T cell composition with improved phenotypic characteristics

Abstract

The present invention provides, in some embodiments, isolated cell compositions suitable for adoptive immunotherapy, methods for making the cell compositions, and therapeutic methods using the cell compositions. The compositions comprise at least about 10 mAbs specific for a target peptide antigen in a pharmaceutically acceptable carrier. 6 The compositions comprise CD8+ T cells and include T memory stem (TSCM) cells. In various embodiments, the compositions are about 1% to about 100% T memory stem cells, providing robust and durable adoptive therapy and providing advances in T cell engineering.

Description

[Technical Field]

[0001] Priority This application claims the interests of U.S. Provisional Patent Application No. 62 / 757,467 filed on 8 November 2018, U.S. Provisional Patent Application No. 62 / 821,031 filed on 20 March 2019, and U.S. Provisional Patent Application No. 62 / 867,499 filed on 27 June 2019, the entirety of which is incorporated herein by reference. [Background technology]

[0002] Immunotherapy underlies cancer therapy, encompassing a wide range of strategies aimed at liberating, inducing, and strengthening a patient's own immune system through adoptive transfer of amplified, naturally occurring, or genetically engineered cytotoxic lymphocytes. Despite recent advances in this field, current adoptive immunotherapy faces several challenges. For example, many adoptive immunotherapies fail to generate sufficient levels of engineered cytotoxic lymphocytes from peripheral blood for clinical or therapeutic value, or to uniformly engineer effector cells, or to provide sustained and long-lasting therapeutic effects to patients, leading to tumor recurrence and other complications. Thus, there is a clear need for cell compositions that provide more effective, durable, and safer adoptive immunotherapy options, including for patients with leukemia or lymphoma (including acute or chronic leukemia), as well as other patients who may benefit from adoptive therapy. In various aspects and embodiments, the present invention addresses these needs. [Overview of the Initiative]

[0003] In various aspects and embodiments, the present invention provides isolated cell compositions suitable for adoptive immunotherapy and / or T cell genetic manipulation. The present invention further provides a method for preparing a cell composition and a therapeutic method using a cell composition. The composition comprises at least about 10 specific to a target peptide antigen(s) in a pharmaceutically acceptable carrier. 6comprising CD8+ T cells and including T memory stem (T SCM ) cells. In various embodiments, at least about 1% of the CD8+ T cells are T memory stem cells. In some embodiments, the T memory stem cells are isolated, thereby preparing a composition substantially comprising T memory stem cells (e.g., 70% - 100% T SCM ). The compositions of the present invention can provide robust and durable adoptive therapies by the presence of significant levels of T SCM . The cell compositions need not include T cells expressing chimeric antigen receptors or recombinant TCRs, and thus, in various embodiments, provide an alternative to these techniques that often produce more exhausted T cell phenotypes, generate less durable responses and greater toxicity. In other embodiments, T SCM is used to recombinantly express a chimeric antigen receptor or heterologous TCR, thereby preparing engineered T cells having high proliferative capacity and few exhausted phenotypes.

[0004] In various embodiments, the cell composition comprises at least 1%, or at least 15% T memory stem cells and CD8+ T cells specific for about 10 6 target peptide antigens, or at least about 10 7 , or at least about 10 8 , or at least about 10 9 , or at least about 10 10 target peptide antigens, providing robust destruction of target cells and long-term persistence in vivo. For example, for the treatment of acute myeloid leukemia (AML) or myelodysplastic syndromes, the cell composition may particularly include T cells specific for WT1, PRAME, survivin, and cyclin A1 peptide antigens.

[0005] In various embodiments, the cell composition comprises about 1% to about 50% T memory stem cells, or about 5% to about 25% T memory stem cells. In various embodiments, the T cells are at least 5% T memory stem cells, or at least about 10% T memory stem cells, or at least 20% T memory stem cells, or at least 25% T memory stem cells, providing adoptive immunotherapy compositions with remarkable proliferative capacity, as well as immune reconstitution capacity and lifespan.

[0006] In various embodiments, more than 95% of the CD8+ T cells in the composition contain the memory phenotype. In various embodiments, the memory phenotype is T SCM In addition, central memory T cells (T CM ), effector memory T cells (T EM ), and effector memory RA+ T cells (T EMRA ) includes one or more (or all) of the following. In some embodiments, at least 80% of the memory representation is T SCM , T CM , and T EM That is the case.

[0007] In various embodiments, CD8+ T cells in the composition are T SCMIn addition, it includes central memory and effector memory T cells. In various embodiments, the T cells (and / or T cells specific to the target antigen) in the composition are at least about 30% central or effector memory T cells, or in some embodiments, at least about 50% central or effector memory T cells, or in some embodiments, at least about 70% central or effector memory T cells, or in some embodiments, at least about 80% central or effector memory T cells, or in some embodiments, at least about 90% central or effector memory T cells. In some embodiments, the CD8+ T cells specific to one or more target antigens are at least 50% central and effector memory T cells, or in some embodiments, at least 80% central and effector memory T cells. In some embodiments, T SCM and T CM This combination accounts for approximately 40% to 70% of CD8+ T cells.

[0008] In some embodiments, the cell composition contains less than 20% or less than 10% terminally differentiated memory T cells (e.g., T EMRA The cells comprise less than 30% naive cells, or in some embodiments less than about 15%, or in some embodiments less than about 5%, or in some embodiments less than 1.5% naive cells. The cellular phenotypes disclosed herein can be created and / or controlled using enrichment and amplification processes with paramagnetic artificial antigen-presenting cells (aAPCs) and recombinant T cell growth factor cocktails.

[0009] In various embodiments, the cell composition consists of at least about 70%, or at least about 80%, or at least about 90% CD8+ or CD4- T cells (e.g., CD3+CD8+ or CD3+CD4- cells). For example, an isolated cell composition may be characterized by containing less than about 10%, or less than 5%, CD4+ T cells. When CD8+ T cells are amplified ex vivo, CD4+ cells tend to overgrow CD8+ cells and compete for growth signals, and exogenous CD4+ cells are not required for a robust and durable in vivo response.

[0010] In various embodiments, antigen-specific T cells exhibit a multifunctional phenotype upon activation. In some embodiments, at least 10% of CD8+ T cells, or at least 20% of CD8+ T cells, or at least 40% of CD8+ T cells, exhibit a multifunctional phenotype upon activation. For example, upon activation, T cells are positive for two or more of the following: intracellular staining for IL-2, IFN-γ production, TNF-α production, and CD107A. In various embodiments, at least 20% of antigen-specific T cells exhibit at least two of these markers. In various embodiments, at least 20% of antigen-specific T cells exhibit at least three of these markers, or in some embodiments, all four of these markers. In various embodiments, at least 5% of CD8+ T cells are polyantigenic, meaning that CD8+ T cells can respond to multiple tumor or viral antigens in vitro or in vivo.

[0011] In various embodiments, the cell composition further comprises γδT cells. γδT cells possess their own T cell receptors (TCRs) on their surface. γδT cells may play a role in the recognition of lipid antigens and phosphoantigens, and can provide HLA-independent anti-pathogen and anti-tumor mechanisms. Furthermore, γδT cells can assist CD8+ cells. The clinical significance of γδT cells has been found in hematopoietic stem cell transplantation (HSCT), and in particular, higher frequencies of γδT cells post-transplant have been associated with desirable outcomes.

[0012] Cell compositions can be prepared by enrichment and amplification processes according to various embodiments. In some embodiments, CD8+ cells specific to a target antigen(s) (e.g., tumor-associated antigen or virus-associated antigen) are enriched. This cell aggregate can be rapidly amplified in culture to reach the cell compositions described herein, even if it is the major naive cell in the source lymphocytes. The enrichment is carried out using paramagnetic beads, which can positively select the cell aggregate and may have the additional advantage of activating naive cells and other T cell aggregates due to strong magnetic clustering of T cell surface receptors. For example, the paramagnetic beads or nanoparticles may contain monomeric or multimeric (e.g., dimeric) HLA ligands that present peptide antigens, along with a co-stimulatory signal on the same or different particles, e.g., a CD28 agonist (e.g., an antibody agonist of CD28). In some embodiments, CD28+ cells are also enriched, which may occur concurrently with antigen-specific enrichment.

[0013] In various embodiments, the target peptide antigen is a tumor or cancer-associated antigen, including tumor-derived, tumor-specific, and neoantigens. Tumor-associated antigen-specific T cells are often very rare and, in many cases, undetectable in the peripheral blood of healthy individuals. This is often the distinction observed between virus-specific T cells and tumor antigen-specific T cells.

[0014] In some embodiments, the target peptide antigen comprises at least one that is associated with or derived from a pathogen, such as a virus, bacterium, fungus, or parasitic pathogen. For example, at least one peptide antigen may be associated with HIV, hepatitis (e.g., B, C, or D), CMV, Epstein-Barr virus (EBV), influenza, herpesvirus (e.g., HSV1 or 2, or varicella-zoster), and adenovirus. For example, CMV is the most common viral pathogen found in organ transplant patients and is a major cause of morbidity and mortality in patients undergoing bone marrow or peripheral blood stem cell transplantation. Viral activation is known to be involved in cancer biology.

[0015] In further embodiments, the cell composition comprises tumor-associated antigen-specific T cells, with pathogen-associated antigen-specific T cells provided as bystander cells. Other bystander cells include γδ T cells. Specifically, bystander cells are enriched by enriching HLA-peptides and anti-CD28, and are amplified, particularly when using a T cell growth factor cocktail capable of inducing some nonspecific amplification of these cells without causing antigen-specific activation. In these embodiments, the majority of the composition consists of target peptide-specific T cells (e.g., 5% to 75%), while the remaining T cells (approximately 0.25% to approximately 25%) provide some reconstitution of the immune system against a common pathogen, which is particularly beneficial post-transplant or in cancers with a viral etiology.

[0016] In some embodiments, T cell growth factors are used during amplification, which affects the proliferation and / or differentiation of T cells. Particularly useful cytokines include MIP-1β, IL-1β, IL-2, IL-4, IL-6, IL-7, IL-10, IL-21, and INF-γ. In these or other embodiments, cells are amplified in a culture in the presence of a cytokine cocktail containing one, two, or three cytokines selected from MIP-1β, IL-1β, and IL-6. In some embodiments, the cytokines further include IL-10. In some embodiments, the growth factors include or are essentially derived from IL-2, IL-4, IL-6, INF-γ, and IL-1β. Cells can be amplified in a culture for 1 to 4 weeks, for example, about 10 to about 21 days.

[0017] In other embodiments, the present invention provides methods for producing cell compositions, including by enrichment and amplification using aAPCs as described herein. Specifically, after depletion of CD4+ cells from a source of lymphocytes (e.g., from a healthy donor or from a patient requiring adoptive immunotherapy), antigen-specific CD8+ T cells are enriched, and in some embodiments, CD28+ cells are enriched, which are T cells specific to the target peptide antigen. The target cells can be enriched using nanoparticles or microparticle aAPCs, for example, superparamagnetic nanoparticles that activate T cells ex vivo by a magnetic field and induce clustering of cell surface receptors. Other materials, including latex or other polymer-based nanoparticles, can also be used to cluster cell surface receptors (without causing magnetic field-induced clustering). The enriched T cells can then be rapidly amplified ex vivo, including by the use of reconstituted T cell growth factors (e.g., factors selected from MIP-1β, IL-1β, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-15, IL-21, and IFN-γ). In some embodiments, cells are amplified in culture in the presence of one, two, or three cytokines selected from MIP-1β, IL-1β, IL-6, and IL-10. In some embodiments, the growth factors include or are essentially derived from IL-2, IL-4, IL-6, INF-γ, and IL-1β. In various embodiments, these cytokines are used in combination with artificial or native antigen-presenting cells to amplified antigen-specific T cells.

[0018] In other embodiments, the present invention provides methods for adoptive cell therapy, including methods for treating cancer patients and / or patients who have undergone allogeneic stem cell transplantation, with or without lymphocyte apheresis, cytoreductive therapy, or immunomodulatory therapy (before cell therapy). Cell therapy may be further provided with or without cytokine-assisted post-therapy. In some embodiments, the patient has a hematological malignancy, and in some embodiments, has relapsed after allogeneic stem cell transplantation. In some embodiments, the patient has acute myeloid leukemia (AML) or myelodysplastic syndrome. For example, in some embodiments, the cell composition comprises T cells specific to WT1, PRAME, Survivin, and cyclin A1 peptide antigen. However, in other embodiments, cancer includes various types of solid tumors, including carcinomas, sarcomas, and lymphomas. Exemplary target peptide antigens are described herein.

[0019] In some embodiments, patients have an infection or are at risk of infection. For example, patients who have undergone HSCT are at particular risk of infection, given their immunocompromised state. Infections that can be treated or prevented include those caused by bacteria, viruses, prions, fungi, parasites, helminths, etc. Such diseases include AIDS, hepatitis B / C, CMV infection, Epstein-Barr virus (EBV) infection, influenza, herpesvirus infections (including shingles), and adenovirus infections.

[0020] In yet another embodiment, the present invention provides a method for producing a γδT cell aggregate. The method comprises amplifying a cell aggregate containing γδT cells in the presence of two or more of IL-2, IL-4, IL-6, INF-γ, and IL-1β. Prior to amplification, the cell aggregate contains less than about 20%, less than about 10%, or less than 8% γδT cells. In some embodiments, the cell aggregate is CD28 enriched. In some embodiments, the cell aggregate is CD4+ depleted. Amplification of cells in culture can be carried out for, for example, 1 to 4 weeks, as described herein. γδT cells can be isolated from other cells using known methods such as cell sorting and can be provided as a cell composition for adoptive transfer or research use, or can be modified to express one or more heterologous or engineered genes, such as heterologous or engineered T cell receptors (e.g., αβTCR), including chimeric antigen receptors (CARs).

