Attorney Docket No.10034-322WO1 SCARLESS ISOLATION OF ANTIGEN-SPECIFIC T CELLS FOR CAR T CELLS FOR CAR T CELL MANUFACTURING VIA DNAGATED SORTING CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of priority to U.S. Provisional Application No. 63/589,814 filed October 12, 2023, which is incorporated by reference herein in its entirety. GOVERNMENT SUPPORT CLAUSE [0002] This invention was made with government support under Grant No. GR00016812 awarded by National Institutes of Health. The Government has certain rights in the invention. REFERENCE TO SEQUENCE LISTING [0003] The sequence listing submitted on October 15, 2024, as an .XML file entitled “10034-322WO1_ST26.xml” created on October 11, 2024, and having a file size of 3,942 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5). BACKGROUND [0004] Currently, chimeric antigen receptor (CAR) T cell therapy expands CD3+ T cells which can lead to the expansion of autoreactive T cells, T regulatory T cells, and effector phenotype T cells possibly resulting in the limited therapeutic effect of CAR T cells for solid tumors. Antigen(Ag)-specific CD8 T cells are key players in adaptive immunity and have transformative applications as cell therapies. Using Ag-specific T cells for CAR T cell therapy has led to enhanced in vivo persistence because of the increased memory phenotype of Ag- specific T cells. Yet isolating these cells by fluorescence-based cytometry is challenging because of their receptor diversity and rarity. Thus, what is needed are new methods for isolating antigen-specific T cells. SUMMARY [0005] Disclosed herein are kits for sorting T cells, the kit comprising: a) a peptide major histocompatibility complex (pMHC) monomer (including, but not limited to class I MHC and class II MHC) and peptide antigen (such as, for example, an immunogenic peptide including, but not limited to a synthetic antigen or an epitope of a cancer, virus, bacteria, fungus, or parasite) complex conjugated to a nucleic acid target probe (target probe)(such as, for example,
Attorney Docket No.10034-322WO1 SEQ ID Nos: 24-28 or any variants thereof); b) a nucleic acid catch probe (catch probe)(such as for example, SEQ ID NOs: 29-33 or any variants thereof); wherein the catch probe is conjugated to a detectable agent (such as, for example a fluorophore or magnetic bead); and c) a nucleic acid release probe (release probe)(such as, for example, SEQ ID NOs: 34-48 or any variants thereof); wherein the nucleic acid target probe is at least partially complementary to each of the catch probe and the release probe. [0006] Also disclosed herein are kits for sorting T cells of any preceding aspect, wherein the peptide epitope is a viral epitope from a virus selected from the group consisting of Herpes Simplex virus-1, Herpes Simplex virus-2, Varicella-Zoster virus, Epstein-Barr virus, Cytomegalovirus, Human Herpes virus-6, Variola virus, Vesicular stomatitis virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Rhinovirus, Coronavirus (such as, for example, a SARS-CoV-2 infection including, but not limited to the SARS-CoV-2 B1.351 variant, SARS-CoV-2B.1.1.7 (alpha), SARS-CoV-2B.1.1.7 variant mutant N501Y (alpha), SARS-CoV-2 delta variant, SARS-CoV-2 P.1 variant, SARS-CoV-2 with T487K, P681R, and L452R mutations in B.1.617.2 (Delta), SARS-CoV-2 with K417N mutation in AY.1/AY.2 (Delta plus), SARS-CoV-2 with D614G, P681H, and D950N mutations in B.1.621 (Mu), SARS-CoV-2 with G75V, T76I, Δ246-252, L452Q, F490S, D614G, and T859N mutations in C.37 (Lambda), SARS-CoV-2 with T478K, Q498R, and H655Y mutations in B.1.1.529 (Omicron)), Influenza virus A, Influenza virus B, Measles virus, Polyomavirus, Human Papilomavirus, Respiratory syncytial virus, Adenovirus, Coxsackie virus, Dengue virus, Mumps virus, Poliovirus, Rabies virus, Rous sarcoma virus, Reovirus, Yellow fever virus, Zika virus, Ebola virus, Marburg virus, Lassa fever virus, Eastern Equine Encephalitis virus, Japanese Encephalitis virus, St. Louis Encephalitis virus, Murray Valley fever virus, West Nile virus, Rift Valley fever virus, Rotavirus A, Rotavirus B, Rotavirus C, Sindbis virus, Simian Immunodeficiency virus, Human T-cell Leukemia virus type-1, Hantavirus, Rubella virus, Simian Immunodeficiency virus, Human Immunodeficiency virus type-1, and Human Immunodeficiency virus type-2. [0007] In one aspect, disclosed herein are kits for sorting T cells of any preceding aspect, wherein the peptide epitope is a bacterial epitope from a bacteria selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium bovis strain BCG, BCG substrains, Mycobacterium avium, Mycobacterium intracellular, Mycobacterium africanum, Mycobacterium kansasii, Mycobacterium marinum, Mycobacterium ulcerans, Mycobacterium avium subspecies paratuberculosis, Nocardia asteroides, other Nocardia species, Legionella pneumophila, other Legionella species, Acetinobacter baumanii,
Attorney Docket No.10034-322WO1 Salmonella typhi, Salmonella enterica, other Salmonella species, Shigella boydii, Shigella dysenteriae, Shigella sonnei, Shigella flexneri, other Shigella species, Yersinia pestis, Pasteurella haemolytica, Pasteurella multocida, other Pasteurella species, Actinobacillus pleuropneumoniae, Listeria monocytogenes, Listeria ivanovii, Brucella abortus, other Brucella species, Cowdria ruminantium, Borrelia burgdorferi, Bordetella avium, Bordetella pertussis, Bordetella bronchiseptica, Bordetella trematum, Bordetella hinzii, Bordetella pteri, Bordetella parapertussis, Bordetella ansorpii other Bordetella species, Burkholderia mallei, Burkholderia psuedomallei, Burkholderia cepacian, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia psittaci, Coxiella burnetii, Rickettsial species, Ehrlichia species, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus agalactiae, Escherichia coli, Vibrio cholerae, Campylobacter species, Neiserria meningitidis, Neiserria gonorrhea, Pseudomonas aeruginosa, other Pseudomonas species, Haemophilus influenzae, Haemophilus ducreyi, other Hemophilus species, Clostridium tetani, other Clostridium species, Yersinia enterolitica, and other Yersinia species. [0008] Also disclosed herein are kits for sorting T cells of any preceding aspect, wherein the peptide epitope is a fungal epitope from a fungi selected from the group consisting of Candida albicans, Cryptococcus neoformans, Histoplama capsulatum, Aspergillus fumigatus, Coccidiodes immitis, Paracoccidiodes brasiliensis, Blastomyces dermitidis, Pneumocystis carnii, Penicillium marneffi, and Alternaria alternata. [0009] In one aspect, disclosed herein are kits for sorting T cells of any preceding aspect, wherein the peptide epitope is a parasitic epitope from a parasite selected from the group consisting of Toxoplasma gondii, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, other Plasmodium species, Entamoeba histolytica, Naegleria fowleri, Rhinosporidium seeberi, Giardia lamblia, Enterobius vermicularis, Enterobius gregorii, Ascaris lumbricoides, Ancylostoma duodenale, Necator americanus, Cryptosporidium spp., Trypanosoma brucei, Trypanosoma cruzi, Leishmania major, other Leishmania species, Diphyllobothrium latum, Hymenolepis nana, Hymenolepis diminuta, Echinococcus granulosus, Echinococcus multilocularis, Echinococcus vogeli, Echinococcus oligarthrus, Diphyllobothrium latum, Clonorchis sinensis; Clonorchis viverrini, Fasciola hepatica, Fasciola gigantica, Dicrocoelium dendriticum, Fasciolopsis buski, Metagonimus yokogawai, Opisthorchis viverrini, Opisthorchis felineus, Clonorchis sinensis, Trichomonas vaginalis, Acanthamoeba species, Schistosoma intercalatum, Schistosoma haematobium, Schistosoma japonicum, Schistosoma mansoni, other Schistosoma species, Trichobilharzia regenti,
