WO2025072081A1 - Enriched t-cell populations - Google Patents

Abstract

Provided herein are systems, kits, and methods for generating an enriched T cell population from an initial peripheral blood mononuclear cell (PBMC) population using at least two types of cell-binding reagents: (e.g., particles conjugated to CD32, CD19, CD244, or CD25 binding agents), where the enriched T cell population is: i) enriched for desired T-cells (e.g., early memory T cells and naïve T cells), and ii) depleted in normal and malignant non-desired cells selected from: CD25hi regulatory T-cells (Tregs), CD25hi CLL cells, CD244 T-cells, CD32+ monocytes, CD32+ myeloid leukemia cells, CD32 basophils, CD19+ and/or CD32+ B cells, CD244+ natural killer (NK) cells, and myeloid cells). In certain embodiments, the enriched T cell populations are used for generating a population of chimeric antigen receptor (CAR) T-cells, T-cell receptor-engineered T cells, or Tumor-infiltrating T lymphocyte (ITL) products.

Description

ENRICHED T-CELL POPULATIONS

The present application claims priority to U.S. Provisional application serial number 63/585,098, filed September 25, 2023, which is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

Provided herein are systems, kits, and methods for generating an enriched T cell population from an initial peripheral blood mononuclear cell (PBMC) population using at least two types of cell-binding reagents: (e.g., particles conjugated to CD32, CD19, CD244, or CD25 binding agents), where the enriched T cell population is: i) enriched for desired T- cells (e.g., early memory T cells and naive T cells), and ii) depleted in normal and malignant non-desired cells selected from: CD25hi regulatory T-cells (Tregs), CD25hi CLL cells, CD244 T-cells, CD32+ monocytes, CD32+ myeloid leukemia cells, CD32 basophils, CD19+ and/or CD32+ B cells, CD244+ natural killer (NK) cells, and myeloid cells). In certain embodiments, the enriched T cell populations are used for generating a population of chimeric antigen receptor (CAR) T-cells, T-cell receptor-engineered T cells, or Tumorinfiltrating T lymphocyte (ITL) products.

BACKGROUND

Chimeric Antigen Receptor (CAR) T-cell therapy can induce complete remissions in B cell leukemias and lymphoma (anti-CD19 CAR T-cells), as well as multiple myeloma (anti-BCMA CAR T-cells), but a significant proportion of patients will not respond or ultimately relapse, often because of the loss of functional CAR T cells. Moreover, CARbased cell therapies have been evaluated in other diseases acute myeloid leukemia (AML) and patients with solid tumors such as breast cancer, glioblastoma, and prostate cancer, but results have been less than satisfactory thus far. Disease-related factors can contribute to the success or failure of CAR-T cell therapy, but T cell-intrinsic factors, identified in CAR- engineered and their apheresed precursors, substantially impact the clinical efficacy of CAR- T cell therapy. Several correlative and mechanistic studies in different hematological malignancies have established that the early expansion of CAR T cells is a key correlate of response, and that memory function is required for persistence and durable remission (Fraietta Nat Med 2018, Deng Nat Med 2020, Bai Sci Adv 2022). Moreover, a higher proportion of regulatory T cells (Treg) has also been associated with a lower response rate (Good Nat Med 2022). In patients with hematological malignancies, the balance of different T cell subsets can be disrupted due to factors such as the disease itself, prior treatments, and age. This imbalance often leads to a reduced proportion of naive and early memory T cells, while favoring a more differentiated and frequently exhausted T cell population. Consequently, this imbalance is reflected in the apheresis product and, ultimately, in the manufactured CAR T cell product.

SUMMARY OF THE INVENTION

Provided herein are systems, kits, and methods for generating an enriched T cell population from an initial peripheral blood mononuclear cell (PBMC) population using at least two types of cell-binding reagents: (e.g., particles conjugated to CD32, CD19, CD244, or CD25 binding agents), where the enriched T cell population is: i) enriched for desired T- cells (e.g., early memory T cells and naive T cells), and ii) depleted in normal and malignant non-desired cells selected from: CD25hi regulatory T-cells (Tregs), CD25hi CLL cells, CD244 T-cells, CD32+ monocytes, CD32+ myeloid leukemia cells, CD32 basophils, CD19+ and/or CD32+ B cells, CD244+ natural killer (NK) cells, and myeloid cells). In certain embodiments, the enriched T cell populations are used for generating a population of chimeric antigen receptor (CAR) T-cells, T-cell receptor-engineered T cells, or Tumorinfiltrating T lymphocyte (ITL) products.

