Co-cultivation

Abstract

The present invention relates to a method for culturing immune cells. In particular, the present invention relates to a method for generating a population of immune cells, comprising co-culturing immune cells and antigen-presenting cells (APCs) in a dynamic suspension. A system used in such a method is also provided.

Description

[Technical Field]

[0001] Technical field The present invention relates to a method for culturing immune cells. In particular, the present invention relates to a method for generating a population of immune cells, comprising co-culturing immune cells and antigen-presenting cells (APCs) in a dynamic suspension. A system used in such a method is also provided. [Background technology]

[0002] background Cell therapy using adoptive transfer of activated immune cells is a powerful clinical tool. Common forms of such therapies include chimeric antigen receptor T (CAR T) cells, natural killer (NK) cells, T cell receptor-transformed T (TCR-T) cells, tumor-infiltrating lymphocytes (TILs), regulatory T (Treg) cells, and cytokine-induced killer (CIK) cells. However, for it to be clinically feasible, it is necessary to generate a sufficient number of these mature, activated immune cells for administration to patients.

[0003] Antigen-presenting cells (APCs) stimulate immature or naive immune cells to activate them; therefore, the presence of APCs is necessary for the maturation of immature or naive immune cells. For this reason, the production of activated immune cells usually involves co-culturing immature or naive immune cells with APCs to promote APC-mediated stimulation of immature or naive immune cells.

[0004] Current production processes for active immune cells involve co-culturing naive or immature immune cells with antigen-presenting cells (APCs) under static conditions. There is a consensus that such static conditions are necessary to maintain close proximity between immune cells and APCs so that APCs can provide the necessary stimulation to activate the immune cells. Existing bioreactors and production protocols for immune cell production, such as G-Rex®, emphasize the importance of static culture and stable interaction between immune cells and APCs for producing mature, active immune cells (as disclosed, e.g., Gotti et al. Cytotherapy (2022) 24:334-343, Lubin et al. (2023), and International Publication No. 2019 / 190579, each incorporated herein by reference). [Overview of the project] [Problems that the invention aims to solve]

[0005] overview The inventors recognize that existing processes for producing activated immune cells involve concerns such as the time, cost, sterility challenges, and other factors associated with the "vein-to-vein" length of the process.

[0006] The object of the present invention is to overcome one or more of these problems and to provide a method and system for producing immune cells, particularly populations of active immune cells, that are suitable for commercial-scale production and GMP compliant, thereby facilitating regulatory approval for use in human patients.

[0007] The inventors have surprisingly revealed that immune cell populations can be effectively co-cultured with APCs in dynamic suspension. In particular, populations of immune cells, especially active immune cells, can be generated by co-culturing immune cells with APCs in dynamic suspension, and the effectiveness of such a method is at least equivalent to that of the static co-culture method, which is the current "gold standard." In particular, the inventors have shown that T cells can be co-cultured with dendritic cells (DCs) in dynamic suspension, as well as reactive CD4 + and CD8 + We demonstrated that the number of T cells was equivalent to that produced using conventional static co-culture.

[0008] The ability to produce immune cell populations using dynamic suspensions offers numerous advantages. In particular, dynamic co-culture can improve the quality and uniformity of immune cells. This is because dynamic culture reduces environmental variability, resulting in improved process reproducibility and consistency of the final product. Dynamic co-culture also facilitates process scale-up / scale-out, as such scalable hardware and software significantly reduces the costs and effort associated with scaling. Dynamic co-culture also reduces risk, as the co-culture process can be more precisely controlled and tuned, while simultaneously reducing the number of human interventions and the risk of batch loss. Furthermore, dynamic co-culture offers improved flexibility, as continuous monitoring enables online, in-situ responses and allows the system to dynamically adjust to compensate for process and / or raw material variations. [Means for solving the problem]

[0009] Accordingly, the present invention provides a method for generating a population of immune cells, wherein the method comprises co-culturing isolated immune cells and antigen-presenting cells (APCs) in a dynamic suspension.

[0010] The dynamic suspension may be generated from the stirring of the coculture or from the flow of fluid, preferably from the stirring of the coculture. The stirring may be (a) continuous; and / or (b) intermittent. The intermittent stirring of the coculture may include stirring for about 5 minutes to about 30 minutes every 1 to 5 hours. Optionally, the intermittent stirring may be (a) every hour for about 5 minutes to about 15 minutes, preferably about 10 minutes; or (b) every 3 hours for about 5 minutes to about 15 minutes, preferably about 10 minutes.

[0011] According to the method of the present invention: (a) the first period of co-culture may be subject to intermittent stirring, wherein the first period is optionally about 0 to about 2 days, preferably about 1 to about 2 days; and / or (b) the first co-culture period may be followed by a second co-culture period of continuous stirring, wherein the second period is optionally about 2 to about 21 days, preferably about 2 to about 10 days.

[0012] According to the present invention, stirring may be (a) mechanical stirring; (b) oscillating stirring; (c) vertical wheel stirring; or (d) pneumatic stirring. Preferably, isolated immune cells and APCs can be co-cultured under mechanical stirring, and more preferably, the co-culture may be carried out in a stirred-tank type bioreactor.

[0013] The stirring may be mechanical stirring and may be carried out at any of the following RPMs: (a) 20 RPM to about 80 RPM, preferably about 20 RPM to about 75 RPM, more preferably about 25 RPM to about 70 RPM; or (b) about 70 RPM to about 150 RPM, preferably about 70 RPM to about 100 RPM.

[0014] The immune cells may be T cells, and optionally tumor-infiltrating lymphocytes (TILs). T cells include: (a) CD8 + T cells; (b) CD4 + T cells, optionally Th1, Th2, Th17, Tfh and / or Th9 cells; (c) NKT cells, optionally non-mutant NKT cells; and / or (d) regulatory T cells (Treg).

[0015] APCs may be: (a) dendritic cells (DCs); (b) B cells; and / or (c) macrophages.

[0016] The antigen may be a tumor antigen, preferably a neoantigen, and more preferably a clonal neoantigen.

[0017] Preferably, the immune cells are T cells, most preferably TILs, and the APCs are DCs.

[0018] In the method of the present invention, one or more of the following parameters can be monitored and maintained at a desired setpoint using a feedback mechanism: (a) pH; (b) dissolved oxygen (DO); (c) temperature; (d) a gas mixture consisting of O2, N2, CO2 and / or compressed air; and / or (e) the concentration of one or more nutrients and / or metabolites. Optionally, the one or more of the above parameters can be monitored and maintained using a control loop mechanism.

[0019] In the method of the present invention, (a) fresh culture medium may be introduced into the co-culture at least once, twice, three times, four times, five times, six times or more during the period of the method; and / or (b) every two to three days during the period of the method.

[0020] In the method of the present invention, (a) the number of immune cells and / or APCs can be measured at least once, twice, three times, four times, five times, six times or more during the period of the method; and / or (b) every two to three days during the period of the method. Optionally, the number of immune cells and / or APCs may be measured using flow cytometry.

[0021] In the method of the present invention, at the start of co-culture, (a) about 1 × 10 6 Immune cells can be seeded at a concentration of immune cells / mL; and / or (b) at an immune cell:APC ratio of approximately 10:1.

[0022] The method of the present invention may further include a step of nonspecifically growing immune cells prior to co-culturing with APC, where optionally (a) the immune cells are T cells; and / or (b) the step of nonspecifically growing immune cells comprises culturing immune cells with one or more of IL-2, IL-21, IL-15, anti-CD3 antibody, anti-CD28 antibody, and anti-CD2 antibody, preferably all of IL-2, IL-21, IL-15, anti-CD3 antibody, anti-CD28 antibody, and anti-CD2 antibody.

[0023] In the method of the present invention, co-cultured cells are harvested after a total co-culture period of about 2 to 21 days, preferably about 2 to 10 days, and immune cells may be used to reseed the culture vessel. In this case, the immune cells may optionally be subjected to about 1 × 10⁶ of cultures in the absence of APC. 6 It can be re-seeded at a density of immune cells / mL.

[0024] After co-culturing, the immune cells may be cultured for a further period in the absence of APC, in which case optionally: (a) during the further period the immune cells are cultured in a dynamic suspension; and / or (b) the further period is optionally about 2 to 21 days, preferably about 2 to 10 days.

[0025] A dynamic suspension of immune cells can be obtained by agitating the immune cells, which can be continuous or intermittent, as desired.

[0026] Intermittent agitation of immune cells may include agitation for about 5 minutes to about 30 minutes every 1 to 5 hours, and optionally, (a) agitation is performed for about 5 minutes to about 15 minutes, preferably about 10 minutes, every hour, or (b) agitation is performed for about 5 minutes to about 15 minutes, preferably about 10 minutes, every 3 hours.

[0027] The stirring of immune cells may be by (a) mechanical stirring; (b) oscillating stirring; (c) vertical wheel stirring; or (d) pneumatic stirring, where preferably the isolated immune cells and APCs are co-cultured under mechanical stirring, and more preferably the co-culture is carried out in a stirred-tank bioreactor.

[0028] The agitation of immune cells may be by (a) 20 RPM to about 80 RPM, preferably about 20 RPM to about 75 RPM, more preferably about 25 RPM to about 70 RPM; or (b) mechanical agitation at about 70 RPM to about 200 RPM, preferably about 70 RPM to about 150 RPM or about 70 RPM to about 100 RPM.

[0029] The method of the present invention may include the following: (a)(i) Co-culture isolated immune cells and APCs for a first period of about 24 hours with intermittent stirring at about 25 RPM for about 10 minutes, followed by about 1 hour without stirring; (ii) Co-culture isolated immune cells and APCs for a second period of about 24 hours with intermittent stirring at about 50 RPM for about 10 minutes, followed by about 1 hour without stirring; (iii) Co-culture isolated immune cells and APCs for a third period of about 1 week with continuous stirring at about 70 RPM; optionally (iv) add immune cells, preferably about 1 × 10⁶ 6 (v) Re-seed to a cell / mL density; and (v) culture the isolated immune cells for a fourth period of about one week in the absence of APC with continuous stirring at about 100 RPM; or (b) (i) Co-culture isolated immune cells and APCs for a first period of about 24 hours with intermittent stirring at about 25 RPM for about 10 minutes, followed by about 3 hours without stirring; (ii) Co-culture isolated immune cells and APCs for a second period of about 24 hours with intermittent stirring at about 50 RPM for about 10 minutes, followed by about 3 hours without stirring; (iii) Co-culture isolated immune cells and APCs for a third period of about 1 week with continuous stirring at about 70 RPM; optionally (iv) Immunocytes, preferably about 1 × 10 6 (v) Re-seed to a cell / mL density; and (v) culture the isolated immune cells for a fourth period of about one week in the absence of APC with continuous stirring at about 100 RPM; or (c)(i) co-culturing the isolated immune cells and APCs for a first period of about 48 hours with intermittent agitation at an RPM of about 100 RPM for about 10 minutes, followed by about 1 hour of no agitation; (ii) co-culturing the isolated immune cells and APCs by continuous agitation at an RPM of about 100 RPM for a second period of about 1 week; optionally (iii) the immune cells are preferably adjusted to about 1×10 6 resuspended and seeded to a density of cells / mL; and (iv) culturing the isolated immune cells for a third period of about 1 week with continuous agitation at an RPM of about 100 RPM in the absence of APCs; or (d)(i) co-culturing the isolated immune cells and APCs for a first period of about 48 hours with intermittent agitation at an RPM of about 100 RPM for about 10 minutes, followed by about 3 hours of no agitation; (ii) co-culturing the isolated immune cells and APCs by continuous agitation at an RPM of about 100 RPM for a second period of about 1 week; optionally (iii) the immune cells are preferably adjusted to about 1×10 6 resuspended and seeded to a density of cells / mL; and (iv) culturing the isolated immune cells for a third period of about 1 week with continuous agitation at an RPM of about 100 RPM in the absence of APCs; or (e)(i) co-culturing the isolated immune cells and APCs by continuous agitation at an RPM of about 100 RPM for a first period of about 9 days; optionally (ii) the immune cells are preferably adjusted to about 1×10 6 resuspended and seeded to a density of cells / mL; and (iii) culturing the isolated immune cells for a second period of about 1 week with continuous agitation at an RPM of about 100 RPM in the absence of APCs.

[0030] The method of the present invention can be carried out using a stirred-tank bioreactor, optionally in combination with a marine impeller.

[0031] The method of the present invention can provide an immune cell yield that is at least equivalent to that obtained by the corresponding method of co-culturing isolated immune cells with APCs without agitation.

[0032] The present invention further provides a method for producing a cell therapy product, which includes (a) generating a population of immune cells in accordance with the method described herein; (b) optionally isolating and / or purifying the immune cell population; and (c) formulating the immune cell population together with a pharmaceutically acceptable carrier.

[0033] The present invention also provides an immune cell population, an immune cell composition comprising the immune cell population, or a cell therapy product obtained or acquired by the present invention, wherein preferably the population, composition, or product comprises at least about 10 × 10 6 The product contains at least 0.2% to 5%, 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 70%, or 70% to 100% reactive immune cells. The immune cells in the immune cell population, immune cell composition, or cell therapy product are preferably antigen-specific T cells.

[0034] The present invention further provides an immune cell population or composition described herein, which is used for the treatment or prevention of cancer in a subject, where preferably the cancer is bladder cancer, gastric cancer, esophageal cancer, breast cancer, colorectal cancer, cervical cancer, ovarian cancer, endometrial cancer, kidney cancer (renal cell carcinoma), lung cancer (small cell lung cancer, non-small cell lung cancer, and mesothelioma), brain tumors (e.g., glioma, astrocytoma, glioblastoma), melanoma, lymphoma, small intestine cancer (duodenal and jejunal cancer), leukemia, pancreatic cancer, hepatobiliary tract tumors, germ cell tumors, prostate cancer, head and neck cancer, thyroid cancer, or sarcoma, and more preferably the subject is human.

[0035] The present invention also provides a system for generating an immune cell population, the system comprising (a) a suspension culture bioreactor; (b) isolated immune cells; (c) APCs; (d) a culture medium; and (e) at least one sensor, where the isolated immune cells are co-cultured with APCs in a dynamic suspension; and optionally, at least one sensor can monitor the following parameters: (i) pH; (ii) dissolved oxygen (DO); (iii) temperature; (iv) a gas mixture of O2, N2, CO2 and / or compressed air; and / or one or more concentrations of one or more nutrients and / or metabolites. The dynamic suspension can be generated by stirring the co-culture of isolated immune cells and APCs. Optionally, the stirring is selected from: (a) mechanical stirring; (b) oscillating stirring; (c) vertical wheel stirring; or (d) pneumatic stirring; preferably, the isolated immune cells and APCs are co-cultured under mechanical stirring, and more preferably, the stirring bioreactor is a stirred-tank type bioreactor. Alternatively, or in addition, the system may incorporate the at least one sensor into a proportional-integral-derivative controller that enables monitoring and maintenance of the one or more parameters using a control loop mechanism. [Brief explanation of the drawing]

[0036] Brief explanation of the drawing [Figure 1] This is a schematic diagram illustrating experimental protocols for various DASbox conditions exemplified herein. [Figure 2] This plot shows the percentage (%) of reactive CD8+ T cells under various DASbox conditions compared to GMF or GSD controls on day 9 or 16. The dotted line indicates the baseline level of required reactivity. Purple crosses = data points on day 9, red crosses = data points on day 16. For D0, baseline reactivity is 92.13%. The variance on day 9 was within approximately 20% of this baseline. The variance on day 16 was within approximately 15% of this baseline. [Figure 3]This graph shows the total number of CD8+ T cells produced on day 9 and day 16 under various DASbox conditions and GMF and GSD controls. [Figure 4] This graph shows the number of reactive CD8+ T cells produced on day 9 and day 16 under various DASbox conditions and GMF and GSD controls. [Figure 5] This graph shows the overall reactive CD8+ T cell population produced on days 9 and 16 under various DASbox conditions and GMF and GSD controls. [Figure 6] This plot shows the percentage of reactive CD4+ T cells under various DASbox conditions, compared to GMF or GSD controls, on either day 9 or day 16. The dotted line indicates the baseline level of required reactivity. Purple crosses = data points on day 9, red crosses = data points on day 16. For D0, baseline reactivity is 12.96.1%. The variance on day 9 was within approximately 25% of this baseline. The variance on day 16 was within approximately 15% of this baseline. [Figure 7] This graph shows the total number of CD4+ T cells produced on day 9 and day 16 under various DASbox conditions and GMF and GSD controls. [Figure 8] This graph shows the number of reactive CD4+ T cells produced on day 9 and day 16 under various DASbox conditions and GMF and GSD controls. [Figure 9] This graph shows the overall reactive CD4+ T cell population produced on days 9 and 16 under various DASbox conditions and GMF and GSD controls. [Figure 10] This is a schematic diagram showing one of the example control groups, namely the "hybrid control," in which the G-Rex100M was set up for the first 48 hours, after which the cells were re-seeded in DASbox containers. [Figure 11] This specification illustrates a schematic diagram showing an experimental protocol and a "hybrid control" for testing the growth and reactivity of patient-derived materials. [Figure 12a] This graph shows cell proliferation in co-culture (D0-9), comparing proliferation in dynamic suspension (AMBR) with that in a static system (G-Rex). As illustrated herein, proliferation data is shown by (a) measuring CD3+ cells using patient-derived material. Cells were harvested on day 9 (D9_H) and re-seeded (D9_S) without APC. [Figure 12b] This graph shows cell proliferation in co-culture (D0-9), comparing proliferation in dynamic suspension (AMBR) with that in a static system (G-Rex). Proliferation data is shown by measuring (b) proliferation ratio using patient-derived material, as illustrated herein. Cells were harvested on day 9 (D9_H) and re-seeded (D9_S) without APC. [Figure 13a] This graph shows cell proliferation (D0-D9) in co-culture, comparing the proliferation of patient-derived material in dynamic suspension (AMBR) with cells derived from a healthy donor (HD) model. As illustrated herein, this proliferation data is indicated by (a) measuring CD3+ cells. Cells were harvested on day 9 (D9_H) and re-seeded (D9_S) without the addition of APC. [Figure 13b] This graph shows cell proliferation (D0-D9) in co-culture, comparing the proliferation of patient-derived material in dynamic suspension (AMBR) with cells derived from a healthy donor (HD) model. As illustrated herein, this proliferation data is indicated by (b) measuring the proliferation ratio. Cells were harvested on day 9 (D9_H) and re-seeded (D9_S) without APC. [Figure 14a] (a) This graph shows the reactivity data of patient-derived materials co-cultured in a dynamic suspension, in line graph format, and measures the percentage of reactive CD8+ cells (TNFα+ and IFNg+). [Figure 14b] (b) This graph shows the reactivity data of patient-derived materials co-cultured in a dynamic suspension, in bar graph format, and measures the percentage of reactive CD8+ cells (TNFa+ and IFNg+). [Modes for carrying out the invention]

[0037] Detailed explanation definition Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art in the field to which this disclosure belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide general dictionaries of many of the terms used herein. The meaning and scope of terms should be clear, but in the event of any potential ambiguity, the definitions set forth herein shall prevail over any dictionary or external definitions.

[0038] It should be understood that the present invention is not limited to the specific methods, protocols, and reagents described herein, and that these are subject to change. In particular, any methods and materials similar or equivalent to those described herein may be used in the practice or testing of embodiments of this disclosure.

[0039] The description of embodiments of this disclosure is not intended to be exhaustive or to limit the disclosure to the exact form disclosed. Specific embodiments and examples of the disclosure are described herein for illustrative purposes, but various equivalent modifications are possible within the scope of the disclosure, as will be apparent to those skilled in the art. For example, while the steps or functions of a method are presented in a predetermined order, in another embodiment the functions may be performed in a different order or substantially simultaneously. The teachings of the disclosure described herein may be applied to other procedures or methods as needed. Various embodiments described herein may also be combined to provide other embodiments. Further embodiments of the disclosure may also be provided by modifying aspects of the disclosure as needed, adopting the compositions, functions, and concepts of the above-mentioned references and applications. Furthermore, several modifications to the protein structure may be made without affecting the type or amount of biological or chemical action, so that equivalence of biological function is taken into consideration. The above and other modifications may be made to the disclosure in light of the detailed description. All such modifications are intended to be included in the appended claims.

[0040] When applied to cells, the term “isolated” in relation to the present invention means that the cells have been removed from their natural environment and are therefore free from other exogenous or undesirable cells or cell types and are in a form suitable for use in the manufacturing methods described herein and / or administration to patients. Such isolated cells are cells that have been separated from their natural environment.

[0041] Numerical ranges include the numerical values ​​that define that range. Where a range of values ​​is described, unless the context clearly indicates otherwise, each intermediate value between the upper and lower limits of that range (up to one-tenth of a unit of the lower limit) is also understood to be specifically disclosed. Each narrower range between any described value, or any intermediate value within any described range, and any other described value or intermediate value within that range is included in this disclosure. The upper and lower limits of these narrower ranges may independently be included in or excluded from that range, and each range in which either or both limits are included in the narrower range, or neither limits are included, is also included in this disclosure, except for limits that are specifically excluded within the described range. If a described range includes either or both of its limit values, the range excluding either or both of those included limit values ​​is also included in this disclosure.

[0042] In this specification, the terms “increased,” “enhance,” “enhance,” or “activate” are all used to mean an increase of a statistically significant amount. The terms “increased,” “enhance,” “enhance,” or “activate” may mean an increase of at least 10% compared to a baseline level, for example, an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% compared to a baseline level, or an increase of 100% or less, or any increase in the range of 10 to 100% compared to a baseline level, or an increase of at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least about 10 times, or any increase of 2 to 10 times or more compared to a baseline level. In relation to yield or potency, “increase” is an observable or statistically significant increase in such level.