[0021] Other aspects and embodiments will become apparent from the following detailed description. [Brief explanation of the drawing]

[0022] [Figure 1] This image shows an artificial immunomodulation (AIM) platform for generating CD8+ antigen-specific T cells. [Figure 2] This image shows AIM ACT (adoptive cell therapy) and the enrichment and amplification (E+E) cell amplification system, which enable rapid in vitro enrichment and amplification of antigen-specific T cells. [Figure 3]The graphs on the left show the enriched and amplified antigen-specific cell products for the AIM ACT platform. The graph on the left shows the total number of CD8+ T cells generated from fresh PBMCs of four healthy donors after T cells were enriched and amplified ex vivo for AML-specific antigens WT137-45, 126-134, PRAME425, and cyclin A1227-235, 341-351. The graph on the right shows the total percentage of the same acute myeloid leukemia (AML)-specific antigens after CD8+ T cells were enriched and amplified ex vivo. [Figure 4A] This shows that CD8+ T cells generated by the AIM ACT platform include memory T cells. The AML antigen-specific CD8+ T cell aggregates are shown after enrichment, but before amplification on day 0 (top) and on day 14 after amplification (bottom). TCM = Central Memory T cells (CD62L+, CD45RA-), TN = Naive T cells (CD62L+, CD45RA+), TEM = Effector Memory T cells (CD62L-, CD45RA-), TEMRA = Effector Memory RA+ T cells (CD62L-, CD45RA+), TSCM = T Memory stem cells (CD62L+, CD45RA+, CD95+). [Figure 4B] This shows that CD8+ T cells generated by the AIM ACT platform include memory T cells. Memory T cell phenotypes for TSCM, TCM, TEM, and TEMRA cells 14 days after AML-specific enrichment and amplification are shown for AML-specific antigens WT137-45, 126-134, PRAME425, and cyclin A1227-235, 341-351. [Figure 5A]Ex vivo enriched and amplified AML-specific T cells exhibit a highly multifunctional phenotype, including intracellular staining of IL-2 (proliferation and memory), IFN-γ (activation of other cells, memory, upregulation of MHC), TNF-α (pro-inflammatory), and CD107A (granzyme release, cytotoxic activity). The majority of AML-specific T cells (i.e., approximately 62%) showed 3-4 effector functions when subjected to nonspecific stimulation (upper side). (Lower side) The graph shows the percentage of T cells expressing IL-2, TNF-α, IFN-γ, and CD107A. T cells were stimulated by nonspecific stimulation of peptide-pulsed T2 cells. [Figure 5B] Ex vivo enriched and amplified AML-specific T cells exhibit a highly multifunctional phenotype, including intracellular staining of IL-2 (proliferation and memory), IFN-γ (activation of other cells, memory, upregulation of MHC), TNF-α (pro-inflammatory), and CD107A (granzyme release, cytotoxic activity). Graphs of T cell-mediated tumor-specific killing of AML cell line U266 are shown using CTLs generated from fresh PBMCs of healthy donors with AML-specific antigens WT137-45, 126-134, PRAME425, and cyclin A1227-235, 341-351, at two effector-to-target (E:T) ratios of 10:1 (left bar) and 20:1 (right bar). [Figure 6] This document consists of four graphs comparing the specificity of Mart-1 specific T cells generated by the enrichment and amplification process between PBMCs derived from melanoma patients (top) and PBMCs derived from healthy donors (bottom). The enrichment and amplification process produces a consistent cell composition regardless of the donor source. The data in this experiment were generated from frozen PBMCs. [Figure 7] This graph shows that the E+E process based on AIM ACT generates a TCR repertoire that mimics a native immune response, thereby providing robust adoptive therapy from a naturally selected native T cell repertoire. The breadth of the polyclonal TCR repertoire enables a native and durable immune response. [Figure 8]The graph shows that the E+E process generated a significant amount of multiple myeloma antigen-specific T memory stem (TSCM) cells ((CD62L+, CD45RA+, CD95+)). This graph shows the phenotype of multiple myeloma-specific antigen T cells before and after amplification from healthy donor leucopak. [Figure 9A] This graph shows the phenotype of T cells enriched and amplified ex vivo during a batch of multiple myeloma antigen-specific T cells from healthy donor leucopak. The graph indicates that the E+E process generated a significant amount of antigen-specific CD8+ T cells (approximately 1.6 × 10⁹ CD8+ T cells based on hinge dimer staining), including T memory stem (TSCM) cells, central memory T cell (TCM), and effector memory T (TEM) cells. [Figure 9B] This graph shows the phenotype of T cells enriched and amplified ex vivo in a batch of multiple myeloma antigen-specific T cells derived from four different clinical multiple myeloma patients. The graph demonstrates that the enrichment and amplification process generated a significant amount of antigen-specific CD8+ T cells, including T memory stem (TSCM) cells, central memory T cell (TCM) cells, and effector memory T (TEM) cells. [Figure 10] This shows the production of γδT cells, including Vδ1 and Vδ2TCR subtypes. [Figure 11] This shows the percentage of γδ T cells on day 14 after amplification of various antigen-specific T cells (AML, MM, EBV, MART-1). The percentage of γδ T cells on day 14 is broadly correlated with the number of γδ T cells on day 0. [Modes for carrying out the invention]

[0023] T cell memory is heterogeneous in its composition, consisting of stable, quiescent, and phenotypic subsets of surface markers capable of unique functional responses upon stimulation. The subsets associated with differentiation include central memory T cells (T CM ), effector memory T cells (T EM ), effector memory RA + T cells (T EMRA), and T memory stem cells (T SCM Memory T cells are formed when antigen-specific naive CD4+ or CD8+ T cells are activated by antigen exposure, and then undergo proliferation, amplification, and differentiation. Therefore, persistent memory is essential for long-term protection against infection and malignancy. SCM Only subset cells are central memory T cells (T CM ), effector memory (T EM ), and terminal effector T cells (T TE It has been shown that they differentiate into T memory stem cells. However, T memory stem cells are rare and represent only a small percentage of circulating lymphocytes. For example, generating a clinically relevant amount of T memory stem cells for adoptive immunotherapy is not currently feasible. Therefore, T memory stem cells against cancer and infectious disease antigens are not feasible. SCM Technologies are needed to generate, amplify, and redirect cells.

[0024] At least approximately 10 specific target peptide antigens (multiple antigens are possible) 6 Contains 1 CD8+ T cell, SCM Isolated cell compositions, including cells, are disclosed herein. SCM These cells express surface markers similar to naive T cells, but they also express elevated levels of the CD95 surface marker. SCM Cells are the memory subset with the least differentiation and amplification. Compared to other memory subsets, the T of this disclosure SCM The cells exhibit enormous amplification capacity and can reconstruct a complete repertoire of memory T cells and effector T cells, resulting in a long-term stable collection of cells with excellent homeostasis and differentiation capabilities. Therefore, they are specific to the target peptide antigen(s) and contain at least approximately 10 cells. 6 Contains 1 CD8+ T cell, SCM The compositions disclosed herein, including cells, are clinically relevant for therapeutic use in amounts that can fight cancer cells. SCM This invention provides a highly effective antitumor composition that enables the generation, amplification, and redirection of cells.

[0025] In various aspects and embodiments, the present invention provides isolated cell compositions, methods for producing cell compositions, and therapeutic methods using cell compositions. In some embodiments, the cell compositions are used in adoptive cell therapy. The compositions contain at least about 10 specific peptide antigens in a pharmaceutically acceptable carrier. 6 Contains 1 CD8+ T cell, SCM Includes cells. In various embodiments, CD8+ T cells are at least about 1% T SCM It is a cell. In some embodiments, T SCM The cells were isolated and thereby contained nearly 100% T memory stem cells (for example, at least 90% were T SCM A cell composition is prepared. In some embodiments, a composition is created in which about 70% to about 100% are T memory stem cells. The compositions of the present invention provide robust and durable adoptive therapy due to the presence of a significant level of T memory stem cells. The cell composition does not need to contain T cells expressing chimeric antigen receptors (CARs) or recombinant TCRs, and therefore provides an alternative to those techniques which in various embodiments often produce more exhausted T cell phenotypes and generate less durable responses. In other embodiments, T SCM Using this method, we recombinantly express chimeric antigen receptors or heterologous TCRs (e.g., αβTCR or γδTCR) to prepare engineered T cells that have higher proliferative capacity and fewer exhausted phenotypes than those already described. SCM Cells can be used to manipulate T cells using CARs or xenogeneic TCRs.

[0026] As used herein, the term “target peptide antigen” or “target antigen” refers to a peptide antigen used ex vivo to enrich and / or amplify a desired CD8+ cell aggregate in combination with, for example, an artificial antigen-presenting cell (aAPC) or professional antigen-presenting cell (pAPC) platform (e.g., dendritic cells). AAPCs or pAPCs are used to activate and amplify CTLs derived from donor or patient lymphocytes. In some embodiments, the target peptide antigen is a peptide epitope loaded onto an aAPC for ex vivo enrichment and amplification of specific CD8+ T cells. Thus, the term “specific to target peptide antigen” means that the T cells are antigens that have experienced the target antigen.

[0027] In various embodiments, the cell composition contains at least about 10 specific target peptide antigens. 6 A number of CD8+ T cells, or at least about 10 specific to the target peptide antigen. 7 A number of CD8+ T cells, or at least about 10 specific to the target peptide antigen. 8 pieces, at least about 10 9 one, or at least about 10 10 It contains 1 × 10⁶ CD8+ T cells and provides robust disruption of target cells. In some embodiments, the cell composition contains 1 × 10⁶ CD8+ T cells specific to the target antigen. 7 pieces~1×10 9 Individual CD8+ T cells, or in some embodiments, 5 × 10⁶ cells specific to the target antigen. 7 pieces~5×10 8 Contains 10 CD8+ T cells. For example, the composition contains approximately 5 × 10 cells per mL in a volume of 50-200 mL. 5 pieces~approx. 5×10 6It may contain a number of cells. In certain embodiments, the volume of the composition is 100 mL or less (e.g., 50-100 mL). The cells of the composition in various embodiments are at least 70% viable, or at least about 80% or about 90% viable, and are provided in a sterile medium, which may be a cryopreservative medium (e.g., 10% DMSO). The medium may be, for example, an aqueous medium suitable for intravenous infusion, and may contain, for example, water and electrolytes. An exemplary medium is PLASMALYTE.

[0028] A cell composition comprising CD8+ cytotoxic lymphocytes (CTLs) and memory T cells is disclosed herein. The CTLs of this disclosure include the following T cell aggregates: naive, T memory stem cells, central memory, effector memory, and terminally differentiated memory cells. According to embodiments of the present invention, T cells specific to a target antigen are present in a significant amount of T SCM The cell composition includes cells. In various embodiments, the target antigen-specific T cells further include central memory T cells and effector memory T cells. In some embodiments, the cell composition provides a durable response, including the in vivo persistence of antigen-specific T cells for at least about 6 months, or at least about 12 months, or at least about 18 months, or at least about 2 years.

[0029] Naive T cells differentiate in the bone marrow and successfully undergo the positive and negative processes of central selection in the thymus. Naive T cells are considered mature and, unlike activated or memory T cells, have not encountered their alloantigen. Naive T cells can be characterized by the surface expression of L-selectin (CD62L) and the absence of activation surface markers. In the naive state, T cells are nearly quiescent and not dividing. According to this disclosure, naive T cells are defined as CD62L+ and CD45RA+.

[0030] Memory T cells include T memory stem cells (T SCMMemory T cells include central memory T cells and effector memory T cells. Memory T cells have previously responded to their allogeneic antigens. Upon a second encounter with the allogeneic antigen, memory T cells can be regenerated to initiate a faster and stronger immune response. Memory T cells include at least T memory stem cells, effector memory T cells, and central memory T cells. Memory T cell subtypes are long-lived and can rapidly amplify into a large number of effector T cells upon re-exposure to their allogeneic antigens.

[0031] T memory stem cells (T scm In this specification, T memory stem cells are defined as CD45RA+ and have at least the following surface markers (CD62L+, CD45RA+, and CD95+). In some embodiments, T memory stem cells disclosed herein are CD62L+, CD45RA+, CD95+ and may have one or more of the following surface markers (CD28+, CD27+, CXCR3+, CD11a+, IL-2Rβ+, CD58+, and CD57-). In some embodiments, T memory stem cells comprise cells that are CD62L+, CD45RA+, CD28+, CD27+, and CD95+. In some embodiments, T memory stem cells comprise cells that are CD62L+, CD45RA+, CD95+, and CXCR3+. In some embodiments, T memory stem cells comprise cells that are CD62L+, CD45RA+, CD95+, and CD11a+. In some embodiments, T memory stem cells include cells that are CD62L+, CD45RA+, CD95+, and IL-2Rβ+. In some embodiments, T memory stem cells include cells that are CD62L+, CD45RA+, CD95+, and CD58+. In some embodiments, T memory stem cells include cells that are CD62L+, CD45RA+, CD95+, and CD57-. This memory subset has stem cell-like ability for self-renewal and pluripotency for reconstitution of memory and effector T cell subsets. SCMThe cells typically represent only a small percentage of circulating T lymphocytes (e.g., more than 5%), and have the ability to rapidly proliferate in response to antigen re-exposure and release inflammatory cytokines. Therefore, T SCM The cells are a subset of a subset of memory T cells. SCM Cells can be created and / or controlled using enrichment and amplification processes with paramagnetic artificial antigen-presenting cells (aAPCs) and recombinant T cell growth factor cocktails, as disclosed herein.

[0032] According to this disclosure, central memory T cells (T CM Effector memory T cells (T) are defined herein as CD62L+ and CD45RA-. These memory subsets are commonly found in lymph nodes and peripheral circulation. EM Memory T cells are defined herein as CD62L- and CD45RA-. These memory T cells lack lymph node homing receptors and are therefore found in peripheral circulation and tissues. TEMRA are terminally differentiated effector memory cells (T) that reexpress CD45RA. emra These cells lack the ability to divide and are CD62L- and CD45RA+.

[0033] T Central Memory (T CM ) Cells exhibit the ability to self-replicate, which is also important for obtaining the effect of prolonged survival according to embodiments of the present invention. EM Cells also possess some degree of self-renewal capabilities and strongly express genes essential for cytotoxic functions. EMRA Cells, while possessing robust cytotoxic functions, do not exhibit the ability to self-replicate.

[0034] The compositions in various embodiments are designed to balance the duration of their potent effect against malignant tumor cells or other target cells, SCM , T CM and T EMThe CTLs are substantially composed of cells. For example, in some embodiments, these cells constitute at least about 75%, or at least about 80%, or at least about 90% of the memory phenotype.

[0035] In various embodiments, the T cells in the composition consist of at least about 30% central and effector memory cells, or at least about 40% central or effector memory cells, or at least about 50% central or effector memory T cells, or in some embodiments, at least about 70% central or effector memory cells, or at least about 80% central or effector memory T cells, or at least about 90% central or effector memory T cells.

[0036] In some embodiments, the cell composition contains less than 20% terminally differentiated memory T cells (e.g., T EMRA CD8+ T cells are or contain less than 10%, less than 5%, or less than 4% terminally differentiated memory T cells. In various embodiments, CD8+ T cells contain less than 30% naive cells, or in some embodiments less than 15% naive cells, or less than 10% naive cells, or less than 5% naive cells, or less than 4% naive cells, or less than 3% naive cells, or less than 2% naive cells, or less than 1.5% naive cells, or less than 1% naive cells.

[0037] In various embodiments, CD8+ T cells contain approximately 1% to 100% T memory stem cells, or in some embodiments, approximately 1% to 50% T memory stem cells, or in some embodiments, approximately 1% to 25% T memory stem cells, or approximately 5% to 25% T memory stem cells, or approximately 5% to 15% T memory stem cells (i.e., naive, T SCM , T CM , T EM T emra (Assuming the total number of cells is 100%).

[0038] In some embodiments, T SCM and T CM Cells constitute approximately 30% to 80% of the memory phenotype, or in some embodiments, approximately 40% to 80% of the memory phenotype, or in some embodiments, approximately 40% to 70% of the memory phenotype.