In some embodiments, provided herein are methods of generating an enriched cell population comprising: a) contacting an initial cell population comprising peripheral blood mononuclear cells (PBMCs) with at least two types of cell binding reagents, wherein said PBMCs comprise: i) desired T cells selected from early memory T cells and naive T-cells and ii) normal and malignant non-desired cells selected from: CD25hi regulatory T-cells (Tregs), CD25hi CLL cells, CD244 T-cells, CD32+ monocytes, CD32+ myeloid leukemia cells, CD32 basophils, CD19+ and/or CD32+ B cells, CD244+ natural killer (NK) cells, and myeloid cells, and wherein said at least two types of cell binding reagents each comprise: i) a different cell-surface binding agent selected from: a CD32 binding agent, a CD 19 binding agent, a CD244 binding agent, and a low-titer CD25 binding agent, and ii) a conjugate partner selected from a label, particle, or solid surface, and b) separating, from said initial cell population, said at least two types of cell binding conjugates that are bound to said non-desired cells, thereby generating an enriched cell population that is: i) enriched for said desired T-cells, and ii) depleted in said non-desired cells. In certain embodiments, the PBMCs are human PBMCs obtained from a human.

In some embodiments, provided herein are methods comprising: a) obtaining an isolated cell population comprising at least 1,000, or at least 10,000, peripheral blood mononuclear cells (PBMCs), wherein: i) at least at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of all of said PBMCs present are early memory T cells and/or naive T- cells, and ii) less than 5%, or less than 1 %, of all of said PBMCs present are non-desired cells which are CD25hi regulatory T-cells (Tregs), CD25hi CLL cells, CD244 T-cells, CD32+ monocytes, CD32+ myeloid leukemia cells, CD32 basophils, CD19+ and/or CD32+ B cells, CD244+ natural killer (NK) cells, and myeloid cells; b) contacting said enriched cell population with a stimulating agent to cause the activation of desired T cells thereby generating an expanded T cell population; c) culturing said expanded T cell population in media to generate a cultured enriched cell population; and d) transfecting or transducing said cultured enriched cell population with: i) a gene expression vector encoding a chimeric antigen receptor (CAR) thereby generating a population of CAR-T cells, or ii) an mRNA sequence that encodes said CAR; or iii) contacting said cultured enriched cell population with a disease relevant antigen presented by an antigen-presenting cell to generated a population of antigen-specific T-cells. In particular embodiments, the methods further comprise: e) administering at least a portion of said population of CAR-T cells, or the population of activated T-cells, to a subject, wherein said subject optionally has cancer, and optionally wherein said cancer is myeloid cancer.

In particular embodiments, the initial cell population comprises: an apheresis product, a non-apheresis peripheral blood collection sample, a bone marrow sample, or a thoracentesis product from a subject. In certain embodiments, the subject is a human (e.g., a human with cancer or other disease). In some embodiments, the at least two types of cell binding conjugates comprises at least three types of cell binding conjugates. In other embodiments, the at least two types of cell binding conjugates comprises all four types of cell binding conjugates.

In additional embodiments, at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.9% of the T-cells in the enriched cell population are the desired T-cells. In other embodiments, less than 5%, or less than 1%, or less than 0.5%, or less than 0.1% of the PBMCs in the enriched cell population are the non-desired cells. In other embodiments, the enriched population is detectably free of macrophages.

In further embodiments, the cell-surface binding agent comprises an antigen-binding agent (e.g., that is labelled) selected from: an antibody, an antigen-binding fragment thereof, a fragment antigen-binding (Fab), two linked Fab fragments F(ab’)2, a single chain variable fragment, a single domain antibody, an affibody, an adnectin, a protein or peptide ligand, or an immunoadhesin. In additional embodiments, the cell- surface binding agent comprises a nanobody or antigen-binding fragment thereof. In other embodiments, the cell-surface binding agent comprises an aptamer or affimer.

In certain embodiments, the at least two types of cell binding conjugates include a first cell binding conjugate that comprises a CD32 binding agent and a second cell binding conjugate that comprises a C19 binding agent. In additional embodiments, the at least two types of cell binding conjugates include a first cell binding conjugate that comprises a CD244 binding agent and a second cell binding conjugate that comprises a low-titer CD25 binding agent. In other embodiments, the separating is performed by a method selected from: magnetic separation, bubble separation, and acoustic sorting.

In some embodiments, the conjugate partner comprises the particle, and optionally the particle comprises a bead or nanoparticle. In further embodiments, the separating comprises magnetic separation of the particle from the initial cell population. In additional embodiments, the separating comprises binding the particle to a surface via a first binding partner on the particle and a corresponding binding partner on the particle. In other embodiments, the first binding partner comprises biotin or avidin, and the corresponding binding partner comprise avidin or biotin. In certain embodiments, the conjugate partner comprises the label, and optionally the label comprises a fluorescent label. In some embodiments, the separating comprises fluorescence-activated cell sorting (FACS).