[0043] In this specification, the terms “reduced,” “reduced,” “reduction,” or “inhibit” are all used to mean a statistically significant reduction. The terms “reduced,” “reduction,” “decrease,” or “inhibit” typically mean a reduction of at least 10% compared to a baseline level (e.g., no given treatment), and may include reductions of at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, and at least about 99% or more. In this specification, “reduction” or “inhibition” includes complete inhibition or reduction compared to a baseline level. “Complete inhibition” is 100% inhibition (i.e., complete disappearance) compared to a baseline level.

[0044] It should be noted that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include multiple referents unless the context clearly indicates otherwise. Therefore, for example, “one immune cell” includes multiple such cells, and “the immune cell” includes one or more immune cells and their equivalents known to those skilled in the art. Furthermore, the use of the term “includes,” and other forms such as “includes” and “contains,” is not limited.

[0045] "Approximately" can generally mean a range of tolerance for the quantity being measured, taking into account the nature or precision of the measurement. Exemplary error ranges are within 20 percent (%) of a given value or range of values, typically within 10%, and more typically within 5%. Preferably, in this specification, the term "approximately" is understood to mean plus or minus (±) 5%, preferably ±4%, ±3%, ±2%, ±1%, ±0.5%, or ±0.1% of the numerical value in which it is used.

[0046] The term "consisting of ~" refers to the compositions, methods, and their respective components described herein, excluding any elements not described in the description of the present invention.

[0047] As used herein, the term "essentially consisting of" refers to the elements required for a given invention. This term permits the presence of elements that do not substantially affect the basic, novel, or functional properties of the invention (i.e., inert or non-immunogenic components).

[0048] Embodiments described herein as "including" one or more features may also be deemed to be disclosures of corresponding embodiments "consisting of" and / or "essentially consisting of" such features.

[0049] Concentrations, quantities, volumes, percentages, and other numerical values ​​may be presented herein in the form of ranges. Such range forms are used solely for convenience and brevity, and should be understood to encompass not only the numerical values ​​explicitly stated as limits of the range, but also all individual numerical values ​​or subranges within that range, as if each numerical value and subrange were explicitly stated.

[0050] In this specification, the terms “individual,” “subject,” and “patient” are used interchangeably to refer to mammalian subjects for whom diagnosis, prognosis, disease monitoring, treatment, therapy, and / or treatment optimization is desired. Mammals may be, for example (but not limited to), humans, non-human primates, mice, rats, dogs, cats, horses, or cattle. In a preferred embodiment, the individual, subject, or patient is human. The “individual” may be an adult, a young person, or an infant. The “individual” may be male or female.

[0051] "Persons in need of treatment for a specific disease" may be individuals who have the disease, have been diagnosed with the disease, or are at risk of developing the disease.

[0052] Subjects may be individuals who have been previously diagnosed or identified as having, or possessing, a condition requiring treatment or one or more complications related to such a condition, and who have optionally already received treatment for the condition or one or more complications related to such condition as defined herein. Alternatively, subjects may be individuals who have not been previously diagnosed with the condition or one or more complications related to such condition as defined herein. For example, subjects may be individuals who present one or more risk factors for the condition or one or more complications related to such condition, or who do not present any risk factors.

[0053] In this specification, "treatment" means reducing, alleviating, or eliminating one or more symptoms of the disease being treated compared to the symptoms before treatment.

[0054] "Prevention" (or preventive measures) refers to delaying or preventing the onset of symptoms of a disease. Prevention may be absolute (a state in which the disease does not occur), or it may be effective only in some individuals or for a limited period of time.

[0055] As used herein, the term “healthy individual” means an individual or population in a healthy state, for example, an individual who does not show any symptoms of the disease, has not been diagnosed with the disease, and / or is unlikely to develop the disease (e.g., cancer or any other disease described herein). Preferably, such healthy individuals are not taking any drugs that affect cancer and have not been diagnosed with any other disease. One or more healthy individuals may have similar sex, age, and / or body mass index (BMI) to the individual under test. By applying standard statistical methods used in medicine, normal levels of expression in healthy individuals and significant deviations from such normal levels can be determined.

[0056] As used herein, the term “population” refers to two or more cells of a given cell type. A population may be homogeneous, i.e., it may include a single cell type, for example, a population of T cells. A population may be heterogeneous, i.e., it may include two or more cell types, for example, T cells and APCs.

[0057] As used herein, the term “peripheral blood cells” refers to the cellular components of blood, such as red blood cells, white blood cells, and platelets, that are present in the circulating blood.

[0058] As used herein, the terms “hematopoietic progenitor cell,” “hematopoietic progenitor cell,” and “HPC” refer to cells committed to the hematopoietic system but capable of further hematopoietic differentiation, including hematopoietic stem cells, pluripotent hematopoietic stem cells (hematopoietic blasts), myeloid progenitor cells, megakaryocyte progenitor cells, erythrocyte progenitor cells, and lymphoid progenitor cells. Hematopoietic stem cells (HSCs) are pluripotent stem cells that give rise to all blood cell types, including myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells) and lymphoid (T cells, B cells, NK cells). Hematopoietic progenitor cells typically express CD34. Hematopoietic progenitor cells may co-express CD133 and / or CD38. Examples of hematopoietic progenitor cells include CD34+ / CD45+ hematopoietic progenitor cells and CD34+ / CD45+ / CD43+ hematopoietic progenitor cells.

[0059] As used herein, the terms “common lymphoid progenitor cells” and “CLP” refer to lymphoid progenitor cells that produce T cells, B cells, and NK cells. Human bone marrow-derived CLPs are typically CD34 + CD38 + Neprilysin + Therefore, umbilical cord blood-derived CLP is typically CD34 + CD38 - CD7 + That is the case.

[0060] As used herein, the terms “proliferate” or “increase” mean increase in number by inducing proliferation of T cells. Immune cells (e.g., T cells) can be proliferated by ex vivo culture under conditions that provide the immune cells (e.g., T cells) with cell division-inducing stimuli.

[0061] As used herein, the term “antigen-specific proliferation” refers to the step of increasing the number of immune cells (e.g., T cells) in the presence of an antigen. The presence of an antigen causes an increase, i.e., proliferation, of immune cells (e.g., T cells) that are specific to the antigen in the entire population. The purpose of this antigen-specific proliferation is to preferentially or selectively proliferate immune cells (e.g., T cells) that bind to and respond to one or more antigens. The conditions of antigen-specific proliferation can be controlled to minimize nonspecific proliferation of immune cells (e.g., T cells). The antigen-specific proliferation step increases the proportion or percentage of immune cells (e.g., T cells) that are specific to the antigen in the entire population of immune cells (e.g., T cells), i.e., the proportion or percentage of immune cells (e.g., T cells) that are not specific to the antigen.

[0062] In this context, "antigen" refers to a molecule that, when presented to the immune system or immune cells in an appropriate manner, can induce an immune response, either by itself or in part.

[0063] As used herein, “cell therapy product” means a biological drug containing or composed of cells or tissues. A cell therapy product has properties intended for the treatment, prevention, or diagnosis of disease through the pharmacological, immunological, or metabolic effects of the cells or tissues of the product, or is used or administered to humans for such purposes. Examples of cell therapy products include cellular immunotherapies, cancer vaccines, and other types of autologous and allogeneic cells for specific therapeutic indications (such as hematopoietic stem cells and adult stem cells, induced pluripotent stem cells, and embryonic stem cells).

[0064] As used herein, “pharmaceutically acceptable carrier or diluent” means any substance suitable for use in administration to animals. Certain such carriers enable the formulation of pharmaceutical compositions as, for example, tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and lozenges for oral administration by subjects. In certain embodiments, the pharmaceutically acceptable carrier or diluent is sterile water, sterile saline, sterile buffer, or sterile artificial cerebrospinal fluid.

[0065] For example, the term "pharmaceutically acceptable" may refer to salts, additives, carriers, diluents, etc., that are approved by federal or state regulatory authorities or listed in the United States Pharmacopeia, the European Pharmacopeia, or other generally accepted pharmacopoeias.

[0066] Other definitions of terms may be provided throughout this specification. Before describing exemplary embodiments in more detail, it should be understood that this disclosure is not limited to the specific embodiments described and is subject to change. It should also be understood that the scope of this disclosure is defined solely by the appended claims, and that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting.

[0067] The publications referenced herein are provided solely for the purpose of their disclosure prior to the filing date of this application. Nothing in this specification should be construed as an acknowledgment that such publications are prior art with respect to the claims attached herein. All references cited herein are incorporated herein by reference with respect to their entire disclosures and the disclosures specifically stated herein.

[0068] The disclosures relating to various methods of the present invention are intended to be equally applicable to other methods, therapeutic uses, or applications, and vice versa.

[0069] immune cells The present invention relates to a method for producing immune cells or populations thereof. As described herein, the method includes co-culturing immune cells and APCs.

[0070] Immune cells may be T cells and / or natural killer (NK) cells as described herein. Preferably, the immune cells are T cells such as tumor-infiltrating lymphocytes (TILs). T cells are CD8 + T cells, CD4 + T cells (optionally Th1, Th2, Th17, Tfh, and / or Th9 cells), NKT cells (optionally non-mutant NKT cells), and / or regulatory T cells (Treg), or any combination thereof.

[0071] As used herein, unless otherwise specified, the term “immune cells” includes innate immune cells, induced immune cells, or adaptive immune cells, and immune cells produced using recombinant DNA technology.

[0072] Immune cells can be characterized based on their marker expression profiles (i.e., the presence or absence of one or more cellular markers). Methods for determining the presence of cellular markers are known in the art, such as flow cytometry.

[0073] The immune cells co-cultured with APC may be isolated immune cells. Therefore, in the method of the present invention, immune cells may be isolated from the sample before co-culturing. The sample can be taken from a subject to be treated with the population of immune cells produced by the method of the present invention; that is, a population of autoimmune cells can also be generated using the method of the present invention. Alternatively, a sample can be taken from an individual other than the individual to be treated with the immune cells produced by the method of the present invention; that is, a population of allogeneic immune cells can also be generated using the method of the present invention. Here, if the immune cells are allogeneic, they may be compatible with the individual to be treated (e.g., HLA compatible).

[0074] Samples can be taken from any suitable tissue or bodily fluid. In non-limiting examples, samples may be taken from tumors, peripheral blood (e.g., peripheral blood mononuclear cells, i.e., PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue from the site of infection, ascites, pleural fluid, splenic tissue, or other tissues of the individual.

[0075] Immune cells can be generated from hematopoietic progenitor cells obtained from a sample. The sample may be a sample taken from an individual to be treated with a population of immune cells generated by the method of the present invention (for generating autoimmune cells), or a sample taken from an individual other than the one to be treated with immune cells produced by the method of the present invention (for generating allogeneic immune cells). In the latter case, the sample can be obtained from a blood or tissue bank. In embodiments of the method of the present invention that generate T cells or NK cells, the hematopoietic progenitor cells are typically hematopoietic progenitor cells (HPCs) or common lymphoid progenitor cells (CLPs), which can then be differentiated into T cells or NK cells for use in the method of the present invention.

[0076] The immune cells (e.g., T cells) may be modified immune cells, such as genetically modified immune cells as described herein.

[0077] T cells Preferably, a population of T cells is generated using the method of the present invention.

[0078] T cells (also called T lymphocytes) express T cell receptors (TCRs) and co-receptors, which may be differentiation antigen group 4 (CD4) or differentiation antigen group 8 (CD8). T cells are found in peripheral blood, lymph nodes, tissues, and tumors. As used herein, the term "T cell" refers to αβ T cells (e.g., CD8). + Alternatively, CD4 + This includes all types of T cells, such as γδT cells, NKT cells, or Treg cells.

[0079] T cells generated by the method of the present invention are CD8 + T cells, CD4 + The T cells may be T cells, NKT cells and / or regulatory T cells, or any combination thereof. Preferably, the T cells produced by the method of the present invention are CD8 + T cells and / or CD4 + T cells, especially CD4 + These are T cells. In other words, the T cell population produced by the method of the present invention is CD8 + T cells and / or CD4 + T cells, especially CD4 + It may contain or be composed of T cells.

[0080] T cell populations can be prepared from T cells in a sample isolated from an individual, or optionally from an individual with a tumor. Samples can be collected from tumors, peripheral blood (e.g., peripheral blood mononuclear cells, i.e., PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymic tissue, tissue from the site of infection, ascites, pleural fluid, splenic tissue, or other tissues of the subject.

[0081] Appropriate techniques for isolating T cells from a sample are known in the art. In non-limiting examples, T cells can be obtained from blood samples taken from a subject using any number of techniques well known to those skilled in the art. For example, density gradient separation methods such as the FlCOLL® separation method and / or apheresis such as leukocyte apheresis can be used. Additional methods for isolating T cells for T cell therapy are disclosed in U.S. Patent Publication 2013 / 0287748, which is incorporated herein by reference in whole.

[0082] CD3 + T cells express CD3, therefore they are CD3-positive or CD3 + This is expressed as follows: All T cells express CD3+, and this coreceptor is involved in the activation of both CD8+ T cells and CD4+ T cells, both of which include the CD3+ T cell population.

[0083] CD8 + T cells CD8 + T cells express CD8, therefore, CD8-positive or CD8 + This may be written as follows: CD8 + T cells are involved in MHC class I-restricted interactions.

[0084] CD8 + T cells are cytotoxic CD8 + T cells and CD8 + It includes both suppressor T cells. To avoid any ambiguity, "CD8 + The term "T cell" and "CD8" as used herein + Any disclosure relating to "T cells" does not refer to cytotoxic CD8 unless otherwise stated. + T cells and CD8 + It includes both suppressor T cell subtypes.

[0085] cytotoxic CD8 + T cells play a major role in killing pathogens and tumor cells, and CD4+ T cells play a crucial role in maintaining the CD8+ response and preventing its depletion. When activated, they trigger cytotoxic CD8+ + T cells secrete cell death-inducing granules containing granzymes, perforins, cathepsin C, and granulosin, which can either fuse with the target cell membrane or be taken up by the target cell via endocytosis. Additionally, cytotoxic CD8 + T cells primarily secrete cytokines such as TNF-α and IFN-γ, which possess antitumor, antiviral, and antibacterial effects. Furthermore, the ligand (FASL) is expressed on CD8+ T cells, and upon binding to the Fas receptor on target cells, it activates the cell death domain (Fas-related protein with a cell death domain: FADD), which in turn activates caspases and endonucleases, leading to fragmentation of target cell DNA. These expressed proteins and other factors are related to CD8 + It can be used as a marker for T cells.

[0086] CD8+ Suppressor T cells are multiple different CD8 cells whose functional characteristic is to downregulate the immune response. + It includes T cell populations. Generally, CD8 + Suppressor cells are Foxp3 + and / or CD122 + This is an example of CD8. + The suppressor cell phenotype is CD8 + CD25 + CTLA-4 + Foxp3 + and CD8 + CD122 + That is the case.

[0087] CD4 + T cells CD4 + T cells express CD4, therefore, CD4-positive or CD4 + This can be written as follows: CD4 + T cells are involved in MHC class II-restricted interactions.

[0088] CD4 + T cells include several different subtypes, such as T helper 1 (Th1), T helper 2 (Th2) cells, T helper 17 (Th17), follicular helper T cells (Tfh), T helper 9 (Th9), and regulatory T cells (Treg). To avoid confusion, the term "CD4" is used. + "T cell" and "CD4" as used herein. + Any disclosure regarding "T cells" is not limited to any CD4 cells unless otherwise stated. + This includes T cell subtypes.

[0089] Naive CD4 + The first step in cell differentiation is antigen stimulation resulting from the interaction between the TCR and CD4 as a co-receptor with the antigen-MHCII complex presented by the APC. The combination of TCR and CD3 activation induces a network of downstream signaling pathways, which ultimately leads to the proliferation of naive cells and further differentiation into specific effector cells.

[0090] Naive CD4 + The differentiation of cells into Th1 cells is driven by IFNγ and IL-12. Th1 cells are involved in the elimination of intracellular pathogens and are also associated with organ-specific autoimmunity. They primarily secrete IFNγ, lymphotoxin α (Lfα), and IL-2. IFNγ is a commonly used marker for Th1 cells.

[0091] Naive CD4 + The differentiation of cells into Th2 cells is driven by IL-4 and IL-2. Th2 cells are involved in the immune response to extracellular parasites such as helminths and play a major role in the development and persistence of asthma and other allergic diseases. Major effector cytokines include IL-4, IL-5, IL-9, IL-13, IL-10, IL-25, and amphiregulin. IL-4 is a commonly used marker for Th2 cells.

[0092] Naive CD4 + Differentiation from cells into Th17 cells is driven by IL-21, IL-6, IL-23, and TGF-β. Th17 cells are involved in immune responses against extracellular bacteria and fungi. They are also involved in the development of autoimmune diseases. Major effector cytokines include IL-17A, IL-17F, IL-21, and IL-22. IL-17 is a commonly used marker for Th17 cells.

[0093] Naive CD4 + Differentiation from cells into Tfh cells is driven by IL-21 and IL-6. Tfh cells play a crucial role in mediating humoral immunity through interactions with B lymphocytes. Tfh cells secrete IFNγ, IL-4, and / or IL-10. CXCR5, ICOS, and / or PD-1 are commonly used as markers for Tfh cells.

[0094] Naive CD4 +Differentiation from other cells into Th9 cells is driven by IL-4 and TGF-β. Th9 cells secrete IL-9 and are involved in the immune response to parasitic infections. IL-9 production, along with the deletion of IL-4, IL-5, and / or IL-13 production, is commonly used as a marker for Th9 cells.

[0095] Treg will be discussed later.

[0096] Tumor-infiltrating lymphocytes (TILs) Immune cells or populations of immune cells, particularly T cells or populations of T cells, can be produced from tumor-derived samples. In other words, populations of immune cells, particularly T cells, can be isolated from samples obtained from tumors of individuals to be treated. Such immune cells are called tumor-infiltrating immune cells, and in particular, when the immune cells are T cells, they are referred to herein as "tumor-infiltrating lymphocytes (TILs)." TILs are T cells that have infiltrated tumor tissue.

[0097] The isolated T cells in the method according to the present invention may be TILs.

[0098] Biopsies and isolation of samples from tumors are common practices in this field and can be carried out according to any appropriate method, which will be well known to those skilled in the art.

[0099] The tumor may be a solid tumor or a non-solid tumor.

[0100] Immune cells (e.g., T cells) can be isolated from tumor samples using methods well known in this field. For example, TILs can be isolated by culturing excised tumor fragments or tumor single-cell suspensions in IL-2-containing medium. T cells can be purified from single-cell suspensions prepared from samples based on CD3, CD4, or CD8 expression. T cells can be enriched from samples by passing them through a density gradient.

[0101] Gamma-delta T cells Gamma delta T cells (γδ T cells) are typically CD8 - and CD4 - These cells express TCR chains encoded by the gamma and delta loci. γδT cells may also express FcγRIII / CD16 and Toll-like receptors. γδT cells typically do not require antigen presentation by MHCI or MHCII for activation, but are instead thought to be involved in the recognition of lipid antigens.

[0102] γδT cells have been shown to play a role in both innate and adaptive immune responses. After recognizing infected or transformed cells, γδT cells exhibit diverse functional plasticity through the production of cytokines (IFN-γ, TNF-α, IL-17) and chemokines (RANTES, IP-10, lymphotactin), cytolysis of infected or transformed target cells (perforin, granzyme, TRAIL), and interactions with other cells, including epithelial cells, monocytes, dendritic cells, neutrophils, and B cells.

[0103] NK T cells (NKT cells) NKT cells possess characteristics of both NK cells and T cells, and are a unique subset of CD1d-restricted T cells that can be subdivided into functional subsets.

[0104] NKT cells recognize lipids and glycolipids presented by CD1d, rather than peptides presented by MHC class I or II. Therefore, NKT cells are important in the recognition of glycolipid antigens. When activated, NKT cells can also produce large amounts of IFN-γ, IL-4, and GM-CSF, as well as several other cytokines and chemokines (such as IL-2, IL-13, IL-17, IL-21, and TNF-α).

[0105] iNKT cells can rapidly respond to danger signals and pro-inflammatory cytokines and can act through a variety of effector functions, including NK transactivation, T cell activation and differentiation, B cell activation, dendritic cell activation and cross-presentation activity, and macrophage activation.

[0106] NKT cells are most commonly found in the liver, but they can also be found in the thymus, spleen, peripheral blood, bone marrow, and adipose tissue.

[0107] Type I NKT cells (also called non-mutant NKT cells or iNKT cells) express a non-mutant TCRα chain and one of a few TCRβ chains. In humans, the highly conserved TCR expressed by iNKT cells is Va24-Ja18 paired with Vb11.

[0108] Type II NKT cells express a wider variety of TCRα chains.

[0109] Regulatory T cells (Treg cells) In this specification, the terms “regulatory T lymphocyte,” “Treg cell,” or “Treg” are used interchangeably and are intended to have the standard definitions used in the art. Treg cells are a specialized subpopulation of T cells that act “regulatorily” to suppress the activation of the immune system, thereby maintaining the homeostasis and tolerance to autoantigens of the immune system.

[0110] Treg cells are sometimes called suppressor T cells. Treg cells are characterized by the expression of Foxp3 (forkhead box p3), a forkhead family transcription factor. They may also express CD4 or CD8 surface proteins. Alternatively, or in addition, Treg cells may express CD25. Therefore, Treg cells express Foxp3 + CD25 + Foxp3 + CD25 - , or Foxp3 - CD25 + It is possible. Treg is CD4+ CD25 + FOXP3 + , CD4 + CD25 + CD127 - , or CD4 + CD25 + FOXP3 + CD127 low It can be. Naturally occurring Treg cells (CD4+CD25+Foxp3+) develop in the thymus like all other T cells. In contrast, adaptive Treg cells (also known as Tr1 cells or Th3 cells) can develop during a normal immune response. Since antigen-specific activation of human effector T cells causes inducible expression of Foxp3 in a subset of activated effector cells, this subset can exhibit a regulatory (Treg) phenotype. One method of inducing Tregs is to expose T effector cells to TGF-β for an extended period of time. In addition, T cells can be converted into Treg cells by transfecting or transducing the Foxp3 gene into a mixed population of T cells. T cells induced to express Foxp3 adopt the Treg phenotype, and such recombinant Tregs are also defined herein as "Tregs".