[0039] In various embodiments, target antigen-specific T cells consist of at least about 30% central and effector memory cells, or at least about 40% central or effector memory cells, or at least about 50% central or effector memory T cells, or in some embodiments, at least about 70% central or effector memory cells, or at least about 80% central or effector memory T cells, or at least about 90% central or effector memory T cells. In some embodiments, these memory cells have a ratio of about 10:90 to about 90:10 central to effector memory cells. In some embodiments, these T cells have a ratio of about 25:75 to about 75:25 central to effector memory cells. In some embodiments, memory T cells have a ratio of about 40:60 to about 60:40 central to effector memory T cells. T cells specific to a target antigen(s) consist of less than 20% terminally differentiated memory T cells (e.g., TEMRA cells), or less than 10%, less than 5%, or less than 4% terminally differentiated memory T cells. In various embodiments, the target antigen-specific T cells consist of less than 30% naive cells, or in some embodiments, less than 20% naive cells, or in some embodiments, less than 15% naive cells, or less than 10% naive cells, or less than 5% naive cells, or less than 2%, or 1.5%, or 1% naive cells. In various embodiments, the antigen-specific T cells consist of about 1% to about 100% T memory stem cells, or in some embodiments, about 1% to about 50% T memory stem cells, or in some embodiments, about 5% to about 25% T memory stem cells, or about 5% to about 15% T memory stem cells. These memory T cells can be created by enrichment and amplification processes using paramagnetic artificial antigen-presenting cells (aAPCs). SCM The cell-containing aggregates are prepared using known techniques, including magnetic concentration or cell sorting. SCM This may be prepared for cells and further isolated or concentrated.

[0040] In various embodiments, the cell composition is at least 90% T cells, or at least 95% T cells, or at least 98%, or at least 99% T cells. For the purposes of this disclosure, T cells are characterized by CD3+ cells. T cells are substantially CD8+ or CD4-. Where used herein, the terms “CD8+” and “CD4-” are interchangeable unless otherwise stated. For example, an isolated cell composition may be characterized by containing less than about 10% or less than 5% CD4+ T cells, or in some embodiments, less than about 2%, less than 1.5%, or less than 1% CD4+ T cells. When CD8+ T cells are amplified ex vivo, CD4+ cells tend to overgrow CD8+ cells and compete for growth signals, and exogenous CD4+ T cells are not required for a robust and durable response during adoptive transfer.

[0041] The presence of multifunctional CD4+ and CD8+ T cells has been reported to correlate with the response to cancer vaccine therapy using peptide neoantigens. (Ott PA, et al., An immunogenic personal neoantigen vaccine for patients with melanoma, Nature 547(7662):217-221 (2017)). It has been further reported that CD4+ and CD8+ T cells are important in mediating tumor cell destruction. (See Tran E, Cancer immunotherapy based on mutation-specific CD4+ T cells in a patient with epithelial cancer, Science 344, 641-645 (2014), Sahin U, et al., Personalized RNA mutanome vaccines mobilize poly-specific therapeutic immunity against cancer, Nature 547(7662):222-226 (2017)). In relation to this disclosure, it is considered necessary for adoptive cell compositions to provide only a considerable number of antigen-specific CD8+ T cells, especially if their phenotype can support a robust and durable response, and especially if a sufficient number of antigen-specific CD8+ T cells are provided.

[0042] In various embodiments, the cell composition is substantially CD28+. For example, in various embodiments, the cell composition is CD28+ by at least about 25%, or at least about 50%, or at least about 75%, or at least about 90%.

[0043] In various embodiments, antigen-specific T cells exhibit a multifunctional phenotype upon activation. For example, upon activation, T cells are positive for two or more of the following: intracellular staining for IL-2, a marker of proliferation and memory; IFN-γ production, which activates other T cells and induces MHC memory and upregulation; TNF-α production, a marker of pro-inflammatory activity; and CD107A, a marker of granzyme release and cytotoxic activity. In various embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% of antigen-specific T cells exhibit at least three of these markers. In various embodiments, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, or at least 80% of antigen-specific T cells exhibit all four of these markers. In some embodiments, multifunctionality is evaluated or quantified using targeted killing assays, which assess the ability of CD8+ cytotoxic T cells to lyse target cells that present peptide antigens in complex with MHC.

[0044] In various embodiments, the cell composition further comprises γδT cells. γδT cells possess their own T cell receptor (TCR) on their surface. In contrast to αβT cells, γδT cells have a TCR composed of one γ chain and one δ chain. γδT cells are thought to not require antigen treatment and presentation of major histocompatibility complex (MHC) peptide epitopes for activation. γδT cells may play a role in the recognition of lipid antigens and phosphoantigens and can play a role in antiviral and antitumor protection. See Kalyan and Kabelitz, Defining the nature of human γδ T cells: a biographical sketch of the highly empathetic, Cellular & Molecular Immunology (2013) 10,21-29. γδT cells can provide support to CD8+ cells by releasing cytokines, for example, contributing to the activation, proliferation, and differentiation of CD8+ cells. Furthermore, the clinical significance of γδ T cells has been found in hematopoietic stem cell transplantation (HSCT), and in particular, a higher frequency of γδ T cells after transplantation was associated with desirable outcomes. See Berglund et al., Expansion of Gammadelta T cells from Cord Blood: A Therapeutic Possibility. Stem Cells International Vol. 2018.

[0045] In various embodiments, the cell composition contains at least about 2% γδT cells, or at least about 5% γδT cells. In some embodiments, the cell composition contains at least about 10% γδT cells, or at least about 20% γδT cells. In some embodiments, the cell composition contains at least about 25% γδT cells, or at least about 30%, or at least about 35%, or at least about 40%, or at least about 45% γδT cells. In these embodiments, the γδT cells may include either or both Vδ1 and Vδ2 cells. In some embodiments, some of the γδT cells are CD8+. In various embodiments, the γδT cells are primarily CD28+.

[0046] According to various embodiments, cell compositions can be prepared by enriching CD8+ cells specific to a target antigen(s) (e.g., tumor-associated antigen or virus-associated antigen). This cell aggregate can be rapidly amplified in a culture, even if it is the major naive cell in the source lymphocytes, to reach the cell compositions described herein. CD4+ cells can be depleted from lymphocytes using CD4+ cell depletion microbeads (before or after antigen-specific enrichment).

[0047] Antigen-specific enrichment of CD8+ cells can be performed using paramagnetic beads, allowing for positive selection of cell aggregates, which may have the additional benefit of activating naive cells due to strong magnetic clustering of T cell surface receptors. For example, the paramagnetic beads or nanoparticles may, in some embodiments, contain monomeric or multimeric (e.g., dimeric) HLA ligands that present peptide antigens, together with a co-stimulatory signal, e.g., a CD28 agonist (e.g., an antibody agonist of CD28). Exemplary methods according to these embodiments are described in WO2016 / 044530, PCT / US2017 / 22663, and US10,908,939, which are incorporated herein by reference in their entirety.

[0048] In some embodiments, CD28+ cells are also enriched, which may occur simultaneously with antigen-specific enrichment. CD28 is expressed on T cells and is a costimulatory signal required for T cell activation and survival. CD28 is the only B7 receptor constitutively expressed on naive T cells. Association of the TCR and MHC antigen complex on naive T cells without CD28 costimulation may result in immune-unresponsive T cells. In some embodiments, CD28+ cells are not enriched, but the CD28 agonist is added in a soluble form during the enrichment process or as conjugated on highly magnetic beads. In some embodiments, CD28 (in conjugated or unconjugated form) is added to cells after antigen-specific enrichment to activate the cells for the amplification phase.

[0049] In various embodiments, T cells specific to a target antigen (e.g., in terms of peptides indicated by aAPC or pAPC) are specific to 1 to about 100 target antigens, or 1 to about 75 target antigens, or 1 to about 50 target antigens, or 1 to about 25 target antigens, or 1 to about 20 target antigens, or 1 to about 15 target antigens, or 1 to 10 target antigens, or 1 to 5 target antigens. In various embodiments, there are at least 3, or at least 4, or at least 5 target antigens. Different target antigens may include overlapping peptide epitopes in some embodiments. These peptide antigen-specific T cells can be enriched and amplified in batches, enabling rapid parallel production of cell compositions. In some embodiments, the composition contains T cells specific to 5 to 15, or 5 to 10 peptide antigens. The T cell specificity to target peptide antigens in the composition is defined by MHC multimer staining (e.g., dimer or tetramer staining), as is well known in the art.

[0050] For example, a cocktail of nano-aAPCs, each presenting a different target antigen, can be used to simultaneously enrich T cells against multiple antigens. For instance, T cells specific to 2 to 10 antigens can be simultaneously enriched from a lymphocyte source. In this embodiment, several different nano-aAPC batches, each having a different MHC peptide, are combined and used to simultaneously enrich T cells against each of the target antigens. The resulting T cell pool is activated against each of these antigens and amplified together in a culture. These antigens may be associated with a single therapeutic intervention, e.g., multiple antigens present in a single tumor or malignant cell.

[0051] The target peptide antigen is generally preferred to be presented as an HLA-A, B, or C molecular complex, and in some embodiments, as an HLA-A2 molecular complex.

[0052] In various embodiments, the target peptide antigen is a tumor or cancer-associated antigen, including tumor-derived or tumor-specific antigens. Tumor-associated antigen-specific T cells are often very rare and, in many cases, undetectable in the peripheral blood of healthy individuals. Furthermore, these cells are often naive phenotypes, especially when using donor T lymphocytes. (See Quintarelli et al., Cytotoxic T lymphocytes directed to the preferentially expressed antigens of melanoma (PRAME) target chronic myeloid leukemia. Blood 2008;112:1876-1885). This is often the distinction observed between virus-specific T cells and tumor antigen-specific T cells.

[0053] "Tumor-associated antigens" or "cancer-specific antigens" include unique tumor or cancer antigens exclusively expressed by the tumor or malignant tumor cells from which they originate, common tumor antigens expressed in many tumors but not in normal adult tissue (oncofetal antigens), and tumor-specific antigens that are also expressed in normal tissue from which tumors arise. Tumor-associated antigens may also be, for example, embryonic antigens, antigens with abnormal post-translational modifications, differentiation antigens, products of mutated oncogenes or tumor suppressor factors, fusion proteins, or oncoviral proteins.

[0054] In some embodiments, the target peptide antigens include one or more that are associated with or derived from hematological malignancies, such as leukemia, lymphoma, or myeloma. For example, hematological malignancies may include acute myeloid leukemia, chronic myeloid leukemia, cyclodysplastic leukemia, non-Hodgkin lymphoma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, myelodysplastic syndrome, malignant cutaneous T cell lymphoma, mycosis fungoides, non-MF cutaneous T cell lymphoma, lymphomatoid papulosis, and cutaneous lymphoid hyperplasia rich in T cells. In other embodiments, the target peptide antigens include one or more that are associated with or derived from solid tumors, including melanoma, colon cancer, duodenal cancer, prostate cancer, breast cancer, ovarian cancer, ductal cancer, liver cancer, pancreatic cancer, kidney cancer, endometrial cancer, testicular cancer, gastric cancer, dysplastic oral mucosa, polyposis, head and neck cancer, invasive oral cancer, non-small cell lung carcinoma, small cell lung cancer, mesothelioma, transitional and squamous cell urinary carcinoma, brain cancer, neuroblastoma, and glioma.

[0055] Various tumor-associated antigens are known in the art. Examples of carcinoembryonic and embryonic antigens include carcinoembryonic antigen and alpha-fetoprotein (usually highly expressed only in developing embryos, but frequently highly expressed in liver and colon tumors, respectively), MAGE-1 and MAGE-3 (expressed in melanoma, breast cancer, and glioma), placental alkaline phosphatase sialyl-Lewis X (expressed in adenocarcinoma), CA-125 and CA-19 (expressed in gastrointestinal, liver, and gynecological tumors), TAG-72 (expressed in colorectal tumors), epithelial glycoprotein 2 (expressed in many carcinomas), pancreatic carcinoembryonic antigen, 5T4 (expressed in gastric carcinoma), alpha-fetoprotein receptor (expressed in multiple tumor types, particularly in breast tumors), and M2A (expressed in germ cell neogenesis).

[0056] Tumor-associated differentiation antigens include tyrosinase (expressed in melanoma) and certain surface immunoglobulins (expressed in lymphoma).

[0057] Mutant oncogenes or tumor suppressor products include Ras and p53, both expressed in many tumor types, Her-2 / neu (expressed in breast and gynecological cancers), EGF-R, estrogen receptor, progesterone receptor, retinoblastoma gene product, myc (associated with lung cancer), ras, p53, non-mutants associated with breast tumors, MAGE-1, and MAGE-3 (associated with melanoma, lung cancer, and other cancers). A fusion protein is BCR-ABL, expressed in chronic myeloid leukemia. Cancer virus proteins include HPV types 16, E6, and E7, found in cervical carcinoma.

[0058] Tissue-specific antigens include melanotransferrin and MUC1 (expressed in pancreatic and breast cancer), CD10 (formerly known as the common acute lymphoblastic leukemia antigen, or CALLA), or surface immunoglobulin (expressed in B-cell leukemia and lymphoma), IL-2 receptor α chain, T-cell receptor, CD45R, CD4+ / CD8+ (expressed in T-cell leukemia and lymphoma), prostate-specific antigen and prostatic acid phosphatase (expressed in prostate carcinoma), GP100, MelanA / Mart-1, tyrosinase, gp75 / brown, BAGE, and S-100 (expressed in melanoma), cytokeratin (expressed in various cancers), and CD19, CD20, and CD37 (expressed in lymphoma).

[0059] Tumor-associated antigens include modified glycolipid and glycoprotein antigens, such as neuraminic acid-containing glycosphingolipids (e.g., GM2 and GD2, expressed in melanoma and some brain tumors), blood group antigens that may be expressed in carcinomas, particularly T and sialylated Tn antigens, and mucins, such as CA-125 and CA-19-9 (expressed in ovarian carcinomas), or insufficiently glycosylated MUC-1 (expressed in breast and pancreatic carcinomas).

