Composition of selected tumor-infiltrating lymphocytes and related methods for their production and use.

Abstract

Various embodiments of the present invention provide compositions of tumor-infiltrating lymphocytes (TILs) enriched with tumor-reactive cells. The embodiments also provide methods for producing tumor-reactive TILs enriched with tumor-reactive cells, and the use of the provided enriched tumor-reactive TILs for treating cancer in humans or other subjects. According to the embodiments, a tumor-reactive T cell enriched pharmaceutical T lymphocyte infiltration (TIL) composition comprises an oligoclonal population of tumor-infiltrating T cells, including tumor-derived CD4+ and CD8+ T cells, with up to 40 clones constituting 40% of the TCR frequency in the population. Embodiments of the present invention are particularly useful for treating tumors that are resistant or refractory to conventional chemotherapy, or that have become resistant or refractory.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application 63 / 489,999, filed on 13 March 2023, entitled "COMPOSITION OF SELECTED TUMOR INFILTRATING LYMPHOCYTES AND RELATED METHODS OF PRODUCING AND USING THE SAME," the contents of which are incorporated in their entirety by reference.

[0002] Embodiments of the present invention relate to compositions of tumor-infiltrating lymphocytes (TILs) enriched with tumor-reactive cells. Embodiments of the present invention also relate to methods for producing tumor-reactive TILs enriched with tumor-reactive cells, and the use of the provided enriched tumor-reactive TILs for treating cancer in a subject. [Background technology]

[0003] Clinical trials have demonstrated that T cells isolated from surgically excised tumors possess T cell receptors (TCRs) that recognize tumor cells, and that proliferating these reactive tumor-infiltrating lymphocyte (TIL) populations and reinjecting them into patients can, in some cases, yield dramatic clinical benefits. However, the main obstacle to the application of such cells in cell therapy is the difficulty in obtaining such cells and compositions with desirable characteristics. For example, existing methods for generating TIL therapies for use in cancer are lengthy, and the compositions may contain a small number of reactive cells that are not suitable for commercial use. Therefore, there is a need for improved TIL compositions and methods for obtaining and producing cell compositions containing tumor-reactive T cells for therapeutic use. Embodiments that meet such needs are provided herein. [Overview of the project]

[0004] In some embodiments, this specification provides a pharmaceutical T lymphocyte infiltration (TIL) composition enriched with tumor-reactive T cells, the composition comprising a population of tumor-infiltrating T cells including tumor-derived CD4+ and CD8+ T cells, wherein at least 90% of the cells in the composition are CD3+ T cells and less than about 5% of the population are regulatory T cells.

[0005] In some embodiments, this specification provides a pharmaceutical T lymphocyte infiltration (TIL) composition enriched with tumor-reactive T cells, the pharmaceutical composition comprising an oligoclonal population of tumor-infiltrating T cells, including tumor-derived CD4+ and CD8+ T cells, with up to 40 clones constituting 40% of the TCR frequency in the population. In any of the several embodiments provided, less than about 5% of the population are regulatory T cells. In any of the several embodiments provided, less than about 3% of the population are regulatory T cells. In any of the several embodiments provided, less than about 1% of the population are regulatory T cells. In any of the several embodiments provided, the regulatory T cell phenotype is characterized by the expression of the surface markers CD4+CD8-CD25+Foxp3+CD127low.

[0006] In any of the several provided embodiments, the T cells in the population express PD-1 and / or CD39. In any of the several provided embodiments, the proportion of cells expressing the surface marker PD-1 in the composition, or the proportion of viable cells thereof, is at least about 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and at least 100% of the cells in the composition. In any of the several provided embodiments, the proportion of cells expressing the surface marker CD39 in the composition, or the proportion of viable cells thereof, is at least about 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and at least 100% of the cells in the composition. In any of the several provided embodiments, the proportion of cells expressing the surface markers PD-1 and CD39 in the composition, or the proportion of viable cells thereof, is at least about 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and at least 100% of the cells in the composition. In any of the several provided embodiments, at least 90% of the cells in the composition are CD3+ T cells. In any of the several provided embodiments, at least 95% of the cells in the population are CD3+ T cells. In any of the several provided embodiments, at least 98% of the cells in the population are CD3+ T cells. In any of the several provided embodiments, at least 80% of the cells in the composition are CD3+CD56- T cells.

[0007] In some provided embodiments, more than 60% of the cells of the population are effector memory T cells. In some provided embodiments, more than 75% of the cells of the population are effector memory T cells. In some provided embodiments, more than 80% of the cells of the population are effector memory T cells. In some provided embodiments, more than 85% of the cells of the population are effector memory T cells. In some provided embodiments, more than 90% of the cells of the population are effector memory T cells. In some provided embodiments, the effector memory phenotype is CD45RA - , CD45RO+, CD62L - , CCR7−, CD28−, and / or CD27− surface marker expression. In some provided embodiments, the effector memory phenotype is CD45RA - , CD45RO+, CD62L - , and CCR7 - surface marker expression. In some provided embodiments, the effector memory phenotype is CD45RA - , CD45RO + , CD62L, CCR7 - , CD28 - , and CD27 - surface marker expression. In some provided embodiments, the effector memory phenotype is CD45RA - CCR7− - surface marker expression.

[0008] In some of the provided embodiments, up to 40 TCR chronotypes constitute at least 50% of the TCR frequency in the population. In some embodiments, the top 40 chronotypes constitute at least 50% of the TCR frequency in the population. In some of the provided embodiments, up to 40 TCR chronotypes constitute at least 60% of the TCR frequency in the population. In some embodiments, the top 40 chronotypes constitute at least 60% of the TCR frequency in the population. In some of the provided embodiments, the TCR chronotypes are reactive to at least one CD8 antigen and at least one CD4 antigen. In some of the provided embodiments, at least 20% of the CD8+ T cells and / or at least 20% of the CD4+ T cells in the composition are reactive to the nascent antigen.

[0009] In any of the several embodiments provided, the TIL composition is characterized by at least one of the following criteria in an in vitro autologous tumor assay: i) production of IFN-γ greater than 2000 pg / mL, ii) production of granzyme B in the supernatant greater than 200 pg / mL, and iii) killing of more than 10% of tumor cells. In some embodiments, the foregoing provides a pharmaceutical T lymphocyte infiltration (TIL) composition enriched with tumor-reactive T cells, wherein the pharmaceutical composition comprises tumor-infiltrating lymphocytes including tumor-derived CD4+ and CD8+ T cells, and at least about 90% of the cells in the composition are CD3+ T cells, and the TIL composition is characterized by at least one of the following criteria in an in vitro autologous tumor assay: i) production of IFN-γ greater than 2000 pg / mL, ii) production of granzyme B greater than 500 pg / mL, and iii) killing of more than 15% of autologous tumor cells. In any of the several embodiments provided, the TIL composition is characterized by criteria (i) and (ii). In any of the provided embodiments, the TIL composition is characterized by criteria (i) and (iii). In any of the provided embodiments, the TIL composition is characterized by criteria (ii) and (iii). In any of the provided embodiments, the TIL composition is characterized by criteria (i), (ii), and (iii). In any of the provided embodiments, the production of IFN-γ is greater than 3000 pg / mL or greater than 4000 pg / mL. In any of the provided embodiments, the production of granzyme B is greater than 400 pg / mL or greater than 500 pg / mL. In any of the provided embodiments, the killing of autologous tumor cells is greater than 40%. In any of the provided embodiments, the composition is characterized by having more CD4+ T cells than CD8+ T cells. In any of the provided embodiments, the ratio of CD4+ T cells to CD8+ T cells in the composition is 5:1 to 1:5.In some of the provided embodiments, the ratio of CD4+ T cells to CD8+ T cells in the composition is 5:1 to 50:1, 5:1 to 25:1, 5:1 to 20:1, 5:1 to 15:1, 5:1 to 10:1, 10:1 to 50:1, 10:1 to 25:1, 10:1 to 20:1, 10:1 to 15:1, 15:1 to 50:1, 15:1 to 25:1, 15:1 to 20:1, 20:1 to 50:1, 20:1 to 25:1, or 25:1 to 50:1. In some of the provided embodiments, the ratio of CD4+ T cells to CD8+ T cells in the composition is 10:1 to 25:1 or about 10:1 to 25:1. In some embodiments, the ratio of CD4+ to CD8+ T cells is 20:1 or about 20:1. In any of the provided embodiments, the number of cells in the composition is a therapeutically effective amount of TIL. In any of the provided embodiments, the number of cells in the composition, or the number of viable cells thereof, is at least 2 × 10. 7 These are cells. In any of the provided embodiments, the number of cells in the composition, or the number of viable cells, is 2 × 10 7 Or approximately 2 x 10 7 cells ~20×10 9 cells, 2 x 10 7 cells ~10×10 9 cells, 2 x 10 7 cells ~2×10 9 cells, 2 x 10 7 cells ~2×10 8 cells, 2 x 10 8 cells ~20×10 9 cells, 2 x 10 8 cells ~10×10 9 cells, 2 x 10 8 cells ~2×10 9 cells, 2 x 10 9 cells ~20×10 9 cells, 2 x 10 9 cells ~10×10 9 Cells, or 10 × 10 9 cells ~20×10 9 It is a cell (including both ends).

[0010] In some of the provided embodiments, the pharmaceutical composition is for the treatment of a patient's tumor. In some of the provided embodiments, the tumor is a colorectal cancer (CRC) tumor, a melanoma tumor, a non-small cell lung cancer (NSCLC) tumor, or an ovarian cancer tumor. In some of the provided embodiments, the tumor is derived from a human subject. In some of the provided embodiments, the pharmaceutical composition is for autologous adoption therapy for a human subject. In some of the provided embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable excipient. In some of the provided embodiments, the pharmaceutical TIL composition comprises a pharmaceutically acceptable excipient and comprises a therapeutically effective amount of tumor-reactive T cells, such as tumor-reactive T cells positive for one or more markers described herein (e.g., PD-1 / CD39), which is sufficient to treat, improve, or otherwise beneficially alter the symptoms of a pathological condition, disorder, or disease, or other indication. In some of the provided embodiments, the pharmaceutical composition comprises a cryoprotective substance. In some of the provided embodiments, the composition is a liquid composition. In any of the several provided embodiments, the TIL composition provided herein is liquid, and the concentration or amount of tumor-reactive T cells in the composition is a therapeutically effective amount sufficient to treat, improve, or otherwise beneficially alter the symptoms of a pathological condition, disorder, or disease or other indication. In any of the several provided embodiments, the composition is frozen and thawed. In any of the several provided embodiments, the volume of the composition is 1 mL to 500 mL. In any of the several provided embodiments, the composition is frozen. In any of the several provided embodiments, the TIL composition containing a therapeutically effective dose or amount of tumor-reactive T cells is frozen.

[0011] In some of the provided embodiments, the composition is prepared by selecting cells surface-positive for PD-1 and CD39 from cells obtained from a donor tumor and growing the cells ex vivo. In some of the provided embodiments, the composition is prepared by a method comprising: a. providing dissociated tumor cells from a tumor obtained from a donor subject, wherein the dissociated tumor cells are a first population of cells comprising CD4+ and CD8+ T cells; b. selecting cells surface-positive for CD45, PD1, and CD39, as well as T cell markers, from the first population of cells to produce a population of selected T lymphocyte-infiltrating cells (TILs); and c. growing the population of selected TILs by culturing the lymphocytes with one or more T cell stimulants under conditions that produce a population of proliferated T cells. In some embodiments, provided herein is a method for producing tumor-reactive T-cell enriched T lymphocyte-infiltrating cells (TILs), the method comprising: a. providing dissociated tumor cells from a tumor obtained from a donor subject, the dissociated tumor cells being a first population of cells comprising CD4+ and CD8+ T cells; b. selecting cells surface-positive for CD45, PD1, and CD39, as well as T cell markers, from the first population of cells to produce a population of selected T lymphocyte-infiltrating cells (TILs); and c. growing the population of selected TILs by culturing lymphocytes with one or more T cell stimulants under conditions that produce a population of proliferated T cells. In some embodiments of the above, the T cell marker is CD4 or CD8.

[0012] In some of the provided embodiments, the dissociated tumor cells are a single-cell suspension obtained by homogenization and enzymatic digestion of one or more tumor fragments derived from an excised tumor. In some of the provided embodiments, one or more T-cell stimulants are selected from one or more allogeneic feeder cells, anti-CD3 antibodies, and recombinant IL-2. In some of the provided embodiments, enzymatic digestion is by incubation with collagenase and hyaluronidase. In some embodiments, the concentration of collagenase is about 10 mg / ml. In some embodiments, the concentration of hyaluronidase is about 10 mg / ml. In some embodiments, the concentration of the first cell population is 5 × 10⁻⁶. 6 cells / mL~50×10 6 The concentration is cells / mL. In some embodiments, the concentration of the first cell population is 20 × 10⁻⁶. 6 The cell volume is cells / mL. In any of the several provided embodiments, cell selection is performed using a microfluidic chip-based cell sorting system including at least four fluorescence detectors. In any of the several provided embodiments, cell selection includes sorting cells positive to at least four fluorescence signals based on a fluorescence minus 1 (FMO) cocktail. In any of the several provided embodiments, sorting is performed at a rate of 5,000 to 10,000 events per second, and optionally at a rate of approximately 6,000 events per second.

[0013] In some embodiments, provided herein is a method for treating a subject having cancer, the method comprising administering to a subject having a tumor any composition provided herein in a therapeutic dose. In any of the provided embodiments, the therapeutically effective dose is about 1 × 10⁻⁶ 9 ~10×10 9These are T cells. In any of the several provided embodiments, the therapeutically effective dose is more than 1 million but less than 100 million T cells per kilogram of body weight. In any of the several provided embodiments, the therapeutically effective dose is more than 1 million but less than 10 million T cells per kilogram of body weight. In any of the several provided embodiments, the therapeutically effective dose is 10 million or about 10 million to 50 million or about 50 million T cells per kilogram of body weight. In any of the several provided embodiments, the cells of the therapeutic composition are autologous to the subject. [Brief explanation of the drawing]

[0014] [Figure 1] This is a schematic diagram of the tumor-infiltrating lymphocyte (TIL) production process using direct selection of TILS co-expressing PD1+CD39+ surface markers. [Figure 2A] The percentages of TIL co-expression PD1+ and CD39+ in fresh or frozen single-cell suspensions derived from different cancer indications are illustrated. Each dot represents a patient sample. [Figure 2B] The graph illustrates the proliferation of PD1+CD39+ selected TILs after a 14-day REP protocol (irradiated PBMCs with an iPBMC:TIL ratio of 200:1, in the presence of 30 ng / ml OKT3 and 3000 IU / ml IL-2). Proliferation was calculated as the increase ratio between the cell count after sampling (day 0) and the cell count collected at the end of the 14-day REP. Each sub-dot represents data from the patient's tumor. [Figure 3] The left image shows the proportion of CD3+ T cells within the CD3+ T cell population in PD1+CD39+ selected TIL products (REP completion), and the right image shows the proportion of CD4+ and CD8+ T cell subsets. Each point represents a tumor from a different patient. [Figure 4A] The diagram illustrates the percentage of cells expressing CD3+CD56- (T cells), CD56+CD3- (NK cells), or CD3+CD56+ cells. Each dot represents a different selected TIL product from the patient sample. [Figure 4B]The proportions of CD4+ T cells, CD8+ T cells, and regulatory T cells (Tregs) within the CD3+ T cell population are illustrated. Each dot represents a selected TIL product derived from a patient sample. Samples from selected TIL products for different indications are represented using different shapes: lung - circle, colon - square, kidney - triangle, melanoma - star, endometrium - diamond. [Figure 5] The proportions of effector memory T cells (Tem; CD45RA-CCR7-), central memory T cells (Tcm; CD45RA-CCR7+), effector memory T cells expressing CD45RA (Temra; CD45RA+CCR7-), and naive / stem cell memory T cells (Tnaive / Scm; CD45RA-CCR7-) within the CD4 (gray bars) and CD8 T cell (white bars) populations are illustrated. Samples from selected TIL products for different indications are represented using different shapes: lung - circle, colon - square, kidney - triangle, melanoma - star, endometrium - diamond. [Figure 6] The results of single-cell RNA sequencing performed on unselected and PD1+CD39+ selected TILs at the end of proliferation for TCR chronotype are illustrated. The graph shows the diversity and abundance of TCR chronotypes in unselected and PD1+CD39+ selected TILs for each patient. Within each sample set, the chronotype of each different TCR is represented by a bar segment. A line connecting the chronotypes between the unselected and PD1+CD39+ samples indicates common TCR chronotypes among TIL products. The frequencies of the top 40 most abundant chronotypes within each sample set are displayed. [Figure 7] The production of IFNγ and granzyme B by PD1+CD39+ selected TIL products and unselected TILs after overnight stimulation with anti-CD3 / CD28 antibody (i.e., polyclonal stimulation (CD3 / CD8)) is illustrated (ns = no significant difference). [Figure 8]The left image shows the production of IFNγ and granzyme B by PD1+CD39+ selected TIL products and unselected TILs after overnight stimulation using autologous tumor cells (CD45-DTC; i.e., autologous tumor substance stimulation), and the percentage of autologous tumor cell killing induced by PD1+CD39+ selected TIL or unselected TIL (right). Cell killing data were normalized based on the viability of tumor cell cultures alone (0% killing). Small dots represent technical replication. Error bars indicate the triple standard deviation. ** = p < 0.01, standard one-way ANOVA with Tukey correction. [Figure 9] The production of IFNγ and granzyme B by PD1+CD39+ selected TIL products and unselected TILs after overnight stimulation with anti-CD3 / CD28 antibody (i.e., polyclonal stimulation (CD3 / CD8)) is illustrated (ns = no significant difference). [Figure 10] The left image shows the production of IFNγ and granzyme B by PD1+CD39+ selected TIL products and unselected TILs after overnight stimulation using autologous tumor cells (CD45-DTC; i.e., autologous tumor substance stimulation), and the percentage of autologous tumor cell killing induced by PD1+CD39+ selected TIL or unselected TIL (right). Cell killing data were normalized based on the viability of tumor cell cultures alone (0% killing). Small dots represent technical replication. Error bars indicate the triple standard deviation. ** = p < 0.01, standard one-way ANOVA with Tukey correction. [Modes for carrying out the invention]

[0015] This specification provides a method for producing T cells. Such a method includes, but is not limited to, (1) selecting cells positive for exhaustion markers, from among PD-1 and / or CD39, from a population of cells containing T lymphocytes obtained from a donor subject, and (2) stimulating the population by incubation or culture of the selected cells with one or more lymphocyte T cell stimulants to produce a population of proliferated T cells. In some embodiments, the methods for selection and / or stimulation are carried out in a closed system. In some embodiments, only a single proliferation step is performed in this method.

[0016] Embodiments of this specification provide a method or process for producing T cell preparations that may be useful in treating patients with pathological diseases or conditions. In contrast to known production methods, the method and process described herein can be completed in a significantly shorter time and can recover more T cells, thereby offering the significant advantage of bringing cells to clinical use in therapeutic doses. Similarly, populations of T cells produced by the method described herein and their pharmaceutical compositions are provided herein.

[0017] The method provided relates to the production of T-cell therapy reactive to tumor-associated antigens. Cancer cells accumulate many different DNA mutations as part of the tumorigenetic process. These mutations can cause amino acid changes in the coding regions of proteins. For mutations to be recognized by the immune system, proteins must be processed intracellularly and presented on the surface along with major histocompatibility complexes (MHC). Peptide neoepitopes (also referred herein as neoepitopes or peptide neoepitopes) are mutant peptides presented by MHC complexes that can be recognized by T cells via TCR binding. For the immune system to recognize the mutation, the mutation must be expressed on the surface of cancer cells via MHC complexes, and T cells must have TCRs that recognize the mutant peptide. These neoepitopes can be presented by MHC class I and MHC class II, and are recognized by CD8+ T cells and CD4+ T cells, respectively.

[0018] In some embodiments, the described method may be used to produce T cells expressing cell surface receptors. The cell surface receptor may be a T cell receptor (TCR) or a novel class of TCRs. In certain embodiments of the provided method, the population of T cells is or includes reactive T cells expressing cell surface receptors, such as T cell receptors (TCRs), which are capable of recognizing peptide antigens on the surface of target cells. In particular, for an antigen to be recognized by the immune system, the protein must be processed into peptide fragments within the cell and then presented on the surface together with major histocompatibility complex (MHC). A TCR has two protein chains, and these two protein chains are designed to bind to specific peptides presented by major histocompatibility complex (MHC) proteins on the surface of specific cells. Because a TCR recognizes peptides in association with MHC molecules expressed on the surface of target cells, a TCR has the potential to recognize antigens not only directly presented on the surface of target cells, such as cancer cells, but also antigens presented by antigen-presenting cells, such as those present in tumors, inflammatory and infected microenvironments, and secondary lymphoid organs. Reactive T cells expressing such cell surface receptors can be used to target and kill any target cells, including but not limited to infected cells, damaged cells, or dysfunctional cells. Accordingly, according to the embodiments described herein, manufactured T cells expressing cell surface receptors can be used to target and kill any target cells, including but not limited to infected cells, damaged cells, or dysfunctional cells. Examples of such target cells include cancer cells, virus-infected cells, bacterial-infected cells, dysfunctionally activated inflammatory cells (e.g., inflammatory endothelial cells), and cells involved in dysfunctional immune responses (e.g., cells involved in autoimmune diseases).

[0019] In some embodiments, a “T cell receptor” or “TCR” is a molecule comprising variable α and β chains (also known as TCRα and TCRβ, respectively) or variable γ and δ chains (also known as TCRγ and TCRδ), or its antigen-binding portion, which can specifically bind to peptides bound to MHC molecules. In some embodiments, the TCR is in the αβ form. Typically, TCRs existing in αβ and γδ forms are generally structurally similar, but the T cells expressing them may have distinct anatomical locations or functions. TCRs can be found on the surface of T cells (or T lymphocytes), where they are generally responsible for recognizing antigens bound to major histocompatibility complex (MHC) molecules.

[0020] In some embodiments, reactive T cells are tumor-reactive T cells that recognize neoplastic antigens. The majority of neoplastic antigens result from passenger mutations, meaning that these neoplastic antigens do not presume any proliferative advantage over cancer cells. A small number of mutations actively promote tumor growth, and these are known as driver mutations. Passenger mutations are unique to each patient and can give rise to neoplastic antigens that may be present in a subset of all cancer cells. Driver mutations can be present in all tumor cells of an individual and give rise to potentially shared neoplastic antigens. In some embodiments of the methods provided, the population of T cells includes tumor-reactive T cells capable of recognizing neoplastic antigens containing passenger and / or driver mutations.

[0021] In certain embodiments, the methods provided may be used for the ex vivo production of T cell therapies, including the ex vivo proliferation of autologous tumor-reactive T cells. In some embodiments, neogenic antigens are ideal targets for immunotherapy because they represent disease-specific targets. For example, such antigens generally do not exist in the body before cancer develops, are truly cancer-specific, are not expressed on normal cells, and are not affected by off-target immunotoxicity. Thus, a unique repertoire of patient-specific neogenic antigens can induce a potent immune response specific to cancer cells, evading normal cells. This is an advantage over other cell therapy targets that may not be disease-specific, as even low levels of target antigens on normal cells can lead to severe and fatal autoimmune toxicity in the context of engineered therapies targeting common antigens. For example, an anti-MAGE-A3-TCR program in melanoma patients was discontinued due to trial-related deaths resulting from cross-reactivity with a similar target, MAGE-A12, which is expressed at low levels in the brain. A key challenge in cancer immunotherapy is the identification of cancer targets.

[0022] Recent clinical trials have demonstrated that T cells isolated from surgically resected tumors possess TCRs that recognize newly generated antigens, and that growing these newly generated antigen-reactive TIL populations and reinjecting them into patients can, in some cases, yield dramatic clinical benefits. This personalized therapy has shown remarkable clinical responses in certain patients with common epithelial tumors.

[0023] Existing methods for obtaining and producing tumor-reactive T cells are not entirely satisfactory. For example, directly isolating tumor-reactive T cells from a target without proliferation is not feasible because it is not possible to obtain therapeutically effective amounts of such cells. As an alternative, attempts have been made to identify TCRs specific to desired nascent antigens for recombination engineering of T cells with TCRs for use in adoptive cell therapy. However, such approaches lack the diversity to recognize a broader repertoire of multiple tumor-specific mutations, as they produce only a single TCR for specific nascent antigens. Other methods involve bulk proliferation of T cells from a tumor source, which carries the risk of proliferating T cells that may contain a large number of bystander cells that are unresponsive to and / or exhibit inhibitory activity against tumor antigens. For example, tumor regulatory T cells (Tregs) are CD4 + Tumor-reactive T cells are a subpopulation of T cells that are specialized in suppressing the immune response and may limit the reactivity of T cell products. These further approaches, which have attempted to proliferate tumor-reactive T cells ex vivo, are not selective because non-reactive T cells preferentially proliferate over reactive T cells in culture, potentially resulting in end products that lack satisfactory reactivity and / or remain insufficient in number of tumor-reactive T cells. Methods for producing tumor-reactive T cells for therapeutic use are needed.

[0024] The provided embodiments relate to improved methods for identifying and proliferating T cells ex vivo, including tumor-reactive T cells, for use in T cell therapy. In some embodiments, the provided methods improve or increase the proliferation and viability of T cells in vitro, e.g., tumor-reactive T cells. In certain embodiments, the methods enrich the proliferation of reactive T cells compared to non-reactive T cells and promote their survival and proliferation in ex vivo culture. In some embodiments, the resulting methods can be carried out in a closed system. In some embodiments, the methods are carried out in an automated or partially automated manner.

[0025] The provided method aims to ensure that the selection of cells during one or more steps of an ex vivo process for producing tumor-reactive T cells based on the expression of CD45, CD4, or CD8 and one or more exhaustion markers PD-1 and / or CD39 results in an improved TIL therapy enriched with tumor-reactive T cells that have high potential for therapeutic efficacy in treating specific cancers. The provided method yields a product containing tumor-reactive T cells that contain an oligoclonal population of TCRs that can target many mutations and / or are reactive to different tumor antigens. Thus, such tumor-reactive T cells offer advantages compared to existing methods of transducing cells to express a single neoepitope-reactive TCR.

[0026] PD-1 and CD39 are checkpoint molecules, and these too can represent markers of exhausted T cells. They are also markers that are activating or upregulating markers in that their expression increases during tumor reactivity, which is a natural mechanism of immunosuppression of the host immune response. For example, the immune system is designed to block itself to avoid excessive immune responses in order to avoid inflammation and autoimmune responses. In this way, an immune response develops against cancer initially, but this can be blocked by upregulation of certain checkpoint molecules such as PD-1 and CD39 that can inhibit the immune response. Since these are markers that are upregulated on cells in which an immune response is unfolding, the method provided is intended to have such markers function as potent markers for specifically enriching tumor-reactive T cells. By specifically selecting tumor-reactive cells based on these activating markers, the method provided would avoid bulk proliferation of T cells from tumor sources that may include a large number of non-tumor-reactive bystander cells or exhibit inhibitory activity such as Treg.

[0027] In some embodiments, the expression of one or more PD-1 and / or CD39 is used to enrich TILs either immediately after tumor dissociation, at the endpoint of tumor fragment culture, or immediately after the mechanical / enzymatic preparation of a single-cell suspension from the tumor fragment. In some embodiments, PD-1 and / or CD39-expressing cells are isolated from either a tumor fragment culture or a single-cell suspension produced by enzymatic digestion, among cells that also express CD45 and CD4 or CD8. In embodiments of the method provided, after selection, the selected cells can be grown in the presence of one or more T cell stimulants. In some embodiments, the T cell stimulant may include any one or more recombinant cytokines IL-2, IL-7, IL-15, IL-21, IL-25, IL-23, IL-27, or IL-25, for example, generally at least IL-2 or IL-15. In some embodiments, the T cell stimulant may include any one or more recombinant cytokines IL-2, IL-7, IL-15, or IL-21. In some embodiments, the T cell stimulant may include the recombinant cytokine IL-2. In some embodiments, the T cell stimulant may further include an anti-CD3 antibody (e.g., OKT3). In some embodiments, the T cell stimulant includes an anti-CD3 antibody (OKT3) and / or recombinant cytokines, e.g., IL-2, IL-7, IL-15, IL-21, IL-25, IL-23.

[0028] Figure 1 shows a schematic diagram of an exemplary process for producing a T-cell therapeutic composition according to the provided method. In the exemplary process, a tumor sample is obtained from a patient. In some cases, TILs are enriched from the sample by selecting cells positive for one or more markers associated with tumor-reactive cells, such as exhaustion markers such as PD-1 / CD39 (hereinafter, "selection markers"). In some cases, for example, a population of patient-derived T cells containing or enriched in tumor-infiltrating lymphocytes (TILs) may be stimulated under conditions for cell proliferation, and tumor-reactive T cells, or T cells positive for one or more markers associated with tumor-reactive cells, such as exhaustion markers such as PD-1 / CD39 (hereinafter, "selection markers"), may be selected and cultured under conditions for proliferation according to the provided method, such as incubation with T-cell stimulants (e.g., recombinant IL-2, anti-CD3). The culture may be carried out in the presence of one or more recombinant cytokines (e.g., IL-2) to support cell proliferation and swelling. The process may be carried out in the presence of serum-free medium containing nutrients.

[0029] One or all of the steps can be carried out in a closed system, for example, without exposing the cells to the environment. Once a therapeutic dose or threshold cell count is reached, the cells can be harvested and formulated, possibly concentrated or cryopreserved, and used for administration to a target, such as by injection.

[0030] The method provided offers advantages over existing methods for producing and proliferating TILs because it includes a step for enriching tumor-reactive cells, for example, by selecting T cells that are likely or suspected to be enriched with tumor-reactive T cells. In some cases, the method can enrich tumor-reactive T cells by selecting CD45+ cells that are PD-1+CD39+, and can also minimize bystander cells such as regulatory T cells. This process enriches the initial small population of tumor-reactive T cells proliferated from a biological sample (e.g., tumor) into cells that are or are likely to be tumor-reactive before a subsequent second proliferation step, thereby promoting the preservation and proliferation of the cells of interest and limiting the proliferation of bystander T cells that may include cells that do not react to and / or exhibit inhibitory activity against tumor antigens. This is in contrast to existing methods, which involve passive proliferation of bulk T cells, where all tumor-derived T cells are subjected to a first initial proliferation at high IL-2 concentrations, followed by a second rapid proliferation of T cells present after the initial proliferation. While other methods can significantly increase the proliferation of whole-cells (TVCs) through these alternative processes, they lack steps that actively ensure the proliferation of tumor-reactive T cells. In aspects of the provided method, all steps of the method are carried out in a closed system.

[0031] The provided method includes one or more features that provide an improved, more efficient, and / or more robust process for generating tumor-reactive T cell therapeutic compositions ex vivo. In particular, the present disclosure relates to a method that offers advantages over available methods for producing TIL therapeutic cell compositions. Such advantages include, for example, cost reduction, streamlining, improved enrichment of tumor-reactive T cells in the therapeutic composition, and increased efficacy of the therapeutic composition for different targets and tumor conditions.

[0032] In embodiments of the provided method, proliferation is carried out with relatively low concentrations of recombinant IL-2 during one or both proliferation steps and is successful. Many existing methods use high concentrations of IL-2 of 6000 IU / mL for T-cell proliferation of TILs. However, high IL-2 concentrations can be limiting, potentially increasing the cost of the process. In some cases, high IL-2 concentrations may have adverse effects on T-cell differentiation by driving the differentiation of effector T cells rather than early memory T cells, which may be more desirable in therapeutic T-cell compositions. The provided method can be carried out at concentrations lower than 6000 IU / mL, for example, less than 3000 IU / mL or less than approximately 3000 IU / mL.

[0033] In embodiments of the provided method, the T cell population is obtained from a biological sample known to contain T cells. In some embodiments, the T cell population is enriched from a biological sample from a subject, particularly a human subject. The biological sample can be any sample containing a bulk population of T cells. In some embodiments, the biological sample is or contains peripheral blood mononuclear cells. In some embodiments, the biological sample is a peripheral blood or serum sample. In some embodiments, the biological sample is a lymph node sample. In some embodiments, the biological sample is a tumor sample. In some embodiments, the bulk T cells may include tumor-infiltrating T cells (TILs). In some embodiments, the subject is a human subject. In some embodiments, the subject is a subject having cancer, a viral infection, a bacterial infection, or an inflammatory condition. In certain embodiments, the subject has cancer.

[0034] In embodiments of the provided method, the cell starting source (input sample) in the method may be a tumor fragment (e.g., a fragment 1–8 mm in diameter) or a single-cell suspension preparation from the enzymatic digestion of a tumor fragment. While certain sources may be superior for certain tumor types, both fragments and single-cell suspensions can support T cell proliferation and tumor-reactive T cell enrichment. In some cases, the tumor cell source may be selected depending on the type of tumor or cancer, for example, to optimize or increase the proliferation and enrichment of tumor-reactive T cells from the tumor. In one example, the cancer is melanoma, and the lymphocyte starting population is, for example, a tumor fragment derived from a resected tumor. In another example, the cancer is colorectal cancer, and the lymphocyte starting population is a single-cell suspension obtained by the enzymatic digestion of a tumor fragment, for example, with collagenase and / or hyaluronidase.

