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WO2025097034A1 - Procédés de fabrication de lymphocytes t modifiés à partir d'échantillons de sang entier - Google Patents

Procédés de fabrication de lymphocytes t modifiés à partir d'échantillons de sang entier Download PDF

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Publication number
WO2025097034A1
WO2025097034A1 PCT/US2024/054236 US2024054236W WO2025097034A1 WO 2025097034 A1 WO2025097034 A1 WO 2025097034A1 US 2024054236 W US2024054236 W US 2024054236W WO 2025097034 A1 WO2025097034 A1 WO 2025097034A1
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WIPO (PCT)
Prior art keywords
cells
population
hours
polyfunctional
car
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PCT/US2024/054236
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English (en)
Inventor
Daniel ANAYA
Brandon Kwong
Ames REGISTER
Ranjita Sengupta
Jeevitha JEEVAN
Nikki KHOSHNOODI
Karen Walker
Sunetra Biswas
Soo Hyun PARK
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Kyverna Therapeutics Inc
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Kyverna Therapeutics Inc
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Publication of WO2025097034A1 publication Critical patent/WO2025097034A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0636—T lymphocytes
    • 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
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/50—Cell markers; Cell surface determinants
    • C12N2501/515—CD3, T-cell receptor complex
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production

Definitions

  • the present disclosure generally relates to methods and compositions for the manufacture of engineered T cells that express a heterologous protein, such as a chimeric antigen receptor (CAR).
  • a heterologous protein such as a chimeric antigen receptor (CAR).
  • Chimeric antigen receptor (CAR) T cell therapy has transformative potential in treating many life-altering diseases and conditions, e.g., cancer and immune disorders.
  • CAR T cell treatments have shown promise in their abilities to selectively target and nullify the underlying causes of a disease, while providing a durable response to prevent recurrence.
  • CAR T cells Despite its potential, several key challenges remain for the commercial viability of CAR T cells.
  • manufacture of CAR T cells involves lengthy ex -vivo cell culture procedures that are costly and result in a high product variability.
  • prolonged ex vivo culture is associated with phenotypic changes (differentiation) that are poorly characterized and potentially detrimental to therapeutic efficacy, with these changes becoming more numerous the longer the manufacturing process extends.
  • exemplary methods for manufacturing CAR T cells of the disclosure may include obtaining T cells from a subject (e.g., a donor or a patient) from whole blood.
  • T cells are obtained from peripheral blood mononuclear cells (PBMC) isolated from the whole blood.
  • PBMC peripheral blood mononuclear cells
  • the present Inventors have discovered that surprisingly, sufficient numbers of T cells for CAR T cell manufacturing may be obtained directly from whole blood, without the need for a leukapheresis step or PBMC isolation step.
  • CAR T cell manufacturing processes of the disclosure that extend for between 6-9 days (generally about 8 days) and the rapid methods, which are able to produce engineered cells in around two-to-three days after receiving a whole blood sample containing donor T cells.
  • certain methods of the disclosure include a concurrent T cell isolation and activation step.
  • T cells are isolated from whole blood or PBMCs and concurrently activated.
  • the concurrent activation/i solation reduce the overall manufacturing time, it also is able to isolate a sufficient number of T cells, at a high purity, to manufacture the CAR T cells from a very small whole blood sample (e.g., samples of 100 mb, or less, of blood).
  • a very small whole blood sample e.g., samples of 100 mb, or less, of blood.
  • Certain exemplary methods for manufacturing CAR T cells of the disclosure are “rapid” processes, and produce CAR T cells significantly faster than other processes. These shortened processes of the disclosure, usually two-to-three days in total, employ a brief culture after T cell isolation/activation before transduction, generally less than or significantly less than a day, and a similarly brief cultivation after transduction before harvesting the desired CAR T cell product. Despite the small starting whole blood samples, the lack of a leukapheresis or other similar T cell isolating step, and the brief cultivation steps, these methods of the disclosure are able to produce a large harvest of CAR T cells — all within 2-3 days of receiving a starting sample.
  • the cells made using the methods of the disclosure unexpectedly show a more favorable phenotype as a result of the condensed processes of the disclosure.
  • the cells were far less differentiated than those using longer processes.
  • the cells produced using the shortened methods of the disclosure included higher levels of stem cell memory T cells (Tscm), which generally only account for 2-3% of circulating T cells and are associated with long-term defensive immunity, anti-tumor activity, and self-renewal. Further, these cells expressed the introduced CARs at a very high percentage, leading to a pure end product that showed targeted cytotoxicity with low T cell exhaustion.
  • Tscm stem cell memory T cells
  • the shorted manufacturing process produces a large number of polyfunctional cells, which simultaneously secrete multiple sets cytokines, chemokines, and/or cytotoxic granules simultaneously. Owing to this polyfunctional behavior, such cells are known to provide a more effective immune response, which is desirable in a therapeutic CAR T cell product.
  • certain methods of the disclosure that use a longer manufacture time (e.g., 6 to 9 days) and a whole blood starting sample, rather than an apheresis sample as used in prior methods, are still able to produce cells with a similar phenotype to those produced from methods using apheresis starting material. Thus, the presently disclosed methods are able to ease the bottlenecks in production associated with obtaining apheresis samples, while delivering an equivalent or better therapeutically effective cellular product.
  • the present disclosure provides methods for producing a population of engineered T cells expressing a heterologous protein, e.g., a chimeric antigen receptor.
  • An exemplary method may include the steps of: obtaining a whole blood sample from a donor; binding T cells from the whole blood sample to one or more anti-CD3 antibodies and one or more anti-CD28 antibodies attached to a support, thereby isolating and activating the T cells from the whole blood sample; after binding the T cells, contacting the activated T cells with a nucleic acid encoding a heterologous protein; cultivating the T cells contacted with said nucleic acid in a serum-free cultivation medium; and harvesting the cultivated T cells, wherein the harvested T cells express the heterologous protein.
  • the present disclosure provides rapid methods for producing a population of engineered T cells expressing a heterologous protein.
  • An exemplary rapid method may include the steps of: obtaining a whole blood sample from a donor; binding T cells from the whole blood sample to one or more anti-CD3 antibodies and one or more anti-CD28 antibodies attached to a support, thereby isolating and activating the T cells from the whole blood sample; between 10 hours and 25 hours after binding the T cells, contacting the activated T cells with a nucleic acid encoding a heterologous protein; cultivating the T cells contacted with said nucleic acid in a serum- free cultivation medium for a period of between 24 hours and 60 hours; and harvesting the cultivated T cells, wherein the harvested T cells express the heterologous protein.
  • the T cells are isolated from the whole blood sample directly without an intervening T cell isolation step and/or leukapheresis.
  • the method comprises isolating peripheral blood mononuclear cells (PBMC) comprising the T cells from the whole blood sample without a leukapheresis step.
  • PBMC peripheral blood mononuclear cells
  • the contacting step occurs between 12 hours and 21 hours after the binding step. In preferred methods of the disclosure, the contacting step occurs between 15 hours and 19 hours after the binding step. In certain preferred aspects, the contacting step occurs between 17 hours and 19 hours after the binding step. In certain methods, the contacting step occurs about 18 hours after the binding step.
  • the cultivating step is for a period of between 29 hours and 59 hours. In preferred methods of the disclosure, the cultivating step is for a period of between 36 hours and 52 hours. In more preferred aspects, the cultivating step is for a period of between 46 hours and 50 hours. In certain aspects, the cultivating step is for a period of about 48 hours.
  • the present disclosure provides methods for producing a population of engineered T cells expressing a heterologous protein.
  • An exemplary method may include the steps of: obtaining a whole blood sample from a donor; binding T cells from the whole blood sample to one or more anti-CD3 antibodies and one or more anti-CD28 antibodies attached to a support, thereby isolating and activating the T cells from the whole blood sample; between 20 hours and 28 hours after binding the T cells, contacting the activated T cells with a nucleic acid encoding a heterologous protein; cultivating the T cells contacted with said nucleic acid in a serum-free cultivation medium for a period of between 4 days and 9 days; and harvesting the cultivated T cells, wherein the harvested T cells express the heterologous protein.
  • the contacting step occurs between about 20 and 28 hours after the binding step and the cultivating step is for a period of 4-9 days. In certain aspects, the contacting step occurs between about 22 and 26 hours after the binding step. In certain aspects, the contacting step occurs between about 23 and 25 hours after the binding step. In certain aspects, the contacting step occurs about 24 hours after the binding step. In certain aspects, the cultivating step is for a period of about 5 to about 7 days. In certain aspects, the cultivating step is for a period of about 6 days. In certain aspects, the cultivating step is for a period of about 8 days.
  • the harvested T cells comprise between about 10% and 60% CD45RO-/CCR7+ T cells (Tnscm). In more preferred methods, the harvested T cells comprise between about 15% and 60% CD45RO-/CCR7+ T cells (Tnscm). In certain aspects, the harvested T cells comprise at least 18% Tnscm. In certain aspects, the harvested T cells comprise at least 22% Tnscm. In certain aspects, the harvested T cells comprise at least 25% Tnscm.
  • the Tnscm may include naive T cells (Tn) and stem cell memory T cells (Tscm).
  • Tnscm naive T cells
  • Tscm stem cell memory T cells
  • the Tnscm comprise more Tscm than Tn.
  • the Tnscm may comprise at least 1.5x more Tscm than Tn; at least twice as many Tscm as Tn; at least 3x more Tscm than Tn; at least 5x more Tscm than Tn; at least lOx more Tscm than Tn; and/or at least 50x more Tscm than Tn.
  • the one or more anti-CD3 antibodies and the one or more anti-CD28 antibodies attached to the same support.
  • the one or more anti- CD3 antibodies and/or the one or more anti-CD28 antibodies are attached to the support via a cleavable linker.
  • the linker may be, for example, an enzymatically cleavable, a hydrolysable linker, a redox cleavable linker, a phosphate-based cleavable linker, an acid cleavable linker, an ester-based cleavable linker, a peptide-based cleavable linker, a disulfide-based cleavable linker, a nitrobenzyl cleavable linker, a methoxymethyl-based cleavable linker and/or photocleavable linker. Accordingly, methods of the disclosure may further include contacting the activated T cells with a stimulus that cleaves the linker, thereby releasing the T cells from the surface.
  • the surface is a solid surface.
  • the solid surface is a bead, well, chip, or microfluidic channel. More preferably, the solid surface is a bead.
  • the surface comprises a polymer.
  • the polymer is a hydrogel. In some methods, the surface comprises a polymer scaffold.
  • the harvested T cells comprise a population of at least 6% of the harvested T cells that are polyfunctional T cells upon specific target-based activation.
  • the polyfunctional T cells and/or a portion of the population thereof simultaneously secrete Granzyme B and TNFb.
  • the polyfunctional T cells and/or a portion of the population thereof simultaneously secrete Granzyme B and IFNg.
  • at least 1% of the harvested T cells and/or a portion of the population thereof simultaneously secrete Granzyme B and IFNg.
  • the polyfunctional T cells and/or a portion of the population thereof simultaneously secrete MIP-la and MIP-lb.
  • the polyfunctional T cells and/or a portion of the population thereof simultaneously secrete IFNgand Granzyme B.
  • the polyfunctional T cells and/or a portion of the population thereof comprise two or more of: cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete Granzyme B and TNFb; cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete Granzyme B and IFNg; cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete MIP- la and MIP-lb; and cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete IFNg and Granzyme B.
  • the polyfunctional T cells and/or a portion of the population of polyfunctional T cells comprise: cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete Granzyme B and TNFb; cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete Granzyme B and IFNg; cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete MIP- la and MIP-lb; and cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete IFNg and Granzyme B.
  • the serum free cultivation medium comprises at least one cytokine.
  • the at least one cytokine comprises one or more of IL-2, IL-21, IL-7, and IL-15.
  • the at least one cytokine comprises one or more of IL-21, IL-7, and IL-15 and does not comprise IL-2.
  • the number of isolated T cells from the whole blood sample is between about IxlO 6 and about IxlO 8 total T cells and the number of harvested T cells is between about IxlO 6 and about 5xl0 8 .
  • the number of isolated T cells from the whole blood sample is between about 1.5xl0 7 and about IxlO 8 total T cells and the number of harvested T cells is between about 7.5xl0 7 and about 1.5xl0 8 .
  • the number of isolated T cells from the whole blood sample is between about 5xl0 7 and about 7.5xl0 7 total T cells and the number of harvested T cells is between about 7.5xl0 7 and about 1.2xl0 8 .
  • the culturing step produces between a 1.0- and a 4-fold expansion of the harvested T cells. In certain aspects, the culturing step produces between a 1.2- and a 4-fold expansion of the harvested T cells.
  • the present disclosure also provides methods for producing a population of engineered T cells expressing a heterologous protein comprising the steps of: obtaining a whole blood sample from a donor; binding T cells from the whole blood sample to one or more anti-CD3 antibodies and one or more anti-CD28 antibodies attached to a support, thereby isolating and activating the T cells from the whole blood sample without an intervening PBMC isolation step or a leukapheresis step; contacting the activated T cells with a nucleic acid encoding a heterologous protein; cultivating the T cells contacted with said nucleic acid in a serum-free cultivation medium; and harvesting the cultivated T cells, wherein the harvested T cells express the heterologous protein.
  • the harvested T cells comprise between about 10% and 60% CD45RO-/CCR7+ T cells (Tnscm). In more preferred methods, the harvested T cells comprise between about 15% and 60% CD45RO-/CCR7+ T cells (Tnscm). In certain aspects, the harvested T cells comprise at least 18% Tnscm. In certain aspects, the harvested T cells comprise at least 22% Tnscm. In certain aspects, the harvested T cells comprise at least 25% Tnscm.
  • the Tnscm may include naive T cells (Tn) and stem cell memory T cells (Tscm).
  • Tnscm naive T cells
  • Tscm stem cell memory T cells
  • the Tnscm comprise more Tscm than Tn.
  • the Tnscm may comprise at least 1.5x more Tscm than Tn; at least twice as many Tscm as Tn; at least 3x more Tscm than Tn; at least 5x more Tscm than Tn; at least lOx more Tscm than Tn; and/or at least 50x more Tscm than Tn.
  • the contacting step occurs between 12 hours and 21 hours after the binding step. In preferred methods of the disclosure, the contacting step occurs between 15 hours and 19 hours after the binding step. In certain preferred aspects, the contacting step occurs between 17 hours and 19 hours after the binding step. In certain methods, the contacting step occurs about 18 hours after the binding step.
  • the cultivating step is for a period of between 29 hours and 59 hours. In preferred methods of the disclosure, the cultivating step is for a period of between 36 hours and 52 hours. In more preferred aspects, the cultivating step is for a period of between 46 hours and 50 hours. In certain aspects, the cultivating step is for a period of about 48 hours.
  • the one or more anti-CD3 antibodies and the one or more anti-CD28 antibodies attached to the same support.
  • the one or more anti- CD3 antibodies and/or the one or more anti-CD28 antibodies are attached to the support via a cleavable linker.
  • the linker may be, for example, an enzymatically cleavable, a hydrolysable linker, a redox cleavable linker, a phosphate-based cleavable linker, an acid cleavable linker, an ester-based cleavable linker, a peptide-based cleavable linker, a disulfide-based cleavable linker, a nitrobenzyl cleavable linker, a methoxymethyl-based cleavable linker and/or photocleavable linker.
  • methods of the disclosure may further include contacting the activated T cells with a stimulus that cleaves the linker, thereby releasing the T cells from the surface.
  • the surface is a solid surface.
  • the solid surface is ahead, well, chip, or microfluidic channel. More preferably, the solid surface is a bead.
  • the surface comprises a polymer.
  • the polymer is a hydrogel.
  • the surface comprises a polymer scaffold.
  • the harvested T cells comprise a population of at least 6% of the harvested T cells that are polyfunctional T cells upon specific target-based activation.
  • the polyfunctional T cells and/or a portion of the population thereof simultaneously secrete Granzyme B and TNFb.
  • the polyfunctional T cells and/or a portion of the population thereof simultaneously secrete Granzyme B and IFNg.
  • at least 1% of the harvested T cells and/or a portion of the population thereof simultaneously secrete Granzyme B and IFNg.
  • the polyfunctional T cells and/or a portion of the population thereof simultaneously secrete MIP-la and MIP-lb.
  • the polyfunctional T cells and/or a portion of the population thereof simultaneously secrete IFNg and Granzyme B.
  • the polyfunctional T cells and/or a portion of the population thereof comprise two or more of: cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete Granzyme B and TNFb; cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete Granzyme B and IFNg; cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete MIP- la and MIP-lb; and cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete IFNg and Granzyme B.
  • the polyfunctional T cells and/or a portion of the population of polyfunctional T cells comprise: cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete Granzyme B and TNFb; cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete Granzyme B and IFNg; cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete MIP- la and MIP-lb; and cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete IFNg and Granzyme B.
  • the serum free cultivation medium comprises at least one cytokine.
  • the at least one cytokine comprises one or more of IL-2, IL-21, IL-7, and IL-15.
  • the at least one cytokine comprises one or more of IL-21, IL-7, and IL-15 and does not comprise IL-2.
  • the serum free cultivation medium comprises no added cytokines.
  • the number of isolated T cells from the whole blood sample is between about lxl0 6 and about IxlO 8 total T cells and the number of harvested T cells is between about 2.5xl0 7 and about 5xl0 8 .
  • the number of isolated T cells from the whole blood sample is between about 1.5xl0 7 and about IxlO 8 total T cells and the number of harvested T cells is between about 7.5xl0 7 and about 1.5xl0 8 .
  • the number of isolated T cells from the whole blood sample is between about 5xl0 7 and about 7.5xl0 7 total T cells and the number of harvested T cells is between about 7.5xl0 7 and about 1.2xl0 8 .
  • the culturing step produces between a 1.0- and a 4-fold expansion of the harvested T cells. In certain aspects, the culturing step produces between a 1.2- and a 4-fold expansion of the harvested T cells.
  • Alternative methods of the disclosure for producing a population of engineered T cells expressing a heterologous protein may include the steps of: obtaining a whole blood sample from a donor; binding T cells from the whole blood sample to one or more anti-CD4 antibodies and one or more anti-CD8 antibodies attached to a support, thereby isolating the T cells from the whole blood sample; between 10 hours and 25 hours after binding the T cells, contacting the activated T cells with a nucleic acid encoding a heterologous protein; cultivating the T cells contacted with said nucleic acid in a serum-free cultivation medium for a period of between 24 hours and 60 hours; and harvesting the cultivated T cells, wherein the harvested T cells express the heterologous protein.
  • the method further includes a step of activating the isolated T cells.
  • the T cells are isolated from the whole blood sample directly without an intervening T cell isolation step and/or leukapheresis.
  • the method comprises isolating peripheral blood mononuclear cells (PBMC) comprising the T cells from the whole blood sample without a leukapheresis step.
  • PBMC peripheral blood mononuclear cells
  • the harvested T cells comprise between about 10% and 60% CD45RO-/CCR7+ T cells (Tnscm).
  • the harvested T cells comprise between about 15% and 60% CD45RO-/CCR7+ T cells (Tnscm).
  • the harvested T cells comprise at least 18% Tnscm.
  • the harvested T cells comprise at least 22% Tnscm.
  • the harvested T cells comprise at least 25% Tnscm.
  • the Tnscm may include naive T cells (Tn) and stem cell memory T cells (Tscm).
  • Tnscm naive T cells
  • Tscm stem cell memory T cells
  • the Tnscm comprise more Tscm than Tn.
  • the Tnscm may comprise at least 1.5x more Tscm than Tn; at least twice as many Tscm as Tn; at least 3x more Tscm than Tn; at least 5x more Tscm than Tn; at least lOx more Tscm than Tn; and/or at least 50x more Tscm than Tn.
  • the contacting step occurs between 12 hours and 21 hours after the binding step. In preferred methods of the disclosure, the contacting step occurs between 15 hours and 19 hours after the binding step. In certain preferred aspects, the contacting step occurs between 17 hours and 19 hours after the binding step. In certain methods, the contacting step occurs about 18 hours after the binding step.
  • the cultivating step is for a period of between 29 hours and 59 hours. In preferred methods of the disclosure, the cultivating step is for a period of between 36 hours and 52 hours. In more preferred aspects, the cultivating step is for a period of between 46 hours and 50 hours. In certain aspects, the cultivating step is for a period of about 48 hours.
  • the one or more anti-CD4 antibodies and the one or more anti-CD8 antibodies attached to the same support.
  • the one or more anti- CD8 antibodies and/or the one or more anti-CD4 antibodies are attached to the support via a cleavable linker.
  • the linker may be, for example, an enzymatically cleavable, a hydrolysable linker, a redox cleavable linker, a phosphate-based cleavable linker, an acid cleavable linker, an ester-based cleavable linker, a peptide-based cleavable linker, a disulfide-based cleavable linker, a nitrobenzyl cleavable linker, a methoxymethyl-based cleavable linker and/or photocleavable linker. Accordingly, methods of the disclosure may further include contacting the activated T cells with a stimulus that cleaves the linker, thereby releasing the T cells from the surface.
  • the surface is a solid surface.
  • the solid surface is ahead, well, chip, or microfluidic channel. More preferably, the solid surface is a bead.
  • the surface comprises a polymer.
  • the polymer is a hydrogel.
  • the surface comprises a polymer scaffold.
  • the harvested T cells comprise a population of at least 6% of the harvested T cells that are polyfunctional T cells upon specific target-based activation. In certain aspects, the polyfunctional T cells and/or a portion of the population thereof simultaneously secrete Granzyme B and TNFb.
  • the polyfunctional T cells and/or a portion of the population thereof simultaneously secrete Granzyme B and IFNg. In preferred aspects, at least 1% of the harvested T cells and/or a portion of the population thereof simultaneously secrete Granzyme B and IFNg. In certain aspects, the polyfunctional T cells and/or a portion of the population thereof simultaneously secrete MLP- la and MIP-lb. In certain methods, the polyfunctional T cells and/or a portion of the population thereof simultaneously secrete IFNg and Granzyme B.
  • the polyfunctional T cells and/or a portion of the population thereof comprise two or more of: cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete Granzyme B and TNFb; cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete Granzyme B and IFNg; cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete MIP- la and MIP-lb; and cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete IFNg and Granzyme B.
  • the polyfunctional T cells and/or a portion of the population of polyfunctional T cells comprise: cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete Granzyme B and TNFb; cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete Granzyme B and IFNg; cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete MIP- la and MIP-lb; and cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete IFNg and Granzyme B.
  • the serum free cultivation medium comprises at least one cytokine.
  • the at least one cytokine comprises one or more of IL-2, IL-21, IL-7, and IL-15.
  • the at least one cytokine comprises one or more of IL-21, IL-7, and IL-15 and does not comprise IL-2.
  • the number of isolated T cells from the whole blood sample is between about lxl0 6 and about IxlO 8 total T cells and the number of harvested T cells is between about 2.5xl0 7 and about 5xl0 8 .
  • the number of isolated T cells from the whole blood sample is between about 1.5xl0 7 and about IxlO 8 total T cells and the number of harvested T cells is between about 7.5xl0 7 and about 1.5xl0 8 .
  • the number of isolated T cells from the whole blood sample is between about 5xl0 7 and about 7.5xl0 7 total T cells and the number of harvested T cells is between about 7.5xl0 7 and about 1.2xl0 8 .
  • the culturing step produces between a 1.0- and a 4-fold expansion of the harvested T cells. In certain aspects, the culturing step produces between a 1.2- and a 4-fold expansion of the harvested T cells.
  • FIG. 1 provides an exemplary workflow for methods of the disclosure used to produce engineered T cells from a whole blood sample.
  • FIG. 2 is a bar graph showing T-cell expansion, represented by the fold increase of the number of CD3+ cells at 72 hours following thawing relative to the number of CD3+ cells upon thawing to compare methods of manufacturing CAR T cells of the disclosure.
  • FIG. 3 is a graph depicting percentage of CAR expression at 72 hours following thawing to compare methods of manufacturing CAR T cells of the disclosure.
  • FIGs. 4A-4B show percentage of TNSCM (naive and stem cell memory T cells (CD45RO- /CCR7+)) upon thawing.
  • “UNTD” denotes untransduced controls.
  • FIGs. 5A-5D are bar graphs depicting memory phenotypes of CAR+ cells upon thawing (FIGS. 5A and 5C) or 72 hours after thawing (FIGS. 5B and 5D). "Post Enrichment" condition was untransduced.
  • FIG. 6 shows percentage of cytolysis of NALM6 target cells when incubated with the CAR-T cells produced by Conditions D and E.
  • D is KYV 6-day condition
  • E is KYV 8-day condition
  • UNTD denoted an untransduced condition.
  • FIGS. 7A-7D are bar graphs depicting secretion frequency of each of 32 cytokines from cells manufactured using the KYV 3-day v2 protocol (“C") (FIGS. 7A and 7C) or the KYV-9- day protocol (“E”) (FIGS. 7B and 7D), after overnight incubation in the presence of CD 19+ NALM6 target cells, "ut” corresponds to untransduced control cells that were otherwise subjected to each of the steps of the 3 -day or 9-day protocol.
  • C KYV 3-day v2 protocol
  • E KYV-9- day protocol
  • FIG. 8 is a bar graph showing polyfunctionality of CAR-T cells produced by Process C.
  • FIG. 9 is a bar graph showing percentage of cells, produced by Process C or E, out of all polyfunctional cells that secreted the cytokines indicated.
  • FIG. 10 is a flow chart illustrating the experimental design and conditions of six CAR-T cell manufacturing processes.
  • FIG. 11 provides data from same-donor studies validate that the X-LAB system provided superior PBMC enrichment from whole blood samples.
