Methods and systems for unbiased microfluidic isolation of antigen-specific t cells

Abstract

Provided herein are methods and systems for phenotype-agnostic isolation of antigen-specific T cells using the ATTACH assay (Assessment of T cells Tethered to Antigen Class I / II Histocompatibility). Also provided are antigen-specific T cells isolated using the ATTACH assay and methods of their use for treatment of a subject.

Description

Attorney Docket No.: 090723-1530732-MDA25-025PCTMETHODS AND SYSTEMS FOR UNBIASED MICROFLUIDIC ISOLATION OF ANTIGEN-SPECIFIC T CELLSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of United States Provisional Patent Application Serial No. 63 / 716,054, filed November 4, 2024, the content of which is incorporated herein by this reference in its entirety as if fully set forth herein.STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0002] This invention was made with government support under grants LC220272 awarded by the Department of Defense. The government has certain rights in the invention.BACKGROUND

[0003] Adoptive cell therapy using tumor-infiltrating lymphocytes (TILs) has proven to be a successful approach to treat solid tumors such as melanoma and non-small cell lung cancer (see, e.g., Rosenberg et al., “Adoptive cell transfer as personalized immunotherapy for human cancer,” 348(6230):62-8, 2015). One of the limitations of TIL therapy is that only a proportion of the TILs exhibit tumor reactivity (see, e.g., Scheper et al., “Low and variable tumor reactivity of the intratumoral TCR repertoire in human cancers,” Nat. Med. 25(l):89-94, 2018). Therefore, there is a concerted effort to identify tumor antigen-specific T cell clonotypes within TIL populations. Current approaches have shed light on neoantigens, tumor-specific mutations, as a key determinant of tumor immunogenicity. However, the unique nature of neoantigens, and the fact neoantigens are rarely shared across patients and HLA-types leads to substantial hurdles in moving these approaches to the mainstream.

[0004] Currently, identification of neoantigen-specific T cells requires several sequential steps of time-consuming and costly approaches, such as whole exome sequencing, RNA sequencing, neoantigen prediction, peptide synthesis and / or plasmid minigene cloning, T cell expansion, dendritic cell generation, functional screening of T cells by flow cytometry and / or ELISpot, synthesis of tetramers presenting the antigen / HLA of interest, and isolation of antigen-specific T cells for this sole, pre-defined antigen. Importantly, each of these steps must be repeated when a new patient presents, as neoantigens, HLAs, and the T cells whichAttorney Docket No.: 090723-1530732-MDA25-025PCT recognize them are hypervariable and rarely conserved across patients. Furthermore, the targets identified are limited to the current understanding of tumor antigens, which is rudimentary and constantly evolving and revealing new potential targets which had previously gone without consideration. The present disclosure provides solutions to this problem, providing more broadly applicable approaches to isolate antigen-specific T cells in an unbiased manner.BRIEF SUMMARY

[0005] The Summary is provided to introduce a selection of concepts that are further described below in the Detailed Description. This Summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used as an aid in limiting the scope of the claimed subject matter.

[0006] In one aspect, methods for the agnostic isolation of antigen-specific T cells are provided. The methods comprise: (a) adhering target cells to a channel or surface of a microfluidic slide; (b) adding a heterogeneous population of cells to the target cells, wherein the heterogeneous population of cells comprises T cells that do not bind to the target cells and a plurality of T cells that bind to a plurality of different target antigens on the target cells, and wherein the target antigen to which each of the plurality of T cells binds is unknown; (c) allowing the T cells of the heterogeneous population to bind to the different target antigens on the target cells; (d) applying a volume of media with constant low shear stress to the channel to displace unbound cells and cells loosely bound to the target cells in a flow-through fraction, wherein the low shear stress is less than about 10 dyne / cm2; (e) removing and discarding the flow-through fraction; and (f) applying mechanical force to collect the bound T cells, thereby isolating antigen-specific T cells.

[0007] In some embodiments, the microfluidic slide comprises glass or a polymer. In some embodiments, about 1 x 104- 5 x 105target cells are allowed to adhere to the channel or surface. In certain embodiments, about 3 x l05- 5 x l05target cells are allowed to adhere to the channel or surface. In some embodiments, the target cells are tumor cells. In some embodiments, the tumor cells are obtained from a human subject. In some embodiments, the tumor cells are obtained from a tissue sample from a solid tumor. In certain embodiments, the tumor is selected from a group consisting of a melanoma, lung cancer, osteosarcoma, Ewing sarcoma, rhabdomyosarcoma, renal cell carcinoma, colorectal carcinoma, thyroid cancer, hepatocellular carcinoma, hepatoblastoma, lymphoma, sarcoma, carcinoma, neuroblastoma,Attorney Docket No.: 090723-1530732-MDA25-025PCT rhabdomyosarcoma. In some embodiments, the tumor cells are from a breast cancer, gynecological cancer, pancreatic cancer, prostate cancer, or head and neck cancer.

[0008] In some embodiments, the step of adhering target cells to the microfluidic slide comprises incubation of the target cells with the substrate for about 8-12 hours. In certain embodiments, the incubation is for about 5-60 minutes or for about 15-20 minutes. In some embodiments, the target cells are biotinylated and the substrate comprises streptavidin. In other embodiments, the target cells comprise streptavidin, and the microfluidic slide is biotinylated. In some embodiments, the heterogeneous population of cells are allowed to bind to the different target antigens on the target cells in presence of dasatinib. In some embodiments, the heterogeneous population of cells are added to the target cells at a ratio of 1 : 1 to 1 :4. In some embodiments, incubation of the heterogeneous population of cells with the target cells is for less than about 60 minutes. In certain embodiments, the incubation is about 5-40 minutes or for about 15-20 minutes. In some embodiments, the incubation comprises agitation of the microfluidic slide.

[0009] In some embodiments, the shear stress is less than about 5 dyne / cm2. In certain embodiments, the shear stress is 1-3 dyne / cm2. In certain embodiments, the shear stress is 2 dyne / cm2. In some embodiments, the steps of applying a volume of media with constant low shear and removing and discarding the flow-through fraction are repeated three times. In some embodiments, the method further comprises adding trypsin and EDTA to the channel or surface.

[0010] In a further aspect, the isolated antigen-specific T cells identified in the disclosed methods are used to treat a patient in need thereof. In some embodiments, the isolated antigenspecific T cells obtained from the disclosed isolation methods are administered to a subject. In some embodiments, the methods further comprise determining the sequence of a T cell receptor of the isolated antigen-specific T cells. In certain embodiments, the methods further comprise introducing a nucleic acid encoding the T cell receptor into a plurality of T cells from a subject ex vivo and administering the resulting plurality of T cells to the subject.

[0011] In another aspect, systems are provided for isolating antigen-specific T cells. The systems comprise: (a) a microfluidic slide with a channel or surface to which cells can bind; (b) a device at a first end of the microfluidic slide for administering a solution at constant low shear stress to the microfluidic slide, wherein the low shear stress is less than about 10 dyne / cm2; (c) a container at a second end of the microfluidic slide for collecting a flowthroughAttorney Docket No.: 090723-1530732-MDA25-025PCT fraction of the solution; (d) optionally a device for agitation of the microfluidic slide; and (e) optionally, a means for analysis of cells. In some embodiments, the device for administering the solution at constant low shear stress is a syringe pump. In some embodiments, the syringe is attached to the microfluidic slide by silicon tubing, the container is attached to the microfluidic slide by silicon tubing, and wherein the container is removable for the discarding the flow-through fraction. In some embodiments, the removable container is attached to the microfluidic slide by silicon tubing.

[0012] In another aspect, methods for isolating antigen-specific T cells are provided. The method steps comprise: (a) adhering target cells to a surface of a flask; (b) adding a heterogeneous population of cells to the target cells, wherein the heterogeneous population of cells comprises T cells that do not bind to the target cells and a plurality of T cells that bind to a plurality of different target antigens on the target cells, and wherein the target antigen to which each of the plurality of T cells binds is unknown; (c) allowing the T cells of the heterogeneous population to bind to the different target antigens on the target cells by centrifugation of the flask; (d) adding a volume of media to displace unbound cells and cells loosely bound to the target cells; (e) removing and discarding the supernatant; and (f) applying mechanical force to collect the bound T cells, thereby isolating antigen-specific T cells.

[0013] In some embodiments, the flask comprises glass or a polymer. In some embodiments, about 1 x 104- 5 x 105target cells are allowed to adhere to the surface of the flask. In certain embodiments, about 3 x l05- 5 x l05target cells are allowed to adhere to the surface of the flask. In some embodiments, the target cells are tumor cells. In some embodiments, the tumor cells are obtained from a human subject. In some embodiments, the tumor cells are obtained from a tissue sample from a solid tumor. In certain embodiments, the tumor is selected from a group consisting of a melanoma, lung cancer, osteosarcoma, Ewing sarcoma, rhabdomyosarcoma, renal cell carcinoma, colorectal carcinoma, thyroid cancer, hepatocellular carcinoma, hepatoblastoma, lymphoma, sarcoma, carcinoma, neuroblastoma, rhabdomyosarcoma. In some embodiments, the tumor cells are from a breast cancer, gynecological cancer, pancreatic cancer, prostate cancer, or head and neck cancer.

