Method for isolating and enhancing a population of tumor-reactive lymphocytes

Abstract

Provided herein is a method for isolating and expanding a population of tumor-reactive lymphocytes (TRLs) from a body fluid sample of a subject in need of isolation and expansion of the population of TRLs. In some embodiments, the method for isolating and expanding a population of TRLs includes flowing the TRLs across a magnetic capture zone of a microfluidic device. Also provided herein is a method for administering a population of TRLs to a subject in need of administration of the population of TRLs. Further provided herein is a composition comprising therapeutically enhanced TRLs.

Description

Technical Field

[0001] Cross-reference This application claims the benefit of U.S. Provisional Patent Application No. 63 / 491,256, filed Mar. 20, 2023; U.S. Provisional Patent Application No. 63 / 390,195, filed Jul. 18, 2022; U.S. Provisional Patent Application No. 63 / 358,513, filed Jul. 5, 2022; and U.S. Provisional Patent Application No. 63 / 344,608, filed May 22, 2022. Each of these provisional patent applications is incorporated herein by reference in its entirety.

Background Art

[0002] Adoptive cell therapy (ACT) is a form of immunotherapy as a cancer treatment that uses cells derived from a patient's immune system, such as T cells. ACT requires isolating and modifying the patient's immune cells and then reinjecting the immune cells into the patient to enhance the ability to combat cancer.

Summary of the Invention

[0003] This specification describes a method for enriching a population of tumor-reactive lymphocytes (TRLs), the method comprising enriching a population of lymphocytes comprising CD103 from a peripheral blood sample of a subject. In some embodiments, enriching comprises magnetically separating by flowing a population of lymphocytes across a magnetic capture zone disposed in a channel of a microfluidic device. In some embodiments, the body fluid sample is a peripheral blood sample. In some embodiments, the body fluid sample comprises pleural effusion. In some embodiments, the body fluid sample comprises ascites. In some embodiments, magnetically separating achieves a recovery rate of at least 80%. In some embodiments, magnetically separating achieves a recovery rate of at least 90%. In some embodiments, the purity of the isolation of the population of TRLs is at least 60%. In some embodiments, the purity of the isolation of the population of TRLs is at least 70%. In some embodiments, the purity of the isolation of the population of TRLs is at least 80%. In some embodiments, magnetically separating comprises contacting the body fluid sample with an antibody capable of binding to a TRL surface protein. In some embodiments, the TRL surface protein is CD3, CD4, CD8, CD39, CD103, SLC6A19, SIDT1, or any combination thereof. In some embodiments, the TRL surface protein is CD103. In some embodiments, the TRL surface protein is CD39. In some embodiments, the TRL surface protein is SLC6A19. In some embodiments, the TRL surface protein is SIDT1. In some embodiments, the antibody is conjugated to magnetic nanoparticles. In some embodiments, magnetically separating comprises contacting the body fluid sample with a second antibody capable of binding to an antibody capable of binding to a TRL surface protein. In some embodiments, the second antibody is conjugated to magnetic nanoparticles. In some embodiments, magnetically separating comprises contacting the body fluid sample, which comprises a peripheral blood sample, with a plurality of major histocompatibility complex (MHC) multimers that mimic tumor epitopes. In some embodiments, at least one of the plurality of MHC multimers is conjugated to a linker molecule. In some embodiments, the linker molecule comprises a fluorophore. In some embodiments, the linker molecule is conjugated to magnetic particles.In some embodiments, the magnetic particles include magnetic nanoparticles. In some embodiments, the microfluidic device includes a plurality of magnetic capture zones, and the plurality of magnetic capture zones are arranged to spatially separate cells having different degrees of magnetization. In some embodiments, the subject has or is suspected of having cancer. In some embodiments, the cancer is in a tissue, and the tissue includes breast tissue, kidney tissue, cervical tissue, lung tissue, ovarian tissue, or skin tissue. In some embodiments, the method further includes culturing the magnetically separated TRLs. In some embodiments, the method includes introducing cells from the cultured TRLs into a nucleotide that expresses a therapeutic enhancing polypeptide under conditions sufficient for the cells to produce the enhancing polypeptide. In some embodiments, the method further includes culturing the cells to produce therapeutically enhanced cells. In some embodiments, the nucleotide encodes a chimeric antigen receptor (CAR). In some embodiments, the CAR includes an antigen-binding domain. In some embodiments, the antigen-binding domain binds to a tumor antigen. In some embodiments, the nucleotide encodes an engineered T cell receptor. In some embodiments, the engineered T cell receptor includes an antigen-binding domain. In some embodiments, the antigen-binding domain binds to a tumor antigen. In some embodiments, the method further includes administering the cultured TRLs to the subject in a therapeutically effective amount. In some embodiments, the subject is treated for cancer or precancer. In some embodiments, the cancer is in a tissue, and the tissue includes breast tissue, kidney tissue, cervical tissue, lung tissue, ovarian tissue, or skin tissue. In some embodiments, the therapeutically enhanced cells are administered with additional cancer therapies. In some embodiments, the additional cancer therapies include immune checkpoint inhibitor therapies. In some embodiments, the additional cancer therapies include co-stimulation therapies. In some embodiments, the co-stimulation therapy includes administering GITR. In some embodiments, the additional cancer therapy is an anti-PD1 therapy or an anti-PD-L1 therapy. In some embodiments, the anti-PD1 therapy or anti-PD-L1 therapy includes an anti-PD1 antibody or an antigen-binding fragment thereof. In some embodiments, the population of tumor-reactive lymphocytes (TRLs) includes a population of circulating tumor-reactive lymphocytes (cTRLs).

[0004] Disclosed herein is a method for enriching a population of tumor-reactive lymphocytes (TRLs), the method comprising enriching a population of lymphocytes comprising SLC6A19, SIDT1, or SLC6A19 and SIDT1 from a bodily fluid sample of a subject. In some embodiments, the population of lymphocytes further comprises CD3, CD4, CD8, CD39, CD103, or any combination thereof. In some embodiments, the population of lymphocytes comprises CD8, CD103, SLC6A19, and SIDT1. In some embodiments, enriching comprises magnetically separating by flowing the population of lymphocytes across a magnetic capture zone disposed in a channel of a microfluidic device. In some embodiments, the bodily fluid sample is a peripheral blood sample. In some embodiments, the bodily fluid sample comprises pleural effusion. In some embodiments, the bodily fluid sample comprises ascites. In some embodiments, magnetically separating achieves a recovery rate of at least 70%. In some embodiments, magnetically separating achieves a recovery rate of at least 80%. In some embodiments, magnetically separating achieves a recovery rate of at least 90%. In some embodiments, the purity of the isolation of the population of TRLs is at least 60%. In some embodiments, the purity of the isolation of the population of TRLs is at least 70%. In some embodiments, the purity of the isolation of the population of TRLs is at least 80%. In some embodiments, magnetically separating comprises contacting the bodily fluid sample with an antibody capable of binding to a TRL surface protein. In some embodiments, the TRL surface protein is CD3, CD4, CD8, CD39, CD103, SLC6A19, SIDT1, or any combination thereof. In some embodiments, the TRL surface protein is CD103. In some embodiments, the TRL surface protein is CD39. In some embodiments, the TRL surface protein is SLC6A19. In some embodiments, the TRL surface protein is SIDT1. In some embodiments, the antibody is conjugated to magnetic nanoparticles. In some embodiments, magnetically separating comprises contacting the bodily fluid sample with a second antibody capable of binding to an antibody capable of binding to a TRL surface protein. In some embodiments, the second antibody is conjugated to magnetic nanoparticles.In some embodiments, magnetic separation comprises contacting a body fluid sample, which includes a peripheral blood sample, with a plurality of major histocompatibility complex (MHC) multimers that mimic tumor epitopes. In some embodiments, at least one of the plurality of MHC multimers is conjugated to a linker molecule. In some embodiments, the linker molecule includes a fluorophore. In some embodiments, the linker molecule is conjugated to magnetic particles. In some embodiments, the magnetic particles include magnetic nanoparticles. In some embodiments, the microfluidic device includes a plurality of magnetic capture zones that are arranged to spatially separate cells having different magnetic susceptibilities. In some embodiments, the subject has or is suspected of having cancer. In some embodiments, the cancer is in tissue, and the tissue includes breast tissue, kidney tissue, cervical tissue, lung tissue, ovarian tissue, or skin tissue. In some embodiments, the method further comprises culturing the magnetically separated TRLs. In some embodiments, the method comprises introducing cells from the cultured TRLs into a nucleotide that expresses a therapeutic enhancing polypeptide under conditions sufficient for the cells to produce the enhancing polypeptide. In some embodiments, the method further comprises culturing the cells to produce therapeutically enhanced cells. In some embodiments, the nucleotide encodes a chimeric antigen receptor (CAR). In some embodiments, the CAR includes an antigen-binding domain. In some embodiments, the antigen-binding domain binds to a tumor antigen. In some embodiments, the nucleotide encodes an engineered T cell receptor. In some embodiments, the engineered T cell receptor includes an antigen-binding domain. In some embodiments, the antigen-binding domain binds to a tumor antigen. In some embodiments, the method further comprises administering the cultured TRLs to the subject in a therapeutically effective amount. In some embodiments, the subject is treated for cancer or precancer. In some embodiments, the cancer is in tissue, and the tissue includes breast tissue, kidney tissue, cervical tissue, lung tissue, ovarian tissue, or skin tissue. In some embodiments, the therapeutically enhanced cells are administered with additional cancer therapies. In some embodiments, the additional cancer therapies include immune checkpoint inhibitor therapies. In some embodiments, the additional cancer therapies include co-stimulation therapies.In some embodiments, the co-stimulatory therapy comprises administering GITR. In some embodiments, the additional cancer therapy is an anti-PD1 therapy or an anti-PD-L1 therapy. In some embodiments, the anti-PD1 therapy or the anti-PD-L1 therapy comprises an anti-PD1 antibody or an antigen-binding fragment thereof. In some embodiments, the population of tumor-reactive lymphocytes (TRL) comprises a population of circulating tumor-reactive lymphocytes (cTRL).

[0005] A method of isolating a population of tumor-reactive lymphocytes (TRLs), comprising magnetically separating a population of CD103+ lymphocytes from a body fluid sample of a subject, wherein the magnetic separation comprises flowing CD103+ lymphocytes across a magnetic capture zone disposed in a channel of a microfluidic device, is described. In some embodiments, the body fluid sample is a peripheral blood sample. In some embodiments, the body fluid sample comprises pleural effusion. In some embodiments, the body fluid sample comprises ascites. In some embodiments, the CD103+ lymphocytes are CD8+CD103+ lymphocytes, CD3+CD103+ lymphocytes, CD4+CD103+ lymphocytes, or CD39+CD103+ lymphocytes. In some embodiments, the CD103+ lymphocytes comprise CD8+CD103+SLC6A19+ lymphocytes, CD3+CD103+SLC6A19+ lymphocytes, or CD4+CD103+SLC6A19+ lymphocytes. In some embodiments, the CD103+ lymphocytes comprise CD8+CD103+SLC6A19+SIDT1+ lymphocytes, CD3+CD103+SLC6A19+SIDT1+ lymphocytes, CD4+CD103+SLC6A19+SIDT1+ lymphocytes. In some embodiments, the magnetic separation achieves a recovery rate of at least 70%. In some embodiments, the magnetic separation achieves a recovery rate of at least 80%. In some embodiments, the magnetic separation achieves a recovery rate of at least 90%. In some embodiments, the purity of the isolation of the population of TRLs is at least 60%. In some embodiments, the purity of the isolation of the population of TRLs is at least 70%. In some embodiments, the purity of the isolation of the population of TRLs is at least 80%. In some embodiments, the magnetic separation comprises contacting the body fluid sample with an antibody capable of binding to a TRL surface protein. In some embodiments, the TRL surface protein is CD3, CD4, CD8, CD39, CD103, SLC6A19, SIDT1, or any combination thereof. In some embodiments, the TRL surface protein is CD103. In some embodiments, the TRL surface protein is CD39. In some embodiments, the TRL surface protein is SLC6A19. In some embodiments, the TRL surface protein is SIDT1.In some embodiments, the antibody is conjugated to magnetic nanoparticles. In some embodiments, magnetic separation comprises contacting the body fluid sample with a second antibody that is capable of binding to an antibody that is capable of binding to the TRL surface protein. In some embodiments, the second antibody is conjugated to magnetic nanoparticles. In some embodiments, magnetic separation comprises contacting the body fluid sample with a plurality of major histocompatibility complex (MHC) multimers that mimic tumor epitopes, and the body fluid sample includes a peripheral blood sample. In some embodiments, at least one of the plurality of MHC multimers is conjugated to a linker molecule. In some embodiments, the linker molecule comprises a fluorophore. In some embodiments, the linker molecule is conjugated to a magnetic particle. In some embodiments, the magnetic particle comprises magnetic nanoparticles. In some embodiments, the microfluidic device comprises a plurality of magnetic capture zones, and the plurality of magnetic capture zones are arranged to spatially separate cells having different magnetic susceptibilities. In some embodiments, the subject has or is suspected of having cancer. In some embodiments, the cancer is in tissue, and the tissue includes breast tissue, kidney tissue, cervical tissue, lung tissue, ovarian tissue, or skin tissue. In some embodiments, the method further comprises culturing the magnetically separated TRL. In some embodiments, the method comprises introducing cells from the cultured TRL into a nucleotide that expresses a therapeutic enhancing polypeptide under conditions sufficient for the cells to produce the enhancing polypeptide. In some embodiments, the method further comprises culturing the cells to produce therapeutically enhanced cells. In some embodiments, the nucleotide encodes a chimeric antigen receptor (CAR). In some embodiments, the CAR comprises an antigen-binding domain. In some embodiments, the antigen-binding domain binds to a tumor antigen. In some embodiments, the nucleotide encodes an engineered T cell receptor. In some embodiments, the engineered T cell receptor comprises an antigen-binding domain. In some embodiments, the antigen-binding domain binds to a tumor antigen. In some embodiments, the method further comprises administering the cultured TRL to the subject in a therapeutically effective amount. In some embodiments, the subject is treated for cancer or precancer.In some embodiments, the cancer is in a tissue, and the tissue includes breast tissue, kidney tissue, cervical tissue, ovarian tissue, lung tissue, or skin tissue. In some embodiments, the therapeutically enhanced cells are administered with an additional cancer therapy. In some embodiments, the additional cancer therapy includes immune checkpoint inhibitor therapy. In some embodiments, the additional cancer therapy includes co-stimulation therapy. In some embodiments, the co-stimulation therapy includes administering GITR. In some embodiments, the additional cancer therapy is anti-PD1 therapy or anti-PD-L1 therapy. In some embodiments, the anti-PD1 therapy or anti-PD-L1 therapy includes an anti-PD1 antibody or an antigen-binding fragment thereof. In some embodiments, the population of tumor-reactive lymphocytes (TRL) includes a population of circulating tumor-reactive lymphocytes (cTRL).

[0006] This specification describes a pharmaceutical preparation comprising a plurality of cells including (i) a population of SLC6A19+ lymphocytes, (ii) a population of SIDT1+ lymphocytes, (iii) a population of CD103+ lymphocytes, or (iv) a combination of (i) to (iii), and a pharmaceutically acceptable excipient, diluent, or vehicle, which is formulated for administration to a subject having or suspected of having cancer. In some embodiments, the pharmaceutical preparation is formulated for administration together with an additional therapeutic agent for the treatment of cancer. In some embodiments, the additional therapeutic agent comprises an anti-PD1 antibody. In some embodiments, the CD103+ lymphocytes are CD8+CD103+ lymphocytes, CD3+CD103+ lymphocytes, CD4+CD103+ lymphocytes, or CD39+CD103+ lymphocytes. In some embodiments, the CD103+ lymphocytes are CD8+CD103+SLC6A19+ lymphocytes, CD3+CD103+SLC6A19+ lymphocytes, or CD4+CD103+SLC6A19+ lymphocytes. In some embodiments, the CD103+ lymphocytes are CD8+CD103+SLC6A19+SIDT1+ lymphocytes, CD3+CD103+SLC6A19+SIDT1+ lymphocytes, or CD4+CD103+SLC6A19+SIDT1+ lymphocytes. In some embodiments, the cancer is in the tissue of the subject, and the tissue includes breast tissue, kidney tissue, cervical tissue, lung tissue, ovarian tissue, or skin tissue. In some embodiments, at least a portion of the plurality of cells further comprises a chimeric antigen receptor (CAR). In some embodiments, the CAR comprises an antigen-binding domain. In some embodiments, the antigen-binding domain binds to a tumor antigen. In some embodiments, at least a portion of the plurality of cells further comprises an engineered T cell receptor. In some embodiments, the engineered T cell receptor comprises an antigen-binding domain. In some embodiments, the antigen-binding domain binds to a tumor antigen.

[0007] Disclosed herein are engineered tumor-reactive lymphocytes (TRLs) comprising (a) lymphocytes comprising cell surface markers including CD3, CD4, CD39, CD103, SLC6A19, or SIDT1, or any combination thereof, and (b) a chimeric antigen receptor (CAR). In some embodiments, the lymphocytes comprise (i) CD8, CD39, and CD103, (ii) CD3, CD39, and CD103, (iii) CD4, CD39, and CD103, (iv) CD8, CD103, SLC6A19, and SIDT1, (v) CD3, CD103, SLC6A19, and SIDT1, or (vi) CD4, CD103, SLC6A19, and SIDT1. In some embodiments, the CAR comprises an antigen-binding domain. In some embodiments, the antigen-binding domain binds to a tumor antigen. In some embodiments, the engineered TRLs disclosed herein are for use in treating cancer in a subject in need thereof.

[0008] Disclosed herein is a method of treating cancer in a subject, the method comprising administering to the subject cells produced by any of the methods disclosed herein. In some embodiments, the cancer is in a tissue of the subject, and the tissue comprises breast tissue, kidney tissue, cervical tissue, lung tissue, ovarian tissue, or skin tissue. In some embodiments, the method further comprises administering to the subject an additional cancer therapy. In some embodiments, the additional cancer therapy comprises immune checkpoint inhibitor therapy. In some embodiments, the additional cancer therapy comprises co-stimulatory therapy. In some embodiments, the co-stimulatory therapy comprises administering GITR. In some embodiments, the additional cancer therapy is anti-PD1 therapy or anti-PD-L1 therapy. In some embodiments, the anti-PD1 therapy or anti-PD-L1 therapy comprises an anti-PD1 antibody or an antigen-binding fragment thereof.

[0009] This specification provides a method for treating cancer in a subject, the method comprising administering to the subject a pharmaceutical formulation disclosed herein or an engineered TRL disclosed herein. In some embodiments, the cancer is in the subject's tissue, and the tissue includes breast tissue, kidney tissue, cervical tissue, lung tissue, ovarian tissue, or skin tissue. In some embodiments, the method further comprises administering to the subject an additional cancer therapy. In some embodiments, the additional cancer therapy includes immune checkpoint inhibitor therapy. In some embodiments, the additional cancer therapy includes co-stimulation therapy. In some embodiments, the co-stimulation therapy includes administering GITR. In some embodiments, the additional cancer therapy is anti-PD1 therapy or anti-PD-L1 therapy. In some embodiments, the anti-PD1 therapy or anti-PD-L1 therapy includes an anti-PD1 antibody or an antigen-binding fragment thereof.

[0010] This specification provides a kit comprising (a) an engineered TRL disclosed herein or a pharmaceutical formulation disclosed herein, and (b) instructions for administering the engineered TRL to a subject in need thereof.

[0011] This specification provides a kit comprising (a) cells produced by any of the methods disclosed herein, and (b) instructions for administering the cells to a subject.

[0012] This specification provides a system comprising a body fluid sample obtained from a subject, the body fluid sample containing a population of cells including (i) CD103+ lymphocytes, (ii) SLC6A19+ lymphocytes, (iii) SIDT1+ lymphocytes, or (iv) any combination of (i) to (iii), and a microfluidic device containing a magnetic capture zone disposed therein, the microfluidic device being configured to magnetically separate the population of cells from the body fluid sample. In some embodiments, the population of SLC6A19+ lymphocytes includes CD103+SLC6A19+SIDT1+ lymphocytes. In some embodiments, the population of SLC6A19+ lymphocytes includes CD8+CD103+SLC6A19+SIDT1+ lymphocytes. In some embodiments, the CD103+ lymphocytes are CD8+CD103+ lymphocytes. In some embodiments, the CD103+ lymphocytes are CD8+CD39+CD103+ lymphocytes. In some embodiments, the CD103+ lymphocytes include CD8+CD103+SLC6A19+ lymphocytes. In some embodiments, the body fluid sample is a peripheral blood sample. In some embodiments, the body fluid sample contains pleural effusion. In some embodiments, the body fluid sample contains ascites. In some embodiments, the system further includes an antibody or an antigen-binding fragment thereof capable of binding to a TRL surface protein. In some embodiments, the TRL surface protein is CD8, CD103, SLC6A19, SIDT1, or a combination thereof. In some embodiments, the antibody or the antigen-binding fragment thereof is conjugated to magnetic nanoparticles. In some embodiments, the system further includes a plurality of major histocompatibility complex (MHC) multimers that mimic tumor epitopes. In some embodiments, at least one of the plurality of MHC multimers is conjugated to a linker molecule containing a fluorophore or a magnetic particle. In some embodiments, the microfluidic device includes a plurality of magnetic capture zones, and the plurality of magnetic capture zones are arranged to spatially separate cells having different magnetization degrees. In some embodiments, the system further includes a cell culture configured to culture therapeutically enhanced cells derived from a population of (i) CD103+ lymphocytes, (ii) SLC6A19+ lymphocytes, or (iii) SIDT1+ lymphocytes.In some embodiments, the system further comprises a nucleic acid molecule encoding a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR). In some embodiments, the CAR or engineered TCR comprises an antigen-binding domain that binds to a tumor antigen. In some embodiments, the system further comprises a pharmaceutically acceptable excipient, diluent, or vehicle, and a population of (i) CD103+ lymphocytes, (ii) SLC6A19+ lymphocytes, or (iii) SIDT1+ lymphocytes is formulated with a pharmaceutically acceptable excipient, diluent, or vehicle for administration to a subject having or suspected of having cancer. In some embodiments, the system further comprises an additional cancer therapy. In some embodiments, the additional cancer therapy comprises an immune checkpoint inhibitor therapy or a co-stimulation therapy. In some embodiments, the additional cancer therapy is an anti-PD1 therapy or an anti-PD-L1 therapy. In some embodiments, the anti-PD1 therapy or anti-PD-L1 therapy comprises an anti-PD1 antibody or an antigen-binding fragment thereof.

[0013] The present specification provides a composition comprising cells derived from a sample enriched with lymphocytes comprising SLC6A19, SIDT1, CD103, or any combination thereof. In some embodiments, the sample is enriched with lymphocytes by a process comprising magnetic separation of lymphocytes from the sample, and magnetic separation comprises flowing lymphocytes across a magnetic capture zone disposed within a channel of a microfluidic device. In some embodiments, the process yields a sample having a purity of about 70% or greater. In some embodiments, the process yields a sample having a purity of about 80% or greater. In some embodiments, the lymphocytes further comprise CD3, CD4, CD8, CD39, CD103, or any combination thereof. In some embodiments, the lymphocytes further comprise CD8, CD103, SLC6A19, and SIDT1. In some embodiments, the sample is a peripheral blood sample. In some embodiments, the lymphocytes further comprise (i) CD8 and CD103, (ii) CD3 and CD103, (iii) CD4 and CD103, (iv) CD39 and CD103. In some embodiments, the lymphocytes further comprise (i) CD8, CD103, and SLC6A19, (ii) CD3, CD103, and SLC6A19, or (iv) CD4, CD103, and SLC6A19. In some embodiments, the lymphocytes further comprise (i) CD8, CD103, SLC6A19, and SIDT1, (ii) CD3, CD103, SLC6A19, and SIDT1, or (iii) CD4, CD103, SLC6A19, and SIDT1. In some embodiments, the lymphocytes comprise at least about 20,000 CD103+ cells per 10 million peripheral blood mononuclear cells. In some embodiments, the lymphocytes comprise at least about 2,000 CD103+ cells per 10 million peripheral blood mononuclear cells. In some embodiments, the lymphocytes comprise at least about 20,000 CD103+ cells, and the CD103+ cells have a purity of about 80% or greater. In some embodiments, the lymphocytes comprise at least about 2,000 CD103+ cells, and the CD103+ cells have a purity of about 80% or greater. In some embodiments, the sample is enriched with lymphocytes by about 70% or greater. In some embodiments, the sample is enriched with lymphocytes by about 80% or greater.In some embodiments, the sample has lymphocytes with a purity of about 70% or more. In some embodiments, the sample has lymphocytes with a purity of about 80% or more.

[0014] Disclosed herein are compositions comprising cells derived from a lymphocyte-enriched sample comprising SLC6A19, SIDT1, CD103, or any combination thereof, wherein the sample is enriched for lymphocytes by a process comprising the methods disclosed herein. In some embodiments, the lymphocytes further comprise CD3, CD4, CD8, CD39, CD103, or any combination thereof. In some embodiments, the lymphocytes further comprise CD8, CD103, SLC6A19, and SIDT1. In some embodiments, the sample is a peripheral blood sample. In some embodiments, the lymphocytes further comprise (i) CD8 and CD103, (ii) CD3 and CD103, (iii) CD4 and CD103, (iv) CD39 and CD103. In some embodiments, the lymphocytes further comprise (i) CD8, CD103, and SLC6A19, (ii) CD3, CD103, and SLC6A19, or (iv) CD4, CD103, and SLC6A19. In some embodiments, the lymphocytes further comprise (i) CD8, CD103, SLC6A19, and SIDT1, (ii) CD3, CD103, SLC6A19, and SIDT1, or (iii) CD4, CD103, SLC6A19, and SIDT1. In some embodiments, the lymphocytes comprise at least about 20,000 CD103+ cells per 10 million peripheral blood mononuclear cells. In some embodiments, the lymphocytes comprise at least about 2,000 CD103+ cells per 10 million peripheral blood mononuclear cells. In some embodiments, the lymphocytes comprise at least about 20,000 CD103+ cells, and the CD103+ cells comprise a purity of about 80% or more. In some embodiments, the lymphocytes comprise at least about 2,000 CD103+ cells, and the CD103+ cells comprise a purity of about 80% or more. In some embodiments, the sample is enriched for lymphocytes by about 70% or more. In some embodiments, the sample is enriched for lymphocytes by about 80% or more. In some embodiments, the sample has lymphocytes with a purity of about 70% or more. In some embodiments, the sample has lymphocytes with a purity of about 80% or more.

[0015] Incorporation by reference All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. If the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, this specification is intended to supersede and / or take precedence over any such conflicting subject matter.

Brief Description of the Drawings

[0016] The novel features of the concepts of the present invention are set forth in detail in the appended claims. The features and advantages of the concepts of the present invention will be better understood by reference to the following detailed description, which describes exemplary embodiments in which the principles of the concepts of the present invention are utilized, and to the accompanying drawings.

[0017]

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[0018] In some embodiments herein, compositions, systems, and kits comprising an isolated and enriched population of cells obtained from a subject sample, and methods for their isolation, enrichment, expansion, and use for the treatment of a disease or disorder disclosed herein are disclosed. In some embodiments, the sample comprises a fluid such as peripheral blood. In some embodiments, the disease or disorder comprises cancer. In some embodiments, the subject is suspected of having cancer. In some embodiments, the isolated and enriched population of cells, when administered to the subject as a treatment, can target and eliminate cancer cells in the subject. In some embodiments, the isolated and enriched population of cells is a tumor-reactive lymphocyte (TRL) or circulating TRL (cTRL) that can recognize cancer antigens of cancer and exhibit anti-cancer activity. Optionally, TRLs are present in the peripheral blood of the subject at a very low frequency, for example, at a frequency of about 0.002% of the peripheral blood T cell population. The isolated and enriched TRLs (e.g., isolated and enriched cTRLs) described herein can be used as an alternative to tumor-infiltrating lymphocytes (TILs) in adoptive cell therapy. The isolated and enriched TRLs disclosed herein can secrete interferon-gamma (IFN-γ). In some embodiments, the isolated and enriched TRLs can invade from the primary tumor into the peripheral blood and accumulate in secondary tumors. In some embodiments, TRLs can be characterized as having reactivity comparable to that of TILs. In some embodiments, TRLs can share a clonotype with TILs. In some embodiments, the population of TRLs can comprise 30% to 85% of the top 50 clones presented in the population of intratumoral TILs. In some embodiments, the TRLs described herein can have a tissue-resident-like (T rm -like) phenotype. In some embodiments, the isolated and enriched population of TRLs can be enhanced to generate a tumor-specific T cell therapy such as chimeric antigen receptor (CAR)-T therapy or T cell receptor (TCR) therapy.

[0019] In some embodiments, the TRLs can express CD8, CD103, CD3, CD4, CD39, SLC6A19, SIDT1, or any combination thereof. In some embodiments, the TRLs can express CD103. In some embodiments, the population of TRLs can include a CD103 signature. In some embodiments, the CD103 signature can define the population of TRLs. In some embodiments, the TRLs can express SLC6A19, SIDT1, or a combination thereof. In some embodiments, the TRLs can exhibit upregulated expression of TCF7, IL7R, LEF1, or a combination thereof. In some embodiments, the population of TRLs can include an SLC6A19+ signature and an SIDT1+ signature. In some embodiments, the CD8+ signature, CD103+ signature, SLC6A19+ signature, and SIDT1+ signature define the population of TRLs. In some embodiments, the TRLs can express CD3 or CD4. In some embodiments, the TRLs can be CD3+ pan-T cells. In some embodiments, the TRLs can be CD4+ helper T cells. In some embodiments, the TRLs can express CD39. In some embodiments, the TRLs can express CD39 and CD103. In some embodiments, the CD39+ signature and CD103+ signature define the population of TRLs. In some embodiments, the TRLs can express CD39, CD103, CD8, or a combination thereof. In some embodiments, the TRLs can express CD8, CD103, SLC6A19, SIDT1, or a combination thereof. In some embodiments, the TRLs can express CD3, CD103, SLC6A19, SIDT1, or a combination thereof. In some embodiments, the TRLs can express CD4, CD103, SLC6A19, SIDT1, or a combination thereof. In some embodiments, the TRLs can express CD4, CD39, CD103, SLC6A19, SIDT1, or a combination thereof.