  • FIG. 12 provides data showing the CD3+, CD4+, CD8+ cell percentage (T cells) in samples, including whole blood samples.
  • FIG. 13 outlines the protocols used to assess a direct-from-blood T cell isolation step.
  • FIG. 14 provides enrichment and expansion data comparing methods of the disclosure that isolate T cells from whole blood for CAR T cell manufacture.
  • FIG. 15 provides CD3+ and CD4+ percentage data comparing methods of the disclosure that isolate T cells from whole blood for CAR T cell manufacture.
  • FIG. 16 provides enrichment and expansion data comparing methods of the disclosure that isolate T cells from whole blood for CAR T cell manufacture.
  • FIG. 17 provides data showing that cells produced using a 3-day manufacturing protocol of the disclosure to produce CAR T cells from T cells isolated directly from whole blood show a less differentiated phenotype compared to mock (untransduced) control cells.
  • FIG. 18 provides data showing the target-specific cytotoxicity of CAR T cells produced using the methods of the disclosure.
  • FIG. 19 provides the results for the target-dependent cytokine release by the CAR T cells derived from healthy donor (HD) whole blood, following 24h in vitro co-culture with CD 19+ NALM6 target cells at the indicated E:T (effectortarget) ratios.
  • FIG. 20 shows data for CD19-targeted CAR-T cells manufactured using methods of the disclosure and CD19+ NALM6 target cells that were co-cultured at the indicated E:T ratios (CAR-T effector:NALM6 target) and % killing of target cells was measured by flow cytometry at each indicated timepoint. At each timepoint, a fresh round of target cells was added to the coculture to assess the serial, repeated cytotoxicity of the CAR-T over time.
  • FIG. 21 provides target-specific expansion data for CAR T cells manufactured using methods of the disclosure.
  • FIG. 22 provides TBNK/memory phenotype of pre- and post-enrichment material for cells produced starting with T cells isolated from whole blood.
  • FIG. 23 provides TBNK/memory phenotype of a final CAR T cell product (e.g., after expansion) produced using an 8-day process.
  • FIG. 24 provides TBNK/memory phenotype of pre- and post-enrichment material for cells produced starting with T cells isolated from whole blood.
  • FIG. 25 provides TBNK/memory phenotype of pre- and post-enrichment material for cells produced starting with T cells isolated from whole blood.
  • FIG. 26 outlines steps of a three-day method of the disclosure starting from whole blood (WB) starting material (SM).
  • WB whole blood
  • SM starting material
  • FIG. 27A shows the T-cell memory phenotype of cells produced using the three-day methods of the disclosure.
  • FIG. 27B shows the cytolytic activity of the cells produced using the the 3-day methods of the disclosure.
  • FIG. 27C shows results of a serial rechallenge assay.
  • FIG. 27D shows that the 3-day process Ingenui-T cells successfully killed autologous primary B cells in a dose-dependent manner.
  • Ingenui-T cells or untransduced T cells derived from whole blood, were co-cultured with autologous (donor-matched) PBMCs at the indicated effector to target (E:T) ratios, representing the ratio of CAR + T cells (effector) to total PBMCs (target).
  • E:T effector to target
  • FIG. 28A shows fold expansion, viability, and T cell purity of CAR-T cells manufactured in the KYV 3-Day process starting from leukapheresis material.
  • KYV 3-Day Conditions “A”, “B”, “C”, “D” indicate different culture cytokine(s) used.
  • FIG. 29A shows % CAR+ expression analyzed by flow cytometry in CAR-T cells manufactured in the KYV 3-Day process starting from leukapheresis material. CAR expression was analyzed within total CD3+ T cells or within CD4+ or CD8+ T cells, at the time of harvest.
  • FIG. 30A shows % CD4+ and CD8+ analyzed within total CD3+ in CAR-T cells manufactured in the KYV 3-Day process starting from leukapheresis material.
  • KYV 3-Day Conditions “Cl”, “C2”, “C3”, “C4” indicate different culture cytokine(s) used.
  • N 4 healthy donors per condition.
  • NT non-transduced controls.
  • Conv 9-day refers to donor-matched CAR-T cells manufactured in a conventional 9-day culture process.
  • Aph SM refers to the leukapheresis donor starting material prior to the KYV 3-Day process.
  • FIG. 31A-31B shows results of T cell memory phenotype analyzed by flow cytometry within CD4+ or CD8+ T cells, in CAR-T cells manufactured in the KYV 3-Day process starting from leukapheresis material.
  • Conv 9-day refers to donor-matched CAR-T cells manufactured in a conventional 9-day culture process.
  • Aph SM refers to the leukapheresis donor starting material prior to the KYV 3-Day process.
  • Tnaive naive (CCR7+CD45RO-CD95-);
  • Tscm stem cell memory (CCR7+CD45RO-CD95+);
  • Tern central memory (CCR7+CD45RO+);
  • FIG. 31C T cell memory phenotype analyzed within total CD3+ T cells, in CAR-T cells manufactured in the KYV 3-Day process starting from freshly collected whole blood, compared to a conventional 9-day process starting from leukapheresis material.
  • N 4 donors combined.
  • WB SM and Aph SM refer to the donor whole blood or leukapheresis starting materials respectively, prior to the culture process.
  • T cell memory subsets were analyzed as defined in Figs. 31A-31B.
  • FIG. 33A shows results of killing or outgrowth of CD 19+ NALM6 target cells during 120h co-culture with anti-CD19 CAR-T cells manufactured in the KYV 3 -Day process from leukapheresis starting material, at the indicated E:T (Effector:Target) ratios of 0.3:1 or 1 : 1.
  • KYV 3-Day Conditions “Cl”, “C2”, “C3”, “C4” indicate different culture cytokine(s) used.
  • NT non-transduced control T cells.
  • One representative donor shown from n 4.
  • FIG. 33B shows results of killing or outgrowth of CD19+ NALM6 target cells during 120h co-culture with anti-CD19 CAR-T cells manufactured in the KYV 3-Day process from freshly collected whole blood, at the indicated E:T (Effector: Target) ratios of 0.3 : 1 or 1 : 1.
  • NT non-transduced control T cells.
  • FIG. 34 shows CAR-mediated cytotoxic activity of anti-CD19 CAR-T cells manufactured from KYV 3-day process against CD 19+ primary human B cells using the measured % Cytolysis of CD 19+ primary human B cells in co-culture with anti-CD19 CAR-T cells manufactured in the KYV 3-Day process from freshly collected whole blood.
  • E:T (Effector: Target) ratios indicate the ratio of CAR+ T cells to total PBMC (peripheral blood mononuclear cells) plated in co-culture.
  • FIG. 36B shows Cytokine release by anti-CD19 CAR-T cells manufactured in the KYV 3-Day process from freshly collected whole blood, in co-culture with CD19+NALM6 target cells at the indicated E:T (EffectorTarget) ratios of 0.3: 1 or 1 : 1. Culture supernatants were collected and the indicated cytokines were analyzed by MSD.
  • FIG. 36C shows cytokine release by anti-CD19 CAR-T cells manufactured in the KYV 3- Day process from freshly collected whole blood, in co-culture with CD 19+ NALM6 target cells at the indicated E:T (EffectorTarget) ratios of 0.3: 1 or 1 : 1. Culture supernatants were collected and the indicated cytokines were analyzed by MSD.
  • FIG. 37 shows results of a long-term serial cytotoxic activity by anti-CD19 CAR-T cells manufactured from KYV 3-day process compared to a conventional 9-day process, in response to CD19+ expressing target cells.
  • Fig. 37 provides the duration of in vitro cytotoxicity by KYV 3- Day or Conv 9-Day anti-CD19 CAR T cells, derived from a healthy donor, in a serial rechallenge assay against CD19+ NALM6 tumor cells.
  • KYV 3-Day CAR T cells were derived from leukapheresis starting material (“APH”, Top panel) or freshly collected whole blood (“WB”, Bottom panel).
  • CAR T cells were co-cultured in triplicate with NALM6 target cells at the indicated Effector: Target (E:T) ratios, and the survival of NALM6 cells was analyzed every 2-3 days by flow cytometry.
  • the time (days) to loss of CAR-mediated cytotoxic activity, defined as the assay timepoint at which >95% survival of target cells was detected, was measured for each individual replicate. Data representative of n 4 donors.
  • FIG. 38 shows results of in vitro expansion by anti -CD 19 CAR-T cells manufactured from KYV 3-day process compared to a conventional 9-day process, in response to CD19+ expressing target cells.
  • CAR T cells were cocultured with mitomycin C-treated REH target cells at a 1 : 1 ratio, and cells were re-plated every 3-4 days with new target cells. Total fold expansion of CAR+ T cells (gated by flow cytometry analysis) was measured at day 16.
  • FIG. 39A shows in vivo activity of anti-CD19 CAR-T cells manufactured from KYV 3- day process compared to conventional 9-day process, in CD 19+ NALM6 tumor-bearing NSG mice.
  • NALM6- luciferase tumor cells were injected i.v. into mice at day -7 prior to T cell transfer. On day 0, mice were given a single i.v. injection of the indicated doses of CAR T cells.
  • FIG. 39B shows individual NALM6 tumor growth curves in NSG mice treated with a le6 CAR+ T cell dose of donor-matched anti-CD19 CAR T cells.
  • FIG. 40 outlines two 9-day processes of the disclosure for manufacturing CAR T cells starting from fresh whole blood starting material.
  • FIGS. 41A-41E provide Flow cytometry data characterizing CAR T cells produced using 9-day methods of the disclosure starting from whole blood starting material.
  • FIG. 42 provides % cytolysis data characterizing CAR T cells produced using 9-day methods of the disclosure starting from whole blood starting material.
  • FIG. 43 provides cytokine secretion data characterizing CAR T cells produced using 9- day methods of the disclosure starting from whole blood starting material.
  • a heterologous protein such as a chimeric antigen receptor (CAR)
  • CAR chimeric antigen receptor
  • CAR chimeric antigen receptor
  • Exemplary methods for manufacturing such cells (e.g., CAR T cells) of the disclosure may include obtaining T cells from a subject (e.g., a donor or a patient) from whole blood.
  • T cells are obtained from peripheral blood mononuclear cells (PBMC) isolated from the whole blood.
  • PBMC peripheral blood mononuclear cells
  • the present Inventors have discovered that surprisingly, sufficient numbers of T cells for CAR T manufacturing may be obtained directly from whole blood, without the need for a leukapheresis step or PBMC isolation step.
  • Methods of the disclosure may include a concurrent T cell isolation and activation step.
  • T cells may be isolated from whole blood or PBMCs and concurrently activated. This reduces the overall manufacturing time, while retaining the ability isolate a sufficient number of T cells, at a high purity, to manufacture the CAR T cells from a very small whole blood sample (e.g., samples of less than 100 mb of blood).
  • Exemplary methods for manufacturing CAR T cells of the disclosure employ a short culture after T cell isolation/activation before transduction, generally less than or significantly less than a day, and a similarly short cultivation after transduction before harvesting the desired CAR T cell product. Accordingly, methods of the disclosure are able to produce a large harvest of CAR T cells — all within 2-3 days of receiving a starting sample.
  • CAR T cells manufactured using the methods of the disclosure have shown a superior T cell expansion in the final CAR T product; a more favorable phenotype as a result of the condensed processes of the disclosure; and less differentiation than with longer processes.
  • the resulting CAR T cells included a very high percentage of stem cell memory T cells (Tscm), which generally only account for 2-3% of circulating T cells and are associated with long-term defensive immunity, anti-tumor activity, selfrenewal, and immune-regulation.
  • Tscm stem cell memory T cells
  • Cells made using methods of the disclosure expressed the introduced CARs at a very high percentage, leading to a pure end product with targeted cytotoxicity and low T cell exhaustion. Even more surprising, the shorted manufacturing process produces a large number of polyfunctional cells, which simultaneously secrete multiple sets cytokines, chemokines, and/or cytotoxic granules simultaneously. Owing to this polyfunctional behavior, such cells are known to provide a more effective immune response, which is desirable in a therapeutic CAR T cell product.
  • reference to a range of 1-5,000 fold includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 fold, etc., as well as 1.1, 1.2, 1.3, 1.4, 1.5-fold, etc., 2.1, 2.2, 2.3, 2.4, 2.5 fold, etc., and so forth.
  • “About” a number refers to range including the number and ranging from 10% below that number to 10% above that number. “About” a range refers to 10% below the lower limit of the range, spanning to 10% above the upper limit of the range.
  • T cell activation when used in the context of T cell activation, encompasses various associated biological processes such as induction of intracellular signaling pathways associated with T cell activation, change in expression of cell surface markers, cytokine release, proliferation, and the like).
  • T cell activation occurs as a result of engagement of a T cell receptor complex or a functional portion thereof (e.g., CD3) and a costimulatory molecule (e.g., CD28) on the T cell by the major histocompatibility complex (MHC) and costimulatory molecules on antigen presenting cells, respectively.
  • MHC major histocompatibility complex
  • Induction of intracellular signaling cascades associated with T cell activation include activation of the P13K pathway, recruitment of PH-domain containing proteins (e.g., PDKI), and eventual cytokine production (e.g., IL-2).
  • Changes in expression of T cell surface markers occur as a result of activation, leading to increases in expression of one or more of CD69, CD71, CD25, CD 137, HLA- DR, CTLA-4, and others.
  • Production and secretion of cytokines, chemokines, and other proteins may also result from T cell activation.
  • the term “basal cultivation medium” refers to a culture medium containing a minimal set of ingredients that are essential for the survival of cells (e.g., T cells).
  • a “basal cultivation medium” typically is an aqueous solution including amino acids (e.g., L-racemers of glycine, arginine, asparagine, aspartate, cysteine, glutamine, histidine, hydroxyproline, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and/or valine), vitamins (e.g., biotin, choline chloride, D-calcium pantothenate, folate, niacinamide, paraaminobenzoic acid, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin Bl 2, and/or i-inositol,
  • amino acids e
  • basal cultivation medium examples include, without limitation, RPM] 1640, Eagle's Minimal Essential Medium (EMEM), Dulbecco 1 s Modified Eagle' s Medium (DMEM), Minimum Essential Medium Eagle (a-MEM), and Glasgow Minimal Essential Medium (Glasgow 1 s MEM), among others.
  • EMEM Eagle's Minimal Essential Medium
  • DMEM Dulbecco 1 s Modified Eagle' s Medium
  • a-MEM Minimum Essential Medium Eagle
  • Glasgow Minimal Essential Medium Glasgow Minimal Essential Medium
  • a "basal cultivation medium” does not include protein additives (e.g., cytokines, growth factors, and/or albumin).
  • a "basal cultivation medium” has a pH of between 7.0 and 7.4, such as, 7.0, 7.1, 7.2, 7.3, or 7.4.
  • a "basal cultivation medium” has an osmolarity of between 290 and 320 mOsmol (e.g., 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, or 320 mOsmol).
  • a "basal cultivation medium” is hypotonic, isotonic, or hypertonic.
  • chimeric antigen receptor or “CAR” refers to a chimeric receptor protein comprising an extracellular domain that has antigen-binding specificity, a transmembrane domain, and an intracellular signaling domain.
  • the extracellular domain can comprise an antigen-binding domain.
  • the transmembrane domain can comprise a transmembrane domain derived from a natural polypeptide obtained from a membrane-binding or transmembrane protein.
  • a transmembrane domain can include, without limitation, a transmembrane domain from a T cell receptor alpha or beta chain, a CD3 zeta chain, a CD28 polypeptide, or a CD8 polypeptide.
  • the intracellular domain can comprise a cytoplasmic signaling domain (e.g., any of the cytoplasmic signaling domains described herein) and one or more costimulatory domains (e.g., any of the exemplary co-stimulatory domains described herein).
  • the term “contact”, “contacting”, “contacted” and the like includes exposing one composition (e.g., a cell or a population of cells, such as T cells) to another composition (e.g., a polynucleotide) by any means such that they can be in direct interaction.
  • a composition e.g., a cell or a population of cells, such as T cells
  • another composition e.g., a polynucleotide
  • an exemplary method of contacting a population of cells with an agent is by mixing an aqueous suspension of the cells with an aqueous solution or suspension of the agent.
  • a substantial amount of the cells e.g., at least 1%, 5% 10% 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more of the cells in the population
  • a substantial amount of the cells e.g., at least 1%, 5% 10% 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or more of the cells in the population
  • cytotoxicity refers to the ability of a cell, such as a T cell engineered to express a chimeric antigen receptor (CAR) according to the methods disclosed herein, to cause cell death (e.g., apoptosis or necrosis) of another cell (e.g., a target cell).
  • a cell such as a T cell engineered to express a chimeric antigen receptor (CAR) according to the methods disclosed herein, to cause cell death (e.g., apoptosis or necrosis) of another cell (e.g., a target cell).
  • CAR chimeric antigen receptor
  • Binding between the CAR' s antigen-binding domain and the target antigen can, in certain embodiments, lead to T cell activation and killing of the target cell .
  • Assays for detecting cytotoxicity induced by CAR-T cells include, without limitation, a chromium release assay, bioluminescence assay (e.g., luciferase-mediated bioluminescence imaging), real-time impedance-based analysis, flow cytometry (e.g., in combination with a viability dye, such as CTV), and CFSE/PI assay.
  • a delivery vehicle refers to any pharmaceutical carrier, diluent, excipient, and the like, which are generally intended for use in connection with administration of biologically active agents, including nucleic acids.
  • a delivery vehicle may include lipid- or polymer-based transfer vehicles for the delivery of nucleic acids, including, but not limited to, a lipid nanoparticle, a liposome, polymer nanoparticle (nanocapsule or nanosphere), and the like.
  • a delivery vehicle is a lipid nanoparticle.
  • a "delivery vehicle” can also include any vector (e.g., viral or non-viral vector) capable of delivering the nucleic acid(s) to the target cell(s).
  • a delivery vehicle is a viral vector, such as a lentiviral vector.
  • the term "engineered,” when referencing a cell (e.g., T cell) that has been contacted with a nucleic acid encoding a heterologous protein (e.g., a CAR), means that the nucleic acid or a fragment thereof encoding the heterologous protein is stably integrated into the cell ' s genome after the contacting.
  • harvesting refers to isolation and/or collection of a cell or population of cells (e.g., T cells) following incubation of said cells under culture conditions.
  • harvesting includes change of one or more conditions, such as temperature, cell culture medium, and/or availability of certain agents (e.g., one or more agents that activate CD3 and/or CD28, one or more cytokines, and/or a polynucleotide encoding a heterologous protein) such that the step immediately prior to the harvesting step is discontinued.
  • agents e.g., one or more agents that activate CD3 and/or CD28, one or more cytokines, and/or a polynucleotide encoding a heterologous protein
  • heterologous refers to a nucleic acid or polypeptide sequence or domain which is not present in its native form or amount in its native environment.
  • a heterologous nucleic acid e g., gene
  • flanking sequences e.g., wherein the heterologous sequence is not found in nature coupled to the nucleic acid or polypeptide sequences occurring at one or both ends.
  • a heterologous protein is entirely absent from a native cell prior to being engineered to expresses the protein.
  • a heterologous protein is present with different post-translational modifications from a protein in a native cell prior to being engineered to expresses the protein. In some embodiments, a heterologous protein is present in a substantially lower amount than a protein in a native cell prior to being engineered to expresses the protein.
  • sample 1 refers to a biological sample, such as a blood sample (e.g., a whole blood sample), obtained from a subject (e.g., a human).
  • the sample is a blood sample processed by conventional methods to isolate a desired blood fraction (e.g., serum or plasma) or one or more cell types of interest (e.g., peripheral blood mononuclear cells (PBMCs), a lymphocyte, such as a T lymphocyte).
  • PBMCs peripheral blood mononuclear cells
  • a lymphocyte such as a T lymphocyte
  • a sample can refer to a leukapheresis sample obtained from the blood of a subject.
  • a “sample” refers to a whole blood sample obtained from a subject.
  • compositions suitable for use in conjunction with the disclosed methods are disclosed.
  • the methods disclosed herein provide certain advantages over prior CAR-T manufacturing methods, including production of more potent CAR-T cells as compared to CAR-T cells produced with longer manufacturing protocols, thereby facilitating the use of lower CAR-T cell dosages for therapeutic use.
  • the disclosed methods preserve T cell "sternness" (i.e., less differentiated phenotype), thereby producing CAR-T cells with higher potential for proliferation.
  • Shorter CAR-T manufacturing time resulting from the disclosed methods also facilitates scaling down of CAR-T cell manufacturing, resulting in reduced costs, reduced "needle-to-needle” time (i.e., time from harvesting of a patient's T cells to delivering autologous engineered T cells back to the patient), and improved patient access.
  • Chimeric antigen receptors are artificially constructed hybrid receptor proteins or polypeptides containing an antigen-binding domain, e.g., an antigen-binding fragment of an antibody which can take various formats such as a single chain variable fragment (scFv), linked to one or more intracellular signaling or activation domains (optionally including a costimulatory domain) via a transmembrane domain.
  • an antigen-binding domain e.g., an antigen-binding fragment of an antibody which can take various formats such as a single chain variable fragment (scFv), linked to one or more intracellular signaling or activation domains (optionally including a costimulatory domain) via a transmembrane domain.
  • scFv single chain variable fragment
  • intracellular signaling or activation domains optionally including a costimulatory domain
  • CARs provide one or more of the following benefits: targeting and destroying target antigen expressing cells, reducing or eliminating the target cells, facilitating infiltration of immune cells to a target tissue, and enhancing/extending anti-cancer responses.
  • CAR-T cells can also be used to reduce an autoimmune response by targeting cells (e.g., B cells) that mediates the autoimmunity.
  • CAR-T cell production has faced substantial obstacles, including, as is relevant to the scope of the present disclosure, long manufacturing times. Prolongation of the CAR-T cell manufacturing process can disadvantageously lead to batch loss, reduced expansion and persistence of CAR-T cells in vivo, increased batch-to-batch variability of the final cell product, increased differentiation and heterogeneity in the final cell product, increased manufacturing costs, and a prolongation in providing potentially life-saving therapy to patients. Accordingly, the present disclosure provides methods and compositions for the rapid of manufacture of CAR-T cells. It is understood that similar methods and compositions can be used for rapid manufacture of T cells that express other heterologous genes.
  • a population of T cells engineered to express a heterologous protein is disclosed.
  • the presently disclosed methods enable rapid manufacture of engineered T cells that express a heterologous protein, such as a chimeric antigen receptor (CAR), under specified conditions.
  • a heterologous protein such as a chimeric antigen receptor (CAR)
  • T cells are leukocytes that have completed maturation in the thymus, can identify certain foreign antigens, and perform various roles in the immune system, including activation and deactivation of other immune cells.
  • a T cell can be any T cell such as a cultured T cell, e.g., a primary T cell, or a T cell derived from a cultured T cell line, e.g., Jurkat, SupTl, etc., or a T cell obtained from a mammal.
  • T cells include, but are not limited to, naive T cells, stimulated T cells, primary T cells (e.g., uncultured), cultured T cells, immortalized T cells, helper T cells, cytotoxic T cells, memory T cells, regulatory T cells, natural killer T cells, combinations thereof, or sub-populations thereof.
  • the T cell can be a CD3+ cell.
  • T cells can be CD4+, CD8+, or CD4+ and CD8+.
  • a T cell can be a CD4+ / CD8+ double positive T cell, CD4 + helper T cell (e.g., Thl or Th2 cell), CD8+ T cell (e.g., a cytotoxic T cell), peripheral cell, including but not limited to a blood mononuclear cell (PBMC), peripheral blood leukocyte (PBL), tumor infdtrating lymphocyte (TIL), memory T cell, naive T cell, regulatory T cell, y8 T cell, etc.
  • PBMC blood mononuclear cell
  • PBL peripheral blood leukocyte
  • TIL tumor infdtrating lymphocyte
  • a T cell can be any T cell at any stage of development.
  • Additional types of helper T cells include Th3 (Treg) cells, Th 17 cells, Th9 cells, or Tfh cells.
  • T cells such as central memory T cells (Tcm cells), effector memory T cells (TEM cells and TEMRA cells).
  • T cell can also refer to a genetically modified T cell (e.g., an engineered T cell), such as a T cell that has been modified to express a heterologous protein, such as a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • T cells can also be differentiated from stem cells or progenitor cells.
  • the disclosed methods relate to the production of engineered T cells expressing a CAR having a binding specificity to a target antigen.
  • the disclosed methods are used, in certain embodiments, to produce a population of engineered T cells from a starting population of cells over a short course (e.g., 2, 3, or 4 days).
  • the methods of the disclosure are capable of producing a population of engineered T cells expressing a heterologous protein (e.g., a CAR) in about 48 hours (hrs), 49 hrs, 50 hrs, 51 hrs, 52 hrs, 53 hrs, 54 hrs, 55 hrs,
  • the methods allow for the production of engineered T cells (e.g., T cells expressing a CAR) in about 2-3 days.
  • engineered T cells e.g., T cells expressing a CAR
  • the disclosed methods include, in certain embodiments, steps for (1) optionally enriching/isolating a biological sample from a subject (e.g., a human subject) containing a starting population of T cells; (2) activating the starting population of T cells with an agent that binds CD3 and an agent that binds a costimulatory molecule (e.g., CD28/CD3); (3) contacting the T cells with a polynucleotide comprising a nucleic acid sequence encoding a heterologous protein (e.g., a CAR) and cultivating the T cells under conditions and for a brief time suitable to facilitate expression of the heterologous protein by the T cells; and (4) harvesting and, optionally, storing the engineered T cells for later use (e.g., therapeutic use or quality control testing).
