T cell populations

Abstract

This document provides methods and materials for generating cell populations enriched for T cells (e.g., CD14negCD127pos or CD14–CD127+ T cells). For example, methods and materials for obtaining a cell population enriched for CD14–CD127+ T cells from a peripheral blood mononuclear cell (PBMC) population are provided. This document also provides CD14negCD127pos T cells designed to express a CAR and methods and materials for using such CARpos T cells to treat cancer (e.g., a B cell cancer) and / or a B cell pathology such as a B cell driven autoimmune disease.

Description

Atomev Docket No. 07039-2373WO1 / 2024-716T CELL POPULATIONSCROSS-REFERENCE TO RELATED APPLICATIONSThis application claims the benefit of U.S. Patent Application Serial No. 63 / 776,415, filed on March 24, 2025. The disclosure of the prior application is considered part of, and is incorporated by reference in, the disclosure of this application.TECHNICAL FIELDThis document relates to methods and materials for generating a cell population enriched for T cells (e.g., CD14negCD127posor CDI4 CDI27 T cells). For example, this document provides methods and materials for obtaining a cell population enriched for CDI4 CDI 27 T cells from a peripheral blood mononuclear cell (PBMC) population (e.g., a PBMC population obtained from a mammal such as a human). In some cases, a cell population enriched for CDI4 CD 127 T cells provided herein can be used to generate chimeric antigen receptor (CAR) T cells. In some cases, CDI 4 CDI 27 CAR T cells produced as described herein can be administered (e.g., in an adoptive cell therapy) to a mammal (e.g., a human) having cancer (e.g., a B cell cancer) and / or a B cell pathology such as a B cell driven autoimmune disease to treat the mammal.BACKGROUNDThe development of CAR T cells generated a new class of immunotherapies that have been life changing and life giving (Korell et al., Med., 3(8):538-564 (2022); Benevolo Savelli et al., Cancers (Basel), 16(1 ):46 (2023); and Lopez-Cantillo et al., Front. Immunol., 13:878209 (2022)). The matriculation of CAR T cell manufacturing has required advances in T cell subpopulation isolation and identification including expansion of CAR T cells while maintaining cytotoxicity, development of quality control testing, and translation of research procedures into Good Manufacturing Practice (GMP) processes. As a result, only six CAR T cell therapies that target either CD 19 or B cell maturation antigen (BCMA), have been approved by the United States (US) Food and Drug Administration (FDA) and the European Medicine Agency (EMA) (Fanciulli et al., Cancers (Basel), 14(16):3991 (2022)). The National Medical Products Administration (NMPA) in China has approved two US CAR TAtomev Docket No. 07039-2373WO1 / 2024-716 cell therapies and has developed four additional CAR T cell products that also target either CD19 or BCMA (Tan et al., J. HematoL Oncol., 17(1 ): 90 (2024)).Several factors influence the clinical benefit of CAR T cell therapies, specifically long-lasting remission; key characteristics are the abilities of infused CAR T cells to expand and persist in the patient (Korell et al., Med., 3(8): 538-564 (2022); Lopez-Cantillo et al., Front. Immunol., 13:878209 (2022); and Kong et al., Front. Immunol., 14:1063454 (2023)). T cell differentiation is influenced by components in their environment such as cytokines, growth factors, antigen, and interactions with other cells. During their differentiation journey from naive T cells (TN) cell to effector T cells (TEFF), the expression of surface markers can ebb and flow, thus allowing for the identification of the specific T cell subpopulations.Through empirical analysis and knowledge of the specific combination of surface markers, TN, stem-like memory T cells (TSCM), and central memory T cells (TCM) have been identified as the preferred starting material for CAR T cell generation (Gattinoni et al., Nat. Rev.Cancer, 12(10):671-84 (2012); Turtle et al., J. Clin. Invest., 126(6):2123-38 (2016);Sommermeyer et al., Leukemia, 30(2):492-500 (2016); McLellan et al., Immunology & Cell Biology’, 97(7) 664-674 (2019); and Golubovskaya et al., Cancers (Basel), 8(3):36 (2016)).CAR T cell products are currently the most expensive cancer therapies (Hay et al., J. Med. Econom., 22(7):613-615 (2019); and Cliff et al., Am. Soc. Clin. Oncol. Educ. Book, 43:e397912 (2023)), with an average cost of approximately $446,200 US dollars (Cliff et al., Am. Soc. Clin. Oncol. Educ. Book, 43:e397912 (2023)). There can be significant additional costs to monitor disease status and to manage the, sometimes serious, side effects (Choi et al., Int. J. Environ. Res. Public Health, 19(19): 12366 (2022)). Production failures and cases of disease relapse damage the image of CAR T cell therapy. Despite the predictions from the pioneers of CAR T cell therapy that costs would eventually decrease (Hay et al., J. Med. Econom., 22(7):613-615 (2019)), they remain unchanged or have increased as product characterization matures.SUMMARYThis document provides methods and materials for generating a cell population enriched for T cells (e.g., CD14 CD127+T cells). In some cases, this document provides methods and materials for obtaining a cell population enriched for CD I4 CD I27 T cellsAtomev Docket No. 07039-2373WO1 / 2024-716 from a PBMC population (e.g., a PBMC population obtained from a mammal such as a human). For example, CD14+cells can be substantially depleted from a PBMC population to obtain a cell population enriched for CD I4 PBMCs, and CD127+cells within that cell population enriched for CDI 4 PBMCs can be substantially enriched to obtain a cell population enriched for CD14 CD127-T cells. In some cases, a cell population enriched for CDI4 CDI 27 T cells can be derived from a PBMC population using a two-step method. For example, a negative selection step can be used to substantially deplete a PBMC population of CD14+cells, and a positive selection step can be used to substantially enrich for CD127+cells within a PBMC population. As demonstrated herein, a cell population enriched for CDI4 CD127 T cells derived from a PBMC population using this two-step method can include, without limitation, naive T cells (TN cells), stem-like memory T cells (TSCM cells), and central memory T cell (TCM cells). Also as demonstrated herein, a cell population enriched for CDI 4 CDI 27 T cells derived from a PBMC population using a two-step method described herein are suitable for generating CAR T cells.The ability to obtain a cell population enriched for CD14 CD127 T cells from a PBMC population as described herein can greatly enhance the CAR T cell manufacturing field. For example, a two-step method described herein can reduce costs and increase yield, as each additional selection step typically results in a loss of cells. In some cases, a cell population enriched for CD14 CDI 27 T cells provided herein can include, without limitation, T cell subtypes that are preferred for CAR T generation (e.g., TN, TSCM, and TCM cells) and exclude or include a minimal number of cells that are less desirable for generating CAR T cells (e.g., B cells and regulatory T cells (Tregs)). In some cases, a cell population enriched for CDI4 CD127 T cells provided herein can be used to generate populations of functional CAR T cells.This document also provides methods and materials for using cell populations enriched for CDI4 CD127 T cells provided herein. For example, this document provides methods and materials for generating CD14 CD I 27 CAR T cells from a cell population enriched for CDI4 CD I 27 T cells provided herein. In some cases, CD14 CD I 27 CAR T cells described herein can be used to treat a mammal (e.g., a human) having cancer (e.g., a B cell cancer) and / or a B cell pathology such as a B cell driven autoimmune disease. For example, CD14 CD 127 CAR T cells described herein can be administered (e.g., in anAtomev Docket No. 07039-2373WO1 / 2024-716 adoptive cell therapy) to a mammal having cancer. For example, CD14 GDI 27 CAR T cells described herein can be administered (e.g., in an adoptive cell therapy) to a mammal having a B cell pathology such as a B cell driven autoimmune disease.In general, one aspect of this document features methods for obtaining a cell population enriched for CD14negCD127posT cells. The methods can include, or consist essentially of, (al) depleting at least some CD14poscells from a starting population of cells to obtain a first cell population enriched for CD14negcells, and (bl) enriching said first cell population for CD127poscells to obtain said cell population enriched for CD14negCD127posT cells, or wherein said method comprises: (a2) enriching a starting population of cells for CD127poscells to obtain a first cell population enriched for CD127poscells, and (b2) depleting at least some CD14poscells from said first population of cells enriched for CD127poscells to obtain said cell population enriched for CD14negCD127posT cells. The method can include or consist of (al) and (bl). The method can include or consist of (a2) and (b2). The starting population can be a population of human cells. The starting population can be a population of PBMCs. Greater than 85 percent of the cells of said cell population enriched for CD14liegCD127posT cells can be CD14neg. For example, about 85 percent to about 99 percent of the cells of said cell population enriched for CD14negCD127posT cells can be CD14neg. Greater than 85 percent of the cells of said cell population enriched for CD14negCD127posT cells can be CD127pos. For example, from about 85 percent to about 99 percent of the cells of said cell population enriched for CD14negCD127posT cells can be CD127pos. Less than 4 percent of the cells of said cell population enriched for CD14negCD127posT cells can be monocytes. For example, from about 2 percent to about 4 percent of the cells of said cell population enriched for CD14negCD127posT cells can be monocytes. Less than 4 percent of the cells of said cell population enriched for CD14negCD127posT cells can be B cells. For example, from about 2 percent to about 4 percent of the cells of said cell population enriched for CD14negCD127posT cells are B cells. Less than 10 percent of the cells of said cell population enriched for CD14negCD127posT cells can be regulatory T (Treg) cells. For example, from about 3 percent to about 10 percent of the cells of said cell population enriched for CD14negCD127posT cells are Tregcells. In another aspect, this document features cell populations enriched for CD14negCD127posT cells produced according to the methods described herein.Atomev Docket No. 07039-2373WO1 / 2024-716 In another aspect, this document features methods for producing a cell that expresses a chimeric antigen receptor CAR. The methods can include, or consist essentially of, introducing nucleic acid encoding a CAR into a CD14negCD127posT cell of a cell population enriched for CD14negCD127posT cells produced according to the methods described herein, wherein said CD14negCD127posT cell expresses said CAR from said nucleic acid. The CAR can target a polypeptide expressed by a cancer cell within a human having cancer. The polypeptide can be a cancer-specific polypeptide. The starting population can be a population of PBMCs obtained from said human having cancer.In another aspect, this document features a CD14negCD127posT cell expressing a CAR, wherein said CD14negCD127posT cell expressing said CAR was produced according to the methods described herein.In another aspect, this document features populations of cells comprising CD14ncgCD127posT cells expressing a CAR, wherein greater than 90 percent (e.g., greater than 95 percent or greater than 90 percent is greater than 99 percent) of the cells of said population are said CD14negCD127posT cells expressing said CAR and (a) were produced according to the methods described herein or (b) were expanded from a cell produced according to the methods described herein.In another aspect, this document features methods for treating a mammal having cancer. The methods can include, or consist essentially of, administering, to a mammal having cancer, a population of cells comprising CD14negCD127posT cells expressing a CAR, wherein greater than 90 percent (e.g., greater than 95 percent or greater than 90 percent is greater than 99 percent) of the cells of said population are said CD14negCD127posT cells expressing said CAR and (a) were produced according to the methods described herein or (b) were expanded from a cell produced according to the methods described herein. The mammal can be a human. The cancer can be a MCL, a DLBCL, a Hodgkin’s lymphoma, a non-Hodgkin lymphoma, an ALL, a CLL, an AML, a germ cell tumor, a hepatocellular carcinoma, a bowel cancer, a lung cancer, a breast cancer, an ovarian cancer, a melanoma, a brain cancer, or a multiple myeloma.Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein canAtomev Docket No. 07039-2373WO1 / 2024-716 be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.BRIEF DESCRIPTION OF THE DRAWINGSFigures 1A-1B. Selective isolation of T cells for CAR T cell production using T cell markers. Figure 1A. During T cell differentiation from naive / stem cell-like memory T cells to highly differentiated effector T cell, the expression of surface markers, such as CD62L and CD127, changes as the T cell matures. Tregs also followed the same differentiation trajectory with different surface markers. Figure IB. (Left) The commonly utilized three-step T cell isolation process for CAR T cell development incorporates CD 14 depletion, followed by CD25 depletion, and concludes with CD62L enrichment. (Right) In some embodiments, the methods provided herein begin with CD14 depletion that is followed by CD127 enrichment to obtain a cell population enriched for CD14 CD127 T cells.Figures 2A-2C. Characterization of the immune cells from the two isolation strategies. Figure 2A. Using two isolation methods to fractionate PBMCs, the CD20+B cell and CD3+T cell subpopulations were differentially identified by flow cytometry. The cell population enriched for CDI4 CDI 27 cells showed a significant reduction in CD20+B cells and enrichment of CD3+T cells, compared to both the original PBMCs and the cell population enriched for CD I4 CD25 CD62L cells. Figure 2B. Quantification of B cell percentages across isolated immune cell populations derived from the two strategies (N=10, *** p < 0.001). Figure 2C. The percentage of CD14+monocytes was significantly reduced in both the cell population enriched for CD I4 CD25 CD62L cells and the cell population enriched for CD I4 CD127 cells, compared to unprocessed PBMCs.Figures 3A-3B. Significant reduction of Tregpopulation in cell populations enriched for CDI4 CDI 27 cells compared to cell populations enriched for CD14 CD62L cells fromAtomev Docket No. 07039-2373WO1 / 2024-716 the same donors. Figure 3A. Tregs, represented by CD25+CD127dimdouble-positive cells (top panels), were reduced in the cell population enriched for CD14 CD127+cells (top right panel) compared to the cell population enriched for GD I 4 CD62L cells (top left panel). These gated populations were further confirmed as Tregs since they are a CD4+FoxP3+population (bottom panels). Figure 3B. Quantification of percentage of Tregsin the cell population enriched for GD I 4 CD62L T cells and the cell population enriched for GD I 4 CD I27 T cells. N = 3,* P < 0.05.Figures 4A-4C. Immunophenotype of T cell products from two isolation protocols. The immunophenotypes of T cells from two isolation strategies were analyzed by flow cytometry. Figure 4A. Cells were gated on lymphocytes, singlets, live cells, and then CD3+T cells. A representative flow plot shows that CD3+T cell enrichment was greater in the cell population enriched for CD 14 GD I 27 cells compared to PBMCs or the cell population enriched for CD14 CD25 CD62L cells. Figure 4B. Percentage of CD3+T cells in either the cell population enriched for CD14 CD I 27 cells or the cell population enriched for CD I4 CD25 CD62L cells (N = 10, **** P < 0.0001). Figure 4C. The naive / memory phenotype of T cells was assessed within CD3 population and further analyzed by using CD45RA and CD45RO expression to further define the T cell subsets. Additional analysis of memory subsets was performed using CCR7 and CD 127 within CD45RA CD45RO (TN and TSCM cells) and CD45R.A CD45RO (Tc\[, TEM, and TEFF cells). The top plots depict TCM, TEM, and TEFF cells within the CD45RA CD45RO population, while the bottom plots represent TN and TSCM cells within the CD45RA CD45RO population. The blank CD127 vs CCR7 boxes are included as a guide for the T cell subpopulations that do or do not express CD 127 and / or CCR7.Figures 5A-5D. In vitro antigen-specific cytotoxicity of B AFF -R-targeted CAR (MC10029) T cells derived from two isolation strategies. Figure 5A. BAFF-R-targeted CAR (MC10029) T cells were manufactured using T cells isolated by two strategies: CD14 CD25 CD62L+(left panel) and CD I4 CD127 (right panel). These CAR T cells were incubated with either Nalm-6 WT (WT) or Nalm-6 BAFF-R KO cells (KO), followed by measurement of CD107a expression in a degranulation assay. Non-CAR T cells isolated by the same methods served as negative controls. Flow cytometry was gated on CD8+T cells or CD4+T cells (Figure 10). Figure 5B. Granzyme B release was detected by ELISA to further compareAtomev Docket No. 07039-2373WO1 / 2024-716 the cytotoxicity of these CAR T cells derived from two T cell isolation strategies. Data represent means from quadruplicate sampling, and the results are representative of three independent experiments. Figure 5C. Direct cytolytic activity was assessed by co-incubating GFP-expressing Nalm-6 cells with MC10029 CAR T cells derived from the cell population enriched for CDI4 CD25 CD62L cells (left panel) and the cell population enriched for CDI4 CDI27 cells (right panel). Figure 5D. Statistical analysis of direct cytolytic activity of MC10029 CAR T cells against Nalm-6 WT or Nalm-6 BAFF-RKO cells. By setting the Non-CAR T cells at 100% for each experiment, the non-CAR T cell group was used to normalize the percentage of live target cells in BAFF-R CAR T cell groups for the same target cells, ns, not significance; *** P < 0.001.Figures 6A-6C. In vivo anti-tumor efficacy of BAFF-R-targeted CAR (MC10029) T cells derived from two isolation strategies. Figure 6A. Bioluminescent imaging monitored the tumor burden in NSG mice that were injected with BAFF-R expressing Nalm-6 cells. Seven days after tumor challenge, mice were randomized into five groups (N = 5 per group) and then received two infusion (IV) treatments on days 7 and 14. The results are representative of two independent experiments using T cells from different donors. Figures 6B-6C. Kaplan-Meier survival plots of mice treated with CAR T cell derived from T cells enriched using the CD14 CD25 CD62L strategy (Figure 6B) and the CD14 CD127 T strategy (Figure 6C). *** p < 0.001; **** P < 0.0001.Figure 7. Evaluation of percentages of NK cells. The percentages of CD56+natural killer (NK) cells were evaluated from both the cell population enriched for CD 14 CD25 CD62L+cells and the cell population enriched for CD14 CD127 cells and compared to unprocessed PBMCs. No significant differences were observed between the populations obtained through the two isolation protocols.Figure 8. Evaluation of the percentages of Tregs. The percentages of Tregs were evaluated from both the cell population enriched for CD14 CD25 CD62L cells and the cell population enriched for CD14 CD I27 cells and compared to unprocessed PBMCs. Low levels were noted, and no significant differences were observed between the two isolation protocols.Figure 9A-9C. Characteristics of BAFF-R-targeted (MC10029) CAR T cell that were manufactured from T cells using two isolation protocols. MC10029 CAR T cell generatedAtomev Docket No. 07039-2373WO1 / 2024-716 from T cells isolated using CDI4 CD25 CD62L or the CD I4 CD I27 methods, along with corresponding non-CAR T cell. Fold expansion (Figure 9A), identity (CD3 phenotype, Figure 9B), and potency (EGFR as a proxy marker, Figure 9C) showed identical properties.Figure 10. Degranulation of CD4 MC10029 CAR T cell. MC10029 CAR T cell were manufactured using starting cell products isolated by two strategies: CDI4 CD25 CD62L (left panel) and CDI4 CD I 27 (right panel). These CAR T cells were incubated with either Nalm-6 WT (WT) or Nalm-6 BAFF-R KO cells (KO), followed by measurement of CD107a expression in degranulation assay of the CD4 T cells. Non-CAR T cell isolated by the same methods served as negative controls.DETAILED DESCRIPTIONThis document provides methods and materials for generating cell populations enriched for T cells (e.g., a CD I4 CDI 27 T cells). In some cases, this document provides methods and materials for obtaining a cell population enriched for CDI4 CD 127 T cells from a PBMC population (e.g., a PBMC population obtained from a mammal such as a human). For example, CD14+cells can be substantially depleted (e.g., to remove or substantially remove monocytes) from a PBMC population (e.g., a PBMC population obtained from a mammal such as a human) to obtain a cell population enriched for CD I 4 cells, and CD127+cells within the cell population enriched for CD I4 cells can be substantially enriched (e.g., to select for T cells) to obtain a cell population enriched for CD I4 CD I27 T cells (e.g., a cell population enriched for CD I4 CD I 27 T cells that can include TN, TSCM, and TCM cells). In some cases, a cell population enriched for CD I 4 CD I 27 T cells provided herein can be used to generate CAR T cells. In some cases, such CAR T cells can be administered to a mammal having a disease or disorder (e.g., a cancer) that can be treated using an adoptive cell therapy.Methods for obtaining a cell population enriched for CD I4 CD I 27 T cells from a PBMC population as provided herein can include any appropriate steps. In some cases, methods for obtaining a cell population enriched for CD14 CD I27 T cells from a PBMC population can involve only two steps. For example, methods for obtaining a cell population enriched for CD14 CD127+T cells from a PBMC population (e.g., a PBMC population obtained from a mammal such as a human) provided herein can include: a first step (e.g., aAtomev Docket No. 07039-2373WO1 / 2024-716 negative selection step) of substantially depleting the PBMC population of CD14+cells, thereby obtaining a cell population enriched for CD 14 cells; and a second step (e.g., positive selection step) of substantially enriching the cell population enriched for CD I4 cells for CD 127 cells, thereby obtaining a cell population enriched for CD14 CDI 27 T cells.Any appropriate method can be used to substantially deplete a cell population (e.g., a PBMC population) of CD14+cells. In some cases, substantially depleting a cell population (e g., a PBMC population) of CD14+cells can be effective to substantially deplete the population of monocytes. In some cases, a negative selection step can be used to substantially deplete a cell population (e.g., a PBMC population) of CD14+cells. In some cases, a cell population (e.g., a PBMC population) can be contacted with a substrate (e.g., a bead such as a magnetic bead) that is coated with one or more polypeptides (e.g., antibodies) that can bind a CD14 polypeptide, such that CD14+cells within the cell population (e.g., the PBMC population) bind to the substrate, and then the substrate can be removed from the cell population, thereby removing the bound CD14+cells from the cell population. In some cases, size-based centrifugation methods, cold aggregation, and / or specialized Percoll density gradients can be used to substantially deplete a cell population of CD14+cells. In some cases, a cell population can be substantially depleted of CD14+cells as described elsewhere (see, e.g., Delireh et al., Cell J., 15(3):218-23 (2013); and Chometon et al., PLoS One, 15(4):e0231132 (2020)).Any appropriate method can be used to substantially enrich a cell population (e.g., a PBMC population) for CD127+cells. In some cases, substantially enriching a cell population (e.g., a PBMC population) for CD127+cells can be effective to substantially enrich the cell population for T cells (e.g., TN, TSCM, and TCM cells). For example, substantially enriching a cell population (e.g., a PBMC population) for CD127+cells can be effective to isolate CDI 27 cells from other cells (e.g., Tregs, B cells, and NK cells) present in the cell population, thereby substantially depleting the cell population (e.g., the PBMC population) of such other cells (e.g., Tregs, B cells, and NK cells). In some cases, a positive selection step can be used to substantially enrich a cell population (e.g., a PBMC population) for CD127+cells. In some cases, a cell population (e.g., a PBMC population) can be contacted with a substrate (e.g., a bead such as a magnetic bead) that is coated with one or more polypeptides (e.g., antibodies) that can bind a CD 127 polypeptide, such that CD127+cells within the cellAtomev Docket No. 07039-2373WO1 / 2024-716 population bind to the substrate, and then the substrate can be isolated from the cell population thereby isolating the bound CD127+cells from the cell population. In some cases, after the substrates bound to the CD127+cells are isolated from the cell population (e.g., the PBMC population), the CD127+cells can be released (e.g., eluted) from the substrates. In some cases, antibody flow cytometry can be used to substantially enrich a cell population for CD127 cellsIn some cases, substantially depleting CD14+cells from a starting cell population (e.g., a PBMC population) can result in less than about 2 percent (e.g., less than about 1.5%, less than about 1%, or less than about 0.5%) of the CD14+cells present in the starting cell population remaining in the resulting cell population enriched in CD14 cells.In some cases, substantially enriching a starting cell population (e.g., a PBMC population or a population enriched for CD I4 cells) for CD127+cells can result in greater than about 10 percent (e.g., greater than about 15%, greater than about 20%, greater than about 25%, greater than about 30%, greater than about 35%, greater than about 40%, or greater than about 45%) of the cells present in the resulting cell population being CD127+cells. For example, substantially enriching a starting cell population (e.g., a PBMC population or a population enriched for CD I4 