[0020] Also disclosed herein is a method for isolating, enriching, and expanding a population of tumor-reactive lymphocytes (TRLs) from a peripheral blood sample. Described herein is a method for isolating and enriching a population of TRLs that are present in a fluid (e.g., peripheral blood) at a very low frequency (e.g., less than 0.002%) using a microfluidic device. In some embodiments, isolating the population of TRLs can include magnetically separating the population of TRLs using a microfluidic device. In some embodiments, microfluidic magnetic cell sorting can rely on immunomagnetic labeling of the population of TRLs, followed by magnetic separation within the microfluidic device.

[0021] Further disclosed herein is a method for enhancing an isolated and enriched population of TRLs comprising CD103, CD39, SLC6A19, and / or SIDT1 by introducing a nucleotide that expresses a therapeutic enhancing polypeptide under conditions sufficient for the cells to produce the enhancing polypeptide. In some embodiments, the nucleotide can encode a T cell receptor (TCR) or a chimeric antigen receptor (CAR). In some embodiments, the TCR or CAR is configured to recognize an antigen associated with cancer.

[0022] Disclosed herein is a method of providing a cell therapy to a subject in need thereof (e.g., adoptive cell therapy), the method comprising: (a) obtaining a population of cells or therapeutically enhanced cells described herein; and (b) providing the cell therapy by administering the population of cells to the subject. Also disclosed herein is a composition comprising a population of TRLs or therapeutically enhanced TRLs (e.g., expressing a CAR). In some embodiments, the composition can comprise a therapeutically effective amount of TRLs produced by any of the methods disclosed herein. In some embodiments, the composition can be for use in treating cancer.

[0023] In some embodiments herein, compositions, systems, and kits for producing or utilizing therapeutically active cells (e.g., enhanced TRLs) described herein are disclosed. The compositions disclosed herein can, in some embodiments, include a microfluidic device for separating a population of TRLs from a body fluid sample disclosed herein. In some embodiments, the composition can include an engineered T cell receptor (TCR), and a nucleic acid molecule encoding the TCR. In some embodiments, the composition can include an engineered chimeric antigen receptor (CAR), and a nucleic acid molecule encoding the CAR. In some embodiments, the fluid compositions disclosed herein are obtained from a subject in need of treatment with therapeutically active cells (e.g., autologous).

[0024] I. Compositions Disclosed herein is an isolated and enriched population of cells (e.g., T cells) from a body fluid (e.g., peripheral blood) of a subject (e.g., an individual suffering from cancer) that can include tumor-reactive lymphocytes (TRLs) capable of targeting and eliminating cancer cells. Also disclosed herein are enhanced TRLs for use in therapeutic applications, such as synthetic chimeric antigen receptors (CARs) that target tumor-specific antigens. The compositions disclosed herein can be, or can include, one or more components of the compositions disclosed herein, such as a polynucleotide encoding a chimeric antigen receptor. The compositions disclosed herein can be included in a pharmaceutical formulation, e.g., a formulation for administration to a subject disclosed herein. In some embodiments, a population of TRLs can be separated from a body fluid sample disclosed herein using the microfluidic devices disclosed herein. In some embodiments, the isolation and enrichment of tumor-reactive lymphocytes can include a process that includes magnetic separation of lymphocytes from the sample. This magnetic separation can include flowing the lymphocytes through a channel of a microfluidic device with one or more magnetic capture zones.

[0025] A. Tumor-reactive lymphocytes This specification discloses a composition comprising isolated and enriched tumor-reactive lymphocytes (TRLs) derived from a body fluid sample (e.g., peripheral blood) that express solute carrier family 6 member 19 (SLC6A19) (NCBI Entrez Gene: 340024; UniProtKB / Swiss-Prot: Q695T7), synthetic RNA interference-deficient (SID) transmembrane family member 1 (SIDT1) (NCBI Entrez Gene: 54847; UniProtKB / Swiss-Prot: Q9NXL6), cluster of differentiation 103 (CD103) (NCBI Entrez Gene: 3682; UniProtKB / Swiss-Prot: P38570), cluster of differentiation 39 (CD39) (NCBI Entrez Gene: 953; UniProtKB / Swiss-Prot: P49961), or any combination thereof. In some embodiments, the TRLs can be circulating tumor-reactive lymphocytes (cTRLs). In some embodiments, a population of TRLs (e.g., cTRLs) can comprise CD103-expressing cells. In some embodiments, a population of TRLs can comprise a CD103+ signature. In some embodiments, a population of TRLs can comprise CD39-expressing cells. In some embodiments, a population of TRLs can comprise a CD39+ signature. In some embodiments, the CD103+ signature can define a population of TRLs. In some embodiments, the TRLs can express SLC6A19, SIDT1, or a combination thereof. In some embodiments, a population of TRLs can comprise an SLC6A19+ signature and / or an SIDT1+ signature. In some embodiments, the SLC6A19+ signature and / or the SIDT1+ signature can define a population of TRLs.

[0026] In some embodiments, the isolated and enriched TRLs can contain about 100 to about 20,000 CD103-expressing cells per 10 million PBMCs (e.g., peripheral blood mononuclear cells). In some embodiments, the isolated and enriched TRLs contain, per 10 million PBMCs, about 100 to about 500, about 100 to about 1,000, about 100 to about 2,000, about 100 to about 3,000, about 100 to about 4,000, about 100 to about 5,000, about 100 to about 6,000, about 100 to about 8,000, about 100 to about 10,000, about 100 to about 15,000, about 100 to about 20,000, about 500 to about 1,000, about 500 to about 2,000, about 500 to about 3,000, about 500 to about 4,000, about 500 to about 5,000, about 500 to about 6,000, about 500 to about 8,000, about 500 to about 10,000, about 500 to about 15,000, about 500 to about 20,000, about 1,000 to about 2,000, about 1,000 to about 3,000, about 1,000 to about 4,000, about 1,000 to about 5,000, about 1,000 to about 6,000, about 1,000 to about 8,000, about 1,000 to about 10,000, about 1,000 to about 15,000, about 1,000 to about 20,000, about 2,000 to about 3,000, about 2,000 to about 4,000, about 2,000 to about 5,000, about 2,000 to about 6,000, about 2,000 to about 8,000, about 2,000 to about 10,000, about 2,000 to about 15,000, about 2,000 to about 20,000, about 3,000 to about 4,000, about 3,000 to about 5,000, about 3,000 to about 6,000, about 3,000 to about 8,000, about 3,000 to about 10,000, about 3,000 to about 15,000, about 3,000 to about 20,000, about 4,000 to about 5,000, about 4,000 to about 6,000, about 4,000 to about 8,000, about 4,000 to about 10,000, about 4,000 to about 15,000, about 4,000 to about 20,000, about 5,000 to about 6,000, about 5,000 to about 8,000, about 5,000 to about 10,000, about 5,000 to about 15,000, about 5,000 to about 20,000, about 6,It may contain 0 to approximately 8,000, approximately 6,000 to approximately 10,000, approximately 6,000 to approximately 15,000, approximately 6,000 to approximately 20,000, approximately 8,000 to approximately 10,000, approximately 8,000 to approximately 15,000, approximately 8,000 to approximately 20,000, approximately 10,000 to approximately 15,000, approximately 10,000 to approximately 20,000, or approximately 15,000 to approximately 20,000 CD103-expressing cells.,

[0027] In some embodiments, the isolated and enriched TRL may contain, per 10 million PBMCs, approximately 100, approximately 500, approximately 1,000, approximately 2,000, approximately 3,000, approximately 4,000, approximately 5,000, approximately 6,000, approximately 8,000, approximately 10,000, approximately 15,000, or approximately 20,000 CD103-expressing cells. In some embodiments, the isolated and enriched TRL may contain, per 10 million PBMCs, at least approximately 100, approximately 500, approximately 1,000, approximately 2,000, approximately 3,000, approximately 4,000, approximately 5,000, approximately 6,000, approximately 8,000, approximately 10,000, or approximately 15,000 CD103-expressing cells. In some embodiments, the isolated and enriched TRL may contain, per 10 million PBMCs, at most approximately 500, approximately 1,000, approximately 2,000, approximately 3,000, approximately 4,000, approximately 5,000, approximately 6,000, approximately 8,000, approximately 10,000, approximately 15,000, or approximately 20,000 CD103-expressing cells.,

[0028] In some embodiments, the isolated and enriched TRLs may contain about 100 to about 20,000 SLA6A19 and / or SIDT1-expressing cells per 10 million PBMCs (e.g., peripheral blood mononuclear cells). In some embodiments, the isolated and enriched TRLs contain, per 10 million PBMCs, about 100 to about 500, about 100 to about 1,000, about 100 to about 2,000, about 100 to about 3,000, about 100 to about 4,000, about 100 to about 5,000, about 100 to about 6,000, about 100 to about 8,000, about 100 to about 10,000, about 100 to about 15,000, about 100 to about 20,000, about 500 to about 1,000, about 500 to about 2,000, about 500 to about 3,000, about 500 to about 4,000, about 500 to about 5,000, about 500 to about 6,000, about 500 to about 8,000, about 500 to about 10,000, about 500 to about 15,000, about 500 to about 20,000, about 1,000 to about 2,000, about 1,000 to about 3,000, about 1,000 to about 4,000, about 1,000 to about 5,000, about 1,000 to about 6,000, about 1,000 to about 8,000, about 1,000 to about 10,000, about 1,000 to about 15,000, about 1,000 to about 20,000, about 2,000 to about 3,000, about 2,000 to about 4,000, about 2,000 to about 5,000, about 2,000 to about 6,000, about 2,000 to about 8,000, about 2,000 to about 10,000, about 2,000 to about 15,000, about 2,000 to about 20,000, about 3,000 to about 4,000, about 3,000 to about 5,000, about 3,000 to about 6,000, about 3,000 to about 8,000, about 3,000 to about 10,000, about 3,000 to about 15,000, about 3,000 to about 20,000, about 4,000 to about 5,000, about 4,000 to about 6,000, about 4,000 to about 8,000, about 4,000 to about 10,000, about 4,000 to about 15,000, about 4,000 to about 20,000, about 5,000 to about 6,000, about 5,000 to about 8,000, about 5,000 to about 10,000, about 5,000 to about 15,000, about 5,000 to about 20,It may contain 0, about 6,000 to about 8,000, about 6,000 to about 10,000, about 6,000 to about 15,000, about 6,000 to about 20,000, about 8,000 to about 10,000, about 8,000 to about 15,000, about 8,000 to about 20,000, about 10,000 to about 15,000, about 10,000 to about 20,000, or about 15,000 to about 20,000 SLA6A19 and / or SIDT1-expressing cells.,

[0029] In some embodiments, the isolated and enriched TRLs may contain about 100, about 500, about 1,000, about 2,000, about 3,000, about 4,000, about 5,000, about 6,000, about 8,000, about 10,000, about 15,000, or about 20,000 SLA6A19 and / or SIDT1-expressing cells per 10 million PBMCs (e.g., peripheral blood mononuclear cells). In some embodiments, the isolated and enriched TRLs may contain at least about 100, about 500, about 1,000, about 2,000, about 3,000, about 4,000, about 5,000, about 6,000, about 8,000, about 10,000, or about 15,000 cells per 10 million PBMCs. In some embodiments, the isolated and enriched TRLs may contain at most about 500, about 1,000, about 2,000, about 3,000, about 4,000, about 5,000, about 6,000, about 8,000, about 10,000, about 15,000, or about 20,000 SLA6A19 and / or SIDT1-expressing cells per 10 million PBMCs.,

[0030] In some embodiments, the purity of the isolated and enriched TRLs (e.g., CD39, CD103, SLC6A19, and / or SIDT1-expressing cells) described herein can be from about 10% to about 95%. In some embodiments, the purity of the isolated and enriched TRLs (e.g., CD103, SLC6A19, and / or SIDT1-expressing cells) described herein can be about 10% to about 15%, about 10% to about 20%, about 10% to about 25%, about 10% to about 30%, about 10% to about 40%, about 10% to about 50%, about 10% to about 60%, about 10% to about 70%, about 10% to about 80%, about 10% to about 90%, about 10% to about 95%, about 15% to about 20%, about 15% to about 25%, about 15% to about 30%, about 15% to about 40%, about 15% to about 50%, about 15% to about 60%, about 15% to about 70%, about 15% to about 80%, about 15% to about 90%, about 15% to about 95%, about 20% to about 25%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to about 60%, about 20% to about 70%, about 20% to about 80%, about 20% to about 90%, about 20% to about 95%, about 25% to about 30%, about 25% to about 40%, about 25% to about 50%, about 25% to about 60%, about 25% to about 70%, about 25% to about 80%, about 25% to about 90%, about 25% to about 95%, about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 70%, about 30% to about 80%, about 30% to about 90%, about 30% to about 95%, about 40% to about 50%, about 40% to about 60%, about 40% to about 70%, about 40% to about 80%, about 40% to about 90%, about 40% to about 95%, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 50% to about 95%, about 60% to about 70%, about 60% to about 80%, about 60% to about 90%, about 60% to about 95%, about 70% to about 80%, about 70% to about 90%, about 70% to about 95%, about 80% to about 90%, about 80% to about 95%, or about 90% to about 95%. In some embodiments, the purity of the isolated and enriched TRLs (e.g., CD39, CD103, SLC6A19, and / or SIDT1-expressing cells) described herein can be about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95%.In some embodiments, the purity of the isolated and enriched TRLs (e.g., CD39, CD103, SLC6A19, and / or SIDT1-expressing cells) described herein can be at least about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%. In some embodiments, the purity of the isolated and enriched TRLs (e.g., CD39, CD103, SLC6A19, and / or SIDT1-expressing cells) described herein can be at most about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 95%.

[0031] In some embodiments, the purity of the isolated and enriched TRLs (e.g., CD39, CD103, SLC6A19, or SIDT1 cells) described herein can be about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99%. In some embodiments, the purity of the isolated and enriched TRLs described herein is at least about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, or about 98%. In some embodiments, the purity of the isolated and enriched TRLs described herein can be at most about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99%.

[0032] In some embodiments, the isolated and enriched TRLs described herein can include at least about 100 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 90% per 10 million peripheral blood mononuclear cells (PBMCs), at least about 1,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 90% per 10 million PBMCs, at least about 2,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 90% per 10 million PBMCs, at least about 3,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 90% per 10 million PBMCs, at least about 4,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 90% per 10 million PBMCs, at least about 5,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 90% per 10 million PBMCs, at least about 6,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 90% per 10 million PBMCs, at least about 8,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 90% per 10 million PBMCs, at least about 10,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 90% per 10 million PBMCs, at least about 15,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 90% per 10 million PBMCs, or at least about 20,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 90% per 10 million PBMCs.

[0033] In some embodiments, the isolated and enriched TRLs described herein may comprise at least about 100 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 80% per 10 million PBMCs, at least about 1,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 80% per 10 million PBMCs, at least about 2,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 80% per 10 million PBMCs, at least about 3,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 80% per 10 million PBMCs, at least about 4,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 80% per 10 million PBMCs, at least about 5,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 80% per 10 million PBMCs, at least about 6,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 80% per 10 million PBMCs, at least about 8,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 80% per 10 million PBMCs, at least about 10,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 80% per 10 million PBMCs, at least about 15,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 80% per 10 million PBMCs, or at least about 20,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 80% per 10 million PBMCs.

[0034] In some embodiments, the isolated and enriched TRLs described herein may include at least about 100 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 70% per 10 million PBMCs, at least about 1,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 70% per 10 million PBMCs, at least about 2,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 70% per 10 million PBMCs, at least about 3,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 70% per 10 million PBMCs, at least about 4,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 70% per 10 million PBMCs, at least about 5,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 70% per 10 million PBMCs, at least about 6,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 70% per 10 million PBMCs, at least about 8,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 70% per 10 million PBMCs, at least about 10,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 70% per 10 million PBMCs, at least about 15,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 70% per 10 million PBMCs, or at least about 20,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 70% per 10 million PBMCs.

[0035] In some embodiments, the isolated and enriched TRLs described herein can comprise at least about 100 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 60% per 10 million PBMCs, at least about 1,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 60% per 10 million PBMCs, at least about 2,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 60% per 10 million PBMCs, at least about 3,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 60% per 10 million PBMCs, at least about 4,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 60% per 10 million PBMCs, at least about 5,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 60% per 10 million PBMCs, at least about 6,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 60% per 10 million PBMCs, at least about 8,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 60% per 10 million PBMCs, at least about 10,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 60% per 10 million PBMCs, at least about 15,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 60% per 10 million PBMCs, or at least about 20,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 60% per 10 million PBMCs.

[0036] In some embodiments, the isolated and enriched TRLs described herein can include at least about 100 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 50% per 10 million PBMCs, at least about 1,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 50% per 10 million PBMCs, at least about 2,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 50% per 10 million PBMCs, at least about 3,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 50% per 10 million PBMCs, at least about 4,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 50% per 10 million PBMCs, at least about 5,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 50% per 10 million PBMCs, at least about 6,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 50% per 10 million PBMCs, at least about 8,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 50% per 10 million PBMCs, at least about 10,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 50% per 10 million PBMCs, at least about 15,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 50% per 10 million PBMCs, or at least about 20,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 50% per 10 million PBMCs.

[0037] In some embodiments, the isolated and enriched TRLs described herein may include at least about 100 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 40% per 10 million PBMCs, at least about 1,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 40% per 10 million PBMCs, at least about 2,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 40% per 10 million PBMCs, at least about 3,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 40% per 10 million PBMCs, at least about 4,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 40% per 10 million PBMCs, at least about 5,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 40% per 10 million PBMCs, at least about 6,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 40% per 10 million PBMCs, at least about 8,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 40% per 10 million PBMCs, at least about 10,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 40% per 10 million PBMCs, at least about 15,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 40% per 10 million PBMCs, or at least about 20,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 40% per 10 million PBMCs.

[0038] In some embodiments, the isolated and enriched TRLs described herein may include at least about 100 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 30% per 10 million PBMCs, at least about 1,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 30% per 10 million PBMCs, at least about 2,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 30% per 10 million PBMCs, at least about 3,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 30% per 10 million PBMCs, at least about 4,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 30% per 10 million PBMCs, at least about 5,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 50% per 10 million PBMCs, at least about 6,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 30% per 10 million PBMCs, at least about 8,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 30% per 10 million PBMCs, at least about 10,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 30% per 10 million PBMCs, at least about 15,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 30% per 10 million PBMCs, or at least about 20,000 cells having a plurality of CD39, CD103, SLC6A19, and / or SIDT1 cells with a purity of at least about 30% per 10 million PBMCs.

[0039] In some embodiments, the population of TRLs can further express CD3, CD4, CD8, CD39, or any combination thereof. For example, the isolated and enriched TRLs can be CD3- and CD103-expressing cells. In some embodiments, the isolated and enriched TRLs can be CD4- and CD103-expressing cells. In some embodiments, the isolated and enriched TRLs can be CD8- and CD103-expressing cells. In some embodiments, the isolated and enriched TRLs can be CD39- and CD103-expressing cells. In some embodiments, the isolated and enriched TRLs can be CD3-, CD39-, and CD103-expressing cells. In some embodiments, the isolated and enriched TRLs can be CD4-, CD39-, and CD103-expressing cells. In some embodiments, the isolated and enriched TRLs can be CD8-, CD39-, and CD103-expressing cells. In some embodiments, the isolated and enriched TRLs can be CD3-, SLC6A19-, and SIDT1-expressing cells. In some embodiments, the isolated and enriched TRLs can be CD4-, SLC6A19-, and SIDT1-expressing cells. In some embodiments, the isolated and enriched TRLs can be CD8-, SLC6A19-, and SIDT1-expressing cells. In some embodiments, the isolated and enriched TRLs can be CD3-, CD103-, and SLC6A19-expressing cells. In some embodiments, the isolated and enriched TRLs can be CD4-, CD103-, and SLC6A19-expressing cells. In some embodiments, the isolated and enriched TRLs can be CD8-, CD103-, and SLC6A19-expressing cells. In some embodiments, the isolated and enriched TRLs can be CD3-, CD103, SLC6A19-, and SIDT1-expressing cells. In some embodiments, the isolated and enriched TRLs can be CD4-, CD103, SLC6A19-, and SIDT1-expressing cells. In some embodiments, the isolated and enriched TRLs can be CD8-, CD103, SLC6A19-, and SIDT1-expressing cells.

[0040] In some embodiments, a population of tumor-reactive lymphocytes (TRLs) can be found in a body fluid sample of a patient suffering from cancer. In some embodiments, the body fluid sample can include a peripheral blood sample. In some embodiments, the body fluid sample can be other biological fluids such as pleural effusion or ascites. In some embodiments, the body fluid sample can also be cord blood, bone marrow, lymph node, hepatic pleura, chest wall, peritoneal cavity, synovial fluid, peritoneum, retroperitoneal space, thymus, and tumor.

[0041] B. Enhanced TRLs In some embodiments herein, a composition is disclosed that includes a population of isolated and enriched TRLs (e.g., isolated and enriched cTRLs) that are therapeutically enhanced (e.g., to express a CAR or TCR). In some embodiments, genetic material encoding either a cloned TCR or a synthetic chimeric antigen receptor (CAR) that targets a tumor-specific antigen can be introduced into the isolated and enriched TRLs described herein.

[0042] Generally, a CAR is an engineered fusion protein constructed from an antigen recognition domain, a signaling domain, and a co-stimulatory domain that can be expressed in T cells to reprogram the T cells to specifically target tumor cells. In some embodiments, a CAR is a recombinant polypeptide construct that includes at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain that includes a functional signaling domain derived from a stimulatory molecule. An exogenous T cell receptor is similar to a CAR in that it can be engineered to recognize an antigen (e.g., a tumor antigen). In some embodiments, a TCR is a recombinant polypeptide.

[0043] In some embodiments, the extracellular antigen-binding domain is an antigen-binding fragment of an antibody, or a functional portion thereof (e.g., scFv) or a functional variant thereof. The specificity of the antigen-binding domain can be modified to treat various different disorders and can be monovalent or multivalent (e.g., divalent, trivalent). In some embodiments, the antigen-binding domain comprises an scFv and multivalent binding is provided by tandem addition of multiple scFvs having different antigen specificities. In some embodiments, the antigen-binding specificity and the intended target are consistent with any of the CAR-T constructs in modern clinical trials. For example, the specificity can include anti-CD19 (e.g., axicabtageneciloleucel in the case of relapsed / refractory diffuse large B-cell lymphoma, or tisagenlecleucel in the case of relapsed / refractory B-cell ALL and non-Hodgkin lymphoma), anti-CD22 (e.g., in the case of relapsed / refractory B-ALL), anti-CD19 / CD22 dual targeting (e.g., in the case of relapsed / refractory ALL), anti-CAIX (carbonic anhydrase 9), anti-PSMA (also known as FOLH1, e.g., in the case of renal cell carcinoma), anti-MUC1 (e.g., in the case of seminal vesicle carcinoma), anti-CD33 (e.g., in the case of acute myeloid leukemia), anti-mesothelin mRNA (e.g., in the case of adenocarcinoma and pleural mesothelioma), anti-FOLR1 (e.g., in the case of metastatic ovarian cancer), anti-carcinoembryonic antigen (also known as CEA, e.g., in the case of liver metastases of CEA-expressing adenocarcinoma), anti-IL13RA2 (e.g., in the case of glioblastoma), anti-HER2 (e.g., in the case of sarcoma), or any combination thereof. In some embodiments, one or more of the following antigens can be bound by the CAR-T construct: 1-40-β-amyloid, 4-1BB, 5AC, 5T4, 707-AP, A kinase anchor protein 4 (AKAP-4), activin receptor type 2B (ACVR2B), activin receptor-like kinase 1 (ALK1), adenocarcinoma antigen, adipophilin, adrenergic receptor beta-3 (ADRB3), AGS-22M6, alpha folate receptor, alpha-fetoprotein (AFP), AIM-2, anaplastic lymphoma kinase (ALK), androgen receptor, angiopoietin 2, angiopoietin 3, angiopoietin-binding cell surface receptor 2 (Tie2), anthrax toxin, AOC3 (VAP-1), B cell maturation antigen (BCMA), B7-H3 (CD276),Bacillus anthracis anthrax, B-cell activating factor (BAFF), B-lymphoma cells, bone marrow stromal cell antigen 2 (BST2), Brother of the Regulator of Imprinted Sites (BORIS), C242 antigen, C5, CA-125, cancer antigen 125 (CA-125 or MUC16), cancer / testis antigen 1 (NY-ESO-1), cancer / testis antigen 2 (LAGE-1a), carbonic anhydrase 9 (CA-IX), carcinoembryonic antigen (CEA), cardiac myosin, CCCTC-binding factor (CTCF), CCL11 (eotaxin-1), CCR4, CCR5, CD11, CD123, CD125, CD140a, CD147 (basigin), CD15, CD152, CD154 (CD40L), CD171, CD179a, CD18, CD19, CD2, CD20, CD200, CD22, CD221, CD23 (IgE receptor), CD24, CD25 (α-chain of IL-2 receptor), CD27, CD274, CD28, CD3, CD3ε, CD30, CD300 molecule-like family member f (CD300LF), CD319 (SLAMF7), CD33, CD37, CD38, CD4, CD40, CD40 ligand, CD41, CD44v7, CD44v8, CD44v6, CD5, CD51, CD52, CD56, CD6, CD70, CD72, CD74, CD79A, CD79B, CD80, CD97, CEA-related antigen, CFD, ch4D5, chromosome X open reading frame 61 (CXORF61), claudin 18.2 (CLDN18.2), claudin 6 (CLDN6), Clostridium difficile, agglutinin A, CLCA2, colony-stimulating factor 1 receptor (CSF1R), CSF2, CTLA-4, C-type lectin domain family 12 member A (CLEC12A), C-type lectin-like molecule-1 (CLL-1 or CLECL1), C-X-C chemokine receptor type 4, cyclin B1, cytochrome P450 1B1 (CYP1B1), cyp-B, cytomegalovirus, cytomegalovirus glycoprotein B, dabigatran, DLL4, DPP4, DR5, Escherichia coli Shiga toxin type 1, Escherichia coli Shiga toxin type 2, ecto-ADP-ribosyltransferase 4 (ART4),EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2), EGF-like domain multiple 7 (EGFL7), mutant elongation factor 2 (ELF2M), endotoxin, Ephrin A2, Ephrin B2, Ephrin A receptor 2, epidermal growth factor receptor (EGFR), epidermal growth factor receptor variant III (EGFRvIII), Epichalin, epithelial cell adhesion molecule (EpCAM), epithelial glycoprotein 2 (EGP-2), epithelial glycoprotein 40 (EGP-40), ERBB2, ERBB3, ERBB4, ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene), Escherichia coli, ETS translocation variant gene 6 located on chromosome 12p (ETV6-AML), respiratory syncytial virus F protein, FAP, Fc fragment of IgA receptor (FCAR or CD89), Fc receptor-like 5 (FCRL5), fetal acetylcholine receptor, fibrin II β chain, fibroblast activation protein α (FAP), fibronectin extra domain-B, FGF-5, Fms-like tyrosine kinase 3 (FLT3), folate-binding protein (FBP), folate hydrolase, folate receptor 1, folate receptor α, folate receptor β, Fos-related antigen 1, Frizzled receptor, fucosyl GM1, G250, G protein-coupled receptor 20 (GPR20), G protein-coupled receptor class C group 5, member D (GPRC5D), ganglioside G2 (GD2), GD3 ganglioside, glycoprotein 100 (gp100), glypican-3 (GPC3), GMCSF receptor α chain, GPNMB, GnT-V, growth differentiation factor 8, GUCY2C, mutant heat shock protein 70-2 (mut hsp70-2), hemagglutinin, hepatitis A virus cell receptor 1 (HAVCR1), hepatitis B surface antigen, hepatitis B virus, HER1, HER2 / neu, HER3, hexasaccharide moiety of globoH glycosphingolipid (GloboH), HGF, HHGFR, high molecular weight melanoma-associated antigen (HMW-MAA), histone complex, HIV-1, HLA-DR, HNGF, Hsp90, HST-2 (FGF6), human papillomavirus E6 (HPV E6), human papillomavirus E7 (HPV E7), human scatter factor receptor kinase, human telomerase reverse transcriptase (hTERT),Human TNF, ICAM-1 (CD54), iCE, IFN-α, IFN-β, IFN-γ, IgE, IgE Fc region, IGF-1, IGF-1 receptor, IGHE, IL-12, IL-13, IL-17, IL-17A, IL-17F, IL-1β, IL-20, IL-22, IL-23, IL-31, IL-31RA, IL-4, IL-5, IL-6, IL-6 receptor, IL-9, immunoglobulin lambda-like polypeptide 1 (IGLL1), influenza A hemagglutinin, insulin-like growth factor 1 receptor (IGF-I receptor), insulin-like growth factor 2 (ILGF2), integrin α4β7, integrin β2, integrin α2, integrin α4, integrin α5β1, integrin α7β7, integrin αllbβ3, integrin αvβ3, interferon α / β receptor, interferon y-induced protein, interleukin 11 receptor α (IL-11Rα), interleukin 13 receptor subunit α-2 (IL-13Ra2 or CD213A2), intestinal carboxylesterase, kinase domain region (KDR), KIR2D, KIT (CD117), L1-cell adhesion molecule (L1-CAM), legumain, leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), Lewis-Y antigen, LFA-1 (CD11A), LINGO-1, lipoteichoic acid, LOXL2, L-selectin (CD62L), lymphocyte antigen 6 complex, locus K9 (LY6K), lymphocyte antigen 75 (LY75), lymphocyte-specific protein tyrosine kinase (LCK), lymphotoxin-α (LT-α) or tumor necrosis factor-β (TNF-β), macrophage migration inhibitory factor (MIF or MMIF), M-CSF, mammary differentiation antigen (NY-BR-1), MCP-1, melanoma cancer testis antigen-1 (MAD-CT-1), melanoma cancer testis antigen-2 (MAD-CT-2), melanoma inhibitor of apoptosis (ML-IAP), melanoma-associated antigen 1 (MAGE-A1), mesothelin, mucin 1, cell surface-associated (MUC1), MUC-2, mucin CanAg, myelin-associated glycoprotein, myostatin, N-acetylglucosaminyl-transferase V (NA17), NCA-90 (granulocyte antigen), nerve growth factor (NGF), nerve apoptosis regulatory protease 1,Neural cell adhesion molecule (NCAM), neurite outgrowth inhibitors (e.g., NOGO-A, NOGO-B, NOGO-C), neuropilin-1 (NRP1), N-glycolylneuraminic acid, NKG2D, Notch receptor, o-acetyl-GD2 ganglioside (OAcGD2), olfactory receptor 51E2 (OR51E2), carcinoembryonic antigen (h5T4), breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl) oncogene fusion protein, Oryctolagus cuniculus, OX-40, oxLDL, p53 variant, paired box protein Pax-3 (PAX3), paired box protein Pax-5 (PAX5), pannexin 3 (PANX3), sodium phosphate cotransporter, phosphatidylserine, placenta-specific 1 (PLAC1), platelet-derived growth factor receptor α (PDGF-Rα), platelet-derived growth factor receptor β (PDGFR-β), polysialic acid, proacrosin-binding protein sp32 (OY-TES1), programmed cell death protein 1 (PD-1), proprotein convertase subtilisin / kexin type 9 (PCSK9), prostase, prostate carcinoma tumor antigen-1 (PCTA-1 or galectin 8), melanoma antigen recognized by T cell 1 (MelanA or MARTI), P15, P53, PRAME, prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), prostate acid phosphatase (PAP), prostate cancer cells, prostasin, protease serine 21 (testisin or PRSS21), proteasome (prososome, Macropain) subunit, beta type, 9 (LMP2), Pseudomonas aeruginosa, rabies virus glycoprotein, RAGE, Ras homolog family member C (RhoC), receptor activator of nuclear factor kappa-B ligand (RANKL), receptor for advanced glycation endproducts (RAGE-1), receptor tyrosine kinase-like orphan receptor 1 (ROR1), renal ubiquitous 1 (RU1), renal ubiquitous 2 (RU2), respiratory syncytial virus, Rh blood group D antigen, rhesus factor, sarcoma translocation breakpoint,Sclerostin (SOST), selectin P, sialyl Lewis adhesion molecule (sLe), sperm protein 17 (SPA17), sphingosine-1-phosphate, squamous cell carcinoma antigens recognized by T cells 1, 2, and 3 (SART1, SART2, and SART3), stage-specific embryonic antigen-4 (SSEA-4), , Staphylococcus aureus, STEAP1, survival, syndecan 1 (SDC1)+A314, SOX10, survivin, survival-2B, synovial sarcoma, X breakpoint 2 (SSX2), T cell receptor, TCRγ alternate reading frame protein (TARP), telomerase, TEM1, tenascin C, TGF-β (e.g., TGF-β1, TGF-β2, TGF-β3), thyroid-stimulating hormone receptor (TSHR), tissue factor pathway inhibitor (TFPI), Tn antigen ((Tn Ag) or (GalNAcα-Ser / Thr)), TNF receptor family member B cell maturation (BCMA), TNF-α, TRAIL-R1, TRAIL-R2, TRG, transglutaminase 5 (TGS5), tumor antigen CTAA16.88, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), tumor protein p53 (p53), tumor-specific glycosylation of MUC1, tumor-associated calcium signal transducer 2, tumor-associated glycoprotein 72 (TAG72). Tumor-associated glycoprotein 72 (TAG-72)+A327, TWEAK receptor, tyrosine kinase, tyrosine kinase-related protein 1 (TYRP1 or glycoprotein 75), tyrosine kinase-related protein 2 (TYRP2), uroplakin 2 (UPK2), vascular endothelial growth factor (e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D, PIGF), vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2 (VEGFR2), vimentin, v-myc avian myelocytomatosis virus oncogene neuroblastoma-derived homolog (MYCN), von Willebrand factor (VWF), Wilms tumor protein (WT1), X antigen family, member 1A (XAGE1), β-amyloid, and κ light chain.