[0035] In some embodiments, the method produces or proliferates T cells for use in adoptive cell therapy to treat diseases or conditions in which cells or tissues associated with the disease or condition are known or suspected to express antigen targets recognized by T cells. In some embodiments, the T cell therapy is autologous to the subject. In some embodiments, the T cell therapy is homogeneous to the subject.

[0036] All publications, including patent documents, scientific articles, and databases, referenced in this application are incorporated herein by reference in their entirety for all purposes, to the same extent that each individual publication is incorporated by reference individually. If any definition contained herein conflicts with or is inconsistent with any definition contained in any patent, application, published application, or other publication incorporated herein by reference, the definition contained herein shall prevail over the definition incorporated herein by reference.

[0037] The section headings used herein are for organizational purposes only and should not be construed as limiting the subjects described.

[0038] I. Compositions and Pharmaceutical Preparations This specification provides TIL compositions enriched with tumor-reactive T cells. In some embodiments, the TIL composition contains primary T cells derived from tumors of subjects selected based on the surface expression of PD-1 and CD39 and grown ex vivo. In some embodiments, the provided TIL composition containing selected and grown T cells may be produced by a provided ex vivo method for generating the TIL composition.

[0039] In some embodiments, the TIL composition provided is a multiclonal population exhibiting TCR diversity and enrichment of T cell receptors (TCRs) that are reactive to tumor antigens. In some embodiments, the TIL composition provided is an oligoclonal population exhibiting TCR diversity and enrichment of different TCR chronotypes. In some embodiments, the TIL composition contains up to 40 different TCR chronotypes (e.g., the top 40 clones) that constitute at least 40% of the TCR frequency in the population. In some embodiments, the TIL composition contains up to 40 different TCR chronotypes that constitute at least 50% of the TCR frequency in the population. In some embodiments, the TIL composition contains up to 40 different TCR chronotypes that constitute at least 60% of the TCR frequency in the population. In some embodiments, the TIL composition contains up to 40 different TCR chronotypes that constitute at least 70% of the TCR frequency in the population. In some of the embodiments described above, the number of TCR chronotypes constituting the proportion is between 10 and 40 different TCR chronotypes. In some of the embodiments described above, the number of TCR chronotypes constituting the proportion is 20 to 40 different TCR chronotypes. In some of the embodiments described above, the number of TCR chronotypes constituting the proportion is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 different TCR chronotypes.

[0040] In some embodiments, the TIL composition contains 20 to 40 different TCR chronotypes that constitute at least 40% of the TCR frequencies in the population. In some embodiments, the TIL composition contains 20 to 40 different TCR chronotypes that constitute at least 50% of the TCR frequencies in the population. In some embodiments, the TIL composition contains 20 to 40 different TCR chronotypes that constitute at least 60% of the TCR frequencies in the population. In some embodiments, the TIL composition contains 20 to 40 different TCR chronotypes that constitute at least 70% of the TCR frequencies in the population.

[0041] In some embodiments, the top 40 chronotypes constitute at least 75% of the TCR frequency in the population. In some embodiments, the top 40 chronotypes constitute at least 80% of the TCR frequency in the population. In some embodiments, the top 40 chronotypes constitute at least 85% of the TCR frequency in the population. In some embodiments, the top 40 chronotypes constitute at least 90% of the TCR frequency in the population.

[0042] In some embodiments, the nascent antigenic reactivity of the TCR chronotype is reactivity to at least one CD4 antigen and at least one CD8 antigen. In some embodiments, the nascent antigenic reactivity of the TCR chronotype is reactivity to at least two peptide antigens, where at least one peptide antigen is a CD4 antigen and at least one peptide antigen is a CD8 antigen.

[0043] Various methods are known for evaluating the TCR repertoire for chronotype identification and TCR repertoire analysis (see, e.g., Rosati et al. (2017) BMC Biotechnology, 17:61; Friedensohn et al. (2016) Trendsin Biotechnology, 35:203-214). In some embodiments, the methods involve high-throughput or next-generation sequencing methods. In some embodiments, the frequency and type of different clones present in a population or composition can be determined. In some embodiments, the composition can be evaluated for clonality, clonal diversity, or clonal heterogeneity of cells in a population of cell compositions based, for example, on the determined frequency and / or type of chronotype present in the population or composition. In some embodiments, single-cell sequencing methods are performed to identify chronotypes on specific cells. In certain embodiments, paired αβTCR sequencing methods are used (see, e.g., WO2017053902A1). In some embodiments, the sequencing method is performed on DNA such as genomic DNA or complementary DNA. In some embodiments, the sequencing method is performed on RNA. In some embodiments, this is done by high-throughput or next-generation sequencing of TCR sequences, or by sequencing the whole genome or transcriptome (e.g., RNAseq). In some embodiments, the method used is an RNAseq-based method.

[0044] In some embodiments, T cell chronotype evaluation, as well as clonality and diversity, in various T cell populations or compositions or samples containing T cells are determined using high-throughput sequencing of all or part of the TCR genes, or based on sequences obtained from high-throughput whole-genome or transcriptome analysis of cell populations or compositions, and / or single-cell analysis. In some embodiments, the provided methods may include various features of the methods described in WO2016 / 044227, WO2016 / 176322, WO2012 / 048340, WO2012 / 048341, WO2014 / 144495, WO2017 / 053902, WO2017 / 053903, or WO2017 / 053905, each of which is incorporated in whole by reference.

[0045] The chronotype of cells, or the chronotype present in a population or composition of cells, can be determined by TCR sequencing in some embodiments. In some embodiments, the sequencing methods that can be used include high-throughput or next-generation sequencing known in the art. In some embodiments, next-generation sequencing methods can be used with T cell-derived genomic DNA or cDNA to evaluate the TCR repertoire, including sequences encoding complementarity-determining region 3 (CDR3). In some embodiments, whole transcriptome sequencing by RNAseq may be used. In some embodiments, TCR repertoire information, such as TCR sequences and relative frequencies, can be constructed or extracted from whole transcriptome sequencing (e.g., by RNAseq). For example, in some embodiments, repertoire TCR sequences or a portion thereof (e.g., CDR3) can be determined from whole transcriptome RNA-seq results using MIXCR (such as those described in Bolotin et al., Nature Methods 12 (2015) 380-381, Bolotin et al., Nature Biotechnology 35 (2017) 908-911) or IMREP (Mangul et al., bioRxiv (2017) 089235). In some embodiments, single-cell sequencing methods may be used. In some embodiments, chronotype can be evaluated or determined by spectral type analysis (measurements of the hypervariable region repertoire of the Vβ, Vα, Vγ, or Vδ chains of the TCR). Chronotype can also be determined by the production and characterization of antigen-specific clones against the antigen of interest.

[0046] In some embodiments, T cell chronotype assessment is determined using high-throughput sequencing of all or part of the TCR gene, or based on sequences obtained from high-throughput whole-genome or transcriptome analysis of a cell population or composition, and / or in a single cell. In some embodiments, chronotypes present in cells within a population or composition can be determined using bulk sequencing of a target sequence (e.g., the TCR chain or a portion thereof), or bulk whole-genome or transcriptome sequencing (e.g., by RNA-seq). In some embodiments, T cell chronotype assessment may involve sequencing of a portion of one or more variable regions of the native TCR chain, such as complementarity-determining region 3 (CDR3). In some embodiments, single-cell sequencing may be used. In some embodiments, the methods provided may include various features of the methods described in WO2016 / 044227, WO2016 / 176322, WO2012 / 048340, WO2012 / 048341, WO2014 / 144495, WO2017 / 053902, WO2017 / 053903, or WO2017 / 053905, each of which is incorporated in whole by reference. In some embodiments, for a target TCR molecule, the gene coding strand of the TCR can be obtained from the genomic DNA or mRNA of an immune cell or T cell.

[0047] In some embodiments, the composition exhibits clonal diversity, i.e., is multiclonal, such as oligoclonal. In some cases, clonal diversity is determined based on the relative frequencies of one or more chronotypes and / or one or more TCR sequences. In some embodiments, determining clonal diversity is expressed as the clonality of each of several samples, Shannon-adjusted clonality, or top 25 clonality. In some embodiments, determining clonal diversity is expressed as the Shannon-adjusted clonality in the composition.

[0048] In some embodiments, the cells of the provided TIL composition exhibit one or more phenotypic or functional markers. In some cases, such cells include cells that are positive or negative for one or more phenotypic markers or functional properties or attributes.

[0049] As used herein, the statement that a cell or population of cells is “positive” for a particular marker, function, or attribute means the detectable presence of a particular marker on or within a cell, such as a surface marker. When referring to a surface marker, the term means the presence of surface expression that is detected by flow cytometry by staining with an antibody that specifically binds to the marker, for example, and detecting the antibody, wherein the stain is detectable by flow cytometry at a level substantially higher than the stain detected by isotype-matched controls and other identical procedures under the same conditions, and / or at a level substantially similar to that of cells known to be positive for the marker, and / or at a level substantially higher than that of cells known to be negative for the marker.

[0050] As used herein, the statement that a cell or population of cells is “negative” for a particular marker, function, or attribute means the absence of a substantial detectable presence of that particular marker on or within the cell, such as a surface marker. When referring to a surface marker, the term means, for example, the absence of surface expression detected by flow cytometry by staining with an antibody that specifically binds to the marker and detecting the antibody, where the staining is not detected by flow cytometry at a level substantially higher than that detected by the same procedure under isotype-matched controls and other identical conditions, and / or at a level substantially similar to that of cells known to be positive for the marker, and / or at a level substantially higher than that of cells known to be negative for the marker.

[0051] Exemplary markers, functions, and attributes of the TIL compositions provided are described below. In some embodiments, the TIL composition is characterized by one or more of such features, for example, two, three, four, or five or more such features. For example, the TIL composition provided may be characterized by the presence or absence of one or more T cell markers, effector memory phenotype markers, exhaustion markers, the ability to produce or secrete cytokines, and / or the ability to produce or secrete cytotoxic factors, for example, as described below. Any two, three, four, five, or more of such features may be present in the described TIL composition.

[0052] In some embodiments, the TIL composition provided contains CD3+ T cells in proportion to more than 85% or about 85%, for example more than 90% or about 90%, for example more than 95% or about 95%, more than 97% or about 97%, or more than 98% or about 98% of the total cells in the population. In some embodiments, the TIL composition provided contains CD3+ T cells in proportion to more than 90% or about 90% of the total cells in the population. In some embodiments, the TIL composition provided contains CD3+ T cells in proportion to more than 95% or about 95% of the total cells in the population. In some embodiments, the TIL composition provided contains CD3+ T cells in proportion to more than 98% or about 98% of the total cells in the population. In some embodiments, the provided TIL composition contains CD4+ T cells and CD8+ T cells in proportion to more than 85% or about 85%, more than 90% or about 90%, more than 95% or about 95%, more than 97% or about 97%, or more than 98% or about 98% as a percentage of the total cells in the population. In some embodiments, the provided TIL composition contains CD3+ T cells in proportion to the total cells in the population in a greater proportion than the amount of CD3+ T cells contained as a percentage of the total cells isolated from the patient.

[0053] In some embodiments, the composition includes a ratio of CD4+ T cells to CD8+ T cells of 5:1 to 50:1 or approximately 5:1 to 50:1, 5:1 to 25:1, 5:1 to 20:1, 5:1 to 15:1, 5:1 to 10:1, 10:1 to 50:1, 10:1 to 25:1, 10:1 to 20:1, 10:1 to 15:1, 15:1 to 50:1, 15:1 to 25:1, 15:1 to 20:1, 20:1 to 50:1, 20:1 to 25:1, or 25:1 to 50:1. In some embodiments, the composition includes a ratio of CD4+ T cells to CD8+ T cells of 10:1 to 25:1 or approximately 10:1 to 25:1. In some embodiments, the composition includes a ratio of CD4+ T cells to CD8+ T cells of approximately 20:1.

[0054] In some embodiments, less than 5% of the CD3+ T cells or their CD4+ and / or CD8+ T cell subsets in the TIL composition express regulatory T cell phenotype markers, for example, less than 4% express regulatory T cell phenotype markers, less than 3% express regulatory T cell phenotype markers, less than 2% express regulatory T cell phenotype markers, and less than 1% express regulatory T cell phenotype markers. In some embodiments, less than 4% of the CD3+ T cells or their CD4+ and / or CD8+ T cell subsets in the TIL composition express regulatory T cell phenotype markers. In some embodiments, less than 3% of the CD3+ T cells or their CD4+ and / or CD8+ T cell subsets in the TIL composition express regulatory T cell phenotype markers. In some embodiments, less than 2% of the CD3+ T cells or their CD4+ and / or CD8+ T cell subsets in the TIL composition express regulatory T cell phenotype markers. In some embodiments, less than 4% of the CD3+ T cells or their CD4+ and / or CD8+ T cell subsets in the TIL composition express regulatory T cell phenotype markers, less than 1% express regulatory T cell phenotype markers, and less than 3% express regulatory T cell phenotype markers. In some embodiments, the regulatory T cell phenotype is characterized by the expression of one or more surface markers among CD25+, Foxp3+, and CD127low. In some embodiments, the regulatory T cell phenotype is characterized by the expression of CD4+ and Foxp3+ surface markers. In some embodiments, the regulatory T cell phenotype is characterized by the expression of CD8-Foxp3+ surface markers. In some embodiments, the regulatory T cell phenotype is characterized as CD4+CD8-CD25+Foxp3+CD127low.

[0055] In some embodiments, the provided TIL composition contains an increased or higher proportion of cells of a particular phenotype or function compared to an unselected TIL composition. In certain embodiments, a reference to an unselected TIL composition refers to a population of TILs grown from an unselected sample using the cell stimulation and growth method described in Section II.C., under the same or substantially the same conditions as the method for growth of the selected TIL composition. In certain embodiments, a reference to an unselected sample refers to a first population of cells prior to selection of PD-1 and / or CD39-positive cells (e.g., PD-1 and CD39-positive cells). In some embodiments, the unselected sample is a first population of cells generated as described in Section II.A. In some embodiments, the unselected sample is a single-cell suspension of dissociated tumor cells. For example, in some embodiments, an unselected TIL composition refers to a TIL composition treated in the same or substantially the same way as the provided TIL composition generated as described in Sections II.A-C, and it is expected that cells from dissociated tumor cells prior to ex vivo growth will not be selected for PD-1 and CD39 cells. Therefore, in some cases, unselected TIL compositions represent a bulk proliferation population of T cells from tumor cells subjected to ex vivo proliferation where tumor-reactive T cells are not enriched.

[0056] In some embodiments, the provided TIL composition includes an increased or higher percentage of PD-1 and / or CD39-positive CD3+ T cells compared to the percentage of PD-1 and / or CD39-positive CD3+ T cells in an unselected TIL composition. In some embodiments, the percentage was increased by at least, or at least about 2x, 3x, 4x, 5x, 10x, 20x or more.

[0057] In some embodiments, the provided composition contains CD3+ T cells that are positive for PD-1 and / or CD39, in amounts of at least 20% or about 20%, at least 30% or about 30%, at least 40% or about 40%, at least 50% or about 50%, at least 60% or about 60%, at least 65% or about 65%, at least 70% or about 70%, at least 75% or about 75%, at least 80% or about 80%, at least 81% or about 81%, at least 82% or about 82%, at least 83% or about 83%, at least 84% or The composition may contain approximately 84%, at least 85% or about 85%, at least 86% or about 86%, at least 87% or about 87%, at least 88% or about 88%, at least 89% or about 89%, at least 90% or about 90%, at least 91% or about 91%, at least 92% or about 92%, at least 93% or about 93%, at least 94% or about 94%, at least 95% or about 95%, at least 96% or about 96%, at least 97% or about 97%, at least 98% or about 98%, at least 99% or about 99%. In some embodiments, the composition contains more than 30% CD3+ T cells that are positive for PD-1 and / or CD39. In some embodiments, the composition contains more than 40% CD3+ T cells that are positive for PD-1 and / or CD39. In some embodiments, the composition contains more than 50% CD3+ T cells that are positive for PD-1 and / or CD39. In some embodiments, the composition contains more than 60% CD3+ T cells that are positive for PD-1 and / or CD39. In some embodiments, the composition contains more than 70% CD3+ T cells that are positive for PD-1 and / or CD39. In some embodiments, the composition contains more than 80% CD3+ T cells that are positive for PD-1 and / or CD39. In some embodiments, the composition contains more than 90% CD3+ T cells that are positive for PD-1 and / or CD39.

[0058] In some embodiments, the provided composition contains more than 30% PD-1 positive CD3+ T cells. In some embodiments, the provided composition contains more than 40% PD-1 positive CD3+ T cells. In some embodiments, the provided composition contains more than 50% PD-1 positive CD3+ T cells. In some embodiments, the provided composition contains more than 60% PD-1 positive CD3+ T cells. In some embodiments, the provided composition contains more than 70% PD-1 positive CD3+ T cells. In some embodiments, the provided composition contains more than 80% PD-1 positive CD3+ T cells. In some embodiments, the provided composition contains more than 90% PD-1 positive CD3+ T cells.

[0059] In some embodiments, the provided composition contains more than 30% CD39-positive CD3+ T cells. In some embodiments, the provided composition contains more than 40% CD39-positive CD3+ T cells. In some embodiments, the provided composition contains more than 50% CD39-positive CD3+ T cells. In some embodiments, the provided composition contains more than 60% CD39-positive CD3+ T cells. In some embodiments, the provided composition contains more than 70% CD39-positive CD3+ T cells. In some embodiments, the provided composition contains more than 80% CD39-positive CD3+ T cells. In some embodiments, the provided composition contains more than 90% CD39-positive CD3+ T cells.

[0060] In some embodiments, the provided composition contains more than 30% CD3+ T cells that are positive for PD-1 and CD39. In some embodiments, the provided composition contains more than 40% CD3+ T cells that are positive for PD-1 and CD39. In some embodiments, the provided composition contains more than 50% CD3+ T cells that are positive for PD-1 and CD39. In some embodiments, the provided composition contains more than 60% CD3+ T cells that are positive for PD-1 and CD39. In some embodiments, the provided composition contains more than 70% CD3+ T cells that are positive for PD-1 and CD39. In some embodiments, the provided composition contains more than 80% CD3+ T cells that are positive for PD-1 and CD39. In some embodiments, the provided composition contains more than 90% CD3+ T cells that are positive for PD-1 and CD39.

[0061] In some embodiments, more than 50% of the CD3+ T cells in the TIL composition, or their CD4+ and / or CD8+ T cell subsets, express the effector memory phenotype marker. In some embodiments, more than 60% of the CD3+ T cells in the TIL composition, or their CD4+ and / or CD8+ T cell subsets, express the effector memory phenotype marker, or more than 70% express the effector memory phenotype marker, or more than 80% express the effector memory phenotype marker, or more than 90% express the effector memory phenotype marker. In some embodiments, more than 60% of the CD3+ T cells in the TIL composition, or their CD4+ and / or CD8+ T cell subsets, express the effector memory phenotype marker. In some embodiments, the effector memory phenotype is CD45RA - The effector memory phenotype is characterized by the expression of one or more surface markers among CD45RO+, CD62L-, CCR7-, CD28-, and CD27-. In some embodiments, the effector memory phenotype is characterized by the expression of surface markers CD45RA- and CCR7-. In some embodiments, the effector memory phenotype is characterized by the expression of CD45RA-CD45RO+, CD62L - , and CCR7- It is characterized by the expression of surface markers. In some embodiments, the effector memory phenotype is CD45RA - CD45RO+, CD62L - , CCR7 - CD28 - and CD27 - It is characterized by the expression of surface markers.

[0062] In some embodiments, of the CD3+ T cells in the TIL composition, or the CD4+ and / or CD8+ T cell subset thereof, more than 50% are CD45RA- and CCR7, more than 60% are CD45RA- and CCR7, more than 70% are CD45RA- and CCR7, more than 80% are CD45RA- and CCR7, or more than 90% are CD45RA- and CCR7.

[0063] In some embodiments, more than 10% of the CD3+ T cells in the TIL composition, or a subset thereof of CD4+ and / or CD8+ T cells, express the central memory T cell marker, or more than 15% express the central memory T cell marker, or more than 20% express the central memory T cell marker, or more than 25% express the central memory T cell marker. In some embodiments, the central memory T cell marker is CD45RA-CCR7+. In some embodiments, more than 10% of the CD3+ T cells in the TIL composition, or a subset thereof of CD4+ and / or CD8+ T cells, express the central memory T cell marker, or more than 15% express the central memory T cell marker, or more than 20% express the central memory T cell marker, or more than 25% express the central memory T cell marker.

[0064] In some embodiments, more than 10% of the CD3+ T cells in the TIL composition, or their CD4+ and / or CD8+ T cell subsets, express a central memory T cell marker (e.g., CD45RA-CCR7+), and more than 60% express an effector memory phenotype marker (e.g., CD45RA-CCR7-). In some embodiments, more than 15% of the CD3+ T cells in the TIL composition, or their CD4+ and / or CD8+ T cell subsets, express a central memory T cell marker (e.g., CD45RA-CCR7+), and more than 60% express an effector memory phenotype marker (e.g., CD45RA-CCR7-). In some embodiments, more than 20% of the CD3+ T cells in the TIL composition, or their CD4+ and / or CD8+ T cell subsets, express a central memory T cell marker (e.g., CD45RA-CCR7+), and more than 60% express an effector memory phenotype marker (e.g., CD45RA-CCR7-). In some embodiments, more than 25% of the CD3+ T cells in the TIL composition, or their CD4+ and / or CD8+ T cell subsets, express a central memory T cell marker (e.g., CD45RA-CCR7+), and more than 60% express an effector memory phenotype marker (e.g., CD45RA-CCR7-).

[0065] In some embodiments, more than 10% of the CD3+ T cells in the TIL composition, or their CD4+ and / or CD8+ T cell subsets, express a central memory T cell marker (e.g., CD45RA-CCR7+), and more than 70% express an effector memory phenotype marker (e.g., CD45RA-CCR7-). In some embodiments, more than 15% of the CD3+ T cells in the TIL composition, or their CD4+ and / or CD8+ T cell subsets, express a central memory T cell marker (e.g., CD45RA-CCR7+), and more than 70% express an effector memory phenotype marker (e.g., CD45RA-CCR7-). In some embodiments, more than 20% of the CD3+ T cells in the TIL composition, or their CD4+ and / or CD8+ T cell subsets, express a central memory T cell marker (e.g., CD45RA-CCR7+), and more than 70% express an effector memory phenotype marker (e.g., CD45RA-CCR7-). In some embodiments, more than 25% of the CD3+ T cells in the TIL composition, or their CD4+ and / or CD8+ T cell subsets, express a central memory T cell marker (e.g., CD45RA-CCR7+), and more than 70% express an effector memory phenotype marker (e.g., CD45RA-CCR7-).

[0066] In some embodiments, more than 10% of the CD3+ T cells in the TIL composition, or their CD4+ and / or CD8+ T cell subsets, express a central memory T cell marker (e.g., CD45RA-CCR7+), and more than 80% express an effector memory phenotype marker (e.g., CD45RA-CCR7-). In some embodiments, more than 15% of the CD3+ T cells in the TIL composition, or their CD4+ and / or CD8+ T cell subsets, express a central memory T cell marker (e.g., CD45RA-CCR7+), and more than 80% express an effector memory phenotype marker (e.g., CD45RA-CCR7-). In some embodiments, more than 20% of the CD3+ T cells in the TIL composition, or the CD4+ and / or CD8+ T cell subset thereof, express a central memory T cell marker (e.g., CD45RA-CCR7+), and more than 80% express an effector memory phenotype marker (e.g., CD45RA-CCR7-).

[0067] In some embodiments, more than 10% of the CD3+ T cells in the TIL composition, or the CD4+ and / or CD8+ T cell subset thereof, express a central memory T cell marker (e.g., CD45RA-CCR7+), and more than 90% express an effector memory phenotype marker (e.g., CD45RA-CCR7-).

[0068] In some embodiments, the TIL composition provided contains about 10-60% tumor-reactive T cells. In some embodiments, the TIL composition provided contains more than about 15%, more than about 20%, more than about 25%, more than about 30%, more than about 40%, or more than about 50%, or any value within any of the aforementioned ranges. In some embodiments, the TIL composition provided containing a therapeutically effective amount of tumor-reactive T cells contains more than about 15%, more than about 20%, more than about 25%, more than about 30%, more than about 40%, or more than about 50%, or any value within any of the aforementioned ranges. In some embodiments, the TIL composition provided contains tumor-reactive T cells present in the composition in a therapeutically effective amount such that the composition exhibits characteristics different from the innate TIL population. In some embodiments, the TIL composition provided contains tumor-reactive T cells present in the composition in a therapeutically effective amount such that the composition exhibits characteristics different from the natural TIL population, and is positive for one or more markers (e.g., PD-1 / CD39) as described herein. In some embodiments, the TIL composition provided containing a therapeutically effective amount of tumor-reactive T cells contains a higher proportion of tumor-reactive T cells in the composition compared to the natural TIL population. In some embodiments, the TIL composition provided contains a certain proportion of tumor-reactive T cells, resulting in an enrichment of tumor-reactive T cells compared to the endogenously present amount (e.g., a proportion) (i.e., a proportion of cells that occur naturally in an individual with cancer, e.g., a tumor). Such an amount can be administered to a cancer patient.

[0069] The TIL compositions provided, concentrated in tumor-reactive cells, exhibit several functional or phenotypic activities demonstrating their reactivity to tumor cells. In some embodiments, cells can be evaluated for any of the functional or phenotypic activities, including but not limited to cytotoxic activity, degranulation, ability to produce or secrete cytokines, and one or more intracellular or surface phenotypic markers. Methods for evaluating such activities are known and illustrated herein and in the examples.

[0070] In some embodiments, TILs may be activated upon recognition of tumor cells. Upon activation, TILs exhibit potent cytolytic activity while simultaneously producing abundant cytokines, chemokines, and other factors. In some embodiments, activation triggers the release of cytoplasmic granules containing granzymes, leading to targeted cell death. Assays for measuring cytokines, chemokines, and other soluble factors are known in the art and include, but are not limited to, ELISA, intracellular cytokine staining, cytometric bead arrays, RT-PCR, ELISPOT, flow cytometry, and bioassays, which test cells responsive to relevant cytokines for responsiveness (e.g., proliferation) in the presence of a test sample.

[0071] In some embodiments, TILs can be evaluated for general functional activity based on the secretion of IFN-γ and / or granzyme B or other cytokines in response to polyclonal stimulation. In some embodiments, the polyclonal stimulation is stimulation with CD3 (e.g., by OKT3). In some embodiments, the in vitro CD3 assay includes OKT3 stimulation. In some embodiments, the in vitro CD3 assay includes seeding TILs on a culture plate pre-coated with OKT3 washed and diluted in phosphate-buffered saline. In some embodiments, the polyclonal stimulation is stimulation with CD3 (e.g., by OKT3) and CD28 to provide a co-stimulatory signal. In some embodiments, the in vitro assay includes stimulation with anti-CD3 and anti-CD28 antibodies, for example, by incubating cells with DynaBeads. After incubation overnight, the supernatant is collected and proteins in the supernatant are measured by ELISA for the cytokine of interest.

[0072] In some embodiments, the TIL composition provided is evaluated for tumor or neogenic antigen reactivity, for example, by an in vitro assay. In some embodiments, the assay may be an in vitro autologous tumor assay. In some embodiments, the assay may be an in vitro co-culture assay. In these and related embodiments, the results from such assays (e.g., in vitro autologous tumor assays or in vitro co-culture assays or similar assays) can be used as criteria for characterizing the TIL composition and / or the cell population comprising the composition. Such criteria may include, but are not limited to, the presence and / or quantity or level of one or more of the following: cytotoxic activity (e.g., tumor cell killing), cell activation and / or reactivity (e.g., to tumor cells), production and / or secretion of one or more cytokines (e.g., secretion of IFN-γ and / or granzyme B), or production or secretion of other compounds related to one or more of cytotoxic activity, cell activation, cell responsiveness, cell viability, or cell exhaustion.

[0073] In some embodiments, TILs may be evaluated for cytokine secretion, e.g., IFN-γ and / or granzyme B secretion, in response to co-culture with autologous tumor digests in an in vitro autologous tumor assay. In some embodiments, the reference to an in vitro autologous tumor assay is understood to be an assay in which TILs are incubated with non-hematopoietic cells derived from autologous primary tumors. In some embodiments, the in vitro autologous tumor assay involves seeding TILs on a culture plate with autologous non-hematopoietic tumor cells (e.g., in a 1:1 ratio). In some embodiments, the autologous tumor cells are a single-cell suspension of CD45-negative (CD45-) cells obtained from a primary tumor. After an incubation period ranging from 12 to 24 hours, the supernatant is collected and factor release can be quantified, for example, by ELISA.

[0074] In some embodiments, TILs may be evaluated for cytokine secretion, such as IFN-γ and / or granzyme B secretion, in response to co-culture with APCs carrying a nascent antigen (i.e., nascent antigen peptide) in an in vitro co-culture assay. In some embodiments, the reference to an in vitro co-culture assay is understood to be an assay in which TILs are incubated with autologous APCs carrying an autologous nascent antigenic peptide (hereinafter also referred to as peptide-carrying autologous APCs). In some embodiments, the in vitro co-culture assay includes seeding TILs onto a culture plate together with autologous irradiated APCs presenting the nascent antigenic peptide. In some embodiments, the APCs are irradiated. In some embodiments, the APCs are blood-derived APCs such as B cells or dendritic cells. In some embodiments, the in vitro co-culture assays referred to herein are assays in which B cells are isolated and proliferated from autologous blood or apheresis, for example by culturing them with CD40L and IL-4 for 14 days before loading nascent antigenic peptides, and then co-cultured with TIL in a ratio ranging from 1:1 to 1:5 TIL:APC. After an incubation period ranging from 12 to 24 hours, the supernatant is collected, and factor release can be quantified, for example, by ELISA. In some embodiments, the in vitro co-culture assays result in potent T cell activation. While we do not wish to be constrained by theory, it is thought that the APCs present in the in vitro co-culture assays, in addition to being pulsed with nascent antigenic peptides that are homologous to TIL TCRs, express potent levels of HLA and costimulatory molecules necessary for optimal T cell activation.

[0075] In some embodiments, the provided TIL composition contains a percentage of cells exhibiting increased or higher nascent antigen reactivity compared to an unselected TIL composition. In some embodiments, the nascent antigen reactivity increases by more than 2-fold, more than 3-fold, more than 4-fold, more than 5-fold, more than 6-fold, more than 7-fold, more than 8-fold, more than 9-fold, more than 10-fold, more than 15-fold, more than 20-fold, more than 30-fold, more than 40-fold, more than 50-fold, or more.

[0076] In some embodiments, the provided TIL composition exhibits higher neonatal antigen reactivity than a selected TIL population or TILs in a neonatal antigen reactivity assay, such as an in vitro co-culture assay or an in vitro autologous tumor assay. In some embodiments, more than 15% or about 15%, more than 20% or about 20%, more than 30% or about 30%, more than 40% or about 40%, or more than 50% or about 50% of CD8+ T cells in the provided TIL composition exhibit neonatal antigen reactivity in an in vitro autologous tumor assay. In some embodiments, more than 15% or about 15%, more than 20% or about 20%, more than 30% or about 30%, more than 40% or about 40%, or more than 50% or about 50% of CD4+ T cells in the provided TIL composition exhibit neonatal antigen reactivity in an in vitro autologous tumor assay. In some embodiments, more than 15% or about 15%, more than 20% or about 20%, more than 30% or about 30%, more than 40% or about 40%, or more than 50% or about 50% of the total T cells in the provided TIL composition exhibit neonatal antigen reactivity in an in vitro autologous tumor assay. In some embodiments, more than 15% or about 15%, more than 20% or about 20%, more than 30% or about 30%, more than 40% or about 40%, or more than 50% or about 50% of the total cells in the provided TIL composition exhibit neonatal antigen reactivity in an in vitro autologous tumor assay. In some embodiments, the TIL composition exhibits more than 2 times, 3 times, 4 times, or 5 times neonatal antigen reactivity in an in vitro autologous tumor assay compared to an unselected TIL composition.

[0077] In some embodiments, of the CD8+ T cells in the provided TIL composition, more than 15% or about 15%, more than 20% or about 20%, more than 30% or about 30%, more than 40% or about 40%, or more than 50% or about 50% exhibit nascent antigen reactivity in an in vitro co-culture assay, for example, after being cultured with autologous APCs (e.g., DC or B cells) that present nascent antigen peptides. In some embodiments, of the CD4+ T cells in the provided TIL composition, more than 15% or about 15%, more than 20% or about 20%, more than 30% or about 30%, more than 40% or about 40%, or more than 50% or about 50% exhibit nascent antigen reactivity in an in vitro co-culture assay, for example, after being cultured with autologous APCs (e.g., DC or B cells) that present nascent antigen peptides. In some embodiments, more than 15% or about 15%, more than 20% or about 20%, more than 30% or about 30%, more than 40% or about 40%, or more than 50% or about 50% of the total T cells in the provided TIL composition exhibit nascent antigen reactivity after being cultured in an in vitro co-culture assay, for example, with autologous APCs (e.g., DC or B cells) presenting a nascent antigen peptide. In some embodiments, the TIL composition exhibits approximately 2x, 3x, 4x, or 5x nascent antigen reactivity in an in vitro co-culture assay, for example, after being cultured with autologous APCs (e.g., DC or B cells) presenting nascent antigen peptides, compared to an unselected TIL composition.