[0060] For example, in some embodiments, one or more target antigens, such as NY-ESO-1, MAGE-A10, and MUC-1 antigens, are associated with bladder cancer. In some embodiments, one or more target antigens are associated with brain cancer and may include one or more of NY-ESO-1, Surivin, and CMV antigens. In some embodiments, one or more target antigens are associated with breast cancer and may include one or more of MUC-1, Surivin, WT-1, HER-2, and CEA antigens. In some embodiments, one or more target antigens are associated with cervical cancer and may include HPV antigens. In some embodiments, one or more target antigens are associated with colorectal cancer and may include one or more of NY-ESO-1, Surivin, WT-1, MUC-1, and CEA antigens. In some embodiments, one or more target antigens are associated with esophageal cancer and may include NY-ESO-1 antigen. In some embodiments, one or more target antigens may be associated with head and neck cancer and may include HPV antigens. In some embodiments, the target antigen is associated with renal or hepatic cancer and may include the NY-ESO-1 antigen. In some embodiments, the target antigen is associated with lung cancer and may include one or more of the following: NY-ESO-1, Survivin, WT-1, MAGE-A10, and MUC-1 antigen. In some embodiments, one or more target antigens are associated with melanoma and may include one or more of the following: NY-ESO-1, Survivin, MAGE-A10, MART-1, and GP-100. In some embodiments, one or more peptide antigens are associated with ovarian cancer and may include one or more of the following: NY-ESO-1, WT-1, and mesothelin antigen. In some embodiments, one or more target antigens are associated with prostate cancer and may include one or more of the following: Survivin, hTERT, PSA, PAP, and PSMA antigen. In some embodiments, the target antigen is associated with sarcoma and may include the NY-ESO-1 antigen. In some embodiments, one or more target antigens are associated with lymphoma and may include the EBV antigen.In some embodiments, one or more target antigens are associated with multiple myeloma and may include one or more of NY-ESO-1, WT-1, XBP1-US, XBP1-SP, CD138, CS1 (SLAMF7), and SOX2 antigens. In some embodiments, the target antigens associated with multiple myeloma are two or more (or three, four, five, or six) of the peptide antigens disclosed in US9,096,681, which is hereby incorporated by reference in its entirety. Exemplary peptides that include antigen epitopes are XBP1 unspliced (UN). 185-193 , XBP1-US 184-192 , XBP1 spliced (SP) 223-231 , XBP1-SP 367-375 , CD138 265-273 , CD138 260-268 , CS1 240-248 , CS1 239-247 , NY-ESO1 157-165A , and SOX2 118-127 are included. In some embodiments, the target antigens include NY-ESO-1, WT-1, SOX-2, CD138, and CS1. In some embodiments, the target antigens include NY-ESO-1, WT-1, SOX-2, CD138, CS1, and XBP1-US and / or XBP1-SP. In some embodiments, the peptide antigens include NY-ESO-1, WT-1, and SOX-2. See Table 2.

[0061] In some embodiments, one or more target antigens are associated with acute myeloid leukemia or myelodysplastic syndrome and may include one or more (including one, two, three, four, or five) of survivin, WT-1, PRAME, RHAMM, PR3, and cyclin A1 antigens. In some embodiments, the target antigens include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or all of the target antigens from Table 1 below.

Table 1

[0062] In some embodiments, one or more target antigens may include one or more of the following: XBP1-US, XBP1-SP, CD138, CS1, NY-ESO1, SOX2, EBV, influenza, CMV, RHAMM, PR3, Mart-1 / Melan A, gp100, CMVpp65, and influenza matrix protein M1 antigen. In some embodiments, the target antigens include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 target antigens from Table 2 below, which are useful for targeting multiple myeloma, melanoma, or various viral diseases or infections. [Table 2]

[0063] In some embodiments, one or more target peptide antigens are neoantigens. For example, in some embodiments, patient-specific neoantigens are identified and synthesized to load aAPCs. In some embodiments, 3 to 10 neoantigens are identified by genetic analysis of the patient's malignant tumor (e.g., by nucleic acid sequencing of malignant tumor cells), followed by predictive bioinformatics. In some embodiments, the antigens are native, unmutated cancer antigens, many of which are known.

[0064] In various embodiments, at least one of the target peptide antigens is recognized by low-frequency precursor T cells. According to these embodiments, the present invention enables rapid activation and amplification of these cells for adoptive therapy.

[0065] In some embodiments, the target peptide antigen comprises at least one that is associated with or derived from a pathogen, such as a virus, bacterium, fungus, or parasitic pathogen. For example, at least one peptide antigen may be associated with HIV, hepatitis (e.g., A, B, C, or D), CMV, Epstein-Barr virus (EBV), influenza, herpesvirus (e.g., HSV1 or 2, or varicella-zoster), and adenovirus. For example, CMV is the most common viral pathogen found in organ transplant patients and is a major cause of morbidity and mortality in patients undergoing bone marrow or peripheral blood stem cell transplantation. This is due to the immunocompromised state of these patients, which allows for reactivation of latent viruses in serologically positive patients or opportunistic infections in serologically negative individuals. In these embodiments, the patient may receive adoptive immunotherapy containing T cells specific to the pathogen antigen. This method may involve the generation of virus-specific CTLs derived from the patient or a suitable donor prior to the commencement of the transplantation procedure.

[0066] In some embodiments, at least one target antigen is a pathogen-associated antigen, including antigens associated with protozoa, bacteria, fungi (both unicellular and multicellular), viruses, prions, intracellular parasites, helminths, and other infectious agents.

[0067] Examples of bacterial antigens include those of Gram-positive cocci, Gram-positive bacilli, Gram-negative bacteria, and anaerobic bacteria, such as organisms belonging to the families Actinomycetaceae, Bacillaceae, Bartonellaceae, Bordetellae, Captophagaceae, Corynebacteriaceae, Enterobacteriaceae, Legionellaceae, Micrococcaceae, Mycobacteriaceae, Nocardiaceae, Pasteurellaceae, Pseudomonadaceae, Spirochaetaceae, and Vibrionaceae, as well as organisms belonging to the genera Acinetobacter, Brucella, Campylobacter, Erysipelothrix, Ewingella, Francisella, Gardnerella, Helicobacter, Levinea, Listeria, Streptobacillus, and Tropheryma.

[0068] Antigens for protozoan infections include those of malarial plasmodia, Leishmania, Trypanosoma, and Schistosoma species.

[0069] Examples of fungal antigens include those from Aspergillus, Blastomyces, Candida, Coccidioides, Cryptococcus, Histoplasma, Paracoccicioides, Sporothrix, organisms of the Mucorales order, organisms that induce black mycosis and mycetoma, and antigens from organisms of the genera Trichophyton, Microsporum, Epidermophyton, and Malassezia.

[0070] Examples of viral peptide antigens include, but are not limited to, those of adenovirus, herpes simplex virus, papillomavirus, polynuclear respiratory virus, poxvirus, HIV, influenza virus, EBV, hepatitis, and CMV. Particularly useful viral peptide antigens include HIV proteins, such as HIV gag protein (including, but not limited to, membrane anchor (MA) protein, core capsid (CA) protein, and nucleocapsid (NC) protein), HIV polymerase, influenza matrix (M1) protein and influenza virus nucleocapsid (NP) protein, hepatitis B surface antigen (HBsAg), hepatitis B core protein (HBcAg), hepatitis protein (HBeAg), hepatitis B DNA polymerase, and hepatitis C antigen.

[0071] In some embodiments, the target peptide antigen comprises one or more tumor-associated antigens and one or more virus-associated antigens (e.g., CMV, EBV, influenza, or adenovirus) to provide an antitumor response while protecting against common pathogens that complicate recovery after HSCT.

[0072] Patients who undergo HSCT are at a special risk of infection due to their immunocompromised state. This immunocompromised state allows for reactivation of latent viruses in serologically positive patients or opportunistic infections in serologically negative individuals. For example, post-transplant lymphoproliferative disorder (PTLD) occurs in a significant proportion of transplant patients and results from Epstein-Barr virus (EBV) infection. EBV infection is estimated to be present in approximately 90% of the adult population in the United States. While active viral replication and infection are suppressed by the immune system, as with CMV, individuals immunocompromised by transplant therapy lose the controlling T cell aggregates, allowing for viral reactivation. This represents a serious flaw in transplant protocols. EBV may also be involved in tumorigenesis in various hematological and non-hematological malignancies.

[0073] In yet another embodiment, the cell composition comprises T cells specific to tumor-associated antigens, with pathogen-associated T cells provided as bystander cells. Specifically, bystander cells are enriched by enriching CD8+ T cells based on selection using both HLA-peptide complexes and anti-CD28, and are amplified, in particular, when using a T cell growth factor cocktail capable of inducing some nonspecific amplification of these cells without causing antigen-specific activation. In these embodiments, the majority of the composition consists of T cells specific to target peptides (e.g., 5% to 75%, or 10% to 50%), while the remaining T cells provide some reconstitution of the immune system against common pathogens, which is particularly beneficial post-transplant. For example, the composition may contain T cells specific to CMV, EBV, influenza, and adenovirus. In each case, pathogen-specific T cells may be present in an amount of 0.1% to about 4% of the composition.

[0074] In various embodiments, the present invention involves compositions prepared by enriching and amplifying antigen-specific CD8+ T cells. Progenitor T cells can be obtained from a patient or a suitable HLA-matched donor. The T cell source may be either a fresh or frozen sample. Progenitor T cells can be obtained from several sources, including WBCs, including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, spleen tissue, buffy coat fraction, and tumors. In some embodiments, progenitor T cells can be obtained from blood units recovered from a subject using any number of techniques known to those skilled in the art. For example, progenitor T cells derived from the circulating blood of an individual can be obtained by apheresis or leukocyte separation. Apheresis products typically contain lymphocytes, including T cells and progenitor T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, erythrocytes, and platelets. Leukocyte separation is an experimental procedure in which leukocytes are separated from a blood sample.

[0075] Cells recovered by apheresis may be washed to remove the plasma fraction, and then placed in a suitable buffer or culture medium for subsequent processing. The washing step can be achieved by methods known to those skilled in the art, for example, by using a semi-automatic "flow-through" centrifuge. After washing, the cells may be resuspended in various biocompatible buffers, such as Ca-free and Mg-free PBS. Alternatively, undesirable components may be removed from the apheresis sample, and the cells may be resuspended directly in the culture medium.

[0076] If desired, precursor T cells can be isolated from peripheral blood lymphocytes by lysing erythrocytes and depleting monocytes, for example, by centrifugation using a PERCOLL® gradient.

[0077] In certain embodiments, leukocytes are recovered by leukocyte separation, and then CD8+ T cells may be enriched, for example, by depleting a sample of CD4+ cells and / or positively enriching CD8+ cells. In some embodiments, other cell types, such as NK cells, are depleted. The CD8-enriched cells may then be further enriched for antigen-specific T cells.

[0078] In various embodiments, a sample containing immune cells (e.g., CD8+ T cells) is brought into contact with artificial antigen-presenting cells (aAPCs) that possess magnetic properties. Paramagnetic materials have a small positive sensitivity to magnetic fields. These materials are attracted to magnetic fields, and when the external magnetic field is removed, the material loses its magnetic properties. Exemplary paramagnetic materials include, but are not limited to, magnesium, molybdenum, lithium, tantalum, and iron oxide. Paramagnetic beads suitable for magnetic enrichment are commercially available (DYNABEADS®, MACS MICROBEADS®, Miltenyi Biotec). In some embodiments, the aAPC particles are iron dextran beads (e.g., dextran-coated iron oxide beads).

[0079] Antigen-presenting complexes include antigen-binding grooves, are generally MHC class I, and can be linked or ligated to provide dimeric or multimeric MHCs. In some embodiments, the MHCs are monomers, but their close association on nanoparticles is sufficient for avidity and activation. In some embodiments, the MHCs are dimeric. Dimeric MHC class I ligands can be constructed by fusion to an immunoglobulin heavy chain sequence, and then by one or more disulfide bonds (with or without the associated light chain). MHC multimers can be made by direct linkage via peptides or chemical linkers, or they may be multimers by association with streptavidin via a biotin moiety. In some embodiments, the antigen-presenting complex is an MHC class I complex with fusion to an immunoglobulin sequence.

[0080] MHC class I molecular complexes having immunoglobulin sequences are described in U.S. Patent No. 6,268,411, which is incorporated herein by reference in its entirety. These MHC class I molecular complexes may be formed in a structurally intact manner at the terminus of an immunoglobulin heavy chain. The MHC class I molecular complex to which the antigen peptide is bound can stably bind to an antigen-specific lymphocyte receptor (e.g., a T cell receptor). In various embodiments, the immunoglobulin heavy chain sequence includes, but is not full-length, an Ig hinge region and one or more of the CH1, CH2, and / or CH3 domains. The Ig sequence may or may not include a variable region, and if a variable region sequence is present, the variable region may be complete or partial. The complex may further include an immunoglobulin light chain. MHC class I ligands (e.g., HLA-Ig) lacking a variable chain sequence (and any light chain) may be used in conjunction with site-specific conjugation to particles, as described in WO2016 / 105542, which is incorporated herein in whole by reference.

[0081] An exemplary MHC class I molecular complex comprises at least two fusion proteins. The first fusion protein comprises a first MHC class I α chain and a first immunoglobulin heavy chain (or a portion thereof including a hinge region), and the second fusion protein comprises a second MHC class I α chain and a second immunoglobulin heavy chain (or a portion thereof including a hinge region). The first and second immunoglobulin heavy chains associate to form an MHC class I molecular complex, which comprises two MHC class I peptide bond grooves. The immunoglobulin heavy chain may be an IgM, IgD, IgG1, IgG3, IgG2β, IgG2α, IgG4, IgE, or IgA heavy chain. In some embodiments, an IgG heavy chain is used to form the MHC class I molecular complex. If a polyvalent MHC class I molecular complex is desired, an IgM or IgA heavy chain can be used to provide a pentavalent or tetravalent molecule, respectively.

[0082] Exemplary Class I molecules include HLA-A, HLA-B, HLA-C, and HLA-E, which can be used individually or in any combination. In some embodiments, the antigen-presenting complex is an HLA-A2 ligand. As used herein, the term MHC may be replaced by HLA in each instance.

[0083] In some embodiments, the immunoglobulin sequence is a humanized monoclonal antibody sequence.

[0084] aAPC may contain a "signal 2" such as an anti-CD28 ligand. Signal 2s are generally molecules that affect T cells, i.e., molecules that have a biological effect on progenitor T cells or antigen-specific T cells. In certain embodiments, signal 2s are T cell costimulatory molecules. T cell costimulatory molecules contribute to the activation of antigen-specific T cells. Such molecules include, but are not limited to, molecules that specifically bind to CD28 (including antibodies), CD80 (B7-1), CD86 (B7-2), B7-H3, 4-1BB, 4-1BBL, CD27, CD30, CD134 (OX-40L), B7h (B7RP-1), molecules that specifically bind to CD40, LIGHT, antibodies that specifically bind to HVEM, antibodies that specifically bind to CD40L, and antibodies that specifically bind to OX40. In some embodiments, the co-stimulatory molecule (signal 2) is an antibody (e.g., a monoclonal antibody) or a part thereof, e.g., F(ab')2, Fab, scFv, or a single-chain antibody, or another antigen-binding fragment. In some embodiments, the antibody is a humanized monoclonal antibody or a part thereof having antigen-binding activity, or a fully human antibody or a part thereof having antigen-binding activity.

[0085] Possible co-stimulatory ligand combinations that can be used (on the same or separate nanoparticles) include anti-CD28 / anti-CD27 and anti-CD28 / anti-41BB. The ratios of these co-stimulatory ligands can be varied to produce amplification.

[0086] Exemplary signal 1 and signal 2 ligands are described in WO2014 / 209868, which describe ligands having free sulfhydryls (e.g., unpaired cysteine) and consequently, a constant region that can be bound to a nanoparticle support having appropriate chemofunctionality.

[0087] Adhesion molecules useful for nano-aAPCs can be used to mediate the adhesion of nano-aAPCs to T cells or T cell precursors. Examples of useful adhesion molecules include ICAM-1 and LFA-3.