[0044] In some embodiments, the transmembrane domain of the CAR is a domain that positions the CAR at the correct membrane location and stabilizes its structure. Suitable transmembrane domains can include the transmembrane regions of the α, β, or ζ chains of the T cell receptor, or transmembrane regions derived from CD28, CD3ε, CD3ζ, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or functional portions or functional variants thereof. Alternatively, the transmembrane domain can be synthetic and can include hydrophobic residues such as leucine and valine. In some embodiments, a triplet of phenylalanine, tryptophan, and valine is found at one or both ends of the synthetic transmembrane domain. Optionally, a short oligonucleotide or polypeptide linker, which in some embodiments is between 2 and 10 amino acids in length, can form a linkage between the transmembrane domain of the CAR and the cytoplasmic signaling domain. In some embodiments, the linker is a glycine-serine linker. In some embodiments, the CAR includes a cytoplasmic signaling domain that includes a functional signaling domain derived from a stimulatory molecule. In some embodiments, the stimulatory molecule is a stimulatory receptor molecule. In some embodiments, the stimulatory receptor molecule is a stimulatory receptor molecule of an adaptive immune cell. In some embodiments, the stimulatory molecule is the ζ chain associated with the T cell receptor complex. In some embodiments, the stimulatory molecule is, for example, FCER1G, FcγRIIa, FcRβ (FcεRib), CD3γ, CD3δ, CD3ε, CD79a, CD79b, DAP10, or DAP1, or functional portions or functional variants thereof. In some embodiments, the intracellular signaling domain includes one or more functional signaling domains derived from at least one costimulatory molecule. In some embodiments, the costimulatory molecule includes a ligand that binds to 4-1BB (i.e., CD137), CD27, CD28 CD30, CD40, PD-1, CD2, CD7, CD258, NKG2C, B7-H3, CD83, ICAM-1, LFA-1 (CD1 Ia / CD18), ICOS, functional portions or functional variants thereof, or combinations thereof.In some embodiments, the CAR comprises a leader sequence at the amino terminus (N-terminus) of the CAR fusion protein. In some embodiments, the CAR comprises a signal peptide sequence at the N-terminus of the extracellular antigen recognition domain, and the signal peptide sequence is optionally cleaved from the antigen recognition domain (e.g., scFv) or a functional portion or functional variant thereof during cellular processing and localization of the CAR to the cell membrane.

[0045] In some embodiments, the CARs disclosed herein are first, second, third, or fourth generation CAR systems, functional variants thereof, or combinations thereof. In some embodiments, a first generation CAR comprises an antigen-binding domain having specificity for a particular antigen (e.g., an antibody or antigen-binding fragment thereof such as an scFv of a heavy-chain only antibody, a Fab fragment, a VHH domain, or a VH domain), a transmembrane domain derived from an adaptive immune receptor (e.g., a transmembrane domain derived from the CD28 receptor or a functional portion or functional variant thereof), and a signaling domain derived from an adaptive immune receptor (e.g., three ITAM domains derived from the intracellular region of the CD3ζ receptor or FcεRIγ or a functional portion or functional variant thereof). In some embodiments, a second generation CAR construct comprises the elements of a first generation CAR and the addition of a co-stimulatory domain to the intracellular signaling domain portion of a CAR (e.g., derived from a co-stimulatory receptor that acts with a T cell receptor such as CD28, CD137 / 4-1BB, and CD134 / OX40 or a functional portion or functional variant thereof). In some embodiments, the co-stimulatory domain obviates the need to administer IL-2 with the first generation CAR. In some embodiments, a third generation CAR comprises the elements of a first generation CAR with multiple co-stimulatory domains added to the intracellular signaling domain portion of the CAR (e.g., CD3ζ-CD28-OX40, or CD3ζ-CD28-41BB, or functional portions or functional variants thereof). In some embodiments, a fourth generation CAR comprises an activating cytokine (e.g., IL-12, IL-23, or IL-27, or functional portions or functional variants thereof) added to the intracellular signaling portion of the CAR (typically between one or more of the co-stimulatory domains and the CD3ζ ITAM domain or a functional portion or functional variant thereof), or elements of a second or third generation CAR under the control of a CAR-inducible promoter (e.g., an NFAT / IL-2 minimal promoter or a functional portion or functional variant thereof).

[0046] Isolated and enriched TRLs may be produced to express CARs by various techniques known to those skilled in the art, which generally include isolating the subject T cells, activating the T cells, transducing the T cells with a CAR transgene, and expanding the transduced T cells to the number required for cell therapy. In some embodiments, T cells (e.g., isolated and enriched TRLs such as CD103, SLC6A19, and / or SIDT1-expressing cells) can be isolated using any of the methods disclosed herein. In some embodiments, transducing the cells with a CAR transgene includes introducing the cells into nucleotides that express the CAR under conditions sufficient for the cells to produce the CAR. Methods for introducing genetically engineered components such as CARs into T cells are well known to those skilled in the art and can be used to generate the CARs disclosed herein. Exemplary methods for introducing a nucleic acid encoding a CAR can include, for example, viral transduction via retroviral transduction or lentiviral transduction, transposons, and electroporation-mediated methods.

[0047] Also provided are polynucleotides encoding the compositions disclosed herein. In some embodiments, the vector can comprise a backbone and a polynucleotide encoding a chimeric antigen receptor (CAR), wherein the CAR comprises (a) an antigen-binding domain, (b) a transmembrane domain, (c) a co-stimulatory signaling domain (e.g., 4-1BB or CD28, or both), and / or (d) a CD3ζ signaling domain. In some embodiments, the vector can comprise one or more of the polynucleotides disclosed herein. In some embodiments, the vector can be a plurality of vectors. In some embodiments, the polynucleotide encoding the CAR can be transferred into TRL (e.g., cTRL) using a lentiviral vector. In some embodiments, the nucleotide encoding the CAR can be transferred into TRL using a retroviral vector. Optionally, the vector can be a non-viral vector. In some embodiments, the non-viral vector can be a Sleeping Beauty transposon. In some embodiments, the vector comprises a plasmid. Optionally, each of the vectors described herein can comprise an expression plasmid.

[0048] In some embodiments, the polynucleotide encoding the CAR can be cloned into a vector comprising a lentiviral backbone component. Exemplary backbone components include, but are not limited to, pFUGW and pSMPUW. The pFUGW lentiviral vector backbone is a self-inactivating (SIN) lentiviral vector backbone from which unnecessary HIV-1 viral sequences have been removed, reducing the likelihood of neoplasia, deleterious mutations, and the regeneration of infectious particles. In some embodiments, the CAR can be under the control of an inducible promoter. Optionally, the inducible promoter can be a two-polypeptide ecdysone receptor-based gene switch that is small molecule ligand-inducible. In some embodiments, the CAR can be under the control of a constitutive promoter.

[0049] Disclosed herein is a system for expressing a CAR during TRL (e.g., cTRL), the system comprising one or more vectors encoding the polynucleotides disclosed herein. Optionally, the system can further comprise a nucleic acid encoding at least one additional gene. In some embodiments, the additional gene can comprise a cytokine. In some embodiments, the cytokine can comprise at least one of IL-2, IL-15, IL-12, IL-21, and a fusion of IL-15 and IL-15Ra, or a functional portion or functional variant thereof. In some embodiments, the cytokine can be secreted. In some embodiments, the cytokine is membrane-bound.

[0050] C. Pharmaceutical Formulations Disclosed herein are pharmaceutical formulations comprising a TRL (e.g., cTRL) or an enhanced TRL (e.g., enhanced cTRL). In some embodiments, the pharmaceutical formulation can comprise a TRL or enhanced TRL comprising a population of SLC6A19+ lymphocytes, SIDT1+ lymphocytes, CD103+ lymphocytes, CD39+ lymphocytes, or a combination thereof. In some embodiments, the pharmaceutical formulation further comprises a pharmaceutically acceptable carrier, excipient, diluent, or nebulizer.

[0051] In some embodiments, the pharmaceutical formulation comprises two or more active agents, or two or more therapeutic agents disclosed herein. In some embodiments, the two or more active agents are included in a single dosage unit, for example, when a TRL or enhanced TRL (e.g., CAR or TCR) comprises or is administered with one or more therapeutic agents. In embodiments, the two or more active agents are included in separate dosage units, such as when a TRL or enhanced TRL (e.g., CAR or TCR) is administered separately from an additional therapeutic agent or adjuvant. In some embodiments, the active agent may be an additional therapeutic agent including, in some embodiments, a chemotherapeutic agent, a cytotoxic agent, a cytokine, a growth inhibitor, an antihormonal agent, an antiangiogenic agent, a cardioprotective agent, and / or a checkpoint inhibitor.Non-limiting checkpoint inhibitors include IMP321 / Eftilagimod α (Immutep), Relatlimab BMS-986016, Ipilimumab (Yervoy), Pembrolizumab (Keytruda), Nivolumab (Opdivo), Cemiplimab (Libtayo), Atezolizumab (Tecentriq), Avelumab (Bavencio), Durvalumab (Imfinzi), Ipilimumab (Yervoy), LAG525, MK-4280, Irinotecan, Oxaliplatin, REGN3767, TSR-033, BI754111, Sym022, FS118 (bispecific anti-LAG3 / PD-L1 antagonistic mAb), MGD013 (bispecific anti-LAG3 / PD-1 antagonistic mAb), TSR-022, Niraparib, Bevacizumab, MBG453, Decitabine, Spartalizumab, Sym023, INCAGN2390, LY3321367, Ramucirumab, Abemaciclib, Melesitnib, BMS-986258, SHR-1702, Camrelizumab, MK-7684, Etigilimab / OMP-313 M32, Tiragolumab (Tiragolumab) / MTIG7192A / RG-6058, BMS-986207, AB-154, ASP-8374, JNJ-61610588, CA-170d, Enoblituzumab / MGA271, MGD009, I-8H9 / Omburtamab, Trastuzumab, MGD013 (anti-PD-1, anti-LAG-3 dual checkpoint inhibitor), BGB-A1217, CM-24 (MK-6018), BMS986178, MEDI6469, PF-04518600, GSK3174998, MOXR0916, Utomilumab (PF-05082566), Urelumab (BMS-663513) ES101, BMS-986156, TRX-518, AMG228, JTX-2011, GSK3359609, BMS-986226, MEDI-570, or Valilumab (CDX-1127).

[0052] The pharmaceutical formulations described herein may be formulated for administration to a subject by a suitable route of administration, including but not limited to intravenous, intraarterial, oral, parenteral, buccal, topical, transdermal, rectal, intramuscular, subcutaneous, intraosseous, transmucosal, inhalation, or intraperitoneal routes. The compositions described herein may include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposome dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast-dissolving formulations, tablets, capsules, pills, delayed release formulations, sustained release formulations, pulsed release formulations, multiparticulate formulations, and immediate release / controlled release hybrid formulations.

[0053] Pharmaceutical formulations containing a therapeutic agent may be manufactured in the conventional manner, for example, by conventional processes of mixing, dissolving, granulating, sugar coating, powdering, emulsifying, encapsulating, entrapping, or compressing.

[0054] The pharmaceutical formulations may contain at least an exogenous therapeutic agent as an active ingredient in the free acid or free base form, or in a pharmaceutically acceptable salt form. Further, the methods and compositions described herein include the use of N-oxides (where appropriate), crystalline forms, amorphous phases, as well as active metabolites of these compounds having the same type of activity. In some embodiments, the therapeutic agent is present in the unsolvated form or in a solvated form with a pharmaceutically acceptable solvent such as water, ethanol. Solvated forms of the therapeutic agent are also contemplated as being disclosed herein.

[0055] In certain embodiments, the pharmaceutical formulations provided herein contain one or more preservatives that inhibit microbial activity. Suitable preservatives include, for example, mercury-containing substances such as merfen and thimerosal, stabilized chlorine dioxide, and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide, and cetylpyridinium chloride.

[0056] In some embodiments, the pharmaceutical formulations described herein benefit from antioxidants, metal chelators, thiol-containing compounds, and other common stabilizers. Examples of such stabilizers include, but are not limited to, (a) glycerol at about 0.5% to about 2% w / v, (b) methionine at about 0.1% to about 1% w / v, (c) monothioglycerol at about 0.1% to about 2% w / v, (d) EDTA at about 1 mM to about 10 mM, ascorbic acid at about 0.01% to about 2% w / v, (f) polysorbate 80 at 0.003% to about 0.02% w / v, (g) polysorbate 20 at 0.001% to about 0.05% w / v, (h) arginine, (i) heparin, (j) dextran sulfate, (k) cyclodextrin, (l) pentosan polysulfate and other heparinoids, (m) divalent cations such as magnesium and zinc, or (n) combinations thereof.

[0057] The pharmaceutical formulations described herein are formulated into any suitable dosage form including, but not limited to, aqueous oral dispersions, liquids, gels, syrups, elixirs, slurries, suspensions, solid oral dosage forms, aerosols, controlled release formulations, fast-dissolving formulations, foaming formulations, lyophilized formulations, tablets, powders, pills, dragees, capsules, delayed release formulations, sustained release formulations, pulsatile release formulations, multiparticulate formulations, and immediate release / controlled release mixed type formulations. In one aspect, the therapeutic agents discussed herein, e.g., the therapeutic agent, are formulated into a pharmaceutical composition suitable for intramuscular, subcutaneous, or intravenous injection. In one aspect, formulations suitable for intramuscular, subcutaneous, or intravenous injection include physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions, and sterile powders for rehydration into sterile injectable solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents, or vehicles include water, ethanol, polyols (such as propylene glycol, polyethylene-glycol, glycerol, Cremophor, etc.), suitable mixtures thereof, vegetable oils (such as olive oil), and injectable organic esters such as ethyl oleate. Appropriate fluidity can be maintained, for example, by the use of coatings such as lecithin, by maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In some embodiments, formulations suitable for subcutaneous injection also contain additives such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the growth of microorganisms can be ensured by various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, etc. In some embodiments, it is desirable to include isotonic agents such as sugars or sodium chloride. Prolonged absorption of injectable pharmaceutical forms can be brought about by the use of agents that delay absorption such as aluminum monostearate or gelatin.

[0058] For intravenous injection, infusion or instillation, the pharmaceutical formulations described herein are formulated in an aqueous solution, preferably in a physiologically compatible buffer such as Hank's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, a penetrant appropriate for the barrier to be permeated is used in the formulation. Such penetrants are generally known in the art. For other parenteral injections, suitable formulations preferably include an aqueous or non-aqueous solution together with a physiologically compatible buffer or excipient. Such excipients are known.

[0059] Parenteral injection may include bolus injection or continuous infusion. The pharmaceutical formulation for injection may be provided in a unit dosage form with a preservative, for example, an ampoule or a multi-dose container. The compositions described herein may be in a form suitable for parenteral injection as a sterile suspension, solution, or emulsion in an oily or aqueous vehicle, and may contain formulatory agents such as suspending agents, stabilizing agents, and / or dispersing agents. In one aspect, the active ingredient is in powder form for constitution together with a suitable vehicle, such as pyrogen-free sterile water, before use.

[0060] In the case of administration by inhalation, the therapeutic agent is formulated for use as an aerosol, mist, or powder. The pharmaceutical formulations described herein are conveniently delivered in a form that provides an aerosol spray from a pressurized pack or nebulizer using a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. By way of example only, capsules and cartridges, such as gelatin for use in an inhaler or insufflator, may be formulated to contain a powder mixture of a therapeutic agent described herein and a suitable powder base such as lactose or starch. Formulations containing the composition are prepared as solutions in saline using benzyl alcohol or other suitable preservatives, fluorocarbons, and / or other solubilizing or dispersing agents known in the art. Preferably, these compositions and formulations are prepared with suitable non-toxic pharmaceutically acceptable ingredients. The choice of suitable carrier depends on the exact nature of the desired nasal dosage form, e.g., solution, suspension, ointment, or gel. Nasal dosage forms generally contain large amounts of water in addition to the active ingredient. Small amounts of other ingredients may optionally be present, such as pH adjusters, emulsifying or dispersing agents, preservatives, surfactants, gelling agents, or buffering and other stabilizing agents, as well as solubilizing agents. Preferably, the nasal dosage form must be isotonic with nasal secretions.

[0061] A pharmaceutical preparation for oral use is obtained by mixing one or more solid excipients with one or more of the compositions described herein, optionally grinding the resulting mixture, adding suitable auxiliaries if desired, and then processing the mixture of granules to obtain tablets or dragee cores. Suitable excipients include, for example, fillers such as sugars including lactose, sucrose, mannitol, or sorbitol, cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methyl cellulose, microcrystalline cellulose, hydroxypropyl methyl cellulose, sodium carboxymethyl cellulose, or polyvinyl pyrrolidone (PVP or povidone) or other such as calcium phosphate. If desired, disintegrants such as cross-linked sodium croscarmellose, polyvinyl pyrrolidone, agar, or alginic acid or its salts (such as sodium alginate) are added. In some embodiments, dyes or pigments are added to the tablet or dragee coating for identification or to characterize different combinations of active therapeutic agent dosages.

[0062] In some embodiments, the pharmaceutical formulation of the exogenous therapeutic agent is in the form of a capsule, including gelatin push-fit capsules, soft-sealed gelatin capsules, and plasticizers such as glycerol and sorbitol. The push-fit capsule contains the active ingredient in a mixture with a filler such as lactose, a binder such as starch, and / or a lubricant such as talc or magnesium stearate, optionally with a stabilizer. In the soft capsule, the active therapeutic agent is dissolved or suspended in a suitable liquid such as fatty oil, liquid paraffin, or liquid polyethylene glycol. In some embodiments, a stabilizer is added. The capsule may be prepared, for example, by disposing a bulk blend of the therapeutic agent formulation inside the capsule. In some embodiments, the formulation (non-aqueous suspensions and solutions) is placed in soft gelatin capsules. In other embodiments, the formulation is placed in non-gelatin capsules such as standard gelatin capsules or capsules containing HPMC. In other embodiments, the formulation is placed in sprinkle capsules, in which case the entire capsule is swallowed or the contents are sprinkled on food prior to eating after opening the capsule.

[0063] A pharmaceutical formulation for oral administration is at a dosage suitable for such administration. In one aspect, a solid oral dosage form prepares the composition by mixing it with one or more of an antioxidant, a flavoring agent, and a carrier material such as a binder, a suspending agent, a disintegrant, a filler, a surfactant, a solubilizing agent, a stabilizer, a lubricant, a wetting agent, and a diluent. In some embodiments, the solid dosage forms disclosed herein are in the form of tablets (including suspension tablets, rapid-dissolving tablets, bite-disintegration tablets, rapidly disintegrating tablets, effervescent tablets, or caplets), pills, powders, capsules, solid dispersions, solid solutions, biodegradable dosage forms, controlled-release formulations, pulse-release dosage forms, multi-particle dosage forms, beads, pellets, granules. In other embodiments, the composition is in the form of a powder. Compressed tablets are solid dosage forms prepared by compressing the bulk blend of the above formulations. In various embodiments, the tablets contain one or more flavoring agents. In other embodiments, the tablets contain a film surrounding the finished compressed tablet. In some embodiments, the film coating can provide a delayed release of the therapeutic agent from the formulation. In other embodiments, the film coating aids patient compliance. The film coating is typically in the range of about 1 wt% to about 3 wt% of the tablet. In some embodiments, solid dosage forms such as tablets, effervescent tablets, and capsules are prepared by mixing the particles of the therapeutic agent with one or more pharmaceutical excipients to form a bulk blend composition. The bulk blend is readily subdivided into equivalently effective unit dosage forms such as tablets, pills, and capsules. In some embodiments, the individual unit dosages contain a film coating. These formulations are manufactured by conventional formulation techniques.

[0064] In another aspect, the dosage form includes a microencapsulated formulation. In some embodiments, one or more other compatible materials are present in the microencapsulating material. Non-limiting examples of materials include pH adjusters, erosion promoters, defoaming agents, antioxidants, flavoring agents, and carrier materials such as binders, suspending agents, disintegrants, fillers, surfactants, solubilizing agents, stabilizers, lubricants, wetting agents, and diluents.

[0065] The dosage form of the liquid formulation for oral administration is, optionally, an aqueous suspension selected from the group consisting of, but not limited to, pharmaceutically acceptable aqueous dispersing agents, emulsions, solutions, elixirs, gels, and syrups. In addition to the therapeutic agent, the liquid dosage form may contain additives, such as, (a) disintegrants, (b) dispersing agents, (c) wetting agents, (d) at least one preservative, (e) viscosity enhancers, (f) at least one sweetening agent, and (g) at least one flavoring agent, as necessary. In some embodiments, the aqueous dispersion further contains a crystal growth inhibitor.

[0066] In some embodiments, the pharmaceutical formulations described herein are self-emulsifying drug delivery systems (SEDDS). An emulsion is a dispersion of one immiscible phase in another immiscible phase, usually in the form of droplets. Generally, emulsions are produced by intense mechanical dispersion. In contrast to emulsions or microemulsions, SEDDS spontaneously form an emulsion when added to an excess of water without the need for any external mechanical dispersion or agitation. The advantage of SEDDS is that only gentle mixing is required to distribute the droplets throughout the solution. Additionally, water or an aqueous phase is optionally added immediately prior to administration, ensuring the stability of the labile or hydrophobic active ingredient. Thus, SEDDS provide an effective delivery system for the oral and parenteral delivery of hydrophobic active ingredients. In some embodiments, SEDDS provide an improvement in the bioavailability of hydrophobic active ingredients.

[0067] Oral formulations are administered using a variety of formulations known in the art. Additionally, the oral dosage forms described herein may further contain a biodegradable (hydrolyzable) polymer carrier that also functions to adhere the dosage form to the oral mucosa. In the case of oral or sublingual administration, the composition may be in the form of tablets, lozenges, or gels formulated in a conventional manner.

[0068] For intravenous injection, the pharmaceutical formulation is optionally formulated in an aqueous solution, preferably in a physiologically compatible buffer such as Hank's solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, a penetrant appropriate for the barrier to be permeated is used in the formulation. For other parenteral injections, the appropriate formulation preferably includes an aqueous or non-aqueous solution together with a physiologically compatible buffer or excipient.

[0069] Parenteral injection optionally includes a bolus injection or continuous infusion. The injectable formulation is optionally provided in a unit dosage form with a preservative, such as an ampoule or a multi-dose container. In some embodiments, the compositions described herein are in a form suitable for parenteral injection as a sterile suspension, solution, or emulsion in an oily or aqueous vehicle, and contain formulating agents such as suspending agents, stabilizing agents, and / or dispersing agents. The composition for parenteral administration includes an aqueous solution of a drug in a water-soluble form that regulates the activity of the carotid body. Further, a suspension of a drug that regulates the activity of the carotid body, such as an oily injection suspension, is optionally prepared.

[0070] Conventional formulation techniques include, for example, one or a combination of the following methods: (1) dry mixing, (2) direct compression, (3) grinding, (4) dry or non-aqueous granulation, (5) wet granulation, or (6) fusion. Other methods include, for example, spray drying, pan coating, melt granulation, granulation, fluidized bed spray drying or coating (such as Wurster coating), tangential coating, top spraying, tableting, extrusion, etc.

[0071] In some embodiments, a composition is provided that includes a therapeutic agent for oral administration to a subject and particles of at least one dispersing or suspending agent. The formulation may be a powder and / or granule for suspension, and when mixed with water, a substantially homogeneous suspension is obtained.

[0072] Furthermore, the pharmaceutical preparation optionally contains one or more pH adjusters or buffering agents, including acids such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid, bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and tris-hydroxymethylaminomethane, and buffering agents such as citrate / dextrose, sodium bicarbonate, and ammonium chloride. Such acids, bases, and buffering agents are included in the amounts necessary to maintain the pH of the composition within an acceptable range.

[0073] Furthermore, the pharmaceutical preparation optionally contains one or more salts in the amounts necessary to bring the weight osmolality of the composition within an acceptable range. Such salts include those having sodium, potassium, or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate, or bisulfite anions, and preferred salts include sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite, and ammonium sulfate.

[0074] Other pharmaceutical preparations optionally contain one or more preservatives that inhibit microbial activity. Suitable preservatives include, for example, mercury-containing substances such as merfen and thimerosal, stabilized chlorine dioxide, and quaternary ammonium compounds such as benzalkonium chloride, cetyltrimethylammonium bromide, and cetylpyridinium chloride.

[0075] In one embodiment, the aqueous suspension and dispersant described herein maintain a homogeneous state for at least 4 hours. In one embodiment, the aqueous suspension is resuspended into a homogeneous suspension by physical agitation that lasts less than 1 minute. In yet another embodiment, agitation is not required to maintain a homogeneous aqueous dispersion.

[0076] Aerosol formulations for nasal administration are generally aqueous solutions designed to be administered to the nasal passages as drops or sprays. Nasal solutions may be isotonic and slightly buffered to maintain a pH of about 5.5 to about 6.5, similar to nasal secretions, although pH values outside this range may also be used. Antimicrobial or preservative agents may also be included in the formulation.

[0077] Inhalation aerosol formulations and inhalants can be designed such that when administered via the nasal or oral breathing route, the drug or combination of drugs is delivered to the target respiratory tree. Inhalation solutions can be administered, for example, by a nebulizer. Inhalation drugs or insufflation drugs containing fine powders or liquid drugs can be delivered to the respiratory system as a pharmaceutical aerosol of a solution or suspension of the drug or combination of drugs in a propellant, for example, to assist in distribution. Propellants can be liquefied gases including hydrocarbons and hydrocarbon ethers, in addition to fluorocarbons such as halocarbons, for example, chlorofluorocarbons, hydrochlorofluorocarbons, and hydrochlorocarbons.

[0078] Halocarbon propellants can include fluorocarbon propellants in which all hydrogens are replaced by fluorine, chlorofluorocarbon propellants in which all hydrogens are replaced by chlorine and at least one fluorine, hydrogen-containing fluorocarbon propellants, and hydrogen-containing chlorofluorocarbon propellants. Useful hydrocarbon propellants include, for example, propane, isobutane, n-butane, pentane, isopentane, and neopentane. Blends of hydrocarbons can also be used as propellants. Ether propellants include, for example, dimethyl ether and other ethers. Aerosol formulations may also contain multiple propellants. For example, an aerosol formulation can contain multiple propellants of the same class, such as two or more fluorocarbons, or more than one, more than two, or more than three propellants of different classes, such as fluorohydrocarbons and hydrocarbons. The compositions of the present disclosure may also be dispensed with a compressed gas, such as an inert gas like carbon dioxide, nitrous oxide, or nitrogen.