Attorney Docket No.10034-322WO1 Trichinella spiralis, Trichinella britovi, Trichinella nelsoni, Trichinella nativa, and Entamoeba histolytica. [0010] In one aspect, disclosed herein are kits for sorting T cells of any preceding aspect, wherein a first end of the target probe is conjugated to the pMHC monomer, and wherein a second end of the target probe is conjugated to the detectable agent. In some aspects, a first end of the catch probe is conjugated to a quencher (such as, for example, IOWA BLACK®, Dabsyl, Black Hole quenchers, or CPα.Q), wherein a second end of the catch probe is conjugated to the detectable agent, and wherein the first end of the catch probe is at least partially complementary to the second end of the target nucleic acid. [0011] Also disclosed herein are kits for sorting T cells of any preceding aspect, wherein the target probe comprises a toehold region to which the release probe binds and the catch probe does not. In some aspects, the toehold region comprises from 3 to 10 nucleotides. [0012] In one aspect, disclosed herein are methods of sorting (i.e., isolating) a plurality of antigen-specific T cells, the method comprising: a) exposing the plurality of T cells to a peptide major histocompatibility complex (pMHC) monomer (including, but not limited to the MHC monomer of any preceding aspect) conjugated to a nucleic acid target probe (target probe) and comprising a peptide epitope which is recognized by antigen-specific T cells; b) exposing the plurality of T cells to a nucleic acid catch probe (catch probe); wherein the catch probe is conjugated to a first detectable agent (such as for example a fluorophore or magnetic bead), and wherein the catch nucleic acid at least partially anneals to the target probe; and c) separating T cells that are positive for the first detectable agent, thereby sorting the antigen-specific T cells. In some aspects, the first detectable agent on the catch probe comprises a magnetic bead, and wherein step c) comprises exposing the plurality of T cells to a magnetic field. In some aspects, the first detectable agent is a first fluorophore, and wherein step c) comprises performing florescence activated cell sorting (FACS) on the T cell population and gating on the T cell population that is positive for the detectable agent. In some aspects, the antigen- specific T cells are not activated by the pMHC monomer. In some aspects, the antigen-specific T cells are chimeric antigen receptor (CAR) T cells. [0013] Also disclosed herein are methods of sorting (i.e., isolating) a plurality of antigen- specific T cells of any preceding aspect, further comprising: d) exposing the antigen- specific T cells to a nucleic acid release probe (release probe)(including, but not limited to the release probe of any preceding aspect), wherein the release probe at least partially anneals to the target probe, thereby displacing the catch probe; wherein the annealing of the target probe
Attorney Docket No.10034-322WO1 and the release probe causes the pMHC monomer to disassociate from the antigen-specific T cells. [0014] In one aspect, disclosed herein are methods of engineering a CAR T cell, the method comprising: a) exposing the plurality of T cells to a peptide major histocompatibility complex (pMHC) monomer (including, but not limited to the MHC monomer of any preceding aspect) conjugated to a nucleic acid target probe (target probe) and comprising a peptide epitope which is recognized by antigen-specific T cells; b) exposing the plurality of T cells to a nucleic acid catch probe (catch probe); wherein the catch probe is conjugated to a first detectable agent (such as, for example a fluorophore or magnetic bead, ), and wherein the catch nucleic acid at least partially anneals to the target probe; c) separating T cells that are positive for the first detectable agent, thereby sorting the antigen-specific T cells; d) exposing the antigen-specific T cells to a nucleic acid release probe (release probe) )(including, but not limited to the release probe of any preceding aspect), wherein the release probe at least partially anneals to the target probe, thereby displacing the catch probe, and wherein the annealing of the target probe and the release probe causes the pMHC monomer to disassociate from the antigen-specific T cells; and e) transducing the antigen-specific T cells with a chimeric antigen receptor (CAR), thereby producing antigen-specific CAR T cells. In some aspects, the first detectable agent on the catch probe comprises a magnetic bead, and wherein step c) comprises exposing the plurality of T cells to a magnetic field. In some aspects, the first detectable agent is a first fluorophore, and wherein step c) comprises performing florescence activated cell sorting (FACS) on the T cell population and gating on the T cell population that is positive for the detectable agent. In some aspects, the antigen-specific T cells are not activated. [0015] Also disclosed herein are methods of performing an adoptive T cell therapy (such as for example, chimeric antigen receptor (CAR) T cell therapy or adoptive transfer of tumor infiltrating lymphocytes (TILs), or adoptive transfer of marrow infiltrating lymphocytes (MILs)), the method comprising: a) exposing the plurality of T cells to a peptide major histocompatibility complex (pMHC) monomer conjugated to a nucleic acid target probe (target probe) and comprising a peptide epitope which is recognized by target antigen-specific T cells; b) exposing the plurality of T cells to a nucleic acid catch probe (catch probe); wherein the catch probe is conjugated to a first detectable agent, and wherein the catch nucleic acid at least partially anneals to the target probe; c) separating T cells that are positive for the first detectable agent, thereby sorting the antigen-specific T cells; d) exposing the antigen-specific T cells to a nucleic acid release probe (release probe), wherein the release probe at least partially anneals to the target probe, thereby displacing the catch probe, and wherein the annealing of the target
Attorney Docket No.10034-322WO1 probe and the release probe causes the pMHC monomer to disassociate from the antigen- specific T cells; and e) administering the antigen-specific T cells to a subject in need thereof. [0016] In some aspects, disclosed herein are methods of performing an adoptive T cell therapy of any preceding aspect, further comprising, before step a) and/or after step c), administering to the subject a peptide pulse or a vaccine, wherein the peptide pulse or vaccine comprises the peptide epitope for which target antigen-specific T cells are specific thereby expanding the antigen-specific T cells. [0017] Other systems, methods, features and/or advantages will be or may become apparent to one with skill in the art upon examination of the following drawings and detailed description. It is intended that all such additional systems, methods, features and/or advantages be included within this description and be protected by the accompanying claims. BRIEF DESCRIPTION OF DRAWINGS [0018] FIGURES 1A-1D depict antigen-specific sorting of Gp33-specific T cells. FIG. 1A is an overview showing DNA Gated Sorting (DGS) process. FIG. 1B shows that DGS captures and elutes Gp33+ T cells. FIG. 1C shows that pMHCI-TP-Cy5 unbinds from cell surface after elution FIG.1D shows that eluted cells do not activate after 3 days of culture. [0019] FIGURE 2 depicts the generation of CAR viral-specific T cells through DNA gated sorting. [0020] FIGURE 3 depicts the expansion of GIL-specific T cells by peptide pulsing PBMCs. [0021] FIGURE 4 depicts that expanded GIL-specific T cells express LDLR. [0022] FIGURE 5 depicts that DGS of GIL-specific T cells results in highly pure eluted fraction. [0023] FIGURE 6 depicts that DGS with pMHC monomer is scarless. [0024] FIGURE 7 depicts that pMHC DNA gated sorting results in scarless isolation of antigen-specific CD8 T cells. [0025] FIGURES 8A-8C depict the complete release of pMHC DNA circuit in under five minutes. [0026] FIGURES 9A-9D depict that the mixture of AgS mouse CD8+ T cells is separated with pMHC DGS. [0027] FIGURES 10A-10C depict that monomeric DGS results in absence of pMHC on T cell surface after isolation.
