Methods of producing memory t cells in vitro and their use in immunotherapy

Abstract

Methods of generating memory T cells in vitro are provided. The methods include culturing a population of T cells with a population of antigen-loaded dendritic cells for at least about 28-48 days in a culture medium and isolating memory T cells (such as CD4+ or CD8+ memory T cells) from the culture. Methods of treating a subject with cancer or infectious disease are also provided. The methods include administering to the subject memory T cells generated by the methods described herein, where the antigen-loaded dendritic cells are loaded with an antigen expressed by the cancer or the infectious agent causing the disease. Also provided are methods of identifying T cell receptors that specifically bind to an antigen recognized by a memory T cell, which include producing a population of memory T cells recognizing an antigen as described herein, and identifying the T cell receptor expressed by the memory T cells.

Description

[0001] METHODS OF PRODUCING MEMORY T CELLS IN VITRO AND THEIR USE IN IMMUNOTHERAPY

[0002] CROSS REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Application No. 63 / 648,422, filed May 16, 2024, which is incorporated by reference in its entirety.

[0004] FIELD

[0005] This disclosure relates to methods of generating memory T cells and their use in immunotherapy, for example for treating cancer or infectious diseases.

[0006] INCORPORATION OF ELECTRONIC SEQUENCE LISTING

[0007] The Sequence Listing is submitted as an XML file in the form of the file named “7213-102390-07_Sequence_Listing.xml” (18,475 bytes), which was created on May 12, 2025, which is incorporated by reference herein.

[0008] BACKGROUND

[0009] Dendritic cell-based immunotherapies have been tested for the treatment of cancer patients for some time with limited success. The limited success is probably due to the ineffective activation of specific T cells by injected dendritic cells and / or apoptosis of T cells by cancer cells. The discovery and the use of PD1 checkpoint inhibitors have solved the latter problem with significant success. These drugs can prevent T cells from undergoing cancer-mediated apoptosis. However, some patients without DNA mismatch repair mutations do not respond well to treatment with the PD-1 inhibitors, suggesting that T cells of cancer patients need to recognize cancer cells with less mutations. In vitro generation of memory T cells that can respond to cancer cells would greatly enhance the efficacy of cancer immunotherapy.

[0010] Memory T cells can also be used to fight bacterial and virus infection. Multidrug resistant (MDR) bacteria are a significant threat to human health, yet systemic infection by MDR bacteria or viruses can only be approached by boosting the immune sytem. Theoretically, if one can prepare memory T cells to an infectious agent, one can control infection by infusing such memory T cells into patients. SUMMARY

[0011] Disclosed herein are methods of generating memory T cells in vitro. In some aspects, the methods include culturing a population of T cells with a population of antigen-loaded dendritic cells for at least about 28-48 days (such as about 30-43 days) in a culture medium and isolating memory T cells from the culture. In some examples, the population is a mixed population of T cells, which in some examples substantially lacks memory T cells. The T cell population may be from whole blood, peripheral blood monocytes, lymphocytes, bone marrow, lymph node tissue, umbilical cord blood, or induced pluripotent stem cells. In some examples, the memory T cells are CD4+memory T cells or CD8+ memory T cells.

[0012] In particular aspects, the population of antigen-loaded dendritic cells is produced by culturing monocytes in culture medium (for example, including one or more of fetal calf serum, GM-CSF, and IL-4) to generate dendritic cells, isolating the dendritic cells, adding one or more antigens and lipopolysaccharide (LPS) (simultaneously or sequentially), and culturing the dendritic cells, antigen, and LPS for about 8-18 hours to generate a population of antigen-loaded dendritic cells. The population of antigen-loaded dendritic cells may be prepared from whole blood, peripheral blood monocytes, lymphocytes, bone marrow, or induced pluripotent stem cells.

[0013] In additional aspects, the memory T cells (such as isolated memory T cells) are contacted with the antigen used to produce the antigen-loaded dendritic cells (for example, for 1-4 days) to produce a population of effector T cells. In some examples, the effector T cells are cytotoxic T cells.

[0014] Also disclosed herein are methods of treating a subject with cancer that include administering to the subject memory T cells (such as CD8+memory T cells and / or CD4+memory T cells) generated by the methods described herein, where the antigen- loaded dendritic cells are loaded with an antigen expressed by the cancer in the subject. In some examples, the antigen is a purified antigen (such as a peptide or polysaccharide antigen) or a homogenate prepared from tumor tissue or tumor cells from the subject. In other examples, effector T cells produced from the memory T cells are administered to the subject. The population of T cells used to generate the memory T cells may be autologous or allogeneic.

[0015] Other aspects include methods of treating a subject with an infectious disease, including administering to the subject memory T cells (such as CD8+memory T cells and / or CD4+memory T cells) generated by the methods described herein, where the antigen- loaded dendritic cells are loaded with an antigen expressed by the agent causing the infectious disease. In other examples, effector T cells produced from the memory T cells are administered to the subject. The agent causing the infectious disease may be a bacterium (such as an antibiotic resistant bacterium), virus, or fungus. The population of T cells used to generate the memory T cells may be autologous or allogeneic.

[0016] In another aspect, the methods include culturing a population of T cells with a population of dendritic cells loaded with a homogenate prepared from tumor tissue or tumor cells from a subject with cancer for at least about 30 days in a culture medium, isolating memory T cells from the culture, and administering the memory T cells (such as CD8+memory T cells and / or CD4+memory T cells) to the subject. In additional examples, effector T cells produced from the memory T cells are administered to the subject. The population of T cells used to generate the memory T cells may be autologous or allogeneic.

[0017] In another aspect, effector T cells are produced by the methods described herein from memory T cells produced using dendritic cells loaded with a homogenate prepared from tumor tissue or tumor cells from a subject with cancer. T cells with cytotoxic activity against the tumor are isolated from the population of the effector cells, the T cell receptor expressed by the cytotoxic T cells is identified (for example, by sequencing), and modified T cells expressing the identified T cell receptor are produced. The modified T cells are administered to the subject with cancer.

[0018] Also provided are methods of identifying T cell receptors that specifically bind to an antigen recognized by a memory T cell. In some aspects, the methods include producing a population of memory T cells recognizing an antigen as described herein, and identifying the T cell receptor expressed by the memory T cells. In some examples, the population of antigen-loaded dendritic cells cultured with the T cells to produce the memory T cells is loaded with a sample comprising a mixture of antigens (such as from a tumor or pathogen). The T cell receptor expressed by the memory T cells is identified (for example by single cell sequencing). The methods may further include activating the memory T cells to produce effector T cells by contacting the memory T cells with the sample used to produce the antigen-loaded dendritic cells and in some examples, identifying the TCR expressed by the effector T cells. In some examples, the methods further include identifying the antigen specifically bound by the identified T cell receptor. The identified antigen may be administered to a subject in need thereof, or used to generate an antibody that specifically binds the antigen.

[0019] The foregoing and other features of the disclosure will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIGS. 1A-1C are a series of phase contrast micrographs of co-incubation of lymphocytes with mouse bone marrow-derived dendritic cells (DCs) stimulated with the OT- 1 peptide at 2 days (FIG. 1A), 14 days (FIG. IB), and 26 days (FIG. 1C) after stimulation by DCs.

[0021] FIGS. 2A-2D show flow cytometry analyses of a mouse T cell-DC mixed culture after 30 days. Cells were first gated to analyze lymphatic cells (FIG. 2A), then, live cells were selected using a Zombie Green™ live / dead dye (FIG. 2B). Both CD3-positive and CD8- positive live lymphocytes (FIG. 2C) were analyzed for the expression of CD44 (FIG. 2D).

[0022] FIGS. 3A and 3B show flow cytometry analysis of T cells after 30 days of culture stimulated by the OT-1 peptide for 2 days and gated for CD3-positive and CD8-positive lymphocytes to analyze expression of OT-1 TCR using PE-labeled OT-1 tetramers. These cells were positive for PE-labeled OT-1 tetramers (FIG. 3A), while naive T cells were not stained (FIG. 3B).

[0023] FIGS. 4A-4H show phase contrast (FIGS. 4A, 4C, 4E, and 4G) of OVA-B16 melanoma and corresponding fluorescence (FIGS. 4B, 4D, 4F, and 4H) staining of OVA-B16 melanoma cells killed by T cells. Without addition of T cells, no cell death was observed (FIGS. 4A and 4B), while effector cells resulted in death of OVA-B16 cells (FIGS. 4C and 4D). Un-activated memory T cells also showed cytotoxicity, though to a lesser extent (FIGS. 4E and 4F). No death was observed when “effector” cells were added to B 16 melanoma cells that do not express OVA (FIGS. 4G and 4H).

[0024] FIGS. 5A and 5B are phase contrast images of mixed human DC cells and autologous human T cells (indicated by arrows) cultured for 1 day (FIG. 5A) and for one month (FIG. 5B). T cells (arrows) survived after one month of culture and some T cells showed direct binding to DCs (arrowheads).

[0025] FIGS. 6A-6E show analysis of a human T cell-DC mixed culture after 30 days. Cells were first gated to analyze lymphatic cells (FIG. 6A), then stained with live-dead dyes to gate live cells (FIG. 6B). Live cells were gated to obtain singlets (FIG. 6C), then gated with CD3 and CD8 to select CD3+CD8+T cells (FIG. 6D). CD8+T cells were gated with memory cell markers CD45RO and CD45RA (FIG. 6E), in which memory T cells are marked by high expression of CD45RO with low expression of CD45RA.

[0026] FIGS. 7A-7H show analysis of activated human memory T cells. Human naive T cells were primed with MART-1 loaded dendritic cells and cultured for 42 days. DC-T cell culture was then activated by addition of MART-1 peptide antigen and cultured for 2 days. Whole cells were gated to select lymphocytes (FIG. 7A) and live lymphocytes were selected by live-dead dye (FIG. 7B). Live lymphocytes were analyzed by AF488-CD3 and PerCp- Cy5.5-CD8 to select CD3+CD8+ T cells (FIG. 7C) and CD3+CD8+ T cells were analyzed by APC-labeled MART-1 tetramer (FIG. 7D). Fig. 7E shows that CD3+CD8+cells gated in FIG. 7B show lower expression of CD4. FIG. 7F shows that live lymphocytes gated from FIG. 7C have two populations, one is high CD8 with lower CD4, the other is low CD8 with higher CD4. FIGS. 7G and 7H show that live lymphocytes with high CD4 expression do not react with MART-1 tetramer, which is expected, as a MART-1 peptide is a MHC-I loaded antigen, not MHC-II loaded antigen.

[0027] FIGS. 8A-8C show flow cytometry of MARTI -antigen-specific CD8+ T cells demonstrating that stimulation of memory T cells with the antigen converted memory T cells into effector T cells. The majority of T cells expressed high levels of CD45RA with low levels of CD45RO, the markers for effector T cells (FIG. 8A). Effector T cells and memory T cells gated in FIG. 8 A were examined for their reactivity to MARTI tetramer (FIG. 8B, effector T cells; FIG. 8C, memory T cells). Both types of T cells showed reactivity to MARTI antigen.