[0111] Natural killer cells In some embodiments, the immune cells may be natural killer (NK) cells.

[0112] NK cells exhibit the highest level of cytotoxic activity in the immune system. NK cells are similar to B cells or T cells, but lack specific cell surface antigen receptors. Instead, NK cells have activating receptors and inhibitory receptors that recognize motifs.

[0113] NK cells circulate in the blood and peripheral lymphoid organs such as lymph nodes and the spleen. They can be activated by cytokines or upon encountering target cells. Recognition and elimination of target cells are based on balancing inhibitory and activating signals. Activating signals are generated by activating receptors (NKG2D, NKp46, NKp30) that bind to ligands, and these ligands can be present not only on cancer cells, pathogen-infected and damaged cells, but also on healthy cells. On the other hand, inhibitory signals are generated when inhibitory receptors (KIR, CD94 / NKG2A) on NK cells bind to major histocompatibility complex (MHC) class I molecules, which are normally present on all healthy cells. Since MHC class I molecules are either absent or significantly downregulated on target cells, they become ideal targets for NK cells. This allows NK cells to distinguish between target cells and healthy cells. For NK cells to recognize and kill target cells, the total activating signal must exceed the inhibitory signal.

[0114] NK cells are part of the innate immune response because they recognize and kill cancer cells, pathogen-infected cells, and damaged cells without prior sensitization. For example, NK cells provide an early response to viral infections, which occurs before T cells kill infected cells. NK cells can kill target cells within minutes. NK cells also secrete cytokines, which "weaponize" other parts of the immune system. For instance, NK cells enhance the effector function of T cells, thereby increasing antibody-dependent cell-mediated cytotoxicity (ADCC).

[0115] NK cells are defined in terms of their marker expression, function / activity, or a combination thereof. Such definitions are standard in the art, and methods for evaluating marker expression and / or NK cell activity are well known. Therefore, those skilled in the art can easily classify cells as NK cells using standard methods and definitions.

[0116] For example, NK cells are CD56 + CD45 +, and / or CD16 + It is possible. The NK cells of the present invention are CD3 - and / or CD19 - It is possible.

[0117] Unlike other immune cells described herein, NK cells do not require co-culture with APCs for activation. However, NK cells used in cell therapy products are typically generated in co-culture systems. Instead of co-culture with APCs, NK cells may be co-cultured with supporting cells.

[0118] As used herein, the term “supporting cells” refers to cells that support the proliferation and / or activation of immune cells such as NK cells. Supporting cells are preferably stromal cells (also called mesenchymal stromal cells or MSCs). Stromal cells are connective tissue cells and may originate from any organ. Non-limiting examples of stromal cells that can be used as supporting cells in the present invention include MSCs, fibroblasts, or pericytes, with MSCs, fibroblasts, and / or fibroblast-like cells being preferred. Examples of commercially available cell lines that can be used as supporting cells include M2-10B4 (an ATCC-derived fibroblast-like cell line), OP9 cells (an MSC cell line), and EL08, particularly EL08.1D2.

[0119] To avoid any doubt, everything disclosed herein with respect to the method of the present invention involving co-culture of immune cells and APCs also applies equally and unconditionally to a method of generating NK cells involving co-culture of NK cells and supporting cells. Accordingly, the present invention provides a method for generating NK cells or a population thereof, wherein the method comprises co-culturing isolated NK cells with supporting cells in a dynamic suspension. As a non-limiting example, the present invention provides a method for generating NK cells or a population thereof, wherein the method comprises co-culturing isolated NK cells with stromal cells (e.g., EL08 cells) in a dynamic suspension. Typically, the dynamic suspension is produced by agitation of the co-culture as described herein.

[0120] antigen presenting cells Antigen-presenting cells (APCs), or accessory cells, are cells that present antigens complexed with MHC on their surface; this process is known as antigen presentation. T cells can recognize these complexes using their T cell receptors (TCRs).

[0121] In one embodiment, an antigen is loaded onto an antigen-presenting cell (APC). Antigen loading can be achieved by methods known in the art. For example, an antigen can be loaded by pulsing of antigen-presenting cells (APCs) with a peptide, or by genetic modification. In relation to the present invention, the term "antigen" refers to one or more antigens.

[0122] The method of antigen loading using pulsed processing of APCs is well known in this field. For example, a protocol for loading antigens into APCs by pulsed processing using peptides containing identified mutations is described in Leko et al. (J Immunol. 2019, 202: 3458-3467).

[0123] Typically, antigen-specific proliferation of immune cells is achieved by loading an antigen onto an APC and co-culturing the loaded APC with isolated immune cells. As used herein, “antigen-specific proliferation” means increasing the number or proportion of immune cells (e.g., T cells) that are specific to a particular antigen within a population of immune cells (e.g., T cells). Optionally, a preliminary proliferation step of nonspecific T cells, as described herein, may be performed prior to antigen-specific proliferation.

[0124] APCs may be selected from dendritic cells (DCs), B cells (also called B lymphocytes), and / or macrophages.

[0125] Preferably, APCs are DCs. DCs can be obtained from monocytes isolated from blood to generate monocyte-derived dendritic cells (MoDCs). DCs can be generated from blood samples collected from the same individual that was used for collecting immune cells, that is, they may be autologous DCs. Preferably, the DCs are autologous MoDCs. Dendritic cells can be generated from isolated monocytes using standard methods in the art. For example, protocols for obtaining PBMC-derived DCs are described in Leko et al. (J. Immunol. 2019, 202: 3458-3467). Furthermore, purification / isolation kits for DCs are commercially available, such as, for example, the EasySep™ DC enrichment kit from StemCell™ Technologies. In addition, CD14 microbeads and related protocols are available from Miltenyi Biotech ( 1781061868570_0.html#130-050-201 available at).

[0126] The APC may be a B cell. Such B cells can be expanded from blood, for example, a blood sample collected from the same individual that is used for generating immune cells, that is, they are autologous B cells.

[0127] B cells can be expanded from CD19 isolated from blood samples + cells. CD19 can be obtained using any suitable method, such as positive selection or negative selection using immunomagnetic particles coated with anti-CD19 antibody +Cells can be isolated. Reagents and kits for CD19 purification / isolation are commercially available, such as CD19 MicroBeads or B Cell Isolation Kit II, for humans (Miltenyi Biotec) and EasySep® Human CD19 Positive Selection Kit (StemCell® Technologies). Another method uses positive screening for CD20 or CD22, for example, using CD20 or CD22 MicroBeads 20 (Miltenyi Biotec).

[0128] Using standard methods known in the art, isolate CD19 + B cells can be generated from monocytes or directly from blood samples or PBMCs. For example, Kotsiou et al. (Blood 2016, 128:72-81) describe a protocol for B cell proliferation using CD40L, F(ab')2 fragment goat anti-IgA+IgG+IgM, CpG, and IL-4. Another typical method is culture using feeder cells expressing CD40L, as taught by Su et al. (JImmunol 2016, 197:4163-4176). B cell proliferation kits are commercially available, such as the ImmunoCult® Human B Cell Expansion Kit from StemCell® Technologies and the B Cell Expansion Kit for humans from Miltenyi Biotec.

[0129] Isolated CD19 + Cells can be cultured with IL-4, CD40L, and CpG to proliferate B cells. Exemplary methods for B cell proliferation, as well as different concentrations of IL-4, CD40L, and CpG in particular, are further described in International Publication No. 2022 / 269250, which is incorporated herein by reference in its entirety.

[0130] The B cell growth medium may contain IL-4 at a concentration of approximately 10 to 100 ng / ml, for example, approximately 25 to 75 ng / ml. In some preferred embodiments, the B cell growth medium contains approximately 50 ng / ml of IL-4.

[0131] Alternatively, or in addition to this, the B cell growth medium may contain CD40L at a concentration of about 0.5 to about 50 IU / mL. In some preferred embodiments, CD40L is present at a concentration of about 12 IU / mL.

[0132] Alternatively, or in addition to this, the B cell growth medium may contain CpG at a concentration of about 0.1 to about 10 μg / mL. In some preferred embodiments, CD40L is present at a concentration of about 4.6 μg / mL.

[0133] APCs may be macrophages. These macrophages may be derived from monocytes isolated from blood and can then be differentiated to produce monocyte-derived macrophages (MoMΦ). Macrophages can be produced from blood samples taken from the same individual used for generating immune cells, i.e., they may be autologous macrophages. Preferably, the macrophages are autologous MoMΦ. Macrophages can be produced from isolated monocytes using methods standard in the art. For example, a protocol for obtaining PBMC-derived macrophages is described in Rios et al. (Hypertension. Methods in Molecular Biology, vol 1527. Humana Press, New York, NY. https: / / doi.org / 10.1007 / 978-1-4939-6625-7_24). Furthermore, macrophage purification / isolation kits are commercially available, such as the EasySep® DC enrichment kit from StemCell® Technologies.

[0134] APCs can be used in a ratio of approximately 2:1 to approximately 1:100 for APCs to immune cells (APC:immune cells), for example, approximately 1:1, 1:2, 1:3, 1:4, 1:5, 1:10, 1:20, 1:50, or 1:75. Preferably, the APC:immune cell ratio is approximately 1:5 to approximately 1:20, particularly approximately 1:10. Therefore, the immune cell:APC ratio is preferably approximately 5:1 to approximately 20:1, particularly approximately 10:1.

[0135] The APC may be loaded with an antigen in peptide form containing one or more identified mutations, either as a single stimulant or as a pool of stimulating peptides, as described herein. For example, a peptide containing a mutation identified as a neoantigen may be loaded into the APC. For example, Leko et al. describe a protocol for loading an antigen into the APC by incubating the APC5 with a pool of up to 12 individual peptides, each containing an identified point mutation flanked on both sides by 12 wild-type amino acids.

[0136] Immature APCs, such as immature DCs, can be matured after being loaded with peptides. Alternatively, or in addition to this, mature APCs (e.g., mature DCs) can also be loaded with peptides. Therefore, an APC (e.g., DC) can be loaded with peptides twice, both in its immature and mature states.

[0137] Alternatively, methods for loading antigens into APCs by modifying them to express antigens are also known in the art. For example, an APC can be modified to express an antigen sequence by transfecting it with mRNA encoding the antigen sequence. The mRNA encoding the antigen sequence may be in the form of a minigene or a tandem minigene. The APC can be transfected with mRNA encoding a peptide containing the identified mutation, either as a construct or as a construct encoding multiple such peptides. For example, Leko et al. described a protocol for loading antigens into APCs by electroporating the APC with a tandem minigene RNA containing up to 12 minigenes, each minigene containing a mutant amino acid coding sequence flanked on both sides by sequences encoding 12 wild-type amino acids.

[0138] APCs may be cells capable of presenting relevant peptides in the correct HLA context, for example. Such cells may be self-cells expressing self-HLA molecules or non-self-cells expressing compatible HLA sequences. Artificial APCs may be irradiated.

[0139] The term "peptide," in its usual sense, refers to a series of residues (typically L-amino acids) that are usually linked to one another by peptide bonds between the amino and carboxyl groups of adjacent amino acids. This term also encompasses modified peptides and synthetic peptide analogs.

[0140] Peptides can be synthesized using chemical methods (Peptide Chemistry, A practical 35 Textbook. Mikos Bodansky, Springer-Verlag, Berlin). For example, peptides can be synthesized by solid-phase methods (Roberge JY et al. (1995) Science 269:202-204), cleaved from resin, and then purified by preparative high-performance liquid chromatography (e.g., Creighton (1983) Proteins Structures And Molecular Principles, WH Freeman and Co, New York NY). Automated synthesis can be achieved, for example, using an ABI 431A Peptide Synthesizer (Perkin Elmer) following instructions provided by the manufacturer.

[0141] Alternatively, this peptide can be prepared by recombinant methods or by cleavage from an antigen or an antigen-containing polypeptide. The peptide composition can be determined by amino acid analysis or sequencing (e.g., Edman degradation).

[0142] As is well known in the art, antigens are presented to T cells when antigen-derived peptides bind to major histocompatibility complexes (MHC). Methods for predicting whether a peptide is likely to bind to a particular MHC molecule and thereby function as an antigen for presentation to T cells are well known in the art. For example, as described below, MHC binding of a peptide can be predicted using the netMHC (Lundegaard et al.) and netMHCpan (Jurtz et al.) algorithms. Thus, an APC can be loaded with a peptide predicted to be likely to be presented by one or more relevant MHC molecules using either of these methods. Alternatively, or in addition to this, antigens can be loaded into an APC using a group of candidate peptides, each containing the desired mutation and having different locations of the desired mutation within the peptide.

[0143] Using equivalent predictive means, non-peptide antigens (e.g., carbohydrates or nucleic acids) that are presented by APCs and can be presented to immune cells that recognize such non-peptide antigens can be identified.

[0144] MHC class I proteins form functional receptors on most nucleated cells in the body. HLA consists of three major MHC class I genes: HLA-A, HLA-B, and HLA-C, and three minor genes: HLA-E, HLA-F, and HLA-G. β2-microglobulin binds to the subunits of the major and minor genes to form heterodimers.

[0145] Peptides that bind to MHC class I molecules are typically 7–13 amino acids long, more commonly 8–11 amino acids long. Peptide binding is stabilized at both ends by the interaction of atoms in the peptide backbone with non-mutant sites in the peptide binding groove of every MHC class I molecule. At both ends of the groove are non-mutant sites that bind the amino and carboxyl terms of the peptide. Variations in peptide length are often regulated by kinking at proline or glycine residues in the peptide backbone, thereby providing the necessary flexibility.

[0146] MHC class II proteins encoded by HLA loci include three major proteins and two minor proteins. When class II genes are combined, heterodimeric (αβ) protein receptors are formed, which are typically expressed on the surface of APCs.

[0147] Peptides that bind to MHC class II molecules are typically 8–20 amino acids long, more commonly 10–17 amino acids, but can be longer (e.g., up to 40 amino acids). These peptides exist in a structure extended along the MHC class II peptide bond groove, which (unlike the MHC class I peptide bond groove) is open at both ends. The peptide is held in place primarily by contact between conserved residues lining the inside of the peptide bond groove and the main chain atoms.

[0148] A peptide may contain mutations (e.g., non-silent amino acid substitutions encoded by SNVs) at any residue position within it. For example, peptides that can bind to MHC class I molecules are typically 7–13 amino acid long. Therefore, in the case of a 13-amino acid peptide, amino acid substitutions may be located at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13.

[0149] In one embodiment, longer peptides, for example, peptides with 27, 28, 29, 30, or 31 amino acids, are used for CD4 + Cells and CD8 + Both cells can be stimulated. The mutation can be located at any position within the peptide. In one embodiment, the mutation is located in the center of the peptide, for example, at positions 12, 13, 14, 15, or 16, or in its vicinity.

[0150] In the method of the present invention, any appropriate number of (peptide or non-peptide) antigens, for example, 10 to 300 antigens, for example, 25 to 250, 50 to 200, 70 to 185, or 100 to 150 antigens, for example, about 10, 20, 50, 75, 100, 125, 150, 175, 200, or 250 antigens can be presented via the APC. Therefore, in the method of the present invention, any appropriate number of (peptide or non-peptide) antigens, for example, 10 to 300 antigens, for example, 25 to 250, 50 to 200, 70 to 185, or 100 to 150 antigens, for example, about 10, 20, 50, 75, 100, 125, 150, 175, 200, or 250 antigens can be used to induce antigen-specific proliferation of immune cells.

[0151] antigen In some embodiments, an isolated immune cell population may be co-cultured with an APC loaded with an antigen (which may be a tumor antigen). More preferably, the antigen may be a tumor-specific antigen such as a neoantigen, more preferably a clonal neoantigen. Thus, the immune cells described herein (e.g., T cells) can target tumor antigens. More preferably, the immune cells described herein (e.g., T cells) can target neoantigens, even more preferably clonal neoantigens.

[0152] The tumor antigens that can be targeted by targeted immune cells (e.g., T cells) according to the present invention are not particularly limited.

[0153] Tumor antigens known in the art include: CEA, immature laminin receptor, TAG-72, HPVE6 and E7, BING-4, calcium-activated chloride channel 2, cyclin B1, 9D7, Ep-CAM, 15 EphA3, Her2 / neu, telomerase, mesothelin, SAP-1, survivin, BAGE family, CAGE family, GAGE ​​family, MAGE family, SAGE family, XAGE family, NY-ESO-1 / LAGE-1, PRAME, SSX-2, Melan-A / MART-1, gp100 / pmel17, tyrosinase, TRP-1 / -2, P. polypeptide, MC1R, prostate-specific antigen, β-catenin, BRCA1 / 2, CDK4, CML66, fibronectin, MART-2, p53, ras, TGF-βRII, and MUC1. Other examples of tumor antigens are described in International Publication No. 2022 / 269250, which is incorporated herein by reference in its entirety.

[0154] Immune cells (e.g., T cells) can target neoantigens. A “neoantigen” is a tumor-specific antigen resulting from mutations within cancer cells. Therefore, neoantigens are not expressed (or are expressed at significantly lower levels) in target healthy (i.e., non-tumor) cells. Neoantigens can be processed to produce unique peptides that can be recognized by immune cells (e.g., T cells) when presented in relation to MHC molecules. As described herein, neoantigens can be used as a basis for cancer immunotherapy. In this specification, “neoantigen” is intended to include neoantigen-derived peptides. As used herein, the term “neoantigen” is intended to encompass all immunogenic portions of neoantigens.

[0155] The binding of neoantigens to specific MHC molecules (encoded by specific HLA alleles) can be predicted using methods known in the art. Examples of methods for predicting MHC binding include those described by Lundegaard et al., O'Donnel et al., and Bullik-Sullivan et al. For example, the MHC binding of neoantigens can be predicted using the netMHC (Lundegaard et al.) and netMHCpan (Jurtz et al.) algorithms. The binding of neoantigens to specific MHC molecules is a prerequisite for the presentation of the neoantigen on the cell surface by the said MHC molecule. Further discussion of neoantigens, particularly the types of mutations that can produce neoantigens, is contained in International Publication No. 2022 / 269250, which is incorporated herein by reference in its entirety.

[0156] Preferably, immune cells (e.g., T cells) can target clonal neoantigens. Clonal neoantigens (also called truncal neoantigens) are neoantigens resulting from clonal mutations. Clonal mutations (also called truncal mutations) are mutations present in substantially all tumor cells in one or more samples from a subject (or mutations that can be presumed to be present in substantially all tumor cells from which the tumor genetic material in the sample originates). Therefore, a clonal mutation may be a mutation present in all tumor cells in one or more samples from a subject. For example, a clonal mutation may be a mutation that occurs early in tumorigenesis.

[0157] A "subclonal neoantigen" (also called a "branched neoantigen") refers to a neoantigen that arises from a subclonal mutation. A "subclonal mutation" (also called a "branched mutation") is a mutation that is present in a subset or a specific proportion of cells in one or more tumor samples from a subject (or a mutation that is presumed to be present in a subset of tumor cells from which the tumor genetic material in the sample originates). For example, a subclonal mutation may be the result of a mutation that occurred in a specific tumor cell in the later stages of tumorigenesis, and this mutation may only be present in cells that are descendants of that cell.

[0158] With respect to one or more samples, the expression "substantially all tumor cells" means at least 70%, at least 75%, at least 80%, at least 85%, 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 tumor cells in one or more samples.

[0159] Thus, clonal neoantigens are neoantigens that are expressed substantially throughout the entire tumor. Subclonal neoantigens are neoantigens that are expressed in a subset of cells or a specific proportion or region within the tumor. "Substantially throughout the tumor" may mean that the clonal neoantigen is expressed throughout the entire region of the tumor from which the sample being analyzed originates.

[0160] The determination that a mutation is "essentially encoded (or expressed) in all tumor cells" refers to a statistical calculation and is therefore subject to statistical analysis and thresholding.

[0161] Similarly, the determination that clonal neoantigens are "effectively expressed throughout the tumor" also involves statistical calculations and is therefore subject to statistical analysis and thresholds.

[0162] Various methods for determining whether a neoantigen is "clonal" are known in the art. For example, clonal neoantigens can be identified using any suitable method, such as those described in Landau et al. (Cell. 2013 Feb 14;152(4):714-26); MacGranahan et al. (Science 2016 March 25;351(6280):1463-1469); or Roth et al. (Nat Methods. 2014 April;11(4):396-398).

[0163] Further discussion of clonal neoantigens and methods for determining whether a neoantigen is a clonal neoantigen are described in International Publication No. 2022 / 269250, which is incorporated herein by reference in its entirety.

[0164] Alternatively, APCs may present non-tumor-specific antigens. These non-tumor-specific antigens may include autoantigens that are important in autoimmune diseases. They may also include donor-derived antigens that are important in transplantation contexts, such as those associated with graft rejection and graft-versus-host disease (GVHD).

[0165] Dynamic suspension According to general teachings in this field, in order to stimulate immune cells with APCs, it is necessary to co-culture APCs and immune cells under static conditions (typically adherent culture) to promote stable interactions between immune cells and APCs.

[0166] As illustrated herein, we have surprisingly demonstrated that a population of immune cells, particularly T cells, can be efficiently produced by co-culturing isolated immune cells (especially T cells) with APC in a dynamic suspension.

[0167] Accordingly, the present invention relates to a method for culturing immune cells, and in particular a method for generating a population of immune cells, wherein isolated immune cells are co-cultured with APCs, and the co-culture is carried out in a dynamic suspension. The immune cells may be any of those described herein.

