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WO2025085680A1 - Methods for manufacturing car t cells - Google Patents

Methods for manufacturing car t cells Download PDF

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Publication number
WO2025085680A1
WO2025085680A1 PCT/US2024/051845 US2024051845W WO2025085680A1 WO 2025085680 A1 WO2025085680 A1 WO 2025085680A1 US 2024051845 W US2024051845 W US 2024051845W WO 2025085680 A1 WO2025085680 A1 WO 2025085680A1
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WIPO (PCT)
Prior art keywords
cells
population
car
day
cell
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French (fr)
Inventor
Kanika CHAWLA
Sum Ying CHIU
Christian HUYNH
Shishir Gadam
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Cargo Therapeutics Inc
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Cargo Therapeutics Inc
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Publication of WO2025085680A1 publication Critical patent/WO2025085680A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/40—Cellular immunotherapy characterised by antigens that are targeted or presented by cells of the immune system
    • A61K40/41—Vertebrate antigens
    • A61K40/42—Cancer antigens
    • A61K40/4202—Receptors, cell surface antigens or cell surface determinants
    • A61K40/421—Immunoglobulin superfamily
    • A61K40/4212—CD22, BL-CAM, siglec-2 or sialic acid binding Ig-related lectin 2
    • 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
    • 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
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2307—Interleukin-7 (IL-7)
    • 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
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/2315—Interleukin-15 (IL-15)
    • 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
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/11—Coculture with; Conditioned medium produced by blood or immune system cells
    • C12N2502/115—Platelets, megakaryocytes
    • 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
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present disclosure relates generally to the fields of immuno-therapeutics and cell therapy, and particularly relates to improved methods of making and/or manufacturing a population of immune cells expressing a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • the disclosure provides populations of CAR-expressing immune cells made and/or manufactured by the improved methods, pharmaceutical compositions comprising the same.
  • the disclosure also describes methods for treating a health condition in a subject in need thereof.
  • BACKGROUND Chimeric antigen receptor (CAR) T-cell therapy has shown promising therapeutic effects in treating various health conditions, including cancers, particularly hematologic cancers.
  • CAR-T cells are generated by genetic engineering of either the patient’s own immune cells (autologous) or immune cells from unrelated human donors (allogenic).
  • CAR-T cells Production of high-quality, GMP-grade CAR-T cells is an essential prerequisite for the wide application of this technology. Successful manufacturing of such high-quality CAR T-cell products for clinical applications has been challenging and costly, with common pitfalls including lengthy turnaround times and unacceptably low manufacturing success rates. [0005] Current manufacturing processes for CAR-T cells have several drawbacks and so there is a pressing need to develop better and more efficient manufacturing processes for large-scale production of CAR-T cells with improved therapeutic activities. SUMMARY [0006] The present disclosure relates generally to, inter alia, improved methods of making and/or manufacturing a population of immune cells expressing a chimeric antigen receptor (CAR).
  • CAR chimeric antigen receptor
  • populations of CAR-expressing immune cells made and/or manufactured by the improved methods, as well as pharmaceutical compositions comprising a population of CAR-expressing immune cells of the disclosure.
  • the disclosure also provides methods for treating a health condition in a subject in need thereof by administering the pharmaceutical compositions of the disclosure.
  • a method of making a population of chimeric antigen receptor (CAR)-expressing immune cells comprising the steps of: (a) obtaining a liquid sample comprising a first population of cells comprising immune cells from a human subject; (b) processing the first population of cells to remove platelets thereby generating a second population of cells, wherein the second population of cells includes at least 1 ⁇ 10 4 total cells and wherein less than 20% of the total number of cells in the second population of cells are platelets; (c) seeding a third population in a volume of a first buffer, wherein the third population of cells is a subset of the second population of cells; (d) transducing the third population of cells with a recombinant polynucleotide encoding a CAR thereby generating a fourth population of cells, wherein the fourth population of cells includes CAR-expressing immune cells; (e) expanding the fourth population of cells yielding a fifth population of cells; and (f)
  • the method further includes removing at least 50% of the volume of the first buffer on or before Day 4 after seeding. In some embodiments, at least 2.4% of cells in the fifth population of cells are CCR7+CD45RA+ immune cells. In various embodiments of the methods disclosed herein, the third population of cells comprising immune cells is seeded in a volume of a first buffer that comprises growth media. In these instances, the first buffer is also referred to as the first media.
  • a method of making a population of CAR-expressing immune cells including the steps of: (a) obtaining a liquid sample including a first population of cells including immune cells from a human subject; (b) processing the first population of cells thereby generating a second population of cells; (c) seeding a third population of cells in a volume of a first buffer (e.g., first media), wherein the third population of cells is a subset of the second population of cells; (d) transducing the third population of cells with a recombinant polynucleotide encoding a CAR thereby generating a fourth population of cells, wherein the fourth population of cells includes CAR-expressing immune cells; (e) expanding the fourth population of cells to yield a fifth population of cells; (f) removing at least 50% of the volume of the first buffer (e.g., first media) on or before Day 4 after the seeding; and (g) harvesting the fifth population of cells on Day 5 or later
  • At least 2.4% of cells in the fifth population of cells are CCR7+CD45RA+ immune cells.
  • a method of making a population of CAR-expressing immune cells including the steps of: (a) obtaining a liquid sample including a first population of cells including immune cells from a human subject; (b) processing the first population of cells thereby generating a second population of cells; (c) seeding a third population of cells in a volume of a first buffer (e.g., first media), wherein the third population of cells is a subset of the second population of cells; (d) transducing the third population of cells with a recombinant polynucleotide encoding a CAR thereby generating a fourth population of cells, wherein the fourth population of cells includes CAR-expressing immune cells; (f) expanding the fourth population of cells yielding a fifth population of cells; and (g) harvesting the fifth population of cells on Day 5 or later after the seeding of
  • a method of making a population of CAR- expressing immune cells including: (a) obtaining a liquid sample including a first population of cells including immune cells from a human subject; (b) processing the first population of cells to remove platelets thereby generating a second population of cells, wherein the second population of cells includes at least 1 ⁇ 10 4 total cells and wherein less than 20% of the total number of cells in the second population of cells are platelets; and (c) seeding a third population of cells in a volume of a first buffer (e.g., first media), wherein the third population of cells is a subset of the second population of cells, wherein the third population of cells include at least 2.0 ⁇ 10 8 cells; (d) transducing the third population of cells with a recombinant polynucleotide encoding a CAR thereby generating a fourth population of cells, wherein the fourth population of cells includes CAR-expressing immune cells; (e) expanding the fourth population of cells yield
  • Non-limiting exemplary embodiments of the disclosed method of making a population CAR-expressing immune cells include one or more of the following features. In some embodiments, less than 18%, less than 15%, less than 12%, less than 10%, less than 8%, or less than 5% of the total number of cells in the second population of cells are platelets. In some embodiments the processing of the first population of cells includes diluting the liquid sample including the first population of cells with a second buffer, thereby generating a diluted liquid sample including the first population of cells. [0012] In some embodiments, the liquid sample including the first population of cells is diluted prior to removing platelets.
  • the liquid sample including the first population of cells has a total volume of at least 50 mL, at least 100 mL, at least 200 mL, at least 250 mL, at least 300 mL, at least 400 mL, at least 500 mL, at least 600 mL, at least 700 mL, at least 800 mL, at least 900 mL, at least 1 L, or at least 2 L.
  • the liquid sample including the first population of cells is a blood sample or a sample obtained by apheresis.
  • the liquid sample including the first population of cells is a leukapheresis or apheresis sample.
  • the diluting includes adding an equal volume of the second buffer to the liquid sample, wherein the volume of the second buffer added to the liquid sample including the first population of cells is the same as the total volume of the liquid sample including the first population of cells prior to diluting.
  • the second buffer includes human serum albumin (HSA).
  • HSA human serum albumin
  • the buffer including human serum albumin is a solution comprising between 1 and 10% (w/v) HSA.
  • the buffer including HSA is a solution comprising 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% HSA.
  • the second buffer includes Plasma-Lyte A and 4% (w/v) human serum albumin in equal volume.
  • the second buffer includes Plasma-Lyte A and 2% (w/v) human serum albumin.
  • the diluted liquid sample has a total volume that is at least two times (2X), at least three times (3X), or at least five times (5X) the total volume of the liquid sample prior to dilution. In some embodiments, the diluted liquid sample has a total volume that is at least three times the total volume of the liquid sample prior to dilution. In some embodiments, the diluted liquid sample has a total volume that is at least five times the total volume of the liquid sample prior to dilution.
  • the processing of the first population of cells includes washing the first population of cells, concentrating the first population of cells, and eluting and/or resuspending the first population of cells, thereby generating the second population of cells.
  • the concentrating of the first population of cells is accomplished using an automated centrifugation system.
  • the automated centrifugation system concentrates the first population of cells by elutriation.
  • the processing of the first population of cells in step (b) further includes adding a third solution to the second population of cells and cryopreserving the second population of cells.
  • the third solution includes one or more of the following: phosphate-buffered saline (“PBS”), dimethyl sulfoxide (“DMSO”), sodium hydroxide, potassium hydroxide, and sucrose.
  • the third buffer includes phosphate-buffered saline (“PBS”), dimethyl sulfoxide (“DMSO”), sodium hydroxide, potassium hydroxide, and sucrose.
  • the cryopreserved second population of cells is thawed before transducing with a recombinant polynucleotide encoding a CAR in step (c).
  • At least 2.4% of cells in the fifth population of cells are CCR7+CD45RA+ immune cells. In some embodiments, at least 2.5%, 2.8%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, or 5.5% of cells in the fifth population of cells are CCR7+ CD45RA+ immune cells.
  • cells in the fifth population of cells are CCR7+ CD45RA+ immune cells.
  • the CCR7+ CD45RA+ immune cells are also CD3+.
  • the CCR7+ CD45RA+CD3+ immune cells are also CD4+.
  • the CCR7+ CD45RA+CD3+ immune cells are also CD8+.
  • At least 78.3%, 78.5%, 79.0%, 79.5%, 80%, 80.5%, 81.0%, 81.4%, 81.5%, 82.0%, 83.0%, 84.0%, 85.0%, 86.0%, or 87.0% of cells in the fifth population of cells are CCR7+ CD45RA- immune cells.
  • At least 78.0%, 78.1%, 78.2%, 78.3%, 78.4%, 78.5%, 78.6%, 78.7%, 78.8%, 78.9%, 79.0%, 79.1%, 79.2%, 79.3%, 79.4%, 79.5%, 79.6%, 79.7%, 79.8%, 79.9%, 80.0%, 80.1%, 80.2%, 80.3%, 80.4%, 80.5%, 80.6%, 80.7%, 80.8%, 80.9%, or 81.0% of cells in the fifth population of cells are CCR7+ CD45RA- immune cells.
  • At least 81.0%, 81.1%, 81.2%, 81.3%, 81.4%, 81.5%, 81.6%, 81.7%, 81.8%, 81.9%, 82.0%, 82.0%, 82.1%, 82.2%, 82.3%, 82.4%, 82.5%, 82.6%, 82.7%, 82.8%, 82.9%, 83.0%, 83.1%, 83.2%, 83.3%, 83.4%, 83.5%, 83.6%, 83.7%, 83.8%, 83.9%, or 84.0% of cells in the fifth population of cells are CCR7+ CD45RA- immune cells.
  • At least 84.0%, 84.1%, 84.2%, 84.3%, 84.4%, 84.5%, 84.6%, 84.7%, 84.8%, 84.9%, 85.0%, 85.0%, 85.1%, 85.2%, 85.3%, 85.4%, 85.5%, 85.6%, 85.7%, 85.8%, 85.9%, 86.0%, 86.1%, 86.2%, 86.3%, 86.4%, 86.5%, 86.6%, 86.7%, 86.8%, 86.9%, or 87.0% of cells in the fifth population of cells are CCR7+ CD45RA- immune cells.
  • At most 16.1%, 16.0%, 15.0%, 14.0%, 13.0%, 12.0%, 11.0%, 10.0%, 9.0%, 8.0%, 7.5%, 7%, 6.5%, 6.0%, 5.5%, 5.0%, 4.5%, 4.0%, 3.5%, or 3.4% of cells in the fifth population of cells are CCR7- CD45RA- immune cells.
  • the efficiency of transducing the third population of cells with a recombinant polynucleotide encoding a CAR is at least 10%, at least 20%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50%.
  • the multiplicity of infection (MOI) used when transducing the third population of cells with a recombinant polynucleotide encoding a CAR is at least 1, at least 1.5, at least 2, at least 2.5, at least 3, at least 3.5, at least 4, at least 4.5, or at least 5.
  • the fifth population of cells is harvested on Day 5, Day 6, Day 7, Day 8, Day 9, Day 10, Day 11, Day 12, Day 13, Day 14, or Day 15.
  • the fifth population of cells is harvested on Day 5, Day 6, Day 7, Day 8, or Day 9.
  • the fifth population of cells is harvested on Day 5, 7, and/or Day 9.
  • the fifth population of cells is harvested on Day 5.
  • the fifth population of cells is harvested on Day 6.
  • the fifth population of cells is harvested on Day 7. In some embodiments, the fifth population of cells is harvested on Day 8. In some embodiments, the fifth population of cells is harvested on Day 9. In some embodiments, the third population of cells is activated by at least one cytokine. In some embodiments, the at least one cytokine includes IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, or a combination thereof. In some embodiments, the at least one cytokine includes a combination of IL-7 and IL-15. In some embodiments, the third population of cells is seeded in the first medium.
  • the first medium includes 12.5 ng/mL IL-7, 12.5 ng/mL IL-15, and 3% (w/v) human serum albumin.
  • the third population of cells includes at least 2.5 ⁇ 10 6 , at least 5 ⁇ 10 6 , at least 7.5 ⁇ 10 6 , at least 1 ⁇ 10 7 , at least 2.5 ⁇ 10 7 , at least 5 ⁇ 10 7 , at least 7.5 ⁇ 10 7 , at least 1 ⁇ 10 8 , 1.1 ⁇ 10 8 , at least 1.2 ⁇ 10 8 , at least 1.4 ⁇ 10 8 , at least 1.6 ⁇ 10 8 , at least 1.8 ⁇ 10 8 , at least 2.0 ⁇ 10 8 , at least 2.2 ⁇ 10 8 , at least 2.4 ⁇ 10 8 , at least 2.6 ⁇ 10 8 , at least 2.8 ⁇ 10 8 , or at least 3.0 ⁇ 10 8 cells comprising a subset of the second population of cells.
  • the third population of cells includes at least about 2 ⁇ 10 8 cells comprising a subset of the second population of cells In some embodiments, the third population of cells includes at least about 3 ⁇ 10 8 cells comprising a subset of the second population of cells. [0017] In some embodiments, the third population of cells is enriched for CD4+CD8+ cells prior to seeding. In some embodiments, the CAR-expressing fourth population of cells produced by transducing the third population of cells with a recombinant polynucleotide encoding a CAR is then expanded to produce a fifth population of cells.
  • the fifth population of cells includes at least 2.5 ⁇ 10 6 , at least 5 ⁇ 10 6 , at least 7.5 ⁇ 10 6 , at least 1 ⁇ 10 7 , at least 2.5 ⁇ 10 7 , at least 5 ⁇ 10 7 , at least 7.5 ⁇ 10 7 , at least 1 ⁇ 10 8 , at least 1.1 ⁇ 10 8 , at least 1.2 ⁇ 10 8 , at least 1.4 ⁇ 10 8 , at least 1.6 ⁇ 10 8 , at least 1.8 ⁇ 10 8 , at least 2.0 ⁇ 10 8 , at least 2.2 ⁇ 10 8 , at least 2.4 ⁇ 10 8 , at least 2.6 ⁇ 10 8 , at least 2.8 ⁇ 10 8 , or at least 3.0 ⁇ 10 8 CAR-expressing immune cells.
  • the human subject from which the liquid sample comprising a first population of cells comprising immune cells was obtained has a disease or health condition.
  • the disease or health condition is cancer or a relapsed/refractory cancer.
  • the cancer is a leukemia or lymphoma. In some embodiments, the cancer is a lymphoma.
  • the lymphoma is selected from a group consisting of diffuse large B cell lymphoma (DLBCL), large B cell lymphoma (LBCL), mantle cell lymphoma (MCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), Burkitt’s lymphoma, anaplastic large-cell lymphoma, angioimmunoblastic T cell lymphoma, and Hodgkin lymphoma.
  • the lymphoma is large B cell lymphoma.
  • the cancer is a leukemia.
  • the leukemia is selected from a group consisting of acute lymphocytic leukemia (ALL), acute lymphoblastic leukemia (ALL), B cell acute lymphocytic leukemia (B-ALL), B cell acute lymphoblastic leukemia (B-ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), B cell prolymphocytic leukemia (B- PLL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), chronic myelomonocytic leukemia (CMML), hairy cell leukemia (HCL), juvenile myelomonocytic leukemia (JMML), large granular lymphocytic leukemia (LGLL), and T cell prolymphocytic leukemia (T-PLL).
  • ALL acute lymphocytic leukemia
  • ALL acute lymphoblastic leukemia
  • B-ALL B cell acute lymphoblastic
  • the leukemia is ALL or B-ALL. In some embodiments, the leukemia is pediatric ALL or B-ALL.
  • the recombinant polynucleotide encoding a CAR is a viral vector. In some embodiments, the viral vector is a lentiviral vector.
  • the CAR-expressing immune cells are T cells. In some embodiments, the T cells are CD4+ or CD8+. In some embodiments, the CD4+ T cells and CD8+ T cells are also CD3+. In some embodiments, the CAR-expressing immune cells are NK cells. In some embodiments, the CAR specifically binds a B cell-specific antigen.
  • the B cell-specific antigen is CD19, CD20, or CD22.
  • the CAR specifically binds the B cell-specific antigen CD22.
  • the CD22-specific CAR includes an anti-CD22 binding domain.
  • the anti-CD22 binding domain is an antibody, an antibody fragment, or an antigen binding domain of thereof.
  • the anti-CD22 binding domain is an antibody fragment.
  • the antibody fragment is an anti-CD22 single chain variable fragment (scFv).
  • the anti-CD22 binding domain includes a VH comprising a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence of SEQ ID NO: 2.
  • the anti-CD22 binding domain includes a VH having the sequence of SEQ ID NO: 2.
  • the anti-CD22 binding domain includes a light chain variable region (VL) comprising a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence of SEQ ID NO: 3.
  • VL light chain variable region
  • the anti-CD22 binding domain includes a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence of SEQ ID NO: 1.
  • the anti-CD22 binding domain includes the sequence of SEQ ID NO: 1.
  • the CD22 CAR includes a sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence of SEQ ID NO: 22.
  • the CD22 CAR includes the sequence of SEQ ID NO: 22.
  • the CD22 CAR includes a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence of SEQ ID NO: 23.
  • the anti-CD22 CAR includes the sequence of SEQ ID NO: 23.
  • the CAR specifically binds the B cell-specific antigen CD19.
  • the CD19-specific CAR includes an anti-CD19 binding domain.
  • the anti-CD19 binding domain is an antibody, an antibody fragment, or an antigen binding domain of thereof.
  • the anti-CD19 binding domain is an antibody fragment.
  • the antibody fragment is an anti-CD19 single chain variable fragment (scFv).
  • the CAR specifically binds the B cell-specific antigen CD20.
  • the CD20-specific CAR includes an anti-CD20 binding domain.
  • the anti-CD20 binding domain is an antibody, an antibody fragment, or an antigen binding domain of thereof.
  • the anti-CD20 binding domain is an antibody fragment.
  • the antibody fragment is an anti-CD20 single chain variable fragment (scFv).
  • a method of making a population of CAR-expressing immune cells comprising: (a) obtaining a liquid sample comprising a first population of cells comprising immune cells from a human subject; (b) processing the first population of cells thereby generating a second population of cells comprising immune cells, wherein the second population of cells includes at least 1 ⁇ 10 4 total cells, wherein less than 20% of the total number of cells in the second population of cells are platelets; (c) cryopreserving the second population of cells; (d) on Day 0, thawing the cryopreserved second population of cells comprising immune cells, processing the thawed second population of cells comprising immune cells, and seeding a third population of cells comprising immune cells with a portion of the processed second population of cells comprising immune cells in a volume of a first media, wherein the third population of cells is a subset of the second population of cells, wherein the third population of cells is seeded into a volume of at least 250
  • Non-limiting exemplary embodiments of the methods in accordance with this aspect and other aspects of the disclosure may include one or more of the following features.
  • the liquid sample including a first population of cells comprises a leukapheresis product.
  • processing of the first population of cells in step (b) comprises a step of washing, concentrating and eluting or resuspending the second population of cells in a buffer.
  • the processing of the first population of cells in step (b) further comprises a step of reducing the number of platelets in the first population of cells.
  • the second population of cells is washed, concentrated, and eluted or resuspended and the number of platelets is reduced by elutriation using a centrifugation system having a molecular weight cutoff sufficient to retain the immune cells.
  • the centrifugal filtration system is a CTS TM Rotea TM Counterflow Centrifugation System.
  • the washed and concentrated second population of cells having a reduced number of platelets compared to the first population of cells is eluted or resuspended in a buffer.
  • the buffer comprises human serum albumin (HSA), Plasma-Lyte A, phosphate buffered saline, sodium chloride, sodium bicarbonate buffer, glutathione, biotin, vitamin B12, inositol, choline, L-glutamine, sodium pyruvate, glucose, or any combination thereof.
  • buffer comprises human serum albumin (HSA).
  • the buffer comprises Plasma-Lyte A.
  • the buffer comprises equal volumes of Plasma-Lyte A and 4% (w/v) human serum albumin.
  • the washed and concentrated second population of cells having a reduced number of platelets compared to the first population of cells is resuspended in a buffer comprising equal volumes of Plasma-Lyte A and 4% (w/v) HSA and further diluted 1:1 with a cryoprotectant.
  • the cryoprotectant is CryoStor R .
  • the processing of the thawed second population of cells in step (d) comprises a step of enriching for CD4+ and CD8+ T cells.
  • the enriching for CD4+ and CD8+ T cells further comprises a step mixing the thawed second population of cells with magnetic beads derivatized with CD4-specific binding agents and CD8-specific binding agents, washing, and eluting the thawed second population of cells enriched for CD4+ and CD8+ cells.
  • the step of enriching the thawed second population of cells for CD4+ and CD8+ T cells is performed on a CliniMACS Prodigy using CliniMACS buffer + 2% (w/v) or 0.5% (w/v) HSA.
  • the third population of cells is seeded with 300 ⁇ 10 6 cells from the processed second population of cells.
  • the third population of cells is seeded with at least 2.5 ⁇ 10 6 cells from the processed second population of cells. In some embodiments, the third population of cells is seeded with between at least 2.5 ⁇ 10 6 cells and at least 300 ⁇ 10 6 cells from the processed second population of cells.
  • the third population of cells is seeded with at least about 2.5 ⁇ 10 6 , 3 ⁇ 10 6 , 3.5 ⁇ 10 6 , 4 ⁇ 10 6 , 4.5 ⁇ 10 6 , 5 ⁇ 10 6 , 5.5 ⁇ 10 6 , 6 ⁇ 10 6 , 6.5 ⁇ 10 6 , 7 ⁇ 10 6 , 7.5 ⁇ 10 6 , 8 ⁇ 10 6 , 8.5 ⁇ 10 6 , 9 ⁇ 10 6 , 9.5 ⁇ 10 6 , 1 ⁇ 10 7 , 1.5 ⁇ 10 7 , 2 ⁇ 10 7 , 2.5 ⁇ 10 7 , 3 ⁇ 10 7 , 3.5 ⁇ 10 7 , 4 ⁇ 10 7 , 4.5 ⁇ 10 7 , 5 ⁇ 10 7 , 5.5 ⁇ 10 7 , 6 ⁇ 10 7 , 6.5 ⁇ 10 7 , 7 ⁇ 10 7 , 7.5 ⁇ 10 7 , 8 ⁇ 10 7 , 8.5 ⁇ 10 7 , 9 ⁇ 10 7 , 9.5 ⁇ 10 7 , 1 ⁇ 10 8 , 1.5 ⁇ 10 8 , 2 ⁇ 10 8 , 2.5 ⁇ 10 8 ,
  • the third population of cells is seeded in modified TexMACS TM medium (MTM).
  • MTM comprises TexMACS TM medium supplemented with 3% human AB plasma (HABS) and 1:1 mixture of recombinant human cytokines IL-7 (hIL-7) and IL-15 (hIL-15).
  • the 1:1 mixture of recombinant human cytokines IL-7 (hIL-7) and IL-15 (hIL-15) comprises 12.5 ng/mL hIL-7 and 12.5 ng/mL hIL-15.
  • the modified TexMACS TM medium further comprises an effective amount of a reagent comprising agonists of CD3 and CD28.
  • the reagent comprising CD3 and CD28 agonists comprises the T cell TransAct TM reagent.
  • the recombinant polynucleotide encoding a CAR further comprises a lentiviral expression vector.
  • the lentiviral expression vector comprises a recombinant polynucleotide encoding a CAR.
  • the lentiviral expression vector is manufactured using an adherent cell culture method or a suspension cell culture method.
  • the transduction in step (e) is performed on Day 1. In some embodiments, the transduction in step (e) is performed 22-26 hours after seeding of the third population of cells comprising immune cells on Day 0.
  • the amount of vector used to transduce the third population of cells comprising immune cells in step (e) is determined based on the infectious titer of the lentiviral vector and the number of cells used to seed the third population of cells comprising immune cells such that the transduction is performed with a multiplicity of infection (MOI) of 2.0.
  • MOI multiplicity of infection
  • the transduction is performed with a MOI of at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2.0, at least 2.1, at least 2.2, at least 2.3, at least 2.4, at least 2.5, at least 2.6, at least 2.7, at least 2.8, at least 2.9, at least 3.0, at least 3.5, at least 4.0, at least 4.5, or at least 5.0.
  • the recombinant polynucleotide encoding a CAR further comprising a lentiviral expression vector is thawed, diluted into modified TexMACS TM medium (MTM), and added to the third population of cells comprising immune cells.
  • MTM modified TexMACS TM medium
  • the MTM comprises TexMACS TM medium supplemented with 3% human AB plasma (HABS) and 1:1 mixture of recombinant human cytokines IL-7 (hIL-7) and IL-15 (hIL-15).
  • 1:1 mixture of recombinant human cytokines IL-7 (hIL-7) and IL-15 (hIL-15) comprises 12.5 ng/mL hIL-7 and 12.5 ng/mL hIL-15.
  • the modified TexMACS TM medium further comprises an effective amount of a reagent comprising agonists of CD3 and CD28.
  • the reagent comprising CD3 and CD28 agonists comprises the T cell TransAct TM reagent.
  • the final volume in which the transduction in step (e) is performed comprises 100 mL.
  • the fifth population of cells in step (f) is expanded in MTM in a volume of 200 mL.
  • the fifth population of cells is washed to remove T cell TransAct TM and residual lentiviral vector.
  • the washing step is first performed on Day 4.
  • the washing step is first performed on Day 4 using the CliniMACS Prodigy program.
  • the washing step comprises removal of cell culture supernatant and resuspension of the fifth population of cells in MTM without T cell TransAct TM reagent.
  • the washing step comprises removal of 65% of the culture volume and resuspension of the fifth population in the desired volume of MTM without T cell TransAct TM .
  • the fifth population of cells is expanded for another day, a sample of the fifth population of cells is taken on Day 6 and the Dose Factor is recalculated assuming a 7% increase in Day 4 Transduction Efficiency.
  • the fifth population of cells is expanded for another day, a sample of the fifth population of cells is taken on Day 7 and the Dose Factor is recalculated assuming a 7% increase in Day 4 Transduction Efficiency.
  • a further media exchange is performed on Day 7.
  • the further media exchange on Day 7 comprises removing 60% of the culture volume and replacing it with fresh MTM.
  • there are not enough viable CD3+CAR+ cells on Day 7 the fifth population of cells is expanded for another day, a sample of the fifth population of cells is taken on Day 8 and the Dose Factor is recalculated assuming a 7% increase in Day 4 Transduction Efficiency.
  • a further media exchange is performed on Day 8.
  • the further media exchange on Day 8 comprises removing 60% of the culture volume and replacing it with fresh MTM.
  • the harvesting step (j) is performed.
  • there are not enough viable CD3+CAR+ cells on Day 8 the fifth population of cells is expanded for another day, and the harvesting step (j) is performed.
  • there are not enough viable CD3+CAR+ cells on Day 8 the fifth population of cells is expanded for another day, and the harvesting step (j) is performed on Day 9.
  • the harvesting step (j) further comprises a step of calculating viable cell density and determining whether the post-harvest viable cell density is ⁇ the minimum transduced viable cell density for the formulation step.
  • the post-harvest viable cell density is ⁇ the minimum transduced viable cell density and the harvested fifth population of cells is formulated for cryopreservation and administration to a human subject.
  • the post-harvest viable cell density is ⁇ the minimum transduced viable cell density and the harvested fifth population of cells is concentrated using a Rotea TM so that the post-harvest viable cell density is ⁇ the minimum transduced viable cell density and the concentrated harvested fifth population of cells is formulated for cryopreservation and administration to a human subject.
  • the minimum transduced viable cell density is 1 ⁇ 10 6 CD3+CAR+ cells/kg.
  • the formulation step is performed manually, and the harvested fifth population of cells or the concentrated harvested fifth population of cells is resuspended to the desired concentration in Final Formulation Medium comprising Plasma- Lyte A + 4% (w/v) HSA, diluted 1:1 with Cryostor R CS10 and frozen.
  • the formulation step is automated.
  • the automated formulation step is performed using a FINIA R Fill and Finish System, and the harvested fifth population of cells or the concentrated harvested fifth population of cells is resuspended to the desired concentration in Final Formulation Medium comprising Plasma-Lyte A+4% (w/v) HSA, diluted 1:1 with Cryostor R CS10 and frozen.
  • the automated formulation step is performed using a Cue ScaleReady Cell Processing System, and the harvested fifth population of cells or the concentrated harvested fifth population of cells is resuspended to the desired concentration in Final Formulation Medium comprising Plasma- Lyte A+4% (w/v) HSA, diluted 1:1 with Cryostor R CS10 and frozen.
  • the fifth population of cells including CAR-expressing immune cells comprises autologous CAR-expressing immune cells.
  • the fifth population of cells including CAR-expressing immune cells comprises autologous T cells expressing a CD22 CAR (i.e., a CD22-specific CAR).
  • the recombinant polynucleotide encoding an autologous CD22 CAR further comprises a lentiviral expression vector.
  • the lentiviral expression vector is manufactured using an adherent cell culture method or a suspension cell culture method.
  • the recombinant polynucleotide encoding a CAR encodes a CD22- specific CAR.
  • the CD22 CAR comprises a CD22-specific binding domain, a transmembrane domain, and an intracellular domain. In some embodiments, the CD22 CAR comprises a CD22-specific binding domain, a hinge domain, a transmembrane domain, a spacer, and an intracellular domain. In some embodiments, the CD22-specific binding domain comprises an antibody capable of binding CD22 or an antigen-binding fragment thereof. In some embodiments, the CD22-specific binding domain comprises an antibody capable of binding human CD22. In some embodiments, the CD22-specific binding domain comprises an antigen-binding fragment of an antibody capable of binding human CD22.
  • the antigen-binding fragment of an antibody capable of binding human CD22 is a single chain variable fragment (scFv) capable of binding CD22.
  • the scFv capable of binding CD22 has the sequence of SEQ ID NO: 1.
  • the scFv capable of binding CD22 comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 1.