[0014] In some embodiments, the step of adhering target cells to the surface of the flask comprises incubation of the target cells with the flask for 8-12 hours. In certain embodiments, the incubation is for 5-60 minutes or for 15-20 minutes. In some embodiments, the target cells are biotinylated and the flask comprises streptavidin. In other embodiments, the target cellsAttorney Docket No.: 090723-1530732-MDA25-025PCT comprise streptavidin, and the flask is biotinylated. In some embodiments, the heterogeneous population of cells are added to the target cells at a ratio of 1 : 1 to 1 :4. In some embodiments, incubation of the T cells with the target cells is for less than 60 minutes. In certain embodiments, the incubation is 5-40 minutes or for 15-20 minutes. In some embodiments, the incubation comprises centrifugation of the flask. In some embodiments, the steps of applying a volume of media to the flask to displace unbound cells and cells loosely bound to the target cells, and discarding the wash are repeated three times. In some embodiments, the method further comprises adding trypsin and EDTA to the flask.

[0015] In a further aspect, the isolated antigen-specific T cells identified in the disclosed methods are used to treat a patient in need thereof. In some embodiments, the isolated antigenspecific T cells are administered to a subject. In some embodiments, the methods further comprise determining the sequence of a T cell receptor of the isolated antigen-specific T cells. In certain embodiments, the methods further comprise introducing a nucleic acid encoding the T cell receptor into a plurality of T cells from a subject ex vivo and administering the resulting plurality of T cells to the subject.

[0016] Moreover, in some embodiments, the technology can be applied to selectively deplete self-reactive T cells by introducing wild type (not cancerous cells) as the target cells within the specified channel. T cells are then added to the system and those that exhibit reactivity towards the wild type cells can be retained and isolated. By retaining these T cells, these methods allow for effective identification and potential depletion of auto-reactive T cells, thereby reducing the likelihood of autoimmunity.BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The present application includes the following figures. The figures are intended to illustrate certain embodiments and / or features of the compositions, systems, and methods, and to supplement any description(s) of the compositions, systems, and methods. The figures do not limit the scope of the compositions and methods, unless the written description expressly indicates that such is the case.

[0018] FIG. 1 is a schematic diagram of a method according to an embodiment of the present disclosure (ATTACH - Assessment of T cells Tethered to Antigen Class I / II Histocompatibility). Target tumor cells are loaded into microfluidic slides (top two panels), and T cells are flowed over at constant low level of shear stress (bottom three panels). HigherAttorney Docket No.: 090723-1530732-MDA25-025PCT avidity T cells (dark grey) bind to their targets and are therefore retained longer while lower avidity T cells (light grey) and non-T cells are discarded. An enlargement of the indicated section of the left middle panel is provided on the right, showing that antigen-specific CD8 T cells bind tightly and remain bound under constant low level of shear stress, while non-specific CD8 T cells only exhibit loose binding initially and then become unbound under constant low level of shear stress.

[0019] FIGS. 2A-2E demonstrate the ability of a method according to an embodiment of the present disclosure (ATTACH) to enrich for antigen specific T cells from a heterogeneous T cell population in a murine system. FIG. 2A provides flow cytometry quantification of the antigen-specific OT-I (mitotracker-negative) and non-specific B6 (mitotracker-positive) splenocytes before performing the disclosed method (left panel) and after performing the method (right two panels). The antigen specific T cells (T cells reactive to the OVA257-264 antigen) are in the top left corner of each plot, and the nonspecific T cells are in the top right corner of each plot. FIG. 2B is a schematic diagram of an embodiment of a microfluidic slide system according to an embodiment of the disclosure, showing top and side views. The left panel of FIG. 2C is a graph showing the number of B6 (bottom line) and OT-I splenocytes (top line) per high power field (HPF) based on shear stress being applied. The right panel of FIG. 2C is a graph showing the percent bound B6 (left bar in each pair) or OT-I cells (right panel in each pair) after the indicated shear stress. FIG. 2D is a graph showing the proportion of B6 (bottom line) and OT-I cells (top line) remaining as a percentage of total T cells per HPF based on shear stress being applied. FIG. 2E provides graphs showing IFN-y production (left panel) and cytotoxicity (right panel) by bulk T cells or T cells following enrichment by ATTACH upon exposure to mouse Lewis lung carcinoma cell line expressing the ovalbumin antigen (LLC-OVA) in vitro.

[0020] FIGS. 3A-3D demonstrate the ability of a method according to an embodiment of the present disclosure (ATTACH) to de-enrich non-specific T cells. FIG. 3A is a schematic overview of the de-enrichment strategy (right panel) in comparison to a classical enrichment strategy (left panel). The different T cell recognizing antigens are shown on the x axis, and the affinity of the T cell for the presented antigen is shown on the y axis. The horizontal dotted line in the left panel indicates the force needed to directly enrich for antigen-specific T cells. The horizontal line in the right panel indicates the lower force needed to indirectly enrich for antigen-specific T cells by depleting non-specific T cells. FIG. 3B is a graph showing the proportion of OT-I cells remaining after ATTACH in LLC-OVA (right bar) as compared toAttorney Docket No.: 090723-1530732-MDA25-025PCTLLC (left bar). FIG. 3C is a graph showing the proportion of OT-I and B6 cells retained after the ATTACH de-enrichment strategy. FIG 3D is a graph showing the proportion of OT-I and B6 cells retained after the ATTACH de-enrichment strategy in LLC-OVA as compared to LLC.

[0021] FIGS. 4A-4F demonstrate the ability of a method according to an embodiment of the present disclosure (ATTACH) to enrich antigen specific human T cells. FIG. 4A shows the flow cytometry quantification of EGFR-TCR-T cells (Cell Tracker CMAC, bottom right quadrant in each FACS plot) and non-transfected CD8+ cells (NT) (Cell Tracker deep red, top left quadrant in each FACS plot)) in the input, wash fraction, and retained fraction (shown in left, middle, and right FACS plots, respectively). FIGS. 4B-4C are graphs showing the relative proportions of EGFR-TCR-T and non-transfected (NT) CD8+ cells in the wash (FIG. 4B) and retained (FIG. 4C) fractions. FIG. 4D is a graph showing the proportion of Foxml-TCR-T cells (left bar in each group) and NT cells (right bar in each group) remaining after performing the ATTACH method after different ratios of Foxml-TCR-T and NT cells are added (1 :5, 1 :2, 1 : 1). FIG. 4E is a graph showing the relative proportions of Foxml-TCR-T (bottom portion of each bar) and NT cells (top portion of each bar) before and after the ATTACH method, showing the increase in the relative proportion of Foxml-TCR-T cells after the ATTACH method. FIG. 4F is a graph macrophage inflammatory protein-1 beta (MIP-ip) concentration after a 24-hour incubation of retained fraction of Foxml-TCR-T after ATTACH (right bar) compared to 24- hour incubation of input only (left bar).DETAILED DESCRIPTION

[0022] The following description recites various aspects and embodiments of the present compositions and methods. No particular embodiment is intended to define the scope of the compositions and methods. Rather, the embodiments merely provide non-limiting examples of various compositions and methods that are at least included within the scope of the disclosed compositions and methods. The description is to be read from the perspective of one of ordinary skill in the art; therefore, information well known to the skilled artisan is not necessarily included.I. INTRODUCTION

[0023] Tumor antigen-specific CD8+ T cells offer a compelling method for therapeutic targeting of solid tumors. However, efforts to identify tumor antigen-specific T cells have been hampered by numerous limitations such as the need to pre-select antigens, inherently biasing such analyses. The sole determinant of a T cell’s ability to bind to an antigen is the interactionAttorney Docket No.: 090723-1530732-MDA25-025PCT between its T cell receptor (TCR) and an MHC-presented antigen presented on a target cell. This has led to an increase in the use of tetramers, synthetic multimers consisting of HLA and antigen complexes which can be used to isolate T cells recognizing these antigens for downstream use. However, the synthesis of tetramers requires prior knowledge of both the antigen of interest and the patient’s expressed HLA, which can be time-consuming, expensive, and most importantly limits the ability to isolate T cells against only known antigens.