[0168] Suspension culture refers to a type of cell culture in which cells are suspended in a culture medium. Dynamic suspension culture refers to a type of suspension culture in which cells are motile while suspended in a culture medium. Dynamic suspension culture refers to a type in which the quiescent phase is reduced or eliminated, i.e., the number or proportion of immune cells and / or antigen-presenting cells (APCs) that settle from the suspension is reduced or removed.

[0169] Typically, in a dynamic suspension, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more, up to 100%, of immune cells and / or APCs are suspended. In other words, less than 20%, less than 15%, less than 10%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or less than 0% of immune cells and / or APCs settle from the suspension culture.

[0170] As will be detailed later, the power input per volume can be set so that the percentage of immune cells and / or APCs remaining in the suspension exceeds a desired threshold or percentage. Determining the appropriate power input per volume to achieve the desired dynamic suspension for a given co-culture of immune cells and APCs is within the ordinary scope of the work of those skilled in the art.

[0171] Typically, the methods of the present invention do not include an initial static co-culture step before co-culture of immune cells and APCs in a dynamic suspension. Therefore, co-culture in a dynamic suspension can be carried out from day 0, for example, from seeding of immune cells and / or APCs into a bioreactor. Starting the dynamic suspension on day 0, particularly from seeding of immune cells and / or APCs into a bioreactor, eliminates the initial static co-culture step. The dynamic suspension can be obtained by stirring (continuous or intermittent) or fluid flow, as described herein.

[0172] stirring Typically, dynamic suspensions are obtained by agitation of the coculture. This agitation can be achieved by any suitable means. In non-limiting examples, agitation may include mechanical agitation; oscillating agitation; vertical wheel agitation; and / or pneumatic agitation. Suitable bioreactors capable of producing these various types of agitation are known in the art, and commercially available bioreactors are also available.

[0173] As described herein, conventional methods for producing immune cell populations rely on co-culture of immune cells and APCs under static conditions. Such conventional methods may include an initial stirring or mixing step to disperse the immune cells and APCs at the start of the co-culture to facilitate contact between them. However, such an initial stirring or mixing step does not result in a dynamic suspension, unlike the stirring described herein. For example, such an initial mixing or stirring step is typically performed only once at the start of the co-culture, and the duration is usually short (e.g., about 5 minutes or less, about 4 minutes or less, about 3 minutes or less, about 2 minutes or less, about 1 minute or less, about 30 seconds or less). Alternatively, or in addition, the power input per volume for stirring or mixing is usually not particularly limited, as long as the step mixes the immune cells and APCs. For example, the cell suspension can be mixed manually using Stripette®. The method of the present invention may include a mixing step, for example, at the start of the co-culture and / or after re-seeding of immune cells as described herein. Alternatively, the method of the present invention may not involve a mixing step, but may utilize a dynamic suspension to disperse immune cells and APCs within the bioreactor used in the method of the present invention.

[0174] The stirring according to the present invention may be continuous and / or intermittent.

[0175] Continuous stirring means that stirring is not interrupted. Stirring can be defined as continuous over a specified period, for example, for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, or at least about 20 days, up to the entire duration of the method (or its co-culture step).

[0176] The stirring may be continuous for at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, or at least about 20 days, up to the entire duration of the method (or its co-culture step).

[0177] "Intermittent stirring" means that stirring is performed at specific intervals. The intervals may be regular or irregular, but are preferably regular. "Regular intervals" means that all intervals in a method or one step of a method are of the same length. "Irregular intervals" means that at least one interval in a method or one step of a method is of a different length from the other intervals.

[0178] The interval includes periods of stirring and periods without stirring. The interval (including stirring and non-stirring) may be any appropriate period. For example, one interval may be about 30 minutes, about 1 hour, about 90 minutes, about 2 hours, about 3 hours, about 4 hours, or longer. As a non-limiting example, a regular interval may be about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, or longer.

[0179] Within a predetermined interval, the duration of stirring may be longer than the time without stirring. For example, stirring can be performed for 45 minutes, followed by 15 minutes of incubation without stirring. Within a predetermined interval, the duration of stirring may be the same as the time without stirring. For example, stirring can be performed for 30 minutes, followed by 30 minutes of incubation without stirring. Typically, within a predetermined interval, the stirring time may be shorter than the time without stirring. As a non-limiting example, stirring can be performed for 30 minutes, followed by 45 minutes of incubation without stirring.

[0180] The stirring time within the interval and the time without stirring may be constant, or may vary from interval to interval. When the intervals are regular, if the stirring time is constant, the time without stirring will be of different lengths. Typically, the stirring time within the interval and the time without stirring are constant. Preferably, the intervals are regular, and the stirring time and the time without stirring within the interval are constant.

[0181] Typically, when the stirring is intermittent, the intervals are regular and have a length of about 1 hour to about 5 hours, for example about 1 hour to about 4 hours, preferably about 1 hour to about 3 hours, more preferably about 1 hour to about 2 hours.

[0182] Intermittent stirring may comprise intervals having a stirring time of from about 5 minutes to about 60 minutes, for example from about 5 minutes to about 45 minutes, preferably from about 5 minutes to about 30 minutes, more preferably from about 5 minutes to about 15 minutes.

[0183] Intermittent stirring may comprise stirring for from about 5 minutes to about 60 minutes, for example from about 5 minutes to about 45 minutes, preferably from about 5 minutes to about 30 minutes, more preferably from about 5 minutes to about 15 minutes, every 1 to 5 hours.

[0184] Intermittent stirring may comprise stirring for from about 5 minutes to about 60 minutes, for example from about 5 minutes to about 45 minutes, preferably from about 5 minutes to about 30 minutes, more preferably from about 5 minutes to about 15 minutes, every 1 to 4 hours.

[0185] Intermittent stirring may comprise stirring for from about 5 minutes to about 60 minutes, for example from about 5 minutes to about 45 minutes, preferably from about 5 minutes to about 30 minutes, more preferably from about 5 minutes to about 15 minutes, every 1 to 3 hours.

[0186] Intermittent stirring may comprise stirring for from about 5 minutes to about 60 minutes, for example from about 5 minutes to about 45 minutes, preferably from about 5 minutes to about 30 minutes, more preferably from about 5 minutes to about 15 minutes, every 1 to 2 hours.

[0187] Intermittent stirring may comprise stirring for from about 5 minutes to about 30 minutes every 1 to 5 hours.

[0188] Intermittent stirring may comprise stirring for from about 5 minutes to about 30 minutes every 1 to 4 hours.

[0189] Intermittent stirring may include stirring for approximately 5 to 30 minutes every 1 to 3 hours.

[0190] Intermittent stirring may include stirring for approximately 5 to 30 minutes every 1 to 2 hours.

[0191] Intermittent stirring may include stirring for approximately 5 to 15 minutes every 1 to 5 hours.

[0192] Intermittent stirring may include stirring for approximately 5 to 15 minutes every 1 to 4 hours.

[0193] Intermittent stirring may include stirring for approximately 5 to 15 minutes every 1 to 3 hours.

[0194] Intermittent stirring may include stirring for approximately 5 to 15 minutes every 1 to 2 hours.

[0195] Typically, the intermittent stirring according to the present invention includes stirring for about 5 minutes to about 30 minutes every 1 to 3 hours. Preferably, the intermittent stirring is performed (i) hourly; or (ii) every 3 hours for about 5 minutes to about 15 minutes (for example, about 5 to about 10 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, or about 15 minutes). More preferably, the intermittent stirring is performed (i) hourly; or (ii) every 3 hours for about 10 minutes.

[0196] Instead of, or in addition to, setting the power input per volume to ensure that the percentage of immune cells and / or APCs remaining in the suspension exceeds a desired threshold or percentage (as described herein), the intermittent stirring according to the present invention may also be set to ensure that the percentage of immune cells and / or APCs remaining in the suspension exceeds a desired threshold or percentage. Determining the appropriate interval for intermittent stirring to achieve a desired dynamic suspension state for a particular co-culture of immune cells and APCs is within the scope of the ordinary work of those skilled in the art.

[0197] The stirring according to the present invention may include a first continuous stirring period and a second intermittent stirring period. Preferably, the stirring according to the present invention may include a first intermittent stirring period and a second continuous stirring period.

[0198] The first intermittent or continuous stirring (preferably intermittent stirring) period may be about 0 to 3 days, for example, about 0 to 2 days, preferably about 1 to 2 days.

[0199] The second intermittent or continuous stirring (preferably continuous stirring) period may be about 0 to about 21 days, for example, about 1 to about 21 days, about 2 to about 21 days, about 2 to about 14 days, preferably about 2 to about 10 days (for example, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days).

[0200] In some preferred embodiments, the stirring according to the present invention may include: (a) a first intermittent or continuous stirring (preferably intermittent stirring) period of about 0 to about 3 days, for example, about 0 to about 2 days, preferably about 1 to about 2 days; and (b) a second intermittent or continuous stirring (preferably continuous stirring) period of about 0 to about 21 days, for example, about 1 to about 21 days, about 2 to about 21 days, about 2 to about 14 days, preferably about 2 to about 10 days (for example, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 days).

[0201] In some preferred embodiments, the method of the present invention may comprise: (a) a first intermittent or continuous stirring (preferably intermittent stirring) period of about 0 to about 3 days, for example, about 0 to about 2 days, preferably about 1 to about 2 days; and (b) a second intermittent or continuous stirring (preferably continuous stirring) period of about 0 to about 21 days, for example, about 1 to about 21 days, about 2 to about 21 days, about 2 to about 14 days, preferably about 2 to about 10 days (for example, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 days).

[0202] In some particularly preferred embodiments, the method of the present invention comprises a first co-culture period subjected to intermittent agitation, followed by a second co-culture period subjected to continuous agitation. Said first period is optionally from about 0 day to about 2 days; preferably from about 1 day to about 2 days; and / or said second period is optionally from about 2 days to about 21 days, preferably from about 2 days to about 10 days.

[0203] As described herein, the methods of the present invention typically do not comprise an initial static co-culture step prior to co-culture of immune cells and APCs in a dynamic suspension. This dynamic suspension can be achieved by continuous agitation. Accordingly, continuous agitation of the co-culture can be performed from day 0, for example, at the time of seeding the immune cells and / or APCs into the bioreactor. By initiating continuous agitation from day 0, particularly at the time of seeding into the bioreactor, the initial static co-culture step is omitted.

[0204] As described herein, alternatively or in addition to the above, the dynamic suspension can also be achieved by intermittent agitation. Accordingly, intermittent agitation of the co-culture can be performed from day 0, for example, at the time of seeding the immune cells and / or APCs into the bioreactor. By initiating intermittent agitation from day 0, particularly at the time of seeding the immune cells and / or APCs into the bioreactor, the initial static co-culture step is omitted. For the avoidance of doubt, the intermittent agitation does not comprise a static step.

[0205] Power input per volume Power input per volume (VPI) refers to the power required to maintain the motion of the culture medium and cells within a culture vessel (e.g., a bioreactor). In the art, VPI is commonly referred to as "power per volume (P / V)", and the two terms (VPI and P / V) can be used interchangeably.

[0206] Power input per volume is a fundamental engineering principle, which forms part of the general common knowledge in the field of bioprocessing. VPI can be calculated using the power input (PI) and the volume of a given reactor.

[0207] The impeller power index (PI) itself can be calculated from the density of the culture medium, the diameter of the impeller, and the impeller power number. The power number can be obtained by using a power number curve corresponding to the impeller type, or by calculating it using known correlations. Many handbooks and texts are available that describe how to calculate power number, PI, and VPI. A non-limiting example is *Bioprocess Engineering Principles* by Pauline M. Doran (2nd ed., Academic Press, Elsevier, Amsterdam), which is incorporated herein by reference in its entirety, particularly Chapter 7, which describes fluid flow and mixing and provides calculations and power number curves for various impeller types. Thus, calculating such values ​​is within the ordinary business of those skilled in the art.

[0208] The VPI can be set so that the percentage of immune cells and / or APCs remaining in the suspension exceeds a desired threshold or percentage. Determining an appropriate VPI to achieve a desired dynamic suspension for a given co-culture of immune cells and APCs is within the ordinary practice of those skilled in the art.

[0209] Many factors affect VPI, including the density of the culture medium, the diameter of the impeller, the power of the impeller, and the volume of the bioreactor.

[0210] Determining the VPI required to achieve the dynamic suspension according to the present invention is within the ordinary scope of work for those skilled in the art. Standard formulas and equations for calculating VPI are known in the art. A non-limiting example is that described in Pauline M. Doran's Bioprocess Engineering Principles, which are incorporated herein by reference in their entirety.

[0211] If the agitation is mechanical, the desired VPI can be achieved by changing the rotation speed of the impeller.

[0212] In the case of mechanical stirring, the impeller can be set to rotate at a low RPM. When used herein, also referred to as "low RPM," means an RPM in the range of about 20 RPM to about 80 RPM (for example, about 20 RPM, about 25 RPM, about 30 RPM, about 35 RPM, about 40 RPM, about 45 RPM, about 50 RPM, about 55 RPM, about 60 RPM, about 65 RPM, about 70 RPM, about 75 RPM, or about 80 RPM), for example, about 20 RPM to about 75 RPM, about 20 RPM to about 70 RPM, about 20 RPM to about 65 RPM, about 20 RPM to about 60 RPM, about 20 RPM to about 55 RPM, about 25 RPM to about 55 RPM, or about 25 RPM to about 50 RPM, preferably about 20 RPM to about 75 RPM, more preferably about 25 RPM to about 70 RPM.

[0213] Alternatively, the impeller can be set to rotate at a high RPM. In this specification, "high RPM" refers to a range of approximately 70 RPM to approximately 200 RPM (for example, approximately 70 RPM, 75 RPM, 80 RPM, 85 RPM, 90 RPM, 95 RPM, 100 RPM, 105 RPM, 110 RPM, 115 RPM, 120 RPM, 125 RPM, 130 RPM, 135 RPM, 140 RPM, 145 RPM, 150 RPM, 155 RPM, 160 RPM, 165 RPM, 1 This means RPMs of approximately 70 RPM, approximately 175 RPM, approximately 180 RPM, approximately 185 RPM, approximately 190 RPM, approximately 195 RPM, or approximately 200 RPM, for example, approximately 70 RPM to approximately 175 RPM, approximately 70 RPM to approximately 150 RPM, approximately 70 RPM to approximately 125 RPM, approximately 70 RPM to approximately 120 RPM, approximately 70 RPM to approximately 110 RPM, approximately 70 RPM to approximately 100 RPM, preferably approximately 70 RPM to approximately 150 RPM, or approximately 70 RPM to approximately 100 RPM.

[0214] The RPMs described above were calculated using 100 mL of BioBlu Single Use DASBox 0.3C in the examples. Using standard calculations and common technical knowledge, those skilled in the art can determine appropriate RPMs for other bioreactors without undue burden. For example, as will be detailed later, RPMs can be calculated based on a desired VPI value.

[0215] The stirring speed (RPM) in mechanical stirring (such as in a stirred-tank bioreactor) can be calculated as follows:

number

[0216] Typically, a low VPI may be used to obtain a low RPM. As used herein, the term “low VPI” means a VPI in the range of about 0.1 to about 1.9 (e.g., about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about 0.6, about 0.7, about 0.8, about 0.9, about 1.0, about 1.1, about 1.2, about 1.3, about 1.4, about 1.5, about 1.6, about 1.7, about 1.8 or about 1.9), for example, a VPI of about 0.1 to about 0.6.

[0217] Typically, a high VPI may be used to obtain a high RPM. As used herein, the term “high VPI” means a VPI in the range of about 1.9 to about 4.6 (e.g., about 1.9, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, or about 4.6).

[0218] As a non-limiting example, for a reactor volume of 100 mL, the exemplary correlation between RPM and VPI is as follows: 25 RPM is equivalent to a VPI of 0.1. 50 RPM is equivalent to 0.6 VPI. 75 RPM corresponds to VPI 1.9. 100 RPM corresponds to VPI 4.6. 200 RPM corresponds to a VPI of 36.6.

[0219] (From a fluid dynamics perspective) the fundamental properties of stirring differ between mechanical stirring (e.g., in a stirred-tank bioreactor) and other forms of stirring such as oscillating stirring. However, methods and calculations for scaling VPI from one form of stirring to another have been described in the Art. As a non-limiting example, Bai et al. (2019) Chem.Eng.Sci.209:115183 (which is incorporated herein by reference in its entirety) compare the PIs of stirred-tank reactors and oscillating reactors and find them to be equivalent. Other parameters that can be used to scale between stirred-tank reactors and oscillating reactors include mixing time (as described in Bartczak et al. (2022), Chem.Eng.J.431(4):133288, incorporated herein) and oxygen mass transfer coefficient (kLa) (as described by Douglas Marsh in Engineering Characterisation of a Rocked Bag Bioreactor for Improved Process Development and Scale-Up (2017); available at https: / / discovery.ucl.ac.uk / id / eprint / 1534658).

[0220] Furthermore, if necessary, VPI values ​​for other forms of agitation can be easily determined empirically by those skilled in the art without undue burden, using standard techniques and / or information provided by the manufacturers of the relevant bioreactor systems.

[0221] Mechanical stirring In some preferred embodiments, stirring is mechanical stirring.

[0222] Mechanically agitated bioreactors typically use a mechanically driven impeller to agitate the culture medium. Common types of impellers include marine impellers (also called pitch-blade impellers), radial flow generators (also called radial flow impellers), angled-pitch blade impellers, and helical impellers.

[0223] Marine impellers generate axial flow when the cell suspension is discharged in one direction and drawn in from the opposite side parallel to the axis. Marine impellers are known for their shear sensitivity and efficient mixing at low impeller tip peripheral speeds. Therefore, in some preferred embodiments, dynamic suspensions are obtained by mechanical agitation using marine impellers. Non-limiting examples of suitable bioreactors equipped with mechanical agitation include: the DASbox® bioreactor system from Eppendorf, the Ambr® bioreactor series from Sartorius, the DynaDrive series from ThermoFisher Scientific, the Eppendorf BioBlu bioreactor (e.g., 0.3L, 3L, 10L, or 50L models), the Mobius® CellReady bioreactor (e.g., 50L and 200L models) from Merck Millipore, and the Univessel® and MA series mechanically agitated bioreactors from Bioreactor Sciences. Preferably, the method of the present invention uses a stirred-tank bioreactor (STR). Typically, the STR provides mechanical agitation by a marine impeller, such as the DASbox® system, Ambr® bioreactor, Eppendorf BioBlu bioreactor, or DynaDrive® bioreactor. In some embodiments, a combination of impellers may be used in the stirred-tank bioreactor. For example, three marine impellers and one radial impeller may be used. The radial flow generator discharges the fluid radially outward and draws it in axially from both sides of the impeller.

[0224] An angled-pitch blade impeller creates a mixed flow, with vertically inclined blades moving the liquid both axially and radially.

[0225] A helical impeller generates dispersed flow, resulting in a uniformly dispersed shear surface and gradients in all directions.

[0226] Alternatively, instead of using a mechanically driven impeller, a mechanically agitated bioreactor can agitate the culture medium using an externally mounted motor (such as a spinner with a mount supporting the bioreactor vessel) so that the vessel itself rotates. One example of such a bioreactor is the LimGROW bioreactor, which is scalable and provides disposable consumables.

[0227] Oscillating stirring Oscillatory stirring (also called wave stirring) generates waves by mechanically rocking the chamber containing the culture back and forth. These waves promote mixing and mass transfer, creating a favorable environment for suspension culture cells.

[0228] Such oscillating bioreactors typically include a chamber partially filled with culture medium and inoculated with cells. The remainder of the chamber is filled with air. During culture mixing, mass transfer is achieved as air continuously passes through the headspace and the chamber oscillates back and forth. This oscillating motion generates waves at the gas-liquid interface, significantly accelerating oxygen transfer.

[0229] Suitable bioreactors using oscillating agitation include the Biostat® RM bioreactor and CultiBag RM bioreactor from Sartorius, the AppliFlex bioreactor from Applikon, the Wave Bioreactor from GE Healthcare, and the HyPerforma Rocker Bioreactor from Thermo Scientific.

[0230] Vertical wheel stirring In a bioreactor using vertical wheel agitation, the culture medium flows in a lemniscate pattern throughout the entire volume of the chamber (usually U-shaped). Typically, the vertical wheel impeller consists of an outer paddle and opposing axial vanes, which combines radial agitation in the vertical plane with axial agitation in the horizontal plane. A non-limiting example of a suitable bioreactor using vertical wheel agitation is the Vertical-Wheel bioreactor from PBS Biotech.

[0231] Pneumatic agitation Pneumatic agitation typically involves the introduction of compressed air or gas, thereby achieving aeration, agitation, and fluid circulation of the bioreactor chamber without the use of moving mechanical parts. The compressed air or gas is usually introduced from the bottom of the bioreactor vessel via a nozzle, perforation plate, or ring sparger.

[0232] Pneumatically agitated bioreactors primarily come in two types: air-lift and bubble column bioreactors. These generally exhibit low shear stress and have simple designs and structures. Air-lift bioreactors feature vertical partitions or loops (inside or outside the reactor chamber) that allow for the circulation of the culture medium. In bubble column bioreactors, compressed air or gas is simply blown into the reaction chamber. Typically, pneumatically agitated bioreactors have a high height-to-diameter ratio. In bubble columns, air is blown in from the bottom of the column, thereby agitating the culture medium.

[0233] A suitable example of a bioreactor using pneumatic agitation is the Air Lift bioreactor manufactured by Electrolab Biotech.

[0234] flow Alternatively, dynamic suspension can be achieved by fluid flow within a bioreactor system, such as a hollow fiber bioreactor. This flow may be continuous or intermittent, but is typically continuous.

[0235] A hollow fiber bioreactor is a high-density continuous perfusion culture system comprising, as its main component, a chamber or cartridge containing multiple semipermeable hollow fibers arranged in parallel, having at least one inlet port and at least one outlet port. The culture medium flowing into the chamber or cartridge flows through the inside of the hollow fibers, thereby diffusing nutrients, gases, and waste products bidirectionally through the walls of the hollow fibers.