[0078] In some embodiments, the provided TIL composition exhibits a higher effector cytokine response than the unselected TIL composition. In some embodiments, more than 15% or about 15%, more than 20% or about 20%, more than 30% or about 30%, more than 40% or about 40%, or more than 50% or about 50% of CD8+ T cells in the provided TIL composition produce IFN-γ in a nascent antigen-responsive assay such as an in vitro autologous tumor assay. In some embodiments, more than 15% or about 15%, more than 20% or about 20%, more than 30% or about 30%, more than 40% or about 40%, or more than 50% or about 50% of CD4+ T cells in the provided TIL composition produce IFN-γ in a nascent antigen-responsive assay such as an in vitro autologous tumor assay. In some embodiments, more than 15% or about 15%, more than 20% or about 20%, more than 30% or about 30%, more than 40% or about 40%, or more than 50% or about 50% of the total T cells in the provided TIL composition produce IFN-γ in a nascent antigen-reactivity assay, such as an in vitro autologous tumor assay. In any of the several provided embodiments, the nascent antigen-reactivity assay is an in vitro co-culture assay. In any of the several provided embodiments, the nascent antigen-reactivity assay is an in vitro autologous tumor assay.

[0079] In some embodiments, the TIL composition produces more than 20 times more IFN-γ in an in vitro autologous tumor assay compared to an unselected TIL composition. In some embodiments, the TIL composition produces more than 30 times, more than 40 times, more than 50 times, more than 60 times, more than 70 times, more than 80 times, more than 90 times, or more than 100 times more IFN-γ in an in vitro autologous tumor assay compared to an unselected TIL composition. In some embodiments, the TIL composition produces approximately 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, or 1000-fold IFN-γ in an in vitro autologous tumor assay compared to an unselected TIL composition.

[0080] In some embodiments, the TIL composition produces IFN-γ after an in vitro autologous tumor assay. In some embodiments, the TIL composition produces IFN-γ at concentrations of 2,000–15,000 pg / mL, 2,000–10,000 pg / mL, or 2,000–5,000 pg / mL. In some embodiments, the TIL composition produces IFN-γ at concentrations of 3,000–15,000 pg / mL, 3,000–10,000 pg / mL, or 3,000–5,000 pg / mL. In some embodiments, the TIL composition produces IFN-γ at concentrations of 5,000–15,000 pg / mL, 5,000–10,000 pg / mL, or 5,000–5,000 pg / mL. In some embodiments, the TIL composition produces 2,000 pg / mL, 5,000 pg / mL, 10,000 pg / mL, or 15,000 pg / mL of IFN-γ. In some embodiments, the TIL composition produces 2,000 mL of IFN-γ. In some embodiments, the TIL composition produces 5,000 pg / mL of IFN-γ. In some embodiments, the TIL composition produces 10,000 pg / mL of IFN-γ. In some embodiments, the TIL composition produces 15,000 mL of IFN-γ.

[0081] In some embodiments, the TIL composition produces IFN-γ after an in vitro co-culture assay, e.g., culture with autologous APCs (e.g., DC or B cells) presenting a nascent antigen peptide. In some embodiments, the TIL composition produces IFN-γ at concentrations of 2,000–1,500,000 pg / mL, 2,000–1,000,000 pg / mL, 2,000–500,000 pg / mL, or 2,000–250,000 pg / mL. In some embodiments, the TIL composition produces IFN-γ at concentrations of 5,000–1,500,000 pg / mL, 5,000–1,000,000 pg / mL, 5,000–500,000 pg / mL, or 5,000–250,000 pg / mL. In some embodiments, the TIL composition produces IFN-γ at concentrations of 5,000 to 1,500,000 pg / mL, 100,000 to 1,000,000 pg / mL, 100,000 to 500,000 pg / mL, or 100,000 to 250,000 pg / mL. In some embodiments, the TIL composition produces IFN-γ at concentrations of 5,000 pg / mL, 10,000 pg / mL, 20,000 pg / mL, 30,000 pg / mL, 40,000 pg / mL, or 50,000 pg / mL. In some embodiments, the TIL composition produces IFN-γ at concentrations of 50,000 pg / mL, 100,000 pg / mL, 200,000 pg / mL, 300,000 pg / mL, 400,000 pg / mL, 500,000 pg / mL, 600,000 pg / mL, 700,000 pg / mL, 800,000 pg / mL, 900,000 pg / mL, 1,000,000 pg / mL, or 1,500,000 pg / mL. In some embodiments, the TIL composition produces IFN-γ at a concentration of 2,000 pg / mL. In some embodiments, the TIL composition produces IFN-γ at a concentration of 5,000 mL. In some embodiments, the TIL composition produces IFN-γ at a concentration of 50,000 mL. In some embodiments, the TIL composition produces 500,000 mL of IFN-γ. In some embodiments, the TIL composition produces 600,000 mL of IFN-γ. In some embodiments, the TIL composition produces 700,000 mL of IFN-γ.In some embodiments, the TIL composition produces 800,000 mL of IFN-γ. In some embodiments, the TIL composition produces 900,000 mL of IFN-γ. In some embodiments, the TIL composition produces 1,000,000 mL of IFN-γ. In some embodiments, the TIL composition produces 1,500,000 mL of IFN-γ.

[0082] In some embodiments, more than 15% or about 15%, more than 20% or about 20%, more than 30% or about 30%, more than 40% or about 40%, or more than 50% or about 50% of the CD8+ T cells in the provided TIL composition produce TNF-α after being cultured in an in vitro co-culture assay, for example, with autologous APCs (e.g., DC or B cells) that present a nascent antigen peptide. In some embodiments, more than 15% or about 15%, more than 20% or about 20%, more than 30% or about 30%, more than 40% or about 40%, or more than 50% or about 50% of the CD4+ T cells in the provided TIL composition produce TNF-α after being cultured in an in vitro co-culture assay, for example, with autologous APCs (e.g., DC or B cells) that present a nascent antigen peptide. In some embodiments, more than 15% or about 15%, more than 20% or about 20%, more than 30% or about 30%, more than 40% or about 40%, or more than 50% or about 50% of the total T cells in the provided TIL composition produce TNF-α after being cultured in an in vitro co-culture assay, for example, with autologous APCs (e.g., DC or B cells) that present a nascent antigen peptide.

[0083] In some embodiments, more than 15% or about 15%, more than 20% or about 20%, more than 30% or about 30%, more than 40% or about 40%, or more than 50% or about 50% of the CD8+ T cells in the provided TIL composition produce TNF-α in an in vitro autologous tumor assay. In some embodiments, more than 15% or about 15%, more than 20% or about 20%, more than 30% or about 30%, more than 40% or about 40%, or more than 50% or about 50% of the CD4+ T cells in the provided TIL composition produce TNF-α in an in vitro autologous tumor assay. In some embodiments, more than 15% or about 15%, more than 20% or about 20%, more than 30% or about 30%, more than 40% or about 40%, or more than 50% or about 50% of the total T cells in the provided TIL composition produce TNF-α in an in vitro autologous tumor assay.

[0084] In some embodiments, the TIL composition produces more than 50 times more TNF-α compared to an unselected TIL composition after being cultured in an in vitro co-culture assay, for example, with autologous APCs (e.g., DC or B cells) presenting a nascent antigen peptide. In some embodiments, the TIL composition produces more than 5 times, more than 10 times, more than 15 times, more than 20 times, more than 25 times, more than 30 times, more than 35 times, or more than 40 times more TNF-α compared to an unselected TIL composition after being cultured in an in vitro co-culture assay, for example, with autologous APCs (e.g., DC or B cells) presenting a nascent antigen peptide. In some embodiments, the TIL composition produces approximately 75-fold, 100-fold, 150-fold, 200-fold, 250-fold, 300-fold, 350-fold, or 400-fold TNF-α compared to an unselected TIL composition after being cultured in, for example, autologous APCs (e.g., DC or B cells) presenting a nascent antigen peptide in an in vitro co-culture assay.

[0085] In some embodiments, the TIL composition produces more than 50 times more TNF-α in an in vitro autologous tumor assay compared to an unselected TIL composition. In some embodiments, the TIL composition produces more than 5 times, more than 10 times, more than 15 times, more than 20 times, more than 25 times, more than 30 times, more than 35 times, or more than 40 times more TNF-α in an in vitro autologous tumor assay compared to an unselected TIL composition. In some embodiments, the TIL composition produces approximately 75 times, 100 times, 150 times, 200 times, 250 times, 300 times, 350 times, or 400 times more TNF-α in an in vitro autologous tumor assay compared to an unselected TIL composition.

[0086] In some embodiments, the TIL composition produces TNF-α in in vitro co-culture assays, e.g., in culture with autologous APCs (e.g., DC or B cells) presenting a nascent antigen peptide. In some embodiments, the TIL composition produces TNF-α at concentrations of 250-2500 pg / mL, 250-2000 pg / mL, 250-1500 pg / mL, 250-1000 pg / mL, and 250-500 pg / mL. In some embodiments, the TIL composition produces TNF-α at concentrations of 250 pg / mL, 500 pg / mL, 1000 pg / mL, 1500 pg / mL, 2000 pg / mL, or 2500 pg / mL. In some embodiments, the TIL composition produces TNF-α at 250 pg / mL. In some embodiments, the TIL composition produces TNF-α at 500 pg / mL. In some embodiments, the TIL composition produces 1000 pg / mL of TNF-α. In some embodiments, the TIL composition produces 1500 pg / mL of TNF-α. In some embodiments, the TIL composition produces 2000 pg / mL of TNF-α. In some embodiments, the TIL composition produces 2500 pg / mL of TNF-α.

[0087] In some embodiments, cytotoxic activity may be determined based on the ability to produce or secrete granzyme B in a nascent antigen-reactivity assay, e.g., an in vitro co-culture assay or an in vitro autologous tumor assay. In some embodiments, a TIL composition produces about 10 times more granzyme B after being cultured in an in vitro co-culture assay, e.g., with autologous APCs (e.g., DC or B cells) presenting a nascent antigen peptide, compared to an unselected TIL composition. In some embodiments, a TIL composition produces about 20 times more granzyme B, 30 times more granzyme B, 40 times more granzyme B, or 50 times more granzyme B after being cultured in an in vitro co-culture assay, e.g., with autologous APCs (e.g., DC or B cells) presenting a nascent antigen peptide, compared to an unselected TIL composition. In some embodiments, the TIL composition produces more than 100 times more granzyme B compared to an unselected TIL composition after being cultured in an in vitro co-culture assay, e.g., with autologous APCs (e.g., DC or B cells) presenting a nascent antigen peptide. In some embodiments, the TIL composition produces more than 200 times, more than 300 times, more than 400 times, or more than 500 times more granzyme B compared to an unselected TIL composition after being cultured in an in vitro co-culture assay, e.g., with autologous APCs (e.g., DC or B cells) presenting a nascent antigen peptide. In some embodiments, the TIL composition produces more than 1000 times more granzyme B compared to an unselected TIL composition after being cultured in an in vitro co-culture assay, e.g., with autologous APCs (e.g., DC or B cells) presenting a nascent antigen peptide. In some embodiments, the TIL composition, after being cultured in an in vitro co-culture assay, e.g., with autologous APCs (e.g., DC or B cells) presenting a nascent antigen peptide, produces approximately 2000-fold or more granzyme B, 3000-fold or more granzyme B, 4000-fold or more granzyme B, or 5000-fold or more granzyme B compared to an unselected TIL composition.In some embodiments, the TIL composition produces approximately 10,000 times more granzyme B compared to an unselected TIL composition after being cultured in an in vitro co-culture assay, for example, with autologous APCs (e.g., DC or B cells) presenting a newly synthesized antigen peptide.

[0088] In some embodiments, the TIL composition produces more than 10 times more granzyme B in an in vitro autologous tumor assay compared to an unselected TIL composition. In some embodiments, the TIL composition produces more than 20 times, more than 30 times, more than 40 times, or more than 50 times more granzyme B in an in vitro autologous tumor assay compared to an unselected TIL composition. In some embodiments, the TIL composition produces more than 100 times more granzyme B in an in vitro autologous tumor assay compared to an unselected TIL composition. In some embodiments, the TIL composition produces more than 200 times, more than 300 times, more than 400 times, or more than 500 times more granzyme B in an in vitro autologous tumor assay compared to an unselected TIL composition. In some embodiments, the TIL composition produces more than 1,000 times more granzyme B in an in vitro autologous tumor assay compared to an unselected TIL composition. In some embodiments, the TIL composition produces more than 2,000 times more granzyme B, more than 3,000 times more granzyme B, more than 4,000 times more granzyme B, or more than 5,000 times more granzyme B in an in vitro autologous tumor assay compared to an unselected TIL composition. In some embodiments, the TIL composition produces more than 10,000 times more granzyme B in an in vitro autologous tumor assay compared to an unselected TIL composition.

[0089] In some embodiments, the TIL composition produces granzyme B in an autologous tumor assay. In some embodiments, the TIL composition produces granzyme B at concentrations of 200-3,000 pg / mL, 200-1,000 pg / mL, or 200-500 pg / mL. In some embodiments, the TIL composition produces granzyme B at concentrations of 300-3,000 pg / mL, 300-1,000 pg / mL, or 300-500 pg / mL. In some embodiments, the TIL composition produces granzyme B at concentrations of 200 pg / mL, 500 pg / mL, 1,000 pg / mL, or 3,000 pg / mL. In some embodiments, the TIL composition produces granzyme B at a concentration of 1,000 pg / mL. In some embodiments, the TIL composition produces granzyme B at a concentration of 3,000 pg / mL.

[0090] In some embodiments, the TIL composition produces granzyme B in in vitro co-culture assays, e.g., in culture with autologous APCs (e.g., DC or B cells) presenting a nascent antigen peptide. In some embodiments, the TIL composition produces granzyme B at concentrations of 50,000–600,000 pg / mL, 50,000–500,000 pg / mL, 50,000–400,000 pg / mL, 50,000–300,000 pg / mL, 200,000–500 pg / mL, and 000,000–500 pg / mL. In some embodiments, the TIL composition produces granzyme B at concentrations of 50,000 pg / mL, 100,000 pg / mL, 200,000 pg / mL, 300,000 pg / mL, 400,000 pg / mL, 500,000 pg / mL, or 600,000 pg / mL. In some embodiments, the TIL composition produces granzyme B at 50,000 pg / mL. In some embodiments, the TIL composition produces granzyme B at 100,000 pg / mL. In some embodiments, the TIL composition produces granzyme B at 200,000 pg / mL. In some embodiments, the TIL composition produces granzyme B at 300,000 pg / mL. In some embodiments, the TIL composition produces granzyme B at 400,000 pg / mL. In some embodiments, the TIL composition produces 500,000 pg / mL of granzyme B. In some embodiments, the TIL composition produces 600,000 pg / mL of granzyme B.

[0091] In some embodiments, the provided TIL compositions exhibit a higher degranulation response than unselected TIL compositions in nascent antigen reactivity assays, such as in vitro co-culture assays or in vitro autologous tumor assays. In some embodiments, more than 15% or about 15%, more than 20% or about 20%, more than 30% or about 30%, more than 40% or about 40%, or more than 50% or about 50% of CD8+ T cells in the provided TIL compositions exhibit degranulation in in vitro co-culture assays, for example, after being cultured with autologous APCs (e.g., DC or B cells) presenting nascent antigen peptides. In some embodiments, degranulation activity can be measured by CD107a expression. In some embodiments, more than 15% or about 15%, more than 20% or about 20%, more than 30% or about 30%, more than 40% or about 40%, or more than 50% or about 50% of the CD8+ T cells in the provided TIL composition express CD107a after being cultured in an in vitro co-culture assay, for example, with autologous APCs (e.g., DC or B cells) that present a nascent antigen peptide. In some embodiments, more than 10% or about 10% of the CD8+ T cells in the provided TIL composition express CD107a after being cultured in an in vitro co-culture assay, for example, with autologous APCs (e.g., DC or B cells) that present a nascent antigen peptide. In some embodiments, more than 20% or about 20% of the CD8+ T cells in the provided TIL composition express CD107a after being cultured in an in vitro co-culture assay, for example, with autologous APCs (e.g., DC or B cells) presenting a nascent antigen peptide. In some embodiments, more than 25% or about 25% of the CD8+ T cells in the provided TIL composition express CD107a after being cultured in an in vitro co-culture assay, for example, with autologous APCs (e.g., DC or B cells) presenting a nascent antigen peptide.

[0092] In some embodiments, more than 15% or about 15%, more than 20% or about 20%, more than 30% or about 30%, more than 40% or about 40%, or more than 50% or about 50% of the CD4+ T cells in the provided TIL composition exhibit degranulation in an in vitro co-culture assay, for example, after being cultured with autologous APCs (e.g., DC or B cells) presenting a nascent antigen peptide. In some embodiments, degranulation activity can be measured by CD107a expression. In some embodiments, more than 15% or about 15%, more than 20% or about 20%, more than 30% or about 30%, more than 40% or about 40%, or more than 50% or about 50% of the CD4+ T cells in the provided TIL composition express CD107a in an in vitro co-culture assay, for example, after being cultured with autologous APCs (e.g., DC or B cells) presenting a nascent antigen peptide. In some embodiments, more than 5% or about 5% of the CD4+ T cells in the provided TIL composition express CD107a after being cultured in an in vitro co-culture assay, for example, with autologous APCs (e.g., DC or B cells) presenting a nascent antigen peptide. In some embodiments, more than 10% or about 10% of the CD4+ T cells in the provided TIL composition express CD107a after being cultured in an in vitro co-culture assay, for example, with autologous APCs (e.g., DC or B cells) presenting a nascent antigen peptide. In some embodiments, more than 15% or about 15% of the CD8+ T cells in the provided TIL composition express CD107a after being cultured in an in vitro co-culture assay, for example, with autologous APCs (e.g., DC or B cells) presenting a nascent antigen peptide.

[0093] In some embodiments, more than 15% or about 15%, more than 20% or about 20%, more than 30% or about 30%, more than 40% or about 40%, or more than 50% or about 50% of the CD8+ T cells in the provided TIL composition exhibit degranulation in an in vitro autologous tumor assay. In some embodiments, degranulation activity can be measured by CD107a expression. In some embodiments, more than 15% or about 15%, more than 20% or about 20%, more than 30% or about 30%, more than 40% or about 40%, or more than 50% or about 50% of the CD8+ T cells in the provided TIL composition express CD107a in an in vitro autologous tumor assay. In some embodiments, more than 10% or about 10% of the CD8+ T cells in the provided TIL composition express CD107a in an in vitro autologous tumor assay. In some embodiments, more than 20% or about 20% of the CD8+ T cells in the provided TIL composition express CD107a in an in vitro autologous tumor assay. In some embodiments, more than 25% or about 25% of the CD8+ T cells in the provided TIL composition express CD107a in an in vitro autologous tumor assay.

[0094] In some embodiments, more than 15% or about 15%, more than 20% or about 20%, more than 30% or about 30%, more than 40% or about 40%, or more than 50% or about 50% of the CD4+ T cells in the provided TIL composition exhibit degranulation in an in vitro autologous tumor assay. In some embodiments, degranulation activity can be measured by CD107a expression. In some embodiments, more than 15% or about 15%, more than 20% or about 20%, more than 30% or about 30%, more than 40% or about 40%, or more than 50% or about 50% of the CD4+ T cells in the provided TIL composition express CD107a in an in vitro autologous tumor assay. In some embodiments, more than 5% or about 5% of the CD4+ T cells in the provided TIL composition express CD107a in an in vitro autologous tumor assay. In some embodiments, more than 10% or about 10% of the CD4+ T cells in the provided TIL composition express CD107a in an in vitro autologous tumor assay. In some embodiments, more than 15% or about 15% of the CD8+ T cells in the provided TIL composition express CD107a in an in vitro autologous tumor assay.

[0095] In some embodiments, the TIL composition is characterized by its ability to kill tumor cells in an in vitro autologous tumor assay. In some embodiments, the TIL composition kills at least 30% of tumor cells in an in vitro autologous tumor assay. In some embodiments, the TIL composition kills at least 40% of tumor cells in an in vitro autologous tumor assay. In some embodiments, the TIL composition kills at least 50% of tumor cells in an in vitro autologous tumor assay. In some embodiments, the TIL composition kills at least 60% of tumor cells in an in vitro autologous tumor assay. In some embodiments, the TIL composition kills at least 70% of tumor cells in an in vitro autologous tumor assay. In some embodiments, the TIL composition kills at least 80% of tumor cells in an in vitro autologous tumor assay.

[0096] In certain embodiments, the number of such cells in the composition is the therapeutically effective dose. The effective dose of cells may vary depending on the patient, as well as the type, severity, and extent of the disease. Thus, a physician can determine what the effective dose is after considering the health status of the subject, the extent and severity of the disease, and other variables. In some embodiments, the dose is the amount that reduces the severity, duration, and / or symptoms associated with cancer in an animal. In some embodiments, the therapeutically effective dose is the dose of cells that reduces cancer growth or spread by at least 2.5%, at least 5%, at least 10%, at least 15%, at least 25%, at least 35%, at least 45%, at least 50%, at least 75%, at least 85%, at least 90%, at least 95%, or at least 99% compared to cancer growth or spread in a patient (or animal) or group of patients (or animals) that has not been administered the composition. In some embodiments, the therapeutically effective dose is the amount that results in cytotoxic activity that inhibits or reduces the growth of cancer cells.

[0097] In some embodiments, the TIL composition provided herein, enriched with tumor-reactive cells, is approximately 10 5 or about 10 5 ~10 12 or about 10 12 It contains a cell volume of about 10. In some embodiments, the TIL composition provided herein, enriched with tumor-reactive cells, is about 10 5 or about 10 5 ~10 8 or about 10 8 It contains a cell volume of about 10. In some embodiments, the TIL composition provided herein, enriched with tumor-reactive cells, is about 10 6 or about 10 6 ~10 12 or about 10 12 It contains a cell volume of about 10. In some embodiments, the TIL composition provided herein, enriched with tumor-reactive cells, is about 10 8 or about 10 8 ~101 or about 10 11 It contains a cell volume of about 10. In some embodiments, the TIL composition provided herein, enriched with tumor-reactive cells, is about 10 9 or about 10 9 ~10 10 or about 10 10 It contains a cell quantity of 10. In some embodiments, the TIL composition provided herein, enriched with tumor-reactive cells, is 10 5 Over or approximately 10 5 Super Cells, 10 6 Over or approximately 10 6 Super Cells, 10 7 Over or approximately 10 7 Super Cells, 10 8 Over or approximately 10 8 Super Cells, 10 9 Over or approximately 10 9 Super Cells, 10 10 Over or approximately 10 10 Super Cells, 10 11 Over or approximately 10 11 Super cells, or 10 12 Over or approximately 10 12 This includes a quantity of supercellular cells. In some embodiments, such a quantity may be administered to subjects with a disease or condition, such as cancer patients.

[0098] In some embodiments, the volume of the composition is at least or at least about 10 mL, 50 mL, 100 mL, 200 mL, 300 mL, 400 mL, or 500 mL, for example, 10 mL or about 10 mL to 500 mL, 10 mL to 200 mL, 10 mL to 100 mL, 10 mL to 50 mL, 50 mL to 500 mL, 50 mL to 200 mL, 50 mL to 100 mL, 100 mL to 500 mL, 100 mL to 200 mL, or 200 mL to 500 mL (including both ends). In some embodiments, the composition is at least or at least about 1 × 10 5 cells / mL, 5×10 5 cells / mL, 1×10 6 cells / mL, 5×10 6 cells / mL, 1×10 7 cells / mL, 5×107 cells / mL, or 1×10 8 has a cell density of cells / mL. In some embodiments, the cell density of the composition is 1×10 or about 1×10 5 cells / mL to 1×10 8 cells / mL, 1×10 5 cells / mL to 1×10 7 cells / mL, 1×10 5 cells / mL to 1×10 6 cells / mL, 1×10 6 cells / mL to 1×10 7 cells / mL, 1×10 6 cells / mL to 1×10 8 cells / mL, 1×10 6 cells / mL to 1×10 7 cells / mL, or 1×10 7 cells / mL to 1×10 8 cells / mL (including both ends).

[0099] The composition includes pharmaceutical compositions and dosage forms for adoptive cell therapy and the like. In some embodiments, the cells are formulated with a pharmaceutically acceptable carrier. In some examples, the pharmaceutical composition containing a pharmaceutically effective carrier is in a therapeutically effective amount sufficient to treat, ameliorate, or otherwise beneficially modify the condition, disorder or disease, or the symptoms of another indication (e.g., cancer).

[0100] Examples of "pharmaceutically acceptable carriers" include any and all solvents, dispersions, coatings, antimicrobial and antifungal agents, isotonic and absorption retardants, etc., suitable for drug administration (Gennaro, 2000, Remington: The science and practice of pharmacy, Lippincott, Williams & Wilkins, Philadelphia, PA). Examples of such carriers or diluents include, but are not limited to, water, physiological saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous media such as liposomes and non-volatile oils may also be used. Supplementary active ingredients may also be incorporated into the composition. The pharmaceutical carrier must be suitable for cells, such as a solution containing physiological saline, dextrose solution, or human serum albumin.

[0101] In some embodiments, a pharmaceutically acceptable carrier or vehicle for such a composition is any non-toxic aqueous solution in which cells can be maintained or remain viable for a sufficient time to allow administration of live cells. For example, a pharmaceutically acceptable carrier or vehicle may be saline or buffered saline. A pharmaceutically acceptable carrier or vehicle may also include various biomaterials that can increase the efficiency of cells. Cell vehicles and carriers include, for example, polysaccharides such as methylcellulose (MCTate, DAShear, SWHoffman, DGStein, MCLaPlaca, Biomaterials 22, 1113, 2001, the whole thereof is incorporated herein by reference), and chitosan (Suh JKF, Matthew HW T. Biomaterials, 21, 2589, 2000; Lahiji A, Sohrabi A, Hungerford DS, et al., J Biomed Mater Res, 51, 586, 2000 (the whole is incorporated herein by reference), N-isopropylacrylamide copolymer P (NIPAM-co-AA) (YHBae, B. Vernon, CK Han, SW Kim, J. Control. Release 53, 249, 1998; H. Gappa, M. Baudys, J. J. Koh, SW Kim, YHBae, Tissue Eng. 7, 35, 2001, the whole is incorporated herein by reference) (Included herein) and poly(oxyethylene) / poly(D,L-lactic acid-coglycolic acid) (B. Jeong, KMLee, A. Gutowska, YHAn, Biomacromolecules 3, 865, 2002, the whole thereof is incorporated herein by reference), P(PF-co-EG) (Suggs LJ, Mikos AG, CellTrans, 8, 345, 1999, the whole thereof is incorporated herein by reference), PEO / PEG (Mann BK, Gobin AS, Tsai AT, Schmedlen RH, West J L., Biomaterials, 22, 3045, 2001; Bryant SJ, Anseth K S.Examples include Biomaterials, 22, 619, 2001 (the entire text of which is incorporated herein by reference), PVA (Chih-Ta Lee, Po-Han Kung and Yu-Der Lee, Carbohydrate Polymers, 61, 348, 2005 (the entire text of which is incorporated herein by reference)), collagen (Lee CR, Grodzinsky AJ, Spector M., Biomaterials 22, 3145, 2001 (the entire text of which is incorporated herein by reference)), and alginates (Bouhadir KH, Lee KY, Alsberg E, Damm KL, Anderson KW, Mooney D J. Biotech Prog 17, 945, 2001; Smidsrd O, Skjak-Braek G., Trends Biotech, 8, 71, 1990 (the entire text of which is incorporated herein by reference)).

[0102] In some embodiments, the composition comprising the pharmaceutical composition is sterile. In some embodiments, cell isolation or concentration is performed in a closed or sterile environment to minimize, for example, errors, user handling, and / or contamination. In some embodiments, sterility can be easily achieved, for example, by filtration through a sterilization filtration membrane.

[0103] This specification also provides compositions suitable for cryopreserving T cells, including tumor-reactive T cells. In some embodiments, the composition comprises a cryoprotective agent. In some embodiments, the cryoprotective agent is or comprises DMSO and / or glycerol. In some embodiments, the composition formulated for cryopreservation can be stored at low temperatures, such as ultra-low temperatures, for example, in a temperature range of -40°C to -150°C, for example, 80°C or about 80°C ± 6.0°C.

[0104] Furthermore, this specification also provides freezing compositions comprising any of the TIL compositions provided and a cryoprotectant.

[0105] In some embodiments, cryopreserved cells are prepared for administration by thawing. In some cases, the cells may be administered to the subject immediately after thawing. In such embodiments, the composition is ready for immediate use without any further processing. In other cases, the cells are further processed after thawing, such as by resuspending with a pharmaceutically acceptable carrier, incubating with an activator or stimulant, or by washing and resuspending in a pharmaceutically acceptable buffer before administration to the subject.

[0106] II. Ex vivo method for producing a composition enriched with tumor-reactive T cells Various embodiments of the provided method involve the ex vivo proliferation and production of T-cell therapeutic compositions, particularly for use in connection with the treatment of cancer. In some embodiments, the production method involves the in vitro proliferation and manipulation of patient cells.

[0107] The provided embodiments relate to a process for preparing a therapeutic TIL composition enriched with tumor-reactive T cells, involving direct selection of cells to yield a further proliferated population of tumor-reactive cells. In the provided method, a TIL tumor sample containing or expected to contain tumor-reactive T cells (e.g., a first population) is obtained. In one embodiment, this population may be treated by digestion to create a single-cell suspension (e.g., a second population). In some embodiments, this second population is separated to select cells enriched with tumor-reactive T cells and to minimize the presence of bystander cells such as regulatory T cells. In some embodiments, the selection is against cells positive for PD-1 and / or CD39. In some embodiments, the selection is against cells positive for both PD-1 and CD39. In the provided method, T cells are separated against cells surface-positive for CD39 and PD1 immediately after tumor digestion or after a short period of cell culture to produce a population of selected T cells. This process removes non-reactive and inhibitory "bystander" cells, resulting in a T cell product enriched with newly generated antigen-reactive T cells. Selection yields a population of selected or isolated cells (e.g., sometimes referred to as a third population), which can then be grown to create a therapeutic composition containing a population of tumor-specific reactive cells (e.g., sometimes referred to as a fourth population). In some embodiments, the cells are then grown into a clinically relevant number of tumor-specific T cells. In embodiments of the provided method, T cells derived from the selected population (third population), e.g., isolated T cells from excised tumor fragments or single-cell suspensions therefrom, are grown by culturing them under T cell-stimulating conditions in the presence of one or more T cell stimulants. In some embodiments, the final grown therapeutic composition is formulated with a cryoprotective agent for cryopreservation.

[0108] AT cell-containing sample The methods provided include selecting or obtaining a T cell input sample derived from a biological specimen that can be used as a source or input of T cells for stimulation with one or more T cell stimulants (e.g., recombinant IL-2 or other T cell stimulating cytokines and / or anti-CD3). In some embodiments, the T cells are derived from a biological specimen from a subject known or likely to contain tumor-reactive T cells. In some embodiments, the biological specimen provides a cell input population that is a single-cell suspension (SCS), which can be used for subsequent selection of TILs for proliferation as described in sections II.B and II.C. In some embodiments, the biological specimen is processed to provide a cell input population that is a single-cell suspension, which can be used for subsequent selection of TILs for proliferation as described in sections II.B and II.C. For example, certain methods include processing tumor fragments by homogenization and / or dissociation using enzymatic methods, and filtering the dissociated cells to prepare a single-cell suspension as a cell input population.

[0109] In any embodiment of the embodiments provided, a suitable biological sample is obtained from a patient of interest, i.e., a patient suspected of having or known to have cancer. In some embodiments, the sample is a sample known or suspected to contain T cells, e.g., T cells expressing or potentially expressing the endogenous T cell receptor (TCR). The biological sample may originate from any initial source containing or suspected of containing such T cells. In some embodiments, the biological sample source of interest may include, but is not limited to, many different physiological sources, e.g., tissue-derived samples, e.g., homogenates, and blood or derivatives thereof.

[0110] Various biological samples can be used as a potential source of reactive T cells. Tumors and downstream lymph nodes may have the highest frequency of reactive T cells (Powell et al., Clin. Cancer. Res., 2014), but other sample sources can also be used. In some cases, the sample may be a tumor sample, a tertiary lymphocyte site, a draining lymph node, peripheral blood, or bone marrow. In some embodiments, the biological sample is a tumor sample. In some embodiments, the biological sample is a lymph sample. In some embodiments, the biological sample is a peripheral blood sample.

[0111] Biological samples include tissues, body fluids, and other samples taken directly from a subject to obtain an input sample, or input samples can be obtained or generated through one or more processing steps such as separation, selection or concentration, centrifugation, washing, and / or incubation. Input samples containing T cells may be samples obtained directly from a biological source or processed samples. Examples of biological samples, but not limited to, include body fluids such as blood, plasma, serum, cerebrospinal fluid, synovial fluid, urine, and sweat, as well as tissue and organ samples, and tumor samples, and processed samples derived therefrom.

[0112] In some embodiments, the sample is blood or a blood-derived sample, or an apheresis or leukocyte apheresis product, or derived therefrom. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumors, leukemia, lymphoma, lymph nodes, intestinal-associated lymphoid tissue, mucosa-associated lymphoid tissue, spleen, other lymphoid tissue, liver, lung, stomach, intestine, colon, kidney, pancreas, breast, bone, prostate, cervix, testis, ovary, tonsil, or other organs, and / or cells derived therefrom. Samples may include autologous and allogeneic source samples in connection with cell therapy, such as adoptive cell therapy.

