+ and / or CD20+.

[0226] 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. When B cells (or any other type of APCs) are used to prepare a population of lymphocytes for autologous cell therapy, the B cells need to be collected from the same patient as the lymphocytes.

[0227] Kits for isolating B cells from PBMCs are known in the art and commercially available. Isolated B cells are preferably activated before adding them to lymphocytes. Preferably, 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, B cells may be activated for 1-48 hours, 8-48 hours, or 12-36 hours. For example, B cell activation may be achieved by contacting B cells with IL-4 and / or CD40L. Additionally, B cells may be expanded in the presence of IL-21.

[0228] When APCs are transfected to express a transgene of interest, this can be done by any means known in the art, including but not limited to electroporation, PEG, lipofection, or Crispr Cas. Alternatively or additionally, APCs can be transfected to express an immune regulator, 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, or STAT-5. Alternatively or additionally, APCs can be transfected with one or more activators of at least one signaling pathway, for example, the JAK / STAT pathway, the Akt / PBK AKT signaling pathway, the BCR signaling pathway, or the BAFF / BAFFR signaling pathway.

[0229] In a non-limiting example, the APC may express human OX40L as set forth in SEQ ID NO:1; or human OX40L encoded by the DNA sequence as set forth in SEQ ID NO:2.

[0230] In another non-limiting example, the APC may express mouse OX40L as set forth in SEQ ID NO:3; or mouse OX40L encoded by the DNA sequence as set forth in SEQ ID NO:4.

[0231] In another non-limiting example, the APC may express human 4-1BBL as set forth in SEQ ID NO:5; or human 4-1BBL encoded by the DNA sequence as set forth in SEQ ID NO:6.

[0232] In another non-limiting example, the APC may express mouse 4-1BBL as set forth in SEQ ID NO:7; or mouse 4-1BBL encoded by the DNA sequence as set forth in SEQ ID NO:8.

[0233] In another non-limiting example, the APC may express human CD80 as set forth in SEQ ID NO:9; or human CD80 encoded by the DNA sequence as set forth in SEQ ID NO:10.

[0234] In another non-limiting example, the APC may express mouse CD80 as set forth in SEQ ID NO:11; or mouse CD80 encoded by the DNA sequence as set forth in SEQ ID NO:12.

[0235] In another non-limiting example, the APC may express human CD86 as set forth in SEQ ID NO:13; or human CD86 encoded by the DNA sequence as set forth in SEQ ID NO:14.

[0236] In another non-limiting example, the APC may express mouse CD86 as set forth in SEQ ID NO:15; or mouse CD86 encoded by the DNA sequence as set forth in SEQ ID NO:16.

[0237] In another non-limiting example, the APC may express human CD83 as set forth in SEQ ID NO:17; or human CD83 encoded by the DNA sequence as set forth in SEQ ID NO:18.

[0238] In another non-limiting example, the APC may express mouse CD83 as set forth in SEQ ID NO:19; or mouse CD83 encoded by the DNA sequence as set forth in SEQ ID NO:20.

[0239] In another non-limiting example, the APC may express human CD70 as set forth in SEQ ID NO:21; or human CD70 encoded by the DNA sequence as set forth in SEQ ID NO:22.

[0240] In another non-limiting example, the APC may express mouse CD70 as set forth in SEQ ID NO:23; or mouse CD70 encoded by the DNA sequence as set forth in SEQ ID NO:24.

[0241] In another non-limiting example, the APC may express human IL7 / CD127 as set forth in SEQ ID NO:25; or human IL7 / CD127 encoded by the DNA sequence as set forth in SEQ ID NO:26.

[0242] In another non-limiting example, the APC may express mouse IL7 / CD127 as set forth in SEQ ID NO:27; or mouse IL7 / CD127 encoded by the DNA sequence as set forth in SEQ ID NO:28.

[0243] In another non-limiting example, the APC may express human CD30L as set forth in SEQ ID NO:29; or human CD30L encoded by the DNA sequence as set forth in SEQ ID NO:30.

[0244] In another non-limiting example, the APC may express mouse CD30L as set forth in SEQ ID NO:31; or mouse CD30L encoded by the DNA sequence as set forth in SEQ ID NO:32.

[0245] In another non-limiting example, the APC may express human LIGHT as set forth in SEQ ID NO:33; or human LIGHT encoded by the DNA sequence as set forth in SEQ ID NO:34.

[0246] In another non-limiting example, the APC may express mouse LIGHT as set forth in SEQ ID NO:35; or mouse LIGHT encoded by the DNA sequence as set forth in SEQ ID NO:36.

[0247] In another non-limiting example, the APC may express human BTLA as set forth in SEQ ID NO:37; or human BTLA encoded by the DNA sequence as set forth in SEQ ID NO:38.

[0248] In another non-limiting example, the APC may express mouse BTLA as set forth in SEQ ID NO:39; or mouse BTLA encoded by the DNA sequence as set forth in SEQ ID NO:40.

[0249] In another non-limiting example, the APC may express human ICOS-L as set forth in SEQ ID NO:41; or human ICOS-L encoded by the DNA sequence as set forth in SEQ ID NO:42.

[0250] In another non-limiting example, the APC may express mouse ICOS-L as set forth in SEQ ID NO:43; or mouse ICOS-L encoded by the DNA sequence as set forth in SEQ ID NO:44.

[0251] In another non-limiting example, the APC may express human CD150 as set forth in SEQ ID NO:45; or human CD150 encoded by the DNA sequence as set forth in SEQ ID NO:46.

[0252] In another non-limiting example, the APC may express mouse CD150 as set forth in SEQ ID NO:47; or mouse CD150 encoded by the DNA sequence as set forth in SEQ ID NO:48.

[0253] In another non-limiting example, the APC may express human IL-12 as set forth in SEQ ID NO:49; or human IL-12 encoded by the DNA sequence as set forth in SEQ ID NO:50.

[0254] In another non-limiting example, the APC may express mouse IL-12 as set forth in SEQ ID NO:51; or mouse IL-12 encoded by the DNA sequence as set forth in SEQ ID NO:52.

[0255] In another non-limiting example, the APC may express human IL-7 as set forth in SEQ ID NO:53; or human IL-7 encoded by the DNA sequence as set forth in SEQ ID NO:54.

[0256] In another non-limiting example, the APC may express mouse IL-7 as set forth in SEQ ID NO:55; or mouse IL-7 encoded by the DNA sequence as set forth in SEQ ID NO:56.

[0257] In another non-limiting example, the APC may express human IL-15 as set forth in SEQ ID NO:57; or human IL-15 encoded by the DNA sequence as set forth in SEQ ID NO:58.

[0258] In another non-limiting example, the APC may express human IL-17 as set forth in SEQ ID NO:59; or human IL-17 encoded by the DNA sequence as set forth in SEQ ID NO:60.

[0259] In another non-limiting example, the APC may express mouse IL-17 as set forth in SEQ ID NO:61; or mouse IL-17 encoded by the DNA sequence as set forth in SEQ ID NO:62.

[0260] In another non-limiting example, the APC may express human IL-21 as set forth in SEQ ID NO:63; or human IL-21 encoded by the DNA sequence as set forth in SEQ ID NO:64.

[0261] In another non-limiting example, the APC may express murine IL-21 set forth in SEQ ID NO:65; or murine IL-21 encoded by the DNA sequence set forth in SEQ ID NO:66.

[0262] In another non-limiting example, the APC may express human IL-1 set forth in SEQ ID NO:67; or human IL-1 encoded by the DNA sequence set forth in SEQ ID NO:68.

[0263] In another non-limiting example, the APC may express mouse IL-1 set forth in SEQ ID NO:69; or mouse IL-1 encoded by the DNA sequence set forth in SEQ ID NO:70.

[0264] In another non-limiting example, the APC may express human BCL-6 as set forth in SEQ ID NO:71; or human BCL-6 encoded by the DNA sequence as set forth in SEQ ID NO:72.

[0265] In another non-limiting example, the APC may express mouse BCL-6 as set forth in SEQ ID NO:73; or mouse BCL-6 encoded by the DNA sequence as set forth in SEQ ID NO:74.

[0266] In another non-limiting example, the APC may express human BCLXL as set forth in SEQ ID NO:75; or human BCLXL encoded by the DNA sequence as set forth in SEQ ID NO:76.

[0267] In another non-limiting example, the APC may express mouse BCLXL as set forth in SEQ ID NO:77; or mouse BCLXL encoded by the DNA sequence as set forth in SEQ ID NO:78.

[0268] In another non-limiting example, the APC may express human BCL 2 as set forth in SEQ ID NO:79; or human BCL 2 encoded by the DNA sequence as set forth in SEQ ID NO:80.

[0269] In another non-limiting example, the APC may express mouse BCL 2 set forth in SEQ ID NO:81; or mouse BCL 2 encoded by the DNA sequence set forth in SEQ ID NO:82.

[0270] In another non-limiting example, the APC may express human MCL 1 as set forth in SEQ ID NO:83; or human MCL 1 encoded by the DNA sequence as set forth in SEQ ID NO:84.

[0271] In another non-limiting example, the APC may express mouse MCL 1 as set forth in SEQ ID NO:85; or mouse MCL 1 encoded by the DNA sequence as set forth in SEQ ID NO:86.

[0272] In another non-limiting example, the APC may express human IL-2 set forth in SEQ ID NO:87; or human IL-2 encoded by the DNA sequence set forth in SEQ ID NO:88.

[0273] In another non-limiting example, the APC may express mouse IL-2 set forth in SEQ ID NO:89; or mouse IL-2 encoded by the DNA sequence set forth in SEQ ID NO:90.

[0274] In another non-limiting example, the APC may express the human CD40L set forth in SEQ ID NO:91; or the human CD40L encoded by the DNA sequence set forth in SEQ ID NO:92.

[0275] In another non-limiting example, the APC may express the murine CD40L set forth in SEQ ID NO:93; or the murine CD40L encoded by the DNA sequence set forth in SEQ ID NO:94.

[0276] In another non-limiting example, the APC may express human GITR-L as set forth in SEQ ID NO:95; or human GITR-L encoded by the DNA sequence as set forth in SEQ ID NO:96.

[0277] In another non-limiting example, the APC may express mouse GITR-L as set forth in SEQ ID NO:97; or mouse GITR-L encoded by the DNA sequence as set forth in SEQ ID NO:98.

[0278] In another non-limiting example, the APC may express human CD66a set forth in SEQ ID NO:99; or human CD66a encoded by the DNA sequence set forth in SEQ ID NO:100.

[0279] In another non-limiting example, the APC may express mouse CD66a set forth in SEQ ID NO:101; or mouse CD66a encoded by the DNA sequence set forth in SEQ ID NO:102.

[0280] In certain embodiments, APCs, particularly B cells, have been engineered to express nucleic acids encoding OX40L (SEQ ID NO: 1), 4-1BBL (SEQ ID NO: 5), and / or IL-12 (SEQ ID NO: 49). In certain embodiments, APCs, particularly B cells, have been engineered to express nucleic acids encoding at least two of OX40L (SEQ ID NO: 1), 4-1BBL (SEQ ID NO: 5), and / or IL-12 (SEQ ID NO: 49). In certain embodiments, APCs, particularly B cells, have been engineered to express nucleic acids encoding OX40L (SEQ ID NO: 1), 4-1BBL (SEQ ID NO: 5), and IL-12 (SEQ ID NO: 49). In certain embodiments, APCs, particularly B cells, have been engineered to express nucleic acids encoding OX40L (SEQ ID NO: 1) and 4-1BBL (SEQ ID NO: 5). In certain embodiments, the nucleic acid encoding OX40L (SEQ ID NO: 1), 4-1BBL (SEQ ID NO: 5) and / or IL-12 (SEQ ID NO: 49) is mRNA transfected into the expanded B cells prior to contact with lymphocytes.

[0281] 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.

[0282] In certain embodiments, lymphocytes may be co-cultured with artificial antigen-presenting cells (aAPCs). The term "aAPC" as used herein includes, but is not limited to, cell-based aAPCs, bead-based APCs, microparticle aAPCs, and nanoparticle aAPCs. Materials that have been used include glass, poly(glycolic acid), poly(lactic-co-glycolic acid), iron oxide, liposomes, lipid bilayers, Sepharose, and polystyrene. The aAPCs contain a stimulatory ligand, for example, a stimulatory ligand that specifically binds to the TCR / CD3 complex to transmit a primary signal. The aAPCs may further contain at least one costimulatory ligand that specifically binds to at least one costimulatory molecule present on T cells. aAPCs are known in the art and are disclosed, inter alia, in WO2013 / 086500, WO2005 / 118788, and WO2015 / 051247. In certain embodiments, aAPCs can comprise any of the costimulatory molecules or immunomodulatory factors disclosed herein. Note that APCs such as B cells can be substituted for aAPCs in any of the embodiments disclosed herein. In certain embodiments, lymphocytes can be co-cultured with a mixture of APCs and aAPCs.

[0283] In certain embodiments, the aAPCs may be aAPCs that comprise the costimulatory molecule OX40L (CD134L).

[0284] In certain embodiments, the aAPC may further comprise one or more costimulatory molecules selected from the group consisting of 4-1BBL (CD137L), CD80 (B7-1), CD86 (B7-2), CD83, CD70 (CD27L), CD40, GITRL, and CD153 (CD30L).