cells) for CD127+cells can result in a cell population having from about 10 percent to about 50 percent (e.g., from about 10% to about 45%, from about 10% to about 40%, from about 10% to about 35%, from about 10% to about 30%, from about 10% to about 25%, from about 10% to about 20%, from about 15% to about 50%, from about 20% to about 50%, from about 25% to about 50%, from about 30% to about 50%, from about 35% to about 50%, from about 40% to about 50%, from about 15% to about 45%, from about 20% to about 40%, from about 25% to about 35%, from about 15% to about 25%, from about 20% to about 30%, from about 30% to about 40%, or from about 35% to about 45%) CD127+cells.In some cases, substantially depleting CD14+cells from a cell population (e.g., a PBMC population) to form a population enriched for CD 14 cells and then substantially enriching the population enriched for CD I4 cells for CD127+cells can result in a cell population enriched for CD14 CD127 T cells where greater than about 85 percent (e.g., greater than about 87%, greater than about 90%, greater than about 92%, greater than about 95%, greater than about 97%, or greater than about 98%) of the cells present in that resultingAtomev Docket No. 07039-2373WO1 / 2024-716 cell population enriched for CD14 CDI 27 T cells are CD14 cells and where greater than about 85 percent (e.g., greater than about 87%, greater than about 90%, greater than about 92%, greater than about 95%, greater than about 97%, or greater than about 98%) of the cells present in that resulting cell population enriched for CD14 CDI 27 T cells are CD127+cells. For example, a depletion / enrichment two-step method according to some embodiments can involve substantially depleting CD14+cells from a cell population (e.g., a PBMC population) to form a population enriched for CD I 4 cells and then substantially enriching the population enriched for CD14 cells for CD127+cells can result in a cell population enriched for CD14 CD I 27 T cells where greater than about 85 percent of the cells present in that resulting cell population enriched for CD14 CD I 27 T cells are CD14 cells and where greater than about 85 percent of the cells present in that resulting cell population enriched for CD14 CD I 27 T cells are CD127+cells. In some cases, a depletion / enrichment two-step method according to some embodiments can result in a cell population enriched for CD I 4 CD I 27 T cells where greater than 80 percent of the cells present in the cell population enriched for CD14 CD I 27 T cells are CD I4 cells and where greater than 80 percent of the cells present in the cell population enriched for CD14 CDI 27 T cells are CD127+cells. In some cases, a depletion / enrichment two-step method according to some embodiments can result in a cell population enriched for CD14 CD I 27 T cells where greater than 85 percent of the cells present in the cell population enriched for CD14 CD127 T cells are CD14 cells and where greater than 85 percent of the cells present in the cell population enriched for CD14 CD I 27 T cells are CD127+cells. In some cases, a depletion / enrichment two-step method according to some embodiments can result in a cell population enriched for CDI4 CD127 T cells where greater than 90 percent of the cells present in the cell population enriched for CD I 4 CD127-T cells are CD I4 cells and where greater than 90 percent of the cells present in the cell population enriched for CD14 CD I 27 T cells are CD127+cells. In some cases, a depletion / enrichment two-step method according to some embodiments can result in a cell population enriched for CD14 CD I 27 T cells where greater than 95 percent of the cells present in the cell population enriched for CD14 CDI 27 T cells are CD14 cells and where greater than 95 percent of the cells present in the cell population enriched for CD14 CD I 27 T cells are CD127+cells. In some cases, a depletion / enrichment two-step method according to some embodiments can result in a cell population enriched for CD I4 CD127 T cellsAtomev Docket No. 07039-2373WO1 / 2024-716 where greater than 98 percent of the cells present in the cell population enriched for CDI 4 CD127 T cells are CD14 cells and where greater than 98 percent of the cells present in the cell population enriched for CD I4 CDI27 T cells are CD127+cells. In some cases, a depletion / enrichment two-step method according to some embodiments can result in a cell population enriched for CD14"CD127-T cells where greater than 99 percent of the cells present in the cell population enriched for CDI4 CDI27 T cells are CDI4 cells and where greater than 99 percent of the cells present in the cell population enriched for CDI4 CDI 27 T cells are CD127+cells. In some cases, a depletion / enrichment two-step method according to some embodiments can result in a cell population enriched for CDI4 CDI27 T cells where greater than about 85 percent of the cells present in the cell population enriched for CDI4 CDI 27 T cells are CDI4 cells and where greater than about 85 percent of the cells present in the cell population enriched for CDI4 CDI27 T cells are CD127+cells.A cell population enriched for CDI4 CDI27 T cells provided herein (e.g., a cell population enriched for CDI4 CDI 27 T cells obtained from a cell population using a two-step method described herein) can be obtained from any appropriate cell population (e.g., a PBMC population). In some cases, a PBMC population can be present in a blood sample (e.g., a blood sample obtained from a mammal such as a human). In some cases, a PBMC population can be derived from (e.g., can be isolated from) a blood sample (e.g., a blood sample obtained from a mammal such as a human). In some cases, a PBMC population is a PBMC population obtained from a mammal (e.g., a human). For example, a PBMC population can be a PBMC population that is obtained from a mammal (e.g., a human) that is to be treated with CAR T cells generated using a cell population enriched for CD I4 CD I 27 T cells as described herein.In some cases, a cell population enriched for CD14 CD127+T cells provided herein (e.g., a cell population enriched for CD I4 CD I 27 T cells obtained from a PBMC population using a two-step method described herein) can include any appropriate amount of T cells (e.g., CD3+T cells). In some cases, from about 85 percent to about 99 percent of the cells present within a cell population enriched for CD I4 CD I 27 T cells provided herein can be T cells. For example, greater than 90% of the cells present within a cell population enriched for CD I 4 CDI 27 T cell population provided herein can be T cells.Atomev Docket No. 07039-2373WO1 / 2024-716 In some cases, a cell population enriched for CDI4 CDI27 T cells provided herein (e.g., a cell population enriched for CD14 CD127+T cells obtained from a PBMC population using a two-step method described herein) can be a cell population enriched for CDI 4 CD 127 T cells where less than about 4 percent (e.g., less than about 3% percent, less than about 3.5% percent, less than about 3% percent, or less than about 2% percent) of the cells of the cell population enriched for CDI4 CDI27 T cells are monocytes. For example, a cell population enriched for CDI4 CDI27 T cells provided herein can be a cell population enriched for CDI4 CDI27 T cells where from about 85 percent to about 99 percent of the cells of cell population enriched for CDI4 CDI27 T cells are CDI4 , where from about 85 percent to about 99 percent of the cells of cell population enriched for CDI4 CDI27 T cells are CD127+, and where less than about 4 percent of the cells of cell population enriched for CDI4 CDI27 T cells are monocytes.In some cases, a cell population enriched for CD14 CD I27 T cells provided herein (e.g., a cell population enriched for CDI4 CDI 27 T cells obtained from a PBMC population using a two-step method described herein) can be a cell population enriched for CD 14 CD127 T cells where less than about 4 percent of the cells of the cell population enriched for CDI4 CDI 27 T cells are B cells. For example, a cell population enriched for CD14 CDI27 T cells provided herein can be a cell population enriched for CD I4 CDI 27 T cells where from about 85 percent to about 99 percent of the cells of cell population enriched for CD I4 CD I27 T cells are CD14 , where from about 85 percent to about 99 percent of the cells of cell population enriched for CD14 CD127+T cells are CD127+, and where less than about 4 percent of the cells of cell population enriched for CD I4 CD I 27 T cells are B cells.In some cases, a cell population enriched for CD I4 CD I27 T cells provided herein (e.g., a cell population enriched for CD I4 CD I27 T cells obtained from a PBMC population using a two-step method described herein) can be a cell population enriched for CD I4 CD I 27 T cells where less than about 5 percent (e.g., less than about 4%, less than about 3%, less than about 2%, or less than about 1%) of the cells of the cell population enriched for CD I4 CDI27 T cells are natural killer (NK) cells. For example, a cell population enriched for CD I4 CD I27 T cells provided herein can be a cell population enriched for CD I4 CDI 27 T cells where from about 85 percent to about 99 percent of the cells of cell population enriched for CD I4 CD I 27 T cells are CD I4 , where from about 85 percent toAtomev Docket No. 07039-2373WO1 / 2024-716 about 99 percent of the cells of cell population enriched for CD14 CD127+T cells are CD127 and where less than about 5 percent of the cells of cell population enriched for CD14 CD127+T cells are NK cells.In some cases, a cell population enriched for CDI4 CD127 T cells provided herein (e.g., a cell population enriched for CD14 CD127+T cells obtained from a PBMC population using a two-step method described herein) can be a cell population enriched for CDI 4 CDI 27 T cells where less than about 10 percent (e.g., less than about 8%, less than about 5%, or less than about 3%) of the cells of the cell population enriched for CD14 CD127 T cells are Tregcells. For example, a cell population enriched for CD I 4 CD 127 T cells provided herein can be a cell population enriched for CD14 CD I 27 T cells where from about 85 percent to about 99 percent of the cells of cell population enriched for CD14 CD I 27 T cells are CD14 , where from about 85 percent to about 99 percent of the cells of cell population enriched for CD14 CD I 27 T cells are CD127+, and where less than about 10 percent of the cells of cell population enriched for CD I 4 CD I 27 T cells are Trcgcells.This document also provides CAR T cells generated using a cell population enriched for CD14 CD127 T cells provided herein (e.g., a cell population enriched for CD14 CD127 T cells obtained from a PBMC population using a two-step method described herein), also referred to as CD I 4 CD I 27 CAR T cells. A CAR T cell generated using a cell population enriched for CD14 CD127 T cells provided herein can express (e.g., can be engineered to express) any appropriate antigen receptor. In some cases, a CAR can be designed to target a tumor antigen (e.g., tumor-specific antigen). For example, a CAR T cell can be engineered to express a tumor-specific CAR that targets a tumor-specific antigen (e.g., a cell surface tumorspecific antigen) expressed by a cancer cell in a mammal having cancer. Examples of antigens that can be recognized by a CAR expressed by a CAR T cell generated using a cell population enriched for CD14 CD I 27 T cells provided herein include, without limitation, cluster of differentiation 19 (CD 19), B cell activating factor receptor (BAFF-R), mucin 1 (MUC-1), human epidermal growth factor receptor 2 (HER-2), estrogen receptor (ER), epidermal growth factor receptor (EGFR), alphafetoprotein (AFP), carcinoembryonic antigen (CEA), CA-125, epithelial tumor antigen (ETA), melanoma-associated antigen (MAGE), CD33, CD123, CLL-1, E-Cadherin, folate receptor alpha, folate receptor beta, IL13R, EGFRviii, CD22, CD20, kappa light chain, lambda light chain, desmopressin, CD44v, CD45,Atomev Docket No. 07039-2373WO1 / 2024-716 CD30, CD5, CD7, CD2, CD38, BCMA, CD 138, FAP, CS-1, and C-met. For example, a T cell of a cell population enriched for CD14 CD127+T cells provided herein can be designed to express CAR targeting a BAFF-R polypeptide. In another example, a T cell of a cell population enriched for CDI4 CDI27 T cells provided herein can be designed to express a CAR targeting a CD 19 polypeptide.A CAR can include an antigen-binding domain, an optional hinge, a transmembrane domain, and one or more signaling domains. Examples of antigen-binding domains include, without limitation, an antigen-binding fragment (Fab), a variable region of an antibody heavy (VH) chain, a variable region of a light (VL) chain, a single chain variable fragment (scFv), and domains from growth factors that can bind to a cancer cell receptor (e.g., domains from EGF, PDGR, FGF, or TGF). In some cases, an antigen-binding domain of a CAR can target (e g., can target and bind to) a cancer antigen or a cancer-specific antigen. In some cases, an antigen-binding domain of a CAR can be as described elsewhere (see, e.g., U.S. Patent Application Publication No. 2017 / 0183418 such as U.S. Patent Application Publication No.2017 / 0183418 at paragraph