[0113] In many embodiments, the sample may originate from a fluid suspected to contain at least the T cells of interest. In many embodiments, a suitable initial source for the sample is blood. In some embodiments, the biological sample is a blood-derived sample. The blood-derived sample may originate from whole blood or a portion thereof, such as serum, plasma, etc., and in many embodiments, the sample originates from blood cells taken from whole blood. In some embodiments, the sample source includes mononuclear cells. For example, the biological sample is or contains peripheral blood mononuclear cells (PBMCs), or originates from PBMCs.

[0114] In some embodiments where the sample is a PBMC-derived sample, the sample is generally a fluid PBMC-derived sample. Any simple methodology for producing a fluid PBMC sample can be employed. In many embodiments, fluid PBMC-derived samples are prepared by separating PBMCs from whole blood, i.e., by collecting PBMCs, for example by centrifugation (e.g., Ficoll-Hypaque density gradient centrifugation), and typical protocols for such separation procedures are disclosed in WO98 / 15646 and U.S. Patent No. 5,985,565.

[0115] In some embodiments, the sample is a tumor sample, thereby providing a source of tumor-infiltrating lymphocytes (TILs). In some embodiments, TILs are T cells that have left the bloodstream of the subject and migrated into or infiltrated the tumor. In certain embodiments, the TILs are reactive to tumor antigens.

[0116] A patient's tumor sample can be obtained by any of the various methods that result in a sample containing a mixture of tumor and TIL cells. In some embodiments, the tumor sample can be obtained by surgical excision. In some embodiments, the tumor sample can be obtained by needle biopsy. Generally, the tumor sample can originate from any solid tumor, including primary tumors, invasive tumors, or metastatic tumors. The tumor sample may also be a liquid tumor, such as a tumor obtained from a hematological malignancy.

[0117] In some embodiments, solid tumors may be any type of cancer, including but not limited to squamous cell carcinoma, basal cell carcinoma, and melanoma, of the ovaries, vulva, endometrium, urothelium, breast, pancreas, prostate, colorectal, lung, brain, kidney, stomach (gastrointestinal), and skin. In some embodiments, tumors originate from cancer patients, including but not limited to cancer patients of the ovaries, vulva, endometrium, urothelium, breast, colorectal, lung, kidney, and skin (including but not limited to melanoma). In some embodiments, tumors originate from ovarian cancer patients. In some embodiments, tumors originate from vulvar cancer patients. In some embodiments, tumors originate from endometrial cancer patients. In some embodiments, tumors originate from urothelial carcinoma patients. In some embodiments, tumors originate from breast cancer patients. In some embodiments, tumors originate from colorectal cancer patients. In some embodiments, tumors originate from lung cancer patients. In some embodiments, tumors originate from kidney patients. In some embodiments, tumors originate from melanoma patients. In certain embodiments, the tumor originates from a patient being treated as described in Section III.

[0118] In certain embodiments, the T cell population includes both CD4+ and CD8+ T cells. Many cancers, including solid tumors such as many common epithelial indications (e.g., GI), express class I and class II restrictive mutations. It is intended that both CD8+ T cells that recognize class I MHC restrictive molecules and CD4+ T cells that recognize class II MHC restrictive molecules are required for T cell products to target such indications, e.g., common epithelial indications.

[0119] Samples may be obtained from a variety of different subjects / patients / hosts. Generally, such hosts are “mammals” or “mammals,” and these terms are used broadly to describe organisms within the class Mammalia, which includes the orders Carnivora (e.g., dogs and cats), Rodentia (e.g., mice, guinea pigs, and rats), and Primates (e.g., humans, chimpanzees, and monkeys). In many embodiments, the host would be a human.

[0120] In some embodiments, the subject is human. Therefore, in some embodiments, the cells are primary cells, e.g., primary human cells. In some embodiments, the sample is autologous to the subject being treated, e.g., a patient who requires a specific therapeutic intervention, e.g., adoptive cell therapy according to the provided method in which the cells are isolated, processed, and / or proliferated. In some embodiments, the sample is allogeneic to the subject being treated.

[0121] In the provided embodiment, the obtained tumor sample is 1 mm 3 ~8mm 3 or about 1 mm 3 ~8mm 3 The size, for example, 1 mm 3 ~3mm 3 or about 1 mm 3 ~about 3mm 3 , 1mm 3 ~4mm 3 or about 1 mm 3 ~about 4mm 3 , 1mm 3 ~2mm 3 or about 1 mm 3 ~approximately 2mm 3 Break it into small fragments. In some embodiments, the tumor fragments are about -3 mm 3 Therefore, in some embodiments, the tumor fragment is approximately 1-3 mm in size. 3 In some embodiments, tumor fragments are obtained by physical fragmentation such as dissection. In some embodiments, tumor specimens are obtained by sharp dissection.

[0122] In some of the provided embodiments, the obtained tumor sample is divided into fine fragments with diameters of 1 mm to 8 mm or about 1 mm to about 8 mm, for example, 1 mm to 6 mm or about 1 mm to about 6 mm, 1 mm to 4 mm or about 1 mm to about 4 mm, or 1 mm to 2 mm or about 1 mm to about 2 mm. In some embodiments, the tumor fragments are about 3 mm in diameter. In some embodiments, the tumor fragments are about 1 to 2 mm in diameter. In some embodiments, the tumor fragments are obtained by physical fragmentation such as dissection. In some embodiments, the tumor fragments are obtained by sharp dissection.

[0123] In some embodiments, tumor samples are cryopreserved before fragmentation. In some embodiments, tumor fragments are cryopreserved.

[0124] In some embodiments, tumor fragments are used as a source for preparing single-cell suspensions for use as input samples for T cells in the provided method. In some embodiments, the provided method includes obtaining cells from the tumor fragments by enzymatic digestion of the tumor fragments to obtain TILs. Enzymatic digestion can be carried out in part using collagenases such as type IV collagenase or type I / II collagenase. Collagenases are enzymes that degrade the collagen network embedded in the extracellular matrix of cells (Eikenes et al. Anticancer Research, 2010). Enzymes such as collagenase may be present in the culture medium for enzymatic digestion at concentrations of 1 mg / mL or approximately 1 mg / mL to 5 mg / mL or approximately 5 mg / mL, for example, 1 mg / mL or approximately 1 mg / mL, 2 mg / mL or approximately 2 mg / mL, 3 mg / mL or approximately 3 mg / mL, 4 mg / mL or approximately 4 mg / mL, 5 mg / mL or approximately 5 mg / mL, 6 mg / mL or approximately 6 mg / mL, 7 mg / mL or approximately 7 mg / mL, 8 mg / mL or approximately 8 mg / mL, 9 mg / mL or approximately 9 mg / mL, 10 mg / mL or approximately 10 mg / mL, or any value within the range of any of the aforementioned values. In some embodiments, collagenase may be present in the culture medium at a concentration of 5 mg / mL or approximately 5 mg / mL to 10 mg / mL or approximately 10 mg / mL. In some embodiments, the concentration of collagenase is approximately 5 mg / mL. In some embodiments, the concentration of collagenase is 10 mg / mL. In some embodiments, the collagenase is type IV collagenase. In some embodiments, the collagenase is type I / II collagenase. In some embodiments, enzymatic digestion is carried out using a medium containing, for example, 5 mg / mL or about 5 mg / mL to 10 mg / mL or about 10 mg / mL of type IV collagenase. In some embodiments, enzymatic digestion is carried out using a medium containing, for example, 5 mg / mL or about 5 mg / mL to 10 mg / mL or about 10 mg / mL of type I / II collagenase. In some embodiments, if milder digestion is desired, 5 mg / mL or about 5 mg / mL of collagenase is used.In some embodiments, if more complete digestion is desired, higher concentrations of collagenase, such as 10 mg / mL or about 10 mg / mL, are used. In some embodiments, the collagenase is type IV collagenase. In some embodiments, the collagenase is type I / II collagenase.

[0125] In some embodiments, enzymatic digestion may be carried out in part using hyaluronidase. Hyaluronidase is a hyaluronic acid metabolic enzyme that subsequently enhances cell membrane permeability (Eikenes et al. Anticancer Research, 2010). Enzymes such as hyaluronidase may be present in the medium for enzymatic digestion at concentrations of 5 mg / mL or about 5 mg / mL to 10 mg / mL or about 10 mg / mL, for example, 5 mg / mL or about 5 mg / mL, 6 mg / mL or about 6 mg / mL, 7 mg / mL or about 7 mg / mL, 8 mg / mL or about 8 mg / mL, 9 mg / mL or about 9 mg / mL, 10 mg / mL or about 10 mg / mL, or any value within any of the aforementioned ranges. In some embodiments, enzymatic digestion is carried out using a medium containing type II hyaluronidase, for example, at a concentration of 5 mg / mL or about 5 mg / mL to 10 mg / mL or about 10 mg / mL. In some embodiments, if milder digestion is desired, 5 mg / mL or about 5 mg / mL of hyaluronidase is used. In some embodiments, if more complete digestion is desired, higher concentrations of hyaluronidase, such as 10 mg / mL or about 10 mg / mL of hyaluronidase, are used.

[0126] In some embodiments, DNase is also present in the culture medium during enzymatic digestion. DNase is an enzyme that degrades any free DNA released into the culture medium as a result of the tumor fragment digestion process. Enzymes such as DNase I may be present in the culture medium for enzymatic digestion at concentrations of 5,000 units / mL or about 5,000 units / mL to 10,000 units / mL or about 10,000 units / mL, for example, 5,000 units / mL or about 5,000 units / mL, 6,000 units / mL or about 6,000 units / mL, 7,000 units / mL or about 7,000 units / mL, 8,000 units / mL or about 8,000 units / mL, 9,000 units / mL or about 9,000 units / mL, 10,000 units / mL or about 10,000 units / mL, or any value within the range of any of the aforementioned values. In some embodiments, enzymatic digestion is carried out using a medium containing DNase I at a concentration of, for example, 5,000 units / mL or about 5,000 units / mL to 10,000 units / mL or about 10,000 units / mL.

[0127] In some embodiments, enzymes from the Miltenyi Human Tumor Dissociation Kit (e.g., catalog O.130-095-929, Miltenyi Biotec) may be used. The enzyme medium containing the enzymes may be a serum-free medium such as any of those described. In certain embodiments, the enzyme medium comprises hyaluronidase and / or collagenase, e.g., Roswell Park Memorial Institute (RPMI) 1640 buffer, 2 mM glutamic acid (e.g., GlutaMAX), 10 mg / mL gentamicin, 10,000 units / mL DNase I, 10 mg / mL collagenase, and 10 mg / mL hyaluronidase.

[0128] Next, the tumor fragments are mechanically dissected and the TILs are dissociated, for example, using a tissue dissociator. An example of a tissue dissociator is GentleMACs® (Miltenyi Biotec) for tissue homogenization. Tumor digests can be produced by placing the tumor in an enzyme medium, mechanically dissociating the tumor for approximately 1 minute, followed by incubation in 5% CO2 at 37°C for 30 minutes, and then repeating the mechanical dissociation and incubation under the aforementioned conditions until only small tissue fragments remain. In some embodiments, the tumor digests are subjected to homogenization and enzymatic digestion by incubation in an enzyme cocktail for 15 minutes to 2 hours, for example, 30 minutes or about 30 minutes to 60 minutes. In some embodiments, the tumor digests are subjected to homogenization and enzymatic digestion by incubation in an enzyme cocktail for about 60 minutes. At the end of this process, if the cell suspension contains a large number of erythrocytes or dead cells, these cells can be removed by performing density gradient separation using FICOLL. In some embodiments, single-cell suspensions are prepared by lining cells with a filter to remove debris, such as a 70 μm strainer, after processing tumor fragments. In some cases, separation can be achieved by centrifugation, in which case the cell pellet can be resuspended and filtered through, for example, a 70 μm strainer to remove debris. Alternative methods known in the art, such as the method described in U.S. Patent Application Publication No. 2012 / 0244133Al, can be used, and their disclosure is incorporated herein by reference. Any of the methods described herein can be used for a method to obtain TIL for use in the provided method.

[0129] In some embodiments, the single-cell suspension to be used as an input sample is 1 × 10⁶ 6 Or approximately 1 x 10 6 Dissociated tumor cells ~1000 × 10 6 Or approximately 1000 x 10 6 Dissociated tumor cells, for example, 1 × 10 6 ~500×10 6 Dissociated tumor cells, 1 × 106 ~100×10 6 Dissociated tumor cells, 1 × 10 6 ~50×10 6 Dissociated tumor cells, 1 × 10 6 ~10×10 6 Dissociated tumor cells, 10 × 10 6 ~1000×10 6 Dissociated tumor cells, 10 × 10 6 ~100×10 6 Dissociated tumor cells, 10 × 10 6 ~500×10 6 Dissociated tumor cells, 10 × 10 6 ~50×10 6 Dissociated tumor cells, 50 × 10 6 ~1000×10 6 Dissociated tumor cells, 50 × 10 6 ~500×10 6 Dissociated tumor cells, 50 × 10 6 ~100×10 6 Dissociated tumor cells, 100 × 10 6 ~1000×10 6 Dissociated tumor cells, 100 × 10 6 ~500×10 6 Dissociative tumor cells, or 500 × 10 6 ~1000×10 6 It contains dissociative tumor cells. In some embodiments, the single-cell suspension for use as an input sample for T cells is 10 × 10 6 Or approximately 10 x 10 6 Or at least 10 x 10 6 Or at least about 10 x 10 6 Dissociated tumor cells, 20 × 10 6 Dissociated tumor cells, 30 × 10 6 Dissociated tumor cells, 40 x 10 6 Dissociated tumor cells, 50 x 10 6 Dissociated tumor cells, 60 x 10 6 Dissociated tumor cells, 70 x 10 6 Dissociated tumor cells, 80 x 10 6 Dissociated tumor cells, 90 x 10 6 Dissociated tumor cells, or 100 × 10 6Contains dissociated tumor cells. In some embodiments, the single-cell suspension for use as an input sample for T cells is 10 × 10 6 Or approximately 10 x 10 6 Dissociated tumor cells ~100 × 10 6 Or approximately 100 x 10 6 Includes dissociated tumor cells.

[0130] B. Cell selection In embodiments of the provided method, the method involves selecting or enriching cells from a first population of T cells (e.g., dissociated tumor cells) that are likely or suspected to be tumor-reactive T cells by selecting or isolating T cells that are surface-positive for the exhaustion markers PD-1 and CD39 from a subset of lymphocytes. In some embodiments, PD-1 and CD39-positive T cells are selected or enriched from a first population of T cells obtained from a biological sample, such as those described in Section II.A. In certain embodiments, the selection is from cells present in a single-cell suspension dissociated tumor cell sample. In some embodiments, the enriched or selected population of cells is used in subsequent processing steps, such as subsequent processing steps involving proliferation according to the provided method.

[0131] Methods for selecting surface receptors on cells may involve one of several techniques, such as antibody binding with an antibody-specific reagent, followed by concentration by magnetic separation or fluorescence-activated cell sorting (FACS).

[0132] In some embodiments, cells are selected directly from an input sample of a single-cell suspension prepared by enzymatic or mechanical digestion of tumor fragments, where selection is performed using CD39 / PD1 positive selection. In some embodiments, the selected cells are then stimulated for proliferation by incubation or culture in the presence of one or more of IL-2, IL-7, IL-15, or IL-21, for example, using the described method. In some embodiments, stimulation does not involve culture with an anti-CD3 antibody (OKT3) or other co-stimulatory molecules. In some embodiments, stimulation may include culture with an anti-CD3 antibody (OKT3) or other co-stimulatory molecules.

[0133] In some embodiments, selection is achieved by enriching a particular cell population through positive selection, or by depleting a particular cell population through negative selection. In some embodiments, positive or negative selection is achieved by expressing, or at relatively high levels of (marker) markers on the cells that have been positively or negatively selected, respectively. 高 (marker) expressed in + This is achieved by incubating cells with one or more antibodies or other conjugates that specifically bind to one or more surface markers.

[0134] In certain embodiments, the T cell population includes both CD4+ and CD8+ T cells. Many cancers, including solid tumors such as many common epithelial indications (e.g., GI), express class I and class II restrictive mutations. It is intended that both CD8+ T cells that recognize class I MHC restrictive molecules and CD4+ T cells that recognize class II MHC restrictive molecules are required for T cell products to target such indications, e.g., common epithelial indications.

[0135] In some embodiments, the method includes selecting PD-1+ and CD39+ cells from a lymphocyte subject of cells including CD4+ and CD8+ T cells. In one method, lymphocytes can be selected by positive selection of CD3+ cells. In another method, lymphocytes can be selected by positive selection of CD4+ and / or CD8+ cells. In some embodiments, positive selection of CD4 and positive selection of CD8 are used. + or CD8 + The selection process is CD4 + Helper and CD8 + It is used to isolate cytotoxic T cells. In some embodiments, such selection is performed simultaneously, and in other embodiments, it is performed sequentially in either order. In some embodiments, the method includes enriching CD4+ and CD8+ T cells by selecting T cells that are surface-positive for CD3, or by sequentially or simultaneously selecting T cells that are surface-positive for CD4 and T cells that are positive for CD8. Such CD3+ T cells, or CD4 + and / or CD8 + The population can be further subdivided into subpopulations by positive or negative selection of markers expressed, or expressed to relatively high degrees, on T cells that have tumor-reactive T cells or T cell markers associated with tumor-reactive T cells (e.g., as described above).

[0136] In some embodiments, the selection produces a population of enriched cells, for example, a population of cells enriched for CD3+ T cells that are further positive for PD-1 and CD39, or for CD4+ and CD8+ cells. In some embodiments, such cells include or are enriched with tumor-reactive T cells or T cells associated with tumor-reactive T cells. In some embodiments, the selection produces a population of enriched cells, for example, a population of cells enriched for CD3+ T cells or for CD4+ and CD8+ cells that are further positive for PD-1 and CD39, and enriched compared to a population of cells isolated from a biological sample derived from a patient (e.g., a cancer patient).

[0137] In some embodiments, enriching T cells that are surface-positive for one or more cell surface markers includes any method for separation based on such markers. In some embodiments, the separation is affinity or immunoaffinity-based. For example, isolation in some embodiments includes the separation of cells or cell populations based on the expression level of one or more markers, typically cell surface markers, by incubation with an antibody or binding partner that specifically binds to such markers, then generally followed by a washing step, and separating cells bound to the antibody or binding partner from cells not bound to the antibody or binding partner. Methods for cell selection include, but are not limited to, bead selection (e.g., serial bead passage for positive / negative cell selection), immunoaffinity chromatography (e.g., serial elution for positive / negative selection), and flow cytometry sorting. When used according to the provided methods, the selection methods meet GMP standards.

[0138] Incubation is generally carried out under detectable conditions for molecules such as antibodies or binding partners, or secondary antibodies or other reagents that specifically bind to such antibodies or binding partners, which are attached to magnetic particles or beads and / or are detectably labeled, and which, if present on cells in the sample, specifically bind to cell surface molecules. In some embodiments, antibody-bound cells can be recovered or separated from unbound cells in the sample.

[0139] In some embodiments, a combination of positive and negative selection is performed during the same selection step, and the positive and negative fractions are retained for further processing or subjected to a further separation step. Such a separation step may be based on positive selection, in which cells bound to the reagent are retained for further use, and / or negative selection, in which cells not bound to the antibody or binding partner are retained. In some embodiments, both fractions are retained for further use. In some embodiments, negative selection may be particularly useful when there are no available antibodies that specifically identify cell types in heterogeneous populations, and as a result, separation is best performed based on markers expressed by cells other than the desired population.

[0140] In some embodiments, T cells for use in connection with the provided method may be concentrated or separated by a variety of methods, including but not limited to magnetic bead separation, fluorescent cell sorting, and disposable sealed cartridge-based cell sorters. In certain embodiments, one or more reagents specific to T cells or subsets thereof may be used, such as reagents specific to T cell activation markers for selecting reactive cells, including but not limited to fluorescent antibodies, nanoparticles, or beads on cell sorters, including but not limited to CliniMACS, Sony FX500, or Tyto cell sorting systems (Miltenyi).

[0141] In certain embodiments, cell selection is performed by flow cytometry-based cell sorting. Compared to other methods, flow cytometry-based cell sorting has the advantage of being able to isolate cells in a single step based on multiple parameters for each cell, thereby achieving higher cell yields and higher purity that may not be possible with bead-based (e.g., magnetic bead-based) separation. Furthermore, multi-parameter cell staining and separation enable simultaneous labeling, identification, and sorting of multiple antigens and characteristic fluorescent signals. Using flow cytometry sorting, specific populations can be removed and isolated based on complex cell surface phenotypes in a single process. Cell selection sorting instruments with sufficiently high throughput to handle large volumes and cell numbers can be used. Non-limiting cell sorting instruments include, for example, the Sony FX500 or the Tyto cell sorting system (Miltenyi). When used in the provided method, the flow cytometer instrument is GMP compliant. Cell sorting methods for achieving multi-parameter sorting of two or more cell surface markers (e.g., CD39 and PD-1) can be carried out using compatible multi-color fluorescent reagents. Selecting appropriate phosphors and reagents, such as by choosing bright phosphors and phosphors with little or no spectral overlap, is within the scope of the skills of those skilled in the art.

[0142] In certain embodiments, the sample is brought into contact with a binder, for example, a detectably labeled binder that specifically binds to a cell surface marker. In certain embodiments, the detectably labeled binder is fluorescently labeled. In certain embodiments, T cells labeled with a cell surface marker-specific binder are identified by flow cytometry. In certain embodiments, the method further includes separating any of the resulting T cells labeled with the binder from other components of the sample to produce a composition enriched with T cells positive for one or more cell surface markers. Cell selection and sorting equipment with sufficiently high throughput to handle large volumes and cell numbers can be used. Non-limiting cell sorting equipment includes, for example, the Sony FX500 or the Tyto cell sorting system (Miltenyi).

[0143] In some embodiments, any antibody reagent used to select cells according to the provided method is a GMP antibody reagent. In some embodiments, the reagent is an analyte-specific reagent (ASR).

[0144] In some embodiments, cells surface-positive for CD39 and PD-1 are selected. In some cases, the staining method may also include selecting CD45, CD4, and / or CD8 T cells from the sample. In some embodiments, multiparameter flow cytometry is performed. In some embodiments, multiparameter flow cytometry is accompanied by a sequential gating strategy. In some cases, CD45-negative cells, such as erythrocytes, can be excluded using CD45 expression, which may be coupled with lateral scattering by choice. In some embodiments, lymphocytes are gated based on CD45 expression and their scattering (e.g., FSClow, SSClow). In some embodiments, T cells may be gated based on positive expression of CD4+ and CD8+ cells. Within the T cell population, cells positive for CD39 and PD-1 can be identified for sorting.

[0145] Methods and antibody reagents for selecting cells positive for these markers are known and commercially available. Various fluorescent materials can conjugate to antibodies and be used in multi-parameter flow cytometry. In some embodiments, multicolor staining or labeling is performed using multiple fluorescent materials, where multiple staining reagents for different cell surface markers are incubated with the cells. In some embodiments, the fluorescent markers conjugated to one or more staining reagents, such as antibodies, are selected to minimize energy transfer between them, for example, by avoiding or minimizing overlapping emission and absorption spectra. In some embodiments, each fluorescent marker has a different emission spectrum. In some embodiments, multiple fluorescent markers can be excited at a single wavelength or multiple wavelengths, but detection occurs in regions where the peak emission spectra do not overlap. In some embodiments, the excitation of one or more fluorescent markers may be by light of a single or the same wavelength, thereby emitting light of different wavelengths.

[0146] In some embodiments, any fluorescent marker or phosphor suitable for use in flow cytometry analysis can be used. Some non-limiting examples of fluorescent markers include fluorescent proteins (e.g., GFP, YFP, RFP), fluorescent moieties (e.g., fluorescein isothiocyanate) (FITC), phycoerythrin (PE), allophycocyanin (APC), Alexa Fluor (AF), nucleic acid stains (e.g., 4',6-diamidino-2-phenylindole (DAPI), SYT016, propidium iodide (PI)), cell membrane stains (e.g., FMI-43), cellular functional dyes (e.g., Fluo-4, Indo-1), and synthetic dyes (e.g., Brilliant Violet (BV)).Exemplary fluorophores include hydroxycoumarin, Cascade Blue, Dyright405 Pacific Orange, Alexa Fluor430, Fluorescein, Oregon Green, Alexa Fluor488, BODIPY493, 2,7-Diochlorofluorescien, ATTO488, Chromeo488, Dylight488, HiLyte488, Alexa Fluor532, Alexa Fluor555, ATTO550, BODIPYTMR-X, CF555, Chromeo546, Cy3, TMR, TRITC, Dy547, Dy548, Dy549, HiLyte555, Dylight550, BODIPY564, Alexa Fluor568, Alexa Fluor594, Rhodamine, Texas Red, Alexa Fluor610, Alexa Fluor633, Dylight633, and Alexa Fluor. Fluor647, APC, ATTO655, CF633, CF640R, Chromeo642, Cy5, Dylight650, Alexa Fluor680, IRDye680, Alexa Fluor700(AF700), Cy5.5, ICG, Alexa Fluor750, Dylight755, IRDye750, Cy7, PE-Cy7, Cy7.5, Alexa Examples include, but are not limited to, Fluor790, Dylight800, IRDye800, BV421, BV510, BV570, BV605, BV650, BV711, BV750, BV785, Qdot(registered trademark) 525, Qdot(registered trademark) 565, Qdot(registered trademark) 605, Qdot(registered trademark) 655, Qdot(registered trademark) 705, or Qdot(registered trademark) 800. In some embodiments, one or more fluorescent markers each individually contain a phosphor selected from the group consisting of PE-Cy7, APC, AF700, BV421, Aqua, and BV605.

[0147] Table 1 lists exemplary antibodies for use in staining and selecting or dispersing cells as described herein. [Table 1] JPEG2026510460000003.jpg38159

[0148] In some embodiments, the antibody or binding partner is labeled with one or more detectable markers to facilitate separation for positive and / or negative selection. For example, separation may be based on binding to a fluorescently labeled antibody. In some embodiments, cell separation based on the binding of an antibody or other binding partner specific to one or more cell surface markers is performed in a fluid stream, which is done, for example, by a preparation-scale fluorescence-activated cell preparatory (FACS) and / or microelectromechanical system (MEMS) chip, including a preparation-scale (FACS) combined with a flow cytometry detection system. In some embodiments, the cell population described herein is collected via flow cytometry, enriched (or depleted), and cells stained for multiple cell surface markers (e.g., with antibody-conjugated fluorescent peptides) are transported in a fluid stream.

[0149] In some embodiments, cell staining involves incubation with an antibody or binding partner that specifically binds to a described marker, followed in some embodiments by a washing step and separation of cells bound to the antibody or binding partner from cells not bound to the antibody or binding partner. In some aspects of such a process, a certain amount of cells is mixed with a certain amount of the desired staining reagent and incubated under conditions for cell staining. In some embodiments, staining or labeling is carried out at a temperature of 0°C to 25°C, for example, 4°C or about 4°C. In some embodiments, staining or labeling is carried out for more than 5 minutes, typically more than 15 minutes. In some embodiments, staining or labeling is carried out for 15 minutes to 6 hours, for example, 30 minutes to 2 hours. In some embodiments, staining or labeling is carried out for, for example, 15 minutes or about 15 minutes, 30 minutes, 1 hour, 1.5 hours, 2 hours, 2.5 hours, 3 hours, or any value within the range of any of the aforementioned values. In some embodiments, labeling with one or more staining reagents is carried out simultaneously. In some embodiments, one or more washing steps are performed before introducing the sample into a flow cytometer for analysis.

[0150] In some embodiments, the cell sample is 1 × 10 to allow the cells to pass through the flow cytometer for reading. 6 ~5×10 7 It is prepared by suspending single cells at a density of cells / ml. In some embodiments, the cell density for preparation is 5 × 10⁻⁶. 6 cells / mL~50×10 6 cells / mL, e.g., 20 × 10 6 Cells / mL or approximately 20 × 10 6 The cell density is cells / mL. In some embodiments, this concentration of cells is called a fluid sheath. In some embodiments, the fluid sheath influences the rate of flow sorting, which typically proceeds at about 2,000–20,000 cells (events) per second. The fluid sheath of a cell sample is typically made from phosphate-buffered saline, but other solutions are available, as is known and understood by those skilled in the art.

[0151] In some embodiments, the flow cytometry sorting rate is 2,000 events / second to 10,000 events / second. In some embodiments, the flow cytometry sorting rate is approximately 2,000 events / second, approximately 3,000 events / second, approximately 4,000 events / second, approximately 5,000 events / second, approximately 6,000 events / second, approximately 7,000 events / second, approximately 8,000 events / second, approximately 9,000 events / second, approximately 10,000 events / second, approximately 15,000 events / second, or approximately 20,000 events / second, or any value within the range of any of the aforementioned values. In some embodiments, the flow cytometry sorting rate is approximately 6,000 events / second.

[0152] In some embodiments, the sample is introduced into a flow cytometer. The cell sample is typically compressed under hydraulic pressure and narrowed into a single flow through a fluid system. This flow then passes through one or more light-scattering or fluorescence-emitting beams. Lasers typically function as the light source for flow cytometers. Lasers generate single-wavelength light, and upon contact with the cell sample, they produce forward-scattered light as a measure of cell size, lateral-scattered light as a measure of cell complexity, and lateral fluorescence proportional to the relative amount of specific cell markers. Fluorescence channels are typically designated as FL1, FL2, FL3, etc., depending on the number of channels in the instrument. Each fluorescence channel has a barrier filter that detects a specific selected dye while filtering out all others. The channel in which a particular dye is primarily detectable is called its primary fluorescence channel, while other fluorescence channels may be called secondary channels. The scattered and fluorescence signals are converted into electron pulses, which are processed by a flow cytometry engine and displayed in a graphical user interface (GUI).

[0153] Methods for analyzing flow cytometry or FACS data may involve data "gating" to separate specific cell populations. Different cell types can be identified by scattering parameters and fluorescence emission resulting from labeling various cellular proteins with dye-labeled antibodies, as described above. Cluster identification, and thereby population identification, can be performed by cell "gating". In some embodiments, gates corresponding to a subset of particles of interest, such as a nascent antigen-reactive TIL expression marker, are defined by the user with the assistance of software operably associated with the flow system, as described above.

[0154] In some embodiments, the gate may be a “threshold” gate, which is a gate for only one optical parameter that defines an open region in multidimensional space. In some embodiments, “threshold” gating can be used for forward light scattering to remove high-frequency low-level signals caused by interference, such as debris in the sample. In some embodiments, “window” gating is used, for example, by defining upper and lower limits on the signal value. In some embodiments, gating is performed on a 2D plot of two parameters, such as side scattering (e.g., vertical axis) and fluorescence signal (e.g., horizontal axis).

[0155] In some embodiments, flow cytometry of cell surface markers includes gating with an "F minus 1" (FMO) control. FMO gating includes a separate portion of the same sample stained with a panel of detectably labeled binders containing all but one of the drugs. The distribution of signals from the removed fluoresces can be used to define the positive threshold for the missing label, since all cells are known to be negative in the control. The position of all gates can be determined using an FMO control in which the antibody against the marker under investigation is replaced with the appropriate isotype control. In an exemplary method, the gate can be drawn using cells stained with an FMO cocktail around cells that are positive for CD45, CD4 and / or CD8, PD1 and CD39. In some embodiments, sequential gates can be used to reach a selected subpopulation. For example, a method can be implemented to gate cells with CD45-positive cells, then CD4 and / or CD8-positive cells, and then cells that are positive for PD-1 and CD39. In some embodiments, a viability dye may also be added. An exemplary viability dye is 7-ADD. In some embodiments, a 7-AAD-negative (7-AAD) neg A gate can be drawn around the cell of ).

[0156] In some embodiments, cells are separated and collected into a population of single cells. The selected population of cells is used as an input for proliferation, as described in Section II.C.

[0157] Selection does not necessarily result in 100% enrichment or removal of a particular cell population or cells expressing a specific marker. For example, positive selection or enrichment of a particular type of cell, such as cells expressing a marker, refers to increasing the number or percentage of such cells, but does not require the complete absence of cells that do not express the marker.

[0158] In some embodiments, the enriched cell population consists of enriched cells derived from the aforementioned starting sample, and the proportion of cells of a particular phenotype in the enriched cell population, e.g., tumor-reactive CD3+ T cells or CD3+ T cells surface-positive for one or more T cell markers, e.g., PD-1 and CD39, is increased by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 500%, 1000%, 5000%, or more than the proportion of such cells in the starting sample. In some embodiments, the purity of tumor-reactive CD3+ T cells or CD3+ T cells surface-positive for PD-1 and CD39 relative to the total cells in the enriched cell population is at least 90%, 91%, 92%, 93%, 94%, and generally at least 95%, 96%, 97%, 98%, 99%, or more.

[0159] C. Stimulation of T cells for proliferation The provided method further comprises growing a selected population of TILs with one or more lymphocyte T-cell stimulants under conditions that produce a population of proliferated T cells. Thus, the provided method involves ex vivo proliferation and production of T-cell therapeutic compositions, particularly for use in connection with the treatment of cancer.

[0160] In some embodiments, incubation or culture with one or more T cell stimulants results in the proliferation or growth of selected T cells, or a desired subset or subtype thereof, or their viable cells, for use in subsequent steps of the provided method. Non-limiting examples of T cell stimulants and conditions for incubation or culture are described herein.