  • the CD22 CAR comprises a CD8 ⁇ hinge domain comprising the sequence of SEQ ID NO: 24 and a CD8 ⁇ transmembrane domain comprising the sequence of SEQ ID NO: 25.
  • the CD22 CAR comprises a CD8 ⁇ hinge domain comprising the sequence of SEQ ID NO: 24, a CD8 ⁇ transmembrane domain comprising the sequence of SEQ ID NO: 25, and a peptide linker having the sequence of SEQ ID NO: 31.
  • the CD22 CAR comprises a CD8 ⁇ hinge domain comprising a sequence comprising 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 24 and a CD8 ⁇ transmembrane domain comprising a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 25.
  • the CD8 ⁇ transmembrane domain further comprises a spacer having the sequence of LYC.
  • the CD8 ⁇ transmembrane domain further comprises a spacer having the sequence of SEQ ID NO: 31.
  • the CD22 CAR comprises an intracellular domain further comprising a primary T cell activating domain comprising an immunoreceptor tyrosine-based activation motif (ITAM) and a costimulatory signaling domain.
  • ITAM immunoreceptor tyrosine-based activation motif
  • the primary T cell activating domain comprising an ITAM comprises a CD3z intracellular signaling domain.
  • the CD3 ⁇ intracellular signaling domain comprises the sequence of SEQ ID NO: 27.
  • the CD3 ⁇ intracellular signaling domain comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 27. In some embodiments, the CD3 ⁇ intracellular signaling domain comprises the sequence of SEQ ID NO: 30. In some embodiments, the CD3 ⁇ intracellular signaling domain comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 30. In some embodiments, the costimulatory signaling domain comprises a 4- 1BB/CD137 signaling domain.
  • the 4-1BB/CD137 signaling domain comprises the sequence of SEQ ID NO: 26.
  • 4-1BB/CD137 costimulatory signaling domain comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 26.
  • the CD22 CAR comprises the sequence of SEQ ID NO: 22.
  • the CD22 CAR comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 22.
  • the CD22 CAR comprises the sequence of SEQ ID NO: 23.
  • the CD22 CAR comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 23..
  • a method of manufacturing a fifth population of cells comprising CAR-expressing immune cells wherein the method of manufacturing includes the method described herein.
  • a method of treating a health condition in a subject including: administering to the subject a therapeutically effective amount of a fifth population of cells comprising CAR-expressing immune cells made according to any of the methods described herein, alone or in combination with an additional therapy, thereby treating the health condition.
  • the health condition is a cancer, wherein the administered population of CAR-expressing immune cells treats or provides anti- tumor immunity to the subject.
  • a fifth population of cells comprising CAR- expressing immune cells made according to any one or more of the methods described herein.
  • a pharmaceutical composition including a fifth population of cells comprising CAR-expressing immune cells as described herein and a pharmaceutically acceptable carrier.
  • FIG.1A shows the version 1 (v1) cell manufacturing protocol for generating a population of chimeric antigen receptor CAR-expressing immune cells.
  • FIG.1B shows the version 2 (v2) cell manufacturing protocol for generating a population of CAR-expressing immune cells.
  • FIGS.2A-2C show three apheresis cryopreservation methods.
  • FIG.2A shows the workflow of the v1 cell manufacturing protocol using a Prodigy concentration instrument, wherein cells were loaded onto the instrument, washed, reformulated, harvested and subsequently frozen.
  • FIG.2B shows the workflow of the v2 cell manufacturing protocol using a Rotea instrument, wherein cells were diluted prior to loading on an instrument, cells were loaded onto the instrument, washed, concentrated, eluted/resuspended and subsequently frozen.
  • FIG.2C shows the workflow of a manual manufacturing protocol using a centrifugation step, wherein cells were loaded into conical tubes, centrifuged, and a platelet layer is manually removed after centrifugation. Subsequently, the sample is diluted with buffer, and frozen.
  • FIGS.3A-3G show post process cell recovery and viability data using different cryopreservation methods.
  • FIG.3A (Table 5) shows leukapheresis hold times of the different cryopreservation methods.
  • FIG.3B shows total viable cell (TVC) input of the different cryopreservation methods.
  • FIG.3C (Table 7) shows run times of the different cryopreservation methods.
  • FIG.3D shows % cell recovery and % viability of the different cryopreservation methods.
  • FIG.3E shows % whole blood cell (WBC) recovery and % viability of the different cryopreservation methods.
  • FIG.3F shows residual platelet levels of the different cryopreservation methods.
  • FIG.3G shows residual platelet levels of the different cryopreservation methods. As shown in FIG.3G, the residual platelet levels using the Rotea v2 method are lowest.
  • FIG.4 (Table 8) shows the post-thaw total recovery and cell viability using 10 mL fill bags.
  • FIG.5A shows % residual platelet, % cell viability and % recovery pre-apheresis cryopreservation using the v2 cell manufacturing protocol.
  • FIG.5B shows % cell viability and % recovery post-thaw of the cryopreserved apheresis using the v2 cell manufacturing protocol.
  • FIGS.6A-6B show results from a cryo-apheresis verification run.
  • FIG.6A (Table 9) shows CD4 and CD8 positive T cell enrichment using the v2 apheresis cryopreservation method.
  • FIG.6B shows high viability and recovery using the v2 apheresis cryopreservation method.
  • FIG.7 shows the total viable cells (TVC), population doubling, post-harvest viability, and CAR+ yield by Day 7 harvest using the different seeding quantities depicted.
  • FIG.8 shows seeding with 200 million cells compensates for contraction post- activation and increases overall CAR+ cell yield compared to seeding with 100 million cells.
  • FIG.9 shows 6 different experimental conditions for evaluating media exchange volumes and timing during the cell manufacturing protocol.
  • FIG.10A shows that media exchange on Day 4 (D4, conditions 4-6) increases cell yield compared to other media exchange protocols.
  • FIG.10B shows that media exchange on D4 results in comparable viability compared to other media exchange protocols.
  • FIG.10C shows that media exchange on D4 results in comparable transduction efficiency compared to other media exchange protocols.
  • FIG.10D shows that media exchange on D4 results in comparable % CD3+CD4+ and % CD3+CD8+ /immunophenotypes compared to other media exchange protocols.
  • FIG.11 shows the media exchange strategy for the v2 cell manufacturing protocol.
  • FIG.12 shows the media exchange protocol for the v1 cell manufacturing protocol.
  • FIG.13A shows that the v2 cell manufacturing protocol media exchange strategy increases cell growth rate compared to the v1 cell manufacturing protocol.
  • FIG.13B shows that the v2 cell manufacturing protocol media exchange strategy results in similar viability compared to the v1 cell manufacturing protocol.
  • FIG.13C shows that the v2 cell manufacturing protocol media exchange strategy results in similar transduction efficiency compared to the v1 cell manufacturing protocol.
  • FIG.14 shows exemplary data that the extended harvest window of the v2 cell manufacturing protocol results in population doublings, viability, and transduction efficiencies which are similar to the v1 cell manufacturing protocol. As shown, the product generated by the v2 cell manufacturing protocol achieves approximately 4 population doublings by D5.
  • FIG.15 shows that the v2 cell manufacturing protocol met final dose fill requirement by Day 5 for both healthy donors and a patient.
  • FIG.16A shows the comparison between CAR-T cells generated using the v2 cell manufacturing protocol using samples from two healthy donors, a sample from a patient and a control verification sample for population doublings.
  • FIG.16B shows the comparison between CAR-T cells generated using the v2 cell manufacturing protocol using samples from two healthy donors, a sample from a patient and a control verification sample for viability %. Both the healthy donor and patient samples met final dose fill requirements by D5.
  • FIG.16C shows the comparison between CAR-T cells generated using the v2 cell manufacturing protocol using samples from two healthy donors, a sample from a patient and a control verification sample for % CAR+ cells. Both the healthy donor and patient samples met final dose fill requirements by D5.
  • FIG.17 shows the T cell memory phenotypes present from six different runs using the v1 and v2 cell manufacturing protocols.
  • FIG.18 shows an increase in stem cell memory T cells (TSCM) and T cell central memory subset (TCM) cells and a decrease in effector memory T cells (TEM) cells generated using the v2 cell manufacturing protocol compared to using the v1 cell manufacturing protocol. Data obtained is from six different runs using the v1 and v2 cell manufacturing protocols.
  • TSCM stem cell memory T cells
  • TCM T cell central memory subset
  • TEM effector memory T cells
  • FIG.19 shows higher levels of T cell activation and exhaustion marker expression in cells using the v2 cell manufacturing protocol compared to using the v1 cell manufacturing protocol., likely due to the proximity of evaluation from the activation step, due to the shorter v2 manufacturing process.
  • FIG.20 shows the experimental design to characterize the performance of the drug products generated by the v1 (D7) and v2 (D5, 7, 9) manufacturing processes, in vivo.
  • FIG.21 shows tumor growth over time (days post injection (DPI)) for mice treated with CAR-T cells generated by harvesting on D7 using the v1 cell manufacturing protocol and harvesting on D5, D7 and D9 using the v2 cell manufacturing protocol.
  • DPI days post injection
  • FIG.22 shows survival curves of mice treated with a low dose of CAR-T cells (1 ⁇ 10 6 CAR-T cells) or a high dose of CAR-T cells (5 ⁇ 10 6 CAR-T cells) generated by harvesting on D7 using the v1 cell manufacturing protocol and harvesting on D5, D7 and D9 using the v2 cell manufacturing protocol. Survival was highest (approximately 80%) for mice treated with D5 cells from the v2 cell manufacturing protocol.
  • FIG.23 shows the number of cells of the depicted phenotypes observed in mice treated with a low dose of CAR-T cells (1 ⁇ 10 6 CAR-T cells) or a high dose of CAR-T cells (5 ⁇ 10 6 CAR-T cells) generated by harvesting on D7 using the v1 cell manufacturing protocol and harvesting on D5, D7 and D9 using the v2 cell manufacturing protocol (top).
  • FIG.24 shows the number of viable cells/mL, viability % and number of population doublings of CAR-T cells generated using the v1 cell manufacturing protocol and the v2 cell manufacturing protocol from two different patient samples.
  • FIG.25 shows IFNgamma (IFN ⁇ ) release ELISPOT assays of CAR-T cells generated using the v1 cell manufacturing protocol and the v2 cell manufacturing protocol from two different patient samples.
  • FIG.26 shows the % frequency of cells of the depicted phenotypes observed in CAR- T cells generated using the v1 cell manufacturing protocol and the v2 cell manufacturing protocol from two different patient samples.
  • FIG.27 shows the % transduction efficiency (CAR+ cells) and the total CAR+ cell observed in CAR-T cells generated using the v1 cell manufacturing protocol and the v2 cell manufacturing protocol from two different patient samples.
  • FIG.28 shows equivalence in cell viability and CD3 expression for CAR-T cells generated using the v1 cell manufacturing protocol and the v2 cell manufacturing protocol.
  • FIG.29 shows equivalence in transduction efficiency and vector copy number per cell for CAR-T cells generated using the v1 cell manufacturing protocol and the v2 cell manufacturing protocol.
  • FIG.30 shows IFNgamma (IFN ⁇ ) release when CAR-T cells generated using the v2 cell manufacturing protocol were co-cultured with CD22 expressing cell lines at the indicated ratios for the indicated amount of time.
  • IFNgamma IFNgamma
  • FIG.31 shows IFNgamma (IFN ⁇ ) release when CAR-T cells generated using the v2 cell manufacturing protocol with a media exchange (MX) step at the indicated day were co- cultured with CD22 expressing cell lines at the indicated ratios. Higher IFNgamma (IFN ⁇ ) release was observed using CAR-T cells generated using the v2 cell manufacturing protocol with a media exchange step on Day 4 compared to CAR-T cells generated using the v2 cell manufacturing protocol with a media exchange on D7, D9 and D11.
  • FIG.32A shows the total number of viable cells in CAR-T cells generated using the v2 cell manufacturing protocol from two different patient samples that were harvested on the indicated days (top).
  • FIG.32B shows IFNgamma (IFN ⁇ ) release levels after co-culturing CAR-T cells generated using the v2 cell manufacturing protocol with CD22 expressing cell lines at the indicated ratios from a patient sample (Donor 1) that were harvested on the indicated days.
  • the data demonstrates earlier harvest Days (5 and 7) exhibit higher potency than later Days (10 and 12) and that growth affects potency.
  • FIG.32C shows IFNgamma (IFN ⁇ ) release levels after co-culturing CAR-T cells generated using the v2 cell manufacturing protocol with CD22 expressing cell lines at the indicated ratios from a patient sample (Donor 2) that were harvested on the indicated days.
  • FIG.33A shows the cytotoxicity of CAR-T cells generated using the v2 cell manufacturing protocol and harvested on D5, D7, and D9. CAR-T cells were co-cultured with CD22 expressing target cells or CD22 knocked out target cells at range between 1:0.03 to 2:1.
  • FIG.33B shows results demonstrating that the cytotoxicity assay was specific to CD22 antigen stimulation and harvesting later resulted in lower E:T EC50.
  • FIG.34A shows the vector copy number (VCN) per cell in the CAR-T cell population generated from two different donors using the v2 cell manufacturing protocol.
  • VCN vector copy number
  • FIG.34B shows the VCN per CAR+ cell in the CAR-T cell population generated from two different donors using the v2 cell manufacturing protocol.
  • the cells were transduced with different multiplicity of infection (MOI) ratio.
  • the results show that the VCN increased as MOI increased.
  • FIG.35 (Table 15) shows the VCN in the CAR-T cell population generated from two donors at two different locations using the v2 cell manufacturing protocol.
  • the VCN was measured by droplet digital polymerase chain reaction (ddPCR). Samples prepared at the two different locations showed similar VCN numbers, suggesting that the ddPCR assay is robust and reproducible.
  • ddPCR droplet digital polymerase chain reaction
  • FIG.36 shows the VCN/cell, % transduction efficiency, and VCN/ CAR+ cell in the CAR-T cells generated using v2 manufacturing protocol and harvested on D5, D7, and D10.
  • FIG.37 shows the experimental design for characterizing T cell memory cell phenotypes in the CAR-T cells generated using the v1 manufacturing protocol and the v2 manufacturing protocol.
  • FIG.38 shows the experimental design for characterizing T cell activation and cell exhaustion in the CAR-T cells generated using the v1 manufacturing protocol and the v2 manufacturing protocol.
  • FIG.39 shows a list of reagents used to characterize activation markers and reagents used to characterize exhaustion markers.
  • FIG.40 shows that greater than 45% of cells in the harvested CAR-T population generated using the v2 cell manufacturing protocol are CD22CAR+CD3+.
  • FIG.41 shows the % cell recoveries and % cell viabilities before and after automated cell concentration.
  • FIG.42 shows the total viable cells, population doubling time, total CAR+ yield over time of CAR-T cells population generated with different seeding density and at two different manufacturing sites. The results demonstrate that seeding with 200 ⁇ 10 6 cells increases CAR+ yield and results in similar percent of contraction and more viable cells on D1.
  • FIG.43 (Tables 18 and 19) shows the % CAR+ cells vs. vector concentration on D7 with two different vectors and different seeding density.
  • FIG.44 shows the projected total number of CAR+ cells generated using the v2 cell manufacturing protocol with media exchange on D5, D6, or D7 and cells harvested on D5, D7, or D9. The result suggests that harvesting on D5 can achieve the 1 ⁇ 10 6 CAR+ cells/kg dosage requirement, assuming a dose factor of 4.1.
  • FIG.45 shows the projected total number of CAR+ cells generated using the v2 cell manufacturing protocol with media exchange on D5, D6, or D7 and cells harvested on D5, D7, or D9.
  • FIG.46 shows a schematic experimental design of the potency assay for CD22 CAR- T cells.
  • FIG.47 shows data from an IL-2 readout via ELISpot to measure the potency of CD22 lentiviral vector transduced in Jurkat T cells.
  • FIG.48 shows a workflow for a co-culture cytotoxicity assay.
  • FIG.49 shows a workflow for a cell preparation and analysis for a cytotoxicity assay.
  • the present disclosure generally relates to, inter alia, improved methods of making and/or manufacturing a population of immune cells expressing a chimeric antigen receptor (CAR). Also provided are populations of CAR-expressing immune cells made and/or manufactured by the improved methods, as well as pharmaceutical compositions comprising a population of CAR-expressing immune cells of the disclosure. The disclosure also provides methods for treating a health condition in a subject in need thereof. [0129] As discussed in greater detail below, the present disclosure provides improved methods that allow more efficient manufacturing processes for rapid clinical-scale production of autologous CAR-T cells with improved therapeutic activities.
  • CAR chimeric antigen receptor
  • some embodiments of disclosure provide a robust manufacturing method allowing the production of drug product from apheresis starting material of varying quality.
  • the optimized media exchange strategy and seeding density allow for an earlier harvest window of 5-9 days compared to the longer windows of comparable processes shown in Figure 13A.
  • the manufactured product is highly enriched in more naive T cell memory subpopulations (central memory T cells (TCM) in particular) (see, e.g., Figure 17).
  • CD45RA and CCR7 human T cells can be divided into four subsets, including CD45RA+CCR7+ naive/stem cell memory (T N /T SCM ), CD45RA ⁇ CCR7+ central memory (TCM), CD45RA ⁇ CCR7 ⁇ effector memory (TEM), and CD45RA+CCR7 ⁇ effector memory re-expressing CD45RA (TEMRA) T cells.
  • T N /T SCM CD45RA ⁇ CCR7+ central memory
  • TEM CD45RA ⁇ CCR7 ⁇ effector memory
  • TEMRA+CCR7 ⁇ effector memory re-expressing CD45RA (TEMRA) T cells CD45RA+CCR7 ⁇ effector memory re-expressing CD45RA
  • some embodiments of the disclosure relate to a method of making a population of CAR-expressing immune cells, the method including the steps of: (a) obtaining a liquid sample including a first population of cells including immune cells from a human subject; (b) processing the first population of cells to remove platelets thereby generating a second population of cells including immune cells, wherein the second population of cells includes at least 1 ⁇ 10 4 total cells and wherein less than 20% of the total number of cells in the second population of cells are platelets; (c) seeding a third population in a volume of a first buffer (e.g., first media), wherein the third population of cells is a subset of the second population of cells; (d) transducing the third population of cells with a recombinant polynucleotide encoding a CAR thereby generating a fourth population of cells, wherein the fourth population of cells includes CAR-expressing immune cells; (e) expanding the fourth population of cells to yield a fifth population of cells comprising C
  • a first buffer
  • some embodiments of the disclosure relate to a method of making a population of CAR-expressing immune cells, the method including the steps of: (a) obtaining a liquid sample including a first population of cells including immune cells from a human subject; (b) processing the first population of cells thereby generating a second population of cells including immune cells; (c) seeding a third population of cells in a volume of a first buffer (e.g., first media), wherein the third population of cells is a subset of the second population of cells; (d) transducing the third population of cells with a recombinant polynucleotide encoding a CAR thereby generating a fourth population of cells, wherein the fourth population of cells includes CAR-expressing immune cells; (e) expanding the fourth population of cells yielding a fifth population of cells; (f) removing at least 50% of the volume of the first buffer (e.g., first media) on or before Day 4 after the seeding; and (g) harvesting the fifth population
  • some embodiments of the disclosure relate to a method of making a population of CAR-expressing immune cells, the method including the steps of: (a) obtaining a liquid sample including a first population of cells including immune cells from a human subject; (b) processing the first population of cells thereby generating a second population of cells including immune cells; (c) seeding a third population of cells in a volume of a first buffer (e.g., first media), wherein the third population of cells is a subset of the second population of cells; (d) transducing the third population of cells with a recombinant polynucleotide encoding a CAR thereby generating a fourth population of cells, wherein the fourth population of cells includes CAR-expressing immune cells; (f) expanding the fourth population of cells yielding a fifth population of cells comprising CAR-expressing immune cells; and (g) harvesting the fifth population of cells on Day 5 or later after the seeding in step (c), wherein at least 2.4% of cells in the
  • some embodiments of the disclosure relate to a method of making a population of CAR-expressing immune cells, the method including: (a) obtaining a liquid sample including a first population of cells including immune cells from a human subject; (b) processing the first population of cells to remove platelets thereby generating a second population of cells including immune cells, wherein the second population of cells includes at least 1 ⁇ 10 4 total cells and wherein less than 20% of the total number of cells in the second population of cells are platelets; and (c) seeding a third population of cells in a volume of a first buffer, wherein the third population of cells is a subset of the second population of cells, wherein the third population of cells include at least 2.0 ⁇ 10 8 cells; (d) transducing the third population of cells with a recombinant polynucleotide encoding a CAR thereby generating a fourth population of cells, wherein the fourth population of cells includes CAR-expressing immune cells; (e) expanding the fourth population of cells yielding
  • some embodiments of the disclosure relate to a method of making a population of CAR-expressing immune cells, the method including: (a) obtaining a liquid sample comprising a first population of cells comprising immune cells from a human subject; (b) processing the first population of cells thereby generating a second population of cells comprising immune cells, wherein the second population of cells includes at least 1 ⁇ 10 4 total cells, wherein less than 20% of the total number of cells in the second population of cells are platelets; (c) cryopreserving the second population of cells; (d) on Day 0, thawing the cryopreserved second population of cells comprising immune cells, processing the thawed second population of cells comprising immune cells, and seeding a third population of cells comprising immune cells with a portion of the processed second population of cells comprising immune cells in a volume of a first media, wherein the third population of cells is a subset of the second population of cells, wherein the third population of cells is seeded into a volume of at
  • the method further includes a step of (k) formulating the fifth population of cells for cryopreservation and administration to patients.
  • the third population of cells comprising immune cells is seeded in a volume of the first buffer that comprises growth media.
  • the first buffer is also referred to as the first media.
  • the term “about” indicates the designated value ⁇ up to 10%, up to ⁇ 5%, or up to ⁇ 1%.
  • the term “about” refers to an amount that is near the stated amount by 10%, 5%, or 1%, including increments therein.
  • the term “about” in reference to a percentage refers to an amount that is greater or less the stated percentage by 10%, 5%, or 1%, including increments therein.
  • Chimeric Antigen Receptor or alternatively a “CAR” refers to a synthetic receptor encoded by one or more polypeptides including at least the following functional domains: an extracellular antigen-binding domain, a transmembrane domain or a hinge and transmembrane domain, and an intracellular or cytoplasmic signaling domain, which when expressed in an immune cell, provides the cell with the ability to specifically bind a target cell expressing a particular antigen, for example a cancer cell, and with the ability to generate a signal (without engaging the T cell receptor) via the intracellular signaling domain following engagement of the antigen-binding domain by its cognate antigen.
  • a CAR includes at least an extracellular antigen binding domain, a transmembrane domain and an intracellular or cytoplasmic signaling domain (also referred to herein as “an intracellular signaling domain”) including a functional signaling domain derived from one or more primary T cell activation molecules and/or one or more costimulatory molecules as defined below.
  • antibody or “antigen-binding domain” herein is used in the broadest sense to encompass monoclonal antibodies, bispecific antibodies, intact antibodies and functional (antigen-binding) antibody fragments thereof, including fragment antigen binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single chain antibody fragments, including single chain variable fragments (sFv or scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments.
  • Fab fragment antigen binding
  • F(ab')2 fragments fragment antigen binding
  • Fab' fragments fragment antigen binding
  • Fv fragments fragment antigen binding
  • rIgG recombinant IgG fragments
  • single chain antibody fragments including single chain variable fragments (sFv or scFv) fragments.
  • single domain antibodies e.g., sdAb, sdF
  • the term encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv.
  • antibody should be understood to encompass functional antibody fragments thereof.
  • the term also encompasses intact or full- length antibodies, including antibodies of any isotype, class or sub-class, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD.
  • the antibody can include a human IgG1 constant region.
  • the antibody can include a human IgG4 constant region.
  • the term “scFv” or “single chain variable fragment” refers to a fusion protein including at least one antibody fragment including a variable region of a light chain and at least one antibody fragment including a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked, e.g., via a synthetic linker, e.g., a short flexible peptide or polypeptide linker, and capable of being expressed as a single chain polypeptide, wherein the scFv retains the specificity of the intact antibody from which it is derived.
  • an scFv may encode the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may include VL-linker-VH or may include VH-linker-VL.
  • the antigen-binding domain of a CAR may exist in a variety of forms where the antigen binding domain is expressed as part of a contiguous polypeptide chain including, for example, a single domain antibody fragment (sdAb), a single chain antibody (scFv) and a humanized antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci.
  • sdAb single domain antibody fragment
  • scFv single chain antibody
  • the antigen binding domain of a CAR includes an antibody fragment.
  • the CAR includes an antibody fragment that includes a scFv.
  • binding domain refers to a protein, e.g., an immunoglobulin polypeptide or fragment thereof, including at least one immunoglobulin variable domain sequence.
  • binding domain or “antibody molecule” encompasses antibodies and antibody fragments.
  • an antibody molecule is a multispecific antibody molecule, e.g., it includes a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope.
  • a multispecific antibody molecule is a bispecific antibody molecule.
  • a bispecific antibody has specificity for no more than two antigens.
  • a bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence which has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope.
  • the term “heavy chain,” refers to the longer of the two types of polypeptides present in antibody molecules in their naturally occurring conformations, and which normally determines the class to which the antibody belongs.
  • the term “light chain,” refers to the shorter of the two types of polypeptides present in antibody molecules in their naturally occurring conformations. There are two major antibody light chain polypeptide isotypes, designated kappa ( ⁇ ) and lambda ( ⁇ ).
  • the term “antigen” or “Ag” refers to a molecule capable of provoking an immune response. This immune response may be innate or adaptive, and may involve either antibody production, the activation of specific immunologically-competent cells, or both.
  • antigens can be derived from (e.g., expressed from) recombinant or genomic DNA.
  • any DNA which includes a nucleotide sequence or a partial nucleotide sequence encoding a protein or protein fragment can elicit an immune response and therefore encodes an “antigen” as that term is used herein.
  • an antigen need not be encoded solely by a full-length nucleotide sequence of a gene.
  • an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample or might be macromolecule besides a polypeptide.
  • a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell, or a fluid including other biological components.
  • cancer refers to a disease characterized by the presence of cells possessing several characteristics typical of cancer-causing cells, such as uncontrolled growth/proliferation of cells (e.g., aberrant cells). Cancer cells can but do not always aggregate into a mass, such as a tumor, or can exist alone within a subject.
  • a tumor can be a solid tumor, a liquid tumor a soft tissue tumor, or a metastatic lesion. Cancers can grow locally or in distant locations following metastasis, a process by which cancer cells are shed from a tumor and spread through the bloodstream and lymphatic system to other parts of the body.
  • cancers may be solid (e.g., cancer of the lung, colon, or breast) or liquid (e.g., cancer of myeloid cells (e.g., myeloma) or B cells (e.g., diffuse large B cell lymphoma).
  • myeloid cells e.g., myeloma
  • B cells e.g., diffuse large B cell lymphoma
  • cancers are described herein and include but are not limited to, lymphoma and leukemia.
  • cancer encompasses solid and liquid, e.g., diffuse or circulating, tumors.
  • cancer includes premalignant, as well as malignant cancers and tumors.
  • “Derived from” as that term is used herein indicates a relationship between a first and a second molecule. It generally refers to structural similarity between the first molecule and a second molecule and does not connote or include a process or source limitation on a first molecule that is derived from a second molecule. For example, in the case of an intracellular signaling domain that is derived from a CD3 ⁇ molecule, the intracellular signaling domain retains sufficient CD3 ⁇ structure such that it has the required function, namely, the ability to generate a signal under the appropriate conditions.
  • relapse refers to reappearance of a disease (e.g., cancer) after an initial period of remission or response to an anti-cancer treatment, e.g., after prior treatment with a therapy, e.g., cancer therapy (e.g., complete response or partial response).
  • a disease e.g., cancer
  • a therapy e.g., cancer therapy
  • the initial period of responsiveness may involve the level of cancer cells falling below a certain threshold, e.g., below 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or below the limit of detection.
  • the reappearance may involve the level of cancer cells rising above a certain threshold, e.g., above 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%.
  • the reappearance may involve, e.g., a reappearance of blasts in the blood, bone marrow (BM) (>5%), or any extramedullary site, after a complete response.
  • BM bone marrow
  • a complete response may involve ⁇ 5% blasts in the bone marrow.
  • a response e.g., complete response or partial response
  • the initial period of responsiveness lasts at least 1, 2, 3, 4, 5, or 6 days; at least 1, 2, 3, or 4 weeks; at least 1, 2, 3, 4, 6, 8, 10, or 12 months; or at least 1, 2, 3, 4, or 5 years.
  • Refractory refers to a disease, e.g., cancer, that does not respond to a treatment.
  • a refractory cancer can be resistant to a treatment before or at the beginning of the treatment.
  • the refractory cancer can become resistant during a treatment.
  • a refractory cancer is also called a resistant cancer.
  • the term “stimulation,” refers to a primary response induced by binding of a stimulatory molecule (e.g., a TCR/CD3 complex or CAR) with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR/CD3 complex or signaling domains of the CAR. Stimulation can mediate altered expression of certain molecules.
  • a stimulatory molecule e.g., a TCR/CD3 complex or CAR
  • Stimulation can mediate altered expression of certain molecules.
  • “Immune cell,” as that term is used herein, refers to cells that are involved in an adaptive immune response, e.g., in the promotion of an immune effector response.
  • Exemplary immune cell types include T cells, e.g., alpha/beta T cells and gamma/delta T cells, regulatory T cells (Tregs) B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, macrophages, and myeloid-derived phagocytes.
  • Tregs regulatory T cells
  • NK natural killer
  • NKT natural killer T
  • mast cells macrophages
  • macrophages e.g., myeloid-derived phagocytes.
  • expression vector refers to a vector including a recombinant polynucleotide to be expressed and various expression control sequences operatively linked to the recombinant nucleotide sequence.
  • An expression vector includes sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system.
  • expression vectors as used herein encompasses those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viral vectors (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) capable of incorporating and expressing the recombinant polynucleotide.
  • cosmids e.g., naked or contained in liposomes
  • viral vectors e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses
  • lentivirus refers to members of a genus in the Retroviridae family.
  • Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a considerable amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses.
  • the term “lentiviral vector” refers to a vector derived from at least a portion of a lentivirus genome, including especially a self-inactivating lentiviral vector as provided in Milone et al., Mol. Ther.17(8): 1453-1464 (2009). Nonclinical types of lentiviral vectors are also available and would be known to one skilled in the art.
  • isolated used in reference to a substance means it has been removed from its natural state.
  • a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.”
  • An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
  • a “subject” or an “individual” includes animals, such as human (e.g., human individuals). In some embodiments, a “subject” or “individual” is a patient under the care of a physician.
  • the subject can be a human patient or an individual who has, is at risk of having, or is suspected of having a health condition of interest (e.g., a cancer) and/or one or more symptoms of the health condition.
  • a health condition of interest e.g., a cancer
  • the subject can also be an individual who is diagnosed with a risk of the health condition of interest at the time of diagnosis or later.
  • the terms “treat”, “treatment” and “treating” refer to the administration of a drug or therapeutic resulting in a reduction or amelioration of the progression, severity and/or duration of a disorder (e.g., a neoplastic disorder such as cancer), or the amelioration of one or more symptoms (e.g., one or more discernible symptoms) of a proliferative disorder resulting from the administration of one or more therapies (e.g., one or more therapeutic agents such as a CAR-expressing immune cell as provided herein) into a subject in need thereof, e.g., a patient.
  • a disorder e.g., a neoplastic disorder such as cancer
  • one or more symptoms e.g., one or more discernible symptoms
  • therapies e.g., one or more therapeutic agents such as a CAR-expressing immune cell as provided herein
  • the terms “treat”, “treatment” and “treating” refer to the amelioration of at least one measurable physical parameter of a neoplastic disorder, such as growth of a tumor, not necessarily discernible by the subject, e.g., patient.