  • a subject e.g., a human subject
  • FIG. 1 provides a schematic for methods of the disclosure used to produce CAR T cells from whole blood samples and/or enriched PBMCs. As will be described in greater detail, the steps of this workflow may include certain variations. One such variation may occur at the isolation and activation step. Certain preferred methods of the disclosure combine these steps in a single, concurrent isolation and activation step using a T cell activating agent (e.g., CD3/CD28 beads) to both isolate and activate the T cells.
  • a T cell activating agent e.g., CD3/CD28 beads
  • a biological sample such as a sample obtained from a subject (e.g., a human subject).
  • biological sample include cells, tissue (e.g., tissue obtained by biopsy), blood, serum, plasma, or any sample derived therefrom.
  • the sample is a whole blood sample obtained from the subject from which T cells are isolated without using an apheresis/leukapheresis step.
  • T cells may be isolated directly, in a single step from the whole blood and/or the whole blood undergoes a step of enriching peripheral blood mononuclear cells (PBMC) from which T cells are isolated.
  • PBMC peripheral blood mononuclear cells
  • T cells are isolated directly from whole blood without using an apheresis/leukapheresis step and without using a step to isolate or enrich PBMCs from which the T cells are obtained.
  • the presently disclosed methods are able to obtain a sufficiently pure and numerous T cell population directly from a small sample of whole blood without a series of intervening steps to isolate/enrich the T cells from the whole blood.
  • the method comprises obtaining the sample from the subject. In certain embodiments, the method comprises having obtained the sample from the subject.
  • the methods disclosed herein include obtaining a starting population of T cells from a biological sample obtained from a subject.
  • the biological sample is a leukapheresis sample.
  • the biological sample is a whole blood sample.
  • the starting population of T cells includes T helper (Th) cells, cytotoxic T (Tc) cells, memory T (TM) cells, regulatory T (Treg) cells, innate like T cells.
  • the Th cells include Thl cells, Th2 cells, Thl7 cells, Th9 cells, Tfh cells, and/or Th22 cells.
  • the TM cells include central memory T cells (TCM) cells, effector memory T (TEM) cells, tissue-resident memory T (TR ) cells, and virtual memory T (TVM) cells.
  • TCM central memory T cells
  • TEM effector memory T
  • TR tissue-resident memory T
  • TVM virtual memory T
  • the innate-like T cells include natural killer T (NKT) cells, mucosal -associated invariant T (MAIT) cells, and y5 T cells.
  • the method includes isolating the starting population of T cells from the sample.
  • Isolation of T cells may include an initial purification of T cells from a mixture of plasma, lymphocytes, platelets, red blood cells, monocytes, and granulocytes.
  • Methods for isolation of T cells from a biological sample such as a whole blood sample, enriched PBMC sample, or a leukapheresis sample, are well-known. Exemplary methods may include elutriation, density gradient centrifugation, enrichment by selection, and the like.
  • the method may include obtaining or having obtained a biological sample, such as a fresh, refrigerated, frozen, or cryopreserved product or alternative source of hematopoietic tissue, such as a whole blood sample, bone marrow sample, or a tumor or organ biopsy or removal (e.g., thymectomy) from an entity, such as a laboratory, hospital, or healthcare provider, and performing the aforementioned isolation steps to produce an enriched population of T cells (e.g., starting population of T cells) suitable for expression of a heterologous protein.
  • a biological sample such as a fresh, refrigerated, frozen, or cryopreserved product or alternative source of hematopoietic tissue, such as a whole blood sample, bone marrow sample, or a tumor or organ biopsy or removal (e.g., thymectomy) from an entity, such as a laboratory, hospital, or healthcare provider, and performing the aforementioned isolation steps to produce an enriched population of T cells (e.g., starting
  • the brief two-to-three-day manufacturing processes of the disclosure are able to produce CAR T cells directly from a whole blood sample and/or an enriched PBMC sample, without use of an initial leukapheresis.
  • methods for producing CAR T cells especially at sufficient throughput and speed, employed leukapheresis to create a concentrated sample of leukocytes from which T cells could be easily isolated.
  • the present Inventors have demonstrated that the presently disclosed methods are able to isolate a sufficient number of T cells directly from whole blood and/or whole blood enriched for PBMCs to produce CAR T cells. This not only avoids the costs and potential complications associated with leukapheresis, but also shortens the time required to produce the desired CAR T cells while expanding the potential availability of obtaining samples due to the reduced logistical complications required for a simple whole blood draw.
  • the purity of the starting population of T cells can be increased by using one or more selection steps, such as negative selection or positive selection.
  • Negative selection typically involves removal of undesired cell types from a mixed population of cells in a sample using one or more agents that selectively bind to the undesired cell type
  • positive selection typically involves isolation of the desired cell population using one or more agents that selectively bind to the desired cell type.
  • Enrichment of a T cell population by negative selection can be accomplished, for example, with a combination of antibodies directed to surface markers unique to the negatively selected cells.
  • One method is cell sorting and/or selection via negative magnetic immuno-adherence or flow cytometry that uses a cocktail of monoclonal antibodies directed to cell surface markers present on the negatively selected cells.
  • a monoclonal antibody cocktail can include antibodies to CD 14, CD20, CDb, CD 16, HLA-DR, and CDR
  • a positive selection step can be used to specifically select for the desired cell type, including in methods of the disclosure in which it may be used to directly isolate T cells from a whole blood or PBMC sample.
  • Positive selection of T cells can, in certain embodiments, include incubation of a mixed population of cells that contains the T cells (e.g., a whole blood/PBMC sample) with an agent having a CD3-binding moiety (e.g., anti-CD3 antibody- conjugated beads) for a time sufficient for positive selection of the desired T cells.
  • the time period is about 30 minutes. In certain embodiments, the time period ranges from 30 minutes to 36 hours or longer and all integer values there between.
  • the time period is at least 1, 2, 3, 4, 5, or 6 hours. In certain embodiments, the time period is 10 to 24 hours, for example, 18 hours. Longer incubation times may be used to isolate T cells in any context where there are few T cells as compared to other cell types.
  • the starting population of T cells comprise CD8+ T cells (e.g., CD8+ cytotoxic T cells).
  • the starting population of T cells further comprise CD4+ T cells (e.g., CD4+ helper T cells).
  • the starting population of T cells comprises 1-10% 1-20%, 1-30%, 1-40%, 1-50%, 1-60% 10-20% 10-30%, 10-40%, 10- 50%, 10-60%, 20-30%, 20-40%, 20-50%, 20-60%, 30-40%, 30-50%, or 30-60% of CD8+ T cells (e.g., CD8+ cytotoxic T cells) out of all T cells in the population.
  • the starting population of T cells further comprises 1-10%, 1-20%, 1-30%, 1-40%, 1-50%, 1-60%, 1- 70%, 10-20%, 10-30%, 10-40%, 10-50%, 10-60%, 10-70%, 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 30-40%, 30-50%, 30-60%, or 30-70% of CD4+ T cells (e.g., CD4+ helper T cells) out of all T cells in the population.
  • CD4+ T cells e.g., CD4+ helper T cells
  • the starting population of T cells comprise CD8+ T cells (e.g., CD8+ cytotoxic T cells) and CD4+ T cells (e.g., CD4+ helper T cells) at a ratio of 1 :5 to 5: 1, 1 :4 to 4: 1, 1 :3 to 3: 1, or 1 :2 to 2:1.
  • CD8+ T cells e.g., CD8+ cytotoxic T cells
  • CD4+ T cells e.g., CD4+ helper T cells
  • the starting population of T cells is produced to achieve a desired degree of purity.
  • the starting population of T cells may include T cells in an amount of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more, of the total number of cells in the population.
  • Purity of the starting population of T cells may be measured using routine methods, such as fluorescence assisted cell sorting (FACS), immuno-panning, microarray-based methods, and the like.
  • FACS fluorescence assisted cell sorting
  • Various known T cell phenotyping methods may also be applied to further increase purity of the starting population of T cells.
  • one or more freezing and thawing cycles can be performed on the starting population of T cells to enrich for the desired cell type.
  • freezing and thawing can improve the purity of a population of T cells by further removing granulocytes and, to some degree, monocytes in a mixed population of cells.
  • Routine and conventional methods for freezing and thawing T cells can be used in conjunction with the methods disclosed herewith.
  • following freezing the frozen cells are thawed, washed, and allowed to rest for, e.g., one hour, at room temperature prior to activation using the disclosed methods.
  • the starting population of T cells may be assayed for viability using known methods.
  • the starting population of T cells may be assayed using one or more known markers of T cell identity and a viability marker (e.g., dye, antibody, and the like), wherein overlap in a signal that indicates both T cell identity and viability is indicative of the viability of the starting population of T cells.
  • the starting population includes a percentage of viable T cells that is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% or more of the total number of T cells in the starting population of T cells.
  • the starting population of T cells may be assayed for exhaustion and/or activation status.
  • the starting population of T cells may be tested for exhaustion status using one or more (e.g., 1, 2, 3, or more) T cell exhaustion markers, including but not limited to overexpression of one or more of LAG-3, PD-1, PD-LI TIM-3, 2B4, CD160, TIGIT, CTLA-4, VISTA, and the like.
  • Activation status of T cells in the starting population can be assessed by testing for overexpression of one or more T cell activation markers (e.g., CD69, CD71, CD25, CD 137, HLA-DR, CTLA-4, L2RA/CD25, IFNy, TNFa, and the like). Additional indicators of T cell activation include, without limitation, T cell proliferation and differentiation.
  • T cell activation markers e.g., CD69, CD71, CD25, CD 137, HLA-DR, CTLA-4, L2RA/CD25, IFNy, TNFa, and the like. Additional indicators of T cell activation include, without limitation, T cell proliferation and differentiation.
  • T cell activation markers e.g., CD69, CD71, CD25, CD 137, HLA-DR, CTLA-4, L2RA/CD25, IFNy, TNFa, and the like. Additional indicators of T cell activation include, without limitation, T cell proliferation and differentiation.
  • the starting population of T cells may be incubated under culture conditions suitable
  • the T cells are concurrently activated when isolated, e.g., through the use of CD3/CD28 beads. In such instances, there is no period of resting prior to activation. However, transduction may occur after a brief culture following activation.
  • transduction occurs at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, at least 24 hours, at least 25 hours, at least 26 hours, at least 27 hours, at least 28 hours, at least 29 hours, at least 30 hours or more after activation.
  • the step of activation and/or contact with activating stimuli may occur for a period occurs of at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours, at least 16 hours, at least 17 hours, at least 18 hours, at least 19 hours, at least 20 hours, at least 21 hours, at least 22 hours, at least 23 hours, at least 24 hours, at least 25 hours, at least 26 hours, at least 27 hours, at least 28 hours, at least 29 hours, at least 30 hours, or more.
  • the time from isolation/activation to transduction is less than 30 hours, less than 25 hours, less than 24 hours, less than 23 hours, less than 22 hours, less than
  • the time from isolation/activation to transduction is about 12-24 hours and more preferably about 14-22 hours, and more preferably about 16-22 hours, and more preferably about 18 hours.
  • the starting population of T cells may be seeded at a desirable density that facilitates T cell transduction (and/or activation if required in certain methods) with a nucleic acid vector encoding a heterologous protein (e.g., a CAR).
  • a heterologous protein e.g., a CAR
  • the starting population of T cells may be seeded in culture at a concentration of IxlO 5 cells/mL to l x 10 7 cells/mL.
  • the starting population of T cells is seeded in culture at a concentration of about 1 X 10 6 cells/mL.
  • the starting population of T cells is seeded in culture at a concentration of l x 10 6 cells/mL.
  • the starting population of T cells is seeded in culture at a concentration of about 2 *10 6 cells/mL. In certain embodiments, the starting population of T cells is seeded in culture at a concentration of 10 6 cells/mL. In certain embodiments, the starting population of T cells is seeded in culture at a concentration of about 5 X 10 6 cells/mL. In certain embodiments, the starting population of T cells is seeded in culture at a concentration of 5xl0 6 cells/mL.
  • the number of isolated T cells from the whole blood sample is between about IxlO 6 and about IxlO 8 total T cells and the number of harvested CAR T cells is between about IxlO 8 and about 5xl0 8 .
  • the number of isolated T cells from the whole blood sample is between about 12.5xl0 7 and about IxlO 8 total CAR T cells and the number of harvested T cells is between about 7.5xl0 7 and about 1.5xl0 8 .
  • the number of isolated T cells from the whole blood sample is between about 5xl0 7 and about 7.5xl0 7 total T cells and the number of harvested CAR T cells is between about 7.5xl0 7 and about 1.2xl0 8 .
  • the starting population of T cells is incubated in a culture medium containing one or more cytokines selected from the group consisting of IL-2, IL-7, IL- 15, and IL-21.
  • the one or more cytokines is IL-2.
  • the one or more cytokines is IL-7 and IL-15.
  • the one or more cytokines is IL-2, IL-7, and IL-15.
  • the one or more cytokines is IL- 21.
  • the one or more cytokines is IL-21, IL-7, and IL-15.
  • the T cells are contacted with 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 ng/mL of IL-2, either alone or in combination with one or more other cytokines.
  • the T cells are contacted with 100 ng/mL of IL -2.
  • the T cells are contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng/mL of IL-7, either alone or in combination with one or more other cytokines.
  • the T cells are contacted with 10 ng/mL of IL-7. In certain embodiments, the T cells are contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng/mL of IL-15, either alone or in combination with one or more other cytokines. In certain embodiments, the T cells are contacted with 10 ng/mL of IL-15. In certain embodiments, the T cells are contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng/mL of IL-21, either alone or in combination with one or more other cytokines.
  • the starting population of T cells prior to activation, is incubated in the absence of any of the cytokines selected from IL-2, IL-7, IL-15, and IL-2L In certain embodiments, prior to activation, the starting population of T cells is incubated in a cytokine-free culture medium.
  • a population of engineered T cells expressing a heterologous protein that include a step for activating a starting population of T cells.
  • activation of the starting population of T cells includes contacting the starting population of T cells with an agent that stimulates a CD3/TCR complex and/or an agent that stimulates a costimulatory molecule on the surface of the T cells.
  • the costimulatory molecule is CD28, ICOS, CD27, HVEM, LIGHT, CD40, 4-1BB, 0X40, DR3, GITR, CD30, TIM1, CD2, CD226, or any combination thereof.
  • the agent that stimulates a CD3/TCR complex is an anti-CD3 antibody or an antigen-binding fragment thereof (e g., full-length IgG, Fab fragment, single domain antibody, scFv, diabody, triabody, and the like).
  • the agent that stimulates a CD3/TCR complex is small molecule or peptide ligand .
  • the costimulatory molecule is CD28.
  • the agent that stimulates a costimulatory molecule is an anti-CD28 antibody or an antigen-binding fragment thereof.
  • the agent that stimulates a CD3/TCR complex or the agent that stimulates a costimulatory molecule comprises a bead (e.g., a magnetic bead).
  • the agent that stimulates a CD3/TCR complex or the agent that stimulates a costimulatory molecule is a solid surface (e.g., bead) comprising an anti-CD3 antibody and/or an anti-CD28 antibody covalently attached thereto.
  • the agent that stimulates a CD3/TCR complex or the agent that stimulates a costimulatory molecule does not comprise a bead.
  • the agent that stimulates a CD3/TCR complex and the agent that stimulates a costimulatory molecule comprise a first agent that stimulates CD3 and a second agent that stimulates the costimulatory molecule.
  • the agent that stimulates a CD3/TCR complex and the agent that stimulates a costimulatory molecule are the same agent.
  • the agent that stimulates a CD3/TCR complex and the agent that stimulates a costimulatory molecule (e.g., CD28) is a bispecific antibody that specifically binds to CD3 and CD28.
  • the agent that stimulates a CD3/TCR complex and the agent that stimulates a costimulatory molecule is a bead (e.g., a magnetic bead) comprising anti-CD3 antibodies and anti-CD28 antibodies covalently attached thereto.
  • the agent that stimulates a CD3/TCR complex and the agent that stimulates a costimulatory molecule is a bead
  • the bead may remain attached with a T cell at the end of the activation step (i.e., the beginning of the transfection step), and the T cell is detached from the bead prior to harvest as a result of natural degradation of the protein part of the agent and/or through the use of a cleavable linker.
  • the stimulating agent e.g., anti-CD3 antibody
  • costimulatory molecule e.g., anti-CD28 antibody
  • the stimulating agent e.g., anti-CD3 antibody
  • costimulatory molecule e.g., anti-CD28 antibody
  • one or more of these activating agents may be selectively removed by providing the cells with a stimulus that cleaves the cleavable linker(s).
  • the stimulating agent(s) e.g., anti-CD3 antibodies
  • the costimulatory molecule(s) e.g., anti-CD28 antibodies
  • the anti-CD4 and anti-CD8 antibodies may likewise be surface attached using cleavable linkers.
  • the anti-CD4/CD8 binding may be stopped through linker cleavage, after which, for example, the cells may be contacted with an activating agent(s).
  • the one or more anti-CD3 antibodies and the one or more anti-CD28 antibodies attached to the same support.
  • the antibodies are bound to different supports, e.g., different beads.
  • one or more surface-bound anti-CD3 antibodies and/or the one or more anti-CD28 antibodies are used to concurrently isolate and activate T cells from a whole blood sample.
  • the anti-CD3 and anti-CD28 antibodies are attached to the support via a cleavable linker.
  • the linker may be, for example, an enzymatically cleavable, a hydrolysable linker, a redox cleavable linker, a phosphate-based cleavable linker, an acid cleavable linker, an ester-based cleavable linker, a peptide-based cleavable linker, a disulfide-based cleavable linker, a nitrobenzyl cleavable linker, a methoxymethyl-based cleavable linker and/or photocleavable linker. Accordingly, methods of the disclosure may further include contacting the activated T cells with a stimulus that cleaves the linker, thereby releasing the T cells from the surface.
  • the surface is a solid surface.
  • the solid surface is a bead, well, chip, or microfluidic channel. More preferably, the solid surface is a bead.
  • the surface comprises a polymer.
  • the polymer is a hydrogel.
  • the surface comprises a polymer scaffold.
  • the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule comprises an anti-CD3 antibody and/or an anti -costimulatory molecule antibody covalently attached to a colloidal polymeric matrix (e.g., nanomatrix).
  • the matrix comprises or consists of a polymeric, for example, biodegradable or biocompatible inert material, for example, which is non-toxic to cells.
  • the matrix is composed of hydrophilic polymer chains, which obtain maximal mobility in aqueous solution due to hydration of the chains.
  • the mobile matrix may be of collagen, purified proteins, purified peptides, polysaccharides, glycosaminoglycans, or extracellular matrix compositions.
  • a polysaccharide may include for example, cellulose ethers, starch, gum arabic, agarose, dextran, chitosan, hyaluronic acid, pectins, xanthan, guar gum or alginate.
  • polymers may include polyesters, polyethers, polyaciylates, polyacrylamides, polyamines, polyethylene imines, polyquatemium polymers, polyphosphazenes, polyvinylalcohols, polyvinylacetates, polyvinylpyrrolidones, block copolymers, or polyurethanes.
  • contacting of the starting population of T cells with an agent that stimulates a CD3/TCR complex and/or an agent that stimulates a costimulatory molecule is performed once at the start of the activation step.
  • contacting of the starting population of T cells with an agent that stimulates a CD3/TCR complex and/or an agent that stimulates a costimulatory molecule is performed concurrently with T cell isolation. In certain embodiments, contacting of the starting population of T cells with an agent that stimulates a CD3/TCR complex and/or an agent that stimulates a costimulatory molecule is performed once at the start of the activation step, and one or more (e.g., 1, 2, 3, or more) times throughout the duration of the activation step.
  • the duration of binding of the agent that stimulates a CD3/TCR complex and/or an agent that stimulates a costimulatory molecule to one or more cells of the starting population of T cells will depend on the specific agent(s) used, the concentration of the agent(s), the concentration of cells seeded in culture, among other factors.
  • the duration of the activation step is 12-24 hours (e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours). In certain embodiments, the duration of the activation step is about 18 hours (e.g., 16, 17, 18, 19, or 20 hours). In certain embodiments, the duration of the activation step is 18 hours.
  • the T cells after the activation step, the T cells remain associated with the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule on the surface of the T cells, such that activation may continue during the subsequent step(s). In certain embodiments, activation effectively continues until detachment of the T cells from the agent (e.g., by natural degradation of the protein part of the agent and/or application of a stimulus that cleaves a cleavable linker).
  • the starting population of T cells is incubated in a culture medium containing no more than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% serum.
  • the starting population of T cells is incubated in a culture medium 2% serum. In certain embodiments, during the activation step, the starting population of T cells is incubated in a cultivation medium comprising a basal cultivation medium and serum (e.g., 2% serum). In certain embodiments, the cultivation medium does not further comprise any added cytokine or growth factor, other than the proteins from the serum. In certain embodiments, the cultivation medium does not further comprise any added protein (e.g., soluble protein), other than the proteins from the serum.
  • the starting population of T cells is incubated in a culture medium containing one or more cytokines selected from the group consisting of IL-2, IL-7, IL-15, and IL-21.
  • the one or more cytokines is IL-2.
  • the one or more cytokines is IL-7 and IL-15.
  • the one or more cytokines is IL-2, IL-7, and IL-15.
  • the one or more cytokines is IL-2L
  • the one or more cytokines is IL-21, IL-7, and IL-15.
  • the T cells are contacted with 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 ng/mL of IL-2, either alone or in combination with one or more other cytokines.
  • the T cells are contacted with 100 ng/mL of IL-2. In certain embodiments, the T cells are contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng/mL of IL-7, either alone or in combination with one or more other cytokines. In certain embodiments, the T cells are contacted with 10 ng/mL of IL-7. In certain embodiments, the T cells are contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng/mL of IL- 15, either alone or in combination with one or more other cytokines. In certain embodiments, the T cells are contacted with 10 ng/mL of IL-15.
  • the T cells are contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng/mL of IL-21, either alone or in combination with one or more other cytokines.
  • the starting population of T cells prior to activation, is incubated in the absence of any of the cytokines selected from IL-2, IL-7, IL-15, and IL-21.
  • Either a serum-free cultivation medium (e.g., a basal cultivation medium) or a serum-supplemented cultivation medium can be provided in the presence or absence of the cytokines and combinations thereof disclosed herein.
  • the cultivation medium is a serum free cultivation medium and comprises at least one cytokine.
  • the at least one cytokine comprises one or more of IL- 2, IL-21, IL-7, and IL-15.
  • the at least one cytokine comprises one or more of IL-21, IL-7, and IL-15 and does not comprise IL-2.
  • the starting population of T cells prior to activation, is incubated in a cytokine-free culture medium or without added cytokine.
  • contacting of the starting population of T cells with the agent that stimulates a CD3/TCR complex and/or an agent that stimulates a costimulatory molecule (e.g., CD28) is performed simultaneously while contacting the population of cells with the one or more cytokines.
  • contacting of the starting population of T cells with the agent that stimulates a CD3/TCR complex and/or an agent that stimulates a costimulatory molecule is performed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 hrs or more prior to contacting with the one or more cytokines.
  • Transfection step delivery of heterologous nucleic acids to T cells
  • a heterologous protein e.g., a CAR
  • methods for producing an engineered population of T cells expressing a heterologous protein including delivering a nucleic acid encoding the heterologous protein to the T cells after the activation step.
  • polynucleotides encoding a heterologous protein e.g., a CAR
  • delivery of the nucleic acid(s) encoding a heterologous protein to the T cells is performed no later than about 18 hours (e.g., no later than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 hours or less) after initiating the activation step. In certain embodiments, delivery of the nucleic acid(s) encoding a heterologous protein to the T cells is performed about 18 hours (e.g., 16, 17, 18, 19, or 20) hours after initiating the activation step. In certain embodiments, delivery of the nucleic acid(s) encoding a heterologous protein to the T cells is performed 18 hours after initiating the activation step.
  • the cells at the end of the activation step are referred to herein as an "activated population of T cells," although at least a portion of the cells in the population may not have been fully activated and further activation can occur during the transfection step.
  • delivery of the nucleic acid(s) encoding a heterologous protein to a starting population of T cells is performed concurrently (e.g., simultaneously) with the activation step.
  • a polynucleotide encoding a heterologous protein includes a codon-optimized nucleic acid sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 35, 45, 55, 65, 75, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, or more) nucleotide differences as compared to the parent (i.e., non-codon optimized) nucleic acid.
  • the parent i.e., non-codon optimized
  • the nucleic acid sequence can be codon-optimized in accordance with various principles, for example, the principle that the frequency of occurrence of synonymous codons (e.g., codons that code for the same amino acid) in coding DNA is biased in different species.
  • Such codon degeneracy allows an identical polypeptide to be encoded by a variety of nucleotide sequences. This process may be performed on any of the sequences described in this specification to enhance expression or stability. Codon optimization may be performed using conventional methods.
  • the sequence surrounding the translational start site can be converted to a consensus Kozak sequence according to known methods.
  • an exogenous gene e.g., a polynucleotide encoding a heterologous polypeptide or a functional fragment thereof
  • stable expression of an exogenous gene in a mammalian cell can be achieved by integration of the polynucleotide containing the gene into the nuclear genome of the mammalian cell.
  • vectors for the delivery and integration of polynucleotides encoding exogenous proteins into the nuclear DNA of a mammalian cell have been developed.
  • Expression vectors for use in the compositions and methods described herein contain a polynucleotide sequence that encodes a heterologous protein (e.g., a CAR), as well as, e.g., additional sequence elements used for the expression of these agents and/or the integration of these polynucleotide sequences into the genome of a mammalian cell.
  • a heterologous protein e.g., a CAR
  • Certain vectors that can be used for the expression of a heterologous protein include plasmids that contain regulatory sequences, such as promoter and enhancer regions, which direct gene transcription.
  • Other useful vectors for expression of heterologous protein contain polynucleotide sequences that enhance the rate of translation of these genes or improve the stability or nuclear export of the mRNA that results from gene transcription.