[0079] Aerosol formulations may also contain other components, such as ethanol, isopropanol, propylene glycol, as well as surfactants, or other components such as oils and detergents. These components can serve to stabilize the formulation and / or lubricate the valve components.

[0080] Aerosol formulations may be packaged under pressure and may be formulated as aerosols using solutions, suspensions, emulsions, powders, and semi-solid preparations. For example, an aerosol formulation solution may contain a solution of an agent, such as a transporter, carrier, or ion channel inhibitor, in a (substantially) pure propellant or as a mixture of a propellant and a solvent. The solvent may be used to dissolve the agent and / or retard the evaporation of the propellant. Examples of solvents can include water, ethanol, and glycols. Any combination of suitable solvents may be used, optionally in combination with preservatives, antioxidants, and / or other aerosol components.

[0081] Aerosol formulations may be dispersions or suspensions. An aerosol formulation suspension contains a suspension of an agent or combination of agents, such as a transporter, carrier, or ion channel inhibitor, and a dispersant. Examples of dispersants can include sorbitan trioleate, oleyl alcohol, oleic acid, lecithin, and corn oil. Aerosol formulation suspensions may also contain lubricants, preservatives, antioxidants, and / or other aerosol components.

[0082] Aerosol formulations may likewise be formulated as emulsions. An aerosol formulation emulsion may contain, for example, an alcohol such as ethanol, a surfactant, water, and a propellant, as well as an agent or combination of agents, such as a transporter, carrier, or ion channel. The surfactant used can be nonionic, anionic, or cationic. An example of an aerosol formulation emulsion contains, for example, ethanol, a surfactant, water, and a propellant. Another example of an aerosol formulation emulsion contains, for example, vegetable oil, glyceryl monostearate, and propane.

[0083] II. Method This specification discloses methods for isolating, enriching, expanding, and using tumor-reactive lymphocytes (TRLs) or enhanced TRLs of the present disclosure. Also provided is a method for producing enhanced TRLs from the TRLs of the present disclosure. This specification discloses a method for isolating and expanding a population of tumor-reactive lymphocytes (TRLs) from a bodily fluid sample (e.g., peripheral blood) of a subject. In some embodiments, the subject may have or be suspected of having cancer. For example, the subject may have cancer in breast tissue, kidney tissue, cervical tissue, lung tissue, ovarian tissue, or skin tissue. The obtained TRLs may be used, for example, in adoptive cell therapy. In some embodiments, the methods disclosed herein include isolating TRLs from the peripheral blood of a subject, and the TRL population includes CD103-expressing lymphocytes (CD103+ lymphocytes). In some embodiments, the methods described herein can include isolating a population of TRLs that express CD103, CD39, SLC6A19, and / or SIDT1 from the peripheral blood of a subject. In some embodiments, isolating the population of TRLs includes magnetically separating the population of TRLs using a microfluidic device. In some embodiments, the method of isolating the population of TRLs includes magnetically separating the population of TRLs using a microfluidic device. In some embodiments, microfluidic magnetic cell sorting relies on immunomagnetic labeling of the population of TRLs, followed by magnetic separation within a microfluidic device.

[0084] This specification also discloses a method for producing a population of enhanced TRLs. In some embodiments, the method of enhancing a population of TRLs includes isolating a population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes and expanding them in a nucleotide that expresses a therapeutic enhancing polypeptide under conditions sufficient for the cells to produce the enhancing polypeptide. In some embodiments, the nucleotide encodes a chimeric antigen receptor (CAR). In some embodiments, the CAR is configured to recognize an antigen associated with cancer.

[0085] Also disclosed herein is a method of providing a cell therapy comprising the TRL or enhanced TRL of the present disclosure to a subject in need of cell therapy (e.g., adoptive cell therapy). In some embodiments, the method comprises (a) obtaining a population of cells or therapeutically enhanced cells described herein, and (b) providing the cell therapy by administering the population of cells to the subject. In some embodiments, the isolated TRL is expanded using any of the methods disclosed herein. In some embodiments, autologous cells are preferred over allogeneic cells because they have an inherent heterogeneity that minimizes off-tissue effects while maximizing tumor recognition T cell receptors (TCRs). In some embodiments, the subject has cancer.

[0086] A. Method for Isolating Tumor-Reactive Lymphocytes In some embodiments herein, a method of isolating a population of TRLs (e.g., cTRLs) comprising obtaining a peripheral blood sample from a subject comprising lymphocytes, and separating from the sample a population of CD103, CD39, SLC6A19, and / or SIDT1-expressing TRLs is disclosed. In some embodiments, separating from the sample a population of CD103, CD39, SLC6A19, and / or SIDT1-expressing TRLs can comprise magnetic separation from the sample. In some embodiments, magnetic separation from the sample of a population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes can comprise immunomagnetically labeling the population of TRLs followed by magnetic separation within a microfluidic device.

[0087] In some embodiments, immunomagnetically labeling a population of TRLs can include attaching a magnetic label directly or indirectly to at least one surface marker of the TRLs. In some embodiments, the surface marker can be CD103, CD39, SLC6A19, SIDT1, or a combination thereof. In some embodiments, the surface marker can further include CD3, CD4, CD8, or a combination thereof. In some embodiments, the surface marker can be CD103. In some embodiments, the surface marker can be CD39. In some embodiments, the surface marker can be SLC6A19. In some embodiments, the surface marker can be SIDT1. In some embodiments, the surface marker can include CD3, CD4, CD8, CD39, CD103, SLC6A19, SIDT1, or a combination thereof. In some embodiments, the TRLs are labeled by an antibody (e.g., an anti-CD103 antibody) that targets the surface marker of interest. In some embodiments, the cells are labeled by a multimer (e.g., an MHC multimer) that targets the surface marker of interest. In some embodiments, the antibody or multimer is conjugated to magnetic microparticles or nanoparticles (MNPs). In some embodiments, the antibody or multimer is labeled by a secondary antibody conjugated to the MNPs. In some embodiments, after labeling, the TRLs obtain a magnetization level as a function of the expression level of the surface marker recognized by the antibody or multimer.

[0088] In some embodiments, the microfluidic device is configured to isolate a population of TRLs from a population of non-TRLs based on the magnetization level indicated by the TRL. For example, the microfluidic device may comprise a sorting chamber that includes several separated zones having different heights. In some embodiments, in each zone, the microstructure is patterned to create capture pockets that create low velocity zones for capturing magnetically labeled TRLs. In some embodiments, during operation, the microfluidic device is clamped by an array of magnets that generate a constant magnetic field within the sorting chamber and is connected to a syringe pump for fluid processing. Without being bound by any theory, when cells are added to the device, they are subject to two main forces: the magnetic force generated by the interaction of the MNPs with the constant magnetic field, and the fluid drag defined by the fluid velocity in a particular zone. When the magnetic force overcomes the drag, the cells acquire sufficient capture force to remain in a particular zone. If the magnetic force does not overcome the drag, the cells are flowed to the next zone with lower drag and, ultimately, if the cells cannot be captured by any zone, they are flowed to the syringe. After sorting, the cells captured in each zone are recovered by removing the external magnet.

[0089] In some embodiments, when capturing a population of rare cells (e.g., a population of TRLs), the microfluidic device exhibits a capture efficiency of about 20% to about 98%. In some embodiments, when capturing a population of rare cells (e.g., a population of TRLs), the microfluidic device exhibits a capture efficiency of about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to about 60%, about 20% to about 70%, about 20% to about 80%, about 20% to about 90%, about 20% to about 92%, about 20% to about 94%, about 20% to about 96%, about 20% to about 98%, about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 70%, about 30% to about 80%, about 30% to about 90%, about 30% to about 92%, about 30% to about 94%, about 30% to about 96%, about 30% to about 98%, about 40% to about 50%, about 40% to about 60%, about 40% to about 70%, about 40% to about 80%, about 40% to about 90%, about 40% to about 92%, about 40% to about 94%, about 40% to about 96%, about 40% to about 98%, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 50% to about 92%, about 50% to about 94%, about 50% to about 96%, about 50% to about 98%, about 60% to about 70%, about 60% to about 80%, about 60% to about 90%, about 60% to about 92%, about 60% to about 94%, about 60% to about 96%, about 60% to about 98%, about 70% to about 80%, about 70% to about 90%, about 70% to about 92%, about 70% to about 94%, about 70% to about 96%, about 70% to about 98%, about 80% to about 90%, about 80% to about 92%, about 80% to about 94%, about 80% to about 96%, about 80% to about 98%, about 90% to about 92%, about 90% to about 94%, about 90% to about 96%, about 90% to about 98%, about 92% to about 94%, about 92% to about 96%, about 92% to about 98%, about 94% to about 96%, about 94% to about 98%, or about 96% to about 98%. In some embodiments, when capturing a population of rare cells (e.g., a population of TRLs), the microfluidic device exhibits a capture efficiency of about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 92%, about 94%, about 96%, or about 98%.In some embodiments, when capturing a population of rare cells (e.g., a population of TRLs), the microfluidic device exhibits a capture efficiency of at least about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 92%, about 94%, or about 96%. In some embodiments, when capturing a population of rare cells (e.g., a population of TRLs), the microfluidic device exhibits a capture efficiency of at most about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 92%, about 94%, about 96%, or about 98%.

[0090] In some embodiments, the method of isolating the population of TRLs described herein achieves a higher cell recovery rate than standard cell sorting techniques (e.g., fluorescence-activated cell sorting, or MACS) performed on the same sample. In some embodiments, the microfluidic technique for cell sorting described herein achieves a higher cell recovery rate than standard cell sorting techniques while maintaining similar purity.

[0091] In some embodiments, the microfluidic approach to cell sorting described herein achieves a cell recovery rate that is at least about 0.5-fold to about 50-fold higher compared to standard cell sorting techniques. In some embodiments, the microfluidic approach to cell sorting described herein, compared to standard cell sorting techniques, is about 0.5-fold to about 1-fold, about 0.5-fold to about 2-fold, about 0.5-fold to about 2.5-fold, about 0.5-fold to about 5-fold, about 0.5-fold to about 7.5-fold, about 0.5-fold to about 10-fold, about 0.5-fold to about 20-fold, about 0.5-fold to about 30-fold, about 0.5-fold to about 40-fold, about 0.5-fold to about 50-fold, about 1-fold to about 2-fold, about 1-fold to about 2.5-fold, about 1-fold to about 5-fold, about 1-fold to about 7.5-fold, about 1-fold to about 10-fold, about 1-fold to about 20-fold, about 1-fold to about 30-fold, about 1-fold to about 40-fold, about 1-fold to about 50-fold, about 2-fold to about 2.5-fold, about 2-fold to about 5-fold, about 2-fold to about 7.5-fold, about 2-fold to about 10-fold, about 2-fold to about 20-fold, about 2-fold to about 30-fold, about 2-fold to about 40-fold, about 2-fold to about 50-fold, about 2.5-fold to about 5-fold, about 2.5-fold to about 7.5-fold, about 2.5-fold to about 10-fold, about 2.5-fold to about 20-fold, about 2.5-fold to about 30-fold, about 2.5-fold to about 40-fold, about 2.5-fold to about 50-fold, about 5-fold to about 7.5-fold, about 5-fold to about 10-fold, about 5-fold to about 20-fold, about 5-fold to about 30-fold, about 5-fold to about 40-fold, about 5-fold to about 50-fold, about 7.5-fold to about 10-fold, about 7.5-fold to about 20-fold, about 7.5-fold to about 30-fold, about 7.5-fold to about 40-fold, about 7.5-fold to about 50-fold, about 10-fold to about 20-fold, about 10-fold to about 30-fold, about 10-fold to about 40-fold, about 10-fold to about 50-fold, about 20-fold to about 30-fold, about 20-fold to about 40-fold, about 20-fold to about 50-fold, about 30-fold to about 40-fold, about 30-fold to about 50-fold, or about 40-fold to about 50-fold higher in cell recovery rate. In some embodiments, the microfluidic approach to cell sorting described herein achieves a cell recovery rate that is about 0.5-fold, about 1-fold, about 2-fold, about 2.5-fold, about 5-fold, about 7.5-fold, about 10-fold, about 20-fold, about 30-fold, about 40-fold, or about 50-fold higher compared to standard cell sorting techniques. In some embodiments, the microfluidic approach to cell sorting described herein achieves a cell recovery rate that is at least about 0.5-fold, about 1-fold, about 2-fold, about 2.5-fold, about 5-fold, about 7.5-fold, about 10-fold, about 20-fold, about 30-fold, or about 40-fold higher compared to standard cell sorting techniques.In some embodiments, the microfluidic approach to cell sorting described herein achieves a cell recovery rate that is up to about 1-fold, about 2-fold, about 2.5-fold, about 5-fold, about 7.5-fold, about 10-fold, about 20-fold, about 30-fold, about 40-fold, or about 50-fold higher compared to standard cell sorting approaches.

[0092] In some embodiments, the methods described herein may include isolating a population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes from a peripheral blood sample by treating the sample with a plurality of major histocompatibility complex (MHC) multimers that mimic defined tumor epitopes. Generally, T cells express a surface T cell receptor (TCR) that enables the T cell to recognize a peptide antigen bound to a major histocompatibility complex (MHC) molecule, and TCR recognition of the MHC-peptide complex results in T cell activation, clonal expansion, and differentiation of T cells into effector, memory, and regulatory T cells. MHC multimers contain multiple copies of the MHC-peptide complex. In some embodiments, the MHC multimer exhibits increased T cell affinity compared to monomers of the same complex. In some embodiments, the MHC molecule is a human MHC molecule. In some embodiments, the MHC molecule is a mouse MHC molecule. In some embodiments, the MHC molecule is a class 1 MHC molecule. In some embodiments, the MHC class 1 molecule is a human HLA-A, HLA-B, or HLA-C molecule. In some embodiments, the MHC class I molecule is a mouse H-2K, H-2D, or H-2L molecule. In some embodiments, the molecule is a class 2 MHC molecule. In some embodiments, the MHC multimer contains at least 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 MHC molecules. In some embodiments, the MHC multimer contains about 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 MHC molecules. In some embodiments, the MHC multimer contains an MHC dimer containing 2 MHC molecules. In some embodiments, the MHC multimer contains an MHC tetramer containing 4 MHC molecules. In some embodiments, the MHC multimer contains an MHC pentamer containing 5 MHC molecules. In some embodiments, the MHC multimer contains a dextramer containing 10 or more MHC molecules.

[0093] In some embodiments, the MHC multimer may contain a peptide. In some embodiments, the MHC multimer may contain a peptide-MHC complex. In some embodiments, the peptide can be any natural or non-natural peptide that can be presented by an MHC molecule. In some embodiments, the peptide-MHC complex can be reactive with a TRL. In some embodiments, the peptide-MHC complex can be reactive with CD8- and CD103-expressing lymphocytes. In some embodiments, the peptide-MHC complex can be reactive with CD39- and CD103-expressing lymphocytes. In some embodiments, the peptide-MHC complex mimics a defined tumor epitope. In some embodiments, the peptide contains an epitope of influenza A hemagglutinin. In some embodiments, the peptide contains amino acid residues 533-541 of influenza A hemagglutinin. In some embodiments, the peptide contains an epitope of chicken ovalbumin. In some embodiments, the peptide contains amino acid residues 257-264 of chicken ovalbumin. In some embodiments, the MHC multimer contains multiple peptide-MHC complexes. In some embodiments, each of the peptide-MHC complexes associates with a multimerization domain. In some examples, the peptide-MHC complex contains a peptide derived from MC-38 with the SIIVFNLL sequence and an H-2Kb molecule. In some embodiments, the MHC multimer is operably linked to magnetic nanoparticles. In some embodiments, the magnetic nanoparticles are bound to the MHC multimer via a fluorophore linker.

[0094] In some embodiments, the linker can include a polymeric linker such as an amino acid linker or a biotin linker. In some embodiments, the linker can be cleavable. In some embodiments, the linker may not be cleavable.

[0095] Microfluidic device for cell sorting In some embodiments, the methods described herein may include separating a population of TRLs from a peripheral blood sample. In some embodiments, the microfluidic devices disclosed herein can be used to separate a population of TRLs (e.g., CD103+ lymphocytes) from a peripheral blood sample. In some embodiments, the isolated TRLs may include CD103+ lymphocytes. In some embodiments, the isolated CD103+ lymphocytes can be CD8+CD103+ lymphocytes, CD3+CD103+ lymphocytes, CD4+CD103+ lymphocytes, or CD39+CD103+ lymphocytes. In some embodiments, the isolated CD103+ lymphocytes can be CD8+CD39+CD103+ lymphocytes, CD3+CD39+CD103+ lymphocytes, or CD4+CD39+CD103+ lymphocytes. In some embodiments, the isolated CD103+ lymphocytes may include CD8+CD103+SLC6A19+ lymphocytes, CD3+CD103+SLC6A19+ lymphocytes, or CD4+CD103+SLC6A19+ lymphocytes. In some embodiments, the isolated CD103+ lymphocytes may include CD8+CD103+SLC6A19+SIDT1+ lymphocytes, CD3+CD103+SLC6A19+SIDT1+ lymphocytes, or CD4+CD103+SLC6A19+SIDT1+ lymphocytes.

[0096] In some embodiments, any of the microfluidic devices disclosed in International Publication No. WO 2014 / 166000, the contents of which are incorporated herein by reference, can be used to separate TRLs from a peripheral blood sample. In some embodiments, once the TRLs are separated, the TRLs can be eluted from the microfluidic device by removing the inducer that acts on the TRLs (e.g., via removal of a magnetic field). In some embodiments, the captured TRLs can then be expanded, enhanced, or a combination thereof, by any of the methods disclosed herein.

[0097] The microfluidic devices disclosed herein can be configured to magnetically sort a population of cells. In some embodiments, the population of cells can include a population of TRLs. In some embodiments, the population of TRLs can be labeled with magnetic nanoparticles. In some embodiments, each of the magnetic nanoparticles can have a diameter of about 0-50 nm, a diameter of 51-100 nm, a diameter of 100-150 nm, or a diameter of 150-200 nm. In some embodiments, each of the magnetic nanoparticles can have a diameter of about 50 nm. In some embodiments, compared to conventional microbeads, the magnetic nanoparticles can have improved colloidal stability and thus can be useful for processing larger samples. In some embodiments, cells labeled with magnetic nanoparticles can be difficult to capture because their capture efficiency is further reduced by several orders of magnitude lower magnetic susceptibility compared to microbeads. Therefore, in some embodiments, the microfluidic devices disclosed herein can include a flow rate reduction structure that creates a localized region of lower flow rate as a sample containing cells flows through the device. In some embodiments, the presence of such a low flow rate region can enable the capture of magnetically labeled cells.

[0098] In some embodiments, the microfluidic device disclosed herein can include a microfluidic chip. In some embodiments, the microfluidic chip can include a sorting chamber. In some embodiments, the sorting chamber can be etched or formed on the chip. In some embodiments, the sorting chamber can communicate with a flow inlet and a flow outlet. In some embodiments, the flow inlet can be configured to receive a sample, such as a peripheral blood sample containing a population of TRLs suspended in a fluid medium, and the flow outlet can be configured to deliver the fluid medium depleted of TRLs. In some examples, a tube can be connected to the inlet such that the fluid medium can be delivered therethrough to the inlet. In some embodiments, a tube can be connected to the outlet such that the fluid medium can be received therethrough from the outlet. In some embodiments, the tube can be a silicone tube. In some embodiments, the microfluidic device can include a syringe pump capable of controlling the flow rate of the fluid medium at the inlet.

[0099] In some embodiments, the sorting chamber can include at least one magnetic capture zone. In some embodiments, the sorting chamber can include a plurality of magnetic capture zones. In some embodiments, the sorting chamber can include at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 magnetic capture zones. In some embodiments, the sorting chamber can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 magnetic capture zones. In some embodiments, at least two of the magnetic capture zones can have variable heights. In some embodiments, the magnetic capture zones can range in height from 50 to 800 μm. In some embodiments, the sorting chamber can include three magnetic capture zones, one having a height of about 100 μm, one having a height of about 200 μm, and one having a height of about 400 μm.

[0100] In some embodiments, the microfluidic device can comprise at least one magnet array located on the outer surface of a microfluidic chip disposed above or below the sorting chamber such that the at least one magnet array can generate a magnetic field within the magnetic capture zone. In some embodiments, the microfluidic device can comprise two magnet arrays located on two outer surfaces of the microfluidic chip above and below the sorting chamber such that the two magnet arrays can generate a magnetic field within the magnetic capture zone. In some embodiments, the magnets can be located in two arrays with alternating polarities on both sides of the sorting chamber. In some embodiments, at least one magnet array can generate a magnetic field strength of 0.1 - 0.5 T, 0.5 - 1 T, or 1 - 1.5 T in the magnetic capture zone. In some embodiments, at least one magnet array can generate a magnetic field strength of 0.5 - 1 T in the magnetic capture zone. In some embodiments, the magnets can comprise neodymium magnets. In some embodiments, the magnets can comprise N52 Nd FeB magnets.

[0101] In some embodiments, the magnetic capture zone can comprise a plurality of microstructures. In some embodiments, the microstructure can be a flow rate reduction structure configured to improve the capture of labeled cells with magnetic nanoparticles in the flow. In some embodiments, the microstructure can generate a localized region of lower flow velocity, enabling particle capture (e.g., the reduction in flow velocity can enable the magnetic force to overcome the drag force on the particles). In some embodiments, this structure can be designed to avoid the capture of non-target particles. For example, despite the lower flow velocity, the region of lower flow velocity can always have a sufficient flow velocity to wash non-target particles out of the device (i.e., the flow rate can be at least non-zero), while target particles can be captured within the low flow velocity region. In some embodiments, the microstructure is X-shaped.

[0102] In some embodiments, the device can include a plurality of magnetic capture zones, where the first zone comprises the inlet of the sorting chamber, the final magnetic capture zone includes the outlet of the sorting chamber, and the plurality of magnetic capture zones are disposed between the first magnetic capture zone and the final magnetic capture zone. In some embodiments, the size or pattern of the microstructure can vary between capture zones. In some embodiments, the height can vary between magnetic capture zones. In some embodiments, the first zone can exhibit the highest linear velocity, such that cells with a high magnetic content can be retained, because the retaining magnetic force can overcome the drag exerted by the locally high flow velocity. In some embodiments, the other magnetic capture zones can exhibit a gradually decreasing linear velocity, and the final magnetic capture zone exhibits the lowest velocity. This design can enable the capture of cells with a high level of magnetization in the first zone of the device, while cells with a lower magnetization can be sorted in subsequent zones depending on the level of magnetization.

[0103] In some of the methods disclosed herein, a population of TRLs suspended in a fluid can be injected through an inlet, across the magnetic capture zones, and through an outlet. In some embodiments, if the magnetic force exerted on the cells is sufficient to overcome the drag that allows the cells to flow through the capture zone, magnetically labeled cells can be captured in the capture zone. If the magnetic force does not overcome the drag, the cells are flushed to the next zone with lower drag and, ultimately, if the cells cannot be captured by any zone, they are flushed into a syringe. After sorting, the cells captured in each zone can be recovered by removing an external magnet.

[0104] In some of the methods disclosed herein, a population of cells can be loaded into a microfluidic device through an inlet at a flow rate of at least 1 milliliter per hour, 3 mL h-1, 6 mL h-1, 9 mL h-1, 12 mL h-1, 15 mL h-1, 18 mL h-1, 21 mL h-1, 24 mL h-1, 27 mL h-1, 30 mL h-1, 35 mL h-1, 40 mL h-1, 45 mL h-1, or 50 mL h-1. The magnetic force exerted on the cells can be determined, for example, by the size of the magnetic nanoparticles, the number of magnetic nanoparticles attached to the cells, the size of the cells, and the strength of the applied magnetic field. In some embodiments, the relationship between drag force and linear flow rate in a microfluidic device capable of magnetically capturing particles by exploiting microstructures where the velocity decreases can be disclosed in International Publication No. WO 2014 / 166000, the contents of which are incorporated herein by reference.

[0105] In some embodiments, the microfluidic approach to cell sorting disclosed herein can yield a population of CD39, CD103, SLC6A19, and / or SIDT1-expressing lymphocytes that is about 2-fold to about 20-fold greater than the population of CD39, CD103, SLC6A19, and / or SIDT1-expressing lymphocytes isolated from a subject's peripheral blood sample using fluorescence-activated cell sorting (FACS). In some embodiments, the microfluidic approach to cell sorting described herein can yield a population of CD39, CD103, SLC6A19, and / or SIDT1-expressing lymphocytes that is about 2-fold to about 4-fold, about 2-fold to about 7-fold, about 2-fold to about 8-fold, about 2-fold to about 10-fold, about 2-fold to about 12-fold, about 2-fold to about 14-fold, about 2-fold to about 16-fold, about 2-fold to about 17-fold, about 2-fold to about 18-fold, about 2-fold to about 19-fold, about 2-fold to about 20-fold, about 4-fold to about 7-fold, about 4-fold to about 8-fold, about 4-fold to about 10-fold, about 4-fold to about 12-fold, about 4-fold to about 14-fold, about 4-fold to about 16-fold, about 4-fold to about 17-fold, about 4-fold to about 18-fold, about 4-fold to about 19-fold, about 4-fold to about 20-fold, about 7-fold to about 8-fold, about 7-fold to about 10-fold, about 7-fold to about 12-fold, about 7-fold to about 14-fold, about 7-fold to about 16-fold, about 7-fold to about 17-fold, about 7-fold to about 18-fold, about 7-fold to about 19-fold, about 7-fold to about 20-fold, about 8-fold to about 10-fold, about 8-fold to about 12-fold, about 8-fold to about 14-fold, about 8-fold to about 16-fold, about 8-fold to about 17-fold, about 8-fold to about 18-fold, about 8-fold to about 19-fold, about 8-fold to about 20-fold, about 10-fold to about 12-fold, about 10-fold to about 14-fold, about 10-fold to about 16-fold, about 10-fold to about 17-fold, about 10-fold to about 18-fold, about 10-fold to about 19-fold, about 10-fold to about 20-fold, about 12-fold to about 14-fold, about 12-fold to about 16-fold, about 12-fold to about 17-fold, about 12-fold to about 18-fold, about 12-fold to about 19-fold, about 12-fold to about 20-fold, about 14-fold to about 16-fold, about 14-fold to about 17-fold, about 14-fold to about 18-fold, about 14-fold to about 19-fold, about 14-fold to about 20-fold, about 16-fold to about 17-fold, about 16-fold to about 18-fold, about 16-fold to about 19-fold, about 16-fold to about 20-fold, about 17-fold to about 18-fold, about 17-fold to about 19-fold, about 17-fold to about 20-fold, about 18-fold to about 19-fold, about 18-fold to about 20-fold, or about 19-fold to about 20-fold greater than the population of CD39, CD103, SLC6A19, and / or SIDT1-expressing lymphocytes.In some embodiments, the microfluidic approach to cell sorting described herein can result in a population of CD39, CD103, SLC6A19, and / or SIDT1-expressing lymphocytes that is about 2-fold, about 4-fold, about 7-fold, about 8-fold, about 10-fold, about 12-fold, about 14-fold, about 16-fold, about 17-fold, about 18-fold, about 19-fold, or about 20-fold greater than the population of CD39, CD103, SLC6A19, and / or SIDT1-expressing lymphocytes isolated from a subject's peripheral blood sample using fluorescence-activated cell sorting. In some embodiments, the microfluidic approach to cell sorting described herein can result in a population of CD39, CD103, SLC6A19, and / or SIDT1-expressing lymphocytes that is at least about 2-fold, about 4-fold, about 7-fold, about 8-fold, about 10-fold, about 12-fold, about 14-fold, about 16-fold, about 17-fold, about 18-fold, or about 19-fold greater than the population of CD39, CD103, SLC6A19, and / or SIDT1-expressing lymphocytes isolated from a subject's peripheral blood sample using fluorescence-activated cell sorting. In some embodiments, the microfluidic approach to cell sorting described herein can result in a population of CD39, CD103, SLC6A19, and / or SIDT1-expressing lymphocytes that is at most about 4-fold, about 7-fold, about 8-fold, about 10-fold, about 12-fold, about 14-fold, about 16-fold, about 17-fold, about 18-fold, about 19-fold, or about 20-fold greater than the population of CD39, CD103, SLC6A19, and / or SIDT1-expressing lymphocytes isolated from a subject's peripheral blood sample using fluorescence-activated cell sorting.

[0106] In some embodiments, the microfluidic approach for cell sorting disclosed herein can achieve a recovery rate of about 40% to about 99% of CD39, CD103, SLC6A19, or SIDT1 lymphocytes. In some embodiments, the microfluidic approach for cell sorting disclosed herein is about 40% to about 50%, about 40% to about 60%, about 40% to about 70%, about 40% to about 80%, about 40% to about 85%, about 40% to about 90%, about 40% to about 95%, about 40% to about 96%, about 40% to about 97%, about 40% to about 98%, about 40% to about 99%, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 85%, about 50% to about 90%, about 50% to about 95%, about 50% to about 96%, about 50% to about 97%, about 50% to about 98%, about 50% to about 99%, about 60% to about 70%, about 60% to about 80%, about 60% to about 85%, about 60% to about 90%, about 60% to about 95%, about 60% to about 96%, about 60% to about 97%, about 60% to about 98%, about 60% to about 99%, about 70% to about 80%, about 70% to about 85%, about 70% to about 90%, about 70% to about 95%, about 70% to about 96%, about 70% to about 97%, about 70% to about 98%, about 70% to about 99%, about 80% to about 85%, about 80% to about 90%, about 80% to about 95%, about 80% to about 96%, about 80% to about 97%, about 80% to about 98%, about 80% to about 99%, about 85% to about 90%, about 85% to about 95%, about 85% to about 96%, about 85% to about 97%, about 85% to about 98%, about 85% to about 99%, about 90% to about 95%, about 90% to about 96%, about 90% to about 97%, about 90% to about 98%, about 90% to about 99%, about 95% to about 96%, about 95% to about 97%, about 95% to about 98%, about 95% to about 99%, about 96% to about 97%, about 96% to about 98%, about 96% to about 99%, about 97% to about 98%, about 97% to about 99%, or about 98% to about 99% recovery rate of 103+ lymphocytes. In some embodiments, the microfluidic approach for cell sorting disclosed herein can achieve a recovery rate of about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of CD39, CD103, SLC6A19, or SIDT1 lymphocytes.In some embodiments, the microfluidic approach to cell sorting disclosed herein can achieve a recovery rate of at least about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, or about 98% of CD39, CD103, SLC6A19, or SIDT1 lymphocytes. In some embodiments, the microfluidic approach to cell sorting disclosed herein can achieve a recovery rate of up to about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% of CD39, CD103, SLC6A19, or SIDT1 lymphocytes.