[0161] Therefore, among the methods provided are those for culturing T cells for the production of tumor-reactive T cells, which involve culturing or incubating T cells in the presence of a T cell stimulant under conditions that promote T cell proliferation.

[0162] In some embodiments, the T cell stimulator(s) comprises recombinant T cell stimulating cytokines, e.g., IL-2, IL-7, IL-15, IL-21, IL-25, IL-23, IL-27, and / or IL-35. In some embodiments, the T cell stimulating cytokine comprises IL-2 alone or in combination with another cytokine from among IL-7, IL-15, IL-21, IL-25, IL-23, IL-27, and / or IL-35. In some embodiments, the T cell stimulator(s) comprises recombinant T cell stimulating cytokines, e.g., IL-2, IL-7, IL-15, IL-21, IL-25, and / or IL-23. In some embodiments, the T cell stimulating cytokine comprises IL-2 alone or in combination with another cytokine from among IL-7, IL-15, IL-21, IL-25, and / or IL-23. In some embodiments, the T cell-stimulating cytokine is one, two, three, or more of IL-2, IL-7, IL-15, and IL-21. In some embodiments, the T cell-stimulating cytokine includes IL-2 alone or in combination with another cytokine from IL-7, IL-15, and / or IL-21. In some embodiments, the T cell-stimulating cytokine includes IL-2 alone or in combination with another cytokine from IL-25, IL-23, IL-27, and / or IL-35. In some embodiments, the T cell-stimulating cytokines are IL-7 and IL-15.

[0163] In some embodiments, the selection of cytokines or combinations of cytokines is within the realm of those skilled in the art, insofar as the cytokines or cytokines provide activity that stimulates T cell proliferation. The activity that stimulates tumor-reactive T cells may be direct or indirect. In some embodiments, one or more cytokines directly stimulate the expansion or proliferate of tumor-reactive T cells. In some embodiments, one or more cytokines suppress T regulatory T cells (Tregs), thereby indirectly stimulating or enhancing the proliferation of desired tumor-reactive T cells.

[0164] In some embodiments, T cell stimulants include anti-CD3 (e.g., anti-CD3 antibody, OKT3, etc.), anti-CD28 reagents (e.g., anti-CD28 antibody), anti-CD3 antibody (e.g., OKT3), and anti-CD28 antibody and / or one or more recombinant cytokines (e.g., IL-2, IL-7, IL-21, and / or IL-15).

[0165] In some embodiments, incubation with T cell stimulants for proliferation does not include incubation with agents(s) that conjugate co-stimulatory molecules such as CD3 and CD28. In some embodiments, incubation with T cell stimulants for proliferation does not include incubation with anti-CD3 antibodies, e.g., OKT3. In some embodiments, incubation with T cell stimulants for proliferation does not include incubation with anti-CD3 antibodies(s) / anti-CD28 antibodies presented by APCs and immobilized on the surface of an organism (e.g., beads), or as soluble antibodies. In some embodiments, incubation with T cell stimulants for proliferation does not include incubation with soluble anti-CD3, e.g., OKT3. In some embodiments, incubation with T cell stimulants for proliferation does not include incubation with anti-CD3 / anti-CD28, including such reagents immobilized on beads, provided as, for example, Dynabeads. In some embodiments, incubation with T cell stimulants for proliferation does not include incubation with APCs, such as irradiated APCs. In some embodiments, incubation with T cell stimulants for proliferation does not include incubation with non-dividing PBMCs, such as irradiated PBMCs.

[0166] In some of the provided embodiments, the T cell stimulant(s) are selected from agents that initiate intracellular TCR / CD3 signaling and agents that initiate signaling via costimulatory receptors. In some of the provided embodiments, the agent that initiates intracellular TCR / CD3 signaling is an anti-CD3 antibody such as OKT3. In some of the provided embodiments, the agent that initiates signaling via costimulatory receptors includes peripheral blood mononuclear cells (PBMCs), optionally undivided or irradiated PBMCs. In some of the provided embodiments, the agent that initiates signaling via costimulatory receptors is an anti-CD28 antibody. In some of the provided embodiments, the T cell stimulant(s) are anti-CD3 antibodies and anti-CD28 antibodies, each being soluble. In certain embodiments, one or more recombinant cytokines are also present as additional T cell stimulants during incubation. In some embodiments, incubation with T cell stimulants(s) includes at least one T cell stimulating recombinant cytokine (e.g., recombinant IL-2, IL-7, IL-21, IL-15, IL-25, IL-23, IL-27, and / or IL-35) as well as further T cell stimulants(s) that bind CD3 and / or co-stimulatory molecules (e.g., CD28) on T cells.

[0167] In embodiments of the provided method, the stimulating condition includes one or more agents, e.g., ligands that turn on or initiate the TCR / CD3 intracellular signaling cascade in T cells and / or costimulatory signals in T cells. Such agents may include antibodies, e.g., those specific to TCR components, e.g., anti-CD3, and / or costimulatory receptors, e.g., anti-CD28 or anti-4-1BB. In some embodiments, such agents are added to the culture medium as soluble antibodies. In other embodiments, such agents are conjugated to a solid support, such as beads. In some embodiments, the T cell stimulant(s) may be anti-CD3 / CD28 conjugate magnetic beads (e.g., DYNABEADS® M-450 CD3 / CD28T Cell Expander).

[0168] Anti-CD3 antibodies may include any antibody directed towards or capable of specifically binding to the CD3 receptor on the surface of T cells, typically human CD3 on human T cells. Anti-CD3 antibodies include OKT3, also known as muromonab. Anti-CD3 antibodies also include UHCTI clones, also known as T3 and CD3E. Other anti-CD3 antibodies include, for example, otelixizumab, teprizumab, and bicilizumab. Anti-CD3 antibodies can be added as soluble reagents or conjugated to beads. In certain embodiments, the anti-CD3 antibody is soluble.

[0169] In certain embodiments, the T cell stimulant(s) comprises an anti-CD3 antibody, which is added to the cell culture medium during incubation. In some embodiments, the anti-CD3 antibody is present in concentrations ranging from 0.1 or about 0.1 ng / mL to 50 ng / mL, for example, 0.5 or about 0.5 ng / mL to 50 or about 50 ng / mL, 0.5 or about 0.5 ng / mL to 30 or about 30 ng / mL, 0.5 or about 0.5 ng / mL to 15 or about 15 ng / mL, 0.5 or about 0.5 ng / mL to 5 or about 5 ng / mL, 0.5 or about 0.5 ng / mL to 1 or about 1 ng / mL, 1 or about 1 ng / mL to 50 or about 50 ng / mL, 1 or about 1 ng / mL to 30 or about 3 It is added at concentrations in the following ranges: 0 ng / mL, 1 or approximately 1 ng / mL to 15 or approximately 15 ng / mL, 1 or approximately 1 ng / mL to 5 or approximately 5 ng / mL, 5 or approximately 5 ng / mL to 50 or approximately 50 ng / mL, 5 or approximately 5 ng / mL to 30 or approximately 30 ng / mL, 5 or approximately 5 ng / mL to 15 or approximately 15 ng / mL, 15 or approximately 15 ng / mL to 50 or approximately 50 ng / mL, 15 or approximately 15 ng / mL to 30 or approximately 30 ng / mL, or 30 or 30 ng / mL to 50 or approximately 50 ng / mL (including both ends).

[0170] In certain embodiments, the anti-CD3 antibody is OKT3. In one embodiment, the cell culture medium contains OKT3 antibody in concentrations of approximately 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 2.5 ng / mL, 5 ng / mL, 7.5 ng / mL, 10 ng / mL, 15 ng / mL, 20 ng / mL, 25 ng / mL, 30 ng / mL, 35 ng / mL, 40 ng / mL, 50 ng / mL, 60 ng / mL, 70 ng / mL, 80 ng / mL, 90 ng / mL, 100 ng / mL, 200 ng / mL, 500 ng / mL, and 1 μg / mL. In one embodiment, the cell culture medium contains OKT3 antibodies in concentrations of 0.1 ng / mL to 1 ng / mL, 1 ng / mL to 5 ng / mL, 5 ng / mL to 10 ng / mL, 10 ng / mL to 20 ng / mL, 20 ng / mL to 30 ng / mL, 30 ng / mL to 40 ng / mL, 40 ng / mL to 50 ng / mL, and 50 ng / mL to 100 ng / mL.

[0171] In some embodiments, the T cell stimulant(s) may include incubation with an anti-CD3 antibody and incubation with a further agent that specifically binds to CD28 in the cells or stimulates or induces CD28-mediated signaling. In some embodiments, the CD28-mediated signaling may be initiated or provided by an anti-CD28 antibody or its antigen-binding fragment. In some embodiments, the CD28-mediated signaling may be provided by antigen-presenting feeder cells (APCs), such as peripheral blood mononuclear cells (PBMCs).

[0172] In some embodiments, the T cell stimulant(s) may be added to a population of T cell feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMCs). In some embodiments, the non-dividing feeder cells may include gamma-irradiated PBMC feeder cells. In some embodiments, the PBMCs are irradiated with gamma rays in the range of about 3000–3600 rad to prevent cell division. In some embodiments, the feeder cells are added to the culture medium before the addition of the T cell population. In some embodiments, the resulting cell population contains at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be proliferated. In some embodiments, the ratio of T cells to PBMCs and / or antigen-presenting cells is about 1–25, about 1–50, about 1–100, about 1–125, about 1–150, about 1–175, about 1–200, about 1–225, about 1–250, about 1–275, about 1–300, about 1–325, about 1–350, about 1–375, about 1–400, or about 1–500.

[0173] In some embodiments, the stimulation does not involve incubation with PBMCs or other feeder cells, such as non-dividing or irradiated PBMCs or other non-dividing or irradiated feeder cells.

[0174] In some embodiments, the T-cell stimulant(s) may include adding an anti-CD28 antibody or its antigen-binding fragment to a population of cells. The anti-CD28 antibody may include any antibody directed to or capable of specifically binding to the CD28 receptor on the surface of T cells. Non-limiting examples of anti-CD28 antibodies include NA / LE (e.g., BD Pharmingen), IM1376 (e.g., Beckman Coulter), or 15E8 (e.g., Miltenyi Biotec). The anti-CD28 antibody may be added as a soluble reagent or conjugated to beads. In certain embodiments, the anti-CD3 antibody is soluble. In some embodiments, the anti-CD28 antibody is added at concentrations ranging from 1 or about 1 ng / mL to 1000 ng / mL, or about 1 ng / mL to 500 ng / mL, 1 or about 1 ng / mL to 100 or about 100 ng / mL, 1 or about 1 ng / mL to 10 or about 10 ng / mL, 10 or about 10 ng / mL to 100 or about 1000 ng / mL, 10 or about 10 ng / mL to 500 or about 500 ng / mL, 10 or about 10 ng / mL to 100 or about 1000 ng / mL, 100 or about 100 ng / mL to 1000 or about 1000 ng / mL, or 500 or about 500 ng / mL to 1000 or about 1000 ng / mL.

[0175] In some embodiments, the T cell stimulant(s) may comprise one or more recombinant cytokines. In some embodiments, the cytokines are added to the culture medium or are exogenous. Thus, in some embodiments, one or more further recombinant cytokines are also included in the culture. In some embodiments, the recombinant cytokines may comprise one or more of IL-2, IL-7, IL-15, IL-21, IL-25, IL-23, IL-27, and / or IL-35. In some embodiments, the recombinant cytokines may comprise one or more of IL-2, IL-7, IL-15, IL-21, IL-25, and / or IL-23. In some embodiments, the culture and incubation are carried out in the presence of recombinant IL-2, IL-15, and IL-7. In some embodiments, the culture is carried out in the presence of IL-2. In some embodiments, the culture is carried out in the presence of IL-15 and IL-17, which, in some embodiments, do not additionally include IL-2. In certain embodiments, the recombinant cytokine(s) are human.

[0176] Recombinant cytokines are generally recombinant human proteins. In certain embodiments, recombinant cytokines are at least 0.5 or at least about 0.5 IU / mL, at least 1.0 or at least about 1.0 IU / mL, at least 5 or at least about 5 IU / mL, at least 10 or at least about 10 IU / mL, at least 100 or at least 100 or at least about 100 IU / mL, at least 1000 or at least 1000 or at least about 1000 IU / mL, at least 1500 or at least 1500 or at least about 1500 IU / mL, at least 2000 or at least 2000 or at least about 2000 IU / mL, at least 2500 or at least 2500 or at least about 2500 IU / mL, at least 3000 or at least 3000 or at least about 3000 IU / mL, at least 3500 or at least 3500 It is present in the cell culture medium during incubation at concentrations of approximately 3500 IU / mL, at least 4000 or 4000 or about 4000 IU / mL, at least 4500 or 4500 or about 4500 IU / mL, at least 5000 or 5000 or about 5000 IU / mL, at least 5500 or 5500 or about 5500 IU / mL, at least 6000 or 6000 or about 6000 IU / mL, at least 6500 or 6500 or about 6500 IU / mL, at least 7000 or 7000 or about 7000 IU / mL, at least 7500 or 7500 or about 7500 IU / mL, or at least 8000 or 8000 or about 8000 IU / mL.In one embodiment, the cell culture medium is 10 or about 10 IU / mL to 100 or about 100 IU / mL, 100 or about 100 IU / mL to 1000 or about 1000 IU / mL, 1000 or about 1000 to 2000 or about 2000 IU / mL, 2000 or about 2000 to 3000 or about 3000 IU / mL, 3000 or about 3000 IU / mL It comprises the following concentrations: ~4000 or approximately 4000 IU / mL, 4000 or approximately 4000~5000 or approximately 5000 IU / mL, 5000 or approximately 5000~6000 or approximately 6000 IU / mL, 6000 or approximately 6000~7000 or approximately 7000 IU / mL, and 7000 or approximately 7000~8000 or approximately 8000 IU / mL (including both ends).

[0177] In some embodiments, recombinant IL-2 is present in the cell culture medium. In some embodiments, IL-2 is the only recombinant cytokine added to the culture. In some embodiments, recombinant IL-2 and one other recombinant regulatory cytokine derived from IL-7, IL-15, IL-21, IL-23, IL-25, IL-27, or IL-35 are added to the culture. IL-2 is a cytokine that supports T cell recovery and proliferation. IL-2 also supports T cell homeostasis, thereby supporting their phenotype, differentiation state, and immune memory. In some cases, the induction of regulatory T cells in the tumor microenvironment may lead to low bioavailability of IL-2. Recombinant IL-2 has been regularly used for widespread T cell proliferation in a variety of contexts. Recombinant IL-2 is commercially available. In certain embodiments, recombinant IL-2 is GMP grade (e.g., MACS GMP Recombinant Human IL-2, Miltenyi Biotec).

[0178] Recombinant IL-2 may be present in the cell culture medium during various stages of the process. In some cases, recombinant IL-2 may be present in T cell proliferation, for example, to promote TIL growth.

[0179] In some embodiments, COMPIL-2 is 10 or about 10 IU / mL to 1000 or about 1000 IU / mL, for example, 10 or about 10 IU / mL to 600 or about 600 IU / mL, 10 or about 10 IU / mL to 400 or about 400 IU / mL, 10 or about 10 IU / mL to 200 or about 200 IU / mL, 10 or about 10 IU / mL to 100 or about 100 IU / mL , 10 or approximately 10 IU / mL to 50 or approximately 50 IU / mL, 50 or approximately 50 IU / mL to 1000 or approximately 1000 IU / mL, 50 or approximately 50 IU / mL to 600 or approximately 600 IU / mL, 50 or approximately 50 IU / mL to 400 or approximately 400 IU / mL, 50 or approximately 50 IU / mL to 200 or approximately 200 IU / mL, 50 or approximately 50 IU / mL to 100 or approximately 10 0 IU / mL, 100 or approximately 100 IU / mL to 1000 or approximately 1000 IU / mL, 100 or approximately 100 IU / mL to 600 or approximately 600 IU / mL, 100 or approximately 100 IU / mL to 400 or approximately 400 IU / mL, 100 or approximately 100 IU / mL to 200 or approximately 200 IU / mL, 200 or approximately 200 IU / mL to 1000 or approximately 1000 IU / mL, 200 or approximately 200 IU / mL It is added to the culture medium at concentrations of approximately 200 IU / mL to 600 or 600 IU / mL, 200 or approximately 200 IU / mL to 400 or approximately 400 IU / mL, 400 or approximately 400 IU / mL to 1000 or approximately 1000 IU / mL, 400 or approximately 400 IU / mL to 600 or approximately 600 IU / mL, or 600 or approximately 600 IU / mL to 1000 or approximately 1000 IU / mL. In some embodiments, recombinant IL-2 is present in amounts of 50 to 400 IU / mL.

[0180] In some embodiments, growth is carried out in the presence of recombinant IL-2 added at a concentration of 200 IU / mL to 5000 or about 5000 IU / mL. In some embodiments, recombinant IL-2 is added to the culture medium at a concentration of 200 or about 200 IU / mL, 300 or about 300 IU / mL, 400 or about 400 IU / mL, 500 or about 500 IU / mL, 600 or about 600 IU / mL, 700 or about 700 IU / mL, 800 or about 800 IU / mL, 900 or about 900 IU / mL, 1000 or about 1000 IU / mL, or any concentration within the aforementioned range. In some embodiments, recombinant IL-2 is added to the culture medium at a concentration of 300 or about 300 IU / mL. In some embodiments, recombinant IL-2 is added to the culture medium at a concentration of 600 or about 600 IU / mL. In some embodiments, recombinant IL-2 is added to the culture medium at a concentration of 1000 IU / mL or about 1000 IU / mL. In some embodiments, at least one other recombinant regulatory cytokine derived from IL-7, IL-15, IL-21, IL-23, IL-25, IL-27, or IL-35 is added to the culture medium.

[0181] In some embodiments, incubation is performed with higher doses of IL-2. In some embodiments, IL-2 is the only recombinant cytokine added to the culture.

[0182] In some embodiments, COMPIL-2 is 1000 or about 1000 IU / mL to 8000 or about 8000 IU / mL, for example, 1000 or about 1000 IU / mL to 7000 or about 7000 IU / mL, 1000 or about 1000 IU / mL to 6000 or about 6000 IU / mL, 1000 or about 1000 IU / mL to 5000 or about 5000 IU / mL, 1000 or about 1000 IU / mL to 4000 or about 4000 IU / mL, 1000 or approximately 1000 IU / mL ~ 2000 or approximately 2000 IU / mL, 2000 IU / mL ~ 8000 or approximately 8000 IU / mL, 2000 or approximately 2000 IU / mL ~ 7000 or approximately 7000 IU / mL, 2000 or approximately 2000 IU / mL ~ 6000 or approximately 6000 IU / mL, 2000 or approximately 2000 IU / mL ~ 5000 or approximately 5000 IU / mL, 2000 or approximately 2000 IU / mL~4000 or approximately 4000 IU / mL, 4000 IU / mL~8000 or approximately 8000 IU / mL, 4000 or approximately 4000 IU / mL~7000 or approximately 7000 IU / mL, 4000 or approximately 4000 IU / mL~6000 or approximately 6000 IU / mL, 4000 or approximately 4000 IU / mL~5000 or approximately 5000 IU / mL, 5000 or approximately 5000 IU / mL~8000 or approximately 8000 IU / mL, 5 It is added to the culture medium at concentrations of 000 or about 5000 IU / mL to 7000 or about 7000 IU / mL, 5000 or about 5000 IU / mL to 6000 or about 6000 IU / mL, 6000 or about 6000 IU / mL to 8000 or about 8000 IU / mL, 6000 or about 6000 IU / mL to 7000 or about 7000 IU / mL, or 7000 or about 7000 IU / mL to 8000 or about 8000 IU / mL. In some embodiments, recombinant IL-2 is present in an amount of 3000 or about 3000 IU / mL.

[0183] In some embodiments, recombinant IL-15 is present in the cell culture medium. IL-15 is a cytokine involved in the homeostasis and activation of memory T cells. In some cases, IL-15 can enhance the effector function of antigen-experienced T cells in the absence of the antigen, preventing their differentiation into an exhausted phenotype. IL-15 is also involved in T cell proliferation. Recombinant IL-15 is commercially available. In certain embodiments, recombinant IL-15 is GMP grade (e.g., MACS GMP Recombinant Human IL-15, Miltenyi Biotec).

[0184] Recombinant IL-15 may be included in the cell culture medium during various stages of the process provided. In some cases, recombinant IL-15 may also be included in the culture to grow tumor-responsive T cells during the proliferation phase. In some cases, recombinant IL-15 can be combined with recombinant IL-7 to result in the activation, survival, and / or proliferation of tumor-responsive T cells in the method provided. In some such embodiments, the combination of recombinant IL-7 and IL-15 is an alternative to the use of recombinant IL-2 in the culture, and the culture medium does not contain recombinant IL-2 in addition.

[0185] In some embodiments, COMPIL-15 is 10 or about 10 IU / mL to 500 IU / mL, for example, 10 or 10 IU / mL to 400 or about 400 IU / mL, 10 or about 10 IU / mL to 300 or about 300 IU / mL, 10 or about 10 IU / mL to 200 or about 200 IU / mL, 10 or about 10 IU / mL to 100 or about 100 IU / mL, 10 or about 10 IU / mL to 70 or about 70 IU / mL, 10 or about 10 IU / mL to 50 or about 50 IU / mL, 10 or Approximately 10 IU / mL to 30 or approximately 30 IU / mL, 30 or approximately 30 IU / mL or 500 IU / mL, 30 or approximately 30 IU / mL to 400 or approximately 400 IU / mL, 30 or approximately 30 IU / mL to 300 or approximately 300 IU / mL, 30 or approximately 30 IU / mL to 200 or approximately 200 IU / mL, 30 or approximately 30 IU / mL to 100 or approximately 100 IU / mL, 30 or approximately 30 IU / mL to 70 or approximately 70 IU / mL, 30 or approximately 30 IU / mL to 50 or approximately 50 IU / mL, 50 Or approximately 50 IU / mL to 400 or approximately 400 IU / mL, 50 or approximately 50 IU / mL to 500 or approximately 500 IU / mL, 50 or approximately 50 IU / mL to 300 or approximately 300 IU / mL, 50 or approximately 50 IU / mL to 200 or approximately 200 IU / mL, 50 or approximately 50 IU / mL to 100 or approximately 100 IU / mL, 50 or approximately 50 IU / mL to 70 or approximately 70 IU / mL, 70 or approximately 70 IU / mL to 500 or approximately 500 IU / mL, 70 or approximately 70 IU / mL to 400 or Approximately 400 IU / mL, 70 or approximately 70 IU / mL to 300 or approximately 300 IU / mL, 70 or approximately 70 IU / mL to 200 or approximately 200 IU / mL, 70 or approximately 70 IU / mL to 100 or approximately 100 IU / mL, 100 or approximately 100 IU / mL to 500 or approximately 500 IU / mL, 100 or approximately 100 IU / mL to 400 or approximately 400 IU / mL, 100 or approximately 100 IU / mL to 300 or approximately 300 IU / mL, 100 or approximately 100 IU / mL to 200 or approximately 200 IU / mL,It is added to the culture medium at a concentration of 200 or about 200 IU / mL to 500 or about 500 IU / mL, 200 or about 200 IU / mL to 400 or about 400 IU / mL, 200 or about 200 IU / mL to 300 or about 300 IU / mL, 300 or about 300 IU / mL to 500 or about 500 IU / mL, 200 or about 200 IU / mL to 400 or about 400 IU / mL, or 400 or about 400 IU / mL to 500 or about 500 IU / mL. In some embodiments, IL-15 is added to the culture medium in an amount of 100 or about 100 IU / mL to 200 or about 200 IU / mL. In some embodiments, IL-15 is added to the culture medium at 180 or about 180 IU / mL.,

[0186] In some embodiments, the incubation is performed with a higher dose of IL-15.

[0187] In some embodiments, EGIL-15 is present in concentrations of 500 or approximately 500 IU / mL to 5000 or approximately 5000 IU / mL, for example, 500 or approximately 500 IU / mL to 4000 or approximately 4000 IU / mL, 500 or approximately 500 IU / mL to 2000 or approximately 2000 IU / mL, 500 or approximately 500 IU / mL to 1500 or approximately 1500 IU / mL, and 500 or approximately 500 IU / mL to 1000 or approximately 1000 IU / mL. , 500 or approximately 500 IU / mL to 750 or approximately 750 IU / mL, 750 or approximately 750 IU / mL to 5000 or approximately 5000 IU / mL, 750 or approximately 750 IU / mL to 4000 or approximately 4000 IU / mL, 750 or approximately 750 IU / mL to 2000 or approximately 2000 IU / mL, 750 or approximately 750 IU / mL to 1500 or approximately 1500 IU / mL, 750 or approximately 750 IU / mL to 1000 or approximately 1000 IU / mL, 1000 or approximately 1000 IU / mL to 5000 or approximately 5000 IU / mL, 1000 or approximately 1000 IU / mL to 4000 or approximately 4000 IU / mL, 1000 or approximately 1000 IU / mL to 2000 or approximately 2000 IU / mL, 1000 or approximately 1000 IU / mL to 1500 or approximately 1500 IU / mL, 1500 or approximately 1500 IU / mL to 5000 or approximately 5000 IU / mL, 1500 as well Alternatively, it is added to the culture medium at concentrations of approximately 1500 IU / mL to 4000 or approximately 4000 IU / mL, 1500 or approximately 1500 IU / mL to 2000 or approximately 2000 IU / mL, 2000 or approximately 2000 IU / mL to 5000 or approximately 5000 IU / mL, for example, 2000 or approximately 2000 IU / mL to 4000 or approximately 4000 IU / mL, or 4000 or approximately 4000 IU / mL to 5000 or approximately 5000 IU / mL.In some embodiments, recombinant IL-15 is added to the cell culture medium at 500 or about 500 IU / mL, 600 or about 600 IU / mL, 700 or about 700 IU / mL, 800 or about 800 IU / mL, 900 or about 900 IU / mL, 1000 or about 1000 IU / mL, 1100 or about 1100 IU / mL, 1200 or about 1200 IU / mL, 1300 or about 1300 IU / mL, 1400 or about 1400 IU / mL, 1500 or about 1500 IU / mL, 1600 or about 1600 IU / mL, 1700 or about 1700 IU / mL, 1800 or about 1800 IU / mL, 1900 or about 1900 IU / mL or 2000 or about 2000 IU / mL, or at any concentration within the range of any of the foregoing concentrations. In some embodiments, recombinant IL-15 is added to the culture medium at a concentration of 1000 or about 1000 IU / mL.

[0188] In some embodiments, proliferation is carried out in the presence of recombinant IL-15 added at a concentration of 500 IU / mL to 2000 IU / mL (e.g., 1000 or about 1000 IU / mL). In some embodiments, proliferation is carried out in the presence of recombinant IL-15 added at a concentration of 1000 or about 1000 IU / mL. In some aspects, at least one other recombinant regulatory cytokine derived from IL-2, IL-7, IL-21, IL-23, IL-25, IL-27 or IL-35 is added to the culture medium.

[0189] In some embodiments, recombinant IL-15 and IL-2 are added to the culture medium. In some embodiments, recombinant IL-15 is added at a concentration of 500 IU / mL to 2000 IU / mL (e.g., 1000 or about 1000 IU / mL), and recombinant IL-2 is added at a concentration of 200 IU / mL to 5000 IU / mL (e.g., 3000 or about 3000 IU / mL). In some embodiments, growth is carried out in the presence of recombinant IL-15 added at 1000 IU / mL and recombinant IL-2 added at 3000 IU / mL. In some embodiments, at least one other recombinant regulatory cytokine derived from IL-7, IL-21, IL-23, IL-25, IL-27, or IL-35 is added to the culture medium.

[0190] In some embodiments, recombinant IL-7 is added to the culture medium. In some embodiments, recombinant IL-7 is added to the culture medium together with either or both IL-2 or IL-15. In some embodiments, recombinant IL-7 and recombinant IL-2 are added to the culture medium. In some embodiments, recombinant IL-7 and recombinant IL-15 are added to the culture medium. In some embodiments, recombinant IL-7 (e.g., in combination with either or both IL-2 or IL-15) and one other recombinant regulatory cytokine derived from IL-23, IL-25, IL-27, or IL-35 are added to the culture medium. IL-7 is a cytokine involved in the maintenance and promotion of homeostasis of T cells. In some cases, IL-7 can boost the survival and proliferation of memory T cells, particularly the central memory compartment. Recombinant IL-7 is commercially available. In certain embodiments, recombinant IL-7 is GMP grade (e.g., MACS GMP Recombinant Human IL-7, Miltenyi Biotec).

[0191] Recombinant IL-7 may be included in the cell culture medium during various stages of the process provided. In some cases, recombinant IL-7 may also be included in the culture to promote the growth of tumor-responsive T cells during the proliferation phase. By including recombinant IL-7 during the process, the proliferation of a subset of memory T cells during the process may be maintained or supported. In some cases, recombinant IL-7 may be combined with recombinant IL-15 to result in the activation, survival, and / or proliferation of tumor-responsive T cells in the method provided. In some such embodiments, the combination of recombinant IL-7 and IL-15 is an alternative to the use of recombinant IL-2 in the culture, and the culture medium does not contain recombinant IL-2 in addition.

[0192] In some embodiments, COMPIL-7 is 100 or about 10 IU / mL to 2000 or about 2000 IU / mL, 100 or about 100 IU / mL to 1500 or about 1500 IU / mL, 100 or about 100 IU / mL to 1000 or about 1000 IU / mL, 100 or about 100 IU / mL to 800 or about 800 IU / mL, 100 or about 100 IU / mL to 600 or about 600 IU / mL, 100 or about 100 IU / mL to 400 or about 400 IU / mL, 100 or about 100 IU / mL~200 or approximately 200 IU / mL, 200 or approximately 200 IU / mL~2000 or approximately 2000 IU / mL, 200 or approximately 200 IU / mL~1500 or approximately 1500 IU / mL, 200 or approximately 200 IU / mL~1000 or approximately 1000 IU / mL, 200 or approximately 200 IU / mL~800 or approximately 800 IU / mL, 200 or approximately 200 IU / mL~600 or approximately 600 IU / mL, 200 or approximately 200 IU / mL~400 or approximately 400 IU / mL, 400 or approximately 400 IU / mL~2000 or approximately 2000 IU / mL, 400 or approximately 400 IU / mL~1500 or approximately 1500 IU / mL, 400 or approximately 400 IU / mL~1000 or approximately 1000 IU / mL, 400 or approximately 400 IU / mL~800 or approximately 800 IU / mL, 400 or approximately 400 IU / mL~600 or approximately 600 IU / mL, 600 or approximately 600 IU / mL~2000 or approximately 2000 IU / mL, 600 or approximately 600 IU / mL~1500 or approximately 1500 IU / mL, 600 or approximately 60 0 IU / mL to 1000 or approximately 1000 IU / mL, 600 or approximately 600 IU / mL to 800 or approximately 800 IU / mL, 800 or approximately 800 IU / mL to 2000 or approximately 2000 IU / mL, 800 or approximately 800 IU / mL to 1500 or approximately 1500 IU / mL, 800 or approximately 800 IU / mL to 1000 or approximately 1000 IU / mL, 1000 or approximately 1000 IU / mL to 2000 or approximately 2000 IU / mL, 1000 or approximately 1000 IU / mL to 1500 or approximately 1500 IU / mL,In some embodiments, IL-7 is added to the culture medium at a concentration of 1500 or approximately 1500 IU / mL to 2000 or approximately 2000 IU / mL. In some embodiments, IL-7 is added to the culture medium in amounts of 1000 or approximately 1000 IU / mL to 2000 or approximately 2000 IU / mL. In some embodiments, IL-7 is added to the culture medium at 600 or approximately 600 IU / mL. In some embodiments, IL-7 is added to the culture medium at 1000 or approximately 1000 IU / mL.

[0193] In some embodiments, recombinant IL-7 and IL-2 are added to the culture medium. In some embodiments, recombinant IL-7 is added at a concentration of 400 IU / mL to 2000 IU / mL (e.g., 600 or about 600 IU / mL or 1000 IU / mL), and recombinant IL-2 is added at a concentration of 200 IU / mL to 5000 IU / mL (e.g., 3000 or about 3000 IU / mL). In some embodiments, growth is carried out in the presence of recombinant IL-7 added at 1000 IU / mL and recombinant IL-2 added at 3000 IU / mL. In some embodiments, growth is carried out in the presence of recombinant IL-7 added at 600 IU / mL and recombinant IL-2 added at 3000 IU / mL. In some embodiments, at least one other recombinant regulatory cytokine derived from IL-15, IL-21, IL-23, IL-25, IL-27, or IL-35 is added to the culture medium.

[0194] In some embodiments, recombinant IL-15 and IL-7 are added to the culture medium. In some embodiments, recombinant IL-15 is added at a concentration of 500 IU / mL to 2000 IU / mL (e.g., 1000 or about 1000 IU / mL), and recombinant IL-7 is added at a concentration of 400 IU / mL to 2000 IU / mL (e.g., 600 or about 600 IU / mL or about 1000 IU / mL). In some embodiments, growth is carried out in the presence of recombinant IL-15 added at 1000 IU / mL and recombinant IL-7 added at 1000 IU / mL. In some embodiments, growth is carried out in the presence of recombinant IL-15 added at 1000 IU / mL and recombinant IL-7 added at 600 IU / mL. In some embodiments, at least one other recombinant regulatory cytokine derived from IL-2, IL-21, IL-23, IL-25, IL-27, or IL-35 is added to the culture medium.