  • sequence elements include, e.g., 5' and 3' untranslated regions, an internal ribosomal entry site ORES), and polyadenylation signal site in order to direct efficient transcription of the gene carried on the expression vector.
  • the expression vectors suitable for use with the compositions and methods described herein may also contain a polynucleotide encoding a marker for selection of cells that contain such a vector. Examples of a suitable marker are genes that encode resistance to antibiotics, such as ampicillin, chloramphenicol, kanamycin, nourseothricin.
  • Expression vectors for use in the compositions and methods described herein may express a heterologous protein (e.g., a CAR) from monocistronic or polycistronic expression cassettes.
  • a monoci stronic expression cassette contains a polynucleotide sequence that encodes a single gene.
  • Host cells described herein can be transfected with multiple vectors, for example, each containing a monocistronic expression cassette, or with a single vector containing more than one monocistronic expression cassette.
  • Polycistronic expression cassettes can be used to simultaneously express two or more proteins from a single transcript.
  • Polycistronic expression cassettes may include bicistronic or tri ci stronic expression cassettes, which can be used to generate two or three proteins, respectively, from a single transcript and may include IRES sequences to recruit ribosomes to initiate translation from a region of the mRNA other than the 5' cap.
  • foot-and-mouth disease virus 2A (FMDV 2A) polynucleotides can be utilized to express two or more genes (e.g., 2 genes, 3 genes, or more), and can be used in polycistronic expression cassettes to produce equimolar levels of multiple genes from the same transcript.
  • FMDV 2A mediates a co-translational cleavage event, which separates proteins linked by 2A sequences, and multiple 2A sequences may be used in one vector.
  • 2A-like sequences from other viruses can also be used in the compositions and methods described herein, including the 2A-like sequences from equine rhinitis A virus (E2A), porcine teschovirus-1 (P2A), and Thosea asigna virus (T2A).
  • Viral genomes provide a rich source of vectors that can be used for the efficient delivery of exogenous genes into a mammalian cell. Viral genomes are particularly useful vectors for gene delivery as the polynucleotides contained within such genomes are typically incorporated into the nuclear genome of a mammalian cell by generalized or specialized transduction. These processes occur as part of the natural viral replication cycle, and do not require added proteins or reagents in order to induce gene integration.
  • viral vectors examples include a retrovirus (e.g., Retroviridae family viral vector, such as a lentiviral vector), adenovirus (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvovirus (e.g., adeno-associated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g., measles and Sendai), positive strand RNA viruses, such as picomavirus and alphavirus, and double stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types I and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, modified vaccinia Ankara (MV A), fowlpox and
  • viruses include Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, human papilloma virus, human foamy virus, and hepatitis virus, for example.
  • retroviruses are: avian leukosissarcoma, avian C-type viruses, mammalian C-type, B-type viruses, D-type viruses, oncoretroviruses, HTLV-BLV group, alpharetrovirus, gammaretrovirus, and spumavirus.
  • murine leukemia viruses murine sarcoma viruses
  • mouse mammary tumor virus bovine leukemia virus
  • feline leukemia virus feline sarcoma virus
  • avian leukemia virus human T-cell leukemia virus
  • baboon endogenous virus Gibbon ape leukemia virus
  • Mason Pfizer monkey virus simian immunodeficiency virus
  • simian sarcoma virus Rous sarcoma virus and lenti viruses.
  • Exemplary lentiviral vectors that may be used in accordance with the present disclosure include vectors derived from human immunodeficiency virus-1 (HIV-1), human immunodeficiency virus-2 (HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), bovine immunodeficiency virus (BIV), Jembrana Disease Virus (JDV), equine infectious anemia virus (EIAV), and caprine arthritis encephalitis virus (CAEV).
  • HSV-1 human immunodeficiency virus-1
  • HV-2 human immunodeficiency virus-2
  • SIV simian immunodeficiency virus
  • FIV feline immunodeficiency virus
  • BIV bovine immunodeficiency virus
  • JDV Jembrana Disease Virus
  • EIAV equine infectious anemia virus
  • CAEV caprine arthritis encephalitis virus
  • Retroviral vectors typically are constructed such that the majority of sequences coding for the structural genes of the virus are deleted and replaced by a gene of interest or expression cassette of interest (e.g., an engineered nucleic acid as described here). Most often, the structural genes (i.e., gag, pol, and env), are removed from the retroviral backbone using genetic engineering techniques known in the art. This may include digestion with the appropriate restriction endonuclease or, in some instances, with Bal 31 exonuclease to generate fragments containing appropriate portions of the packaging signal.
  • a gene of interest or expression cassette of interest e.g., an engineered nucleic acid as described here.
  • the structural genes i.e., gag, pol, and env
  • This may include digestion with the appropriate restriction endonuclease or, in some instances, with Bal 31 exonuclease to generate fragments containing appropriate portions of the packaging signal.
  • a minimum retroviral vector comprises from 5' to 3' a 5' long terminal repeat (LTR), a packaging signal, an optional exogenous promoter and/or enhancer, an exogenous gene of interest (or engineered nucleic acid), and a 3' LTR.
  • LTR long terminal repeat
  • gene expression may be driven by the 5' LTR, which is a weak promoter and requires the presence of Tat to activate expression.
  • structural genes can be provided in separate vectors for manufacture of the lentivirus, rendering the produced virions replication-defective.
  • the packaging system may comprise a single packaging vector encoding the Gag, PO1, Rev, and Tat genes, and a third, separate vector encoding the envelope protein Env (usually VSV-G due to its wide infectivity).
  • the packaging vector can be split, expressing Rev from one vector, Gag and POI from another vector.
  • Tat can also be eliminated from the packaging system by using a retroviral vector comprising a chimeric 5' LTR, wherein the U3 region of the 5' LTR is replaced with a heterologous regulatory element.
  • Nucleic acids (e.g., genes) to be packaged into a retrovirus can be incorporated into the proviral backbone in several general ways.
  • the most straightforward constructions are ones in which the structural genes of the retrovirus are replaced by a single gene which then is transcribed under the control of the viral regulatory sequences within the LTR.
  • Retroviral vectors have also been constructed which can introduce more than one gene into target cells. Usually, in such vectors one gene is under the regulatory control of the viral LTR, while the second gene is expressed either off a spliced message or is under the regulation of its own, internal promoter.
  • nucleic acids e.g., genes
  • LTR long terminal repeat
  • the term "long terminal repeat” or “LTR” refers to domains of base pairs located at the ends of retroviral DNAs which, in their natural sequence context, are direct repeats and contain U3, R and U5 regions. LTRs generally provide functions fundamental to the expression of retroviral genes (e.g., promotion, initiation and polyadenylation of gene transcripts) and to viral replication.
  • the LTR contains numerous regulatory signals including transcriptional control elements, polyadenylation signals, and sequences needed for replication and integration of the viral genome.
  • the U3 region contains the enhancer and promoter elements.
  • the U5 region is the sequence between the primer binding site and the R region and contains the polyadenylation sequence.
  • the R (repeat) region is flanked by the U3 and U5 regions.
  • the R region comprises a trans-activation response (TAR) genetic element, which interacts with the trans-activator (tat) genetic element to enhance viral replication. This element is not required in embodiments wherein the U3 region of the 5' LTR is replaced by a heterologous promoter.
  • a retroviral vector comprises a modified 5' LTR and/or 3 ' LTR. Modifications of the 3 ' LTR are often made to improve the safety of lenti viral or retroviral systems by rendering viruses replication-defective.
  • a retroviral vector is a selfinactivating (SIN) vector.
  • SIN retroviral vector refers to a replication defective retroviral vector in which the 3 ' LTR U3 region has been modified (e.g., by deletion or substitution) to prevent viral transcription beyond the first round of viral replication.
  • a 3' LTR is modified such that the U5 region is replaced, for example, with an ideal polyadenylation sequence. It should be noted that modifications to the LTRs such as modifications to the 3' LTR, the 5' LTR, or both 3 ' and 5' LTRs, are also included in some embodiments of the present disclosure.
  • the U3 region of the 5' LTR is replaced with a heterologous promoter to drive transcription of the viral genome during production of viral particles.
  • heterologous promoters which can be used include, for example, viral simian virus 40 (SV40) (e.g., early or late), cytomegalovirus (CMV) (e.g., immediate early), Moloney murine leukemia virus (MoMLV), Rous sarcoma virus (RSV), and herpes simplex virus (HSV) [0195] (thymidine kinase) promoters. Typical promoters are able to drive high levels of transcription in a Tat-independent manner. This replacement reduces the possibility of recombination to generate replication-competent virus, because there is no complete U3 sequence in the virus production system.
  • SV40 viral simian virus 40
  • CMV cytomegalovirus
  • MoMLV Moloney murine leukemia virus
  • RSV Rous sarcoma virus
  • Adjacent to a 5' LTR are sequences necessary for reverse transcription of the genome (the tRNA primer binding site) and for efficient packaging of viral RNA into particles (the Psi site).
  • the term "packaging signal” or "packaging sequence” refers to sequences located within the retroviral genome which are required for encapsidation of retroviral RNA strands during viral particle formation (see e.g., Clever et al., 1995 J. Virology, 69(4):2101-09).
  • the packaging signal may be a minimal packaging signal (also referred to as the psi [yr] sequence) needed for encapsidation of the viral genome.
  • a retroviral vector (e.g., lentiviral vector) further comprises a FLAP
  • FLAP refers to a nucleic acid whose sequence includes the central polypurine tract and central termination sequences (CPP T and CTS) of a retrovirus, e.g., HIV-1 or HIV-2.
  • CCPP T and CTS central polypurine tract and central termination sequences
  • Suitable FLAP elements are described in U.S. Patent No. 6,682,907 and in Zennou el al. (2000) Cell 101 : 173.
  • central initiation of the plus-strand DNA at the cPPT and central termination at the CTS lead to the formation of a three-stranded DNA structure: a central DNA flap.
  • the DNA flap may act as a cis-active determinant of lentiviral genome nuclear import and/or may increase the titer of the virus.
  • retroviral vector backbones comprise one or more FLAP elements upstream or downstream of the heterologous genes of interest in the vectors.
  • a transfer plasmid includes a FLAP element.
  • a vector of the present disclosure comprises a FLAP element isolated from HIV-1.
  • a retroviral vector (e.g., lentiviral vector) further comprises an export element.
  • retroviral vectors comprise one or more export elements.
  • the term "export element" refers to a cis-acting post-transcriptional regulatory element which regulates the transport of an RNA transcript from the nucleus to the cytoplasm of a cell.
  • Examples of RNA export elements include, but are not limited to, the human immunodeficiency virus (HIV) RRE (see e.g., Cullen el al., (1991) J. Virol. 65: 1053; and Cullen et al., (1991) Cell 58: 423) and the hepatitis B virus post-transcriptional regulatory element (HPRE).
  • HIV human immunodeficiency virus
  • HPRE hepatitis B virus post-transcriptional regulatory element
  • RNA export element is placed within the 3' UTR of a gene, and can be inserted as one or multiple copies.
  • a retroviral vector (e.g., lentiviral vector) further comprises a posttranscriptional regulatory element.
  • posttranscriptional regulatory elements can increase expression of a heterologous nucleic acid, e.g., woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) (see Zufferey et al., (1999) J. Virol., 73:2886); the posttranscriptional regulatory element present in hepatitis B virus (HPRE) (Huang et al., Mol. Cell.
  • the posttranscriptional regulatory element is generally positioned at the 3' end the heterologous nucleic acid sequence. This configuration results in synthesis of an mRNA transcript whose 5' portion comprises the heterologous nucleic acid coding sequences and whose 3' portion comprises the posttranscriptional regulatory element sequence.
  • vectors of the present disclosure lack or do not comprise a posttranscriptional regulatory element such as a WPRE or HPRE, because in some instances these elements increase the risk of cellular transformation and/or do not substantially or significantly increase the amount of mRNA transcript or increase mRNA stability. Therefore, in certain embodiments, vectors of the present disclosure lack or do not comprise a WPRE or HPRE as an added safety measure.
  • a posttranscriptional regulatory element such as a WPRE or HPRE
  • a retroviral vector e.g., lentiviral vector
  • a retroviral vector further comprises a polyadenylation signal.
  • polyadenylation signal or "polyadenylation sequence” as used herein denotes a DNA sequence which directs both the termination and polyadenylation of the nascent RNA transcript by RNA polymerase H. Efficient polyadenylation of the recombinant transcript is desirable as transcripts lacking a polyadenylation signal are unstable and are rapidly degraded.
  • polyadenylation signals that can be used in a vector of the present disclosure, include an ideal polyadenylation sequence (e.g., AATAAA, ATTAAA AGTAAA), a bovine growth hormone polyadenylation sequence (BGHpA), a rabbit B-globin polyadenylation sequence (rBgpA), or another suitable heterologous or endogenous polyadenylation sequence known in the art.
  • an ideal polyadenylation sequence e.g., AATAAA, ATTAAA AGTAAA
  • BGHpA bovine growth hormone polyadenylation sequence
  • rBgpA rabbit B-globin polyadenylation sequence
  • a retroviral vector further comprises an insulator element.
  • Insulator elements may contribute to protecting retrovirus-expressed sequences, e.g., therapeutic genes, from integration site effects, which may be mediated by cis-acting elements present in genomic DNA and lead to deregulated expression of transferred sequences (i.e., position effect; see, e.g., Burgess-Beusse et al., (2002) Proc. Natl. Acad. Sci., USA, 99: 16433; and Zhan et al., 2001, Hum. Genet., 109:471).
  • a retroviral vector comprises an insulator element in one or both LTRs, or elsewhere in the region of the vector that integrates into the cellular genome.
  • Suitable insulators for use in the present disclosure include, but are not limited to, the chicken B-globin insulator (see Chung et al., (1993). Cell 74: 505; Chung et al., (1997) Proc. Natl. Acad, sci., USA 94:575; and Bell al., 1999. Cell 98:387).
  • Examples of insulator elements include, but are not limited to, an insulator from a B-globin locus, such as chicken HS4.
  • Non-limiting examples of lentiviral vectors include pLVX-EF I alpha-AcGFPl-C I (Clontech Catalog 1984), pLVX-EF lalpha-IRES-mCherry (Clontech Catalog 1987), pLVX-Puro (Clontech Catalog #632159), pLVX-IRES-Puro (Clontech Catalog #632186), pLenti6/V5- DESTTM (Thermo Fisher), pLenti6.2/V5-DESTTM (Thermo Fisher), pLKO.
  • lentiviral vectors can be modified to be suitable for therapeutic use.
  • a selection marker e.g., puro, EGFP, or mCherry
  • a second exogenous gene of interest e.g., puro, EGFP, or mCherry
  • lentiviral vectors are disclosed in U. S. Patent Nos. 7,629,153, 7,198,950, 8,329,462, 6,863,884, 6,682,907, 7,745,179, 7,250,299, 5,994,136, 6,287,814, 6,013,516, 6,797,512, 6,544,771, 5,834,256, 6,958,226, 6,207,455, 6,531,123, 5,352,694, and PCT Publication No. WO 2017/091786.
  • a nucleic acid vector such as a viral vector, encoding a heterologous protein disclosed herein (e.g., a CAR) is administered to a population of cells (e.g., a starting population of T cells) at a multiplicity of infection (MOI) of between 0 and 24 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24).
  • MOI multiplicity of infection
  • a nucleic acid vector encoding a heterologous protein is administered to a population of cells (e.g., a starting population of T cells) at a MOI of about 4.
  • a nucleic acid vector encoding a heterologous protein is administered to a population of cells (e.g., a starting population of T cells) at a MOI of about 5.
  • a nucleic acid vector encoding a heterologous protein is administered to a population of cells (e.g., a starting population of T cells) at a MOI of about 6.
  • a nucleic acid vector encoding a heterologous protein is administered to a population of cells (e.g., a starting population of T cells) at a MOI of about 7.
  • a nucleic acid vector encoding a heterologous protein is administered to a population of cells (e.g., a starting population of T cells) at a MOI of about 8.
  • a nucleic acid vector encoding a heterologous protein is administered to a population of cells (e.g., a starting population of T cells) at a MOI of about 9.
  • a nucleic acid vector encoding a heterologous protein is administered to a population of cells (e.g., a starting population of T cells) at a MOI of about 10.
  • a nucleic acid vector encoding a heterologous protein is administered to a population of cells (e.g., a starting population of T cells) at a MOI of about 12.
  • a nucleic acid vector encoding a heterologous protein is administered to a population of cells (e.g., a starting population of T cells) at a MOI of about 14.
  • transfer vehicles described herein encapsulate nucleic acids encoding the heterologous protein from degradation and provide for effective delivery of the nucleic acid(s) to target cells in vivo and in vitro.
  • contacting of the activated population of T cells with polynucleotides encoding a heterologous protein is performed in a culture medium containing no more than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% serum (e.g., human serum).
  • contacting of the activated population of T cells with polynucleotides encoding a heterologous protein is performed in a culture medium containing 2% serum.
  • the activated population of T cells is incubated in a cultivation medium comprising a basal cultivation medium and serum (e.g., 2% serum).
  • the basal cultivation medium is a serum-free medium.
  • the activated T cells are cultured in a serum-free cultivation medium for a period of between 24 hours and 60 hours.
  • the cultivating step is for a period of between 29 hours and 59 hours.
  • the cultivating step is for a period of between 36 hours and 52 hours.
  • the cultivating step is for a period of between 46 hours and 50 hours.
  • the cultivating step is for a period of about 48 hours.
  • Methods of the disclosure may further include harvesting the cultivated T cells, wherein the harvested T cells express the heterologous protein.
  • the cultivation medium does not further comprise any added cytokine or growth factor, other than the proteins from the serum.
  • the cultivation medium does not further comprise any added protein, other than the proteins from the serum.
  • contacting of the activated population of T cells with polynucleotides encoding a heterologous protein is performed in a serum-free culture medium.
  • contacting of the activated population of T cells with polynucleotides encoding a heterologous protein is performed in a basal cultivation medium not supplemented by serum.
  • the basal cultivation medium does not comprise any cytokine or growth factor.
  • the basal cultivation medium does not comprise any added protein.
  • the cells in the cultivation medium may secrete proteins.
  • the agent that stimulates a CD3/TCR complex and/or the agent that stimulates a costimulatory molecule may naturally degrade overtime, releasing fragments into the cultivation medium. None of these proteins are considered as “added proteins”.
  • contacting of the activated population of T cells with polynucleotides encoding a heterologous protein is performed in a culture medium containing one or more cytokines selected from the group consisting of IL-2, IL-7, IL- 15, and IL- 21.
  • the one or more cytokines is IL-2.
  • the one or more cytokines is IL-7 and IL-15.
  • the one or more cytokines is IL-2, IL-
  • the one or more cytokines is IL-21. In certain embodiments, the one or more cytokines is IL-21, IL-7, and IL-15. In certain embodiments, the T cells are contacted with 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 ng/mL of IL-2, either alone or in combination with one or more other cytokines. In certain embodiments, the T cells are contacted with 100 ng/mL of IL-2. In certain embodiments, the T cells are contacted with 1, 2, 3, 4, 5, 6, 7,
  • the T cells are contacted with 10 ng/mL of IL-7. In certain embodiments, the T cells are contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng/mL of IL-15, either alone or in combination with one or more other cytokines. In certain embodiments, the T cells are contacted with 10 ng/mL of IL-15.
  • the T cells are contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng/mL of IL-21, either alone or in combination with one or more other cytokines. In certain embodiments, the T cells are contacted with 20 ng/mL of IL-21, either alone or in combination with one or more other cytokines. In certain embodiments, the T cells are cultivated in the absence of any of the cytokines selected from IL-2, IL-7, IL- 15, and IL-21. Either a serum-free cultivation medium (e.g., a basal cultivation medium) or a serum supplemented cultivation medium can be provided in the transfection step, in the presence or absence of the cytokines and combinations thereof disclosed herein.
  • a serum-free cultivation medium e.g., a basal cultivation medium
  • a serum supplemented cultivation medium can be provided in the transfection step, in the presence or absence of the cytokines and combinations thereof disclosed herein.
  • contacting of the activated population of T cells with polynucleotides encoding a heterologous protein is performed in a cytokine-free culture medium or without added cytokine.
  • cytokines were added to the cultivation medium at the initiation of the previous step (activation step). It is understood that at the beginning of the transfection step, the effective concentration of the cytokines may have decreased as a result of degradation or have increased as a result of secretion from the cells (e.g., T cells) in the culture.
  • additional cytokines are added at the beginning of the transfection step (e.g., of the same kind and amount as added at the beginning of the activation step).
  • contacting of the activated population of T cells with a polynucleotide encoding a heterologous protein is performed simultaneously with contacting with the one or more cytokines.
  • contacting of the activated population of T cells with a polynucleotide encoding a heterologous protein is performed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 hours or more after contacting with the one or more cytokines.
  • contacting of the activated population of T cells with a polynucleotide encoding a heterologous protein is performed at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 hours or more prior to contacting with the one or more cytokines.
  • a heterologous protein e.g., a CAR
  • the culturing step produces between a 1.0- and a 4-fold expansion of the harvested T cells. In certain aspects, the culturing step produces between a 1.2- and a 4-fold expansion of the harvested T cells.
  • the cells When the T cells are contacted with polynucleotides encoding a heterologous protein of the disclosure (e.g., a CAR), the cells may be cultivated in culture under conditions and for a time sufficient to facilitate integration of the nucleic acid into the genome of the T cells for stable expression of the heterologous protein in the T cells, and/or attainment of a desired T cell phenotype (e.g., TNSCM cell phenotype).
  • a desired T cell phenotype e.g., TNSCM cell phenotype
  • the present Inventors have discovered that the two-to-three-day methods for manufacture of the disclosure obtain a very high CAR+ percentage after only a brief culture (generally about 48 hours).
  • the T cells contacted with polynucleotides encoding a heterologous protein of the disclosure are cultivated in culture between 24 and 72 hours (e.g., 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, or 72 hours).
  • 24 and 72 hours e.g., 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53
  • the T cells contacted with polynucleotides encoding a heterologous protein of the disclosure are cultivated in culture for about 48 hours (e.g., about 44, 45, 46, 47, 48, 49, 50, 51, hours 52 hours). In certain embodiments, the T cells contacted with polynucleotides encoding a heterologous protein of the disclosure (e.g., a CAR) are cultivated in culture for 48 hours.
  • the population of T cells may be harvested for storage and subsequent therapeutic use.
  • the T cells are harvested for storage no later than 120, 119, 118, 117, 116, 115, 114, 113, 112, 111, 110, 109, 108, 107, 106, 105, 104, 103, 102, 101, 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44,
  • the T cells are harvested for storage no later than about 64 (e.g., 62, 63, 64, 65, or 66) hours after initiation of the activation step. In certain embodiments, the T cells are harvested for storage no later than 64 hours after initiation of the activation step. In certain embodiments, the T cells are harvested for storage no later than about 72 (e.g., 70, 71, 72, 73, or 74) hours after initiation of the activation step. In certain embodiments, the T cells are harvested for storage no later than 72 hours after initiation of the activation step.
  • 64 e.g., 62, 63, 64, 65, or 66
  • the T cells are harvested for storage no later than 64 hours after initiation of the activation step.
  • the T cells are harvested for storage no later than about 72 (e.g., 70, 71, 72, 73, or 74) hours after initiation of the activation step. In certain embodiments, the T cells are harvested for storage no later than 72 hours after initiation of the activation
  • the T cells are harvested for storage no later than 72, 71, 70, 69, 68, 67, 66, 65, 64, 63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30 hours after completion of the activation step (i.e., the beginning of the transfection step).
  • the T cells are harvested for storage about 48 (e.g., 46, 47, 48, 49, or 50) hours after contacting the T cells with a polynucleotide encoding a heterologous protein (e.g., a CAR).
  • a heterologous protein e.g., a CAR
  • the harvesting step is accompanied by one or more assays intended to test for one or more parameters of cell viability, cell count, purity (e.g., fraction of T cells in the total population of cells), fraction of cells expressing the heterologous protein, T cell phenotype, T cell activation/exhaustion status, amount of proliferation of T cells relative to the starting population, T cell cytotoxicity, cytokine release, among others.
  • assays intended to test for one or more parameters of cell viability, cell count, purity (e.g., fraction of T cells in the total population of cells), fraction of cells expressing the heterologous protein, T cell phenotype, T cell activation/exhaustion status, amount of proliferation of T cells relative to the starting population, T cell cytotoxicity, cytokine release, among others.
  • the harvesting step may be followed by a storage step, whereby the T cells produced according to the disclosed method are maintained under conditions suitable to preserve the cells, including their viability, as well as functional and molecular profiles, until later therapeutic application or quality control testing.
  • the storage step comprises one or more of: (1) reformulating the population of cells in a storage medium (e.g., a refrigeration medium, freezing medium, or cry opreservation medium); (2) transfer of the cells to a suitable container means for storage under appropriate storage conditions; and (3) maintenance of the cells under suitable conditions.
  • a storage medium e.g., a refrigeration medium, freezing medium, or cry opreservation medium
  • the T cells are engineered (e.g., genetically manipulated) to express a heterologous protein, such as a CAR.
  • a heterologous protein such as a CAR.
  • an engineered T cell or a population of engineered T cells stably express a CAR, [0228] e g., by genomic integration of a heterologous nucleic acid sequence encoding the CAR in the T cell.
  • the brief two-to-three-day methods for manufacturing CAR T cells disclosed herein including those starting from whole blood and/or PBMC samples, produce a population of engineered T cells expressing a heterologous protein (e.g., a CAR) from a starting population of T cells at a very high percentage such that at 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more of the cells of the starting population of T cells express the heterologous protein (e.g., CAR).