[0024] The disclosed approach aims to overcome these limitations and streamline the process of isolating antigen-specific T cells against any MHC-presented antigen, known or not. The present disclosure provides a method called ATTACH (Assessment of T cells Tethered to Antigen Class I / II Histocompatibility) to enrich for tumor antigen-specific T cells using tumor cells as de facto “tetramer pools,” thereby allowing for rapid, versatile, and unbiased isolation of antigen-specific T cells. The disclosed methods and systems involve the co-culture of target cells (e.g., tumor cells) and T cells followed by a shear stress application, resulting in selection and recovery of the population of T cells that bind most strongly to their tumor targets (see FIG. 1). This approach provides a rapid and unbiased manner to isolate tumor-specific T cells using the same method in all patients at a fraction of the cost. The method of isolation is a “deenrichment” method, focusing on excluding cells (e.g., TILs) that are non-tumor reactive and preserving most of the TILs that show reactivity towards the tumor regardless of whether they have the highest avidity. Table 1 provides a comparison of the classical approach and the ATTACH approach to isolating antigen-specific T cells.Attorney Docket No.: 090723-1530732-MDA25-025PCTTABLE 1II. TERMINOLOGY

[0025] Unless otherwise defined, all terms of art, notations, and other scientific or medical terms or terminology used herein are intended to have the meanings commonly understood by those of ordinary skill in the art. In some cases, terms with commonly understood meanings are defined herein for clarity and / or for ready reference, and the inclusion of such definitions herein should not be construed as representing a substantial difference over the definition of the term as generally understood in the art.

[0026] Articles “a” and “an” are used herein to refer to one or to more than one (i.e., at least one) of the grammatical object of the article. By way of example, “an element” means at least one element and can include more than one element.

[0027] The use herein of the terms “including,” “comprising,” or “having,” and variations thereof, is meant to encompass the elements listed thereafter and equivalents thereof as well as additional elements. Embodiments recited as “including,” “comprising,” or “having” certain elements are also contemplated as “consisting essentially of’ and “consisting of those certain elements.” As used herein, “and / or” refers to and encompasses any and all possibleAttorney Docket No.: 090723-1530732-MDA25-025PCT combinations of one or more of the associated listed items, as well as the lack of combinations where interpreted in the alternative (“or”).

[0028] As used herein, the transitional phrase “consisting essentially of’ (and grammatical variants) is to be interpreted as encompassing the recited materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention. See, e.g., In re Herz, 537 F.2d 549, 551-52, 190 U.S.P.Q. 461, 463 (CCPA 1976) (emphasis in the original); see also MPEP § 2111.03. Thus, the term “consisting essentially of’ as used herein should not be interpreted as equivalent to “comprising.”

[0029] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is stated as 1% to 50%, it is intended that values such as 2% to 40%, 10% to 30%, or 1% to 3%, etc., are expressly enumerated in this specification. These are only examples of what is specifically intended, and all possible combinations of numerical values between and including the lowest value and the highest value enumerated are to be considered to be expressly stated in this disclosure.

[0030] The terms “about” and “approximately” as used herein shall generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20%; preferably, within 10%; and more preferably, within 5% of a given value or range of values. Any reference to “about X” or “approximately X” specifically indicates at least the values X, 0.95X, 0.96X, 0.97X, 0.98X, 0.99X, 1.01X, 1.02X, 1.03X, 1.04X, and 1.05X. Thus, expressions “about X” or “approximately X” are intended to teach and provide written support for a claim limitation of, for example, “0.98X.” Numerical quantities given herein are approximate unless stated otherwise, meaning that the term “about” or “approximately” can be inferred when not expressly stated. When “about” is applied to the beginning of a numerical range, it applies to both ends of the range.

[0031] As used throughout, “subject” can be a vertebrate, more specifically a mammal (e.g., a human, monkey, horse, cat, dog, cow, pig, sheep, camel, goat, mouse, rabbit, rat, and guinea pig), birds, reptiles, amphibians, fish, and any other animal. The term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered. As used herein, “patient” or “subject” may be used interchangeably and includes human and veterinary subjects.Attorney Docket No.: 090723-1530732-MDA25-025PCTIII. Methods and Systems for Agnostic Isolation of Antigen-Specific T cellsA. Microfluidic methods

[0032] In one aspect, methods for the agnostic isolation of antigen-specific T cells are provided. The methods comprise: (a) adhering target cells to a channel or surface of a microfluidic slide; (b) adding a heterogeneous population of cells to the target cells, wherein the heterogeneous population of cells comprises cells that do not bind to the target cells and a plurality of T cells that bind to a plurality of different target antigens on the target cells, and wherein the target antigen to which each of the plurality of T cells binds is unknown; (c) allowing the T cells of the heterogeneous population to bind to the different target antigens on the target cells; (d) applying a volume of media with constant low shear stress to the channel to displace unbound cells and cells loosely bound to the target cells in a flow-through fraction, wherein the low shear stress is less than 10 dyne / cm2; (e) removing and discarding the flow- through fraction; and (f) collecting the bound T cells, thereby isolating antigen-specific T cells.

[0033] In some embodiments, the microfluidic slide comprises glass or a polymer. In some embodiments, about 1 x 102- 5 x 105target cells are allowed to adhere to the channel or surface. For example, the number of target cells that are provided to the channel or surface may be about 1 x 102- 5 x 105cells, 1 x 102- l x 105cells, 1 x 102- l x 104cells, 1 x 102- l x 103cells, 1 x 103- 5 x 105cells, 1 x 103- l x 105cells, 1 x 103- l x 104cells, 1 x 104- 5 x 105cells, 1 x 104- 5 x 105cells, 1 x 104- 4 x 105cells, 1 x 104- 3 x 105cells, 1 x 104- 2 x 105cells, 1 x 104- 1 x 105cells, 1 x 105- 5 x 105cells, 1 x 102- 4 x 105cells, 1 x 105- 3 x 105cells, or 1 x 105- 2 x 105cells, as well as any range included therein. In certain embodiments, about 3 x 105- 5 x 105target cells are allowed to adhere to the channel or surface. The number of target cells used in a particular ATTACH method may depend on the sample from which the cells are derived. Accordingly, if the number of target cells obtained is fewer than required for the disclosed methods, the target cells may be cultured under suitable conditions to obtain a culture with a sufficient number of target cells. Suitable samples include, but are not limited to, any tissue, solid or semisolid sample, or fluid sample that contains the target cells, such as a resected tissue or tumor sample, needle biopsy, blood, serum, urine, saliva, cerebrospinal fluid, and the like. In some embodiments, the target cells are cancer cells. In some embodiments, the tumor cells are obtained from a human subject. In some embodiments, the tumor cells are obtained from a sample from a solid tumor. In certain embodiments, the tumor is selected from a group consisting of a melanoma, lung cancer, osteosarcoma, Ewing sarcoma,Attorney Docket No.: 090723-1530732-MDA25-025PCT rhabdomyosarcoma, renal cell carcinoma, colorectal carcinoma, thyroid cancer, hepatocellular carcinoma, hepatoblastoma, lymphoma, sarcoma, carcinoma, neuroblastoma, and rhabdomyosarcoma. In some embodiments, the tumor cells are from a breast cancer, gynecological cancer, pancreatic cancer, prostate cancer, or head and neck cancer.

[0034] In some embodiments, the step of adhering target cells to the microfluidic slide comprises incubation of the target cells with the slide overnight under suitable conditions to allow the cells to adhere to the slide. For example, in some embodiments, the target cells are incubated with the slide for about 6-14 hours, about 7-13 hours, about 8-12 hours, about 9-10 hours, or any range therein. In some embodiments, the incubation period is about 8-12 hours at 37°C and 5% CO2.

[0035] In other embodiments, the step of adhering the target cells to the slide is a rapid adhesion step. The incubation in such methods may be conducted for about 5-60 minutes. For example, the incubation may be conducted for about 5-60, 5-50, 5-40, 5-30, 5-20, 10-50, 10- 40, 10-30, 10-20, 15-50, 15-40, 15-30, or 15-20 minutes, or any range therein. In some embodiments, the incubation period is about 15-20 minutes at 37°C and 5% CO2. In some embodiments, the target cells and the slide are attached to an affinity agent and its affinity binding partner, respectively, to facilitate the rapid adherence of the target cells to the slide. In certain embodiments, the target cells are biotinylated and the slide comprises streptavidin. In other embodiments, the target cells comprise streptavidin, and the microfluidic slide is biotinylated. In some embodiments, the target cells are bound to a plurality of magnetic beads which may be immobilized by a magnet. In certain embodiments, the magnetic beads are conjugated to a binding agent (e.g., an antibody) that specifically binds a molecule on the surface of the target cells.