[0015] and the sequence listing; U.S. Patent Application Publication No. 2017 / 0183413 such as U.S. Patent Application Publication No.2017 / 0183413 at paragraph

[0049] , Figure 2, Table 9, and the sequence listing; U.S. Patent Application Publication No. 2018 / 0291079 such as U.S. Patent Application Publication No.2018 / 0291079 at paragraphs

[0041] -

[0045] , and Table 4; U.S. Patent Application Publication No. 2020 / 0289563 such as U.S. Patent Application Publication No.2020 / 0289563 at paragraphs

[0006] -

[0053] ,

[0186] -

[0189] , and Table 1; and U.S. Patent Application Publication No. 2003 / 0211097 such as U.S. Patent Application Publication No.2003 / 0211097 at paragraphs

[0081] and [0211-0215] and the sequence listing.In some cases, a CAR can include an optional hinge region. In some cases, a hinge region can be located between an antigen-binding domain and a transmembrane domain of a CAR. In some cases, a hinge region can provide a CAR with increased flexibility for the antigen-binding domain. For example, a hinge region can reduce spatial limitations of an antigen-binding domain of a CAR and its target antigen (e.g., to increase binding between an antigen-binding domain of a CAR and its target antigen). Examples of hinge regions that can be used as described herein include, without limitation, a membrane-proximal region from an IgG, a membrane-proximal region from CD8, and a membrane-proximal region from CD28.Atomev Docket No. 07039-2373WO1 / 2024-716 In some cases, a hinge region of a CAR can be as described elsewhere (see, e.g., U.S. Patent Application Publication No. 2018 / 0000914 such as U.S. Patent Application Publication No.2018 / 0000914 at paragraph

[0168] , and Table 1; U.S. Patent Application Publication No. 2017 / 0183418 such as U.S. Patent Application Publication No. 2017 / 0183418 at paragraphs

[0034] ,

[0037] ,

[0040] , and Table 2; U.S. Patent Application Publication No. 2017 / 0183413 such as U.S. Patent Application Publication No. 2017 / 0183413 at paragraph

[0116] ; and U.S. Patent Application Publication No. 2017 / 0145094 such as U.S. Patent Application Publication No. 2017 / 0145094 at paragraph

[0104] ,A CAR described herein can include any appropriate transmembrane domain. A transmembrane domain can be located between an antigen-binding domain and a signaling domain of a CAR and / or located between a hinge and a signaling domain of a CAR. In some cases, a transmembrane domain can provide structural stability for the CAR. For example, a transmembrane domain can include a structure (e.g., a hydrophobic alpha helix structure) that can span a cell membrane and can anchor the CAR to the plasma membrane. Examples of transmembrane domains that can be used as described herein include, without limitation, CD3 transmembrane domains, CD4 transmembrane domains, CD8 (e.g., a CD8a) transmembrane domains, CD28 transmembrane domains, CD 16 transmembrane domains, and erythropoietin receptor transmembrane domains. In some cases, a transmembrane domain of a CAR can be as described elsewhere (see, e.g., U.S. Patent Application Publication No. 2016 / 0120906 such as U.S. Patent Application Publication No.2016 / 0120906 at paragraphs

[0155] ,

[0161] ,

[0269] , Figure 4, and Figure 11; U.S. Patent Application Publication No. 2019 / 0209616 such as U.S. Patent Application Publication No.2019 / 0209616 at paragraph

[0026] ; U.S. Patent Application Publication No. 2018 / 0000914 such as U.S. Patent Application Publication No. 2018 / 0000914 at paragraphs

[0168] -

[0171] ; U.S. Patent Application Publication No. 2017 / 0183418 such as U.S. Patent Application Publication No. 2017 / 0183418 at paragraphs

[0116] -

[0118] ; U.S. Patent Application Publication No. 2017 / 0183413 such as U.S. Patent Application Publication No.2017 / 0183413 at paragraphs

[0116] -

[0118] ; and U.S. Patent Application Publication No. 2017 / 0145094 such as U.S. Patent Application Publication No. 2017 / 0145094 at paragraphs

[0104] -

[0107] ,Atomev Docket No. 07039-2373WO1 / 2024-716 A CAR described herein can include any appropriate signaling domain or combination of signaling domains (e.g., a combination of two, three, or four signaling domains). In some cases, a signaling domain of a CAR can be an intracellular signaling domain normally found within T cells or NK cells. Examples of signaling domains that can be used as described herein include, without limitation, CD2 signaling domains, CD3 signaling domains, CD28 signaling domains, Toll-like receptor (TLR) signaling domains (e g., TLR3 or TLR4 signaling domains), CD27 intracellular signaling domains, 0X40 (CD134) intracellular signaling domains, 4-1BB (CD137) intracellular signaling domains, CD278 intracellular signaling domains, DAP10 intracellular signaling domains, DAP12 intracellular signaling domains, CD278 intracellular signaling domains, CD 122 intracellular signaling domains, CD 132 intracellular signaling domains, CD70 intracellular signaling domains, cytokine receptor intracellular signaling domains, and CD40 intracellular signaling domains. In some cases, a CAR for use as described herein can be designed to be a first-generation CAR having a CD3^ intracellular signaling domain. In some cases, a CAR for use as described herein can be designed to be a second-generation CAR having a CD28 intracellular signaling domain followed by a CD3(^ intracellular signaling domain. In some cases, a CAR for use as described herein can be designed to be a third generation CAR having (a) a CD28 intracellular signaling domain followed by (b) a CD27 intracellular signaling domain, an 0X40 intracellular signaling domains, or a 4-1BB intracellular signaling domain followed by (c) a CD3(^ intracellular signaling domain. In some cases, the intracellular signaling domain(s) of a CAR can be as described elsewhere (see, e.g., U.S. Patent Application Publication No. 2018 / 0000914 such as U.S. Patent Application Publication No. 2018 / 0000914 at paragraphs

[0164] -

[0167] ; and U.S. Patent Application Publication No. 2017 / 0183413 such as U.S. Patent Application Publication No.2017 / 0183413 at paragraphs