[0195] In some embodiments, recombinant IL-21 is added to the culture medium. In some embodiments, recombinant IL-21 is added to the culture medium along with one or both of IL-2, IL-7, or IL-15. In some embodiments, recombinant IL-21 and recombinant IL-2 are added to the culture medium. In some embodiments, recombinant IL-21 and recombinant IL-15 are added to the culture medium. In some embodiments, recombinant IL-21 (e.g., in combination with one or more of IL-2, IL-7, or IL-15) and one other recombinant regulatory cytokine derived from IL-23, IL-25, IL-27, or IL-35 are added to the culture medium. IL-21 is a cytokine that supports broad-spectrum T cell activation without increasing regulatory T cell signaling. In some cases, IL-21 may support memory cell stabilization, effector function, and proliferation of antigen-experienced T cells. IL-21 can induce upregulation of effector molecules in both CD4 and CD8 T cells. Recombinant IL-21 is commercially available. In certain embodiments, the recombinant IL-21 is GMP grade (e.g., MACS GMP Recombinant Human IL-21, Miltenyi Biotec).

[0196] Recombinant IL-21 may be included in cell culture media during various stages of the supplying process. In some cases, recombinant IL-21 may also be included in cultures for growing tumor-reactive T cells during the proliferative phase to support the proliferation and stabilization of memory phenotypes.

[0197] In some embodiments, COMPIL-21 is expressed in concentrations of 0.5 or about 0.5 IU / mL to 20 or about 20 IU / mL, 0.5 or about 0.5 IU / mL to 15 or about 15 IU / mL, 0.5 or about 0.5 IU / mL to 10 or about 10 IU / mL, 0.5 or about 0.5 IU / mL to 5 or about 5 IU / mL, 0.5 or about 0.5 IU / mL to 2.5 or about 2.5 IU / mL, 0.5 or about 0.5 IU / mL to 1 or about 1 IU / mL, 1 or about 1 IU / mL to 20 or about 20 IU / mL, 1 or about 1 IU / mL to 15 or about 15 IU / mL, 1 or about 1 IU / mL to 10 or about 10 IU / mL, 1 or about 1 IU / mL to 5 or about 5 IU / mL, and 1 or about 1 IU / mL. It is added to the culture medium at concentrations of ~2.5 or approximately 2.5 IU / mL, 2.5 or approximately 2.5 IU / mL to 20 or approximately 20 IU / mL, 2.5 or approximately 2.5 IU / mL to 15 or approximately 15 IU / mL, 2.5 or approximately 2.5 IU / mL to 10 or approximately 10 IU / mL, 2.5 or approximately 2.5 IU / mL to 5 or approximately 5 IU / mL, 5 or approximately 5 IU / mL to 20 or approximately 20 IU / mL, 5 or approximately 5 IU / mL to 15 or approximately 15 IU / mL, 5 or approximately 5 IU / mL to 10 or approximately 10 IU / mL, 10 or approximately 10 IU / mL to 20 or approximately 20 IU / mL, 10 or approximately 10 IU / mL to 15 or approximately 15 IU / mL, or 15 or approximately 15 IU / mL to 20 or approximately 20 IU / mL. In some embodiments, IL-21 is added to the culture medium in an amount of 0.5 or about 0.5 IU / mL to 2.5 or about 2.5 IU / mL. In some embodiments, IL-21 is added to the culture medium in an amount of 1 or about 1 IU / mL.

[0198] In some embodiments, incubation is performed with higher doses of IL-21.

[0199] In some embodiments, EGIL-21 is present in concentrations of 500 or approximately 500 IU / mL to 5000 or approximately 5000 IU / mL, for example, 500 or approximately 500 IU / mL to 4000 or approximately 4000 IU / mL, 500 or approximately 500 IU / mL to 2000 or approximately 2000 IU / mL, 500 or approximately 500 IU / mL to 1500 or approximately 1500 IU / mL, or 500 or approximately 500 IU / mL to 1000 or approximately 1000 IU / mL. , 500 or approximately 500 IU / mL to 750 or approximately 750 IU / mL, 750 or approximately 750 IU / mL to 5000 or approximately 5000 IU / mL, 750 or approximately 750 IU / mL to 4000 or approximately 4000 IU / mL, 750 or approximately 750 IU / mL to 2000 or approximately 2000 IU / mL, 750 or approximately 750 IU / mL to 1500 or approximately 1500 IU / mL, 750 or approximately 750 IU / mL to 1000 or approximately 1000 IU / mL, 1000 or approximately 1000 IU / mL to 5000 or approximately 5000 IU / mL, 1000 or approximately 1000 IU / mL to 4000 or approximately 4000 IU / mL, 1000 or approximately 1000 IU / mL to 2000 or approximately 2000 IU / mL, 1000 or approximately 1000 IU / mL to 1500 or approximately 1500 IU / mL, 1500 or approximately 1500 IU / mL to 5000 or approximately 5000 IU / mL, 1500 as well Alternatively, it is added to the culture medium at concentrations of approximately 1500 IU / mL to 4000 or approximately 4000 IU / mL, 1500 or approximately 1500 IU / mL to 2000 or approximately 2000 IU / mL, 2000 or approximately 2000 IU / mL to 5000 or approximately 5000 IU / mL, for example, 2000 or approximately 2000 IU / mL to 4000 or approximately 4000 IU / mL, or 4000 or approximately 4000 IU / mL to 5000 or approximately 5000 IU / mL.In some embodiments, COMPIL-21 is 500 or about 500 IU / mL, 600 or about 600 IU / mL, 700 or about 700 IU / mL, 800 or about 800 IU / mL, 900 or about 900 IU / mL, 1000 or about 1000 IU / mL, 1100 or about 1100 IU / mL, 1200 or about 1200 IU / mL, 1300 or about 1300 It is added to the cell culture medium at a concentration of IU / mL, 1400 or about 1400 IU / mL, 1500 or about 1500 IU / mL, 1600 or about 1600 IU / mL, 1700 or about 1700 IU / mL, 1800 or about 1800 IU / mL, 1900 or about 1900 IU / mL, or 2000 or about 2000 IU / mL, or any concentration within any of the aforementioned concentration ranges. In some embodiments, recombinant IL-21 is added to the culture medium at a concentration of 1000 or about 1000 IU / mL.

[0200] In some embodiments, recombinant IL-21 and IL-2 are added to the culture medium. In some embodiments, recombinant IL-21 is added at a concentration of 500 IU / mL to 2000 IU / mL (e.g., 1000 or about 1000 IU / mL), and recombinant IL-2 is added at a concentration of 200 IU / mL to 5000 IU / mL (e.g., 3000 or about 3000 IU / mL). In some embodiments, growth is carried out in the presence of recombinant IL-21 added at 1000 IU / mL and recombinant IL-2 added at 3000 IU / mL. In some embodiments, at least one other recombinant regulatory cytokine derived from IL-7, IL-15, IL-23, IL-25, IL-27, or IL-35 is added to the culture medium.

[0201] In one embodiment of the method provided, tumor-reactive T cells are directly isolated after tumor digestion and subjected to only a single proliferation step, in which the proliferated T cell population is collected as a therapeutic TIL composition. In such embodiments, tumor fragments are digested into a single-cell suspension and provided as an input sample for isolation / selection of its tumor-reactive T cells. The selected cells are then proliferated and collected as a therapeutic TIL composition. In some embodiments, proliferation is carried out over a period of time to achieve a therapeutic dose. In some embodiments, proliferation is carried out to achieve a cell proliferation ratio of 200 or about 200 times to 3000 or about 3000 times. In some embodiments, proliferation is carried out to achieve a therapeutic dose of 500 million or about 500 million or more than 500 million total cells. In some embodiments, propagation takes place over 1 to 28 days, for example, 7 or about 7 to 28 days, 7 to 21 days, 7 to 14 days, for example, 7 or about 7 days, 8 days, 9 days, 10 days, 11 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, or 28 days.

[0202] In some embodiments, provided herein are methods for producing tumor-responsive T cells, the method comprising (a) selecting cells surface-positive for PD-1 and / or CD39 from an input sample containing T cells derived from a subject having a tumor to obtain the selected cells from the sample, and (b) carrying out proliferation by culturing the selected cells with one or more T cell stimulants for lymphocytes under conditions that produce a population of proliferated T cells. In some embodiments, the method comprises harvesting the population of proliferated T cells produced by the method for formulation as a therapeutic composition.

[0203] In some embodiments, provided herein are methods for producing tumor-responsive T cells, the method comprising (a) selecting cells surface-positive for PD-1 and CD39 from an input sample containing T cells derived from a subject having a tumor to obtain the selected cells from the sample, and (b) carrying out proliferation by culturing the selected cells with one or more T cell stimulants of lymphocytes under conditions that produce a population of proliferated T cells. In some embodiments, the method comprises harvesting the population of proliferated T cells produced by the method for formulation as a therapeutic composition.

[0204] In certain embodiments, the T cell stimulant(s) present during incubation for cell proliferation, for example, includes recombinant IL-2. In some embodiments, it may include one or more recombinant cytokines derived from IL-7, IL-15, IL-21, IL-25, and / or IL-23, or one or more other stimulants such as an anti-CD3 antibody (e.g., OKT-3). In the case of an anti-CD3 antibody (e.g., OKT-3), the T cell stimulant(s) may also include a co-stimulant, such as one provided by antigen-presenting feeder cells such as PBMCs, or a soluble anti-CD28 antibody.

[0205] In certain embodiments, for example, the T-cell stimulant(s) present during incubation for cell proliferation may include recombinant IL-2 and an anti-CD3 antibody.

[0206] In certain embodiments, the T-cell stimulant(s) present during incubation for cell proliferation may include recombinant IL-2, anti-CD3 antibodies, such as OKT-3, and antigen-presenting feeder cells such as PBMCs.

[0207] In certain embodiments, the T cell stimulant(s) present during incubation, for example for cell proliferation, contain recombinant IL-2, anti-CD3 antibodies such as OKT-3, and anti-CD28 antibodies. In some embodiments, the anti-CD3 antibody and / or the anti-CD28 antibody are soluble. In some embodiments, one or both of the anti-CD3 antibody and the anti-CD28 antibody are bound to a solid surface such as beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander).

[0208] In certain embodiments, the T cell stimulant(s) present during incubation, for example for cell proliferation, contain recombinant IL-2, anti-CD3 antibodies such as OKT-3, and antigen presenting feeder cells such as PBMC.

[0209] In certain embodiments, the incubation or culture of T cells is also carried out using a nutrient-containing medium so that the cells can survive ex vivo. In embodiments of the provided methods, one or more of the steps can be carried out in a serum-containing medium such as a medium containing human AB serum. The culture medium containing the T cell stimulant(s) can be a serum-free medium.

[0210] In one embodiment, the serum-free medium is OpTmizer CTS (LifeTech), Immunocult XF (Stemcell technologies), CellGro (CellGenix), TexMacs (Miltenyi), Stemline (Sigma), XVivo15 (Lonza), PrimeXV (Irvine Scientific), or Stem XVivo (Rand Dsystems).

[0211] The serum-free medium can be supplemented with a serum substitute, such as LifeTech's ICSR (Immune Cell Serum Substitute). The level of the serum substitute (e.g., ICSR) may be, for example, up to 5%, for example, about 1%, 2%, 3%, 4%, or 5%. In some embodiments, the serum-free medium contains 0.5 mM to 5 mM of L-glutamine in dipeptide form, for example, L-alanyl-L-glutamine (Glutamax®). In some embodiments, the concentration of L-glutamine in dipeptide form, such as L-alanyl-L-glutamine, is 0.5 or about 0.5 mM to 5 mM, 0.5 mM to 4 mM, 0.5 mM to 3 mM, 0.5 mM to 2 mM, 0.5 mM to 1 mM, 1 mM to 5 mM, 1 mM to 4 mM, 1 mM to 3 mM, 1 mM to 2 mM, 2 mM to 5 mM, 2 mM to 4 mM, 2 mM to 3 mM, 3 mM to 5 mM, 3 mM to 4 mM, or 4 mM to 5 mM (including both ends). In some embodiments, the concentration of L-glutamine in dipeptide form, such as L-alanyl-L-glutamine, is 2 or about 2 mM.

[0212] In some embodiments, the cells are cultured at approximately 37°C with approximately 5% CO2.

[0213] In some embodiments, incubation with T-cell stimulants(s) is performed for 1 day or approximately 1 day, for example, generally 2 days or approximately 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, or any period within the range of any of the aforementioned periods. In some embodiments, incubation with T-cell stimulants(s) is performed for 7 to 21 days, for example, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, or any period within the range of any of the aforementioned periods. In some embodiments, incubation is performed for 7 to 14 days. In some embodiments, incubation is performed for 7 to 10 days. In some embodiments, incubation is performed for 7 days or approximately 7 days. In some embodiments, incubation is performed for 8 days or approximately 8 days. In some embodiments, incubation is carried out for 9 days or approximately 9 days. In some embodiments, incubation is carried out for 10 days or approximately 10 days.

[0214] In some embodiments, incubation with T cell stimulants(s) is minimal, such that it does not result in downregulation of T cell activation markers (e.g., PD-1 and / or CD39). For example, incubation with T cell stimulants(s) in proliferation is short, such that markers CD39 and / or PD1 are still present during the sorting process and the cells do not downregulate those markers.

[0215] In some embodiments, for example, incubation with T cell stimulants (multiple) for the proliferation of T cells in the input sample is performed for 1 day or about 1 day. In some embodiments, for example, incubation with T cell stimulants (multiple) for the proliferation of T cells in the input sample is performed for 2 days or about 2 days. In some embodiments, for example, incubation with T cell stimulants (multiple) for the proliferation of T cells in the input sample is performed for 3 days or about 3 days. In some embodiments, for example, incubation with T cell stimulants (multiple) for the proliferation of T cells in the input sample is performed for 4 days or about 4 days. In some embodiments, for example, incubation with T cell stimulants (multiple) for the proliferation of T cells in the input sample is performed for 5 days or about 5 days. In some embodiments, for example, incubation with T cell stimulants (multiple) for the proliferation of T cells in the input sample is performed for 6 days or about 6 days. In some embodiments, for example, incubation with a T cell stimulant(s) for the proliferation of T cells in the input sample is carried out for 7 days or approximately 7 days.

[0216] Incubation for T cell proliferation in the input sample may be carried out under GMP conditions. In some embodiments, incubation is carried out in a closed system, and in some embodiments, it may be a closed automated system. In some embodiments, the culture medium containing the T cell stimulant(s) may be serum-free medium. In some embodiments, incubation is carried out in a closed automated system using serum-free medium.

[0217] In some embodiments, cell growth under one or more stimulating conditions is carried out in a culture vessel suitable for cell growth. In some embodiments, the culture vessel is a gas-permeable culture vessel such as a G-Rex system (e.g., G-Rex10, G-Rex10M, G-Rex100M / 100M-CS, or G-Rex500M / 500M-CS). In some embodiments, the culture vessel is a microplate, flask, bar, or other culture vessel suitable for cell growth in a closed system. In some embodiments, growth may be carried out in a bioreactor. In some embodiments, growth may be carried out using a cell growth system, for example, by transferring cells to a gas-permeable bag connected to a bioreactor (e.g., Xuri Cell Expansion System W25 (GE Healthcare)). In one embodiment, the cell proliferation system includes a culture vessel such as a bag, e.g., a gas-permeable cell bag, having a volume of approximately 50 mL, approximately 100 mL, approximately 200 mL, approximately 300 mL, approximately 400 mL, approximately 500 mL, approximately 600 mL, approximately 700 mL, approximately 800 mL, approximately 900 mL, approximately 1 L, approximately 2 L, approximately 3 L, approximately 4 L, approximately 5 L, approximately 6 L, approximately 7 L, approximately 8 L, approximately 9 L, and approximately 10 L, or any value within the range of any of the aforementioned values. In some embodiments, the process is automated or semi-automated. Suitable bioreactors for automated perfusion proliferation include, but are not limited to, the GE Xuri W25, GE Xuri W5, Sartorius BioSTAT RM20|50, Finesse Smart Rocker Bioreactor Systems, and Pall XRS Bioreactor Systems, or Miltenyi Prodigy. In some embodiments, the proliferation culture is carried out under static conditions. In some embodiments, the proliferation culture is carried out under shaking conditions. The culture medium can be added in a bolus or in a perfusion schedule.In some embodiments, the bioreactor maintains a temperature of 37°C or near 37°C and a CO2 level of 5% or near 37°C with an airflow of 0.01 or about 0.01 or at least 0.01 L / min, 0.05 L / min, 0.1 L / min, 0.2 L / min, 0.3 L / min, 0.4 L / min, 0.5 L / min, 1.0 L / min, 1.5 L / min, or 2.0 L / min or greater than 2.0 L / min. In certain embodiments, at least a portion of the culture is carried out using perfusion at rates of, for example, 290 ml / day, 580 ml / day, and / or 1160 ml / day.

[0218] In some embodiments, the cells are 0.5 × 10 6 cells / mL~1.5×10 6 The cells are seeded in a suitable culture vessel (e.g., a gas-permeable bag) at a density of cells / mL. In some embodiments, the density is 0.5 × 10⁻⁶. 6 Or approximately 0.5 × 10 6 cells / mL, 0.75×10 6 Alternatively, approximately 0.75 × 10 6 cells / mL, 1×10 6 Or approximately 1 x 10 6 cells / mL, 1.25×10 6 Or approximately 1.25 × 10 6 cells / mL, or 1.5 × 10⁶ 6 Or approximately 1.5 x 10 6 This is cells / mL, or any value within the range of any of the aforementioned values.

[0219] In some embodiments, cells are grown in an automated closed growth system that allows for perfusion. Perfusion ensures that the optimal growth rate is achieved by continuously adding culture medium to the cells.

[0220] The growth method can be carried out using serum-free medium under GMP conditions, including in a closed automated system. In some embodiments, any one or more steps of the method can be carried out in a closed system or under GMP conditions. In certain embodiments, all process operations are performed in a GMP suite. In some embodiments, the closed system is used to carry out one or more other processing steps of a method for manufacturing, generating, or producing cell therapy. In some embodiments, one or more of the processing steps, e.g., isolation, selection and / or concentration, processing, culture steps including incubation related to cell proliferation, and formulation steps, are carried out within an integrated or built-in system and / or using a system, device, or apparatus in an automated or programmable manner. In some embodiments, the system or apparatus includes a computer and / or computer program that communicates with the system or apparatus, thereby allowing the user to program, control, evaluate results, and / or adjust various aspects of the processing, separation, engineering, and formulation steps.

[0221] In some embodiments, stimulated cells are harvested and frozen. In some embodiments, for cryopreservation, the stimulated cells are formulated as a composition having a cryoprotective agent. In some embodiments, the cryoprotective agent is or comprises DMSO and / or glycerol. In some embodiments, the composition formulated for cryopreservation can be stored at low temperatures, such as ultra-low temperatures, for example, in a temperature range of -40°C to -150°C, for example, 80°C or about 80°C ± 6.0°C.

[0222] In some embodiments, cryopreserved cells are prepared for subsequent steps by thawing.

[0223] III. Treatment methods and application of treatment Provided herein are compositions and methods relating to the therapeutic cell compositions provided herein for use in the treatment of diseases or conditions in subjects such as cancer. Such methods and uses include, for example, therapeutic methods and uses involving the administration of therapeutic cells, or compositions containing them, to a subject having a disease, condition, or disorder. In some cases, the disease or disorder is a tumor or cancer. In some embodiments, the cells or pharmaceutical compositions thereof are administered in an amount effective to treat the disease or disorder. Uses include the use of cells or pharmaceutical compositions thereof in such methods and treatments, and in the preparation of agents for carrying out such therapeutic methods. In some embodiments, the disease, condition, or disorder in the subject is treated by the method.

[0224] In some embodiments, the subject is the same subject from which a biological sample was obtained to produce a therapeutic cell composition. In some such embodiments, the therapeutic method provided is adoptive cell therapy using a therapeutic composition containing autologous T cells applied to the subject.

[0225] In some embodiments, the cell compositions provided herein are derived from the subject to be treated. In such embodiments, the starting cells for proliferation are isolated directly from a biological sample derived from the subject described herein and optionally comprise an enrichment of T cells positive for one or more selection markers described herein, and are cultured under the conditions for proliferation provided herein. In some embodiments, the biological sample from the subject is or comprises a tumor or lymph node sample, and such sample tumor, and the amount of such tissue is obtained by excision or biopsy (e.g., core needle biopsy or fine needle aspiration). In some embodiments, following culturing under the conditions for proliferation according to the method provided, the cells are formulated for subsequent administration to the same subject to treat cancer and optionally cryopreserved. In some embodiments, the therapeutic method comprises administering an effective amount of a composition containing tumor-reactive CD3+ T cells or CD3+ T cells positive for one or more markers described herein (e.g., PD-1 / CD39). In some embodiments, the use of a therapeutic method or composition involves administering a therapeutically effective amount of a composition containing tumor-reactive CD3+ T cells or CD3+ T cells positive for one or more markers (e.g., PD-1 / CD39) as described herein, which is sufficient to treat, improve, or otherwise beneficially alter the symptoms of any condition, disorder, or disease described herein, or other indication. Such compositions may include those described herein, including compositions produced by the methods provided.

[0226] In some embodiments, the subject (e.g., home) is 10 5 Or about 10 5 ~10 12 Or about 10 12 CD3+ T cells produced by any of the methods provided, or 10 5 Or about 10 5 ~10 8 Or about 10 8 CD3+ T cells produced by any of the methods provided, or 10 6 Or about 106 ~10 12 Or about 10 12 CD3+ T cells produced by any of the methods provided, or 10 8 Or about 10 8 ~10 11 Or about 10 11 CD3+ T cells produced by any of the methods provided, or 10 9 Or about 10 9 ~10 10 Or about 10 10 CD3+ T cells produced by one of the provided methods are administered. In some embodiments, the therapeutically effective dose for administration is 10 5 Over or approximately 10 5 CD3+ T cells produced by any of the methods provided, 10 6 Or about 10 6 CD3+ T cells produced by any of the methods provided, 10 7 Or about 10 7 CD3+ T cells produced by any of the methods provided, 10 8 Or about 10 8 CD3+ T cells produced by any of the methods provided, 10 9 Or about 10 9 CD3+ T cells produced by any of the methods provided, 10 10 Or about 10 10 CD3+ T cells produced by any of the methods provided, 10 11 Or about 10 11 CD3+ T cells produced by any of the methods provided, or 10 12 Or about 10 12 The present invention comprises CD3+ T cells produced by any of the methods provided. In some embodiments, such amounts may be administered to subjects with a disease or condition, e.g., cancer patients. In some embodiments, the number of T cells administered is viable T cells.

[0227] In some embodiments, the dose is administered as a flat dose. In other embodiments, the dose is administered per kilogram of body weight of the subject. In some embodiments, the dose administered is a therapeutically effective dose sufficient to treat, improve, or otherwise beneficially alter the symptoms of a condition, disorder, or disease, or other indication. For example, a pharmaceutical T lymphocyte infiltration (TIL) composition contains a therapeutically effective dose (e.g., a flat dose or a dose per kilogram of body weight of the subject) of tumor-reactive T cells or T cells positive for a selection marker (e.g., PD-1 / CD39) to treat a disease or condition, such as cancer, for example, a tumor.

[0228] In some embodiments, a composition comprising tumor-reactive T cells or T cells positive for a selection marker (e.g., PD-1 / CD39), produced by, for example, one of the methods provided, is administered to an individual immediately after proliferation according to the method provided. In other embodiments, the proliferated T cells, e.g., proliferated tumor-reactive T cells or T cells positive for a selection marker, are cryopreserved before administration, for example, by the method described above. For example, T cells, e.g., tumor-reactive T cells or T cells positive for a selection marker, may be stored for more than 6, 12, 18, or 24 months before administration to an individual. Such cryopreserved cells may be thawed before administration.

[0229] In some embodiments, the provided composition, provided by any of the provided methods or containing tumor-reactive T cells or T cells positive for a T cell selection marker, can be administered to a subject by any convenient route, including parenteral routes such as subcutaneous, intramuscular, intravenous, and / or epidural administration routes.

[0230] In some embodiments, a composition provided, for example, by one of the methods provided, or containing tumor-reactive T cells or T cells positive for a selection marker, may be administered in a single dose. Such administration may be by injection, for example, intravenous injection. In some embodiments, tumor-reactive T cells or T cells positive for a selection marker may be administered in multiple doses. Dosage may be once, twice, three, four, five, six times, or six or more times per year. Dosage may be once a month, once every two weeks, once a week, or every other day. Administration of such compositions and cells may be continued for as long as necessary. In some embodiments, a composition provided, for example, by one of the methods provided, or containing tumor-reactive T cells or T cells positive for a T cell selection marker, may be administered in a therapeutically effective dose, or an amount sufficient to treat, improve, or otherwise beneficially alter the symptoms of a condition, disorder, or disease, or other indication.

[0231] In some embodiments, the subject is administered lymphatic depletion therapy prior to the administration of a dose of cells derived from a prenatalized composition containing tumor-reactive T cells or T cells positive for a selection marker, for example, produced by one of the methods provided. Lymphatic depletion therapy may include the administration of fludarabine and / or cyclophosphamide (the active form is referred to as maphosphamide) and combinations thereof. Such methods are described in Gassner et al. (Cancer Immunol Immunother. 2011, 60(l):75-85, Muranski, et al., Nat Clin Pract Oncol, 2006 3(12):668-681, Dudley, et al., J Clin Oncol 2008, 26:5233-5239, and Dudley, et al., J Clin These are described in Oncol. 2005, 23(10):2346-2357, all of which are incorporated herein by reference in their entirety. In some embodiments, fludarabine is administered in doses of 10 mg / kg / day, 15 mg / kg / day, 20 mg / kg / day, 25 mg / kg / day, 30 mg / kg / day, 35 mg / kg / day, 40 mg / kg / day, or 45 mg / kg / day, or any dose within the aforementioned range. In some embodiments, fludarabine is administered for 2 to 7 days, for example, 3 to 5 days, for example, 3 days or about 3 days, 4 days or about 4 days, or 5 days or about 5 days. In some embodiments, cyclophosphamide is administered in doses of 100 mg / m² / day, 150 mg / m² / day, 175 mg / m² / day, 200 mg / m² / day, 225 mg / m² / day, 250 mg / m² / day, 275 mg / m² / day, or 300 mg / m² / day. In some embodiments, cyclophosphamide is administered intravenously (i.e., iv). In some embodiments, cyclophosphamide treatment lasts for 2 to 7 days, for example, 3 to 5 days, 3 or about 3 days, 4 or about 4 days, or 5 or about 5 days. Lymphatic depletion therapy is administered before the provided cell composition. In some embodiments, lymphatic depletion therapy is performed within one week of administration of the provided cell composition, for example, 5 to 7 days before administration of the cell dose.

[0232] The compositions described herein may be used in methods for treating hyperproliferative disorders. In preferred embodiments, they are for use in the treatment of cancer. In some embodiments, the types of cancer may include, but are not limited to, those of the ovaries, vulva, endometrium, urothelium, breast, pancreas, prostate, colorectal, lung, brain, kidney, stomach (gastrointestinal), and skin (including, but not limited to, squamous cell carcinoma, basal cell carcinoma, and melanoma). In some embodiments, the types of cancer may include, but are not limited to, those of the ovaries, vulva, endometrium, urothelium, breast, colorectal, lung, kidney, and skin (including, but not limited to, melanoma). In some embodiments, the types of cancer may include, but are not limited to, those of the ovaries. In some embodiments, the types of cancer may include, but are not limited to, those of the vulva. In some embodiments, the types of cancer may include, but are not limited to, those of the endometrium. In some embodiments, the types of cancer may include, but are not limited to, those of the urothelium. In some embodiments, the types of cancer may include, but are not limited to, those of the breast. In some embodiments, the type of cancer may include, but is not limited to, colorectal cancer. In some embodiments, the type of cancer may include, but is not limited to, lung cancer. In some embodiments, the type of cancer may include, but is not limited to, kidney cancer. In some embodiments, the type of cancer may include, but is not limited to, skin cancer (including, but not limited to, melanoma). In some embodiments, the cancer is epithelial cancer. In some embodiments, the cancer is selected from non-small cell lung cancer (NSCLC), CRC, ovarian cancer, breast cancer, esophageal cancer, gastric cancer, pancreatic cancer, cholangiocarcinoma, and endometrial cancer. In some embodiments, the breast cancer is HR+ / Her2- breast cancer. In some embodiments, the breast cancer is triple-negative breast cancer (TNBC). In some embodiments, the breast cancer is HER2+ breast cancer.

[0233] In some embodiments, the subject has cancer that is a hematological malignancy. Non-limiting examples of hematological malignancies include leukemias, including acute leukemia (such as 11q23-positive acute leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythrocytes), chronic leukemia (such as chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin lymphoma (slowly progressive and high-grade types), multiple myeloma, Waldenström macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

[0234] In some embodiments, the subject has solid tumors. Non-limiting examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, malignant lymphoma, pancreatic cancer, breast cancer (including basal breast cancer, ductal breast cancer, and lobular breast cancer), lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, and papilloma. Examples include cancer, pheochromocytoma, seborrheic carcinoma, papillary carcinoma, papillary carcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocellular carcinoma, cholangiocarcinoma, choriocarcinoma, Welms' tumor, cervical cancer, testicular tumor, seminoma, bladder cancer, and CNS tumors (such as glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroma, meningioma, melanoma, neuroblastoma, and retinoblastoma). In some examples, the tumor is melanoma, lung cancer, lymphoma, breast cancer, or colon cancer.

[0235] In some embodiments, the cancer is skin cancer. In certain embodiments, the cancer is a melanoma, such as cutaneous melanoma. In some embodiments, the cancer is Merkel cell carcinoma or metastatic cutaneous squamous cell carcinoma (CSCC).

[0236] In some embodiments, the tumor is a carcinoma, which is a cancer arising from or of epithelial origin from epithelial cells. In some embodiments, the cancer arises from epithelial cells, including but not limited to breast cancer, basal cell carcinoma, adenocarcinoma, gastrointestinal cancer, lip cancer, oral cancer, esophageal cancer, small intestine cancer and stomach cancer, colon cancer, liver cancer, bladder cancer, pancreatic cancer, ovarian cancer, cervical cancer, lung cancer, breast cancer and skin cancer, for example squamous cell carcinoma and basal cell carcinoma, prostate cancer, renal cell carcinoma, and other known cancers affecting epithelial cells throughout the body.

[0237] In some embodiments, the subject is gastrointestinal cancer, which is cancer of the upper or lower digestive tract, or of the digestive tract (GI tract), including the esophagus, stomach, biliary tract, pancreas, small intestine, large intestine, rectum, or anus. In some embodiments, the cancer is esophageal cancer, gastric (stomach) cancer, pancreatic cancer, liver cancer (hepatocellular carcinoma), gallbladder cancer, mucosal-associated lymphoid tissue cancer (MALT lymphoma), bile tree cancer, colorectal cancer (including colon cancer, rectal cancer, or both), anal cancer, or gastrointestinal carcinoid tumor. In certain embodiments, the cancer is colorectal cancer.

[0238] In some embodiments, the cancer is colorectal cancer. Colorectal cancer (CRC) is a common tumor with increasing incidence and often does not respond to checkpoint inhibitors or other immunotherapies. This is true despite the fact that such cancers have a well-established association between prognosis and response-related characteristics, such as fairly high mutation rates and levels of T-cell infiltration.

[0239] In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is triple-negative breast cancer (TNBC).

[0240] In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is Merkel cell carcinoma. In some embodiments, the cancer is metastatic cutaneous squamous cell carcinoma (CSCC). In some embodiments, the cancer is melanoma.

[0241] In some embodiments, the subjects are those whose cancer is resistant to treatment with checkpoint blockers, such as anti-PD1 or anti-PD-L1 therapy, or who have relapsed after such treatment.

[0242] In some embodiments, the cell compositions provided herein are allogeneic to the subject to be treated. In some embodiments, the subject from which the cells are derived or isolated is a healthy subject or is not known to have a disease or condition such as cancer. In such embodiments, the starting cells for proliferation are isolated directly from a biological sample derived from such subject as described herein, optionally comprising an enrichment of T cells positive for one or more selection markers described herein, and are cultured under the conditions for proliferation provided herein. In some embodiments, the biological sample from the subject is or comprises a tumor or lymph node sample, and such sample tumors and quantities of such tissue are obtained by excision or biopsy (e.g., core needle biopsy or fine needle aspiration). In some embodiments, following culture under conditions for proliferation, the cells are formulated for subsequent administration to treat different cancers of different subjects relating to different subjects and are optionally cryopreserved.

[0243] In some embodiments, the provided method can be carried out using one or more other immunotherapies. In some embodiments, the immunotherapy is an immunomodulator, which is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor specifically binds to molecules selected from CD25, PD-1, PD-L1, PD-L2, CTLA-4, LAG-3, TIM-3, 4-1BB, GITR, CD40, CD40L, OX40, OX40L, CXCR2, B7-H3, B7-H4, BTLA, HVEM, CD28, TIGIT, and VISTA. In some embodiments, the immune checkpoint inhibitor is an antibody or antigen-binding fragment, a small molecule, or a polypeptide. In some embodiments, the immune checkpoint inhibitor is selected from nivolumab, pembrolizumab, pizilizumab, MK-3475, BMS-936559, MPDL3280A, ipilimumab, tremelimumab, IMP31, BMS-986016, uremab, TRX518, dasetuzumab, lucatumumab, SEQ-CD40, CP-870, CP-893, MED16469, MEDI4736, MOXR0916, AMP-224, and MSB001078C, or their antigen-binding fragments.