  • the terms “treat”, “treatment” and “treating” refer to the inhibition of the progression of a proliferative disorder, either physically by, e.g., stabilization of a discernible symptom, physiologically by, e.g., stabilization of a physical parameter of the disease or disorder, or both.
  • the terms “treat”, “treatment” and “treating” refer to the reduction or stabilization of tumor size or cancer cell count.
  • transfected or “transformed” or “transduced” refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell.
  • a “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid, e.g., a lentiviral expression vector encoding a CAR.
  • the term “transduced”, “transfected”, or “transformed” cell includes the primary subject cell and its progeny.
  • the methods described herein may include the steps shown in Figure 1.
  • the methods may include one or more of the following steps: cell collection, cell processing, cell cryopreservation, cell thawing, cell seeding, cell transduction, cell expansion, cell washing, media exchange, cell harvesting, cell packaging, and cell storing.
  • Day 0 is the day on which a processed, cryopreserved leukapheresis or apheresis product is thawed and the method is initiated.
  • a method of making a population of chimeric antigen receptor CAR-expressing immune cells including the steps of: (a) obtaining a liquid sample including a first population of cells including immune cells from a human subject; (b) processing the first population of cells to remove platelets thereby generating a second population of cells including immune cells, wherein the second population of cells includes at least 1 ⁇ 10 4 total cells and wherein less than 20% of the total number of cells in the second population of cells are platelets; (c) seeding a third population of cells including immune cells in a volume of a first buffer, wherein the third population of cells is a subset of the second population of cells; (d) transducing the third population of cells including immune cells with a recombinant polynucleotide encoding a CAR thereby generating a fourth population of cells, wherein the fourth population of cells includes CAR- expressing immune cells; (e) expanding the fourth population of cells to yield a fifth population of cells
  • the method further includes a step of removing at least 50% of the volume of the first buffer on or before Day 4 after seeding. In some embodiments, at least 2.4% of cells in the fifth population of cells are CCR7+CD45RA+ immune cells. In some embodiments, the liquid sample including a first population of cells including immune cells from a human subject comprises a leukapheresis product or an apheresis product.
  • a method of making a population of CAR- expressing immune cells including the steps of: (a) obtaining a liquid sample including a first population of cells including immune cells from a human subject; (b) processing the first population of cells thereby generating a second population of cells including immune cells; (c) seeding a third population of cells including immune cells in a volume of a first buffer, wherein the third population of cells is a subset of the second population of cells; (d) transducing the third population of cells with a recombinant polynucleotide encoding a CAR thereby generating a fourth population of cells, wherein the fourth population of cells includes CAR-expressing immune cells; (e) expanding the fourth population of cells including CAR-expressing immune cells yielding a fifth population of cells including CAR-expressing immune cells; (f) removing at least 50% of the volume of the first buffer on or before Day 4 after the seeding in step (c); and (g) harvesting the fifth
  • At least 2.4% of cells in the fifth population of cells are CCR7+CD45RA+ immune cells.
  • the liquid sample including a first population of cells including immune cells from a human subject comprises an apheresis product.
  • a method of making a population of CAR- expressing immune cells including the steps of: (a) obtaining a liquid sample including a first population of cells including immune cells from a human subject; (b) processing the first population of cells thereby generating a second population of cells including immune cells; (c) seeding a third population of cells including immune cells in a volume of a first buffer, wherein the third population of cells is a subset of the second population of cells; (d) transducing the third population of cells with a recombinant polynucleotide encoding a CAR thereby generating a fourth population of cells, wherein the fourth population of cells includes CAR-expressing immune cells; (f) expanding the fourth population of cells to yield a fifth population of cells comprising CAR-expressing immune cells; and (g) harvesting the fifth population of cells on Day 5 or later after the seeding of step (c), wherein at least 2.4% of cells in the fifth population of cells are CCR7
  • the liquid sample including a first population of cells including immune cells from a human subject comprises a leukapheresis product or an apheresis product.
  • a method of making a population of CAR- expressing immune cells including: (a) obtaining a liquid sample including a first population of cells including immune cells from a human subject; (b) processing the first population of cells to remove platelets thereby generating a second population of cells including immune cells, wherein the second population of cells includes at least 1 ⁇ 10 4 total cells and wherein less than 20% of the total number of cells in the second population of cells are platelets; and (c) seeding a third population of cells including immune cells in a volume of a first buffer, wherein the third population of cells is a subset of the second population of cells, wherein the third population of cells includes at least 2.0 ⁇ 10 8 cells; (d) transducing the third population of cells with a recombinant polynucleot
  • the liquid sample including a first population of cells including immune cells from a human subject comprises an apheresis product.
  • METHODS OF MAKING CAR-EXPRESSING IMMUNE CELLS WITH REDUCED AMOUNT OF PLATELETS [0168]
  • described herein is a method of making a population of chimeric antigen receptor CAR-expressing immune cells, the method including the steps of: (a) obtaining a liquid sample including a first population of cells including immune cells from a human subject; (b) processing the first population of cells to remove platelets thereby generating a second population of cells including immune cells, wherein the second population of cells includes at least 1 ⁇ 10 4 total cells and wherein less than 20% of the total number of cells in the second population of cells are platelets.
  • the processing of the first population of cells in step (b) includes diluting the liquid sample including the first population of cells with a first buffer, thereby generating a diluted liquid sample including the first population of cells. In some embodiments, the liquid sample is diluted prior to removing platelets.
  • the liquid sample has a total volume of at least 50 mL, e.g., about 50 mL to 10 L, about 50 mL to 1 L, about 50 mL to 500 mL, about 100 mL to 600 mL, about 200 mL to 700 mL, about 300 mL to 800 mL, about 400 mL to 900 mL, about 500 mL to 1 L, about 600 mL to 1.5 L, or about 600 mL to 2 L.
  • 50 mL e.g., about 50 mL to 10 L, about 50 mL to 1 L, about 50 mL to 500 mL, about 100 mL to 600 mL, about 200 mL to 700 mL, about 300 mL to 800 mL, about 400 mL to 900 mL, about 500 mL to 1 L, about 600 mL to 1.5 L, or about 600 mL to 2 L.
  • the liquid sample has a total volume of at least 50 mL, at least 100 mL, at least 200 mL, at least 250 mL, at least 300 mL, at least 400 mL, at least 500 mL, at least 600 mL, at least 700 mL, at least 800 mL, at least 900 mL, at least 1 L, or at least 2 L.
  • the step of diluting the liquid sample including the first population of cells includes adding a volume of the first buffer to the liquid sample, wherein the volume of the first buffer added to the liquid sample is at least the same as the total volume of liquid sample prior to diluting (i.e., the dilution is at least 1:1).
  • the dilution buffer may include human serum albumin (HSA), Plasma-Lyte A, phosphate buffered saline, sodium chloride, sodium bicarbonate buffer, glutathione, biotin, vitamin B12, inositol, choline, L-glutamine, sodium pyruvate, glucose, or a combination thereof.
  • the first buffer includes human serum albumin (HSA).
  • the first buffer includes Plasma-Lyte A.
  • the first buffer includes equal volumes of Plasma-Lyte A and 4% (w/v) human serum albumin.
  • the concentration of HSA may be between about 0.25-2% (w/v), about 1-4% (w/v), about 2-6% (w/v), about 4-8% (w/v), about 5-10% (w/v), or about 0.25% (w/v), about 0.5% (w/v), about 0.75% (w/v), about 1% (w/v), about 1.25% (w/v), about 1.5% (w/v), about 1.75% (w/v), about 2% (w/v), about 2.25% (w/v), about 2.5% (w/v), about 2.75% (w/v), about 3% (w/v), about 3.25% (w/v), about 3.5% (w/v), about 3.75% (w/v), about 4% (w/v), about 4.25% (w/v), about 4.5% (w/v), about 4.75% (w/v), about 5% (w/v), about 5% (w/v), about 5.25% (w/v), about 5.5% (w/v), about 5.75% (w/
  • the volume of the first buffer added to the liquid sample may be at least about 5mL, at least about 10mL, at least about 15mL, at least about 20mL, at least about 25mL, at least about 30mL, at least about 40mL, at least about 50mL, at least about 75mL, at least about 100mL, at least about 150mL, at least about 200mL, at least about 250mL, at least about 300mL, at least about 400mL, at least about 500mL, at least about 600mL, at least about 700mL, at least about 800mL, at least about 900mL, at least about 1L, at least about 1.5L, at least about 2L, at least about 2.5L, at least about 3L, at least about 3.5L, at least about 4L, at least about 4.5L, or at least about 5L.
  • the diluted liquid sample has a total volume that is at least twice (2X) the total volume of the liquid sample prior to diluting, e.g., at least 2X to 10X, at least 2X to 5X, at least 2X to 4X, at least 2X to 3X, at least 3X to 7X, at least 4X to 8X, at least 5X to 9X, at least 6X to 10X, at least 3X to 5X, or at least 4X to 6X.
  • the diluted liquid sample has a total volume that is at least two times (2X), at least three times (3X), or at least five times (5X) the total volume of the liquid sample prior to diluting.
  • the diluted liquid sample has a total volume that is at least three times (3X) the total volume of the liquid sample prior to diluting. In some embodiments, the diluted liquid sample has a total volume that is at least four (4X) times the total volume of the liquid sample prior to diluting. In some embodiments, the diluted liquid sample has a total volume that is at least five (5X) times the total volume of the liquid sample prior to diluting. [0174] In some embodiments, the processing of the first population of cells includes the steps of washing the first population of cells, concentrating the first population of cells, and eluting and/or resuspending the first population of cells, thereby generating the second population of cells.
  • concentrating the first population of cells is accomplished using an automated centrifugation system.
  • the automated centrifugation system concentrates the first population of cells by elutriation.
  • the particle size filter has a size cutoff sufficient to retain immune cells on the filter.
  • concentrating the first population of cells is accomplished using elutriation to keep larger immune cells in the centrifuge chamber and elute out smaller particles.
  • the wash buffer may include HSA, Plasma-Lyte A, phosphate buffered saline, sodium chloride, sodium bicarbonate buffer, glutathione, biotin, vitamin B12, inositol, choline, L-glutamine, sodium pyruvate, glucose, or a combination thereof.
  • the cell population may be concentrated after washing. The washing step may result in a higher concentration of cells than the concentration of cells on the automated instrument.
  • the cell population may be eluted and/or resuspended from the automated cell processing instrument after concentration.
  • the cell population is transferred to a sterile container after elution/resuspension.
  • the transfer is performed manually. In some embodiments, the transfer is automated.
  • the sterile container may include a bag, a bottle, a tube, a glass jar, or a plastic container. In some embodiments, before transfer to a sterile container, the cell population may be diluted after elution/resuspension into a dilution buffer.
  • the dilution buffer may include dimethyl sulfoxide, sucrose, sodium hydroxide, potassium hydroxide, fructose, HSA, Plasma-Lyte A, phosphate buffered saline, sodium chloride, sodium bicarbonate buffer, glutathione, biotin, vitamin B12, inositol, choline, L-glutamine, sodium pyruvate, glucose, or a combination thereof.
  • the volume of dilution buffer added to the cell population after elution and/or resuspension may be at least about 5mL, at least about 10mL, at least about 15mL, at least about 20mL, at least about 25mL, at least about 30mL, at least about 40mL, at least about 50mL, at least about 75mL, at least about 100mL, at least about 150mL, at least about 200mL, at least about 250mL, at least about 300mL, at least about 400mL, at least about 500mL, at least about 600mL, at least about 700mL, at least about 800mL, at least about 900mL, at least about 1L, at least about 1.5L, at least about 2L, at least about 2.5L, at least about 3L, at least about 3.5L, at least about 4L, at least about 4.5L, or at least about 5L.
  • the volume of dilution buffer added to the cell population after elution and/or resuspension is more than the volume of the cell population. In some embodiments, the volume of dilution buffer added to the cell population is less than the volume of the cell population. In some embodiments, the volume of the dilution buffer added to the cell population is the same as the volume of the cell population.
  • the cell population may be cryopreserved after transfer to a sterile container. In some embodiments, the cell population may be cryopreserved after dilution and transfer to a sterile container. In some embodiments, the sterile packaged cell population is cryopreserved using a controlled rate freezer.
  • the sterile packaged cell population is cryopreserved by immersion in liquid nitrogen.
  • the processing of the first population of cells in step (b) further includes a step of adding a second buffer to the second population of cells and cryopreserving the second population of cells.
  • the second buffer includes one or more of the following: phosphate-buffered saline, dimethyl sulfoxide, sodium hydroxide, potassium hydroxide, and sucrose.
  • the second buffer includes phosphate buffered saline, dimethyl sulfoxide, sodium hydroxide, potassium hydroxide, and sucrose.
  • the cryopreserved second population of cells is thawed before it is transduced with a recombinant polynucleotide encoding a CAR in step (d).
  • Day 0 is the day on which a processed, cryopreserved leukapheresis or apheresis product is thawed and the method is initiated.
  • the recombinant polynucleotide comprises a lentiviral expression vector encoding a chimeric antigen receptor.
  • the lentiviral expression vector is manufactured in an adherent cell culture.
  • the lentiviral expression vector is manufactured in a suspension cell culture.
  • the second population of cells after processing may include fewer platelets compared to the cell sample before processing.
  • the amount of platelets in the second cell population after processing may comprise less than 60%, less than 55%, less than 50%, less than 45%, less than, 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2% of the amount of platelets present in the sample before processing.
  • the amount of viable cells left in the sample after processing may be at least about 65%, at least about 68%, at least about 70%, at least about 72%, at least about 74%, at least about 76%, at least about 78%, at least about 80%, at least about 82%, at least about 84%, at least about 86%, at least about 88%, at least about 90%, at least about 92%, at least about 94%, at least about 96%, at least about 98%, or at least about 100% of the amount of viable cells in the sample before processing.
  • the cell recovery after processing may be at least about 65%, at least about 68%, at least about 70%, at least about 72%, at least about 74%, at least about 76%, at least about 78%, at least about 80%, at least about 82%, at least about 84%, at least about 86%, at least about 88%, at least about 90%, at least about 92%, at least about 94%, at least about 96%, at least about 98%, or at least about 100% of the cells in the sample before processing.
  • one aspect of the disclosure relates to a method of making a population of CAR-expressing immune cells, the method including the steps of: (a) obtaining a liquid sample including a first population of cells including immune cells from a human subject; (b) processing the first population of cells thereby generating a second population of cells including immune cells; (c) seeding a third population of cells in a volume of a first buffer, wherein the third population of cells is a subset of the second population of cells; (d) transducing the third population of cells with a recombinant polynucleotide encoding a CAR thereby generating a fourth population of cells, wherein the fourth population of cells includes CAR-expressing immune cells; (e) expanding the fourth population of cells to yield a fifth
  • Non-limiting exemplary embodiments of the methods in accordance with this aspect and other aspects of the disclosure may include one or more of the following features.
  • the processing of the first population of cells in step (b) includes a step of adding a second buffer to the second population of cells and cryopreserving the second population of cells.
  • at least 2.4% of cells in the fifth population of cells are CCR7+CD45RA+ immune cells.
  • the liquid sample including a first population of cells including immune cells from a human subject comprises a leukapheresis product or an apheresis product.
  • Another aspect of the disclosure relates to a method of making a population of CAR- expressing immune cells, the method including the steps of: (a) obtaining a liquid sample including a first population of cells including immune cells from a human subject; (b) processing the first population of cells thereby generating a second population of cells including immune cells and optionally cryopreserving the second population of cells; (c) seeding a third population of cells in a volume of a first buffer, wherein the third population of cells is a subset of the second population of cells; (d) transducing the third population of cells with a recombinant polynucleotide encoding a CAR thereby generating a fourth population of cells, wherein the fourth population of cells includes CAR-expressing immune cells; (f) expanding the fourth population of cells yielding a fifth population of cells; and (g) harvesting the fifth population of cells on Day 5 or later after the seeding in step (c), wherein at least 2.4% of cells in the fifth population of cells are CCR7
  • the processing of the first population of cells in step (b) includes a step of adding a second buffer to the second population of cells and cryopreserving the second population of cells.
  • the liquid sample including a first population of cells including immune cells from a human subject comprises an apheresis product.
  • Another aspect of the disclosure relates a method of making a population of CAR- expressing immune cells, the method including: (a) obtaining a liquid sample including a first population of cells including immune cells from a human subject; (b) processing the first population of cells to remove platelets thereby generating a second population of cells including immune cells, wherein the second population of cells includes at least 1 ⁇ 10 4 total cells, wherein less than 20% of the total number of cells in the second population of cells are platelets and optionally cryopreserving the second population of cells; and (c) seeding a third population of cells with a portion of the second population of cells in a volume of a first media or thawing the cryopreserved second population of cells on Day 0 and seeding a third population of cells with a portion of the thawed cryopreserved second population of cells in a volume of a first media, wherein the third population of cells is a subset of the second population of cells, wherein the third population of cells includes at least 2.0
  • the processing of the first population of cells in step (b) includes a step of adding a second buffer to the second population of cells and cryopreserving the second population of cells.
  • the liquid sample including a first population of cells including immune cells from a human subject comprises an apheresis product.
  • at least 2.4% of cells in the fifth population of cells are CCR7+CD45RA+ immune cells.
  • at least 2.5%, 2.8%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, or 5.5% of cells in the fifth population of cells are CCR7+CD45RA+ immune cells.
  • At least 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.9%, 5.0%, 5.1%, 5.2%, 5.3%, 5.4%, or 5.5% of cells in the fifth population of cells are CCR7+ CD45RA+ immune cells.
  • the CCR7+ CD45RA+ immune cells are also CD3+.
  • the CCR7+CD45RA+CD3+ immune cells are also CD8+ (i.e., CCR7+CD45RA+CD3+CD8+).
  • At least 78.3%, 78.5%, 79.0%, 79.5%, 80%, 80.5%, 81.0%, 81.4%, 81.5%, 82.0%, 83.0%, 84.0%, 85.0%, 86.0%, or 87.0% of the cells in the fifth population of cells are CCR7+CD45RA- immune cells.
  • the CCR7+CD45RA+CD3+ immune cells are also CD4+ (i.e., CCR7+CD45RA+CD3+CD4+).
  • At least 78.0%, 78.1%, 78.2%, 78.3%, 78.4%, 78.5%, 78.6%, 78.7%, 78.8%, 78.9%, 79.0%, 79.1%, 79.2%, 79.3%, 79.4%, 79.5%, 79.6%, 79.7%, 79.8%, 79.9%, 80.0%, 80.1%, 80.2%, 80.3%, 80.4%, 80.5%, 80.6%, 80.7%, 80.8%, 80.9%, or 81.0% of cells in the fifth population of cells are CCR7+ CD45RA- immune cells.
  • At least 81.0%, 81.1%, 81.2%, 81.3%, 81.4%, 81.5%, 81.6%, 81.7%, 81.8%, 81.9%, 82.0%, 82.0%, 82.1%, 82.2%, 82.3%, 82.4%, 82.5%, 82.6%, 82.7%, 82.8%, 82.9%, 83.0%, 83.1%, 83.2%, 83.3%, 83.4%, 83.5%, 83.6%, 83.7%, 83.8%, 83.9%, or 84.0% of cells in the fifth population of cells are CCR7+ CD45RA- immune cells.
  • At least 84.0%, 84.1%, 84.2%, 84.3%, 84.4%, 84.5%, 84.6%, 84.7%, 84.8%, 84.9%, 85.0%, 85.0%, 85.1%, 85.2%, 85.3%, 85.4%, 85.5%, 85.6%, 85.7%, 85.8%, 85.9%, 86.0%, 86.1%, 86.2%, 86.3%, 86.4%, 86.5%, 86.6%, 86.7%, 86.8%, 86.9%, or 87.0% of cells in the fifth population of cells are CCR7+ CD45RA- immune cells.
  • At most 16.1%, 16.0%, 15.0%, 14.0%, 13.0%, 12.0%, 11.0%, 10.0%, 9.0%, 8.0%, 7.5%, 7%, 6.5%, 6.0%, 5.5%, 5.0%, 4.5%, 4.0%, 3.5%, or 3.4% of the cells in the fifth population of cells are CCR7-CD45RA- immune cells.
  • the harvested immune cells in the fifth population in (e) include at least 2.3%, at least 2.4%, at least 2.5%, at least 2.6%, at least 2.7%, at least 2.8%, at least 2.9%, at least 3%, at least 3.1%, at least 3.2%, at least 3.3%, at least 3.4%, at least 3.5%, at least 3.6%, at least 3.7%, at least 3.8%, at least 3.9%, at least 4%, at least 4.1%, at least 4.2%, at least 4.3%, at least 4.4%, at least 4.5%, at least 4.6%, at least 4.7%, at least 4.8%, at least 4.9%, at least 5%, at least 5.1%, at least 5.2%, at least 5.3%, at least 5.4%, at least 5.5%, at least 5.6%, at least 5.7%, at least 5.8%, at least 5.9%, at least 6%, at least 6.1%, at least 6.2%, at least 6.3%, at least 6.4%, at least 6.5%, at least 6.6%, at least 6.7%, at least 6.8%, at least 6.9%, at least 6%, at
  • the harvested immune cells in the fifth population in (e) include at least 78.3%, at least 78.5%, at least 79.0%, at least 79.5%, at least 80.0%, at least 80.5%, at least 81.0%, at least 81.4%, at least 81.8%, at least 82.4%, at least 82.8%, at least 83.4%, at least 83.8%, at least 84.4%, at least 84.8%, at least 85.4%, at least 85.8%, at least 86.4%, at least 86.8%, at least 87.4%, at least 87.8%, at least 88.4%, at least 88.8%, at least 89.4%, at least 89.8%, at least 90.4%, at least 90.8%, at least 91.4%, at least 91.8%, at least 92.4%, at least 92.8%, at least 93.4%, at least 93.8%, at least 94.4%, at
  • the harvested immune cell population in (e) includes fewer than 16%, fewer than 15.8%, fewer than 15.6%, fewer than 15.4%, fewer than 15.2%, fewer than 15%, fewer than 14.8%, fewer than 14.6%, fewer than 14.4%, fewer than 14.2%, fewer than 14%, fewer than 13.8%, fewer than 13.6%, fewer than 13.4%, fewer than 13.2%, fewer than 13%, fewer than 12.8%, fewer than 12.6%, fewer than 12.4%, fewer than 12.2%, fewer than 12%, fewer than 11.8%, fewer than 11.6%, fewer than 11.4%, fewer than 11.2%, fewer than 11%, fewer than 10.8%, fewer than 10.6%, fewer than 10.4%, fewer than 10.2%, fewer than 10%, fewer than 9.8%, fewer than 9.6%, fewer than 9.4%, fewer than 9.2%, fewer than 9%, fewer than 8.8%, fewer than 8.6%, fewer than 8.4%, fewer than 8.
  • less than 60%, less than 55%, less than 50%, less than 45%, less than, 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 18%, less than 15%, less than 12%, less than 10%, less than 8%, less than 5%, or less than 2% of the total number of cells in the second population of cells are platelets.
  • the efficiency of transduction of the third population of cells is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%.
  • the efficiency of transduction of the third population of cells is at most 60%, at most 55%, at most 50%, at most 45%, at most 40%, at most 35%, at most 30%, at most 25%, at most 20%, at most 15%, at most 10%.
  • the third population of cells is transduced at a multiplicity of infection (MOI) of at least 0.5, at least 1, at least 1.5, at least 2, at least 2.5, at least 3, at least 3.5, at least 4.0, at least 4.5, at least 5.0, or at least 5.5.
  • MOI multiplicity of infection
  • the third population of cells is transduced at an MOI of at most 0.5, at most 1, at most 1.5, at most 2, at most 2.5, at most 3, at most 3.5, at most 4.0, at most 4.5, at most 5.0, or at most 5.5.
  • the third population of cells is enriched for CD3+CD4+ T cells before seeding.
  • the third population of cells is enriched for CD3+CD8+ T cells before seeding.
  • the third population of cells is enriched for CD4+CD8+ T cells before seeding.
  • the third population of cells is activated by at least one cytokine.
  • the at least one cytokine includes IL-2, IL-4, IL-7, IL-9, IL- 15, IL-21, or a combination thereof. In some embodiments, the at least one cytokine includes a combination of IL-7 and IL-15.
  • the third population of cells is seeded in a first buffer. In some embodiments, the first buffer includes 12.5ng/mL IL-7, 12.5ng/mL IL-15, Plasma-Lyte A, and 4% human serum albumin. [0196] In some embodiments, the third population of cells includes at least 1.1 ⁇ 10 8 cells from the second population of cells.
  • the third population of cells includes at least at least 2.5 ⁇ 10 6 , at least 5 ⁇ 10 6 , at least 7.5 ⁇ 10 6 , at least 1 ⁇ 10 7 , at least 2.5 ⁇ 10 7 , at least 5 ⁇ 10 7 , at least 7.5 ⁇ 10 7 , at least 1 ⁇ 10 8 , 1.2 ⁇ 10 8 , at least 1.4 ⁇ 10 8 , at least 1.6 ⁇ 10 8 , at least 1.8 ⁇ 10 8 , at least 2.0 ⁇ 10 8 , at least 2.2 ⁇ 10 8 , at least 2.4 ⁇ 10 8 , at least 2.6 ⁇ 10 8 , at least 2.8 ⁇ 10 8 , or at least 3.0 ⁇ 10 8 cells from the second population of cells.
  • the third population of cells includes at least about 2 ⁇ 10 8 cells from the second population of cells. In some embodiments, the third population of cells includes at least about 3 ⁇ 10 8 cells from the second population of cells. In some embodiments, the third population of cells includes at least about 1 ⁇ 10 9 cells, at least about 1.05 ⁇ 10 9 cells, at least about 1.1 ⁇ 10 9 cells, at least about 1.15 ⁇ 10 9 cells, at least about 1.2 ⁇ 10 9 cells, at least about 1.25 ⁇ 10 9 cells, at least about 1.3 ⁇ 10 9 cells, at least about 1.35 ⁇ 10 9 cells, at least about 1.4 ⁇ 10 9 cells, at least about 1.45 ⁇ 10 9 cells, at least about 1.5 ⁇ 10 9 cells, at least about 1.55 ⁇ 10 9 cells, at least about 1.6 ⁇ 10 9 cells, at least about 1.65 ⁇ 10 9 cells, at least about 1.7 ⁇ 10 9 cells, at least about 1.75 ⁇ 10 9 cells, at least about 1.8 ⁇ 10 9 cells, at least about 1.85 ⁇ 10 9 cells, at least about 1.9 ⁇ 10 9 cells, at least about 1.
  • the fifth population of cells is harvested on Day 5, Day 6, Day 7, Day 8, Day 9, Day 10, Day 11, Day 12, Day 13, Day 14, or Day 15. In some embodiments, the fifth population of cells is harvested on Day 5, Day 6, Day 7, Day 8, or Day 9. In some embodiments, the fifth population of cells is harvested on Day 5, 7, and/or Day 9. In some embodiments, the fifth population of cells is harvested on Day 5. In some embodiments, the fifth population of cells is harvested on Day 6. In some embodiments, the fifth population of cells is harvested on Day 7. In some embodiments, the fifth population of cells is harvested on Day 8. In some embodiments, the fifth population of cells is harvested on Day 9.
  • the harvested fifth population of cells includes at least 1 ⁇ 10 3 , at least 1 ⁇ 10 4 , at least 1 ⁇ 10 5 , at least 1 ⁇ 10 6 , at least 1 ⁇ 10 7 , at least 1 ⁇ 10 8 , at least 2.2 ⁇ 10 8 , at least 2.4 ⁇ 10 8 , at least 2.6 ⁇ 10 8 , at least 2.8 ⁇ 10 8 , or at least 3.0 ⁇ 10 8 CAR-expressing immune cells.
  • at least 70%, 75%, 80%, 85%, or 90% of the cells in the fifth population of cells are viable.
  • the fifth population of cells is cryopreserved after harvesting.
  • the number of CAR+ cells harvested in step (f) or step (g) may be 1.0 ⁇ 10 8 cells, 1.5 ⁇ 10 8 cells, at least about 2 ⁇ 10 8 cells, at least about 2.5 ⁇ 10 8 cells, at least about 3 ⁇ 10 8 cells, at least about 3.5 ⁇ 10 8 cells, at least about 4 ⁇ 10 8 cells, at least about 4.5 ⁇ 10 8 cells, at least about 5 ⁇ 10 8 cells, at least about 5.5 ⁇ 10 8 cells, at least about 6 ⁇ 10 8 cells, at least about 6.5 ⁇ 10 8 cells, at least about 7 ⁇ 10 8 cells, at least about 7.5 ⁇ 10 8 cells, at least about 8 ⁇ 10 8 cells, at least about 8.5 ⁇ 10 8 cells, at least about 9 ⁇ 10 8 cells, at least about 9.5 ⁇ 10 8 cells, at least about 1 ⁇ 10 9 cells, at least about 1.05 ⁇ 10 9 cells, at least about 1.1 ⁇ 10 9 cells, at least about 1.15 ⁇ 10 9 cells, at least about 1.2 ⁇ 10 9 cells, at least about 1.25 ⁇ 10 9 cells, at least about
  • the percentage of CAR+ cells within the total harvested cell population may be at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 57%, at
  • the harvested CAR-T cells may include different cell subsets.
  • multiple T-cell subsets may be present within the harvested CAR-T cell sample.
  • the T cell subsets may be CD4+, CD8+, CD3+, CD5+, CD2+, CD7+, or a combination thereof.
  • the T cell subsets may be CD45RA+, CCR7+, CD45RO+, or a combination thereof.
  • the T cell subsets may be CD45RA-, CCR7-, CD45RO-, or a combination thereof.
  • the T cell subsets may include stem cell-like memory T cells (TSCM), central memory T cells (TCM), transitional memory T cells (T TM ), effector T cells (T EFF ), or a combination thereof.
  • T SCM cells may include CD45RA+, CD45RO- and CCR7+ cells.
  • TCM cells may include CD45RA-, CD45RO+ and CCR7+ cells.
  • TTM cells may include CD45RA+, CD45RO- and CCR7- cells.
  • T EFF cells may include CD45RA-, CD45RO+ and CCR7- cells.
  • marker expression is analyzed using flow cytometry.
  • a determination of positive or negative expression for a given marker may be determined by a gating analysis of flow cytometry data.
  • the gating analysis is performed manually.
  • the gating analysis is performed automatically.
  • the method comprises the steps of: (a) obtaining a liquid sample comprising a first population of cells including immune cells from a human subject; (b) processing the first population of cells thereby generating a second population of cells including immune cells, wherein the second population of cells includes at least 1 ⁇ 10 4 total cells, wherein less than 20% of the total number of cells in the second population of cells are platelets; (c) cryopreserving the second population of cells; (d) on Day 0, thawing the cryopreserved second population of cells including immune cells, processing the thawed second population of cells including immune cells, and seeding a third population of cells including immune cells with a portion of the processed second population of cells including immune cells in a volume of a first media, wherein the third population of cells is a subset of the second population of cells, wherein the third population of cells is seeded into a volume of at least 250 mL of media with at least 2.0 ⁇ 10 8 cells from the second population of cells; (e) transducing the third
  • the third population of cells is a subset of the second population of cells and include at least 3.0 ⁇ 10 8 cells from the second population of cells. In some embodiments, the third population of cells is a subset of the second population of cells, wherein the third population of cells is seeded into a volume of at least 250 mL of media with at least 3.0 ⁇ 10 8 cells from the second population of cells.
  • the liquid sample including a first population of cells including immune cells from a human subject comprises a leukapheresis product or an apheresis product.