[0285] In certain embodiments, the aAPCs may comprise the costimulatory molecules OX40L (CD134L) and 4-1BBL (CD137L).

[0286] In certain embodiments, the aAPC further comprises an antigen-binding protein, preferably an antibody or antibody fragment, capable of specifically binding to a lymphocyte-stimulating receptor, preferably the lymphocyte-stimulating receptor is selected from the group consisting of CD28, CD40L (CD154), OX40 (CD134), and 4-1BB (CD137).

[0287] 5.5 Expansion and culture media Lymphocyte culture is an expansion culture, i.e., the selective expansion of 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). Cell selection and collection 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.

[0288] It is preferred herein that the methods of the present invention are performed in a "single, controlled culture vessel." That is, 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. To this end, the total volume of the cell culture must be adjustable based on the cell expansion rate. That is, the patient sample can be initially cultured in a small volume, and once the cells contained in the patient sample begin to expand, the culture volume can be increased to maintain optimal culture conditions. Furthermore, it is preferred that the medium composition be adjusted throughout the process to maintain optimal culture conditions.

[0289] In the present invention, the single culture vessel is preferably a 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 for 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 to a large scale without the need to switch to a larger vessel.

[0290] 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 as necessary. Preferably, one or more of the culture medium parameters disclosed herein can be monitored and adjusted in a controlled single culture vessel according to the present invention.

[0291] 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), AIM V (Gibco), CTS Optimizer (Thermo Fisher), LymphoOne T Cell Medium (Takara), Stemline, ATCC Media (LGC Standards), and ImmunoCult™-XF T Cell Expansion Medium. The expansion medium can contain IL-2 or variant IL-2, which 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). Additionally, the IL-2 variant can be any of the IL-2 variants disclosed in WO2011 / 063770 or U.S. Pat. No. 8,759,486, which are incorporated herein by reference in their entireties.

[0292] 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).

[0293] Preferably, the lymphocytes are cultured in an ADVA bioreactor, in particular an ADVA X3 bioreactor.

[0294] 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, and most preferably primary human T cells (TILs)) over tumor and other cells. In some embodiments, the IL-2 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 of IL-2, about 9,000 IU / mL of IL-2, about 8,000 IU / mL of IL-2, about 7,000 IU / mL of IL-2, about 6,000 IU / mL of IL-2, about 5,000 IU / mL of IL-2, about 4,000 IU / mL, about 3,000 IU / mL of IL-2, or about 1,000 IU / mL of 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 3,000 IU / mL. In certain embodiments, IL-2 is added to the culture medium at a final concentration of about 6,000 IU / mL during batch mode and at a final concentration of about 3,000 IU / mL during later stages of the process.

[0295] 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 hAB substitute, such as human serum (huS) or platelet lysate (hPL), may be used, or any synthetic hAB variant known in the art may be used.

[0296] 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 of IL-15, about 400 IU / mL of IL-15, about 300 IU / mL of IL-15, about 200 IU / mL of IL-15, about 180 IU / mL of IL-15, about 160 IU / mL of IL-15, about 140 IU / mL of IL-15, about 120 IU / mL of IL-15, or about 100 IU / mL of IL-15.

[0297] 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, 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.

[0298] It is preferred herein that the APCs in the culture medium are genetically engineered to produce IL-12. However, rather than using genetically engineered APCs, IL-12 can also be added to the culture medium as a supplement at any suitable concentration to support lymphocyte expansion.

[0299] The cell culture medium may also contain one or more TNFRSF agonists. In some embodiments, the TNFRSF agonist includes a 4-1BB agonist, which may be, for example, urelumab, utomilumab, 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.

[0300] Additionally or alternatively, the culture medium can contain Tora-dol (ketorolac) at about 0.1 to about 1000 μM, about 1 to about 100 μM, about 5 to about 50 μM, or about 11 μM.

[0301] As described herein, the culture may also include feeder cells known in the art, such as B cells, dendritic cells, T cells, macrophages, and / or PBMCs, which may be autologous or allogeneic. In certain embodiments, the feeder cells are irradiated cells. In certain embodiments, the feeder cells are irradiated PBMCs. The feeder cells may be added at the initiation of the culture, i.e., together with the tumor sample and APCs, or on any day of the expansion culture. In certain embodiments, the feeder cells, particularly irradiated feeder cells, may be added to the lymphocytes in the culture 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 days after the initiation of the culture. In certain embodiments, the feeder cells, particularly irradiated feeder cells, may be added to the lymphocytes in the culture 5 to 15 days, preferably 7 to 12 days, and more preferably 10 days after the initiation of the culture. The ratio of lymphocytes to feeder cells may range from 10:1 to 1:100. In certain embodiments, 1 to 3 mm 3 10 per tumor fragment 7 ~10 9 Between individual feeder cells, in particular irradiated feeder cells, are added to the culture.

[0302] 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. No or very limited lymphocyte expansion occurs during this typically static expansion step. Preferably, pH and dissolved oxygen (DO) concentrations are monitored and controlled during the expansion initiation step, adjusting to predefined ranges as needed. To ensure a sufficient supply of oxygen and nutrients during batch mode, the medium within the growth chamber may be agitated. For example, the medium within the growth chamber may be circulated within the growth chamber, e.g., the medium may be removed from the side of the growth chamber and reintroduced at the bottom. Preferably, the medium is circulated within the growth chamber at a flow rate of 1 to 5 mL / min. Circulation within the growth chamber may begin any time between days 1 and 7 of the process, preferably between days 1 and 5, more preferably on day 1. In the ADVA X3 bioreactor, circulation during batch mode may be achieved by activating a small loop.

[0303] b) Fed-batch mode: As lymphocytes expand 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 lactic acid will accumulate. To maintain the glucose and lactic acid concentrations within a defined range, fresh medium (containing glucose and no lactic acid) is supplied to the growth chamber to increase the glucose concentration and decrease the lactic acid concentration in the culture medium. During fed-batch mode, the pH, DO concentration, glucose concentration, and lactic acid concentration of the culture medium are preferably monitored and adjusted as needed. Due to the addition of culture medium during fed-batch mode, the culture volume will increase. The fed-batch mode is preferably continued until the bioreactor reaches a defined volume.

[0304] 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.

[0305] d) Perfusion mode: When glucose and / or lactate concentrations are no longer within predefined tolerances, the bioreactor switches to perfusion mode. That is, growth medium is constantly or gradually removed from the growth chamber (or an attached conditioning chamber) to waste, while fresh culture medium is simultaneously added. During perfusion mode, the pH, DO concentration, glucose concentration, and lactate concentration of the culture medium are preferably monitored. pH and DO concentration can be adjusted to meet defined values ​​as needed. Glucose and lactate concentrations can be fine-tuned by adjusting the perfusion rate.

[0306] 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 specific nutrients to the growth medium).

[0307] In the present invention, tumor samples are preferably seeded together with antigen-presenting cells in the growth chamber of a bioreactor and initially cultured in batch mode. Preferably, cell culture medium is circulated within the growth chamber during batch mode to improve oxygen and nutrient supply. In the ADVA X3 bioreactor, this can be achieved by small-loop circulation. After a certain number of days and / or when the lactate concentration within the growth chamber reaches a threshold value, e.g., 10 mM, an activating agent, such as an activating anti-CD3 antibody, is added to the growth chamber. That is, the activating anti-CD3 antibody is preferably added while the cells are still in batch mode.

[0308] After the activation step, fresh medium can be added to the growth chamber to initiate fed-batch mode. In certain embodiments, fed-batch mode will be initiated 1, 2, 3, 4, or 5 days after the activation step, preferably 2 days after the activation step. In the ADVA X3 bioreactor, this can be achieved by starting a large circulation loop. Once the final volume of the growth chamber is reached, medium can be circulated between the growth chamber and a reservoir containing fresh medium or a conditioning chamber (circulation mode). Medium can be circulated between the growth chamber and the conditioning chamber until one or more parameters of the growth medium fall outside of a predefined range. At that point, perfusion mode can be initiated by constantly removing growth medium from the conditioning chamber and replacing the removed medium in the conditioning chamber with fresh medium.

[0309] That is, in certain embodiments, fed-batch, circulation, and / or perfusion modes can include adding culture medium from a reservoir or conditioning chamber to the growth chamber. The flow rate at which culture medium is pumped from the reservoir / conditioning chamber to the growth chamber can depend on the conditions in the growth chamber and / or the expansion rate of the cells and can be adjusted accordingly. Typically, the flow rate is higher near the end of the process when cells are expanding rapidly. In certain embodiments, the flow rate at which culture medium is pumped into the growth chamber ranges from 1 to 20 mL / min. To maintain a constant volume within the growth chamber, medium is preferably removed from the growth chamber at a similar rate.

[0310] 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 growth medium is simultaneously removed from the bioreactor and / or associated conditioning chamber. Preferably, perfusion of the lymphocytes is performed as disclosed in WO2018 / 037402, which is incorporated herein by reference in its entirety.

[0311] 5.6 TIL Expansion In a first step, the tumor sample is cultured in a growth chamber of a bioreactor in batch mode for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days. During this time, TILs contained in the tumor sample will migrate out of the tumor sample. However, it should be understood that lymphocytes may expand, at least to some extent, even in batch mode, for example, by activation by APCs.

[0312] It is preferred herein that the batch mode be performed immediately prior to the subsequent expansion step in the same bioreactor. However, the batch mode may be omitted or shortened if the tumor sample is processed / before it is added to the bioreactor. For example, tumor fragments can be enzymatically digested, and the resulting TILs can then be transferred to the bioreactor for the expansion step.

[0313] Expansion of lymphocytes requires the presence of an activation signal. In the methods of the present invention, lymphocytes are preferably first activated by a population of antigen-presenting cells (APCs) or artificial antigen-presenting cells that are co-cultured with the lymphocytes. It is preferred herein that the lymphocytes are co-cultured with APCs for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days, more preferably between 7 and 15 days. The APCs are preferably activated B cells as disclosed herein.

[0314] APCs are preferably added to the growth chamber along with the tumor sample and, optionally, the pool of antigenic peptides during batch mode, although APCs can also be added to the TILs at a later time point.

[0315] In a preferred embodiment, lymphocytes are co-cultured with antigen-presenting cells (APCs), particularly B cells. The lymphocytes and APCs can be mixed in a ratio that allows sufficient availability of MHC-presented antigen peptides by the lymphocytes.

[0316] Furthermore, it is known that APCs, and particularly 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 and costimulation of lymphocytes by lymphocytes.

[0317] In certain embodiments, B cells are cultured with tumor fragments known or suspected to contain lymphocytes, particularly TILs. 3 One tumor fragment with a size of approximately 1 x 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 individual B cells.

[0318] 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 10 ...

[0319] In certain embodiments, 1 to 3 mm 3 Between 50 and 100 tumor fragments with a size of 10-200 × 10 6 In certain embodiments, the B cells are contacted with 1 to 3 mm 3 Between 50 and 100 tumor fragments with a size of 50-150 x 10 6 In certain embodiments, the B cells are contacted with 1 to 3 mm 360 tumor fragments with a size of 100 x 10 6 The antibody is contacted with a single B cell.

[0320] In certain embodiments, 1 to 3 mm 3 Between 50 and 100 tumor fragments with a size of 1-100 × 10 6 In certain embodiments, the B cells are contacted with 1 to 3 mm 3 Between 50 and 100 tumor fragments with a size of 5-75 x 10 6 In certain embodiments, the B cells are contacted with 1 to 3 mm 3 60 tumor fragments with a size of 60 x 10 6 The antibody is contacted with a single B cell.

[0321] 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.

[0322] It is preferred herein that lymphocytes are initially co-cultured with B cells in a suitable cell culture medium supplemented with IL-2 (preferably at a concentration of 6000 IU / mL) and optionally Tora-dol (ketorolac) preferably at a concentration of 11.7 μM.

[0323] It is 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.

[0324] 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 methods of the present invention. Preferably, the anti-CD3 antibody OKT-3 is used to activate lymphocytes in culture. However, any other suitable activating anti-CD3 antibody can be used within the scope of the present invention.

[0325] Preferably, the APCs are added to the tumor sample in the growth chamber at the beginning of the process, thus prior to the addition of the activating anti-CD3 antibody. Preferably, the APCs and optionally the peptide antigen are added to the tumor sample at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days prior to the activating anti-CD3 antibody. Thus, the activating anti-CD3 antibody or any other suitable activator can be added to the culture while in batch mode, fed-batch mode, recirculating mode, and / or perfusion mode.

[0326] 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 OKT-3 antibody 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. The cell culture medium may contain OKT-3 antibody at a concentration of 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. In another preferred embodiment, the OKT-3 antibody is added to the culture medium to obtain a final concentration of about 30 ng / mL.

[0327] It is preferred herein that the anti-CD3 antibody, particularly the OKT-3 antibody, is added to the cell culture after the addition of APCs. Preferably, the anti-CD3 antibody, particularly the OKT-3 antibody, is added to the culture after the lymphocytes have been cultured in the presence of APCs for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days. In a particularly preferred embodiment, the anti-CD3 antibody, particularly the OKT-3 antibody, is added to the culture after the lymphocytes have been cultured in the presence of APCs for 8 to 12 days, even more preferably 9 to 11 days, and most preferably 10 days.