[0244] In some embodiments, the methods provided include combination therapy of the described cell therapy with a PD-1 or PD-L1 inhibitor. The PD-1 or PD-L1 inhibitor may include a conjugated antibody, an antagonist, or an inhibitor (i.e., a blocker).

[0245] In one embodiment, the PD-I inhibitor is nivolumab (marketed as OPDIVO by Bristol-Myers Squibb Co.), or its biosimilar, antigen-binding fragment, conjugate, or variant. Nivolumab is a fully human IgG4 antibody that blocks the PD-I receptor. In one embodiment, the anti-PD-I antibody is immunoglobulin G4 kappa, anti-(human CD274) antibody. Nivolumab is assigned Chemical Abstracts Service (CAS) registry number 946414-94-4 and is also known as 5C4, BMS-936558, IDX-1106, and ONO-4538. The preparation and characterization of nivolumab are described in U.S. Patent No. 8,008,449 and International Patent Publication No. WO2006 / 121168.

[0246] In another embodiment, the PD-1 inhibitor includes pembrolizumab (marketed as KEYTRUDA by Merck & Co., Inc., Kenilworth, NJ, USA), or its antigen-binding fragment, conjugate, or variant. Pembrolizumab is assigned CAS Registry No. 1374853-91-4 and is also known as lambrolizumab, MK-3475, and SCH-900475. The characterization, use, and preparation of pembrolizumab are described in International Patent Publication WO2008 / 156712A1, U.S. Patent No. 8,354,509, and U.S. Patent Application Publications US2010 / 0266617A1, US2013 / 0108651A1, and US2013 / 0109843A2.

[0247] In one embodiment, the PD-LI inhibitor is durvalumab, also known as MEDI4736 (marketed by Medimmune, LLC, Gaithersburg, JVIaryland, a subsidiary of AstraZeneca plc.), or its antigen-binding fragment, conjugate, or variant. In one embodiment, the PD-LI inhibitor is an antibody disclosed in U.S. Patent No. 8,779,108 or U.S. Patent Application Publication No. 2013 / 0034559.

[0248] In one embodiment, the PD-LI inhibitor is avelumab (commercially available from Merck KGaA / EMDSerono), also known as MSB0010718C, or its antigen-binding fragment, conjugate, or variant. The preparation and characterization of avelumab are described in U.S. Patent Application Publication US2014 / 0341917A1.

[0249] In one embodiment, the PD-LI inhibitor is atezolizumab, also known as MPDL3280A or RG7446 (marketed as TECENTRIQ by Genentech, Inc., a subsidiary of Roche Holding AG, Basel, Switzerland), or its antigen-binding fragment, conjugate, or variant. In one embodiment, the PD-LI inhibitor is the antibody disclosed in U.S. Patent No. 8,217,149, the disclosure of which is specifically incorporated herein by reference. In one embodiment, the PD-LI inhibitor is the antibody disclosed in U.S. Patent Publication No. 2010 / 0203056A1, No. 2013 / 0045200A1, No. 2013 / 0045201A1, No. 2013 / 0045202A1, or No. 2014 / 0065135A1. The preparation and characterization of atezolizumab are described in U.S. Patent No. 8,217,149.

[0250] IV. Kits and Products This specification provides products and kits comprising compositions, such as compositions containing T cells produced by any of the methods provided, or compositions containing or enriching a described selection marker (e.g., PD-1 / CD39). In some embodiments, the compositions are produced by any of the methods provided.

[0251] The kit may optionally include one or more components such as instructions for use, devices and additional reagents (e.g., sterile water or saline for diluting compositions and / or reconstitution of lyophilized proteins), as well as components such as tubes, containers and syringes for carrying out the method. In some embodiments, the kit includes reagents for sample collection, sample preparation and processing, and / or reagents for quantifying the amount of one or more surface markers in the sample, for example, but not limited to detection reagents, such as antibodies, buffers, substrates for enzyme staining, chromogens, or other materials, such as slides, containers, microtiter plates, and optionally, instructions for carrying out the method. Those skilled in the art will recognize many other possible containers and plates and reagents that may be used according to the provided method.

[0252] In some embodiments, a kit may be provided as a product comprising packaging material for packaging cells, antibodies or reagents, or compositions thereof, or one or more other components. For example, a kit may include containers, bottles, tubes, vials, and any packaging material suitable for separating or organizing the components of the kit. One or more containers may be formed from a variety of materials, such as glass or plastic. In some embodiments, one or more containers hold a composition containing cells or antibodies or other reagents for use in the method. The products or kits herein may contain cells, antibodies or reagents in separate containers or in the same container.

[0253] In some embodiments, one or more containers holding the composition may be single-use or multi-use vials, which may, in some cases, allow for repeated use of the composition. In some embodiments, the product or kit may further include a second container containing a suitable diluent. The product or kit may further include other materials desirable from a commercial, therapeutic and user perspective, including other buffers, diluents, filters, needles, syringes, a package insert having therapeutic agents and / or instructions for use.

[0254] In some embodiments, the kit optionally includes instructions. Instructions typically include tangible representations describing the cell composition, optionally other components included in the kit, and methods for using them. In some embodiments, the instructions illustrate methods for using the cell composition for administration to a subject to treat a disease or condition according to any of the embodiments provided. In some embodiments, the instructions are provided as a label or accompanying document on or associated with the container. In some embodiments, the instructions may provide instructions for recomposing and / or using the composition.

[0255] V. Definition Unless otherwise defined, all technical terms, symbols, and other technical and scientific terms used herein are intended to have the same meaning as those generally understood by those skilled in the art in the field to which the subject matter of the patent application pertains. In some cases, terms that have a generally understood meaning are defined herein for clarity and / or for immediate reference, and the inclusion of such definitions herein should not necessarily be construed as representing a substantial difference from the generally understood meaning in the art.

[0256] As used herein, unless otherwise clearly indicated by the context, the singular forms “a,” “an,” and “the” include multiple references. For example, “a” or “an” means “at least one” or “one or more.” The embodiments and variations described herein are understood to include “consisting of” and / or “essentially consisting of” the embodiments and variations.

[0257] Through this disclosure, various aspects of the subject matter of a patent application may be presented in range form. It should be understood that the range form is merely for convenience and brevity and should not be interpreted as immutably limiting the scope of the subject matter of the patent application. Therefore, a range description should be considered to specifically disclose not only the individual numerical values ​​within that range, but also all possible secondary ranges. For example, if a range of values ​​is provided, it should be understood that each intermediate value between the upper and lower limits of that range, as well as any other indicated values ​​or intermediate values ​​within that range, are included in the subject matter of the patent application. These upper and lower limits of smaller ranges may be independently included within those smaller ranges and are also included in the subject matter of the patent application, according to any specific exclusion limits within the indicated range. If an indicated range includes one or both limiting values, the range excluding one or both of those limiting values ​​is also included in the subject matter of the patent application. This applies regardless of the breadth of the range.

[0258] As used herein, the term “about” refers to the normal range of error for each value, as readily understood by those skilled in the art. References to “about” values ​​or parameters herein include (and describe) embodiments relating to that value or parameter itself. For example, a statement referring to “about X” includes a statement of “X”.

[0259] As used herein, the term “allogeneic” means cells or tissues that are taken from one organism and subsequently injected into or adopted into another genetically different organism of the same species.

[0260] As used herein, the term “autologous” means cells or tissues taken from the same organism that will be subsequently injected or adopted into.

[0261] In this specification, the term “antibody” is used in its broadest sense and includes polyclonal and monoclonal antibodies, intact antibodies and functional (antigen-binding) antibody fragments, including fragment antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single-chain antibody fragments including single-chain variable fragments (scFv), and single-domain antibody (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses intrabody, peptide-body, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecificity, e.g., bispecificity, antibodies, diabody, triabody, and tetrabody, tandem di-scFv, tandem tri-scFv, and other immunomodulatory and / or otherwise modified forms of immunoglobulins. Unless otherwise specified, the term “antibody” should be understood to encompass its functional antibody fragments. This term also encompasses intact or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses, IgM, IgE, IgA, and IgD. Some of the antibodies offered may be antibody fragments.

[0262] An "antibody fragment" or "antigen-binding fragment" refers to a molecule other than a conventional or intact antibody that contains at least a variable region that binds to an antigen, or a portion of an intact antibody. Examples of antibody fragments include Fv, single-chain Fv(sdFv), Fab, Fab', Fab'-SH, F(ab')2; diabody; linear antibody; V H Examples include, but are not limited to, single-domain antibodies containing only the VHH region.

[0263] As used herein, “to bind,” “bound,” or its grammatical variations thereof refer to the participation of a molecule in any attractive interaction with another molecule, resulting in a stable association in close proximity between the two molecules. Examples of binding include, but are not limited to, non-covalent bonds and covalent bonds (such as reversible and irreversible covalent bonds), and intermolecular interactions, such as those between small molecules including, but not limited to, proteins, nucleic acids, carbohydrates, lipids, and chemical substances including drugs.

[0264] The term "biological sample" refers to a quantity of material that is living or formerly living. Such material includes, but is not limited to, blood (e.g., whole blood), plasma, serum, urine, amniotic fluid, synovial fluid, endothelial cells, leukocytes, monocytes, other cells, organs, tissues, bone marrow, lymph nodes, and spleen.

[0265] As used herein, “concentrate” means increasing the number or proportion of one or more specific cell types or populations of a cell type or population, for example, by comparison to the total number of cells in the composition or volume, or by comparison to other cell types, for example, by positive selection based on markers expressed by the population or cells, or by negative selection based on markers not present in the cell population or cells to be depleted. The term does not require the complete removal of other cells, cell types, or populations from the composition, nor does it require that the thus concentrated cells constitute 100% or nearly 100% of the concentrated composition.

[0266] The term “simultaneously” is used herein to refer to a procedure, such as incubation, selection, concentration, or administration, that involves two or more drugs, where at least a portion of a particular procedure with one drug temporally overlaps with at least a second drug.

[0267] The term “intermittently” is used herein to refer to procedures such as incubation, selection, concentration, or administration involving two or more drugs, where the specific procedures involving each drug are not performed at regular intervals, are not continuous, or involve repeated pauses and starts.

[0268] The term “sequential” is used herein to refer to procedures such as incubation, selection, concentration, or administration involving two or more drugs, where the specific procedures in which each drug is involved do not overlap in time.

[0269] As used herein, “isolated” or “purified” with respect to peptides, proteins, or polypeptides means a molecule that is substantially free of all other polypeptides, contaminants, starting reagents, or other materials, or that is substantially free of chemical precursors or other chemicals when chemically synthesized. A preparation can be determined to be substantially free of impurities if it appears to be free of readily detectable impurities by standard analytical methods such as high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC), or capillary electrophoresis (CE), which are used by those skilled in the art to assess whether such purity, or purity sufficient so that the physical and chemical properties of the substance are not detectably altered by further purification.

[0270] As used herein, the term “recombinant” refers to a cell, microorganism, nucleic acid molecule, or vector modified by the introduction of an exogenous element such as a heterogeneous nucleic acid molecule, or a cell or microorganism in which the expression of an endogenous nucleic acid molecule or gene has been altered to be controlled, uncontrolled, or constitutive, and such alteration or modification may be introduced by genetic engineering. Genetic modifications may include, for example, modifications that introduce a nucleic acid molecule encoding one or more proteins or enzymes (which may include expression regulatory elements such as promoters), or the addition, deletion, substitution, or other functional disruption or addition to the genetic material of a cell. Exemplary modifications include those in the coding region or functional fragment of a heterogeneous or homologous polypeptide derived from a reference molecule or parent molecule. The term “recombinant” may also refer to a protein product expressed from such a nucleic acid molecule or vector, or from such a cell or microorganism into which an exogenous nucleic acid has been introduced or thereby modified.

[0271] As used herein, “composition” means any mixture of two or more products, substances, or compounds, including cells. This may be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof.

[0272] As used herein, “optional” or “optionally” means whether or not the event or situation described therein occurs, and that the description includes examples of the event or situation occurring and examples of it not occurring. For example, an optionally substituted base means that the base is either unsubstituted or substituted.

[0273] The term "pharmaceutical composition" refers to a composition suitable for pharmaceutical use in mammals, often in humans. A pharmaceutical composition typically comprises an effective amount of an active agent (e.g., cells that grow according to the method provided) and a carrier, excipient, or diluent. The carrier, excipient, or diluent is typically a pharmaceutically acceptable carrier, excipient, or diluent, respectively. A "pharmaceutically acceptable carrier" refers to a conventional, non-toxic solid, semi-solid, or liquid filler, diluent, encapsulating material, formulation aid, or carrier used in conjunction with a therapeutic agent containing a "pharmaceutical composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to the recipient at the dose and concentration used and is compatible with the other components of the formulation.

[0274] In this specification, the term “population of cells” refers to several cells that share common traits. A population of cells generally includes several cells, for example, more than 100 or about 100 cells, 1000 or about 1000 cells, and is typically 1 × 10⁶ 4 ~1 × 10 10 It is a number within the range of [a certain range].

[0275] When used herein in relation to proteins, the term "soluble" means that a protein does not bind, immobilize, or adhere to cells or solid supports, such as particles like beads. For example, soluble proteins include proteins that are not bound to the cell membrane of a cell as transmembrane proteins. In some cases, the solubility of a protein may be improved by direct or indirect linker-mediated linking or attachment to another molecule, such as an Fc domain, which may also improve the stability and / or half-life of the protein. In some embodiments, a soluble protein is an Fc fusion protein.

[0276] As used herein, the term “specifically binding” means the ability of a protein to bind to a target protein under specific binding conditions, such that its affinity or binding activity is at least 10 times, but arbitrarily 50, 100, 250, or 500 times, or even at least 1000 times, the average affinity or binding activity of the same protein to a sufficiently statistically sized assortment of random peptides or polypeptides. A specifically binding protein does not need to bind to only a single target molecule; it may bind specifically to multiple target molecules. In some cases, a specifically binding protein may bind to a protein (e.g., a paralog or ortholog) that has structural similarity to the target protein. Those skilled in the art will recognize that specific binding to molecules with the same function in different animal species (i.e., orthologs), or to molecules with substantially similar epitopes to the target molecule (e.g., paralogs), is possible without compromising the specificity of binding determined to a statistically valid assortment of intrinsic non-targets (e.g., random polypeptides). Specific binding between two proteins can be determined using solid-phase ELISA immunoassays, ForteBio Octet, or Biacore measurements. Generally, the interaction between two binding proteins is approximately 1 × 10⁻⁶. -5 Less than M, often about 1 × 10 -12 It has a dissociation constant (Kd) as low as M. In certain aspects of this disclosure, the interaction between the two binding proteins is approximately 1 × 10⁻⁶.-6 Less than M, 1 x 10 -7 M, 1×10 -8 M, 1×10 -9 M, 1×10 -10 M, or 1 × 10 -11 It has a dissociation constant less than or equal to M.

[0277] As used herein, the statement that a cell or population of cells is “positive” for a particular marker means the detectable presence of a particular marker, typically a surface marker, on or within a cell. When referring to a surface marker, the term means the presence of surface expression, for example, detected by flow cytometry by staining with an antibody that specifically binds to the marker and detecting the antibody, wherein the stain is detectable by flow cytometry at a level substantially higher than that detected by isotype-matched controls and by performing the same procedure under otherwise identical conditions, and / or at a level substantially similar to that of cells known to be positive for the marker, and / or at a level substantially higher than that of cells known to be negative for the marker.

[0278] As used herein, the statement that a cell or population of cells is “negative” for a particular marker, function, or attribute means the absence of a substantial detectable presence of that particular marker on or within the cell, such as a surface marker. When referring to a surface marker, the term means the absence of surface expression, for example, that can be detected by flow cytometry by staining with an antibody that specifically binds to the marker and detecting the antibody, where the staining is not detected by flow cytometry at a level substantially higher than that detected by the same procedure under isotype-matched controls and other identical conditions, and / or at a level substantially similar to that of cells known to be positive for the marker, and / or at a level substantially higher than that of cells known to be negative for the marker.

[0279] As used herein, “Subject” means a mammal such as a human or other animal, typically a human. A subject may be male or female and may be of any appropriate age, including infants, juveniles, adolescents, adults, and elderly subjects.

[0280] The terms “effective dose” or “therapeutic effective dose” refer to the amount and / or concentration of a therapeutic composition, such as, for example, cells grown according to the method provided, which, when administered to a patient, improves or otherwise beneficially alters the symptoms of a pathological condition, disorder, or disease or other indication. An effective dose for treating a disease or disorder may be an amount that alleviates, reduces, or reduces at least one symptom or biological response or effect associated with the disease or disorder, prevents the progression of the disease or disorder, or improves the patient’s physical function. In certain embodiments, for example, there is a statistically significant inhibition of disease progression by improving or eliminating the symptoms and / or causes of the disease. In the case of cell therapy, the effective dose is the effective dose or number of cells administered to the patient. In some embodiments, the patient is a human patient.

[0281] As used herein, “disease,” “disorder,” or “pathological condition” means a pathological condition of an organism resulting from a cause or condition including, but not limited to, infection, acquired condition, or genetic condition, and characterized by identifiable symptoms. In particular, it is a condition for which treatment is required and / or desired.

[0282] As used herein, the terms “to treat,” “to cure,” or “to therapy” of a disease or disorder mean to slow, halt, or reverse the progression of the disease or disorder, as demonstrated by reducing, stopping, or eliminating any of the clinical or diagnostic symptoms, and include administration of the immunomodulatory proteins or engineered cells of the present invention alone or in combination with other compounds described herein. “To treat,” “to cure,” or “to therapy” also means a reduction in the severity of symptoms in an acute or chronic disease or disorder, or a reduction in the relapse rate in the course of a relapsing or remission autoimmune disease, for example, or a reduction in inflammation in the inflammatory aspect of an autoimmune disease. As used in the context of the present invention, “to prevent,” “prophylaxis,” or “prevention” of a disease or disorder means the administration of the immunomodulatory proteins or engineered cells expressing the immunomodulatory proteins of the present invention, either alone or in combination, to prevent the onset or development of a disease or disorder, or some or all of the symptoms of a disease or disorder, or to reduce the likelihood of the development of a disease or disorder. For example, in the context of cancer, the terms “treatment” or “inhibition” of cancer mean at least one of the following, as measured by standard criteria, such as, but not limited to, the Solid Tumor Response Assessment Criteria (RECIST): a statistically significant reduction in tumor growth rate, cessation of tumor growth, or a reduction in tumor size, mass, metabolic activity, or volume, or a statistically significant increase in progression-free survival (PFS) or overall survival (OS).

[0283] The term "antigen" refers to a molecule that can induce an immune response. Typically, an antigen is a molecule that can be bound by a recognition site on an immune molecule, such as an antibody or a T cell receptor, when presented by a major histocompatibility complex (MHC) molecule. An antigen may have one or more epitopes, each epitope that is part of the antigen, which can be bound by an antibody recognition site or a TCR / MHC complex. In some embodiments, an antigen can induce a humoral or cellular immune response that results in the activation of B lymphocytes and / or T lymphocytes.

[0284] As used herein, “tumor-associated antigen” or “tumor-specific antigen” refers to a protein or other molecule found only on cancer cells and not on normal cells.

[0285] The term "in vivo" refers to events that occur within the body of mammals.

[0286] The term "ex vivo" refers to an event that occurs on or within tissues or cells derived from a mammalian subject, but takes place outside the mammalian subject. Typically, the event is performed in an external environment. In certain embodiments, ex vivo procedures include the collection of organs, cells, or tissues from a subject, typically a living organism, for treatment or procedure, and subsequent return to the subject.

[0287] The term "in vitro" refers to events that occur in testing systems such as laboratories.

[0288] When used herein, a kit is a packaged combination that optionally includes additional reagents and other elements such as instructions for use of the combination or its components.

[0289] The term “package insert” is used to refer to the instruction sheet that is customarily included on the market packaging of a therapeutic drug, containing information about indications, uses, dosage, administration, combination therapies, contraindications, and / or warnings regarding the use of such a drug.

[0290] Where used herein, “product” refers to a product that is manufactured and, if applicable, can be sold. In some embodiments, this term may refer to a composition contained in a packaged article, such as in a container.

[0291] It is understood that the aspects and embodiments of the present invention described herein include "including," "consisting of," and "essentially consisting of."

[0292] VI. Exemplary Embodiments The embodiments provided are as follows: 1. A pharmaceutical T lymphocyte infiltration (TIL) composition enriched with tumor-reactive T cells, wherein the composition comprises a population of tumor-infiltrating T cells including tumor-derived CD4+ and CD8+ T cells, wherein at least 90% of the cells in the composition are CD3+ T cells, and less than 5% of the population are regulatory T cells.

[0293] 2. A pharmaceutical T lymphocyte infiltration (TIL) composition enriched with tumor-reactive T cells, wherein the pharmaceutical composition comprises an oligoclonal population of tumor-infiltrating T cells, including tumor-derived CD4+ and CD8+ T cells, and up to 40 clones constitute 40% of the TCR frequency in the population.

[0294] 3. The pharmaceutical composition according to Embodiment 2, wherein less than approximately 5% of the population consists of regulatory T cells.

[0295] 4. The pharmaceutical composition according to any one of Embodiments 1 to 3, wherein less than approximately 3% of the population consists of regulatory T cells.

[0296] 5. The pharmaceutical composition according to any one of Embodiments 1 to 4, wherein less than approximately 1% of the population consists of regulatory T cells.

[0297] 6. A pharmaceutical composition according to any one of Embodiments 1 and 3 to 5, wherein the regulatory T cell phenotype is characterized by the expression of the surface marker CD4+CD8-CD25+Foxp3+CD127low.

[0298] 7. The pharmaceutical composition according to any one of Embodiments 1 to 6, wherein the T cells of the population express PD-1 and / or CD39.

[0299] 8. The pharmaceutical composition according to Embodiment 7, wherein the proportion of cells expressing the surface marker PD-1 in the composition, or the proportion of viable cells expressing the surface marker PD-1, is at least about 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of the cells in the composition.

[0300] 9. The pharmaceutical composition according to Embodiment 7 or Embodiment 8, wherein the proportion of cells expressing the surface marker CD39 in the composition, or the proportion of such viable cells, is at least about 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of the cells in the composition.

[0301] 10. The pharmaceutical composition according to Embodiment 7, wherein the proportion of cells expressing the surface markers PD-1 and CD39 in the composition, or the proportion of such viable cells, is at least about 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and at least 100% of the cells in the composition.

[0302] 11. The pharmaceutical composition according to any one of Embodiments 2 to 10, wherein at least 90% of the cells in the composition are CD3+ T cells.

[0303] 12. The pharmaceutical composition according to any one of Embodiments 1 to 11, wherein at least 95% of the cells in the population are CD3+ T cells.

[0304] 13. The pharmaceutical composition according to any one of Embodiments 1 to 11, wherein at least 98% of the cells in the population are CD3+ T cells.

[0305] 14. The pharmaceutical composition according to any one of Embodiments 1 to 13, wherein at least 80% of the cells in the composition are CD3+CD56-T cells.

[0306] 15. The pharmaceutical composition according to any one of Embodiments 1 to 14, wherein more than 60% of the cells in the population are T-effector memory cells.

[0307] 16. The pharmaceutical composition according to any one of Embodiments 1 to 15, wherein more than 75% of the cells in the population are T-effector memory cells.

[0308] 17. The pharmaceutical composition according to any one of Embodiments 1 to 15, wherein more than 80% of the cells in the population are T-effector memory cells.

[0309] 18. The pharmaceutical composition according to any one of Embodiments 1 to 15, wherein more than 85% of the cells in the population are T-effector memory cells.

[0310] 19. The pharmaceutical composition according to any one of Embodiments 1 to 15, wherein more than 90% of the cells in the population are T-effector memory cells.

[0311] 20. The effector memory representation type is CD45RA - CD45RO+, CD62L - A pharmaceutical composition according to any one of embodiments 15 to 19, characterized by the expression of one or more surface markers among CCR7-, CD28-, and CD27-.

[0312] 21. The effector memory representation type is CD45RA - CD45RO+, CD62L - , and CCR7 - A pharmaceutical composition according to any one of embodiments 15 to 20, characterized by the expression of a surface marker.

[0313] 22. The effector memory representation type is CD45RA - CD45RO +CD62L, CCR7 - CD28 - , and CD27 - A pharmaceutical composition according to any one of embodiments 15 to 20, characterized by the expression of a surface marker.

[0314] 23. The effector memory representation type is CD45RA - CCR7- - A pharmaceutical composition according to any one of embodiments 15 to 20, characterized by the expression of a surface marker.

[0315] 24. A pharmaceutical composition according to any one of embodiments 2 to 23, wherein up to 40 TCR chronotypes constitute at least 50% of the TCR frequencies in the population, and optionally, the top 40 chronotypes constitute at least 50% of the TCR frequencies in the population.

[0316] 25. A pharmaceutical composition according to any one of embodiments 2 to 23, wherein up to 40 TCR chronotypes constitute at least 60% of the TCR frequencies in the population, and optionally, the top 40 chronotypes constitute at least 60% of the TCR frequencies in the population.

[0317] 26. The pharmaceutical composition according to any one of Embodiments 1 to 25, wherein the TCR chronotype is reactive to at least one CD8 antigen and at least one CD4 antigen.

[0318] 27. The pharmaceutical composition according to any one of Embodiments 1 to 26, wherein at least 20% of the CD8+ T cells and / or at least 20% of the CD4+ T cells in the composition exhibit reactivity to the newly generated antigen.

[0319] 28. The pharmaceutical composition according to any one of Embodiments 1 to 27, wherein the TIL composition is characterized by at least one of the following criteria in an in vitro autologous tumor assay: i) Production of IFN-γ exceeding 2000 pg / mL; ii) Production of granzyme B in the supernatant at concentrations exceeding 200 pg / mL; iii) Killing more than 10% of tumor cells.

[0320] 29. A pharmaceutical T lymphocyte infiltration (TIL) composition enriched with tumor-reactive T cells, wherein the pharmaceutical composition comprises tumor-infiltrating lymphocytes including tumor-derived CD4+ and CD8+ T cells, wherein at least about 90% of the cells in the composition are CD3+ T cells, and the TIL composition is characterized by at least one of the following criteria in an in vitro autologous tumor assay: i) Production of IFN-γ exceeding 2000 pg / mL; ii) Production of granzyme B at concentrations exceeding 500 pg / mL; iii) Over 15% of autologous tumor cells are killed.

[0321] 30. The pharmaceutical composition according to Embodiment 28 or Embodiment 29, wherein the TIL composition is characterized by criteria (i) and (ii).

[0322] 31. The pharmaceutical composition according to Embodiment 28 or Embodiment 30, wherein the TIL composition is characterized by criteria (i) and (iii).

[0323] 32. The pharmaceutical composition according to Embodiment 28 or Embodiment 30, wherein the TIL composition is characterized by criteria (ii) and (iii).

[0324] 33. The pharmaceutical composition according to Embodiment 28 or Embodiment 30, wherein the TIL composition is characterized by criteria (i), (ii), and (iii).

[0325] 34. A pharmaceutical composition according to any one of Embodiments 28 to 33, wherein the production of IFN-γ is greater than 3000 pg / mL or greater than 4000 pg / mL.

[0326] 35. A pharmaceutical composition according to any one of Embodiments 28 to 34, wherein the production of granzyme B is greater than 400 pg / mL or greater than 500 pg / mL.

[0327] 36. The pharmaceutical composition according to any one of embodiments 28 to 35, wherein the killing of autologous tumor cells exceeds 40%.

[0328] 37. The pharmaceutical composition according to any one of Embodiments 1 to 36, characterized in that the composition has a greater number of CD4+ T cells than CD8+ T cells.

[0329] 38. The pharmaceutical composition according to any one of Embodiments 1 to 37, wherein the ratio of CD4+ T cells to CD8+ T cells in the composition is 5:1 to 1:5.

[0330] 39. The pharmaceutical composition according to any one of Embodiments 1 to 38, wherein the ratio of CD4+ T cells to CD8+ T cells in the composition is 5:1 to 50:1, 5:1 to 25:1, 5:1 to 20:1, 5:1 to 15:1, 5:1 to 10:1, 10:1 to 50:1, 10:1 to 25:1, 10:1 to 20:1, 10:1 to 15:1, 15:1 to 50:1, 15:1 to 25:1, 15:1 to 20:1, 20:1 to 50:1, 20:1 to 25:1, or 25:1 to 50:1.

[0331] 40. The pharmaceutical composition according to any one of Embodiments 1 to 39, wherein the ratio of CD4+ T cells to CD8+ T cells in the composition is 10:1 to 25:1 or about 10:1 to 25:1, and optionally 20:1 or about 20:1.

[0332] 41. The pharmaceutical composition according to any one of Embodiments 1 to 40, wherein the number of cells in the composition is a therapeutically effective amount of TIL.

[0333] 42. The pharmaceutical composition according to any one of Embodiments 1 to 41, wherein the number of cells in the composition, or the number of viable cells thereof, is at least 2 × 10⁷ cells.

[0334] 43. The number of cells in the composition, or the number of surviving cells, is 2 × 10 7 Or approximately 2 x 10 7 cells ~20×10 9 cells, 2 x 10 7 cells ~10×109 cells, 2 x 10 7 cells ~2×10 9 cells, 2 x 10 7 cells ~2×10 8 cells, 2 x 10 8 cells ~20×10 9 cells, 2 x 10 8 cells ~10×10 9 cells, 2 x 10 8 cells ~2×10 9 cells, 2 x 10 9 cells ~20×10 9 cells, 2 x 10 9 cells ~10×10 9 Cells, or 10 × 10 9 cells ~20×10 9 A pharmaceutical composition according to any one of Embodiments 1 to 42, which is a cell (including both ends).

[0335] 44. The pharmaceutical composition according to any one of Embodiments 1 to 43, wherein the pharmaceutical composition is for the treatment of a patient's tumor.

[0336] 45. The pharmaceutical composition according to any one of Embodiments 1 to 44, wherein the tumor is a colorectal cancer (CRC) tumor, a melanoma tumor, a non-small cell lung cancer (NSCLC) tumor, or an ovarian cancer tumor.

[0337] 46. ​​The pharmaceutical composition according to any one of Embodiments 1 to 45, wherein the tumor is derived from a human subject.

[0338] 47. The pharmaceutical composition according to Embodiment 46, wherein the pharmaceutical composition is for self-adoption therapy for the human subject.

[0339] 48. A pharmaceutical composition according to any one of Embodiments 1 to 47, comprising a pharmaceutically acceptable excipient.

[0340] 49. A pharmaceutical composition according to any one of Embodiments 1 to 48, comprising a cryoprotective substance.

[0341] 50. The pharmaceutical composition according to any one of Embodiments 1 to 49, wherein the composition is a liquid composition.

[0342] 51. The pharmaceutical composition according to Embodiment 50, wherein the composition has been frozen and thawed.

[0343] 52. The pharmaceutical composition according to any one of Embodiments 1 to 51, wherein the volume of the composition is 1 mL to 500 mL.

[0344] 53. The pharmaceutical composition according to any one of Embodiments 1 to 52, wherein the composition is frozen.

[0345] 54. The pharmaceutical composition according to any one of Embodiments 1 to 53, wherein the composition is prepared by selecting cells that are surface-positive for PD-1 and CD39 from cells obtained from a donor tumor, and by growing the cells ex vivo.

[0346] 55. The pharmaceutical composition according to any one of Embodiments 1 to 53, wherein the composition is prepared by a method comprising the following: a. To provide dissociated tumor cells obtained from a tumor from a donor subject, wherein the dissociated tumor cells are a first population of cells including CD4+ and CD8+ T cells; b. From the aforementioned population of cells, select cells that are surface-positive for CD45, PD1, and CD39, as well as a T cell marker, to produce a population of selected T lymphocyte-infiltrating cells (TILs), wherein the T cell marker is optionally CD4 or CD8; and c. Proliferate the selected TIL population by culturing it with one or more lymphocyte T cell stimulants under conditions that produce a population of proliferated T cells.

[0347] 56. A method for producing a T lymphocyte infiltration (TIL) composition enriched with tumor-reactive T cells, wherein the method is: a. To provide dissociated tumor cells obtained from a tumor from a donor subject, wherein the dissociated tumor cells are a first population of cells including CD4+ and CD8+ T cells; b. From the aforementioned population of cells, select cells that are surface-positive for CD45, PD1, and CD39, as well as a T cell marker, to produce a population of selected T lymphocyte-infiltrating cells (TILs), wherein the T cell marker is optionally CD4 or CD8; and c. The method comprising growing the selected population of TILs together with one or more lymphocyte T cell stimulants under conditions that produce a population of proliferated T cells.

[0348] 57. The method according to Embodiment 55 or Embodiment 56, wherein the one or more T cell stimulants are selected from one or more allogeneic feeder cells, anti-CD3 antibodies, and recombinant IL-2.

[0349] 58. The method according to any one of embodiments 55 to 57, wherein the dissociated tumor cells are a single-cell suspension obtained by homogenization and enzymatic digestion of one or more tumor fragments derived from an excised tumor.

[0350] 59. The method according to Embodiment 58, wherein the enzymatic digestion is carried out by incubation with collagenase at an optional concentration of approximately 10 mg / ml and hyaluronidase at an optional concentration of approximately 10 mg / ml.