[0028] FIGS. 9A-9E show flow cytometry analysis of mouse memory T-cells generated against B16 / F10 melanoma homogenates as antigens. Mouse dendritic cells were loaded with homogenates of B 16 / F10 melanoma cells, then cultured with lymphocytes for 30 days. Whole cells were gated to select lymphocytes (FIG. 9A) and CD3 positive, live lymphocytes were selected by live-dead dye and Alexa Fluor 700 (FIG. 9B). CD3+live lymphocytes were analyzed by FITC-CD8 and PerCep-Cy5.5-CD4 to select CD3+CD8+T cells and CD3+CD4+T cells (FIG. 9C). CD3+CD8+T-cells were analyzed by PE-CD44 and FITC-CD8 to detect CD44 positive memory CD8 T-cells (FIG. 9D). CD3+CD4+T-cells were analyzed by PE- CD44 and PerCp-Cy5.5-CD4 to detect CD44 positive CD4 T-cells (FIG. 9E). FIG. 9C indicates that memory T-cells generated in vitro with B 16 / F10 homogenates contained both CD4+T-cells and CD8+memory T-cells, as evidenced by the expression of memory T-cell markers of CD44 cells (FIGS 9D & E).

[0029] FIG. 10 is a plot showing cytotoxicity of “memory” T cells against Bl 6 / F10 melanoma. Melanoma cells labeled with CSFE were incubated with memory T cells and 2 days later labeled with far-red dead / live dye. Addition of memory T cells increased the fraction of dead cells compared with control (untreated) melanoma cells. FIG. 11 shows the clonality of three independent samples of mouse memory T-cells generated in vitro using OT-1 peptide as an antigen. CD8+T-cells were selected by positive magnetic selection and subjected to next generation sequencing for immunoprofiling of the CDR3 of the TCRp chain (Adaptive Biotech). The figure displays the distributions of the top 30 rearrangements of thee in vitro generated memory T-cells (Samples 1-3), as well as naive T-cells and T-cells immunized in vivo with OVA. The percentage of the top 30 clones is indicated at the top of the bar graph. The figure indicates that memory T-cells show twice higher clonality than in vivo immunized T-cells. As expected, naive T-cell show very low clonality.

[0030] FIG. 12 is a graph showing clonal enrichment by in vitro generation of memory T- cells. Frequencies of productive rearrangements were compared between naive T-cells and in vitro generated memory T-cells. T-cell clones with the highest frequencies found in the in vitro generated memory T-cell preparation (right) show near zero frequencies in the naive T- cell preparation indicating that memory T-cells were highly enriched. T-cell clones with the highest frequencies in the naive T cell preparation (left) showed near zero frequencies in the preparation of in vitro generated T-cells, indicating that they were not selected during in vitro generation of memory T-cells.

[0031] FIGS. 13A and 13B show expression of CD8 on in vitro generated memory T-cells in the presence (FIG. 13A) or absence (FIG. 13B) of IL-15 starting at day 14 of co-culture with antigen-loaded dendritic cells.

[0032] FIGS. 14A-14E show generation of CD8 memory T cells using OT-2 peptide. After 30 days, T-cells were analyzed by flow cytometry. FIG. 14A: live CD3+T-cells were analyzed by CD4 and CD8 markers. FIG. 14B: CD3+CD4+T-cells were analyzed by CD44 marker, a memory T-cell marker. FIG. 14C: CD3+CD4+CD44+T-cells were analyzed for antigen specificity using OT-2 tetramer assay. FIG. 14D: CD44-negative CD4 T-cells showed less than 1% positivity toward OT-2 tetramer. FIG. 14E: CD3+CD4+CD44+T-Cells were analyzed for CD62L marker.

[0033] FIGS. 15A-15C show that sequential addition of OT-1 antigen peptide and LPS to DCs induces higher oligomerization of antigen-loaded MHC than simultaneous addition. Proximity ligation assay showed more and stronger fluorescence (indicating oligomer formation of antigen-loaded MHC-I) with sequential addition (FIG. 15 A) than simultaneous addition of antigen and LPS (FIG. 15B). FIG. 15C is a graph showing quantitative analysis of the data from FIGS. 15A and 15B. FIGS. 16A-16B show cytotoxicity of human memory T-cells toward MART-1 expressing MEL526 melanoma. MEL526 cells labeled with CSFE were incubated without (FIG. 16A) or with human memory T cells specific to MART-1 (FIG. 16B). Two days later, dead cells were labeled with far-red dead / live dye. Addition of human memory T cells killed the majority of MART-1 expressing melanoma cells.

[0034] SEQUENCE LISTING

[0035] The nucleic and amino acid sequences listed in the accompanying sequence listing are shown using standard letter abbreviations for nucleotide bases, and single letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand.

[0036] SEQ ID NOs: 1-20 are exemplary CDR3 amino acid sequences from in vitro generated memory T cells.

[0037] DETAILED DESCRIPTION

[0038] Disclosed herein are methods to generate memory T cells in vitro. The OT-1 peptide (a short peptide sequence of ovalbumin, OVA) was used as a model antigen. OT-1 peptide is known to be complexed with MHC-I of dendritic cells, which is recognized by a unique TCR of CD8+T cells, resulting in the activation of CD8+T cells. Both DCs and T cells from wild type C57 / BL6 mice were prepared. After in vitro priming of DCs with the OT-1 antigen, T cells from C57 / BL6 were co-cultured in vitro with DCs for one month. While most T cells died within two weeks, a small population of T cells survived. These surviving T cells had the phenotypes of memory T cells. First, they expressed CD3, CD8, and CD44, the markers for memory CD8+T cells in mice. Second, an OT-1 tetramer assay revealed that these cells showed antigen- specific responses toward the OT-1 peptide antigen. Third, these cells exhibited cytotoxicity toward B16 melanoma cells expressing OVA, but not control B16 without OVA expression. This system has also been extended to demonstrate successful generation of human memory T cells using the MART-1 antigen from melanoma cells.

[0039] I. Terms

[0040] Unless otherwise noted, technical terms are used according to conventional usage. Definitions of many common terms in molecular biology may be found in Krebs et al. (eds.), Lewin’s genes XII, published by Jones & Bartlett Learning, 2017. As used herein, the singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, the term “an antigen” includes singular or plural antigens and can be considered equivalent to the phrase “at least one antigen.” As used herein, the term “comprises” means “includes.” It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various aspects, the following explanations of terms are provided:

[0041] Antigen: A molecule that stimulates an immune response. Antigens are usually proteins, polysaccharides, or fragments thereof. An epitope is an antigenic determinant. These are particular chemical groups or peptide sequences on a molecule that are antigenic, such that they elicit a specific immune response.

[0042] Antigen-specific T cell: A CD8+or CD4+T cell (such as a CD8+or CD4+memory T cell) that recognizes a particular antigen, such as a target antigen. Generally, antigen-specific T cells specifically bind to a particular antigen, but not other antigens. A target antigenspecific T cell specifically binds to a particular target antigen, such as a tumor antigen or an antigen expressed by an infectious agent.

[0043] Cancer: A malignancy characterized by abnormal or uncontrolled cell growth. Other features often associated with cancer include metastasis, interference with the normal functioning of neighboring cells, release of cytokines or other secretory products at abnormal levels and suppression or aggravation of inflammatory or immunological response, invasion of surrounding or distant tissues or organs, such as lymph nodes, etc. “Metastatic disease” refers to cancer cells that have left the original tumor site and migrated to other parts of the body, for example via the bloodstream or lymph system.

[0044] Culturing or Cell culture: Growth or maintenance of a population of cells in a defined set of conditions (such as culture medium, extracellular matrix, temperature, and / or time of culture) in vitro. In some examples, a cell culture includes a substantially pure culture (such as T cells or dendritic cells). In other examples a cell culture includes a mixed culture, such as co-culture of two or more types of cells (for example a culture of T cells with dendritic cells). In further examples, a cell culture may include cells grown in contact with an extracellular matrix.

[0045] Culture Medium: A synthetic set of culture conditions with the nutrients necessary to support the viability, function, and / or growth of a specific population of cells, such as T cells and / or dendritic cells. Culture media generally include components such as a carbon source, a nitrogen source, and a buffer to maintain pH. Additional components in culture media also may include one or more of serum, cytokines, hormones, growth factors, protease inhibitors, protein hydrolysates, shear force protectors, proteins, vitamins, glutamine, trace elements, inorganic salts, minerals, lipids, and / or attachment factors.

[0046] Dendritic cell (DC): Dendritic cells are the principal antigen presenting cells (APCs) involved in primary immune responses. DCs include plasmacytoid dendritic cells and myeloid dendritic cells. Immature DCs originate in the bone marrow and reside in the periphery as immature cells. In the case of injury or infection, the immature DCs capture antigens, which are processed by endosomal or proteosomal pathways for presentation on the cell surface. Antigens processed by the proteosomal pathway bind to major histocompatibility complex (MHC) class 1 molecules for cell surface presentation to stimulate CD8+cytotoxic T cells (CTLs). Antigens processed by the endosomal pathway bind to MHC class II molecules for presentation on the cell surface and stimulation of CD4+helper T cells. DCs also express costimulatory molecules on their cell surface, such as members of the B7 family, TNF family, and intracellular adhesion molecules which participate in activation of T cells.

[0047] Infectious disease: Also known as transmissible disease or communicable disease, infectious diseases are illnesses resulting from an infection. Infections are caused by infectious (or pathogenic agents), including viruses, viroids, bacteria, fungi, nematodes, and other macroparasites, such as helminths.

[0048] Isolated: An “isolated” biological component, such as a nucleic acid, protein (including antigens) or cell, has been substantially separated or purified away from other biological components, e.g., chromosomal and extra-chromosomal DNA and RNA, proteins, and / or cells. Nucleic acids, proteins, and cells that have been “isolated” include nucleic acids, proteins, and / or cells purified by standard purification methods. The term also embraces nucleic acids and proteins prepared by recombinant expression in a host cell as well as chemically synthesized nucleic acids.

[0049] Pharmaceutically acceptable carrier: Remington: The Science and Practice of Pharmacy, Adejare (Ed.), Academic Press, London, United Kingdom, 23rdEdition (2021) describes compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic compositions, such as one or more of memory T cells, effector T cells generated from the memory T cells, and / or additional pharmaceutical agents.

[0050] In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral formulations usually comprise injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. In addition to biologically-neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.

[0051] Purified: The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified protein, nucleic acid, or cell preparation is one in which the protein, nucleic acid, or cell is more enriched than the protein or nucleic acid is in its natural environment. In one aspect, a preparation is purified such that the protein, nucleic acid, or cell represents at least 50% of the total protein, nucleic acid, or cell content of the preparation. Substantial purification denotes purification from other proteins or cellular components. A substantially purified protein, nucleic acid, or cell is at least 60%, 70%, 80%, 90%, 95% or 98% pure. Thus, in one specific, non-limiting example, a substantially purified cell population is at least 90% free of other components.

[0052] Subject: A living multi-cellular vertebrate organism, a category that includes both human and veterinary subjects, including human and non-human mammals.

[0053] T cell: A white blood cell (lymphocyte) that is an important mediator of the immune response. T cells include, but are not limited to, CD4+T cells and CD8+T cells. A CD4+T lymphocyte is an immune cell that carries a marker on its surface known as “cluster of differentiation 4” (CD4). These cells, also known as helper T cells, help orchestrate the immune response, including antibody responses as well as killer T cell responses. CD8+T cells carry the “cluster of differentiation 8” (CD8) marker. In one aspect, a CD8+T cell is a cytotoxic T lymphocyte (CTL). In another aspect, a CD8+cell is a suppressor T cell. Activated T cells can be detected by an increase in cell proliferation and / or expression of or secretion of one or more cytokines (such as IL-2, IL-4, IL-6, IFNy, or TNFa). Activation of CD8+T cells can also be detected by an increase in cytolytic activity in response to an antigen. Memory T cells are antigen- specific CD8+or CD4+T cells that persist long-term after an immune response. Upon re-exposure to the antigen, memory T cells expand and become effector T cells. Memory T cells may include effector memory T cells, central memory T cells, effector memory Treg cells, and stem cell memory T cells.