In further embodiments, the methods further comprise: c) contacting the enriched cell population with a stimulating agent to cause the activation of desired T cells. In particular embodiments, wherein: i) the stimulating agent comprises an agonistic agent selected from: antibodies specific for the T cell receptor, CD3, CD2, CD28, 4-1BB, and ICOS; and/or ii) wherein the enriched cell population is present in a culture medium, and optionally wherein the culture medium is supplemented with at least one cytokine selected from: interleukin-4 (IL-4), IL-5, IL-6, IL-7, IL-10, IL-15, IL-21 , and IL- 22. In additional embodiments, the methods further comprise: culturing the expanded T cell population in media comprising at least one of the following base media: RPMI 1640, X VIVO-15, AIM-V, OpTimizer, ImmunoCult, supplemented with human serum, AB serum, serum substitute, or a combination of serum and serum substitute. In other embodiments, the methods further comprise: d) transfecting or transducing the cultured enriched cell population with: i) a gene expression vector, or other nucleic acid expression vector, encoding a chimeric antigen receptor (CAR) thereby generating a population of CAR-T cells, or ii) an mRNA sequence that encodes the CAR; or d) contacting said cultured enriched cell population with a disease relevant antigen presented by an antigen-presenting cell to generated a population of activated T-cells. In particular embodiments, the gene acid expression vector is a retroviral vector which is optionally an integrating gammaretrovirus (RV) or a lentiviral (LV) vector. In some embodiments, the methods further comprise: culturing the population of CAR-T cells in media comprising at least one of the following: RPMI 1640, AIM-V, Optimizer, ImmunoCult, human serum, AB serum, serum substitute, or a combination of serum and serum substitute.

In certain embodiments, the methods further comprise: administering at least a portion of the population of CAR-T cells (or population of antigen activated T-cells) to a subject, and optionally wherein the subject has cancer; is the source of the initial cell population; or wherein the subject has an autoimmune disease; or wherein the subject has a condition manageable using CAR-engineered T cells (or antigen activated T-cells) selected from: graft versus host disease, graft rejection, and cardiac malfunction. In other embodiments, the cancer comprises a hematological or non-hematological cancer, lymphoid cancer, or myeloid cancer.

In particular embodiments, provided herein are compositions comprising: a) a carrier liquid and/or culture media, and b) an isolated cell population comprising at least 1,000, or at least 10,000, peripheral blood mononuclear cells (PBMCs), wherein: i) at least at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of all of said PBMCs present are early memory T cells and/or naive T-cells, and ii) less than 5%, or less than 1%, of all of said PBMCs present are non-desired cells which are CD25hi regulatory T-cells (Tregs), CD25hi CLL cells, CD244 T-cells, CD32+ monocytes, CD32+ myeloid leukemia cells, CD32 basophils, CD 19+ and/or CD32+ B cells, CD244+ natural killer (NK) cells, and myeloid cells. In some embodiments, the isolated cell population is detectably free of macrophages.

In some embodiments, most or all of the early memory T cells and/or naive T-cells are antigen activated T-cells, or are CAR-T cells that comprise: i) a gene expression vector (or other nucleic acid vector) encoding a chimeric antigen receptor, or ii) an exogenous mRNA encoding the chimeric antigen receptor. In some embodiments, the carrier fluid comprises an IV solution and/or a buffer. In other embodiments, the culture media comprises at least one of the following: RPMI 1640, AIM-V, Optimizer, ImmunoCult, human serum, AB serum, serum substitute, or a combination of serum and serum substitute. In particular embodiments, the less than 1% is less than 0.1%.

In certain embodiments, provides herein are systems and kits comprising at least three types of cell binding conjugates which each comprise: a) a different cell-surface binding agent selected from: a CD32 binding agent, a CD 19 binding agent, a CD244 binding agent, and a low-titer CD25 binding agent, and b) a conjugate partner selected from a label, particle, or solid surface. In particular embodiments, the at least three types of cell binding conjugates comprises at least four types of cells binding conjugates. In other embodiments, the cellsurface binding agent comprises an antibody antigen-binding fragment thereof. In additional embodiments, the cell-surface binding agent comprises a nanobody or antigen-binding fragment thereof. In further embodiments, the cell-surface binding agent comprises an aptamer or affimer. In some embodiments, the conjugate partner comprises the particle, and optionally the particle comprises a bead or nanoparticle. In additional embodiments, the conjugate partner comprises the label, and optionally the label comprises a fluorescent label.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Flow cytometry analysis of the phenotype of lymphocytes from healthy donors and CLL patients (data depicted in UMAP displays).

Figure 2. Schematic representation of an exemplary depletion process. This exemplary process allows the selective depletion of monocytes (CD32+), B cells (CD19+, CD32+), and some NK cells (CD244+), as well as Treg (CD25hi) and exhausted T cells (CD244+), while leaving untouched (e.g., not linked to any antibody) the highly functional T cell population (e.g., that may used for CAR T cell manufacturing).