[0328] In certain embodiments, lymphocytes are first cultured with B cells and a pool of peptides for 7 to 15 days, preferably 8 to 12 days, even more preferably 9 to 11 days, and most preferably 10 days, before an anti-CD3 antibody, particularly an OKT-3 antibody, is added to the culture.

[0329] Alternatively or additionally, the activating anti-CD3 antibody can be added to the cell culture medium when a certain lactate concentration is reached in the cell culture medium. That is, the activating anti-CD3 antibody can be added to the cell culture medium when the lactate concentration in the medium reaches at least 5 mM, at least 6 mM, at least 7 mM, at least 8 mM, at least 9 mM, at least 10 mM, at least 11 mM, at least 12 mM, at least 13 mM, at least 14 mM, or at least 15 mM. Note that the cell culture medium initially does not contain any lactate, and all lactate in the cell culture medium will be formed by the metabolic activity of the cells. In a preferred embodiment, the activating anti-CD3 antibody is added to the cell culture medium when the lactate concentration in the medium reaches at least 10 mM.

[0330] In certain embodiments, an activator, such as an anti-CD3 antibody, can be added to lymphocytes more than once. That is, in certain embodiments, an anti-CD3 antibody, such as OKT-3, can be added to lymphocytes twice, with the second dose of antibody being given 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after the first dose. In certain embodiments, an anti-CD3 antibody, such as OKT-3, can be added to lymphocytes multiple times, for example, at intervals of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days.

[0331] In addition to the CD3 agonist, further activators, such as CD28 agonists, may be added to the culture.

[0332] 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 be adjusted (transition from batch to fed-batch mode) based on the rate of lymphocyte expansion. To this end, certain parameters of the culture medium must be continuously monitored.

[0333] The batch mode is followed by a fed-batch mode, during which fresh culture medium is added to the growth chamber 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. These parameters include, but are not limited to, pH, dissolved oxygen (DO), glucose, lactate, glutamine, 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. pH and / or DO can be adjusted by adjusting oxygen and / or carbon dioxide in the headspace of the growth chamber. The temperature of the culture medium can be adjusted with a heating element.

[0334] When fresh culture medium is added to the growth chamber during fed-batch mode, it is preferred that the fresh culture medium be added near the bottom of the growth chamber so 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.

[0335] The fed-batch mode ultimately results in an increased culture volume. Because the rate at which fresh culture medium is added to the growth chamber depends on the composition of nutrients (particularly glucose) and / or the production of metabolites (particularly 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 adjusting the volume of the culture medium according to the expansion rate of lymphocytes in the growth chamber.

[0336] In certain embodiments, the culture volume will be increased by at least 2, 3, 4, 5, or 6 fold during fed-batch mode. Preferably, fed-batch mode is performed until the maximum or defined volume of the growth chamber is reached.

[0337] Once a defined cell culture volume in the growth chamber, e.g., the maximum volume of the growth chamber, is reached, the bioreactor can be set to a circulation mode, i.e., 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 within the growth chamber.

[0338] 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 during recirculation mode after the culture has reached its final volume. However, pH (by CO), DO (by O), and temperature (by a heating element) can be adjusted during recirculation mode.

[0339] It should be noted that circulation is primarily performed to reduce consumption of fresh medium, however, the circulation mode may be omitted and instead the fed-batch mode may be immediately followed by the perfusion mode.

[0340] Finally, during the perfusion mode, medium is constantly or gradually removed from the growth chamber and replaced with fresh medium. As with 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 comes into contact with the lymphocytes within the growth chamber.

[0341] During perfusion mode, the same parameters as discussed above for fed-batch and circulation modes are preferably monitored. The perfusion rate can be adjusted according to the composition of nutrients (especially glucose) or the formation of metabolites (especially lactate).

[0342] The expansion phase can last from 5 to 35 days. The expansion phase can be from 5 to 30 days, from 5 to 25 days, from 5 to 20 days, or from 5 to 15 days. In certain embodiments, the expansion phase is only 15 days. In certain embodiments, the expansion phase can be from 25 to 50 days, from 25 to 45 days, from 25 to 40 days, or from 25 to 35 days. It is more 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.

[0343] That is, once obtained from a 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 x 10 9 Preferably, the cells are not frozen at any point until they reach a density of at least 1 x 10 cells. 9 , 2 × 10 9 , 3×10 9 , 4×10 9 , 5×10 9 , 6×10 9 , 7×10 9 , 8×10 9 , 9×10 9 , 10×10 9 , 11×10 9 , 12×10 9 , 13×10 9 , 14×10 9 , 15×10 9 , 16×109 , 17×10 9 , 18×10 9 , 19×10 9 , or at least 20 × 10 9 This can be continued until 100 T cells are obtained.

[0344] If desired, the first expansion phase described herein above can be combined with a second expansion phase to reach even higher cell numbers, in which case the cells can be harvested from the first culture vessel and transferred to a second culture vessel when the desired cell number is reached in the first culture vessel.

[0345] Thus, in a particular embodiment, the invention relates to a method according to the invention, wherein the cells are harvested from the culture vessel after expansion and, optionally, transferred to a second culture vessel for a second expansion.

[0346] In certain embodiments, the cells are at least 1 x 10 9 , 2 × 10 9 , 3×10 9 , 4×10 9 , or 5 × 10 9 When cells are obtained in the first culture vessel, they are collected from the first culture vessel. Thus, in certain embodiments, the present invention provides a method for producing a culture of at least 1 x 10 cells. 9 The present invention relates to a method according to the invention, wherein the cells are transferred from a first culture vessel to a second culture vessel.

[0347] The second culture vessel contains lymphocytes, preferably at approximately 1 x 10 9 Starting with a cell count of at least 1 x 10 10 cells, preferably 2 x 10 10 cells, more preferably 3 x 10 10 cells, most preferably 4 x 10 10 Any culture vessel that allows for expansion to reach a certain amount of cells may be used.

[0348] In a particular embodiment, the present invention relates to a method according to the present invention, wherein the culture medium in the second container is supplemented with human or synthetic AB serum, IL-2, IL-15, nicotinamide and / or nicotinamide mononucleotide.

[0349] That is, the lymphocytes can be cultured in the second culture vessel in any cell culture medium suitable for lymphocyte expansion, particularly any of the cell culture media disclosed elsewhere herein. In certain embodiments, the cell culture medium used in the second culture vessel is supplemented with human or synthetic AB serum, IL-2, IL-15, nicotinamide and / or nicotinamide mononucleotide.

[0350] In certain embodiments, the cell culture medium used in the second culture vessel is supplemented with human or synthetic AB serum at a concentration ranging from 0-20%, preferably 1-10%, more preferably 2.5-7.5%, and most preferably 5%.

[0351] In certain embodiments, the cell culture medium used in the second culture vessel is supplemented with IL-2 at a concentration ranging from 1000 to 10000 IU / mL, preferably 2000 to 8000 IU / mL, more preferably 3000 to 6000 IU / mL, and most preferably 3000 IU / mL.

[0352] In certain embodiments, the cell culture medium used in the second culture vessel is supplemented with human or synthetic AB serum at a concentration ranging from 0 to 20% and IL-2 at a concentration ranging from 1000 to 10000 IU / mL.

[0353] In certain embodiments, the cell culture medium used in the second culture vessel is supplemented with human or synthetic AB serum at a concentration ranging from 1-10% and IL-2 at a concentration ranging from 2000-8000 IU / mL.

[0354] In certain embodiments, the cell culture medium used in the second culture vessel is supplemented with human or synthetic AB serum at a concentration ranging from 2.5 to 7.5% and IL-2 at a concentration ranging from 3000 to 6000 IU / mL.

[0355] In certain embodiments, the cell culture medium used in the second culture vessel is supplemented with human or synthetic AB serum at a concentration of 5% and IL-2 at a concentration of 3000 IU / mL.

[0356] In certain embodiments, the cell culture medium used in the second culture vessel further comprises nicotinamide and / or nicotinamide mononucleotide. In certain embodiments, nicotinamide and / or nicotinamide mononucleotide is added to the cell culture medium used in the second culture vessel at a concentration of 0.1 to 100 mM, preferably 0.5 to 50 mM, more preferably 1 to 25 mM, even more preferably 2 to 10 mM, and most preferably 4 mM.

[0357] In certain embodiments, nicotinamide and / or nicotinamide mononucleotide is added to the cell culture medium in the second culture vessel only during seeding of the lymphocytes, while fresh medium added to the lymphocytes during culture, i.e., by perfusion, is free or substantially free of nicotinamide and / or nicotinamide mononucleotide.

[0358] In a particular embodiment, the invention relates to a method according to the invention, wherein the second expansion comprises a step of dynamic culture of the lymphocytes, in particular a step of perfusion.

[0359] That is, the second expansion preferably includes a step in which the used medium from the second culture vessel is replaced with fresh culture medium. Preferably, the second expansion includes a step of perfusion as described in more detail elsewhere herein.

[0360] It is preferred herein that the lymphocytes collected from the first culture vessel are seeded into a second culture vessel in fresh culture medium. During expansion of the lymphocytes in the second culture vessel, fresh medium can be added to the cells until the final volume of the second culture vessel is reached.

[0361] Preferably, the medium is added to the second culture vessel at a rate that allows a certain cell concentration to be maintained in the second culture vessel. Preferably, the fresh culture medium is added ... 6 , preferably 2 x 10 6 The cells are added to the second culture vessel at a rate that allows the cell concentration in the second culture vessel to be maintained at a rate of at least 1 x 10 cells / mL. Thus, in certain embodiments, the present invention provides a method for maintaining a cell concentration in the second culture vessel of at least 1 x 10 cells / mL. 6 , preferably 2 x 10 6 cells / mL.

[0362] When the final volume of the second culture vessel is reached, the used medium can be removed from the second culture vessel and replaced with fresh culture medium (perfusion). The culture medium from the second culture vessel can be constantly or gradually replaced with fresh culture medium.

[0363] In certain embodiments, the culture medium in the second culture vessel is constantly exchanged at a fixed perfusion rate. In certain embodiments, the culture medium in the second culture vessel is exchanged at a perfusion rate of 0.5 to 10 L / day. More preferably, the culture medium in the second culture vessel is exchanged at a perfusion rate of 1 to 6 L / day.

[0364] The second expansion can be carried out until a desired cell number is reached. In particular, the second expansion can be carried out until a desired cell number is reached. 10 cells, preferably at least 2 x 10 10 cells, more preferably at least 3 x 10 10 cells, most preferably at least 4 x 10 10 This can be done until cells are obtained in the second culture vessel.

[0365] To reach this cell number, approximately 1 x 10 cells were harvested from the first culture vessel. 9 ~5×10 9 When starting from cells, the second expansion may be carried out for at least 2, 3, 4, 5, 6, or 7 days. Thus, in a particular embodiment, the invention relates to a method according to the invention, wherein the second expansion lasts for at least 2, 3, 4, 5, 6, or 7 days.

[0366] For the second expansion, a XURI bioreactor with a volume of at least 5 L is preferably used. The cells collected from the first culture vessel can be inoculated into approximately 2 L of suitable cell culture medium in the XURI bioreactor. Preferably, 1 to 5 x 10 cells from the first culture vessel are inoculated into approximately 2 L of suitable cell culture medium in the XURI bioreactor. 9 After the filtration step, the cells can be inoculated into 1 to 3 L, preferably 1.5 to 2.5 L, of cell culture medium.

[0367] It is understood that other bioreactors, particularly those having similar dimensions and suitable for perfusion, can be used as the second culture vessel.

[0368] In a preferred embodiment, the first and / or second expanded populations are at least 90% CD3+, contain at least 15% cells reactive with the desired antigen, e.g., neoantigen recovered / identified in the patient, contain a majority of CD8+ cells, and have a viability of at least 70%. It is further preferred that at least half of the T cells responding to stimulation with the neoantigen peptide produce a sustained response in the patient. To this end, peripheral lymphocytes can be recovered from the patient and tested for the presence of the neoantigen in an ELISpot assay.

[0369] 5.7 CAR-T cell expansion In certain embodiments, the method of the present invention is used to expand CAR-T cells.To this end, T cells isolated from a subject can first be cultured in a suitable culture medium, such as one of the T cell culture media disclosed herein, in a batch mode.In certain embodiments, the T cells are enriched CD4+ and / or CD8+ T cells.

[0370] Preferably, the isolated T cells are activated during the initial culture step. T cell activation can be achieved using a CD3 and / or CD28 agonist. In certain embodiments, the CD3 agonist is an anti-CD3 antibody, and the CD28 agonist is an anti-CD28 antibody. In certain embodiments, the anti-CD3 antibody and / or the anti-CD28 antibody are immobilized on solid particles such as microbeads or nanobeads. Alternatively, T cells can be activated with antigen-presenting cells, or more preferably, with artificial antigen-presenting cells (aAPCs). Those skilled in the art will be aware of the ratio of T cells to activators to achieve sufficient T cell activation.

[0371] Activation of T cells can be accomplished in batch mode, however, the batch mode may be omitted for production of CAR-T cells, and the T cells may be activated while the reactor is in fed-batch mode.

[0372] Preferably, the activation medium is supplemented with IL-2 or an active variant thereof disclosed herein. Alternatively or additionally, T cells can be activated in the presence of IL-7 and / or IL-15. Activation can be performed for 3 to 16 days.