[0054] Treating or ameliorating a disease: “Treating” refers to a therapeutic intervention that decreases or inhibits a sign or symptom of a disease or pathological condition after it has begun to develop, such as a reduction in tumor size or tumor burden. “Ameliorating” refers to the reduction in the number or severity of signs or symptoms of a disease, such as cancer.

[0055] IL Methods of Generating Memory T Cells In Vitro

[0056] Disclosed herein are methods of generating memory T cells in vitro. In some aspects, the memory T cells are CD8+memory T cells. In other aspects, the memory T cells are CD4+memory T cells. Also disclosed are methods of producing effector T cells from the memory T cells, for example, by re-exposing the memory T cells to the antigen used to generate the memory T cells.

[0057] In some aspects, the methods include culturing a population of T cells with a population of antigen-loaded dendritic cells for at least about 30 days in a culture medium and isolating memory T cells from the culture. In particular examples, no further cells (such as antigen-loaded dendritic cells) or antigen are added to the population of T cells after initiation of the culture.

[0058] In some examples, the population of T cells cultured with the antigen- loaded dendritic cells is a mixed population of T cells. In some examples, the population of T cells is part of a mixed population of cells, such as total lymphocytes, which may include T cells, B cells, and other types of cells. Thus in some examples, the population of T cells is not isolated or purified prior culturing with antigen-loaded dendritic cells. In other examples, the population of T cells (such as a mixed population of T cells) is isolated prior to use. For example, the population of T cells may be total T cells (e.g., CD3+cells) or a mixture of CD4+and CD8+T cells from a starting sample (such as whole blood or other types of samples described below). Methods of purifying or isolating T cells include positive selection of T cells (such as CD3 selection) and / or negative selection to remove non-T cells and gradient separation, immunomagnetic separation, flow cytometry, or other separation methods. Exemplary, nonlimiting examples of methods for preparing a population of T cells are described in Examples 2 and 11. In some examples, the population of T cells includes CD4+T cells and CD8+T cells. In some aspects, the population of T cells substantially lacks or does not include memory T cells, such as CD45RO+cells. For example, the population of T cells may be processed to remove memory T cells (for example by negative selection of CD45RO+cells) prior to culture with antigen-loaded dendritic cells. In some examples, the population substantially lacks CD45RO+CD45RA" cells. For example, the population may include less than 5% (e.g., less than 4%, 3%, 2%, 1.5%, 1%, 0.5%, 0.2%, or 0.1%) memory T cells. In some examples, the preparation includes less than 1% or less than 0.5% memory T cells.

[0059] The disclosed methods include culturing a population of T cells with a population of antigen-loaded dendritic cells for at least about 30 days in a culture medium. In some examples, the population of T cells and the population of antigen-loaded dendritic cells are co-cultured for about 28-48 days (such as about 28-35 days, about 30-42 days, or about 36-45 days). In some examples, the population of T cells and the population of antigen-loaded dendritic cells are co-cultured for about 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 days. In particular examples, the population of T cells and the population of antigen-loaded dendritic cells are co-cultured for about 30 days or about 42 days. In some examples, no further cells (such as antigen-loaded dendritic cells) or antigen are added to the population of T cells after initiation of the culture. In some examples, the cells are cultured in a 35 mm cell culture dish, a 60 mm cell culture dish, or a 6-well cell culture dish. An appropriate size vessel can be selected based on the number of cells being cultured. In some examples, the cell culture dish is at least 35 mm size or larger.

[0060] The population of T cells and the population of antigen-loaded dendritic cells are cultured in an appropriate cell culture medium. In some examples, the cell culture medium is Iscove’s modified Dulbecco’s medium. In other examples, the medium is RPMI medium (also known as RPMI 1640 medium). Other appropriate cell culture media can be selected. In particular examples, the cell culture medium includes serum, for example fetal calf serum (such as about 10% fetal calf serum). In other examples, the medium does not include animal serum and in some examples is a serum-free medium formulation. In some examples, the cell culture medium also includes antibiotics (such as 100 U / ml penicillin and 100 U / ml streptomycin). In some examples, at least a portion of the cell culture medium is periodically removed (such as at least one-half, one-third, one-quarter, one-fifth, or one-tenth of the medium) and replaced with fresh medium, taking care not to disturb the surviving T cells. Fresh medium is added about every 2-5 days during the culture period (such as every 2, 3, 4, or 5 days). In some aspects, one or more exogenous growth factors or cytokines are added to the culture medium for at least a portion of the culture of the population of T cells with the population of antigen-loaded dendritic cells. In some examples, the cytokine or growth factor is added to the culture medium around day 6-24 (e.g., day 6-12, day 10-15, day 14-18, day 16-20, or day 20-24) of the culture of the population of T cells with the population of antigen- loaded dendritic cells. In some examples, the cytokine is added around day 14-18 (such as day 14, 15, 16, 17, or 18) of the co-culture. In one example, the cytokine is added at day 14 of the co-culture. In other examples, the cytokine is added to the co-culture after the death of substantially all naive and effector T-cells in the population. In some examples, the cytokine is interleukin- 15 (IL- 15). The IL- 15 may be added to the culture medium at a final concentration of about 20-100 ng / ml (such as about 20-40 ng / ml, 30-50 ng / ml, 40-60 ng / ml, 50-70 ng / ml, 60-80 ng / ml, 70-90 ng / ml, or 80-100 ng / ml), for example, about 50 ng / ml. In some examples, a single addition of cytokine (such as IL- 15) is added to the culture medium during the co-culture.

[0061] In some examples, the cell culture medium does not include additional exogenous growth factors or cytokines. This indicates that growth factors or cytokines are not added to the culture medium; however, growth factors and / or cytokines may be present in the culture medium (for example, present in components of the medium, such as serum). In addition, growth factors and / or cytokines may accumulate in the medium over the course of the culture or co-culture. For example, the dendritic cells may secrete cytokines during the co-culture with the T cells.

[0062] In some examples, at least a portion of the cell culture medium is removed (such as at least one-half, one-third, one-quarter, one-fifth, or one-tenth of the medium) and replaced with fresh medium. Fresh medium may be added about every 1 -3 days (for example, every 1 , 2, or 3 days) during the culture period. In other examples, the culture medium is not changed during the culture period.

[0063] The ratio of T cells to antigen- loaded dendritic cells is about 1 : 1 to about 20: 1 (for example, about 1:1 to about 5:1, about 5: 1 to about 10:1, or about 10:1 to about 20:1). In particular examples, the ratio of T cells to antigen-loaded dendritic cells is about 1: 1, about 2:1 , about 3:1, about 4:1, about 5:1, about 6:1 , about 7:1, about 8:1, about 9:1, about 10:1, about 15:1, or about 20:1. In some examples, about 500,000 to 5 million T cells (for example, about 500,000 to 2 million, about 1 to 4 million, or about 3 to 5 million T cells) are cultured with the population of antigen-loaded dendritic cells. In non-limiting examples, about 1 million T cells, about 1.5 million T cells, or about 2 million T cells are cultured with the antigen-loaded dendritic cells.

[0064] Following the culture of the population of T cells and the population of antigen- loaded dendritic cells, memory T cells are isolated. In some examples, the memory T cells are floating cells. Memory T cells can also be identified by measuring expression of one or more cell surface markers. In some examples, human CD8+memory T cells express CD3, CD8, and CD45RO. Human CD8+memory T cells also have low expression of CD45RA (e.g., are CD45RA ). In other examples, mouse CD8+memory T cells express CD3, CD8, and CD44. In other examples, human CD4+memory T cells express CD3, CD4, and CD45RO. Human CD4+memory T cells also have low expression of CD45RA (e.g., are CD45RA ). Additional memory T cell markers can be identified and may include CD62L and / or CCR7 (e.g., for central memory T cells).

[0065] The memory T cells can be further isolated or purified, for example using positive- and / or negative-selection with one or more cell surface markers and separation (for example, immunomagnetic separation, flow cytometry, or gradient separation). The memory T cells may be washed one or more times and resuspended in an appropriate buffer or other pharmaceutically acceptable carrier, for example, for administration to a subject. In some examples, the cells are harvested and washed (for example in a buffer, such as phosphate buffered saline). The memory T cells may be resuspended in a medium containing PLASMA-LYTE™ multiple electrolytes injection (Baxter Healthcare), autologous plasma, or a pharmaceutically acceptable carrier (for example, a balanced salt solution).

[0066] In some examples, the methods also include preparing a population of antigen- loaded dendritic cells. Dendritic cells can be prepared by culturing monocytes with a culture medium including one or more cytokines, such as granulocyte-macrophage colonystimulating factor (GM-CSF) and / or interleukin-4 (IL-4) for a sufficient period of time to produce dendritic cells. In one example, monocytes are obtained by elutriation. Elutriation systems are commercially available and include ELUTRA® Cell Separation System (TerumoBCT, Lakewood, CO) and JE-50 Elutriation System (Beckman Coulter, Indianapolis, IN). In another example, monocytes are obtained using negative selection to deplete non-monocytic cells (for example, RosetteSep™ Human Monocyte Enrichment Cocktail, Stemcell Technologies, Cambridge, MA) or by positive selection using anti-CD14 magnetic beads (such as Classical Monocyte Isolation kit, Miltenyi Biotec, Auburn, CA). In another example, PBMCs are prepared by centrifugation over a Ficoll-Paque (Pharmacia, Uppsala, Sweden) density gradient and the monocytes are separated from lymphocytes by counterflow centrifugation (for example using an elutriator system) or centrifugation on a continuous Percoll (Pharmacia, Piscataway, NJ) density gradient.

[0067] In particular examples, dendritic cells are prepared by culturing monocytes with one or more cytokines for 5-10 days, such as 5-8 days, 7-10 days, or 7-8 days (for example, 5, 6, 7, 8, 9, or 10 days), until dendritic cells are produced. The dendritic cells are isolated, for example by pipetting floating dendritic cells and transferring the dendritic cells to fresh cell culture medium, for example to produce a population of purified dendritic cells. The culture medium may include GM-CSF and / or IL-4. In some examples, the culture medium includes GM-CSF (e.g., about 10-100 ng / ml, such as about 10 ng / ml, about 20 ng / ml, about 30 ng / ml, about 40 ng / ml, about 50 ng / ml, about 60 ng / ml, about 70 ng / ml, about 80 ng / ml, about 90 ng / ml, or about 100 ng / ml) and IL-4 (e.g. , about 10-100 ng / ml, such as about 10 ng / ml, about 20 ng / ml, about 30 ng / ml, about 40 ng / ml, about 50 ng / ml, about 60 ng / ml, about 70 ng / ml, about 80 ng / ml, about 90 ng / ml, or about 100 ng / ml). In non-limiting examples the medium includes 50 ng / ml GM-CSF and 50 ng / ml IL-4. In some examples, the cell culture medium is Iscove’s modified Dulbecco’s medium or RPMI medium. In particular examples, the cell culture medium includes serum, for example fetal calf serum (such as about 10% fetal calf serum). In additional examples, the cell culture medium also includes antibiotics, such as penicillin (e.g., 100 U / ml) and / or streptomycin (e.g., 100 U / ml). During the culturing of the monocytes to produce dendritic cells, fresh cell culture medium is added periodically. In some examples, at least a portion of the cell culture medium is removed (such as at least one- half, one-third, one-quarter, one-fifth, or one-tenth of the medium) and replaced with fresh medium. Fresh medium is added about every 1 -3 days (for example, every 1 , 2, or 3 days) during the culture period.