Attorney Docket No.10034-322WO1 [0028] FIGURES 11A-11B depict that flow sorting antigen-specific T cells leads to expression of activation markers. C [0029] FIGURES 12A-12B depict that DNA gated sorted antigen-specific T cells have similar expression of activation markers as unstained T cells. DETAILED DESCRIPTION [0030] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate aspects, can also be provided in combination with a single aspect. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single aspect, can also be provided separately or in any suitable subcombination. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure. DEFINITIONS [0031] In this specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings: [0032] As used herein, “comprising” is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Moreover, each of the terms “by”, “comprising,” “comprises”, “comprised of,” “including,” “includes,” “included,” “involving,” “involves,” “involved,” and “such as” are used in their open, non-limiting sense and may be used interchangeably. Further, the term “comprising” is intended to include examples and aspects encompassed by the terms “consisting essentially of” and “consisting of.” Similarly, the term “consisting essentially of” is intended to include examples encompassed by the term “consisting of. [0033] As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a compound”, “a composition”, or “a cancer”, includes, but is not limited to, two or more such compounds, compositions, or cancers, and the like. [0034] It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It can be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that
Attorney Docket No.10034-322WO1 each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it can be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed. [0035] When a range is expressed, a further aspect includes from the one particular value and/or to the other particular value. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g. the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g. ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘less than x’, less than y’, and ‘less than z’. Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’. In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”. [0036] It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the sub- ranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range. [0037] As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably
Attorney Docket No.10034-322WO1 determined. In such cases, it is generally understood, as used herein, that “about” and “at or about” mean the nominal value indicated ±10% variation unless otherwise indicated or inferred. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise. [0038] As used herein, the term “effective amount” refers to an amount that is sufficient to achieve the desired modification of a physical property of the composition or material. For example, an “effective amount” of a monomer refers to an amount that is sufficient to achieve the desired improvement in the property modulated by the formulation component, e.g. desired antioxidant release rate or viscoelasticity. The specific level in terms of wt% in a composition required as an effective amount will depend upon a variety of factors including the amount and type of monomer, amount and type of polymer, e.g., acrylamide, amount of antioxidant, and desired release kinetics. [0039] As used herein, the term “therapeutically effective amount” refers to an amount that is sufficient to achieve the desired therapeutic result or to have an effect on undesired symptoms but is generally insufficient to cause adverse side effects. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and like factors within the knowledge and expertise of the health practitioner and which may be well known in the medical arts. In the case of treating a particular disease or condition, in some instances, the desired response can be inhibiting the progression of the disease or condition. This may involve only slowing the progression of the disease temporarily. However, in other instances, it may be desirable to halt the progression of the disease permanently. This can be monitored by routine diagnostic methods known to one of ordinary skill in the art for any particular disease. The desired response to treatment of the disease or condition also can be delaying the onset or even preventing the onset of the disease or condition. [0040] For example, it is well within the skill of the art to start doses of a compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose can
Attorney Docket No.10034-322WO1 be divided into multiple doses for purposes of administration. Consequently, single dose compositions can contain such amounts or submultiples thereof to make up the daily dose. The dosage can be adjusted by the individual physician in the event of any contraindications. It is generally preferred that a maximum dose of the pharmacological agents of the invention (alone or in combination with other therapeutic agents) be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons. [0041] A response to a therapeutically effective dose of a disclosed drug delivery composition can be measured by determining the physiological effects of the treatment or medication, such as the decrease or lack of disease symptoms following administration of the treatment or pharmacological agent. Other assays will be known to one of ordinary skill in the art and can be employed for measuring the level of the response. The amount of a treatment may be varied for example by increasing or decreasing the amount of a disclosed compound and/or pharmaceutical composition, by changing the disclosed compound and/or pharmaceutical composition administered, by changing the route of administration, by changing the dosage timing and so on. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. Guidance can be found in the literature for appropriate dosages for given classes of pharmaceutical products. [0042] As used herein, the term “prophylactically effective amount” refers to an amount effective for preventing onset or initiation of a disease or condition. [0043] As used herein, the terms “optional” or “optionally” means that the subsequently described event or circumstance can or cannot occur, and that the description includes instances where said event or circumstance occurs and instances where it does not. [0044] An "increase" can refer to any change that results in a greater amount of a symptom, disease, composition, condition or activity. An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount. Thus, the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% increase so long as the increase is statistically significant. [0045] A "decrease" can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity. A substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance. Also for example, a decrease
Attorney Docket No.10034-322WO1 can be a change in the symptoms of a disorder such that the symptoms are less than previously observed. A decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount. Thus, the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease so long as the decrease is statistically significant. [0046] "Inhibit," "inhibiting," and "inhibition" mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels. [0047] By “reduce” or other forms of the word, such as “reducing” or “reduction,” is meant lowering of an event or characteristic (e.g., tumor growth). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. For example, “reduces tumor growth” means reducing the rate of growth of a tumor relative to a standard or a control. [0048] By “prevent” or other forms of the word, such as “preventing” or “prevention,” is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented. Likewise, something could be prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed. [0049] The term “subject” refers to any individual who is the target of administration or treatment. The subject can be a vertebrate, for example, a mammal. In one aspect, the subject can be human, non-human primate, bovine, equine, porcine, canine, or feline. The subject can also be a guinea pig, rat, hamster, rabbit, mouse, or mole. Thus, the subject can be a human or veterinary patient. The term “patient” refers to a subject under the treatment of a clinician, e.g., physician. [0050] As used herein, the terms "treating" and "treatment" can refer generally to obtaining a desired pharmacological and/or physiological effect. The effect can be, but does not necessarily have to be, prophylactic in terms of preventing or partially preventing a disease,