[0373] After activation, the T cells are transduced with a vector encoding a CAR. Suitable vectors for introducing the genetic information of a CAR include viral vectors, such as retroviral, lentiviral, or AAV vectors, or transposon vectors, such as Sleeping Beauty transposon vectors. Those skilled in the art can identify the appropriate amount of vector to achieve sufficient transduction of T cells. In certain embodiments, the vector is added to activated T cells at an MOI ranging from 1 to 5, more preferably from 2 to 3.

[0374] In certain embodiments, the transduction of T cells occurs while the reactor is still in batch mode. In certain embodiments, the transduction of T cells occurs while the reactor is in fed-batch mode. In certain embodiments, the transduction of T cells occurs while the reactor is already in circulation or perfusion mode.

[0375] After the transduction step, the CAR-T cells can be expanded in a culture medium to obtain a large number of cells. To this end, the CAR-T cells can be cultured as described herein, i.e., in a single controlled culture vessel in which the culture volume is adjusted to the cell expansion rate and one or more parameters of the culture medium are controlled and continuously adjusted. It is preferred herein that during expansion, the T cells are continuously supplemented with IL-2 or its active variant, or alternatively, IL-7 and IL-15.

[0376] The expansion can be carried out for any period of time, but preferably for at least 10 9 This may be carried out until a cell number of 1000 is reached.

[0377] 5.8 Bioreactor design for the first expansion step The bioreactor herein preferably includes 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 includes at least one inlet that can pump the medium from the growth chamber into the conditioning chamber. The conditioning chamber, which is connected to the growth chamber via at least one inlet and at least one outlet, can be used to circulate the culture medium within the growth chamber.

[0378] 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.

[0379] The conditioning chamber and / or growth chamber preferably include one or more sensors that allow monitoring of one or more parameters of the culture medium. That is, the conditioning chamber may include 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, glutamate concentration, and temperature. However, the bioreactor may also include 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 constantly or at defined intervals. In certain embodiments, glucose and lactate concentrations, and optionally glutamate / glutamine concentrations, are measured in the analysis unit by any suitable method known in the art.

[0380] A tolerance range can be defined for each parameter of the culture medium. 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 that parameter. Certain parameters, such as pH, DO, or temperature, can be constantly monitored. However, determination of other parameters, such as glucose or lactate concentration, can take longer and therefore can be performed at certain intervals. For example, without limitation, a certain parameter can be determined every 5 seconds, every 10 seconds, every 30 seconds, every minute, every 5 minutes, every 10 minutes, every 15 minutes, every 30 minutes, or every 60 minutes.

[0381] Lymphocyte expansion leads to the consumption of medium components (e.g., glucose, glutamate, 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 can 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.

[0382] It will be appreciated that a bioreactor for the above process is equipped with at least a growth chamber connected to a supply of fresh medium and a waste container, and further includes the pumps necessary to add fresh medium to the growth chamber and to remove used medium from the growth chamber.

[0383] However, it is preferred herein that the bioreactor for the above process further comprises a conditioning chamber and a pump necessary for circulating 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 for monitoring parameters of the culture medium throughout the process. Suitable devices for the above single-step expansion of lymphocytes 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 incorporated herein by reference in its entirety.

[0384] The growth chamber is a chamber suitable for culturing lymphocytes, particularly T cells. It is preferred herein that the growth chamber is suitable for culturing lymphocytes in 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 culture medium from the growth chamber (either to a waste container or to a conditioning chamber).

[0385] 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 the culture medium can be removed from near the surface of the culture medium within the growth chamber. Adding culture medium at the bottom of the growth chamber and removing it from the top of the growth chamber creates a flow of culture medium along the lymphocytes to adequately provide nutrients to the lymphocytes.

[0386] In certain embodiments, the growth chamber can include multiple outlets at a top portion of the growth chamber, the outlets being 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 culture medium near the surface of the culture medium within the growth chamber.

[0387] Preferably, the cells are separated from the inlet at the bottom of the growth chamber by a perforated barrier. Growth chambers that can be used in the methods of the invention for culturing lymphocytes are disclosed in WO2018037402, which is incorporated herein by reference in its entirety.

[0388] When lymphocytes are provided in a recirculated 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. Furthermore, the conditioning chamber may include one or more sensors for monitoring parameters of the culture medium within the conditioning chamber. Furthermore, the conditioning chamber may include an agitator to facilitate mixing of the culture medium and supplements within the conditioning chamber. The conditioning chamber may further include a heating element to maintain the culture medium at a predefined temperature.

[0389] As described above, the bioreactor may include multiple sensors for monitoring parameters in the culture medium. The sensors are preferably located within the growth chamber and / or the conditioning chamber. Alternatively or additionally, one or more sensors may be located at 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.

[0390] 5.9 Control of culture conditions It is preferred herein that one or more parameters of the culture medium in the growth chamber are maintained within a predefined range. The conditioned culture medium may be based on any culture medium suitable for culturing lymphocytes. In particular, the conditioned growth medium may be based on any culture medium suitable for culturing T cells. In particular, the conditioned growth medium may be based on any T cell medium disclosed herein.

[0391] It is preferred herein that at least one of the parameters disclosed herein is monitored throughout the process and maintained within a defined range. However, it should be understood that the target ranges may be different for the different modes disclosed herein (batch mode, fed-batch mode, recirculation mode, perfusion mode). That is, the target range for a parameter may be different, for example, but not limited to, between batch mode and perfusion mode.

[0392] 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 to 8, preferably 6.5 to 7.5, and more preferably 7.0 to 7.4. Maintaining the 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.

[0393] 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 ranging from 10% to 100% DO, preferably 20% to 90% DO, and more preferably 30% to 80% DO. Maintaining the DO concentration in the culture medium can be achieved by sparging air or oxygen into the culture medium.

[0394] 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 ranging from 0.5 to 10 g / L of glucose, preferably from 1 to 8 g / L of glucose, and more preferably from 2 to 6 g / L of glucose. Maintaining 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 be maintained by supplementing the culture medium with fresh glucose-containing culture medium.

[0395] 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. Maintaining the 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 be maintained by supplementing the culture medium with fresh glutamate-containing culture medium.

[0396] In certain embodiments, a defined glutamine concentration is maintained in the conditioned growth medium. Sensors or methods for continuously measuring glutamine concentration in a fluid are known in the art and are commonly used in bioreactors. Maintaining the 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 be maintained by supplementing the culture medium with fresh glutamine-containing culture medium.

[0397] 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 so that the lactate concentration is maintained at less than 15 mM g / L lactate, preferably 10 mM g / L lactate, and 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 lactate-free culture medium.

[0398] In certain embodiments, the conditioned growth medium is maintained at a defined temperature. Sensors for continuously measuring temperature in fluids are known in the art and are commonly used in bioreactors. The culture medium according to the present invention is preferably maintained at a temperature in the range of 35-39°C, preferably 36-38°C, and more preferably 36.5-37.5°C. Maintaining the temperature of the culture medium within the defined range can be achieved by heating means included within the bioreactor.

[0399] While it is possible to replenish the growth medium in the growth chamber, it is preferable to replenish the growth medium in the conditioning chamber to prevent direct contact between lymphocytes and highly concentrated supplements. Rather than replenishing the medium in the conditioning chamber, it is preferable to replace the medium in the conditioning chamber with fresh medium whenever necessary or at predefined intervals. During perfusion mode, if medium is removed from the growth chamber and becomes waste and fresh medium is added from the conditioning chamber to the growth chamber, it may be necessary to add fresh medium to the growth chamber to ensure a constant medium supply. DO and pH are preferably regulated directly in the growth chamber by adjusting the CO2 and O2 composition in the headspace of the growth chamber.

[0400] 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.

[0401] 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 the defined ranges disclosed herein.

[0402] 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 the defined ranges disclosed herein.

[0403] 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 the defined ranges disclosed herein.

[0404] 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 the defined ranges disclosed herein.

[0405] 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 the defined ranges disclosed herein.

[0406] 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.

[0407] 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.

[0408] 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 incorporated herein by reference in its entirety.

[0409] 5.10 Antigens and Neoantigens In certain embodiments, it is preferred that the T cells contained within the population of lymphocytes, particularly TILs, specifically recognize one or more predetermined antigens. This 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 those antigens.

[0410] As described in more detail above, antigens are preferably presented to lymphocytes by antigen-presenting cells, particularly B cells. Methods for achieving specific antigen presentation by APCs are disclosed herein and include genetic engineering of APCs, adding synthetic peptides to APCs, or adding antigen-containing tissues, such as tumor samples, to APCs. Alternatively, homogenized tumor samples can be added to APCs.

[0411] Neoantigens arise as a result of somatic mutations in tumor cells and are therefore expressed only by tumor cells, not by 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 mutational context to achieve therapeutic effects.

[0412] In certain embodiments, the presented antigen is recovered by sequencing the tumor or peripheral blood cells or other potential antigen source (for example, tumor sample or infected tissue sample) of the patient to be treated, and identified by suitable algorithm.Such algorithms are well known in the art, and include, for example, Neon (Neon Therapeutics) and Achilles (Achilles Therapeutics).The identification of neoantigen in tumor sample 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.

[0413] Neo-antigenic 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.

[0414] In the method according to the invention, lymphocytes, preferably APCs, are preferably contacted with a pool of chemically synthesized peptides.

[0415] The pool of chemically synthesized peptides can be specifically designed for the subject to be treated with the lymphocyte population. 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 from which the subject is suffering.

[0416] Alternatively, the pool of peptides may be personalized for a subject with cancer, i.e., the pool of peptides may include antigen and / or neo-antigenic peptides identified as present in the subject's tumor.

[0417] The pool of peptides may also comprise a mixture of "known" and "personalized" antigenic and / or neo-antigenic peptides.

[0418] Preferably, the pool of chemically synthesized peptides consists of or comprises neo-antigenic peptides, and more preferably, the neo-antigenic peptides contained in the pool of chemically synthesized peptides are those identified in a tumor sample from the same subject from which lymphocytes for the culture process were obtained.

[0419] The identified neo-antigens can range in length from 6 to 20 amino acids or from 9 to 25 amino acids. Alternatively, complete MHC complexes (maximum size 45 KDa) bearing the neo-antigen peptide can be contacted with a population of cells. In certain embodiments, the present invention also encompasses the use of antigens described herein (whether known or identified according to the methods of the present invention) to attract and collect peripheral immune cells (including T cells, B cells, NK cells, or macrophages).

[0420] In certain embodiments, neoantigens are not individually identified, but rather are presented by adding a sample of tumor or infected tissue, particularly an encapsulated sample, to lymphocyte cultures.

[0421] 5.11 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 constitutive depending on the system used, as is known in the art. The encoding nucleic acid may or may not be stably integrated into the genome of the engineered cell.

[0422] 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. Those skilled in the art can easily determine the appropriate method for a given cell type and intended use using common general knowledge. Such methods for genetically engineering cells by introducing a nucleic acid molecule / sequence encoding a polypeptide of interest (e.g., in an expression vector) include, but are not limited to, chemical methods, electroporation, calcium phosphate, cationic lipid, and liposome methods. The nucleic acid molecule / sequence to be transduced can be conventionally and highly efficiently transduced using commercially available transfection reagents and / or any suitable method known in the art or described herein. In addition to methods for genetically engineering cells using nucleic acid molecules comprising or consisting of DNA sequences, the methods 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.

[0423] Physical methods for introducing polynucleotides into host cells 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.

[0424] The biological method for introducing target polynucleotide into host cell includes the use of DNA and RNA vector.Virus vector, especially retrovirus vector, has become the most widely used method for inserting gene into mammalian cell.Therefore, retrovirus vector is preferred for use in the method and cell disclosed herein.Virus vector can be derived from various different viruses, including but not limited to lentivirus, poxvirus, herpes simplex virus type I, adenovirus and adeno-associated virus; for example, see United States Patent No. 5,350,674 and United States 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 83 (1986), 6563-6567), 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. 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, the PL-SIN lentiviral vector (Hotta et al., Nat Methods. 6 (2009), 370-376), p156RRL-sinPPT-CMV-GFP-PRE / NheI (Campeau et al., PLoS One 4 (2009), e6529), and 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 number 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).

[0425] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., artificial membrane vesicle). Other cutting-edge targeted delivery methods for nucleic acids are available, such as delivery of polynucleotides in targeted nanoparticles or other suitable submicron-sized delivery systems.

[0426] 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 These include detecting 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 the expression and / or endogenous activity of endogenously expressed proteins, for example, to assess and / or sort populations based on endogenous function.

[0427] 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 relative to the endogenous / wild-type sequence), and / or endogenous cytokine receptors having a sequence modified relative to the wild-type sequence (i.e., modified endogenous cytokine receptors). Alternatively, or in addition, one or more of the T cells in the populations 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 lacking such modification.

[0428] 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 be capable of endogenously expressing 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 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.