[0088] In some embodiments, signal 1 is provided by a peptide-HLA-A2 complex, and signal 2 is provided by B7.1-Ig or anti-CD28. An exemplary anti-CD28 monoclonal antibody is 9.3 mAb (Tan et al., J.Exp.Med. 1993 177:165) and may be humanized in certain embodiments and / or conjugated to beads as a complete intact antibody or its antigen-binding fragment.

[0089] Magnetic activation may be performed for 2 minutes to 5 hours, or 5 minutes to 2 hours, in some embodiments, and then amplified in culture for at least 5 days, up to 2 weeks, or up to 3 weeks. In some embodiments, magnetic activation occurs for at least 2 minutes, but less than 30 minutes, or less than 15 minutes (e.g., about 5 or 10 minutes). The resulting CD8+ T cells are phenotypically characterized and are T memory stem cells (T scm The presence of ) and a high central and effector memory phenotype may also be confirmed.

[0090] Some embodiments utilize T cell growth factors during amplification, thereby influencing T cell proliferation and / or differentiation. Examples of T cell growth factors include cytokines (e.g., interleukins, interferons) and superantigens. If desired, cytokines may be present in molecular complexes containing fusion proteins, encapsulated by aAPCs, or provided in soluble form. Particularly useful cytokines include MIP-1β, IL-1β, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-15, IL-21, IFN-γ, and CXCL10. In some embodiments, the growth factors include three, four, five, or six from MIP-1β, IL-1β, IL-2, IL-4, IL-6, IL-7, IL-10, IL-15, IL-21, and INF-γ. In these or other embodiments, cells are amplified in a culture in the presence of cytokines, including one, two, or three cytokines selected from MIP-1β, IL-1β, IL-6, and IL-10. In some embodiments, cells are not cultured in the presence of IL-7 and / or IL-21 and / or IL-15. Cells can be amplified in the culture for 1 to 4 weeks, for example, about 2 weeks (about 14 days), or about 3 weeks.

[0091] In some embodiments, cells are amplified in culture in the presence of 4 to 8 cytokines to achieve a balance between T cell amplification (including antigen-specific T cell amplification), activation, and memory phenotype. In some embodiments, cells are amplified in the presence of IL-4. In some embodiments, cells are amplified in the presence of IL-4 and IL-6. In some embodiments, cells are amplified in the presence of IL-4 and IL-1β. In some embodiments, cells are amplified in the presence of IL-4, IL-6, and IL-1β. In some embodiments, cells are amplified in the presence of IL-2, IL-4, and IL-6. In some embodiments, cells are amplified in culture in the presence of IL-2, IL-4, IL-6, INF-γ, and IL-1β. In some embodiments, cells are further amplified in the presence of IL-10. In various embodiments, these cytokines are used in combination with artificial or native antigen-presenting cells to amplify antigen-specific T cells.

[0092] In some embodiments, the growth factors consist of IL-2, IL-4, IL-6, INF-γ, IL-1β, and optionally IL-10, or essentially consist of these.

[0093] In some embodiments, IL-2 is present at the start of culture at 10–200 international units (IU) per mL, e.g., about 20–100 IU / mL, or about 20–60 IU / mL. In some embodiments, IL-2 is present at the start of culture at about 30–50 IU / mL (e.g., about 40 IU / mL). The IU (86 / 500 NIBSC) of IL-2 can be determined using a growth assay (e.g., using the CTLL-2 cell line), as described, for example, in Gearing and Bird (1987), Lymphokines and Interferons, A Practical Approach. Clemens, MJ et al. (eds.): IRL Press. 295. In some embodiments, IL-2 is present at the start of culture at about 2–25 ng / mL, or about 2–15 ng / mL, e.g., about 5–15 ng / mL.

[0094] In these or independent embodiments, IL-4 is present at the start of culture at 0.2 to 25 international units (IU) per mL, e.g., about 0.5 to about 10 IU / mL, or about 0.5 to about 5 IU / mL. In some embodiments, IL-4 is present at the start of culture at about 1 IU / mL. The IU (88 / 656 NIBSC) of IL-4 can be determined using a proliferation assay (e.g., using the TF-1 cell line), as described, for example, by Kitamura T. et al., (1991) IL-1 up-regulates the expression of cytokine receptors on a factor-dependent human hemopoietic cell line, TF-1. Int. Immunol. 3:571-577. In some embodiments, IL-4 is present at the start of culture at about 0.2 to about 2 ng / mL, e.g., about 0.2 to about 1 ng / mL (e.g., about 0.5 ng / mL).

[0095] In these or independent embodiments, IL-6 may be present at the start of culture at 10–200 international units (IU) per mL, e.g., about 25–100 IU / mL, e.g., 25–75 IU / mL. In some embodiments, IL-6 is present at the start of culture at about 40–60 IU / mL (e.g., about 50 IU / mL). The IU (89 / 548 NIBSC) of IL-6 can be defined using a growth assay (e.g., using a B9 cell line), as described, for example, Gaines-Das RE and Poole S. (1993) The international standard for interleukin-6. Evaluation in an international collaborative study. J.Immunol.Methods 160:147–153. In some embodiments, IL-6 is present at the start of culture in a concentration of approximately 0.2 to 10 ng / ml, for example, approximately 0.2 to 5 ng / ml (e.g., approximately 0.2 to 1 ng / ml, or approximately 0.5 to 2 ng / ml).

[0096] In these or independent embodiments, interferon-gamma (INF-γ) may be present at the start of culture at 10–200 international units (IU) per mL, e.g., about 20–100 IU / mL, e.g., 20–60 IU / mL. In some embodiments, INF-γ is present at the start of culture at about 30–50 IU / mL (e.g., about 40 IU / mL). The IU (87 / 586 NIBSC) of INF-γ can be defined using an antiviral assay (e.g., using EMC-infected HeLa cells), as described, for example, in Meager A. (1987), Lymphokines and interferons, a Practical Approach. Clemens, MJ, et al. (eds.): IRL Press. 129. In some embodiments, INF-γ is present at the start of culture in concentrations of approximately 0.5 to 20 ng / ml, for example, approximately 0.5 to 10 ng / ml, or approximately 0.5 to 5 ng / ml, or approximately 1 to 10 ng / ml (for example, 1 to 5 ng / ml).

[0097] IL-1β may be present at the start of culture at 5 to 100 international units (IU) per mL, for example, about 10 to about 50 IU / mL, for example, about 10 to about 30 IU / mL. In some embodiments, IL-1β is present at the start of culture at about 10 to about 20 IU / mL (for example, about 15 IU / mL). The IU (86 / 680 NIBSC) of IL-1β can be defined using a growth assay (e.g., using the D.10.G4.1 cell line), as described, for example, Poole, S. and Gaines-Das, RE (1991) The international standards for interleukin-1 alpha and interleukin-1 beta. Evaluation in an international collaborative study. J.Immunol.Methods 142:1-13. In some embodiments, IL-1β is present at the start of culture at a concentration of approximately 0.1–5 ng / ml, or approximately 0.2–5 ng / ml, for example, approximately 0.2–2 ng / ml, or approximately 0.2–1 ng / ml.

[0098] In various embodiments, cells are cultured in the presence of a growth factor cocktail containing or comprising IL-2, IL-4, IL-6, INF-γ, and IL-1β. In some embodiments, the relative activity of IL-2 and INF-γ (defined by their respective IUs) is about 0.5:1 to about 1:0.5 (e.g., about 1:1). In these or independent embodiments, the relative activity of IL-2 and IL-6 (defined by their respective IUs) is about 0.5:1 to about 1:0.5. In these or independent embodiments, the relative activity of IL-1β to IL-2, IL-6, and / or IFN-γ (defined by their respective IUs) is 1:4 to 1:2 (e.g., about 1:3). In these or independent embodiments, the relative activity of IL-4 to IL-2, IL-6, and / or IFN-γ (defined by their respective IUs) is 1:30 to 1:60. In these or independent embodiments, the relative activity of IL-4 to IL-1β (defined by their respective IU) is about 1:5 to about 1:25, for example, about 1:10 to about 1:20.

[0099] In some embodiments, the specific activity (in units of IU) of each growth factor (IL-2, IL-4, IL-6, INF-γ, and IL-1β) at the start of culture can be expressed as a percentage when the total IU of all growth factors in the culture is considered to be 100%. For example, in some embodiments, the percentages of each growth factor in the culture may be as follows: 20% to 40% IL-2 (for example, 20% to 30% IL-2), 0.5% to 5% IL-4 (for example, 1% to 3% IL-4), 25% to 50% IL-6 (for example, 30% to 40% IL-6), 20% to 40% IFN-γ (for example, 20% to 30% IFN-γ), and 5% to 20% IL-1β (for example, 5% to 15% IL-1β).

[0100] aAPC nanoparticles can be made from any material, which can be appropriately selected for the desired magnetic properties and may include metals such as iron, nickel, cobalt, or alloys of rare earth metals. Paramagnetic materials also include magnesium, molybdenum, lithium, tantalum, and iron oxide. Suitable paramagnetic beads for enriching materials (including cells) are commercially available and include iron dextran beads, e.g., dextran-coated iron oxide beads. In embodiments of the present invention where magnetic properties are not required, nanoparticles can also be made from non-metallic or organic (e.g., polymer) materials such as cellulose, ceramics, glass, nylon, polystyrene, rubber, plastics, or latex. Exemplary materials for the preparation of nanoparticles include poly(lactic acid-coglycolic acid) (PLGA) or PLA, and copolymers thereof, which may be used in connection with these embodiments. Other materials, including polymers and copolymers, that may be used include those described in PCT / US2014 / 25889, which is incorporated herein by reference in whole.

[0101] In various embodiments, the particles have a size (e.g., average diameter) within approximately 10 to 500 nm, or within approximately 40 to 400 nm, or within approximately 40 nm to 200 nm. For magnetic clustering, it is preferable that the nanoparticles have a size (e.g., average diameter) in the range of 10 to 250 nm, or 50 to 200 nm, or 80 to 200 nm, or 20 to 100 nm, in some embodiments. Receptor-ligand interactions at the cell-nanoparticle interface are not well understood. However, nanoparticle binding and cell activation are sensitive to membrane space tissue, which is particularly important during T cell activation, and activation can be enhanced by manipulating clustered nanoparticles using a magnetic field. For example, T cell activation induces a state of sustainedly enhanced nanoscale TCR clustering, and nanoparticles are sensitive to this clustering in a way that larger particles are not.

[0102] Furthermore, nanoparticle interactions with TCR clusters can be utilized to enhance receptor triggering. T cell activation is mediated by the aggregation of signaling proteins, with “signaling clusters” existing over hundreds of nanometers, initially forming around T cell-APC contact sites and then migrating inward. As described herein, an external magnetic field can be used to enrich antigen-specific T cells (including rare naive cells) and promote the aggregation of magnetic nano-aAPCs bound to the TCR, resulting in the aggregation of TCR clusters and enhanced activation of naive T cells. Magnetic fields can exert a sufficiently strong force on paramagnetic particles but are otherwise biologically inert, making them a powerful tool for controlling particle behavior. T cells bound to paramagnetic nano-aAPCs are activated in the presence of an externally applied magnetic field. The nano-aAPCs themselves are magnetized and attracted to the magnetic field source and nearby nanoparticles within the magnetic field, inducing bead aggregation, and thus TCR aggregation, promoting aAPC-mediated activation.

[0103] Using activation chemistry, specific and stable attachment of molecules to the surface of nanoparticles can be enabled. There are many methods that can be used to attach proteins to functional groups. For example, using glutaraldehyde, a common crosslinking agent, protein amine groups can be attached to the surface of amination nanoparticles in a two-step process. The resulting bond is stable to hydrolysis. Other methods include crosslinking agents containing n-hydrosuccinimide (NHS) esters that react with amines on proteins, crosslinking agents containing active halogens that react with proteins containing amines, sulfhydryls, or histidines, crosslinking agents containing epoxides that react with amine or sulfhydryl groups, bonding between maleimide groups and sulfhydryl groups, and the use of periodic oxidation of the pendant sugar moiety followed by reductive amination to form protein aldehyde groups.

[0104] The effectiveness of nanoparticle or co-stimulatory ligand presentation in antigens can be increased by changing the ratio of specific ligands when used simultaneously on the same or different particles. For example, nanoparticles can be bound to HLA-A2-Ig and anti-CD28 (or other signal 2 ligands) in various ratios such as approximately 30:1, approximately 25:1, approximately 20:1, approximately 15:1, approximately 10:1, approximately 5:1, approximately 3:1, approximately 2:1, approximately 1:1, approximately 0.5:1, approximately 0.3:1, approximately 0.2:1, approximately 0.1:1, or approximately 0.03:1. In some embodiments, this ratio is 2:1 to 1:2. The total amount of protein bound to the support may be, for example, approximately 250 mg / ml, approximately 200 mg / ml, approximately 150 mg / ml, approximately 100 mg / ml, or approximately 50 mg / ml of particles. Effector functions such as cytokine release and growth may have different requirements for signal 1 in response to signal 2 than T cell activation and differentiation, and therefore these functions can be determined separately.

[0105] In certain embodiments, the aAPCs are paramagnetic particles in the range of 50–150 nm, with a PDI (size distribution) of less than 0.2, or less than 0.1 in some embodiments. The aAPCs may have a surface charge of 0–-10 mV, e.g., about -2–-6 mV. The aAPCs may have 10–120 ligands per particle, e.g., about 25–100 ligands per particle, and the ligands are conjugated to the particles via free cysteine ​​introduced into the Fc region of the immunoglobulin sequence. The particles may contain HLA dimer:anti-CD28 in a ratio of about 1:1, which may be present in the same or different aggregates of particles. The nanoparticles do not exhibit stimulation of non-allogeneic TCRs while providing potent amplification of allogeneic T cells, even if passive loading of the peptide antigen is present. The particles are stable in lyophilized form for at least 2 or 3 years.

[0106] After enrichment and amplification, the antigen-specific T cell component of the sample will consist of at least about 5%, or at least about 10%, or at least about 15%, or at least about 20%, or at least about 25% antigen-specific T cells. Furthermore, these T cells may include T memory stem cells and also include central and effector memory T cells. From the original sample isolated from a patient or donor, in various embodiments, antigen-specific T cells are amplified about 100-10,000 times, for example, at least about 100 times, or at least about 200 times (in about 7 days). After 2 weeks, antigen-specific T cells are amplified at at least 1,000 times, or at least about 2,000 times, at least about 3,000 times, at least about 4,000 times, or at least about 5,000 times, in various embodiments. In some embodiments, antigen-specific T cells are amplified more than 5,000 times, or more than 10,000 times, after 2 weeks. After one or two weeks of amplification, at least about 10 6 one, or at least about 10 7 one, or at least about 10 8 one, or at least about 10 9 Individual antigen-specific T cells are obtained.

[0107] Suitable incubation conditions (culture medium, temperature, etc.) include those used for culturing T cells or T cell precursors, as well as those known in the art for inducing the formation of antigen-specific T cells using DCs or artificial antigen-presenting cells.