Attorney Docket No.10034-322WO1 symptom or condition thereof. The effect can be therapeutic in terms of a partial or complete cure of a disease, condition, symptom or adverse effect attributed to the disease, disorder, or condition. The term "treatment" as used herein can include any treatment of a disease disorder in a subject, particularly a human and can include any one or more of the following: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., mitigating or ameliorating the disease and/or its symptoms or conditions. The term "treatment" as used herein can refer to both therapeutic treatment alone, prophylactic treatment alone, or both therapeutic and prophylactic treatment. Those in need of treatment (subjects in need thereof) can include those already with the disorder and/or those in which the disorder is to be prevented. As used herein, the term "treating", can include inhibiting the disease, disorder or condition, e.g., impeding its progress; and relieving the disease, disorder, or condition, e.g., causing regression of the disease, disorder and/or condition. Treating the disease, disorder, or condition can include ameliorating at least one symptom of the particular disease, disorder, or condition, even if the underlying pathophysiology is not affected, e.g., such as treating the pain of a subject by administration of an analgesic agent even though such agent does not treat the cause of the pain. [0051] As used herein, “dose,” “unit dose,” or “dosage” can refer to physically discrete units suitable for use in a subject, each unit containing a predetermined quantity of a disclosed compound and/or a pharmaceutical composition thereof calculated to produce the desired response or responses in association with its administration. [0052] As used herein, “therapeutic” can refer to treating, healing, and/or ameliorating a disease, disorder, condition, or side effect, or to decreasing in the rate of advancement of a disease, disorder, condition, or side effect. COMPOSITION [0053] Currently, chimeric antigen receptor (CAR) T cell therapy expands CD3+ T cells which can lead to the expansion of autoreactive T cells, T regulatory T cells, and effector phenotype T cells possibly resulting in the limited therapeutic effect of CAR T cells for solid tumors. Antigen(Ag)-specific CD8 T cells are key players in adaptive immunity and have transformative applications as cell therapies. Using Ag-specific T cells for CAR T cell therapy has led to enhanced in vivo persistence because of the increased memory phenotype of Ag- specific T cells. Additionally, most all patients have viral-specific T cells present (i.e. influenza, EBV, and CMV) and these T cells can be expanded in vivo through vaccination (i.e. DC vaccination). Yet isolating these cells by fluorescence-based cytometry is challenging
Attorney Docket No.10034-322WO1 because of their receptor diversity and rarity. A study was conducted which generated CAR T cells from Ag-specific T cells isolated using DNA gated sorting (DGS). The study developed DNA-gated sorting for scarless and multiplexed sorting of Ag-specific CD8 T cells to overcome current challenges. DGS includes a molecular DNA circuit that couples a magnetic bead to pMHCI molecules through DNA hybridization, and that functions as a sorting ‘gate’ to capture, release, and recover Ag-specific T cells by toehold-mediated strand displacement (FIG. 1A). Unlike cell isolation with pMHC tetramers, this approach results in reversible clustering of pMHCI such that DGS isolation results in ‘scarless’ CD8 T cells to prevent TCR activation. These advances can be validated using CD8 T cells from transgenic mice (OT1, Pmel, P14), viral infection models (LCMV), and healthy human samples (EBV, CMV, Influenza). [0054] Disclosed herein are kits for sorting T cells, the kit comprising: a) a peptide major histocompatibility complex (pMHC) monomer (including, but not limited to class I MHC and class II MHC) and peptide antigen (such as, for example, an immunogenic peptide including, but not limited to a synthetic antigen or an epitope of a cancer, virus, bacteria, fungus, or parasite) complex conjugated to a nucleic acid target probe (target probe)(such as, for example, SEQ ID Nos: 24-28 or any variants thereof); b) a nucleic acid catch probe (catch probe)(such as for example, SEQ ID NOs: 29-33 or any variants thereof); wherein the catch probe is conjugated to a detectable agent (such as, for example a fluorophore or magnetic bead, a membrane-bound reporter, a member of a binding pair, such as biotin/streptavidin, a metal (e.g., gold), or an epitope tag that can specifically interact with a molecule that can be detected, such as by producing a colored substrate or fluorescence); and c) a nucleic acid release probe (release probe)(such as, for example, SEQ ID NOs: 34-48 or any variants thereof); wherein the nucleic acid target probe is at least partially complementary to each of the catch probe and the release probe. [0055] The disclosed kits use detectable agents to enable the sorting (i.e., separation or isolation) of T cells. Examples of detectable agents for use in sorting techniques like magnetic bead sorting, a fluorescence activated cell sorting (FACS) are well known in the art and utilize detectable agents such as a fluorophore, a membrane-bound reporter, a member of a binding pair, such as biotin/streptavidin, a metal (e.g., gold), or an epitope tag that can specifically interact with a molecule that can be detected, such as by producing a colored substrate or fluorescence. [0056] Fluorophores are compounds or molecules that luminesce. Typically fluorophores absorb electromagnetic energy at one wavelength and emit electromagnetic energy at a second
Attorney Docket No.10034-322WO1 wavelength. Representative fluorophores include, but are not limited to, 1,5 IAEDANS; 1,8- ANS; 4- Methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; 5-Carboxyfluorescein (5- FAM); 5-Carboxynapthofluorescein; 5-Carboxytetramethylrhodamine (5-TAMRA); 5- Hydroxy Tryptamine (5-HAT); 5-ROX (carboxy-X-rhodamine); 6-Carboxyrhodamine 6G; 6- CR 6G; 6-JOE; 7-Amino-4-methylcoumarin; 7-Aminoactinomycin D (7-AAD); 7-Hydroxy-4- I methylcoumarin; 9-Amino-6-chloro-2-methoxyacridine (ACMA); ABQ; Acid Fuchsin; Acridine Orange; Acridine Red; Acridine Yellow; Acriflavin; Acriflavin Feulgen SITSA; Aequorin (Photoprotein); AFPs - AutoFluorescent Protein - (Quantum Biotechnologies) see sgGFP, sgBFP; Alexa Fluor 350 ^; Alexa Fluor 430 ^; Alexa Fluor 488 ^; Alexa Fluor 532 ^; Alexa Fluor 546 ^; Alexa Fluor 568 ^; Alexa Fluor 594 ^; Alexa Fluor 633 ^; Alexa Fluor 647 ^; Alexa Fluor 660 ^; Alexa Fluor 680 ^; Alizarin Complexon; Alizarin Red; Allophycocyanin (APC); AMC, AMCA-S; Aminomethylcoumarin (AMCA); AMCA-X; Aminoactinomycin D; Aminocoumarin; Anilin Blue; Anthrocyl stearate; APC-Cy7; APTRA- BTC; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R; Astrazon Red 6B; Astrazon Yellow 7 GLL; Atabrine; ATTO- TAG ^ CBQCA; ATTO-TAG ^ FQ; Auramine; Aurophosphine G; Aurophosphine; BAO 9 (Bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH); Berberine Sulphate; Beta Lactamase; BFP blue shifted GFP (Y66H); Blue Fluorescent Protein; BFP/GFP FRET; Bimane; Bisbenzemide; Bisbenzimide (Hoechst); bis- BTC; Blancophor FFG; Blancophor SV; BOBO ^ -1; BOBO ^-3; Bodipy492/515; Bodipy493/503; Bodipy500/510; Bodipy; 505/515; Bodipy 530/550; Bodipy 542/563; Bodipy 558/568; Bodipy 564/570; Bodipy 576/589; Bodipy 581/591; Bodipy 630/650-X; Bodipy 650/665-X; Bodipy 665/676; Bodipy Fl; Bodipy FL ATP; Bodipy Fl-Ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-X conjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE; BO-PRO ^ -1; BO-PRO ^ -3; Brilliant Sulphoflavin FF; BTC; BTC- 5N; Calcein; Calcein Blue; Calcium Crimson - ; Calcium Green; Calcium Green-1 Ca