[0107] In some embodiments, the purity of the cells isolated using the methods described herein can be from about 10% to about 99% or essentially pure (e.g., 100%). In some embodiments, the purity of the cells isolated using the methods described herein can be about 10% to about 15%, about 10% to about 20%, about 10% to about 25%, about 10% to about 30%, about 10% to about 40%, about 10% to about 50%, about 10% to about 60%, about 10% to about 70%, about 10% to about 80%, about 10% to about 90%, about 10% to about 99%, about 15% to about 20%, about 15% to about 25%, about 15% to about 30%, about 15% to about 40%, about 15% to about 50%, about 15% to about 60%, about 15% to about 70%, about 15% to about 80%, about 15% to about 90%, about 15% to about 99%, about 20% to about 25%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to about 60%, about 20% to about 70%, about 20% to about 80%, about 20% to about 90%, about 20% to about 99%, about 25% to about 30%, about 25% to about 40%, about 25% to about 50%, about 25% to about 60%, about 25% to about 70%, about 25% to about 80%, about 25% to about 90%, about 25% to about 99%, about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 70%, about 30% to about 80%, about 30% to about 90%, about 30% to about 99%, about 40% to about 50%, about 40% to about 60%, about 40% to about 70%, about 40% to about 80%, about 40% to about 90%, about 40% to about 99%, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 50% to about 99%, about 60% to about 70%, about 60% to about 80%, about 60% to about 90%, about 60% to about 99%, about 70% to about 80%, about 70% to about 90%, about 70% to about 99%, about 80% to about 90%, about 80% to about 99%, or about 90% to about 95%. In some embodiments, the purity of the cells isolated using the methods described herein can be about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99%. In some embodiments, the purity of the cells isolated using the methods described herein can be at least about 10%, about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, or about 90%.In some embodiments, the purity of the cells isolated using the methods described herein can be up to about 15%, about 20%, about 25%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 99%.

[0108] B. Culture In some embodiments herein, methods for enriching and expanding a population of TRLs (e.g., cTRLs) are disclosed. In some embodiments, the method can include magnetically separating the population of TRLs described using any of the methods disclosed herein, and culturing the magnetically separated population of TRLs.

[0109] In some embodiments, an initial cell population comprising from about 1,000 to about 20,000 magnetically separated CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes can be seeded into the culture system described herein. In some embodiments, about 1,000 to about 2,000, about 1,000 to about 3,000, about 1,000 to about 4,000, about 1,000 to about 5,000, about 1,000 to about 6,000, about 1,000 to about 7,000, about 1,000 to about 8,000, about 1,000 to about 9,000, about 1,000 to about 10,000, about 1,000 to about 15,000, about 1,000 to about 20,000, about 2,000 to about 3,000, about 2,000 to about 4,000, about 2,000 to about 5,000, about 2,000 to about 6,000, about 2,000 to about 7,000, about 2,000 to about 8,000, about 2,000 to about 9,000, about 2,000 to about 10,000, about 2,000 to about 15,000, about 2,000 to about 20,000, about 3,000 to about 4,000, about 3,000 to about 5,000, about 3,000 to about 6,000, about 3,000 to about 7,000, about 3,000 to about 8,000, about 3,000 to about 9,000, about 3,000 to about 10,000, about 3,000 to about 15,000, about 3,000 to about 20,000, about 4,000 to about 5,000, about 4,000 to about 6,000, about 4,000 to about 7,000, about 4,000 to about 8,000, about 4,000 to about 9,000, about 4,000 to about 10,000, about 4,000 to about 15,000, about 4,000 to about 20,000, about 5,000 to about 6,000, about 5,000 to about 7,000, about 5,000 to about 8,000, about 5,000 to about 9,000, about 5,000 to about 10,000, about 5,000 to about 15,000, about 5,000 to about 20,000, about 6,000 to about 7,000, about 6,000 to about 8,000, about 6,000 to about 9,000, about 6,000 to about 10,000, about 6,000 to about 15,000, about 6,000 to about 20,000, about 7,000 to about 8,000, about 7,000 to about 9,000, about 7,000 to about 10,000, about 7,000 to about 15,000, about 7,000 to about 20,000, about 8,000 to about 9,000, about 8,000 to about 10,000, about 8,000 to about 15,000, about 8,000 to about 20,000, about 9,000 to about 10,000, about 9,An initial cell population comprising from 0 to about 15,000, about 9,000 to about 20,000, about 10,000 to about 15,000, about 10,000 to about 20,000, or about 15,000 to about 20,000 magnetically separated CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes can be seeded into the culture system described herein. In some embodiments, an initial cell population comprising about 1,000, about 2,000, about 3,000, about 4,000, about 5,000, about 6,000, about 7,000, about 8,000, about 9,000, about 10,000, about 15,000, or about 20,000 magnetically separated CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes can be seeded into the culture system described herein. In some embodiments, an initial cell population comprising at least about 1,000, about 2,000, about 3,000, about 4,000, about 5,000, about 6,000, about 7,000, about 8,000, about 9,000, about 10,000, or about 15,000 magnetically separated CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes can be seeded into the culture system described herein. In some embodiments, an initial cell population comprising at most about 2,000, about 3,000, about 4,000, about 5,000, about 6,000, about 7,000, about 8,000, about 9,000, about 10,000, about 15,000, or about 20,000 magnetically separated CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes can be seeded into the culture system described herein.,

[0110] In some embodiments, culturing magnetically separated CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes can include growing the CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes in a cell culture to expand a population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes. In some embodiments, the population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes can expand at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, or at least 20-fold. In some embodiments, the population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes can expand at least 0-fold, 100-fold, 200-fold, 300-fold, 400-fold, 500-fold, 600-fold, 700-fold, 800-fold, 900-fold, 1000-fold, 1100-fold, 1200-fold, 1300-fold, 1400-fold, 1500-fold, 1600-fold, 1700-fold, 1800-fold, 1900-fold, 2000-fold, 2100-fold, 2200-fold, 2300-fold, 2400-fold, 2500-fold, 2600-fold, 2700-fold, 2800-fold, 2900-fold, 3000-fold, 3100-fold, 3200-fold, 3300-fold, 3400-fold, 3500-fold, 3600-fold, 3700-fold, 3800-fold, 3900-fold, 4000-fold, 4100-fold, 4200-fold, 4300-fold, 4400-fold, 4500-fold, 4600-fold, 4700-fold, 4800-fold, 4900-fold, or 5000-fold.

[0111] In some embodiments, magnetically separated lymphocytes can be cultured using the rapid expansion protocol disclosed in Dudley, M.E., Wunderlich, J.R., Shelton, T.E., Even, J., and Rosenberg, S.A., Generation of Tumor-Infiltrating Lymphocyte Cultures for Use in Adoptive Transfer Therapy for Melanoma Patients: J. Immunother. 26, 332-342 (2003), which is incorporated herein by reference in its entirety. In some embodiments, the culture can be achieved in a culture flask or other container known in the art using feeder cells, T cell growth factors, and monoclonal antibodies capable of inducing T cell activation. In some embodiments, the culture can be performed using a culture flask or container known to those of ordinary skill in the art. In some embodiments, the culture can be performed for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 11, 12, 13, or 14 days.

[0112] In some embodiments, the culture can be grown in the presence of a culture medium. In some embodiments, the culture medium can contain a T cell growth factor. In some embodiments, the T cell growth factor can include Il-2, Il-7, Il-9, or Il-15. In some embodiments, the T cell growth factor can include ll-2. In some embodiments, the culture medium can contain at least about 6,000 IU / mL of IL-2. In some embodiments, the culture medium can contain at least about 5 - 50 IU / mL, 50 - 500 IU / mL, 500 - 1000 IU / mL, 1000 - 1500 IU / mL, about 1500 - 2000 IU / mL, about 2000 - 2500 IU / mL, about 2500 - 3000 IU / mL, about 3000 - 3500 IU / mL, about 3500 - 4000 IU / mL, about 4000 - 4500 IU / mL, about 4500 - 5000 IU / mL, about 5000 - 5500 IU / mL, about 5000 - 6000 IU / mL, about 6000 - 6500 IU / mL, about 6500 - 7000 IU / mL, about 7000 - 7500 IU / mL, about 7500 - 8000 IU / mL, or about 8000 - 8500 IU / mL of IL-2.

[0113] In some embodiments, the culture medium can contain an antibody (e.g., a monoclonal antibody) capable of inducing T cell activation. In some embodiments, the culture medium can contain an OKT-3 antibody. In some embodiments, the culture medium can contain about 30 ng / mL of the OKT-3 antibody. In some embodiments, the culture medium can contain an antibody (e.g., a monoclonal antibody) specific for CD2, CD3, CD28, or any combination thereof. In some embodiments, the culture medium can contain multiple antibodies disclosed herein, such as anti-OKT-3 antibody, anti-CD2 antibody, anti-CD3 antibody, and anti-CD28 antibody, or any combination thereof. In some embodiments, the culture medium can be one or more, or each, of the antibodies at about 0.1 - 5 ng / mL, about 5 - 10 ng / mL, about 10 - 15 ng / mL, about 15 - 20 ng / mL, about 20 - 25 ng / mL, about 25 - 30 ng / mL, about 30 - 35 ng / mL, about 35 - 40 ng / mL, about 40 - 45 ng / mL, or about 45 - 50 ng / mL.

[0114] In some embodiments, the feeder cells can be allogeneic. In some embodiments, the feeder cells can be peripheral blood mononuclear cells (PBMCs). In some embodiments, the PBMCs can be obtained from standard whole blood from a donor. In some embodiments, the ratio of TRL to feeder cells can be about 1:25, about 1:50, about 1:100, about 1:125, about 1:150, about 1:175, about 1:200, about 1:225, about 1:250, about 1:275, about 1:300, about 1:325, about 1:350, about 1:375, or about 1:400.

[0115] In some embodiments, the TRL population recovered from the magnetic separation method described herein can be cultured using the growth protocol described herein. In some embodiments, irradiated PBMC feeder cells, OKT3 antibody, and TRL can be combined, mixed, and aliquoted into tissue culture flasks or tissue culture plates. In some embodiments, the TRL population can be cultured in a feeder-free system. In some embodiments, the flasks can be incubated upright at 37 °C in 5% CO2. In some embodiments, IL-2 is added to the culture flask at 6000 IU / mL on day 2. In some embodiments, IL-2 can be added to the culture flask at a concentration in the range of 5 - 50 IU / mL. In some embodiments, IL-2 can be added to the culture flask at a concentration in the range of 5 - 6000 IU / mL. In some embodiments, on day 5, the culture supernatant can be removed by aspiration and the culture medium can be exchanged with a 1:1 mixture of CM / AIM V containing 6000 IU / mL IL-2. In some embodiments, on day 6 and daily thereafter, the cell concentration can be determined and the cells are either split into additional flasks or transferred to a culture bag containing additional medium containing 6000 IU / mL of IL-2 as needed to maintain the cell density at approximately 1 x 106 cells / mL. In some embodiments, at approximately 14 days after the start of the culture, the cells can be harvested from the culture bag. In some embodiments, the harvest can be achieved using a Baxter / Fenwal continuous centrifugal cell harvest system. In some embodiments, the harvested cells can be washed in sodium chloride. In some embodiments, the cells can be resuspended in sodium chloride with human albumin. In some embodiments, the resulting cell population may be suitable for administration to a subject in need thereof.

[0116] C. Method for enhancing production of TRL cells In some embodiments herein, a method of generating a population of enhanced TRLs (e.g., cTRLs) is disclosed. In some embodiments, the method includes isolating a population of TRLs as described using any of the methods disclosed herein, culturing the TRLs isolated using any of the methods disclosed herein, and introducing the cultured TRL cells into a nucleotide that expresses a therapeutic enhancing polypeptide under conditions sufficient for the cells to produce the enhancing polypeptide. In some embodiments, the nucleotide can encode a chimeric antigen receptor (CAR). In some embodiments, the methods described herein can include culturing the therapeutic enhancing cells using any of the culturing methods disclosed herein.

[0117] In some embodiments, at least one cell of the population of TRLs disclosed herein can be enhanced to express a chimeric antigen receptor (CAR) or an exogenous T cell receptor (TCR). In some embodiments, at least one cell of the enriched population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes disclosed herein can be used to produce CAR-T cells. In some embodiments, at least 5 cells of the enriched population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes disclosed herein can be used to produce CAR-T cells. In some embodiments, at least 10 cells of the enriched population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes disclosed herein can be used to produce CAR-T cells. In some embodiments, at least 50 cells of the enriched population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes disclosed herein can be used to produce CAR-T cells. In some embodiments, at least 100 cells of the enriched population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes disclosed herein can be used to produce CAR-T cells. In some embodiments, at least 500 cells of the enriched population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes disclosed herein can be used to produce CAR-T cells. In some embodiments, at least 1,000 cells of the enriched population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes disclosed herein can be used to produce CAR-T cells. In some embodiments, at least 2,000 cells of the enriched population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes disclosed herein can be used to produce CAR-T cells. In some embodiments, at least 5,000 cells of the enriched population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes disclosed herein can be used to produce CAR-T cells.In some embodiments, CAR-T cells can be produced using at least 10,000 cells of a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes disclosed herein. In some embodiments, CAR-T cells can be produced using at least 15,000 cells of a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes disclosed herein. In some embodiments, CAR-T cells can be produced using at least 20,000 cells of a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes disclosed herein. In some embodiments, CAR-T cells can be produced using at least 25,000 cells of a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes disclosed herein.

[0118] In some embodiments, CAR-T cells can be produced using at least one cell from a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 90% as disclosed herein. In some embodiments, CAR-T cells can be produced using at least five cells from a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 90% as disclosed herein. In some embodiments, CAR-T cells can be produced using at least ten cells from a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 90% as disclosed herein. In some embodiments, CAR-T cells can be produced using at least fifty cells from a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 90% as disclosed herein. In some examples, CAR-T cells can be produced using at least one hundred cells from a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 90% as disclosed herein. In some embodiments, CAR-T cells can be produced using at least five hundred cells from a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 90% as disclosed herein. In some embodiments, CAR-T cells can be produced using at least one thousand cells from a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 90% as disclosed herein. In some embodiments, CAR-T cells can be produced using at least two thousand cells from a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 90% as disclosed herein.In some embodiments, CAR-T cells can be produced using at least 5,000 cells of a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 90% as disclosed herein. In some embodiments, CAR-T cells can be produced using at least 10,000 cells of a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 90% as disclosed herein. In some embodiments, CAR-T cells can be produced using at least 15,000 cells of a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 90% as disclosed herein. In some embodiments, CAR-T cells can be produced using at least 20,000 cells of a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 90% as disclosed herein. In some embodiments, CAR-T cells can be produced using at least 25,000 cells of a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 90% as disclosed herein.

[0119] In some embodiments, CAR-T cells can be produced using at least one cell from a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 80% as disclosed herein. In some embodiments, CAR-T cells can be produced using at least five cells from a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 80% as disclosed herein. In some embodiments, CAR-T cells can be produced using at least ten cells from a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 80% as disclosed herein. In some embodiments, CAR-T cells can be produced using at least fifty cells from a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 80% as disclosed herein. In some examples, CAR-T cells can be produced using at least one hundred cells from a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 80% as disclosed herein. In some embodiments, CAR-T cells can be produced using at least five hundred cells from a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 80% as disclosed herein. In some embodiments, CAR-T cells can be produced using at least one thousand cells from a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 80% as disclosed herein. In some embodiments, CAR-T cells can be produced using at least two thousand cells from a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 80% as disclosed herein.In some embodiments, CAR-T cells can be produced using at least 5,000 cells of a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 80% as disclosed herein. In some embodiments, CAR-T cells can be produced using at least 10,000 cells of a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 80% as disclosed herein. In some embodiments, CAR-T cells can be produced using at least 15,000 cells of a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 80% as disclosed herein. In some embodiments, CAR-T cells can be produced using at least 20,000 cells of a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 80% as disclosed herein. In some embodiments, CAR-T cells can be produced using at least 25,000 cells of a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 80% as disclosed herein.

[0120] In some embodiments, CAR-T cells can be produced using at least one cell from a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 70% as disclosed herein. In some embodiments, CAR-T cells can be produced using at least five cells from a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 70% as disclosed herein. In some embodiments, CAR-T cells can be produced using at least ten cells from a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 70% as disclosed herein. In some embodiments, CAR-T cells can be produced using at least fifty cells from a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 70% as disclosed herein. In some examples, CAR-T cells can be produced using at least one hundred cells from a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 70% as disclosed herein. In some embodiments, CAR-T cells can be produced using at least five hundred cells from a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 70% as disclosed herein. In some embodiments, CAR-T cells can be produced using at least one thousand cells from a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 70% as disclosed herein. In some embodiments, CAR-T cells can be produced using at least two thousand cells from a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 70% as disclosed herein.In some embodiments, CAR-T cells can be produced using at least 5,000 cells of a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 70% as disclosed herein. In some embodiments, CAR-T cells can be produced using at least 10,000 cells of a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 70% as disclosed herein. In some embodiments, CAR-T cells can be produced using at least 15,000 cells of a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 70% as disclosed herein. In some embodiments, CAR-T cells can be produced using at least 20,000 cells of a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 70% as disclosed herein. In some embodiments, CAR-T cells can be produced using at least 25,000 cells of a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 70% as disclosed herein.

[0121] In some embodiments, CAR-T cells can be produced using at least one cell from a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 60% as disclosed herein. In some embodiments, CAR-T cells can be produced using at least five cells from a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 60% as disclosed herein. In some embodiments, CAR-T cells can be produced using at least ten cells from a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 60% as disclosed herein. In some embodiments, CAR-T cells can be produced using at least fifty cells from a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 60% as disclosed herein. In some examples, CAR-T cells can be produced using at least one hundred cells from a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 60% as disclosed herein. In some embodiments, CAR-T cells can be produced using at least five hundred cells from a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 60% as disclosed herein. In some embodiments, CAR-T cells can be produced using at least one thousand cells from a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 60% as disclosed herein. In some embodiments, CAR-T cells can be produced using at least two thousand cells from a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 60% as disclosed herein.In some embodiments, CAR-T cells can be produced using at least 5,000 cells of a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 60% as disclosed herein. In some embodiments, CAR-T cells can be produced using at least 10,000 cells of a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 60% as disclosed herein. In some embodiments, CAR-T cells can be produced using at least 15,000 cells of a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 60% as disclosed herein. In some embodiments, CAR-T cells can be produced using at least 20,000 cells of a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 60% as disclosed herein. In some embodiments, CAR-T cells can be produced using at least 25,000 cells of a concentrated population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes having a purity of at least 60% as disclosed herein.

[0122] In some embodiments, the TRLs described herein can be engineered to express a T cell receptor (TCR) or a chimeric antigen receptor (CAR), and the cells exhibit anti-tumor properties. In some embodiments, the TRLs can be transformed using a TCR or a CAR, and at least a portion of the TCR or CAR can be expressed on the cell surface. In some embodiments, the TRLs can be transduced with a viral vector encoding a TCR or a CAR. In some embodiments, the viral vector can be a retroviral vector. In some embodiments, the viral vector can be a lentiviral vector. In some such embodiments, the cells can stably express a TCR or a CAR. In another embodiment, the TRLs can be transfected with a nucleic acid encoding a TCR or a CAR, such as mRNA, cDNA, DNA. In some such embodiments, the cells can transiently express a TCR or a CAR. In one aspect, the antigen-binding domain of the TCR or CAR comprises a mouse (e.g., rat or mouse) antibody or antibody fragment.

[0123] In some embodiments, the TRLs can be engineered to express a chimeric antigen receptor (e.g., CART), and the cells (e.g., "CART") exhibit anti-tumor properties. In some embodiments, the methods disclosed herein can include a recombinant DNA construct comprising a sequence encoding a CAR, and the CAR comprises an antigen-binding domain (e.g., an antibody, antibody fragment) that binds to an antigen of interest (e.g., a tumor antigen). The intracellular signaling domain can comprise a co-stimulatory signaling domain and / or a primary signaling domain, such as a zeta chain. The co-stimulatory signaling domain can refer to a portion of the CAR that comprises at least a portion of the intracellular domain of a co-stimulatory molecule.

[0124] In some embodiments, the enhanced TRL can be an allogeneic immune effector cell lacking the expression of a functional T cell receptor (TCR) and / or human leukocyte antigen (HLA), such as HLA class I and / or HLA class II. An enhanced TRL lacking a functional TCR can be engineered, for example, to not express any functional TCR on its surface, to not express one or more subunits containing a functional TCR, or to produce only very low levels of functional TCR on its surface. Alternatively, the TRL can express a substantially impaired TCR, for example, by the expression of one or more mutant or truncated forms of the subunits of the TCR. The term "substantially impaired TCR" means that this TCR does not induce a harmful immune response in the host. Such cells can be generated by the use of one or more gene editing systems. The enhanced TRLs described herein can be engineered, for example, to not express functional HLA on their surface. For example, the enhanced TRLs described herein can be engineered such that cell surface expression of HLA, such as HLA class 1 and / or HLA class II, is downregulated. Such cells can be generated by the use of one or more gene editing systems described herein. In embodiments, the gene editing system targets a sequence encoding a component of one or more HLA molecules. In embodiments, the gene editing system can target a sequence encoding a factor that affects the expression of one or more HLA molecules. In embodiments, the gene editing system can target a sequence encoding a regulator of MHC class I expression, such as β-2 microglobulin (B2M). In embodiments, the gene editing system can target a sequence encoding a regulator of MHC class II molecule expression, such as CIITA. In embodiments, the gene editing system can target both a regulator of MHC class I expression (e.g., B2M) and a regulator of MHC class II molecule expression (e.g., CIITA) such that at least MHC class I molecule and at least one MHC class II molecule expression are downregulated.Modified T cells lacking functional TCR and / or HLA expression can be obtained by any suitable means including knockout or knockdown of one or more subunits of TCR or HLA. For example, the T cells can include knockdown of TCR and / or HLA using siRNA, shRNA, clustered regularly interspaced short palindromic repeats (CRISPR) transcription activator-like effector nucleases (TALEN), or zinc finger endonucleases (ZFN).

[0125] This specification provides delivery systems (e.g., virus-based systems or non-virus-based systems) that can insert polynucleotides encoding the TCR or CAR disclosed herein. Representative viral expression vectors include, but are not limited to, adenovirus-based vectors (e.g., the adenovirus-based Per.C6 system available from Crucell, Inc. (Leiden, The Netherlands)), lentivirus-based vectors (e.g., the lentivirus-based pLPI from Life Technologies (Carlsbad, Calif.)), and retroviral vectors (e.g., pFB-ERV + pCFB-EGSH), herpes virus. In some embodiments, the viral vector can be a lentiviral vector. Vectors derived from retroviruses such as lentiviruses can be used to achieve long-term gene transfer. In some embodiments, lentiviruses can transduce non-proliferating cells. In some embodiments, lentiviruses can have low immunogenicity. In some embodiments, suitable vectors can include an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers. Other suitable vectors can include integration expression vectors, which can be randomly integrated into the DNA of the host cell or can include recombination sites that allow for specific recombination between the expression vector and the host cell chromosome. Such integration expression vectors can utilize the endogenous expression control sequences of the host cell chromosome to effect the expression of the desired protein. Examples of vectors that perform site-specific integration can be found, for example, in the flp-in system (e.g., pcDNA™5 / FRT) from Invitrogen (Carlsbad, Calif.), or components of the cre-lox system such as those found in the pExchange-6 Core Vectors from Stratagene (La Jolla, CA).Examples of vectors that are randomly integrated into the host cell chromosome include, for example, pcDNA3.1 from Invitrogen (Carlsbad, CA) (when introduced in the absence of the T antigen), and pCI or pFNIOA(ACT)FLEXI(trademark) from Promega (Madison, Wis.). Additional promoter elements, such as enhancers, can regulate the frequency of transcription initiation. Non-limiting examples of suitable promoters can be the cytomegalovirus (CMV) promoter sequence, which can constitutively drive high-level expression of any operably linked polynucleotide sequence. Another non-limiting example of a suitable promoter can be human elongation growth factor 1α1 (hEFlal). In some embodiments, the vector constructs containing the CARs described herein can include functional variants of hEFlal. In some embodiments, the vector can include constitutive promoter sequences including, but not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein - Barr virus immediate early promoter, Rous sarcoma virus promoter, and can further include human gene promoters such as, but not limited to, the actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, the present invention should not be limited to the use of constitutive promoters. In some embodiments, inducible promoters can be used. Examples of inducible promoters include, but are not limited to, the metallothionein promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter.

[0126] In some embodiments, the expression vector introduced into the cells can also contain a selectable marker gene, a reporter gene, or both, to facilitate the identification and selection of the expressing cells from a population of cells that are to be transfected or infected via a viral vector. In some embodiments, the selectable marker is carried on a separate DNA fragment and can be used in co-transfection procedures. Useful selectable markers include, for example, antibiotic resistance genes such as the neomycin resistance gene (neo) and the ampicillin resistance gene. In some embodiments, a truncated epidermal growth factor receptor (HER1 or HER1-1) tag can be used as a selectable marker gene. Reporter genes can be used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. Generally, a reporter gene is a gene that encodes a polypeptide whose expression is revealed by some easily detectable property, such as enzymatic activity, and that is not present or expressed by the recipient organism or tissue. Suitable reporter genes include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein.

[0127] In some embodiments, the polynucleotide encoding the TCR or CAR described herein can also be introduced into T cells using a non-viral delivery system such as the "Sleeping Beauty (SB) transposon system", which refers to a synthetic DNA transposon system for introducing DNA sequences into the chromosomes of vertebrates. The Sleeping Beauty transposon system consists of the Sleeping Beauty (SB) transposase and the SB transposon. In some embodiments, the Sleeping Beauty transposon system can include the SB11 transposon system, the SB100X transposon system, or the SB110 transposon system.

[0128] In some embodiments, a non-viral-based delivery system (e.g., a delivery vehicle) can include a lipid-based delivery system, a polymer delivery system, inorganic compound-based nanoparticles, or extracellular vesicle-based delivery. In some embodiments, an exemplary delivery vehicle can be a liposome. Lipid formulations can be used for the introduction of polynucleotides into host cells (in vitro, ex vivo, or in vivo). In some embodiments, a polynucleotide can be associated with a lipid. The polynucleotide associated with the lipid can be encapsulated within the aqueous interior of the liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linking molecule associated with both the liposome and the oligonucleotide, trapped within the liposome, complexed with the liposome, dispersed in a lipid-containing solution, mixed with the lipid, combined with the lipid, contained as a suspension in the lipid, contained with or complexed with micelles, or otherwise associated with the lipid. Lipid, lipid / DNA, or lipid / expression vector association compositions are not limited to any particular structure in solution. For example, they can exist in bilayer structures, as micelles, or in a "folded" structure.

[0129] In some embodiments, an exemplary delivery vehicle can be a lipid nanoparticle (LNP). In some embodiments, the polynucleotide encoding the compositions disclosed herein can be incorporated into or associated with one or more LNPs. In some embodiments, the LNP can be mixed with 1,2-dioleoyl-3-trimethylammonium propane (DOTAP), dioleoylphosphatidylethanolamine (DOPE), a cationic cholesterol derivative (DC-Chol) mixed with dimethylaminoethane-carbamoyl, phosphatidylcholine (PC), triolein (glyceryl trioleate), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-2000] (DSPE-PEG), 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2K), and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and / or can include one or more molecules selected from polyethyleneimine (PEI), poly(lactic-co-glycolic acid) (PLGA), and N-acetylgalactosamine (GalNAc). The LNP can include one or more of a structural lipid (e.g., DSPC), a PEG-conjugated lipid (CDM-PEG), a cationic lipid (MC3), cholesterol, and a targeting ligand (e.g., GalNAc). In some embodiments, the nanoparticles described herein can be particles with a diameter of less than about 1000 nm. In some embodiments, the nanoparticles can have a maximum dimension (e.g., diameter) of about 500 nm or less, or about 400 nm or less, or about 300 nm or less, or about 200 nm or less, or about 100 nm or less. In some embodiments, the nanoparticles described herein can have a maximum dimension in the range between about 50 nm and about 150 nm, or about 70 nm and about 130 nm, or about 80 nm and about 120 nm, or about 90 nm and about 110 nm. In some embodiments, the nanoparticles described herein can have a maximum dimension (e.g., diameter) of about 100 nm.

[0130] In some embodiments, TRLs transduced with nucleic acids encoding TCR or CAR can be expanded, for example, by the methods described herein. In some embodiments, expanded TRLs (e.g., TRLs transduced with nucleic acids encoding CAR) can be grown in culture for several hours (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 18, 21 hours) to about 14 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days). In one embodiment, the cells can be grown for 4 to 9 days. In one embodiment, the cells can be grown for 8 days or less, e.g., 7, 6, or 5 days.

[0131] Conditions suitable for T cell cultures (e.g., expanded TRLs) can include a suitable medium (e.g., Minimum Essential Medium or RPMI Medium 1640 or X-vivo15 (Lonza)) containing factors necessary for growth and survival, including serum (e.g., fetal bovine serum or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ, and TNF-α, or any other additives known to those of skill in the art for cell growth. Other additives for cell growth include, but are not limited to, surfactants, Plasmanate, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol. The medium can include RPMI1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo15, and X-Vivo20, Optimizer, with amino acids, sodium pyruvate, and vitamins added, and can be serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or cytokines in an amount sufficient for T cell growth and expansion. Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures and not in cultures of cells to be injected into a subject. Target cells are maintained under conditions necessary to support growth, e.g., at an appropriate temperature (e.g., 37° C.) and atmosphere (e.g., air + 5% CO2).