[0429] The lymphocyte populations of the present invention (preferably including human lymphocytes, more preferably primary human lymphocytes, and most preferably primary human T cells ((TILs)) can be further modified to express chimeric antigen receptors (also referred to as "CARs") 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 contains 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., a specific antigen associated with cancer, e ... The chimeric antigen receptor may be in the form of a single-chain variable fragment (scFv) derived from a monoclonal antibody, providing specificity for an antigen (epitope or antigen) preferentially expressed on the surface of cancer cells or other disease-causing cells. The extracellular ligand-binding domain may be specific for the antigen or epitope of interest. The intracellular stimulatory domain typically comprises the intracellular 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 CD3ζ, CD28, 4-1BB, OX40, or combinations thereof. Chimeric antigen receptors may further comprise additional structural elements, including a transmembrane domain connected to the extracellular ligand-binding domain via a hinge or spacer sequence.

[0430] One or more lymphocytes in the population of lymphocytes of the present 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 altered compared to that of the endogenous / wild-type sequence) and / or one or more endogenous cytokine receptors having a sequence that is altered 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 to 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 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 ligand-specific activity.

[0431] 5.12 Non-alloreactive T cells Populations 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 pharmaceuticals, for example, in the treatment of cancer. They, and treatments based on their use, can be either part of autoimmunotherapy or part of allogeneic immunotherapy treatments. As understood in the art, "autologous" in the context of immunotherapy methods refers to a situation in which the origin of the population used in 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 a situation in which the origin of the lymphocytes or population of lymphocytes used in immunotherapy is from a donor that is genetically distinct from the patient.

[0432] The lymphocyte population of the present invention and / or obtainable by the methods disclosed herein can be genetically modified before, during, or after expansion so that they can be used in allogeneic treatment. As is 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 responses. In 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 can be 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 obtaining a sample known or suspected to contain lymphocytes from a donor (especially T cells (preferably TILs)), and inactivating their genes involved in MHC recognition, as is well known in the art. Such methods generally rely on the destruction of endogenous TCRs. TCRs contain 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 a TCR consists of immunoglobulin-like N-terminal variable (V) and constant (C) regions, a hydrophobic transmembrane domain, and a short cytoplasmic region. As with immunoglobulin molecules, the variable regions of the alpha and beta chains are generated by V(D)J recombination, thereby generating a great 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 MHC by T cell receptors between donors and recipients contribute to T cell proliferation and the potential development of graft-versus-host immune responses, which 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 gene (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.

[0433] Alternatively, non-allo-reactive engineering methods 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, which 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.

[0434] Similarly, lymphocyte populations 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 to eliminate or reduce their recognition by the host immune system. This is an effort to minimize or eliminate host-versus-graft immune responses. Similar to non-alloreactive engineering, engineering 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 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 expression of endogenous major histocompatibility complexes.

[0435] 5.13 Pharmaceutical Compositions In a particular embodiment, the present invention relates to a pharmaceutical composition comprising a population of lymphocytes according to the present invention.

[0436] The lymphocyte populations of the present invention are 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 suitable for injection into the human body. Suitable liquids for suspending the cells contained in the lymphocyte population include, but are not limited to, pharmaceutically acceptable buffers.

[0437] In certain embodiments, the pharmaceutically acceptable buffer may be a sodium chloride buffer. In certain embodiments, the pharmaceutically acceptable buffer may be a 0.9% NaCl buffer. In certain embodiments, the pharmaceutically acceptable buffer may be supplemented with at least 5%, 10%, 15%, or 20% DMSO to enable freezing of the lymphocyte population. In certain embodiments, the pharmaceutically acceptable buffer may contain between 0 and 15% DMSO. That is, the pharmaceutically 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.

[0438] Preferably, the pharmaceutical composition is substantially free of bacterial contaminants, particularly mycoplasma. The absence of bacteria / mycoplasma can be tested using devices or kits known in the art, such as, but not limited to, the BacTec device and / or the MycoSeq kit. Furthermore, the pharmaceutical composition is preferably substantially free of endotoxin.

[0439] 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. Accordingly, 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., a recipient that is a different individual from the one 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 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.

[0440] In preparing pharmaceutical formulations according to the present invention, expanded populations of lymphocytes (preferably human lymphocytes, more preferably primary human lymphocytes, most preferably comprising primary human T cells (TILs)) are typically mixed with a pharmaceutically 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 ingredients 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, e.g., 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 can be formulated according to well-known conventional methods. Pharmaceutical compositions of the present invention may further comprise one or more additional agents useful for treating disease in a subject. Pharmaceutical compositions of the present invention may be administered in The population of lymphocytes of the present invention may further include biomolecules known to be beneficial to lymphocyte function or activity, including, but not limited to, cytokines that promote in vivo cell proliferation and engraftment (e.g., IL-2, IL-7, IL-15, and / or IL-21). The population of lymphocytes 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.

[0441] 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), 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), immunomodulatory agents, such as thalidomide or thalidomide derivatives (e.g., lenalidomide).

[0442] Common chemotherapy drugs 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 riboflavin 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.

[0443] Anti-cancer agents for use in combination with the lymphocyte populations of the present invention include, but are not limited to, anthracyclines; alkylating agents; antimetabolites; 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.

[0444] 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.

[0445] Exemplary alkylating agents include, but are not limited to, nitrogen mustard, uracil mustard, ethyleneimine derivatives, alkylsulfonate esters, nitrosoureas, triazenes, chlormethine, cyclophosphamide, ifosfamide, melphalan, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, temozolomide, thiotepa, busulfan, carmustine, lomustine, streptozocin, dacarbazine, oxaliplatin, temozolomide, dactinomycin, melphalan, altretamine, carmustine, bendamustine, busulfan, carboplatin, lomustine, cisplatin, chlorambucil, cyclophosphamide, dacarbazine, altretamine, ifosfamide, prednumustine, procarbazine, meclomidine, methylparaben ... These include rituximab, streptozocin, thiotepa, cyclophosphamide, and bendamustine HCl.

[0446] 5.14 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 pharmaceutical 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. Because the unique characteristics of a cancer / proliferative disease or precancerous condition are not relevant to the methods disclosed herein, i.e., because the population of lymphocytes is specifically expanded to be selective for a desired antigen, e.g., a neoantigen of a particular cancer, cancer / proliferative diseases that can be treated according to the methods disclosed herein and with the population of lymphocytes include all types of tumors, lymphomas, and carcinomas.

[0447] 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.

[0448] 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 a disease or its symptoms are completely or partially prevented, and / or therapeutic, in that a disease or condition and / or adverse effects resulting from the disease or condition are partially or completely cured. 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 cancer progression; (c) alleviating the disease, i.e., causing regression of the disease, e.g., suppressing 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.

[0449] A 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 an appropriate treatment protocol known in the art for a particular disease or condition. 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 its symptoms. Thus, a treatment regimen disclosed herein encompasses administering a population of lymphocytes as disclosed herein or obtainable by a method disclosed herein, together with one or more treatment protocols suitable for treating or preventing either the disease, condition, or its symptoms, without any of the treatment protocols described herein or known in the art. Administration "in combination" with or use "together with" other known therapies encompasses administering a medicament / pharmaceutical composition of the present invention before, during, after, or concurrently with any of the combination therapies described herein or known in the art. The pharmaceutical compositions / medicaments disclosed herein can be administered alone or in combination with other therapies or treatments during active disease or during periods of remission or low disease activity.

[0450] When administered in combination, the lymphocyte populations of the present invention, or obtainable by the methods of the present invention, can be administered in amounts or dosages that are greater than, less than, or the same as the amount or dosage of each treatment or agent that 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.

[0451] The lymphocyte populations of the present invention and / or obtainable by the methods disclosed herein can further be made resistant to chemotherapeutic agents used as standard of care as described herein or known in the art. Engineering the lymphocyte populations of the present invention to be so resistant can be used to improve in vivo survival in patients undergoing chemotherapy or immunosuppression. It is anticipated that this will aid in the selection and expansion of such engineered lymphocytes in vivo.

[0452] The lymphocyte populations of the present invention and / or obtainable by the methods 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 1 week, more preferably 2 weeks, and even more preferably at least 1 month. The lymphocyte populations of the present invention and / or obtainable by the methods disclosed herein can also be further manipulated with a safety switch, allowing for potential administration of cell therapy. Such safety switches potentially useful in cell therapy are known in the art and include (but are not limited to) engineering cells to express targets that allow 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).

[0453] Administration of the lymphocyte populations of the present invention can be by any conventional method, including aerosol inhalation, injection, oral ingestion, transfusion, implantation, or transplantation. The medicaments and compositions described herein can be administered subcutaneously, intradermally, intratumorally, intranodal, intramedullary, intramuscularly, intravenously, or intralymphaticly, or intraperitoneally. The lymphocytes, medicaments, and / or compositions of the present invention are preferably administered by intravenous injection.

[0454] 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 concomitantly. For example, the lymphocyte population of the present invention and / or obtainable by the methods disclosed herein can be administered to a subject in a dose 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 approximately 10 T cells / kg body weight. 5 ~10 6 Starting with a target dose of T cells / kg body weight, then 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.

[0455] 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:

[0456] 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.

[0457] When used in autologous cell therapy, it is preferable that the lymphocytes in the lymphocyte composition specifically attack the target tumor. To do this, at least a portion of the lymphocytes in the lymphocyte population must recognize antigens present in the target tumor. To ensure that at least a portion of the lymphocytes in the lymphocyte population recognize antigens present in the target tumor, it is preferable that the lymphocytes are expanded in the presence of antigen peptides previously identified as present in the target tumor.

[0458] 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 a 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:

[0459] The term "tumor antigen" as used throughout this specification refers to an antigen that is uniquely or differentially expressed by tumor cells, whether intracellularly or on the surface of tumor cells (preferably on the surface of tumor cells), 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 the 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, this term includes tumor-specific antigens (TSAs), including tumor-specific membrane antigens, tumor-associated antigens (TAAs), including tumor-associated membrane antigens, tumor-embryonic antigens, growth factor receptors, growth factor ligands, etc. This term also includes cancer / testis (CT) antigens.

[0460] 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 leukemia virus 2 (LEV), and leukemia virus 2 (LEV). These include melanoma antigen 1 (MUC1), melanoma-associated antigen (MAGE) family antigens, high molecular weight melanoma-associated antigen (HMW-MAA), T cell-recognized melanoma antigen 1 (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, for example, U.S. Patent No. 9,115,402, and International Patent Application Publication Nos. WO2016 / 100977, WO2014 / 168874, WO2015 / 085233 and WO2015 / 095811).

[0461] In a preferred embodiment, the population of lymphocytes for use in cancer treatment 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 a tumor sample, or T cells obtained from a tumor sample, 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 containing neo-TILs.

[0462] In the foregoing detailed description of the present 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. Accordingly, to avoid excessively repetitive and lengthy passages, the present description refrains from reiterating every possible combination and permutation. However, whether explicitly stated or not, such combinations are fully within the scope of the presently disclosed subject matter.

[0463] Technical and scientific terms used herein, unless otherwise defined, are intended to have the same meaning as commonly understood by those skilled in the art. 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 those skilled in the art. [Brief explanation of the drawings]

[0464] 6. Brief description of the drawings [Figure 1] FIG. 1 shows expression of IFNγ and TNFα in the presence of different antigens by TILs produced in ADVA or G-REX bioreactors. [Figure 2] Overview of the batch. Batch CE_101_001 was terminated on day 34 after a positive BacT (sterility) test. For all batches, tumor biopsies were performed on D-1. Abbreviations: TIL: tumor infiltrating lymphocytes. [Figure 3] FIG. 10 shows cell viability, diameter, and viable cells / mL of T cells (before Lovo washing and final formulation) throughout culture expansion in the XURI bioreactor. [Figure 4]Figure 1 shows the distribution of T cells, NKT cells, NK cells, and B cells among CD45+ cells obtained from tumor digestion and cultured in ADVA X3 (IP) and XURI (FP) bioreactors. Results from analysis of monocytes, CD3+CD14+ and CD16-CD56+CD14+, among CD45+ cells, were <1.7%; therefore, they are not reported here. Abbreviations: IP: intermediate product; FP: final product; NK: natural killer cells; NKT: NK T cells. [Figure 5] Figure 1. CD4 and CD8 T cells obtained from CD3+ cells cultured in ADVA X3 and XURI bioreactors. Abbreviations: FP: final product; IP: intermediate product. [Figure 6] Figure 1. Expanded T cell populations with CD3, CD4 and CD8 markers in ADVA X3 and XURI. Abbreviations: FP: final product; IMP: intermediate product. [Figure 7] Schematic of expanded CD8 memory cell subsets in ADVA X3 and XURI. Abbreviations: FP: end product; IP: intermediate product; TEMRA: T effector memory re-expression; CD45RA; TSCM: memory T stem cells. [Figure 8] Diagram of differentiation markers (gating on CD8) in cells produced in ADVA X3 and XURI bioreactors. Abbreviations: FP: final product; IP: intermediate product. [Figure 9] Activation markers (gating on CD8) in cells produced in ADVA X3 and XURI bioreactors. Abbreviations: FP: final product; IP: intermediate product. [Figure 10] Proliferation markers (gated on CD8) in cells produced in ADVA X3 and XURI bioreactors. Abbreviations: FP: final product; IMP: intermediate product. [Figure 11] Figure 1. Apoptosis assay (gating on total cells) of cells produced in ADVA X3 and XURI bioreactors. Apoptosis assay was performed using AnnV and 7AAD markers. Abbreviations: FP: final product; IP: intermediate product. [Figure 12] Figure 1. Ratio of CD45+ / CD45- cells from digested tumor fragments. [Figure 13] Results in a T cell activation assay: IFN-g secretion in response to anti-CD3 (OKT3). Abbreviations: FP: final product; IP: intermediate product. [Figure 14] Figure 1. Results in a T cell activation assay: IFN-γ secretion in response to an activation cocktail (PMA / ionomycin). Abbreviations: FP: end product; IMP: intermediate product. [Figure 15] TuRA results: Percentage of 4-1BB (CD137) positive cells (gated on CD8+ T cells) expanded in ADVA X3 and XURI bioreactors. Abbreviations: AC: activation cocktail; FP: end product; IMP: intermediate product; PHA: phytohemagglutinin. [Figure 16] Figure 1. Results in TuRA: IFN-γ secretion in cells produced in ADVA X3 and XURI bioreactors. Abbreviations: AC: activation cocktail; FP: end product; IMP: intermediate product; PHA: phytohemagglutinin. [Example]

[0465] 7. Working Example 7.1 Example 1: Expansion of TILs in ADVA Bioreactors 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).