2+ Dye; Calcium Green-2 Ca
2+; Calcium Green-5N Ca
2+; Calcium Green-C18 Ca
2+; Calcium Orange; Calcofluor White; Carboxy-X-rhodamine (5-ROX); Cascade Blue ^; Cascade Yellow; Catecholamine; CCF2 (GeneBlazer); CFDA; CFP (Cyan Fluorescent Protein); CFP/YFP FRET; Chlorophyll; Chromomycin A; Chromomycin A; CL-NERF; CMFDA; Coelenterazine; Coelenterazine cp; Coelenterazine f; Coelenterazine fcp; Coelenterazine h; Coelenterazine hcp; Coelenterazine ip; Coelenterazine n; Coelenterazine O; Coumarin Phalloidin; C-phycocyanine; CPM I Methylcoumarin; CTC; CTC Formazan; Cy2 ^; Cy3.18; Cy3.5 ^; Cy3 ^; Cy5.18; Cy5.5 ^; Cy5 ^; Cy7 ^; Cyan GFP; cyclic AMP Fluorosensor (FiCRhR); Dabcyl; Dansyl;
Attorney Docket No.10034-322WO1 Dansyl Amine; Dansyl Cadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl fluoride; DAPI; Dapoxyl; Dapoxyl 2; Dapoxyl 3’DCFDA; DCFH (Dichlorodihydrofluorescein Diacetate); DDAO; DHR (Dihydorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS (non-ratio); DiA (4-Di 16-ASP); Dichlorodihydrofluorescein Diacetate (DCFH); DiD- Lipophilic Tracer; DiD (DilC18(5)); DIDS; Dihydorhodamine 123 (DHR); Dil (DilC18(3)); I Dinitrophenol; DiO (DiOC18(3)); DiR; DiR (DilC18(7)); DM-NERF (high pH); DNP; Dopamine; DsRed; DTAF; DY-630-NHS; DY-635-NHS; EBFP; ECFP; EGFP; ELF 97; Eosin; Erythrosin; Erythrosin ITC; Ethidium Bromide; Ethidium homodimer-1 (EthD-1); Euchrysin; EukoLight; Europium (111) chloride; EYFP; Fast Blue; FDA; Feulgen (Pararosaniline); FIF (Formaldehyd Induced Fluorescence); FITC; Flazo Orange; Fluo-3; Fluo-4; Fluorescein (FITC); Fluorescein Diacetate; Fluoro-Emerald; Fluoro-Gold (Hydroxystilbamidine); Fluor-Ruby; FluorX; FM 1- 43 ^; FM 4-46; Fura Red ^ (high pH); Fura Red ^/Fluo-3; Fura-2; Fura-2/BCECF; Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink 3G; Genacryl Yellow 5GF; GeneBlazer; (CCF2); GFP (S65T); GFP red shifted (rsGFP); GFP wild type’ non-UV excitation (wtGFP); GFP wild type, UV excitation (wtGFP); GFPuv; Gloxalic Acid; Granular blue; Haematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS; Hydroxycoumarin; Hydroxystilbamidine (FluoroGold); Hydroxytryptamine; Indo-1, high calcium; Indo-1 low calcium; Indodicarbocyanine (DiD); Indotricarbocyanine (DiR); Intrawhite Cf; JC-1; JO JO-1; JO-PRO-1; LaserPro; Laurodan; LDS 751 (DNA); LDS 751 (RNA); Leucophor PAF; Leucophor SF; Leucophor WS; Lissamine Rhodamine; Lissamine Rhodamine B; Calcein/Ethidium homodimer; LOLO-1; LO-PRO-1; ; Lucifer Yellow; Lyso Tracker Blue; Lyso Tracker Blue-White; Lyso Tracker Green; Lyso Tracker Red; Lyso Tracker Yellow; LysoSensor Blue; LysoSensor Green; LysoSensor Yellow/Blue; Mag Green; Magdala Red (Phloxin B); Mag-Fura Red; Mag-Fura-2; Mag-Fura-5; Mag-lndo-1; Magnesium Green; Magnesium Orange; Malachite Green; Marina Blue; I Maxilon Brilliant Flavin 10 GFF; Maxilon Brilliant Flavin 8 GFF; Merocyanin; Methoxycoumarin; Mitotracker Green FM; Mitotracker Orange; Mitotracker Red; Mitramycin; Monobromobimane; Monobromobimane (mBBr-GSH); Monochlorobimane; MPS (Methyl Green Pyronine Stilbene); NBD; NBD Amine; Nile Red; Nitrobenzoxedidole; Noradrenaline; Nuclear Fast Red; i Nuclear Yellow; Nylosan Brilliant lavin E8G; Oregon Green ^; Oregon Green ^ 488; Oregon Green ^ 500; Oregon Green ^ 514; Pacific Blue; Pararosaniline (Feulgen); PBFI; PE-Cy5; PE-Cy7; PerCP; PerCP-Cy5.5; PE-TexasRed (Red 613); Phloxin B (Magdala Red); Phorwite AR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; PhotoResist; Phycoerythrin B [PE];
Attorney Docket No.10034-322WO1 Phycoerythrin R [PE]; PKH26 (Sigma); PKH67; PMIA; Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO- I PRO-3; Primuline; Procion Yellow; Propidium lodid (Pl); PyMPO; Pyrene; Pyronine; Pyronine B; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Resorufin; RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5 GLD; Rhodamine 6G; Rhodamine B; Rhodamine B 200; Rhodamine B extra; Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine; Rhodamine: Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal; R-phycocyanine; R-phycoerythrin (PE); rsGFP; S65A; S65C; S65L; S65T; Sapphire GFP; SBFI; Serotonin; Sevron Brilliant Red 2B; Sevron Brilliant Red 4G; Sevron I Brilliant Red B; Sevron Orange; Sevron Yellow L; sgBFP ^ (super glow BFP); sgGFP ^ (super glow GFP); SITS (Primuline; Stilbene Isothiosulphonic Acid); SNAFL calcein; SNAFL-1; SNAFL-2; SNARF calcein; SNARF1; Sodium Green; SpectrumAqua; SpectrumGreen; SpectrumOrange; Spectrum Red; SPQ (6-methoxy- N-(3 sulfopropyl) quinolinium); Stilbene; Sulphorhodamine B and C; Sulphorhodamine Extra; SYTO 11; SYTO 12; SYTO 13; SYTO 14; SYTO 15; SYTO 16; SYTO 17; SYTO 18; SYTO 20; SYTO 21; SYTO 22; SYTO 23; SYTO 24; SYTO 25; SYTO 40; SYTO 41; SYTO 42; SYTO 43; SYTO 44; SYTO 45; SYTO 59; SYTO 60; SYTO 61; SYTO 62; SYTO 63; SYTO 64; SYTO 80; SYTO 81; SYTO 82; SYTO 83; SYTO 84; SYTO 85; SYTOX Blue; SYTOX Green; SYTOX Orange; Tetracycline; Tetramethylrhodamine (TRITC); Texas Red ^; Texas Red-X ^ conjugate; Thiadicarbocyanine (DiSC3); Thiazine Red R; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TON; Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TIER; TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITC TetramethylRodaminelsoThioCyanate; True Blue; Tru Red; Ultralite; Uranine B; Uvitex SFC; wt GFP; WW 781; X-Rhodamine; XRITC; Xylene Orange; Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO- PRO 3; YOYO-1;YOYO-3; Sybr Green; Thiazole orange (interchelating dyes); semiconductor nanoparticles such as quantum dots; or caged fluorophore (which can be activated with light or other electromagnetic energy source), or a combination thereof. [0057] In some aspects, the kits for sorting T cells can comprise a synthetic antigen (i.e., a modified or non-naturally occurring peptide). [0058] Also disclosed herein are kits for sorting T cells, wherein the peptide epitope is a viral epitope from a virus selected from the group consisting of Herpes Simplex virus-1, Herpes Simplex virus-2, Varicella-Zoster virus, Epstein-Barr virus, Cytomegalovirus, Human Herpes virus-6, Variola virus, Vesicular stomatitis virus, Hepatitis A virus, Hepatitis B virus, Hepatitis
Attorney Docket No.10034-322WO1 C virus, Hepatitis D virus, Hepatitis E virus, Rhinovirus, Coronavirus (such as, for example, a SARS-CoV-2 infection including, but not limited to the SARS-CoV-2 B1.351 variant, SARS- CoV-2B.1.1.7 (alpha), SARS-CoV-2B.1.1.7 variant mutant N501Y (alpha), SARS-CoV-2 delta variant, SARS-CoV-2 P.1 variant, SARS-CoV-2 with T487K, P681R, and L452R mutations in B.1.617.2 (Delta), SARS-CoV-2 with K417N mutation in AY.1/AY.2 (Delta plus), SARS-CoV-2 with D614G, P681H, and D950N mutations in B.1.621 (Mu), SARS-CoV- 2 with G75V, T76I, Δ246-252, L452Q, F490S, D614G, and T859N mutations in C.37 (Lambda), SARS-CoV-2 with T478K, Q498R, and H655Y mutations in B.1.1.529 (Omicron)), Influenza virus A, Influenza virus B, Measles virus, Polyomavirus, Human Papilomavirus, Respiratory syncytial virus, Adenovirus, Coxsackie virus, Dengue virus, Mumps virus, Poliovirus, Rabies virus, Rous sarcoma virus, Reovirus, Yellow fever virus, Zika virus, Ebola virus, Marburg virus, Lassa fever virus, Eastern Equine Encephalitis virus, Japanese Encephalitis virus, St. Louis Encephalitis virus, Murray Valley fever virus, West Nile virus, Rift Valley fever virus, Rotavirus A, Rotavirus B, Rotavirus C, Sindbis virus, Simian Immunodeficiency virus, Human T-cell Leukemia virus type-1, Hantavirus, Rubella virus, Simian Immunodeficiency virus, Human Immunodeficiency virus type-1, and Human Immunodeficiency virus type-2. [0059] In one aspect, disclosed herein are kits for sorting T cells, wherein the peptide epitope is a bacterial epitope from a bacteria selected from the group consisting of Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium bovis strain BCG, BCG substrains, Mycobacterium avium, Mycobacterium intracellular, Mycobacterium africanum, Mycobacterium kansasii, Mycobacterium marinum, Mycobacterium ulcerans, Mycobacterium avium subspecies paratuberculosis, Nocardia asteroides, other Nocardia species, Legionella pneumophila, other Legionella species, Acetinobacter baumanii, Salmonella typhi, Salmonella enterica, other Salmonella species, Shigella boydii, Shigella dysenteriae, Shigella sonnei, Shigella flexneri, other Shigella species, Yersinia pestis, Pasteurella haemolytica, Pasteurella multocida, other Pasteurella species, Actinobacillus pleuropneumoniae, Listeria monocytogenes, Listeria ivanovii, Brucella abortus, other Brucella species, Cowdria ruminantium, Borrelia burgdorferi, Bordetella avium, Bordetella pertussis, Bordetella bronchiseptica, Bordetella trematum, Bordetella hinzii, Bordetella pteri, Bordetella parapertussis, Bordetella ansorpii other Bordetella species, Burkholderia mallei, Burkholderia psuedomallei, Burkholderia cepacian, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia psittaci, Coxiella burnetii, Rickettsial species, Ehrlichia species, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes,