[0132] In one embodiment, the enhanced TRL can be expanded in a suitable medium (e.g., the media described herein) containing one or more interleukins that result in at least a 200-fold (e.g., 200-fold, 250-fold, 300-fold, 350-fold) increase in the cells over a 14-day expansion period when measured by methods described herein such as flow cytometry. In one embodiment, the enhanced TRL is expanded in the presence of IL-15 and / or IL-7 (e.g., IL-15 and IL-7).

[0133] Once a CAR (e.g., enhanced TRL) is constructed, various assays can be used to evaluate the activity of the molecule, e.g., but not limited to, the ability to expand T cells after antigen stimulation, the ability to maintain T cell expansion in the absence of restimulation, and anti-cancer activity in appropriate in vitro and animal models. For example, Western blot analysis of CAR expression in primary T cells can be used to detect the presence of monomers and dimers. In vitro expansion of enhanced CAR cells after antigen stimulation can be measured by flow cytometry. CART activity can also be measured using animal models. For example, imaging techniques can be used to evaluate the specific trafficking and expansion of CAR in animal models bearing tumors.

[0134] D. Treatment Methods Disclosed herein are methods of treating a disease or disorder in a subject by administering to the subject a composition or pharmaceutical formulation disclosed herein. In some embodiments, the composition comprises a TRL (e.g., cTRL) or enhanced TRL (e.g., enhanced cTRL) disclosed herein. In some embodiments, multiple TRLs are therapeutically enhanced to express, for example, a chimeric antigen receptor (CAR) having antigen specificity for any of the cancer antigens disclosed herein. In some embodiments, the cells are isolated from the subject's peripheral blood using any of the methods disclosed herein. In some embodiments, the cells are autologous to the subject. In some embodiments, the cells are isolated from the subject's peripheral blood using any of the methods of isolating a TRL disclosed herein. In some embodiments, the isolated TRLs are expanded using any of the methods disclosed herein. In some embodiments, autologous cells are preferred over allogeneic cells because they have an inherent heterogeneity that maximizes tumor-recognizing T cell receptors (TCRs) while minimizing off-tissue effects.

[0135] In some embodiments, the TRL (e.g., enhanced TRL) is injected into a body part of the subject (e.g., a patient's vein, bone marrow, etc.). In some embodiments, the cells are administered by intradermal, subcutaneous, intramuscular, intravenous, intraosseous, intraperitoneal, intrathecal, epidural, intracardiac, intraarticular, intracavernous, and / or intravitreal injection.

[0136] In some embodiments, the administration of the population of cells disclosed herein is performed by a single administration at continuous or separate intervals, which can be readily determined by one of ordinary skill in the art. In some embodiments, the subject is injected with a fixed dose of cells at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. In some embodiments, the subject is injected with a fixed dose of cells at most about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 time. In some embodiments, the subject is injected with a fixed dose of cells at a frequency of once every at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 60, 90, 180, 360 days, or more. In some embodiments, the subject is injected with a fixed dose of cells at a frequency of once every at most 360, 180, 90, 60, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day. In some embodiments, the subject is injected with a fixed dose of at least about 1×10 4 cells / kg, 1×10 5 cells / kg, 1×10 6 cells / kg, 1×10 7 cells / kg, 1×10 8 cells / kg, 1×10 9 cells / kg, 1×10 10 cells / kg, 1×10 11 cells / kg, or more cells. In some embodiments, the subject is injected with a fixed dose of at most at least about 1×10 4 cells, 1×10 5 cells, 1×10 6 cells, 1×10 7 cells, 1×10 8 cells, 1×10 9 cells, 1×10 10 cells, 1×10 11 cells, 1×10 12 cells, or more cells are injected.

[0137] In some embodiments, the cells can be useful for a variety of applications including, but not limited to, immunotherapy for treating diseases and disorders. Diseases and disorders that can be treated using the cells of the present disclosure include, but are not limited to, inflammatory diseases, cancer, infectious diseases, autoimmune diseases, and neurodegenerative diseases. In some embodiments, the cell therapy disclosed herein is used to treat cancer. In some embodiments, the cell therapy is used to treat tumors that are not optimal raw materials for tumor-infiltrating lymphocyte (TIL) isolation. In some embodiments, the cell therapy described herein is used to treat tumors that are not easily accessible by large, resectable lesions (e.g., less than 3 cm in diameter). In some embodiments, the cell therapy described herein is used to treat patients for whom resection surgery is not an option for the patient (e.g., due to substantial risk or rapid tumor progression). In some embodiments, the cell therapy described herein is used to treat tumors that have shown a decreased response rate to adoptive cell therapy using TILs. In some embodiments, the cell therapy is used to treat solid tumors such as renal cancer, cervical cancer, and breast cancer, which have been demonstrated to be less responsive to TIL therapy in some embodiments. In some embodiments, the cells of the present disclosure are used to treat cancer. In some embodiments, the cancer is in tissue. In some embodiments, the tissue includes bone tissue, muscle tissue, breast tissue, epithelial tissue, connective tissue, brain tissue, lung tissue, kidney tissue, liver tissue, pancreatic tissue, prostate tissue, lymphoid tissue, bone marrow tissue, or bladder tissue. Non-limiting examples of cancer include the following: acanthoma, acinar cell carcinoma, acoustic neuroma, acral lentiginous melanoma, acrospiroma, acute eosinophilic leukemia, acute lymphoblastic leukemia, acute megakaryoblastic leukemia, acute monocytic leukemia, acute myeloblastic leukemia with maturation, acute myeloid dendritic cell leukemia, acute myeloid leukemia, acute promyelocytic leukemia, adamantinoma, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenomatoid odontogenic tumor, adrenocortical carcinoma, adult T-cell leukemia, aggressive NK-cell leukemia, AIDS-related cancer, AIDS-related lymphoma, alveolar soft part sarcoma, blastic plasmacytoid dendritic cell neoplasm, anal cancer, anaplastic large cell lymphoma, anaplastic thyroid carcinoma, angioimmunoblastic T-cell lymphoma, angiosarcoma, appendiceal cancer,Astrocytoma, typical teratoid / rhabdoid tumor, basal cell carcinoma, basal cell-like carcinoma, B-cell leukemia, B-cell lymphoma, Bellini duct carcinoma, biliary tract cancer, bladder cancer, blastoma, bone cancer, bone tumor, brainstem glioma, brain tumor, breast cancer, Brenner tumor, bronchial tumor, bronchioloalveolar carcinoma, brown tumor, Burkitt lymphoma, cancer of unknown primary origin, carcinoid tumor, carcinoma, carcinoma in situ, penile cancer, cancer of unknown primary origin, carcinosarcoma, Castleman disease, central nervous system embryonal tumor, cerebellar astrocytoma, cerebral astrocytoma, cervical cancer, cholangiocarcinoma, chondroma, chondrosarcoma, chorioma, choriocarcinoma, choroid plexus papilloma, chronic lymphocytic leukemia, chronic monocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorder, chronic neutrophilic leukemia, clear cell tumor, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, Degos disease, dermatofibrosarcoma protuberans, dermoid cyst, desmoplastic small round cell tumor, diffuse large B-cell lymphoma, embryonal dysplastic neuroepithelial tumor, fetal carcinoma, endodermal sinus tumor, endometrial cancer, endometrial carcinoma, endometrial tumor, enteropathy-associated T-cell lymphoma, epithelioblastoma, epithelioma, epitheloid sarcoma, erythroleukemia, esophageal cancer, ganglioneuroblastoma, Ewing family tumor, Ewing family sarcoma, Ewing sarcoma, extracranial primitive neuroectodermal tumor, extragonadal primitive neuroectodermal tumor, extrahepatic cholangiocarcinoma, extramammary Paget disease, fallopian tube cancer, fetiform teratoma, fibroma, fibrosarcoma, follicular lymphoma, follicular thyroid cancer, gallbladder cancer, gallbladder carcinoma, glioma, ganglioneuroma, gastric cancer, gastric lymphoma, gastrointestinal cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gastrointestinal stromal tumor, germ cell tumor, germinoma, gestational choriocarcinoma, gestational trophoblastic tumor, giant cell tumor of bone, glioblastoma multiforme, glioma, cerebral gliomatosis, glomus tumor, glucagonoma, gonadoblastoma, granulosa cell tumor, hairy cell leukemia, hairy cell leukemia, head and neck cancer, head and neck carcinoma, heart cancer, hemangioblastoma, hemangiopericytoma, angiosarcoma, hematologic malignancy, hepatocellular carcinoma, hepatosplenic T-cell lymphoma, hereditary breast and ovarian cancer syndrome, Hodgkin lymphoma, Hodgkin lymphoma, hypopharyngeal cancer, hypothalamic glioma, inflammatory breast cancer, intraocular melanoma, islet cell carcinoma, islet cell tumor, juvenile myelomonocytic leukemia, Kaposi sarcoma, Kaposi sarcoma, kidney cancer, Krukenberg tumor, laryngeal cancer, laryngeal carcinoma, malignant melanoma, leukemia, leukemia, lip and oral cavity cancer, liposarcoma, lung cancer, luteoma, lymphangioma, lymphangiosarcoma, lymphoepithelioma, lymphocytic leukemia, lymphoma,Macroglobulinemia, malignant fibrous histiocytoma, malignant fibrous histiocytoma, malignant fibrous histiocytoma of bone, malignant glioma, malignant mesothelioma, malignant peripheral nerve sheath tumor, malignant rhabdoid tumor, malignant Triton tumor, MALT lymphoma, mantle cell lymphoma, mast cell leukemia, spinal embryonal cell tumor, mediastinal tumor, spinal thyroid cancer, medulloblastoma, medulloblastoma, medulloepithelioma, melanoma, melanoma, meningioma, Merkel cell carcinoma, mesothelioma, mesothelioma, obstructive primary metastatic squamous cell carcinoma of the neck, metastatic transitional cell carcinoma, malignant mesodermal Müllerian duct mixed tumor, monocytic leukemia, oral cancer, mucinous tumor, multiple endocrine neoplasia syndrome, multiple myeloma, multiple myeloma, mycosis fungoides, mycosis fungoides, myelodysplastic disorder, myelodysplastic syndrome, myeloid leukemia, myeloid sarcoma, myeloproliferative disorder, myxoma, nasal cancer, nasopharyngeal cancer, nasopharyngeal cancer, neoplasm, nerve tumor, neuroblastoma, neuroblastoma, neurofibroma, nerve tumor, nodular melanoma, non-Hodgkin lymphoma, non-Hodgkin lymphoma, non-melanoma skin cancer, non-small cell lung cancer, eye tumor, anaplastic astrocytoma, anaplastic glioma, oncocytoma, optic nerve sheath meningioma, oral cancer, oral cancer, pharyngeal cancer, osteosarcoma, osteosarcoma, ovarian cancer, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, low malignant potential ovarian tumor, Paget's disease of the breast, Pancoast tumor, pancreatic cancer, pancreatic cancer, papillary thyroid cancer, papillomatosis, paraganglioma, paranasal sinus cancer, parathyroid cancer, senile cancer, perivascular epithelioid cell tumor, pharyngeal cancer, pheochromocytoma, moderately differentiated pineal parenchymal tumor, pineoblastoma, pituitary cell tumor, pituitary adenoma, pituitary tumor, plasma cell neoplasm, splenopulmonary blastoma, multiple embryonal tumor, precursor T lymphoblastic lymphoma, primary central nervous system lymphoma, primary effusion lymphoma, primary hepatocellular carcinoma, primary liver cancer, primary peritoneal cancer, primitive neuroectodermal tumor, prostate cancer, peritoneal pseudomyxoma, rectal cancer, renal cell carcinoma, respiratory tract cancer with NUT gene on chromosome 15, retinoblastoma, rhabdomyoma, rhabdomyosarcoma, Richter transformation, sacrococcygeal teratoma, salivary gland cancer, sarcoma, schwannoma, sebaceous gland carcinoma, secondary tumor, seminoma, serous tumor, Sertoli-Leydig cell tumor, sex cord-stromal tumor, Sézary syndrome, signet ring cell carcinoma, skin cancer, small round blue cell tumor, small cell carcinoma, small cell lung cancer, small cell lymphoma, small intestine cancer, soft tissue sarcoma, somatostatinoma, keloid, spinal tumor, spinal cord tumor, splenic marginal zone lymphoma, squamous cell carcinoma, gastric cancer, superficial melanoma, undifferentiated neuroectodermal tumor, surface epithelial stromal tumor, synovial sarcoma, T cell acute lymphoblastic leukemia,T-cell large granular lymphocytic leukemia, T-cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia, teratoma, peripheral lymphoma, testicular cancer, granulosa cell tumor, laryngeal cancer, thymic cancer, thymoma, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, transitional cell carcinoma, urothelial carcinoma, urethral cancer, urogenital neoplasm, uterine sarcoma, choroidal melanoma, vaginal cancer, Verner Morrison syndrome, verrucous carcinoma, glioma of the visual pathway, vulvar cancer, Waldenström macroglobulinemia, Warthin tumor, Wilms tumor, and combinations thereof.

[0138] In some embodiments, the compositions and methods disclosed herein are administered in combination with other cancer immunotherapies or chemotherapies. In some embodiments, a method of providing a cell therapy to a subject in need thereof further comprises administering at least one additional therapy. In some embodiments, administering at least one additional therapy comprises administering an additional therapeutic agent to the subject. In some embodiments, the additional therapeutic agent can be an immunosuppressive agent. In some embodiments, the immunosuppressive agent can include a monoclonal antibody. In some embodiments, the monoclonal antibody can deplete endogenous lymphocytes. In some embodiments, anti-CD3, anti-CD2, and / or anti-CD52 can be used to deplete endogenous lymphocytes. In some embodiments, the additional therapeutic agent is an anti-tumor agent. In some embodiments, the anti-tumor agent is a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is a lymphocyte-depleting chemotherapeutic agent. In some embodiments, the anti-tumor agent is an immunotherapeutic agent. In some embodiments, the anti-tumor agent is an immune checkpoint inhibitor, a chemotherapeutic agent, or any combination thereof. In some embodiments, the anti-tumor agent is a co-stimulatory molecule. In some embodiments, the co-stimulatory molecule is glucocorticoid-induced tumor necrosis factor receptor (GITR). In some embodiments, the co-stimulatory molecule can include CD28, CD137 (4-1BB), CD134 (OX40), inducible T cell co-stimulation (ICOS), CD27, or any combination thereof. In some embodiments, the anti-tumor agent can be a cytokine that stimulates survival, proliferation, and activation. In some embodiments, the cytokine can be IL-2, IL-7, IL-15, IL-21, or any combination thereof. In some embodiments, the anti-tumor agent is an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is an anti-PD1 antibody, an anti-PD-L1 antibody, or a variant or functional fragment thereof. In some embodiments, the anti-PD1 antibody is selected from pidilizumab, BMS-936559, nivolumab, pembrolizumab, or a variant or functional fragment thereof. In some embodiments, the anti-PD-L1 antibody is selected from atezolizumab, avelumab, durvalumab, MDX-1105, or a variant or functional fragment thereof.

[0139] In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody, or a variant or functional fragment thereof. The anti-CTLA-4 antibody of the present invention can bind to human CTLA-4 and interfere with the interaction between CTLA-4 and the human B7 receptor. Since the interaction between CTLA-4 and B7 converts the signal that leads to the inactivation of T cells carrying the CTLA-4 receptor, interference with the interaction effectively induces the activation of these T cells. An exemplary and clinical anti-CTLA-4 Ab is human mAb 10D1 (currently known as ipilimumab and commercially available as YERVOY®). In some embodiments, the anti-CTLA-4 Ab is an mAb. In certain other embodiments, the anti-CTLA-4 antibody is a chimeric, humanized, or human antibody. In certain embodiments, the anti-CTLA-4 antibody is ipilimumab.

[0140] In some embodiments, the additional therapeutic agent is administered prior to the administration of the cell population. For example, anti-CD3 can deplete endogenous lymphocytes by being administered prior to the administration of TRL (e.g., enhanced TRL). In some embodiments, the additional therapeutic agent is administered after the administration of the cell population. For example, IL-2 can stimulate the proliferation (e.g., cell proliferation, activation, survival) of the administered TRL by being administered after the administration of TRL (e.g., enhanced TRL). In some embodiments, the additional therapeutic agent is administered simultaneously with the administration of the cell population. In some embodiments, the additional therapeutic agent is administered 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 times, or more. In some embodiments, the additional therapeutic agent is administered up to about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 time. In some embodiments, the additional therapeutic agent is administered at a frequency of once every at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 60, 90, 180, 360 days, or more. In some embodiments, the additional therapeutic agent is administered at a frequency of once every up to 360, 180, 90, 60, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day.

[0141] In practicing the methods of treatment or use provided herein, a therapeutically effective amount of a pharmaceutical formulation described herein is administered to a mammal suffering from a disease, disorder, or illness, such as cancer, being treated. In some embodiments, the mammal is a human. The therapeutically effective amount can vary widely depending on the severity of the disease, the age and relative health of the subject, the potency of the therapeutic agent being used, as well as other factors. In some embodiments, the pharmaceutical formulations described herein may be used alone or in combination with one or more therapeutic agents as components of a mixture.

[0142] III. SYSTEM In some embodiments herein, a system is described that includes one or more compositions or devices disclosed herein. In some embodiments, the system includes a microfluidic device and instructions on how to use the microfluidic device to perform magnetic separation to isolate tumor-reactive lymphocytes (TRLs) from a sample of a subject's body fluid. In some embodiments, the microfluidic device comprises a magnetic capture zone disposed within its channels, and the microfluidic device is configured to magnetically separate a population of cells from a body fluid sample. In some embodiments, the system includes a body fluid sample obtained from a subject, and the body fluid sample includes a population of cells that includes (i) CD103 lymphocytes, (ii) CD39 lymphocytes, (iii) SLC6A19+ lymphocytes, (iv) SIDT1+ lymphocytes, or (v) any combination of (i)-(iv). In some embodiments, the system includes enhanced TRLs provided in Section I (Compositions) of this disclosure. In some embodiments, the system includes a kit provided in Section IV (Kits) of this disclosure.

[0143] In some embodiments, the system may include a population of isolated and enriched tumor-reactive lymphocytes derived from a sample (e.g., peripheral blood) that expresses SLC6A19, SIDT1, CD103, CD39, or any combination thereof. In some embodiments, the TRLs express CD103. In some embodiments, the population of TRLs includes a CD103 signature. In some embodiments, the CD103 signature defines the population of TRLs. In some embodiments, the TRLs express CD39. In some embodiments, the population of TRLs includes a CD39 signature. In some embodiments, the CD39 signature defines the population of TRLs. In some embodiments, the TRLs express SLC6A19, SIDT1, or a combination thereof. In some embodiments, the population of TRLs includes an SLC6A19+ signature, an SIDT1+ signature. In some embodiments, the population of TRLs can further include CD3, CD4, CD8, or any combination thereof. For example, the isolated and enriched TRLs can be CD3 and CD103 cells. In some embodiments, the isolated and enriched TRLs can be CD4 and CD103 cells. In some embodiments, the isolated and enriched TRLs can be CD8 and CD103 cells. In some embodiments, the isolated and enriched TRLs can be CD39 and CD103 cells. In some embodiments, the isolated and enriched TRLs can be CD3, CD39, and CD103 cells. In some embodiments, the isolated and enriched TRLs can be CD4, CD39, and CD103 cells. In some embodiments, the isolated and enriched TRLs can be CD8, CD39, and CD103 cells. In some embodiments, the isolated and enriched TRLs can be CD3, SLC6A19, and SIDT1 cells. In some embodiments, the isolated and enriched TRLs can be CD4, SLC6A19, and SIDT1 cells. In some embodiments, the isolated and enriched TRLs can be CD8, SLC6A19, and SIDT1 cells. In some embodiments, the isolated and enriched TRLs can be CD3, CD103, and SLC6A19 cells.In some embodiments, the isolated and enriched TRLs can be CD4, CD103, and SLC6A19 cells. In some embodiments, the isolated and enriched TRLs can be CD8, CD103, and SLC6A19 cells. In some embodiments, the isolated and enriched TRLs can be CD3, CD103, SLC6A19, and SIDT1 cells. In some embodiments, the isolated and enriched TRLs can be CD4, CD103, SLC6A19, and SIDT1 cells. In some embodiments, the isolated and enriched TRLs can be CD8, CD103, SLC6A19, and SIDT1 cells.

[0144] In some embodiments, a population of tumor-reactive lymphocytes (TRLs) can be found in a body fluid sample of a patient suffering from cancer. In some embodiments, the body fluid sample can include a peripheral blood sample. In some embodiments, the body fluid sample can be other biological fluids such as pleural effusion or ascites. In some embodiments, the body fluid sample can also be cord blood, bone marrow, lymph nodes, liver ascites, chest wall, abdominal cavity, synovial fluid, peritoneum, retroperitoneal space, thymus, and tumor.

[0145] In some embodiments, the systems described herein include isolating or enriching a population of CD103, CD39, SLC6A19, and / or SIDT1-expressing lymphocytes from a peripheral blood sample by treating the sample with a plurality of major histocompatibility complex (MHC) multimers that mimic defined tumor epitopes. Generally, T cells express a surface T cell receptor (TCR) that enables the T cell to recognize a peptide antigen bound to a major histocompatibility complex (MHC) molecule, and TCR recognition of the MHC-peptide complex results in T cell activation, clonal expansion, and differentiation of the T cell into effector, memory, and regulatory T cells. An MHC multimer contains multiple copies of the MHC-peptide complex. In some embodiments, the MHC multimer exhibits increased T cell affinity compared to monomers of the same complex. In some embodiments, the MHC molecule is a human MHC molecule. In some embodiments, the MHC molecule is a mouse MHC molecule. In some embodiments, the MHC molecule is a class 1 MHC molecule. In some embodiments, the class 1 MHC molecule is a human HLA-A, HLA-B, or HLA-C molecule. In some embodiments, the class I MHC molecule is a mouse H-2K, H-2D, or H-2L molecule. In some embodiments, the molecule is a class 2 MHC molecule. In some embodiments, the MHC multimer contains at least 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 MHC molecules. In some embodiments, the MHC multimer contains about 1, 2, 3, 4, 5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 MHC molecules. In some embodiments, the MHC multimer contains an MHC dimer containing 2 MHC molecules. In some embodiments, the MHC multimer contains an MHC tetramer containing 4 MHC molecules. In some embodiments, the MHC multimer contains an MHC pentamer containing 5 MHC molecules. In some embodiments, the MHC multimer contains a dextramer containing 10 or more MHC molecules.

[0146] In some embodiments, the MHC multimer comprises a peptide. In some embodiments, the MHC multimer comprises a peptide-MHC complex. In some embodiments, the peptide is any natural or non-natural peptide that can be presented by an MHC molecule. In some embodiments, the peptide-MHC complex is one to which a TRL is reactive. In some embodiments, the peptide-MHC complex is one to which CD8+CD103+ lymphocytes are reactive. In some embodiments, the peptide-MHC complex is one to which CD103+CD39+ lymphocytes are reactive. In some embodiments, the peptide-MHC complex is one to which CD8+CD103+CD39+ lymphocytes are reactive. In some embodiments, the peptide-MHC complex mimics a defined tumor epitope. In some embodiments, the peptide comprises an epitope of influenza A hemagglutinin. In some embodiments, the peptide comprises amino acid residues 533-541 of influenza A hemagglutinin. In some embodiments, the peptide comprises an epitope of chicken ovalbumin. In some embodiments, the peptide comprises amino acid residues 257-264 of chicken ovalbumin. In some embodiments, the MHC multimer comprises a plurality of peptide-MHC complexes. In some embodiments, each of the peptide-MHC complexes associates with a multimerization domain. In some examples, the peptide-MHC complex comprises a peptide derived from MC-38 of the SIIVFNLL sequence and an H-2Kb molecule. In some embodiments, the MHC multimer is operably linked to magnetic nanoparticles. In some embodiments, the magnetic nanoparticles are bound to the MHC multimer via a fluorophore linker.

[0147] In some embodiments, the linker comprises a polymeric linker such as an amino acid linker or a biotin linker. In some embodiments, the linker is cleavable. In some embodiments, the linker is non-cleavable.

[0148] In some embodiments, the systems described herein may include separating, isolating, or enriching a population of TRLs from a peripheral blood sample. In some embodiments, the microfluidic devices disclosed herein are used to separate a population of TRLs from a peripheral blood sample. In some embodiments, any of the microfluidic devices disclosed in International Publication No. WO 2014 / 166000, the contents of which are incorporated herein by reference, are used to separate TRLs from a peripheral blood sample. In some embodiments, once the TRLs are separated, the TRLs may be eluted from the microfluidic device by removing the primers that act on the TRLs (e.g., via removal of a magnetic field). In some embodiments, the captured TRLs are then grown, enhanced, or a combination thereof, by any of the methods disclosed herein.

[0149] The microfluidic devices disclosed herein are configured to magnetically sort a population of cells. In some embodiments, the microfluidic device is provided in U.S. Patent No. 10,073,079, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the population of cells includes a population of TRLs. In some embodiments, the population of TRLs is labeled with magnetic nanoparticles. In some embodiments, each of the magnetic nanoparticles has a diameter of about 0 to 50 nanometers (nm), a diameter of 51 to 100 nm, a diameter of 100 to 150 nm, or a diameter of 150 to 200 nm. In some embodiments, each of the magnetic nanoparticles has a diameter of about 50 nm. In some embodiments, compared to conventional microbeads, magnetic nanoparticles may be useful for processing larger samples because they have improved colloidal stability. In some embodiments, cells labeled with magnetic nanoparticles are difficult to capture because their capture efficiency is further reduced by several orders of magnitude in magnetic susceptibility compared to microbeads. Thus, in some embodiments, the microfluidic devices disclosed herein include a flow rate reduction structure that creates a localized region of lower flow rate as a sample containing cells flows through the device. In some embodiments, the presence of such a low flow rate region enables the capture of magnetically labeled cells.

[0150] In some embodiments, the microfluidic device disclosed herein includes a microfluidic chip. In some embodiments, the microfluidic chip includes a sorting chamber. In some embodiments, the sorting chamber is etched or formed on the chip. In some embodiments, the sorting chamber communicates with a flow inlet and a flow outlet. In some embodiments, the flow inlet is configured to receive a sample, such as a peripheral blood sample containing a population of TRLs suspended in a fluid medium, and the flow outlet is configured to deliver the fluid medium depleted of TRLs. In some examples, a tube is connected to the inlet such that the fluid medium can be delivered therethrough to the inlet. In some embodiments, a tube is connected to the outlet such that the fluid medium can be received therethrough from the outlet. In some embodiments, the tube is a silicone tube. In some embodiments, the microfluidic device includes a syringe pump capable of controlling the flow rate of the fluid medium at the inlet.

[0151] In some embodiments, the sorting chamber includes at least one magnetic capture zone. In some embodiments, the sorting chamber includes a plurality of magnetic capture zones. In some embodiments, the sorting chamber includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 magnetic capture zones. In some embodiments, the sorting chamber includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 magnetic capture zones. In some embodiments, at least two of the magnetic capture zones vary in height. In some embodiments, the magnetic capture zones range in height from 50 to 800 μm. In some embodiments, the sorting chamber includes three magnetic capture zones, one having a height of about 100 μm, one having a height of about 200 μm, and one having a height of about 400 μm.

[0152] In some embodiments, the microfluidic device comprises at least one magnet array located on the outer surface of a microfluidic chip disposed above or below the sorting chamber so as to generate a magnetic field within the magnetic capture zone by means of at least one magnet array. In some embodiments, the microfluidic device comprises two magnet arrays located on two outer surfaces of the microfluidic chip above and below the sorting chamber so as to generate a magnetic field within the magnetic capture zone by means of the two magnet arrays. In some embodiments, the magnets are located in two arrays with alternating polarities on both sides of the sorting chamber. In some embodiments, at least one magnet array generates a magnetic field strength of 0.1 to 0.5 Tesla (T), 0.5 to 1 T, or 1 to 1.5 T in the magnetic capture zone. In some embodiments, at least one magnet array generates a magnetic field strength of 0.5 to 1 T in the magnetic capture zone. In some embodiments, the magnets include neodymium magnets. In some embodiments, the magnets include N52 Nd FeB magnets.

[0153] In some embodiments, the magnetic capture zone includes a plurality of microstructures. In some embodiments, the microstructure is a flow rate reduction structure configured to improve captured cells labeled with magnetic nanoparticles in the flow. In some embodiments, the microstructure generates a localized region of lower flow velocity so that particles can be captured (e.g., the reduction in flow velocity can enable the magnetic force to overcome the drag force on the particles). In some embodiments, this structure is designed to avoid capturing non-target particles. For example, even though the flow velocity is lower, the region of lower flow velocity can always have a sufficient flow velocity to wash non-target particles out of the device (i.e., the flow rate may be at least non-zero), while target particles can be captured within the low flow velocity region. In some embodiments, the microstructure is X-shaped.

[0154] In some embodiments, the device comprises a plurality of magnetic capture zones, where the first zone comprises the inlet of the sorting chamber, the final magnetic capture zone comprises the outlet of the sorting chamber, and the plurality of magnetic capture zones are disposed between the first magnetic capture zone and the final magnetic capture zone. In some embodiments, the size or pattern of the microstructure varies between capture zones. In some embodiments, the height varies between magnetic capture zones. In some embodiments, the first zone exhibits the highest linear velocity and thus holds cells with a high magnetic content because the holding magnetic force overcomes the drag exerted by the locally high flow velocity. In some embodiments, the other magnetic capture zones exhibit a gradually decreasing linear velocity, and the final magnetic capture zone exhibits the lowest velocity. This design enables capturing cells with a high level of magnetization in the first zone of the device, while cells with lower magnetization are sorted in subsequent zones according to their level of magnetization.