[0036] In some embodiments, the heterogeneous population of cells are allowed to bind to the different target antigens on the target cells in presence of dasatinib, a tyrosine kinase inhibitor, to prevent activation of T cells. As used herein, a heterogeneous population of cells includes non-T cells, T cells that do not bind specifically to an MHC -presented antigen on the target cells (e.g., bystander T cells), and T cells that specifically bind an MHC-presented antigen on the target cells. In some embodiments, the heterogeneous population of T cells comprise tumor-infiltrating lymphocytes (TILs). The heterogeneous population of cells may be obtained from a subject. In some embodiments, the subject has cancer. In some embodiments, the cells of the heterogeneous population of cells are added to the target cells atAttorney Docket No.: 090723-1530732-MDA25-025PCT a ratio of about 1 : 1 to about 1 :5 (e.g., about 1 : 1, 1 :2, 1 :3, 1:4, or 1 :5). In certain embodiments, the cells of the heterogeneous population of cells are added to the target cells at a ratio of 1 : 1. In some embodiments, incubation of the cells of the heterogeneous population of cells with the target cells is for less than about 60 minutes (e.g., less than about 60, 50, 40, 30, 20, 15, 10, or 5 minutes). In some embodiments, the incubation is about 5-40, 5-30, 5-20, 10-40, 10-30, 10- 20, 15-40, 15-30, or 15-20 minutes, or any range therein. In certain embodiments, the incubation is about 5-40 minutes or for about 15-20 minutes at 37°C, 5% CO2. In some embodiments, the incubation comprises agitation of the microfluidic slide.

[0037] As used herein, “shear stress” refers to the force that acts on a surface when a liquid flows across a surface. In some embodiments, the shear stress is less than about 10 dyne / cm2. For example, the shear stress may be less than about 10, 9, 8, 7, 6, 5, 4, 3, or 2 dyne / cm2. In some embodiments, the shear stress is less than about 5 dyne / cm2. In certain embodiments, the shear stress is 1-3 dyne / cm2. In certain embodiments, the shear stress is about 2 dyne / cm2. In some embodiments, application of shear stress is for less than about 20 minutes (e.g., less than about 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 minutes). In certain embodiments, the application of shear stress is about 5 minutes. In some embodiments, the steps of applying a volume of media with constant low shear stress and removing and discarding the flow-through fraction are repeated (e.g., repeated two or three times). As used herein, the flowthrough fraction refers to the media that has been flowed over the target cells, as well as the “de-enriched cells” (i.e., cells from the heterogeneous population of cells that have not bound or have not remained bound to the target cells under low shear stress conditions).

[0038] In some embodiments, the antigen-specific T cells are collected by mechanical force. In certain conditions, the method further comprises adding trypsin and EDTA to the channel or surface. In some embodiments, the depletion of bystander T cells and recovery of antigenspecific T cells is at a ratio of about 1 : 10, 1 :50, 1 : 100, 1 :500, 1 : 1,000, or greater. In some embodiments, the depletion of bystander T cells and recovery of antigen-specific T cells is at a ratio of about 1 : 1,000.

[0039] Methods of Treatment

[0040] In a further aspect, the isolated antigen-specific T cells identified in the disclosed methods are used to treat a patient in need thereof. In some embodiments, a therapeutically effective amount of the isolated antigen-specific T cells in a pharmaceutically acceptable composition are administered to a subject. In certain embodiments, the cells are administeredAttorney Docket No.: 090723-1530732-MDA25-025PCT to the subject from which the heterogeneous population of cells and / or the target cells were obtained. In other embodiments, the cells are administered to a different subject in need thereof. In some embodiments, the therapeutically effective amount of the pharmaceutical composition is administered via intravenous injection, subcutaneous injection, or direct injection into a diseased tissue to the subject. As used herein, the term “therapeutically effective amount” or “effective amount” refers to an amount of a therapeutic composition that, when administered to a subject, is effective to treat a disease or disorder such that the symptoms of the disease or disorder are ameliorated, or the likelihood of the disease or disorder developing or progressing is decreased. A therapeutically effective amount is not, however, a dosage so large as to cause adverse side effects, such as hyperviscosity syndromes, pulmonary edema, congestive heart failure, and the like.

[0041] “Treating,” “treatment,” and the like may refer to any indicia of success in the treatment or amelioration of cancer. Treating or treatment of cancer refers to ameliorating cancer in a subject or any one or more symptoms thereof. The term ameliorating refers to any therapeutically beneficial result in the treatment of cancer, lessening in the severity or progression, promoting remission or durations of remission, or curing thereof. Thus, treating or treatment includes ameliorating at least one physical parameter or symptom. Treating or treatment includes modulating the cancer, either physically (e.g., stabilization of a discernible symptom) or physiologically (e.g., stabilization of a physical parameter) or both. For example, “treating” or “treatment” with respect to cancer includes the administration of an agent to impede growth of a cancer, to do one or more of the following: cause a cancer to shrink by weight or volume (i.e., shrink from a first weight or volume to a second weight or volume, wherein the second weight or volume is less than the first), delay or prevent metastasis, extend the expected survival time of the subject, or extend the expected time to progression of the tumor, or the like. Thus, in the disclosed methods, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of the cancer. For example, a method for treating a cancer in a subject by administering a pharmaceutical composition as described in this disclosure is considered to be a treatment if there is a 10% reduction in one or more symptoms of the cancer in a subject as compared to a control. Thus the reduction can be a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percent reduction in between 10% and 100% as compared to native or control levels. The effect of treatment can be compared to an individual or pool of individuals not receiving the treatment, or to the same patient prior to treatment or at a different time during treatment. It is understood that treatmentAttorney Docket No.: 090723-1530732-MDA25-025PCT does not necessarily refer to a cure or complete ablation of the cancer or symptoms of the cancer.

[0042] In some embodiments, the methods comprise determining the sequence of a T cell receptor of the isolated antigen-specific T cells. In certain embodiments, the methods further comprise introducing a nucleic acid encoding the T cell receptor into a plurality of T cells from a subject ex vivo and administering a therapeutically effective amount of the resulting plurality of T cells to the subject.

[0043] In other embodiments, the disclosed methods are used for patients being treated with checkpoint blockade or other therapies, by repeating the method steps on samples taken at different timepoints to determine the frequency of tumor-specific T cells over time and how they relate to patient outcome.

[0044] In other embodiments, the disclosed methods are used to identify and eliminate self- reactive T cells in a subject. For example, the disclosed methods are performed using cells from normal / healthy tissue as the target cells, isolating and eliminating the self-reactive T cells.

[0045] Systems

[0046] In another aspect, systems are provided for use in the disclosed methods for isolating antigen-specific T cells. The systems comprise: (a) a microfluidic slide with a channel or surface to which cells can bind; (b) a device at a first end of the microfluidic slide for administering a solution at constant low shear stress to the microfluidic slide, wherein the low shear stress is less than about 10 dyne / cm2; (c) a container at a second end of the microfluidic slide for collecting a flowthrough fraction of the solution; (d) optionally a device for agitation of the microfluidic slide; and (e) optionally, a means for analysis of cells. In some embodiments, the device for administering the solution at constant low shear stress is a syringe pump. In some embodiments, the syringe is attached to the microfluidic slide by silicon tubing, the container is attached to the microfluidic slide by silicon tubing, and the container is removable for the discarding the flow-through fraction. In some embodiments, the removable container is attached to the microfluidic slide by silicon tubing.B. Centrifugation methods

[0047] In another aspect, methods for the agnostic isolation of antigen-specific T cells are provided which utilize a centrifugation step for introducing a heterogeneous population of cells to target cells. If not specifically described below in this section, the parameters may be theAttorney Docket No.: 090723-1530732-MDA25-025PCT same as described above with respect to the microfluidic methods. The centrifugation method steps include: (a) adhering target cells to a surface of a flask; (b) adding a heterogeneous population of cells to the target cells, wherein the heterogeneous population of cells comprises cells that do not bind to the target cells and a plurality of T cells that bind to a plurality of different target antigens on the target cells, and wherein the target antigen to which each of the plurality of T cells binds is unknown; (c) allowing the T cells of the heterogeneous population to bind to the different target antigens on the target cells by centrifugation of the flask; (d) adding a volume of media to displace unbound cells and cells loosely bound to the target cells; (e) removing and discarding the supernatant; and (f) applying mechanical force to collect the bound T cells, thereby isolating antigen-specific T cells.

[0048] In some embodiments, the flask comprises glass or a polymer. In some embodiments, about 1 X 102- 5 X 105target cells are allowed to adhere to the surface of the flask. For example, the number of target cells that are provided to the flask may be about 1 x 102- 5 x 105cells, 1 x 102- 1 x 105cells, 1 x 102- 1 x 104cells, 1 x 102- l x 103cells, 1 x 103- 5 x 105cells, 1 x 103- 1 x 105cells, 1 x 103- 1 x 104cells, 1 x 104- 5 x 105cells, 1 x 104- 5 x 105cells, 1 x 104- 4 x 105cells, 1 x 104- 3 x 105cells, 1 x 104- 2 x 105cells, 1 x 104- l x 105cells, 1 x 105- 5 x 105cells, 1 x 102- 4 x 105cells, 1 x 105- 3 x 105cells, or 1 x 105- 2 x 105cells, as well as any range included therein. In certain embodiments, about 3 x 105- 5 x 105target cells are allowed to adhere to the surface of the flask. In some embodiments, the target cells are tumor cells. In some embodiments, the tumor cells are obtained from a human subject. In some embodiments, the tumor cells are obtained from a tissue sample from a solid tumor. In certain embodiments, the tumor is selected from a group consisting of a melanoma, lung cancer, osteosarcoma, Ewing sarcoma, rhabdomyosarcoma, renal cell carcinoma, colorectal carcinoma, thyroid cancer, hepatocellular carcinoma, hepatoblastoma, lymphoma, sarcoma, carcinoma, neuroblastoma, rhabdomyosarcoma. In some embodiments, the tumor cells are from a breast cancer, gynecological cancer, pancreatic cancer, prostate cancer, or head and neck cancer.