[0112] -

[0115] ,In some cases, a CAR can be as set forth in Table 1.Table 1. Exemplary C ARs for targeting tumor antigens.Antigen scFv antibody Reference(s)nameAtomev Docket No. 07039-2373WO1 / 2024-716 CD19 FMC63 Milone et al., Mol. Ther., 17(8): 1453-64 (2009) (PMID: 26330164)MOR208 Kellner et al., Leukemia, 27(7): 1595-8 (2013)(PMID: 23277329)Humanized International Patent Application Publication No. scFv WO20151572524G7 EP Patent No. EP2997141Low affinity Chinese Patent No. CN107406517 scFvFMC63 CAR Kang et al., hit. J. Mol. Sei., 27(23):9163 (2020) KHYG-1 and4G7 CARKHYG-1MUC-1 5E5 Posey et al., Immunity, 45(5):947-948 (2016)(PMID: 27851918)HER-2 4D5 Ohnishi et al., Br. J. Cancer, 71(5):969-73 (1995) (PMID: 7734322)Forsberg et al., Cancer Res., 79(5):899-904 (2019) (PMID: 30622115)Nellan et al., J. Immunother. Cancer, 6(l):30 (2018) (PMID: 29712574)Priceman et al., Clin Cancer Res., 24( 1): 95- 105 (2018) (PMID: 29061641)FRP5 Bielamowics et al., Neuro. Oncol., 20(4):506-518 (2018) (PMID: 29016929)K70H-28z, Zhou et al., Front. Cell Develop. Biol., 10 :(2022). K70L-28z,KSAbH-28zand KSAbL-28zEGFR M27 Jiang et al., Cancer Immunol. Res., 6(11): 1314- 1326 (2018) (PMID: 30201736)Cetuximab Caruso et al., Cancer Res., 75(17): 3505-18 (2015) (PMID: 26330164)Folate receptor C4 based Ao et al., J. Immunother., 42(8):284-296 (2019) alpha (PMID: 31261167)M0vl9 Song et al., J. Hematol. Oncol., 9(1): 56 (2016)(PMID: 27439908)Mesothelin SSI Haas et al., Mol. Ther., S1525-0016(19)30328-4 (2019) (PMID: 31420241)Atomev Docket No. 07039-2373WO1 / 2024-716 M clone Adusumilli et al., Sci. Transl. Med.,6(261):261RA151 (2014) (PMID: 25378643) AFP ET1402L1 Liu et al., Clin. Cancer Res., 23(2):478-488 (2017)(PMID: 27535982)CEA Anti-CEA scFv Chi et al., Cancer Med., 8(10):4753-4765 (2019) (PMID: 31237116)CEACAM5 Ehistlethwaite et al., Cancer Immunol.Immunother., 66(11): 1425-1436 (2017) (PMID: 28660319)hMN14 Katz et al., Clin. Cancer Res., 21(14):3149-59(2015) (PMID: 25850950)CD 123 22172,22176 Gill et al., Blood, 123(15):2343-54 (2014) (PMID:24596416)Humanized US Patent Application Publication No.scFv 2016 / 0068601Humanized EP Patent No. EP2968415scFvTagrazofusp Pemmaraju et al. N. Engl. J. Med., 380( 17): 1628- based 1637 (2019) (PMID: 31018069) CD33 MY96 Kenderian et al., Leukemia, 29(8): 1637-47 (2015) (PMID: 25721896)Humanized International Patent Application Publication No. scFv WO2016014576CLEC12A Humanized US Patent Application Publication No.scFv US20160051651CLL1 scFv International Patent Application Publication No.W02016120219CD22 M0971 Fry et al., Nat. Med., 24(l):20-28 (2018) (PMID:29155426)Humanized International Patent Application Publication No. scFv clones WO / 2016 / 164731Inotuzumab Kantarjian et al., N. Engl. J. Med., 375(8):740-53 based (2016) (PMID: 27292104)Moxetumomab Kreitman et al., Leukemia, 32(8): 1768-1777 (2018) based (PMID: 30030507)CD20 Rituximab Zhang et al., Signal Transduct. Target Then,1:16002 (2016) (PMID: 29263894)Leu 16 Lee et al., J. Immunother., 41(1): 19-31 (2018)(PMID: 29176334)Atomev Docket No. 07039-2373WO1 / 2024-716 CD20 scFvs International Patent Application Publication No.WO / 2016 / 164731BCMA BCMA-02 Raje et al., N. Engl. J. Med., 380(18): 1726-1737 (2019) (PMID: 31042825)LCAR38 Zhao et al., J. Hematol. Oncol., 11(1): 141 (2018) (PMID: 30572922)BCMA scFv Smith et al., Cancer Immunol. Res., 7(7): 1047-1053(2019) (PMID: 31113804)Biepitopic Xu et al., Proc. Natl. Acad. Sci. USA,116(19):9543-9551 (2019) (PMID: 30988175) NVS BCMA Cohen etal., J. Clin. Invest., 129(6):2210-2221(2019) (PMID: 30896447)CS-1 CS1R Wang et al., Clin. Cancer Res., 24(1): 106-119(2018) (PMID: 29061640)CS1 ScFv Chu et al., Leukemia, 28(4):917-27 (2014) (PMID:24067492)Elotuzumab Dimopoulos et al., N. Engl. J. Med., 379(19): 1811- based 1822 (2018) (PMID: 30403938) CD138 ScFv Sun et al., Oncotarget., 10(24):2369-2383 (2019) (PMID: 31040928)CD44v6 Humanized Leuci et al., Oncoimmunology, 7(5):el423167scFv (2018) (PMID: 29721373)cMAb U36 Sandstrom et al., Int. J. Oncol., 40(5): 1525-32(2012) (PMID: 22307465)NKG2D scFv Yang et al., J. Immunother. Cancer, 7(1): 171 (2019)(PMID: 31288857)NKG2Dg scFv Parihar et al., Cancer Immunol. Res., 7(3):363-375(2019) (PMID: 30651290)CD38 Nanobody An et al., Mol. Pharm., 15(10):4577-4588 (2018) CD38 (PMID: 30185037)Humanized Yoshida et al., Clin. Transl. Immunology, scFv 5(12):ell6 (2016) (PMID: 28090317) GPRC5D Humanized Smith et al., Sci. Transl. Med., Il(485)eaau7746 scFv (2019) (PMID: 30918115)CD79b scFv (L-H) and Ormhoj et al., Clin. Cancer Res., (2019) (PMID:(H-L) 31439577)CD 103 M290 Zhang et al., Am. J. Transplant., 9(9):2012-23(2009) (PMID: 19645708)Atomev Docket No. 07039-2373WO1 / 2024-716 FAP FAP5 Wang et al., Cancer Immunol. Res., 2(2): 154-66 (2014) (PMID: 24778279)CD70 Human CD70 Park et al., Oral Oncol., 78:145-150 (2018) (PMID:29496042)MUC16 4H11 Koneru et al., Oncoimmunol., 4(3):e994446 (2015)(PMID: 25949921)IL13Ra2 IL13R Kong et al., Clin. Cancer Res., 18(21):5949-60(2012) (PMID: 22966020)Any appropriate method can be used to express a CAR on a CDI 4 CDI 27 T cell provided herein (e.g., a CD14 CD127 T cell of a cell population enriched for CD14 CD127 T cells provided herein). For example, nucleic acid encoding a CAR can be introduced into one or more T cells of a cell population enriched for CDI4 CD127 T cells provided herein. In some cases, viral transduction can be used to introduce nucleic acid encoding a CAR into a non-dividing a cell. Nucleic acid encoding a CAR can be introduced in a CDI 4 CD I 27 T cell of a cell population enriched for CD14 CD I 27 T cells provided herein using any appropriate method. In some cases, nucleic acid encoding a CAR can be introduced into a CD 14 CD127 T cell provided herein by transduction (e.g., viral transduction using a retroviral vector such as a lentiviral vector) or transfection. In some cases, nucleic acid encoding a CAR can be introduced ex vivo into one or more T cells of a cell population enriched for CD14 CD127 T cells from a PBMC population obtained from a mammal to be treated with CAR T cells. For example, ex vivo engineering of one or more T cells of a cell population enriched for CD14 CD I 27 T cells provided herein to express a CAR can include transducing isolated a CD14 CD I 27 T cell with a lentiviral vector encoding a CAR.This document also provides methods and materials for using a cell population enriched for CD14 CD127 T cells provided herein. For example, this document provides methods and materials for using a cell population enriched for CD14 CD1271T cells provided herein to generate CD14 CD I 27 CAR T cells. In some cases, CD14 CD I 27 CAR T cells can be used to treat a mammal (e.g., a human) having cancer. For example, CD I 4 CD127 CAR T cells can be administered (e.g., in an adoptive cell therapy) to a mammal having cancer to treat the mammal. In some cases, CD I4 CD I 27 CAR T cells provided herein (e.g., those produced from a cell population enriched for CD14 CD127 T cellsAtomev Docket No. 07039-2373WO1 / 2024-716 provided herein) can be administered to a mammal (e.g., a human) to reduce the number of cancer cells within the mammal and / or to increase the survival of the mammal.In some cases, methods of treating a mammal (e.g., a human) having cancer as described herein (e.g., by administering CD14 CD127 CAR T cells provided herein) can be effective to reduce the size of cancer within the mammal. For example, a mammal (e.g., a human) having cancer and in need of treatment thereof can be administered CD14 CD I 27 CAR T cells provided herein to reduce the number of cancer cells (e.g., cancer cells expressing a tumor antigen) in the mammal by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. In another example, a mammal (e.g., a human) having cancer and in need of treatment thereof can be administered CD14 CDI 27 CAR T cells provided herein to reduce the volume of one or more solid tumors (e.g., one or more tumors including cancer cells expressing a tumor antigen) in the mammal by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.In some cases, methods of treating a mammal (e.g., a human) having cancer as described herein (e.g., by administering CD14 CD127 CAR T cells provided herein) can be effective to improve survival of the mammal. For example, a mammal (e.g., a human) having cancer and in need of treatment thereof can be administered CD 14 CDI 27 CAR T cells provided herein to improve the survival of a mammal having cancer by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. In another example, a mammal (e.g., a human) having cancer and in need of treatment thereof can be administered CD I 4 CD I 27 CAR T cells provided herein to improve the survival of a mammal having cancer by, for example, at least 6 months (e.g., about 6 months, about 8 months, about 10 months, about 1 year, about 1.5 years, about 2 years, about 2.5 years, about 3 years, about 4 years, about 5 years, or more).Any appropriate amount (e.g., number) of CD14 CD 127 CAR T cells provided herein can be administered (e.g., in an adoptive cell therapy) to a mammal (e.g., a human) having cancer. In some cases, from about 0.1 million cells per kilogram body weight (cells / kg) to about 10 million cells / kg (e.g., from about 0.2 million cells / kg to about 2 million cells / kg) of CD14 CD I 27 CAR T cells provided herein can be administered to a mammal having cancer to treat the mammal.Atomev Docket No. 07039-2373WO1 / 2024-716 Any appropriate mammal (e.g., a human) having a cancer can be treated as described herein. Examples of mammals that can be treated as described herein include, without limitation, humans, non-human primates (e.g., monkeys), dogs, cats, horses, cows, pigs, sheep, mice, and rats. For example, a human having a cancer can be treated with CD14 CD127-CAR T cells provided herein in, for example, an adoptive T cell therapy using the methods and materials described herein.When treating a mammal (e.g., a human) having a cancer as described herein (e.g., by administering CD14 CD127 CAR T cells provided herein), the cancer can be any appropriate cancer. In some cases, a cancer treated as described herein can include one or more solid tumors. In some cases, a cancer treated as described herein can be a blood cancer. In some cases, a cancer treated as described herein can be a primary cancer. In some cases, a cancer treated as described herein can be a metastatic cancer. In some cases, a cancer treated as described herein can be a refractory cancer. In some cases, a cancer treated as described herein can be a relapsed cancer. In some cases, a cancer treated as described herein can express a tumor-associated antigen (e.g., an antigenic substance produced by a cancer cell). Examples of cancers that can be treated as described herein include, without limitation, mantle cell lymphomas (MCLs), diffuse large B cell lymphomas (DLBCLs), Hodgkin’s lymphomas, non-Hodgkin lymphomas, acute lymphoblastic leukemias (ALLs), chronic lymphocytic leukemias (CLLs), acute myeloid leukemias (AMLs), germ cell tumors, hepatocellular carcinomas, bowel cancers, lung cancers, breast cancers, ovarian cancers, melanomas, brain cancers, and multiple myelomas.A cancer that can be treated as described herein can include cancer cells expressing one or more antigens. For example, a cancer that can be treated as described herein can include cancer cells that express an antigen targeted by CD I4 CD 127 CAR T cells provided herein. A cancer that can be treated as described herein can include cancer cells expressing any appropriate one or more antigens (e.g., a tumor antigen targeted by the CAR T cells). In some cases, an antigen can be a tumor-associated antigen (e.g., an antigenic substance produced by a cancer cell). In some cases, a cancer that can be treated as described herein can be a B cell cancer. Examples of tumor-associated antigens that can be targeted by an adoptive T cell therapy provided herein include, without limitation, CD 19 (associated with DLBCL, ALL, and CLL), AFP (associated with germ cell tumors and / or hepatocellularAtomev Docket No. 07039-2373WO1 / 2024-716 carcinoma), CEA (associated with bowel cancer, lung cancer, and / or breast cancer), CA-125 (associated with ovarian cancer), MUC-1 (associated with breast cancer), ETA (associated with breast cancer), MAGE (associated with malignant melanoma), CD33 (associated with AML), CD 123 (associated with AML), CLL-1 (associated with AML), E-Cadherin (associated with epithelial tumors), folate receptor alpha (associated with ovarian cancers), folate receptor feta (associated with ovarian cancers and AML), IL13R (associated with brain cancers), EGFRviii (associated with brain cancers), CD22 (associated with B cell cancers), CD20 (associated with B cell cancers), kappa light chain (associated with B cell cancers), lambda light chain (associated with B cell cancers), CD44v (associated with AML), CD45 (associated with hematological cancers), CD30 (associated with Hodgkin lymphomas and T cell lymphomas), CD5 (associated with T cell lymphomas), CD7 (associated with T cell lymphomas), CD2 (associated with T cell lymphomas), CD38 (associated with multiple myelomas and AML), BCMA (associated with multiple myelomas), CD138 (associated with multiple myelomas and AML), FAP (associated with solid tumors), CS-1 (associated with multiple myeloma), and c-Met (associated with breast cancer). For example, CD14 CD 127 CAR T cells provided herein can be used in CAR T cell therapy targeting BAFF-R to treat cancer as described herein. For example, CD14 CD I 27 CAR T cells provided herein can be used in CAR T cell therapy targeting CD19 (e.g., a CART19 cell therapy) to treat cancer as described herein.In some cases, the methods described herein can include identifying a mammal (e.g., a human) as having a cancer. Any appropriate method can be used to identify a mammal having cancer. For example, imaging techniques and biopsy techniques can be used to identify mammals (e.g., humans) having cancer.In some cases, CD 14 CD 127 CAR T cells provided herein can