Attorney Docket No.10034-322WO1 Streptococcus agalactiae, Escherichia coli, Vibrio cholerae, Campylobacter species, Neiserria meningitidis, Neiserria gonorrhea, Pseudomonas aeruginosa, other Pseudomonas species, Haemophilus influenzae, Haemophilus ducreyi, other Hemophilus species, Clostridium tetani, other Clostridium species, Yersinia enterolitica, and other Yersinia species. [0060] Also disclosed herein are kits for sorting T cells, wherein the peptide epitope is a fungal epitope from a fungi selected from the group consisting of Candida albicans, Cryptococcus neoformans, Histoplama capsulatum, Aspergillus fumigatus, Coccidiodes immitis, Paracoccidiodes brasiliensis, Blastomyces dermitidis, Pneumocystis carnii, Penicillium marneffi, and Alternaria alternata. [0061] In one aspect, disclosed herein are kits for sorting T cells, wherein the peptide epitope is a parasitic epitope from a parasite selected from the group consisting of Toxoplasma gondii, Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, other Plasmodium species, Entamoeba histolytica, Naegleria fowleri, Rhinosporidium seeberi, Giardia lamblia, Enterobius vermicularis, Enterobius gregorii, Ascaris lumbricoides, Ancylostoma duodenale, Necator americanus, Cryptosporidium spp., Trypanosoma brucei, Trypanosoma cruzi, Leishmania major, other Leishmania species, Diphyllobothrium latum, Hymenolepis nana, Hymenolepis diminuta, Echinococcus granulosus, Echinococcus multilocularis, Echinococcus vogeli, Echinococcus oligarthrus, Diphyllobothrium latum, Clonorchis sinensis; Clonorchis viverrini, Fasciola hepatica, Fasciola gigantica, Dicrocoelium dendriticum, Fasciolopsis buski, Metagonimus yokogawai, Opisthorchis viverrini, Opisthorchis felineus, Clonorchis sinensis, Trichomonas vaginalis, Acanthamoeba species, Schistosoma intercalatum, Schistosoma haematobium, Schistosoma japonicum, Schistosoma mansoni, other Schistosoma species, Trichobilharzia regenti, Trichinella spiralis, Trichinella britovi, Trichinella nelsoni, Trichinella nativa, and Entamoeba histolytica. [0062] In one aspect, disclosed herein are kits for sorting T cells, wherein a first end of the target probe is conjugated to the pMHC monomer, and wherein a second end of the target probe is conjugated to the detectable agent. In some aspects, a first end of the catch probe is conjugated to a quencher (such as, for example, IOWA BLACK®, Dabsyl, Black Hole quenchers, or CPα.Q), wherein a second end of the catch probe is conjugated to the detectable agent, and wherein the first end of the catch probe is at least partially complementary to the second end of the target nucleic acid. [0063] Also disclosed herein are kits for sorting T cells, wherein the target probe comprises a toehold region to which the release probe binds and the catch probe does not. Toehold regions (i.e., domains) are known in the art and comprise a short single chain exposed expanse of
Attorney Docket No.10034-322WO1 nucleotides that is able to be bound by a complementary strand of nucleic acid. In some aspects the toehold region is formed from the nonoverlapping portion of DNA remaining from a target probe when a target probe and a catch probe bind. In such instances the toehold region is at the opposite end of the target probe from the catch probe. In some aspects, the toehold region comprises from 3 to 10 nucleotides (such as, for example, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides). In some aspects, the toehold region serves as the target for the release probe to initiate strand displacement of the catch probe. [0064] In one aspect, disclosed herein are methods of sorting (i.e., selecting or isolating) a plurality of antigen-specific T cells, the method comprising: a) exposing the plurality of T cells to a peptide major histocompatibility complex (pMHC) monomer (including, but not limited to the MHC monomer of any of the pMHC monomers disclosed herein) conjugated to a nucleic acid target probe (target probe) (such as, for example, SEQ ID Nos: 24-28 or any variants thereof) and comprising a peptide epitope which is recognized by antigen-specific T cells; b) exposing the plurality of T cells to a nucleic acid catch probe (catch probe) (such as for example, SEQ ID NOs: 29-33 or any variants thereof); wherein the catch probe is conjugated to a first detectable agent (such as for example a fluorophore or magnetic bead), and wherein the catch nucleic acid at least partially anneals to the target probe; and c) separating T cells that are positive for the first detectable agent, thereby sorting the antigen-specific T cells. In some aspects, the first detectable agent on the catch probe comprises a magnetic bead, and wherein step c) comprises exposing the plurality of T cells to a magnetic field. In some aspects, the first detectable agent is a first fluorophore, and wherein step c) comprises performing florescence activated cell sorting (FACS) on the T cell population and gating on the T cell population that is positive for the detectable agent. In some aspects, the antigen-specific T cells are not activated by the pMHC monomer. In some aspects, the antigen-specific T cells are chimeric antigen receptor (CAR) T cells. [0065] Also disclosed herein are methods of sorting (i.e., isolating) a plurality of antigen- specific T cells, further comprising: d) exposing the antigen-specific T cells to a nucleic acid release probe (release probe)(including, but not limited to any of the release probes disclosed herein such as, for example, SEQ ID NOs: 34-48 or any variants thereof), wherein the release probe at least partially anneals to the target probe, thereby displacing the catch probe; wherein the annealing of the target probe and the release probe causes the pMHC monomer to disassociate from the antigen-specific T cells. [0066] In one aspect, disclosed herein are methods of engineering a CAR T cell, the method comprising: a) exposing the plurality of T cells to a peptide major histocompatibility complex