  • a heterologous protein e.g., a CAR
  • expression of the heterologous protein is measured in the T cells 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days after the harvesting step. In certain embodiments, expression of the heterologous protein (e.g., CAR) is measured in the T cells 3 days after the harvesting step. In certain embodiments, expression of the heterologous protein (e.g., CAR) is measured in the T cells at least 3, 4, 5, 6, 7, 8, 9, 10, or more days after initiation of the transfection step, such that transient expression from unintegrated vector is not significantly detected.
  • Methods for quantifying expression of a heterologous protein at the genomic, transcriptomic, and proteomic levels in cells are well known in the art and include, without limitation, flow cytometry (e.g., fluorescence assisted cell sorting; FACS), quantitative (q)PCR, digital (d)PCR, fluorescence imaging, integration site analysis, RNA sequencing, in situ hybridization, immunoprecipitation, and Topanga assay, among others.
  • the present Inventors have discovered that methods of the disclosure for manufacturing CAR T cells produce cells exhibiting an improved CAR+ percentage relative to other, existing methods.
  • This improved CAR+ percentage generally greater than at least 50-60%, was obtainable from methods of the disclosure using: (i) whole blood as a starting sample; (ii) isolation of T cells directly from whole blood; (iii) concurrent T cell isolation and activation; (iv) a transduction step approximately 10-25 hours (preferably about 18 hours) after T cells isolation; and (v) a brief culture step after transduction (generally about 40-60 hours and preferably around 48 hours).
  • the methods disclosed herein produce a population of engineered T cells expressing a CAR that secretes increased amounts of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32) proteins selected from the group consisting of IFNg, Granzyme B, IL-113, IL -2, IL-4, IL-5, IL-6, IL-7, IL- 8, IL-9, IL- 10, IL- 12, IL-13, IL- 15, IL-17A, IL-17F, IL-21, IL-22, IP- 10, MCP1, MCP4, TNFa, TNF13, TGF13, GM-CSF, MIPla, MIPU3, CCL11, perform, RANTES, sCD137, and VEGF after being contacted with a target cell that expresses an antigen (e.g., CD 19) bound by the CAR, as compared to a T
  • an antigen e.
  • secretion of the one or more proteins after contact with the target cell is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500%, or higher as compared to secretion of the one or more proteins in the absence of the target cell or in the presence of a control target cell that does not express the antigen.
  • cytokine secretion is measured in the T cells 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days after the harvesting step.
  • Methods for quantifying cytokine release from T cells include, without limitation, ELISA, flow cytometry (e.g., cytometric bead array assay), and proteomic analysis (e.g., multiplexed single cell chip analysis), among others.
  • the short manufacturing time afforded by the presently disclosed methods produce CAR T cells with an improved polyfunctional phenotype upon contact with a target (e.g., CD 19).
  • the methods disclosed herein produce a population of engineered T cells expressing a CAR of which at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, or more are a population of polyfunctional CAR T cells that secrete increased amounts of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32) proteins selected from the group consisting of IFNg, Granzyme B, IL-113, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,
  • secretion of the one or more proteins after contact with the target cell is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500%, or higher as compared to secretion of the one or more proteins in the absence of the target cell or in the presence of a control target cell that does not express the antigen.
  • cytokine secretion is measured in the T cells 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days after the harvesting step.
  • the harvested T cells comprise a population of at least 6% of the harvested T cells that are polyfunctional T cells upon specific target-based activation.
  • the polyfunctional T cells and/or a portion of the population thereof simultaneously secrete Granzyme B and TNFb.
  • the polyfunctional T cells and/or a portion of the population thereof simultaneously secrete Granzyme B and IFNg.
  • at least 1% of the harvested T cells and/or a portion of the population thereof simultaneously secrete Granzyme B and IFNg.
  • the polyfunctional T cells and/or a portion of the population thereof simultaneously secrete MIP-la and MIP-lb.
  • the polyfunctional T cells and/or a portion of the population thereof simultaneously secrete IFNg and Granzyme B.
  • the polyfunctional T cells and/or a portion of the population thereof comprise two or more of cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete Granzyme B and TNFb; cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete Granzyme B and IFNg; cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete MIP- la and MIP-lb; and cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete IFNg and Granzyme B.
  • the polyfunctional T cells and/or a portion of the population of polyfunctional T cells comprise: cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete Granzyme B and TNFb; cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete Granzyme B and IFNg; cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete MIP- la and MIP-lb; and cells and/or a portion of the population of polyfunctional T cells that simultaneously secrete IFNg and Granzyme B.
  • the methods disclosed herein produce a population of engineered T cells expressing a CAR that exhibit increased expression of one or more T cell activation markers selected from the group consisting of CD69, CD25, and CD 137 after being contacted with a target cell that expresses an antigen (e.g., CD 19) bound by the CAR, as compared to expression of the one or more T cell activation markers in the absence of the target cell or in the presence of a control target cell that does not express the antigen.
  • an antigen e.g., CD 19
  • expression of the one or more T cell activation markers selected from the group consisting of CD69, CD25, and CD137 is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500%, or more, as compared to expression of the one or more T cell activation markers in the absence of the target cell or in the presence of a control target cell that does not express the antigen.
  • expression of the one or more T cell activation markers is measured 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days after the harvesting step.
  • Methods for quantifying expression of T cell activation markers are well known in the art and include, without limitation ELISA, flow cytometry, quantitative (q)PCR, digital (d)PCR, fluorescence imaging, in situ hybridization, and proteomic analysis, among others.
  • the methods disclosed herein produce a population of engineered T cells expressing a CAR that exhibit increased cytotoxicity against a target cell expressing an antigen (e.g., CD 19) bound by the CAR, as compared to cytotoxicity against a cell that does not express the antigen.
  • cytotoxicity is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500%, or more, as compared to cytotoxicity against a cell that does not express the antigen.
  • cytotoxicity of the engineered T cells against a target cell expressing an antigen is measured 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days after the harvesting step.
  • Methods of assessing cytotoxicity of engineered T cells expressing a heterologous protein (e.g., a CAR) against a target cell include, without limitation, chromium release assay, bioluminescence assay (e.g., luciferase-mediated bioluminescence imaging), real-time impedance-based analysis, flow cytometry (e.g., in combination with a viability dye, such as CTV), CFSE/PI assay, among others.
  • the methods disclosed herein produce a population of engineered T cells expressing a CAR that exhibit increased proliferation after being contacted with a target cell that expresses an antigen (e.g., CD 19) bound by the CAR, as compared to proliferation in the absence of the target cell or in the presence of a control target cell that does not express the antigen.
  • an antigen e.g., CD 19
  • proliferation is increased by at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, or more, as compared to proliferation in the absence of the target cell or in the presence of a control target cell that does not express the antigen.
  • proliferation of the T cells following contact with a target cell expressing an antigen is measured 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days after the harvesting step.
  • an antigen e.g., CD19
  • Methods of assessing proliferation of engineered T cells expressing a heterologous protein are well-known in the art and include, without limitation, MTT assay, MTS assay, cell counting (e.g., via flow cytometry), CFSE/flow cytometric analysis, and [3H] thymidine incorporation, among others.
  • the methods disclosed herein produce a population of engineered T cells various phenotypes, such as naive T (TN) cells characterized as CD45RO- CCR7+, and CD95-, central memory T (TCM) cells characterized as CD45RO+ and CCR7+, effector memory T (TEM) cells characterized as CD45RO+ and CCR7-, stem memory T (TSCM) cells characterized as CD45RO-, CCR7+, and CD95+, and effector memory cells re-expressing CD45RA T (TEMRA) cells characterized as CD45RO- and CCR7-.
  • TN naive T
  • TCM central memory T
  • TEM effector memory T
  • TSCM stem memory T
  • TEMRA effector memory cells re-expressing CD45RA T
  • T cell subsets include but are not limited to naive T (TN) cells characterized as CD45RA+, CCR7+, and CD95, central memory T (TCM) cells characterized as CD45RA- and CCR7+, effector memory T (TEM) cells characterized as CD45RA- and CCR7-, stem memory T (TSCM) cells characterized as CD45RA+, CCR7+, and CD95+, and effector memory cells re-expressing CD45RA T (TEMRA) cells characterized as CD45RA+ and CCR7-.
  • TN naive T
  • TCM central memory T
  • TEM effector memory T
  • TSCM stem memory T
  • the methods disclosed herein are able to produce a population of engineered T cells expressing a heterologous protein (e.g., a CAR) that include an increased amount of naive and stem cell memory T (TNSC ) cells (identified by markers such as CD45RA+/CD45RO-/CCR7+/CD62L+; CD45RA+/CCR7+; or CD45RO-/CCR7+) cells as compared to the starting population of T cells.
  • a heterologous protein e.g., a CAR
  • TNSC naive and stem cell memory T
  • the T cells produced according to the disclosed methods comprise TNSCM cells in an amount higher by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 2-fold, 2.5-fold, 3-fold, 3.5- fold, 4-fold, or more), as compared to the number of TNSCM cells in the starting population of T cells.
  • the T cells produced according to the disclosed methods comprise stem cell memory T (TSCM) cells (identified by markers such as CD45RA+/CD45RO- /CCR7+/CD62L+/CD95+; CD45RA+/CCR7+/CD95+; or CD45RO-/CCR7+/CD95+) in an amount higher by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, or more, as compared to the number of TSC cells in the starting population of T cells.
  • TSCM stem cell memory T
  • the T cells produced according to the disclosed methods comprise at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more of TNSC cells, out of all the T cells harvested. In certain embodiments, the T cells produced according to the disclosed methods comprise at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more of TSCM cells, out of all the T cells harvested. In certain embodiments, the amount of TNSCM cells in the engineered population of T cells is measured 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days after the harvesting step. Methods for quantifying TNSCM cells are well known in the art and include, without limitation, flow cytometry (e.g., FACS) and fluorescence microscopy, among others.
  • flow cytometry e.g., FACS
  • fluorescence microscopy among others.
  • the methods disclosed herein produce a population of engineered T cells expressing a heterologous protein (e g., a CAR) that include a decreased amount of effector memory (TEM) T cells (identified by markers such as D45RA-/CD45RO+/CCR7-/CD62L-; D45RA-/CCR7-; or CD45RO+/CCR7-) as compared to the staffing population of T cells.
  • a heterologous protein e e a CAR
  • TEM effector memory
  • the T cells produced according to the disclosed methods include TEM cells in an amount lower by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more, as compared to the number of TEM cells in the starting population of T cells.
  • the amount of TEM cells in the engineered population of T cells is measured 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days after the harvesting step.
  • Methods for quantifying TEM cells are well known in the art and include, without limitation, flow cytometry (e.g., FACS) and fluorescence microscopy, among others.
  • the heterologous protein is a chimeric antigen receptor (CAR).
  • the CAR comprises: (1) an extracellular domain containing an antigen-binding site that specifically binds to a target antigen, (2) a transmembrane domain; (3) an intracellular signaling domain; and, optionally, (4) a costimulatory domain.
  • a CAR disclosed herein further comprises a hinge region.
  • the CAR is a human CAR, comprising fully human sequences, e.g., naturally-occurring human sequences.
  • the extracellular domain is, in certain embodiments, linked to one or more intracellular signaling domains that, in certain embodiments, can mediate cell activation through an antigen receptor complex.
  • the transmembrane domain is linked to the extracellular domain.
  • the transmembrane domain that is naturally associated with one of the domains in the CAR is used.
  • the transmembrane domain is selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.
  • a CAR disclosed herein can include any number of amino acids, provided that the CAR retains its biological activity, e.g., the ability to specifically bind to antigen, mediate cytotoxic activity, detect diseased cells in a mammal, or treat or prevent disease in a mammal, etc.
  • the CAR includes 50 or more (e.g., 60 or more, 100 or more, or 500 or more) amino acids, but less than 1,000 (e.g., 900 or less, 800 or less, 700 or less, or 600 or less) amino acids.
  • the CAR is about 50 to about 700 amino acids (e.g., about 300 to about 1,000 amino acids (e.g., about 300 to about 800, about 300 to about 600, or about 400 to about 600 amino acids), or a range defined by any two of the foregoing values.
  • a CAR contains additional amino acids at the amino or carboxy terminus of the portion, or at both termini, which additional amino acids are not found in the amino acid sequence of the parent CAR.
  • the additional amino acids do not interfere with the biological function of the CAR, e.g., recognize target cells, mediate cytotoxic activity, treat or prevent a disease or disorder, etc. More desirably, the additional amino acids enhance the biological activity of the CAR, as compared to the biological activity of the parent CAR.
  • the CAR disclosed herein comprises an extracellular antigen binding domain comprising an antibody or an antigen-binding fragment thereof.
  • Anticalins or other alternative scaffolds are also contemplated.
  • the antigen binding domain of the CAR can be a whole antibody or an antibody fragment (e.g., scFv).
  • a whole antibody typically consists of four polypeptides: two identical copies of a heavy (H) chain polypeptide and two identical copies of a light (L) chain polypeptide.
  • Each of the heavy chains contains one N-terminal variable (VH) region and three C-terminal constant (CHI, CH2 and CH3) regions, and each light chain contains one N- terminal variable (VL) region and one C-terminal constant (CL) region.
  • variable regions of each pair of light and heavy chains form the antigen-binding site of an antibody.
  • the VH and VL regions have similar general structures, with each region including three complementarity determining regions (CDRs).
  • CDRs complementarity determining regions
  • the three CDRs known as CDR1, CDR2, and CDR3, form the "hypervariable region" of an antibody, which is responsible for antigen recognition and binding.
  • the three CDR regions are connected by four framework regions, whose sequences are relatively conserved.
  • the antigen-binding fragment of the antibody retains the ability of the antibody to specifically bind to its antigen.
  • the antibody fragment desirably includes, for example, one or more CDRs or the variable region (or portions thereof).
  • antibody fragments include, but are not limited to: (i) a Fab fragment, which is a monovalent fragment consisting of the VL, VH, CL, and CHI domains; (ii) a Fab' fragment, which is monovalent fragment consisting of the VL, VH, CL, CHI domains, and a disulfide bridge thiol (iii) a F(ab')2 fragment, which is a bivalent fragment including two Fab fragments linked by a disulfide bridge at the hinge region; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a single chain Fv (scFv), which is a monovalent molecule consisting of the two domains of the Fv
  • antibody or antigen-binding fragment thereof in the extracellular domain of the CAR can be obtained or derived from a mammal, including but not limited to, a mouse, a rat, or a human.
  • the antigen binding domain includes a variable region of a mouse or human monoclonal antibody or antigen-binding fragment thereof that binds to an antigen.
  • the antigen binding domain includes a light chain variable region, a heavy chain variable region, or both a light chain variable region and a heavy chain variable region of a mouse or human monoclonal antibody or antigen-binding fragment thereof that binds to an antigen.
  • an extracellular domain of a CAR disclosed herein includes a signal sequence.
  • the signal sequence may be positioned at the amino terminus of the antigen recognition domain (e.g., the variable region of the antibody or antigen-binding fragment thereof).
  • the signal sequence may include any suitable signal sequence.
  • the signal sequence is a human granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor signal sequence or a CD8a signal sequence.
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • a CAR including a murine scFv can include a GM-CSF signal sequence
  • a CAR including a human scFv can include a CD8a signal sequence.
  • N-terminal signal sequences are typically cleaved from the CAR protein after being expressed, but a nucleic acid encoding the CAR generally includes a sequence encoding the signal sequence.
  • the antigen-binding domain binds to a target antigen (e.g., a polypeptide). In some embodiments, the antigen-binding domain binds specifically to a target antigen (e.g., a polypeptide).
  • the CAR further includes a hinge or spacer between the antigen binding domain and the transmembrane domain.
  • the hinge or spacer may be or include at least a portion of an immunoglobulin constant region or variant or modified version thereof, such as a hinge region, e.g., a CD8a hinge, an IgG4 hinge region, and/or a CH1/CL and/or FC region.
  • the constant region or portion is of a human IgG, such as IgG4 or IgGl
  • the portion of the constant region serves as a spacer region between the antigen-recognition component, e.g., scFv, and transmembrane domain.
  • the spacer can be of a length that provides for increased responsiveness of the cell following antigen binding, as compared to in the absence of the spacer.
  • the spacer is at or about 12 amino acids in length or is no more than 12 amino acids in length.
  • Exemplary spacers include those having at least about 10 to 229 amino acids, about 10 to 200 amino acids, about 10 to 175 amino acids, about 10 to 150 amino acids, about 10 to 125 amino acids, about 10 to 100 amino acids, about 10 to 75 amino acids, about 10 to 50 amino acids, about 10 to 40 amino acids, about 10 to 30 amino acids, about 10 to 20 amino acids, or about 10 to 15 and including any integer between the endpoints of any of the listed ranges.
  • a spacer region has about 12 amino acids or less, about 119 amino acids or less, or about 229 amino acids or less.
  • Exemplary spacers include CD8a hinge, IgG4 hinge alone, IgG4 hinge linked to CH2 and CH3 domains, or IgG4 hinge linked to the CH3 domain.
  • the CAR hinge comprises a CD8a, truncated CD8a, or CD28 hinge domain.
  • the hinge region is a short sequence of amino acids that can facilitate structural flexibility between polypeptide domains, e.g., between an extracellular domain and a transmembrane domain (see, e.g., Woof et al., Nal. Rev. Immunol. 4(2):89-99 (2004)).
  • a hinge region may include all, or a portion of, an extracellular region of any suitable transmembrane protein (e.g., CD8a).
  • the hinge region is derived from a CD8a protein or a CD28 protein. In some embodiments, a hinge region is derived from a CD8a protein. In some embodiments, the hinge region is derived from a CD28 protein. In some embodiments, a hinge region is or comprises a hinge region or functional fragment thereof from a CD28 protein. In some embodiments, the hinge region is or comprises a hinge region or functional fragment thereof from a CD8a protein. In some embodiments, the hinge region is derived from a human CD8a protein or a human CD28 protein. In some embodiments, the hinge region is derived from a human CD8a protein. In some embodiments, the hinge region is derived from a human CD28 protein. In some embodiments, the hinge region is or comprises a hinge region or functional fragment thereof from a human CD28 protein. In some embodiments, the hinge region is or comprises a hinge region or functional fragment thereof from a human CD8a protein.
  • a hinge region comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 28.
  • a hinge region comprises an amino acid sequence as set forth in SEQ ID NO: 28.
  • a hinge region is derived from the same polypeptide as a transmembrane domain.
  • a hinge region and a transmembrane domain are derived from a CD8 polypeptide.
  • a hinge region and a transmembrane domain are derived from a CD8a polypeptide.
  • a hinge region and transmembrane domain comprise an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 19.
  • a hinge region and transmembrane domain comprise an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 19.
  • a hinge region and transmembrane domain comprise an amino acid sequence as set forth in SEQ ID NO: 19.
  • a hinge region and transmembrane domain are encoded by nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 20.
  • a hinge region and transmembrane domain are encoded by a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 20.
  • a hinge region and transmembrane domain are encoded by a nucleic acid sequence as set forth in SEQ ID NO: 20.
  • transmembrane domain operably connected to an extracellular domain and an intracellular signaling domain of the CAR.
  • the transmembrane domain can be any transmembrane domain derived or obtained from any molecule (e.g., type I transmembrane protein) known in the art.
  • the transmembrane domain of the CAR is derived from a natural source (e.g., a natural or wild-type polypeptide).
  • the transmembrane domain as used in accordance with the present disclosure, is derived from any suitable transmembrane protein or polypeptide known in the art.
  • a transmembrane domain is derived from a CD3 epsilon polypeptide, a CD4 polypeptide, a CD5 polypeptide, a CD8 polypeptide, a CD9 polypeptide, a CD 16 polypeptide, a CD22 polypeptide, a CD28 polypeptide, a CD33 polypeptide, a CD37 polypeptide, a CD45 polypeptide, a CD64 polypeptide, a CD80 polypeptide, a CD86 polypeptide, a CD 134 polypeptide, a CD137 polypeptide, a CD154 polypeptide, a T cell receptor alpha chain polypeptide, a T cell receptor beta chain polypeptide, a T cell receptor zeta chain polypeptide, or any derivatives thereof and/or combination thereof.
  • a transmembrane is or comprises a transmembrane domain or functional fragment thereof from a CD3 epsilon polypeptide, a CD4 polypeptide, a CD5 polypeptide, a CD8 polypeptide, a CD9 polypeptide, a CD 16 polypeptide, a CD22 polypeptide, a CD28 polypeptide, a CD33 polypeptide, a CD37 polypeptide, a CD45, polypeptide, a CD64 polypeptide, a CD80 polypeptide, a CD86 polypeptide, a CD 134 polypeptide, a CD 137 polypeptide, a CD 154 polypeptide, a T cell receptor alpha chain polypeptide, a T cell receptor beta chain polypeptide, a T cell receptor zeta chain polypeptide, or any combination thereof.
  • a transmembrane is synthetically derived, or engineered.
  • a synthetically derived or engineered transmembrane domain comprises predominantly hydrophobic residues (e.g., leucine, valine, etc.).
  • an engineered transmembrane domain is or comprises any engineered transmembrane domain known in the field.
  • the transmembrane domain is synthetic.
  • the synthetic transmembrane domain comprises predominantly hydrophobic residues such as leucine and valine.
  • a triplet of phenylalanine, tryptophan and valine will be found at each end of a synthetic transmembrane domain.
  • the linkage is by linkers, spacers, and/or transmembrane domain(s).
  • CD8 is a transmembrane glycoprotein that functions as a co-receptor for the T-cell receptor (TCR), and is expressed primarily on the surface of T cells, e g., cytotoxic T-cells.
  • a transmembrane domain is derived from a CD8a protein.
  • a transmembrane protein comprises an amino acid sequence having at 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 11.
  • a transmembrane protein comprises an amino acid sequence as set forth in SEQ LD NO: 11.
  • a CAR of the present disclosure comprises a CD28 transmembrane domain.
  • the transmembrane protein comprises an amino acid sequence having at least 770%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 12.
  • a transmembrane protein comprises an amino acid sequence as set forth in SEQ ID NO: 12.
  • the cytoplasmic domain or intracellular signaling domain of the receptor activates at least one of the normal effector functions or responses of the immune effector cell, e.g., T cell engineered to express the receptor.
  • the receptor induces a function of a T cell, such as cytolytic activity or T-helper activity, such as secretion of cytokines or other factors.
  • a truncated portion of an intracellular signaling domain of an antigen receptor component or costimulatory molecule is used in place of an intact immunostimulatory chain, for example, if it transduces the effector function signal.
  • the intracellular signaling domain or domains include the cytoplasmic sequences of the T cell receptor (TCR), and in some aspects also those of co-receptors that in the natural context act in concert with such receptor to initiate signal transduction following antigen receptor engagement, and/or any derivative or variant of such molecules, and/or any synthetic sequence that has the same functional capability.
  • TCR T cell receptor
  • the receptor includes a primary cytoplasmic signaling sequence that regulates primary activation of the TCR complex.
  • Primary cytoplasmic signaling sequences that act in a stimulatory manner may contain signaling motifs which are known as immunoreceptor tyrosine-based activation motifs (ITAMs).
  • ITAMs immunoreceptor tyrosine-based activation motifs
  • ITAM-containing primary cytoplasmic signaling sequences include those derived from TCR or CD3 zeta, FcR gamma, 4-1BB, B7-H3, CD2, CD27, CD28, CD30, CD40, FceRl (e.g., an FceRl gamma chain polypeptide), FcyRI, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD8, CD22, CD79a, CD79b, LIGHT, NKG2C, 0X40, PD-1, CD66d, or any derivatives or any combination thereof.
  • the receptor includes an intracellular component of a TCR complex. It is understood that the most common intracellular signaling domain used in CAR therapies is an intracellular signaling domain of CD3 zeta (CD3Q. CD3 zeta associates with T cell receptors to produce a signal and contains ITAMs.
  • intracellular signaling molecule(s) in the CAR contain(s) an intracellular signaling domain, portion thereof, or sequence derived from CD3 zeta.
  • the intracellular signaling domain comprises a CD3( ⁇ domain.
  • the intracellular signaling domain comprises a human CD3 zeta stimulatory signaling domain or functional fragment thereof, such as a 112AA cytoplasmic domain of isoform 3 of human CD3 zeta. (UniProt Accession No.: P20963.2).
  • an intracellular signaling domain comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 23.
  • an intracellular signaling domain comprises an amino acid sequence as set forth in SEQ ID NO: 23.
  • an intracellular signaling domain is encoded by nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 24.
  • an intracellular signaling domain is encoded by a nucleic acid sequence as set forth in SEQ ID NO: 24.
  • the intracellular signaling domain comprises at least one intracellular signaling domain or functional fragment thereof from a 4-1BB polypeptide, a B7H3 polypeptide, a CD2 polypeptide, a CD3 gamma polypeptide, a CD3 delta polypeptide, a CD3 zeta polypeptide, a CD7 polypeptide, a CD27 polypeptide, a CD28 polypeptide, a CD30 polypeptide, a CD40 polypeptide, an FCERI polypeptide (e.g., an FceRI gamma chain polypeptide), an FcyRI polypeptide, LIGHT polypeptide, NKG2C polypeptide, 0X40 polypeptide, PD-1 polypeptide, or any derivatives thereof or any combination thereof.
  • FCERI polypeptide e.g., an FceRI gamma chain polypeptide
  • FcyRI polypeptide LIGHT polypeptide
  • NKG2C polypeptide 0X40 polypeptide
  • the intracellular signaling domain comprises an intracellular signaling domain or functional fragment thereof from a CD3 zeta polypeptide. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain or functional fragment thereof from a CD28 polypeptide. In some embodiments, the intracellular signaling domain comprises an intracellular signaling domain or functional fragment thereof from a CD28 polypeptide and an intracellular signaling domain or functional fragment thereof from a CD3 zeta polypeptide. In some embodiments, the intracellular signaling domain comprises, from N-terminus to C-terminus, an intracellular signaling domain or functional fragment thereof from a CD28 polypeptide and an intracellular signaling domain or functional fragment thereof from a CD3 zeta polypeptide.