  • at least 2.4% of cells in the fifth population of cells are CCR7+CD45RA+ immune cells.
  • At least 2.5%, 2.8%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, or 5.5% of cells in the fifth population of cells are CCR7+CD45RA+ immune cells. In some embodiments, at least 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.9%, 5.0%, 5.1%, 5.2%, 5.3%, 5.4%, or 5.5% of cells in the fifth population of cells are CCR7+CD45RA+ immune cells. In some embodiments, the CCR7+CD45RA+ immune cells are also CD3+.
  • the CCR7+CD45RA+CD3+ immune cells are also CD8+ (i.e., CCR7+CD45RA+CD3+CD8+). In some embodiments, at least 78.3%, 78.5%, 79.0%, 79.5%, 80%, 80.5%, 81.0%, 81.4%, 81.5%, 82.0%, 83.0%, 84.0%, 85.0%, 86.0%, or 87.0% of the cells in the fifth population of cells are CCR7+CD45RA- immune cells. In some embodiments, the CCR7+CD45RA+CD3+ immune cells are also CD4+ (i.e., CCR7+CD45RA+CD3+CD4+).
  • At least 78.0%, 78.1%, 78.2%, 78.3%, 78.4%, 78.5%, 78.6%, 78.7%, 78.8%, 78.9%, 79.0%, 79.1%, 79.2%, 79.3%, 79.4%, 79.5%, 79.6%, 79.7%, 79.8%, 79.9%, 80.0%, 80.1%, 80.2%, 80.3%, 80.4%, 80.5%, 80.6%, 80.7%, 80.8%, 80.9%, or 81.0% of cells in the fifth population of cells are CCR7+CD45RA- immune cells.
  • At least 81.0%, 81.1%, 81.2%, 81.3%, 81.4%, 81.5%, 81.6%, 81.7%, 81.8%, 81.9%, 82.0%, 82.0%, 82.1%, 82.2%, 82.3%, 82.4%, 82.5%, 82.6%, 82.7%, 82.8%, 82.9%, 83.0%, 83.1%, 83.2%, 83.3%, 83.4%, 83.5%, 83.6%, 83.7%, 83.8%, 83.9%, or 84.0% of cells in the fifth population of cells are CCR7+CD45RA- immune cells.
  • At least 84.0%, 84.1%, 84.2%, 84.3%, 84.4%, 84.5%, 84.6%, 84.7%, 84.8%, 84.9%, 85.0%, 85.0%, 85.1%, 85.2%, 85.3%, 85.4%, 85.5%, 85.6%, 85.7%, 85.8%, 85.9%, 86.0%, 86.1%, 86.2%, 86.3%, 86.4%, 86.5%, 86.6%, 86.7%, 86.8%, 86.9%, or 87.0% of cells in the fifth population of cells are CCR7+CD45RA- immune cells.
  • At most 16.1%, 16.0%, 15.0%, 14.0%, 13.0%, 12.0%, 11.0%, 10.0%, 9.0%, 8.0%, 7.5%, 7%, 6.5%, 6.0%, 5.5%, 5.0%, 4.5%, 4.0%, 3.5%, or 3.4% of the cells in the fifth population of cells are CCR7-CD45RA- immune cells.
  • the harvested immune cells in the fifth population in (j) include at least 2.3%, at least 2.4%, at least 2.5%, at least 2.6%, at least 2.7%, at least 2.8%, at least 2.9%, at least 3%, at least 3.1%, at least 3.2%, at least 3.3%, at least 3.4%, at least 3.5%, at least 3.6%, at least 3.7%, at least 3.8%, at least 3.9%, at least 4%, at least 4.1%, at least 4.2%, at least 4.3%, at least 4.4%, at least 4.5%, at least 4.6%, at least 4.7%, at least 4.8%, at least 4.9%, at least 5%, at least 5.1%, at least 5.2%, at least 5.3%, at least 5.4%, at least 5.5%, at least 5.6%, at least 5.7%, at least 5.8%, at least 5.9%, at least 6%, at least 6.1%, at least 6.2%, at least 6.3%, at least 6.4%, at least 6.5%, at least 6.6%, at least 6.7%, at least 6.8%, at least 6.9%, at least 6%, at
  • the harvested immune cells in the fifth population in (j) include at least 78.3%, at least 78.5%, at least 79.0%, at least 79.5%, at least 80.0%, at least 80.5%, at least 81.0%, at least 81.4%, at least 81.8%, at least 82.4%, at least 82.8%, at least 83.4%, at least 83.8%, at least 84.4%, at least 84.8%, at least 85.4%, at least 85.8%, at least 86.4%, at least 86.8%, at least 87.4%, at least 87.8%, at least 88.4%, at least 88.8%, at least 89.4%, at least 89.8%, at least 90.4%, at least 90.8%, at least 91.4%, at least 91.8%, at least 92.4%, at least 92.8%, at least 93.4%, at least 93.8%, at least 94.4%, at
  • the harvested immune cell population in (j) includes fewer than 16%, fewer than 15.8%, fewer than 15.6%, fewer than 15.4%, fewer than 15.2%, fewer than 15%, fewer than 14.8%, fewer than 14.6%, fewer than 14.4%, fewer than 14.2%, fewer than 14%, fewer than 13.8%, fewer than 13.6%, fewer than 13.4%, fewer than 13.2%, fewer than 13%, fewer than 12.8%, fewer than 12.6%, fewer than 12.4%, fewer than 12.2%, fewer than 12%, fewer than 11.8%, fewer than 11.6%, fewer than 11.4%, fewer than 11.2%, fewer than 11%, fewer than 10.8%, fewer than 10.6%, fewer than 10.4%, fewer than 10.2%, fewer than 10%, fewer than 9.8%, fewer than 9.6%, fewer than 9.4%, fewer than 9.2%, fewer than 9%, fewer than 8.8%, fewer than 8.6%, fewer than 8.4%, fewer than 8.
  • the processing of the first population of cells in step (b) comprises a step of washing, concentrating and eluting or resuspending the second population of cells in a buffer.
  • the processing of the first population of cells further comprises a step of reducing the number of platelets in the first population of cells.
  • the second population of cells is washed, concentrated, and eluted or resuspended and the number of platelets is reduced by elutriation using a centrifugation system having a molecular weight cutoff sufficient to retain the immune cells.
  • the centrifugal filtration system is a CTS TM Rotea TM Counterflow Centrifugation System.
  • the concentrated second population of cells is resuspended in a buffer comprising human serum albumin (HSA), Plasma-Lyte A, phosphate buffered saline, sodium chloride, sodium bicarbonate buffer, glutathione, biotin, vitamin B12, inositol, choline, L-glutamine, sodium pyruvate, glucose, or any combination thereof.
  • the buffer comprises human serum albumin (HSA).
  • the buffer comprises Plasma-Lyte A.
  • the buffer comprises equal volumes of Plasma-Lyte A and 4% (w/v) human serum albumin.
  • the first population of cells is resuspended in a buffer comprising equal volumes of Plasma-Lyte A and 4% (w/v) HSA and further diluted 1:1 with a cryoprotectant.
  • the cryoprotectant is CryoStor R .
  • the processing of the thawed second population of cells in step (d) comprises a step of enriching for CD4+ and CD8+ T cells.
  • the enriching for CD4+ and CD8+ T cells comprises a step of measuring the total viable cells and determining the percentage (%) of cells that are CD3+ (%CD3+), CD4+CD8- (%CD4+CD8- ), CD8+CD4- (%CD8+CD4-), and CD4+CD8+ (%CD4+CD8+) in the thawed second population of cells.
  • the enriching for CD4+ and CD8+ T cells further comprises a step mixing the thawed second population of cells with magnetic beads derivatized with CD4-specific binding agents and CD8-specific binding agents, washing, and eluting the thawed second population of cells enriched for CD4+ and CD8+ cells.
  • the number of aliquots of cryopreserved Apheresis Product to be thawed for use in the enrichment step is determined to be the amount that results in at least 1.0 ⁇ 10 9 CD3+ cells and the closest in absolute value to 3.0 ⁇ 10 9 CD3+ cells.
  • the step of enriching the thawed second population of cells for CD4+ and CD8+ T cells is performed on a CliniMACS Prodigy using CliniMACS buffer + 2% (w/v) or 0.5% (w/v) HSA.
  • the third population of cells is seeded with 300 ⁇ 10 6 cells from the processed second population of cells.
  • the third population of cells is seeded with at least 2.5 ⁇ 10 6 cells from the processed second population of cells. In some embodiments, the third population of cells is seeded with between at least 2.5 ⁇ 10 6 cells and at least 300 ⁇ 10 6 cells from the processed second population of cells.
  • the third population of cells is seeded with at least about 2.5 ⁇ 10 6 , 3 ⁇ 10 6 , 3.5 ⁇ 10 6 , 4 ⁇ 10 6 , 4.5 ⁇ 10 6 , 5 ⁇ 10 6 , 5.5 ⁇ 10 6 , 6 ⁇ 10 6 , 6.5 ⁇ 10 6 , 7 ⁇ 10 6 , 7.5 ⁇ 10 6 , 8 ⁇ 10 6 , 8.5 ⁇ 10 6 , 9 ⁇ 10 6 , 9.5 ⁇ 10 6 , 1 ⁇ 10 7 , 1.5 ⁇ 10 7 , 2 ⁇ 10 7 , 2.5 ⁇ 10 7 , 3 ⁇ 10 7 , 3.5 ⁇ 10 7 , 4 ⁇ 10 7 , 4.5 ⁇ 10 7 , 5 ⁇ 10 7 , 5.5 ⁇ 10 7 , 6 ⁇ 10 7 , 6.5 ⁇ 10 7 , 7 ⁇ 10 7 , 7.5 ⁇ 10 7 , 8 ⁇ 10 7 , 8.5 ⁇ 10 7 , 9 ⁇ 10 7 , 9.5 ⁇ 10 7 , 1 ⁇ 10 8 , 1.5 ⁇ 10 8 , 2 ⁇ 10 8 , 2.5 ⁇ 10 8 ,
  • the third population of cells is seeded in modified TexMACS TM medium (MTM).
  • MTM comprises TexMACS TM medium supplemented with 3% human AB plasma (HABS) and 1:1 mixture of recombinant human cytokines IL-7 (hIL-7) and IL-15 (hIL-15).
  • the modified TexMACS TM medium further comprises an effective amount of a reagent comprising agonists of CD3 and CD28.
  • the reagent comprising CD3 and CD28 agonists comprises the T cell TransAct TM reagent.
  • the recombinant polynucleotide encoding a CAR further comprises a lentiviral expression vector (i.e., it comprises a lentiviral expression vector comprising a recombinant polynucleotide encoding a CAR).
  • the lentiviral expression vector is manufactured using a suspension cell culture method.
  • the lentiviral expression vector is manufactured using an adherent cell culture method.
  • the transduction in step (e) is performed on Day 1. In some embodiments, the transduction in step (e) is performed 22-26 hours after seeding of the third population of cells comprising immune cells on Day 0.
  • the amount of vector used to transduce the third population of cells comprising immune cells in step (e) is determined based on the infectious titer of the lentiviral vector and the number of cells used to seed the third population of cells comprising immune cells such that the transduction is performed with a multiplicity of infection (MOI) of 2.0.
  • MOI multiplicity of infection
  • the transduction is performed with a MOI of at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2.0, at least 2.1, at least 2.2, at least 2.3, at least 2.4, at least 2.5, at least 2.6, at least 2.7, at least 2.8, at least 2.9, at least 3.0, at least 3.5, at least 4.0, at least 4.5, or at least 5.0.
  • the recombinant polynucleotide encoding a CAR further comprising a lentiviral expression vector is thawed, diluted into modified TexMACS TM medium (MTM), and added to the third population of cells comprising immune cells.
  • MTM modified TexMACS TM medium
  • the MTM comprises TexMACS TM medium supplemented with 3% human AB plasma (HABS) and 1:1 mixture of recombinant human cytokines IL-7 (hIL-7) and IL-15 (hIL-15).
  • the modified TexMACS TM medium further comprises an effective amount of a reagent comprising agonists of CD3 and CD28.
  • the reagent comprising CD3 and CD28 agonists comprises the T cell TransAct TM reagent.
  • the final volume in which the transduction in step (e) is performed comprises 100 mL.
  • the fifth population of cells in step (f) is expanded in MTM in a volume of 200 mL.
  • the fifth population of cells is washed to remove T cell TransAct TM and residual lentiviral vector.
  • the washing step is performed on Day 4.
  • the washing step is performed using the automated CliniMACS Prodigy program.
  • the washing step is performed on Day 4 using the CliniMACS Prodigy program.
  • the washing step comprises removal of cell culture supernatant and resuspension of the fifth population of cells in MTM without T cell TransAct TM reagent.
  • the washing step comprises removal of 65% of the culture volume and resuspension of the fifth population in the desired volume of MTM without T cell TransAct TM .
  • the fifth population of cells is expanded for another day, a sample of the fifth population of cells is taken on Day 6 and the Dose Factor is recalculated assuming a 7% increase in Day 4 Transduction Efficiency.
  • the fifth population of cells is expanded for another day, a sample of the fifth population of cells is taken on Day 7 and the Dose Factor is recalculated assuming a 7% increase in Day 4 Transduction Efficiency.
  • a further media exchange is performed on Day 7.
  • the further media exchange on Day 7 comprises removing 60% of the culture volume and replacing it with fresh MTM.
  • there are not enough viable CD3+CAR+ cells on Day 7 the fifth population of cells is expanded for another day, a sample of the fifth population of cells is taken on Day 8 and the Dose Factor is recalculated assuming a 7% increase in Day 4 Transduction Efficiency.
  • a further media exchange is performed on Day 8.
  • the further media exchange on Day 8 comprises removing 60% of the culture volume and replacing it with fresh MTM.
  • the harvesting step (j) is performed.
  • there are not enough viable CD3+CAR+ cells on Day 8 the fifth population of cells is expanded for another day, and the harvesting step (j) is performed.
  • there are not enough viable CD3+CAR+ cells on Day 8 the fifth population of cells is expanded for another day, and the harvesting step (j) is performed on Day 9.
  • the harvesting step (j) further comprises a step of calculating viable cell density and determining whether the post-harvest viable cell density is ⁇ the minimum transduced viable cell density for the formulation step.
  • the post-harvest viable cell density is ⁇ the minimum transduced viable cell density and the harvested fifth population of cells is formulated for cryopreservation and administration to patients.
  • the post-harvest viable cell density is ⁇ the minimum transduced viable cell density and the harvested fifth population of cells is concentrated using a Rotea TM so that the post-harvest viable cell density is ⁇ the minimum transduced viable cell density and the concentrated harvested fifth population of cells is formulated for cryopreservation and administration to patients.
  • the minimum transduced viable cell density is 1 ⁇ 10 6 CD3+CAR+ cells/kg.
  • the formulation step is performed manually, and the harvested fifth population of cells or the concentrated harvested fifth population of cells is resuspended to the desired concentration in Final Formulation Medium comprising Plasma-Lyte A + 4% (w/v) HSA, diluted 1:1 with Cryostor R CS10 and frozen.
  • the formulation step is automated.
  • the automated formulation step is performed using a FINIA R Fill and Finish System, and the harvested fifth population of cells or the concentrated harvested fifth population of cells is resuspended to the desired concentration in Final Formulation Medium comprising Plasma-Lyte A+4% (w/v) HSA, diluted 1:1 with Cryostor R CS10 and frozen.
  • the automated formulation step is performed using a Cue ScaleReady Cell Processing System, and the harvested fifth population of cells or the concentrated harvested fifth population of cells is resuspended to the desired concentration in Final Formulation Medium comprising Plasma-Lyte A+4% (w/v) HSA, diluted 1:1 with Cryostor R CS10 and frozen.
  • the fifth population of cells including CAR-expressing immune cells comprises autologous CAR-expressing immune cells.
  • the fifth population of cells including CAR-expressing immune cells comprises autologous T cells expressing a CD22 CAR (i.e., a CD22-specific CAR).
  • the recombinant polynucleotide encoding an autologous CD22 CAR further comprises a lentiviral expression vector.
  • the lentiviral expression vector is manufactured using a suspension cell culture platform.
  • the lentiviral expression vector is manufactured using an adherent cell culture platform.
  • the recombinant polynucleotide encoding a CAR encodes a CD22-specific CAR.
  • the CD22 CAR comprises a CD22-specific binding domain, a transmembrane domain, and an intracellular domain.
  • the CD22 CAR comprises a CD22-specific binding domain, a hinge domain, a transmembrane domain, a spacer, and an intracellular domain,
  • the CD22-specific binding domain comprises an antibody capable of binding CD22 or an antigen-binding fragment thereof.
  • the CD22-specific binding domain comprises an antibody capable of binding human CD22.
  • the CD22- specific binding domain comprises an antigen-binding fragment of an antibody capable of binding human CD22.
  • the anti-CD22 binding domain includes a VH comprising a heavy chain CDR1 (HCDR1) sequence with at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an HCDR1 sequence in Table 1.
  • the anti- CD22 binding domain comprises a VH that comprises an HCDR1 sequence in Table 1.
  • the anti-CD22 binding domain includes a VH that includes a heavy chain CDR2 (HCDR2) sequence with at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an HCDR2 sequence in Table 1.
  • the anti-CD22 binding domain includes a VH that includes an HCDR2 sequence in Table 1.
  • the anti-CD22 binding domain includes a heavy chain variable region (VH) comprising a heavy chain CDR3 (HCDR3) sequence with at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an HCDR3 sequence in Table 1.
  • the anti- CD22 binding domain includes a VH that includes an HCDR3 sequence in Table 1.
  • the anti-CD22 binding domain includes the heavy chain CDRs 1, 2, and 3 (HCDR1, HCDR2, HCDR3) sequences as set forth in SEQ ID NOs: 4, 5, and 6, respectively.
  • the anti-CD22 binding domain includes the heavy chain HCDR1, HCDR2, HCDR3 sequences as set forth in SEQ ID NOs: 10, 11, and 12, respectively. In some embodiments, the anti-CD22 binding domain includes the heavy chain HCDR1, HCDR2, HCDR3 sequences as set forth in SEQ ID NOs: 16, 17, and 18, respectively. [0219] In some embodiments, the anti-CD22 binding domain includes a VL comprising a light chain CDR1 (LCDR1) sequence with at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an LCDR1 sequence in Table 1. In some embodiments, the anti-CD22 binding domain includes a VL comprising an LCDR1 sequence in Table 1.
  • LCDR1 light chain CDR1
  • the anti-CD22 binding domain comprises a VL that comprises a light chain CDR2 (LCDR2) sequence with at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an LCDR2 sequence in Table 1.
  • the anti-CD22 binding domain comprises a VL that comprises an LCDR2 sequence in Table 1.
  • the anti-CD22 binding domain comprises a VL that comprises a light chain CDR3 (LCDR3) sequence with at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an LCDR3 sequence in Table 1.
  • the anti-CD22 binding domain comprises a VL that comprises an LCDR3 sequence in Table 1.
  • the anti-CD22 binding domain includes the light chain CDRs 1, 2, and 3 (LCDR1, LCDR2, LCDR3) sequences as set forth in SEQ ID NOs: 7, 8, and 9, respectively. In some embodiments, the anti-CD22 binding domain includes the light chain LCDR1, LCDR2, LCDR3 sequences as set forth in SEQ ID NOs: 13, 14, and 15, respectively. In some embodiments, the anti-CD22 binding domain includes the light chain LCDR1, LCDR2, LCDR3 sequences as set forth in SEQ ID NOs: 19, 20, and 21, respectively.
  • the anti-CD22 binding domain includes the heavy chain CDRs 1, 2, and 3 (HCDR1, HCDR2, HCDR3) sequences and the light chain CDRs 1, 2, and 3 (LCDR1, LCDR2, LCDR3) sequences as set forth in SEQ ID NOs: 4, 5, 6, 7, 8, and 9, respectively.
  • the anti-CD22 binding domain includes the heavy chain HCDR1, HCDR2, HCDR3 sequences and the light chain LCDR1, LCDR2, LCDR3 sequences as set forth in SEQ ID NOs: 10, 11, 12, 13, 14, and 15, respectively.
  • the anti-CD22 binding domain includes the heavy chain HCDR1, HCDR2, HCDR3 sequences and the light chain LCDR1, LCDR2, LCDR3 sequences as set forth in SEQ ID NOs: 16, 17, 18, 19, 20, and 21, respectively.
  • the antigen-binding fragment of an antibody capable of binding human CD22 is a single chain variable fragment (scFv) capable of binding CD22.
  • the scFv capable of binding CD22 has the sequence of SEQ ID NO: 1.
  • the scFv capable of binding CD22 comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 1.
  • the CD22 CAR comprises a CD8 ⁇ hinge domain comprising the sequence of SEQ ID NO: 24 and a CD8 ⁇ transmembrane domain comprising the sequence of SEQ ID NO: 25.
  • the CD22 CAR comprises a CD8 ⁇ hinge domain comprising the sequence of SEQ ID NO:24, a CD8 ⁇ transmembrane domain comprising the sequence of SEQ ID NO:25, and a peptide linker having the sequence of SEQ ID NO:31.
  • the CD22 CAR comprises a CD8 ⁇ hinge domain comprising a sequence comprising 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 24 and a CD8 ⁇ transmembrane domain comprising a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 25.
  • the CD8 ⁇ transmembrane domain further comprises a spacer having the sequence of LYC.
  • the CD8 ⁇ transmembrane domain further comprises a spacer having the sequence of SEQ ID NO:31.
  • the CD22 CAR comprises an intracellular domain further comprising a primary T cell activating domain comprising an immunoreceptor tyrosine-based activation motif (ITAM) and a costimulatory signaling domain.
  • ITAM immunoreceptor tyrosine-based activation motif
  • the primary T cell activating domain comprising an ITAM comprises a CD3 ⁇ intracellular signaling domain.
  • the CD3 ⁇ intracellular signaling domain comprises the sequence of SEQ ID NO: 27.
  • the CD3 ⁇ intracellular signaling domain comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 27. In some embodiments, the CD3 ⁇ intracellular signaling domain comprises the sequence of SEQ ID NO: 30. In some embodiments, the CD3 ⁇ intracellular signaling domain comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 30. In some embodiments the costimulatory signaling domain comprises a 4-1BB/CD137 signaling domain.
  • the 4-1BB/CD137 signaling domain comprises the sequence of SEQ ID NO: 26.
  • the 4-1BB/CD137 costimulatory signaling domain comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 26.
  • the CD22 CAR comprises the sequence of SEQ ID NO: 22. In some embodiments, the CD22 CAR comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 22.
  • the CD22 CAR comprises the sequence of SEQ ID NO: 23. In some embodiments, the CD22 CAR comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 23. See, e.g., Table 2 and paragraph [0198] above.
  • the anti-CD22 binding domain includes a VH comprising a heavy chain CDR1 (HCDR1) sequence with at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an HCDR1 sequence in Table 1.
  • the anti- CD22 binding domain comprises a VH that comprises an HCDR1 sequence in Table 1.
  • the anti-CD22 binding domain includes a VH that includes a heavy chain CDR2 (HCDR2) sequence with at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an HCDR2 sequence in Table 1.
  • the anti-CD22 binding domain includes a VH that includes an HCDR2 sequence in Table 1.
  • the anti-CD22 binding domain includes a heavy chain variable region (VH) comprising a heavy chain CDR3 (HCDR3) sequence with at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an HCDR3 sequence in Table 1.
  • the anti- CD22 binding domain includes a VH that includes an HCDR3 sequence in Table 1.
  • the anti-CD22 binding domain includes the heavy chain CDRs 1, 2, and 3 (HCDR1, HCDR2, HCDR3) sequences as set forth in SEQ ID NOs: 4, 5, and 6, respectively.
  • the anti-CD22 binding domain includes the heavy chain HCDR1, HCDR2, HCDR3 sequences as set forth in SEQ ID NOs: 10, 11, and 12, respectively.
  • the anti-CD22 binding domain includes the heavy chain HCDR1, HCDR2, HCDR3 sequences as set forth in SEQ ID NOs: 16, 17, and 18, respectively.
  • the anti-CD22 binding domain includes a VL comprising a light chain CDR1 (LCDR1) sequence with at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an LCDR1 sequence in Table 1.
  • the anti-CD22 binding domain includes a VL comprising an LCDR1 sequence in Table 1.
  • the anti-CD22 binding domain comprises a VL that comprises a light chain CDR2 (LCDR2) sequence with at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an LCDR2 sequence in Table 1.
  • the anti-CD22 binding domain comprises a VL that comprises an LCDR2 sequence in Table 1.
  • the anti-CD22 binding domain comprises a VL that comprises a light chain CDR3 (LCDR3) sequence with at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an LCDR3 sequence in Table 1.
  • the anti-CD22 binding domain comprises a VL that comprises an LCDR3 sequence in Table 1.
  • the anti-CD22 binding domain includes the light chain CDRs 1, 2, and 3 (LCDR1, LCDR2, LCDR3) sequences as set forth in SEQ ID NOs: 7, 8, and 9, respectively.
  • the anti-CD22 binding domain includes the light chain LCDR1, LCDR2, LCDR3 sequences as set forth in SEQ ID NOs: 13, 14, and 15, respectively.
  • the anti-CD22 binding domain includes the light chain LCDR1, LCDR2, LCDR3 sequences as set forth in SEQ ID NOs: 19, 20, and 21, respectively. [0224] In some embodiments, the anti-CD22 binding domain includes the heavy chain CDRs 1, 2, and 3 (HCDR1, HCDR2, HCDR3) sequences and the light chain CDRs 1, 2, and 3 (LCDR1, LCDR2, LCDR3) sequences as set forth in SEQ ID NOs: 4, 5, 6, 7, 8, and 9, respectively. In some embodiments, the anti-CD22 binding domain includes the heavy chain HCDR1, HCDR2, HCDR3 sequences and the light chain LCDR1, LCDR2, LCDR3 sequences as set forth in SEQ ID NOs: 10, 11, 12, 13, 14, and 15, respectively.
  • the anti-CD22 binding domain includes the heavy chain HCDR1, HCDR2, HCDR3 sequences and the light chain LCDR1, LCDR2, LCDR3 sequences as set forth in SEQ ID NOs: 16, 17, 18, 19, 20, and 21, respectively.
  • CHIMERIC ANTIGEN RECEPTOR the CAR-expressing immune cells of the disclosure express a CAR.
  • the CAR comprises an antigen-binding domain, optionally a hinge domain, a transmembrane domain, optionally a peptide linker, and an intracellular signaling domain.
  • the CAR includes an antigen-binding domain having binding affinity for one or more antigens.
  • the one or more antigens are selected from the group consisting of CD22, CD20, and CD19.
  • the CAR includes an anti-CD22 antigen-binding domain.
  • the CAR includes an anti-CD20 antigen-binding domain.
  • the CAR includes an anti-CD19 antigen-binding domain.
  • the CAR includes a bispecific binding domain having binding affinity for two different antigens.
  • the CAR includes an anti-CD19 antigen-binding domain and an anti-CD20 antigen-binding domain.
  • the CAR includes an anti-CD19 antigen-binding domain and an anti-CD22 antigen binding domain.
  • the CAR includes an anti-CD20 antigen-binding domain and an anti-CD22 antigen-binding domain.
  • Anti-CD22 binding domain can be any molecule having a binding affinity and/or specificity to CD22.
  • the anti-CD22 binding domain is an antibody or an antibody derivative, such as an scFv, single domain antibody (sdAb), Fab' fragment, (Fab')2 fragment, nanobody, diabody, or the like.
  • the anti-CD22 binding domain can be a receptor or a receptor fragment that binds specifically to CD22.
  • the anti-CD22 binding domain can be attached to the rest of the receptor directly (covalently) or indirectly (for example, through the noncovalent binding of two or more binding partners).
  • Antibody derivatives are molecules that resemble antibodies in their mechanism of ligand binding, and include, for example, nanobodies, duobodies, diabodies, triabodies, minibodies, F(ab')2 fragments, Fab fragments, single chain variable fragments (scFv), single domain antibodies (sdAb), and functional fragments thereof. See, for example, D.L. Porter et al., N Engl J Med ( 2011) 365(8):725-33 (scFv); E.L.
  • Antibody derivatives can also be prepared from therapeutic antibodies, for example without limitation, by preparing a nanobody, duobody, diabody, triabody, minibody, F(ab')2 fragment, Fab fragment, single chain variable fragment (scFv), or single domain antibody (sdAb) based on a therapeutic antibody.
  • Antibody derivatives can also be identified using phage display techniques (see, e.g., E.
  • the anti-CD22 binding domain is a single chain variable fragment (scFv).
  • the anti-CD22 scFv comprises an antibody heavy chain variable domain polypeptide (VH) covalently linked to an antibody light chain variable domain polypeptide (VL).
  • the anti-CD22 scFv further comprises a peptide linker disposed between the VH and VL polypeptides.
  • the anti-CD22 scFv comprises, from N-terminus to C-terminus, a VH polypeptide, a polypeptide linker, and a VL polypeptide.
  • the anti-CD22 scFv comprises, from N- terminus to C-terminus, a VL polypeptide, a polypeptide linker, and a VH polypeptide.
  • the polypeptide linker comprises a polypeptide having the sequence GGGGS (SEQ ID NO: 29).
  • the anti-CD22 binding domain includes a VH comprising a heavy chain CDR1 (HCDR1) sequence with at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an HCDR1 sequence in Table 1.
  • the anti- CD22 binding domain comprises a VH that comprises an HCDR1 sequence in Table 1.
  • the anti-CD22 binding domain includes a VH that includes a heavy chain CDR2 (HCDR2) sequence with at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an HCDR2 sequence in Table 1.
  • the anti-CD22 binding domain includes a VH that includes an HCDR2 sequence in Table 1.
  • the anti-CD22 binding domain includes a heavy chain variable region (VH) comprising a heavy chain CDR3 (HCDR3) sequence with at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an HCDR3 sequence in Table 1.
  • the anti- CD22 binding domain includes a VH that includes an HCDR3 sequence in Table 1.
  • the anti-CD22 binding domain includes the heavy chain CDRs 1, 2, and 3 (HCDR1, HCDR2, HCDR3) sequences as set forth in SEQ ID NOs: 4, 5, and 6, respectively. In some embodiments, the anti-CD22 binding domain includes the heavy chain HCDR1, HCDR2, HCDR3 sequences as set forth in SEQ ID NOs: 10, 11, and 12, respectively. In some embodiments, the anti-CD22 binding domain includes the heavy chain HCDR1, HCDR2, HCDR3 sequences as set forth in SEQ ID NOs: 16, 17, and 18, respectively.
  • the anti-CD22 binding domain includes a VL comprising a light chain CDR1 (LCDR1) sequence with at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an LCDR1 sequence in Table 1.
  • the anti-CD22 binding domain includes a VL comprising an LCDR1 sequence in Table 1.
  • the anti-CD22 binding domain comprises a VL that comprises a light chain CDR2 (LCDR2) sequence with at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an LCDR2 sequence in Table 1.
  • the anti-CD22 binding domain comprises a VL that comprises an LCDR2 sequence in Table 1.
  • the anti-CD22 binding domain comprises a VL that comprises a light chain CDR3 (LCDR3) sequence with at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an LCDR3 sequence in Table 1.
  • the anti-CD22 binding domain comprises a VL that comprises an LCDR3 sequence in Table 1.
  • the anti-CD22 binding domain includes the light chain CDRs 1, 2, and 3 (LCDR1, LCDR2, LCDR3) sequences as set forth in SEQ ID NOs: 7, 8, and 9, respectively.
  • the anti-CD22 binding domain includes the light chain LCDR1, LCDR2, LCDR3 sequences as set forth in SEQ ID NOs: 13, 14, and 15, respectively.