Attorney Docket No.10034-322WO1 (pMHC) monomer (including, but not limited to the any of the MHC monomers disclosed herein) conjugated to a nucleic acid target probe (target probe) (such as, for example, SEQ ID Nos: 24-28 or any variants thereof) and comprising a peptide epitope which is recognized by antigen-specific T cells; b) exposing the plurality of T cells to a nucleic acid catch probe (catch probe) (such as for example, SEQ ID NOs: 29-33 or any variants thereof); wherein the catch probe is conjugated to a first detectable agent (such as, for example a fluorophore or magnetic bead), and wherein the catch nucleic acid at least partially anneals to the target probe; c) separating T cells that are positive for the first detectable agent, thereby sorting the antigen- specific T cells; d) exposing the antigen-specific T cells to a nucleic acid release probe (release probe) )(including, but not limited to any of release probes disclosed herein such as, for example, SEQ ID NOs: 34-48 or any variants thereof), wherein the release probe at least partially anneals to the target probe, thereby displacing the catch probe, and wherein the annealing of the target probe and the release probe causes the pMHC monomer to disassociate from the antigen-specific T cells; and e) transducing the antigen-specific T cells with a chimeric antigen receptor (CAR), thereby producing antigen-specific CAR T cells. In some aspects, the first detectable agent on the catch probe comprises a magnetic bead, and wherein step c) comprises exposing the plurality of T cells to a magnetic field. In some aspects, the first detectable agent is a first fluorophore, and wherein step c) comprises performing florescence activated cell sorting (FACS) on the T cell population and gating on the T cell population that is positive for the detectable agent. In some aspects, the antigen-specific T cells are not activated. [0067] Also disclosed herein are methods of performing an adoptive T cell therapy, the method comprising: a) exposing the plurality of T cells to a peptide major histocompatibility complex (pMHC) monomer conjugated to a nucleic acid target probe (target probe) (such as, for example, SEQ ID Nos: 24-28 or any variants thereof) and comprising a peptide epitope which is recognized by target antigen-specific T cells; b) exposing the plurality of T cells to a nucleic acid catch probe (catch probe) (such as for example, SEQ ID NOs: 29-33 or any variants thereof); wherein the catch probe is conjugated to a first detectable agent, and wherein the catch nucleic acid at least partially anneals to the target probe; c) separating T cells that are positive for the first detectable agent, thereby sorting the antigen-specific T cells; d) exposing the antigen-specific T cells to a nucleic acid release probe (release probe), wherein the release probe at least partially anneals to the target probe, thereby displacing the catch probe, and wherein the annealing of the target probe and the release probe causes the pMHC monomer to
Attorney Docket No.10034-322WO1 disassociate from the antigen-specific T cells; and e) administering the antigen-specific T cells to a subject in need thereof. [0068] In some aspects, disclosed herein are methods of performing an adoptive T cell therapy, further comprising, before step a) and/or after step c), administering to the subject a peptide pulse or a vaccine, wherein the peptide pulse or vaccine comprises the peptide epitope for which target antigen-specific T cells are specific thereby expanding the antigen-specific T cells. EXAMPLES Example 1 [0069] Results and Discussion: Sorting with Gp33-Db pMHCI-TP-Cy5 increased Gp33+ T cell population from 28.7% to 95.5% (FIG.1B). Sorted Gp33+ T cell had similar percentage of pMHC-TP-Cy5 on the cell surface with three-fold higher Cy5 MFI over unsorted (FIG.1C). After sorting, Gp33+ T cells were cultured for three days after which were stained for expression of activation makers CD69 (early-activation) and CD25 (late-activation). Sorting resulted in no significant expression of CD69 and CD25 over MACS sorted CD8+ T cells signifying that the pMHC-TP sorting does not impact T cell product (FIG.1D). [0070] Conclusion: This study demonstrates an approach to capture and elute Ag-specific T cells using pMHCI-TP without significant impact on T cell product. Looking forward, pMHC-TP can be used to sort out defined Ag-specific T cells to manufacture defined T cell therapy products. Example 2 [0071] FIG. 2 depicts the generation of CAR viral-specific T cells through DNA gated sorting. Currently, chimeric antigen receptor (CAR) T cell therapy expands CD3+ T cells which can lead to the expansion of autoreactive T cells, T regulatory T cells, and effector phenotype T cells possibly resulting in the limited therapeutic effect of CAR T cells for solid tumors. Using antigen-specific T cells for CAR T cell therapy has led to enhanced in vivo persistence because of the increased memory phenotype of antigen-specific T cells. Additionally, most all patients have viral-specific T cells present (i.e. influenza, EBV, and CMV) and these T cells can be expanded in vivo through vaccination (i.e. DC vaccination). A study was conducted which generated CAR T cells from antigen-specific T cells isolated using DNA gated sorting (DGS). For perform this, after isolation of peripheral blood mononuclear cells (PBMCs) from patient’s blood, PBMCs are pulsed with a viral-specific peptide. Antigen presenting cells present the viral peptide on major histocompatibility complex class I (pMHC), resulting in the activation and expansion of cognate viral-specific T cells. DGS can be
Attorney Docket No.10034-322WO1 performed to isolate activated viral-specific T cells, which can be transduced with CAR to generate CAR viral-specific T cells. After infusion, CAR viral-specific T cells can be expanded in vivo through vaccination of dendritic cells pulsed with viral-specific peptide. [0072] FIG. 3 depicts the expansion of GIL-specific T cells by peptide pulsing PBMCs. After isolation from healthy human donor, PBMCs were pulsed with GILGFVFTL (SEQ ID NO: 23) (GIL) peptide, resulting in expansion of cognate GIL-specific T cells from 0.22% to 27.2% of PBMCs. [0073] FIG. 4 depicts that expanded GIL-specific T cells express LDLR. After peptide pulsing PBMCs, GIL-specific T cells are 87% on day 6 and 47% on day 9 positive for low density lipoprotein receptor (LDLR), which is responsible for transduction of lentivirus in standard chimeric antigen receptor manufacturing processes. [0074] FIG. 5 depicts that DGS of GIL-specific T cells results in highly pure eluted fraction. After peptide pulsing PBMCs, GIL-specific T cells were isolated through DNA gated sorting (DGS), resulting in over 90% pure eluted fraction. [0075] FIG. 6 depicts that DGS with pMHC monomer is scarless. DNA gated sorting (DGS) was performed with pMHC streptavidin (Stv) tetramers and peptide major histocompatibility complex (pMHC) monomers, which has a lower avidity that pMHC Stv tetramers. After performing DGS with Cy5 conjugated to the DNA gates results in nearly 100% of Stv eluted T cells to be Cy5+, while pMHC eluted T cells were near 0% Cy5+. The recombinant pMHC used for DGS is refolded with human beta-2 microglobulin (β2m). Staining for human β2m after performing DGS results in nearly 100% of Stv eluted T cells to be Cy5+, while pMHC eluted cells is around 10%. Example 3 [0076] FIG. 7 depicts that pMHC DNA gated sorting results in scarless isolation of antigen-specific CD8 T cells. A magnetic bead coated with pMHC DNA gates captures antigen-specific CD8+ T cell by binding to T cell receptor (TCR). After addition of complementary release probe (RP), bead is displaced from pMHC target probe (pMHC.TP), leading to dissociation of pMHC.TP from T cell surface due to loss of avidity and low affinity between pMHC and TCR. Due to absence of pMHC-TCR interaction, isolated T cells are absent of T cell activation markers (i.e. CD69 and CD25). [0077] FIGS. 8A-8C depict the complete release of pMHC DNA circuit in under five minutes. FIG. 8A shows the target probe α conjugated to Cy5 (TPα.Cy5) was annealed to complementary catch probe α conjugated to quencher (CPα.Q), resulting in no fluorescence. After five minutes, complementary release probe α (RPα) was added, resulting in displacement
Attorney Docket No.10034-322WO1 of CPα.Q and full return of TPα.Cy5 signal in five minutes. FIG.8B shows that five CP.Q and TP.Cy5 pairs (α-ε) were annealed, mixed with five RPs (α-ε), and normalized fluorescence was quantified after 15 minutes, exhibiting creation of orthogonal DNA circuits. FIG. 8C shows that, after conjugating TPα.Cy5 to pMHC (pMHC.TPα.Cy5) and annealing to CPα.Q, addition of RPα once again results in full release of pMCH.TPα.Cy5 in five minutes. The sequences used in FIGS.8A-8B are provided in TABLE 1. The sequences used in FIG.8C are provided in TABLE 2. TABLE 1. Sequences of fluorescent DNA probes used for fluorescent experiments in FIGS. 8A-8B.
TABLE 2. Sequences of pMHC conjugatable fluorescent DNA probes used for fluorescent experiments in FIG.8C.