[0108] The cell composition can be administered to the patient by any suitable route, including intravenous infusion, intra-arterial administration, intra-lymphatic administration, and intratumoral administration.

[0109] In some embodiments, the patient receives or initiates immunotherapy with one or more checkpoint inhibitors before (or optionally after) receiving the cell composition by adoptive transfer. In various embodiments, the checkpoint inhibitor(s) target one or more of CTLA-4 or PD-1 / PD-L1 and may include antibodies against such targets, e.g., monoclonal antibodies, a portion thereof, or humanized or fully human embodiments thereof. In some embodiments, the checkpoint inhibitor therapy includes ipilimumab or Keytruda (pembrolizumab), or an equivalent monoclonal antibody. In some embodiments, the patient has previously received PD1 blockade therapy and was refractory or only partially responsive to that treatment. In such embodiments, the cell composition described herein can be optionally combined with a second round of immunotherapy (e.g., anti-CTLA4 or PD-1 blockade therapy) to reactivate a robust T cell response.

[0110] In some embodiments, the patient receives approximately 1 to 5 rounds of adoptive immunotherapy (e.g., 1, 2, 3, 4, or 5 rounds). In some embodiments, each dose of adoptive immunotherapy is administered concurrently with or after one round of checkpoint inhibitor therapy (e.g., approximately 1 day or 1 week later). In some embodiments, adoptive immunotherapy is provided approximately 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 1 week after the administration of the checkpoint inhibitor. In some embodiments, the patient receives only a single dose of the cell composition.

[0111] In some embodiments, the present invention provides a method for personalized cancer immunotherapy. The method is achieved using aAPCs to identify the antigen to which the patient responds, and then administering the appropriate peptide-loaded aAPC to the patient, or subsequently enriching and amplifying antigen-specific T cells ex vivo.

[0112] Genome sequencing has dramatically altered our understanding of cancer biology. Cancer sequencing has provided crucial data on the molecular processes involved in the development of many human cancers. The mutations induced have been identified in key genes involved in pathways that regulate three major cellular processes: (1) cell fate, (2) cell survival, and (3) genome maintenance. Vogelstein et al., Science 339, 1546-58 (2013).

[0113] Genome-wide sequencing also has the potential to revolutionize the inventors' approach to cancer immunotherapy. Sequencing data can provide information on both shared and personalized targets for cancer immunotherapy. In principle, mutant proteins are foreign to the immune system and are putative tumor-specific antigens. Indeed, sequencing efforts have defined hundreds, if not thousands, of potentially relevant immune targets. Limited studies have shown that T-cell responses to these neoepitopes may be found in cancer patients or induced by cancer vaccines. However, the frequency of such responses for specific cancers and the extent to which such responses are shared among patients are not well known. One of the main reasons for the limited understanding of tumor-specific immune responses is that current approaches to verifying potentially immunologically relevant targets are cumbersome and time-consuming.

[0114] Central tolerance invalidates T cell responses to self-proteins, while oncogenic mutations induce neoepitopes that can form T cell responses. A mutation catalog derived from whole-exome sequencing provides a starting point for identifying such neoepitopes. Using an HLA-binding prediction algorithm (Srivastava, PLoS One 4, e6094 (2009)), it has been predicted that each cancer may have up to 7–10 neoepitopes. A similar approach has estimated hundreds of tumor neoepitopes. However, such algorithms may have low accuracy in predicting T cell responses, with only 10% of predicted HLA-binding epitopes predicted to bind in terms of HLA (Lundegaard C, Immunology 130, 309-18 (2010)). Therefore, predicted epitopes must be validated for the presence of T cell responses to their potential neoepitopes.

[0115] In certain embodiments, a nano-aAPC system is used to screen for neoepitopes that induce T cell responses in various cancers or in cancers of specific patients. The cancers can be genetically analyzed, for example, by whole exome sequencing.

[0116] A list of candidate peptides can be generated by superimposing nine amino acid windows in the mutant protein. All nine AA windows containing the mutated amino acids, along with two unmutated "controls" from each protein, are selected. These candidate peptides are computationally evaluated for MHC binding using consensus MHC binding prediction algorithms, including Net MHC and Stabilized Matrix Method (SMM). Nano-aAPC and MHC binding algorithms have been developed primarily for the HLA-A2 allele. The sensitivity cutoff of the consensus prediction can be adjusted until a manageable number of peptides containing mutations (approximately 500) and unmutated control peptides (approximately 50) are identified.

[0117] In an exemplary embodiment, the cell composition comprises, in a pharmaceutically acceptable carrier, at least 70%, at least 80% or at least 90% CD8+ or CD4− T cells, less than 5% CD4+ T cells, and at least 5% T SCM cells, wherein the CD8+ cells comprise at least 10 6 T cells specific for 1 - 10 target peptide antigens. Optionally, the CD8+ or CD4− T cells may comprise T cells specific for bacterial, viral, fungal and / or parasitic pathogens. In various embodiments, at least 30% of the CD8+ or CD4− T cells are T SCM , central, and effector memory T cells, less than 10% of the CD8+ or CD4− T cells are terminally differentiated T cells, and less than 10% of the CD8+ or CD4− T cells are naive cells. In various embodiments, at least 50% of the CD8+ or CD4− T cells specific for the target peptide antigen are T SCM , central and effector memory T cells. In some embodiments, the cell composition comprises about 5% - about 25% T memory stem cells (T scm ), or about 5% - about 20% T memory stem cells.

[0118] In various embodiments, the cell composition further comprises γδ T cells. For example, the cell composition may comprise at least about 2% γδ T cells, or at least about 5% γδ T cells. In some embodiments, the cell composition comprises at least about 10% γδ T cells, or at least about 20% γδ T cells. In some embodiments, the cell composition comprises at least about 25% γδ T cells, or at least about 30%, or at least about 35%, or at least about 40% γδ T cells. In these embodiments, the γδ T cells may comprise one or both of Vδ1 and Vδ2 cells. In some embodiments, the γδ T cells are predominantly Vδ2 (e.g., at least about 60%, or at least about 75%). In some embodiments, a portion of the γδ T cells are CD8+. In various embodiments, the γδ T cells are predominantly CD28+.

[0119] In some embodiments, the cell composition further comprises a pharmaceutically acceptable carrier suitable for intravenous injection and which may be suitable as a cryoprotective agent. An exemplary carrier is DMSO (e.g., about 10%). The cell composition may be supplied in unit vials or bags and stored frozen until use. The unit dose is 50–200 mL in volume, with about 5 × 10¹⁴ per mL. 5 pieces~approx. 5×10 6 It may contain a number of cells. In certain embodiments, the volume of the composition is 100 mL or less (e.g., 50 to 100 mL).

[0120] In some embodiments, the present invention provides a method for treating cancer patients, comprising administering the cell compositions described herein to patients in need.

[0121] In some embodiments, the patient has a hematological malignancy, and in some embodiments, the malignancy has relapsed after allogeneic stem cell transplantation. In some embodiments, the patient has acute myeloid leukemia (AML) or myelodysplastic syndrome.

[0122] Other cancers that may be treated in accordance with this disclosure include cancers with a historically poor immune response or a high recurrence rate. Exemplary cancers include various types of solid tumors, including carcinomas, sarcomas, and lymphomas. In various embodiments, the cancer is melanoma (including metastatic melanoma), colon cancer, duodenal cancer, prostate cancer, breast cancer, ovarian cancer, ductal cancer, liver cancer, pancreatic cancer, kidney cancer, endometrial cancer, testicular cancer, gastric cancer, dysplastic oral mucosa, polyposis, head and neck cancer, invasive oral cancer, non-small cell lung carcinoma, small cell lung cancer, mesothelioma, transitional and squamous cell urinary carcinoma, brain cancer, neuroblastoma, and glioma. In various embodiments, the cancer is stage I, stage II, stage III, or stage IV. In some embodiments, the cancer is metastatic and / or recurrent and / or unresectable.

[0123] In some embodiments, the patient is refractory to chemotherapy and / or checkpoint inhibitor therapy.

[0124] In some embodiments, the patient receives further low-dose cytokine therapy that can improve the persistence and in vivo response.

[0125] In some embodiments, the cancer is a hematological malignancy and includes leukemia, lymphoma, or myeloma. For example, the hematological malignancies may be acute myeloid leukemia, chronic myeloid leukemia, cyclodicytic leukemia, non-Hodgkin lymphoma, acute lymphoblastic leukemia, chronic lymphocytic leukemia, myelodysplastic syndrome, malignant cutaneous T-cell lymphoma, mycosis fungoides, non-MF cutaneous T-cell lymphoma, lymphomatoid papulosis, and T-cell-rich cutaneous lymphoid hyperplasia. In exemplary embodiments, the patient has a hematological cancer such as acute myeloid leukemia (AML) or myelodysplastic syndrome, and in some embodiments, the patient has relapsed after allogeneic stem cell transplantation. In some embodiments, this therapy does not induce GVHD.

[0126] In some embodiments, in addition to allogeneic stem cell transplantation, the patient also receives lymphocyte apheresis, cytoreductive therapy, or immunomodulatory therapy (before cell therapy is administered). In some embodiments, cell therapy may be further provided with or without cytokine-assisted post-treatment.

[0127] In some embodiments, patients have an infection or are at risk of infection. For example, patients undergoing HSCT are at particular risk of infection given their immunocompromised state. Infections that can be treated or prevented include those caused by bacteria, viruses, prions, fungi, parasites, helminths, etc. Such diseases include AIDS, hepatitis B / C, CMV infection, Epstein-Barr virus (EBV) infection, influenza, herpesvirus infections (including herpes zoster), and adenovirus infection. For example, CMV is the most common viral pathogen found in organ transplant patients and is a major cause of morbidity and mortality in patients undergoing bone marrow or peripheral blood stem cell transplants. This is due to the immunocompromised state of these patients, which allows for reactivation of latent viruses in serologically positive patients or opportunistic infections in serologically negative individuals. In these embodiments, patients may receive adoptive immunotherapy containing T cells specific to the pathogen antigen. This method may involve the generation of virus-specific CTLs derived from the patient or a suitable donor prior to the commencement of the transplantation procedure.

[0128] Post-traumatic lung disease (PTLD) occurs in a significant proportion of transplant patients and results from Epstein-Barr virus (EBV) infection. EBV infection is estimated to be present in approximately 90% of the adult population in the United States. Active viral replication and infection are suppressed by the immune system, but, as with CMV, individuals immunocompromised by transplant therapy lose the regulatory T cell aggregates, allowing for viral reactivation. This represents a serious flaw in transplant protocols. EBV may also be involved in tumor promotion in various hematological and non-hematological malignancies.

[0129] In further embodiments, the present invention provides a method for producing a γδ T cell aggregate. The method comprises amplifying the T cell aggregate in the presence of two or more of IL-2, IL-4, IL-6, INF-γ, and IL-1β. The T cell aggregate may be enriched with CD28+ enriched cells and may be positively selected using anti-CD28-containing beads or particles, for example, aAPC as described herein. In some embodiments, the cell aggregate is CD4+ depleted or CD8+ selected. In various embodiments, the starting composition contains less than about 20%, less than about 10%, less than about 8%, or less than 5% γδ T cells. In some embodiments, the source cells are derived from peripheral blood.

[0130] In various embodiments, T cell aggregates are amplified in the presence of IL-4, or in the presence of IL-4 and IL-6. In some embodiments, cells are amplified in the presence of IL-4 and IL-1β. In some embodiments, cells are amplified in the presence of IL-4, IL-6, and IL-1β. In some embodiments, cells are amplified in the presence of IL-2, IL-4, and IL-6. In some embodiments, cells are amplified in a culture in the presence of IL-2, IL-4, IL-6, INF-γ, and IL-1β. The amplification of cells in a culture may be carried out for, for example, 1 to 4 weeks, as described herein. After the amplification period, the percentage of cells that are γδ may be about 5% to about 60%, for example, about 10% to about 60%, or about 15% to about 60%, and this number of γδ T cells is amplified to at least about 100, or at least about 1000, or at least about 10,000, compared to the initial aggregate of cells.

[0131] γδT cells can be isolated from other cells using known methods, such as FACS or magnetic cell sorting. γδT cells may be provided as a cell composition for adoptive transfer or research use, or they may be engineered to express one or more heterologous genes (e.g., T cell receptors, optionally αβTCR). In some embodiments, γδT cells are engineered to heterologously express chimeric antigen receptors (CARs).