  • the anti-CD22 binding domain includes the light chain LCDR1, LCDR2, LCDR3 sequences as set forth in SEQ ID NOs: 19, 20, and 21, respectively.
  • the anti-CD22 binding domain comprises a VH with a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRS KWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIW GQGTMVTVSS (SEQ ID NO: 2).
  • the anti-CD22 binding domain comprises a VH with the sequence QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRS KWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIW GQGTMVTVSS (SEQ ID NO: 2).
  • the anti-CD22 binding domain comprises a VL with a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence DIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGV PSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIK (SEQ ID NO: 3).
  • the anti-CD20 binding domain comprises a VL with the sequence DIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGV PSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIK (SEQ ID NO: 3).
  • the anti-CD22 binding domain includes the heavy chain CDRs 1, 2, and 3 (HCDR1, HCDR2, HCDR3) sequences and the light chain CDRs 1, 2, and 3 (LCDR1, LCDR2, LCDR3) sequences as set forth in SEQ ID NOs: 4, 5, 6, 7, 8, and 9, respectively.
  • the anti-CD22 binding domain includes the heavy chain HCDR1, HCDR2, HCDR3 sequences and the light chain LCDR1, LCDR2, LCDR3 sequences as set forth in SEQ ID NOs: 10, 11, 12, 13, 14, and 15, respectively.
  • the anti-CD22 binding domain includes the heavy chain HCDR1, HCDR2, HCDR3 sequences and the light chain LCDR1, LCDR2, LCDR3 sequences as set forth in SEQ ID NOs: 16, 17, 18, 19, 20, and 21, respectively.
  • the anti-CD22 binding domain is an scFv.
  • the anti-CD22 scFv comprises a peptide linker between the VH and the VL domains.
  • the peptide linker comprises the sequence GGGGS (SEQ ID NO: 29).
  • the anti-CD22 binding domain comprises a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRS KWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIW GQGTMVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPG KAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQ GTKLEIK (SEQ ID NO: 29).
  • the anti-CD22 binding domain comprises the sequence QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRS KWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIW GQGTMVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPG KAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQ GTKLEIK (SEQ ID NO: 1).
  • the antigen binding domain is a mouse anti-human CD22 binding domain.
  • the antigen binding domain is a chimeric mouse anti- human CD22 binding domain. In some embodiments, the antigen binding domain is a humanized anti-human CD22 binding domain. In some embodiments, the antigen binding domain is a fully human anti-human CD22 binding domain. T ABLE 1: Exemplary anti-CD22 scFv Constructs Constructs Amino Acid Sequence SEQ ID NO. Constructs Amino Acid Sequence SEQ ID NO. Constructs Amino Acid Sequence SEQ ID NO. [0235] In some embodiments, the anti-CD22 binding domain is a humanized binding domain.
  • a humanized anti-CD22 binding domain may include heavy and light chain variable region sequences from a non-human species (e.g., a mouse) but in which at least a portion of the VH and/or VL sequence has been altered to be more “human-like”, i.e., more similar to the corresponding human sequences.
  • a non-human species e.g., a mouse
  • One type of humanized anti-CD22 binding domain is a CDR-grafted binding domain in which human CDR sequences are introduced into non-human VH and VL sequences to replace the corresponding non-human CDR sequences.
  • Another type of humanized antibody is a framework region (FWR)-grafted antibody in which human FWR sequences are introduced into non-human VH and VL sequences to replace corresponding non-human FWR sequences.
  • FWR framework region
  • the anti-CD22 binding domain of the CD22 CARs disclosed herein is or comprises a scFv derived from a fully human anti-CD22 antibody.
  • the anti-CD22 CAR includes a hinge domain.
  • hinge domains suitable for the methods disclosed herein include hinge domains from LFA-1 (CD11a/CD18), LFA-2 (CD2), CD4, CD5, CD8 ⁇ , CD8 ⁇ , CD27 (TNFRSF7), CD28, CD70, 4-1BB (CD137), OX40 (CD134), CD152 (CTLA4), ICOS (CD278), and the IgG1 Fc region, IgG4 Fc region.
  • the hinge domain is from CD8 ⁇ or CD28. In some embodiments, the hinge domain is from CD28. In some embodiments, the hinge domain is from CD8 ⁇ . In some embodiments, the anti-CD22 CAR further comprises a linker positioned between the anti-CD22 binding domain and the hinge domain. In some embodiments, the linker between the anti-CD22 binding domain and the hinge domain comprises the sequence AAA. In some embodiments, the hinge domain is from CD8 ⁇ and the linker includes the sequence AAA. In some embodiments, the CD8 ⁇ hinge domain of the anti-CD22 CAR comprises the sequence TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 24).
  • the anti-CD22 CAR further includes a transmembrane domain.
  • the transmembrane domain is from CD8 ⁇ .
  • the CD8 ⁇ transmembrane domain of the anti-CD22 CAR comprises the sequence IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 25).
  • the anti-CD22 CAR comprises a peptide linker (e.g., spacer) disposed between the transmembrane domain and the intracellular signaling domain.
  • the peptide linker (e.g., spacer) comprises a portion of a CD8 ⁇ cytoplasmic domain.
  • the peptide linker comprising a portion of a CD8 ⁇ cytoplasmic domain of the anti-CD22 CAR comprises the sequence LYC (SEQ ID NO: 31).
  • the anti-CD22 CAR includes an intracellular signaling domain.
  • the intracellular signaling domain comprises a signaling domain from an immunoreceptor tyrosine-based activation motif (“ITAM”)-containing protein and a signaling domain from a co-stimulatory protein.
  • ITAM immunoreceptor tyrosine-based activation motif
  • Non-limiting exemplary ITAM-containing proteins having ITAM-containing signaling domains suitable for use in the intracellular signaling domain of the anti-CD22 CAR used in the methods of the disclosure include the T cell receptor-associated proteins CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , and CD3 ⁇ , the B cell receptor- associated proteins Ig ⁇ and Ig ⁇ , the Fc ⁇ RI ⁇ polypeptide, the Fc ⁇ RI-, Fc ⁇ RII-, and Fc ⁇ RIII ⁇ polypeptide, DAP12, Dectin-1, CLEC-1, CD28, and CD27.
  • the signaling domain from an ITAM-containing protein is an ITAM-containing signaling domain from a CD3 ⁇ polypeptide.
  • the CD3 ⁇ intracellular signaling domain comprises the sequence RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR (SEQ ID NO: 27).
  • the CD3 ⁇ intracellular signaling domain comprises the sequence RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR (SEQ ID NO: 30)
  • Non-limiting exemplary co-stimulatory proteins having co-stimulatory signaling domains suitable for use in the intracellular signaling domain of the anti-CD22 CAR used in the methods of the disclosure include co-stimulatory domains from 4-1BB (CD137), CD27 (TNFRSF7), CD28, OX40 (CD134), CD70, LFA-2 (CD2), CD5, ICAM-1 (CD54), LFA-1 (CD11a/CD18), DAP10, and DAP12.
  • the signaling domain from a co- stimulatory protein is a co-stimulatory signaling domain from 4-1BB (CD137).
  • the 4-1BB co-stimulatory domain includes the sequence KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 26).
  • the anti-CD22 CAR further includes a signal peptide sequence.
  • the signal peptide sequence of the anti-CD22 CAR comprises the sequence MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 28).
  • the anti-CD22 CAR includes (i) an anti- CD22 scFv, (ii) a CD8 ⁇ hinge domain, (iii) CD8 ⁇ transmembrane (TM) domain; (iv) a 4- 1BB co-stimulatory domain, and (v) a CD3 ⁇ intracellular signaling domain.
  • the anti-CD22 CAR includes (i) anti-CD22 scFv of SEQ ID NO: 1, (ii) a CD8 ⁇ hinge domain of SEQ ID NO: 24, (iii) CD8 ⁇ transmembrane (TM) domain of SEQ ID NO: 25; (iv) a 4-1BB co-stimulatory domain of SEQ ID NO: 26, and (v) a CD3 ⁇ intracellular signaling domain of SEQ ID NO: 27 or CD3 ⁇ intracellular signaling domain of SEQ ID NO: 30.
  • the anti-CD22 CAR includes (i) a signal peptide sequence, (ii) an anti-CD22 scFv, (iii) a CD8 ⁇ hinge domain, (iv) CD8 ⁇ transmembrane (TM) domain; (v) a 4-1BB co-stimulatory domain, and (vi) a CD3 ⁇ intracellular signaling domain.
  • the anti-CD22 CAR includes (i) a signal peptide sequence of SEQ ID NO: 28, (ii) an anti-CD22 scFv of SEQ ID NO: 1, (iii) a CD8 ⁇ hinge domain of SEQ ID NO: 24, (iv) CD8 ⁇ transmembrane (TM) domain of SEQ ID NO: 25; (v) a 4-1BB co-stimulatory domain of SEQ ID NO: 26, and (vi) a CD3 ⁇ intracellular signaling domain of SEQ ID NO: 27 or SEQ ID NO: 30.
  • anti-CD22 scFv includes an anti-CD22 VH and an anti-CD22 VL linked to one another by a linker.
  • anti-CD22 scFv includes an anti-CD22 VH of SEQ ID NO: 2 and an anti-CD22 VL of SEQ ID NO: 3 linked to one another by a linker of SEQ ID NO: 29.
  • I MMUNE C ELLS [0243] In another aspect, the methods disclosed herein are used to manufacture CAR- expressing immune cells for use as human therapeutics.
  • the immune cell is an immune system cell, e.g., a lymphocyte (for example without limitation, a T cell, natural killer cell or NK cell, natural killer T cell or NKT cell, a B cell, a plasma cell, tumor- infiltrating lymphocyte (TIL)), a monocyte or macrophage, or a dendritic cell.
  • a lymphocyte for example without limitation, a T cell, natural killer cell or NK cell, natural killer T cell or NKT cell, a B cell, a plasma cell, tumor- infiltrating lymphocyte (TIL)), a monocyte or macrophage, or a dendritic cell.
  • TIL tumor- infiltrating lymphocyte
  • the immune system cell is selected from the group consisting of B cells, T cells, monocytes, dendritic cells, and epithelial cells.
  • the immune system cell is a T lymphocyte.
  • the T cell is a CD8-positive T cell, a CD4-positive T cell, a regulatory T cell, a cytotoxic T cell, or a tumor infiltrating lymphocyte.
  • the immune cell can also be a precursor cell, e.g., a cell that is capable of differentiating into an immune cell.
  • the immune cell is from a blood sample.
  • the immune cell is obtained by apheresis.
  • an expression vector e.g., a lentiviral expression vector, comprising a recombinant polynucleotide acid encoding a CAR are known in the art and described in the technical and scientific literature.
  • the nucleic acid molecule is introduced into a host cell by transduction, or electroporation.
  • a sample comprising a population of immune cells is transformed with an expression vector encoding a CAR construct to produce a population of cells for use in any of the methods described herein.
  • the expression vector is a lentiviral expression vector.
  • Lentiviral vectors and various lentiviral components useful for the production of a lentiviral vector are known in the art.
  • an expression cassette encoding a CAR construct may be incorporated into a lentiviral vector backbone, which may then be packaged using a packaging system known in the art.
  • a number of techniques suitable for lentiviral manufacturing are also generally known in the art and described in the technical and scientific literature.
  • the lentiviral expression vector comprising an expression cassette encoding a CAR construct is manufactured using an adherent cell-based process.
  • the lentiviral expression vector comprising an expression cassette encoding a CAR construct is manufactured using a suspension cell-based process, which may help ease lentiviral manufacturing at scale with the use of stirred tank bioreactors and, in some instances, provides significantly better transduction efficiency.
  • immune cells are transduced with a lentiviral expression vector encoding a CAR construct produced using an adherent cell-based lentiviral vector manufacturing platform.
  • immune cells are transduced with a lentiviral expression vector encoding a CAR construct produced using a suspension cell culture-based LV manufacturing platform.
  • the CAR construct comprises a CD22 CAR.
  • Methods and systems suitable for generating and maintaining cell cultures are known in the art.
  • the population of CAR-expressing immune cells described herein may be autologous (e.g., obtained from the same subject who will be treated with them) or allogeneic (e.g., obtained from a healthy donor and administered to multiple subjects, e.g., patients).
  • the cells are T cells obtained from a mammal.
  • the T cells obtained from a mammal are CD8-positive T cells, CD4- positive T cells, regulatory T cells (Tregs), cytotoxic T cells (CTLs), or tumor infiltrating lymphocytes (TILs).
  • the mammal is a primate.
  • the primate is a human.
  • T cells can be obtained from a number of sources including, but not limited to, peripheral blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors.
  • T cells are obtained from a unit of blood collected from an individual using any number of known techniques such as sedimentation, e.g., FICOLLTM separation or apheresis.
  • an isolated or purified population of T cells is used.
  • TCTL and TH lymphocytes are purified from PBMCs.
  • the T cells are obtained directly from the patient by leukapheresis and/or apheresis.
  • the TCTL and TH lymphocytes are sorted into naive (TN), memory (T MEM ), stem cell memory (TSCM), central memory (TCM), effector memory (TEM), and effector (TEFF) T cell subpopulations either before or after activation, expansion, and/or genetic modification.
  • Suitable approaches for such sorting include, e.g., magnetic-activated cell sorting (MACS), where TN are CD45RA+ CD62L+ CD95-; TSCM are CD45RA+CCR7+; TCM are CD45RA-CCR7+; and TEM are CCR7-CD45RA-.
  • MCS magnetic-activated cell sorting
  • TN are CD45RA+ CD62L+ CD95-
  • TSCM are CD45RA+CCR7+
  • TCM are CD45RA-CCR7+;
  • TEM effector
  • a specific subpopulation of T cells expressing one or more of the following markers: CD3, CD4, CD8, CD28, CD45RA, CD45RO, CD62, CD127, and HLA-DR can be further isolated by positive or negative selection techniques.
  • a specific subpopulation of T cells, expressing one or more of the markers selected from the group consisting of CD62L, CCR7, CD28, CD27, CD122, CD127, CD197; or CD38 or CD62L, CD127, CD197, and CD38 is further isolated by positive or negative selection techniques.
  • the manufactured T cell compositions do not express one or more of the following markers: CD57, CD244, CD 160, PD-1, CTLA4, TIM3, and LAG3.
  • the manufactured T cell compositions do not substantially express one or more of the following markers: CD57, CD244, CD 160, PD-1, CTLA4, TIM3, and LAG3.
  • the cell is a mammalian cell.
  • the mammalian cell is a primate cell or a human cell.
  • the mammalian cell is a human cell.
  • the human cell is a blood cell.
  • the cell can be a human cell.
  • the cell can be a blood cell.
  • the blood cell is a lymphocyte.
  • the lymphocyte is a T cell.
  • the T cell obtained from a mammal is a CD8-positive T cell, a CD4-positive T cell, a regulatory T cell (Treg), a cytotoxic T cells (CTL), or a tumor infiltrating lymphocyte (TIL).
  • the cell is a population of cells.
  • the population of cells is a population of blood cells.
  • the blood cells can be lymphocytes.
  • the lymphocytes can be T cells.
  • the T cells are CD8- positive T cells, CD4-positive T cells, regulatory T cells (Tregs), cytotoxic T cells (CTLs), or tumor infiltrating lymphocytes (TILs).
  • the population of cells is a homogeneous mixture of cells of the same cell type. In some instances, the population of cells is a heterogeneous mixture of cells of different cell types. In some instances, the population of cells comprises at least about 1 ⁇ 10 3 cells. In some instances, the population of cells comprises at least about 1 ⁇ 10 4 cells. In some instances, the population of cells comprises at least about 1 ⁇ 10 5 cells. In some instances, the population of cells comprises at least about 1 ⁇ 10 6 cells. In some instances, the population of cells comprises at least about 1 ⁇ 10 7 cells. In some instances, the population of cells comprises at least about 1 ⁇ 10 8 cells. In some instances, the population of cells comprises at least about 1 ⁇ 10 9 cells.
  • the population of cells comprises from about 1 ⁇ 10 3 cells to about 1 ⁇ 10 9 cells. In some embodiments, the population of cells comprises from about 1 ⁇ 10 3 cells to about 1 ⁇ 10 8 cells. In some embodiments, the population of cells comprises from about 1 ⁇ 10 3 cells to about 1 ⁇ 10 7 cells. In some embodiments, the population of cells comprises from about 1 ⁇ 10 3 cells to about 1 ⁇ 10 6 cells. In some embodiments, the population of cells comprises from about 1 ⁇ 10 3 cells to about 1 ⁇ 10 5 cells. In some embodiments, the population of cells comprises from about 1 ⁇ 10 3 cells to about 1 ⁇ 10 4 cells. In some embodiments, the population of cells comprises from about 1 ⁇ 10 4 cells to about 1 ⁇ 10 9 cells.
  • the population of cells comprises from about 1 ⁇ 10 4 cells to about 1 ⁇ 10 8 cells. In some embodiments, the population of cells comprises from about 1 ⁇ 10 4 cells to about 1 ⁇ 10 7 cells. In some embodiments, the population of cells comprises from about 1 ⁇ 10 4 cells to about 1 ⁇ 10 6 cells. In some embodiments, the population of cells comprises from about 1 ⁇ 10 4 cells to about 1 ⁇ 10 5 cells. In some instances, the population of cells comprises from about 1 ⁇ 10 5 cells to about 1 ⁇ 10 9 cells. In some instances, the population of cells comprises from about 1 ⁇ 10 5 cells to about 1 ⁇ 10 8 cells. In some instances, the population of cells comprises from about 1 ⁇ 10 5 cells to about 1 ⁇ 10 7 cells.
  • the population of cells comprises from about 1 ⁇ 10 5 cells to about 1 ⁇ 10 6 cells.
  • P HARMACEUTICAL C OMPOSITIONS the CAR-expressing immune cells of the disclosure can be formulated as compositions, including pharmaceutical compositions. Such compositions generally include the CAR-expressing immune cells as described herein and a pharmaceutically acceptable carrier. Accordingly, one aspect of the present disclosure is related to pharmaceutical compositions comprising a population of CAR-expressing immune cells made according to the methods described herein. [0252] The pharmaceutical compositions generally include a therapeutically effective amount of cells.
  • terapéuticaally effective amount is meant a number of cells sufficient to produce a desired result, e.g., an amount sufficient to effect beneficial or desired therapeutic (including preventative) results, such as a reduction in a symptom of a disease (e.g., cancer) or disorder associated, e.g., with the target cell or a population thereof (e.g., cancer cells), as compared to a control.
  • An effective amount can be administered in one or more administrations.
  • the amount of CAR-expressing immune cells that comprises a “therapeutically effective amount” may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the cells to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the cells are outweighed by the therapeutically beneficial effects.
  • the term “therapeutically effective amount” includes an amount that is effective to “treat” an individual, e.g., a patient.
  • a therapeutic amount is indicated, the precise amount of the compositions contemplated in particular embodiments to be administered, can be determined by a physician in view of the specification and with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (individual).
  • a pharmaceutical composition of the present disclosure includes from 1 ⁇ 10 3 to 5 ⁇ 10 10 of the cells of the present disclosure.
  • the CAR- expressing immune cells of the present disclosure can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable excipients or diluents.
  • Formulations of the cells suitable for administration to a patient are generally sterile and may further be free of detectable pyrogens or other contaminants contraindicated for administration to a patient according to a selected route of administration.
  • the cells may be formulated for parenteral (e.g., intravenous, intra-arterial, intraosseous, intramuscular, intracerebral, intracerebroventricular, intrathecal, subcutaneous, etc.) administration, or any other suitable route of administration.
  • compositions that include the cells of the present disclosure may be prepared by mixing the cells having the desired degree of purity with optional physiologically acceptable carriers, excipients, stabilizers, surfactants, buffers and/or tonicity agents.
  • Acceptable carriers, excipients and/or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine and citric acid; preservatives (such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, or combinations thereof); amino acids such as arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine
  • An aqueous formulation of the recombinant polypeptides, proteases, nucleic acids, expression vectors, and/or cells may be prepared in a pH-buffered solution, e.g., at pH ranging from about 7.0 to 8.0, 4.0 to about 7.0, or from about 5.0 to about 6.0, or alternatively about 5.5.
  • buffers that are suitable for a pH within this range include phosphate-, histidine-, citrate-, succinate-, acetate-buffers and other organic acid buffers.
  • the buffer concentration can be from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending, e.g., on the buffer and the desired tonicity of the formulation.
  • a tonicity agent may be included in the formulation to modulate the tonicity of the formulation.
  • Exemplary tonicity agents include sodium chloride, potassium chloride, glycerin and any component from the group of amino acids, sugars as well as combinations thereof.
  • the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be suitable.
  • the term “isotonic” denotes a solution having the same tonicity as some other solution with which it is compared, such as physiological salt solution or serum.
  • Tonicity agents may be used in an amount of about 5 mM to about 350 mM, e.g., in an amount of 100 mM to 350 mM.
  • a surfactant may also be added to the formulation to reduce aggregation and/or minimize the formation of particulates in the formulation and/or reduce adsorption.
  • Example surfactants include polyoxyethylensorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenylpolyoxyethylene ethers (Triton-X), polyoxyethylene- polyoxypropylene copolymer (Poloxamer, Pluronic), and sodium dodecyl sulfate (SDS).
  • Suitable polyoxyethylenesorbitan-fatty acid esters are polysorbate 20, (sold under the trademark Tween 20TM) and polysorbate 80 (sold under the trademark Tween 80TM).
  • suitable polyethylene-polypropylene copolymers are those sold under the names Pluronic® F68 or Poloxamer 188TM.
  • suitable Polyoxyethylene alkyl ethers are those sold under the trademark BrijTM.
  • Exemplary concentrations of surfactant may range from about 0.001% to about 1% w/v.
  • the pharmaceutical composition includes CAR-expressing immune cells of the present disclosure, and one or more of the above-identified agents (e.g., a surfactant, a buffer, a stabilizer, a tonicity agent) and is essentially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, and combinations thereof.
  • a preservative is included in the formulation, e.g., at concentrations ranging from about 0.001 to about 2% (w/v).
  • the pharmaceutical composition comprises the harvested fifth population of cells comprising CAR-expressing immune cells resuspended to the desired concentration in Final Formulation Medium comprising Plasma-Lyte A+4% (w/v) HSA, diluted 1:1 with Cryostor R CS10 and frozen.
  • Final Formulation Medium comprising Plasma-Lyte A+4% (w/v) HSA, diluted 1:1 with Cryostor R CS10 and frozen.
  • a disease, disorder, or health condition in a subject in need thereof comprising administering a therapeutically effective amount of a pharmaceutical composition comprising a therapeutically effective amount of CAR- expressing immune cells as disclosed herein, wherein the pharmaceutical composition comprising a therapeutically effective amount of CAR-expressing immune cells is administered to the subject individually as a single therapy (monotherapy) or as a first therapy in combination with at least one additional therapies (e.g., second therapy).
  • the health condition is a cancer, e.g., a hematologic cancer.
  • the administered CAR-expressing immune cells or the pharmaceutical composition is administered to or provide anti-tumor immunity to the subject.
  • the first therapy and the second therapy are administered concomitantly. In some embodiments, the first therapy is administered at the same time as the second therapy. In some embodiments, the first therapy and the second therapy are administered sequentially. In some embodiments, the first therapy is administered before the second therapy. In some embodiments, the first therapy is administered after the second therapy. In some embodiments, the first therapy is administered before and/or after the second therapy. In some embodiments, the first therapy and the second therapy are administered in rotation. In some embodiments, the first therapy and the second therapy are administered together in a single formulation.
  • the CAR-expressing immune cells and pharmaceutical compositions of the disclosure can be administered alone or in combination with other agents (e.g., an antibody or an antigen binding fragment thereof, or a molecule).
  • agents e.g., an antibody or an antigen binding fragment thereof, or a molecule.
  • a vaccine, an oncoloytic virus, a checkpoint inhibitor, a T cell agonist antibody, chemotherapy, and/or a bispecific antibody can be combined with the pharmaceutical composition disclosed herein.
  • the pharmaceutical composition is administered with other cells (e.g., CAR T cells or other adoptively transferred T cells).
  • Administration “in combination with” one or more additional therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.
  • the one or more additional therapeutic agents, chemotherapeutics, anti-cancer agents, or anti-cancer therapies is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy, and surgery.
  • chemotherapy radiotherapy
  • immunotherapy immunotherapy
  • hormonal therapy hormonal therapy
  • toxin therapy and surgery.
  • anti-cancer agent are used interchangeably herein.
  • Various classes of anti-cancer agents can be used.
  • Non-limiting examples of anti- cancer agents include: alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, podophyllotoxin, antibodies (e.g., monoclonal or polyclonal), checkpoint inhibitors, immunomodulators, cytokines, nanoparticles, radiation therapy, tyrosine kinase inhibitors (for example, imatinib mesylate), hormone treatments, soluble receptors and other antineoplastics.
  • the disease, disorder, or condition is a cancer, an inflammatory disease, a neuronal disorder, HIV/AIDS, diabetes, a cardiovascular disease, an infectious disease, or an autoimmune disease.
  • the disease, disorder, or condition is cancer.
  • the cancer is lymphoma or leukemia.
  • the disease, disorder, or condition is a hyperproliferative disorder.
  • Hyperproliferative disorders include cancers and hyperplasia characterized by the unregulated overgrowth of cells. Hyperproliferative disorders frequently display loss of genetic regulatory mechanisms and may express native proteins inappropriately (including expression of proteins from other cell types or developmental stages, expression of mutated proteins, and expression of proteins at levels higher or lower than normal).
  • B-cell hyperproliferative disorders include B-cell leukemias and lymphomas such as, but not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), B-cell prolymphocytic leukemia, precursor B lymphoblastic leukemia, hairy cell leukemia, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, marginal zone lymphoma, mantle cell lymphoma, Burkitt’s lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, Waldenstrom’s macroglobulinemia, and/or other disorders characterized by the overgrowth of B-lineage cells.
  • ALL acute lymphoblastic leukemia
  • CLL chronic lymphocytic leukemia
  • B-cell prolymphocytic leukemia precursor B lymphoblastic leukemia
  • hairy cell leukemia hairy cell leukemia
  • DLBCL diffuse large B-cell lympho
  • the B-cell hyperproliferative disorder is a lymphoma.
  • the lymphoma is selected from a group consisting of diffuse large B cell lymphoma (DLBCL), large B cell lymphoma (LBCL), mantle cell lymphoma (MCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), Burkitt’s lymphoma, anaplastic large-cell lymphoma, angioimmunoblastic T cell lymphoma, and Hodgkin lymphoma.
  • the lymphoma is large B cell lymphoma.
  • the B- cell hyperproliferative disorder is a leukemia.
  • the leukemia is selected from a group consisting of acute lymphocytic leukemia (ALL), acute lymphoblastic leukemia (ALL), B cell acute lymphocytic leukemia (B-ALL), B cell acute lymphoblastic leukemia (B- ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), B cell prolymphocytic leukemia (B-PLL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), chronic myelomonocytic leukemia (CMML), hairy cell leukemia (HCL), juvenile myelomonocytic leukemia (JMML), large granular lymphocytic leukemia (LGLL), and T cell prolymphocytic leukemia (T-PLL).
  • ALL acute lymphocytic leukemia
  • ALL acute lymphoblastic leukemia
  • B-ALL B cell acute lymphoblastic
  • the leukemia is ALL or B-ALL. In some embodiments, the leukemia is pediatric ALL or B- ALL.
  • Hyperproliferative disorders include diseases such as, but not limited to, bladder cancer, including upper tract tumors and urothelial carcinoma of the prostate; bone cancer, including chondrosarcoma, Ewing's sarcoma, and osteosarcoma; breast cancer, including noninvasive, invasive, phyllodes tumor, Paget's disease, and breast cancer during pregnancy; central nervous system cancers, adult low-grade infiltrative supratentorial astrocytoma/oligodendroglioma, adult intracranial ependymoma, anaplastic astrocytoma/anaplastic oligodendroglioma/glioblastoma multiforme, carcinomatous lymphomatous meningitis, non-immunosuppressed primary CNS lymphoma, and metastatic spine tumors; cervical cancer; colon cancer, rectal cancer,
  • compositions comprising CAR-expressing immune cells are known and may be used in connection with the provided methods and compositions.
  • adoptive T cell therapy methods are described in US 2003/0170238; US 4690915; S.A. Rosenberg, Nat Rev Clin Oncol (2011) 8(10):577-85. See also M. Themeli et al., Nat Biotechnol (2013) 31(10):928-33; and T. Tsukahara et al., Biochem Biophys Res Commun (2013) 438(l):84-89.
  • this administering step can be accomplished using any method of delivery known in the art.
  • the CAR-expressing immune cells can be infused intravenously directly into the subject’s bloodstream or otherwise administered to the subject.
  • the step of administering which term is used interchangeably with the terms “introducing,” implanting,” and “transplanting,” CAR-expressing immune cells into an individual, by a method or route such that a desired effect(s) is/are produced.
  • the CAR- expressing immune cells, or their differentiated progeny can be administered by any appropriate route for the disease being treated that results in at least a portion of the administered cells or components of the cells remaining viable.
  • the period of viability of the cells after administration to a subject can be as short as a few hours, e.g., twenty-four hours, to a few days, to as long as several years, or even the lifetime of the individual, e.g., long-term engraftment.
  • the CAR-expressing immune cells described herein can be administered to a subject in advance of any symptom of a disease or health condition to be treated. Accordingly, in some embodiments the prophylactic administration of a pharmaceutical composition comprising a population of CAR-expressing immune cells prevents the occurrence of symptoms of the disease or health condition.
  • CAR-expressing immune cells are provided at (or after) the onset of a symptom or indication of a disease or health condition, e.g., upon the onset of disease or health condition.
  • a therapeutically effective amount of CAR-expressing immune cells can be at least 10 2 cells, at least 5 ⁇ 10 2 cells, at least 10 3 cells, at least 5 ⁇ 10 3 cells, at least 10 4 cells, at least 5 ⁇ 10 4 cells, at least 10 5 cells, at least 2 ⁇ 10 5 cells, at least 3 ⁇ 10 5 cells, at least 4 ⁇ 10 5 cells, at least 5 ⁇ 10 5 cells, at least 6 ⁇ 10 5 cells, at least 7 ⁇ 10 5 cells, at least 8 ⁇ 10 5 cells, at least 9 ⁇ 10 5 cells, at least 1 ⁇ 10 6 cells, at least 2 ⁇ 10
  • the pharmaceutical compositions include a therapeutically effective amount of CD22 CAR T cells as disclosed herein, which can be at least 10 2 cells, at least 5 ⁇ 10 2 cells, at least 10 3 cells, at least 5 ⁇ 10 3 cells, at least 10 4 cells, at least 5 ⁇ 10 4 cells, at least 10 5 cells, at least 2 ⁇ 10 5 cells, at least 3 ⁇ 10 5 cells, at least 4 ⁇ 10 5 cells, at least 5 ⁇ 10 5 cells, at least 6 ⁇ 10 5 cells, at least 7 ⁇ 10 5 cells, at least 8 ⁇ 10 5 cells, at least 9 ⁇ 10 5 cells, at least 1 ⁇ 10 6 cells, at least 2 ⁇ 10 6 cells, at least 3 ⁇ 10 6 cells, at least 4 ⁇ 10 6 cells, at least 5 ⁇ 10 6 cells, at least 6 ⁇ 10 6 cells, at least 7 ⁇ 10 6 cells, at least 8 ⁇ 10 6 cells, at least 9 ⁇ 10 6 cells, or multiples thereof.
  • CD22 CAR T cells as disclosed herein, which can be at least 10
  • the pharmaceutical compositions include a therapeutically effective amount of CD22 CAR T cells as disclosed herein, which may be about 1 ⁇ 10 6 cells/kg or 1 ⁇ 10 6 cells/kg.