be administered to a mammal (e.g., a human) having cancer as the sole active agents to treat the cancer. In some cases, an adoptive cell therapy that includes CD14 CDI 27 CAR T cells provided herein can be administered to a mammal (e.g., a human) having cancer as the sole active agents to treat the cancer.In some cases, methods for treating a mammal (e.g., a human) as described herein (e.g., by administering CD I4 CD I 27 CAR T cells provided herein) also can include administering to the mammal one or more (e.g., one, two, three, or more) additional agentsAtomev Docket No. 07039-2373WO1 / 2024-716 used to treat cancer and / or performing one or more (e.g., one, two, three, or more) therapies used to treat cancer. For example, a combination therapy used to treat a mammal (e.g., a human) having cancer can include administering to the mammal CDI4 CDI 27 CAR T cells provided herein and administering to the mammal one or more (e.g., one, two, three, or more) additional anti-cancer agents. In some cases, an additional anti-cancer agent that can be administered to a mammal can be a chemotherapeutic agent. In some cases, an additional anti-cancer agent that can be administered to a mammal can be a cytotoxic agent. In some cases, an additional anti -cancer agent that can be administered to a mammal can be an immune-checkpoint inhibitor (e.g., anti-PD-1 antibodies, PD-1 inhibitors, anti-PD-Ll antibodies, PD-L1 inhibitors, and anti-CTLA-4 antibodies). Examples of additional anticancer agents that can be administered to a mammal (e.g., a human) having cancer (e.g., a cancer including one or more solid tumors) to treat the mammal include, without limitation, BCL2 inhibitors, Mcl-1 inhibitors, BRAF inhibitors, MEK inhibitors, cyclooxygenase-2 inhibitors, BTK inhibitors, ITK inhibitors, dual BTK / ITK inhibitors, cytokines (e.g., IL-15 and / or IL-7), anti-CD20 antibodies, and any combinations thereof. In cases where CDI4 CD127 CAR T cells provided herein are used in combination with one or more additional anti-cancer agents, the one or more additional anti-cancer agents can be administered at the same time (e.g., in a single composition) or independently.In some cases, a combination therapy used to treat a mammal (e.g., a human) having cancer can include administering to the mammal CDI4 CDI27 CAR T cells provided herein, and can include performing one or more (e.g., one, two, three, or more) anti-cancer therapies. Examples of anti-cancer therapies that can be used to treat a mammal (e.g., a human) having cancer include, without limitation, radiation therapies and / or surgeries. In cases where CD14 CD127+CAR T cells provided herein are used in combination with one or more anti-cancer therapies to treat a mammal (e.g., a human) having cancer, the one or more anti-cancer therapies can be performed at the same time or independently of the administration of the CDI4 CDI27 CAR T cells provided herein. For example, CD 14 CDI 27 CAR T cells provided herein can be administered before, during, or after the one or more anti-cancer therapies are performed.In some cases, CD I4 CDI 27 CAR T cells provided herein (e.g., those produced from a cell population enriched for CD I4 CD I 27 T cells provided herein) can be used toAtomev Docket No. 07039-2373WO1 / 2024-716 treat a mammal (e.g., a human) having a disease or disorder other than cancer. For example, CD14 CD127+CAR T cells provided herein can be used to treat a mammal having a B cell pathology such as a B cell driven autoimmune disease. For example, CDI4 CDI 27 CAR T cells can be administered (e.g., in an adoptive cell therapy) to a mammal having a B cell pathology to treat the mammal. In some cases, CD 14 CD 127 CAR T cells provided herein (e.g., those produced from a cell population enriched for CD14 CD I27 T cells provided herein) can be administered to a mammal (e.g., a human) to reduce the number of B cells (e.g., self-activated B cells) within the mammal.The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.EXAMPLESExample 1: Isolating CD 14- -CD 127+ T cells for CAR T cell manufactureThis Example describes the development of a two-step isolation method for enriching CD14 CD127 T cells from PBMCs. This method effectively excluded unwanted or minimized the number of B cells, yet maintained the naive, stem-like central memory, and central memory T cells that can be used to produce functional CAR T cell.Materials and MethodsCell linesNalm-6 cells were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, Braunschweig, Germany) and cultured with 90% RPMI 1640 (Thermo Fisher, Waltham, MA) contained with 10% heat-inactivated fetal bovine serum (HI-FBS; Thermo Fisher, Waltham, MA). 293FT and Jurkat cell lines were purchased from American Type Culture Collection and cultured with either 90% Dulbecco’s modified Eagle medium (Thermo Fisher, Waltham, MA) or RPMI 1640 containing 10% HI-FBS. A BAFF-R-KO of the Nalm-6 cell line (Nalm-6 BAFF-R-KO) and GFP expressing Nalm-6 cell lines were generated as described elsewhere (Luo et al., Cancer Immunol Immunother 72(12):4031-4047 (2023); and Qie et al., MedComm., (2020) 5(9):e716 (2024)) to investigateAtomev Docket No. 07039-2373WO1 / 2024-716 the cytotoxic function of CAR T cell. Routine mycoplasma contamination testing was performed on all cell lines.PBMCs isolation from blood samples from health donorsPBMCs from healthy donors were isolated via leukapheresis using leukocyte reduction system (LRS) cones, as described elsewhere (Luo et al., Cancer Immunol Immunother., 72(12):4031-4047 (2023); Qie et A , MedComm ., (2020) 5(9):e716 (2024); and Dietz et al., Transfusion, 46(12):2083-9 (2006)).Isolation of CD 14 CD25 CD62L , CD 14 CD127 , or CD14 CD62L+T cells from PBMCs CD14 CD25 CD62L+, CD14 CD127+, or CD14~CD62L+T cells were isolated from PBMCs using targeted enrichment protocols for downstream applications. CD 14 CD25 CD62L+T cells were isolated using a three-step procedure (Luo et al., Cancer Immunol Immunother., 72(12):4031-4047 (2023); Qie et A., MedComm., (2020) 5(9):e716 (2024); and Wang et al., Leukemia, 36(4): 1015-1024 (2022)): negative selection of CD14 and then CD25, followed by positive selection of CD62L. CD14 (catalogue number: 130-050-201), CD25 (catalogue number: 130-092-983), and CD62L (catalogue number: 130-091-758) microbeads were used, according to the manufacturer’s protocol (Miltenyi Biotech, Germany).CD14 CD127 or CD14 CD62L T cells were isolated using a two-step procedure of CD14 negative selection followed by enrichment of CD127+or CD62L+T cells. The CD127 MicroBead kit (catalogue number: 130-094-945) and CD62L microbeads were utilized according to the manufacturer’s protocol (Miltenyi Biotech, Germany).LS columns (catalogue number: 130-042-401; Miltenyi Biotech, Germany) were used for positive selection, while LD columns (catalogue number: 130-042-901; Miltenyi Biotech, Germany) were utilized for negative selection.Antibodies and flow cytometryFor flow cytometry analysis, cells were harvested, washed with phosphate-buffered saline (PBS), and resuspended in FACS buffer (PBS supplemented with 2% fetal bovine serum and 1 mM EDTA). Surface staining was performed by incubating cells with fluorochrome-conjugated antibodies for 30 minutes at 4°C in the dark. The antibodies that were used are listed in Table 2.Atomev Docket No. 07039-2373WO1 / 2024-716Table 2. Antibodies used for flow cytometry.Marker Fluorophore Clone Company providerCD3 BV605 HIT3a BDCD4 PE-Cy7 SK3 BDCD4 BUV496 SK3 BDCD8 APC-Cy7 SK1 BDCD14 BV650 M5E2 BDCD20 BUV395 2H7 BDCD25 BV-421 2A3 BDCD56 BUV496 NCAM16.2 BDCD107a APC H4A3 BDCD127 PE HIL-7R-M21 BDCD45RA BV711 HI100 BDCD45RO Alexa 700 UCHL1 BDCCR7 BV785 2-L1-A BDEGFR APC AY13 BDFoxP3 A647 259D / C7 BDViability Sytox Blue NA Thermo FisherBD = BD Biosciences; NA = not applicableFor intracellular staining, cells were fixed and permeabilized using the eBioscience™ Foxp3 / Transcription Factor Staining Buffer Kit (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions, followed by staining with intracellular FoxP3 antibody (Table 2). The cell surface markers were identified using antibodies with the listed fluorophores from the provided vendors; however, these reagents can be sourced from other vendors along with other fluorophores for detection.Flow cytometry data were acquired using the Fortessa flow cytometer (BD Biosciences) and analyzed with FlowJo™ version 10 software. Compensation was performed using single-stained compensation beads (BD Biosciences) for each fluorochrome. Gating strategies were defined based on fluorescence minus one (FMO) controls and isotype controls to ensure accurate identification of populations.CAR T cell manufactureA second-generation BAFF-R CAR (MC10029) was engineered as described elsewhere (Luo et al., Cancer Immunol. Immunother ., 72(12):4031-4047 (2023)). CAR-lentiviruses were produced in 293FT cells, concentrated, and titered using Jurkat cells.Atomev Docket No. 07039-2373WO1 / 2024-716 CDI4 CD25 CD62L+or CD I4 CD127+T cells were isolated as starting populations for CAR T cell generation. In brief, the isolated T cells were divided into two aliquots. For the aliquot destined for CAR T cell generation: T cells were activated with Dynabeads Human T-Activator CD3 / CD28 (Thermo Fisher, Waltham, MA) for 24 hours, transduced with CAR-encoding lentivirus at a multiplicity of infection (MOI) of 1, and cultured with CD3 / CD28 beads for 6 days. After bead removal, the CAR T cells were expanded for an additional 7 days. The second aliquot served as non-CAR T cells in which the T cells were expanded using the same protocol but without lentiviral transduction.In vitro functional assaysCD 107a Degranulation assayThis assay was performed as described elsewhere (Luo et al., Cancer Immunol Immunother., 72(12):4031-4047 (2023); Qie et al., MedComm., (2020) 5(9):e716 (2024); and Luo et al., Molecular Therapy Oncology, 32(4):200891 (2024)). Briefly, CAR T cells were co-incubated with target cells in medium containing GolgiStop™ reagent and CD107a antibody for 4-6 hours. After incubation, the cells were surface-stained with antibodies against CD3, CD4, CD8, and EGFR.The samples were analyzed using a flow cytometer, and data processing was conducted using FlowJo™ software. Non-CAR T cells derived from the same healthy donors were used as negative controls for the assay.Granzyme B release assayCAR T cells and target cells were co-cultured at an effector-to-target (E:T) ratio of 4:1 for 72 hours. After the incubation period, the supernatant was collected and analyzed for granzyme B levels using an enzyme-linked immunosorbent assay (ELISA).Direct killing assayTo evaluate the cytolytic activity of CAR T cells against tumor cells, GFP-positive target cells were co-incubated with CAR T cells at an empirically optimized effector-to-target (E:T) ratio of 20:1 for 24 hours. The proportion of viable GFP-positive tumor cells was determined using Sytox Blue live dye (Thermo Fisher, Waltham, MA) to exclude dead cells,Atomev Docket No. 07039-2373WO1 / 2024-716 followed by analysis on a flow cytometer. The percentages of live GFP-positive tumor cells were quantified using a gating strategy that first identified live cells based on Sytox Blue exclusion and subsequently gated for GFP-positive cells.In vivo anti-tumor efficacyNSG mouse breeding pairs (The Jackson Laboratory, stock no. 005557) were used to establish a breeding colony. Mice (8-12 weeks old) were engrafted with luciferaseexpressing Nalm-6 cells and randomized into test groups (5 mice per group). Animals received two intravenous (IV) treatment doses on day 7 and day 14 of either PBS, non-CAR T cells, or MC10029 CAR T cells that were engineered from T cells isolated via CD14 CD25~CD62L+or CDI4 CDI27+isolation strategies. Tumor burden was monitored weekly using bioluminescent imaging after injecting the mice with D-luciferin (150 mg / g body weight) 10 minutes before whole body imaging (TVIS®, PerkinElmer, Waltham, MA).Survival data were analyzed and displayed as Kaplan-Meier plots.Statistical analysisThe Student’s t-test was conducted using GraphPad Prism software (GraphPad data are presented as mean ± standard error of the mean (SEM). Statistical significance between non-CAR T and CAR T cell groups was indicated as follows: P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), and E < 0.0001 (****). A bar placed above a pair of data sets indicates the statistical comparison.ResultsSelection of CD 127 for isolating T cells for CAR T cell manufacturingLymphoid progenitor cells within the bone marrow receive signals from cytokines and Notch ligands to develop into T cells. In humans, these cells migrate to the thymus during early life to yield naive T cells. These naive T cells circulate in the blood-lymphatic system until their T cell receptor recognizes an antigen, triggering differentiation into effector T cells. During T cell differentiation, naive (TN), stem-like memory (TSCM), central memory (TCM), effector memory (TEM), effector (TEFF) cells, and various regulatory T (Treg) cells emerge, each characterized by distinct surface markers, as outlined in Figure 1A.Atomev Docket No. 07039-2373WO1 / 2024-716 Great strides in understanding T cell subpopulations have advanced adoptive cell therapies, specifically CAR T cells. Translational studies linking clinical outcomes to CAR T cell design revealed that utilizing minimally differentiated T cells, which are capable of sustained proliferation and survival, resulted in CAR T cells with an enhanced anti-tumor activity. This insight established the early-stage T cells of TN, TSCM, and TC as the primary precursors for CAR T cell generation. A well-established protocol uses CD62L to capture TN, TSCM, and TCM cells, while excluding more differentiated effector T cells (Wang et al., J. Immimother ., 35(9):689-701 (2012); and Beke Debreceni et al., Cytometry B Clin. Cytom., 96(2): 149-157 (2019)). This approach also includes the depletion of CD14+monocytes, which hinder CAR T cells expansion (Hollyman et al., J. Immunother ., 32(2): 169-80 (2009); and Stroncek et al., Cytotherapy, 18(7):893-901 (2016)), and CD25+Tregs, which may impair the anti -turn or efficacy of the final CAR T cell product (Hosseinalizadeh et al., Frontiers in Medicine, 10:1244298 (2023); Verma et al., eBioMedicine . 