[0236] Hollow fiber bioreactors are commercially available. Non-exclusive examples include the Quantum® bioreactor system from Terumo BCT and hollow fiber bioreactors from FiberCellSystems.

[0237] As described above with respect to dynamic suspensions resulting from agitation, the method of the present invention for obtaining a dynamic suspension from a fluid flow may include, for example, a mixing step at the start of co-culture and / or after re-seeding of immune cells as described herein. Alternatively, such a method of the present invention may not include a mixing step and may rather rely on the dynamic suspension to disperse the immune cells and APCs in the bioreactor used in the method.

[0238] Methods for generating immune cells The present invention relates to a method for culturing immune cells and / or generating a population of immune cells, the method comprising co-culturing isolated immune cells and APCs in a dynamic suspension.

[0239] As described herein, a common instruction in the prior art is that co-culture of immune cells and APCs must be carried out under static conditions, because it is necessary to keep the immune cells and APCs in close proximity to form a stable interaction that promotes stimulation by APCs and activation of immune cells.

[0240] In contrast, the inventors have surprisingly demonstrated that co-culturing immune cells and APCs in a dynamic suspension is at least as efficient as the static co-culturing method, which is the current "gold standard," while also offering numerous advantages.

[0241] While not bound by theory, it is believed that immune cells can still come into contact with APCs even in dynamic suspension, thereby allowing APCs to stimulate immune cells and promote their proliferation. "Communication" means that the immune cells and APCs are in direct contact for a sufficient amount of time for the activation signal from the APCs to stimulate the activation of the immune cells. For example, the contact time could be up to 48 hours, up to 36 hours, up to 24 hours, up to 12 hours, up to 8 hours, up to 4 hours, up to 2 hours, up to 1 hour, or up to 30 minutes. The immune cells and APCs may be in direct contact throughout the entire contact period, or only for a portion of the contact period.

[0242] As a non-limiting example, it has been reported that contact with APCs (through the formation of immune synapses between TCRs and MHCs) ranging from approximately 1 minute to 1 hour (e.g., approximately 1 minute to 30 minutes, or approximately 10 minutes to 30 minutes) is required to induce activation-leading signaling within T cells. Contact time may be the result of contact between a single immune cell and an APC, or it may be cumulative (e.g., multiple short contacts are repeated until the total contact time is sufficient to trigger activation).

[0243] During the dynamic suspension process, immune cells may come into contact with one or more APCs one or more times. Any particular immune cell may come into contact with the same APC or different APCs.

[0244] When manufacturing cell therapy products, the starting material is typically a sample derived from the subject described herein. The sample is typically heterogeneous, i.e., contains multiple different cell types (e.g., at least two, at least three, at least four, at least five or more cell types). During the course of the method of the present invention, the composition of cells in the co-culture may change over time. For example, the method of the present invention may begin with a heterogeneous sample or population in the co-culture, but during its implementation, one or more immune cell types may increase and / or one or more different cell types may decrease. The final product of the method is a homogeneous immune cell population (e.g., a TIL population) or a desired immune cell type (e.g., CD4 + T cells and CD8 + This may be a population concentrated with T cells, or consisting solely of T cells. Therefore, the method of the present invention can be described interchangeably as a method for generating immune cells and a method for generating an immune cell population.

[0245] As described herein, co-culturing immune cells (e.g., T cells) with APCs results in activated immune cells. In other words, the immune cells produced by the method of the present invention typically respond to restimulation with antigens. Thus, the method of the present invention typically produces activated immune cells. Therefore, the method of the present invention can be described interchangeably as a method for producing activated immune cells and a method for producing a population of activated immune cells.

[0246] The immune cells may be any immune cells as described herein. The APC may be any APC as described herein, insofar as the APC supplies one or more stimulating or activating signals to the immune cells.

[0247] As a non-limiting example, immune cells include T cells or certain subtypes of T cells as described herein (e.g., CD4 + T cells and / or CD8 + APC may be a T cell, and APC may be a DC. As another non-limiting example, immune cells may be T cells or certain subtypes of T cells as described herein (e.g., CD4+ T cells and / or CD8 + The immune cell may be a T cell, and the APC may be a B cell. In yet another non-limiting example, the immune cell may be a TIL, and the APC may be a DC.

[0248] Accordingly, the present invention provides a method for culturing T cells, wherein the method comprises co-culturing isolated T cells with DCs in a dynamic suspension. The present invention also provides a method for generating a population of T cells, wherein the method comprises co-culturing isolated T cells with DCs in a dynamic suspension. The T cells may be any T cell type or combination thereof, such as those described herein. As a non-limiting example, the present invention provides CD4 + T cells and / or CD8 + A method for culturing T cells is also provided, wherein the method involves isolating CD4 + T cells and / or CD8 + This invention includes co-culturing T cells with DCs in a dynamic suspension. As another non-limiting example, the present invention relates to CD4 + T cells and / or CD8 + This invention provides a method for generating a population of T cells, wherein the method involves isolating CD4 + T cells and / or CD8 + This includes co-culturing T cells with DCs in a dynamic suspension. In yet another non-limiting example, the present invention provides a method for culturing TILs, wherein the method includes co-culturing isolated TIL cells with DCs in a dynamic suspension. In yet another non-limiting example, the present invention provides a method for generating a population of TILs, wherein the method includes isolated CD4 + T cells and / or CD8 + This includes co-culturing T cells with DCs in a dynamic suspension.

[0249] Typically, a dynamic suspension is produced by stirring the co-culture. Such stirring may be continuous and / or intermittent, as described herein. In non-limiting examples, intermittent stirring may include stirring for about 5 minutes to about 30 minutes every 1 to 5 hours, for example, (i) about 5 minutes to about 15 minutes per hour, preferably about 10 minutes; or (ii) about 5 minutes to about 15 minutes every 3 hours, preferably about 10 minutes.

[0250] To avoid any doubt, any continuous and / or intermittent stirring described herein can be used in combination with any immune cell and / or APC cell type described herein. For example, the present invention provides a method for culturing T cells or generating populations thereof, the method comprising co-culturing isolated T cells with DCs in a dynamic suspension, the dynamic suspension being obtained by stirring, particularly continuous and / or intermittent stirring. As another non-limiting example, the present invention relates to CD4 + T cells and / or CD8 + The present invention also provides a method for culturing T cells or generating a population thereof, wherein the method involves single CD4 + T cells and / or CD8 +The co-culture method comprises co-culturing T cells with DCs in a dynamic suspension, wherein (a) intermittent stirring is performed during a first co-culture period, the first period being optionally about 0 to about 2 days, preferably about 1 to about 2 days; and (b) a second co-culture period is followed by continuous stirring, the second period being optionally about 2 to about 21 days, preferably about 7 to about 21 days, more preferably about 10 to about 18 days, and even more preferably about 14 to about 16 days. As yet another non-limiting example, the present invention also provides a method for culturing TILs or generating populations thereof, the method comprising co-culturing isolated TILs with DCs in a dynamic suspension, in which (a) intermittent stirring is performed during a first co-culturing period, the first period being optionally about 0 to about 2 days, preferably about 1 to about 2 days; and (b) a second co-culturing period is followed by continuous stirring, the second period being optionally about 2 to about 21 days, preferably about 7 to about 21 days, more preferably about 10 to about 18 days, and even more preferably about 14 to about 16 days.

[0251] Many advantages are expected from culturing immune cells and / or APCs in dynamic suspension. Typically, dynamic suspension culture is suitable for repeated or continuous monitoring. This allows for real-time monitoring of conditions within the bioreactor and adjustments to ensure that one or more culture parameters are maintained within the desired range or at the desired value.

[0252] Thus, the method of the present invention may include monitoring one or more of the following parameters: (a) pH; (b) dissolved oxygen (DO); (c) temperature; (d) a gas mixture consisting of O2, N2, CO2 and / or compressed air; and / or (e) the concentration of nutrients and / or metabolites. Any combination of these parameters can be monitored, for example, one, two, three, four, or all five of these five parameters can be monitored. Preferably, the method of the present invention may include monitoring (a) pH; (b) dissolved oxygen (DO); (c) temperature; and (d) the concentration of nutrients and / or metabolites.

[0253] One or more sensors can be used to monitor one or more of the aforementioned parameters. When monitoring two or more parameters, each parameter can be monitored using a separate sensor. Alternatively, the sensor may be capable of monitoring multiple parameters, such as pH and DO; pH and temperature; or a gas mixture, pH and temperature.

[0254] The optimal values ​​or ranges of one or more of these parameters may depend on several factors, such as the immunocytotype, APC type, type of agitation, type of culture vessel (e.g., bioreactor), and the specific cell medium and / or additives used. In the prescribed method of the present invention, determining the optimal ranges for any / all of these parameters is within the scope of the ordinary practice of those skilled in the art.

[0255] For example, pH can be maintained within a range of approximately 7 to 7.4. For example, DO can be maintained within a range of approximately 10% to 100% (e.g., approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%), typically between approximately 20% and 90% (e.g., approximately 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%).

[0256] For example, the temperature can be maintained in the range of approximately 36.5°C to approximately 37.5°C, preferably at 37.0°C.

[0257] For example, the gas mixture may be a mixture of oxygen, nitrogen, and CO2, with the oxygen content maintained in the range of approximately 4.2% to 21.0%, the CO2 content in the range of approximately 0% to 5%, and the remainder adjusted with nitrogen.

[0258] One or more parameters to be monitored can be monitored and maintained at a desired setpoint using a feedback mechanism. Thus, one or more of the following can be monitored: (a) pH; (b) dissolved oxygen (DO); (c) temperature; (d) a gas mixture consisting of O2, N2, CO2 and / or compressed air; and / or (e) the concentration of nutrients and / or metabolites. If a parameter is determined to be deviating from a desired range / value, appropriate adjustments can be made to bring the parameter back to the desired range or value. For example, DO can be controlled by using an oxygen blend passed through the bioreactor's headspace, in which case the DO is increased by increasing the proportion of oxygen in the blend. As yet another non-limiting example, pH can be controlled using CO2 and / or a base (e.g., sodium bicarbonate).

[0259] The one or more of the aforementioned parameters can be monitored and maintained using a control loop mechanism. The control loop mechanism typically includes, or comprises, sensors, control functions, and a final control element (FCE) that controls the processes necessary to automatically adjust the values ​​of the given parameters to be equal to or within a desired setpoint. The control loop can be an open loop (where the control function is independent of the parameters) or a closed loop (where the control function depends on the parameters). Preferably, the control loop is a closed control loop.

[0260] At the start of the method of the present invention (or its co-culture step), a predetermined number of immune cells (e.g., T cells) and / or APCs (e.g., DCs) are typically seeded in a culture vessel (e.g., a bioreactor). An appropriate number of immune cells (e.g., T cells) and / or APCs (e.g., DCs) can be easily selected by those skilled in the art without undue burden. As a non-limiting example, commercially available bioreactors often have standard operating conditions and parameters (SOC and SOP) published by the manufacturer, which may include the initial number or density of cells seeded. The number of immune cells (e.g., T cells) and / or APCs (e.g., DCs) can be measured by any suitable technique, preferably flow cytometry, such as cell counting (e.g., hemocytometer or Cedex HiRes (Roche CustomBiotech, Germany), Vi-CELL (Beckman-Coulter, USA), Eve (NanoEntek, South Korea)) or flow cytometry.

[0261] At the start of the method of the present invention (or the co-culture step thereof), immune cells (e.g., T cells) are raised to approximately 0.5 × 10⁻⁶ 6 cells / mL ~ approx. 5×10 6 cells / mL, e.g., approximately 0.5 × 10⁻⁶ 6 cells / mL ~ approx. 2.5×10 6 cells / mL, approximately 0.5×10 6 cells / mL ~ approx. 2×10 6 cells / mL, approximately 0.5×10 6 cells / mL ~ approx. 1.5×10 6 Cells / mL, or approximately 0.5 × 10⁻⁶ 6 cells / mL ~ approx. 1×10 6 Cells / mL can be seeded. Typically, immune cells (e.g., T cells) are seeded at approximately 0.5 × 10⁶ cells / mL. 6 cells / mL ~ approx. 1.5×10 6 Cells / mL, or approximately 0.5 × 10⁻⁶ 6 cells / mL ~ approx. 1×10 6 Cells are seeded at a rate of cells / mL. Preferably, immune cells (e.g., T cells) are seeded at approximately 0.25 × 10⁻⁶ cells / mL. 6 cells / mL, 0.5×10 6 cells / mL, 0.6×10 6cells / mL, 0.7×10 6 cells / mL, 0.8×10 6 cells / mL, 0.9×10 6 cells / mL, 1×10 6 cells / mL, 1.1×10 6 cells / mL, 1.2×10 6 cells / mL, 1.3×10 6 cells / mL, 1.4×10 6 cells / mL, or 1.5 × 10⁶ 6 cells / mL, most preferably about 1 × 10⁶ 6 Cells can be seeded at a rate of cells / mL.

[0262] Alternatively, or in addition, at the start of the method of the present invention (or the co-culture step thereof), immune cells (e.g., T cells) can be seeded in a ratio of approximately 20:1 immune cells:APC to approximately 1:1 immune cells:APC, for example, approximately 15:1 immune cells:APC to approximately 5:1 immune cells:APC, approximately 20:1 immune cells:APC to approximately 5:1 immune cells:APC, approximately 15:1 immune cells:APC to approximately 5:1 immune cells:APC, approximately 10:1 immune cells:APC to approximately 1:1 immune cells:APC, or approximately 10:1 immune cells:APC to approximately 5:1 immune cells:APC. Typically, immune cells (e.g., T cells) are seeded in a ratio of approximately 15:1 immune cells:APC to approximately 5:1 immune cells:APC, or approximately 10:1 immune cells:APC to approximately 5:1 immune cells:APC. Preferably, immune cells (e.g., T cells) are seeded in ratios of approximately 15:1 immune cells:APC, approximately 14:1 immune cells:APC, approximately 13:1 immune cells:APC, approximately 12:1 immune cells:APC, approximately 11:1 immune cells:APC, approximately 10:1 immune cells:APC, approximately 9:1 immune cells:APC, approximately 8:1 immune cells:APC, approximately 7:1 immune cells:APC, approximately 6:1 immune cells:APC, approximately 5:1 immune cells:APC, and most preferably approximately 10:1 immune cells:APC.

[0263] Generally, as cells are cultured, nutrients in the culture medium are consumed and waste products accumulate. As a result, to ensure continuous / optimal cell proliferation, it is generally necessary to introduce or replace the culture medium. Therefore, the method of the present invention may include introducing fresh culture medium into a co-culture or replacing the co-culture with fresh culture medium. In this specification, the terms "culture medium" and "culture medium" are used interchangeably. In this specification, the terms "fresh culture medium" and "fresh culture medium" are used to refer to a culture medium that has not previously come into contact with cells (e.g., immune cells and / or APCs). Therefore, fresh culture medium is usually nutrient-free and contains little to no waste products.

[0264] Therefore, in the method of the present invention, fresh culture medium can be introduced into the culture vessel (e.g., bioreactor) at least once during the implementation of the method. Typically, the fresh culture medium is introduced during the co-culture stage. Fresh culture medium can be introduced at least once, twice, three times, four times, five times, six times, or more times during the implementation of the method (or its co-culture step). Alternatively, or in addition, fresh culture medium may be introduced every two to three days or every other day during the implementation of the method (or its co-culture step).

[0265] The number of immune cells (e.g., T cells) and / or APCs (e.g., DCs) may be measured at one or more time points during the implementation of the method of the present invention. During the implementation of the method of the present invention (or its co-culture step), the number of immune cells (e.g., T cells) and / or APCs (e.g., DCs) may be measured at least once, twice, three, four, five, six or more times. Alternatively, or in addition, the number of immune cells (e.g., T cells) and / or APCs (e.g., DCs) may be measured every two to three days during the implementation of the method.

[0266] Any suitable technique can be used to measure the number of immune cells (e.g., T cells) and / or antigen-presenting cells (e.g., DCs). Suitable techniques are well known in the art and can be easily selected without undue burden to those skilled in the art. As a non-limiting example, the number of immune cells (e.g., T cells) and / or APCs (e.g., DCs) can be determined by cell counting (e.g., using a hemocytometer or image-based automated cell counter such as Cedex HiRes (Roche CustomBiotech, Germany), Vi-CELL (Beckman-Coulter, USA), Eve (NanoEntek, South Korea)), flow cytometry, in situ microscopy, Raman spectroscopy, and / or capacitance methods (e.g., using Viamass from Sartorius Biotech GmbH), and non-invasive acoustic quantification based on ultrasonic pulsed Doppler (USPD). Preferably, the number of immune cells (e.g., T cells) and / or APCs (e.g., DCs) can be measured using flow cytometry.

[0267] Once the number of immune cells (e.g., T cells) and / or APCs (e.g., DCs) is measured, this information can be used to feed back into the method. For example, the number of immune cells (e.g., T cells) and / or APCs (e.g., DCs) can be used to determine when to introduce fresh culture medium and / or the timing of intermittent stirring (length of interval and / or stirring time within the interval).

[0268] As will be described later, once the number of immune cells (e.g., T cells) and / or APCs (e.g., DCs) reaches a threshold, the immune cells (e.g., T cells) can be purified from the antigen-presenting cells (e.g., DCs) and used for re-inoculation into a culture vessel (e.g., bioreactor). This culture vessel (e.g., bioreactor) may be the same one used in the co-culture step, or a different one.

[0269] Additional steps The method of the present invention may include one or more additional steps.

[0270] For example, the method of the present invention may include one or more of the following optional steps: • A step to isolate immune cells (e.g., T cells) from the sample; For example, a step of modifying at least some immune cells (e.g., T cells) through gene editing; • The step of isolating APCs (e.g., DCs) from a sample (which may be the same sample used for immune cells, or a different sample, and may optionally be from the same patient); • A step to non-specifically proliferate immune cells (T cells) before co-culturing with APCs (also called a pre-proliferation step); • A final step at the end of this method to purify and / or formulate immune cells (e.g., T cells); and / or • A step of culturing immune cells (e.g., T cells) in the absence of APC.

[0271] Modification of immune cells The method for generating immune cells (e.g., T cells) according to the present invention may further include the step of modifying at least a portion of the immune cells (e.g., T cells) by, for example, gene editing.

[0272] Immune cells (e.g., T cells) can be modified by gene editing methods. Gene editing methods are known in the art and can be selected from CRISPR, TALE, zinc finger, and combinations thereof.

[0273] The gene editing method is described in its entirety in International Publication No. 2021 / 081378, which is incorporated herein by reference.

[0274] A non-limiting example of preferred modifications to immune cells (e.g., T cells) is described in International Publication No. 2022 / 269250, which is incorporated herein by reference in its entirety.

[0275] Pre-propagation step In embodiments of this method, which includes a nonspecific pre-proliferation step before co-culture and in which the immune cells are T cells, the pre-proliferation step may include culturing the T cells in the presence of IL-2 and IL-21.

[0276] Such pre-proliferation steps may further include culturing T cells in the presence of anti-CD3 antibody, anti-CD28 antibody, anti-CD2 antibody, and / or IFNy.

[0277] Such pre-proliferation steps may include culturing T cells in the presence of IL-2, IL-15, IL-21, anti-CD3 antibody, anti-CD28 antibody, and anti-CD2 antibody, as described in International Publication No. 2022 / 269250, which is incorporated herein by reference in its entirety.

[0278] Culture of immune cells in the absence of APC In some preferred embodiments, the method of the present invention comprises, as a first step of the present invention, co-culturing isolated immune cells with APCs in a dynamic suspension, and a subsequent step of culturing immune cells (e.g., T cells) in the absence of APCs (e.g., DCs).

[0279] Typically, in a method that includes a subsequent step of culturing immune cells (e.g., T cells) in the absence of APCs (e.g., DCs), the co-cultured immune cells (e.g., T cells) and APCs (e.g., DCs) are harvested when thresholds are reached for the total number of immune cells / cell density (e.g., T cells) and APCs (e.g., DCs), and the immune cells (e.g., T cells) are re-seed into a culture vessel (e.g., bioreactor). The culture vessel to which the cells are re-seeded may be the same culture vessel (e.g., bioreactor) used in the co-culture step, or a different culture vessel (e.g., bioreactor).

[0280] After the recovery of immune cells (e.g., T cells) and APCs (e.g., DCs), it may be necessary to purify the immune cells (e.g., T cells) from the APCs (e.g., DCs) before reseeding the culture vessel (e.g., bioreactor). Purification of immune cells (e.g., T cells) can be carried out by any suitable method, examples of which are known in the art and described herein. Alternatively, after the co-culture step, the APCs may have died and become fragmented and may no longer be present in the culture medium, in which case it is not necessary to separate the immune cells (e.g., T cells) from the APCs (e.g., DCs) before reseeding.

[0281] The threshold cell count required before purifying immune cells (e.g., T cells) from APCs (e.g., DCs) may be expressed as the total number of immune cells (e.g., T cells) and APCs (e.g., DCs). The threshold cell count required before purifying immune cells (e.g., T cells) from APCs (e.g., DCs) may be expressed as the number of immune cells (e.g., T cells) only (excluding APCs (e.g., DCs)).

