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WO2025081196A1 - Isolement sans trace de lymphocytes t spécifiques d'un antigène pour lymphocytes t car afin de produire des lymphocytes t car par tri régulé par adn - Google Patents

Isolement sans trace de lymphocytes t spécifiques d'un antigène pour lymphocytes t car afin de produire des lymphocytes t car par tri régulé par adn Download PDF

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Publication number
WO2025081196A1
WO2025081196A1 PCT/US2024/051467 US2024051467W WO2025081196A1 WO 2025081196 A1 WO2025081196 A1 WO 2025081196A1 US 2024051467 W US2024051467 W US 2024051467W WO 2025081196 A1 WO2025081196 A1 WO 2025081196A1
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WIPO (PCT)
Prior art keywords
probe
cells
virus
specific
catch
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Inventor
Gabriel A. Kwong
Aaron D. SILVA TRENKLE
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Georgia Tech Research Institute
Georgia Tech Research Corp
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Georgia Tech Research Institute
Georgia Tech Research Corp
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6804—Nucleic acid analysis using immunogens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70539—MHC-molecules, e.g. HLA-molecules
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles

Definitions

  • 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.
  • kits for sorting T cells 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
  • 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
  • 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,
  • 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.
  • Candida albicans Candida albicans
  • Cryptococcus neoformans Histoplama capsulatum
  • Aspergillus fumigatus Coccidiodes immitis
  • Paracoccidiodes brasiliensis Paracoccidiodes brasiliensis
  • Blastomyces dermitidis Blastomyces dermitidis
  • Pneumocystis carnii Penicillium marneffi
  • 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 granulo
  • 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.
  • a first end of the catch probe is conjugated to a quencher (such as, for example, IOWA BLACK®, Dabsyl, Black Hole quenchers, or CP ⁇ .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.
  • a quencher such as, for example, IOWA BLACK®, Dabsyl, Black Hole quenchers, or CP ⁇ .Q
  • 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.
  • the toehold region comprises from 3 to 10 nucleotides.
  • pMHC peptide major histocompatibility complex
  • a nucleic acid release probe release probe
  • 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.
  • 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.
  • FACS florescence activated cell sorting
  • the antigen-specific T cells are not activated.
  • 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
  • pMHC peptide major his
  • peptide pulse or a vaccine comprises the peptide epitope for which target antigen-specific T cells are specific thereby expanding the antigen-specific T cells.
  • 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.
  • FIGURE 2 depicts the generation of CAR viral-specific T cells through DNA gated sorting.
  • FIGURE 3 depicts the expansion of GIL-specific T cells by peptide pulsing PBMCs.
  • FIGURE 4 depicts that expanded GIL-specific T cells express LDLR.
  • FIGURE 5 depicts that DGS of GIL-specific T cells results in highly pure eluted fraction.
  • FIGURE 6 depicts that DGS with pMHC monomer is scarless.
  • FIGURE 7 depicts that pMHC DNA gated sorting results in scarless isolation of antigen-specific CD8 T cells.
  • FIGURES 8A-8C depict the complete release of pMHC DNA circuit in under five minutes.
  • FIGURES 9A-9D depict that the mixture of AgS mouse CD8+ T cells is separated with pMHC DGS.
  • 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.
  • 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.
  • 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.
  • a further aspect includes from the one particular value and/or to the other particular value.
  • 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’.
  • 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’.
  • the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values includes “about ‘x’ to about ‘y’”.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • "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.
  • 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.
  • 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.
  • “reduces tumor growth” means reducing the rate of growth of a tumor relative to a standard or a control.
  • 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.
  • 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.
  • the subject can be a human or veterinary patient.
  • patient refers to a subject under the treatment of a clinician, e.g., physician.
  • 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.
  • treatment 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.
  • 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 can include those already with the disorder and/or those in which the disorder is to be prevented.
  • 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.
  • dose 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • DGS DNA gated sorting
  • CD8 T cells from transgenic mice (OT1, Pmel, P14), viral infection models (LCMV), and healthy human samples (EBV, CMV, Influenza).
  • kits for sorting T cells 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.
  • a detectable agent such as, for example
  • kits use detectable agents to enable the sorting (i.e., separation or isolation) of T cells.
  • 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.
  • FACS fluorescence activated cell sorting
  • Fluorophores are compounds or molecules that luminesce.
  • 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
  • kits for sorting T cells can comprise a synthetic antigen (i.e., a modified or non-naturally occurring peptide).
  • 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
  • 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 haemolytic
  • 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.
  • Candida albicans Candida albicans
  • Cryptococcus neoformans Histoplama capsulatum
  • Aspergillus fumigatus Coccidiodes immitis
  • Paracoccidiodes brasiliensis Paracoccidiodes brasiliensis
  • Blastomyces dermitidis Blastomyces dermitidis
  • Pneumocystis carnii Penicillium marneffi
  • 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, Echi
  • 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.
  • a first end of the catch probe is conjugated to a quencher (such as, for example, IOWA BLACK®, Dabsyl, Black Hole quenchers, or CP ⁇ .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.
  • a quencher such as, for example, IOWA BLACK®, Dabsyl, Black Hole quenchers, or CP ⁇ .Q
  • 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
  • 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.
  • 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.
  • pMHC peptide major histocompatibility complex
  • 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.
  • 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.
  • FACS florescence activated cell sorting
  • the antigen-specific T cells are not activated by the pMHC monomer.
  • the antigen-specific T cells are chimeric antigen receptor (CAR) T cells.
  • a nucleic acid release probe 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
  • 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.
  • 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.
  • FACS florescence activated cell sorting
  • the antigen-specific T cells are not activated.
  • Also disclosed herein are methods of performing an adoptive T cell therapy 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
  • pMHC
  • peptide pulse or vaccine comprises the peptide epitope for which target antigen-specific T cells are specific thereby expanding the antigen-specific T cells.
  • 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).
  • 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).
  • This study demonstrates an approach to capture and elute Ag-specific T cells using pMHCI-TP without significant impact on T cell product.
  • FIG. 2 depicts the generation of CAR viral-specific T cells through DNA gated sorting.
  • CAR chimeric antigen receptor
  • FIG. 2 depicts the generation of CAR viral-specific T cells through DNA gated sorting.
  • CAR chimeric antigen receptor
  • CAR T cells from antigen-specific T cells isolated using DNA gated sorting (DGS).
  • DGS DNA gated sorting
  • PBMCs peripheral blood mononuclear cells
  • pMHC major histocompatibility complex class I
  • 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.
  • 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.
  • FIG. 4 depicts that expanded GIL-specific T cells express LDLR.
  • FIG. 5 depicts that DGS of GIL-specific T cells results in highly pure eluted fraction.
  • GIL-specific T cells were isolated through DNA gated sorting (DGS), resulting in over 90% pure eluted fraction.
  • FIG. 6 depicts that DGS with pMHC monomer is scarless.
  • DNA gated sorting was performed with pMHC streptavidin (Stv) tetramers and peptide major histocompatibility complex (pMHC) monomers, which has a lower avidity that pMHC Stv tetramers.
  • Stv streptavidin
  • pMHC peptide major histocompatibility complex
  • 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).
  • TCR T cell receptor
  • RP complementary release probe
  • 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.
  • FIGS. 8A-8C depict the complete release of pMHC DNA circuit in under five minutes.
  • FIG. 8A shows the target probe ⁇ conjugated to Cy5 (TP ⁇ .Cy5) was annealed to complementary catch probe ⁇ conjugated to quencher (CP ⁇ .Q), resulting in no fluorescence. After five minutes, complementary release probe ⁇ (RP ⁇ ) was added, resulting in displacement Attorney Docket No.10034-322WO1 of CP ⁇ .Q and full return of TP ⁇ .Cy5 signal in five minutes.
  • FIG.8B shows that five CP.Q and TP.Cy5 pairs ( ⁇ - ⁇ ) were annealed, mixed with five RPs ( ⁇ - ⁇ ), and normalized fluorescence was quantified after 15 minutes, exhibiting creation of orthogonal DNA circuits.
  • FIG. 8C shows that, after conjugating TP ⁇ .Cy5 to pMHC (pMHC.TP ⁇ .Cy5) and annealing to CP ⁇ .Q, addition of RP ⁇ once again results in full release of pMCH.TP ⁇ .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.
  • 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.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.TP ⁇ , Db-Gp100.TP ⁇ , and Kb-OVA.TP ⁇ 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.
  • 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 ⁇ 2m 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.
  • 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.
  • 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.