[0155] In some of the methods disclosed herein, a population of TRLs suspended in a fluid is injected through an inlet, across magnetic capture zones, and through an outlet. In some embodiments, magnetically labeled cells are captured in a capture zone if the magnetic force exerted on the cells is sufficient to overcome the drag that allows the cells to flow through the capture zone. If the magnetic force does not overcome the drag, the cells are flushed to the next zone with lower drag and ultimately to a syringe if the cells cannot be captured by any zone. After sorting, the cells captured in each zone are recovered by removing an external magnet.

[0156] In some of the methods disclosed herein, a population of cells is loaded into a microfluidic device through an inlet at a flow rate of at least 1 milliliter per hour (mL h-1), 3 mL h-1, 6 mL h-1, 9 mL h-1, 12 mL h-1, 15 mL h-1, 18 mL h-1, 21 mL h-1, 24 mL h-1, 27 mL h-1, 30 mL h-1, 35 mL h-1, 40 mL h-1, 45 mL h-1, or 50 mL h-1. The magnetic force exerted on the cells will be determined, for example, by the size of the magnetic nanoparticles, the number of magnetic nanoparticles attached to the cells, the size of the cells, and the strength of the applied magnetic field. In some embodiments, the relationship between drag force and linear flow rate in a microfluidic device capable of magnetically capturing particles by leveraging velocity-reducing microstructures is disclosed in U.S. Patent No. 10,073,079, which is incorporated herein by reference in its entirety.

[0157] In some embodiments herein, a system for generating a population of enhanced TRLs is disclosed. In some embodiments, the system includes isolating a population of TRLs as described using any of the systems disclosed herein, culturing the isolated TRLs using any of the systems disclosed herein, and introducing the cultured TRL cells into a nucleotide that expresses a therapeutic enhancing polypeptide under conditions sufficient for the cells to produce the enhancing polypeptide. In some embodiments, the nucleotide encodes a chimeric antigen receptor (CAR). In some embodiments, the system of the inventive concept includes culturing therapeutic enhancing cells using any of the culture systems disclosed herein.

[0158] Disclosed herein are pharmaceutical formulations comprising the enhanced TRLs or compositions described herein. In some embodiments, the pharmaceutical formulation further comprises a pharmaceutically acceptable carrier, excipient, diluent, or nebulized inhalant.

[0159] In some embodiments, the pharmaceutical formulation comprises two or more active agents, or two or more therapeutic agents disclosed herein. In some embodiments, the two or more active agents are included in a single dosage unit, for example, when the enhanced TRL (e.g., CAR or TCR) comprises two or more therapeutic agents. In embodiments, the two or more active agents are included in separate dosage units, such as when the enhanced TRL (e.g., CAR or TCR) is administered separately from an additional therapeutic agent or adjuvant. In some embodiments, the active agent, which may be an additional therapeutic agent in some embodiments, includes a chemotherapeutic agent, a cytotoxic agent, a cytokine, a growth inhibitor, an antihormonal agent, an antiangiogenic agent, a cardioprotective agent, and / or a checkpoint inhibitor.

[0160] IV. Kit In some embodiments herein, a kit is provided that includes a composition or system disclosed herein, together with instructions on how to produce or use the composition or system. In some embodiments, the kit includes the microfluidic device disclosed herein, together with instructions on how to use the microfluidic device to isolate TRL from a body fluid sample obtained from a subject. In some embodiments, the kit further includes reagents for isolating, concentrating, or expanding TRL from a body fluid sample. Such reagents may include antibodies (e.g., magnetic nanoparticles), buffers, and / or conditioned media.

[0161] In some embodiments, the kit comprises a vector comprising a polynucleotide encoding a CAR component described herein. The kit can comprise a plurality of vectors, each encoding a different protein or subset of proteins. These vectors can be viral, non-viral, episomal, or integrative. In some embodiments, the vector is a transposon. In some embodiments, the kit further comprises reagents or devices for TRL transfection or transduction, such as calcium phosphate DNA coprecipitation, DEAE-dextran, electroporation, cationic lipid-mediated transfection, tungsten particle-facilitated microparticle bombardment, and strontium phosphate DNA coprecipitation.

[0162] In some embodiments, the instructions further include instructions on how to manipulate the TRL to produce the enhanced TRL. The instructions may also include instructions for cryopreserving, lyophilizing, or cryopausing the compositions disclosed herein during storage and / or transportation. The instructions may also include instructions for thawing or otherwise restoring the biological activity of the compositions disclosed herein prior to administration to a subject. In some embodiments, the instructions may direct a medical laboratory to isolate the TRL from a body fluid sample (e.g., peripheral blood) disclosed herein using a system component (e.g., a microfluidic device) disclosed herein. For example, the instructions may include a method that includes separating a population of SLC6A19+SIDT1+ lymphocytes from a body fluid sample of a subject. In some embodiments, the instructions may include a method that includes magnetically separating a population of CD103 lymphocytes from a body fluid sample of a subject, and magnetically separating may include flowing the CD103 lymphocytes across a magnetic capture zone disposed in a channel of a microfluidic device. In some embodiments, the instructions include a method for administering to a subject a pharmaceutical formulation disclosed herein or an engineered TRL disclosed herein. In some embodiments, the method further includes treating cancer in a subject using a pharmaceutical formulation or an engineered TRL disclosed herein. Overall, the instructions may include any of the methods disclosed herein.

[0163] In some embodiments, the kits disclosed herein may be used to treat a subject's disease or disorder. For example, the kit may include a population of TRLs or enhanced TRLs in a pharmaceutical formulation for administration to a subject disclosed herein. In some embodiments, the kit includes instructions on how to formulate a population of TRLs or enhanced TRLs to produce a pharmaceutical formulation prior to administration to a subject. In some embodiments, the kit further includes additional therapeutic agents or active agents disclosed herein for the treatment of a disease or disorder. In some embodiments, the disease or disorder includes cancer. In some embodiments, the kit described herein includes a pharmaceutical formulation disclosed herein that includes a population of TRLs disclosed herein. In some embodiments, the TRLs can be further engineered to express the T cells or chimeric antigen receptors (e.g., enhanced TRLs) described herein. In some embodiments, the kit further includes additional therapeutic agents (e.g., anti-PD1 antibodies) such as those disclosed herein. In some embodiments, the kit further includes instructions for administering the pharmaceutical formulation and / or additional therapeutic agents to a subject to treat a disease or disorder such as cancer in the subject. In some embodiments, the kit includes instructions that can instruct a healthcare provider on how to treat a subject disclosed herein using the components of the kit at a healthcare facility or a point of care capacity. In some embodiments, the kit includes instructions for administering a composition to a subject in need of administration. In some embodiments, the kit includes instructions for further engineering a composition to express a biomolecule (e.g., a therapeutic agent). In some embodiments, the kit includes instructions for thawing or otherwise recovering the biological activity of a composition that can be cryopreserved, lyophilized, or cryopaused during storage or transportation. In some embodiments, the kit includes instructions for measuring the viability of the recovered composition to ensure its effectiveness for its intended purpose (e.g., therapeutic effectiveness when used to treat a subject).

[0164] In some embodiments, the kit may include one or more of the compositions disclosed herein, such as a microfluidic device, a culture and / or medium, a body fluid sample obtained from a subject, or an engineered TCR (e.g., a CAR), or any combination thereof. The kits disclosed herein may include instructions for separating TRLs from body fluid samples and / or for utilizing TRLs for therapeutic uses (e.g., treatment of cancer).

[0165] Optionally, the kit may also contain other useful components such as diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring tools, dressing materials, or other useful equipment. The materials or components assembled in the kit can be provided to medical personnel in any convenient and suitable form that retains their operability and usefulness. For example, the components may be in dissolved form, dehydrated form, or lyophilized form, and may be provided at room temperature, refrigerated temperature, or frozen temperature. The components are typically enclosed in suitable packaging materials. As used herein, the term "packaging material" refers to one or more physical structures used to contain the contents of the kit, such as a composition. The packaging material is preferably constructed by well-known methods to provide a sterile and contaminant-free environment. The packaging materials used in the kits are those conventionally utilized in the practice of gene expression assays and treatments. As used herein, the term "package" refers to a suitable solid matrix or material such as glass, plastic, paper, foil, etc. that is capable of holding the individual kit components. Thus, for example, the package may be a glass vial or a pre-filled syringe used to contain an appropriate amount of a pharmaceutical composition. The packaging material has an external label indicating the contents and / or purpose of the kit and its components.

[0166] V. Definitions Unless otherwise defined, all technical terms, notations, and other technical and scientific terms or terminology used in this specification are intended to have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter belongs. In some embodiments, terms with commonly understood meanings are defined in this specification for clarity and / or for ease of reference, and including such definitions in this specification should not necessarily be construed as representing a substantial difference from what is generally understood in the art.

[0167] Throughout this application, various embodiments may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, a description of a range should be considered to specifically disclose all the possible sub-ranges within that range, as well as the individual numerical values within that range. For example, a description of a range such as 1 - 6 should be considered to specifically disclose sub-ranges such as 1 - 3, 1 - 4, 1 - 5, 2 - 4, 2 - 6, 3 - 6, etc., as well as the individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the range.

[0168] As used in this specification and the claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "sample" includes plural samples, including mixtures thereof.

[0169] The terms "determining", "measuring", "evaluating", "assessing", "assaying", and "analyzing" are often used interchangeably herein to refer to forms of measurement. These terms include determining whether an element is present (e.g., detecting). These terms can include quantitative, qualitative, or both quantitative and qualitative determinations. An evaluation can be relative or absolute. "Detecting the presence of" can, depending on the context, include determining the amount of what is present in addition to determining whether it is present or not.

[0170] The terms "subject", "individual", or "patient" are often used interchangeably herein. A "subject" can be a biological entity that contains expressed genetic material. The biological entity can be a plant, an animal, or a microorganism, including, for example, bacteria, viruses, fungi, and protozoa. A subject can be a tissue, a cell, and their progeny of a biological entity obtained in vivo or cultured in vitro. A subject can be a mammal. The mammal can be a human. A subject can be diagnosed or suspected of being at high risk of a disease. In some embodiments, a subject is not necessarily diagnosed or suspected of being at high risk of a disease.

[0171] The term "in vivo" is used to describe an event that occurs within the body of a subject.

[0172] The term "ex vivo" is used to describe an event that occurs outside the body of a subject. An ex vivo assay is not performed on a subject. Rather, it is performed on a sample isolated from a subject. An example of an ex vivo assay performed on a sample is an "in vitro" assay.

[0173] The term "in vitro" is used to describe events that occur in a container holding laboratory reagents so that the materials are separated from the biological source from which they are obtained. An in vitro assay may include cell-based assays in which live or dead cells are utilized. An in vitro assay may also include cell-free assays that do not utilize intact cells.

[0174] As used herein, the term "about" refers to a number that is plus or minus 10% of that number. The term "about" refers to a range that subtracts 10% from the lowest value and adds 10% to the highest value.

[0175] Nucleic acids and / or nucleic acid sequences are "homologous" if they are naturally or artificially derived from a common ancestral nucleic acid or nucleic acid sequence. Proteins and / or protein sequences are homologous if the DNA encoding them is naturally or artificially derived from a common ancestral nucleic acid or nucleic acid sequence. Homologous molecules can be referred to as homologs. For example, any naturally occurring protein described herein can be modified by any available mutagenesis method. Upon expression, this mutagenized nucleic acid encodes a polypeptide that is homologous to the protein encoded by the original nucleic acid. Homology is generally inferred from sequence identity between two or more nucleic acids or proteins (or their sequences). The exact percentage of identity between sequences useful for establishing homology varies depending on the nucleic acids and proteins of interest, but a sequence identity of as little as 25% is conventionally used to establish homology. Higher levels of sequence identity, such as 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% or more, can also be used to establish homology. Methods for determining the percentage of sequence identity (e.g., BLASTP and BLASTN using default parameters) are described herein and are generally available.

[0176] The terms "identity" or "sequence identity" in the context of two nucleic acid or amino acid sequences of a polypeptide refer to residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window. A "comparison window", as used herein, refers to a segment of at least about 20 contiguous positions, usually about 50 to about 200, more usually about 100 to about 150, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of sequence alignment for comparison are well known in the art.Optimal array alignment for comparison may be performed by the local homology algorithm of Smith and Waterman, Adv. Appl. Math., 2:482 (1981), the alignment algorithm of Needleman and Wunsch, J. Mol. Biol, 48:443 (1970), the investigation of similarity methods by Pearson and Lipman, Proc. Nat. Acad. Sci U.S.A., 85:2444 (1988), computer-type implementations of these algorithms (including, but not limited to, CLUSTAL, GAP, BESTFIT, BLAST, FASTA, of the PC / Gene program by Intelligentics, Mountain View Calif, and TFASTA by the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis., U.S.A.), the CLUSTAL program fully described by Higgins and Sharp, Gene, 73:237-244 (1988) and Higgins and Sharp, CAB OS, 5:151-153 (1989), Nucleic Acids Res., 16:10881-10890 (1988) by Corpet et al., Computer Applications in the Biosciences, 8:155-165 (1992) by Huang et al., and Methods in Molecular Biology, 24:307-331 (1994) by Pearson et al. Alignment may also be performed by inspection and manual alignment.

[0177] In some embodiments, the polypeptides herein are, for example, at least 80%, 85%, 90%, 98%, 99%, or 100% identical to a reference polypeptide or fragment thereof, as measured by BLASTP (or CLUSTAL, or any other available alignment software) using default parameters. Similarly, nucleic acids can be described with reference to a starting nucleic acid and can be, for example, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 98%, 99%, or 100% identical to the reference nucleic acid or fragment thereof, as measured by BLASTP (or CLUSTAL, or any other available alignment software) using default parameters. When one molecule is said to have a certain percentage sequence identity to a larger molecule, this means that when the two molecules are optimally aligned, the aforementioned percentage of residues in the smaller molecule will be found in the larger molecule in the order in which the two molecules are optimally aligned, matching residues in the larger molecule.

[0178] The "functional variant" of the protein disclosed in this specification may include, for example, the amino acid sequence of a reference protein having at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 conservative amino acid substitutions. The phrase "conservative amino acid substitution" or "conservative mutation" refers to the substitution of one amino acid by another amino acid having common characteristics. A functional way to define the common characteristics between individual amino acids is to analyze the normalized frequency of amino acid changes between corresponding proteins of homologous organisms (Principles of Protein Structure by Schulz, G.E. and Schirmer, R.H., Springer-Verlag, New York (1979)). According to such analysis, groups of amino acids can be defined such that the amino acids within the group preferentially exchange with each other and are thus most similar to each other in terms of their impact on the overall protein structure. Examples of conservative mutations include amino acid substitutions within the above subgroups, for example, substitution from lysine to arginine such that the positive charge can be maintained, and vice versa, substitution from glutamic acid to aspartic acid such that the negative charge can be maintained, and vice versa, substitution from serine to threonine such that the free -OH can be maintained, and substitution from glutamine to asparagine such that the free -NH2 can be maintained.

[0179] Alternatively or additionally, the functional variant may include the amino acid sequence of a reference protein having at least one non - conservative amino acid substitution. "Non - conservative mutation" includes amino acid substitutions between different groups, for example, substitution from lysine to tryptophan, or substitution from phenylalanine to serine, etc. In this case, it is preferred that the non - conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. The non - conservative amino acid substitution may enhance the biological activity of the functional variant such that the biological activity of the functional variant is increased compared to the parental CAR.

[0180] The proteins disclosed herein (including their functional portions and functional variants) may contain synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino-n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine β-hydroxy-phenylalanine, phenylglycine, a-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, a-aminocyclopentanecarboxylic acid, a-aminocyclohexanecarboxylic acid, a-aminocycloheptanecarboxylic acid, a-(2-amino-2-norbornene)-carboxylic acid, α,γ-diaminobutyric acid, α,β-diaminopropionic acid, homophenylalanine, and a-tert-butylglycine.

[0181] As used herein, "polynucleotide" or "oligonucleotide" refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, this term includes double-stranded and single-stranded DNA, triple-stranded DNA, as well as double-stranded and single-stranded RNA. It also includes, for example, modifications such as by methylation and / or capping, as well as the unmodified form of the polynucleotide. This term also means that it includes molecules containing nucleotide analogs in addition to non-naturally occurring nucleotides or synthetic nucleotides.

[0182] "Polypeptide" is used interchangeably with the terms "polypeptide", "peptide", and "protein" and refers to a polymer of amino acid residues.

[0183] As used herein, "Synthetic" refers to a compound that is formed or expressed through a chemical process and / or by human intervention, as contrasted with a compound of natural origin.

[0184] As used herein, the terms "treatment" or "treating" are used with respect to a pharmaceutical regimen or other intervention regimen to obtain a beneficial or desired result in a recipient. Beneficial or desired results include, but are not limited to, therapeutic and / or prophylactic benefits. A therapeutic benefit may refer to the eradication or alleviation of a treated symptom or underlying disease. Also, a therapeutic benefit can be achieved by the eradication or alleviation of one or more of the physiological symptoms associated with an underlying disorder such that improvement is observed in a subject, even if the subject still suffers from the underlying disorder. Prophylactic effects include delaying, preventing, or eliminating the onset of a disease or illness, delaying or eliminating the onset of symptoms of a disease or illness, slowing, stopping, or reversing the progression of a disease or illness, or any combination thereof. In the case of a prophylactic benefit, a subject at risk of developing a particular disease, or a subject reporting one or more of the physiological symptoms of a disease, may be treated even if the subject has not been diagnosed with the disease.

[0185] As used herein, the term "tumor-reactive lymphocyte" refers to an immune cell that can recognize and target tumor cells. "Circulating tumor-reactive lymphocytes" refers to tumor-reactive lymphocytes found in a patient's body fluid sample (e.g., peripheral blood).

[0186] As used herein, the term "engineered" as used herein, and grammatical equivalents thereof, can refer to one or more modifications of a nucleic acid (e.g., a nucleic acid within the genome of an organism) or a polypeptide. The one or more modifications can include modification, addition, and / or deletion of a gene. An engineered cell can refer to a cell in which a gene has been added, deleted, and / or modified.

[0187] The term "isolate" refers to the removal of an entity (e.g., a cell, protein, nucleic acid) from its natural environment. However, it should be understood that a nucleic acid, protein, or cell can be formulated with a diluent or adjuvant and still be isolated for practical purposes. For example, a nucleic acid is typically mixed with an acceptable carrier or diluent when used for introduction into a cell.

[0188] The term "purify" refers to the case where the purity of an entity is increased, and "purity" is a relative term rather than "absolute purity". For example, the purity can be at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or can be 100%. However, it should be understood that a nucleic acid and a protein can be formulated with a diluent or adjuvant and still be purified for practical purposes. For example, a nucleic acid is typically mixed with an acceptable carrier or diluent when used for introduction into a cell.

[0189] As used herein, the terms "enrich" or "enriching" with respect to the cells of the present disclosure refer to the isolation of a particular cell population from a larger heterogeneous cell sample. For example, a system, method, and kit may include enrichment of CD103+ circulating TRLs (cTRLs) from a heterogeneous cell sample. Enrichment of CD103+ cTRLs can increase the number or percentage of CD103+ cTRLs compared to the total number of cells in the composition or the volume of the composition, or compared to other cell types, by positive selection based on a marker expressed by the population or cell, or negative selection based on a marker absent from the depleting cell population or cell. This term should not be construed to require complete removal of other cells, cell types, or populations from the composition, nor does it require that the cells so enriched be present at 100% or near 100% in the enriched composition.

[0190] The term "sorting" refers to the process of identifying, selecting, and separating a target cell or cell population from other components in a sample, such as other cells that are not of interest.

[0191] As used herein, a description that a cell or population of cells is "positive" or "+" for a particular marker refers to the detectable presence of the particular marker, typically a surface marker, on or in the cell. When referring to a surface marker, the term refers to the presence of surface expression. Surface expression can be detected by devices (e.g., flow cytometry, MACS, microfluidic devices) used to analyze the characteristics of cells or particles by staining with an antibody that specifically binds to the marker and detecting the antibody, where the staining is at a level substantially above that detected by performing the same procedure using an isotype-matched control under otherwise identical conditions, and / or at a level substantially similar to that of cells known to be positive for the marker, and / or at a level substantially higher than that of cells known to be negative for the marker, and is detectable by the device.

[0192] As used herein, the term "effective amount" refers to an amount or concentration of a composition sufficient to bring about the intended result. An effective amount can be a "therapeutically effective amount" that refers to an amount or concentration of a composition sufficient to bring about a therapeutic result (e.g., delay the onset of symptoms of a disorder being treated by the methods of the present disclosure, halt its progression, or alleviate or reduce at least one of its symptoms). The intended result can be the desired activity of the composition upon administration of the composition to a subject disclosed herein.

[0193] As used herein, the terms "pharmaceutically acceptable carrier", "pharmaceutically acceptable excipient", "physiologically acceptable carrier", or "physiologically acceptable excipient" refer to a pharmaceutically acceptable material, composition, or vehicle such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material. A component can be "pharmaceutically acceptable" in the sense of being compatible with the other components of a pharmaceutical formulation. It can also be suitable for use in contact with human and animal tissues or organs without undue toxicity, irritation, allergic response, immunogenicity, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0194] As used herein, the terms "administration", "administering", and variations thereof refer to introducing a composition or agent to a subject. Administration can include simultaneous and sequential introduction of a composition or agent. Introduction of a composition or agent to a subject can be by any suitable route including oral, pulmonary, intranasal, parenteral (intravenous, intramuscular, intraperitoneal, or subcutaneous), rectal, intralymphatic, or topical. Administration includes self-administration and administration by another person. A suitable route of administration enables the composition or agent to perform its intended function. For example, if the suitable route is intravenous, the composition is administered by introducing the composition or agent into a vein of the subject. Administration can be by any suitable route. In some embodiments, administration is intravenous administration. In some embodiments, administration is pulmonary administration. In some embodiments, administration is by inhalation.

[0195] As used herein, the term "pharmaceutical preparation" refers to the compositions disclosed herein and a diluent or carrier (e.g., a pharmaceutically acceptable inert ingredient) such as a carrier, excipient, binder, filler, suspending agent, flavoring agent, sweetening agent, disintegrant, dispersing agent, surfactant, lubricant, coloring agent, diluent, solubilizing agent, humectant, plasticizer, stabilizer, penetration enhancer, wetting agent, defoaming agent, antioxidant, preservative, or any combination thereof. A pharmaceutical preparation may facilitate the administration of the composition to a subject or cell. There are multiple techniques in the art for administering a compound, including but not limited to oral, injection, aerosol, parenteral, and topical administration.

[0196] An "expression vector" or "vector" is any genetic element capable of introducing an exogenous nucleic acid sequence into a cell or organism. Expression vectors can be, for example, plasmids, chromosomes, viruses, transposons, bacteriophages, or cosmids. An expression vector can act as an autonomous unit of polynucleotide replication within a cell (i.e., capable of replicating under its own control) to effect replication and / or expression of the exogenous nucleic acid sequence, or can be made replicable by insertion into a host cell chromosome to which another nucleic acid sequence is attached. A vector can contain polynucleotide sequences necessary to achieve ligation or insertion of the vector into a desired host cell and to affect expression of the joined portion. Such sequences vary depending on the host organism. Sequences include promoter sequences to achieve transcription, enhancer sequences to increase transcription, ribosome binding site sequences, and transcription / translation termination sequences. Alternatively, an expression vector may be capable of directly expressing the nucleic acid sequence product encoded therein without ligation or integration of the vector into the host cell DNA sequence.

[0197] The vector can also contain a "selectable marker gene". As used herein, the term "selectable marker gene" refers to a nucleic acid sequence that enables a cell expressing the nucleic acid sequence to be specifically selected in the presence of, or selected against, the corresponding selection agent.

[0198] The nucleic acid sequences and vectors disclosed or contemplated herein can be introduced into cells by "transfection", "transformation", or "transduction". As used herein, "transfection", "transformation", or "transduction" refers to the introduction of one or more exogenous polynucleotides into a host cell using physical or chemical methods. Many transfection techniques are known in the art, including, for example, calcium phosphate DNA co-precipitation, DEAE-dextran, electroporation, cationic liposome-mediated transfection, tungsten particle-facilitated microparticle bombardment, and strontium phosphate DNA co-precipitation. Phage vectors or viral vectors can be introduced into host cells after growing infectious particles in suitable packaging cells, and many of the packaging cells are commercially available.

[0199] In some embodiments, the vector is an "episomal expression vector" or "episome" that can replicate in a host cell and persists as an extrachromosomal portion of DNA within the host cell in the presence of appropriate selection pressure. Representative commercially available episomal expression vectors include, but are not limited to, episomal plasmids that utilize Epstein-Barr nuclear antigen 1 (EBNA1) and the Epstein-Barr virus (EBV) origin of replication (oriP). Vectors pREP4, pCEP4, pREP7, and pcDNA3.1 from Invitrogen (Carlsbad, Calif.), and pBK-CMV from Stratagene (La Jolla, Calif.) represent non-limiting examples of episomal vectors that use the T antigen and the SV40 origin of replication instead of EBNA1 and oriP.

[0200] As used herein, "Operably linked" refers to physically and / or functionally linking a nucleic acid (e.g., DNA) portion to another nucleic acid (e.g., DNA) portion such that the portion functions in its intended manner. In non-limiting examples, a DNA sequence encoding a gene product is operably linked to a regulatory sequence (e.g., a promoter, enhancer, and / or silencer) when the regulatory sequence is capable of directly or indirectly regulating the transcription of the DNA sequence. In another non-limiting example, a DNA sequence is operably linked to a promoter when it is ligated to a promoter downstream of the transcription start site of the promoter in the correct reading frame with respect to the transcription start site such that transcription elongation is permitted through the DNA sequence. In yet another non-limiting example, an enhancer or silencer is operably linked to a DNA sequence encoding a gene product when it is ligated to the DNA sequence to increase or decrease the transcription of the DNA sequence, respectively. Enhancers and silencers may be located upstream or downstream of the coding region of the DNA sequence or may be embedded within the coding region. When a signal sequence is expressed as a protein precursor involved in the secretion of a polypeptide, the DNA of the signal sequence can be operably linked to the DNA encoding the polypeptide. Ligation of a DNA sequence to a regulatory sequence is typically accomplished by ligation at a suitable restriction site using restriction endonucleases known to those of skill in the art or via an adapter or linker inserted into the sequence.

[0201] As used herein, the term "sample" can include any material from which nucleic acids, cells, and / or proteins can be obtained. Non-limiting examples of samples include, but are not limited to, whole blood, peripheral blood, plasma, serum, saliva, mucus, urine, semen, lymph fluid, fecal extracts, buccal swabs, cells or other body fluids or tissues including tissue obtained by surgical biopsy. In some embodiments, the sample includes tissue derived from a tumor. In some embodiments, the sample is a body fluid sample. In some embodiments, the sample includes tissue derived from a tumor. In some embodiments, the sample is whole blood, or plasma or serum derived from whole blood. In some embodiments, the sample is a peripheral blood sample, or plasma or serum derived from peripheral blood. Alternatively, the sample can be obtained via a primary patient-derived cell line, or a stored patient sample in the form of a preserved sample, or a fresh frozen sample.

[0202] "CD103" is also known as integrin subunit alpha E or ITGAE and refers to a cell surface protein such as the protein provided by GenBank: AAI13437.1. This belongs to the integrin family of cell adhesion molecules. Integrins are heterodimeric transmembrane proteins composed of an alpha chain and a beta chain.

[0203] "CD3" is also known as cluster of differentiation 3 and refers to a cell surface protein expressed on the surface of immune cells. The CD3 complex consists of several subunits including CD3γ, CD3δ, CD3ε, and CD3ζ.

[0204] "CD4" is also known as cluster of differentiation 4 and refers to a cell surface protein expressed on the surface of immune cells. The CD4 antigen functions as a co-receptor with the T cell receptor on T lymphocytes and recognizes antigens presented by antigen-presenting cells in the context of class II MHC molecules. This is expressed not only on T lymphocytes but also on B cells, macrophages, granulocytes, and various regions of the brain.

[0205] "CD8", also known as cluster of differentiation 8, refers to cell surface proteins expressed on the surface of immune cells. The CD8 antigen is a cell surface glycoprotein found on most cytotoxic T lymphocytes that mediates efficient cell-cell interactions within the immune system. The CD8 antigen functions as a co-receptor with the T cell receptor on T lymphocytes and recognizes antigens presented by antigen-presenting cells in the context of class I MHC molecules. The co-receptor functions as a homodimer composed of two α chains or as a heterodimer composed of one α chain and one β chain. Both the α chain and the β chain share significant homology with the immunoglobulin variable light chain. Multiple transcript variants encoding different isoforms have been found for this gene.

[0206] "CD39", also known as ectonucleoside triphosphate diphosphohydrolase 1 (ENTPD1), refers to cell surface enzymes expressed on various immune cells. CD39 is a plasma membrane protein that hydrolyzes extracellular ATP and ADP to AMP. Inhibition of the activity of this protein may result in anti-cancer benefits. Several transcript variants encoding different isoforms have been found for this gene.

[0207] "SLC6A19", also known as solute carrier family 6 member 19, is a gene encoding a system B(0) transmembrane protein that actively transports most neutral amino acids across the apical membrane of epithelial cells.

[0208] "SIDT1", also known as SID1 transmembrane family member 1, is involved in the transport of small interfering RNA (siRNA) across the cell membrane. SIDT1 belongs to the SID1 family of transmembrane dsRNA-dependent channels. Family members transport dsRNA into cells and are required for systemic RNA interference.

[0209] The section headings used in this specification are for organizational purposes only and should not be construed as limiting the subject matter described.

Example

[0210] The following examples are included for illustrative purposes only and are not intended to limit the scope of the inventive concept.