[0282] Alternatively, or in addition to this, immune cells (e.g., T cells) can be reseeded to obtain the desired cell density, thereby increasing the number of immune cells to approximately 0.5 × 10⁻⁶. 6 cells / mL ~ approx. 5×10 6 cells / mL, e.g., approximately 0.5 × 10⁻⁶ 6 cells / mL ~ approx. 2.5×10 6 cells / mL, approximately 0.5×10 6 cells / mL ~ approx. 2×10 6 cells / mL, approximately 0.5×10 6 cells / mL ~ approx. 1.5×10 6 Cells / mL, or approximately 0.5 × 10⁻⁶ 6 cells / mL ~ approx. 1×10 6 They can be re-seeded at a rate of cells / mL. Typically, immune cells (e.g., T cells) are about 0.5 × 10⁶ 6 cells / mL ~ approx. 1.5×10 6 Cells / mL, or approximately 0.5 × 10⁻⁶ 6 cells / mL ~ approx. 1×10 6The cells are re-seed at a concentration of cells / mL. Preferably, immune cells (e.g., T cells) are re-seed at a concentration of 0.5 × 10⁻⁶. 6 cells / mL, 0.6×10 6 cells / mL, 0.7×10 6 cells / mL, 0.8×10 6 cells / mL, 0.9×10 6 cells / mL, 1×10 6 cells / mL, 1.1×10 6 cells / mL, 1.2×10 6 cells / mL, 1.3×10 6 cells / mL, 1.4×10 6 cells / mL, or 1.5 × 10⁶ 6 cells / mL, most preferably about 1 × 10⁶ 6 Re-seeding can be performed at a cell / mL rate. The total number of immune cells re-inoculated typically depends on the reactor volume. As a non-limiting example based on the cell density described above, for a reactor volume of 100 mL, immune cells (e.g., T cells) can be approximately 50 × 10⁶. 6 Cells ~ approx. 500×10 6 Cells, for example, about 50 x 10 6 Cells ~ approx. 250×10 6 cells, approximately 50 x 10 6 Cells ~ approx. 200×10 6 cells, approximately 50 x 10 6 Cells ~ approx. 150 x 10 6 Cells, or approximately 50 x 10 6 Cells ~ approx. 100×10 6 The total cell density can be reseeded. Typically, immune cells (e.g., T cells) can be reseeded at approximately 50 × 10⁶ in the absence of APCs. 6 Cells ~ approx. 150×10 6 Cells, or approximately 50 x 10 6 Cells ~ approx. 100×10 6 The cells are reseed. Preferably, immune cells (e.g., T cells) are reseed in the absence of APCs at approximately 50 × 10⁻⁶ 6 cells, approximately 60 x 10 6 cells, approximately 70 x 10 6 cells, approximately 80 x 10 6 cells, approximately 90 x 10 6 cells, approximately 100 x 10 6 cells, approximately 110 x 10 6cells, approximately 120 x 10 6 Cells, approximately 130 x 10 6 cells, approximately 140 x 10 6 Cells, or approximately 150 x 10 6 Cells, most preferably about 100 × 10 6 Cells can be reseeded.

[0283] If the method of the present invention includes a subsequent step of culturing immune cells for a certain period of time in the absence of APC after co-culturing, this culturing of immune cells is usually carried out in a dynamic suspension.

[0284] This dynamic suspension may be as defined herein in relation to the co-culture step. To avoid doubt, unless expressly stated otherwise, all disclosures herein describing and defining a dynamic suspension (including disclosures of dynamic suspensions in relation to co-culture) apply similarly and unconditionally to subsequent steps of culturing immune cells in the dynamic suspension for a further period in the absence of APC.

[0285] The additional period for culturing immune cells (e.g., T cells) in the absence of APCs (e.g., DCs) is at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, or at least about 20 days or longer. Preferably, the additional period for culturing immune cells (e.g., T cells) in the absence of antigen-presenting cells (e.g., DCs) is about 2 to 21 days, about 2 to 14 days, preferably about 2 to 10 days (e.g., about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 days).

[0286] A dynamic suspension of immune cells (e.g., T cells) cultured in the absence of APCs (e.g., DCs) can be generated by agitation. The agitation may be continuous and / or intermittent. Preferably, if the method of the present invention includes a subsequent step of culturing the immune cells for a further period in the absence of APCs after co-culturing, the immune cell culture may be subjected to continuous agitation.

[0287] "Continuous stirring" may be as defined herein in relation to the co-culture step. To avoid any doubt, unless otherwise expressly stated, any disclosure describing and defining continuous stirring herein, including disclosures relating to continuous stirring in relation to co-culture, applies similarly and unconditionally to a subsequent step of culturing immune cells for a further period of time with continuous stirring in the absence of APC.

[0288] As a non-limiting example, continuous agitation of immune cells (e.g., T cells) in the absence of APCs may last at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 11 days, at least about 12 days, at least about 13 days, at least about 14 days, at least about 15 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, or at least about 20 days or longer. Preferably, continuous agitation of immune cells (e.g., T cells) in the absence of APCs may last from about 2 to about 21 days, from about 2 to about 14 days, preferably from about 2 to about 10 days (e.g., about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, or about 10 days).

[0289] This additional step of culturing immune cells for a further period in the absence of APC after co-culture can be performed with continuous stirring as an alternative to, or in addition to, continuous stirring during co-culture. Therefore, the co-culture may be subjected to intermittent stirring, and the additional step of culturing immune cells for a further period in the absence of APC after co-culture may be performed with continuous stirring. Alternatively, the co-culture may be subjected to a first intermittent stirring period and a second continuous stirring period, and the additional step of culturing immune cells for a further period in the absence of APC after co-culture may be performed with continuous stirring. As yet another example, the co-culture may be subjected to continuous stirring, and the additional step of culturing immune cells for a further period in the absence of APC after co-culture may also be performed with continuous stirring.

[0290] Intermittent stirring may be the same as that defined herein in relation to the co-culture step. To avoid any doubt, unless otherwise expressly stated, any disclosures describing and defining intermittent stirring herein, including disclosures of intermittent stirring in relation to co-culture, apply equally and unconditionally to subsequent steps of culturing immune cells for a further period with intermittent stirring in the absence of APC.

[0291] As a non-limiting example, a subsequent step of culturing immune cells for a further period with intermittent stirring in the absence of APC may include stirring for about 5 to 60 minutes, about 5 to 45 minutes, preferably about 5 to 30 minutes, and more preferably about 5 to 15 minutes, every 1 to 5 hours. Typically, the intermittent stirring may include stirring for about 5 to 30 minutes every 1 to 3 hours. Preferably, the intermittent stirring may include stirring for about 5 to 15 minutes, more preferably about 10 minutes, every 1 to 3 hours. For example, the intermittent stirring may include stirring for about 5 to 15 minutes, more preferably about 10 minutes, every hour. As yet another example, the intermittent stirring may include stirring for about 5 to 15 minutes, more preferably about 10 minutes, every 3 hours.

[0292] As discussed in relation to the co-culture described above, the subsequent step of culturing immune cells for a further period in the absence of APC may include a period of continuous stirring followed by a period of intermittent stirring. The subsequent step of culturing immune cells for a further period in the absence of APC may include a period of continuous stirring followed by a period of intermittent stirring. Preferably, the subsequent step of culturing immune cells for a further period in the absence of APC includes continuous stirring without culturing the immune cells in the absence of APC and under intermittent stirring. In other words, preferably, if the method of the present invention includes a step of culturing immune cells for a further period in the absence of APC, this culture of immune cells is preferably subjected to continuous stirring.

[0293] As discussed above in relation to co-culture, instead of setting the input power per volume so that the percentage of immune cells remaining in the suspension reliably exceeds a desired threshold or percentage (as described herein), the intermittent stirring according to the present invention can also be set so that the percentage of immune cells remaining in the suspension reliably exceeds a desired threshold or percentage. Determining the appropriate interval for intermittent stirring to achieve the desired dynamic suspension state for a given immune cell culture is within the scope of the ordinary work of those skilled in the art.

[0294] The disclosures relating to VPIs in this specification (including the disclosure of VPIs relating to the co-culture step) also apply similarly and unconditionally to subsequent steps of culturing immune cells for a further period of time in the absence of APC and under intermittent agitation.

[0295] As a non-limiting example, if the agitation of immune cells in the absence of APC is mechanical agitation, the desired VPI can be achieved by changing the rotational speed of the impeller. For example, a low RPM of about 20 RPM to about 70 RPM, preferably about 20 RPM to about 60 RPM, more preferably about 25 RPM to about 50 RPM can be used. Alternatively, a high RPM in the range of about 70 RPM to about 200 RPM, preferably about 70 RPM to about 150 RPM, or about 70 RPM to about 100 RPM may be used.

[0296] Typically, when culturing immune cells in the absence of APC, high RPM and / or high VPI are used for stirring. To avoid any doubt, the disclosures of RPM and / or high VPI described herein with respect to co-culture stirring are equally and unconditionally applicable to stirring immune cells in the absence of APC. As a non-limiting example, high VPIs of about 1.9 to about 36.6 or higher (e.g., about 1.9, about 2.5, about 5, about 10, about 15, about 20, about 25, about 30, about 35, about 36, or about 36.6), for example, about 1.9 to about 4.6 or higher (e.g., about 1.9, about 2.0, about 2.5, about 3.0, about 3.5, about 4.0, about 4.5, or about 4.6), preferably about 4.6 VPI or about 36.6, can be used. When higher VPIs are used (for example, to support up to 200 RPM with 100 mL in the BioBlu Single Use DASBox0.3C), these can be easily calculated as described herein without requiring excessive technical skill or burden.

[0297] The various agitation disclosures described above (including the disclosure of various agitation methods related to the co-culture step) also apply similarly and unconditionally to subsequent steps of culturing immune cells for a further period of time in the absence of APC and under intermittent agitation. Therefore, mechanical agitation, oscillating agitation, vertical wheel agitation, or pneumatic agitation can be used. Preferably, the agitation is mechanical. The same type of agitation may be used for both co-culture and the culture of immune cells in the absence of APC. The types of agitation used for co-culture and the culture of immune cells in the absence of APC may differ. Preferably, the same type of agitation can be used for both co-culture and the culture of immune cells in the absence of APC.

[0298] In some embodiments, the present invention provides a method for generating a population of immune cells, the method comprising: (a) co-culturing isolated immune cells with APCs over a first period of about 24 hours by intermittent stirring at an RPM of about 25 to about 50 RPM (e.g., about 25 RPM) for about 5 to about 15 minutes (e.g., about 10 minutes), followed by no stirring for about 1 hour; (b) co-culturing isolated immune cells with APCs over a second period of about 24 hours by intermittent stirring at an RPM of about 25 to about 50 RPM (e.g., about 50 RPM) for about 5 to about 15 minutes (e.g., about 10 minutes), followed by no stirring for about 1 hour; and (c) co-culturing isolated immune cells with APCs over a third period of about 1 week by continuous stirring at an RPM of about 25 to about 80 (e.g., about 70). After this third period, the method further: (d) in the absence of APC, preferably about 0.5 × 10 6 Immune cells / mL ~ approx. 2×10 6 Immune cells / mL (for example, approximately 1 × 10⁻⁶) 6 The process may include (e) reseeding immune cells at a density of immune cells / mL; and (f) culturing isolated immune cells for a fourth period of about one week by continuous stirring at an RPM of about 70 to about 100 RPM (e.g., about 100 RPM) in the absence of APCs. The immune cells may preferably be T cells, particularly TILs. The APCs may be DCs.

[0299] Preferably, the present invention provides a method for generating a population of immune cells, the method comprising: (a) co-culturing isolated immune cells with APCs for a first period of about 24 hours by intermittent stirring at about 25 RPM for about 10 minutes, followed by about 1 hour without stirring; (b) co-culturing isolated immune cells with APCs for a second period of about 24 hours by intermittent stirring at about 50 RPM for about 10 minutes, followed by about 1 hour without stirring; and (c) co-culturing isolated immune cells with APCs for a third period of about 1 week by continuous stirring at about 70 RPM. After this third period, the method further comprises: (d) preferably about 1 × 10 in the absence of APCs. 6The process may include (e) re-seeding immune cells at a density of immune cells / mL, and (f) culturing isolated immune cells for a fourth period of about one week by continuous stirring at about 100 RPM in the absence of APCs. The immune cells may preferably be T cells, particularly TILs. The APCs may be DCs.

[0300] In some embodiments, the present invention provides a method for generating a population of immune cells, the method comprising: (a) co-culturing isolated immune cells with APCs over a first period of about 24 hours by intermittent stirring for about 5 minutes to about 15 minutes (e.g., about 10 minutes) at an RPM of about 25 to about 50 RPM (e.g., about 25 RPM), followed by no stirring for about 3 hours; (b) co-culturing isolated immune cells with APCs over a second period of about 24 hours by intermittent stirring for about 5 minutes to about 15 minutes (e.g., about 10 minutes) at an RPM of about 25 to about 50 RPM (e.g., about 50 RPM), followed by no stirring for about 3 hours; and (c) culturing isolated immune cells with APCs over a third period of about 1 week by continuous stirring at an RPM of about 25 to about 80 RPM (e.g., about 70 RPM). After this third period, the method further: (d) in the absence of APC, preferably about 0.5 × 10 6 Immune cells / mL ~ approx. 2×10 6 Immune cells / mL (for example, approximately 1 × 10⁻⁶) 6 The process may include (e) reseeding immune cells at a density of immune cells / mL; and co-culturing isolated immune cells for a fourth period of about one week by continuous stirring at an RPM of about 70 to about 100 RPM (e.g., about 100 RPM) in the absence of APCs. The immune cells may preferably be T cells, particularly TILs. The APCs may be DCs.

[0301] Preferably, the present invention provides a method for generating a population of immune cells, the method comprising: (a) co-culturing isolated immune cells with APCs for a first period of about 24 hours by intermittent stirring at about 25 RPM for about 10 minutes, followed by about 3 hours without stirring; (b) co-culturing isolated immune cells with APCs for a second period of about 24 hours by intermittent stirring at about 50 RPM for about 10 minutes, followed by about 3 hours without stirring; (c) culturing isolated immune cells with APCs for a third period of about 1 week by continuous stirring at about 70 RPM. After this third period, the method further comprises: (d) preferably about 1 × 10 in the absence of APCs. 6 The process may include (e) re-seeding immune cells at a density of immune cells / mL; and co-culturing isolated immune cells for a fourth period of about one week by continuous stirring at about 100 RPM in the absence of APCs. The immune cells may preferably be T cells, particularly TILs. The APCs may be DCs.

[0302] In some embodiments, the present invention provides a method for generating a population of immune cells, the method comprising: (a) co-culturing isolated immune cells with APCs for a first period of about 48 hours by performing intermittent stirring for about 10 minutes at an RPM of about 70 to about 100 RPM (e.g., about 100 RPM), followed by no stirring for about 1 hour; and (b) co-culturing isolated immune cells with APCs for a second period of about 1 week by continuous stirring at an RPM of about 70 to about 100 RPM (e.g., about 100 RPM). After this second period, the method further comprises: (c) in the absence of APCs, preferably about 0.5 × 10 6 Immune cells / mL ~ approx. 2×10 6 Immune cells / mL (for example, approximately 1 × 10⁻⁶) 6 The method may include (d) re-seeding immune cells at a density of immune cells / mL; and culturing isolated immune cells for a third period of about one week by continuous stirring at an RPM of about 70 to about 100 RPM (e.g., about 100 RPM) in the absence of APCs. The immune cells may preferably be T cells, particularly TILs. The APCs may be DCs.

[0303] Preferably, the present invention provides a method for generating a population of immune cells, the method comprising: (a) co-culturing isolated immune cells with APCs for a first period of about 48 hours by performing intermittent stirring at about 10 minutes at about 100 RPM, followed by about 1 hour without stirring; and (b) co-culturing isolated immune cells with APCs for a second period of about 1 week by continuous stirring at about 100 RPM. After this second period, the method further comprises: (c) in the absence of APCs, preferably about 1 × 10⁻⁶ 6 The process may include (d) re-seeding immune cells at a density of immune cells / mL; and culturing isolated immune cells for a third period of about one week by continuous stirring at about 100 RPM in the absence of APCs. The immune cells may preferably be T cells, particularly TILs. The APCs may be DCs.

[0304] In some embodiments, the present invention provides a method for generating a population of immune cells, the method comprising: (a) co-culturing isolated immune cells with APCs for a first period of about 48 hours by performing intermittent stirring for about 10 minutes at an RPM of about 70 to about 100 RPM (e.g., about 100 RPM), followed by about 3 hours without stirring; and (b) co-culturing isolated immune cells with APCs for a second period of about 1 week by continuous stirring at an RPM of about 70 to about 100 RPM (e.g., about 100 RPM). After this second period, the method further comprises: (c) in the absence of APCs, preferably about 0.5 × 10 6 Immune cells / mL ~ approx. 2×10 6 Immune cells / mL (for example, approximately 1 × 10⁻⁶) 6 The method may include (d) re-seeding immune cells at a density of immune cells / mL; and culturing isolated immune cells for a third period of about one week by continuous stirring at an RPM of about 70 to about 100 RPM (e.g., about 100 RPM) in the absence of APCs. The immune cells may preferably be T cells, particularly TILs. The APCs may be DCs.

[0305] Preferably, the present invention provides a method for generating a population of immune cells, the method comprising: (a) co-culturing isolated immune cells with APCs for a first period of about 48 hours by performing intermittent stirring at about 100 RPM for about 10 minutes, followed by about 3 hours without stirring; and (b) co-culturing isolated immune cells with APCs for a second period of about 1 week by continuous stirring at about 100 RPM. After this second period, the method further comprises: (c) in the absence of APCs, preferably about 1 × 10⁻⁶ 6 The process may include (d) re-seeding immune cells at a density of immune cells / mL; and culturing isolated immune cells for a third period of about one week by continuous stirring at about 100 RPM in the absence of APCs. The immune cells may preferably be T cells, particularly TILs. The APCs may be DCs.

[0306] In some embodiments, the present invention provides a method for generating a population of immune cells, the method comprising: (a) co-culturing isolated immune cells with APC by continuous stirring at an RPM of about 70 to about 100 RPM (e.g., about 100 RPM) for a first period of about 9 days. After this period, the method further comprises: (b) preferably about 0.5 × 10 6 Immune cells / mL ~ approx. 2×10 6 Immune cells / mL (for example, approximately 1 × 10⁻⁶) 6 The method may include (c) reseeding immune cells at a density of immune cells / mL; and (d) culturing isolated immune cells for a second period of about one week by continuous stirring at an RPM of about 70 to about 100 RPM (e.g., about 100 RPM) in the absence of APCs. The immune cells may preferably be T cells, particularly TILs. The APCs may be DCs.

[0307] Preferably, the present invention provides a method for generating a population of immune cells, the method comprising: (a) co-culturing isolated immune cells with APC by continuous stirring at about 100 RPM for a first period of about 9 days. After this period, the method further comprises: (b) preferably about 1 × 10 6The process may include (c) re-seeding immune cells at a density of immune cells / mL; and (d) culturing isolated immune cells for a second period of about one week by continuous stirring at about 100 RPM in the absence of APCs. The immune cells may preferably be T cells, particularly TILs. The APCs may be DCs.

[0308] In some particularly preferred embodiments, the method of the present invention is carried out using a stirred-tank type bioreactor, optionally in combination with a marine impeller.

[0309] As described and illustrated herein, the method of the present invention may offer numerous advantages compared to conventional “gold standard” methods for co-culturing immune cells and APCs that require static culture.

[0310] For example, the method of the present invention may yield an immune cell yield at least equivalent to that obtained by a suitable control method. Suitable control methods may include co-culturing isolated immune cells with APC in a static culture medium, and / or co-culturing isolated immune cells with APC without agitation. Non-limiting examples of suitable control methods include the GMF method and the GSD method, which are detailed in the examples herein.

[0311] As used herein, the term "equivalent" is defined as a method in which the use of dynamic suspension and / or agitation does not significantly reduce the yield of immune cells compared to a method using a suitable control method. In non-limiting examples, the methods of the present invention yield immune cells at or below 2 times, 1.5 times, 1.0 times, 0.5 times, 0.25 times, or less, compared to a method using a suitable control method. The term "equivalent" may be defined as a method in which the yield of immune cells produced by a method using dynamic suspension is statistically no different (e.g., p<0.05, p<0.01) from the yield of immune cells produced by a method using a suitable control method.

[0312] Preferably, the method of the present invention produces an increased yield of immune cells compared to the yield of immune cells produced by a suitable control method. The yield of immune cells may be at least 1.5 times, at least 2 times, or at least 2.5 times greater than the yield of immune cells produced by a suitable control method.

[0313] Regardless of whether the method of the present invention produces at least equivalent or increased yields of immune cells compared to a suitable control method, the method of the present invention may offer other advantages as a result of the use of dynamic suspension. As a non-limiting example, immune cells produced by the method of the present invention may have improved quality and consistency compared to immune cells produced by a suitable control method. Alternatively, or in addition, the method of the present invention may be easier to scale up / scale out and / or significantly reduce the costs and effort associated with scaling compared to a suitable control method. Alternatively, or in addition, the method of the present invention may be more precisely controlled and regulated compared to a suitable control method, while simultaneously reducing the risk of batch loss due to fewer human interventions. Alternatively, or in addition, the method of the present invention is suitable for continuous monitoring and allows for online, in situ response, enabling the system to be dynamically adjusted to compensate for variations in the process and / or starting materials.

[0314] Culture medium and additives Any suitable culture medium can be used for co-culturing immune cells (e.g., T cells) with APCs (e.g., DCs), and / or culturing immune cells (e.g., T cells) in the absence of APCs (e.g., DCs). The same culture medium can be used for co-culturing immune cells (e.g., T cells) with APCs (e.g., DCs), and for culturing immune cells (e.g., T cells) in the absence of APCs (e.g., DCs). Alternatively, different culture media may be used for co-culturing immune cells (e.g., T cells) with APCs (e.g., DCs), and for culturing immune cells (e.g., T cells) in the absence of APCs (e.g., DCs).

[0315] In cell culture, it is common to add one or more additional components to the culture medium. For example, the culture medium used in the method of the present invention may contain one or more cytokines; serum (e.g., fetal bovine serum) or preferably a serum substitute (e.g., human serum albumin or a GMP-compliant synthetic equivalent); one or more antibodies; amino acids; sodium pyruvate; and glutamine; or any combination thereof.