[0068] A population of purified dendritic cells (for example, dendritic cells produced as described above) is contacted with an antigen for which memory T cells are desired e.g., antigen-specific memory T cells) and activated. In some examples, the dendritic cells are contacted with an antigen and an activator simultaneously. In other examples, the dendritic cells are contacted with the antigen for a period of time and then subsequently contacted with an activator e.g. , in the presence of the antigen) for a period of time.

[0069] In particular examples, the dendritic cells are contacted with antigen (e.g., a peptide or a protein) in a cell culture medium (e.g., Iscove’s DMEM) including GM-CSF (e.g., 50 ng / ml) and IL-4 (e.g., 50 ng / ml). In some examples, the dendritic cells are contacted with about 1 pg / ml to about 200 pg / ml of antigen (for example, about 1-10 pg / ml, about 5-50 pg / ml, about 20-100 pg / ml, about 50-150 pg / ml, or about 100-200 pg / ml antigen), such as a purified peptide. In certain non-limiting examples, the dendritic cells are contacted with about 1-2 pg / ml of a peptide antigen. In other non-limiting examples, the dendritic cells are contacted with about 20-200 pg / ml of a protein or cell homogenate. In some examples, the antigen is a tumor associated antigen or an antigen from an infectious agent (such as a bacterium, virus, or fungus). In some non-limiting examples, the antigen is a melanoma antigen, such as MART-1. In other examples, the dendritic cells are contacted with a mixture or sample that may contain more than one antigen, such as a tumor homogenate or a mixture including tumor cells. In one non-limiting example, the mixture is a homogenate prepared from a melanoma tumor or melanoma cells.

[0070] The purified dendritic cells are incubated with the antigen or mixture for about 15-90 minutes (for example, about 15 minutes, about 30 minutes, about 45 minutes, about 60 minutes, or about 90 minutes). Following contacting the dendritic cells with the antigen or mixture, the dendritic cells are activated, for example by adding lipopolysaccharide (LPS), and the dendritic cells are incubated about 8-18 hours (for example about 8-12, about 10-14, about 12-16, or about 15-18 hours), for example overnight, to produce a population of antigen-loaded dendritic cells. In some examples, the dendritic cells are activated by adding about 1-2 pg / ml LPS to the population of dendritic cells that has been contacted with the antigen(s).

[0071] Exemplary antigens that can be used in the methods described herein include tumor associated antigens. In some non-limiting examples, the tumor associated antigen is MART- 1, gplOO, carcinoembryonic antigen (CEA), CD19, NY-ESO-1, MAGE (such as MAGE-A3), hTERT, epidermal growth factor receptor (EGFR), mesothelin, Mum-1, [Lcatenin, CDK4, ERBB2. In other examples, the antigen is a tumor homogenate or tumor cells, for example, from a tumor or hematological malignancy listed in Section III below. In further examples, the antigen is a viral, bacterial, or fungal antigen, such as an antigen from one of the infectious agents listed in Section III below.

[0072] In some examples, the dendritic cells and / or T cells utilized to produce the memory T cells described herein are obtained from peripheral blood (e.g., whole blood), peripheral blood monocytes, lymphocytes, umbilical cord blood, lymph node tissue, bone marrow, or induced pluripotent stem (iPS) cells. Preparation of dendritic cells from iPS cells is described in, e.g., Senju et al. , Gene Therapy 18:874-883, 2011.

[0073] In additional aspects, the memory T cells are stimulated with the antigen used to produce the antigen- loaded dendritic cells to produce a population of effector T cells. In some examples, the memory T cells are contacted with the antigen in cell culture medium for about 1-4 days (such as about 1, 2, 3, or 4 days), resulting in proliferation of the memory T cells into effector T cells. In other examples, the memory T cells are stimulated with antigen- loaded dendritic cells to convert them into effector T cells. The effector cells exhibit cytotoxic activity to cells expressing the antigen. Cytotoxicity can be measured using live- dead staining,51Cr release assay, or other methods known to one of ordinary skill in the art. In other examples, effector T cells are identified by expression of CD45RA and loss of CD45RO expression in human T cells or loss of CD44 expression (and possibly CCR7) in mouse T cells. In particular examples, the memory T cells are contacted with the antigen in the same cell culture medium used to produce the memory T cells (such as Iscove’s modified Dulbecco’s medium containing 10% FCS). In some examples, the memory T cells are contacted with about 1 pg / ml to about 200 pg / ml of antigen (for example, about 1-10 pg / ml, about 5-50 pg / ml, about 20-100 pg / ml, about 50-150 pg / ml, or about 100-200 pg / ml antigen), such as a purified peptide. In certain non- limiting examples, the dendritic cells are contacted with about 1-2 pg / ml of a peptide antigen. In other non-limiting examples, the dendritic cells are contacted with about 20-200 pg / ml of a protein or cell homogenate.

[0074] Any of the populations of cells described herein (e.g., T cells, dendritic cells, memory T cells, antigen-loaded dendritic cells, and effector T cells) can be cryopreserved for later use. In other examples, the cells can be maintained in culture. For example, both mouse and human memory T cells can be maintained in culture for more than one month.

[0075] III. Methods of Immunotherapy

[0076] Provided are methods of treating cancer or infectious disease in a subject with memory T cells (for example, CD4+or CD8+memory T cells) or effector cells produced as disclosed herein. In some aspects, the methods include administering to the subject a composition including the memory T cells and a pharmaceutically acceptable carrier. In other aspects, the methods include administering to the subject a composition including effector T cells produced by activating the memory T cells with an antigen (such as the antigen used to generate the memory T cells) and a pharmaceutically acceptable carrier. In some examples, the T cells are autologous or allogeneic to the subject being treated.

[0077] In some aspects, a subject with cancer is administered memory T cells produced by the methods disclosed herein. The memory T cells are produced using dendritic cells that are loaded with one or more antigens expressed by the cancer in the subject. In other aspects, a subject with cancer is administered effector T cells produced by the methods described herein. For example, effector T cells are generated from memory T cells are produced using dendritic cells that are loaded with one or more antigens expressed by the cancer in the subject prior to administering the effector T cells to the subject.

[0078] In other aspects, the methods include producing memory T cells from a homogenate of tumor tissue or tumor cells prepared from the tumor or hematological malignancy from the subject. T cells are cultured with dendritic cells loaded with the homogenate to produce memory T cells. The memory T cells are isolated and then administered to the subject. In some examples, the memory T cells are activated with the homogenate to produce effector T cells, which are then administered to the subject. In additional examples, cytotoxic T cells are isolated from the population of effector cells, the T cell receptor expressed by the cytotoxic T cells is identified (for example, by sequencing), and the identified T cell receptor is expressed in T cells (for example, by transduction) to produce modified T cells. The modified T cells expressing the T cell receptor are then administered to the subject.

[0079] In other aspects, a subject with an infectious disease is administered memory T cells produced by the methods disclosed herein. The memory T cells are produced using dendritic cells that are loaded with one or more antigens expressed by the agent causing the infectious disease. In other aspects, a subject with an infectious disease is administered effector T cells produced by the methods described herein. For example, effector T cells are generated from memory T cells are produced using dendritic cells that are loaded with one or more antigens expressed by the agent causing the infectious disease prior to administering the effector T cells to the subject.

[0080] The memory T cells and / or effector T cells described herein can be incorporated into pharmaceutical compositions. Such compositions typically include a population of cells and a pharmaceutically acceptable carrier. A “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, buffering, preservative, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration (see, e.g., Remington: The Science and Practice of Pharmacy, Adejare (Ed.), Academic Press, London, United Kingdom, 23rdEdition (2021)). Examples of such carriers or diluents include, but are not limited to, water, saline, Ringer’s solutions, dextrose solution, balanced salt solutions, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. Supplementary active compounds can also be incorporated into the compositions. Actual methods for preparing administrable compositions include those provided in Remington: The Science and Practice of Pharmacy, Adejare (Ed.), Academic Press, London, United Kingdom, 23rdEdition (2021). In some examples, the composition includes about 104to 1012of the memory T cells (for example, about 104- 108cells, about 106- 10scells, or about 106-l 012cells). In other examples, the composition includes about 104to 1012of the activated memory T cells (effector cells, for example, about 104- 108cells, about 106- 10scells, or about 106- 1012cells). For example, the composition may be prepared such that about 104to 108memory or effector T cells / kg (such as about 104, 10s, 106, 107, or 108cells / kg) are administered to a subject. In specific examples, the composition includes at least 104, 10s, 106, 107, 10s, 109, or IO10memory T cells or effector T cells. The memory T cells or effector T cells are typically administered parenterally, for example intravenously; however, injection or infusion to a tumor or close to a tumor (local administration) or administration to the peritoneal cavity can also be used. Appropriate routes of administration can be determined based on the subject being treated, the condition being treated, and other factors.

[0081] Multiple doses of the composition including the memory T cells or effector T cells can be administered to a subject. For example, the memory or effector T cells can be administered daily, every other day, twice per week, weekly, every other week, every three weeks, monthly, or less frequently. A skilled clinician can select an administration schedule based on the subject, the condition being treated, the previous treatment history, and other factors. In some examples, the subject is also administered one or more cytokines to support survival of the administered T cells, such as IL-2, IL-7, and / or IL-15.

[0082] In some examples, the subject being treated has a solid tumor, for example, a solid tumor expressing an antigen used to generate the memory T cells and / or effector T cells administered to the subject. Examples of solid tumors, include sarcomas (such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, soft tissue sarcoma, and other sarcomas), synovioma, mesothelioma, Ewing sarcoma, leiomyosarcoma, rhabdomyosarcoma, colon cancer, colorectal cancer, peritoneal cancer, esophageal cancer (such as esophageal squamous cell carcinoma), pancreatic cancer, breast cancer (including basal breast carcinoma, ductal carcinoma and lobular breast carcinoma), endometrial cancer, lung cancer (such as non-small cell lung cancer), ovarian cancer, prostate cancer, liver cancer (including hepatocellular carcinoma), gastric cancer, squamous cell carcinoma (including head and neck squamous cell carcinoma), basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, Wilms tumor, cervical cancer, fallopian tube cancer, testicular tumor, seminoma, bladder cancer (such as renal cell cancer), melanoma, and CNS tumors (such as a glioma, glioblastoma, astrocytoma, medulloblastoma, craniopharyrgioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma and retinoblastoma). Solid tumors also include tumor metastases (for example, metastases to the lung, liver, brain, or bone). In some examples, the subject has hepatocellular carcinoma, neuroblastoma, breast cancer, gastric cancer, endometrial cancer, bladder cancer (such as renal cell carcinoma), lung cancer (such as non-small cell lung cancer), cervical cancer, medulloblastoma, esophageal cancer (such as esophageal squamous cell carcinoma), prostate cancer, seminoma, glioblastoma, osteosarcoma, astrocytoma, or soft tissue sarcoma. In particular examples, the subject has melanoma, hepatocellular carcinoma, or neuroblastoma.

[0083] In other examples, the subject has a hematological malignancy, for example, a hematological malignancy expressing an antigen used to generate the memory T cells and / or effector T cells administered to the subject. Examples of hematological malignancies include leukemias, including acute leukemias (such as llq23 -positive acute leukemia, acute lymphocytic leukemia (ALL), T cell ALL, acute myelocytic leukemia, acute myelogenous leukemia (AML), and myeloblastic, promyelocytic, myelomonocytic, monocytic and erythroleukemia), chronic leukemias (such as chronic myelocytic (granulocytic) leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), lymphoblastic leukemia, polycythemia vera, lymphoma, diffuse large B cell lymphoma, Burkitt lymphoma, T cell lymphoma, follicular lymphoma, mantle cell lymphoma, Hodgkin disease, non-Hodgkin lymphoma, multiple myeloma, Waldenstrom macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia. In particular examples, the subject has acute lymphocytic leukemia (ALL), T cell ALL, acute myelocytic leukemia, or acute myelogenous leukemia (AML).