  • the maximum dose will be fixed at the dose for a subject of 100 kg for any subjects weighing >100 kg at time of treatment.
  • a pharmaceutical composition comprising a therapeutically effective amount of CAR-expressing immune cells can be administered by any appropriate route that results in effective treatment in the subject, e.g., administration results in delivery to a desired location in the subject where at least a portion of the composition delivered, e.g., at least 1 ⁇ 10 5 cells, at least 10 5 cells, at least 2 ⁇ 10 5 cells, at least 3 ⁇ 10 5 cells, at least 4 ⁇ 10 5 cells, at least 5 ⁇ 10 5 cells, at least 6 ⁇ 10 5 cells, at least 7 ⁇ 10 5 cells, at least 8 ⁇ 10 5 cells, at least 9 ⁇ 10 5 cells, at least 1 ⁇ 10 6 cells, at least 2 ⁇ 10 6 cells, at least 3 ⁇ 10 6 cells, at least 4 ⁇ 10 6 cells, at least 5 ⁇ 10 6 cells, at least 6 ⁇ 10 6 cells, at least 7 ⁇ 10 6 cells, at least 8 ⁇ 10 6 cells, or at least 9 ⁇ 10 6 cells, is delivered to the desired site for a period of time.
  • the CAR-expressing immune cells are administered systemically, e.g., via intravenous infusion or injection.
  • the efficacy of a treatment including any of the compositions provided herein for the prevention or treatment of a disease or health condition can be determined by a skilled clinician. However, one skilled in the art will appreciate that a prevention or treatment is considered effective if any one or all of the signs or symptoms or markers of disease are improved or ameliorated. Efficacy can also be measured by failure of a subject to worsen as assessed by decreased hospitalization or need for medical interventions (e.g., progression of the disease is halted or at least slowed).
  • Treatment includes any treatment of a disease in a subject or an animal (some non-limiting examples include a human, or a mammal) and includes: (1) inhibiting the disease, e.g., arresting, or slowing the progression of symptoms; or (2) relieving the disease, e.g., causing regression of symptoms, slowing tumor growth or otherwise reducing tumor burden; and (3) preventing or reducing the likelihood of the development of symptoms.
  • Measurement of the degree of efficacy is based on parameters selected with regard to the disease being treated and the symptoms experienced.
  • a parameter is selected that is known or accepted as correlating with the degree or severity of the disease, such as a parameter accepted or used in the medical community.
  • suitable parameters can include reduction in the number and/or size of metastases, number of months of progression-free survival, overall survival, stage or grade of the disease, the rate of disease progression, the reduction in diagnostic biomarkers (for example without limitation, a reduction in circulating tumor DNA or RNA, a reduction in circulating cell-free tumor DNA or RNA, and the like), and combinations thereof.
  • diagnostic biomarkers for example without limitation, a reduction in circulating tumor DNA or RNA, a reduction in circulating cell-free tumor DNA or RNA, and the like
  • Therapeutic methods of the disclosure reduce symptoms and/or disease severity and/or disease biomarkers by at least about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% as compared to a reference subject that has not been administered with the CAR-expressing immune cells of the disclosure.
  • a therapeutically effective amount of a pharmaceutical composition can be an amount of the pharmaceutical composition that is sufficient to promote a particular beneficial effect when administered to a subject, such as one who has, is suspected of having, or is at risk for a disease or health condition.
  • an effective amount includes an amount sufficient to prevent or delay the development of a symptom of the disease or health condition, alter the course of a symptom of the disease or health condition (for example but not limited to, slow the progression of a symptom of the disease), or reverse a symptom of the disease or health condition.
  • Examples 1-18 below describe, inter alia, two different protocols of manufacturing T cells expressing a CD22 CAR (i.e., a CD22-specific CAR) in accordance with some non- limiting embodiments of the disclosure.
  • a CD22 CAR i.e., a CD22-specific CAR
  • Process 1 the lentiviral vector encoding CD22 CAR was generated in an adherent cell-based lentiviral vector manufacturing process.
  • the step of processing and cryopreserving the samples comprising immune cells obtained from human subject is optional (see, e.g., Example 1).
  • Protocol 2 the lentiviral vector encoding CD22 CAR was generated in an suspension cell- based-based lentiviral vector manufacturing process, which produces a much higher concentration of vector as compared to the adherent cell-based manufacturing process in Process 1.
  • Protocol v2 the step of processing and cryopreserving the samples comprising immune cells obtained from human subject is mandatory.
  • Protocol 2 also includes an expansion step where the cell media undergoes one or more media exchange steps, wherein a portion of the culture volume was removed and replaced with new media between Day 4 and Day 6 after seeding (see, e.g., Example 2 and 5).
  • CD22 CAR T cells (protocol v1)
  • a population of CD22 CAR-T cells was generated from a blood sample collected from a patient using a protocol for manufacturing CD22 CAR T cells, termed the v1 protocol, as illustrated in Figure 1A.
  • the patient leukapheresis (also referred to interchangeably as “apheresis”) sample was collected by standard protocol, and CD4+ and CD8+ T-cell enrichment was performed using beads that enrich for CD4+ and CD8+ cells.
  • Post-enrichment cells were washed and resuspended in a buffer in a process shown in Figure 2A. A subset of the cells (1 ⁇ 10 8 cells) was seeded.
  • transduction was performed by adding a solution of lentiviral vector encoding CRG-022, a CD22-specific CAR, to the cell sample to produce CAR-expressing cells. After transduction, the culture was expanded. The media used in the seeding step was the same media for expansion, e.g., TexMACS TM culture medium supplemented with appropriate cytokines (IL-7 and IL-15).
  • the lentiviral vector was generated using an adherent cell-based lentiviral vector manufacturing process. As shown in Figure 12, cells were subsequently washed and actively shaken on Day 3. On Day 6, 125mL of buffer was removed and 125mL of new buffer was added. The transduction efficiency on Day 6 was analyzed.
  • CD22 CAR T cells (protocol v2)
  • a population of CD22 CAR-T cells was generated from a blood sample collected from a patient using a protocol for manufacturing CD22 CAR T cells, termed the v2 protocol as illustrated in Figure 1B.
  • a patient apheresis sample was collected by standard protocol and used to manufacture a CD22 CAR-T cell population.
  • the cells were filtered (e.g., concentrated the sample into a smaller volume), washed and cryopreserved prior to enrichment using beads. Briefly, the washing and cryopreservation steps were performed on an automated cell processing instrument. The details of this process are shown in Figure 2B. Apheresis samples were diluted in Plasma-Lyte A and 4% (w/v) human serum albumin (HSA). The diluted samples were then washed, concentrated, eluted/resuspended, and filled in a FINIA 250 tubing set with Cryostor 10 buffer. Cells were cryopreserved in a controlled rate freezer until use.
  • HSA human serum albumin
  • Cells were then thawed using a dry heating device at 37°C, after which cells were enriched for CD4+ T cells and CD8+ T cells using beads that bind to these corresponding targets.
  • a subset of the enriched cells (2 ⁇ 10 8 cells) were activated by cytokines IL-7 and IL- 15 and seeded on Day 0.
  • transduction was performed by adding a solution of lentiviral vector encoding CRG-022, a CD22-specific CAR, was added to the cell sample to produce CAR-expressing cells. After transduction, the culture was expanded.
  • the media used in the seeding step was the same media for expansion, e.g., TexMACS TM culture medium supplemented with appropriate cytokines (IL-7 and IL-15).
  • the lentiviral vector encoding CRG-022 was generated in a suspension cell-based lentiviral vector manufacturing process. As shown in Figure 11, cells were subsequently washed and actively shaken on Day 3. On Day 4, 130mL of buffer was removed and 180mL of new buffer was added. Cells were harvested between Day 5 and Day 9 after seeding, at which point the cells were washed, concentrated, and automatically filled and finished in CryoMACS 50 bags and QC vials. The packaged cells were stored at -150° Celsius or colder.
  • FIG.2A-2C Three different methods for processing apheresis product for cryopreservation were compared (Fig.2A-2C).
  • the second method, used in the v2 cell manufacturing protocol described in Example 2 comprised a workflow using a Rotea instrument, wherein cells were diluted prior to loading on an instrument, cells were loaded onto the instrument, washed, concentrated, eluted/resuspended and subsequently frozen (Fig.2B).
  • the third method comprised a centrifugation step, wherein cells were loaded into conical tubes, centrifuged at 1936 ⁇ g for 15 minutes, and the platelet layer manually removed after centrifugation. Subsequently, the sample was diluted with buffer, and frozen (Fig.2C). For each of the three methods, healthy donor derived leukapheresis samples were used. [0288] After processing, cell viability, cell recovery and residual platelet levels were measured using methods described herein. Residual platelet levels were measured by the Sysmex instrument. The results for each of these metrics for each method are shown in Figure 3. The number of input cells is listed in Figures 3A-3C.
  • Cells were analyzed before (thawed cryoapheresis) and after CD4+/CD8+ enrichment (enriched fraction) by flow cytometry.
  • the sample analyzed before enrichment was designated the “thawed cryoapheresis” sample.
  • the sample analyzed after enrichment was designated the “enriched fraction” sample.
  • the enriched fraction contained 94% CD3+CD4+ and CD3+CD8+ cells, compared to just 50% in the thawed cyroapheresis samples.
  • the enriched fraction also had a higher percentage of viable cells (93.3%) compared to the thawed cryoapheresis samples (83.2%).
  • the second method used in the v2 manufacturing protocol generated a cell population with substantially lower residual platelets compared to the first method used in the v1 manufacturing protocol (the Prodigy method).
  • the cryopreservation apheresis preparation using v2 method also yielded similar or higher levels of viability and recovery compared to the other two methods.
  • EXAMPLE 4 Cell seeding evaluation [0292] Blood samples from donors were processed according to the methods described in Example 3 and transduced with lentiviral vectors. Thereafter, a population of the transfected cells were used to seed growth to generate a larger population of CAR-expressing cells. Two different seed quantities were tested: 100 ⁇ 10 6 and 200 ⁇ 10 6 .
  • Cells were expanded and generated at two different manufacturing sites using the manufacturing protocols described in Example 1 and Example 2. Cells were harvested on Day 7 and analyzed to compare cell characteristics between samples initially seeded with different CAR T quantities. As shown in Figure 7, the overall number of viable cells was comparable between the two seeding conditions, but the yield of CAR+ cells was slightly higher in the 200 ⁇ 10 6 condition compared to the 100 ⁇ 10 6 condition. Both the percentage of cell viability on Day 7 and the percentage contraction on Day 1 were similar between the two seed conditions ( Figure 8). The post-harvest transduction efficiency on D7 was slightly higher in the 200 ⁇ 10 6 condition compared to the 100 ⁇ 10 6 condition, as measured by the % CD3+/CAR+ cells.
  • the CD22 CAR-T manufacturing processes described herein comprised an expansion step, where activated, transfected CAR-T cells were cultured in growth medium for multiple days after seeding. The cells were grown in suspension. During this growth period, the cell media might be supplemented with additional volume and/or exchanged, wherein volume was removed and replaced with new media between Day 4 and Day 6 after seeding.
  • Six different media exchange protocols (G-Rex 1, G-Rex 2, G-Rex 3, G-Rex 4, G-Rex 5, G-Rex 6) were tested to determine the optimal media exchange protocol. The details of each of the conditions are listed in Figure 9.
  • CAR-expression products were prepared according to the v1 or v2 protocol described in examples 1 and 2. Cells were harvested on Days 3-11 and analyzed for population doubling, viability and CAR expression (CAR+). As shown in Figure 13A, the cumulative population doubling was higher for the v2 protocol on Days 5-11 compared with the v1 protocol. As shown in Figure 13B, the v2 protocol showed cell viability percentages between the two samples were similar and the v2 protocol had slightly higher cell viability on Day 6 and 7 compared with the v1 protocol.
  • FIG. 13C showed that transduction efficiency between samples collected on different days for the two different protocols were similar.
  • the data suggested that media exchange on Day 4 increased yield and resulted in comparable cell viability and transduction efficiency.
  • E XAMPLE 6 T cell harvesting time optimization [0297] CD22-specific CAR T cells were prepared according to the v1 or v2 protocol described in Examples 1 and 2. Cells were harvested between Day 5 and 9. Population doubling time and transduction efficiency were measured. Samples with the highest population doublings and highest transduction efficiencies enable the harvesting of cells earlier in the process. As shown in Figure 14, v2 product harvested on Day 5 achieved similar number of population doublings and transduction efficiency as the v1.0 product harvested on Day 6 and v2 product harvested on Day 7.
  • CD22 CAR-T products were generated for two healthy donors (4224BW and 4267BW) and a patient sample (CCT-5029-047) according to the v2 method described in Example 2.
  • CD22 CAR-T products were harvested on Day 4, 5, 7, or 9.
  • Total CAR+ yield, cumulative population doublings, viability, and transduction efficiencies were measured.
  • Figure 15 and Figure 16A-C show that CAR+ T cell yield in the cells harvested on Day 5 met the final dose fill requirement for both healthy donor and patient.
  • E XAMPLE 7 Characterization of T cell subtypes [0299] Six CAR-T products were prepared according to the v1 or v2 cell manufacturing protocol described in Examples 1 and 2.
  • T cell memory phenotypes were analyzed using flow cytometry analysis based on the gating scheme described in Figure 38.
  • An equivalence test with matched-pairs Two One-sided Test (TOST) was used to compare the results from the v1 and v2 protocol with respect to the production of certain T cell memory phenotypes.
  • TOST Two One-sided Test
  • a more juvenile, less differentiated phenotype (with increased TSCM and T CM , and decreased T EM ) for the v2 protocol was produced compared to the v1 protocol.
  • EXAMPLE 8 Characterization of T-cell exhaustion and activation [0300] Six preparations of CAR-T products were prepared according to the v1 or v2 protocol described in Examples 1 and 2.
  • T-cell exhaustion and activation phenotypes were analyzed using a flow cytometry analysis measuring 4-1BB, CD69 and PD-1 expression (Figure 39), to assess the quality of the starting apheresis product. Since apheresis are collected taken from sick patients that have already been treated with other B cell-specific immunotherapies, their T cells are often poor quality and already close to exhausted. Therefore, measuring 4- 1BB, CD69 and PD-1 expression is a well-accepted methodology to evaluate the starting material for manufacturing. [0301] An equivalence test with matched-pairs TOST was used to compare the results from the v1 and v2 protocol related to expression of markers of T cell activation/exhaustion.
  • Figure 19 showed higher levels of T cell activation/exhaustion markers were observed in cells generated by the v2 protocol, where levels of 4-1BB, CD69 and PD-1 were all increased in the drug product made with the v2 protocol as compared to the v1 protocol.
  • EXAMPLE 9 In vivo tumor clearance study [0302] A cohort of 17 NSG (NOD.Cg-Prkdc scid Il2rgamma tm1Wjl/SzJ) mice were used to analyze in vivo tumor clearance using the CD22 CAR-T cell drug product described herein. All mice were infused with 1 ⁇ 10 6 Nalm6 tumor cells.
  • Nalm6 is a B cell precursor leukemia cell line used as a xenograft model of acute lymphoblastic leukemia.
  • the mice were infused with CD22 CAR+ cells, un-transduced T cell controls or no cells according to the workflow scheme shown in Figure 20.
  • Ten mice in each group received CD22 CAR-T cells generated using the v2 protocol and harvested on Day 5, Day 7 or Day 9, respectively, and ten mice received CAR-T cells generated using the v1 protocol and harvested on Day 7.
  • two different dosing schemes were used: five mice were infused with a low dose (1 ⁇ 10 6 cells) and five mice were infused with a high dose (5 ⁇ 10 6 cells).
  • mice treated with T cells generated by harvesting on D5 using the v2 cell manufacturing protocol continued to limit tumor burden out to Day 53 post injection. In contrast, tumor progression was much faster in other groups. Tumors in both the mice infused with un-transduced cells and the mice with tumor only reached termination size on Day 12, and tumors in mice treated with CD22 CAR+ T cells generated using the v1 protocol harvested on D7 and using the v2 protocol but harvested on D7 or D9 reached termination size around Day 53.
  • Figure 22 shows that more than 50% survival was observed at the end of study in mice treated with the low dose of CD22 CAR-T cells generated by harvesting on D7 using the v1 cell manufacturing protocol and CD22 CAR-T cells generated by harvesting on D5 using the v2 cell manufacturing protocol. More than 50% survival was observed at the end of study in mice treated with the high dose of CAR-T cells generated by harvesting on D5, D7 and D9 using the v2 cell manufacturing protocol, but less than 50% survival was observed at the end of study in mice treated with the high dose of CAR-T cells generated by harvesting on D7 using the v1 cell manufacturing protocol. Body weight was maintained throughout the study because, in these studies, tumor growth did not appear to significantly change body weight.
  • Peripheral mouse blood was sampled at various timepoints during the course of the experiment from each animal. Blood cells were analyzed by flow cytometry to detect CD3+/CD19- cells, CD3+/CD19-/CD22CAR+ cells, CD3+/CD19-/CD22CAR+CD4+ cells, and CD3+/CD19-/CD22CAR+CD8+ cells. Additionally, VCN per CD22 CAR+ per cell was analyzed by measuring the number of UC462 copies.
  • mice treated with the CAR-T cells generated by the v2 cell manufacturing protocol and harvested on Day 5 resulted in the highest levels of detected CD3+ T cells, CD22 CAR+ T cells, CD3+/CD19-/CD22CAR+ cells, and CD3+/CD19-/CD22CAR+/CD4+ cells in the blood compared to the other CAR-T cell drug products.
  • the mice treated with the high dose of the CAR-T cells generated by the v2 protocol and harvested on Day 5 resulted in a much higher VCN on Day 28 compared to any other analyzed data point.
  • CD22 drug products were also manufactured from excess patient apheresis for two patients (CCT-5029-050 and CCT-5029-047) using the v1 and v2 cell manufacturing protocols described herein.
  • Cell viability %, number of viable cells/mL and population doublings over time were measured ( Figure 24). Both patient-derived CAR-T drug products yielded high % viability when harvested on Day 5 and onward. Additionally, the generated cell density was similar for both samples.
  • Cell samples were harvested on Day 5, Day 7 and Day 9 for the v2 protocol and Day 7 for the v1 protocol. Cells were analyzed using the IFNgamma (IFN ⁇ ) ELISpot assay described previously ( Figure 25).
  • CAR-T cells generated from the CCT-5029-050 sample showed similar IFNgamma (IFN ⁇ ) secretion, whereas cells generated from the CCT-5029-047 sample showed higher IFNgamma (IFN ⁇ ) secretion by CAR-T cells generated using v2 cell manufacturing protocol.
  • CAR-T cells for each time point and protocol were also analyzed using flow cytometry to assess the frequency of different T cell activation and exhaustion markers using the flow cytometry protocol previously described.
  • Figure 26 showed that CAR-T cells generated using the v2 protocol and harvested on Day 5 had higher levels of CD3+CAR+PD1+ and CD3+CAR+TIGIT+ cells and therefore showed higher T-cell activation in the population compared to CAR-T cells generated using the v1 protocol and CAR-T cells generated using the v2 protocol and harvested on D7 or D9.
  • Transduction efficiencies and total CAR+ yield were analyzed for each of the process Days 5, 7 and 9.
  • Figure 27 showed that transduction efficiency was similar in CAR-T cells harvested at different time points, and the total CAR+ yield increased as cells were harvested later.
  • E XAMPLE 10 v1 and v2 products equivalency [0306] A comparability experiment was performed between products generated by the v1 and v2 protocols. The experiment used a prospective side-by-side (matched pairs) evaluation of the drug product from 6 split batches of healthy donor apheresis collections. This number of split paired healthy donor runs was determined to ensure the study was powered sufficiently based on equivalence acceptance criteria. Apheresis was collected from normal peripheral blood of healthy donors and was split approximately equally into six samples. CAR-T products were generated using the v1 or v2 cell manufacturing protocol. Cells were harvested and then evaluated for cell viability, CD3 expression, transduction efficiency and vector copy number using the protocols described herein.
  • IFNgamma (IFN ⁇ ) secretion was measured by the ELISpot cytokine release assay according to the protocol described herein.
  • CAR-T cells were co-cultured with CD22-expressing target cells at a range of E:T ratios between 0:1 to 2:1 on an ELISpot IFNg plate. The plates were washed and substrate was added. IFNgamma (IFN ⁇ ) positive spots were scored by CTL Immunospot.
  • Figure 30 shows that CAR-T cells harvested on Day 5 released similar amount of IFNgamma (IFN ⁇ ) as the CAR-T cells harvested on Day 6 and Day 7.
  • CAR-T cells were prepared by v2 process with media exchange according to methods described herein, and cells were harvested on Days 5, 7, 9 or 11.
  • IFNgamma (IFN ⁇ ) secretion was measured by the ELISpot cytokine release assay according to the protocol described herein.
  • CAR-T cells were co-cultured with CD22-expressing target cells at a range of E:T ratios between 0:1 to 2:1 on an ELISpot IFNgamma plate. The plates were washed and substrate was added. IFNgamma (IFN ⁇ ) positive spots were scored by CTL Immunospot.
  • Figure 31 shows that CAR-T cells prepared with media exchange and harvested on Day 5 induced the highest amount of IFNgamma (IFN ⁇ ) release.
  • CD22 CAR-T products were prepared from two donors (donor 1 and donor 2) according to the v2 method described in Example 2. CAR-T cells were harvested on Days 5, 6, 7, 10, or 12 after seeding. CAR-T cells were co-cultured with CD22-expressing target cells at a range of E:T ratios between 0:1 to 2:1 on an ELISpot IFNgamma plate. The plates were washed and substrate was added. IFNgamma (IFN ⁇ ) positive spots were scored by CTL Immunospot. Total viable cells were measured at different time points.
  • Figure 32A showed that the number of total viable cells increased as the cells expanded in culture.
  • Figures 32B and 32C demonstrated that CAR-T cells harvested between Days 5 and 7 released more IFNgamma (IFN ⁇ ) than cells harvested on Day 10 and Day 12.
  • Cytotoxicity [0312] CAR-T products were prepared according to the v2 protocol described in Example 2. Cells were harvested on Days 5, 7 and 9. CAR-T cells were co-cultured with CD22 expressing target cells or CD22 knocked out target cells at a range of E:T ratios between 1:0.03 to 2:1. Cell toxicity was measured by cytotoxicity assay as described in Figure 49.
  • EXAMPLE 13 Measurement of CD22CAR-T transduction efficiency and CD3+ T cells by flow cytometry [0314] A single flow cytometry method, designated “Transduction Efficiency and T Cell Phenotype for CD22-CAR”, was used to measure several different product quality attributes for drug product release as follows. [0315] Cryopreserved CD22 CAR-T cells were thawed, counted by NC-200, and stained with anti-human CD3 PE-Cy7, recombinant human CD22 Fc Chimera Protein AF647, and 7AAD viability dye. Cells that are negative for the 7AAD dye are viable.
  • CD22 CAR-T drug product potency was evaluated by measuring the production of interferon gamma (IFN ⁇ or IFNgamma) produced by CD22 CAR-T drug product upon stimulation with a Nalm6 cell line engineered to express CD22.
  • IFN ⁇ or IFNgamma interferon gamma
  • CD22 CAR-T drug product CD22 CAR-T drug product
  • untransduced CD3P T cells CCG ⁇ 022 ⁇ NEG
  • CD22 CAR-T drug product system suitability (previously characterized) CD22-CAR T-Cells (CD22 CAR-T-SS) were thawed, counted, and cultured in flasks for 18-24 hours prior to initiating the ELISpot assay to allow effector cells to recover from the effects of cryopreservation.
  • CD22 CAR-T drug product and CD22 CAR-T-PC cells the concentration of CD22 CAR-TP cells in suspension were calculated by normalizing the viable cell concentration to their respective %7AAD-/CD3P/CD22 CAR P value. All three sample effector cells were then separately plated in quadruplicate and serially diluted across a range of effector:target ratios. Additionally, effector cell control samples such as effector cells alone and effector cells alone stimulated with a pre-mixed cocktail containing phorbol 12- myristate-13-acetate (PMA) and ionomycin were also plated in quadruplicate. All ELISpot plates were then incubated for 18-24 hours.
  • PMA phorbol 12- myristate-13-acetate
  • ELISpot plates were decanted, washed, and stained with anti-human-IFN- ⁇ (biotin) detection antibody.
  • a biotin-conjugated secondary antibody was used for detection using alkaline phosphatase linked to streptavidin and a colorimetric substrate (BCIP; 5-bromo-4-chloro-3’- indolylphosphate p-toluidine).
  • BCIP colorimetric substrate
  • Stained plates were then imaged and analyzed using the CTL Immunospot® S6 Universal Analyzer and the Immunospot® Single ⁇ Color Enzymatic Suite Analysis Software.
  • Cytotoxic T-cells secreting IFN- ⁇ are visualized as individual spots (spot- forming units, SFU) with each spot representing a single CAR-T cell.
  • EXAMPLE 15 Measuring vector copy number in transduced cells [0319] In this Example, a method for measuring vector copy number per cell using droplet digital PCR (ddPCR) is described. [0320] The vector copy number assay, Vector Copy Number Determination, targets the product-specific sequence presented in the transduced cells to determine the number of gene copies integrated into the T cell genome in the CD22 CAR-T drug product.
  • DNA extracted from the drug product was used in a Droplet Digital TM PCR (Bio-Rad QX200TM) reaction to detect a portion of the CD22 CAR DNA sequence and multiplexed with an assay for the human albumin sequence to determine the cell number.
  • Non-transduced DNA serves as a negative control
  • linearized CD22 gBlock plasmid (Integrated DNA Technologies) diluted in human gDNA served as a positive control for the assay. The number of CD22 CAR DNA copies per transduced cell is reported.
  • CD22 CAR-T cells were prepared according to v2 method described in Example 2 and harvested on Days 5, 7, and 10 post seeding. The vector copy number per cell, per CAR+ cells, and transduction efficiency were measured. As shown in Figure 36, VCN per transduced cell was well below 5 copies/CAR+ cells (generally accepted limit for VCN in autologous CAR-T products). There was a small trend observed of decreasing VCN with increased day of harvest.
  • EXAMPLE 17 Confirmation of CD22 CAR potency using Jurkat cells
  • Jurkat cells were transduced using a lentiviral vector encoding a CD22 CAR. The Jurkat cells were expanded, isolated and confirmed to express the CD22 CAR using a flow cytometry analysis.
  • CD22 CAR-expressing Jurkat cells were then co-cultured in the presence of Nalm6 cells expressing CD22.
  • An ELISpot analysis was performed to measure IL-2 release as a function of CAR-Jurkat cell function.
  • the level of IL-2 released by the CAR-expressing Jurkat cells was proportional to the ratio of Jurkat cell:Nalm6 cell and also highest for the Nalm6 expressing high levels of CD22 and lowest for the Nalm6 without CD22 expression. This result demonstrates the ability of the CD22 CAR to recognize CD22 and initiate the relevant immune signaling pathways that lead to potency in a clinical context.
  • EXAMPLE 18 Manufacturing CD22 CAR T cells (protocol v2)
  • This Example describes the manufacturing of a population of CD22 CAR-T cells from a cryopreserved apheresis sample in accordance with some non-limiting embodiments of the disclosure.
  • the cryopreserved apheresis sample was thawed and the manufacturing process proceeded continuously for approximately 5 - 9 days.
  • T cells were isolated from the cryopreserved apheresis sample, transduced with a lentiviral vector expressing an anti-CD22 CAR comprising a leader sequence (SEQ ID NO: 28), an anti-CD22 scFv (SEQ ID NO: 1), a peptide linker (SEQ ID NO: 29), a CD8 ⁇ hinge domain (SEQ ID NO: 24), a CD8 ⁇ transmembrane domain (SEQ ID NO: 25), a peptide linker (e.g., spacer) LYC (SEQ ID NO: 31) comprising a portion of the CD8 ⁇ cytoplasmic domain, a 4- 1BB(CD137) co-stimulatory signaling domain (SEQ ID NO: 26) , and a CD3 ⁇ primary T cell activation domain (SEQ ID NO: 27) expressed under the control of an EF1 ⁇ promoter, and expanded to produce a dose of 1 ⁇ 10 6 viable CD3+CAR+ cells/
  • TexMACSTM medium An aliquot of TexMACSTM medium was added to a vial of lyophilized IL-7 and a vial of lyophilized IL-15, respectively. Once the cytokines were resuspended in solution, the entire amount was removed from each vial and added to the mixing assembly along with TexMACSTM and 3% HABS. This medium was referred to as modified TexMACSTM Medium (MTM). The supplemented medium was then terminally filtered using a customized XLM Media Filtration Assembly that consisted of 0.2/0.1 ⁇ m Polysulfone (PES) double layer filter membranes. Aliquots were filled as shown in Table 4 and were stored at 2 - 8°C with light protective cover until use.
  • PES Polysulfone
  • Apheresis sample receipt and cryopreservation [0338] Apheresis sample was collected at an approved blood collection center and transported to the manufacturing site in a temperature-controlled shipping container at 2 - 8°C for manufacturing within 48 hours from the end of collection to spiking of the apheresis bag. At the manufacturing site, the apheresis material was initially quarantined and assessed for label integrity, bag integrity, and verification of chain of identity and chain of custody.
  • FINIA FINIA Fill and Finish System
  • the processed apheresis sample was loaded on a FINIA, an automated formulation and fill system, where final formulation involved a 1:1 volume dilution with the cryoprotectant CryoStor® 10.
  • the total viable cells post-Rotea wash determines the volume and number of CS250 bags that were formulated. In instances where there were >12.59 ⁇ 10 9 WBCs post-Rotea wash, three bags were formulated at a volume of 54 mL per bag. In instances where ⁇ 12.59 ⁇ 10 9 WBCs post-Rotea wash, two bags were formulated at 70 mL per bag.
  • a 12 mL QC bag was also filled with cell suspension on the FINIA and aliquoted in the biosafety cabinet (BSC) at 1 mL/vial for further characterization and retains.
  • the FINIA inputs for 2 or 3 bags are summarized in Table 22 and Table 23, respectively.
  • T ABLE 22 FINIA inputs for 2 apheresis bags Parameter Value Units QC Bag Fill Volume (mL) 12
  • TABLE 23 FINIA inputs for 3 apheresis bags Parameter Value Units
  • the formulated cell suspension was placed inside the CRF before the sample vials to ensure cells experience shorter DMSO exposure (e.g., addition of DMSO to placement the inside CRF) hold time than the sample vials.
  • TABLE 24 Key process setpoints on Day -X Parameter Target Values Apheresis dilution by Rotea 1:1 (v/v) in Wash/Resuspension Buffer . b Applicable when transporting cryopreserved apheresis and samples on dry ice.
  • T ABLE 25 CRF profile for apheresis Step Rate of Cooling/Warming Temperature [0342] Identification of the storage cryogenic freezer and rack were generally performed prior to the completion of the cryopreservation.
  • cryopreserved cells were removed from the CRF before the sample vials to minimize CRF to cryogenic freezer transfer hold time. The cells were then placed on dry ice or inside a CryoPod TM and transferred to be stored in a temperature-monitored cryogenic freezer at ⁇ - 130°C until ready for manufacturing.
  • C. T cell enrichment and activation (Day 0) [0343] On Day 0, prior to the thaw of the cryopreserved apheresis sample, the CliniMACS buffer with 2% v/v or 0.5% w/v HSA was prepared in the BSC. A transfer set was spiked into the CliniMACS buffer bag and HSA was transferred into the buffer bag.