[0316] Cytokine The culture medium used in the method of the present invention may contain one or more types of cytokines, such as those described herein.

[0317] In particular, the culture medium may contain IL-2 if the immune cells are T cells.

[0318] The term "IL-2" refers to the T cell growth factor known as interleukin-2 and includes all forms of IL-2, including human and mammalian types, conserved amino acid substitutions, glycoforms, biosimilars, and their variants. For example, the term IL-2 encompasses human recombinant IL-2 such as aldesleukin (trade name: PROLEUKIN®). Aldesleukin (des-alanyl-I, serine-125 human IL-2) is a non-glycosylated human recombinant IL-2 with a molecular weight of approximately 15 kDa. The term IL-2 also encompasses pegylated forms of IL-2, as described in International Publication No. 2012 / 065086.

[0319] When included in the method of the present invention, the concentration of IL-2 used in the antigen-specific proliferation step may be described as "lower" or "reduced" compared, for example, to the concentration of IL-2 used in the nonspecific pre-proliferation step.

[0320] Alternatively, or in addition to this, the culture medium may contain IL-15, especially if the immune cells are T cells.

[0321] The term "IL-15" refers to the immunomodulatory cytokine interleukin-15 and includes all forms of IL-15, such as human and mammalian types, conserved amino acid substitutions, glycoforms, biosimilars, and their variants. For example, the term IL-15 encompasses the human recombinant form of IL-15.

[0322] Furthermore, in lieu of or in addition to the above, the culture medium may contain IL-21, especially when the immune cells are T cells.

[0323] The term "IL-21" refers to the immunomodulatory cytokine interleukin-21 and includes all forms of IL-21, such as human and mammalian types, conserved amino acid substitutions, glycoforms, biosimilars, and their variants. For example, the term IL-21 encompasses the human recombinant form of IL-21.

[0324] The use of IL-2, IL-15, and IL-21 in T cell culture, and their appropriate concentrations, are described in International Publication No. 2022 / 266250, which is incorporated herein by reference in its entirety.

[0325] serum substitute In vitro cultures of cells are generally supplemented with serum, such as human or bovine serum, to aid in cell proliferation and maintenance. However, for GMP purposes in the manufacture of therapeutic formulations intended for human administration, it is desirable to avoid the inclusion of human or bovine serum whenever possible.

[0326] Serum substitutes derived from humans or cattle (e.g., CTS® Immune Cell SR (Gibco)) are commercially available.

[0327] A further option for serum substitutes is the use of platelet lysates. Platelet lysates are an alternative additive to fetal bovine serum (FBS) in cell cultures. They are obtained from platelets after lysing them by a freeze / thaw cycle, releasing growth factors that support cell proliferation. FBS-free cell culture media containing platelet lysates are commercially available in GMP quality and can be used in the manufacture of cell therapies. Platelet lysates are preferably derived from human blood and are referred to herein as human platelet lysates (hPL).

[0328] Platelet lysates can be included in cell culture media as defined herein. Platelet lysates may be present at a concentration of about 1% to about 10%, for example, about 5%.

[0329] The use of serum substitutes, particularly hPL, in T cell culture, and their appropriate concentrations is described in International Publication No. 2022 / 266250, which is incorporated herein by reference in its entirety.

[0330] antibody The culture medium used in the method of the present invention may contain one or more types of antibodies, such as those described herein.

[0331] The term "CD3" refers to differentiation antigen group 3. CD3 is a protein complex and a co-receptor for T cells involved in T cell activation. CD3 consists of a CD3γ chain, a CD3δ chain, and two CD3ε chains. These chains bind to the T cell receptor and the ζ (zeta) chain to generate an activation signal in T lymphocytes.

[0332] When an anti-CD3 antibody binds to CD3, it stimulates T cell activation. Anti-CD3 30 antibodies are known in the art. For example, suitable anti-CD3 antibodies include OKT3 (muromab), TRX4 (otelixizumab), PRV-031 (teprizumab), and bicilizumab. Preferably, the anti-CD3 antibody is OKT3.

[0333] The term "CD28" refers to differentiation antigen group 28. CD28 is constitutively expressed on naive T cells. For example, stimulation of CD28 with an anti-CD28 antibody provides a co-stimulatory signal necessary for T cell activation and survival. Appropriate anti-CD28 antibodies are known in the art.

[0334] The term "CD2" refers to differentiation antigen group 2. CD2 is a cell adhesion molecule present on the surface of T cells and natural killer (NK) cells. In addition to its adhesion properties, CD2 also functions as a costimulatory molecule in T cells and NK cells. Appropriate anti-CD2 antibodies are known in the art.

[0335] Antibodies can be supplied as soluble tetrameric antibody conjugates. Binding of the tetrameric antibody conjugate results in cross-linking of cell surface ligands, thereby providing the primary and co-stimulatory signals necessary for the activation of immune cells (particularly T cells). Such antibody conjugates are designed to activate and proliferate human immune cells (particularly human T cells) in the absence of magnetic beads, feeder cells, or antigens.

[0336] In one embodiment, a CD3 / CD28 tetrameric antibody conjugate is used. Such conjugates are commercially available (for example, ImmunoCult® Human CD3 / CD28 T cell Activator from STEMCELL Technologies, Inc.).

[0337] The use of antibodies in T cell culture and their appropriate concentrations are described in International Publication No. 2022 / 266250, which is incorporated herein by reference in its entirety.

[0338] System for immune cell production The present invention also provides a system for culturing immune cells and / or producing immune cell populations, said system being compatible with the methods of the present invention.

[0339] Accordingly, the present invention provides a system comprising: (a) a suspension culture bioreactor; (b) isolated immune cells; (c) APC; (d) culture medium; and (e) at least one sensor; wherein the isolated immune cells are co-cultured with APC in a dynamic suspension.

[0340] Examples of bioreactors suitable for use in the system of the present invention are known in the art and are commercially available. Non-limiting examples are described herein. Preferably, the suspension culture bioreactor is a stirred-tank type bioreactor.

[0341] Typically, the dynamic suspension in the system of the present invention is achieved by stirring the co-culture of isolated immune cells and APCs. This stirring may be of any type, such as those described herein. In non-limiting examples, the stirring may be selected from mechanical stirring, oscillating stirring, vertical wheel stirring, or pneumatic stirring. Examples of suitable bioreactors compatible with and / or providing these various types of stirring are known and commercially available in the art. Non-limiting examples of such bioreactors are described herein. Preferably, the isolated immune cells and APCs are co-cultured under mechanical stirring.

[0342] This system may include at least one, at least two, at least three, at least four, at least five, or more sensors. This system may include one, two, three, four, five, six, seven, eight, or more sensors.

[0343] The at least one sensor can monitor one or more parameters that may affect the co-culture of immune cells and APCs. Non-limiting examples of such parameters are described herein. In some preferred embodiments, the at least one sensor can monitor one or more of the following parameters: (i) pH; (ii) DO; (iii) temperature; (iv) a gas mixture consisting of O2, N2, CO2 and / or compressed air; and / or (v) the concentration of nutrients and / or metabolites.

[0344] The at least one of the sensors may be incorporated into a proportional-integral-derivative (PID) controller, which enables monitoring and maintenance of one or more of the parameters using a control loop mechanism. Such a control loop mechanism is described herein.

[0345] Immune cell population The present invention also provides an immune cell population that can be obtained or acquired by the method of the present invention. The immune cell population may be a T cell population, particularly antigen-specific T cells. Preferably, the immune cell population may be a TIL population.

[0346] The immune cell population (e.g., T cell population) generated according to the present invention may be enriched with immune cells (e.g., T cells) that are specific to, i.e., target a given antigen. In other words, the immune cell population (e.g., T cell population) generated according to the present invention has an increased number of immune cells (e.g., T cells) that target one or more given antigens. For example, the immune cell population (e.g., T cell population) of the present invention typically has a greater number of immune cells (e.g., T cells) that target the given antigen compared to immune cells (e.g., T cells) in a sample isolated from a subject. In other words, the composition of the immune cell population (e.g., T cell population) differs from the composition of a “native” immune cell population (e.g., a population not described herein) in that the percentage or proportion of immune cells (e.g., T cells) that target the given antigen is increased.

[0347] A population of immune cells (e.g., T cells) according to the present invention may have at least about 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% T cells that target a given antigen or set of antigens. For example, a population of immune cells (e.g., T cells) may have about 0.2% to 5%, 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 70%, or 70% to 100% of immune cells (e.g., T cells) that target a given antigen or set of antigens. In one embodiment, the immune cell (e.g., T cell) population has at least about 1, 2, 3, 4, or 5% of immune cells (e.g., T cells) that target the antigen, for example, at least about 2% or at least 2% of immune cells (e.g., T cells) that target the antigen.

[0348] In other words, a population of immune cells (e.g., T cells) may contain at most approximately 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, or 99.8% of immune cells (e.g., T cells) that do not target a given antigen. For example, an immune cell population (e.g., T cells) may have at most about 95% to 20%, 99.8%, 90% to 95%, 80% to 90%, 70% to 80%, 60% to 70%, 50% to 60%, 30% to 50%, or 0% to 30% of immune cells (e.g., T cells) that do not target a given antigen. In one embodiment, an immune cell population (e.g., T cells) may have at most about 99%, 98%, 97%, 96%, or 95% of immune cells (e.g., T cells) that do not target the antigen, for example, at most about 98% or 95% of immune cells (e.g., T cells) that do not target the antigen.

[0349] A proliferating population of antigen-reactive immune cells (e.g., T cells) may have higher activity than, for example, a population of immune cells (e.g., T cells) that were not proliferated using an antigen. "Activity" can refer to the response of the immune cell population (e.g., T cells) to restimulation with an antigen peptide (e.g., a peptide corresponding to the peptide used for proliferation, or a mixture of antigen-derived peptides). Appropriate methods for testing this response are known in the art. For example, cytokine production can be measured (e.g., in the case of T cells, IL-2 or IFN-γ production can be measured). The expression "higher activity" includes, for example, increases of 1-5 times, 5-10 times, 10-20 times, 20-50 times, 50-100 times, 100-500 times, and 500-1000 times. In one embodiment, activity can be more than 1000 times higher.

[0350] The present invention further provides a plurality of immune cells (e.g., T cells), where the plurality of immune cells (e.g., T cells) includes immune cells (e.g., T cells) that recognize a given antigen and immune cells (e.g., T cells) that recognize a different antigen. Thus, the present invention provides a plurality of immune cells (e.g., T cells) that recognize different antigens. Alternatively, different immune cells (e.g., T cells) included in the plurality of immune cells or population may have different receptors (e.g., TCRs in the case of T cells) that recognize the same antigen.

[0351] Immune cells (e.g., T cells) produced by the method of the present invention may possess one or more desirable functional properties.

[0352] As a non-restrictive example, T cells may have increased CD25 expression, CD27 expression, and / or IFNγ expression.

[0353] The term "CD25" refers to the interleukin-2 receptor α chain (IL2RA). The interleukin-2 receptor α and β (IL2RB) chains, together with the common γ chain (IL2RG), constitute the high-affinity IL2 receptor. The homodimeric α chain (IL2RA) produces the low-affinity receptor, while the homodimeric β chain (IL2RB) produces the medium-affinity receptor. CD25 is expressed in regulatory T cells along with CD4. CD27 is a member of the tumor necrosis factor receptor superfamily. CD27 binds to CD70 and induces T cell differentiation and clonal proliferation. CD27 plays a specific role in the formation of T cell memory.

[0354] As another non-limiting example, or in addition to the above, T cells may have reduced CD57 expression. The CD57 antigen is present on subsets of peripheral blood mononuclear cells, NK lymphocytes, and T lymphocytes. While CD57 expression in human lymphocytes may indicate non-proliferative (senescence), CD57-positive cells may also exhibit high cytotoxicity, memory-like characteristics, and potent effector function.

[0355] The advantageous functional characteristics of T cells are described in International Publication No. 2022 / 266250, which is incorporated herein by reference in its entirety.

[0356] In embodiments where the immune cells are T cells, the T cell population may consist entirely or mainly of CD8+ T cells, or a mixture of entirely or mainly of CD8+ T cells and CD4+ T cells, or entirely or mainly of CD4+ T cells.

[0357] Helper T cells (TH cells) assist other leukocytes in immunological processes, including the maturation of B cells into plasma cells and memory B cells, as well as the activation of cytotoxic T cells and macrophages. TH cells express CD4 on their surface (i.e., they are CD4+ T cells). TH cells are activated when they are presented with peptide antigens by MHC class II molecules on the surface of antigen-presenting cells (APCs). These cells can differentiate into one of several subtypes, including TH1, TH2, TH3, TH17, Th9, or TFH, each secreting different cytokines to promote different types of immune responses.

[0358] The present invention further provides an immune cell (e.g., T cell) composition comprising a population of immune cells (e.g., T cells) according to the present invention, as described herein.

[0359] The immune cell (e.g., T cell) composition may be a pharmaceutical composition comprising a plurality of immune cells (e.g., T cells) as defined herein. This pharmaceutical composition may further contain pharmaceutically acceptable carriers, diluents, or additives. The pharmaceutical composition may optionally contain one or more further pharmaceutically active polypeptides and / or compounds. Such formulations may be in a form suitable for, for example, intravenous infusion.

[0360] Preferably, the group or composition comprises at least about 10 × 10 6 The composition comprises 1 reactive immune cell, or at least about 0.2% to 5%, 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 70%, or 70% to 100% reactive immune cells. The immune cell composition may be a T cell composition, particularly an antigen-specific T cell composition. Preferably, the composition is a TIL composition.

[0361] The present invention also provides an immune cell population or composition for use in the treatment or prevention of a target cancer. Preferably, the cancer is bladder cancer, gastric cancer, esophageal cancer, breast cancer, colorectal cancer, cervical cancer, ovarian cancer, endometrial cancer, kidney cancer (renal cell carcinoma), lung cancer (small cell lung cancer, non-small cell lung cancer, and mesothelioma), brain tumors (e.g., glioma, astrocytoma, glioblastoma), melanoma, lymphoma, small intestine cancer (duodenal cancer, and jejunal cancer), leukemia, pancreatic cancer, hepatobiliary tract tumors, germ cell tumors, prostate cancer, head and neck cancer, thyroid cancer, or sarcoma, and more preferably, the target is human.

[0362] The present invention also provides a method for treating or preventing a target cancer, the method comprising administering a therapeutically effective dose of the immune cell population or composition of the present invention to a subject in need thereof. Preferably, the cancer is bladder cancer, gastric cancer, esophageal cancer, breast cancer, colorectal cancer, cervical cancer, ovarian cancer, endometrial cancer, kidney cancer (renal cell carcinoma), lung cancer (small cell carcinoma, non-small cell carcinoma, and mesothelioma), brain tumors (e.g., glioma, astrocytoma, glioblastoma), melanoma, lymphoma, small intestine cancer (duodenal cancer, and jejunal cancer), leukemia, pancreatic cancer, hepatobiliary tract tumors, germ cell tumors, prostate cancer, head and neck cancer, thyroid cancer, or sarcoma, and more preferably, the subject is human.

[0363] The present invention further provides the use of an immune cell population or composition of the present invention in the manufacture of a pharmaceutical product for the prevention or treatment of cancer. Preferably, the cancer is bladder cancer, gastric cancer, esophageal cancer, breast cancer, colorectal cancer, cervical cancer, ovarian cancer, uterine cancer, kidney cancer (renal cell carcinoma), lung cancer (small cell lung cancer, non-small cell lung cancer, and mesothelioma), brain tumors (e.g., glioma, astrocytoma, glioblastoma), malignant melanoma, lymphoma, small intestine cancer (duodenal cancer, and jejunal cancer), leukemia, pancreatic cancer, hepatobiliary tract tumors, germ cell tumors, prostate cancer, head and neck cancer, thyroid cancer, or sarcoma, and more preferably, the subject is human.

[0364] The present invention also provides a method for producing a cell therapy product, which comprises carrying out a method for culturing immune cells and / or generating an immune cell population as described herein, and then formulating the immune cells and / or immune cell population together with a pharmaceutically acceptable carrier to produce a cell therapy product. The method may optionally include one or more steps of isolating and / or purifying the immune cells and / or immune cell population before formulation. Suitable methods for isolating and / or purifying immune cells / immune cell populations, and suitable carriers for formulating immune cells for the purpose of producing a cell therapy product, are known in the art. [Examples]

[0365] Examples The present invention will be described with reference to the following examples. These examples are not intended to limit the scope of the invention, and those skilled in the art will understand that suitable equivalents can be used within the scope of the invention. Accordingly, the examples can be considered as part of the present invention, and the individual embodiments described therein can be considered as disclosed independently or in any combination.

[0366] Example 1 - Dynamic suspension co-culture produced at least the same level of reactive CD8 as that produced by static co-culture. + Generate cell yield To investigate whether dynamic suspension co-culture can support the proliferation of targeted immune cell types, we used dynamic suspension co-culture in a 100 ml BioBlu Single Use DASbox 0.3C system to study reactive CD4 + / CD8 + We designed an experiment to generate T cells and compared it to a conventional static co-culture system, namely the G-Rex system (a standard existing system).

[0367] To enable a comparison between DASbox conditions and the G-Rex manufacturing process, we established the G-Rex10M MFG control (GMF), a representative example of standard static co-culture manufacturing. The feeding regime in G-Rex10M was a batch-fed approach every 2-3 days. Due to the capacity limitations of the DASbox, a new feeding regime with a different capacity than the standard feeding regime (batch-fed) was applied. Therefore, we used a second G-Rex10M, which implemented a scaled-down model (GSD) of the DASbox feeding regime, along with the MFG G-Rex10M, to enable a more direct comparison between dynamic DASbox conditions and static co-culture.

[0368] Immature dendritic cells (iDCs) and TIL-like cells derived from healthy donors (HD) were generated from leukocyte apheresis from healthy donors. iDCs were derived from isolated monocytes (CD14) from healthy donors. + TIL-like cells were differentiated from ). TIL-like cells were created by initially co-culturing CD14- cells with iDCs for 9-10 days, followed by a further 6-7 days of activation and proliferation, to mimic the phenotype of patient TIL cells. In this experiment, on day -1 of co-culture, cryopreserved aliquots of HDiDCs and TIL-like cells were thawed in parallel and incubated at 37°C for 20-22 hours. After differentiating iDCs into mature dendritic cells (mDCs) using a cytokine cocktail, they were pulsed with commercially available peptides on day 0 to form antigen-presenting cells (APCs). Finally, TIL-like cells were co-cultured with the newly formed APCs and grown for 16 days. The TIL-like cells and iDCs used in this experiment were 1 × 10 in a 10:1 ratio (TIL-like cells:DC ratio). 6 Cells were seeded at a concentration of cells / mL.

[0369] On day 0, the MFG and GSD G-Rex10M were 10x10 6 From the cells, G-Rex100M and DASbox were 10x10 7 We started with cells. On day 9, the MFG control was 20 × 10 6 In contrast to the cell seeding, the GSD control group had its seeding density increased again to 10 × 10 6To maintain cells / mL, use lower starting seeding (20 × 10 6 Not cells, but 10x10 6 Cells were seeded. On day 9, all DASbox containers were 100 × 10 6 The cells were reseeded.

[0370] TIL-like models derived from healthy donors Peripheral blood T cells derived from healthy donors were cultured in the presence of dendritic cells (DCs) and selected antigens, and then subjected to one round of polyclonal proliferation. Cells obtained after polyclonal proliferation were selected as "TIL-like" cells based on their proliferative capacity, pluripotency, and expression of inhibitory receptors (e.g., PD-1, TIM-3, TIGIT, etc.). The cells were restimulated with antigens, and cytokine profiles were evaluated by flow cytometry.

[0371] As shown in Figure 1, dynamic suspension co-culture was performed under various stirring conditions.

[0372] At the end of this procedure, the number of CD8+ T cells was quantified, and the overall proliferation rate of CD8+ reactive T cells was calculated as follows:

number

[0373] Next, these values ​​were compared to the GMF / GSD control. From a commercial standpoint, dynamic co-culture / DASbox conditions yielded reactivity CD4 equivalent to or better than that of the GMF and / or GSD control. + / CD8 + Supporting cell proliferation is desirable. This is expected to enable the production of commercially viable cell numbers while providing other advantages associated with dynamic suspension culture (e.g., continuous monitoring, real-time parameter adjustment, cost reduction, etc.).

[0374] As shown in Figure 2, on either day 9 or day 16, the reactivity CD8 between different DASbox conditions was compared to the GMF or GSD control group. + There was no significant decrease in the number of T cells.

[0375] As shown in Figure 3, on day 9, CD8 + The total number of T cells increased significantly under continuous (high) agitation conditions and two intermittent (high) agitation conditions. A slight decrease was observed between two low-intermittent agitation conditions and between GSD and GMF controls.

[0376] On the 16th day, CD8 + The total number of T cells was compared with the CD8 generated in the GSD control. + The total number of T cells was comparable to that of the GMF control, with only a slight (but not statistically significant) decrease compared to the GMF control (also shown in Figure 3). Since cells were reseeded on day 9 in all cases, the total number of cells decreased on day 16 compared to day 9 under various DASbox conditions and controls.

[0377] Reactive CD8 under different DASbox conditions and GMF and GSD controls + The number of T cells was quantified. As shown in Figure 4, on day 9, reactive CD8 was observed under continuous (high) stirring conditions and two intermittent (high) stirring conditions. + T cell counts were significantly increased. A slight decrease was observed between the two low-intermittent stirring conditions and between the GSD and GMF control groups.

[0378] On day 16, reactive CD8 + The total number of T cells is measured by reactive CD8 generated under GSD or GFM control. + No statistically significant difference was observed in the number of T cells (as shown in Figure 4). Since cells were reseeded on day 9 in all cases, the total number of cells under various DASbox conditions and controls decreased on day 16 compared to day 9.