[0049] In some embodiments, the step of adhering target cells to the surface of the flask comprises incubation of the target cells with the flask for 8-12 hours, about 6-14 hours, about 7-13 hours, about 8-12 hours, about 9-10 hours, or any range therein. In some embodiments, the incubation period is about 8-12 hours at 37°C and 5% CO2. In other embodiments, the step of adhering the target cells to the flask is a rapid adhesion step. The incubation in such methods may be conducted for about 5-60 minutes. For example, the incubation may be conducted forAttorney Docket No.: 090723-1530732-MDA25-025PCT about 5-60, 5-50, 5-40, 5-30, 5-20, 10-50, 10-40, 10-30, 10-20, 15-50, 15-40, 15-30, or 15-20 minutes, or any range therein. In some embodiments, the target cells are biotinylated and the surface of the flask comprises streptavidin. In other embodiments, the target cells comprise streptavidin, and the surface of the flask is biotinylated. In some embodiments, the heterogeneous population of cells are allowed to bind to the different target antigens on the target cells in presence of dasatinib, a tyrosine kinase inhibitor, to prevent activation of T cells. In some embodiments, the cells of the heterogeneous population of cells are added to the target cells at a ratio of 1 : 1 to about 1 :5 (e.g., about 1: 1, 1 :2, 1 :3, 1 :4, or 1 :5). In certain embodiments, the cells of the heterogeneous population of cells are added to the target cells at a ratio of 1 : 1. In some embodiments, incubation of the cells of the heterogeneous population of cells with the target cells is for less than about 60 minutes (e.g., less than about 60, 50, 40, 30, 20, 15, 10, or 5 minutes). In some embodiments, the incubation is about 5-40, 5-30, 5-20, 10-40, 10-30, 10- 20, 15-40, 15-30, or 15-20 minutes, or any range therein. In certain embodiments, the incubation is about 5-40 minutes or for about 15-20 minutes at 37°C and 5% CO2. In some embodiments, the incubation comprises agitation of the flask. In some embodiments, the incubation comprises centrifugation of the flask. In some embodiments, the steps of applying a volume of media to the flask to displace unbound cells and cells loosely bound to the target cells, and discarding the wash are repeated (e.g., repeated two or three times). In some embodiments, the method further comprises adding trypsin and EDTA to the flask to collect the antigen-specific T cells.

[0050] In another aspect, the isolated antigen-specific T cells identified in the disclosed methods are used to treat a patient in need thereof as described above. For example, in some embodiments, the isolated antigen-specific T cells are administered to a subject. In certain embodiments, the cells are administered to the subject from which the heterogeneous population of cells and / or the target cells were obtained. In other embodiments, the cells are administered to a different subject in need thereof.

[0051] In some embodiments, the methods further comprise determining the sequence of a T cell receptor of the isolated antigen-specific T cells. In certain embodiments, the methods further comprise introducing a nucleic acid encoding the T cell receptor into a plurality of T cells from a subject ex vivo and administering the resulting plurality of T cells to the subject.IV. Exemplary Embodiments

[0052] Embodiment 1. A method for isolating antigen-specific T cells, comprising:Attorney Docket No.: 090723-1530732-MDA25-025PCT(a) adhering target cells to a channel or surface of a microfluidic slide;(b) adding a heterogeneous population of cells to the target cells, wherein the heterogeneous population of cells comprises T cells that do not bind to the target cells and a plurality of T cells that bind to a plurality of different target antigens on the target cells, and wherein the target antigen to which each of the plurality of T cells binds is unknown;(c) allowing the plurality of T cells of the heterogeneous population to bind to the plurality of different target antigens on the target cells;(d) applying a volume of media with constant low shear stress to the channel to displace unbound cells and cells loosely bound to the target cells in a flow-through fraction, wherein the low shear stress is less than 10 dyne / cm2;(e) removing and discarding the flow-through fraction; and(f) applying mechanical force to collect the bound T cells, thereby isolating antigen-specific T cells.

[0053] Embodiment 2. The method of embodiment 1, wherein the microfluidic slide comprises glass or a polymer.

[0054] Embodiment 3. The method of embodiments 1 or 2, wherein 1 x 104- 5 x 105target cells are allowed to adhere to the channel or surface.

[0055] Embodiment 4. The method of any one of embodiments 1-3, wherein 3 x 105- 5 x 105target cells are allowed to adhere to the channel or surface.

[0056] Embodiment 5. The method of any one of embodiments 1-4, wherein the target cells are tumor cells.

[0057] Embodiment 6. The method of embodiment 5, wherein the tumor cells are obtained from a human subject.

[0058] Embodiment 7. The method of embodiment 6, wherein the tumor cells are obtained from a tissue sample from a solid tumor.

[0059] Embodiment 8. The method of embodiment 7, wherein the tumor is selected from a group consisting of a melanoma, lung cancer, osteosarcoma, Ewing sarcoma, rhabdomyosarcoma, renal cell carcinoma, colorectal carcinoma, thyroid cancer, hepatocellular carcinoma, hepatoblastoma, lymphoma, sarcoma, carcinoma, neuroblastoma, rhabdomyosarcoma, breast cancer, gynecological cancer, pancreatic cancer, prostate cancer, or head and neck cancer.Attorney Docket No.: 090723-1530732-MDA25-025PCT

[0060] Embodiment 9. The method of any one of embodiments 1-6, wherein step (a) comprises incubation of the target cells with the substrate for 8-12 hours.

[0061] Embodiment 10. The method of any one of embodiments 1-6, wherein step (a) comprises incubation of the target cells with the substrate for 5-60 minutes.

[0062] Embodiment 11. The method of any one of embodiments 1-6, wherein step (a) comprises incubation of the target cells with the substrate for 15-20 minutes.

[0063] Embodiment 12. The method of any one of embodiments 1-11, wherein the target cells are biotinylated and the microfluidic slide comprises streptavidin.

[0064] Embodiment 13. The method of any one of embodiments 1-12, wherein step (c) is performed in presence of dasatinib.

[0065] Embodiment 14. The method of any one of embodiments 1-13, wherein step (b) comprises adding the heterogeneous population of cells to the target cells at a ratio of 1 : 1 to 1 :4.

[0066] Embodiment 15. The method of any one of embodiments 1-14, wherein step (c) comprises incubation of the T cells with the target cells for less than 60 minutes.

[0067] Embodiment 16. The method of any one of embodiments 1-14, wherein step (c) comprises incubation of the T cells with the target cells for 5-40 minutes.

[0068] Embodiment 17. The method of any one of embodiments 1-14, wherein step (c) comprises incubation of the T cells with the target cells for 15-20 minutes.

[0069] Embodiment 18. The method of any one of embodiments 1-17, wherein step (c) comprises agitation of the microfluidic slide.

[0070] Embodiment 19. The method of any one of embodiments 1-18, wherein the shear stress is less than 5 dyne / cm2.

[0071] Embodiment 20. The method of any one of embodiments 1-18, wherein the shear stress is 1-3 dyne / cm2.

[0072] Embodiment 21. The method of any one of embodiments 1-18, wherein the shear stress is 2 dyne / cm2.

[0073] Embodiment 22. The method of any one of embodiments 1-21, wherein steps (d) and (e) are repeated three times.Attorney Docket No.: 090723-1530732-MDA25-025PCT

[0074] Embodiment 23. The method of any one of embodiments 1-22, wherein step (f) comprises adding trypsin and EDTA to the channel.

[0075] Embodiment 24. The method of any one of embodiments 1-23, wherein the isolated antigen-specific T cells are administered to a subject.

[0076] Embodiment 25. The method of any one of embodiments 1-24, further comprising determining the sequence of a T cell receptor of the isolated antigen-specific T cells.

[0077] Embodiment 26. The method of embodiment 25, further comprising introducing a nucleic acid encoding the T cell receptor into a plurality of T cells from a subject ex vivo and administering the resulting plurality of T cells to the subject.

[0078] Embodiment 27. A system for isolating antigen-specific T cells, comprising:(a) a microfluidic slide with a channel or surface to which cells can bind;(b) a device at a first end of the microfluidic slide for administering a solution at constant low shear stress to the microfluidic slide, wherein the low shear stress is less than about 10 dyne / cm2;(c) a container at a second end of the microfluidic slide for collecting a flowthrough fraction of the solution;(d) optionally a device for agitation of the microfluidic slide; and(e) optionally, a means for analysis of cells.