  • the intracellular signaling domain of the present disclosure comprises at least one signaling sequence from a 4-1BB polypeptide, a B7-H3 polypeptide, a CD2 polypeptide, a CD3 gamma polypeptide, a CD3 delta polypeptide, a CD3 zeta polypeptide, a CD7 polypeptide, a CD27 polypeptide, a CD28 polypeptide, a CD30 polypeptide, a CD40 polypeptide, an FceRI polypeptide (e.g., an FceRI gamma chain polypeptide), an FcyRI polypeptide, LIGHT polypeptide, NKG2C polypeptide, 0X40 polypeptide, PD-1 polypeptide, or any combination thereof.
  • an FceRI polypeptide e.g., an FceRI gamma chain polypeptide
  • FcyRI polypeptide LIGHT polypeptide
  • NKG2C polypeptide 0X40 polypeptide
  • PD-1 polypeptide or
  • the intracellular signaling domain comprises at least one signaling sequence from a CD3 zeta polypeptide. In some embodiments, the intracellular signaling domain comprises at least one signaling sequence from a CD28 polypeptide. In some embodiments, the intracellular signaling domain comprises at least one signaling sequence from a CD28 polypeptide and at least one signaling sequence from a CD3 zeta polypeptide. In some embodiments, the intracellular signaling domain comprises, from N-terminus to C-terminus, at least one signaling sequence from a CD28 polypeptide and at least one signaling sequence from a CD3 zeta polypeptide. Costimulatory domain
  • a CAR of the disclosure contains an intracellular domain of a T cell costimulatory molecule, e.g., positioned between the transmembrane domain and intracellular signaling domain.
  • the intracellular domain comprises an intracellular costimulatory signaling domain of CD28 or 4-1BB, or a functional variant or portion thereof, such as a 41 -amino acid cytoplasmic domain of a human CD28 (UniProt Accession No. Pl 0747.1) or a 42-amino acid cytoplasmic domain of a human 4-1BB (UniProt Accession No. Q07011.1) or functional variant or portion thereof.
  • the receptor encompasses one or more, e.g., two or more, costimulatory domains in addition to an activation domain, e.g., primary activation domain, in the cytoplasmic portion.
  • exemplary receptors include intracellular components of CD3-zeta and CD28, or intracellular components of CD3-zeta and 4-1BB.
  • the receptor includes a signaling domain and/or transmembrane portion of a costimulatory receptor, such as CD28, 4-1BB, 0X40, DAP10, and ICOS.
  • a costimulatory receptor such as CD28, 4-1BB, 0X40, DAP10, and ICOS.
  • the same receptor includes both the activating and costimulatory components.
  • the intracellular signaling domain comprises a CD8a transmembrane and signaling domain linked to a CD3 (e.g., CD3-zeta) intracellular domain, and further comprises a CD28 or 4- IBB (CD TNFRSF9) co-stimulatory domain, linked to a CD3 zeta intracellular domain.
  • a CD3 e.g., CD3-zeta
  • a CD28 or 4- IBB CD TNFRSF9 co-stimulatory domain
  • an intracellular signaling domain comprises a CD28 intracellular signaling domain. In some embodiments, an intracellular signaling domain comprises an intracellular signaling domain or a functional fragment thereof from a CD28 polypeptide. In some embodiments, a CD28 polypeptide intracellular signaling domain or functional fragment thereof comprises a co-stimulatory domain.
  • an intracellular signaling domain disclosed herein comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 21.
  • the intracellular signaling domain comprises an amino acid sequence as set forth in SEQ ID NO: 21.
  • an intracellular signaling domain is encoded by nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 22.
  • an intracellular signaling domain is encoded by a nucleic acid sequence as set forth in SEQ ID NO: 22.
  • a CAR of the present disclosure comprises an extracellular domain, a hinge domain, a transmembrane domain, and an intracellular signaling domain.
  • a CAR of the present disclosure comprises a signal peptide sequence (also referred to as a targeting signal, localization signal, localization sequence, leader sequence, or leader peptide), an extracellular domain, a hinge domain, a transmembrane domain, and an intracellular signaling domain.
  • a CAR of the present disclosure comprises, from N- terminus to C-terminus, an extracellular domain, a hinge domain, a transmembrane domain, and an intracellular signaling domain.
  • a CAR of the present disclosure comprises, from N-terminus to C-terminus, a signal peptide sequence, an extracellular domain, a hinge domain, a transmembrane domain, and an intracellular signaling domain.
  • the signal peptide sequence is cleaved from the CAR during or after its insertion into a membrane (e.g., ER membrane) during synthesis of the CAR protein.
  • domains or components e.g., extracellular domains, hinge regions, transmembrane domains, intracellular signaling domains, etc.
  • domains or components of a CAR are directly linked, or are contiguous. In some embodiments, domains or components of a CAR are not-directly linked, or are non-contiguous.
  • a CAR as described herein comprises an intracellular signaling domain, wherein the intracellular signaling domain comprises: (a) a CD3 zeta intracellular signaling domain or functional fragment thereof; and (b) at least one of a 4-1BB, an 0X40, or a CD28 intracellular signaling domain or functional fragment thereof.
  • a 4- 1BB intracellular signaling domain or functional fragment thereof, an 0X40 intracellular signaling domain, and/or a CD28 intracellular signaling domain or functional fragment thereof is or comprises a co-stimulatory domain.
  • a CAR of the present disclosure comprises: (a) a CD28 transmembrane domain; and (b) an intracellular signaling domain comprising: (i) a CD3 ⁇ intracellular signaling domain or functional fragment thereof; and (ii) a CD28 intracellular signaling domain or functional fragment thereof.
  • a CD28 intracellular signaling domain or functional fragment thereof is or comprises a CD28 co-stimulatory domain.
  • a CAR of the present disclosure comprises: (a) a CD8a transmembrane domain; (b) an intracellular signaling domain comprising: (i) a CD3 ⁇ intracellular signaling domain or functional fragment thereof; and (ii) a CD28, an FceRI gamma chain, and/or a 4-1BB intracellular signaling domain or functional fragment thereof.
  • a CAR of the present disclosure comprises: (a) a CD8a transmembrane domain; (b) an intracellular signaling domain comprising: (i) a CD3( ⁇ intracellular signaling domain or functional fragment thereof; and (ii) a CD28, an FceRI gamma chain, and a 4- IBB intracellular signaling domain or functional fragment thereof.
  • a CAR of the present disclosure comprises: (a) a CD8a transmembrane domain; (b) an intracellular signaling domain comprising: (i) a CD3( ⁇ intracellular signaling domain or functional fragment thereof; and (ii) an FceRI gamma chain intracellular signaling domain or functional fragment thereof.
  • a CAR of the present disclosure comprises: (a) a CD8a transmembrane domain; (b) an intracellular signaling domain comprising: (i) a CD3 ⁇ intracellular signaling domain or functional fragment thereof; and (ii) a 4- IBB intracellular signaling domain or functional fragment thereof.
  • a CD28 intracellular signaling domain or functional fragment thereof is or comprises a CD28 costimulatory domain.
  • a FceRI intracellular signaling domain or functional fragment thereof is or comprises a FceRI costimulatory domain.
  • a 4-1BB intracellular signaling domain or functional fragment thereof is or comprises a 4- IBB costimulatory domain.
  • a CAR of the present disclosure comprises (a) a CD8a transmembrane domain, and (b) an intracellular signaling domain comprising: (i) a CD3 ⁇ intracellular signaling domain or functional fragment thereof, and (ii) a CD27 and/or a CD28 intracellular signaling domain or functional fragment thereof.
  • a CD27 intracellular signaling domain or functional fragment thereof is or comprises a CD27 costimulatory domain.
  • a CD28 intracellular signaling domain or functional fragment thereof is or comprises a CD28 co-stimulatory domain.
  • a CAR of the present disclosure comprises (a) a CD28 transmembrane domain, and (b) an intracellular signaling domain comprising: (i) a CD3( ⁇ intracellular signaling domain or functional fragment thereof; and (ii) a CD27, a 4- IBB, and/or an FceRI gamma chain intracellular signaling domain or functional fragment thereof.
  • a CAR of the present disclosure comprises (a) a CD28 transmembrane domain, and (b) an intracellular signaling domain comprising: (i) a CD3 ⁇ intracellular signaling domain or functional fragment thereof; and (ii) a 4- IBB intracellular signaling domain or functional fragment thereof.
  • a CAR of the present disclosure comprises (a) a CD28 transmembrane domain, and (b) an intracellular signaling domain comprising: (i) a CD3 ⁇ intracellular signaling domain or functional fragment thereof; and (ii) an FceRI gamma chain intracellular signaling domain or functional fragment thereof.
  • a CD27 intracellular signaling domain or functional fragment thereof is or comprises a CD27 costimulatory domain.
  • an intracellular signaling domain or functional fragment thereof is or comprises a FceRI costimulatory domain.
  • a 4- IBB intracellular signaling domain or functional fragment thereof is or comprises a 4- IBB costimulatory domain.
  • the present disclosure further provides for CARs comprising an extracellular domain directed to any target molecule of interest (e.g., comprising any of known antigen-binding domain, e.g., antibody, scFv, etc.), and further comprising any transmembrane domain described herein (including any hinge domain described herein), any intracellular signaling domain described herein (including any signal sequences or motifs, any co-stimulatory domains, etc., described herein), present in any combination.
  • target molecule of interest e.g., comprising any of known antigen-binding domain, e.g., antibody, scFv, etc.
  • transmembrane domain described herein including any hinge domain described herein
  • any intracellular signaling domain described herein including any signal sequences or motifs, any co-stimulatory domains, etc., described herein
  • a CAR comprises: (a) a hinge region, (b) a transmembrane domain derived from a human CD8a polypeptide, (c) an intracellular signaling domain comprising: (i) a human CD3( ⁇ intracellular signaling domain or fragment thereof; and (ii) a human CD28 intracellular signaling domain or fragment thereof, wherein the CD28 intracellular signaling domain or fragment thereof is or comprises a co-stimulatory domain.
  • a CAR comprises: (a) a hinge region derived from a human CD8a polypeptide, (b) a transmembrane domain derived from a human CD8a polypeptide, (c) an intracellular signaling domain comprising: (i) a human CD3 intracellular signaling domain; and (ii) a human CD28 intracellular signaling domain.
  • a CAR comprises a sequence as set forth in SEQ ID NO: 27.
  • a CAR comprises: (a) a hinge region, (b) a transmembrane domain derived from a human CD8a polypeptide, (c) an intracellular signaling domain comprising: (i) a human CD3 ⁇ intracellular signaling domain or fragment thereof; and (ii) a CD27 and/or a CD28 intracellular signaling domain or fragment thereof, wherein the CD27 and/or CD28 intracellular signaling domain or fragment thereof is or comprises a co-stimulatory domain.
  • a CAR comprises: (a) a hinge region, (b) a transmembrane domain derived from a human CD8a polypeptide, (c) an intracellular signaling domain comprising: (i) a human CD3 ⁇ intracellular signaling domain or fragment thereof; and (ii) a human CD28, a human CD27, and/or an FceRl gamma chain intracellular signaling domain or fragment thereof, wherein the human CD28, the human CD27, and/or the FceRl gamma chain intracellular signaling domain or fragment thereof are or comprise a co-stimulatory domain.
  • a CAR can comprises: (a) a hinge region, (b) a transmembrane domain derived from a human CD8a polypeptide, (c) an intracellular signaling domain comprising: (i) a human CD3 ⁇ intracellular signaling domain; and (ii) a human CD28 and/or an FceRl gamma chain intracellular signaling domain, wherein the CD28 and/or the FceRl gamma chain intracellular signaling domain or fragment thereof are or comprise a co-stimulatory domain.
  • a CAR as described herein further comprises a signal peptide sequence.
  • a signal peptide is positioned at the amino terminus of an extracellular domain (e.g., at the N-terminus of an antigen-binding domain).
  • a signal peptide as used in accordance with the present disclosure may comprise any suitable signal peptide sequence.
  • a signal peptide sequence is a human granulocyte-macrophage colonystimulating factor (GM-CSF) receptor signal peptide sequence or a CD8a signal peptide sequence.
  • GM-CSF granulocyte-macrophage colonystimulating factor
  • a CAR provided herein comprises a human or humanized scFv comprising a CD8a signal peptide sequence.
  • a signal peptide sequence comprises an amino acid sequence as set forth in SEQ ID NO: 15.
  • a provided CAR comprises: (a) a CD8a hinge region comprising SEQ ID NO: 28, (b) a CD8a transmembrane domain comprising SEQ ID NO: 11, (c) a CD28 intracellular signaling domain comprising SEQ ID NO: 21, and (d) a CD3( ⁇ intracellular signaling domain comprising SEQ ID NO: 23.
  • a provided CAR comprises, from N- terminus to C-terminus: (a) a CD8a hinge region comprising SEQ ID NO: 28, (b) a CD8a transmembrane domain comprising SEQ ID NO: 11, (c) a CD28 intracellular signaling domain comprising SEQ ID NO: 21, and (d) a CD3 ⁇ intracellular signaling domain comprising SEQ ID NO: 23.
  • a provided CAR comprises: (a) an antigen-binding domain comprising SEQ ID NO: 17, (b) a CD8a hinge region comprising SEQ ID NO: 28, (c) a CD8a transmembrane domain comprising SEQ ID NO: 11, (d) a CD28 intracellular signaling domain comprising SEQ ID NO: 21, and (e) a CD3( ⁇ intracellular signaling domain comprising SEQ ID NO: 23.
  • a provided CAR comprises, from N-terminus to C-terminus: (a) an antigen-binding domain comprising SEQ ID NO: 17, (b) a CD8a hinge region comprising SEQ ID NO: 28, (c) a CD8a transmembrane domain comprising SEQ ID NO: 11, (d) a CD28 intracellular signaling domain comprising SEQ ID NO: 21, and (e) a CD3( ⁇ intracellular signaling domain comprising SEQ ID NO: 23.
  • a provided CAR comprises: (a) a CD8a signal peptide sequence comprising SEQ ID NO: 15, (b) an antigen-binding domain comprising SEQ ID NO: 17, (c) a CD8a hinge region as set forth in SEQ ID NO: 28, (d) a CD8a transmembrane domain as set forth in SEQ ID NO: 11, (e) a CD28 intracellular signaling domain as set forth in SEQ ID NO: 21, and (f) a CD3( ⁇ intracellular signaling domain as set forth in SEQ ID NO: 23.
  • a provided CAR comprises, from N-terminus to C-terminus: (a) a CD8a signal peptide sequence comprising SEQ ID NO: 15, (b) an antigen-binding domain comprising SEQ ID NO: 17, (c) a CD8a hinge region as set forth in SEQ ID NO: 28, (d) a CD8a transmembrane domain as set forth in SEQ ID NO: 11, (e) a CD28 intracellular signaling domain as set forth in SEQ ID NO: 21, and (I) a CD3( ⁇ intracellular signaling domain as set forth in SEQ ID NO: 23.
  • a CAR of the present disclosure comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 10. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 10. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 10. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 10.
  • a CAR of the present disclosure comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 10. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO 10. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 10. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 10. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 10. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence as set forth in SEQ ID NO: 10.
  • a CAR of the present disclosure comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, a least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 13.
  • a CAR of the present disclosure comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 13.
  • a CAR of the present disclosure comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 13.
  • a CAR of the present disclosure comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 13.
  • a CAR of the present disclosure comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 13. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 96% sequence identity to SEQ ID NO: 13. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 97% sequence identity to SEQ ID NO: 13. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 98% sequence identity to SEQ ID NO: 13. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence having at least 99% sequence identity to SEQ ID NO: 13. In some embodiments, a CAR of the present disclosure comprises an amino acid sequence as set forth in SEQ ID NO: 13.
  • a CAR of the present disclosure is encoded by nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater sequence identity to SEQ ID NO: 14. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence having at least 80% sequence identity to SEQ ID NO: 14. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence having at least 85% sequence identity to SEQ ID NO 14.
  • a CAR of the present disclosure is encoded by a nucleic acid sequence having at least 90% sequence identity to SEQ ID NO: 14 In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence having at least 95% sequence identity to SEQ ID NO: 14. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence having at least 96% sequence identity to SEQ ID NO: 14. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence having at least 97% sequence identity to SEQ ID NO: 14.
  • a CAR of the present disclosure is encoded by a nucleic acid sequence having at least 98 0 0 sequence identity to SEQ ID NO: 14. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence having at least 99% sequence identity to SEQ ID NO: 14. In some embodiments, a CAR of the present disclosure is encoded by a nucleic acid sequence as set forth in SEQ ID NO: 14.
  • the CAR may comprise one or modified synthetic amino acids in place of one or more naturally-occurring amino acids.
  • modified amino acids include, but are not limited to, aminocyclohexane carboxylic acid, norleucine, a-amino n-decanoic acid, homoserine, Sacetylaminomethylcysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4nitrophenyl alanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, (3-phenylserine (3- hydroxyphenylalanine, phenylglycine, a-naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, l,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzylic acid,
  • the CAR (including functional portions and functional variants thereof) can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized via, e.g., a disulfide bridge, or converted into an acid addition salt and/or optionally dimerized or polymerized, or conjugated.
  • the engineered T cells disclosed herein can be incorporated into a pharmaceutical composition.
  • These compositions can comprise, in addition to the engineered T cells disclosed herein, a pharmaceutically acceptable excipient, carrier, buffer, stabilizer, or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • the precise nature of the carrier or other material can depend on the intended route of administration, e g., intravenous, cutaneous, or subcutaneous, nasal, intramuscular, intraperitoneal routes.
  • the pharmaceutical composition may comprise one or more pharmaceutical excipients. Any suitable pharmaceutical excipient may be used, and one of ordinary skill in the art is capable of selecting suitable pharmaceutical excipients.
  • the pharmaceutical excipients provided below are intended to be illustrative, and not limiting. Additional pharmaceutical excipients include, for example, those described in the Handbook of Pharmaceutical Excipients, Rowe et al. (Eds.) 6th Ed. (2009), incorporated by reference in its entirety.
  • kits that include one or more doses of a population of engineered T cells (e.g., CAR-T cells) produced or obtainable according to the methods disclosed herein in a suitable container means.
  • the kit comprises a container means comprising the engineered T cells described herein.
  • the container means is any suitable container which houses, e.g., a liquid or lyophilized composition including, but not limited to, a vial, syringe, bottle, and an intravenous (IV) bag or ampoule.
  • a syringe holds any volume of liquid suitable for injection into a subject, including, but not limited to, 0.5 cc, 1 cc, 2 cc, 5 cc, 10 cc, or more.
  • packages and kits include a label specifying information required by US FDA or similar regulatory authority, e.g., a product description, amount and mode of administration, and/or indication of treatment.
  • packages provided herein include any of the compositions as described herein.
  • packages and kits additionally include a buffering agent, a preservative, and/or a stabilizing agent in a pharmaceutical formulation.
  • each component of the kit is enclosed within an individual container and all of the various containers are within a single package.
  • disclosure kits are designed for cold storage or room temperature storage.
  • the preparations contain stabilizers to increase the shelf-life of the kits and include, e.g., bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • the kit contains, in certain embodiments, further preparations of solutions to reconstitute the lyophilized preparations.
  • Acceptable reconstitution solutions are well known in the art and include, e.g., pharmaceutically acceptable phosphate buffered saline (PBS).
  • a kit includes a compound in a pack or dispenser together with instructions for administering the compound in a method described herein.
  • the disclosure provides a method of manufacturing a population of engineered T cells expressing a heterologous protein, the method comprising, in the following order: (a) contacting a starting population of T cells with one or more agents that activate CD3 and CD28; (b) contacting the T cells with a polynucleotide comprising a nucleic acid sequence that encodes the heterologous protein, and cultivating the T cells in a serum-free cultivation medium comprising an interleukin-2 (IL-2) protein for 29-71 hours; and (c) harvesting the T cells; optionally wherein step (b) is performed at least 1 hour after initiation of step (a); and wherein the harvested T cells comprise one or more T cells engineered to express the heterologous protein.
  • IL-2 interleukin-2
  • the disclosure provides a method of manufacturing a population of engineered T cells expressing a heterologous protein, the method comprising, in the following order:(a) contacting a starting population of T cells with one or more agents that activate CD3 and CD28; (b) contacting the T cells with a polynucleotide comprising a nucleic acid sequence that encodes the heterologous protein, and cultivating the T cells in a cultivation medium comprising interleukin 7 (IL-7) and interleukin- 15 (IL- 15) proteins for 31-71 hours; and (c) harvesting the T cells; wherein the harvested T cells comprise one or more T cells engineered to express the heterologous protein.
  • IL-7 interleukin 7
  • IL- 15 interleukin- 15
  • the disclosure provides a method of manufacturing a population of engineered T cells expressing a heterologous protein, the method comprising, in the following order: (a) contacting a starting population of T cells with one or more agents that activate CD3 and CD28; (b) contacting the T cells with a polynucleotide comprising a nucleic acid sequence that encodes the heterologous protein, and (c) cultivating the T cells in a serum-free cultivation medium comprising interleukin-7 (IL-7) and/or interleukin- 15 (IL-15) proteins for 30-60 hours; and (c) harvesting the T cells; wherein the harvested T cells comprise one or more T cells engineered to express the heterologous protein.
  • IL-7 interleukin-7
  • IL-15 interleukin- 15
  • the disclosure provides a method of manufacturing a population of engineered T cells expressing a heterologous protein, the method comprising, in the following order: (a) contacting a starting population of T cells with one or more agents that activate CD3 and CD28 in the absence of a cytokine; (b) contacting the T cells with a polynucleotide comprising a nucleic acid sequence that encodes the heterologous protein, and cultivating the T cells in a cultivation medium comprising at least one cytokine for 24-72 hours; and (c) harvesting the T cells; wherein step (b) is performed about 18 hours after step (a); and wherein the harvested T cells comprise one or more T cells engineered to express the heterologous protein.
  • the at least one cytokine in step (c) comprises one or more (e.g., 1, 2, 3, or 4) of IL-2, IL-7, IL-15, and/or IL- 21.
  • the at least one cytokine in step (c) comprises IL-2.
  • the at least one cytokine in step (c) comprises IL-21.
  • the at least one cytokine in step (c) comprises IL-7 and IL-15.
  • the at least one cytokine in step (c) comprises IL-2, IL-7, and IL-15.
  • the at least one cytokine in step (c) comprises IL-21, IL-7, and IL-15.
  • the disclosure provides a method of manufacturing a population of engineered T cells expressing a heterologous protein, the method comprising, in the following order: (a) contacting a starting population of T cells with one or more agents that activate CD3 and CD28; (b) contacting the T cells with a polynucleotide comprising a nucleic acid sequence that encodes the heterologous protein, and cultivating the T cells in a cultivation medium comprising interleukin-21 (IL-21) protein for 31-71 hours; and (c) harvesting the T cells; wherein the harvested T cells comprise one or more T cells engineered to express the heterologous protein.
  • the cultivation medium further comprises IL-7 and/or IL- 15.
  • the disclosure provides a method of manufacturing a population of engineered T cells expressing a heterologous protein, the method comprising, in the following order: (a) contacting a starting population of T cells with one or more agents that activate CD3 and CD28;
  • step (c) harvesting the T cells; wherein the cultivation medium does not comprise IL-2, IL-7, IL- 15, or IL-21; and wherein the harvested T cells comprise one or more T cells engineered to express the heterologous protein.
  • the cultivation medium is cytokine free.
  • step (a) does not comprise use of IL-2, IL-7, IL- 15, or IL-21.
  • step (a) is performed in the absence of a cytokine.
  • step (c) does not comprise use of IL-2, IL-7, IL-15, or IL-2L
  • step (c) is performed in the absence of a cytokine.
  • the method is performed in the absence of a cytokine.
  • the cultivation medium is a basal cultivation medium.
  • the cultivation medium comprises serum.
  • the cultivation medium does not further comprise an added cytokine or growth factor.
  • the cultivation medium is serum-free.
  • the starting population of T cells is seeded in culture at a concentration of about lx 10 6 cells per mL. In certain embodiments, the starting population of T cells is seeded in culture at a concentration of about 2xl0 6 cells per mL. In certain embodiments, the starting population of T cells is seeded in culture at a concentration of about 5xl0 6 cells per mL. In certain embodiments, (a) the starting population of T cells is seeded in culture at a concentration of about IxlO 6 cells per mL; and (b) the cultivation medium comprises IL -2.
  • the starting population of T cells is seeded in culture at a concentration of about IxlO 6 cells per mL; and (b) the cultivation medium comprises IL-7 and IL-15. In certain embodiments, (a) the starting population of T cells is seeded in culture at a concentration of about IxlO 6 cells per mL; and (b) the cultivation medium comprises IL-7, IL- 15, and IL-21. In certain embodiments, (a) the starting population of T cells is seeded in culture at a concentration of about IxlO 6 cells per mL; and (b) the cultivation medium comprises IL-21.
  • the starting population of T cells is seeded in culture at a concentration of about IxlO 6 cells per mL; and (b) the cultivation medium does not comprise IL-2, IL-7, IL-15, or IL-21 (i.e., does not comprise any one of IL-2, IL-7, IL-15, and IL-21).
  • the starting population of T cells is seeded in culture at a concentration of about IxlO 6 cells per mL; and (b) the cultivation medium comprises IL -2.