  • each bag contained 1.2 ⁇ 10 9 CD3+ cells and 2.5 ⁇ 10 9 target cells, only one bag would be thawed.
  • the cryopreserved apheresis sample was retrieved from vapor phase of liquid nitrogen storage and transported on dry ice or in a CryoPod TM . Prior to thawing, the cryopreserved apheresis sample was placed in a secondary bag. The bag(s) were thawed using the “plasma” protocol at 37 ⁇ 1°C with mixing via the Plasmatherm for target thaw duration of 4 minutes ⁇ 30 seconds.
  • the post-thaw apheresis bag(s) were placed on cold gel pack(s) and transferred into a BSC. In instances where more than one bag was needed, the bags were combined into a transfer pack prior to dilution with 2-8°C MTM to minimize the DMSO toxicity during process hold time.
  • a sample was taken from the diluted apheresis sample for cell count and viability. This sample, in combination with the apheresis flow phenotype results on Day -X (CD3/4/8), were used to generate the number of input target cells for the subsequent step.
  • CD4+/CD8+ cells Up to 3.0 ⁇ 10 9 CD4+/CD8+ cells were loaded on the CliniMACS Prodigy for co- enrichment of CD4+/CD8+ cells.
  • the cells were washed with CliniMACS buffer + 2% v/v or 0.5% w/v HSA, co-labeled with CliniMACS CD4/CD8 selection beads, and co-enriched via an integrated magnetic cell selection column.
  • T cell enrichment the volume of the post-selection positive fraction containing the enriched CD4+/CD8+ cells was determined. A sample was taken from the positive fraction bag through the integrated closed-system sample pouch. The sample pouch was heat sealed and removed.
  • Samples were also aliquoted in the BSC into 5 cryovials for phenotype at 1 ⁇ 10 7 cells/vial and retained at 3 ⁇ 10 6 cells/mL for use as negative controls for VCN.
  • the phenotype vials were cryopreserved and stored in LN2 while VCN retains were stored at -80°C.
  • the negative fraction (non-target cells) was also aliquoted into 4 ⁇ 1 mL cryovials and stored in the vapor phase of LN2 for future characterization testing or investigational purposes if needed to troubleshoot the enrichment step.
  • T ABLE 26 Prodigy T Cell Transduction (TCT) activity matrix P D i f Total a Value is entered based on Day 0 activation time T ABLE 27: Key process setpoint on Day 10 Parameter Target Values D. Transduction (Day 1) [0353] On Day 1, approximately 22 - 26 hours after Day 0 culture initiation, activated T cells were transduced with lentiviral vector (CD22.BB.Z) produced by Oxford Biomedica. Vector volume consumption was calculated based on the official infectious titer of the vector batch and number of cells seeded on Day 0 (up to 200 ⁇ 10 6 co-enriched CD4+/CD8+ cells) to achieve a multiplicity of infection (MOI) of 2.0 TU/cell.
  • MOI multiplicity of infection
  • the lentiviral vector vials were removed from the storage freezer and thawed at room temperature for ⁇ 60 minutes.
  • the required vector volume was diluted into MTM using appropriately syringe(s) that can measure volume to the hundredth of a unit.
  • the medium served as a carrier for the vector formulation during the Prodigy’s automated vector addition step.
  • the Prodigy rinsed the bag with additional MTM to ensure all vector volume is added, which brought the total volume to 100 mL and cell culture continued at 37°C with 5% CO2 until Day 3.
  • TABLE 28 Key process setpoint on Day 1 Parameter Target Values Incubation temperature 37 ⁇ 0.5°C E.
  • the dose factor In order to proceed with harvest, the dose factor must be ⁇ 4.1 using the equation below. [0357] If there were sufficient viable CD3+CAR+ cells to meet the dose requirements during Day 5 sampling, harvest would proceed. If total viable CD3+CAR+ cell numbers were not sufficient, cell counts were taken on Day 6, and the viable CD3+CAR+ cell numbers were calculated with Day 4 transduction efficiency (TE) added with a projected 7% increase based on historical patient runs to determine if cells could be harvested on Day 7. If viable CD3+CAR+ cell numbers were still not sufficient on Day 7 using the Day 4 TE with projected 7% increase, the culture could be extended up to Day 9.
  • TE Day 4 transduction efficiency
  • Harvest (Day 5, 7, or 9) [0359] Harvest determination: [0360] On Day 5, a pre-harvest cell count was performed to confirm target viable CD3+CAR+ cell number was achieved based on Day 5 cell count and Day 4 TE before harvest was initiated. If target viable CD3+CAR+ cell number for harvest was not achieved based on the pre-harvest cell count and additional culture was needed, a 50% media exchange was performed on both Day 5 and 6. A portion of spent media was removed (125 mL) via centrifugation of the CentriCult Unit (CCU) and replaced with supplemented media (125 mL).
  • CCU CentriCult Unit
  • TCT Prodigy T Cell Transduction
  • transduced cells were harvested out of the CCU via centrifugation of the CCU, followed by media exchange and elution/resuspension into Plasma-Lyte A + 4% HSA (w/v).
  • the cell suspension was filled into one to two of the 50 mL size product bag(s) at a target fill volume of 20 mL per bag, and one QC bag was filled at a volume of 24 mL. If 2 final bags were filled, Table 36 was used to complete a 3 Material Fill on the FINIA. If only 1 bag was filled, Table 37 and Table 38 were used to complete a 2 Material Fill on the FINIA and adjust the inputs accordingly. If there were any unexpected cell recovery losses, the QC bag fill could be reduced to as low as 20 mL without sacrificing volume needed for QC testing and retains.

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Abstract

The present disclosure relates generally to methods of making a population of immune cells engineered to express a chimeric antigen receptor (CAR) that provide several improvements over existing manufacturing methods, thereby enabling production of a robust supply of clinically useful CAR T-cell therapies.

Description

Attorney Docket: 063384-518001WO METHODS FOR MANUFACTURING CAR T CELLS CROSS-REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of priority to U.S. Provisional Patent Application Serial No.63/591,026, filed on October 17, 2023. The disclosure of the above-referenced application is herein expressly incorporated by reference in its entirety, including any drawings. INCORPORATION OF THE SEQUENCE LISTING [0002] The material in the accompanying Sequence Listing is hereby incorporated by reference into this application. The accompanying Sequence Listing XML file, named Sequence_Listing_ST26063384-518001WO.xml, was created on October 16, 2024, and is 40,960 bytes. FIELD [0003] The present disclosure relates generally to the fields of immuno-therapeutics and cell therapy, and particularly relates to improved methods of making and/or manufacturing a population of immune cells expressing a chimeric antigen receptor (CAR). The disclosure provides populations of CAR-expressing immune cells made and/or manufactured by the improved methods, pharmaceutical compositions comprising the same. The disclosure also describes methods for treating a health condition in a subject in need thereof. BACKGROUND [0004] Chimeric antigen receptor (CAR) T-cell therapy has shown promising therapeutic effects in treating various health conditions, including cancers, particularly hematologic cancers. Typically, CAR-T cells are generated by genetic engineering of either the patient’s own immune cells (autologous) or immune cells from unrelated human donors (allogenic). Production of high-quality, GMP-grade CAR-T cells is an essential prerequisite for the wide application of this technology. Successful manufacturing of such high-quality CAR T-cell products for clinical applications has been challenging and costly, with common pitfalls including lengthy turnaround times and unacceptably low manufacturing success rates. [0005] Current manufacturing processes for CAR-T cells have several drawbacks and so there is a pressing need to develop better and more efficient manufacturing processes for large-scale production of CAR-T cells with improved therapeutic activities. SUMMARY [0006] The present disclosure relates generally to, inter alia, improved methods of making and/or manufacturing a population of immune cells expressing a chimeric antigen receptor (CAR). Also provided are populations of CAR-expressing immune cells made and/or manufactured by the improved methods, as well as pharmaceutical compositions comprising a population of CAR-expressing immune cells of the disclosure. The disclosure also provides methods for treating a health condition in a subject in need thereof by administering the pharmaceutical compositions of the disclosure. [0007] In one aspect of the disclosure, provided herein is a method of making a population of chimeric antigen receptor (CAR)-expressing immune cells, the method comprising the steps of: (a) obtaining a liquid sample comprising a first population of cells comprising immune cells from a human subject; (b) processing the first population of cells to remove platelets thereby generating a second population of cells, wherein the second population of cells includes at least 1×104 total cells and wherein less than 20% of the total number of cells in the second population of cells are platelets; (c) seeding a third population in a volume of a first buffer, wherein the third population of cells is a subset of the second population of cells; (d) transducing the third population of cells with a recombinant polynucleotide encoding a CAR thereby generating a fourth population of cells, wherein the fourth population of cells includes CAR-expressing immune cells; (e) expanding the fourth population of cells yielding a fifth population of cells; and (f) harvesting the fifth population of cells on Day 5 or later after the seeding in step (c). In some embodiments, the method further includes removing at least 50% of the volume of the first buffer on or before Day 4 after seeding. In some embodiments, at least 2.4% of cells in the fifth population of cells are CCR7+CD45RA+ immune cells. In various embodiments of the methods disclosed herein, the third population of cells comprising immune cells is seeded in a volume of a first buffer that comprises growth media. In these instances, the first buffer is also referred to as the first media. [0008] In one aspect, provided herein is a method of making a population of CAR-expressing immune cells, the method including the steps of: (a) obtaining a liquid sample including a first population of cells including immune cells from a human subject; (b) processing the first population of cells thereby generating a second population of cells; (c) seeding a third population of cells in a volume of a first buffer (e.g., first media), wherein the third population of cells is a subset of the second population of cells; (d) transducing the third population of cells with a recombinant polynucleotide encoding a CAR thereby generating a fourth population of cells, wherein the fourth population of cells includes CAR-expressing immune cells; (e) expanding the fourth population of cells to yield a fifth population of cells; (f) removing at least 50% of the volume of the first buffer (e.g., first media) on or before Day 4 after the seeding; and (g) harvesting the fifth population of cells on Day 5 or later after the seeding in step (c). In some embodiments, at least 2.4% of cells in the fifth population of cells are CCR7+CD45RA+ immune cells. [0009] In one aspect, provided herein is a method of making a population of CAR-expressing immune cells, the method including the steps of: (a) obtaining a liquid sample including a first population of cells including immune cells from a human subject; (b) processing the first population of cells thereby generating a second population of cells; (c) seeding a third population of cells in a volume of a first buffer (e.g., first media), wherein the third population of cells is a subset of the second population of cells; (d) transducing the third population of cells with a recombinant polynucleotide encoding a CAR thereby generating a fourth population of cells, wherein the fourth population of cells includes CAR-expressing immune cells; (f) expanding the fourth population of cells yielding a fifth population of cells; and (g) harvesting the fifth population of cells on Day 5 or later after the seeding of step (c), wherein at least 2.4% of cells in the fifth population of cells are CCR7+ CD45RA+ immune cells. [0010] In another aspect, provided herein is a method of making a population of CAR- expressing immune cells, the method including: (a) obtaining a liquid sample including a first population of cells including immune cells from a human subject; (b) processing the first population of cells to remove platelets thereby generating a second population of cells, wherein the second population of cells includes at least 1×104 total cells and wherein less than 20% of the total number of cells in the second population of cells are platelets; and (c) seeding a third population of cells in a volume of a first buffer (e.g., first media), wherein the third population of cells is a subset of the second population of cells, wherein the third population of cells include at least 2.0 ^108 cells; (d) transducing the third population of cells with a recombinant polynucleotide encoding a CAR thereby generating a fourth population of cells, wherein the fourth population of cells includes CAR-expressing immune cells; (e) expanding the fourth population of cells yielding a fifth population of cells; (f) removing at least 50% of the volume of the first buffer (e.g., first media) on or before Day 4 after the seeding; and (g) harvesting the fifth population of cells on or before Day 9 after the seeding, wherein at least 2.4% of cells in the fifth population of cells are CCR7+CD45RA+ immune cells. [0011] Non-limiting exemplary embodiments of the disclosed method of making a population CAR-expressing immune cells include one or more of the following features. In some embodiments, less than 18%, less than 15%, less than 12%, less than 10%, less than 8%, or less than 5% of the total number of cells in the second population of cells are platelets. In some embodiments the processing of the first population of cells includes diluting the liquid sample including the first population of cells with a second buffer, thereby generating a diluted liquid sample including the first population of cells. [0012] In some embodiments, the liquid sample including the first population of cells is diluted prior to removing platelets. In some embodiments, the liquid sample including the first population of cells has a total volume of at least 50 mL, at least 100 mL, at least 200 mL, at least 250 mL, at least 300 mL, at least 400 mL, at least 500 mL, at least 600 mL, at least 700 mL, at least 800 mL, at least 900 mL, at least 1 L, or at least 2 L. In some embodiments, the liquid sample including the first population of cells is a blood sample or a sample obtained by apheresis. In some embodiments, the liquid sample including the first population of cells is a leukapheresis or apheresis sample. In some embodiments, the diluting includes adding an equal volume of the second buffer to the liquid sample, wherein the volume of the second buffer added to the liquid sample including the first population of cells is the same as the total volume of the liquid sample including the first population of cells prior to diluting. In some embodiments, the second buffer includes human serum albumin (HSA). In some embodiments, the buffer including human serum albumin is a solution comprising between 1 and 10% (w/v) HSA. In some embodiments, the buffer including HSA is a solution comprising 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% HSA. In some embodiments, the second buffer includes Plasma-Lyte A and 4% (w/v) human serum albumin in equal volume. In some embodiments, the second buffer includes Plasma-Lyte A and 2% (w/v) human serum albumin. In some embodiments, the diluted liquid sample has a total volume that is at least two times (2X), at least three times (3X), or at least five times (5X) the total volume of the liquid sample prior to dilution. In some embodiments, the diluted liquid sample has a total volume that is at least three times the total volume of the liquid sample prior to dilution. In some embodiments, the diluted liquid sample has a total volume that is at least five times the total volume of the liquid sample prior to dilution. In some embodiments, the processing of the first population of cells includes washing the first population of cells, concentrating the first population of cells, and eluting and/or resuspending the first population of cells, thereby generating the second population of cells. In some embodiments, the concentrating of the first population of cells is accomplished using an automated centrifugation system. In some embodiments, the automated centrifugation system concentrates the first population of cells by elutriation. In some embodiments, the processing of the first population of cells in step (b) further includes adding a third solution to the second population of cells and cryopreserving the second population of cells. In some embodiments, the third solution includes one or more of the following: phosphate-buffered saline (“PBS”), dimethyl sulfoxide (“DMSO”), sodium hydroxide, potassium hydroxide, and sucrose. In some embodiments, the third buffer includes phosphate-buffered saline (“PBS”), dimethyl sulfoxide (“DMSO”), sodium hydroxide, potassium hydroxide, and sucrose. In some embodiments, the cryopreserved second population of cells is thawed before transducing with a recombinant polynucleotide encoding a CAR in step (c). [0013] In some embodiments, at least 2.4% of cells in the fifth population of cells are CCR7+CD45RA+ immune cells. In some embodiments, at least 2.5%, 2.8%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, or 5.5% of cells in the fifth population of cells are CCR7+ CD45RA+ immune cells. In some embodiments, at least 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.9%, 5.0%, 5.1%, 5.2%, 5.3%, 5.4%, or 5.5% of cells in the fifth population of cells are CCR7+ CD45RA+ immune cells. In some embodiments, the CCR7+ CD45RA+ immune cells are also CD3+. In some embodiments, the CCR7+ CD45RA+CD3+ immune cells are also CD4+. In some embodiments, the CCR7+ CD45RA+CD3+ immune cells are also CD8+. In some embodiments, at least 78.3%, 78.5%, 79.0%, 79.5%, 80%, 80.5%, 81.0%, 81.4%, 81.5%, 82.0%, 83.0%, 84.0%, 85.0%, 86.0%, or 87.0% of cells in the fifth population of cells are CCR7+ CD45RA- immune cells. In some embodiments, at least 78.0%, 78.1%, 78.2%, 78.3%, 78.4%, 78.5%, 78.6%, 78.7%, 78.8%, 78.9%, 79.0%, 79.1%, 79.2%, 79.3%, 79.4%, 79.5%, 79.6%, 79.7%, 79.8%, 79.9%, 80.0%, 80.1%, 80.2%, 80.3%, 80.4%, 80.5%, 80.6%, 80.7%, 80.8%, 80.9%, or 81.0% of cells in the fifth population of cells are CCR7+ CD45RA- immune cells. In some embodiments, at least 81.0%, 81.1%, 81.2%, 81.3%, 81.4%, 81.5%, 81.6%, 81.7%, 81.8%, 81.9%, 82.0%, 82.0%, 82.1%, 82.2%, 82.3%, 82.4%, 82.5%, 82.6%, 82.7%, 82.8%, 82.9%, 83.0%, 83.1%, 83.2%, 83.3%, 83.4%, 83.5%, 83.6%, 83.7%, 83.8%, 83.9%, or 84.0% of cells in the fifth population of cells are CCR7+ CD45RA- immune cells. In some embodiments, at least 84.0%, 84.1%, 84.2%, 84.3%, 84.4%, 84.5%, 84.6%, 84.7%, 84.8%, 84.9%, 85.0%, 85.0%, 85.1%, 85.2%, 85.3%, 85.4%, 85.5%, 85.6%, 85.7%, 85.8%, 85.9%, 86.0%, 86.1%, 86.2%, 86.3%, 86.4%, 86.5%, 86.6%, 86.7%, 86.8%, 86.9%, or 87.0% of cells in the fifth population of cells are CCR7+ CD45RA- immune cells. [0014] In some embodiments, at most 16.1%, 16.0%, 15.0%, 14.0%, 13.0%, 12.0%, 11.0%, 10.0%, 9.0%, 8.0%, 7.5%, 7%, 6.5%, 6.0%, 5.5%, 5.0%, 4.5%, 4.0%, 3.5%, or 3.4% of cells in the fifth population of cells are CCR7- CD45RA- immune cells. In some embodiments, at most 16.5%, 16.0%, 15.5%, 15.0%, 14.5%, 14.0%, 13.5%, 13.0%, 12.5%, 12.0%, 11.5%, 11.0%, 10.5%, 10.0%, 9.5%, 9.0%, 8.5%, 8.0%, 7.5%, 7.0%, 6.5%, 6.0%, 5.5%, 5.0%, 4.5%, 4.0%, 3.5%, or 3.5% of cells in the fifth population of cells are CCR7- CD45RA- immune cells. [0015] In some embodiments, the efficiency of transducing the third population of cells with a recombinant polynucleotide encoding a CAR is at least 10%, at least 20%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50%. In some embodiments, the multiplicity of infection (MOI) used when transducing the third population of cells with a recombinant polynucleotide encoding a CAR is at least 1, at least 1.5, at least 2, at least 2.5, at least 3, at least 3.5, at least 4, at least 4.5, or at least 5. In some embodiments, the fifth population of cells is harvested on Day 5, Day 6, Day 7, Day 8, Day 9, Day 10, Day 11, Day 12, Day 13, Day 14, or Day 15. In some embodiments, the fifth population of cells is harvested on Day 5, Day 6, Day 7, Day 8, or Day 9. In some embodiments, the fifth population of cells is harvested on Day 5, 7, and/or Day 9. In some embodiments, the fifth population of cells is harvested on Day 5. In some embodiments, the fifth population of cells is harvested on Day 6. In some embodiments, the fifth population of cells is harvested on Day 7. In some embodiments, the fifth population of cells is harvested on Day 8. In some embodiments, the fifth population of cells is harvested on Day 9. In some embodiments, the third population of cells is activated by at least one cytokine. In some embodiments, the at least one cytokine includes IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, or a combination thereof. In some embodiments, the at least one cytokine includes a combination of IL-7 and IL-15. In some embodiments, the third population of cells is seeded in the first medium. In some embodiments, the first medium includes 12.5 ng/mL IL-7, 12.5 ng/mL IL-15, and 3% (w/v) human serum albumin. [0016] In some embodiments, the third population of cells includes at least 2.5 ^106, at least 5 ^106, at least 7.5 ^106, at least 1 ^107, at least 2.5 ^107, at least 5 ^107, at least 7.5 ^107, at least 1 ^108, 1.1 ^108, at least 1.2 ^108, at least 1.4 ^108, at least 1.6 ^108, at least 1.8 ^108, at least 2.0 ^108, at least 2.2 ^108, at least 2.4 ^108, at least 2.6 ^108, at least 2.8 ^108, or at least 3.0 ^108 cells comprising a subset of the second population of cells. In some embodiments, the third population of cells includes at least about 2×108 cells comprising a subset of the second population of cells In some embodiments, the third population of cells includes at least about 3×108 cells comprising a subset of the second population of cells. [0017] In some embodiments, the third population of cells is enriched for CD4+CD8+ cells prior to seeding. In some embodiments, the CAR-expressing fourth population of cells produced by transducing the third population of cells with a recombinant polynucleotide encoding a CAR is then expanded to produce a fifth population of cells. In some embodiments, the fifth population of cells includes at least 2.5 ^106, at least 5 ^106, at least 7.5 ^106, at least 1 ^107, at least 2.5 ^107, at least 5 ^107, at least 7.5 ^107, at least 1 ^108, at least 1.1 ^108, at least 1.2 ^108, at least 1.4 ^108, at least 1.6 ^108, at least 1.8 ^108, at least 2.0 ^108, at least 2.2 ^108, at least 2.4 ^108, at least 2.6 ^108, at least 2.8 ^108, or at least 3.0 ^108 CAR-expressing immune cells. [0018] In some embodiments, at least 70%, 75%, 80%, 85%, or 90% of the cells in the fifth population of cells are viable. In some embodiments, the fifth population of cells is cryopreserved after harvesting. [0019] In some embodiments, the human subject from which the liquid sample comprising a first population of cells comprising immune cells was obtained has a disease or health condition. In some embodiments, the disease or health condition is cancer or a relapsed/refractory cancer. In some embodiments, the cancer is a leukemia or lymphoma. In some embodiments, the cancer is a lymphoma. In some embodiments, the lymphoma is selected from a group consisting of diffuse large B cell lymphoma (DLBCL), large B cell lymphoma (LBCL), mantle cell lymphoma (MCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), Burkitt’s lymphoma, anaplastic large-cell lymphoma, angioimmunoblastic T cell lymphoma, and Hodgkin lymphoma. In some embodiments, the lymphoma is large B cell lymphoma. In some embodiments, the cancer is a leukemia. In some embodiments, the leukemia is selected from a group consisting of acute lymphocytic leukemia (ALL), acute lymphoblastic leukemia (ALL), B cell acute lymphocytic leukemia (B-ALL), B cell acute lymphoblastic leukemia (B-ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), B cell prolymphocytic leukemia (B- PLL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), chronic myelomonocytic leukemia (CMML), hairy cell leukemia (HCL), juvenile myelomonocytic leukemia (JMML), large granular lymphocytic leukemia (LGLL), and T cell prolymphocytic leukemia (T-PLL). In some embodiments, the leukemia is ALL or B-ALL. In some embodiments, the leukemia is pediatric ALL or B-ALL. [0020] In some embodiments, the recombinant polynucleotide encoding a CAR is a viral vector. In some embodiments, the viral vector is a lentiviral vector. In some embodiments, the CAR-expressing immune cells are T cells. In some embodiments, the T cells are CD4+ or CD8+. In some embodiments, the CD4+ T cells and CD8+ T cells are also CD3+. In some embodiments, the CAR-expressing immune cells are NK cells. In some embodiments, the CAR specifically binds a B cell-specific antigen. In some embodiments, the B cell-specific antigen is CD19, CD20, or CD22. [0021] In some embodiments, the CAR specifically binds the B cell-specific antigen CD22. In some embodiments, the CD22-specific CAR includes an anti-CD22 binding domain. In some embodiments, the anti-CD22 binding domain is an antibody, an antibody fragment, or an antigen binding domain of thereof. In some embodiments, the anti-CD22 binding domain is an antibody fragment. In some embodiments, the antibody fragment is an anti-CD22 single chain variable fragment (scFv). [0022] In some embodiments, the anti-CD22 binding domain includes a VH comprising a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence of SEQ ID NO: 2. In some embodiments, the anti-CD22 binding domain includes a VH having the sequence of SEQ ID NO: 2. [0023] In some embodiments, the anti-CD22 binding domain includes a light chain variable region (VL) comprising a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence of SEQ ID NO: 3. [0024] In some embodiments, the anti-CD22 binding domain includes a VL having the sequence of SEQ ID NO: 3. [0025] In some embodiments, the anti-CD22 binding domain includes a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence of SEQ ID NO: 1. [0026] In some embodiments, the anti-CD22 binding domain includes the sequence of SEQ ID NO: 1. [0027] In some embodiments, the CD22 CAR includes a sequence having at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence of SEQ ID NO: 22. [0028] In some embodiments, the CD22 CAR includes the sequence of SEQ ID NO: 22. [0029] In some embodiments, the CD22 CAR includes a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence of SEQ ID NO: 23. [0030] In some embodiments, the anti-CD22 CAR includes the sequence of SEQ ID NO: 23. [0031] In some embodiments, the CAR specifically binds the B cell-specific antigen CD19. In some embodiments, the CD19-specific CAR includes an anti-CD19 binding domain. In some embodiments, the anti-CD19 binding domain is an antibody, an antibody fragment, or an antigen binding domain of thereof. In some embodiments, the anti-CD19 binding domain is an antibody fragment. In some embodiments, the antibody fragment is an anti-CD19 single chain variable fragment (scFv). [0032] In some embodiments, the CAR specifically binds the B cell-specific antigen CD20. In some embodiments, the CD20-specific CAR includes an anti-CD20 binding domain. In some embodiments, the anti-CD20 binding domain is an antibody, an antibody fragment, or an antigen binding domain of thereof. In some embodiments, the anti-CD20 binding domain is an antibody fragment. In some embodiments, the antibody fragment is an anti-CD20 single chain variable fragment (scFv). [0033] In one aspect of the disclosure, provided herein is a method of making a population of CAR-expressing immune cells, the method comprising: (a) obtaining a liquid sample comprising a first population of cells comprising immune cells from a human subject; (b) processing the first population of cells thereby generating a second population of cells comprising immune cells, wherein the second population of cells includes at least 1×104 total cells, wherein less than 20% of the total number of cells in the second population of cells are platelets; (c) cryopreserving the second population of cells; (d) on Day 0, thawing the cryopreserved second population of cells comprising immune cells, processing the thawed second population of cells comprising immune cells, and seeding a third population of cells comprising immune cells with a portion of the processed second population of cells comprising immune cells in a volume of a first media, wherein the third population of cells is a subset of the second population of cells, wherein the third population of cells is seeded into a volume of at least 250 mL of first media with at least 3.0´108 cells from the second population of cells; (e) transducing the third population of cells comprising immune cells with a recombinant polynucleotide encoding a CAR thereby generating a fourth population of cells, wherein the fourth population of cells comprises CAR-expressing immune cells; (f) expanding the fourth population of cells in media to yield a fifth population of cells comprising CAR-expressing immune cells; (g) removing at least 50% of the volume of the media on or before Day 4 after the seeding in step (d); (h) determining the number of viable CD3+ CAR-expressing T cells; and either (i) continuing to expand the fifth population of cells and exchanging the media, or (j) harvesting the fifth population of cells comprising CAR-expressing immune cells on or before Day 9 after the seeding in step (d), wherein at least 2.4% of cells in the fifth population of cells including CAR-expressing immune cells are CCR7+CD45RA+ immune cells; and (k) formulating the fifth population of cells for cryopreservation and administration to patients. [0034] Non-limiting exemplary embodiments of the methods in accordance with this aspect and other aspects of the disclosure may include one or more of the following features. In some embodiments, the liquid sample including a first population of cells comprises a leukapheresis product. In some embodiments, processing of the first population of cells in step (b) comprises a step of washing, concentrating and eluting or resuspending the second population of cells in a buffer. In some embodiments, the processing of the first population of cells in step (b) further comprises a step of reducing the number of platelets in the first population of cells. [0035] In some embodiments, the second population of cells is washed, concentrated, and eluted or resuspended and the number of platelets is reduced by elutriation using a centrifugation system having a molecular weight cutoff sufficient to retain the immune cells. In some embodiments, the centrifugal filtration system is a CTSTM RoteaTM Counterflow Centrifugation System. In some embodiments, the washed and concentrated second population of cells having a reduced number of platelets compared to the first population of cells is eluted or resuspended in a buffer. In some embodiments, the buffer comprises human serum albumin (HSA), Plasma-Lyte A, phosphate buffered saline, sodium chloride, sodium bicarbonate buffer, glutathione, biotin, vitamin B12, inositol, choline, L-glutamine, sodium pyruvate, glucose, or any combination thereof. In some embodiments, buffer comprises human serum albumin (HSA). In some embodiments, the buffer comprises Plasma-Lyte A. In some embodiments, the buffer comprises equal volumes of Plasma-Lyte A and 4% (w/v) human serum albumin. In some embodiments, the washed and concentrated second population of cells having a reduced number of platelets compared to the first population of cells is resuspended in a buffer comprising equal volumes of Plasma-Lyte A and 4% (w/v) HSA and further diluted 1:1 with a cryoprotectant. In some embodiments, the cryoprotectant is CryoStorR. [0036] In some embodiments, the processing of the thawed second population of cells in step (d) comprises a step of enriching for CD4+ and CD8+ T cells. In some embodiments, the enriching for CD4+ and CD8+ T cells further comprises a step mixing the thawed second population of cells with magnetic beads derivatized with CD4-specific binding agents and CD8-specific binding agents, washing, and eluting the thawed second population of cells enriched for CD4+ and CD8+ cells. In some embodiments, the step of enriching the thawed second population of cells for CD4+ and CD8+ T cells is performed on a CliniMACS Prodigy using CliniMACS buffer + 2% (w/v) or 0.5% (w/v) HSA. In some embodiments, the third population of cells is seeded with 300×106 cells from the processed second population of cells. In some embodiments, the third population of cells is seeded with at least 2.5×106 cells from the processed second population of cells. In some embodiments, the third population of cells is seeded with between at least 2.5×106 cells and at least 300×106 cells from the processed second population of cells. In some embodiments, the third population of cells is seeded with at least about 2.5×106, 3×106, 3.5×106, 4×106, 4.5×106, 5×106, 5.5×106, 6×106, 6.5×106, 7×106, 7.5×106, 8×106, 8.5×106, 9×106, 9.5×106, 1×107, 1.5×107, 2×107, 2.5 ×107, 3×107, 3.5×107, 4×107, 4.5×107, 5×107, 5.5×107, 6×107, 6.5×107, 7×107, 7.5×107, 8×107, 8.5×107, 9×107, 9.5×107, 1×108, 1.5×108, 2×108, 2.5×108, 3×108, 3.5×108, 4×108, 4.5×108, or 5×108 cells from the processed second population of cells. [0037] In some embodiments, the third population of cells is seeded in modified TexMACSTM medium (MTM). In some embodiments, the MTM comprises TexMACSTM medium supplemented with 3% human AB plasma (HABS) and 1:1 mixture of recombinant human cytokines IL-7 (hIL-7) and IL-15 (hIL-15). In some embodiments, the 1:1 mixture of recombinant human cytokines IL-7 (hIL-7) and IL-15 (hIL-15) comprises 12.5 ng/mL hIL-7 and 12.5 ng/mL hIL-15. In some embodiments, the modified TexMACSTM medium further comprises an effective amount of a reagent comprising agonists of CD3 and CD28. In some embodiments, the reagent comprising CD3 and CD28 agonists comprises the T cell TransActTM reagent. [0038] In some embodiments, the recombinant polynucleotide encoding a CAR further comprises a lentiviral expression vector. In some embodiments, the lentiviral expression vector comprises a recombinant polynucleotide encoding a CAR. In some embodiments, the lentiviral expression vector is manufactured using an adherent cell culture method or a suspension cell culture method. In some embodiments, the transduction in step (e) is performed on Day 1. In some embodiments, the transduction in step (e) is performed 22-26 hours after seeding of the third population of cells comprising immune cells on Day 0. In some embodiments, the amount of vector used to transduce the third population of cells comprising immune cells in step (e) is determined based on the infectious titer of the lentiviral vector and the number of cells used to seed the third population of cells comprising immune cells such that the transduction is performed with a multiplicity of infection (MOI) of 2.0. In some embodiments, the transduction is performed with a MOI of at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2.0, at