[0132] The present invention encompasses embodiments described in the following sections. [Section 1] An isolated cell composition suitable for adoptive immunotherapy, wherein the composition contains at least 10 specific target peptide antigens in a pharmaceutically acceptable carrier. 6 Contains individual CD8+ T cells and T memory stem (T SCM The isolated cell composition comprising ) cells. [Section 2] The isolated cell composition according to item 1, wherein the CD8+ T cells are specific to 1 to 100 target peptide antigens. [Section 3] The isolated cell composition according to item 1, wherein the T cell specificity for the target peptide antigen in the composition is defined by MHC multimer staining. [Section 4] The isolated cell composition according to item 1, wherein the target peptide antigen is a tumor-associated antigen. [Section 5] The isolated cell composition according to item 4, wherein one or more target peptide antigens are tumor-derived neoantigens or tumor-specific neoantigens. [Section 6] The isolated cell composition according to item 1, wherein one or more target peptide antigens are bacterial, viral, fungal, or parasitic antigens. [Section 7] An isolated cell composition according to any one of claims 1 to 6, comprising CD8+ T cells specific to at least five target peptide antigens. [Section 8] The isolated cell composition according to item 7, wherein at least one of the target peptide antigens is recognized by low-frequency precursor T cells. [Section 9] The cell composition is an isolated cell composition according to any one of claims 1 to 8, wherein at least 90% of the cell composition consists of T cells. [Section 10] The isolated cell composition according to item 9, wherein at least 5% of the CD8+ T cells are specific to the target peptide antigen. [Section 11] The isolated cell composition according to item 10, wherein at least 10% of the CD8+ T cells are specific to the target peptide antigen. [Section 12] The isolated cell composition according to item 11, wherein at least 15% of the CD8+ T cells are specific to the target peptide antigen. [Section 13] The isolated cell composition according to item 12, wherein at least 30% of the CD8+ T cells are specific to the target peptide antigen. [Section 14] The isolated cell composition according to item 13, wherein at least 50% of the CD8+ T cells are specific to the target peptide antigen. [Section 15] The isolated cell composition according to any one of claims 4 or 5, wherein the cell composition further comprises CD8+ T cells specific to bacteria, viruses, and / or fungal pathogens. [Section 16] The isolated cell composition according to claim 15, wherein the CD8+ T cells specific to bacterial, viral, and / or fungal pathogens include T cells specific to the antigens of influenza, CMV, EBV, and / or adenovirus. [Section 17] The isolated cell composition according to any one of items 1 to 16, wherein the CD8+ T cells are approximately 1% to approximately 100% T memory stem cells. [Section 18] The isolated cell composition according to item 17, wherein the CD8+ T cells consist of approximately 1% to approximately 50% T memory stem cells. [Section 19] The isolated cell composition according to item 18, wherein the CD8+ T cells consist of approximately 5% to 25% T memory stem cells. [Section 20] The isolated cell composition according to item 18, wherein the CD8+ T cells are at least 5% T memory stem cells. [Section 21] The isolated cell composition according to item 18, wherein the CD8+ T cells are at least 20% T memory stem cells. [Section 22] The isolated cell composition according to item 18, wherein the CD8+ T cells are at least 25% T memory stem cells. [Section 23] The isolated cell composition according to any one of claims 1 to 22, wherein the T cells specific to one or more target antigens are at least 15% T memory stem cells. [Section 24] An isolated cell composition according to any one of claims 1 to 23, wherein more than 95% of the CD8+ T cells contain a memory phenotype. [Section 25] The isolated cell composition according to item 24, wherein the CD8+ T cells include central memory and effector memory T cells. [Section 26] The isolated cell composition according to item 25, wherein at least 30% of the CD8+ T cells are central and effector memory T cells. [Section 27] The isolated cell composition according to item 25, wherein at least 50% of the CD8+ T cells are central and effector memory T cells. [Section 28] The isolated cell composition according to item 25, wherein at least 70% of the CD8+ T cells are central and effector memory T cells. [Section 29] The isolated cell composition according to item 25, wherein at least 80% of the CD8+ T cells are central and effector memory T cells. [Section 30] The isolated cell composition according to item 25, wherein at least 90% of the CD8+ T cells are central and effector memory T cells. [Section 31] The isolated cell composition according to item 25, wherein the CD8+ T cells specific to one or more target antigens are at least 50% central and effector memory T cells. [Section 32] The isolated cell composition according to item 31, wherein the T cells specific to one or more target antigens are at least 80% central and effector memory T cells. [Section 33] The isolated cell composition according to any one of items 1 to 32, wherein less than 20% of the CD8+ T cells are terminally differentiated. [Section 34] The isolated cell composition according to item 33, wherein less than 10% of the T cells are terminally differentiated. [Section 35] The composition contains less than 30% naive T cells (T N An isolated cell composition according to any one of items 1 to 34, including ). [Section 36] The isolated cell composition according to claim 35, wherein the composition contains less than 15% naive cells. [Section 37] The isolated cell composition according to claim 35, wherein the composition contains less than 5% naive cells. [Section 38] The isolated cell composition according to claim 35, wherein the composition contains less than 1.5% naive cells. [Section 39] An isolated cell composition according to any one of claims 1 to 38, wherein at least 10% of the CD8+ T cells exhibit a multifunctional phenotype upon activation. [Section 40] An isolated cell composition according to any one of claims 1 to 39, wherein at least 20% of the CD8+ T cells exhibit a multifunctional phenotype upon activation. [Section 41] The isolated cell composition according to item 40, wherein at least 40% of the CD8+ T cells exhibit a multifunctional phenotype upon activation. [Section 42] The cell composition is an isolated cell composition according to any one of claims 1 to 41, wherein less than 10% are CD4+ T cells. [Section 43] The isolated cell composition according to item 42, wherein less than 5% of the cell composition are CD4+ T cells. [Section 44] The isolated cell composition according to item 42, wherein less than 2% of the cell composition are CD4+ T cells. [Section 45] The isolated cell composition according to item 42, wherein less than 1.5% of the cell composition is CD4+ T cells. [Section 46] The isolated cell composition according to item 42, wherein less than 1% of the cell composition are CD4+ T cells. [Section 47] The isolated cell composition according to any one of claims 1 to 42, wherein the composition further comprises γδT cells. [Section 48] An isolated cell composition according to item 47, comprising at least about 2% γδT cells. [Section 49] An isolated cell composition according to item 47, comprising at least about 5% γδT cells. [Section 50] An isolated cell composition as described in item 47, comprising at least about 10% γδT cells. [Section 51] An isolated cell composition as described in item 47, comprising at least about 20% γδT cells. [Section 52] An isolated cell composition according to item 47, comprising at least about 25% γδT cells. [Section 53] The isolated cell composition according to any one of claims 47 to 52, wherein the γδT cells include Vδ1 and Vδ2 cells. [Section 54] The isolated cell composition according to claim 17, wherein the composition is substantially composed of T memory stem cells, and the cells are manipulated to express a chimeric antigen receptor or recombinant TCR. [Section 55] The isolated cell composition according to any one of claims 1 to 54, wherein the composition is produced by enrichment of CD8+ T cells specific to a target peptide antigen derived from source cells, and / or amplification of CD8+ T cells specific to a target peptide antigen derived from source cells. [Section 56] The isolated cell composition according to item 55, wherein the source cells are derived from a patient or an HLA-matched donor. [Section 57] The isolated cell composition according to item 56, wherein donor cells are isolated by leukocyte separation. [Section 58] The isolated cell composition according to item 55, wherein the source cells are isolated from the patient's tumor. [Section 59] The isolated cell composition according to item 55, wherein the source cells are the buffy coat fraction. [Section 60] An isolated cell composition according to any one of items 55 to 59, wherein the cell source is depleted of CD4+ T cells before enrichment or amplification. [Section 61] The source cells are an isolated cell composition according to any one of claims 55 to 59, which is enriched with CD8+. [Section 62] The cell source is an isolated cell composition according to any one of claims 55 to 59, in which NK cells are depleted. [Section 63] The isolated cell composition according to any one of claims 55 to 62, wherein the antigen-specific T cells are enriched with aAPC having an MHC class I ligand and optionally a costimulatory ligand. [Section 64] The isolated cell composition according to item 63, wherein the aAPC comprises a costimulatory ligand which is a ligand that binds to CD28. [Section 65] The isolated cell composition according to item 64, wherein the costimulatory ligand is a monoclonal antibody that is a CD28 agonist, or a portion thereof. [Section 66] The isolated cell composition according to any one of claims 1 to 65, wherein the concentration is magnetic concentration using a paramagnetic aAPC, and the cells and aAPC are optionally incubated in the presence of a magnetic field for at least 1 minute. [Section 67] The isolated cell composition according to item 66, wherein the cells and aAPC are incubated in the presence of a magnetic field for about 5 hours or less. [Section 68] The isolated cell composition according to item 67, wherein the enriched antigen-specific T cells are amplified without the use of a magnetic field. [Section 69] The isolated cell composition according to any one of claims 66 to 68, wherein the concentrated cells are amplified in a culture for 1 to 4 weeks. [Section 70] The isolated cell composition according to item 69, wherein the cells are amplified in a culture in the presence of one or more cytokines or growth factors selected from MIP-1β, IL-1β, IL-2, IL-4, IL-6, IL-7, IL-10, IL-15, IL-21, and INF-γ. [Section 71] The isolated cell composition according to claim 70, wherein IL-15 is not included for amplification of the cells in the culture. [Section 72] The isolated cell composition according to item 70, wherein the cells are amplified in a culture in the presence of two, three, four, or five cytokines or growth factors selected from MIP-1β, IL-1β, IL-2, IL-4, IL-6, IL-7, IL-10, IL-15, IL-21, and INF-γ. [Section 73] The isolated cell composition according to item 72, wherein IL-15 is not included for amplification of the cells in the culture. [Section 74] The isolated cell composition according to any one of claims 70 to 73, wherein the cells are amplified in a culture in the presence of at least one cytokine or growth factor selected from MIP-1β, IL-1β, IL-6, and IL-10. [Section 75] The isolated cell composition according to any one of claims 70 to 74, wherein the cells are amplified in the presence of IL-4. [Section 76] The isolated cell composition according to any one of claims 70 to 74, wherein the cells are amplified in the presence of IL-4 and IL-6. [Section 77] The isolated cell composition according to any one of claims 70 to 74, wherein the cells are amplified in the presence of IL-4 and IL-1β. [Section 78] The isolated cell composition according to any one of claims 70 to 74, wherein the cells are amplified in the presence of IL-4, IL-6, and IL-1β. [Section 79] The isolated cell composition according to any one of claims 70 to 74, wherein the cells are amplified in the presence of Il-2, IL-4, and IL-6. [Section 80] The isolated cell composition according to any one of claims 70 to 74, wherein the cells are amplified in a culture in the presence of IL-2, IL-4, IL-6, INF-γ, and IL-1β. [Section 81] An isolated cell composition according to any one of claims 1 to 80, wherein one or more target peptide antigens are selected from the peptide epitopes of Survivin, WT-1, PRAME, and Cyclin A1. [Section 82] An isolated cell composition according to any one of claims 1 to 81, wherein one or more target peptide antigens are selected from the peptide epitopes of PR3, XBP1-US, XBP1-SP, CD138, CS1, NY-ESO1, SOX2, EBV, influenza, CMV, RHAMM, Mart-1 / Melan A, gp100, CMVpp65, and influenza matrix protein M1. [Section 83] An isolated cell composition suitable for adoptive immunotherapy, wherein the composition is contained in a pharmaceutically acceptable carrier. It contains at least 70% CD8+ T cells and less than 5% CD4+ T cells. At least 5% of the CD8+ cells are T memory stems (T SCM ) These are cells, At least 30% of the aforementioned CD8+ cells are central and effector memory T cells. Less than 10% of the aforementioned CD8+ T cells are terminally differentiated T cells. The isolated cell composition wherein less than 10% of the CD8+ cells are naive cells. [Section 84] at least 10 6 The isolated cell composition according to item 83, wherein each CD8+ T cell is specific to the target peptide antigen. [Section 85] at least 10 6 The isolated cell composition according to item 84, wherein each CD8+ T cell is specific to 1 to 20 or 1 to 10 target peptide antigens. [Section 86] At least 10 specific to the target peptide antigen 7 or 10 8 An isolated cell composition according to any one of items 83 to 85, comprising 1 CD8+ T cell. [Section 87] An isolated cell composition according to any one of claims 83 to 86, comprising 5% to 25% T memory stem cells. [Section 88] An isolated cell composition according to any one of claims 83 to 87, comprising less than 5% terminally differentiated T cells and / or less than 5% naive cells. [Section 89] The isolated cell composition according to any one of claims 83 to 88, wherein the target peptide antigen is a tumor or cancer-associated antigen and optionally associated with hematological malignancies. [Section 90] The isolated cell composition according to item 89, wherein the target peptide antigen is associated with hematological malignancies. [Section 91] An isolated cell composition according to item 89 or 90, wherein one or more target peptide antigens are selected from the peptide epitopes of Survivin, WT-1, PRAME, Cyclin A1, and PR3, and the peptide epitopes optionally comprise one or more selected from Table 1. [Section 92] An isolated cell composition according to item 89 or 90, wherein one or more target peptide antigens are selected from the peptide epitopes XBP1-US, XBP1-SP, CD138, CS1, NY-ESO1, and SOX2, and the peptide epitopes optionally comprise one or more selected from Table 2. [Section 93] The isolated cell composition according to any one of claims 83 to 92, wherein the composition further comprises γδT cells. [Section 94] An isolated cell composition according to item 93, comprising at least about 5% γδT cells. [Section 95] An isolated cell composition according to item 93, comprising at least about 10% γδT cells. [Section 96] An isolated cell composition according to item 93, comprising at least about 25% γδT cells. [Section 97] The isolated cell composition according to any one of claims 93 to 96, wherein the γδT cells include Vδ1 and Vδ2 cells. [Section 98] A method for treating a cancer patient, comprising administering a cell composition described in any one of items 1 to 97 to a patient in need. [Section 99] The patient has a blood cancer, as described in paragraph 98. [Section 100] The method according to paragraph 99, wherein the hematological cancer recurs after allogeneic stem cell transplantation. [Section 101] The method according to paragraph 99 or 100, wherein the patient has acute myeloid leukemia (AML) or myelodysplastic syndrome. [Section 102] The method according to any one of paragraphs 98 to 101, wherein the patient has also received lymphocyte apheresis, cytoreductive therapy, or immunomodulatory therapy prior to cell therapy. [Section 103] The method according to any one of claims 98 to 102, wherein the cell therapy may be further provided with or without cytokine-assisted posttherapy. [Section 104] The method according to any one of items 98 to 103, wherein antigen-specific T cells persist for at least 6 months in the patient. [Section 105] A method for producing a cell composition according to any one of claims 1 to 97, comprising amplifying an aggregate of CD4+ depleted T cells in the presence of two or more of IL-2, IL-4, IL-6, INF-γ, and IL-1β. [Section 106] The T cells are enriched with CD28+ cells, as described in item 105. [Section 107] The method according to section 105 or 106, wherein the cells are amplified in the presence of IL-4. [Section 108] The method according to item 107, wherein the cells are amplified in the presence of IL-4 and IL-6. [Section 109] The method according to item 107, wherein the cells are amplified in the presence of IL-4 and IL-1β. [Section 110] The method according to item 107, wherein the cells are amplified in the presence of IL-4, IL-6, and IL-1β. [Section 111] The method according to item 107, wherein the cells are amplified in the presence of IL-2, IL-4, and IL-6. [Section 112] The method according to item 107, wherein the cells are amplified in a culture in the presence of IL-2, IL-4, IL-6, INF-γ, and IL-1β. [Section 113] The method according to any one of claims 93 to 112, wherein the cells are amplified in a culture for 1 to 4 weeks. [Section 114] A method for producing an aggregate of γδ T cells, comprising amplifying the T cell aggregate in the presence of two or more of IL-2, IL-4, IL-6, INF-γ, and IL-1β. [Section 115] The T cell aggregate is CD28+ enriched, as described in item 114. [Section 116] The aggregate of CD28+ cells is positively selected using anti-CD28 beads or particles, as described in item 115. [Section 117] The T cell aggregate described above is CD4+ depleted, according to the method described in any one of items 114 to 116. [Section 118] The method according to any one of items 114 to 117, wherein the cells are amplified in the presence of IL-4. [Section 119] The method according to item 118, wherein the cells are amplified in the presence of IL-4 and IL-6. [Section 120] The method according to item 118, wherein the cells are amplified in the presence of IL-4 and IL-1β. [Section 121] The method according to item 118, wherein the cells are amplified in the presence of IL-4, IL-6, and IL-1β. [Section 122] The method according to item 118, wherein the cells are amplified in the presence of IL-2, IL-4, and IL-6. [Section 123] The method according to item 118, wherein the cells are amplified in a culture in the presence of IL-2, IL-4, IL-6, INF-γ, and IL-1β. [Section 124] The method according to any one of items 117 to 123, wherein the cells are amplified in a culture for 1 to 4 weeks. [Section 125] The method according to any one of claims 114 to 124, further comprising isolating the γδ+ aggregate of cells from γδ cells. [Section 126] The method according to item 125, further comprising heterologously expressing an αβTCR T cell receptor in an optional manner. [Section 127] The method according to item 125, further comprising heterologous expression of a chimeric antigen receptor (CAR). Other aspects and embodiments of the present invention will be obvious to those skilled in the art. [Examples]

[0133] To generate antigen-specific CD8+ T cells, fresh PBMCs were obtained from a donor by leukocyte isolation, as schematically shown in Figure 2. CD4+ cells were depleted by negative selection with anti-CD4 microbeads. Antigen-specific T cells were enriched by incubation of the resulting cells with paramagnetic nanoparticles (i.e., dextran-coated iron oxide nanoparticles or PLGA-PEG nanoparticles, in the range of approximately 80–200 nm in diameter). As shown in Figure 1, the nanoparticles have a dimeric HLA ligand conjugated to a surface (presenting the target peptide antigen) capable of incorporating multiple tumor-specific antigen peptides. The dimeric HLA ligand contains two HLA-A2 domains, each containing a peptide-binding groove, fused to an arm of the Ig hinge region. Dimeric HLA-Ig is expressed together with β2 microglobulin. Dimeric HLA ligands, such as the HLA-IgG4 hinge dimer, can be readily modified for multiple HLA subtypes and can provide direct engagement with target T cells. As shown in Figure 1, a co-stimulatory ligand or inhibitory ligand, such as an anti-CD28 monoclonal antibody, is also conjugated to the nanoparticle. The co-stimulatory ligand or inhibitory ligand provides specific instructions (e.g., activation, inhibition) for targeting T cells (i.e., naive T cells or memory T cells) for therapeutic purposes. The ligands and aAPC constructs are disclosed in WO2016 / 044530 and WO2016 / 105542, which are incorporated herein by reference in their entirety.