[0351] 60. The concentration of the first cell population is 5 × 10 6 cells / mL~50×10 6 Cells / mL, optional, approximately 20 × 10 6 The method according to any one of embodiments 55 to 59, wherein the cell density is cells / mL.

[0352] 61. The method according to any of embodiments 55-60, wherein in any of the several embodiments provided, cell selection is performed using a microfluidic chip-based cell sorting comprising at least four fluorescence detectors.

[0353] 62. The method according to Embodiment 61, wherein the selection of the cells comprises separating the cells that are positive for the at least four fluorescent signals based on a fluorescent minus 1 (FMO) cocktail.

[0354] 63. The method according to Embodiment 62, wherein the parsing is performed at a rate of 5,000 events per second to 10,000 events per second, and optionally at a rate of approximately 6,000 events per second.

[0355] 64. A method for treating a subject having cancer, the method comprising administering to a subject having a tumor a therapeutically effective dose of a composition described in any of Embodiments 1 to 55.

[0356] 65. The above-mentioned therapeutically effective dose is approximately 1 × 10 9 ~10×10 9 The method according to embodiment 64, wherein the T cells are...

[0357] 66. The method according to Embodiment 65, wherein the therapeutically effective dose is more than 1 million but less than 100 million T cells per kilogram of body weight.

[0358] 67. The method according to Embodiment 65, wherein the therapeutically effective dose is more than 1 million but less than 10 million T cells per kilogram of body weight.

[0359] 68. The method according to Embodiment 65, wherein the therapeutically effective dose is 10 million or about 10 million to 50 million or about 50 million T cells per kilogram of body weight.

[0360] 69. The method according to any one of Embodiments 64 to 68, wherein the cells in the pharmaceutical composition are autologous to the subject. [Examples]

[0361] VII. Examples The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention.

[0362] Example 1: Recovery of PD1+CD39+ tumor-infiltrating lymphocytes (TILs) via cell sorting from tumor samples Fresh or frozen tumors from patients with colorectal cancer (CRC), ovarian cancer, non-small cell lung cancer (NSCLC), kidney, breast, vulva, urothelial, endometrial, or melanoma were processed as described below, and the resulting invasive T cell populations were analyzed for cell number viability. Tumors were supplied from primary tumors in cancer patients and transported overnight in HypoThermosol at 4°C. Tumors were processed as single-cell suspension (SCS) cultures.

[0363] To generate SCS, the tumor was cut into fragments approximately 3 mm in diameter. Next, the fragments were homogenized in a closed system using MiltenyiGentleMACS in 4 mL of CTSOptimizer medium (Life Technologies, #A10221-01) supplemented with CTS Optimizer growth additive (Life Technologies, #A10484-02), CTS Immune CellSR (Life Technologies, #A2596101), gentamicin, and GlutaMax (Thermo Fisher, #35050-061) (hereinafter referred to as CTS medium), in the presence of an enzyme cocktail for tumor digestion containing collagenase (Sigma, #C5138, 10 mg / ml), DNase I (Sigma, #D5025, 10000 IU / ml), and hyaluronidase (Sigma, #H2126, 10 mg / ml). Fragments designated for SCS were incubated with an enzyme cocktail for a total of 60 minutes, followed by homogenization and enzymatic digestion. The tumor contents were then processed into a single suspension using a 70-micron cell strainer. Immediately after SCS formation, cell count and viability were assessed using an NC-200 Automated Cell Counter (ChemoMetec).

[0364] TILs derived from tumor samples were isolated by fluorescence-activated cell sorting (FACS) as described below.

[0365] Single-cell suspensions of various tumor samples were prepared as described above. When frozen SCS cells were used, the SCS cells were thawed and washed in CTS medium containing benzoase to avoid cell aggregation. Live cells were then transferred and incubated with Fc blocks at room temperature for 10 minutes. In the preparative process using an FX500 sorter, cells were washed and either cocktail-washed with commercially available antibodies including anti-CD45APC-Cy7 (BD), anti-CD4PE-CF594 (BD), anti-CD8PE (BD), anti-PD-1 BB515 (BD), anti-CD39 AF647 (BD), and a fluorescent minus one (FMO) cocktail, or left unstained. Cells were approximately 20 × 10⁶ 6 Cells were divided at a concentration of cells / mL and a dispensing rate of approximately 6,000 events per second. A live / dead stain (7-AAD) was used in the dispensing process with the MA900. Cell viability was measured 10 minutes before dispensing by adding 1 μl of 7-AAD stain per 100 μl of cell solution. Cells were positive for CD45, CD4 and / or CD8, PD1 and CD39, and negative for 7-AAD (7-AAD neg We used cells stained with an FMO cocktail around the cells of ) to guide the gate and sort them into a single population (Table E1). This was the population that was sorted with living positive cells. Furthermore, the unselected cells were sorted into another population and 7-AAD neg CD45 pos PD1 pos CD39 pos , 7-AAD neg CD45 pos PD1 pos CD39 neg and / or 7-AAD neg CD45posPD1 neg CD39 neg Cells lacking both 7-AAD and CD45 expression were isolated into separate populations and identified as a mixture of tumor and stromal cells for use in the reactivity assays described in Examples 4 and 5. After isolation, the isolated positive and negative (also referred to as unselected) populations and the unselected population were analyzed to verify purity and evaluate recovery. In the exemplary process, the PD-1+CD39+ purity after selection was approximately 80%. [Table 2]

[0366] The proportion of PD1+CD39+ cells within the CD45+, CD4+, and / or CD8+ TIL cell populations is shown in Figure 2A with respect to tumor type. PD1+CD39+ TILs were detected at varying frequencies (ranging from 7 to 76%) in tumors across all indications (e.g., the tumor types tested).

[0367] Example 2: Proliferation of activated tumor-derived T cells after sorting. T cells supplied from the primary tumor were treated as described in Example 1 and then selectively isolated as described in Example 2. The cells were then grown in vitro using the Rapid Expansion Protocol (REP) described below.

[0368] Freshly separated PD1+CD39+ cells or their unselected counterparts were seeded at 250,000–1,000,000 cells / cm2 in serum-free OpTmizer medium supplemented with 10 μg / mL gentamicin and 2.0 mM L-alanyl-L-glutamine dipeptide glutamine (GlutaMAX Supplement; Thermo Fisher) in gas-permeable 24-well culture plates. Alternatively, this culture process can be carried out in the presence of human AB serum. Cells were stimulated with 30 ng / ml anti-CD3 antibody (OKT3 clone) and 3000 IU / ml recombinant human IL-2 (proleukin) in a G-Rex flask in the presence of allogeneic feeder cells at a feeder (irradiated PBMC or iPBMC):TIL ratio of 200:1. Cells were incubated for a total of 14 days, with medium changes and divisions to maintain optimal cell density and growth. Cell counting was performed on each culture day using an NC-200 automated cell counter (Chemometec). Cells were divided on day 7. After completion of the proliferation phase, cells were washed with PBS and then cryopreserved in the presence of cryoprotective agents. Cryopreservation was performed using a CoolCell device (Corning) or VIA Freeze (GE Healthcare).

[0369] As shown in Figure 2B as polyploidy, each tumor-infiltrating lymphocyte (TIL) T cell population tested underwent measurable proliferation. PD1+CD39+TILs were successfully isolated from 16 of these samples (indicated for ovarian, NSCLC, CRC, melanoma, breast, endometrium, urothelial, and kidney) and proliferated in vitro with 14 days of REP, resulting in an average 1000-fold proliferation across the different samples treated.

[0370] Example 3: Determination of the phenotype and clonality of the propagated PD1+CD39+TIL The composition and phenotype of PD1+CD39+ selected TIL products derived from lung, colorectal, kidney, endometrial, and melanoma tumors were further analyzed by flow cytometry. After proliferation using REP as described in Example 2, positively selected cells were harvested and stained for various markers using the Aurora TIL-ACT Antibody Panel. The Aurora TIL-ACT Antibody Panel includes Live / Dead Blue viability marker (Thermo Fisher), anti-CD3B V510 (BioLegend), anti-CD4 BUV395 (BD), anti-CD8 BUV805 (BD), anti-CD25 PE-Cy7 (BD), anti-FOXP3A F647 (BioLegend), and anti-TCRdelta / gamma. PerCP-Vio700 (Miltenyi), anti-CD56 BV570 (BioLegend), anti-CCR 7APC-Fire810 (BioLegend), anti-CD27 APC-H7 (BD), anti-CD28 BV650 (BioLegend), anti-CD45RA BUV563 (BD), anti-CD45RP eFluor450 (Thermo Fisher), anti-CD95 BUV737(BD), anti-CD127 APC-R700(BD), anti-CD62L BV480(BD), anti-TCF-1 BV421(BD), anti-CXCR6 BB700(BD), anti-CD57 FITC(BioLegend), anti-KLRG1 PE-Fire810(BioLegend), anti-CD39 BUY661(BD), anti-CD69 Spark NIR 685 (BioLegend), anti-CD103 (PE-Dazzle594 (BioLegend), anti-CD137 APC (BioLegend), anti-OX40 BV605 (BioLegend), anti-PD-1 BV785 (BioLegend), anti-TIGIT BV711 (BioLegend), anti-TIM3 PE (BioLegend), anti-LAG-3 Contains PE-Cy5 (Thermo Fisher).

[0371] As shown in Figure 3, flow cytometry analysis revealed that the PD1+CD39+ selected TIL product was mainly composed of CD3+ T cells (>92%), with variable proportions of CD4 and CD8 T cells. CD3+CD56- T cells accounted for the majority of the PD1+CD39+ selected TIL product (>85%), while CD3+CD56- T cells ranged from 1 to 11%, and CD3-CD56+ NK cells were less than 0.1% (Figure 4A). High variability was observed in the proportion of CD4 and CD8 T cell subsets across the entire PD1+CD39+ selected TIL product. Regulatory T cells (Treg), identified as CD4+CD8-CD25+Foxp3+CD127low, were consistently detected at low frequencies (0.1-2%) (Figure 4B).

[0372] To evaluate T cell phenotypes, the expression of CD45RA and the chemokine receptor CCR7 was analyzed (Figure 5). CD45RA-CCR7- cells were defined as effector memory T cells (Tem), CD45RA-CCR7+ cells as central memory T cells (Tcm), CD45RA+CCR7- cells as effector memory T cells, and CD45RA-CCR7- cells as naive / stem cell memory T cells (T-naive / SCM). This analysis revealed that CD4 and CD8 T cells in PD1+CD39+ selected TIL products were primarily effector memory T cells (Tem). Central memory T cells (Tcm), a phenotype associated with increased in vivo persistence in animal models, were also detected at varying levels in different samples.

[0373] In addition to phenotypic characterization by flow cytometry, single-cell RNA (scRNA) sequencing was performed on PD1+CD39+ selected and unselected (PD1-CD39+; PD1+CD39- and PD1-CD39-) TIL products (two colorectal cancers, two melanomas, one non-small cell lung cancer, and one ovarian cancer) to evaluate the TCR repertoire. To generate single-cell sequencing data, cells were processed according to the manufacturer's instructions to produce a library (10× genomics), which was then sequenced to obtain information on the sequence and expression of genes expressed by individual cells based on the detected sequences. These T cell clones were then defined as cells sharing a unique association of TCR alpha and beta CDR3 sequences. Approximately 1e5 viable cells per group were washed and filtered through a Flowmi 40 μM cell strainer to a final sequencing concentration of 700–1300 cells / μl (for 2000 cells).

[0374] The smallest overlap among the top 40 most highly expressed chronotypes was observed between the TCR sequences of unselected TILs and PD1+CD39+ selected TIL products, demonstrating that PD1+CD39+ direct selection enriches a unique subset of tumor-responsive TCRs (Figure 6). PD1+CD39+ selected TILs are oligoclonal and exhibit reduced chronotype diversity compared to unselected TILs, as indicated by a higher proportion of the repertoire comprised of the top 40 most frequently expressed chronotypes.

[0375] Example 4: Evaluation of the functionality of PD1+CD39+ cells derived from ovarian tumors and their autologous tumor material. The functionality of PD1+CD39+ selected and unselected TILs derived from ovarian tumors was evaluated using two different functional assays in which cells were stimulated either with polyclonal stimulation (anti-CD3 and anti-CD28 antibody-mediated stimulation) or antigen-specific stimulation (autologous tumor cells). The results are described below.

[0376] A. Polyclonal stimulation Polyclonal stimulation provides information about the general functional capacity of T cells because anti-CD3 and anti-CD28 antibodies activate all viable T cells regardless of their specificity. Tumor-derived PD-1+CD39+ selected and unselected TILs were grown and cryopreserved as described in Example 2. The cells were washed in OpTmizer cell medium supplemented with 300 IU / mL recombinant IL-2, 10 μg / mL gentamicin, 5% Immune Cell Serum Replacement (Thermo Fisher), and glutamine in the form of L-alanyl-L-glutamine dipeptide (GlutaMAX Supplement; Thermo Fisher) at a final concentration of 2.0 mM. Next, an anti-CD3 / CD28 activator mix (final concentration: 25 μl / mL) was added, and the cells were placed in 96-well culture plates in 1 × 10⁶ wells. 6 Cells were seeded at a final cell density of cells / mL. Cells were stimulated overnight, and the supernatant was collected for cytokine analysis. Cytokine concentrations were measured using a LegendPlex bead-based immunoassay according to the manufacturer's instructions.

[0377] PD1+CD39+ selected TIL products produced from ovarian tumor (PD039) produced IFNγ and granzyme B after polyclonal stimulation at levels comparable to their unselected TIL counterparts (Figure 7), demonstrating that PD1+CD39+ selected TIL products function after in vitro proliferation, even when selected based on the expression of terminal differentiation / exhaustion markers.

[0378] B. Antigen-specific stimulation Next, the response to antigen-specific stimuli was evaluated to determine the responsiveness of TILs to the tumor. Autologous tumor cells (target cells) and TIL samples were thawed and diluted in OpTmizer cell medium supplemented with 300 IU / mL recombinant IL-2, 10 μg / mL gentamicin, 5% Immune Cell Serum Replacement (Thermo Fisher), and glutamine in the form of L-alanyl-L-glutamine dipeptide (GlutaMAX Supplement; Thermo Fisher) to a final concentration of 2.0 mM. The cells were then co-cultured in 96-well plates in the following combinations: negative control: TIL sample + anti-CD28 (2 ug / ml) + anti-PD-1 (20 ug / ml); positive control: TIL sample + anti-CD3 / CD28 activator mix (25 ug / ml); co-culture: TIL sample + target cells + anti-CD28 (2 ug / ml) + anti-PD-1 (20 ug / ml). Cells were incubated at 37°C and 5% CO2 for 20–24 hours. After co-culture overnight, the supernatant was collected and cytokine concentrations were assessed using Legendplex as described above. The cells were also washed and collected. The viability of CD45-negative cells in co-culture (including tumor cells) was evaluated by flow cytometry and used to calculate tumor cell toxicity.

[0379] When co-cultured with autologous tumor cells, PD1+CD39+ selected TIL products showed a significant increase in tumor-specific responsiveness compared to unselected TILs (PD1-CD39- double-negative or PD1+CD39- or PD1-CD39+ single-negative cells), as evidenced by a significant increase in IFNγ and granzyme B production compared to unselected TILs (Figure 8). Notably, PD1+CD39+ selected TILs also showed superior tumor cell killing compared to unselected TILs, demonstrating that PD1+CD39+ selection effectively enriches tumor-responsive TILs.

[0380] Example 5: Evaluation of the functionality of PD1+CD39+ cells derived from colorectal tumors and their autologous tumor material. The functionality of PD1+CD39+ selected and unselected TILs was evaluated using two different functional assays, as described above, in which cells were stimulated either with polyclonal stimulation (anti-CD3 and anti-CD28 antibody-mediated stimulation) or antigen-specific stimulation (autologous tumor cells).

[0381] A. Polyclonal stimulation Tumor-derived PD-1+CD39+ selected and unselected TILs were grown and cryopreserved as described in Example 2. The cells were washed in OpTmizer cell medium supplemented with 300 IU / mL recombinant IL-2, 10 μg / mL gentamicin, 5% Immune Cell Serum Replacement (Thermo Fisher), and glutamine in the form of L-alanyl-L-glutamine dipeptide (GlutaMAX Supplement; Thermo Fisher) at a final concentration of 2.0 mM. Next, an anti-CD3 / CD28 activator mix (final concentration: 25 μl / mL) was added, and the cells were placed in 1 × 10⁶ wells of a 96-well culture plate. 6 Cells were seeded at a final cell density of cells / mL. Cells were stimulated overnight, and the supernatant was collected for cytokine analysis. Cytokine concentrations were measured using a LegendPlex bead-based immunoassay according to the manufacturer's instructions.

[0382] PD1+CD39+ selected TIL products produced from ovarian tumor (PD039) produced IFNγ and granzyme B after polyclonal stimulation at levels comparable to their unselected TIL counterparts (Figure 9), demonstrating that PD1+CD39+ selected TIL products function after in vitro proliferation, even when selected based on the expression of terminal differentiation / exhaustion markers.

[0383] B. Antigen-specific stimulation Next, the response to antigen-specific stimuli was evaluated to determine the responsiveness of TILs to the tumor. Autologous tumor cells (target cells) and TIL samples were thawed and diluted in OpTmizer cell medium supplemented with 300 IU / mL recombinant IL-2, 10 μg / mL gentamicin, 5% Immune Cell Serum Replacement (Thermo Fisher), and glutamine in the form of L-alanyl-L-glutamine dipeptide (GlutaMAX Supplement; Thermo Fisher) to a final concentration of 2.0 mM. The cells were then co-cultured in 96-well plates in the following combinations: negative control: TIL sample + anti-CD28 (2 ug / ml) + anti-PD-1 (20 ug / ml); positive control: TIL sample + anti-CD3 / CD28 activator mix (25 ug / ml); co-culture: TIL sample + target cells + anti-CD28 (2 ug / ml) + anti-PD-1 (20 ug / ml). Cells were incubated at 37°C and 5% CO2 for 20–24 hours. After co-culture overnight, the supernatant was collected and cytokine concentrations were assessed by Legendplex as described above. The cells were also washed and collected. The viability of CD45-negative cells in co-culture (including tumor cells) was evaluated by flow cytometry and used to calculate tumor cell toxicity.

[0384] When co-cultured with autologous tumor cells, PD1+CD39+ selected TIL products showed a significant increase in tumor-specific responsiveness compared to unselected TILs (PD1-CD39- double-negative or PD1+CD39- or PD1-CD39+ single-negative cells), as evidenced by a significant increase in IFNγ and granzyme B production compared to unselected TILs (Figure 10). Notably, PD1+CD39+ selected TILs also showed superior tumor cell killing compared to unselected TILs, demonstrating that PD1+CD39+ selection effectively enriches tumor-responsive TILs.

[0385] VIII. Conclusion The foregoing description of embodiments is presented for illustrative and explanatory purposes only. It is not intended to limit the invention to the exact forms disclosed. Many modifications, variations, and improvements will be apparent to those skilled in the art. For example, embodiments of the pharmaceutical T lymphocyte infiltration (TIL) compositions described herein, including those enriched with tumor-reactive T cells, can be applied to solid tumors such as GI, breast, bone, and melanoma, as well as various liquid / hematopoietic malignancies such as leukemia, lymphoma, multiple myeloma, and related diseases. Furthermore, those skilled in the art will be able to recognize or confirm numerous equivalents to the specific procedures described herein using only routine experiments. Such equivalents are within the scope of the invention and are encompassed by the appended claims.

[0386] Elements, features, or actions from one embodiment can be readily rearranged or replaced with one or more elements, features, or actions from other embodiments to form numerous additional embodiments within the scope of the invention. Furthermore, elements shown or described as being combined with other elements may exist as independent elements in various embodiments. Moreover, embodiments of the invention also intend to exclude or negatively enumerate elements, features, chemicals, therapeutics, properties, values, and processes where such elements, features, chemicals, therapeutics, properties, values, and processes are explicitly stated. Accordingly, the scope of the invention is not limited to the details of the embodiments described, but rather is limited only by the appended claims.

Claims

1. A pharmaceutical T lymphocyte infiltration (TIL) composition enriched with tumor-reactive T cells, wherein the composition comprises a population of tumor-infiltrating T cells including tumor-derived CD4+ and CD8+ T cells, wherein at least 90% of the cells in the composition are CD3+ T cells, and less than about 5% of the population are regulatory T cells.

2. A pharmaceutical T lymphocyte infiltration (TIL) composition enriched with tumor-reactive T cells, wherein the pharmaceutical composition comprises an oligoclonal population of tumor-infiltrating T cells, including tumor-derived CD4+ and CD8+ T cells, and up to 40 clones constitute 40% of the TCR frequency in the population.

3. The pharmaceutical composition according to claim 2, wherein less than approximately 5% of the population consists of regulatory T cells.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein less than approximately 3% of the population are regulatory T cells.

5. The pharmaceutical composition according to any one of claims 1 to 4, wherein less than approximately 1% of the population consists of regulatory T cells.

6. The pharmaceutical composition according to any one of claims 1 and 3 to 5, wherein the regulatory T cell phenotype is characterized by the expression of the surface marker CD4+CD8-CD25+Foxp3+CD127low.

7. The pharmaceutical composition according to any one of claims 1 to 6, wherein the T cells of the population express PD-1 and / or CD39.

8. The pharmaceutical composition according to claim 7, wherein the proportion of cells expressing the surface marker PD-1 in the composition, or the proportion of viable cells expressing the surface marker PD-1, is at least about 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of the cells in the composition.

9. The pharmaceutical composition according to claim 7 or claim 8, wherein the proportion of cells expressing the surface marker CD39 in the composition, or the proportion of viable cells expressing the surface marker CD39, is at least about 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of the cells in the composition.

10. The pharmaceutical composition according to claim 7, wherein the proportion of cells expressing the surface markers PD-1 and CD39 in the composition, or the proportion of such viable cells, is at least about 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of the cells in the composition.

11. The pharmaceutical composition according to any one of claims 2 to 10, wherein at least 90% of the cells in the composition are CD3+ T cells.

12. The pharmaceutical composition according to any one of claims 1 to 11, wherein at least 95% of the cells in the population are CD3+ T cells.

13. The pharmaceutical composition according to any one of claims 1 to 12, wherein at least 98% of the cells in the population are CD3+ T cells.

14. The pharmaceutical composition according to any one of claims 1 to 13, wherein at least 80% of the cells in the composition, and optionally at least 85%, are CD3+CD56-T cells.

15. The pharmaceutical composition according to any one of claims 1 to 14, wherein more than 60% of the cells in the population are T-effector memory cells.

16. The pharmaceutical composition according to any one of claims 1 to 15, wherein more than 75% of the cells in the population are T-effector memory cells.

17. The pharmaceutical composition according to any one of claims 1 to 15, wherein more than 80% of the cells in the population are T-effector memory cells.

18. The pharmaceutical composition according to any one of claims 1 to 15, wherein more than 85% of the cells in the population are T-effector memory cells.

19. The pharmaceutical composition according to any one of claims 1 to 15, wherein more than 90% of the cells in the population are T-effector memory cells.

20. The aforementioned effector memory representation type is CD45RA － , CD45RO+, CD62L － The pharmaceutical composition according to any one of claims 15 to 19, characterized by the expression of one or more surface markers selected from CCR7-, CD28-, and CD27-.

21. The aforementioned effector memory representation type is CD45RA － , CD45RO+, CD62L － , and CCR7 － A pharmaceutical composition according to any one of claims 15 to 20, characterized by the expression of a surface marker.

22. The effector memory phenotype is CD45RA － , CD45RO ＋ , CD62L, CCR7 － , CD28 － , and CD27 － The pharmaceutical composition according to any one of claims 15 to 20, characterized by the expression of surface markers thereof.

23. The aforementioned effector memory representation type is CD45RA － CCR7- － A pharmaceutical composition according to any one of claims 15 to 20, characterized by the expression of a surface marker.

24. The pharmaceutical composition according to any one of claims 2 to 23, wherein up to 40 TCR chronotypes constitute at least 50% of the TCR frequencies in the population, and optionally, the top 40 chronotypes constitute at least 50% of the TCR frequencies in the population.

25. The pharmaceutical composition according to any one of claims 2 to 23, wherein up to 40 TCR chronotypes constitute at least 60% of the TCR frequencies in the population, and optionally, the top 40 chronotypes constitute at least 60% of the TCR frequencies in the population.

26. The pharmaceutical composition according to any one of claims 1 to 25, wherein the TCR chronotype is reactive to at least one CD8 antigen and at least one CD4 antigen.

27. The pharmaceutical composition according to any one of claims 1 to 26, wherein at least 20% of the CD8+ T cells and / or at least 20% of the CD4+ T cells in the composition exhibit reactivity to the newly generated antigen.

28. The pharmaceutical composition according to any one of claims 1 to 27, wherein the TIL composition is characterized by at least one of the following criteria in an in vitro autologous tumor assay: i) Production of IFN-γ exceeding 2000 pg / mL; ii) Production of granzyme B in the supernatant at concentrations greater than 200 pg / mL; and iii) Kills over 10% of tumor cells.

29. A pharmaceutical T lymphocyte infiltration (TIL) composition enriched with tumor-reactive T cells, wherein the pharmaceutical composition comprises tumor-infiltrating lymphocytes including tumor-derived CD4+ and CD8+ T cells, wherein at least about 90% of the cells in the composition are CD3+ T cells, and the TIL composition is characterized by at least one of the following criteria in an in vitro autologous tumor assay: i) Production of IFN-γ exceeding 2000 pg / mL; ii) Production of granzyme B at concentrations exceeding 500 pg / mL; and iii) Over 15% of autologous tumor cells are killed.

30. The pharmaceutical composition according to claim 28 or claim 29, wherein the TIL composition is characterized by criteria (i) and (ii).

31. The pharmaceutical composition according to claim 28 or claim 29, wherein the TIL composition is characterized by criteria (i) and (iii).

32. The pharmaceutical composition according to claim 28 or claim 29, wherein the TIL composition is characterized by criteria (ii) and (iii).

33. The pharmaceutical composition according to claim 28 or claim 29, wherein the TIL composition is characterized by criteria (i), (ii), and (iii).

34. The pharmaceutical composition according to any one of claims 28 to 33, wherein the production of IFN-γ is greater than 3000 pg / mL or greater than 4000 pg / mL.

35. The pharmaceutical composition according to any one of claims 28 to 34, wherein the production of granzyme B is greater than 400 pg / mL or greater than 500 pg / mL.

36. The pharmaceutical composition according to any one of claims 28 to 35, wherein the killing of the autologous tumor cells exceeds 40%.

37. The pharmaceutical composition according to any one of claims 1 to 36, characterized in that the composition has a greater number of CD4+ T cells than CD8+ T cells.

38. The pharmaceutical composition according to any one of claims 1 to 37, wherein the ratio of CD4+ T cells to CD8+ T cells in the composition is 5:1 to 1:

5.

39. The pharmaceutical composition according to any one of claims 1 to 38, wherein the ratio of CD4+ T cells to CD8+ T cells in the composition is 5:1 to 50:1, 5:1 to 25:1, 5:1 to 20:1, 5:1 to 15:1, 5:1 to 10:1, 10:1 to 50:1, 10:1 to 25:1, 10:1 to 20:1, 10:1 to 15:1, 15:1 to 50:1, 15:1 to 25:1, 15:1 to 20:1, 20:1 to 50:1, 20:1 to 25:1, or 25:1 to 50:

1.

40. The pharmaceutical composition according to any one of claims 1 to 39, wherein the ratio of CD4+ T cells to CD8+ T cells in the composition is 10:1 to 25:1 or about 10:1 to 25:1, and optionally 20:1 or about 20:

1.

41. The pharmaceutical composition according to any one of claims 1 to 40, wherein the number of cells in the composition is a therapeutically effective amount of TIL.

42. The number of cells in the composition, or the number of surviving cells, is at least 2 × 10 ７ A pharmaceutical composition according to any one of claims 1 to 41, wherein the composition is a cell.

43. The number of cells in the composition, or the number of surviving cells, is 2 × 10 ７ Or approximately 2 x 10 ７ Cell ~20×10 ９ cells, 2 x 10 ７ Cell ~10×10 ９ cells, 2 x 10 ７ cells ~2×10 ９ cells, 2 x 10 ７ cells ~2×10 ８ cells, 2 x 10 ８ Cell ~20×10 ９ cells, 2 x 10 ８ Cell ~10×10 ９ cells, 2 x 10 ８ cells ~2×10 ９ cells, 2 x 10 ９ Cell ~20×10 ９ cells, 2 x 10 ９ Cell ~10×10 ９ Cells, or 10 x 10 ９ Cell ~20×10 ９ A pharmaceutical composition according to any one of claims 1 to 42, wherein the composition is a cell (including both ends).

44. The pharmaceutical composition according to any one of claims 1 to 43, wherein the pharmaceutical composition is for the treatment of a patient's tumor.

45. The pharmaceutical composition according to any one of claims 1 to 44, wherein the tumor is a colorectal cancer (CRC) tumor, a melanoma tumor, a non-small cell lung cancer (NSCLC) tumor, or an ovarian cancer tumor.

46. The pharmaceutical composition according to any one of claims 1 to 45, wherein the tumor is derived from a human subject.

47. The pharmaceutical composition according to claim 46, wherein the pharmaceutical composition is for self-adoption therapy for the human subject.

48. A pharmaceutical composition according to any one of claims 1 to 47, comprising a pharmaceutically acceptable excipient.

49. A pharmaceutical composition according to any one of claims 1 to 48, comprising a cryoprotective substance.

50. The pharmaceutical composition according to any one of claims 1 to 49, wherein the composition is a liquid composition.

51. The pharmaceutical composition according to claim 50, wherein the composition is frozen and thawed.

52. The pharmaceutical composition according to any one of claims 1 to 51, wherein the volume of the composition is 1 mL to 500 mL.

53. The pharmaceutical composition according to any one of claims 1 to 52, wherein the composition is frozen.

54. The pharmaceutical composition according to any one of claims 1 to 53, wherein the composition is prepared by selecting cells that are surface-positive for PD-1 and CD39 from cells obtained from a tumor of a donor subject, and by proliferating the cells ex vivo.

55. The pharmaceutical composition according to any one of claims 1 to 53, wherein the composition is prepared by a method comprising the following: a. Providing dissociated tumor cells obtained from a tumor of a donor subject, wherein the dissociated tumor cells are a first group of cells including CD4+ and CD8+ T cells; b. To produce a population of selected T lymphocyte-infiltrating cells (TILs) by selecting cells that are surface-positive for CD45, PD1, and CD39, as well as a T cell marker, from the first population of cells, wherein the T cell marker is optionally CD4 or CD8; and c. The method comprising growing the selected population of TILs together with one or more lymphocyte T cell stimulants under conditions that produce a population of proliferated T cells.

56. A method for producing a T lymphocyte infiltration (TIL) composition enriched with tumor-reactive T cells, wherein the method is: a. Providing dissociated tumor cells obtained from a tumor of a donor subject, wherein the dissociated tumor cells are a first group of cells including CD4+ and CD8+ T cells; b. To produce a population of selected T lymphocyte-infiltrating cells (TILs) by selecting cells that are surface-positive for CD45, PD1, and CD39, as well as a T cell marker, from the first population of cells, wherein the T cell marker is optionally CD4 or CD8; and c. The method comprising growing the selected population of TILs together with one or more lymphocyte T cell stimulants under conditions that produce a population of proliferated T cells.

57. The method according to claim 55 or claim 56, wherein the one or more T cell stimulants are selected from one or more allogeneic feeder cells, anti-CD3 antibodies, and recombinant IL-2.

58. The method according to any one of claims 55 to 57, wherein the dissociated tumor cells are a single-cell suspension obtained by homogenization and enzymatic digestion of one or more tumor fragments derived from an excised tumor.

59. The method according to claim 58, wherein the enzymatic digestion is carried out by incubation with collagenase at an optional concentration of about 10 mg / ml and hyaluronidase at an optional concentration of about 10 mg / ml.

60. The concentration of the first cell population is 5 × 10 ６ cells / mL~50×10 ６ Cells / mL, optional, approximately 20 x 10 ６ The method according to any one of claims 55 to 59, wherein the amount is cells / mL.

61. The method according to any one of claims 55 to 60, wherein in any of the several provided embodiments, cell selection is performed using a microfluidic chip-based cell sorting comprising at least four fluorescence detectors.

62. The method according to claim 61, wherein the selection of the cells comprises separating the cells that are positive for the at least four fluorescent signals based on a fluorescence minus 1 (FMO) cocktail.

63. The method according to claim 62, wherein the parsing is performed at a rate of 5,000 events per second to 10,000 events per second, and optionally at a rate of approximately 6,000 events per second.

64. A method for treating a subject having cancer, the method comprising administering to a subject having a tumor a therapeutically effective dose of the composition described in any of claims 1 to 55.

65. The aforementioned effective therapeutic dose is approximately 1 × 10 ９ ~10 x 10 ９ The method according to claim 64, wherein the T cell is a T cell.

66. The method according to claim 65, wherein the therapeutically effective dose is more than 1 million but less than 100 million T cells per kilogram of body weight.

67. The method according to claim 65, wherein the therapeutically effective dose is more than 1 million but less than 10 million T cells per kilogram of body weight.

68. The method according to claim 65, wherein the therapeutically effective dose is 10 million or about 10 million to 50 million or about 50 million T cells per kilogram of body weight.

69. The method according to any one of claims 64 to 68, wherein the cells in the pharmaceutical composition are autologous to the subject.

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