Figure 3 A. Example of PBMC depletion (from a healthy donor) using either the pan T cell isolation kit, or an exemplary CD32/19/25/244 depletion process of the present disclosure (data depicted in UMAP displays). Figure 3B shows an example of PBMC depletion and selection (from a healthy donor) using either CD4/CD8 selection or an exemplary CD32/19/244 depletion process of the present disclosure.

Figure 4. Flow cytometry analysis of the phenotype of lymphocytes after the exemplary depletion process (data depicted in UMAP displays; pooled data from 2 healthy donors).

Figure 5. Comparison of cell proliferation during CAR T cell manufacturing (cells from the same healthy donor, 3 replicates with different culture conditions).

Figure 6A. Comparison of the T cell phenotype after 9 days of CAR T cell manufacturing (cells from the same healthy donor, 6 replicates with different culture conditions) when either the pan T cell isolation kit, or an exemplary CD32/ 19/25/244 depletion process of the present disclosure are used. Figure 6B shows a comparison of the T cell phenotype after 9 days of CAR T cell manufacturing (cells from the same healthy donor, 6 replicates with different culture conditions) when either CD4/CD8 selection or an exemplary CD32/19/244 depletion process of the present disclosure are used.. DEFINITIONS

The terms "individual," "host," "subject," and "patient" are used interchangeably herein, and generally refer to a mammal, including, but not limited to, primates, including simians and humans, equines (e.g., horses), canines (e.g., dogs), felines, various domesticated livestock (e.g., ungulates, such as swine, pigs, goats, sheep, and the like), as well as domesticated pets and animals maintained in zoos. In some embodiments, the subject is specifically a human subject.

The term “transfection” as used herein refers to the introduction of foreign DNA or RNA into eukaryotic cells. Transfection may be accomplished by a variety of means known to the art including, for example, calcium phosphate-DNA co-precipitation, DEAE-dextran- mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection, and biolistics.

DETAILED DESCRIPTION OF THE INVENTION

Provided herein are systems, kits, and methods for generating an enriched T cell population from an initial peripheral blood mononuclear cell (PBMC) population using at least two types of cell-binding reagents: (e.g., particles conjugated to CD32, CD19, CD244, or CD25 binding agents), where the enriched T cell population is: i) enriched for desired T- cells (e.g., early memory T cells and naive T cells), and ii) depleted in normal and malignant non-desired cells selected from: CD25hi regulatory T-cells (Tregs), CD25hi CLL cells, CD244 T-cells, CD32+ monocytes, CD32+ myeloid leukemia cells, CD32 basophils, CD19+ and/or CD32+ B cells, CD244+ natural killer (NK) cells, and myeloid cells). In certain embodiments, the enriched T cell populations are used for generating a population of chimeric antigen receptor (CAR) T-cells, T-cell receptor-engineered T cells, or Tumorinfiltrating T lymphocyte (ITL) products.

In certain embodiments, provided herein are methods of using a unique cocktail of CD32/CD19/CD244/CD25 antibodies for negative selection of the target population. The enrichment methods herein allow, for example, enrichment of highly functional T cells (naive and early memory CD8 T cells), and depletion of Treg. Further, the negative selection herein allows, in some embodiments, the selection of an untouched T cell population (e.g., no antibody linked to the selected cells) for CAR T cell manufacturing.

In patients with hematological malignancies, the balance of different T cell subsets can be disrupted due to factors such as the disease itself, prior treatments, and age. This imbalance often leads to a reduced proportion of naive and early memory T cells, while favoring a more differentiated and frequently exhausted T cell population. Consequently, this imbalance is reflected in the apheresis product and, ultimately, in the manufactured CAR T cell product. The methods, compositions, and kits herein allow an enhancement of such manufacturing processes to enrich the final product with the cells responsible for efficacy, particularly the early memory T cells, while depleting those associated with lower efficiency, such as regulatory T (Treg) cells. In other embodiments, provided herein is a practical approach to eliminate non-T cells expressing the Fc receptor CD32. In certain embodiments, such as in cases where the product contains a high abundance of B cells, the methods herein can also remove these cells through CD19 depletion.

In work conducted during the development of embodiments herein, we observed that the more differentiated/exhausted CD8+ cells broadly express CD244 on their surface, while naive and early memory CD8+ cells, as well as most CD4+ cells, do not (Figure 1). Moreover, CD25 is expressed on activated cells, but only Treg have a very high CD25 expression (allowing a selective depletion of Treg by using low titer of CD25 antibody or other low titer CD25 binding agent) (Figure 1). Therefore, embodiments of the present disclosure were developed to enrich the T cell product in early memory T cells and deplete Tregs, using a T cell negative selection (e.g., using magnetic cell separation or other suitable art known methods). For example, starting from the apheresis product (peripheral blood mononuclear cells, or PBMC), in exemplary embodiments, we used CD32, CD19, CD244 and low titer of CD25 antibodies coupled to magnetic beads to deplete PBMC from unwanted cells (monocytes, B cells, NK cells, exhausted T cells, regulatory T cells), therefore only keeping the most functional T cells, which are untouched (not linked to any antibody or other binding agent) (Figure 2). These selected T cells can then, for example, be manufactured in CAR T cells using a manufacturing process with limited T cell activation, to avoid inducting exhaustion. In case of circulating tumor cells, other antibodies (or other binding agents) may be added to the cocktail to ensure that we completely remove all tumor cells.