[0084] In some examples, the subject is also treated with one or more of surgery, radiation therapy and chemotherapeutic agents. Exemplary chemotherapeutic agents include (but are not limited to) alkylating agents, such as nitrogen mustards (such as mechlorethamine, cyclophosphamide, melphalan, uracil mustard or chlorambucil), alkyl sulfonates (such as busulfan), nitrosoureas (such as carmustine, lomustine, semustine, streptozocin, or dacarbazine); antimetabolites such as folic acid analogs (such as methotrexate), pyrimidine analogs (such as 5-FU or cytarabine), and purine analogs, such as mercaptopurine or thioguanine; or natural products, for example vinca alkaloids (such as vinblastine, vincristine, or vindesine), epipodophyllotoxins (such as etoposide or teniposide), antibiotics (such as dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, or mitocycin C), and enzymes (such as L-asparaginase). Additional agents include platinum coordination complexes (such as cis-diamine-dichloroplatinum II, also known as cisplatin), substituted ureas (such as hydroxyurea), methyl hydrazine derivatives (such as procarbazine), and adrenocrotical suppressants (such as mitotane and aminoglutethimide); hormones and antagonists, such as adrenocorticosteroids (such as prednisone), progestins (such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and magestrol acetate), estrogens (such as diethylstilbestrol and ethinyl estradiol), antiestrogens (such as tamoxifen), and androgens (such as testosterone proprionate and fluoxymesterone). Examples of the most commonly used chemotherapy drugs include adriamycin, melphalan (Alkeran®) Ara-C (cytarabine), carmustine, busulfan, lomustine, carboplatinum, cisplatinum, cyclophosphamide (Cytoxan®), daunorubicin, dacarbazine, 5-fluorouracil, fludarabine, hydroxyurea, idarubicin, ifosfamide, methotrexate, mithramycin, mitomycin, mitoxantrone, nitrogen mustard, paclitaxel (or other taxanes, such as docetaxel), vinblastine, vincristine, VP-16, while newer drugs include gemcitabine (Gemzar®), trastuzumab (Herceptin®), irinotecan (CPT-11), leustatin, navelbine, rituximab (Rituxan®) imatinib (STI-571), Topotecan (Hycamtin®), capecitabine, ibritumomab (Zevalin®), and calcitriol. A skilled clinician can select appropriate additional therapies (from those listed here or other current therapies) for the subject, depending on factors such as the subject, the cancer being treated, treatment history, and other factors.

[0085] In other aspects, the subject has an infectious disease, for example, an infectious disease caused by an agent expressing an antigen used to generate the memory T cells and / or effector T cells administered to the subject. Infectious diseases are caused by pathogenic agents, such as bacteria, viruses, fungi, protozoa and parasites.

[0086] In some examples, viruses include, but are not limited to human immunodeficiency virus (HIV), polio virus, hepatitis A virus, hepatitis C virus, enteroviruses, human coxsackie viruses, rhinoviruses, echoviruses, foot-and-mouth disease virus, Norwalk virus, chikungunya virus, equine encephalitis viruses, Simliki Forest virus, Sindbis virus, Ross River virus, rubella viruses, dengue viruses, yellow fever viruses, West Nile virus, St. Louis encephalitis virus, Japanese encephalitis virus, Powassan virus, Zika virus, coronaviruses (e.g., alpha coronaviruses, beta coronaviruses, MERS-CoV, SARS-CoV, or SARS-CoV-2), rabies viruses, Ebola virus, Marburg virus, parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus, influenza viruses, hepatitis B virus, papilloma viruses, polyoma viruses, adenoviruses, herpes simplex viruses, cytomegalovirus, Epstein-Barr virus, varicella zoster virus, and others. Examples of bacteria include, but are not limited to: Helicobacter pylori, Escherichia coli, Vibrio cholerae, Borrelia burgdorferi, Legionella pneumophilia, Mycobacteria sp. (such as. M. tuberculosis, M. avium, M. intracellulare, M. kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (yiridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcus pneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilus influenzae, Bacillus anthracis, Corynebacterium diphtheriae, Corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasturella multocida, Bacteroides sp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponema pertenue, Leptospira, Bordetella pertussis, Shigella flexnerii, Shigella dysenteriae, Actinomyces israelii, and Acinetobacter sp. In some examples, the bacterium is a drug resistant bacterium, including but not limited to methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Staphylococcus aureus (VRSA), vancomycin-resistant Enterococci (VRE), extended spectrum beta- lactamase-producing gram-negative bacilli (ESBL), multidrug-resistant Streptococcus pneumoniae (MDRSP), carbapenem-resistant Enterobacteriaceae (CRE), multidrug-resistant Acinetobacter, and multidrug-resistant tuberculosis (MDR TB or XDR TB).

[0087] Examples of fungi include, but are not limited to Candida (such as Candida albicans), Aspergillus (such as Aspergillus fumigatus and Aspergillus flavus), Cryptococcus (such as Cryptococcus neoformans and Cryptococcus gattii), Histoplasma (such as Histoplasma capsulatum), Pneumocystis (such as Pneumocystis jirovecii), and Stachybotrys (such as Stachybotrys chartarum).

[0088] In some examples, the subject with an infectious disease is also administered one or more anti-infection agents (e.g., antibiotics, antifungals, antivirals, and / or antiparasitics). A skilled clinician can select appropriate additional therapies for the subject, depending on factors such as the subject, the cancer being treated, treatment history, and other factors.

[0089] IV. Methods of Identifying T Cell Receptors and Cognate Antigens Recognized by In Vitro Generated Memory T Cells and Their Use

[0090] Methods of identifying TCRs and cognate antigens recognized by in vitro generated memory T cells are provided herein. In some aspects, the methods further include producing antibodies (such as monoclonal antibodies) that specifically bind the identified antigen(s). The antibody can be administered to a subject in need thereof, or can be used to produce a chimeric antigen receptor (CAR)-T cell, which is administered to a subject in need thereof. In other aspects, the methods further include administering the identified antigen to a subject in need thereof to induce a memory T cell response. In additional aspects, the methods further include using the identified TCR to produce a TCR-T cell, which is administered to a subject in need thereof.

[0091] In some aspects, the methods include producing a population of memory T cells recognizing an antigen as described herein, and identifying the T cell receptor expressed by the memory T cells. The T cell receptor (such as a T cell a chain and P chain) expressed by the memory T cells is identified (for example, by sequencing, such as single cell RNA or DNA sequencing). In some examples, TCRs of memory T-cells are analyzed by single cell RNA or DNA sequencing, for example to determine whether in vitro expansion of effector T- cells results in bias for one or more particular TCRs. In some examples, the methods further include activating the memory T cells to produce effector T cells by contacting the memory T cells with the sample used to produce the antigen-loaded dendritic cells, and in some examples, identifying the TCR expressed by the effector T cells.

[0092] In some examples, the population of antigen-loaded dendritic cells cultured with the T cells to produce the memory T cells is loaded with a sample comprising a one or more antigens (such as a mixture of antigens from a tumor or pathogen). In some examples, memory T cells are produced using a sample including at least one antigen, which may be known or unknown (such as a tumor antigen or a pathogen antigen). In some examples, the antigen is a tumor antigen and the methods utilize cancer cells or a sample from a cancer or tumor, for example, a tumor biopsy, tumor homogenate, a population of tumor cells, homogenate of tumor cells, or recombinant tumor antigen proteins. In other examples, the antigen is a pathogen antigen and the methods utilize a pathogen, for example, a culture or suspension of pathogen cells, fungus, inactivated virus or bacteria, recombinant pathogen proteins, or pathogen DNAs (via electroporation) or RNAs (using lipid-based transfection reagents).

[0093] In some aspects, the methods further include identifying the antigen recognized by the TCR. In some examples, the methods include using a yeast display library of peptide-MHC complexes combined with bioinformatics. In some examples, an expression library of 8mer to 1 Imer peptides with random sequences is generated in yeast. A truncated HLA is coexpressed in yeast so that HLA can present the peptide antigen library on surface. The yeast library is then screened multiple times with a soluble, biotinylated TCR molecule that is coupled with a streptavidin-conjugated magnetic bead column. Yeast clones showing strong binding to a TCR molecule of interest are deep sequenced to determine candidate antigen peptide sequences. The antigen specificity is examined using algorithms that predict the binding between the TCR and candidate peptides, together with activation of T-cells with the candidate antigens. See e.g., Gee et al., Cell 172:549-563, 2018. The cognate antigen can also be searched by using a database of TCR sequences with known antigen specificities (e.g., VDJdb; Shugay et al., Nucleic Acids Res. 46:D419-427, 2017).

[0094] In some examples, the identified antigen is administered to a subject in need thereof. In some examples, the antigen is conjugated to an adjuvant prior to administration to the subject. Exemplary adjuvants include aluminum, monophosphoryl lipid A (ASOIB or AS04), MF59 and CpG 1080. Other adjuvants for peptide antigens include polymer-based adjuvants (such as lactide-co-glycolide) acid (PLGA), chitosan and dendritic polymers), virus like- particles, immunostimulatory complex (including cholesterol, phospholipids and saponin), self-assembling peptides and lipopeptides.

[0095] In other aspects, the identified antigen is used to generate antibodies (such as monoclonal antibodies). Methods of generating antibodies are known to one of ordinary skill in the art. In additional aspects, the generated antibody, or a fragment thereof can be used to produce a modified T cell, such as a CAR-T cell, which can be administered to a subject in need thereof. Methods of producing CAR-T cells are known to one of ordinary skill in the art.

[0096] In some aspects, the methods are carried out with cancer cells or a sample from a cancer or tumor and the identified antigen, antibody, or modified T cell is administered to a subject with the same type of cancer. Exemplary cancers include the solid tumors and hematological malignancies described in Section III.

[0097] In other aspects, the methods are carried out with a pathogen and the identified antigen, antibody, or modified T cell is administered to a subject infected with the same pathogen. Exemplary pathogens include those described in Section III.

[0098] V. Additional Aspects

[0099] Aspect 1. A method of producing memory T cells in vitro, comprising: culturing a population of T cells with a population of antigen-loaded dendritic cells for at least about 28-48 days in a culture medium; and isolating memory T cells from the culture. Aspect 2. The method of aspect 1 , wherein the population of T cells is a mixed population of T cells.

[0100] Aspect 3. The method of aspect 1 or 2, wherein the population of T cells comprises CD8+T cells and / or CD4+T cells.

[0101] Aspect 4. The method of any one of aspects 1 to 3, wherein the population of T cells substantially lacks memory T cells.

[0102] Aspect 5. The method of aspect 4, wherein the population of T cells is negatively selected for CD45RO expression prior to culturing the population of T cells with the antigen- loaded dendritic cells.

[0103] Aspect 6. The method of any one of aspects 1 to 5, wherein the culture medium is Iscove’ s modified Dulbecco’ s medium.

[0104] Aspect 7. The method of any one of aspects 1 to 6, wherein the culture medium includes fetal calf serum.

[0105] Aspect 8. The method of any one of aspects 1 to 7, further comprising adding a cytokine to the culture medium at about day 14-18 of the culture.

[0106] Aspect 9. The method of aspect 8, wherein the cytokine is interleukin- 15.

[0107] Aspect 10. The method of any one of aspects 1 to 7, wherein the culture medium does not include additional exogenous growth factors and / or cytokines.