[0466] After the activation step and before contacting with T lymphocytes, B cells are transfected with mRNA encoding 4-1BBL, OX40L, and IL-12 by mixing the B cells with the mRNA and transfecting the cells using an electroporation device and a suitable electroporation buffer.

[0467] Electroporated B cells are resuspended in medium supplemented with 200 μg / mL Pen-Strep and 10% human AB serum (hABS). Resuspended B cells are stored or used directly as antigen-presenting cells (APCs) for T lymphocyte expansion.

[0468] The goal is 100 x 10 6 Prepare 100 B cells in a volume of 40 mL.

[0469] 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.

[0470] 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.

[0471] Preparation of peptide solutions Prepare a stock solution 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.

[0472] T lymphocyte expansion 60 tumor fragments or equivalent and electroporated B cells are seeded in appropriate medium into an ADVA bioreactor (ADVA biotechnology).

[0473] 100 x 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).

[0474] Sixty tumor fragments (1-2 mm) were cultured 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).

[0475] B cells and tumor fragments are cultured in an ADVA X3 bioreactor for 1 day in batch mode. (pH and dO are monitored and CO / O in the headspace of the growth chamber are adjusted as needed.) After 24 hours, peptides are added to the ADVA X3 bioreactor.

[0476] 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.

[0477] 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.

[0478] Thereafter, IL-2 is added every 3 days to maintain high IL-2 concentrations.

[0479] Continue increasing 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.

[0480] Cells are harvested with ADVA X3, the medium is changed, and the cells are prepared for final formulation. The formulated cells are dispensed / aliquoted and frozen for storage until analysis.

[0481] 7.2 Example 2: Expansion of TILs in ADVA and G-REX Bioreactors Cell product manufacturing was performed in a sterile, sealed ADVA bioreactor (ADVA biotechnology). Dissolved oxygen, pH, glucose, lactate, and temperature were monitored and adjusted as needed during cell expansion. Handling of starting materials, raw materials, and all open steps was performed in a conventional biosafety cabinet.

[0482] B cells were isolated from peripheral blood mononuclear cells (PBMCs) obtained by leukapheresis, expanded with IL-21 in a cell culture incubator, activated with IL-4 and CD40L, and electroporated with OX40L, 4-1BBL (CD137L), and IL-12 mRNA.

[0483] Tumor samples were cut into pieces and incubated in a bioreactor in the presence of electroporated autologous B cells.

[0484] After seeding tumor and B cells in the ADVA bioreactor, irradiated allogeneic feeder cells were added to the culture along with anti-CD3 (OKT3). The feeder cells were used to support T cell activation and expansion.

[0485] Cells were expanded until day 18, harvested, and cryopreserved.

[0486] Tumor cells, autologous B cells and irradiated allogeneic feeder cells were also seeded and expanded in G-Rex 10M bioreactors as cell growth controls.

[0487] Analytical experiments were performed on the cryopreserved cells to characterize them.

[0488] 7.2.1 B Cell Isolation, Expansion, and Electroporation Day -11: Frozen PBMCs from patients who provided tumor specimens were thawed in RPMI medium (Thermofisher) containing 10% human AB serum (Access Biological). After a washing step, cells were resuspended in EasySep human B cell buffer (Stemcells) at 5 × 10 7 B cells were negatively isolated from PBMCs using the EasySep Human B Cell Isolation Kit (Stemcells).

[0489] Isolated B cells were cultured at 1 × 10 in RPMI Glutamax medium containing 8% human AB serum (Access Biologicals), 100 μg / mL penicillin / streptomycin (Thermofisher), 2 mM L-glutamine (Gibco), 10 mM HEPES (Gibco), 0.05 mM 2-beta-mercaptoethanol (Gibco), 1 mM sodium pyruvate (Gibco), and 1 × Minimal Essential Medium Non-Essential Amino Acids (Gibco). 6 Cells were seeded at 1000 cells / mL. B cell medium was supplemented with 40 ng / mL IL-4 (Miltenyi), 50 ng / mL IL-21 (Miltenyi), and 200 ng / mL CD40L (AdipoGen) for B cell expansion and activation. Cells were cultured in a standard incubator at 37°C with 5.0% carbon dioxide and >90.0% humidity.

[0490] Days -9, -7 and -5: B cells were cultured at 0.15 × 10 in supplemented B cell medium (as described for day −11) in a standard incubator at 37°C with 5.0% carbon dioxide and >90.0% humidity. 6 cells / mL.

[0491] Day -3: B cells were cultured at 0.075 × 10 in supplemented B cell medium (as described for day −11) in a standard incubator at 37°C with 5.0% carbon dioxide and >90.0% humidity. 6 cells / mL.

[0492] Day 0: B cells were electroporated with 30 μg / mL OX-40L mRNA (Trilink), 30 μg / mL 4-1BBL mRNA (Trilink), and 40 μg / mL IL-12 mRNA (Trilink) using a MaxCyte device.

[0493] 7.2.2 Tumor cell preparation On day 0, frozen tumor specimens were cut into 1-2 mm pieces. 3 were cut into small pieces (66 pieces: 60 pieces for ADVA and 6 pieces for G-Rex) and placed in a thawing buffer consisting of DPBS (VWR), 10% human AB serum (Access Biological), 100 μg / ml DNAse (Sigma), and 200 μg / ml penicillin / streptomycin (Thermofisher).

[0494] Tumor specimens were resuspended in RPMI Glutamax medium, 10% human AB serum (Access Biologicals), 100 μg / mL penicillin / streptomycin (Thermofisher).

[0495] 7.2.3 Cell seeding and expansion in sterile closed ADVA bioreactors Day 0: 4.23×10 7 Electroporated B cells and 60 tumor fragments were seeded in a sealed bioreactor in RPMI Glutamax medium containing 10% human AB serum (Access Biologicals), 100 μg / mL penicillin / streptomycin (Thermofisher), supplemented with 6000 IU / mL high-dose IL-2 (Peprotech).

[0496] Day 10: 5 x 10 collected from buffy coats from healthy donors 8 Frozen PBMCs were thawed, washed, and irradiated (two 30 Gy doses) before being added to the cells in the bioreactor.

[0497] The cells were activated by adding AIM V medium supplemented with 30 ng / mL anti-CD3 (OKT3; Biolegend) and 3000 IU / mL IL-2 (Miltenyi Biotech) into the bioreactor.

[0498] Day 13: The ADVA bioreactor was switched from batch mode to recirculation mode.

[0499] Day 17: 3000 IU / mL of IL-2 (Miltenyi Biotech) was added to the cells in the bioreactor.

[0500] Day 18: 1.87×10 9 Viable cells were collected and cryopreserved in Cryostor CS5 or CS10 (StemCell).

[0501] 7.2.4 Cell Seeding and Expansion in G-Rex Bioreactors Day 0: 4.22×10 6 Electroporated B cells and six tumor fragments were seeded in 50 mL of RPMI complete medium, 10% human AB serum (Access Biologicals), 200 μg / mL penicillin / streptomycin (Thermofisher), and high-dose IL-2 at 6000 IU / mL (Peprotech) (50% fill volume) in a G-Rex 10M. The G-Rex was placed in a standard incubator at 37°C with 5.0% carbon dioxide and >90.0% humidity.

[0502] Day 10: 7.448 x 10 counted in G-Rex on day 10 7 2 x 10 cells 65 × 10 cells were set aside for culture in RPMI complete medium + AIM V medium (1:1) supplemented with 3000 IU / mL IL-2 (Peprotech) and 30 ng / mL anti-CD3 (OKT3; Biolegend) in a total of 100 mL of medium (100% filled volume) in the same G-Rex. 7 Irradiated feeder cells (PBMCs isolated from the buffy coat of a healthy donor and irradiated with 2 x 30 Gy) were thawed, washed, and then added to the cells in G-Rex 10M.

[0503] Day 17: 3.259×10 8 Viable cells were harvested and cultured in a Cryostor CS10 (StemCell) culture medium at 15 × 10 6 Cells / mL were cryopreserved.

[0504] 7.3 Example 3: Analysis of TILs The cells expanded and collected in Example 2 were characterized using a variety of analytical methods: 7.3.1 Analysis of antigen recognition Day 0 of the analysis experiment: Cryopreserved cells were thawed and washed with RPMI medium (Gibco), 20% FBS (Gibco), 1% penicillin / streptomycin (Bio-Concept).

[0505] They were cultured overnight (at 37°C with 5.0% carbon dioxide) in 24-well plates with RPMI medium (Gibco), 10% FBS (Gibco), 1% penicillin / streptomycin (Bio-Concept), and IL-2 at 3000 IU / mL.

[0506] Day 1 of the analysis experiment: Cells were plated in 24-well plates with the same medium but without IL-2 for 48 hours at 37°C.

[0507] Day 3 of the analysis experiment: Tumor fragments were thawed and washed with RPMI medium (Gibco), 20% FBS (Gibco), and 1% penicillin / streptomycin (Bio-Concept). They were enzymatically digested with a digestion solution consisting of 0.3 PZ activity units / mL of collagenase NB6 GMP grade (Witec) and 30 IU / mL of Pulmozyme (Roche) in RPMI (Gibco) at room temperature. Tumor fragments were incubated at 37°C for 4 hours at 100 rpm using a rotating mixer (digital orbital shaker, VWR:HEAT120460).

[0508] After filtration and washing steps, tumor cells (a total of 10 × 10 6 Cells) were counted and resuspended in RPMI (Gibco), 8% hABS (BIOWEST), 1% penicillin / streptomycin (Bio-Concept), L-glutamine 2 mM (Thermo Fisher Scientific), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) 10 mM (Thermo Fisher Scientific AG), 2-beta-mercaptoethanol 0.05 mM (Thermo Fisher Scientific), sodium pyruvate 1 mM (Thermo Fisher Scientific), and minimal essential medium non-essential amino acids 1 / 100 (Thermo Fisher Scientific).

[0509] Tumor cells were sorted using CD45 microbeads, miniMACS kit from Miltenyi biotec.

[0510] Count the production cells from the ADVA and G-Rex bioreactors and plate them at 2 x 10 in the above medium containing BD GolgiPlug (1:1000) in a 48-well plate. 6 Resuspended at 1 × 10 cells / mL 6 To test the reactivity of the production cells against tumor cells, the following experimental conditions were considered: Control: Production cells only Manufacturing cells + tumor cells (2:1) Manufacturing cells + CEF peptide pool (1 μg / mL) Manufacturing cells + Staphylococcal enterotoxin B (100 ng / mL; Sigma-Aldrich) Manufacturing cells + phytohemagglutinin (1 μg / mL) The cells were incubated overnight at 37°C.

[0511] Day 4 of the analysis experiment: Cells were stained with the following intracellular staining mix:

[0512] [Table 1]

[0513] Cells were resuspended in CytoFix / Cytoperm kit (BD) buffer and analyzed by flow cytometry (Fortessa).

[0514] G-REX

[0515] [Table 2]

[0516] ADVA

[0517] [Table 3]

[0518] 7.3.2 Analysis of T cell phenotype Cryopreserved cells were thawed and washed with RPMI medium (Gibco), 20% FBS (Gibco), 1% penicillin / streptomycin (Bio-Concept).

[0519] Cells were resuspended in CytoFix / Cytoperm Kit (BD) buffer and stained for T cell phenotypic characterization by flow cytometry (Fortessa) for the following markers: CD3, CD4, CD8, CD19, CD27, CD28, CD45RA, CD57, and KLRG1.

[0520] Flow Jo software was used for analysis.

[0521] Recovery and viability

[0522] [Table 4]

[0523] Cell population composition

[0524] [Table 5]

[0525] Marker expression

[0526] [Table 6]

[0527] apoptosis

[0528] [Table 7]

[0529] 7.4 Example 4: Expansion of TILs in ADVA Bioreactors Followed by an Additional Cell Culture Expansion Phase in XURI Bioreactors The objective of batches TL_101_017, TL_101_020, CE_101_001, and CE_101_002 manufacturing was to isolate, activate, and expand tumor-infiltrating lymphocytes (TILs) in the ADVA X3 bioreactor, followed by an additional cell culture expansion phase in the XURI bioreactor. An overview of the process steps is provided in Figure 2.