In general, in the art known methods for T cell selection for commercial CAR T cell manufacturing, all T cells are isolated from the apheresis product with anti-CD4 and with anti-CD8 magnetic beads, including exhausted T cells and regulatory T cells. These cells have been associated in correlative studies with lack of efficacy of CAR T cell therapy. Therefore, removing these cells from the apheresis product prior to CAR T cell manufacturing (or other uses) is expected to improve efficacy. Moreover, the proportion of these subsets of cells are highly variable amongst patients, and this party explains the heterogeneity of the results in the clinical setting. This enrichment in early memory T cells with the methods herein results in a product that has a more conserved composition between patients, which is expected to result in more predictable results. Other groups in the art have developed process to select subsets of T cells using magnetic cell separation. However, they either use a positive selection (selecting CD62L+ cells, which results in an enrichment in other cell types expressing CD62L, including monocytes, and in a final T cell population used for manufacturing that is linked to the antibody used for selection) (clinical trials, Wang Blood 2016, Aldoss Clin Cancer Res 2022, Larson Cancer Discov 2023), or select only naive T cells (by a negative selection, using the commercial Miltenyi kit “human naive pan T cell isolation kit”), therefore removing all other cells, including early memory T cells (preclinical study, Meyran Sci Trans! Med 2023). This might impair the efficacy, and also the feasibility of the process, since many patients have very low numbers of naive T cells. The methods, kits, and compositions herein allow, in certain embodiments, the selective depletion of monocytes and monocytic leukemia cells (CD32+), B cells (CD19+, CD32+), and some NK cells (CD244+), as well as Treg (CD25hi) and exhausted T cells (CD244+), while leaving untouched (e.g., not linked to bind to any of the mentioned binding agent) the highly functional T cell population (e.g., that can be used for CAR T cell manufacturing).

Exemplary work was conducted during development of embodiments of the present disclosure. For example, we validated the feasibility of an exemplary process on normal donor cells, yielding a similar T cell purity as the commercial T cell isolation kit (Miltenyi) (Figure 3). We also confirmed, by analyzing the phenotype of the T cells before (PBMC) and after the depletion (negative fraction), that such methods significantly enriched for central memory T cells (by depleting effector memory and effector cells) and successfully depleted Treg (Figure 4).

In other exemplary work conducted during the development of embodiments of the present disclosure, we confirmed that CAR T cell could be successfully manufactured from T cells selected by the methods herein, and that the proliferation of this population during a 9- day manufacturing process was similar to the proliferation of the whole population of T cell (isolated using the T cell isolation kit) (Figure 5). Transduction efficiency was acceptable, though slightly lower in the cells selected by our depletion process versus the standard isolation process. We also observed that the proportion of CD244+ cells among CD8+ cells was still lower after 9 days of in vitro culture. Similarly, the enrichment in early memory (CD45RO-CD27+) cells was preserved among the CD8+ T cells, and the Treg subset was still lower among the CD4+ T cells (Figure 6).

In some embodiments, the enriched T cells are anti-tumor T cells (e.g., T cells with activity against a tumor (e.g., an autologous tumor) that become activated and expand in response to antigen). Anti-tumor T cells (e.g., useful for adoptive T cell transfer) include, in one embodiment, peripheral blood derived T cells genetically modified with receptors that recognize and respond to tumor antigens. Such receptors are generally composed of extracellular domains comprising a single-chain antibody (scFv) specific for tumor antigen, linked to intracellular T cell signaling motifs (See, e.g., Westwood, J. A. et al, 2005, Proc. Natl. Acad. Sci., USA, 102(52):19051-19056). Other anti-tumor T cells include T cells obtained from resected tumors or tumor biopsies (e.g., tumor infiltrating lymphocytes (TILs). In another embodiment, the T cell is a polyclonal or monoclonal tumor-reactive T cell (e.g., obtained by apheresis, expanded ex vivo against tumor antigens presented by autologous or artificial antigen-presenting cells). In another embodiment, the enriched T cells are engineered to express a T cell receptor of human or murine origin that recognizes a tumor antigen. The invention is not limited by the type of tumor antigen so recognized. Indeed, any T cell containing a receptor that recognizes a tumor antigen finds use in the compositions and methods of the invention. Examples include, but are not limited to, T cells expressing a receptor (e.g., a native or naturally occurring receptor, or a receptor engineered to express a synthetic receptor such as an engineered T cell receptor or a CAR) that recognize an antigen selected from CD19, CD20, CD22, receptor tyrosine kinase-like orphan receptor 1 (ROR1), disialoganglioside 2 (GD2), Epstein-Barr Virus (EBV) protein or antigen, folate receptor, mesothelin, human carcinoembryonic antigen (CEA), CD33/IL3Ra, tyrosine protein kinase Met (c-Met) or hepatocyte growth factor receptor (HGFR), prostate-specific membrane antigen (PSMA), Glycolipid F77, epidermal growth factor receptor variant III (EGFRvIII), NY-ESO-1, melanoma antigen gene (MAGE) Family Member A3 (MAGE-A3), melanoma antigen recognized by T cells 1 (MART-1), GP1000, p53, or other tumor antigen described herein.