[0379] Finally, regarding reactive CD8 under various DASbox conditions and GMF and GSD controls, +The entire T cell population was quantified, as shown in Figure 5. + The overall T cell population was clearly increased under continuous (high) agitation conditions compared to both GSD and GMF controls. Reactive CD8 under two intermittent (high) agitation conditions. + The total number of T cells was comparable to that of the GMF and GSD controls. A slight decrease was observed between the two low-intermittent stirring conditions and the GMF and GSD controls.

[0380] Therefore, these results indicate that under DASbox conditions with intermittent stirring using high RPM, the reactivity of CD8 is at least equivalent to that obtained using the current gold standard (GMF) method and GSD controls. + This demonstrates that the entire T cell population can be obtained. Furthermore, under DASbox conditions with continuous agitation at high RPM, reactive CD8 cells were obtained compared to both the current gold standard (GMF) method and the GSD control. + The overall T cell population increased significantly.

[0381] Example 2 - Dynamic suspension co-culture yielded at least the same level of reactive CD4 as that produced by static co-culture. + Yield is generated The same co-culture reaction verified in Example 1 was used with reactive CD4 + Further analysis was conducted regarding cell generation.

[0382] Specifically, CD4 + The number of T cells was quantified, and reactive CD4 + The overall proliferation of T cells was calculated as follows:

number

[0383] Next, these values ​​were compared to the GMF / GSD control. From a commercial standpoint, it is desirable that dynamic co-culture / DASbox conditions support reactive CD4+ / CD8+ cell proliferation equivalent to or better than that of the GMF and / or GSD control. This is expected to enable the production of commercially viable cell numbers while providing other advantages associated with dynamic suspension culture (continuous monitoring, real-time parameter adjustment, cost reduction, etc.).

[0384] As shown in Figure 6, CD4 was generated under all DASbox conditions except DB3 and DB4 (intermittent stirring, low RPM). + The cells were above the required baseline responsiveness, and there was no statistically significant difference in responsiveness compared to the GMF / GSD control at either day 9 or day 16.

[0385] As shown in Figure 7, on the 9th day, CD4 + The total number of T cells was clearly increased under continuous (high) agitation conditions, and the two intermittent (high) agitation conditions were comparable to the GSD and GMF controls. A slight decrease was observed between the two low intermittent agitation conditions and the GSD and GMF control groups.

[0386] On the 16th day, CD4 + The total number of T cells was the same as the CD4 generated under GSD and GMF controls in all DASbox conditions. + The total number of T cells increased compared to the total number of T cells, with the greatest increase observed under continuous agitation conditions (also shown in Figure 7). Since cells were reseeded on day 9 in all cases, the total number of cells decreased on day 16 compared to day 9 under various DASbox conditions and controls.

[0387] Reactive CD4 under different DASbox conditions and GMF and GSD controls + The number of T cells was quantified. As shown in Figure 8, on day 9, under continuous (high) stirring conditions, reactive CD4 +T cell counts were comparable to those under GMF and GSD controls, as well as under the two intermittent (high) stirring conditions. A slight decrease was observed between the two low intermittent stirring conditions and the GSD and GMF controls.

[0388] On day 16, reactive CD4 + The total number of T cells is equal to the number of reactive CD4 cells generated under GSD or GFM control. + The number of T cells increased compared to the total number of T cells (also shown in Figure 8). Since cells were reseeded on day 9 in all cases, the total number of cells under various DASbox conditions and controls decreased on day 16 compared to day 9.

[0389] Finally, regarding reactive CD4 under various DASbox conditions and GMF and GSD controls, + The entire T cell population was quantified, as shown in Figure 9. + The overall T cell population increased under all DASbox conditions compared to both GSD and GMF controls. Furthermore, reactive CD4 under continuous (high) agitation conditions was also increased. + The overall T cell population was significantly increased compared to GMF and GSD controls.

[0390] Therefore, these results indicate that DASbox conditions, particularly continuous stirring at high RPM, yield CD4 reactivity equivalent to or greater than that obtained using the current gold standard (GMF) method and GSD controls. + It has been demonstrated that this results in the entire T cell population. Furthermore, under DASbox conditions with continuous agitation, reactive CD4 + The overall T cell population increased significantly.

[0391] Example 3 - Comparison of Dynamic Suspension Co-culture and Hybrid Controls As a further validation of the dynamic co-culture protocols verified in Examples 1 and 2, these dynamic suspension culture protocols were compared with a third set of control conditions. This third control was a "hybrid control," in which static culture was performed using G-Rex100M for the first 48 hours, followed by re-seeding of cells into DASbox containers and subsequent co-culture in dynamic suspension (Figure 10).

[0392] These additional experiments demonstrated that the results observed in Examples 1 and 2 were comparable to those of the GMF and GSD controls (data not shown). This also indicates that the dynamic co-culture protocol tested is commercially desirable for CD4 reactivity. + / CD8 + This supports the conclusion that it supports cell proliferation.

[0393] Example 4 - Preparation of patient-derived cNeT in a dynamic system Prior to initiating patient-derived co-culture in a dynamic system, patient-derived autointermediates, TILs and mDCs, and related patient-specific peptides were prepared as described below:

[0394] TIL fabrication Tumor fragments measuring 4 x 4 mm were weighed and transferred to appropriately sized sealed G-Rex containers. TexMACs medium containing 22.5 U / mL IL-21, 6000 U / mL IL-2, IL-15, 5% platelet lysate, and 1% antibiotic / antimitotic agent was added to the G-Rex containers. The containers were then incubated at 37°C for 15–17 days.

[0395] After 15-17 days, the growth containers were removed from the incubator, and the TIL / CD3+ cells were counted using flow cytometry. Subsequently, the TILs were transferred to cryopreservation medium in cryopreservation bags, frozen in a controlled-speed freezer, and then transferred to liquid nitrogen for long-term storage.

[0396] Peptide production Before initiating co-culture, patient-derived clonal neoantigens (sequenced blood and tumor-derived) were prepared using Achilles' proprietary PELEUS® platform, following the procedure below: (i) Identifying patient-specific somatic mutations (including single nucleotide polymorphisms (SNVs), multinucleotide polymorphisms (MNVs), and insertions / deletions (indels)) by comparing DNA sequence data from germ cell (blood) samples and corresponding tumor samples with each other and with a reference genome; (ii) Using a Bayesian approach (see, for example, McGranahan et al., Science Vol 135:6280, p.1463-1469; Roth et al., Nat Methods. 2014 April; 11(4):396-398) to identify a set of mutations that are likely to be clones based on patient-derived sequence data; and (iii) Design a set of peptides that include a set of somatic mutations identified as likely to be clones.

[0397] The resulting set of candidate antigenic peptides was prepared using standard peptide synthesis methods. These were pooled together with commercially available tumor-associated antigens to create a patient-specific peptide pool. The peptide pool may be a master pool of long-chain peptides (LMP) and / or a master pool of short-chain peptides.

[0398] Patient-derived co-cultures in dynamic systems Co-culture was initiated by mixing patient-derived dendritic cells and TILs pulsed in a pre-prepared patient-derived peptide pool, as described herein. Using the dynamic stirring conditions outlined in Figure 11, the cells were compared to the GSD / G-Rex control as previously described for the healthy donor model (Figures 1 and 10). As before, cells were harvested on day 9 (D9_H) and re-seeded (D9_S) without APC. In this experiment, a Sartorius AMBR® 250 container was used as an alternative bioreactor to the DASbox bioreactor used in the above examples. Therefore, the stirring conditions used for the DASbox were transferred to the AMBR® 250 using the P / V described above.

[0399] Analysis method Flow cytometry To monitor the proliferation process, flow cytometry was used to count the phenotype of T cells at specific intervals (days 0, 2, 5, 7, 9, 12, 14, and 16). Because the patient-derived dynamic co-culture experiment was performed at a different facility than previously used for healthy donor cells, and using slightly different peptides and flow cytometer configurations, in this example, the complete CD3+ T cell population, rather than CD4+ and CD8+ cells, was used as the performance indicator, as shown in Figures 12a and 12b. Since the combined CD4+ and CD8+ cells encompass the entire CD3+ cell population, these performance indicators are considered equivalent. Figures 12a and 12b show stable CD3+ cell proliferation (also known as the second expansion phase) under AMBR (dynamic) conditions, particularly for polyclonal cells. This demonstrates that the results obtained in the HD model are applicable to patient-derived materials.

[0400] Intracellular cytokine staining (ICS) Intracellular staining (ICS) assays are flow cytometry techniques used to identify cells that produce cytokines and other cytotoxic molecules in response to peptide stimulation. Cells are treated overnight with a Golgi inhibitor to induce cytokine retention within the cells. Simultaneously, the cells are stimulated with an antigen-specific peptide pool generated during co-culture. This leads to the accumulation of cytokines within the stimulated cells.

[0401] Next, the cells are fixed, permeabilized, and fluorescently labeled, and then incubated with antigen-specific antibodies alongside peptide-stimulated cells. This allows for the fluorescent detection of phenotypes (CD3+, CD4+, CD8+) that produce the target cytokines. Cells are then acquired using a flow cytometer, antigens present on the cell surface are identified, and the presence of intracellular cytokines is determined. Cells that produce the target cytokines (IFNγ and TNFα) are called "reactive" cells, and their frequency is expressed as a percentage of the total cell population.

[0402] The reactivity of CD8+ cells is shown in Figures 14a and 14b.

[0403] Figures 14a and 14b show that a higher percentage of CD8+ cells were observed under AMBR (dynamic) conditions from day 0 to 9 compared to static G-Rex / GSD (static) controls. This demonstrates that patient-derived reactive T cells can be successfully proliferated during the co-culture phase under dynamic conditions and that they function even better than G-Rex / GSD controls.

Claims

1. A method for generating a population of immune cells, the method comprising co-culturing isolated immune cells and antigen-presenting cells (APCs) in a dynamic suspension.

2. The method according to claim 1, wherein the dynamic suspension is obtained by stirring the co-culture.

3. The aforementioned stirring is as follows: (a) continuous; and / or (b) intermittent The method according to claim 2.

4. The intermittent stirring of the co-culture includes stirring for approximately 5 to 30 minutes every 1 to 5 hours, and optionally, (a) The stirring is carried out every hour for about 5 minutes to about 15 minutes, preferably about 10 minutes; or (b) every three hours, for about 5 to 15 minutes, preferably about 10 minutes, The method according to claim 3.

5. (a) The first period of the co-culture is subjected to intermittent stirring, wherein the first period is arbitrarily about 0 to about 2 days, preferably about 1 to about 2 days; and (b) Following the first co-culture period, a second co-culture period is followed by continuous stirring, where the second period is arbitrarily about 2 to about 21 days, preferably about 2 to about 10 days. The method according to any one of claims 2 to 4.

6. The aforementioned stirring is as follows: (a) Mechanical stirring; (b) Oscillating stirring; (c) Vertical wheel stirring; or (d) Pneumatic agitation The method according to any one of claims 2 to 5, wherein preferably the isolated immune cells and APCs are co-cultured under mechanical stirring, and more preferably the co-culture is carried out in a stirred-tank type bioreactor.

7. The aforementioned stirring is as follows: (a) RPM of 20 RPM to about 80 RPM, preferably about 20 RPM to about 75 RPM, more preferably about 25 RPM to about 70 RPM; or (b) RPM of about 70 RPM to about 150 RPM, preferably about 70 RPM to about 100 RPM The method according to any one of claims 2 to 6, wherein mechanical stirring is performed.

8. The method according to any one of claims 1 to 7, wherein the immune cells are T cells, and optionally tumor-infiltrating lymphocytes (TILs).

9. The T cells are: (a) CD8 ＋ T cells; (b) CD4 ＋ T cells, optionally Th1, Th2, Th17, Tfh, and / or Th9 cells; (c) NKT cells, optionally non-mutant NKT cells; and / or (d) Regulatory T (Treg) cells, The method according to claim 8.

10. The aforementioned APC is as follows: (a) Dendritic cells (DCs); (b) B cells; and / or (c) Macrophages, The method according to any one of claims 1 to 9.

11. The method according to any one of claims 1 to 10, wherein the antigen is a tumor antigen, preferably a neoantigen, and more preferably a clonal neoantigen.

12. The method according to any one of claims 1 to 11, wherein the immune cells are T cells, preferably tumor-infiltrating lymphocytes (TILs), and the APCs are dendritic cells (DCs).

13. Using a feedback mechanism, the following parameters: (a) pH; (b) Dissolved oxygen (DO); (c) temperature; (d) O ２ , N ２ CO ２ and / or a gas mixture consisting of compressed air; and / or (e) Nutrient and / or metabolite concentrations Monitor one or more of these and maintain them at the desired settings. The method according to any one of claims 1 to 12, wherein, optionally, one or more of the parameters are monitored and maintained using a control loop mechanism.

14. During the aforementioned co-culture, fresh culture medium was added. (a) at least once, twice, three times, four times, five times, six times or more during the period of the method; and / or (b) Every 2 to 3 days during the period of the above method The method according to any one of claims 1 to 13, which is introduced.

15. The number of immune cells and / or APCs is as follows: (a) at least once, twice, three times, four times, five times, six times or more during the period of the method; and / or (b) Every 2 to 3 days during the period of the above method Measured, The method according to any one of claims 1 to 14, wherein optionally, the number of immune cells and / or APCs is measured using flow cytometry.

16. At the start of the aforementioned co-culture, the following: (a) Approximately 1 x 10 ６ At a concentration of immune cells / mL; and / or (b) With an immune cell:APC ratio of approximately 10:1, The method according to any one of claims 1 to 15, wherein the immune cells are seeded.

17. The process further includes a step of nonspecifically growing the immune cells before co-culturing with the APC, wherein optionally, (a) The immune cells are T cells; and / or (b) The method according to any one of claims 1 to 16, wherein the step of nonspecifically growing the immune cells comprises culturing the immune cells together with one or more of the following: IL-2, IL-21, IL-15, anti-CD3 antibody, anti-CD28 antibody, and anti-CD2 antibody, preferably all of IL-2, IL-21, IL-15, anti-CD3 antibody, anti-CD28 antibody, and anti-CD2 antibody.

18. The co-cultured cells are harvested after a total co-culture period of approximately 2 to 21 days, preferably approximately 2 to 10 days, and the immune cells are used to re-seed the culture vessel, in which case optionally: The aforementioned immune cells, in the absence of APC, are approximately 1 × 10⁶ ６ The method according to any one of claims 1 to 17, wherein the immune cells are reseeded at a density of immune cells / mL.

19. After co-culturing, the immune cells are cultured for a certain period in the absence of APC, in which case optionally: (a) During the further period, the immune cells are cultured in a dynamic suspension; and / or (b) The further period is optionally about 2 to 21 days, preferably about 2 to 10 days. The method according to any one of claims 1 to 18.

20. The method according to claim 19, wherein the dynamic suspension is obtained by stirring the immune cells.

21. The aforementioned stirring is as follows: (a) continuous; and / or (b) intermittent The method according to claim 20.

22. The intermittent stirring of the co-culture includes stirring for approximately 5 to 30 minutes every 1 to 5 hours, and optionally, (a) The stirring is carried out every hour for about 5 minutes to about 15 minutes, preferably about 10 minutes; or (b) every three hours, for about 5 to 15 minutes, preferably about 10 minutes, The method according to claim 21.

23. The aforementioned stirring is as follows: (a) Mechanical stirring; (b) Oscillating stirring; (c) Vertical wheel stirring; or (d) Pneumatic agitation The method according to any one of claims 20 to 22, wherein preferably the isolated immune cells and APCs are co-cultured under mechanical stirring, and more preferably the co-culture is carried out in a stirred-tank type bioreactor.

24. The agitation of the aforementioned immune cells is as follows: (a) RPM of 20 RPM to about 80 RPM, preferably about 20 RPM to about 75 RPM, more preferably about 25 RPM to about 70 RPM; or (b) RPM of about 70 RPM to about 200 RPM, preferably about 70 RPM to about 150 RPM or about 70 RPM to about 100 RPM The method according to any one of claims 20 to 23, wherein mechanical stirring is performed.

25. The aforementioned method is as follows: (a)(i) Co-culture the isolated immune cells and APCs for a first period of approximately 24 hours, with intermittent stirring at approximately 25 RPM for approximately 10 minutes, followed by approximately 1 hour without stirring; (ii) Co-culture the isolated immune cells and APCs for a second period of approximately 24 hours, with intermittent stirring at approximately 50 RPM for approximately 10 minutes, followed by approximately 1 hour without stirring; (iii) Co-culture the isolated immune cells and APCs for a third period of about one week with continuous stirring at approximately 70 RPM; at will (iv) The immune cells are preferably about 1 × 10 ６ Reseeding to a cell / mL density; and (v) Culturing the isolated immune cells for a fourth period of about one week in the absence of APC, with continuous stirring at about 100 RPM; or (b) (i) Co-culture the isolated immune cells and APCs for a first period of about 24 hours, with intermittent stirring at about 25 RPM for about 10 minutes, followed by about 3 hours without stirring; (ii) Co-culture the isolated immune cells and APCs for a second period of approximately 24 hours, with intermittent stirring at approximately 50 RPM for approximately 10 minutes, followed by approximately 3 hours without stirring; (iii) Co-culture isolated immune cells and APCs for a third period of approximately one week with continuous stirring at approximately 70 RPM; at will (iv) resuspending said immune cells, preferably at about 1×10 ６ cells / mL to reseed at this density; and (v) Culturing the isolated immune cells for a fourth period of about one week in the absence of APC, with continuous stirring at about 100 RPM; or (c) (i) Co-culture the isolated immune cells and APCs for a first period of approximately 48 hours, with intermittent stirring at approximately 100 RPM for approximately 10 minutes, followed by approximately 1 hour without stirring; (ii) Co-culture the isolated immune cells and APCs for a second period of about one week by continuous stirring at about 100 RPM; at will (iii) The immune cells are preferably about 1 × 10 ６ Reseeding to a cell / mL density; and (iv) Culturing the isolated immune cells for a third period of about one week in the absence of APC, with continuous stirring at about 100 RPM; or (d) (i) Co-culture the isolated immune cells and APCs for a first period of approximately 48 hours, with intermittent stirring at approximately 100 RPM for approximately 10 minutes, followed by approximately 3 hours without stirring; (ii) Co-culture the isolated immune cells and APCs for a second period of about one week with continuous stirring at about 100 RPM; at will (iii) The immune cells are preferably about 1 × 10 ６ Reseeding to a cell / mL density; and (iv) Culturing the isolated immune cells for a third period of about one week in the absence of APC, with continuous stirring at about 100 RPM; or (e) (i) Co-culture the isolated immune cells and APCs for a first period of about nine days with continuous stirring at about 100 RPM; at will (ii) The immune cells are preferably about 1 × 10 ６ Reseeding to a cell / mL density; and (iii) For a second period of about one week, the isolated immune cells are cultured in the absence of APC and with continuous stirring at approximately 100 RPM. including, The method according to any one of claims 1 to 24.

26. The method according to any one of claims 1 to 25, wherein a stirred-tank type bioreactor is used and optionally a marine impeller is used in combination.

27. The method according to any one of claims 1 to 26, wherein the yield of the immune cells is at least equivalent to that produced by the corresponding method of co-culturing the isolated immune cells with APC without agitation.

28. A method for manufacturing cell therapy products, the following: (a) generating a population of immune cells according to the method described in any one of claims 1 to 27; (b) optionally, isolating and / or purifying the immune cell population; and (c) Formulating the immune cell population together with a pharmaceutically acceptable carrier. Methods that include...

29. Preferably, the group, composition, or product is at least about 10 × 10 ６ A population of immune cells obtained or obtainable by the method of any one of claims 1 to 27, comprising at least 0.2% to 5%, 5% to 10%, 10% to 20%, 20% to 30%, 30% to 40%, 40% to 50%, 50% to 70%, or 70% to 100% reactive immune cells, an immune cell composition comprising the immune cell population, or a cell therapy product obtained or obtainable by the method of claim 28.

30. The immune cell population, immune cell composition, or cell therapy product according to claim 29, wherein the immune cells are antigen-specific T cells.

31. An immune cell population or composition according to claim 29 or 30, used for the treatment or prevention of cancer in a subject, wherein the cancer is preferably bladder cancer, gastric cancer, esophageal cancer, breast cancer, colorectal cancer, cervical cancer, ovarian cancer, endometrial cancer, kidney cancer (renal cell carcinoma), lung cancer (small cell lung cancer, non-small cell lung cancer and mesothelioma), brain tumor (e.g., glioma, astrocytoma, glioblastoma), melanoma, lymphoma, small intestine cancer (duodenal and jejunal cancer), leukemia, pancreatic cancer, hepatobiliary tract tumor, germ cell tumor, prostate cancer, head and neck cancer, thyroid cancer or sarcoma, and more preferably the subject is human.

32. A system for generating a population of immune cells, wherein the system is as follows: (a) Suspension culture bioreactor; (b) Isolated immune cells; (c) APC; (d) Culture medium; and (e) at least one sensor Including; Here, the isolated immune cells are co-cultured with APC in a dynamic suspension; Optionally, at least one of the sensors has the following parameters: (i) pH; (ii) Dissolved oxygen (DO); (iii) temperature; (iv) O ２ , N ２ CO ２ and / or gas mixtures of compressed air; and / or (v) Concentration of nutrients and / or metabolites A system that can monitor one or more of these.

33. The dynamic suspension is generated from stirring the co-culture of the isolated immune cells and APC, and optionally, (a) The stirring described above is as follows: (i) Mechanical stirring; (ii) Oscillating stirring; (iii) Vertical wheel stirring; or (iv) Pneumatic agitation Selected from; Preferably, the isolated immune cells and APCs are co-cultured under mechanical agitation, and more preferably, the agitated bioreactor is a agitated tank type bioreactor; and / or (b) The system according to claim 32, wherein at least one sensor is incorporated into a proportional, integral, and differential controller that enables monitoring and maintenance of one or more parameters using a control loop mechanism.

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