[0079] Embodiment 28. The system of embodiment 27, wherein the device for administering the solution at constant low shear stress is a syringe pump.

[0080] Embodiment 29. The system of embodiment 28, wherein the syringe is attached to the microfluidic slide by silicon tubing.

[0081] Embodiment 30. The system of embodiment 27, wherein the container is attached to the microfluidic slide by silicon tubing, and wherein the container is removable for the discarding the flow-through fraction.

[0082] Embodiment 3 E The system of any one of embodiments 27-30, wherein the removable container is attached to the microfluidic slide by silicon tubing.

[0083] Embodiment 32. A method for isolating antigen-specific T cells, comprising:(a) adhering target cells to a surface of a flask;Attorney Docket No.: 090723-1530732-MDA25-025PCT(b) adding a heterogeneous population of cells to the target cells, wherein the heterogeneous population of cells comprises T cells that do not bind to the target cells and a plurality of T cells that bind to a plurality of different target antigens on the target cells, and wherein the target antigen to which each of the plurality of T cells binds is unknown;(c) allowing the T cells of the heterogeneous population to bind to the different target antigens on the target cells by centrifugation of the flask;(d) adding a volume of media to the flask to displace unbound cells and cells loosely bound to the target cells;(e) removing and discarding the supernatant; and(f) applying mechanical force to collect the bound T cells, thereby isolating antigen-specific T cells.

[0084] Embodiment 33. The method of embodiment 32, wherein 1 x 104- 5 x 105target cells are allowed to adhere to the surface of the flask.

[0085] Embodiment 34. The method of embodiments 32 or 33, wherein 3 x 105- 5 x 105target cells are allowed to adhere to the surface of the flask.

[0086] Embodiment 35. The method of any one of embodiments 32-34, wherein the target cells are tumor cells.

[0087] Embodiment 36. The method of embodiment 35 wherein the tumor cells are obtained from a human subject.

[0088] Embodiment 37. The method of embodiment 36, wherein the tumor cells are obtained from a tissue sample from a solid tumor.

[0089] Embodiment 38. The method of embodiment 37, wherein the tumor is selected from a group consisting of a melanoma, lung cancer, osteosarcoma, Ewing sarcoma, rhabdomyosarcoma, renal cell carcinoma, colorectal carcinoma, thyroid cancer, hepatocellular carcinoma, hepatoblastoma, lymphoma, sarcoma, carcinoma, neuroblastoma, rhabdomyosarcoma, breast cancer, gynecological cancer, pancreatic cancer, prostate cancer, or head and neck cancer.

[0090] Embodiment 39. The method of any one of embodiments 32-38, wherein step (a) comprises incubation of the target cells with the flask for 8-12 hours.Attorney Docket No.: 090723-1530732-MDA25-025PCT

[0091] Embodiment 40. The method of any one of embodiments 32-39, wherein step (a) comprises incubation of the target cells with the flask for 5-60 minutes.

[0092] Embodiment 41. The method of any one of embodiments 32-40, wherein step (a) comprises incubation of the target cells with the flask for 15-20 minutes.

[0093] Embodiment 42. The method of any one of embodiments 32-41, wherein the target cells are biotinylated and the flask comprises streptavidin.

[0094] Embodiment 43. The method of any one of embodiments 32-42, wherein step (b) comprises adding the heterogeneous population of cells to the target cells at a ratio of 1 : 1 to 1 :4.

[0095] Embodiment 44. The method of any one of embodiments 32-43, wherein step (c) is performed in presence of dasatinib.

[0096] Embodiment 45. The method of any one of embodiments 32-43, wherein step (c) comprises incubation of the T cells with the target cells for less than 60 minutes.

[0097] Embodiment 46. The method of any one of embodiments 32-43, wherein step (c) comprises incubation of the T cells with the target cells for 5-40 minutes.

[0098] Embodiment 47. The method of any one of embodiments 32-43, wherein step (c) comprises incubation of the T cells with the target cells for 15-20 minutes.

[0099] Embodiment 48. The method of any one of embodiments 32-47, wherein steps (d) and (e) are repeated three times.

[0100] Embodiment 49. The method of any one of embodiments 32-48, wherein step (f) comprises adding trypsin and EDTA to the flask.

[0101] Embodiment 50. The method of any one of embodiments 32-49, wherein the isolated antigen-specific T cells are administered to a subject.

[0102] Embodiment 51. The method of any one of embodiments 32-50, further comprising determining the sequence of a T cell receptor of the isolated antigen-specific T cells.

[0103] Embodiment 52. The method of embodiment 51, further comprising introducing a nucleic acid encoding the T cell receptor into a plurality of T cells from a subject ex vivo and administering the resulting plurality of T cells to the subject.Attorney Docket No.: 090723-1530732-MDA25-025PCTEXAMPLES

[0104] The following examples are offered to illustrate, but not to limit, the claimed invention.Example 1. Materials and Methods

[0105] Cells and Compositions

[0106] In some experiments, Mouse OT-I T cells (recognize OVA antigen) and B6 T cells (lack OVA TCR, do not recognize OVA) were used, and mouse LLC-OVA (express OVA antigen) and LLC (do not express OVA) were used as target cells.

[0107] The ATTACH method was tested in a human antigen system using TCR transduced T cells. Previously validated TCRs (HLA-A*03:01 recognizing the neoantigen EGFR(L858R) and HLA-A*02:01 recognizing the Foxml tumor associated antigen) were used in the assay.

[0108] Immunofluorescence staining

[0109] Adherent LLC, LLC-SIINFEKL EGFR specific cells (LLC-OVA), and Foxml specific cells were resuspended to < 5.33 x 105cells / mL in 1 x PBS and stained with 6 pM of CellTracker™ blue CMAC dye (Invitrogen, catalog #C2110) following manufacturer’s instructions. Then, cells where resuspended in complete Roswell Park Memorial Institute (RPMI) media and immediately used for ATTACH. OT-I CD8+ T cells were thawed and washed prior to staining. Cells were resuspended to < 3 x 105cells / mL in lx PBS + 0.1% FBS and stained with 0.7 pM of CFSE dye (Invitrogen, catalog #65-0850-85) for 8 minutes at room temperature, obscured from light. An equal volume of 100% FBS was added to quench staining before transferring cells to a 37°C water bath and incubating for 10 minutes. After staining, cells were washed with 1 x PBS + 2% FBS and resuspended to IxlO6cells / mL in complete RPMI media for immediate use in ATTACH. C57BL / 6 and NT CD8+ T cells were resuspended to < 1 x 106 cells / mL in 1 x PBS followed by staining with 500 nM of CellTracker™ Deep Red dye (Invitrogen, catalog #C34565) or 250 nM MitoTracker™ Deep Red dye (Invitrogen, catalog # M22426) for 30 minutes at 37°C, 5% CO2, washed, and resuspended in complete RPMI media for immediate use in ATTACH.

[0110] A TTA CH microfluidic set-up

[0111] Sterilized ibiTreat: #1.5 polymer coverslip p-Slide 10.4 Luer tissue culture-treated channel slides (catalog #80176), luer lock female connectors (catalog #10825), elbow luer maleAttorney Docket No.: 090723-1530732-MDA25-025PCT connectors (catalog #10802), and 1.6 mm silicone tubing (catalog #10842) were obtained directly from the manufacturer ibidi. Target cells were seeded in separate channel slides with complete RPMI and were incubated at 37°C, 5% CO2 overnight to allow target cells to adhere to the channel. The following day, slides were flushed by pipetting 125 pL of complete RPMI through channels to remove dead and detached cells prior to loading T cells. After fluorescently staining, CD8+ effector cells were loaded into channels at a 1:4 ratio of effector to target cells followed by a 15-minute incubation for murine system experiments. For human system experiments, a 1 : 1 ratio of effector to target cells was used.

[0112] Shear stress was applied to channels by infusion via 140 mL syringes filled with prewarmed complete RPMI media connected to a Harvard Apparatus PHD ULTRA™ automated syringe pump. For ramped “enrichment” experiments, applied shear stress increased linearly with time and ranged from 0.5-20 dyne / cm2(0.53- 21.1 mL / min) over the course of 12 minutes. For de-enrichment experiments where a low shear stress was constantly applied to wash off non-specific CD8+ T cells, a shear stress of 1 dyne / cm2 (1.05 mL / min) was selected based on the point at which OT-I CD8+ became detached from negative control slides containing LLC cells (no antigen). For the EGFR specific T cells 1, 2, and 3 dyne / cm2 were tested to optimize best conditions for enrichment. 2 dyne / cm2 was used for experiments using foxml TCR. Slides were visualized under Nikon AlRSi (Center for Advanced Microscopy, Department of Integrative Biology & Pharmacology at McGovern Medical School, UTHealth), Nikon ECLIPSE Ti confocal microscopes using Nikon NIS-Elements AR 5.21.03 and Zeiss Axio Observer 7. Fluorescent images were captured via time lapse, 1 image captured / second, within 1 region of interest (ROI) in the middle of the channel. The ROI was consistent across all slides within an experiment.