In some embodiments, the enriched T cells are engineered to express a CAR. The invention is not limited by the type CAR. Indeed, any CAR that binds with specificity to a desired antigen (e.g., tumor antigen) may be employed. In certain embodiments, the CAR comprises an antigen-binding domain. In certain embodiments, the antigen-binding domain is a single-chain variable fragment (scFv) containing heavy and light chain variable regions that bind with specificity to the desired antigen. In some embodiments, the CAR further comprises a transmembrane domain (e.g., a T cell transmembrane domain (e.g., a CD28 transmembrane domain)) and a signaling domain comprising one or more immunoreceptor tyrosine-based activation motifs (IT AMs) (e.g., a T cell receptor signaling domain (e.g., TCR zeta chain). In some embodiments, the CAR comprises one or more co- stimulatory domains (e.g., domains that provide a second signal to stimulate T cell activation). The invention is not limited by the type of co- stimulatory domain. Indeed, any co-stimulatory domain known in the art may be used including, but not limited to, CD28, OX40/CD134, 4- 1BB/CD137/TNFRSF9, the high affinity immunoglobulin E receptor-gamma subunit (FcERIy, ICOS/CD278, interleukin 2 subunit beta (ILR[3) or CD122, cytokine receptor common subunit gamma (IL-2Ry) or CD 132, and CD40. In one embodiment, the costimulatory domain is 4- IBB.

The CAR may be engineered to target a tumor antigen of interest by way of engineering a desired antigen binding moiety that specifically binds to an antigen on a tumor cell. As used herein, a “tumor antigen” or “hyperproliferative disorder antigen” or “antigen associated with a hyperproliferative disorder” or “cancer antigen,” refers to antigens that are common to specific hyperproliferative disorders such as cancer. Exemplary antigens mentioned herein are included by way of example. Tumor antigens are proteins that are produced by tumor cells that elicit an immune response, particularly T-cell mediated immune responses. Thus, an antigen binding moiety can be selected based on the particular type of cancer to be treated. Tumor antigens are well known in the art and include, for example, a glioma-associated antigen, carcinoembryonic antigen (CEA), beta-human chorionic gonadotropin, alphafetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, prostein, PSMA, Her2/neu, survivin and telomerase, prostate-carcinoma tumor antigen- 1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrinB2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF-I receptor and mesothelin.

A tumor antigen may comprise one or more antigenic cancer antigens/epitopes associated with a malignant tumor. Malignant tumors express a number of proteins that can serve as target antigens for an immune attack. These molecules include but are not limited to tissue-specific antigens such as MART-1, tyrosinase and GP100 in melanoma and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-related molecules such as the oncogene HER-2/Neu/ErbB-2. Still another group of target antigens are onco-fetal antigens such as carcinoembryonic antigen (CEA). In B-cell lymphoma the tumor- specific idiotype immunoglobulin constitutes a truly tumor-specific immunoglobulin antigen that is unique to the individual tumor. B-cell differentiation antigens such as CD19, CD20 and CD37 are other candidates for target antigens in B-cell lymphoma.

The tumor antigen may also be a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). A TSA is unique to tumor cells and does not occur on other cells in the body. A TAA is not unique to a tumor cell and instead is also expressed on some normal cells under conditions that fail to induce a state of immunologic tolerance to the antigen. The expression of the antigen on the tumor may occur under conditions that enable the immune system to respond to the antigen. TA As may be antigens that are expressed on normal cells during fetal development when the immune system is immature and unable to respond or they may be antigens that are normally present at extremely low levels on normal cells but which are expressed at much higher levels on tumor cells. Examples of TSA or TAA include, but are not limited to, differentiation antigens such as MART-l/MelanA (MART-1), gplOO (Pmel 17), tyrosinase, TRP-1 , TRP-2 and tumor-specific multilineage antigens such as MAGE-1 , MAGE-3, BAGE, GAGE-1, GAGE-2, pl5; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutated tumor- suppressor genes such as p53, Ras, HER-2/neu; unique tumor antigens resulting from chromosomal translocations; such as BCR-ABL, E2A- PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as the Epstein Barr virus antigens EBVA and the human papillomavirus (HPV) antigens E6 and E7. Other large, protein-based antigens include TSP- 180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO-1, pl85erbB2, pl80erbB-3, c-met, nm-23Hl, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alphafetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3\CA 27.291\BCAA, CA 195, CA 242, CA-50, CAM43, CD68\P1, CO-029, FGF-5, G250, Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV 18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP, and TPS.