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Abstract

Sont divulgués des procédés de tri de lymphocytes T spécifiques à un antigène sans activation ; des procédés utilisant lesdits procédés de tri pour concevoir des lymphocytes T porteurs d'un récepteur antigénique chimérique (CAR) ; des procédés utilisant lesdits procédés de tri dans des thérapies cellulaires adoptives et des kits comprenant les composants pour mettre en œuvre les procédés divulgués.
PCT/US2024/051467 2023-10-12 2024-10-15 Isolement sans trace de lymphocytes t spécifiques d'un antigène pour lymphocytes t car afin de produire des lymphocytes t car par tri régulé par adn Pending WO2025081196A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009126828A2 (fr) * 2008-04-09 2009-10-15 California Institute Of Technology Agents de capture, et procédés et systèmes en rapport pour détecter et/ou trier des cibles
CN110791589A (zh) * 2019-10-18 2020-02-14 张家口健垣科技有限公司 一种用于rna恒温扩增检测诺如病毒的引物和探针、试剂盒及检测方法
US20200407781A1 (en) * 2018-08-28 2020-12-31 10X Genomics, Inc. Method for transposase-mediated spatial tagging and analyzing genomic dna in a biological sample
US20210087605A1 (en) * 2018-02-07 2021-03-25 Georgia Tech Research Corporation Methods for Multiplexed Cell Isolation Using DNA Gates
WO2022162191A2 (fr) * 2021-01-28 2022-08-04 Fwd Holding Aps Multiplexeurs pmhc par addition de mhc2 et affichage multimère de peptide par cercle roulant

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009126828A2 (fr) * 2008-04-09 2009-10-15 California Institute Of Technology Agents de capture, et procédés et systèmes en rapport pour détecter et/ou trier des cibles
US20210087605A1 (en) * 2018-02-07 2021-03-25 Georgia Tech Research Corporation Methods for Multiplexed Cell Isolation Using DNA Gates
US20200407781A1 (en) * 2018-08-28 2020-12-31 10X Genomics, Inc. Method for transposase-mediated spatial tagging and analyzing genomic dna in a biological sample
CN110791589A (zh) * 2019-10-18 2020-02-14 张家口健垣科技有限公司 一种用于rna恒温扩增检测诺如病毒的引物和探针、试剂盒及检测方法
WO2022162191A2 (fr) * 2021-01-28 2022-08-04 Fwd Holding Aps Multiplexeurs pmhc par addition de mhc2 et affichage multimère de peptide par cercle roulant

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