[0113] After shear, mechanical force was applied to the microfluidic channels to remove the remaining T cells and used for flow cytometry experiments.

[0114] Flow Cytometry

[0115] Samples were immediately acquired by the University of Texas MD Anderson Flow Cytometry & Cellular Imaging Facility and collected on BD FACSCanto™ and Beckman Coulter Gallios and Gallios 561 cytometers. Cytek Aurora was used as well. Acquired data were analyzed using FlowJo™ vl0.8.0 software (BD Biosciences), gating on live single cells.Attorney Docket No.: 090723-1530732-MDA25-025PCT

[0116] MIP-1B ELISA

[0117] Retained T cells were incubated in the channels overnight. After incubation, supernatant was collected and Human MIP-ip concentration was measured using Invitrogen’s Human MIP-lb (CCL4) Instant ELISA™ Kit following manufacturer’s instructions. Absorbance values were obtained using Accouris smart reader 96 and concentrations of unknown samples were interpolated using the standard curve’s equation of the line and absorbance values.

[0118] Live-cell imaging

[0119] Retained T cells were incubated in the channels overnight on a live cell imaging evos microscope taking pictures every 3 minutes for 24 hours. Propidium iodide (PI) was added to the medium to observe viability of the cells.Example 2. ATTACH method in murine system.

[0120] Adherent LLC, LLC-OVA, and Foxml specific cells were immunofluorescently labeled as described in Example 1. For analysis of the ATTACH method in a murine system, 4 x 105target cells (LLC or LLC-OVA) were added to p-slide I Luer channel slides in 105 pL total volume. Then 125 pL total volume of complete media was added to the channel by dispensing into the wells dropwise, alternating sides. Slides were placed at 37°C, 5% CO2 overnight to allow target cells to adhere to channel slides. The following morning, slides were flushed with media 3x to remove dead / unattached target cells. All media was removed from the channel slides and 1 x 105T cells (OT-1 (OVA antigen-specific T cells) or B6 cells (nonspecific T cells) (1 :4 effector: target ratio) were loaded into each channel (105 pL final volume). Then 125 pL total volume of complete media was added to the channel by dispensing into the wells dropwise, alternating sides. Slides were incubated at 37°C, 5% CO2 for 15 minutes. Syringes filled with complete media (30C) were mounted onto the syringe pump. Slides were attached to syringe pump via Luer connectors and silicone tubing. On the side of the slide opposite of the pump, silicone tubing was attached and placed in a collection container to collect flow-through. The syringe pump infused complete media at a constant rate of 1 dyne / cm2through the channel for 5 minutes. Flow-through (de-enriched product) was collected in collection container. At 5 minutes, the pump was stopped, and the slide was removed. Cells in the channel were detached with trypsin + EDTA, and enrichment of target T cell population was confirmed by fluorescent microscopy and functional assays (FIGS. 2A- 2E).Attorney Docket No.: 090723-1530732-MDA25-025PCT

[0121] Enumeration of fluorescent cells pre- and post-ATTACH confirmed a 5-fold antigendependent enrichment of OT-I in presence versus absence of OVA (23.25% vs 4.36%, p=0.0004) (FIGS. 2B-2D and data not shown). CD8 co-receptor blockade confirmed retention was mediated through TCR / pMHC binding (8.79% vs 2.86% p=0.075) (data not shown). With a 1 :4 ratio of OT-I to C57BL / 6 CD8+ T cells, a 2-fold enrichment of antigen-specific T cells (16.40% vs. 7.7%, p=0.0151) was achieved. Furthermore, enriched antigen-specific populations recovered using ATTACH exhibited a 12-fold increase in IFN-y secretion and 3- fold higher cytotoxic potential as measured by cleaved caspase-3 / 7 (FIG. 2E).Example 3. ATTACH de-enrichment strategy.

[0122] The ATTACH method was analyzed to demonstrate its ability to de-enrich nonspecific T cells (FIG. 3A) This figure shows a comparison of the de-enrichment strategy (right panel) to a classical enrichment strategy (left panel). The different T cell recognizing antigens are shown on the x axis, and the affinity of the T cell for the presented antigen is shown on the y axis. The horizontal dotted line in the left panel indicates the force needed to directly enrich for antigen-specific T cells. By contrast, the horizontal line in the right panel indicates the lower force needed to indirectly enrich for antigen-specific T cells by depleting non-specific T cells.

[0123] Quantification of CD8+ T cell proportions pre- and post-ATTACH confirmed a 5- fold antigen-dependent enrichment of OT-I CD8+ T cells in LLC-OVA257-264 slides compared to LLC slides (p=0.004) (FIG. 3B). To further elucidate the specificity of T cell enrichment, a heterogeneous CD8+ T cell population was used for de-enrichment experiments. A 1 : 1 ratio of OT-I to C57BL / 6 (B6) CD8+ T cells was added to channels containing LLC- OVA257-264 cells and 1 dyne / cm2 of shear stress was applied via infusion for 5 minutes. Quantification of fluorescent images pre- and post-ATTACH revealed a 2-fold enrichment of OT-I CD8+ T cells (p=0.0151) with up to 25% of input OT-I CD8+ T cells retained (FIG. 3C). Retention of C57BL / 6 CD8+ T was consistently only approximately 8% (p=0.7561) in channels seeded with either LLC or LLC- OVA257-264 (FIG. 3D).Example 4. ATTACH method in human system.

[0124] The ATTACH method was analyzed to demonstrate its ability to enrich antigenspecific T cells in a human system. T cells were transfected with previously validated TCRs (HLA-A*03:01 recognizing the neoantigen EGFR(L858R) and HLA-A*02:01 recognizing the Foxml tumor associated antigen) to produce EGFR-TCR-T cells and Foxml-TCR-T cells.Attorney Docket No.: 090723-1530732-MDA25-025PCTControl T cells (NT; normal HLA-A*03:01 or normal HLA-A*02:01) were not transfected. Cell lines overexpressing EGFR or Foxml were used as the target cells.

[0125] Flow cytometry analysis of EGFR-TCR-T cells (Cell Tracker CMAC, bottom right quadrant in each FACS plot) and non-transfected CD8+ cells (NT) (Cell Tracker deep red, top left quadrant in each FACS plot)) was performed for the input, wash fraction, and retained fraction as shown in FIG. 4A (left, middle, and right FACS plots, respectively). Analysis of EGFR-TCR-T in the retained fractions, after ATTACH (with 1-3 dyne / cm2), revealed an average 40% enrichment (p=0.0068) of EGFR-TCR-T (FIGS. 4B-4C). Live cell imaging of channels showed different ROIs where TCR-T killing of tumor cells was observed (data not shown). Quantification of fluorescent images before and after ATTACH revealed that 1.9, 1.2, and 2.1 times more Foxml-TCR-T cells were retained in channels where Foxml-TCR-T to NT ratios of 5: 1, 2: 1, and 1 : 1 were added, respectively (FIGS. 4D-4E). Further functional analysis of the fractions demonstrated that macrophage inflammatory protein- 1 beta (MIP-ip) concentration increased after a 24-hour incubation of the retained fraction of Foxml-TCR-T after ATTACH (right bar) as compared to a 24-hour incubation of the input cells only (left bar) (FIG. 4F)

[0126] These data demonstrate the feasibility of enriching for antigen-specific T cells recognizing solid tumor antigens using a microfluidic platform.Example 5. Additional ATTACH modifications.

[0127] Agitation method. In other embodiments, the disclosed ATTACH methods for the unbiased isolation of antigen-specific T cells were performed as described in the previous examples. In addition, the microfluidic slides were subjected to agitation during the step in which the effector cells (plurality of non-specific cells and antigen-specific T cells) were incubated with the target cells to allow binding of the effector cells to their target antigens on the target cells. In some embodiments, the agitation allowed for a reduction in the time required for the incubation step.

[0128] Centrifugation method. In other embodiments of the disclosed ATTACH methods for the unbiased isolation of antigen-specific T cells (from mouse OT-I T cells and B6 T cells), the target cells (mouse LLC-OVA and LLC) were seeded in a flask. The supernatant was decanted from the flask, and effector cells (plurality of non-specific cells and antigen-specific T cells (mouse OT-I T cells and B6 T cells)) were added to the target cells in media at a ratio of 1 : 1 to 1 :4 effectortarget cell. The flasks were centrifuged to force contact of the effector cells withAttorney Docket No.: 090723-1530732-MDA25-025PCT their target antigens on the target cells. Cells were co-incubated for 15 minutes to 4 hours, and supernatant was aspirated (non-specific cells were removed along with the supernatant), followed by a wash with PBS, and trypsinization to collect tumor cells and other adherent cells (e.g., antigen-specific T cells, for which the target antigen of each T cell was unknown). These experiments revealed that adherent cells were strongly enriched for antigen-specific OT-I T cells (but not B6 T cells) and that CD4 T cells (which do not bind to OVA) were almost entirely depleted (found in the supernatant and washed fraction) (data not shown). Forty minute incubation offered the greatest enrichment without increasing non-specific binding, and forcing cell-cell contact through centrifugation increased antigen-specific binding (data not shown). Importantly, using the LLC cell line which does not express OVA resulted in the same amount of enrichment between OT-I and B6 T cells suggesting this enrichment is in fact antigenspecific.