[0134] Cells were incubated for approximately 5 minutes in the presence of paramagnetic aAPCs, and then in the presence of a magnetic field. Cells associated with the particles were then harvested and amplified ex vivo for varying lengths of time (generally 1–2 weeks). Amplification was carried out in the presence of growth factors. Growth factors were added on days 1 and 7 during a 2-week culture period. Cells were re-stimulated with nano-aAPCs on day 7. Amplification to therapeutic levels of tumor-specific CD8+ T cells was observed within 2 weeks of donor cell isolation. In some embodiments, the enrichment and amplification processes can be carried out in a closed, automated cell amplification system. Such a system can be simple, scalable, and cost-effective in manufacturing, and can provide consistent and rapid generation of antigen-specific CD8+ T cells from different antigen peptide cocktails (i.e., derived from patient or donor PBMCs).

[0135] Table 3 shows the composition of cytokines used for amplification. [Table 3]

[0136] Cellular phenotypes utilizing enrichment and amplification processes, including cytokines during amplification, are disclosed in PCT / US2018 / 051971 (title: CELL COMPOSITIONS COMPRISING ANTIGEN-SPECIFIC T CELLS FOR ADOPTIVE THERAPY). PCT / US2018 / 051971 is incorporated herein by reference in its entirety.

[0137] Figure 3 shows the enrichment and amplification of acute myeloid leukemia (AML)-specific T cells using the method disclosed herein. The graph on the left of Figure 3 shows AML-specific antigen WT1 37-45 WTI 126-134 PRAME 425 Cyclin A1 227-235 , and Cyclin A1 341-351Figure 3 shows the total number of CD8+ T cells generated from fresh PBMCs of four healthy donors after T cells were enriched and amplified ex vivo. The graph on the right of Figure 3 shows the total percentage of acute myeloid leukemia (AML) specific antigens after CD8+ T cells were enriched and amplified ex vivo. The results in Figure 3 show that a significantly higher percentage of AML-specific CD8+ T cells are generated by the method of this disclosure compared to cell compositions derived from other endogenous T cell therapies.

[0138] Next, in Figures 4A and 4B, the cells are shown, along with their phenotypes, and naive T cells (T N )(CD62L+, CD45RA+), central memory T cells (T CM )(CD62L+, CD45RA-), effector memory T cells (T EM )(CD62L-, CD45RA-), effector memory RA+ T cell (T EMRA )(CD62L-, CD45RA+), and T memory stem cells (T SCM Characterization was performed for one of the following: (CD62L+, CD45RA+, CD95+). More than 95% of AML-specific T cells enriched and amplified ex vivo from donor lymphocytes had the memory T cell phenotype. More than 60% of T cells generated by E+E had the T memory stem cell phenotype and the central memory T cell phenotype. In addition, as shown in Figure 4B, the E+E system generated a consistent memory T cell phenotype across all donors. The E+E process also generated a significant amount of multiple myeloma-specific T memory stem cells from two different healthy donor leucopaks. (See Figures 8 and 9A.) In Figures 9A and 9B, multiple myeloma-specific antigen T cells were enriched in batches based on hinge dimer staining, reaching approximately 1.6 × 10⁶. 9 It was amplified into individual CD8+ T cells.

[0139] Figures 5A and 5B show that ex vivo enriched and amplified AML-specific T cells exhibit a highly multifunctional phenotype, including intracellular staining of IL-2 (proliferation and memory), IFN-γ (activation of other cells, memory, upregulation of MHC), TNF-α (pro-inflammatory), and CD107A (granzyme release, cytotoxic activity). The majority of AML-specific T cells (i.e., approximately 62%) showed 3-4 effector functions when subjected to nonspecific stimulation (Figure 5A, upper). In Figure 5A (lower), the graph shows the percentage of T cells expressing IL-2, TNF-α, IFN-γ, and CD107A. In Figure 5A, T cells were generated by nonspecific stimulation of peptide-pulsed T2 cells. The results of this experiment show that at least 3 or 4 cytokine effector functions were observed in the majority of AML-specific CD8+ T cells generated by E+E. In Figure 5B, T cell-mediated tumor-specific killing of AML cell line U266 is shown for AML-specific antigens from fresh PBMCs of healthy donors with two effector-target (E:T) ratios of 10:1 (left bar) and 20:1 (right bar). The results of this experiment demonstrate that CD8+ T cell compositions generated by E+E from healthy donors exhibit robust killing activity across multiple E:T ratios.

[0140] Figure 6 consists of four graphs comparing the specificity of Mart-1 specific T cells produced by the enrichment and amplification processes disclosed herein, between PBMCs derived from melanoma patients (top) and PBMCs derived from healthy donors (bottom). The enrichment and amplification processes produce a consistent cell composition regardless of the donor source. The data in this experiment were generated from frozen PBMCs.

[0141] Figure 7 is a graph showing that the AIM ACT-based E+E process generates a TCR repertoire that mimics the innate immune response, thereby providing robust adoptive therapy from a naturally selected innate T cell repertoire. The breadth of the polyclonal TCR repertoire enables a naturally robust immune response.

[0142] Figure 8 shows that the E+E process generated a significant amount of multiple myeloma antigen-specific T memory stem (TSCM) cells ((CD62L+, CD45RA+, CD95+)). This graph shows the phenotype of multiple myeloma-specific antigen T cells before and after amplification from healthy donor leucopak. Figure 9A shows the phenotype of T cells enriched and amplified ex vivo in a batch for multiple myeloma antigen-specific T cells from healthy donor leucopak. This graph shows that the E+E process generated a significant amount of antigen-specific CD8+ T cells (approximately 1.6 × 10⁶ based on hinge dimer staining), including T memory stem (TSCM) cells, central memory T cell (TCM), and effector memory T (TEM) cells. 9 This shows that it generated (100 CD8+ T cells). Figure 9B shows the phenotype of T cells enriched and amplified ex vivo in a batch for multiple myeloma antigen-specific T cells derived from four different clinical multiple myeloma patients. This graph shows that the E+E process is responsible for the T memory stem (T SCM ) cells, central memory T cells CM , and effector memory T(T EM This indicates that a significant amount of antigen-specific CD8+ T cells, including ) cells, were generated. This data shows that patient-derived PBMCs have the same phenotypic features as donor-derived PBMCs, and notably, that the E+E process is effective in generating a significant amount of antigen-specific CD8+ T cells, including T memory cells (e.g., T memory stem (TSCM) cells, central memory T cell (TCM) cells, and effector memory T (TEM) cells).

[0143] Figure 10 shows the production of γδT cells using the E+E process. Both Vδ1 and Vδ2TCR subtypes were observed. The clinical significance of γδT cells in terms of hematopoietic stem cell transplantation (HSCT) has been reported, and in particular, a higher frequency of γδT cells after transplantation is associated with desirable outcomes. See Berglund et al., Expansion of Gammadelta T cells from Cord Blood: A Therapeutic Possibility. Stem Cells International Vol. 2018. As shown in Figure 11, the %γδT cell count on day 14 varied, with an average of approximately 15% to 50% γδT cells. The number of γδT cells on day 14 was broadly correlated with the number of γδT cells on day 0.

[0144] In the following experiment, the characteristics of amplified T cells will be evaluated in terms of identity, purity, phenotype, and specificity. A summary of the characteristics of lots enriched with anti-multiple myeloma (MM) antigen peptide and lots enriched with anti-leukemia antigen peptide is shown below. [Table 4-1] [Table 4-2]

[0145] In the following experiment, phenotype is a measure of the total percentage of memory T cells relative to CD3+ cells. The characterized memory T cell aggregates include T stem cell memory (Tscm) and T central memory (Tcm) aggregates, both of which retain the ability to proliferate and self-replicate, as well as T effector memory (Tem) cells. The remaining aggregates to be characterized include Temra and T naive cells. [Table 5-1] [Table 5-2]

[0146] As demonstrated by the experiments described above, the CD8+ T cell compositions disclosed herein directly engage with T cell receptors on naive and memory T cells to trigger desired immune responses. The CD8+ T cell compositions produced by the enrichment and amplification processes consist of multi-antigen-specific CD8+ restricted T cells derived from the endogenous repertoire. Antigen-specific CD8+ T cell compositions include T memory stem cells. T memory stem cells and central memory T cells are useful and important for both early and long-term clinical responses. Antigen-specific CD8+ T cell compositions have a multifunctional phenotype, as assessed by effector cytokine production and target cell killing. The E+E process also generated innate immune responses triggered by a diverse TCR repertoire. In addition to robust aggregates of αβ T cells, significant aggregates of γδ T cells were also present in the amplified aggregates, including both Vδ1 and Vδ2 cells. γδ T cells are thought to provide a further mechanism for pathogen or cancer cell killing, which is not HLA-dependent.

Claims

1. An isolated cell composition for use in adoptive immunotherapy, wherein the cell composition contains at least 10 specific peptide antigens in a pharmaceutically acceptable carrier. ６ It contains CD8+ T cells and has central memory and effector memory phenotypes, and has a T memory stem (T ＳＣＭ ) containing cells, wherein the target peptide antigen is WT1 ３７－４５ (Sequence ID 3), WT-1 １２６－１３４ (Sequence ID 1), PRAME ４２５ (Sequence ID 9), Cyclin A1 ２２７－２３５ (Sequence ID 12), and Cyclin A1 ３４１－３５１ The isolated cell composition comprising (SEQ ID NO: 13), wherein at least 15% of the CD8+ T cells are specific to the one or more target peptide antigens.

2. The isolated cell composition according to claim 1, wherein the T cell specificity for the target peptide antigen in the cell composition is defined by MHC multimer staining.

3. The isolated cell composition according to claim 1 or 2, wherein at least 90% of the cell composition consists of T cells, and at least 30% of the CD8+ T cells are specific to the target peptide antigen.

4. The isolated cell composition according to claim 1, wherein the cell composition further comprises CD8+ T cells specific to bacteria, viruses, and / or fungal pathogens.

5. The isolated cell composition according to claim 4, wherein the CD8+ T cells specific to bacteria, viruses, and / or fungal pathogens include T cells specific to the antigens of influenza, CMV, EBV, and / or adenovirus.

6. The isolated cell composition according to any one of claims 1 to 5, wherein at least 1% of the T cells specific to the target peptide antigen are T memory stem cells.

7. More than 95% of the aforementioned CD8+ T cells exhibit a memory phenotype. The CD8+ T cells are such that at least 30% are central and effector memory T cells, or The CD8+ T cells are such that at least 50% are central and effector memory T cells, or The CD8+ T cells are, at least 70% of which are central and effector memory T cells, or The CD8+ T cells are, at least 80% central and effector memory T cells, or At least 90% of the CD8+ T cells are central and effector memory T cells, or The CD8+ T cells specific to the target peptide antigen are such that at least 50% are central and effector memory T cells, or The isolated cell composition according to any one of claims 1 to 6, wherein at least 80% of the T cells specific to the target peptide antigen are central and effector memory T cells.

8. The isolated cell composition according to any one of claims 1 to 7, wherein less than 20% of the CD8+ T cells are terminally differentiated, or less than 10% are terminally differentiated, and less than 15% of the T cells are naive cells, or less than 5% are naive cells, or less than 1.5% are naive cells.

9. The isolated cell composition according to any one of claims 1 to 8, wherein at least 10% of the CD8+ T cells exhibit a multifunctional phenotype upon activation, or at least 20% of the CD8+ T cells exhibit a multifunctional phenotype upon activation, or at least 40% of the CD8+ T cells exhibit a multifunctional phenotype upon activation.

10. The isolated cell composition according to any one of claims 1 to 9, wherein the cell composition comprises less than 10% CD4+ T cells, or less than 5% CD4+ T cells, or less than 2% CD4+ T cells, or less than 1.5% CD4+ T cells, or less than 1% CD4+ T cells.

11. The isolated cell composition according to any one of claims 1 to 10, wherein the cell composition further comprises γδ T cells.

12. A method for producing an isolated cell composition according to any one of claims 1 to 11, comprising enriching CD8+ T cells specific to the target peptide antigen derived from source cells, and then amplifying the CD8+ T cells specific to the target peptide antigen derived from the enriched cells.

13. The method according to claim 12, wherein the concentration is magnetic concentration using paramagnetic aAPC, the cells and aAPC are incubated for at least 1 minute in the presence of a magnetic field, the concentrated cells are amplified in a culture for 1 to 4 weeks, and the cells are amplified in a culture in the presence of IL-2, IL-4, IL-6, IFN-γ, and IL-1β.

14. The method according to claim 13, wherein the cells and aAPC are incubated in the presence of a magnetic field for about 5 hours or less.

15. The method according to claim 14, wherein the enriched antigen-specific T cells are amplified without the use of a magnetic field.

16. The method according to any one of claims 13 to 15, wherein the concentrated cells are amplified in a culture for 1 to 4 weeks.

17. An isolated cell composition according to any one of claims 1 to 11 for treating a patient having acute myeloid leukemia.

18. The isolated cell composition according to claim 17, wherein the patient has also received lymphocyte apheresis, cytoreductive therapy, or immunomodulatory therapy prior to cell therapy.

19. The isolated cell composition according to claim 17 or 18, wherein the cell therapy may be further provided with or without cytokine-assisted post-treatment.

20. The isolated cell composition according to any one of claims 17 to 19, wherein antigen-specific T cells persist for at least six months in the patient.

21. A method for producing the cell composition according to any one of claims 1 to 11 and 17 to 20, comprising amplifying an aggregate of CD4+ depleted T cells in a culture in the presence of an aggregate of artificial antigen-presenting cells that present the target peptide antigen, and in the presence of IL-2, IL-4, IL-6, IFN-γ, and IL-1β.

22. The method according to claim 21, wherein the T cells are enriched with CD28+ cells.

23. The method according to claim 21 or 22, wherein the cells are amplified in a culture for 1 to 4 weeks.

Images