83:104216 (2022); Good et al., Nature Medicine, 28(9):1860-1871 (2022); and Li et al., J. Cancer, 7(7):784-93 (2016)). The left panel of Figure IB schematically represents this pathway.Incidentally, CD62L is highly expressed on B cells, including malignant ones, which poses a risk of tumor cell contamination during CAR T cell manufacturing for B cell malignancies (Figure 1A). To address this, a two-step isolation strategy was developed where CD14+monocytes were removed in the first step, followed by CD127 enrichment to isolate TN, TSCM, and TC lymphocytes while excluding or minimizing the number of B cells (Figure IB, right panel).Depletion of B cells by CD 127 enrichmentPBMCs from a single healthy donor were divided into two aliquots: one applied to the existing isolation procedure to yield CD14 CD25 CD62L T cells and the other using the new method to yield a cell population enriched for CD I4 CD I27 T cells. These isolations were performed side-by-side with PBMCs from ten different donors. The resulting enriched T cell populations were first evaluated for residual contaminating cell populations. The presence of B cells was identified using an antibody against CD20 (Figure 2A). The CD I4 CD I27 T cell isolation procedure consistently removed nearly all CD20+B cells, whereas the CD14 CD25 CD62L T cell isolation procedure retained significant donor-dependentAtomev Docket No. 07039-2373WO1 / 2024-716 CD20+B cell populations, averaging 34.55% with a range of 15.4% to 64% (Figure 2B). Both methods effectively culled CD 14 positive cells from the desired T cell subpopulations (Figure 2C). Further analysis revealed similarly low levels of NK and NKT cells (Figure 7) as well as Tregs (Figure 8) using both methods.CD 127 enrichment significantly reduced Tregs without the need for CD25 depletion.Tregs play a critical role in maintaining immunosuppression in cancer by suppressing the activity of effector T cells. To improve the final CAR T cell product, the residual Tregs in the cell population enriched for CD14 CDI 27 cells isolated with this protocol were specifically quantified. The existing CD62L-based protocol uses CD25 depletion to remove Tregs, which also depletes activated T cells that upregulate CD25, resulting in a reduced yield of T cells for CAR T cell production. Given that CD127 is deficient in most Treg subpopulations, it was evaluated whether CD127 enrichment could sufficiently exclude Tregs, thus removing the need for a specific CD25 depletion step.The CD I 4 CDI 27 protocol was compared to a modified CD62L protocol without CD25 depletion. In the absence of CD25 depletion, CD62L enrichment alone was insufficient to exclude the CD127dim'7CD25+Treg subpopulation, identified as CD4+ / FoxP3+double positive Tregs. CD127 enrichment efficiently eliminated the majority of the CD127dim / 7CD25+Tregs (Figure 3A). This analysis was replicated (N = 3) and confirmed that the CD I 4 CD I 27 method significantly reduced the Treg population, whereas the modified CD62L without CD25 depletion showed variable Treg content (Figure 3B).CD 127 enrichment captures naive and memory T cellsThe CD14"CD127+method effectively removed undesired cell populations. Next, the exact profile of retained T cells was determined. Both the CD I4 CD I 27 method and the three-step methods were adept at enriching the CD3 positive T cell population (Figure 4A, representative data). As previously noted, the CD I 4 CD25 CD62L T cell isolation procedure showed a donor-dependent range of CD3 positive T cells with an average of 52.29% and a range of 20.1% to 78.6% (N = 10) (Figure 4B). The CD14~CD127+T cell isolation method, however, yielded a remarkable average of 97.19% of isolated cells, having a CD3 positive phenotype with a very modest range of 92.4% to 99.2% (N = 10) (Figure 4B).Atomev Docket No. 07039-2373WO1 / 2024-716 Phenotypical analysis of the isolated CD3+T cells revealed a comparable distribution of T cell subsets between the two isolation methods. A predominant subset was made up of CD45RA CD45RO naive T cells / stem cell-like memory T cells (TN / TSCM), which was characterized by the expression of CCR7 and CD127. The second major subset was made up of CD45RA CD45RO cells, most of which exhibited the CCR7+CD127+central memory phenotype (TCM). A smaller proportion displayed an effector memory phenotype (TEM), characterized by CCR7 CD I27 expression. Effector T cells (CD45RA CD45RO+CCR7 CD127 ) were absent in both isolation methods (Figure 4C). These results demonstrated that CD 127 enrichment, similar to CD62L enrichment, effectively enriched the early-stage T cell subsets of TN, TSCM, and TCM.CAR T cells manufactured from CD14 CD127+T cells demonstrated the anticipated antitumor effectsHaving successfully isolated TN, TSCM, and TC cells with CD127 enrichment, T cells isolated from this method were used to engineer MC10029 CAR T cells, a BAFF-R targeted CAR T cell therapy. These CAR T cells were compared to those produced with the exiting method to evaluate functional differences. Both MCI 0029 CAR T cell products displayed similar fold expansion (Figure 9A), identity (CD3 phenotype, Figure 9B), and potency (EGFR as a proxy marker, Figure 9C). Non-CAR T cells were also generated via each isolation method served as controls. The cytotoxic functions of the MC10029 CAR T cells, generated from CD I4 CD25 CD62L T cells or CD I4 CD I27 T cells, were evaluated using a CD107a degranulation assay. The assay was performed against Nalm-6 and BAFF-R KO Nalm-6 cells and gated on CD8 (Figure 5 A) or CD4 (Figure 10). Granzyme B release was also measured (Figure 5B). Non-CAR T cells showed no significant activity. In these two indirect activity assays, no significant differences were observed between the MCI 0029 CAR T cells generated from CD I4 CD25 CD62L T cells and those generated from CD I4 CD127 T cells. In addition, in a direct killing assay monitoring GFP-labeled Nalm-6 and BAFF-R KO Nalm-6 target cells, both MC10029 CAR T cells effectively lysed the target cells (Figure 5C and 5D).MC10029 CAR T cells generated from CD14 CD25 CD62L+T cells and from CD I4 CD I27 T cells (characteristics in Table 3) were also compared using an in vivo anti-Atomev Docket No. 07039-2373WO1 / 2024-716 tumor model in which NSG mice were first challenged with luciferase-labeled Nalm-6 tumor cells. Once tumor was established, the mice received two infusions of either PBS, one of the MC 10029 CAR T cells, and its corresponding non-CAR T cells, seven days apart (Figure 6). Bioluminescent imaging of the mice was used to monitor the time dependent changes in tumor burden in response to the various treatments. The survival data were plotted in Kaplan- Meier survival plots and showed that that the MC10029 CAR T cells generated from CD I4 CD25 CD62L T cells and from CD14 CD127 T cells were indistinguishable in this model, with both CAR T cells providing excellent anti-tumor effects (Figure 6B and 6C).Table 3. Quality control measurements for MCI 0029 CAR T cells derived using different isolation methods to evaluate in vivo anti-tumor efficacy.NoCAR-T CAR-T NoCAR-T CAR-T Folds expansion 103.5 110 95 92 ViabiHty(%^70%@D14 88 87 90 86 ldentity(%)(CD3+%)>80% 99.6 99.4 99.6 99.7 Potency (%) (EGFR+%)n w 7 n on QTogether, these results demonstrate that the two-step method described herein can be used to obtain cell populations enriched for CDI4 CD I27 T cell including TN, TSCM, and TCM cells, which can be used in turn to generate CAR T cells.Example 2: Treating CancerA PBMC population is obtained from a human having cancer.The PBMC population is contacted with magnetic beads coated with an anti-CD14 antibody, and the beads are washed away to obtain a cell population enriched for CD14 cells. The cell population enriched for CD14 cells is contacted with magnetic beads coated with an anti-CD127 antibody, and the beads are collected and the cells are eluted to obtain a cell population enriched for CD I4 CD 127 T cells. T cells of the cell population enriched forAtomev Docket No. 07039-2373WO1 / 2024-716 CD I4 CD I27 T cells are engineered to express a CAR that can target an antigen expressed by cancer cells within that human to obtain CD14 CD127+CAR T cells.The engineered CD I4 CD I 27 CAR T cells are administered to that human to treat the cancer.Example 3: Exemplary EmbodimentsEmbodiment 1. A method for obtaining a cell population enriched for CD14negCD127posT cells, wherein said method comprises:(al) depleting at least some CD14poscells from a starting population of cells to obtain a first cell population enriched for CD14negcells, and(bl) enriching said first cell population for CD127poscells to obtain said cell population enriched for CD14negCD127posT cells, orwherein said method comprises:(a2) enriching a starting population of cells for CD127poscells to obtain a first cell population enriched for CD127poscells, and(b2) depleting at least some CD14poscells from said first population of cells enriched for CD127poscells to obtain said cell population enriched for CD14negCD127posT cells.Embodiment 2. The method of embodiment 1, wherein said method comprises or consists of (al) and (bl).Embodiment 3. The method of embodiment 1, wherein said method comprises or consists of (a2) and (b2).Embodiment 4. The method of any one of embodiments 1-3, wherein said starting population is a population of human cells.Embodiment 5. The method of any one of embodiments 1-4, wherein said starting population is a population of peripheral blood mononuclear cells (PBMCs).Atomev Docket No. 07039-2373WO1 / 2024-716Embodiment 6. The method of any one of embodiments 1-5, wherein greater than 85 percent of the cells of said cell population enriched for CD14negCD127posT cells are CD14neg.Embodiment 7. The method of any one of embodiments 1-5, wherein from about 85 percent to about 99 percent of the cells of said cell population enriched for CD14negCD127posT cells are CD14neg.Embodiment 8. The method of any one of embodiments 1-7, wherein greater than 85 percent of the cells of said cell population enriched for CD14negCD127posT cells are CD127pos.Embodiment 9. The method of any one of embodiments 1-7, wherein from about 85 percent to about 99 percent of the cells of said cell population enriched for CD14negCD127posT cells are CD127pos.Embodiment 10. The method of any one of embodiments 1-9, wherein less than 4 percent of the cells of said cell population enriched for CD14negCD127posT cells are monocytes.Embodiment 11. The method of any one of embodiments 1-9, wherein from about 2 percent to about 4 percent of the cells of said cell population enriched for CD14negCD127posT cells are monocytes.Embodiment 12. The method of any one of embodiments 1-11, wherein less than 4 percent of the cells of said cell population enriched for CD14negCD127posT cells are B cells.Embodiment 13. The method of any one of embodiments 1-11, wherein from about 2 percent to about 4 percent of the cells of said cell population enriched for CD14negCD127posT cells are B cells.Atomev Docket No. 07039-2373WO1 / 2024-716 Embodiment 14. The method of any one of embodiments 1-13, wherein less than 10 percent of the cells of said cell population enriched for CD14negCD127posT cells are regulatory T (Treg) cells.Embodiment 15. The method of any one of embodiments 1-13, wherein from about 3 percent to about 10 percent of the cells of said cell population enriched for CD14negCD127posT cells are Tregcells.Embodiment 16. A cell population enriched for CD14negCD127posT cells produced according to the method of any one of embodiments 1-15.Embodiment 17. A method for producing a cell that expresses a chimeric antigen receptor (CAR), wherein said method comprises introducing nucleic acid encoding said CAR into a CD14negCD127posT cell of said cell population enriched for CD14negCD127posT cells of embodiment 16, wherein said CD14negCD127posT cell expresses said CAR from said nucleic acid.Embodiment 18. The method of embodiment 17, wherein said CAR targets a polypeptide expressed by a cancer cell within a human having cancer.Embodiment 19. The method of embodiment 18, wherein said polypeptide is a cancerspecific polypeptide.Embodiment 20. The method of any one of embodiments 18-19, wherein said starting population is a population of PBMCs obtained from said human having cancer.Embodiment 21. A CD14negCD127posT cell expressing a CAR, wherein said CD14negCD127posT cell expressing said CAR was produced according to the method of any one of embodiments 17-20.Atomev Docket No. 07039-2373WO1 / 2024-716 Embodiment 22. A population of cells comprising CD14negCD127posT cells expressing a CAR, wherein greater than 90 percent of the cells of said population are said CD14negCD127posT cells expressing said CAR and (a) were produced according to the method of any one of embodiments 17-20 or (b) were expanded from a cell produced according to the method of any one of embodiments 17-20.Embodiment 23. The population of embodiment 22, wherein said greater than 90 percent is greater than 95 percent.Embodiment 24. The population of embodiment 22, wherein said greater than 90 percent is greater than 99 percent.Embodiment 25. A method for treating a mammal having cancer, wherein said method comprises administering, to said mammal, the population of cells of any one of embodiments 22-24.Embodiment 26. The method of embodiment 25, wherein said mammal is a human.Embodiment 27. The method of any one of embodiments 25-26, wherein said cancer is selected from the group consisting of a MCL, a DLBCL, a Hodgkin’s lymphoma, a nonHodgkin lymphoma, an ALL, a CLL, an AML, a germ cell tumor, a hepatocellular carcinoma, a bowel cancer, a lung cancer, a breast cancer, an ovarian cancer, a melanoma, a brain cancer, and a multiple myeloma.OTHER EMBODIMENTSIt is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