[0129] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

Attorney Docket No.: 090723-1530732-MDA25-025PCTWHAT IS CLAIMED IS:

1. A method for isolating antigen-specific T cells, comprising:(a) adhering target cells to a channel or surface of a microfluidic slide;(b) adding a heterogeneous population of cells to the target cells, wherein the heterogeneous population of cells comprises T cells that do not bind to the target cells and a plurality of T cells that bind to a plurality of different target antigens on the target cells, and wherein the target antigen to which each of the plurality of T cells binds is unknown;(c) allowing the plurality of T cells of the heterogeneous population to bind to the plurality of different target antigens on the target cells;(d) applying a volume of media with constant low shear stress to the channel to displace unbound cells and cells loosely bound to the target cells in a flow-through fraction, wherein the low shear stress is less than 10 dyne / cm2;(e) removing and discarding the flow-through fraction; and(f) applying mechanical force to collect the bound T cells, thereby isolating antigen-specific T cells.

2. The method of claim 1, wherein the microfluidic slide comprises glass or a polymer.

3. The method of claim 1, wherein 1 x 104- 5 x 105target cells are allowed to adhere to the channel or surface.

4. The method of claim 1, wherein 3 x 105- 5 x 105target cells are allowed to adhere to the channel or surface.

5. The method of any one of claims 1-4, wherein the target cells are tumor cells.

6. The method of claim 5, wherein the tumor cells are obtained from a human subject.

7. The method of claim 6, wherein the tumor cells are obtained from a tissue sample from a solid tumor.

8. The method of claim 7, wherein the tumor is selected from a group consisting of a melanoma, lung cancer, osteosarcoma, Ewing sarcoma, rhabdomyosarcoma, renal cellAttorney Docket No.: 090723-1530732-MDA25-025PCT carcinoma, colorectal carcinoma, thyroid cancer, hepatocellular carcinoma, hepatoblastoma, lymphoma, sarcoma, carcinoma, neuroblastoma, rhabdomyosarcoma, breast cancer, gynecological cancer, pancreatic cancer, prostate cancer, or head and neck cancer.

9. The method of claim 1, wherein step (a) comprises incubation of the target cells with the substrate for 8-12 hours.

10. The method of claim 1, wherein step (a) comprises incubation of the target cells with the substrate for 5-60 minutes.

11. The method of claim 1, wherein step (a) comprises incubation of the target cells with the substrate for 15-20 minutes.

12. The method of claim 1, wherein the target cells are biotinylated and the microfluidic slide comprises streptavidin.

13. The method of claim 1, wherein step (c) is performed in presence of dasatinib.

14. The method of claim 1, wherein step (b) comprises adding the heterogeneous population of cells to the target cells at a ratio of 1 : 1 to 1 :4.

15. The method of claim 1, wherein step (c) comprises incubation of the T cells with the target cells for less than 60 minutes.

16. The method of claim 1, wherein step (c) comprises incubation of the T cells with the target cells for 5-40 minutes.

17. The method of claim 1, wherein step (c) comprises incubation of the T cells with the target cells for 15-20 minutes.

18. The method of claim 1, wherein step (c) comprises agitation of the microfluidic slide.

19. The method of claim 1, wherein the shear stress is less than 5 dyne / cm2.

20. The method of claim 1, wherein the shear stress is 1-3 dyne / cm2.

21. The method of claim 1, wherein the shear stress is 2 dyne / cm2.Attorney Docket No.: 090723-1530732-MDA25-025PCT22. The method of claim 1, wherein steps (d) and (e) are repeated three times.

23. The method of claim 1, wherein step (f) comprises adding trypsin and EDTA to the channel.

24. The method of claim 1, wherein the isolated antigen-specific T cells are administered to a subject.

25. The method of claim 1, further comprising determining the sequence of a T cell receptor of the isolated antigen-specific T cells.

26. The method of claim 25, further comprising introducing a nucleic acid encoding the T cell receptor into a plurality of T cells from a subject ex vivo and administering the resulting plurality of T cells to the subject.

27. A system for isolating antigen-specific T cells, comprising:(a) a microfluidic slide with a channel or surface to which cells can bind;(b) a device at a first end of the microfluidic slide for administering a solution at constant low shear stress to the microfluidic slide, wherein the low shear stress is less than about 10 dyne / cm2;(c) a container at a second end of the microfluidic slide for collecting a flowthrough fraction of the solution;(d) optionally a device for agitation of the microfluidic slide; and(e) optionally, a means for analysis of cells.

28. The system of claim 27, wherein the device for administering the solution at constant low shear stress is a syringe pump.

29. The system of claim 28, wherein the syringe is attached to the microfluidic slide by silicon tubing.

30. The system of claim 27, wherein the container is attached to the microfluidic slide by silicon tubing, and wherein the container is removable for the discarding the flow-through fraction.

31. The system of any one of claims 27-30, wherein the removable container is attached to the microfluidic slide by silicon tubing.Attorney Docket No.: 090723-1530732-MDA25-025PCT32. A method for isolating antigen-specific T cells, comprising:(a) adhering target cells to a surface of a flask;(b) adding a heterogeneous population of cells to the target cells, wherein the heterogeneous population of cells comprises T cells that do not bind to the target cells and a plurality of T cells that bind to a plurality of different target antigens on the target cells, and wherein the target antigen to which each of the plurality of T cells binds is unknown;(c) allowing the T cells of the heterogeneous population to bind to the different target antigens on the target cells by centrifugation of the flask;(d) adding a volume of media to the flask to displace unbound cells and cells loosely bound to the target cells;(e) removing and discarding the supernatant; and(f) applying mechanical force to collect the bound T cells, thereby isolating antigen-specific T cells.

33. The method of claim 32, wherein 1 x 104- 5 x 105target cells are allowed to adhere to the surface of the flask.

34. The method of claim 33, wherein 3 x 105- 5 x 105target cells are allowed to adhere to the surface of the flask.

35. The method of any one of claims 32-34, wherein the target cells are tumor cells.

36. The method of claim 35 wherein the tumor cells are obtained from a human subject.

37. The method of claim 36, wherein the tumor cells are obtained from a tissue sample from a solid tumor.

38. The method of claim 37, wherein the tumor is selected from a group consisting of a melanoma, lung cancer, osteosarcoma, Ewing sarcoma, rhabdomyosarcoma, renal cell carcinoma, colorectal carcinoma, thyroid cancer, hepatocellular carcinoma, hepatoblastoma, lymphoma, sarcoma, carcinoma, neuroblastoma, rhabdomyosarcoma, breast cancer, gynecological cancer, pancreatic cancer, prostate cancer, or head and neck cancer.Attorney Docket No.: 090723-1530732-MDA25-025PCT39. The method of claim 32, wherein step (a) comprises incubation of the target cells with the flask for 8-12 hours.

40. The method of claim 32, wherein step (a) comprises incubation of the target cells with the flask for 5-60 minutes.

41. The method of claim 32, wherein step (a) comprises incubation of the target cells with the flask for 15-20 minutes.

42. The method of claim 32, wherein the target cells are biotinylated and the flask comprises streptavidin.

43. The method of claim 32, wherein step (b) comprises adding the heterogeneous population of cells to the target cells at a ratio of 1 : 1 to 1 :4.

44. The method of claim 32, wherein step (c) is performed in presence of dasatinib.

45. The method of claim 32, wherein step (c) comprises incubation of the T cells with the target cells for less than 60 minutes.

46. The method of claim 32, wherein step (c) comprises incubation of the T cells with the target cells for 5-40 minutes.

47. The method of claim 32, wherein step (c) comprises incubation of the T cells with the target cells for 15-20 minutes.

48. The method of claim 32, wherein steps (d) and (e) are repeated three times.

49. The method of claim 32, wherein step (f) comprises adding trypsin and EDTA to the flask.

50. The method of claim 32, wherein the isolated antigen-specific T cells are administered to a subject.

51. The method of claim 32, further comprising determining the sequence of a T cell receptor of the isolated antigen-specific T cells.Attorney Docket No.: 090723-1530732-MDA25-025PCT52. The method of claim 51, further comprising introducing a nucleic acid encoding the T cell receptor into a plurality of T cells from a subject ex vivo and administering the resulting plurality of T cells to the subject.

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