  • the starting population of T cells is seeded in culture at a concentration of about 2xl0 6 cells per mL; and (b) the cultivation medium comprises IL-7 and IL-15. In certain embodiments, (a) the starting population of T cells is seeded in culture at a concentration of about 2xl0 6 cells per mL; and (b) the cultivation medium comprises IL-7, IL- 15, and IL-21. In certain embodiments, (a) the starting population of T cells is seeded in culture at a concentration of about 2xl0 6 cells per mL; and (b) the cultivation medium comprises IL-21.
  • the starting population of T cells is seeded in culture at a concentration of about 2xl0 6 cells per mL; and (b) the cultivation medium does not comprise IL-2, IL-7, IL-15, or IL-21 (i.e., does not comprise any one of IL-2, IL-7, IL-15, and IL-21).
  • the starting population of T cells is seeded in culture at a concentration of about 5xl0 6 cells per mL; and (b) the cultivation medium comprises IL-2.
  • the starting population of T cells is seeded in culture at a concentration of about 5xl0 6 cells per mb; and (b) the cultivation medium comprises IL-7 and IL-15. In certain embodiments, (a) the starting population of T cells is seeded in culture at a concentration of about 5xl0 6 cells per mL; and (b) the cultivation medium comprises IL-7, IL-15, and IL-21. In certain embodiments, (a) the starting population of T cells is seeded in culture at a concentration of about 5xl0 6 cells per mL; and (b) the cultivation medium comprises IL-21.
  • the starting population of T cells is seeded in culture at a concentration of about 5xl0 6 cells per mL; and (b) the cultivation medium does not comprise IL-2, IL-7, IL-15, or IL-21 (i.e., does not comprise any one of IL-2, IL-7, IL-15, and IL -21).
  • the method further comprises (d) maintaining the T cells harvested in step (c) at a temperature of no greater than 38° C (e.g., no greater than 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9° C, or less), between 2-8° C (e.g., 2, 3, 4, 5, 6, 7, or 8° C), or no greater than -80° C (e.g., no greater than -80, - 81, -82, -83, -84, -85, -86, -87, -88, -89, -90, -91, -92, -93, -94, -95, -96, -97, -98, -99, -100, or less).
  • 38° C e.g., no greater than 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14,
  • the one or more agents that activate CD3 and/or CD28 comprise an anti-CD3 antibody, anti-CD28 antibody, or both.
  • the one or more agents that activate CD3 and/or CD28 comprise a first agent that binds CD3 and a second agent that binds CD28.
  • the first agent and the second agent are the same agent (e.g., a bispecific antibody that specifically binds to CD3 and CD28).
  • the method comprises, prior to step (a), obtaining a sample comprising the starting population of T cells from a subject. In certain embodiments, the method comprises, prior to step (a), having obtained a sample comprising the starting population of T cells from a subject. In certain embodiments, the sample is a whole blood sample obtained from the subject.
  • the starting population of T cells comprises T helper (Th) cells, cytotoxic T (Tc) cells, memory T (LM) cells, regulatory T (Treg) cells, innate-like T cells.
  • the Th cells comprise Thl cells, Th2 cells, Thl7 cells, Th9 cells, Tfh cells, and/or Th22 cells.
  • the memory T cells comprise central memory T cells (TCM) cells, effector memory T (TEM) cells, tissue-resident memory T (TRM) cells, and virtual memory T (TVM) cells.
  • the innate-like T cells are natural killer T (NKT) cells, mucosal-associated invariant T (MAIT) cells, and y5 T cells.
  • the polynucleotide is comprised in a delivery vehicle.
  • the delivery vehicle is a lipid nanoparticle.
  • the delivery vehicle is a nucleic acid vector.
  • the nucleic acid vector is a viral vector.
  • the viral vector is a lentiviral vector.
  • the method results in expansion of the starting population of T cells that is no greater than 10-fold, 9-fold, 8-fold, 7-fold, 6-fold, 5-fold, 4-fold, 3-fold, 2-fold, 1.9- fold, 1.8-fold, 1.7-fold, 1.6-fold, 1.5-fold, 1.4-fold, 1.3-fold, 1.2-fold, 1.1-fold, or less, following step (c).
  • at least 20%, at least 30%, at least 40%, at least 50%, or at least 75%, of the cells of the starting population of T cells, following step (c), are engineered to express the heterologous protein.
  • the heterologous protein comprises a chimeric antigen receptor (CAR).
  • the CAR comprises: (a) an antigen-binding fragment of an antiCD 19 antibody; (b) a transmembrane domain; (c) an intracellular T cell signaling domain from human CD3 ⁇ , and (d) an intracellular T cell signaling domain from human CD28.
  • one or more of the T cells harvested in step (c) secretes increased amounts of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, or 32) proteins selected from the group consisting of IFNg, Granzyme B, IL-113, IL -2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL-17A, IL-17F, IL-21, IL-22, IP -IO, MCPI, MCP4, TNFa, TNF13, TGF13, GMCSF, MIPla, MIP113, CCL11, perforin, RANTES, sCD137, and VEGF after being contacted with a target cell that expresses CD 19, as compared to a T cell not contacted with the target cell
  • secretion of the one or more e.g., 1, 2, 3, 4,
  • one or more of the T cells harvested in step (c) exhibit increased expression of one or more T cell activation markers selected from the group consisting of CD69, CD25, and CD 137 after being contacted with a target cell that expresses a target antigen, as compared to expression of the one or more T cell activation markers in the absence of the target cell.
  • expression of the one or more T cell activation markers selected from the group consisting of CD69, CD25, and CD137 is increased by at least 55%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450% or, 500% higher, or more, as compared to expression of the one or more T cell activation markers in the absence of the target cell.
  • one or more of the T cells harvested in step (c) exhibit increased cytotoxicity against a target cell that expresses a target antigen (e.g., antigen related to a disease such as CD 19), as compared to cytotoxicity against a cell that does not express the antigen.
  • cytotoxicity is increased by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450% or, 500% higher, or more, as compared to cytotoxicity against a cell that does not express antigen.
  • one or more of the T cells harvested in step (c) exhibit increased proliferation after being contacted with a target cell that expresses a target, as compared to proliferation in the absence of the target cell.
  • proliferation is increased by at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15- fold, 20-fold, 30-fold, 40-fold, 50-fold, or more, as compared to proliferation in the absence of the target cell.
  • proliferation is measured between 0 and 240 hrs after contact with the target cell.
  • proliferation is measured at 0 hr, 1 hr, 2 hrs, 3 hrs, 4 hrs, 5 hrs, 6 hrs, 7 hrs, 8 hrs, 9 hrs, 10 hrs, 11 hrs, 12 hrs, 13 hrs, 14 hrs, 15 hrs, 16 hrs, 17 hrs, 18 hrs, 19 hrs, 20 hrs, 21 hrs, 22 hrs, 23 hrs, 24 hrs, 30 hrs, 36 hrs, 42 hrs, 48 hrs, 72 hrs, 96 hrs, 120 hrs, 144 hrs, 168 hrs, 192 hrs, 216 hrs, and/or 240 hrs after contact with the target cell.
  • proliferation is measured as the fold-change in the number of CD3-positive (CD3+) cells in the T cells of step (c) as compared to the number of CD3+ cells in the starting population of T cells. In certain embodiments, proliferation is measured as the fold-change in the number of T cells of step (c) expressing the heterologous protein after being contacted with a target cell that expresses a target antigen, as compared to the number of T cells of step (c) expressing the heterologous protein in the absence of the target cell.
  • the T cells harvested in step (c) exhibit an increased amount of naive and stem cell memory T (collectively TNSCM) cells (e.g., TSCM cells) as compared to the starting population of T cells.
  • TNSCM naive and stem cell memory T
  • the T cells harvested in step (c) comprise TNSCM cells (e.g., TSCM cells) in an amount of at least 6%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450% or, 500% higher, or more, as compared to the starting population of T cells.
  • the T cells harvested in step (c) exhibit substantially the same percentage of naive and stem cell memory T (TNSCM) cells (e.g., TSCM cells) as compared to the starting population of T cells (e.g., the percentage of TNSCM cells in the T cells of step (c) is increased or decreased by no more than 4%, 3%, 2%, or 1%, or less (absolute difference between the percentages of TNSCM cells in the T cells), as compared to the number of TNSCM cells in the starting population of T cells).
  • the T cells harvested in step (c) comprise a decreased amount of T effector memory (TEM) cells as compared to the starting population of T cells.
  • TEM T effector memory
  • the T cells harvested in step (c) comprise TEM cells in an amount that is at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% lower, or more, as compared to the starting population of T cells.
  • the T cells harvested in step (c) exhibit substantially the same percentage of TEM cells as compared to the starting population of T cells (e.g., the percentage of TEM cells in the T cells of step (c) is increased or decreased by no more than 4% 3%, 2%, 1%, or less (absolute difference between the percentages of TEM cells in the T cells), as compared to the number of TEM cells in the starting population of T cells).
  • the method is performed ex vivo.
  • Example 1 Preparation of T cells engineered to express a chimeric antigen receptor using 3-day process
  • T-cells were extracted from the peripheral blood mononuclear cells (PBMCs) isolated from donor I (27 year old Asian male, with the BMI of 25.4, and a smoker), or using leukapheresis from donor 2 (31 year old Caucasian male, with the BMI of 42.0, and not a smoker), or donor 3 (52 year old male of mixed ethnicity, with the BMI of 78.1, and not a smoker).
  • PBMCs peripheral blood mononuclear cells isolated from donor I (27 year old Asian male, with the BMI of 25.4, and a smoker), or using leukapheresis from donor 2 (31 year old Caucasian male, with the BMI of 42.0, and not a smoker), or donor 3 (52 year old male of mixed ethnicity, with the BMI of 78.1, and not a smoker).
  • the T cells in the samples were isolated using Miltenyi StraightFrom CD3 Microbeads (Miltenyi Biotec: cat. no. 130-090- 874) and stored at
  • the isolated T cells were then independently processed, using the various manufacturing methods outlined in FIG. 10, which includes the following protocols: A (“KYV 3-Day Alternative”), which relies on leukapheresis and combines activation and transduction; B (“KYV 3-Day vl), which may use PBMCs/whole blood; C (“KYV 3-Day v2”), which may use PBMCs/whole blood; D (“KYV 6 Day”) which may use PBMCs/whole blood; E (“KYV 8 day”) which may use PBMCs/whole blood; and F (“KYV 3-Day v3”), which may use PBMCs/whole blood.
  • the initial starting population cells was 6xl0 7 cells (enriched T cell population containing 85%-95% T cells) and for protocols D and E IxlO 7 cells (enriched T cell population containing 85%-95% T cells) in Conditions D and E.
  • the cells were seeded at the density of 3xl0 6 /cm 2 (Conditions B, C, and F) or lxl0 6 /cm 2 (Conditions A, D and E) in a G-Rex bioreactor.
  • TransAct T Cell TransAct, human; Miltenyi Biotec; cat. No. 130-111-160 activation reagent was added to the cells in Conditions A, B, C, D, and E.
  • Cytokines were added together with the activation reagent in Conditions B (100 ng/mE human IL -2), condition C (10 ng/mL human IL-7 and 10 ng/mL human IL-15), condition D (10 ng/mL human IL-7 and 10 ng/mL human IL- 15), and condition E (10 ng/mL human IL-7 and 10 ng/mL human IL- 15).
  • the cells were incubated in cultivation media (TexMACS Medium supplemented with CTS Immune Cell SR) in a CO2 incubator.
  • the T cells were transduced with KL-hl98a28z, a self-inactivating (SIN) vesicular stomatitis virus (VSV)-G pseudotyped 3rd generation lentiviral vector encoding a chimeric antigen receptor that binds CD19.
  • This CAR construct named Hul9-CD828Z, has the amino acid sequence set forth in SEQ ID NO: 13.
  • the lentiviral vector contained an MSCV promoter and other regulatory factors, including a central polypurine tract/central termination sequence upstream of the promoter, and a post-transcriptional regulatory element (PRE) downstream of the CAR expression sequence.
  • PRE post-transcriptional regulatory element
  • the lentiviral vector KL-hl98a28z was manufactured using a HEK 293T cell line transiently transfected with a state-of-the-art four-plasmid system.
  • the envelope protein encoding plasmid (pLTG1292) expresses a heterologous spike protein, the VSV-G protein, under control of the cytomegalovirus (CMV) promoter.
  • CMV cytomegalovirus
  • the cells were incubated with the lenti virus vector for various lengths of time: 30 hours in Condition A; 48 hours in Conditions B, C, and E; 5 days in Condition D; and 8 days in Condition E.
  • Conditions A-C and F yielded approximately 3xl0 7 cells each
  • condition D yielded approximately IxlO 8 cells
  • condition E yielded approximately 3xl0 8 cells.
  • the transduced T cells were frozen using a standard protocol. The cells were thawed at a later time and characterized according to Example 2 below.
  • Example 2 Characterization of the lentivirus vector-transduced T cells
  • Viability and cell count were measured using a NucleoCounter NC-200TM (ChemoMetec A/S, Allerod, Denmark), an automated cell counter that utilizes fluorescence detection to distinguish between viable and non-viable cells. Each test article was loaded into a proprietary cassette which contains two separate dyes that stain for total nucleated cells and non-viable cells. The software then calculated the percent viability and cell count. The total cell count was performed at 0 hours and 72 hours post thaw.
  • each batch manufactured using the activated KYV 3 -Day processes (“vl” and “v2”) showed better overall T cell expansion during the 72 hours post-thaw than the 8- day process, which was at least comparable to the KYV Alternative 3-day process, which requires leukapheresis.
  • Donor 1 showed least expansion overall, possibly due to the low viability of the starting cell population.
  • T cells were next assessed for CAR expression and memory T cell phenotype by flow cytometry using a Cytoflex LX (Beckman Coulter) cytometer, using fluorescent antibodies recognizing the CD19 CARs and cell-surface markers associated Tn, Tscm, Tcm, Tern, and Temra memory T cell subsets. Specifically, Tn cells were identified as CD45RO-/CCR7+/CD95-; Tscm cells were identified as CD45RO-/CCR7+/CD95+; Tcm cells were identified as CD45RO+/CCR7+; Tern cells were identified as CD45RO+/CCR7-; and Temra cells were identified as CD45RO-/CCR7-.
  • KYV Alternative 3-Day cells displayed similar percentage of Tnscm as KYV 3-Day vl and KYV 3-Day v2 cells.
  • the "Post Enrichment" sample was untransduced with lenti virus.
  • T cells processed using the 3-day processes showed comparable or greater CAR expression than T cells manufactured using the KYV Alternative 3 -Day process, which relies on leukapheresis. Additionally, taking donor-to-donor variability and CAR expression into account, the three 3-day processes showed similar percentage of Tnscm among each other and higher percentage of Tnscm than the longer KYV 8-day and KYV 6-Day processes.
  • the activity of the CAR-T cells of killing CD 19+ target cells was measured by flow cytometry using a Cytoflex LX (Beckman Coulter) cytometer.
  • CD19+ Raji or Nalm6 cells were labeled with cell trace violet (CTV) and co-cultured with effector CAR-T cells at several effector-to-target (E:T) cell ratios for 18-20 hours. Percent cell killing was determined by the ratio of live CTV positive cells cocultured with effector cells to live CTV positive cells cultured in the absence of effector cells, at each E: T ratio. Data analysis was performed using FlowJo (BD Biosciences). [0348] The percentage cytolysis was measured and plotted vs. E:T ratio, as shown in FIG. 6 for Nalm6 cells. As provided in Table 1 below, cytotoxic activity at E:T ratio of 1: 1 was comparable between cells manufactured using the Processes A-E.
  • the human adaptive immune panel included the following cytokines: CCL-11, GM-CSF, Granzyme B, IFNg, IL-113, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-15, IL- 17A, IL-17F, IL-21, IL-22, IP- 10, MCP-I, MCP-4, MIP-la, MIP-113, Perforin, RANTES, sCD137, TNF-a, TNF-13, TGF-13, and VEGF.
  • FIGS. 7A-7D illustrate percentage of cells secreting each of the 32 cytokines, and show higher levels of IFNg (IFNg) and TNF-13 observed in cells manufactured using the KYV 3-day v2 process as compared to the KYV 9-day process.
  • IFNg IFNg
  • TNF-13 TNF-13 observed in cells manufactured using the KYV 3-day v2 process as compared to the KYV 9-day process.
  • Polyfunctionality represented the percentage of subsets of highly polyfunctional cells that simultaneously secrete multiple sets of cytokines, following co-culture with CD19+ target cells.
  • the CAR-T cells manufactured using the KYV 3 -day v2 process showed higher polyfunctionality than the CAR-T cells manufactured using the 9-day process. Further, the percentage of polyfunctional cells that simultaneously secreted multiple sets of cytokines was assessed with respect to each cytokine.
  • FIG. 9 shows the top 15 cytokines, with the cells manufactured using the KYV 3-day v2 processes displaying highest percentage of polyfunctional cells that simultaneously secrete multiple sets of cytokines.
  • Donors provided whole blood samples from which PBMCs were isolated using either a Rotea Counterflow Centrifugation System (ThermoFisher Scientific) or the X-LAB System (Coming Life Sciences).
  • the Rotea system applies a counterflow centrifugation method for a broad range of cell processing applications, including PBMC isolation.
  • the Corning X-LAB System is a sedimentation-based process that isolates mononuclear cells (MNCs) in a closed, sterile, semi-automated fashion — without the need for density gradient media or manual transfer steps.
  • the CD3+, CD4+, CD8+ cell percentage (T cells) in the whole blood samples was stable at 72 hours, while the percentage of CD45+ cells decreased over time.
  • samples obtained within 24 hours gave a better yield than those obtained within 48 hours of sample draw.
  • whole blood contains a sufficient number of T cells for CAR T manufacture, with fresh whole blood providing the best starting product. Coupled with the shortened CAR T manufacturing methods of the disclosure, using fresh blood should produce a superior, polyfunctional end product.
  • Example 4 Based on the promising whole blood composition and stability results from Example 4, a set of experiments to assess the CAR T cell manufacturing processes of the disclosure in which the PBMC step is omitted in favor of isolating T cell directly from a whole blood sample. Further, this Example not only assesses the feasibility of using a direct-from-blood T cell isolation step, but pairing that step with a concurrent activation of the isolated T cells.
  • FIG. 13 outlines the protocols used to assess the direct-from-blood T cell isolation step. Briefly, whole blood samples were obtained from three donors. The samples were used either 24 hours or 48 post-sample draw. For the concurrent T cell isolation and activation protocol, whole blood samples were run through a ThermoFisher DynaCellect Cell Isolator/WasherSystem (ThermoFisher) using anti-CD3/anti-CD28 Dynabeads, which captured and activated T cells from whole blood. The bead-bound T cells were collected using a G-RexlOM cell sorter. As a comparison, PBMCs were extracted from whole blood samples using the X-LAB system or the Rotea System.
  • ThermoFisher DynaCellect Cell Isolator/WasherSystem ThermoFisher
  • anti-CD3/anti-CD28 Dynabeads which captured and activated T cells from whole blood.
  • the bead-bound T cells were collected using a G-RexlOM cell
  • T cells were extracted from the PBMCs using a CliniMACS Plus Cell Isolator (Miltenyi Biotec) and activated using TransACT.
  • a CliniMACS Plus Cell Isolator Miltenyi Biotec
  • TransACT TransACT
  • the DynaCellect Cell isolation/activation protocol was also used on PBMCs extracted from Rotea to assess the relative efficacy of the DynaCellect system’s and the CliniMACS system’s enrichment comparative enrichment capability and to determine whether concurrent activation/i solation impacts enrichment.
  • the DynaCellect provides a higher yield of enriched and final product relative to CliniMACS. This indicates that concurrent activation and T cell isolation appears to provide a higher yield of isolated T cells from a sample and a resulting higher yield of CAR T cells produced using said T cells. As shown, DynaCellect gives an average fold expansion of 1.2 compared to 0.56 from CliniMACS.
  • the DynaCellect concurrent isolation/activation protocols produced a high level of CD3+ cells across all materials (whole blood, enriched PBMCs, and final product), a more consistent CD4+/CD8+ ratio across all donors from starting material to final product.
  • the concurrent activation/i solation method appears superior to a split isolation and activation protocol.
  • this set of experiments clearly validates methods of the disclosures, that omit not only a leukapheresis step, but also a PBMC enrichment step when obtaining T cells from a whole blood sample.
  • Example 6 Phenotype of CAR T cells engineered from T cells isolated directly from whole blood.
  • Tn cells were identified as CD45RO-/CCR7+/CD95- ; Tscm cells were identified as CD45RO-/CCR7+/CD95+; Tcm cells were identified as CD45RO+/CCR7+; Tern cells were identified as CD45RO+/CCR7-; and Temra cells were identified as CD45RO-/CCR7-.
  • Example 7 Functional characteristics of CAR T cells engineered from T cells isolated directly from whole blood.
  • Anti-CD19 CAR T cells were produced using the KYV 3 -Day process as outlined in Examples 1 and 2, but by isolating T cells directly from whole blood from healthy donors rather than PBMCs and omitting the TransACT activation step in favor of concurrent activation and isolation using CD3/CD28 Dynabeads.
  • the T cells were transduced with KL-hl98a28z, a self-inactivating (SIN) vesicular stomatitis virus (V SV)-G pseudotyped 3rd generation lentiviral vector encoding a chimeric antigen receptor that binds CD19.
  • This CAR construct named Hul9-CD828Z, has the amino acid sequence set forth in SEQ ID NO: 13.
  • the lentiviral vector contained an MSCV promoter and other regulatory factors, including a central polypurine tract/central termination sequence upstream of the promoter, and a post-transcriptional regulatory element (PRE) downstream of the CAR expression sequence.
  • PRE post-transcriptional regulatory element
  • the lentiviral vector KL-hl98a28z was manufactured using a HEK 293T cell line transiently transfected with a state-of-the-art four-plasmid system.
  • the envelope protein encoding plasmid (pLTG1292) expresses a heterologous spike protein, the VSV-G protein, under control of the cytomegalovirus (CMV) promoter.
  • CMV cytomegalovirus
  • FIG. 19 provides the results for the target-dependent cytokine release by the CAR T cells derived from healthy donor (HD) whole blood, following 24h in vitro co-culture with CD 19+ NALM6 target cells at the indicated E:T (effectortarget) ratios. Supernatants were collected and analyzed by ELLA assay. Mock untransduced (UT) cells, which do not express a CAR, exhibited low background levels of cytokine release. In contrast, the CAR T cells exhibit a clear target-dependent cytokine release, indicative of their target-dependent cytotoxic behavior.
  • the CD19-targeted CAR-T cells and CD 19+ NALM6 target cells were co-cultured at the indicated E:T ratios (CAR-T effector:NALM6 target) and % killing of target cells was measured by flow cytometry at each indicated timepoint. At each timepoint, a fresh round of target cells was added to the co-culture to assess the serial, repeated cytotoxicity of the CAR-T over time. Mock transduced (“UT”) cells, which do not express a CAR, did not demonstrate any cytotoxic activity. In contrast, the CAR-T cells showed a durable immune response and provided recurring cytotoxic response upon rechallenge for at least 30-days post first exposure.
  • cells produced using the two-to-three-day methods of the disclosure which include methods in which the T cells are isolated and activated directly from whole blood, provide an exhaustion-resistant phenotype that exhibits the desired target-specific cytotoxic activity.
  • the resulting CAR T cells were shown to expand upon contact with target cells at a higher rate than CAR T cells made using the "KYV 8 day” or “KYV 6 Day” process, outlined in FIG. 10.
  • the 8-day process uses T cells obtained from an enriched population (e g., from a leukapheresis sample or isolated PBMCs); (2) a separated T cell isolation and activation step; and (3) longer periods of time for culture and expansion.
  • the anti-CD19 CAR-T cells were generated from the conventional 8-day manufacturing process (“Conv”) or the KYV 3 -day process outlined in this example. Both sets of CAR T cells were derived from healthy donor (HD) whole blood starting material. CAR-T cells were stimulated by co-culturing with mitomycin C-treated CD 19+ NALM6 target cells added at a 1 : 1 ratio every 3-4 days. Expansion of viable T cells was calculated at each indicated timepoint using Vicell cell count analysis.
  • the results are provided in FIG. 21.
  • the cells made using the 3-day process showed a greater rate of target-specific expansion, which indicates a desirable hi vivo immune profile.
  • the short CAR T manufacturing methods of the disclosure which include those using direct-from-whole-blood T cell isolation, concurrent activation and T cell isolation, and brief periods of culture and cultivation produces cells with favorable phenotypes, target-specific cytotoxicity, durable immune responses, and a high rate of expansion upon target stimulus.
  • the CAR T cells manufactured using the methods of the disclosure provide a high CAR expression, including a higher CAR expression within CD4 and CD8 subsets relative to other existing methods. Moreover, the cells exhibited a high proportion of the desired Tcm and Tscm cell subsets.
  • Single cell cytokine analyses revealed cell produced using the methods of the disclosure have higher levels of IFNg and TNF-0 co pared to traditional processes, including other shortened processes such as the KYV Alternative 3 day, which relies on leukapheresis.
  • the cells produced using the methods of the disclosure exhibited a higher level of polyfunctionality relative to CAR T cells made using alternative methods, which is likely due to the brief period of time between isolating the T cells from whole blood and harvesting the finale CAR T product. Further, the resulting CAR T product was shown to have an increased, relative to other methods as described, level of antigen-induced proliferation.
  • the methods of the disclosure provide the shortest path from sample to CAR T product, while providing cells of higher quality than existing CAR T manufacturing processes.
  • Example 8 T cell subpopulations from whole blood samples
  • three sets of CAR T cells were produced using a whole blood starting sample: “AR037” which used isolation of T cells from whole blood (1 week old at 2-8 °C) and in which cells were processed downstream in the classic 9-day process from Examples 1-2; “AR039” did not use any downstream processing of the fresh whole blood material; and “AR050” which applied the 3-day v2 (CPD-23-007) process to isolate and engineer T cells from fresh whole blood.