[0530] Briefly, we produced TIL-based products in an ADVA X3 bioreactor (ADVA Biotechnology) in a sterile, sealed, disposable kit, followed by expansion in an XURI bioreactor (Cytiva). During cell expansion in the ADVA X3, we monitored, controlled, and managed dissolved oxygen, pH, glucose, lactate, and temperature as needed.

[0531] We handled starting materials, raw materials and all other open steps in conventional biosafety cabinets or isolators.

[0532] Expanded, activated, and electroporated autologous B cells (ACT-EP-B cells) were used to stimulate T cells during the experiment. B cells were isolated from peripheral blood mononuclear cells (PBMCs) from the patient who provided the tumor specimen. They were expanded in a cell culture incubator with IL-21 and activated with IL-4 and CD40L. Finally, they were electroporated with mRNA encoding OX40L (CD134L) and 4-1BBL (CD137L).

[0533] Tumor samples were mechanically dissociated into fragments and incubated in the presence of ACT-EP-B cells and IL-2 in an ADVA X3 bioreactor (day 0 [D0]).

[0534] On D9, D10, D11, or D12, i.e., two days after activation, we switched the medium circulation mode of the ADVA X3 from small loop to large circulation. In the small loop, the medium flows out the side of the culture cone and returns directly to the bottom of the culture cone, homogenizing the medium composition with respect to metabolites, by-products, pH, and oxygen. The circulation flow starts at 1 ml / min and can increase to 4 ml / min. In the large circulation, the medium circulates from the reservoir to the culture cone.

[0535] On D7, D9 or D10, T cells were activated by adding anti-CD3 (OKT3), IL-2, and irradiated allogeneic feeder cells to the culture.

[0536] Cells were expanded in the ADVA X3 bioreactor until harvest. On the same day, cells harvested from the ADVA X3 were seeded into the XURI bioreactor, where they were cultured until D27-D28.

[0537] Cells produced in the XURI bioreactor were harvested on days 27-28. They were washed with and maintained in a NaCl-human albumin solution supplemented with IL-2 using the Lovo device. Analytical experiments were performed using cryopreserved cells to characterize the intermediate product collected from the ADVA X3 bioreactor and the final product from the XURI bioreactor.

[0538] 7.4.1 Preparation of B cells Isolation of B cells and culture prior to electroporation PBMCs were isolated from whole blood samples taken from the same patients who provided the tumor specimens. B cells from batch TL_101_017 were positively isolated from PBMCs using CD19 microbeads (Miltenyi). For other experiments, B cells were negatively isolated using the Easy Sep Human B Cell Isolation Kit (Stemcells).

[0539] B cell electroporation For batch TL_101_017, B cells were electroporated with OX40L mRNA (30 μg / ml), 4-1BBL mRNA (30 μg / ml), and IL-12 mRNA (40 μg / ml). For the other batches, B cells were electroporated with only OX40L mRNA (30 μg / ml) and 4-1BBL mRNA (30 μg / ml).

[0540] As expected, a significant decrease in cell number and a small decrease in viability was observed after electroporation.

[0541] Analysis of electroporated B cells The expression levels of CD19, 4-1BBL, and OX40L analyzed by flow cytometry are presented in the table below, along with the survival rates before and after electroporation.

[0542] The percentage of CD19 cells before and after electroporation was similar among different batches. In addition, the transfection efficiency of both 4-1BBL and OX40L was high (87–98% positive cells) for all batches. The viability of B cells before and after electroporation in batches TL_101_017 and CE_101_001 was significantly low. The low viability observed in batch TL_101_017 may be due to adverse transportation conditions between collection and electroporation, as B cell culture and electroporation were performed at different locations. The low viability of B cells in batch CE_101_001 reflects their low viability at the time of collection.

[0543] Analysis of electroporated B cells

[0544] [Table 8]

[0545] 7.4.2 Tumor dissociation The tumor type and number of fragments used in each batch are listed in the table below. The number of fragments used to produce each batch in ADVA X3 was similar across experiments. The remaining fragments were frozen in LN2 for analytical testing.

[0546] Tumor fragments

[0547] [Table 9]

[0548] 7.4.3 T Cell Manufacturing in ADVA X3 and XURI Bioreactors ADVA X3 Bioreactor Seeding and Expansion Seeding (D0-batch mode) On D0, electroporated and activated B cells were seeded together with tumor fragments into the cone of an ADVA X3 bioreactor.

[0549] For batches TL_101_017, TL_101_020, and CE_101_001, fresh ACT-EP-B cells were seeded. For batch CE_101_002, thawed ACT-EP-B cells were used. These last ones were seeded at 200.00 × 10 cells per well in culture. 6 Because the target number of cells was reached, they were freeze-dried after electroporation. Additionally, this decision was made to reduce the risk of B cells differentiating into plasma cells and losing their antigen-presenting cell capacity after 14–16 days of culture.

[0550] The different seeding volumes and media compositions used across the different batches are displayed in the table below. The volumes used to seed ACT-EP-B cells and tumor fragments varied between batches.

[0551] ADVA X3 Seeding Volume

[0552] [Table 10]

[0553] Small Loop The small loop was started from D5 for batch TL_101_017 and from D1 for batches TL_101_020, CE_101_001 and CE_101_002. This mode circulates 200 mL of corn medium from the side of the cone to the bottom of the cone, homogenizing the medium composition with respect to metabolites, by-products, pH and oxygen.

[0554] activation Once the lactate concentration reached approximately 10 mM, we initiated cell activation by replacing the TIL complete medium with activation medium (a 50:50 mix of AIM-V medium and TIL complete medium) supplemented with anti-CD3 (clone OKT3; 30 ng / mL) and IL-2 (3000 U / mL) (final concentrations in a total of 360 mL in the cone).

[0555] Batches TL_101_017 were activated on D10, CE_101_001 on D7, and TL_101_020 and CE_101_001 on D9. Activation of TILs began when lactate levels in the cell culture reached approximately 10 mM.

[0556] Cell expansion After 24 hours of activation, we switched from a small loop to a large circulation (i.e., medium circulation between the cone and reservoir (Error! Reference not found)) in the ADVA X3.

[0557] The overall medium addition and exchange strategy after activation was similar across batches: medium circulation was initiated when lactate was >9 mM. Medium circulation was primarily managed manually by pumps 1 and 3. For batch TL_101_020, medium circulation was automated using a macro.

[0558] The total volume of medium used during expansion ranged from 7500 to 10150 mL. The larger medium volume used in batch TL_101_017 was consistent with the higher FP cell numbers obtained in this batch.

[0559] Harvesting intermediate products from the ADVA X3 bioreactor Given the increased accumulation of lactate, cells were harvested on D19–D26 using an ADVA X3 macro to transfer cells from the cone into a collection bag.

[0560] Seeding, expansion and collection of TILs using the XURI bioreactor 2.61×10 9 ~4.06×10 9 Between cells (of the intermediate product) were filtered and then seeded into the XURI bioreactor to further expand their numbers after expansion in ADVA X3.

[0561] Cells were seeded in different media at different volumes. During expansion, 2.00 × 10 6 Culture medium was added to the XURI bag to maintain cell concentration with a minimum target of 100 cells / mL. Once the culture volume reached 5 L, the culture volume was kept constant at 5 L. Perfusion was then initiated, and the perfusion rate ranged from 1.25 L / day to 5.5 L / day, depending on the lactate concentration.

[0562] The cell viability, diameter, and number of viable cells / mL of T cells throughout culture expansion in the XURI bioreactor were measured with an NC202 cell counter (Figure 3). Two technical replicates were tested (to increase accuracy) for batches CE_101_002 and TL_101_020.

[0563] Over time, viable cell numbers increased for all batches. In most batches, viability dropped to 75-80% after inoculation of the XURI bioreactor and subsequent harvest, except for batch CE_101_002, which showed a drop in viability from D23 until harvest.

[0564] Cell diameter varied, with cells from TL_101_017 being the largest throughout the experiment.

[0565] 7.4.4 Analysis test Overview of the analysis tests conducted An overview of the analytical studies performed on cells cultured in ADVA X3 (IMP) and XURI (FP) bioreactors is presented in the table below.

[0566] Overview of the analysis conducted

[0567] [Table 11]

[0568] Summary of analytical results from the ADVA X3 / XURI bioreactor

[0569] [Table 12]

[0570] Cell population measured by Accellix (fresh cells) Aliquots of fresh cells obtained from digested tumor, ADVA X3 culture collection, and XURI culture collection were tested for the frequency of cell populations.

[0571] CD45+ cell population The percentages of leukocytes (CD45+), T cells (CD3+CD16- / CD56-), B cells (CD19+), NKT cells (CD3+CD16+ / CD56+), and NK cells (CD3-CD16+ / CD56+) are presented in Figure 4 and the table below (Accellix TBNK-16 NL cartridge). The majority of cells were T cells with the CD3+CD16- / CD56- phenotype, regardless of the sample considered (digested tumor, ADVA X3-collected cells, XURI-collected cells).

[0572] The percentage of T cells from digested tumors was lower in batch TL_101_017 compared to the other three batches.

[0573] For all completed experiments, the proportion of T cells was greater in FP compared to IMP and digested tumors.

[0574] The proportion of NKT cells was low (<5%) in all samples.

[0575] The proportion of NK cells was variable and was at low levels (<5%) in batches TL_101_020 and CE_101_002, unlike batches TL_101_017 (15.6%) and CE_101_001 (16.5%).

[0576] The percentage of B cells in the digested tumor ranged from 1.5% to 9.4%, but almost no B cells were observed in either the IMP or the FP. B cells seeded with tumor fragments on D0 probably underwent apoptosis during X3 expansion.

[0577] Leukocytes, B cells, T cells, and NKT cells cultured in ADVA X3 and XURI bioreactors (identified using Accellix TBNK-16 NL cartridges)

[0578] [Table 13]

[0579] T cell population Analysis of T cell populations (CD8+ and / or CD4+ cells) is presented in Figure 5 and the table below. All batches had a large number of CD8+ cells except for TL_101_020.

[0580] In both IMP and FP, the majority of cells were CD8+ cells. The proportion of CD4+ cells was significantly higher in batch TL_101_020, while the proportion of double-negative cells was more pronounced in batch CE_101_001.

[0581] The CD4+ / CD8+ ratio showed a higher CD8+ cell content in the FP of batch TL_101_017.

[0582] CD3, CD4 and CD8 T cell populations (identified using Accellix T cell NL cartridges)

[0583] [Table 14]

[0584] T cell phenotyping by flow cytometry (thawed cells) Because the panel of markers available in Accellix was limited, T cell phenotypes were further studied using flow cytometry, an approach that allowed for an unbiased exploration of T cell characteristics.

[0585] T cell population Results from flow cytometry analysis of cryopreserved cells produced in the ADVA X3 and XURI bioreactors are presented in Figures 6 and 7.

[0586] The majority of TIL products were at least 80% CD3+ cells, ≥69% CD8+ cells, and ≤12% CD4+ cells (Figure 6). Batch TL_101_020 showed a different ratio of CD8+ and CD4+ cells, with 45-50% of cells being CD8+ and 35-45% being CD4+. These results confirmed those obtained using the Accellix T Cell NL

[0102] cartridge.

[0587] The majority of CD8+ cells (>80%) were effector memory cells (Figure 7). Interestingly, the ADVA X3 products contained more central memory and fewer TEMRA T cells than their XURI counterparts. FP cells from batch TL_101_020 contained a higher percentage of TEMRA T cells than the other batches.

[0588] Differentiation markers Results from differentiation marker CD27, CD28, CD62L, TCF1, TOX, CD57, KLRG1, TIM3 and GZMK expression in cells produced in ADVA X3 and XURI bioreactors are presented in Figure 8.

[0589] Activation markers Results from activation markers CD39, CD69, CD25, CD103, CXCR3, HLA-DR, PD1, LAG3, TIGIT, and BTLA expression in cells produced in ADVA X3 and XURI bioreactors are presented in Figure 9.

[0590] CD25, HLA-DR, BTLA and LAG3 expression was consistent across cell populations expanded within the same bioreactor.

[0591] HLA-DR and CXCR3 markers were highly expressed (>99% and >67%, respectively), whereas BTLA and LAG3 remained low (<5%) for both ADVA X3 and XURI products.

[0592] In the ADVA X3 product, expression of PD1, TIGIT, CXCR3, CD39, and CD103 was inconsistent across batches. CD69 expression was lowest in batch CE_101_002 (39%) compared to other batches, which ranged from 53% to 55%.

[0593] In the XURI product, CD39 and CD103 expression was inconsistent across batches. CD69 expression was lowest in batch TL_101_017 (29%) compared to other batches (47% and 48% in batches TL_101_020 and CE_101_002, respectively). TIGIT expression was approximately 38% except for batch CE_101_002 (86%).

[0594] Proliferation markers Results from proliferation marker Ki67 expression in cells produced in ADVA X3 and XURI bioreactors are presented in FIG.

[0595] Ki67 expression in FPs was similar across batches (79.9–81.4%).

[0596] Apoptosis markers Viability is a critical attribute of cell therapy products and is part of the release specifications. Results from an apoptosis assay of cells produced in the ADVA X3 and XURI bioreactors are presented in Figure 11.