[0108] Aspect 11. The method of any one of aspects 1 to 10, wherein at least a portion of the culture medium is replaced every 2-5 days.

[0109] Aspect 12. The method of any one of aspects 1 to 11 , wherein additional cells or antigens are not added to the culture medium during the culturing.

[0110] Aspect 13. The method of any one of aspects 1 to 12, wherein the T cell population is prepared from whole blood, peripheral blood monocytes, lymphocytes, bone marrow, lymph node tissue, umbilical cord blood, or induced pluripotent stem cells.

[0111] Aspect 14. The method of any one of aspects 1 to 13, wherein the memory T cells are human memory T cells and express CD3, CD8, and CD45RO or are mouse memory T cells and express CD3, CD8, and CD44.

[0112] Aspect 15. The method of any one of aspects 1 to 14, wherein the memory T cells are CD4+memory T cells or CD8+ memory T cells.

[0113] Aspect 16. The method of any one of aspects 1 to 15, wherein the population of antigen-loaded dendritic cells is produced by: culturing monocytes in culture medium comprising fetal calf serum, GM-CSF, and IL-4 to generate dendritic cells; isolating the dendritic cells; adding one or more antigen for a period of time; adding lipopolysaccharide (LPS); and culturing the dendritic cells, antigen, and LPS for 8-18 hours to generate a population of antigen-loaded dendritic cells.

[0114] Aspect 17. The method of any one of aspects 1 to 16, wherein the population of antigen-loaded dendritic cells is prepared from whole blood, peripheral blood monocytes, lymphocytes, bone marrow, or induced pluripotent stem cells.

[0115] Aspect 18. The method of any one of aspects 1 to 17, further comprising: activating the memory T cells to produce effector T cells by contacting the memory T cells with the antigen used to produce the antigen-loaded dendritic cells.

[0116] Aspect 19. The method of aspect 18, wherein the effector T cells are cytotoxic T cells.

[0117] Aspect 20. A method of treating a subject with cancer, comprising administering to the subject the memory T cells generated by the method of any one of aspects 1 to 19, wherein the antigen- loaded dendritic cells are loaded with an antigen expressed by the cancer in the subject.

[0118] Aspect 21. A method of treating an infectious disease in a subject, comprising administering to the subject the memory T cells generated by the method of any one of aspects 1 to 19, wherein the antigen-loaded dendritic cells are loaded with an antigen expressed by the agent causing the infectious disease.

[0119] Aspect 22. The method of aspect 21, wherein the agent causing the infectious disease is a bacterium, virus, or fungus.

[0120] Aspect 23. The method of aspect 22, wherein the bacterium is an antibiotic resistant bacterium.

[0121] Aspect 24. The method of any one of aspects 20 to 23, wherein the memory T cells are activated to produce effector T cells prior to administering the cells to the subject.

[0122] Aspect 25. The method of any one of aspects 20 to 24, wherein the population of T cells used to generate the memory T cells is autologous or allogeneic.

[0123] Aspect 26. A method of treating a subject with a tumor, comprising: culturing a population of T cells with a population of dendritic cells loaded with a homogenate prepared from the tumor from the subject for at least about 30 days in a culture medium; isolating memory T cells from the culture; and administering the memory T cells to the subject.

[0124] Aspect 27. The method of aspect 26, wherein the memory T cells are CD8+memory T cells and / or CD4+memory T cells.

[0125] Aspect 28. The method of aspect 26 or aspect 27, further comprising adding a cytokine to the culture medium at about day 14-18 of the culture.

[0126] Aspect 29. The method of aspect 28, wherein the cytokine is interleukin- 15.

[0127] Aspect 30. The method of any one of aspects 26 to 29, further comprising: activating the memory T cells to produce a population of effector T cells; and administering the effector T cells to the subject.

[0128] Aspect 31. The method of aspect 30, further comprising: isolating T cells with cytotoxic activity against the tumor from the population of the effector cells; identifying the T cell receptor expressed by the cytotoxic T cells; expressing the identified T cell receptor in T cells; and administering the T cells expressing the identified T cell receptor to the subject with cancer.

[0129] Aspect 32. A method, comprising: producing a population of memory T cells recognizing an antigen according to the method of any one of aspects 1 to 17; and identifying the T cell receptor expressed by the memory T cells.

[0130] Aspect 33. The method of aspect 32, wherein the population of antigen-loaded dendritic cells cultured with the T cells is loaded with a sample comprising a mixture of antigens.

[0131] Aspect 34. The method of aspect 33, wherein the sample comprising a mixture of antigens is a tumor sample or a pathogen sample.

[0132] Aspect 35. The method of any one of aspects 32 to 34, further comprising activating the population of memory T cells to produce a population of effector T cells and identifying the T cell receptor expressed by the effector T cells.

[0133] Aspect 36. The method of aspect 35, wherein activating the memory T cells to produce effector T cells comprises contacting the memory T cells with the sample used to produce the antigen-loaded dendritic cells.

[0134] Aspect 37. The method of aspect 36, wherein the effector T cells are CD8+and / or CD4+T cells. Aspect 38. The method of any one of aspects 32 to 37, wherein identifying the T cell receptor expressed by the memory T cells or effector T cells comprises single cell sequencing.

[0135] Aspect 39. The method of any one of aspects 32 to 38, further comprising identifying the antigen specifically bound by the identified T cell receptor.

[0136] Aspect 40. The method of aspect 39, further comprising administering the identified antigen to a subject in need thereof.

[0137] Aspect 41. The method of aspect 40, wherein the antigen is conjugated to an adjuvant.

[0138] Aspect 42. The method of aspect 41, further comprising producing an antibody that specifically binds the identified antigen.

[0139] Aspect 43. The method of aspect 42, further comprising administering the antibody or a chimeric antigen receptor comprising the antibody or a fragment thereof to a subject in need thereof.

[0140] EXAMPLES

[0141] The following examples are provided to illustrate particular features of certain aspects of the disclosure, but the scope of the claims should not be limited to those features exemplified.

[0142] Example 1

[0143] In Vitro Generation of Mouse Memory T Cells

[0144] In an in vivo situation, memory T cells are generated 3-4 weeks after stimulation by antigen-presenting cells such as dendritic cells (DCs). To generate memory T cells in vitro, such long-term culture was reconstituted by co-incubation of T cells with DCs. Mouse bone marrow-derived DCs were stimulated with the OT-1 peptide (which is known to stimulate a specific set of CD8 T cells that react to the OT-1 antigen), and then mixed with cells from lymphocytes. Total lymphatic cells were used because it has been reported that memory CD8+T cell generation in vivo is greatly stimulated by the presence of CD4+T cells, and also because a crude system is well suited for reconstitution. FIGS. 1 A-1C show phase contrast micrographs of such mixed culture at 2 days, 14 days, and 26 days after stimulation by DCs. While many T cells were still alive at 2 days (FIG. 1A), most T cells died at 14 days with a small portion of live T cells (FIG. IB). These cells survived at 26 days (FIG. 1C), suggesting the presence of memory T cells. Whether these surviving cells could represent memory CD8 T cells was then examined. To this end, cells were stained with antibodies against CD3 (a T cell marker), CD8, and CD44 (a marker for both memory and effector T cells). FIGS. 2A-2D show flow cytometry analyses of a T cell-DC mixed culture after 30 days. Cells were first gated to analyze lymphatic cells (FIG. 2A), then, live cells were selected using a Zombie Green™ live / dead dye (FIG. 2B). Both CD3-positive and CD8-positive live lymphocytes (FIG. 2C) were analyzed for the expression of CD44 (FIG. 2D). This gating strategy revealed that CD3 and CD8 positive live lymphocytes expressed high CD44. Because CD44 is expressed in both effector and memory T cells, and because the DC-T cell mixture was cultured for 30 days, the CD44+, CD3+, CD8+cells are likely to represent memory CD8 T cells.

[0145] If these cells indeed are memory CD8 T cells, they should show specificity to the OT- 1 antigen. This was examined by OT-1 tetramer assay, in which cells expressing the T cell receptor (TCR) specific to OT-1 antigen are stained. Memory CD8 T cells at 30 days were stimulated by the OT-1 peptide for 2 days. Then, CD3- and CD8-positive lymphocytes were gated to analyze expression of OT-1 TCR using PE-labeled OT-1 tetramers. As FIG. 3A shows, these cells were positive for PE-labeled OT-1 tetramers. As a control, naive T cells were stained by PE-labeled tetramers. As expected, no staining of these cells was observed by the tetramer assay (FIG. 3B).

[0146] Memory CD8 T cells should have cytotoxic activity. To determine the cytotoxicity, ovalbumin (OVA)-expressing B16 melanoma cells were used. Because memory T cells are specific to OT-1 (a part of the OVA protein sequence), these memory T cells should show cytotoxicity toward OVA-B16 melanoma, but not to the control Bl 6 parental melanoma cells that express no OVA. Memory T cells were first activated into effector T cells by the addition of the OT-1 peptide, and then added to melanoma cells. After 2 days, cells were stained with a Zombi Green live / dead dye. Both phase (FIGS. 4 A, 4C, 4E, 4F) and fluorescence (FIGS. 4B, 4D, 4F, 4H) micrographs were taken to determine cytotoxicity. The addition of effector cells resulted in cell death of OVA-B16 melanoma (FIGS. 4C and 4D) while control B16 cells showed no death FIGS. 4G and 4H). Without addition of T cells, melanoma cells showed no sign of death (FIGS. 4A and 4B). The addition of un-activated memory T cells also showed, though to a lower extent, cytotoxicity (FIGS. 4E and 4F). This is because memory T cells need to be first activated by OVA expressed by OVA-B16 cells.

[0147] In summary, these results indicate that (1) the culture conditions allow long-term culture of a DC-T cell mixture over 30 days; (2) after 30 days, cells showed markers of memory T cells; (3) the memory T cells showed antigen- specificity; and (4) the memory T cells had cytotoxic activity toward the antigen-expressing cells. These results suggest that memory T cells were successfully generated in vitro.

[0148] Example 2

[0149] In Vitro Generation of Human Memory T Cells

[0150] Preparation of Human Memory T cells

[0151] Human monocytes were prepared from whole blood using RosetteSep™ human monocyte enrichment cocktail (Stemcell), according to the manufacturer’s protocol and frozen until use. Thawed human monocytes from the serotype A2 (HLA-A*02) were plated in a 65 mm Falcon tissue culture dish in Iscove’s medium containing 10% FCS, 100 U / ml penicillin, 100 U / ml streptomycin, 50 ng / ml GM-CSF, and 50 ng / ml IL-4. Every two days, one-third of the medium was replaced with fresh Iscove’s medium containing FCS, GM-CSF and IL-4. The cells were cultured for about 7-8 days until dendritic cells were generated. Floating dendritic cells were isolated by gentle pipetting, centrifuged, and suspended in 4 ml per 65 mm dish. MART-1 antigen was added for 30-60 minutes, then LPS was added to activate the DCs. The cells were cultured overnight.

[0152] Human T cells were prepared from whole blood using RosetteSep™ human T cell enrichment cocktail according to the manufacturer’s protocol and frozen until use. Human T cells were thawed one day before the isolation of dendritic cells and cultured overnight. T cells were isolated by centrifugation and suspended in Iscove’s medium containing 10% FCS. Biotinylated CD45RO antibody was added and incubated for 20 minutes on ice. Streptavidin microbeads were added for 20 minutes, added 5 ml of Iscove’s medium, and centrifuged to isolate T cells. The T cells were loaded on LS magnetic column to remove pre-existing memory T cells. The flow through fraction was isolated, centrifuged, and suspended in Iscove’s medium.