Attorney Docket No. 07039-2373WO1 / 2024-716 WHAT IS CLAIMED IS:

1. A method for obtaining a cell population enriched for CD14negCD127posT cells, wherein said method comprises:(al) depleting at least some CD14poscells from a starting population of cells to obtain a first cell population enriched for CD14negcells, and(bl) enriching said first cell population for CD127poscells to obtain said cell population enriched for CD14negCD127posT cells, orwherein said method comprises:(a2) enriching a starting population of cells for CD127poscells to obtain a first cell population enriched for CD127poscells, and(b2) depleting at least some CD14poscells from said first population of cells enriched for CD127poscells to obtain said cell population enriched for CD14negCD127posT cells.

2. The method of claim 1, wherein said method comprises or consists of (al) and (bl).

3. The method of claim 1, wherein said method comprises or consists of (a2) and (b2).

4. The method of any one of claims 1-3, wherein said starting population is a population of human cells.

5. The method of any one of claims 1-3, wherein said starting population is a population of peripheral blood mononuclear cells (PBMCs).

6. The method of any one of claims 1-3, wherein greater than 85 percent of the cells of said cell population enriched for CD14negCD127posT cells are CD14neg.

7. The method of any one of claims 1-3, wherein from about 85 percent to about 99 percent of the cells of said cell population enriched for CD14negCD127posT cells are CD14neg.Attorney Docket No. 07039-2373WO1 / 2024-716 8. The method of any one of claims 1-3, wherein greater than 85 percent of the cells of said cell population enriched for CD14negCD127posT cells are CD127pos.

9. The method of any one of claims 1-3, wherein from about 85 percent to about 99 percent of the cells of said cell population enriched for CD14negCD127posT cells are CD127pos.

10. The method of any one of claims 1-3, wherein less than 4 percent of the cells of said cell population enriched for CD14negCD127posT cells are monocytes.

11. The method of any one of claims 1-3, wherein from about 2 percent to about 4 percent of the cells of said cell population enriched for CD14negCD127posT cells are monocytes.

12. The method of any one of claims 1-3, wherein less than 4 percent of the cells of said cell population enriched for CD14negCD127posT cells are B cells.

13. The method of any one of claims 1-3, wherein from about 2 percent to about 4 percent of the cells of said cell population enriched for CD14negCD127posT cells are B cells.

14. The method of any one of claims 1-3, wherein less than 10 percent of the cells of said cell population enriched for CD14negCD127posT cells are regulatory T (Treg) cells.

15. The method of any one of claims 1-3, wherein from about 3 percent to about 10 percent of the cells of said cell population enriched for CD14negCD127posT cells are Tregcells.

16. A cell population enriched for CD14negCD127posT cells produced according to the method of any one of claims 1-15.Attorney Docket No. 07039-2373WO1 / 2024-716 17. A method for producing a cell that expresses a chimeric antigen receptor (CAR), wherein said method comprises introducing nucleic acid encoding said CAR into a CD14negCD127posT cell of said cell population enriched for CD14negCD127posT cells of claim 16, wherein said CD14negCD127posT cell expresses said CAR from said nucleic acid.

18. The method of claim 17, wherein said CAR targets a polypeptide expressed by a cancer cell within a human having cancer.

19. The method of claim 18, wherein said polypeptide is a cancer-specific polypeptide.

20. The method of claim 18, wherein said starting population is a population of PBMCs obtained from said human having cancer.

21. A CD14negCD127posT cell expressing a CAR, wherein said CD14negCD127posT cell expressing said CAR was produced according to the method of any one of claims 17-20.

22. A population of cells comprising CD14negCD127posT cells expressing a CAR, wherein greater than 90 percent of the cells of said population are said CD14negCD127posT cells expressing said CAR and (a) were produced according to the method of any one of claims 17-20 or (b) were expanded from a cell produced according to the method of any one of claims 17-20.

23. The population of claim 22, wherein said greater than 90 percent is greater than 95 percent.

24. The population of claim 22, wherein said greater than 90 percent is greater than 99 percent.

25. A method for treating a mammal having cancer, wherein said method comprises administering, to said mammal, the population of cells of any one of claims 22-24.Attorney Docket No. 07039-2373WO1 / 2024-716 26. The method of claim 25, wherein said mammal is a human.

27. The method of claim 25, wherein said cancer is selected from the group consisting of a MCL, a DLBCL, a Hodgkin’s lymphoma, a non-Hodgkin lymphoma, an ALL, a CLL, an AML, a germ cell tumor, a hepatocellular carcinoma, a bowel cancer, a lung cancer, a breast cancer, an ovarian cancer, a melanoma, a brain cancer, and a multiple myeloma.

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