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8. Wang et al., (2016) Phase 1 studies of central memory-derived CD19 CAR T-cell therapy following autologous HSCT in patients with B-cell NHL. Blood, 127, 2980-90.

Although only a number exemplary embodiments have been described in detail, those skilled in the art will readily appreciate that many modifications are possible in the exemplary embodiments without materially departing from the novel teachings and advantages of this disclosure. Accordingly, all such modifications and alternative are intended to be included within the scope of the invention as defined in the following claims. Those skilled in the art should also realize that such modifications and equivalent constructions or methods do not depart from the spirit and scope of the present disclosure, and that they may make various changes, substitutions, and alterations herein without departing from the spirit and scope of the present disclosure.

Claims

We claim:

1. A method of generating an enriched cell population comprising: a) contacting an initial cell population comprising peripheral blood mononuclear cells (PBMCs) with at least two types of cell binding reagents, wherein said PBMCs comprise: i) desired T cells selected from early memory T cells and naive T-cells and ii) normal and malignant non-desired cells selected from: CD25hi regulatory T-cells (Tregs), CD25hi CLL cells, CD244 T-cells, CD32+ monocytes, CD32+ myeloid leukemia cells, CD32 basophils, CD 19+ and/or CD32+ B cells, CD244+ natural killer (NK) cells, and myeloid cells, and wherein said at least two types of cell binding reagents each comprise: i) a different cell-surface binding agent selected from: a CD32 binding agent, a CD 19 binding agent, a CD244 binding agent, and a low-titer CD25 binding agent, and ii) a conjugate partner selected from a label, particle, or solid surface, and b) separating, from said initial cell population, said at least two types of cell binding conjugates that are bound to said non-desired cells, thereby generating an enriched cell population that is: i) enriched for said desired T-cells, and ii) depleted in said non-desired cells.

2. The method of claim 1, wherein said initial cell population comprises: an apheresis product, a non-apheresis peripheral blood collection sample, a bone marrow sample, or a thoracentesis product from a subject.

3. The method of claim 2, wherein said subject is a human.

4. The method of claim 1, wherein said at least two types of cell binding conjugates comprises at least three, or at least four, types of cell binding conjugates.

5. The method of claim 1, wherein said separating is performed by a method selected from: magnetic separation, bubble separation, and acoustic sorting.

6. The method of claim 1, wherein at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the T-cells in said enriched cell population are said desired T-cells.

7. The method of claim 1, wherein less than 5%, or less than 1%, of said PBMCs in said enriched cell population are said non-desired cells, and/or wherein said enriched population is detectably free of macrophages.

8. The method of claim 1 , wherein said cell-surface binding agent comprises an antigenbinding agent selected from: an antibody, an antigen-binding fragment thereof, a fragment antigen-binding (Fab), two linked Fab fragments F(ab’)2, a single chain variable fragment, a single domain antibody, an aflibody, an adnectin, a protein or peptide ligand, or an immunoadhesin.

9. The method of claim 1 , wherein said cell-surface binding agent comprises a nanobody or antigen-binding fragment thereof.

10. The method of claim 1, wherein said cell-surface binding agent comprises an aptamer or affimer.

11. The method of claim 1 , wherein said at least two types of cell binding conjugates include a first cell binding conjugate that comprises a CD32 binding agent and a second cell binding conjugate that comprises a C19 binding agent.

12. The method of claim 1, wherein said at least two types of cell binding conjugates include a first cell binding conjugate that comprises a CD244 binding agent and a second cell binding conjugate that comprises a low-titer CD25 binding agent.

13. The method of claim 1, wherein said conjugate partner comprises said particle, and optionally said particle comprises a bead or nanoparticle.

14. The method of claim 13, wherein said separating comprises magnetic separation of said particle from said initial cell population.

15. The method of claim 13, wherein said separating comprises binding said particle to a surface via a first binding partner on said particle and a corresponding binding partner on said particle.

16. The method of claim 15, wherein said first binding partner comprises biotin or avidin, and said corresponding binding partner comprise avidin or biotin.

17. The method of claim 1 , wherein said conjugate partner comprises said label, and optionally said label comprises a fluorescent label.

18. The method of claim 17, wherein said separating comprises fluorescence-activated cell sorting (FACS).

19. The method of claim 1, further comprising: c) contacting said enriched cell population with a stimulating agent to cause the activation of desired T cells.