[0153] One million T cells were added per 65 mm dish of human antigen-loaded dendritic cells and cultured in Iscove’s medium containing 10% FCS. Every 3-4 days, a portion of the medium was removed and fresh medium was added. The culture was continued for 30 days. Then, floating T cells were isolated for analyses of memory T cells. For antigen-specificity, cells were stimulated by MART-1 and cultured for 2-3 days to analyze antigen-specific effector T cells.

[0154] Results

[0155] Long-term cultures (more than 30 days) were successfully achieved by mixing human dendritic cells (DCs) and autologous T cells. DCs were generated from monocytes and then loaded with the MART-1 (melanA) antigen peptide. After overnight activation with LPS, DCs were mixed together with autologous human T cells (FIG. 5A). The cultures were maintained for over one month (FIG. 5B). While many cells died, a small fraction of T celllike cells survived. Without DCs, the number of surviving T cells was much lower.

[0156] To eliminate a possibility of the presence of pre-existing memory T cells in the original T cell population, naive T cells were used for a mixed DC-T cell culture. Naive T cells were isolated with negative selection with a CD45RO antibody, because pre-existing memory cells with high CD45RO expression were trapped in a magnetic column. Phase contrast microscopy revealed that T cell-like cells still survived after one month.

[0157] Surviving T cells expressed memory T cell markers. All cells were isolated (FIG. 6A) and stained with live-dead dyes to gate live cells (FIG. 6B). Live cells were gated to obtain singlets (FIG. 6C) and singlet cells were gated with CD3 and CD8 to select CD3+CD8+T cells (FIG. 6D). CD8+T cells were finally gated with memory cell markers of CD45RO and CD45RA (FIG. 6E). Table 1 shows population hierarchy of the memory T cells, which indicates cell populations of lymphocytes (corresponding to FIG. 6A), live lymphocytes (FIG. 6B), singlets (FIG. 6C), CD3+CD8+lymphocytes (FIG. 6D) and CD3+CD8+CD45ROlughCD45RAlowmemory T cells. Such analyses revealed that most of the CD3+CD8+T cells expressed high CD45RO and low CD45RA, the expression pattern which is characteristic of memory CD8+T cells. Thus, the surviving T cells are likely to be memory T cells.

[0158] Table 1. Population hierarchy of the memory T cells MART- 1 -specific MHC-I tetramer was used to determine whether the memory T cells showed specificity to MART-1 antigen. Memory T cells were activated for 2 days with MART-1 peptide antigen, which converted them into effector T cells. Effector T cells were then analyzed by cytometry using CD3, CD4, CD8 and MHC-I Tetramer specific to the MART-1 antigen. As FIGS. 7A-7F show, CD3+CD8+effector T cells were found to be specific to MART-1, indicating that the protocol was able to generate in vitro memory T cells with the antigen specificity. Importantly, about 9,000 antigen-specific effector T cells were obtained, which should be enough to obtain TCR sequencing information. About 36% of live lymphocytes were antigen- specific effector T cells, produced from 1 million naive T cells (Table 2). CD4+ T cells did not show MART-1 tetramer staining (FIGS. 7G and 7H). CD3+CD8+CD4 T lymphocytes gated in FIG. 7E expressed high CD45RA but low CD45RO, indicating that they are effector T cells (FIG. 8A). FIGS. 8B and C show that effector T cells and memory T cells gated in Fig. 8A both reacted with MART-1 tetramer, respectively. This confirms that the CD3+CD8+CD4" cells were antigen specific.

[0159] Table 2. Population hierarchy of the activated T cells

[0160] Example 3

[0161] Generating Memory T Cells Against Cancer Antigens

[0162] This technology was tested to determine if it could be applied to generate memory T cells against weak cancer antigens. To this end, DCs were loaded with homogenates of parental B16 / F10 mouse melanoma cells and cultured with naive T cells. After 30 days, T cells survived. Flow cytometry analyses revealed that these surviving “memory” T cells consist of both CD4+T cells and CD8+T cells. CD3+CD8+T-cells expressed memory marker of CD44 (FIGS. 9A-9E). Table 3 shows population hierarchy of memory T cells.

[0163] Importantly, surviving T cells were able to kill B16 / F10 melanoma, an antigen used for immunization. To quantitate the cytotoxicity, melanoma cells were first labeled with CSFE and then incubated with memory T cells for 2 days. Dead cells were detected by a fixable viability red dye. As shown in FIG. 10, the addition of memory T cells increased the fraction of dead cells compared with control melanoma. This is a promising result, suggesting that this technology can generate cytotoxic memory T cells against weak tumor antigens.

[0164] Table 3. Population hierarchy of memory T cells.

[0165] Example 4

[0166] Use of In Vitro Generated Memory T Cells to Identify T Cell Receptors

[0167] TCR repertoire analyses were performed using ImmunoSEQ Analyzer (Adoptive Biotechnologies) with mouse CD8+memory T-cells generated with a model antigen of OT-1. Naive CD8+T-cells, as well as T-cells immunized in vivo with OVA, were prepared and their respective TCR repertoires were compared. Genomic DNAs were prepared from these T- cells and CDR3 sequences of the TCRP chain were analyzed using ImmunoSEQ Analyzer (Adoptive Biotechnologies). FIG. 11 shows a clonal analysis of TCRs from in vitro generated OT-1 -specific memory CD8+T-cells, naive CD8+T-cells and in vivo immunized CD8+T-cells. As expected, the clonality of in vitro generated memory T-cells was found to be the highest while that of naive T-cells is the lowest, indicating that in vitro generated memory T-cells are polyclonal (FIG. 12). Table 4 shows the CDR3 amino acid sequences and productive frequencies (productive means functional V(D)J recombination) of the top 20 rearrangements of in vitro generated memory T-cells. Table 4 also lists the productive frequencies of naive T-cells with the corresponding T-cell clones (clone with the same amino acid sequences), for comparison. By dividing the frequencies of in vitro generated T-cell clones with those of naive T-cells, it is shown that in vitro generated memory T-cells are greatly enriched particular T-cell clones (as much as 1.7%), which would be expected to react the epitope, in this case OT-1 model antigen. These data demonstrate that in vitro generation of memory T-cells shows enrichment of a particular set of T-cell clone. Indeed, an amino acid sequence corresponding to the CD3 region known to be reactive to OT-1 was identified in this screen. These results indicate that in vitro generated memory T-cells are capable of producing the TCR sequence specific to the OT-1 peptide antigen.

[0168] Table 4. CDR3 sequences and frequencies of top 20 productive clones of in vitro generated memory T-cells and naive T-cells Example 5

[0169] Use of Cytokines for In Vitro Generation of Memory T-Cells

[0170] The effect of addition of IL- 15 to the culture medium during in vitro generation of memory T-cells was tested. Mouse naive T-cells were co-cultured with OT-l-loaded dendritic cells as described in Example 1. Both naive T-cells and effector T-cells died after 2 weeks. After most T-cells died, IL- 15 was added once to the culture medium at a concentration of 50 ng / ml. At day 30, surviving T-cells were stimulated with OT-1 antigen for 2 days. Stimulated T-cells were then gated to analyze by flow cytometry to see the expression of CD3, CD4 and CD8. CD3- and CD8-positive lymphocytes were gated to analyze expression of OT-1 TCR using PE-labeled tetramers (FIG. 13 A). For comparison, flow cytometry analysis of CD3- and CD8-positive T-cells without addition of IL-15 is shown in FIG. 13B. Addition of IL- 15 two weeks after stimulation of T-cells with antigen- loaded dendritic cells greatly increased CD8 expression on the memory T-cells (compare FIG. 13A with FIG. 13B). Addition of IL-15 at the beginning (days 1-5) or end (days 25-30) of the T-cell-dendritic cell co-culture did not alter CD8 expression. IL-2 addition did not significantly affect the maintenance and development of memory T-cells. The increase in CD8 expression is important for association between TCRs and antigen-loaded MHC class I complex, thus increased CD8 expression should improve recognition between cytotoxic T- cells and target cells (such as cancer cells).

[0171] Example 6 Generation of OT-2 Antigen- Specific Memory CD4+ T Cells

[0172] OT-2 peptide antigen was used to determine whether CD4 memory T-cells were generated by the in vitro system. The procedure for generation of CD8 memory T-cells described in Example 1 was used, except that OT-2 peptide was used instead of OT-1. After 30 days, T-cells were analyzed by flow cytometry. Live CD3+T-cells were analyzed by CD4 and CD8 markers (FIG. 14A). About 53% of CD3+CD4+ T-cells were CD44 positive (FIG. 14B). CD44 is a memory T-cell marker. Using OT-2 tetramer assay, about 25% of CD3+CD4+CD44+T-Cells showed reactivity to OT-2 antigen (FIG. 14C). In contrast to CD44+CD4 T-cells, CD44-negative CD4 T-cells showed less than 1% positivity toward OT- 2 tetramer (FIG. 14D). CD3+CD4+CD44+T-Cells were analyzed for CD62L marker. About 24% CD3+CD4+CD44+T-cells showed CD62L positive (central memory T-cells) while the rest (76%) showed CD62L negative (effector memory T-cells) (FIG. 14E). Example 7

[0173] Oligomerization of OT-1 Peptide- Loaded MHC

[0174] DCs were immunized first with an antigen (OT-1) and then activated DCs with LPS (sequential addition: first antigen, followed by LPS). Using PLA (proximity ligation assay that detects association among molecules), it was determined that this protocol induced more oligomerization of antigen-loaded MHC-1 on the surface of DCs. As shown in FIG. 15 A, this protocol showed more and stronger fluorescent speckles (indicating oligomer formation of antigen-loaded MHC-I), indicating that this protocol gave more oligomerization of antigen-loaded MHC-I complexes than simultaneous addition of antigen and LPS (FIG. 15B). This was confirmed by quantitative analyses (FIG. 15C). Because receptor oligomerization generally enhances the extent of signaling, the result suggests that sequential addition favors higher interactions between DCs and T-cells.

[0175] Example 8

[0176] Cytotoxicity of human memory T-cells specific to MART-1 cancer antigen

[0177] Human memory T cells specific to the MART-1 cancer antigen (generated as shown in Example 2) were able to kill MART-1 expressing human melanoma cells (MEL526). To quantitate the cytotoxicity, human memory T cells from the serotype A2 were incubated with MEL526 cells with the same A2 serotype. MEL 526 cells were first labeled with CSFE and then incubated with memory T cells for 2 days. Dead cells were detected by fixable viability red dye eFluor™ 780. As shown in FIG. 16A, 69% of CSFE-labeled MEL526 were killed by human memory T cells. In contrast, only 28% of control MEL526 cells were dead without memory T cells (FIG. 16B). This result, together with the cytotoxicity of mouse memory T cells (see FIG. 4 of Example 1 and FIG. 10 of Example 3), suggests that this technology can generate cytotoxic memory T cells for immunotherapy against cancer cells.

[0178] Example 9

[0179] Use of In Vitro Generated Memory T Cells to Identify Cognate Antigen and their Induction of Memory T Cell Responses

[0180] Following identification of the TCRs (e.g., as described in Example 4), cognate antigen is identified using yeast display libraries of peptide-mouse MHC combined with bioinformatics (Gee el al., Cell 172:549-563, 2018) and / or using publicly available databases for searching cognate antigens for TCRs. Once the antigen is identified, antigen-adjuvant complexes are injected to determine whether the antigen induces memory T cell reactions.