  • the TBNK/memory phenotype of Pre- and post-enrichment material and TBNK/memory phenotype of the final CAR T cell product (e.g., after expansion) for AR037 is provided in FIGS. 22-23.
  • the TBNK/memory phenotype of Pre- and post-enrichment material for AR037 is provided in FIG. 24.
  • the TBNK/memory phenotype of Pre- and post-enrichment material for AR050 is provided in FIG. 25.
  • Tnscm viable CAR T cells of which a high percentage are Tnscm.
  • FIG. 23 the number of Tnscm is shown to increase upon expansion of the cells.
  • the initial number of Tnscm surpasses that of the 8-day process.
  • the 3-day process should produce a final product with an extraordinarily high percentage of Tnscm cells.
  • apheresis has been the source of cellular starting material for T-cell therapy products due to the large numbers of T-cells required to go through the conventional manufacturing process and generate sufficient modified T-cells for a therapeutic dose to treat oncology patients.
  • the associated burden on patients due to the length and invasiveness of the apheresis cell collection procedure and the logistical constraints of transporting the apheresis product to the manufacturing location are challenges currently associated with CAR T-cell products that limit accessibility and necessitate a new approach.
  • Ingenui-T is a next-generation CAR T-cell manufacturing platform initially being developed for autoimmune disease, utilizing the same fully human anti-CD19 CAR construct as KYV-101.
  • KYV-101 is an investigational autologous anti-CD19 CAR T-cell therapy (manufactured using conventional methods) under investigation for patients with B-cell-driven autoimmune diseases, including lupus nephritis, systemic sclerosis, myasthenia gravis, multiple sclerosis, and other diseases with a strong rationale for B-cell involvement in the disease pathology.
  • This example provides an exemplary use of the novel Ingenui-T manufacturing platform of the disclosure, which highlights the platform’s ability to generate high-purity and functional CAR T cells.
  • the Ingenui-T platform yields CAR T cells with a potent functional profile and a less differentiated phenotype compared to CAR T cells generated in a conventional manufacturing process that uses apheresis-derived cellular starting material.
  • the Ingenui-T platform offers a promising avenue for enhancing the efficiency and accessibility of CAR T-cell therapy, lowering costs, and ultimately advancing its application in the realm of autoimmune diseases.
  • Fig. 26 provides an overview of the exemplary use of the presently disclosed Ingenui-T manufacturing platform to produce engineered immune cells starting from whole blood.
  • T-cells were transduced using a lentiviral vector encoding the Hul9-CD828Z anti- CD19 CAR construct at a fixed multiplicity of infection (MOI). This is the same construct used in KYV-101-a first-in-class, fully human autologous anti-CD19 CAR T-cell therapy (Kyverna Therapeutics, Emeryville, C A).
  • the cell product was manufactured to represent the KYV-101 product.
  • Cryopreserved leukapheresis material was thawed, washed, and subjected to antibody-driven T-cell isolation using magnetic beads (Miltenyi Biotec).
  • the isolated T cells were counted, analyzed by flow cytometry for CD3 + T cell purity, and activated using T Cell TransAct (Miltenyi Biotec) in the presence of supporting cytokines (human IL-2, IL-21, IL-15, IL-7, or a combination thereof).
  • T cells were transduced at a fixed MOI using the same lentiviral vector incorporating the Hul9-CD828Z anti-CD19 CAR construct (Kyvema Therapeutics, Emeryville, CA). Cells were then cultured for 8- 10 days before harvesting, formulation, and cry opreservation of the final product.
  • CAR T cells were co-cultured with CD 19+ NALM6 target cells expressing an mCherry fluorescent reporter protein at the indicated effector to target (E:T) ratios for 120 hours.
  • Target-specific cytotoxic activity was assessed by imaging co-cultures using an Incucyte Sx5 (Sartorius) instrument and calculating the survival or outgrowth of fluorescent target cells over time, normalized to the signal intensity at the start of co-culture.
  • CAR T cells To evaluate the long-term functionality of CAR T cells, donor-matched Ingenui-T cells or conventional CAR T cells were serially rechallenged every 2-3 days with CD19+ NALM6 target cells at the indicated E:T ratios. At each time point, samples were split in half to assess the percent cytotoxicity and to re-plate with fresh target cells. Percent cytotoxicity was calculated at each timepoint by measuring the target cell survival using flow cytometry and was normalized to the survival of target cells in the absence of CAR T effector cells.
  • Ingenui- T cells were co-cultured with peripheral blood mononuclear cells (PBMCs) obtained from donor-matched leukapheresis material. Effector-to-target (E:T) ratios were defined according to the number of CAR + Ingenui-T cells (effector) to total PBMCs (targets). After 48 hours, target-specific cytolytic activity against B cells was measured by flow cytometry. B cells were defined by surface expression of either CD19 or CD20, within gated CD3' cells, to ensure proper detection of B cells even in the presence of interactions with anti-CD19 CAR T cells. Percent cytolysis against B cells was calculated by normalizing to the survival of B cells in PBMC-only cultures.
  • the objective was to demonstrate the technical feasibility of generating anti-CD19 CAR T cells starting from fresh whole blood material in a shortened manufacturing process.
  • fresh whole blood from healthy donors was loaded onto the DynaCellect platform for simultaneous isolation and activation of T cells using anti-CD3/CD28 Dynabeads at a defined bead: cell ratio.
  • the isolated/activated T cells were sampled to confirm the isolation purity (>95% CD3+) by flow cytometry and subsequently seeded into culture with media containing the cytokines IL-2, IL-7, IL- 15, IL-21, or a combination thereof.
  • Transduction using a lentiviral vector encoding the anti-CD19 CAR construct occurred at a fixed MOI within the first 24 hours of culture, followed by a brief period in culture to allow for cell recovery and integration of the transgene ( ⁇ 72 hours post-seeding). Following this brief culture period, CAR T cells were collected for bead removal and subsequent formulation in cryopreservation media. T-cell purity analysis of the final Ingenui-T cell product showed a T-cell percentage of 93.9 ⁇ 1.6%, obtained from a starting T-cell frequency of 42.3 ⁇ 6.8% in whole blood.
  • Ingenui-T cells Given the short culture time, Ingenui-T cells demonstrated minimal expansion during the manufacturing process, resulting in a 0.68 ⁇ 0.09-fold change of the total T-cell number from the time of culture seeding to final formulation (including any losses due to washing and bead removal procedures). Nevertheless, the final yield of Ingenui-T cell product was 38.5 ⁇ 6.6> ⁇ 10 6 T cells per 100 mL of starting whole blood. Product attributes were tested upon harvest and after 72 hours of post-thaw culture to simulate product performance in the patient.
  • CAR+ expression ranged between 45.1% 54.5% in Ingenui-T cells, which was statistically similar to the 37.4%-56.3% CAR+ expression obtained from the conventional CAR T-cell manufacturing process derived from apheresis (Table 1). This demonstrates that using the Ingenui-T platform, anti-CD19 CART cells can be successfully manufactured directly from whole blood, in a shortened manufacturing process, at a scale sufficient for therapeutic dosing of B-cell- driven autoimmune disease patients.
  • Ingenui-T cells comprised a less differentiated T cell memory phenotype than CAR T cells generated in a conventional manufacturing process.
  • Whole blood-derived Ingenui-T cells preserved a T-cell memory phenotype that closely resembled the phenotype observed in the starting material, with slight increases in the overall effector/memory compartment (combined TCM, TEM, and TE populations).
  • the effector/memory compartment shifted from a mean of 48.8 ⁇ 5.6% to 69.4 ⁇ 4.8% within the CD4+ T cell fraction, and from 42.9 ⁇ 3.9% to 46.6 ⁇ 5.5% within the CD8+ T cell fraction, while maintaining a substantial proportion of cells within the TN+TSCM compartment (Fig. 27A).
  • CAR T cells obtained from the traditional manufacturing process had mostly converted to the effector/memory compartment, shifting from a mean of 58.4 ⁇ 3.5% to 94.0 ⁇ 2.8% and from 40.1 ⁇ 5.2% to 86.2 ⁇ 4.9% within the CD4+ and CD8+ T cell fractions, respectively.
  • Ingenui-T cells The functional activity of Ingenui-T cells was assessed in both short-term and long-term in vitro preclinical assays in order to demonstrate target-specific cytotoxicity against CD 19- expressing cells.
  • the cytotoxic activity of Ingenui-T cells and donor-matched, apheresis-derived conventional CAR T cells were compared in a short-term cytotoxicity assay against CD19+ NALM6 tumor cells, as a representative target cell line.
  • Ingenui-T cells controlled the outgrowth of NALM6 target cells over a period of 120 hours, at lower E:T ratios than conventional CAR T cells. This reflects the expected increase in CAR T-cell potency and target-mediated CAR T-cell proliferation due to the less differentiated memory phenotype of Ingenui-T cells. Minimal cytotoxicity was observed against a control CD19-negative target cell line (CEM/C1), nor was any cytotoxic activity observed in untransduced Ingenui-T cells (i.e., without CAR expression; against CD 19-positive targets data not shown). These results confirmed the anti-CD19 target-specific activity of Ingenui-T cells and their increased functional potency relative to conventional CAR T cells that had been generated in a conventional manufacturing process.
  • CEM/C1 CD19-negative target cell line
  • Ingenui-T cells continued to kill target cells for a significantly longer period at a given E:T ratio, and required a >4-fold lower E:T ratio than donor-matched CAR T cells generated from a 9-day culture process to maintain the same duration of killing.
  • Ingenui-T cells were co-cultured for 48 hours with autologous total peripheral blood mononuclear cells (PBMCs), B cells were eliminated in a specific and dosedependent manner (Fig. 27D).
  • PBMCs peripheral blood mononuclear cells
  • Ingenui-T cells demonstrated greater potency of B cell killing than conventional CAR T cells when tested at dose-limiting E:T ratios (e.g., at 0.011 :1; Fig. 27D and data not shown).
  • This example provides further data related to cells manufactured from leukapheresis or WB SM using the Ingenui-T platform using methods as disclosed herein.
  • the objective was to characterize anti-CD19 CAR T cells starting from fresh whole blood material in a shortened manufacturing process as compared with those made using a longer, 9-day process and/or starting from a leukapheresis SM.
  • manufactured CAR T cells were prepared using the method outlined in Fig. 26, in which the starting material obtained from the subject in Fig. 26 was from a leukapheresis sample. [0415] Peripheral whole blood and leukapheresis SM was obtained from healthy donors, which was collected and transported fresh for immediate processing.
  • a 100 mL whole blood or leukapheresis sample was collected and an aliquot was added directly to the GibcoTM CTSTM DynaCellectTM Magnetic Separation System (Thermo Fisher Scientific, Waltham, MA), and anti-CD3/CD28 Dynabeads (CTSTM Detachable DynabeadsTM CD3/CD28 Kit; Thermo Fisher Scientific, Waltham, MA) were used at a defined ratio for enrichment and activation of T cells.
  • GibcoTM CTSTM DynaCellectTM Magnetic Separation System Thermo Fisher Scientific, Waltham, MA
  • CTSTM Detachable DynabeadsTM CD3/CD28 Kit Thermo Fisher Scientific, Waltham, MA
  • T-cells were transduced using a lentiviral vector encoding the Hul9-CD828Z anti-CD19 CAR construct at a fixed multiplicity of infection (MOI). This is the same construct used in KYV-101, which was described above. After a targeted in vitro cell culture, beads were removed from the culture, and cells were harvested, formulated into final product containers, and cryopreserved. In parallel, untransduced cells were also generated through the same manufacturing process in the absence of lentiviral transduction for use as control cells.
  • cytokines human interleukin 2 [IL-2], IL-21, IL- 15, IL-7, or a combination thereof.
  • MOI multiplicity of infection
  • FIG. 28A flow cytometry was used to analyze the overall T cell expansion, T cell viability, and T cell purity of CAR-T cells manufactured using the 3-day process of the Ingenui-T platform using a starting leukapheresis sample. Across a variety of different cytokine cultures, the 3-day process consistently produced T cells with over a 1-fold expansion, with over 90% viability, and over 95% purity. Importantly, these results remained consistent whether or not the T-cells were transduced with an exogenous immune receptor. [0418] Similarly, as shown in Fig. 28B, when starting with a whole blood sample, a high final concentrations of expanded T cells were produced, with a very high T-cell purity, using the 3-day process of the disclosure.
  • Figure 29A shows the % CAR+ expression analyzed by flow cytometry in CAR-T cells manufactured in the KYV 3-Day process starting from leukapheresis material.
  • CAR expression was analyzed within total CD3+ T cells or within CD4+ or CD8+ T cells, at the time of harvest.
  • KYV 3-Day Conditions “A”, “B”, “C”, “D” indicate different culture cytokine(s) used.
  • N 4 healthy donors per condition.
  • CAR+ expression in Ingenui-T cells was statistically similar to the CAR+ expression obtained from the conventional CAR T-cell manufacturing process.
  • CD4 and CD8 T-cell memory phenotypes of the Ingenui-T final product were compared to the T- cell memory populations in whole blood/ leukapheresis starting material based on the expression of CD45RO, CCR7, and CD95 surface markers.
  • the T-cell memory phenotypes in conventional CAR T cells were compared to the T-cell memory populations in the leukapheresis starting material (donor-matched with whole blood Ingenui-T cells). CAR expression was analyzed at 0 hour and 72 hours post-thaw of the final drug product, to ensure an accurate determination of stably integrated expression.
  • Figure 30A shows the CD4+ to CD8+ ratio for CAR-T cells using a leukapheresis SM and the 3-day process compared to similar cells produced using a traditional 9-day process. As shown, the 3-day process produces CAR-T cells with a CD4:CD8 ratio that is similar to the far-longer 9- day process.
  • Figure 30B shows similar results for cells manufactured using the 3-day process, but starting from whole blood (WB).
  • the 3-day processes of the disclosure using a leukapheresis SM produced T cells with fewer effector T cells and a larger proportion of TNSCM cells, particularly, Tscm cells.
  • the 3-day method of the disclosure produced T-cells with an even higher proportion of TNSCM cells.
  • these TNSCM cells included a much larger proportion of naive T cells relative to methods starting with a leukapheresis SM and a 9-day method starting with WB SM.
  • Ingenui-T cells The functional activity of Ingenui-T cells was assessed using both WB SM and leukapheresis SM in order to demonstrate target-specific cytotoxicity against CD19-expressing cells.
  • the cytotoxic activity of Ingenui-T cells and donor-matched, apheresis-derived conventional CAR T cells were compared in a short-term cytotoxicity assay against CD 19+ NALM6 tumor cells, as a representative target cell line.
  • Fig. 33A shows the results of killing or outgrowth of CD19+ NALM6 target cells during 120h co-culture with anti-CD19 CAR-T cells manufactured in the KYV 3-Day process from leukapheresis starting material, at the indicated E:T (Effector:Target) ratios of 0.3: 1 or 1 : 1.
  • KYV 3-Day Conditions “Cl”, “C2”, “C3”, “C4” indicate different culture cytokine(s) used.
  • NT non-transduced control T cells.
  • One representative donor shown from n 4.
  • 33B provides analogous results of killing or outgrowth of CD19+ NALM6 target cells during 120h co-culture with anti-CD19 CAR-T cells manufactured in the KYV 3-Day process from freshly collected whole blood, at the indicated E:T (Effector:Target) ratios of 0.3: 1 or 1 :1.
  • NT non-transduced control T cells.
  • Ingenui-T cells (leukapheresis SM) controlled the outgrowth of NALM6 target cells over a period of 120 hours, at lower E:T ratios than conventional CAR T cells. Similar results were found for cells produced using WB SM (Fig. 33B). T-cells produced using the three-day process, whether starting from WB SM or a leukapheresis sample produced a durable, target-specific cytotoxic response that surpassed the efficacy of comparable cells produced using a longer (e.g., 9-day) process. Cells produced using a WB SM produce a more effective and durable cytotoxic response even when compared to cells produced using a leukapheresis sample.
  • Ingenui-T cells The in vitro cytolytic activity of Ingenui-T cells was assessed against autologous primary B cells, which are the cells targeted for depletion in the treatment of patients with B-cell-driven autoimmune diseases.
  • autologous primary B cells To evaluate cytolytic activity of CAR T cells against autologous primary B cells, Ingenui-T cells, or control untransduced T cells were co-cultured with peripheral blood mononuclear cells (PBMCs) obtained from donor-matched leukapheresis material. Effector-to- target (E:T) ratios were defined according to the number of CAR + Ingenui-T cells (effector) to total PBMCs (targets). After 48 hours, target-specific cytolytic activity against B cells was measured by flow cytometry.
  • PBMCs peripheral blood mononuclear cells
  • B cells were defined by surface expression of either CD 19 or CD20, within gated CD3' cells, to ensure proper detection of B cells even in the presence of interactions with anti-CD19 CAR T cells. Percent cytolysis against B cells was calculated by normalizing to the survival of B cells in PBMC-only cultures.
  • Ingenui-T cells made from WB SM were co-cultured for 48 hours with autologous total peripheral blood mononuclear cells (PBMCs), B cells were eliminated in a specific and dosedependent manner (Fig. 34). Ingenui-T cells demonstrated greater potency of B cell killing than conventional CAR T cells when tested at dose-limiting E:T ratios.
  • PBMCs peripheral blood mononuclear cells
  • Fig 36A shows cytokine release by anti-CD19 CAR-T cells manufactured in the KYV 3- Day process from leukapheresis starting material, in co-culture with CD19+ NALM6 target cells at the indicated E:T (Effector: Target) ratios of 0.3 : 1 or 1 : 1.
  • Figs. 36B-36C shows cytokine release by anti -CD 19 CAR-T cells manufactured in the KYV 3-Day process from freshly collected whole blood, in co-culture with CD19+ NALM6 target cells at the indicated E:T (Effector: Target) ratios of 0.3 : 1 or 1 : 1. Culture supernatants were collected and the indicated cytokines were analyzed by MSD.
  • KYV 3-Day Conditions “Cl”, “C2”, “C3”, “C4” indicate different culture cytokine(s) used in the manufacturing process.
  • Conv 9-day refers to donor-matched CAR-T cells manufactured in a conventional 9-day culture process, derived from leukapheresis starting material.
  • cells produced using the 3-day methods of the disclosure provide an effector dose-dependent, CAR-mediated cytokine release in response to CD19+ expressing target cells.
  • Fig. 37 shows the duration of in vitro cytotoxicity by KYV 3-Day or Conv 9-Day anti-CD19 CAR T cells, derived from a healthy donor, in a serial rechallenge assay against CD19+ NALM6 tumor cells.
  • KYV 3-Day CAR T cells were derived from leukapheresis starting material (“APH”, Top panel) or freshly collected whole blood (“WB”, Bottom panel).
  • CAR T cells were co-cultured in triplicate with NALM6 target cells at the indicated Effector: Target (E:T) ratios, and the survival of NALM6 cells was analyzed every 2-3 days by flow cytometry.
  • the time (days) to loss of CAR-mediated cytotoxic activity, defined as the assay timepoint at which >95% survival of target cells was detected, was measured for each individual replicate. Data representative of n 4 donors. As shown, the WB cells produced a more durable a long-lasting cytotoxic response.
  • Fig. 38 shows data comparing in vitro expansion by anti-CD19 CAR-T cells manufactured from KYV 3-day process compared to a conventional 9-day process, in response to CD19+ expressing target cells.
  • the data provides the responses of KYV 3-Day or conventional (“Conv”) 9-Day anti-CD19 CAR T cells to repeated stimulation in co-culture with CD 19+ expressing REH target cells.
  • CAR T cells were co-cultured with mitomycin C-treated REH target cells at a 1 : 1 ratio, and cells were re-plated every 3-4 days with new target cells. Total fold expansion of CAR+ T cells (gated by flow cytometry analysis) was measured at day 16. As shown, the cells show the potential to expand upon contact with an appropriate target.
  • Fig. 39A-39B show data pertaining to the in vivo activity of anti-CD19 CAR-T cells manufactured from KYV 3 -day process compared to conventional 9-day process, in CD19+ NALM6 tumor-bearing NSG mice.
  • Fig. 39A shows mean NALM6 tumor growth in NSG mice treated with the indicated doses of donor-matched anti-CD19 CAR T cells manufactured from the KYV 3-day process or a conventional (“Conv”) 9-day process, both derived from leukapheresis (“APH”) starting material.
  • NALM6-luciferase tumor cells were injected intravenously into mice at day -7 prior to T cell transfer.
  • mice were given a single intravenous injection of the indicated doses of CAR T cells.
  • Fig. 39B shows individual NALM6 tumor growth curves in NSG mice treated with a le6 CAR+ T cell dose of donor-matched anti-CD19 CAR T cells.
  • anti-CD19 CAR-T drug products were engineered from T cells isolated directly from whole blood (WB) obtained from healthy donors (HD), using one of the two 9-day culture process.
  • WB whole blood
  • HD healthy donors
  • T-cells were transduced using a lentiviral vector encoding the Hul9-CD828Z anti-CD19 CAR construct at a fixed multiplicity of infection (MOI).
  • MOI multiplicity of infection
  • IB provides flow cytometry data showing the % CAR+ cells within the DP.
  • Fig. 41 C provides flow cytometry data showing % T cell purity within the DP compared to the WB starting material (SM).
  • SM WB starting material
  • the CD4/8 fraction is similar to the 3-day methods.
  • the T-cell memory phenotype shows a different final drug product relative to both the 3-day whole blood methods and the conventional 9-day processes (not starting from whole blood) described above.
  • these 9-day methods Compared to the “conventional” 9-day process, these 9-day methods based on the whole-blood 3-day methods, produce a final drug product in which the memory or effector phenotype of the T cells (e.g., predominantly Tcm cells) is similar to that seen in the “conventional” 9-day process that starts with apheresis material (e.g., Fig. 31C). Accordingly, the 9-day whole blood processes also produced a lower TNSCM component relative to the 3-day processes (e.g., Fig. 31C), which had higher proportions of Tscm cells and naive T cells.
  • apheresis material e.g., Fig. 31C
  • the presently disclosed 9-day whole-blood methods offer an alternative to methods that, while requiring 9-days at the manufacturing step, must still rely on obtaining apheresis starting material.
  • the presently disclosed methods are able to ease the bottlenecks in production associated with obtaining apheresis samples, while delivering an equivalent or better therapeutically effective cellular product.
  • the CD19-dependent, CAR-mediated cytotoxic activity of the cells was evaluated by co-culturing the CAR T-cells derived from WB starting material, with CD19+ NALM6 target cells or CD19- CEMC1 control cells, or co-culturing non-transduced control T cells with CD 19+ NALM6 target cells.
  • the % cytolysis of target cells was evaluated by luminescence after 24h co-culture.
  • Fig. 42 provides the % cytolysis results.
  • Fig. 43 provides corresponding cytokine secretion data. As shown, cells produced from whole blood using the 9-day methods described herein provide a target-dependent cytotoxic response.
  • Whole blood-derived Ingenui-T cells preserve a T-cell memory phenotype that more closely resembles the phenotypes observed in the starting materials, with slight increases in the overall effector/memory compartment (combined TCM, TEM, and TE populations) as the culture process lengthens. Both the 3-day and 9-day processes improved over the existing, conventional 9-day process. Thus, although the cells were transduced with the same exogenous CARs, they nevertheless are fundamentally different final products.
  • the Ingenui-T platform is focused on enhancing patient experience and reducing the cost of manufacturing CAR T-cell therapies.
  • the next-generation manufacturing process starts from autologous whole blood and uses a rapid ( ⁇ 3 day) manufacturing process, resulting in a potent CAR T-cell product with demonstrated target-specific killing activity. This manufacturing process marks a significant departure from traditional methods that necessitate apheresis, a laborious and resource-intensive process, and extended cell culture.
  • the Ingenui-T platform reduces the differentiation of the cells in vitro by minimizing the culture time, resulting in both a shorter process and a more potent product that can potentially provide equivalent therapeutic benefit with a lower dose, while enabling the feasibility of using a limited volume of whole blood rather than apheresis as starting material.
  • Streamlining the process through a combination of collecting up to 300 mb of whole blood, and minimizing the culture time not only reduces and optimizes resource utilization, but also reduces the time spent in specialized facilities and reduces involvement of highly skilled personnel, enhancing the costefficiency of CAR T-cell therapy.
  • This reduction in the overall cost of goods to manufacture and decreased burden on patients holds promise for broader accessibility and affordability
  • This optimization also aligns with the goal of scalability of CAR T-cell therapies, addressing a critical need in the field.

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Abstract

L'invention concerne des procédés de fabrication de lymphocytes T modifiés qui expriment une protéine hétérologue, telle qu'un récepteur antigénique chimérique (CAR), dans laquelle des lymphocytes T sont isolés à partir d'échantillons de sang entier.
PCT/US2024/054236 2023-11-03 2024-11-01 Procédés de fabrication de lymphocytes t modifiés à partir d'échantillons de sang entier Pending WO2025097034A1 (fr)

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Citations (2)

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US20200239910A1 (en) * 2017-08-09 2020-07-30 Juno Therapeutics, Inc. Methods and compositions for preparing genetically engineered cells
US20230256017A1 (en) * 2020-02-27 2023-08-17 Jennifer Brogdon Methods of making chimeric antigen receptor-expressing cells

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20200239910A1 (en) * 2017-08-09 2020-07-30 Juno Therapeutics, Inc. Methods and compositions for preparing genetically engineered cells
US20230256017A1 (en) * 2020-02-27 2023-08-17 Jennifer Brogdon Methods of making chimeric antigen receptor-expressing cells

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