20. The method of claim 19, wherein: i) said stimulating agent comprises an agonistic agent selected from: antibodies specific for the T cell receptor, CD3, CD2, CD28, 4-1BB, and ICOS; and/or ii) wherein said enriched cell population is present in a culture medium, and optionally wherein said culture medium is supplemented with at least one cytokine selected from: interleukin-4 (IL-4), IL-5, IL-6, IL-7, IL-10, IL-15, IL-21, and IL-22.

21. The method of claim 19, further comprising: culturing said expanded T cell population in media comprising at least one of the following: RPMI 1640, AIM-V, Optimizer, ImmunoCult, human serum, X VIVO- 15, AB serum, serum substitute, or a combination of serum and serum substitute.

22. The method of claim 19, further comprising: d) transfecting or transducing said cultured enriched cell population with: i) a gene expression vector encoding a chimeric antigen receptor (CAR) thereby generating a population of CAR-T cells, or ii) an mRNA sequence that encodes said CAR; or d) contacting said cultured enriched cell population with a disease relevant antigen presented by an antigen-presenting cell to generated a population of antigen-specific T-cells.

23. The method of claim 22, said gene acid expression vector is a retroviral vector which is optionally an integrating gammaretrovirus (RV) or a lentiviral (LV) vector.

24. The method of claim 22, further comprising culturing said population of CAR-T cells or antigen-specific T cells in media comprising at least one of the following: RPMI 1640, AIM-V, Optimizer, ImmunoCult, X- VIVO- 15, human serum, AB serum, serum substitute, or a combination of serum and serum substitute.

25. The method of claim 22, further comprising: e) administering at least a portion of said population of CAR-T cells, or the population of activated T-cells, to a subject, and optionally wherein said subject has cancer; is the source of the initial cell population; or wherein said subject has an autoimmune disease; or wherein said subject has a condition manageable using CAR-engineered T cells selected from: graft versus host disease, graft rejection, and cardiac malfunction.

26. The method of claim 25, wherein said cancer comprises a hematological or non- hematological cancer, lymphoid cancer, or myeloid cancer.

27. A composition comprising: a) a carrier liquid and/or culture media, and b) an isolated cell population comprising at least 1,000, or at least 10,000, peripheral blood mononuclear cells (PBMCs), wherein: i) at least at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of all of said PBMCs present are early memory T cells and/or naive T-cells, and ii) less than 5%, or less than 1%, of all of said PBMCs present are nondesired cells which are CD25hi regulatory T-cells (Tregs), CD25hi CLL cells, CD244

T-cells, CD32+ monocytes, CD32+ myeloid leukemia cells, CD32 basophils, CD19+ and/or CD32+ B cells, CD244+ natural killer (NK) cells, and myeloid cells.

28. The composition of claim 27, wherein most or all of said early memory T cells and/or naive T-cells are antigen-specific T-cells, or are CAR-T cells that comprise: i) a gene expression vector encoding a chimeric antigen receptor, or ii) an exogenous mRNA encoding said chimeric antigen receptor.

29. The composition of claim 27, wherein said carrier fluid comprises an IV solution and/or a buffer.

30. The composition of claim 27, wherein said culture media comprises at least one of the following: RPMI 1640, AIM-V, Optimizer, ImmunoCult, human serum, AB serum, serum substitute, or a combination of serum and serum substitute. 1 . The composition of claim 27, wherein said 99% is at least 99.9%.

32. The composition of claim 27, wherein said less than 1% is less than 0.1%.

33. The composition of claim 27, wherein said isolated cell population is detectably free of macrophages.

34. A method comprising: a) obtaining an isolated cell population comprising at least 1 ,000, or at least 10,000, peripheral blood mononuclear cells (PBMCs), wherein: i) at least at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of all of said PBMCs present are early memory T cells and/or naive T-cells, and ii) less than 5%, or less than 1%, of all of said PBMCs present are nondesired cells which are CD25hi regulatory T-cells (Tregs), CD25hi CLL cells, CD244

T-cells, CD32+ monocytes, CD32+ myeloid leukemia cells, CD32 basophils, CD19+ and/or CD32+ B cells, CD244+ natural killer (NK) cells, and myeloid cells, and b) contacting said enriched cell population with a stimulating agent to cause the activation of desired T cells thereby generating an expanded T cell population; c) culturing said expanded T cell population in media to generate a cultured enriched cell population; and d) transfecting or transducing said cultured enriched cell population with: i) a gene expression vector encoding a chimeric antigen receptor (CAR) thereby generating a population of CAR-T cells, or ii) an mRNA sequence that encodes said CAR; or iii) contacting said cultured enriched cell population with a disease relevant antigen presented by an antigen-presenting cell to generated a population of antigen-specific T-cells.

35. The method of claim 34, further comprising: e) administering at least a portion of said population of CAR-T cells, or the population of activated T-cells, to a subject, wherein said subject optionally has cancer, and optionally wherein said cancer is myeloid cancer.