[0211] Example 1: Profiling and Isolation of Circulating Tumor-Reactive Lymphocytes in Blood for Cancer Immunotherapy Isolation and expansion of tumor-reactive lymphocytes (TRLs) are essential procedures for adoptive cell therapy. However, existing expansion methods are invasive as they require the use of tumor tissue as a starting material and cannot be applied to patients with inoperable tumors or recurrent metastases. In this example, it is shown that TRLs are present in peripheral blood circulation at low frequency during tumor progression and can be selectively isolated using a high-performance microfluidic device. Transcriptome analysis and tetramer-binding experiments on circulating TRLs (cTRLs) revealed that the expression of CD103 almost exclusively defines the tumor reactivity of CD8 T cells in peripheral blood. In this example, it is further demonstrated that expanded CD8+CD103 cTRLs have therapeutic efficacy comparable to that of tumor-infiltrating lymphocytes. Additionally, CD8+CD103 cTRLs isolated from patient samples resulted in a significant enrichment of intratumoral TCR clonotypes and IFN-γ-secreting populations during co-culture. This example demonstrates that TRLs can be isolated using minimally invasive strategies for therapeutic use.

[0212] Introduction Autologous transplantation of tumor-infiltrating lymphocytes (TILs) expanded from resected tumors has emerged as a promising therapeutic modality in the clinic. Adoptive cell therapy using TILs has significant advantages over other allogeneic and engineered cell therapies due to its unique heterogeneity, which maximizes tumor-recognizing T cell receptors (TCRs) while minimizing off-tissue effects. Clinical outcomes are extremely promising, and long-term complete responses have been observed in subsets of melanoma patients to date.

[0213] Despite the positive outcomes from pioneering clinical trials, the applicability of TIL-mediated ACT has mainly been demonstrated in metastatic melanoma with resectable metastatic lesions that are often large (greater than 3 cm in diameter), providing an optimal source material for TIL isolation. 4 However, in other solid tumors, such large lesions are not readily accessible. Additionally, in some embodiments, resection surgery may not be an option for patients due to substantial risk or rapid tumor progression. Recent trials have explored the potential of applying TIL ACT to other solid tumors such as renal cancer, cervical cancer, and breast cancer. However, limitations in TIL functionality and reduced response rates have been observed. Thus, existing TIL expansion workflows are considered to have low compatibility with treatment modalities for other types of solid tumors.

[0214] Advances in organoid development and deep sequencing have facilitated the generation of TIL-like tumor-reactive lymphocytes (TRLs) by co-culturing peripheral blood lymphocytes with tumor-derived organoids or peptide pools derived from tumor neoantigens. 10 However, these techniques still require resection of primary tumor cells obtained using invasive surgical procedures. Additionally, the establishment of organoids 9 and the synthesis of neoantigen-derived peptides 10 take several weeks to complete. Overall, the need for tumor biopsies and long workflows limits the translational value of these techniques as alternatives to TILs.

[0215] Subsets of peripheral lymphocytes share a clonotype with TILs, and their expansion is highly correlated with response to treatment. Trials also show the presence of very low-frequency TRLs in circulation in melanoma patients even before immunotherapy. However, all existing techniques rely on neoantigen-derived multimers to identify and isolate such rare populations from circulation, thus requiring characterization of tumor neoantigens by invasive biopsy. Biomarkers that provide clear discrimination between cTRLs and their non-tumor-reactive counterparts remain under investigation.

[0216] In most molecular approaches, at least thousands of cells are required as input, and millions of cells need to be tested for therapeutic efficacy. Therefore, rarity remains a significant challenge in the comprehensive analysis and therapeutic applications of cTRL. Given the low level of 0.002% in the peripheral T cell population, it is extremely difficult to enrich cTRL to high purity and recover it for downstream analysis. This example focuses on the development of a microfluidic platform that enables the analysis and enrichment of extremely rare cells, allowing for the accurate profiling of their phenotypes under conditions of limited cell numbers.

[0217] A systematic identification of surface markers for isolating cTRL using a microfluidic platform was performed. An ATCR-mediated labeling workflow was established to convert the level of tumor reactivity into the degree of magnetization in immunomagnetic cell sorting. cTRL for transcriptome and clone comparison was purified from an animal model with a defined epitope. The CD8+CD103 signature was found to define almost exclusively the circulating tumor-reactive population. This population has a tissue-resident-like (Trm-like) phenotype and the ability to re-enter the bloodstream from the primary tumor and accumulate in secondary tumors. cTRL has been demonstrated to have strong potential as a therapeutic agent using multiple adoptive cell transfer models. Enrichment based on CD8+CD103 was also confirmed to result in higher tumor reactivity in patient cohorts by comparing the levels of interferon gamma (IFN-γ) secretion and clone similarity.

[0218] High-performance cell isolation enabling cTRL profiling The overall workflow of the tumor-reactive mediator-based cell labeling and sorting strategy is illustrated in Fig. 1A. Lymphocytes are treated with major histocompatibility complex (MHC) multimers that mimic defined tumor epitopes, and a subset of TRLs with putative tumor reactivity is selectively isolated. The multimer is conjugated to a fluorophore used as a linker for attaching magnetic nanoparticles (MNPs). To separate multimer-bound lymphocytes or tumor-reactive lymphocytes, the magnetically labeled cell mixture is processed in a microfluidic device clamped by an array of magnets (Figs. 11A - 11E). This device contains multiple capture zones that can spatially separate cells by varying degrees of magnetization, with higher magnetization resulting in capture in the compartment closer to the inlet (Fig. 12). In the case of multimer-mediated labeling, TRLs are captured here, while the non-TRL population is captured in a different compartment. The captured cells can be easily and efficiently recovered from the device compartments when the external magnet is removed. The recovered cells are highly viable and suitable for downstream culture and analysis. As previously reported, microfluidic cell sorting is superior to commercially available cell sorting techniques when recovering rare cell populations. In multimer-mediated labeling and sorting, our microfluidic approach achieved a cell recovery rate up to 10-fold higher while maintaining similar purity (Fig. 13).

[0219] This approach was pursued for the isolation of cTRLs using animal models with two highly immunogenic epitopes, chicken ovalbumin (OVA257 - 264, SIINFEKL) in the C57BL6 model and influenza A hemagglutinin (HA533 - 541, IYSTVASSL) in the Balb / c model. Tumor cells with / without the expression of these defined epitopes were injected subcutaneously. Blood and tumors were collected at the intermediate to late stages of tumorigenesis (300 - 800 mm 3(determined by tumor size), appropriately labeled with multimers and antibodies corresponding to CD8+ T cells (FIGS. 1B and 7A-7B). By flow cytometry analysis, it was found that mice with tumors expressing immunogenic epitopes showed a higher degree of immune response and slower tumor growth (FIGS. 8A-8C). The upregulated immune response produced a significantly higher fraction of OVA / HA-reactive T cells presented in the tumor and blood (FIGS. 1C, 9A-9C), which is consistent with past observations in melanoma patients. In addition to cytometry analysis, the inventors utilized the corresponding multimers together with MNP and microfluidics to purify tumor-reactive T cells. The purity of isolation was 76% and 84% with OVA and HA multimers, respectively (FIG. 1D).

[0220] Due to the high yield and purity by microfluidic sorting, the inventors were able to perform direct TCR sequencing on rare cTRL populations (FIG. 1E). The inventors identified over 1,000 clonotypes (defined by unique CDR sequences) in cTRL. By using V-J mapping, a significant similarity between cTRL and intratumoral TIL, rather than the non-reactive part of PBMC, was revealed. From the perspective of the TCR repertoire, cTRL covers 30%-85% of the top 50 clones presented in TIL, which is 3-8 times higher than the coverage observed in PBMC (about 10%, FIG. 1F). Collectively, these data suggest that cTRL exists in circulation during disease progression and shares a higher degree of clonal similarity with intratumoral TIL. It is thus recognized that cTRL may originate from or be derived from the same precursor as intratumoral TIL.

[0221] High yields and purity by microfluidic sorting also enabled RNAseq to be performed on rare cTRL populations. In OVA-reactive cTRLs isolated from melanoma models, a memory phenotype was detected along with upregulated expression of TCF7, IL7R, and LEF1 (Figure 2A). Furthermore, cTRLs had high expression of ITGAE (CD103) and moderate to high expression of CD69. Among lineage-specific transcription factors, cTRLs upregulated the expression of ZFP683 (also known as Hobit), but not TBX21 (also known as T-bet) and EOMES (Figure 2A, right).

[0222] Therefore, cTRLs have a phenotype similar to that of tissue-resident memory T (Trm) cells. Trm cells are regarded as the front line for protecting the host at the site of pathogenesis. 26 。Gene set enrichment analysis (GSEA) further suggests that cTRLs have a statistically significant upregulation of T cell activation and TCR signaling pathways (Figure 2B, Figure 14). A similar trend was observed in marker expression in HA-reactive cTRLs isolated from a colon cancer model (Figure 15). By overlaying differentially expressed genes, 26 shared and upregulated genes were identified (Figure 2C). Genes known to be transiently or permanently upregulated during TCR-MHC interaction (e.g., CRTAM27, GGT128, and CD8A29) were excluded from this subset. One of the most potent hits observed was ITGAE, the gene encoding CD103. Since this factor is associated with a migratory phenotype and shows low expression across PBMCs 30 , it is recognized that it may represent a specific biomarker relevant to cTRLs.

[0223] To further evaluate CD103 as a marker for cTRL isolation, circulating CD8+ lymphocytes were isolated from mice bearing B16F10 and AE17 cancer cell lines expressing the OVA epitope. Flow cytometry analysis revealed that OVA-reactive cTRLs were almost exclusively CD103 (Figure 2D). Collectively, since approximately 30% of CD8+CD103 cells in circulation are OVA-reactive (Figure 2E), up to a 50-fold enrichment of OVA-reactive T cells is achieved. Thus, CD103 serves to define the tumor-reactive population in bulk T cells, either 31、32 alone or in combination with other markers 33 to define T cells with enhanced efficacy for adoptive cell therapy and immune checkpoint blockade. However, it is interesting that such a Trm-like phenotype, thought to be present in non-lymphoid tissues, exists in the circulation.

[0224] To investigate the mechanisms underlying the presence of CD8+CD103 cTRL in the circulation, we used a tumor transplantation model to visualize the trafficking of these cells. In this case, OVA-expressing tumors from donor CD45.2 mice were orthotopically transplanted into host CD45.1 mice bearing secondary (second) tumors with or without the OVA epitope. Two tumors were grown simultaneously, and the second tumor was harvested last for flow cytometry analysis (Figure 2F, Figures 10A–10B). For all specimens analyzed, we observed a small fraction of CD45.2+ cells presented in the second tumor. Since the only source of CD45.2+ T cells is TILs within the transplanted tumor, this observation provides direct evidence that TILs can enter the circulation and migrate to distant organs. Furthermore, the second tumor with the OVA epitope attracted at least five-fold higher percentages of CD45.2+ T cells compared to the WT tumor without OVA (Figures 2F–2G, Figures 10C–10F). This suggests that the migration and accumulation of cTRL are antigen-driven. Sites with similar infection patterns (e.g., the same immunogenic epitope) attract more cTRL and allow it to re-infiltrate and persist. We also examined the molecular signature of migratory cTRL in the second tumor by CyTOF. CD45.2+ cTRL had higher expression of CD103, CD69+, and PD-1+ compared to CD45.1+ endogenous T cells (Figures 2H–2I), suggesting that they retained a Trm-like activated phenotype after migration.

[0225] In summary, it was found that from the collected data, Trm-like cTRLs in the blood migrate towards distant tumors and specifically accumulate in those tumors. This observation is consistent with new evidence indicating that Trm cells enter the circulation to enhance the overall immune response. Mechanistically, CD103 is an integrin protein that binds to E-cadherin and controls the formation of cell protrusions / filopodia, an essential component for initiating cell migration. This points to an important role of CD103 in cell motility. Additionally, CD103+ TRLs have been reported to have an elevated energy potential and enhanced migratory ability. Thus, the phenotypic characteristics of cTRLs are consistent with previous observations of CD103+ T cells.

[0226] Proliferation and administration of cTRLs for testing therapeutic activity Due to the "homing" behavior of cTRLs towards specific tumor microenvironments, cTRLs are attractive candidates for adoptive cell therapy. Therefore, a rapid expansion protocol (REP) for rare tumor-reactive TILs was adopted, achieving up to 2000-fold cTRL expansion in 10 days, resulting in a final number of approximately 100,000 - 4...

Claims

1. A method for enriching a population of tumor-reactive lymphocytes (TRLs), the method comprising enriching a population of lymphocytes comprising CD103 from a peripheral blood sample of a subject.

2. A method for enriching a population of tumor-reactive lymphocytes (TRLs), the method comprising enriching a population of lymphocytes comprising SLC6A19, SIDT1, or SLC6A19 and SIDT1 from a body fluid sample of a subject.

3. The method according to claim 2, wherein the population of lymphocytes further comprises CD3, CD4, CD8, CD39, CD103, or any combination thereof.

4. The method according to claim 2, wherein the population of lymphocytes comprises CD8, CD103, SLC6A19, and SIDT1.

5. The method according to any one of claims 1 to 4, wherein the enriching comprises magnetic separation by flowing the population of lymphocytes across a magnetic capture zone disposed in a channel of a microfluidic device.

6. A method for isolating a population of tumor-reactive lymphocytes (TRLs), the method comprising magnetically separating a population of CD103+ lymphocytes from a body fluid sample of a subject, wherein the magnetically separating comprises flowing the CD103+ lymphocytes across a magnetic capture zone disposed in a channel of a microfluidic device.

7. The method according to any one of claims 2 to 6, wherein the body fluid sample is a peripheral blood sample.

8. The method according to any one of claims 2 to 6, wherein the body fluid sample comprises pleural effusion.

9. The method according to any one of claims 2 to 6, wherein the body fluid sample comprises ascites.

10. The method according to claim 6, wherein the CD103+ lymphocytes are CD8+CD103+ lymphocytes, CD3+CD103+ lymphocytes, CD4+CD103+ lymphocytes, or CD39+CD103+ lymphocytes.

11. The method according to claim 6, wherein the CD103+ lymphocytes comprise CD8+CD103+SLC6A19+ lymphocytes, CD3+CD103+SLC6A19+ lymphocytes, or CD4+CD103+SLC6A19+ lymphocytes.

12. The method according to claim 6, wherein the CD103+ lymphocytes comprise CD8+CD103+SLC6A19+SIDT1+ lymphocytes, CD3+CD103+SLC6A19+SIDT1+ lymphocytes, or CD4+CD103+SLC6A19+SIDT1+ lymphocytes.

13. The method according to any one of claims 5 to 12, wherein the magnetic separation achieves a recovery rate of at least 70%.

14. The method according to any one of claims 5 to 12, wherein the magnetic separation achieves a recovery rate of at least 80%.

15. The method according to any one of claims 5 to 12, wherein the magnetic separation achieves a recovery rate of at least 90%.

16. The method according to any one of claims 1 to 15, wherein the purity of the isolation of the population of the TLRs is at least 60%.

17. The method according to any one of claims 1 to 15, wherein the purity of the isolation of the population of the TLRs is at least 70%.

18. The method according to any one of claims 1 to 17, wherein the purity of the isolation of the population of the TLRs is at least 80%.

19. The method according to any one of claims 5 to 18, wherein the magnetic separation comprises contacting the body fluid sample with an antibody capable of binding to the TLR surface protein.

20. The method according to claim 19, wherein the TLR surface protein is CD3, CD4, CD8, CD39, CD103, SLC6A19, SIDT1, or any combination thereof.

21. The method according to claim 20, wherein the TLR surface protein is CD103.

22. The method according to claim 20, wherein the TLR surface protein is CD39.

23. The method according to claim 20, wherein the TLR surface protein is SLC6A19.

24. The method according to claim 20, wherein the TLR surface protein is SIDT1.

25. The method according to any one of claims 20 to 24, wherein the antibody is conjugated to magnetic nanoparticles.

26. The method according to any one of claims 5 to 25, wherein the magnetic separation comprises contacting the body fluid sample with a second antibody capable of binding to an antibody capable of binding to the TLR surface protein.

27. The method according to claim 26, wherein the second antibody is conjugated to magnetic nanoparticles.

28. The method according to any one of claims 5 to 27, wherein the magnetic separation comprises contacting the body fluid sample, which comprises a peripheral blood sample, with a plurality of major histocompatibility complex (MHC) multimers that mimic tumor epitopes.

29. The method according to claim 28, wherein at least one of the plurality of MHC multimers is conjugated to a linker molecule.

30. The method according to claim 29, wherein the linker molecule comprises a fluorophore.

31. The method according to claim 30, wherein the linker molecule is conjugated to a magnetic particle.

32. The method according to claim 31, wherein the magnetic particle comprises a magnetic nanoparticle.

33. The method according to any one of claims 5 to 32, wherein the microfluidic device comprises a plurality of magnetic capture zones, and the plurality of magnetic capture zones are arranged to spatially separate cells having different degrees of magnetization.

34. The method according to any one of claims 1 to 33, wherein the subject has cancer or is suspected of having cancer.

35. The method according to claim 34, wherein the cancer is in a tissue, and the tissue comprises breast tissue, kidney tissue, cervical tissue, lung tissue, ovarian tissue, or skin tissue.

36. The method according to any one of claims 1 to 35, comprising culturing the magnetically separated TRL.

37. The method according to any one of claims 1 to 36, comprising introducing cells from the cultured TRL into a nucleotide that expresses a therapeutic enhancing polypeptide under conditions sufficient for the cells to produce the enhancing polypeptide.

38. The method according to claim 37, comprising culturing the cells to produce therapeutically enhanced cells.

39. The method according to claim 38, wherein the nucleotide encodes a chimeric antigen receptor (CAR).

40. The method according to claim 39, wherein the CAR comprises an antigen-binding domain.

41. The method according to claim 40, wherein the antigen-binding domain binds to a tumor antigen.

42. The method according to claim 37, wherein the nucleotide encodes an engineered T cell receptor.

43. The method according to claim 42, wherein the engineered T cell receptor comprises an antigen-binding domain.

44. The method according to claim 43, wherein the antigen-binding domain binds to a tumor antigen.

45. The method according to any one of claims 1 to 44, further comprising administering the cultured TRL to the subject in a therapeutically effective amount.

46. The method according to claim 45, wherein the subject is treated for cancer or precancer.

47. The method according to claim 46, wherein the cancer is in a tissue, and the tissue comprises breast tissue, kidney tissue, cervical tissue, ovarian tissue, lung tissue, or skin tissue.

48. The method according to any one of claims 45 to 47, wherein the therapeutically enhanced cells are administered together with an additional cancer therapy.

49. The method according to claim 48, wherein the additional cancer therapy comprises an immune checkpoint inhibitor therapy.

50. The method according to claim 48, wherein the additional cancer therapy comprises a co-stimulation therapy.

51. The method according to claim 50, wherein the co-stimulation therapy comprises administering GITR.

52. The method according to claim 49 or 50, wherein the additional cancer therapy is an anti-PD1 therapy or an anti-PD-L1 therapy.

53. The method according to claim 52, wherein the anti-PD1 therapy or the anti-PD-L1 therapy comprises an anti-PD1 antibody or an antigen-binding fragment thereof.

54. The method according to any one of claims 1 to 53, wherein the population of tumor-reactive lymphocytes (TRL) comprises a population of circulating tumor-reactive lymphocytes (cTRL).

55. A pharmaceutical formulation comprising: a plurality of cells comprising (i) a population of SLC6A19+ lymphocytes, (ii) a population of SIDT1+ lymphocytes, (iii) a population of CD103+ lymphocytes, or (iv) a combination of (i) to (iii); and a pharmaceutically acceptable excipient, diluent, or vehicle. The pharmaceutical formulation is formulated for administration to a subject having or suspected of having cancer.

56. The pharmaceutical formulation according to claim 55, which is formulated for administration together with an additional therapeutic agent for the treatment of cancer.

57. The pharmaceutical formulation according to claim 56, wherein the additional therapeutic agent comprises an anti-PD1 antibody.

58. The pharmaceutical formulation according to any one of claims 55 to 57, wherein the CD103+ lymphocytes are CD8+CD103+ lymphocytes, CD3+CD103+ lymphocytes, CD4+CD103+ lymphocytes, or CD39+CD103+ lymphocytes.

59. The pharmaceutical formulation according to any one of claims 55 to 57, wherein the CD103+ lymphocytes are CD8+CD103+SLC6A19+ lymphocytes, CD3+CD103+SLC6A19+ lymphocytes, or CD4+CD103+SLC6A19+ lymphocytes.

60. The pharmaceutical preparation according to any one of claims 55 to 57, wherein the CD103+ lymphocyte is a CD8+CD103+SLC6A19+SIDT1+ lymphocyte, a CD3+CD103+SLC6A19+SIDT1+ lymphocyte, or a CD4+CD103+SLC6A19+SIDT1+ lymphocyte.

61. The pharmaceutical preparation according to any one of claims 55 to 60, wherein the cancer is in the tissue of the subject, and the tissue includes breast tissue, kidney tissue, cervical tissue, lung tissue, ovarian tissue, or skin tissue.

62. The pharmaceutical preparation according to any one of claims 55 to 61, wherein at least a part of the plurality of cells further comprises a chimeric antigen receptor (CAR).

63. The pharmaceutical preparation according to claim 62, wherein the CAR includes an antigen-binding domain.

64. The pharmaceutical preparation according to claim 63, wherein the antigen-binding domain binds to a tumor antigen.

65. The pharmaceutical preparation according to any one of claims 55 to 61, wherein at least a part of the plurality of cells further comprises an engineered T cell receptor.

66. The pharmaceutical preparation according to claim 65, wherein the engineered T cell receptor includes an antigen-binding domain.

67. The pharmaceutical preparation according to claim 66, wherein the antigen-binding domain binds to a tumor antigen.

68. (a) lymphocytes containing cell surface markers including CD3, CD4, CD39, CD103, SLC6A19, or SIDT1, or any combination thereof, and (b) a chimeric antigen receptor (CAR) An engineered tumor-reactive lymphocyte (TRL).

69. The tumor-reactive lymphocyte is (i) CD8, CD39, and CD103, (ii) CD3, CD39, and CD103, (iii) CD4, CD39, and CD103, (iv) CD8, CD103, SLC6A19, and SIDT1, (v) CD3, CD103, SLC6A19, and SIDT1, or (vi) CD4, CD103, SLC6A19, and SIDT1, and the engineered TRL according to claim 68.

70. The engineered TRL according to claim 68, wherein the CAR includes an antigen-binding domain.

71. The engineered TRL according to claim 68, wherein the antigen-binding domain binds to a tumor antigen.

72. The engineered TRL according to claim 71, for use in treating cancer in a subject in need of treatment for cancer.

73. A method of treating cancer in a subject, the method comprising administering to the subject cells produced by any of the methods according to claims 1 to 54.

74. A method of treating cancer in a subject, the method comprising administering to the subject a pharmaceutical preparation according to any one of claims 55 to 67 or an engineered TLR according to any one of claims 68 to 72.

75. The method according to claim 73 or 74, wherein the cancer is in the tissue of the subject, and the tissue comprises breast tissue, kidney tissue, cervical tissue, lung tissue, ovarian tissue, or skin tissue.

76. The method according to claim 73 or 74, further comprising administering an additional cancer therapy to the subject.

77. The method according to claim 76, wherein the additional cancer therapy comprises immune checkpoint inhibitor therapy.

78. The method according to claim 76, wherein the additional cancer therapy comprises co-stimulation therapy.

79. The method according to claim 78, wherein the co-stimulation therapy comprises administering GITR.

80. The method according to claim 76 or 77, wherein the additional cancer therapy is anti-PD1 therapy or anti-PD-L1 therapy.

81. The method according to claim 80, wherein the anti-PD1 therapy or the anti-PD-L1 therapy comprises an anti-PD1 antibody or an antigen-binding fragment thereof.

82. A kit comprising: (a) an engineered TLR according to any one of claims 68 to 72, or a pharmaceutical preparation according to any one of claims 55 to 67; and (b) instructions for administering the engineered TLR to a subject in need thereof.

83. A kit comprising: (a) cells produced by any of the methods according to claims 1 to 54; and (b) instructions for administering the cells to a subject.

84. A body fluid sample obtained from a subject, the body fluid sample comprising a population of cells comprising (i) CD103+ lymphocytes, (ii) SLC6A19+ lymphocytes, (iii) SIDT1+ lymphocytes, or (iv) any combination of (i) to (iii); and A microfluidic device comprising a magnetic capture zone disposed therein, the microfluidic device configured to magnetically separate the population of cells from the body fluid sample.

85. The system according to claim 84, wherein the population of SLC6A19+ lymphocytes comprises CD103+ SLC6A19+ SIDT1+ lymphocytes.

86. The system according to claim 84, wherein the population of SLC6A19+ lymphocytes comprises CD8+ CD103+ SLC6A19+ SIDT1+ lymphocytes.

87. The system according to claim 84, wherein the CD103+ lymphocytes are CD8+ CD103+ lymphocytes.

88. The system according to claim 84, wherein the CD103+ lymphocytes are CD8+ CD39+ CD103+ lymphocytes.

89. The system according to claim 84, wherein the CD103+ lymphocytes comprise CD8+ CD103+ SLC6A19+ lymphocytes.

90. The system according to any one of claims 84 to 89, wherein the body fluid sample is a peripheral blood sample.

91. The system according to any one of claims 84 to 89, wherein the body fluid sample comprises pleural effusion.

92. The system according to any one of claims 84 to 89, wherein the body fluid sample comprises ascites.

93. The system according to any one of claims 84 to 92, further comprising an antibody or an antigen-binding fragment thereof capable of binding to a TLR surface protein.

94. The system according to claim 93, wherein the TLR surface protein is CD8, CD103, SLC6A19, SIDT1, or a combination thereof.

95. The system according to claim 93, wherein the antibody or antigen-binding fragment is conjugated to magnetic nanoparticles.

96. The system according to any one of claims 84 to 95, further comprising a plurality of major histocompatibility complex (MHC) multimers mimicking tumor epitopes.

97. The system according to claim 96, wherein at least one of the plurality of MHC multimers is conjugated to a linker molecule comprising a fluorophore or magnetic particles.

98. The system according to any one of claims 84 to 97, wherein the microfluidic device comprises a plurality of magnetic capture zones, and the plurality of magnetic capture zones are arranged to spatially separate cells having different magnetic susceptibilities.

99. The system according to any one of claims 84 to 98, further comprising a cell culture configured to culture therapeutic enhancing cells derived from a population of (i) CD103+ lymphocytes, (ii) SLC6A19+ lymphocytes, or (iii) SIDT1+ lymphocytes.

100. The system according to any one of claims 84 to 99, further comprising a nucleic acid molecule encoding a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR).

101. The system according to claim 100, wherein the CAR or the engineered TCR comprises an antigen-binding domain that binds to a tumor antigen.

102. Further comprising a pharmaceutically acceptable excipient, diluent, or vehicle, wherein a population of (i) CD103+ lymphocytes, (ii) SLC6A19+ lymphocytes, or (iii) SIDT1+ lymphocytes is formulated with the pharmaceutically acceptable excipient, diluent, or vehicle for administration to a subject having or suspected of having cancer. The system according to any one of claims 84 to 101.

103. The system according to any one of claims 84 to 102, further comprising an additional cancer therapy.

104. The system according to claim 103, wherein the additional cancer therapy comprises an immune checkpoint inhibitor therapy or a co-stimulation therapy.

105. The system according to claim 103, wherein the additional cancer therapy is an anti-PD1 therapy or an anti-PD-L1 therapy.

106. The system according to claim 105, wherein the anti-PD1 therapy or the anti-PD-L1 therapy comprises an anti-PD1 antibody or an antigen-binding fragment thereof.

107. A composition comprising cells derived from a sample enriched for lymphocytes comprising SLC6A19, SIDT1, CD103, or any combination thereof.

108. The sample is enriched for the lymphocytes by a process comprising magnetic separation of the lymphocytes from the sample, and the magnetic separation comprises flowing the lymphocytes across a magnetic capture zone disposed within a channel of a microfluidic device. The composition according to claim 107.

109. The composition according to any one of claims 107 to 108, wherein the process yields a sample having a purity of about 70% or greater.

110. The composition according to any one of claims 106 to 108, wherein the process yields a sample having a purity of about 80% or greater.

111. A composition comprising cells derived from a sample enriched with lymphocytes comprising SLC6A19, SIDT1, CD103, or any combination thereof, wherein the sample is enriched with the lymphocytes by a process comprising the method according to any one of claims 1 to 81.

112. The composition according to any one of claims 107 to 111, wherein the lymphocytes further comprise CD3, CD4, CD8, CD39, CD103, or any combination thereof.

113. The composition according to any one of claims 107 to 112, wherein the lymphocytes further comprise CD8, CD103, SLC6A19, and SIDT1.

114. The composition according to any one of claims 107 to 113, wherein the sample is a peripheral blood sample.

115. The composition according to claim 114, wherein the lymphocytes further comprise (i) CD8 and CD103, (ii) CD3 and CD103, (iii) CD4 and CD103, (iv) CD39 and CD103.

116. The composition according to claim 114, wherein the lymphocytes further comprise (i) CD8, CD103, and SLC6A19, (ii) CD3, CD103, and SLC6A19, or (iv) CD4, CD103, and SLC6A19.

117. The composition according to claim 114, wherein the lymphocytes further comprise (i) CD8, CD103, SLC6A19, and SIDT1, (ii) CD3, CD103, SLC6A19, and SIDT1, or (iii) CD4, CD103, SLC6A19, and SIDT1.

118. The composition according to any one of claims 115 to 117, wherein the lymphocytes comprise at least about 20,000 CD103+ cells per 10 million peripheral blood mononuclear cells.

119. The composition according to claim 118, wherein the lymphocytes comprise at least about 2,000 CD103+ cells per 10 million peripheral blood mononuclear cells.

120. The composition according to any one of claims 115 to 119, wherein the lymphocytes comprise at least about 20,000 CD103+ cells, and the CD103+ cells comprise a purity of about 80% or more.

121. The composition according to claim 120, wherein the lymphocytes comprise at least about 2,000 CD103+ cells, and the CD103+ cells comprise a purity of about 80% or more. Claim 122 The method according to any one of claims 107 to 121, wherein the sample is concentrated with about 70% or more of the lymphocytes. Claim 123 The method according to any one of claims 107 to 121, wherein the sample is concentrated with about 80% or more of the lymphocytes. Claim 124 The method according to any one of claims 107 to 123, wherein the sample has lymphocytes with a purity of about 70% or more. Claim 125 The method according to any one of claims 107 to 123, wherein the sample has lymphocytes with a purity of about 80% or more.

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