[0597] Apoptosis can be measured by the binding of annexin V (AnnV) to phosphatidylserine, which flips from the inside to the outside of the cell membrane early during apoptosis. When combined with a DNA-binding fluorescent dye such as 7-aminoactinomycin D (7AAD), early and late stages of apoptosis, as well as necrosis, can be detected in cells.

[0598] Viable cells are AnnV-7AAD-, early apoptotic are AnnV+7AAD-, late apoptotic are AnnV+7AAD+, and necrotic are AnnV-7AAD+. The percentages of viable, apoptotic, and necrotic cells in IP and FP were similar across batches.

[0599] Characterization of digested tumors Tumor fragments from different batches were enzymatically digested using collagenase (1 mg / mL) and DNase (0.25 mg / mL). CD45+ and CD45- cells were isolated by MACS. After counting the number of cells in each fraction using a cell counter NC202, the CD45+ / CD45- ratio was calculated (Figure 12). This ratio provides an indication of the amount of infiltrating lymphocytes in the tumor; samples with a higher ratio (high CD45+ / CD45-) are likely to be "hot tumors," while a lower ratio (low CD45+ / CD45-) is likely to indicate "cold tumors."

[0600] The higher CD45+ / CD45- ratio in batches CE_101_001 and CE_101_002 indicates that they were more likely to have "hot tumors."

[0601] Interestingly, despite some variability in the CD45+ / CD45- ratio observed in hot tumors, which may be related to the number of tumor fragments used to digest tumor heterogeneity, the CD45+ / CD45- ratio appeared to be consistent between fresh and frozen tumor fragments. This ratio could be an indicator of tumor lymphocyte infiltration status (hot versus cold tumors). Therefore, the tumor fragments used in batches CE_101_001 and CE_101_002 could be considered hot tumors, although this has yet to be confirmed by immunohistochemical staining.

[0602] T cell activation assay A T cell activation assay is performed as part of the characterization of IMP and FP. The assay will show the sensitivity of cells to nonspecific stimulation by CD3 and the subsequent response of cells to release IFN-gamma (IFNg). Four (4) different levels of CD3 stimulation are given, and a response curve is plotted according to the concentration of IFNg released. The maximum ability of cells to release IFNg is achieved by the activation cocktail (PMA and ionomycin).

[0603] IMP and FP cells were thawed and seeded in AIM-V cell culture medium with anti-CD3 (OKT3) (Figure 13) or activation cocktail (Figure 14). After 16-20 hours of incubation at 37°C and 5% CO2, cell culture supernatants were collected and stored at -80°C. IFNg in the supernatants was quantified using the Lumit assay.

[0604] In response to CD3 stimulation, cells from batch TL_101_020 showed the highest secretion of IFN-γ, and batch CE_101_002 showed the lowest secretion. Cell stimulation with the activation cocktail induced high levels of IFN-γ secretion in both IMP and FP, with quantified IFN-γ levels >100,000 pg / ml (Figure 14).

[0605] Tumor Recognition Assay (TuRA) To test the reactivity of the manufactured cells against autologous tumor cells, the following experimental conditions were considered: Manufacturing cells + tumor cells Negative control: Manufacturing cells only (unstimulated) Positive control: Manufacturing cells + phytohemagglutinin (PHA) Positive control: manufacturing cells + anti-CD3 (OKT3) Positive control: Manufacturing cells + activation cocktail After tumor recognition function assay, CD8+ T cells from all batches increased CD137 membrane expression after stimulation with PHA, activation cocktail, or anti-CD3 (OKT3), thus indicating that the cells responded to the stimulation and upregulated CD137.

[0606] The activation cocktail was the stronger stimulus tested, with >90% of CD8+ cells expressing CD137, a finding observed in all batches. Coculture of TILs with autologous digested tumor or tumor cell lines induced CD137 expression, particularly in batch CE_101_002 (both IMP and FP samples; see Figure 15). Compared to the CD137 marker, IFN-γ secretion assays yielded similar results for all batches tested (Figure 16). In this case, the activation cocktail induced the highest IFN-γ secretion by TILs from all batches. In addition, all samples responded to PHA and anti-CD3 (OKT3) stimulation. TILs from the FP of CE_101_002 secreted IFN-γ after coculture with autologous digested tumor or tumor cell lines. These expected results were consistent with the highly invasive tumor "hot tumor" (CD45 ratio) described above.

Claims

1. 1. A method for expanding a population of lymphocytes 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; or b) culturing lymphocytes, which are lymphocytes isolated from a tissue or blood sample from the subject; Including; expanding the lymphocytes in a culture medium in which at least one of the following parameters is monitored and adjusted to a predefined value or range: pH, dissolved oxygen (DO) concentration, glucose concentration, lactate concentration, and / or temperature; The method comprises adjusting the culture volume to match the expansion rate of the lymphocytes.

2. 10. The method of claim 1, wherein the culture medium is a culture medium in which pH, ​​dissolved oxygen (DO) concentration, glucose concentration, lactate concentration, and temperature are monitored and adjusted to predefined values ​​or ranges.

3. 3. The method of claim 1 or 2, wherein the culture volume is increased by at least 2, 3, 4, 5, or 6 fold during expansion of the lymphocytes.

4. 4. The method according to claim 1, further comprising a step of dynamic culturing of the lymphocytes.

5. The method of claim 1 , wherein the tissue sample is a tumor sample.

6. The method of claim 5, wherein the tumor sample contains at least one neoantigen.

7. 7. The method of any one of claims 1 to 6, wherein the population of lymphocytes comprises tumor-infiltrating lymphocytes, in particular, the tumor-infiltrating lymphocytes are T cells.

8. 8. The method of any one of claims 1 to 7, wherein the lymphocytes are expanded in the presence of one or more antigens.

9. The method of claim 8, wherein the one or more antigens are contained in a tumor sample.

10. 10. The method of claim 9, wherein the tumor sample is the same tumor sample from which the lymphocytes were obtained.

11. 11. The method of any one of claims 8 to 10, wherein the one or more antigens are added to the culture medium in the form of peptides.

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

13. 13. The method of claim 1, wherein the culturing step comprises co-culturing the lymphocytes with antigen-presenting cells (APCs) or artificial antigen-presenting cells (aAPCs).

14. 14. The method of claim 13, wherein the antigen-presenting cells (APCs) comprise or consist of B cells.

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

16. 16. The method of claim 14 or 15, wherein the B cells are activated prior to addition to the lymphocytes.

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

18. 18. The method of any one of claims 13 to 17, wherein the antigen-presenting cells (APCs) are genetically engineered to express one or more transgenes.

19. 19. The method of claim 18, wherein the genetically engineered APC is obtained by introducing nucleic acids encoding one or more transgenes into the APC.

20. 20. The method of claim 18 or 19, wherein at least one of the one or more transgenes encodes an immunomodulatory factor.

21. 21. The method of 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), CD62L, 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 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).

22. 22. The method of claim 20 or 21, wherein the immunomodulatory factor is one or more of OX40L, 4-1BBL and / or interleukin-12.

23. 23. The method of any one of claims 8 to 22, wherein the presence of at least one of the one or more antigens has been confirmed in a tumor sample taken from the subject.

24. 24. The method of any one of claims 8 to 23, wherein at least one of the one or more antigens is a neoantigen, the presence of which has been confirmed in a tumor sample taken from the subject.

25. 25. The method of 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.

26. 5. The method of claim 1, wherein the lymphocytes are isolated from a blood sample.

27. 27. The method of claim 26, wherein the lymphocytes are genetically engineered to express a transgene.

28. 28. The method of claim 27, wherein the transgene encodes a chimeric antigen receptor.

29. 29. The method of any one of claims 1 to 28, wherein the lymphocytes are expanded in the presence of feeder cells.

30. 30. The method of claim 29, wherein the feeder cells are autologous or allogeneic cells.

31. The method of claim 29 or 30, wherein the feeder cells are B cells, dendritic cells, T cells, macrophages and / or PBMCs.

32. 32. The method of any one of claims 29 to 31, wherein the feeder cells are irradiated cells.

33. 33. The method of any one of claims 1 to 32, comprising activating the lymphocytes during culture.

34. 34. The method of claim 33, wherein the activation step comprises the addition of a CD3 agonist and / or a CD28 agonist to the culture medium.

35. 35. The method of claim 34, wherein the CD3 agonist is an agonist anti-CD3 antibody and / or the CD28 agonist is an agonist anti-CD28 antibody.

36. 36. The method of claim 35, wherein the anti-CD3 antibody and / or the anti-CD28 antibody are immobilized on solid particles.

37. 37. The method of any one of claims 34 to 36, wherein the CD3 agonist and / or the CD28 agonist is added to the culture medium 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 days after the initiation of culture.

38. 38. The method of any one of claims 1 to 37, wherein the culture medium is supplemented with human or synthetic AB serum, IL-2 and / or IL-15.

39. The step of culturing comprises culturing the population of T cells to a concentration of at least 10 ９ 39. The method of any one of claims 1 to 38, wherein the method is continued until a cell is reached.

40. 40. The method of any one of claims 1 to 39, wherein the culturing step is carried out at a temperature above 0°C.

41. 41. The method of any one of claims 1 to 40, wherein the sample or the lymphocytes are maintained at a temperature above 0°C after isolation from the subject and prior to culturing.

42. 42. The method of any one of claims 1 to 41, wherein the cells are harvested from the culture vessel after expansion and, optionally, transferred to a second culture vessel for a second expansion.

43. At least 1 x 10 ９ 43. The method of claim 42, wherein the cells are transferred from a first culture vessel to the second culture vessel.

44. 44. The method of claim 42 or 43, wherein the culture medium in the second container is supplemented with human or synthetic AB serum, IL-2, IL-15, nicotinamide and / or nicotinamide mononucleotide.

45. 45. The method according to any one of claims 42 to 44, wherein the second expansion comprises a step of dynamic culture, in particular a step of perfusion, of the lymphocytes.

46. 46. ​​The method of claim 45, wherein the perfusion rate is in the range of 0.5 to 10 L / day, preferably 1 to 6 L / day.

47. The cell concentration in the second culture vessel is at least 1 x 10 ６ cells / mL, preferably 2 x 10 ６ 47. The method of any one of claims 42 to 46, wherein the concentration is cells / mL.

48. 48. The method of any one of claims 42 to 47, wherein the second expansion is carried out for at least 2, 3, 4, 5, 6 or 7 days.

49. A second expansion is performed to obtain at least 1 x 10 cells in the second culture vessel. １０ 49. The method of any one of claims 42 to 48, wherein the method is stopped when the total cell number of cells is reached.

50. 50. A population of lymphocytes obtainable by the method of any one of claims 1 to 49.

51. 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 optionally less than 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 10% are triple positive for CD45RA, CD57 and KLRG1.

52. 52. The population of lymphocytes of claim 51, wherein the T cells are specific for one or more antigens.

53. 53. The population of lymphocytes of claim 51 or 52, wherein less than 15% of said T cell portion secrete IL-4 and / or IL-5 in response to an antigen.

54. 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 ＋ 54. A population of lymphocytes according to any one of claims 51 to 53, which are T cells.

55. A population of lymphocytes according to any one of claims 51 to 54, comprising CAR-T cells.

56. 55. A population of lymphocytes according to any one of claims 51 to 54, wherein at least two T cells in the T cell portion are directed against different antigens.

57. 57. The population of lymphocytes of claim 56, wherein at least one antigen is a neoantigen.

58. The T cell portion comprises at least 10 ９ 58. A population of lymphocytes according to any one of claims 51 to 57, comprising T cells.

59. 59. A pharmaceutical composition comprising a population of lymphocytes according to any one of claims 50 to 58.

60. 60. The pharmaceutical composition of claim 59, wherein the lymphocytes are suspended in a pharmaceutically acceptable buffer.

61. 61. The pharmaceutical composition of claim 60, wherein the pharmaceutically acceptable buffer comprises about 0.9% NaCl, and optionally up to 15% DMSO.

62. 62. A population of lymphocytes according to any one of claims 50 to 58 or a pharmaceutical composition according to any one of claims 59 to 61 for use as a medicament.

63. 62. A population of lymphocytes according to any one of claims 50 to 58 or a pharmaceutical composition according to any one of claims 59 to 61 for use in cancer treatment.

64. 64. The population of lymphocytes or pharmaceutical composition for use according to claim 63, wherein said cancer treatment is adoptive cell therapy.

65. 65. The population of lymphocytes or pharmaceutical composition for use according to claim 63 or 64, wherein the cancer treatment is an autologous cell therapy.

66. 1. A method of treating cancer, comprising: a) providing a population of lymphocytes according to any one of claims 50 to 58 or a pharmaceutical composition according to any one of claims 59 to 61; and b) injecting said population of lymphocytes or said pharmaceutical composition into a subject suffering from cancer. A method comprising:

67. 1. 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 the method of any one of claims 1 to 49, 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; d) injecting the expanded lymphocytes obtained in step (c) into the subject from whom the tumor sample was taken. A method comprising:

68. 68. The method of claim 67, wherein the tumor antigen is a tumor-associated antigen or a tumor-specific antigen.

69. 69. The method of any one of claims 66 to 68, wherein the lymphocytes comprise tumor infiltrating lymphocytes (TILs).

70. 70. The method of claim 69, wherein the TIL specifically recognizes one or more tumor antigens.

71. 71. The method of claim 70, wherein at least one tumor antigen is a neoantigen.

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