Attorney Docket No.10034-322WO1 [0078] FIGS.9A-9D depict that the mixture of AgS mouse CD8+ T cells is separated with pMHC DGS. FIG.9A shows that transgenic P14 mouse splenocytes are stained similarly with Db-Gp33 tetramerized with traditional biotinylated pMHCs and pMHC.DNA gates. FIG.9B shows that magnetic beads coated with Db-Gp33.DNA gates bind to CD8 T cells from transgenic P14 mouse and are released after addition of release probe. FIG. 9C shows that Gp33-specific T cells are isolated from transgenic P14 splenocytes using pMHC DNA gated sorting (DGS) at over 90% purity. FIG. 9D shows that the mixture of transgenic P14, pmel, and OT-1 antigen-specific CD8+ T cells were isolated into individual parts using DNA-gates Db-Gp33.TPα, Db-Gp100.TPβ, and Kb-OVA.TPγ at near 90% purity, respectively. The sequences used in FIGS.9A-9D are provided in TABLE 3. TABLE 3. Sequences of pMHC conjugatable DNA probes for DGS. DNA probes used to perform DGS on antigen-specific T cells in FIGS.9A-9D, FIGS.11A-11B, and FIGS.12A- 12B.
[0079] FIGS.10A-10C depict that monomeric DGS results in absence of pMHC on T cell surface after isolation. FIG. 10A shows that Gp33-specific CD8+ T cells were isolated from transgenic P14 splenocytes using tetramer DGS or monomer DGS, resulting in over 90% pure fraction for both. FIG.10B shows that the remaining recombinant pMHC on the Gp33-specific CD8+ T cell surface was stained for using anti-human β2m antibody after isolation, indicating that pMHC-TCR interaction remains for tetramer isolated cells (98.8%). FIG.10C shows that the signal of Cy5 conjugated to TP (TP.Cy5) is absent from monomer isolated cells (0.92%), unlike tetramer isolated cells, indicating no continued TCR-pMHC interaction. The sequences used in FIGS.10A-10C are provided in TABLE 4.
Attorney Docket No.10034-322WO1 TABLE 4. Sequenecs of pMHC and StvC conjugatable fluorescent DNA probes for DGS. DNA probes used to perform DGS on antigen-specific T cells and visualize residual MHC binding in FIGS.10A-10C.
[0080] FIGS.11A-11B depict that flow sorting antigen-specific T cells leads to expression of activation markers. CD25 (FIG.11A) and CD69 (FIG.11B) expression was measured of Gp33-specific CD8+ T cells cultured in 60 IU/ml IL-2 in mouse T cell media over two days that were initially unstained, dynabead activated, Gp33 tetramer stained, and Gp33+ tetramer flow sorted on day 0, resulting Gp33+ tetramer flow sorted cells to have similar activation patterns as dynabead activated CD8+ T cells. The sequences used in FIGS. 11A-11B are provided in TABLE 3. [0081] FIGS.12A-12B depict that DNA gated sorted antigen-specific T cells have similar expression of activation markers as unstained T cells. CD25 (FIG.12A) and CD69 (FIG.12B) expression was measured of Gp33-specific CD8+ T cells cultured in 60 IU/ml IL-2 in mouse T cell media over two days that were initially unstained, dynabead activated, Gp33 tetramer stained, and Gp33.TP sorted on day 0, resulting similar low expression of activation markers between unstained and DGS eluted CD8+ T cells. The sequences used in FIGS.12A-12B are provided in TABLE 3. [0082] Any patents, applications and publications as listed throughout this document are hereby incorporated by reference in their entirety herein.
Attorney Docket No.10034-322WO1 SEQUENCES SEQ ID NO: 1 5’ AGTTGGAGAGTTGTGAGGGAGTATG-Cy5 3’ SEQ ID NO: 2 5’ , IOWA BLACK®-CATACTCCCTCACAACTCTC 3’ SEQ ID NO: 3 (RP alpha) 5’ CATACTCCCTCACAACTCTCCAACTC 3’ SEQ ID NO: 4 5’ GTTGAGGTGAGATGGAAGGATGTTGG-Cy5 3’ SEQ ID NO: 5 5’ , IOWA BLACK®-CCAACATCCTTCCATCTCAC 3’ SEQ ID NO: 6 (RP beta) 5’ CCAACATCCTTCCATCTCACCTCAAC 3’ SEQ ID NO: 7 5’ GTGTAGGGAGGGTTGTAGTAGGAATG-Cy5 3’ SEQ ID NO: 8 5’ , IOWA BLACK®-CATTCCTACTACAACCCTCC 3’ SEQ ID NO: 9 (RP gamma) 5’ CATTCCTACTACAACCCTCCCTACAC 3’ SEQ ID NO: 10 5’ GATGTGGGTGGTGTAATGAGTGAGAG-Cy5 3’ SEQ ID NO: 11
Attorney Docket No.10034-322WO1 5’ , IOWA BLACK®-CTCTCACTCATTACACCACC 3’ SEQ ID NO: 12 (RP delta) 5’ CTCTCACTCATTACACCACCCACATC 3’ SEQ ID NO: 13 5’ GGATAGGTGGAGAAGGTTGAGGTTAG-Cy5 3’ SEQ ID NO: 14 5’ , IOWA BLACK®-CTAACCTCAACCTTCTCCAC 3’ SEQ ID NO: 15 (RP e) 5’ CTAACCTCAACCTTCTCCACCTATCC 3’ SEQ ID NO: 16 5’ NH2-AGTTGGAGAGTTGTGAGGGAGTATG-Cy5 3’ SEQ ID NO: 17 5’ NH2-AGTTGGAGAGTTGTGAGGGAGTATG 3’ SEQ ID NO: 18 5’ Biotin-CATACTCCCTCACAACTCTC 3’ SEQ ID NO: 19 5’ NH2-GTTGAGGTGAGATGGAAGGATGTTGG 3’ SEQ ID NO: 20 5’ Biotin-CCAACATCCTTCCATCTCAC 3’ SEQ ID NO: 21 5’ NH2-GTGTAGGGAGGGTTGTAGTAGGAATG 3’ SEQ ID NO: 22 5’ Biotin-CATTCCTACTACAACCCTCC 3’
Attorney Docket No.10034-322WO1 SEQ ID NO: 23 GILGFVFTL SEQ ID NO: 24 (TP alpha) 5’ AGTTGGAGAGTTGTGAGGGAGTATG 3’ SEQ ID NO: 25 (TP beta) 5’ GTTGAGGTGAGATGGAAGGATGTTGG 3’ SEQ ID NO: 26 (TP gamma) 5’ GTGTAGGGAGGGTTGTAGTAGGAATG 3’ SEQ ID NO: 27 (TP delta) 5’ GATGTGGGTGGTGTAATGAGTGAGAG 3’ SEQ ID NO: 28 (TP e) 5’ GGATAGGTGGAGAAGGTTGAGGTTAG 3’ SEQ ID NO: 29 (CP alpha) 5’ CATACTCCCTCACAACTCTC 3’ SEQ ID NO: 30 (CP beta) 5’ CCAACATCCTTCCATCTCAC 3’ SEQ ID NO: 31 (CP gamma) 5’ CATTCCTACTACAACCCTCC 3’ SEQ ID NO: 32 (CP delta) 5’ CTCTCACTCATTACACCACC 3’ SEQ ID NO: 33 (CP e) 5’ CTAACCTCAACCTTCTCCAC 3’
Attorney Docket No.10034-322WO1 SEQ ID NO: 34 (RP alpha) 5’ CATACTCCCTCACAACTCTCCAACTC 3’ SEQ ID NO: 35 (RP beta) 5’ CCAACATCCTTCCATCTCACCTCAAC 3’ SEQ ID NO: 36 (RP gamma) 5’ CATTCCTACTACAACCCTCCCTACAC 3’ SEQ ID NO: 37 (RP delta) 5’ CTCTCACTCATTACACCACCCACATC 3’ SEQ ID NO: 38 (RP e) 5’ CTAACCTCAACCTTCTCCACCTATCC 3’