[0181] Memory T-cells reactive to B16 / F10 melanoma are generated as described in Example 3 above. Expression of an identified cognate antigen is compared between normal melanocytes (isolated from C57BL6 mice) and B16 / F10 melanoma. Any antigens that show higher expression in B16 / F10 melanoma or mutations are selected for immunization. Such antigen peptides are conjugated with KLH (keyhole limpet hemocyanin) and injected into the mice with a subsequent booster at one week. Alternatively, dendritic cells are loaded with the antigen peptide and injected into mice. After one month, T-cells are analyzed to find whether they express memory T-cell marker of CD44 with the specificity to the original antigen.

[0182] The antigen specificity is assessed by several methods including tetramer assays, antigen-specific proliferation, and cytokine release (such as IFNy). If immunization of certain antigens induces memory T-cell responses in mice, a mouse model of melanoma is used to determine whether immunization with such antigens inhibits melanoma growth or metastasis (see, e.g., Ya et al., Curr. Protoc. Immunol. 108:20.1.1-20.1.43, 2015; Overwijk et al., Curr. Protoc. Immunol. Chapter 20:Unit 20.1, 2001). Furthermore, if such antigens are found to be a surface protein on melanoma cells, an antibody can be produced against such antigens to see whether injection of the antibody would block melanoma development and metastasis

[0183] Example 10

[0184] Use of In Vitro Generated Memory T Cells to Identify T Cell Receptors and Cognate Antigens from Human Cancer Cells

[0185] Memory T-cells are generated in vitro using human cancer cells as antigens, using protocols as described in Example 3 and 4. For human studies, HLA types of human cancer antigens are required to match those of human T-cells and monocytes that are used to generate memory T-cells. Numerous human cancer cell lines with known HLA types are available for cancer antigens (see, e.g., Scholtalbers et al., Genome Med. 7: 118, 2015; Pollack et al., J. Natl. Cancer Inst. 66:1003-1012, 1981). Alternatively, a cancer antigen is a biopsy specimen from cancer patients together with patients’ own blood. The authenticity of in vitro generated human memory T-cells is examined by memory marker expression and by cytotoxicity assay. For cytotoxicity, the protocol described in Example 3 is used, except that target cells are human cancer cells used as antigens. As a control, normal human cells (with a matched tissue type) obtained from a commercial source (such as PromoCell) are used.

[0186] As described in Example 4 above, memory T-cells with particular expression markers (such as CD45RO+) are prepared by magnetic bead selection and / or FACS cell sorter for TCR analyses. The sequence of TCRs are determined by single cell RNA or DNA sequencing. Following identification of the TCRs, cognate antigen is identified with a method using yeast display libraries of peptide-human leukocyte antigen (pHLA) combined with bioinformatics. Once the antigen is identified, their expression levels are analyzed by public database (such as the cancer genome atlas program available at cancer.gov / about- nci / organization / ccg / research / structural-genomics / tcga) to determine whether they are overexpressed in particular cancer types or any mutations are known. The candidate cancer antigens are conjugated with adjuvant, and antigen-adjuvant complexes are injected to determine whether the antigen induces memory T cell reactions.

[0187] Identification of cognate antigens of TCRs on CD4+ helper T cells will allow production of corresponding antibodies for use in pre-clinical and potential clinical trials for treating the cancer from which the TCR and antigen were identified.

[0188] Example 11

[0189] Generation of Human Memory T cells from a Limited Amount of PBMC

[0190] One goal of the disclosed technology is to generate memory T-cells from a very low amount of blood sample from a subject (such as a cancer patient). This Example describes a method for the simultaneous isolation of monocytes and T cells from 5-20 million PBMCs (from 5-10 ml of blood). Monocytes isolated by a negative selection are important for the optimal development of human DCs.

[0191] EasySep™ Release Human CD3 Positive Selection kit (Stem Cell Technologies catalog #17751) and EasySep™ Human Monocyte Isolation kit (Stem Cell Technologies catalog #19359) were used to achieve the simultaneous isolation of monocytes and T cell. Five million PBMC from colon or pancreatic cancer patients (5 million cells / 0.2 ml) in a well of a 96 well plate were mixed with 20 pl of the antibody selection cocktail of the CD3 Positive Selection kit. After incubation for 3 min, 20 pl of Releasable RapidSpheres™ were added. Then, 10 pl of the Monocyte Isolation Antibody Cocktail (#19359) were added. The plate was placed on the magnet for 5 min, which separated CD3 positive T cells attached on the bottom from the monocytes remaining in the supernatant (called here the first supernatant containing monocytes). After saving the first supernatant, CD3 positive T cells attached to the magnet were washed 3 times. The well was removed from the magnet and incubated with Release Buffer to release CD3 positive T cells from the Releasable RapidSpheres™ Particles. The well was then placed on the magnet for 5 min to separate T cells in the supernatant (the second supernatant) from the Releasable RapidSpheres™ Particle. About 1.2 -1.5 million T cells were recovered. They were frozen in liquid nitrogen until use.

[0192] The first supernatant containing monocytes was negatively separated by the addition of EasySep™ D Magnetic Particles. After incubation for 5 min, the supernatant was the desired human monocytes. About 0.5-1 million monocytes were recovered. They were cultured in a 24 well plate in the presence of GM-CSF and IL-4, resulting in the differentiation into human DCs as described in Example 2.

[0193] Human DCs were loaded with antigens using cancer tissue homogenates. Cancer tissue homogenates were prepared by homogenization of minced tissues with a Dounce homogenizer, followed by the incubation at 55°C for 20 min. The homogenates were filtered through a 70 pm filter and stored at -75°C. Human DCs were loaded with cancer tissue homogenates for 30 min followed by the activation with the addition of LPS. The cells were cultured overnight. Autologous T-cells were then added to antigen-loaded DCs and cultured for at least about 30 days to generate memory T cells as described in Example 2. Surviving T-cells were present, which are likely to be memory T-cells.

[0194] It will be apparent that the precise details of the methods or compositions described may be varied or modified without departing from the spirit of the described aspects of the disclosure. We claim all such modifications and variations that fall within the scope and spirit of the claims below.

Claims

1. We claim:

1. A method of producing memory T cells in vitro, comprising: culturing a population of T cells with a population of antigen-loaded dendritic cells for at least about 28-48 days in a culture medium; and isolating memory T cells from the culture.

2. The method of claim 1 , wherein the population of T cells is a mixed population of T cells.

3. The method of claim 1, wherein the population of T cells comprises CD8+T cells and / or CD4+T cells.

4. The method of claim 1 , wherein the population of T cells substantially lacks memory T cells.

5. The method of claim 4, wherein the population of T cells is negatively selected for CD45RO expression prior to culturing the population of T cells with the antigen-loaded dendritic cells.

6. The method of claim 1 , wherein the culture medium is Iscove’ s modified Dulbecco’ s medium.

7. The method of claim 1, wherein the culture medium includes fetal calf serum.

8. The method of claim 1 , further comprising adding a cytokine to the culture medium at about day 14-18 of the culture.

9. The method of claim 8, wherein the cytokine is interleukin- 15.

10. The method of claim 1, wherein the culture medium does not include additional exogenous growth factors and / or cytokines.

11. The method of claim 1 , wherein at least a portion of the culture medium is replaced every 2-5 days.

12. The method of claim 1, wherein additional cells or antigens are not added to the culture medium during the culturing.

13. The method of claim 1, wherein the T cell population is prepared from whole blood, peripheral blood monocytes, lymphocytes, bone marrow, lymph node tissue, umbilical cord blood, or induced pluripotent stem cells.

14. The method of claim 1, wherein the memory T cells are human memory T cells and express CD3, CD8, and CD45RO or are mouse memory T cells and express CD3, CD8, and CD44.

15. The method of claim 1, wherein the memory T cells are CD4+memory T cells or CD8+ memory T cells.

16. The method of claim 1, wherein the population of antigen- loaded dendritic cells is produced by: culturing monocytes in culture medium comprising fetal calf serum, GM-CSF, and IL-4 to generate dendritic cells; isolating the dendritic cells; adding one or more antigen for a period of time; adding lipopolysaccharide (LPS); and culturing the dendritic cells, antigen, and LPS for 8-18 hours to generate a population of antigen-loaded dendritic cells.

17. The method of claim 1, wherein the population of antigen- loaded dendritic cells is prepared from whole blood, peripheral blood monocytes, lymphocytes, bone marrow, or induced pluripotent stem cells.

18. The method of claim 1, further comprising: activating the memory T cells to produce effector T cells by contacting the memory T cells with the antigen used to produce the antigen-loaded dendritic cells.

19. The method of claim 18, wherein the effector T cells are cytotoxic T cells.

20. A method of treating a subject with cancer, comprising administering to the subject the memory T cells generated by the method of claim 1, wherein the antigen-loaded dendritic cells are loaded with an antigen expressed by the cancer in the subject.

21. A method of treating an infectious disease in a subject, comprising administering to the subject the memory T cells generated by the method of claim 1 , wherein the antigen- loaded dendritic cells are loaded with an antigen expressed by the agent causing the infectious disease.

22. The method of claim 21 , wherein the agent causing the infectious disease is a bacterium, virus, or fungus.

23. The method of claim 22, wherein the bacterium is an antibiotic resistant bacterium.

24. The method of claim 20, wherein the memory T cells are activated to produce effector T cells prior to administering the cells to the subject.

25. The method of claim 20, wherein the population of T cells used to generate the memory T cells is autologous or allogeneic.

26. A method of treating a subject with a tumor, comprising: culturing a population of T cells with a population of dendritic cells loaded with a homogenate prepared from the tumor from the subject for at least about 30 days in a culture medium; isolating memory T cells from the culture; and administering the isolated memory T cells to the subject.

27. The method of claim 26, wherein the memory T cells are CD8+memory T cells and / or CD4+memory T cells.

28. The method of claim 26, further comprising adding a cytokine to the culture medium at about day 14-18 of the culture.

29. The method of claim 28, wherein the cytokine is interleukin- 15.

30. The method of claim 26, further comprising: activating the memory T cells to produce a population of effector T cells; and administering the effector T cells to the subject.

31. The method of claim 30, further comprising: isolating T cells with cytotoxic activity against the tumor from the population of the effector cells; identifying the T cell receptor expressed by the cytotoxic T cells; expressing the identified T cell receptor in T cells; and administering the T cells expressing the identified T cell receptor to the subject with cancer.

32. A method, comprising: producing a population of memory T cells recognizing an antigen according to the method of claim 1 ; and identifying the T cell receptor expressed by the memory T cells.

33. The method of claim 32, wherein the population of antigen-loaded dendritic cells cultured with the T cells is loaded with a sample comprising a mixture of antigens.

34. The method of claim 33, wherein the sample comprising a mixture of antigens is a tumor sample or a pathogen sample.

35. The method of claim 32, further comprising activating the population of memory T cells to produce a population of effector T cells and identifying the T cell receptor expressed by the effector T cells.

36. The method of claim 35, wherein activating the memory T cells to produce effector T cells comprises contacting the memory T cells with the sample used to produce the antigen- loaded dendritic cells.

37. The method of claim 36, wherein the effector T cells are CD8+and / or CD4+T cells.

38. The method of claim 32, wherein identifying the T cell receptor expressed by the memory T cells or effector T cells comprises single cell sequencing.

39. The method of claim 32, further comprising identifying the antigen specifically bound by the identified T cell receptor.

40. The method of claim 39, further comprising administering the identified antigen to a subject in need thereof.

41. The method of claim 40, wherein the antigen is conjugated to an adjuvant.

42. The method of claim 41 , further comprising producing an antibody that specifically binds the identified antigen.

43. The method of claim 42, further comprising administering the antibody or a chimeric antigen receptor comprising the antibody or a fragment thereof to a subject in need thereof.

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