least 2.1, at least 2.2, at least 2.3, at least 2.4, at least 2.5, at least 2.6, at least 2.7, at least 2.8, at least 2.9, at least 3.0, at least 3.5, at least 4.0, at least 4.5, or at least 5.0. [0039] In some embodiments, the recombinant polynucleotide encoding a CAR further comprising a lentiviral expression vector is thawed, diluted into modified TexMACSTM medium (MTM), and added to the third population of cells comprising immune cells. In some embodiments, the MTM comprises TexMACSTM medium supplemented with 3% human AB plasma (HABS) and 1:1 mixture of recombinant human cytokines IL-7 (hIL-7) and IL-15 (hIL-15). In some embodiments, 1:1 mixture of recombinant human cytokines IL-7 (hIL-7) and IL-15 (hIL-15) comprises 12.5 ng/mL hIL-7 and 12.5 ng/mL hIL-15. In some embodiments, the modified TexMACSTM medium further comprises an effective amount of a reagent comprising agonists of CD3 and CD28. In some embodiments, the reagent comprising CD3 and CD28 agonists comprises the T cell TransActTM reagent. [0040] In some embodiments, the final volume in which the transduction in step (e) is performed comprises 100 mL. In some embodiments, the fifth population of cells in step (f) is expanded in MTM in a volume of 200 mL. In some embodiments, the fifth population of cells is washed to remove T cell TransActTM and residual lentiviral vector. In some embodiments, the washing step is first performed on Day 4. In some embodiments, the washing step is first performed on Day 4 using the CliniMACS Prodigy program. In some embodiments, the washing step comprises removal of cell culture supernatant and resuspension of the fifth population of cells in MTM without T cell TransActTM reagent. In some embodiments, the washing step comprises removal of 65% of the culture volume and resuspension of the fifth population in the desired volume of MTM without T cell TransActTM. [0041] In some embodiments, the step (h) of determining the number of viable CD3+ CAR- expressing T cells is performed on Day 5 by taking a sample of the fifth population of cells and calculating the Dose Factor = Total Viable Cells (cell number)×Day 4 Transduction Efficiency (%) / Dose Target (1.0×106 viable CD3+CAR+ cells/kg)×Patient weight (kg). In some embodiments, there are enough viable CD3+CAR+ cells to meet the dose requirements on Day 5 and the harvesting step (j) is performed. In some embodiments, there are not enough viable CD3+CAR+ cells on Day 5, the fifth population of cells is expanded for another day, a sample of the fifth population of cells is taken on Day 6 and the Dose Factor is recalculated assuming a 7% increase in Day 4 Transduction Efficiency. In some embodiments, there are enough viable CD3+CAR+ cells to meet the dose requirements on Day 6 and the harvesting step (j) is performed. In some embodiments, there are not enough viable CD3+CAR+ cells on Day 6, the fifth population of cells is expanded for another day, a sample of the fifth population of cells is taken on Day 7 and the Dose Factor is recalculated assuming a 7% increase in Day 4 Transduction Efficiency. [0042] In some embodiments, a further media exchange is performed on Day 7. In some embodiments, the further media exchange on Day 7 comprises removing 60% of the culture volume and replacing it with fresh MTM. In some embodiments, there are enough viable CD3+CAR+ cells to meet the dose requirements on Day 7 and the harvesting step (j) is performed. In some embodiments, there are not enough viable CD3+CAR+ cells on Day 7, the fifth population of cells is expanded for another day, a sample of the fifth population of cells is taken on Day 8 and the Dose Factor is recalculated assuming a 7% increase in Day 4 Transduction Efficiency. In some embodiments, a further media exchange is performed on Day 8. In some embodiments, the further media exchange on Day 8 comprises removing 60% of the culture volume and replacing it with fresh MTM. In some embodiments, there are enough viable CD3+CAR+ cells to meet the dose requirements on Day 8 and the harvesting step (j) is performed. In some embodiments, there are not enough viable CD3+CAR+ cells on Day 8, the fifth population of cells is expanded for another day, and the harvesting step (j) is performed. In some embodiments, there are not enough viable CD3+CAR+ cells on Day 8, the fifth population of cells is expanded for another day, and the harvesting step (j) is performed on Day 9. [0043] In some embodiments, the harvesting step (j) further comprises a step of calculating viable cell density and determining whether the post-harvest viable cell density is ≥ the minimum transduced viable cell density for the formulation step. In some embodiments, the post-harvest viable cell density is ≥ the minimum transduced viable cell density and the harvested fifth population of cells is formulated for cryopreservation and administration to a human subject. In some embodiments, the post-harvest viable cell density is < the minimum transduced viable cell density and the harvested fifth population of cells is concentrated using a RoteaTM so that the post-harvest viable cell density is ≥ the minimum transduced viable cell density and the concentrated harvested fifth population of cells is formulated for cryopreservation and administration to a human subject. [0044] In some embodiments, the minimum transduced viable cell density is 1×106 CD3+CAR+ cells/kg. In some embodiments, the formulation step is performed manually, and the harvested fifth population of cells or the concentrated harvested fifth population of cells is resuspended to the desired concentration in Final Formulation Medium comprising Plasma- Lyte A + 4% (w/v) HSA, diluted 1:1 with CryostorR CS10 and frozen. In some embodiments, the formulation step is automated. In some embodiments, the automated formulation step is performed using a FINIAR Fill and Finish System, and the harvested fifth population of cells or the concentrated harvested fifth population of cells is resuspended to the desired concentration in Final Formulation Medium comprising Plasma-Lyte A+4% (w/v) HSA, diluted 1:1 with CryostorR CS10 and frozen. In some embodiments, the automated formulation step is performed using a Cue ScaleReady Cell Processing System, and the harvested fifth population of cells or the concentrated harvested fifth population of cells is resuspended to the desired concentration in Final Formulation Medium comprising Plasma- Lyte A+4% (w/v) HSA, diluted 1:1 with CryostorR CS10 and frozen. [0045] In some embodiments, the fifth population of cells including CAR-expressing immune cells comprises autologous CAR-expressing immune cells. In some embodiments, the fifth population of cells including CAR-expressing immune cells comprises autologous T cells expressing a CD22 CAR (i.e., a CD22-specific CAR). In some embodiments, the recombinant polynucleotide encoding an autologous CD22 CAR further comprises a lentiviral expression vector. In some embodiments, the lentiviral expression vector is manufactured using an adherent cell culture method or a suspension cell culture method. In some embodiments, the recombinant polynucleotide encoding a CAR encodes a CD22- specific CAR. In some embodiments, the CD22 CAR comprises a CD22-specific binding domain, a transmembrane domain, and an intracellular domain. In some embodiments, the CD22 CAR comprises a CD22-specific binding domain, a hinge domain, a transmembrane domain, a spacer, and an intracellular domain. In some embodiments, the CD22-specific binding domain comprises an antibody capable of binding CD22 or an antigen-binding fragment thereof. In some embodiments, the CD22-specific binding domain comprises an antibody capable of binding human CD22. In some embodiments, the CD22-specific binding domain comprises an antigen-binding fragment of an antibody capable of binding human CD22. In some embodiments, the antigen-binding fragment of an antibody capable of binding human CD22 is a single chain variable fragment (scFv) capable of binding CD22. In some embodiments, the scFv capable of binding CD22 has the sequence of SEQ ID NO: 1. In some embodiments, the scFv capable of binding CD22 comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 1. In some embodiments, the CD22 CAR comprises a CD8 ^ hinge domain comprising the sequence of SEQ ID NO: 24 and a CD8 ^ transmembrane domain comprising the sequence of SEQ ID NO: 25. In some embodiments, the CD22 CAR comprises a CD8 ^ hinge domain comprising the sequence of SEQ ID NO: 24, a CD8 ^ transmembrane domain comprising the sequence of SEQ ID NO: 25, and a peptide linker having the sequence of SEQ ID NO: 31. In some embodiments, the CD22 CAR comprises a CD8 ^ hinge domain comprising a sequence comprising 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 24 and a CD8 ^ transmembrane domain comprising a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 25. In some embodiments, the CD8 ^ transmembrane domain further comprises a spacer having the sequence of LYC. In some embodiments, the CD8 ^ transmembrane domain further comprises a spacer having the sequence of SEQ ID NO: 31. [0046] In some embodiments, the CD22 CAR comprises an intracellular domain further comprising a primary T cell activating domain comprising an immunoreceptor tyrosine-based activation motif (ITAM) and a costimulatory signaling domain. In some embodiments, the primary T cell activating domain comprising an ITAM comprises a CD3z intracellular signaling domain. In some embodiments, the CD3 ^ intracellular signaling domain comprises the sequence of SEQ ID NO: 27. In some embodiments, the CD3 ^ intracellular signaling domain comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 27. In some embodiments, the CD3 ^ intracellular signaling domain comprises the sequence of SEQ ID NO: 30. In some embodiments, the CD3 ^ intracellular signaling domain comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 30. In some embodiments, the costimulatory signaling domain comprises a 4- 1BB/CD137 signaling domain. In some embodiments, the 4-1BB/CD137 signaling domain comprises the sequence of SEQ ID NO: 26. In some embodiments, 4-1BB/CD137 costimulatory signaling domain comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 26. In some embodiments, the CD22 CAR comprises the sequence of SEQ ID NO: 22. In some embodiments, the CD22 CAR comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 22. In some embodiments, the CD22 CAR comprises the sequence of SEQ ID NO: 23. In some embodiments, the CD22 CAR comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 23.. [0047] In another aspect of the disclosure, provided herein is a method of manufacturing a fifth population of cells comprising CAR-expressing immune cells, wherein the method of manufacturing includes the method described herein. [0048] In another aspect, provided herein is a method of treating a health condition in a subject, the method including: administering to the subject a therapeutically effective amount of a fifth population of cells comprising CAR-expressing immune cells made according to any of the methods described herein, alone or in combination with an additional therapy, thereby treating the health condition. In some embodiments, the health condition is a cancer, wherein the administered population of CAR-expressing immune cells treats or provides anti- tumor immunity to the subject. [0049] In another aspect, provided herein is a fifth population of cells comprising CAR- expressing immune cells made according to any one or more of the methods described herein. [0050] In another aspect, provided herein is a pharmaceutical composition including a fifth population of cells comprising CAR-expressing immune cells as described herein and a pharmaceutically acceptable carrier. [0051] The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative embodiments and features described herein, further aspects, embodiments, objects and features of the disclosure will become fully apparent from the drawings and the detailed description and the claims. [0052] Each of the aspects and embodiments described herein are capable of being used together, unless excluded either explicitly or clearly from the context of the embodiment or aspect. [0053] Throughout this specification, various patents, patent applications and other types of publications (e.g., journal articles, electronic database entries, etc.) are referenced. The disclosure of all patents, patent applications, and other publications cited herein are hereby incorporated by reference in their entirety for all purposes. BRIEF DESCRIPTION OF THE DRAWINGS [0054] FIG.1A shows the version 1 (v1) cell manufacturing protocol for generating a population of chimeric antigen receptor CAR-expressing immune cells. [0055] FIG.1B shows the version 2 (v2) cell manufacturing protocol for generating a population of CAR-expressing immune cells. [0056] FIGS.2A-2C show three apheresis cryopreservation methods. [0057] FIG.2A shows the workflow of the v1 cell manufacturing protocol using a Prodigy concentration instrument, wherein cells were loaded onto the instrument, washed, reformulated, harvested and subsequently frozen. [0058] FIG.2B shows the workflow of the v2 cell manufacturing protocol using a Rotea instrument, wherein cells were diluted prior to loading on an instrument, cells were loaded onto the instrument, washed, concentrated, eluted/resuspended and subsequently frozen. [0059] FIG.2C shows the workflow of a manual manufacturing protocol using a centrifugation step, wherein cells were loaded into conical tubes, centrifuged, and a platelet layer is manually removed after centrifugation. Subsequently, the sample is diluted with buffer, and frozen. [0060] FIGS.3A-3G show post process cell recovery and viability data using different cryopreservation methods. [0061] FIG.3A (Table 5) shows leukapheresis hold times of the different cryopreservation methods. [0062] FIG.3B (Table 6) shows total viable cell (TVC) input of the different cryopreservation methods. [0063] FIG.3C (Table 7) shows run times of the different cryopreservation methods. [0064] FIG.3D shows % cell recovery and % viability of the different cryopreservation methods. [0065] FIG.3E shows % whole blood cell (WBC) recovery and % viability of the different cryopreservation methods. [0066] FIG.3F shows residual platelet levels of the different cryopreservation methods. [0067] FIG.3G shows residual platelet levels of the different cryopreservation methods. As shown in FIG.3G, the residual platelet levels using the Rotea v2 method are lowest. [0068] FIG.4 (Table 8) shows the post-thaw total recovery and cell viability using 10 mL fill bags. [0069] FIG.5A shows % residual platelet, % cell viability and % recovery pre-apheresis cryopreservation using the v2 cell manufacturing protocol. [0070] FIG.5B shows % cell viability and % recovery post-thaw of the cryopreserved apheresis using the v2 cell manufacturing protocol. [0071] FIGS.6A-6B show results from a cryo-apheresis verification run. [0072] FIG.6A (Table 9) shows CD4 and CD8 positive T cell enrichment using the v2 apheresis cryopreservation method. The bottom flow cytometry gating results show higher levels of CD3+CD4+ and CD3+CD8+ cells in the enriched fraction compared to the thawed cryo-apheresis prior to the v2 method. [0073] FIG.6B (Table 10) shows high viability and recovery using the v2 apheresis cryopreservation method. [0074] FIG.7 shows the total viable cells (TVC), population doubling, post-harvest viability, and CAR+ yield by Day 7 harvest using the different seeding quantities depicted. [0075] FIG.8 shows seeding with 200 million cells compensates for contraction post- activation and increases overall CAR+ cell yield compared to seeding with 100 million cells. The chart at the bottom shows the Day 7 (D7) post-harvest transduction efficiency as % CD3+/CAR+ population. [0076] FIG.9 (Table 11) shows 6 different experimental conditions for evaluating media exchange volumes and timing during the cell manufacturing protocol. [0077] FIG.10A shows that media exchange on Day 4 (D4, conditions 4-6) increases cell yield compared to other media exchange protocols. [0078] FIG.10B shows that media exchange on D4 results in comparable viability compared to other media exchange protocols. [0079] FIG.10C shows that media exchange on D4 results in comparable transduction efficiency compared to other media exchange protocols. [0080] FIG.10D shows that media exchange on D4 results in comparable % CD3+CD4+ and % CD3+CD8+ /immunophenotypes compared to other media exchange protocols. [0081] FIG.11 (Table 12) shows the media exchange strategy for the v2 cell manufacturing protocol. [0082] FIG.12 (Table 13) shows the media exchange protocol for the v1 cell manufacturing protocol. [0083] FIG.13A shows that the v2 cell manufacturing protocol media exchange strategy increases cell growth rate compared to the v1 cell manufacturing protocol. [0084] FIG.13B shows that the v2 cell manufacturing protocol media exchange strategy results in similar viability compared to the v1 cell manufacturing protocol. [0085] FIG.13C shows that the v2 cell manufacturing protocol media exchange strategy results in similar transduction efficiency compared to the v1 cell manufacturing protocol. [0086] FIG.14 shows exemplary data that the extended harvest window of the v2 cell manufacturing protocol results in population doublings, viability, and transduction efficiencies which are similar to the v1 cell manufacturing protocol. As shown, the product generated by the v2 cell manufacturing protocol achieves approximately 4 population doublings by D5. [0087] FIG.15 shows that the v2 cell manufacturing protocol met final dose fill requirement by Day 5 for both healthy donors and a patient. [0088] FIG.16A shows the comparison between CAR-T cells generated using the v2 cell manufacturing protocol using samples from two healthy donors, a sample from a patient and a control verification sample for population doublings. [0089] FIG.16B shows the comparison between CAR-T cells generated using the v2 cell manufacturing protocol using samples from two healthy donors, a sample from a patient and a control verification sample for viability %. Both the healthy donor and patient samples met final dose fill requirements by D5. [0090] FIG.16C shows the comparison between CAR-T cells generated using the v2 cell manufacturing protocol using samples from two healthy donors, a sample from a patient and a control verification sample for % CAR+ cells. Both the healthy donor and patient samples met final dose fill requirements by D5. [0091] FIG.17 shows the T cell memory phenotypes present from six different runs using the v1 and v2 cell manufacturing protocols. [0092] FIG.18 (Table 14) shows an increase in stem cell memory T cells (TSCM) and T cell central memory subset (TCM) cells and a decrease in effector memory T cells (TEM) cells generated using the v2 cell manufacturing protocol compared to using the v1 cell manufacturing protocol. Data obtained is from six different runs using the v1 and v2 cell manufacturing protocols. [0093] FIG.19 shows higher levels of T cell activation and exhaustion marker expression in cells using the v2 cell manufacturing protocol compared to using the v1 cell manufacturing protocol., likely due to the proximity of evaluation from the activation step, due to the shorter v2 manufacturing process. [0094] FIG.20 shows the experimental design to characterize the performance of the drug products generated by the v1 (D7) and v2 (D5, 7, 9) manufacturing processes, in vivo. [0095] FIG.21 shows tumor growth over time (days post injection (DPI)) for mice treated with CAR-T cells generated by harvesting on D7 using the v1 cell manufacturing protocol and harvesting on D5, D7 and D9 using the v2 cell manufacturing protocol. Mice treated with CAR-T cells generated by harvesting on D5 using the v2 cell manufacturing protocol continued to limit tumor burden out to Day 53 post injection. [0096] FIG.22 shows survival curves of mice treated with a low dose of CAR-T cells (1×106 CAR-T cells) or a high dose of CAR-T cells (5×106 CAR-T cells) generated by harvesting on D7 using the v1 cell manufacturing protocol and harvesting on D5, D7 and D9 using the v2 cell manufacturing protocol. Survival was highest (approximately 80%) for mice treated with D5 cells from the v2 cell manufacturing protocol. Survival was noted, although to a less degree (40-60%) for mice that were treated with D7 cells generated from the v1 cell manufacturing protocol or D7 and D9 from the v2 cell manufacturing protocol. [0097] FIG.23 shows the number of cells of the depicted phenotypes observed in mice treated with a low dose of CAR-T cells (1×106 CAR-T cells) or a high dose of CAR-T cells (5×106 CAR-T cells) generated by harvesting on D7 using the v1 cell manufacturing protocol and harvesting on D5, D7 and D9 using the v2 cell manufacturing protocol (top). Also shown is the vector copy number (VCN) of the transduced anti-CD22 CAR construct in mouse blood samples harvested 14 days or 28 days post infusion with a low dose of CAR-T cells (1×106 CAR-T cells) or a high dose of CAR-T cells (5×10^6 CAR-T cells) generated by
Figure imgf000021_0001
using the v1 cell manufacturing protocol and harvesting on D5, D7 and D9 using the v2 cell manufacturing protocol. [0098] FIG.24 shows the number of viable cells/mL, viability % and number of population doublings of CAR-T cells generated using the v1 cell manufacturing protocol and the v2 cell manufacturing protocol from two different patient samples. [0099] FIG.25 shows IFNgamma (IFN ^) release ELISPOT assays of CAR-T cells generated using the v1 cell manufacturing protocol and the v2 cell manufacturing protocol from two different patient samples. [0100] FIG.26 shows the % frequency of cells of the depicted phenotypes observed in CAR- T cells generated using the v1 cell manufacturing protocol and the v2 cell manufacturing protocol from two different patient samples. [0101] FIG.27 shows the % transduction efficiency (CAR+ cells) and the total CAR+ cell observed in CAR-T cells generated using the v1 cell manufacturing protocol and the v2 cell manufacturing protocol from two different patient samples. [0102] FIG.28 shows equivalence in cell viability and CD3 expression for CAR-T cells generated using the v1 cell manufacturing protocol and the v2 cell manufacturing protocol. [0103] FIG.29 shows equivalence in transduction efficiency and vector copy number per cell for CAR-T cells generated using the v1 cell manufacturing protocol and the v2 cell manufacturing protocol. [0104] FIG.30 shows IFNgamma (IFN ^) release when CAR-T cells generated using the v2 cell manufacturing protocol were co-cultured with CD22 expressing cell lines at the indicated ratios for the indicated amount of time. [0105] FIG.31 shows IFNgamma (IFN ^) release when CAR-T cells generated using the v2 cell manufacturing protocol with a media exchange (MX) step at the indicated day were co- cultured with CD22 expressing cell lines at the indicated ratios. Higher IFNgamma (IFN ^) release was observed using CAR-T cells generated using the v2 cell manufacturing protocol with a media exchange step on Day 4 compared to CAR-T cells generated using the v2 cell manufacturing protocol with a media exchange on D7, D9 and D11. [0106] FIG.32A shows the total number of viable cells in CAR-T cells generated using the v2 cell manufacturing protocol from two different patient samples that were harvested on the indicated days (top). [0107] FIG.32B shows IFNgamma (IFN ^) release levels after co-culturing CAR-T cells generated using the v2 cell manufacturing protocol with CD22 expressing cell lines at the indicated ratios from a patient sample (Donor 1) that were harvested on the indicated days. The data demonstrates earlier harvest Days (5 and 7) exhibit higher potency than later Days (10 and 12) and that growth affects potency. [0108] FIG.32C shows IFNgamma (IFN ^) release levels after co-culturing CAR-T cells generated using the v2 cell manufacturing protocol with CD22 expressing cell lines at the indicated ratios from a patient sample (Donor 2) that were harvested on the indicated days. The data demonstrates earlier harvest Days (5-7) exhibit higher potency than later Days (10) and that growth affects potency. [0109] FIG.33A shows the cytotoxicity of CAR-T cells generated using the v2 cell manufacturing protocol and harvested on D5, D7, and D9. CAR-T cells were co-cultured with CD22 expressing target cells or CD22 knocked out target cells at range between 1:0.03 to 2:1. [0110] FIG.33B shows results demonstrating that the cytotoxicity assay was specific to CD22 antigen stimulation and harvesting later resulted in lower E:T EC50. [0111] FIG.34A shows the vector copy number (VCN) per cell in the CAR-T cell population generated from two different donors using the v2 cell manufacturing protocol. The cells were transduced with different multiplicity of infection (MOI) ratio. The results show that the VCN increased as MOI increased. [0112] FIG.34B shows the VCN per CAR+ cell in the CAR-T cell population generated from two different donors using the v2 cell manufacturing protocol. The cells were transduced with different multiplicity of infection (MOI) ratio. The results show that the VCN increased as MOI increased. [0113] FIG.35 (Table 15) shows the VCN in the CAR-T cell population generated from two donors at two different locations using the v2 cell manufacturing protocol. The VCN was measured by droplet digital polymerase chain reaction (ddPCR). Samples prepared at the two different locations showed similar VCN numbers, suggesting that the ddPCR assay is robust and reproducible. [0114] FIG.36 (Table 16) shows the VCN/cell, % transduction efficiency, and VCN/ CAR+ cell in the CAR-T cells generated using v2 manufacturing protocol and harvested on D5, D7, and D10. [0115] FIG.37 shows the experimental design for characterizing T cell memory cell phenotypes in the CAR-T cells generated using the v1 manufacturing protocol and the v2 manufacturing protocol. [0116] FIG.38 shows the experimental design for characterizing T cell activation and cell exhaustion in the CAR-T cells generated using the v1 manufacturing protocol and the v2 manufacturing protocol. [0117] FIG.39 (Table 17) shows a list of reagents used to characterize activation markers and reagents used to characterize exhaustion markers. [0118] FIG.40 shows that greater than 45% of cells in the harvested CAR-T population generated using the v2 cell manufacturing protocol are CD22CAR+CD3+. [0119] FIG.41 shows the % cell recoveries and % cell viabilities before and after automated cell concentration. [0120] FIG.42 shows the total viable cells, population doubling time, total CAR+ yield over time of CAR-T cells population generated with different seeding density and at two different manufacturing sites. The results demonstrate that seeding with 200×106 cells increases CAR+ yield and results in similar percent of contraction and more viable cells on D1. [0121] FIG.43 (Tables 18 and 19) shows the % CAR+ cells vs. vector concentration on D7 with two different vectors and different seeding density. The result demonstrates that vector concentration, in the range evaluated, and seeding density do not affect transduction efficiency. [0122] FIG.44 (Table 20) shows the projected total number of CAR+ cells generated using the v2 cell manufacturing protocol with media exchange on D5, D6, or D7 and cells harvested on D5, D7, or D9. The result suggests that harvesting on D5 can achieve the 1×106 CAR+ cells/kg dosage requirement, assuming a dose factor of 4.1. [0123] FIG.45 (Table 21) shows the projected total number of CAR+ cells generated using the v2 cell manufacturing protocol with media exchange on D5, D6, or D7 and cells harvested on D5, D7, or D9. The result suggests that harvesting on D5 can achieve the 1×106 CAR+ cells/kg dosage requirement, assuming a dose factor of 3.4. [0124] FIG.46 shows a schematic experimental design of the potency assay for CD22 CAR- T cells. [0125] FIG.47 shows data from an IL-2 readout via ELISpot to measure the potency of CD22 lentiviral vector transduced in Jurkat T cells. [0126] FIG.48 shows a workflow for a co-culture cytotoxicity assay. [0127] FIG.49 shows a workflow for a cell preparation and analysis for a cytotoxicity assay. DETAILED DESCRIPTION OF THE DISCLOSURE [0128] The present disclosure generally relates to, inter alia, improved methods of making and/or manufacturing a population of immune cells expressing a chimeric antigen receptor (CAR). Also provided are populations of CAR-expressing immune cells made and/or manufactured by the improved methods, as well as pharmaceutical compositions comprising a population of CAR-expressing immune cells of the disclosure. The disclosure also provides methods for treating a health condition in a subject in need thereof. [0129] As discussed in greater detail below, the present disclosure provides improved methods that allow more efficient manufacturing processes for rapid clinical-scale production of autologous CAR-T cells with improved therapeutic activities. In particular, some embodiments of disclosure provide a robust manufacturing method allowing the production of drug product from apheresis starting material of varying quality. For example, the optimized media exchange strategy and seeding density allow for an earlier harvest window of 5-9 days compared to the longer windows of comparable processes shown in Figure 13A. The manufactured product is highly enriched in more naive T cell memory subpopulations (central memory T cells (TCM) in particular) (see, e.g., Figure 17). [0130] On the basis of the expression of two surface molecules, CD45RA and CCR7, human T cells can be divided into four subsets, including CD45RA+CCR7+ naive/stem cell memory (TN/TSCM), CD45RA−CCR7+ central memory (TCM), CD45RA−CCR7−effector memory (TEM), and CD45RA+CCR7− effector memory re-expressing CD45RA (TEMRA) T cells. See, e.g., Y. Tian et al., “Unique phenotypes and clonal expansions of human CD4 effector memory T cells re-expressing CD45RA”, Nat. Commun.8:1473 (2017). [0131] In one aspect, some embodiments of the disclosure relate to a method of making a population of CAR-expressing immune cells, the method including the steps of: (a) obtaining a liquid sample including a first population of cells including immune cells from a human subject; (b) processing the first population of cells to remove platelets thereby generating a second population of cells including immune cells, wherein the second population of cells includes at least 1 ^104 total cells and wherein less than 20% of the total number of cells in the second population of cells are platelets; (c) seeding a third population in a volume of a first buffer (e.g., first media), wherein the third population of cells is a subset of the second population of cells; (d) transducing the third population of cells with a recombinant polynucleotide encoding a CAR thereby generating a fourth population of cells, wherein the fourth population of cells includes CAR-expressing immune cells; (e) expanding the fourth population of cells to yield a fifth population of cells comprising CAR-expressing immune cells; and (f) harvesting the fifth population of cells on Day 5 or later after the seeding in step (c). [0132] In another aspect, some embodiments of the disclosure relate to a method of making a population of CAR-expressing immune cells, the method including the steps of: (a) obtaining a liquid sample including a first population of cells including immune cells from a human subject; (b) processing the first population of cells thereby generating a second population of cells including immune cells; (c) seeding a third population of cells in a volume of a first buffer (e.g., first media), wherein the third population of cells is a subset of the second population of cells; (d) transducing the third population of cells with a recombinant polynucleotide encoding a CAR thereby generating a fourth population of cells, wherein the fourth population of cells includes CAR-expressing immune cells; (e) expanding the fourth population of cells yielding a fifth population of cells; (f) removing at least 50% of the volume of the first buffer (e.g., first media) on or before Day 4 after the seeding; and (g) harvesting the fifth population of cells on Day 5 or later after the seeding of step (c). [0133] In another aspect, some embodiments of the disclosure relate to a method of making a population of CAR-expressing immune cells, the method including the steps of: (a) obtaining a liquid sample including a first population of cells including immune cells from a human subject; (b) processing the first population of cells thereby generating a second population of cells including immune cells; (c) seeding a third population of cells in a volume of a first buffer (e.g., first media), wherein the third population of cells is a subset of the second population of cells; (d) transducing the third population of cells with a recombinant polynucleotide encoding a CAR thereby generating a fourth population of cells, wherein the fourth population of cells includes CAR-expressing immune cells; (f) expanding the fourth population of cells yielding a fifth population of cells comprising CAR-expressing immune cells; and (g) harvesting the fifth population of cells on Day 5 or later after the seeding in step (c), wherein at least 2.4% of cells in the fifth population of cells are CCR7+ CD45RA+ immune cells. [0134] In another aspect, some embodiments of the disclosure relate to a method of making a population of CAR-expressing immune cells, the method including: (a) obtaining a liquid sample including a first population of cells including immune cells from a human subject; (b) processing the first population of cells to remove platelets thereby generating a second population of cells including immune cells, wherein the second population of cells includes at least 1 ^104 total cells and wherein less than 20% of the total number of cells in the second population of cells are platelets; and (c) seeding a third population of cells in a volume of a first buffer, wherein the third population of cells is a subset of the second population of cells, wherein the third population of cells include at least 2.0 ^108 cells; (d) transducing the third population of cells with a recombinant polynucleotide encoding a CAR thereby generating a fourth population of cells, wherein the fourth population of cells includes CAR-expressing immune cells; (e) expanding the fourth population of cells yielding a fifth population of cells; (f) removing at least 50% of the volume of the first buffer on or before Day 4 after the seeding in step (c); and (g) harvesting the fifth population of cells on or before Day 9 after the seeding in step (c), wherein at least 2.4% of cells in the fifth population of cells are CCR7+CD45RA+ immune cells. [0135] Yet in another aspect, some embodiments of the disclosure relate to a method of making a population of CAR-expressing immune cells, the method including: (a) obtaining a liquid sample comprising a first population of cells comprising immune cells from a human subject; (b) processing the first population of cells thereby generating a second population of cells comprising immune cells, wherein the second population of cells includes at least 1 ^104 total cells, wherein less than 20% of the total number of cells in the second population of cells are platelets; (c) cryopreserving the second population of cells; (d) on Day 0, thawing the cryopreserved second population of cells comprising immune cells, processing the thawed second population of cells comprising immune cells, and seeding a third population of cells comprising immune cells with a portion of the processed second population of cells comprising immune cells in a volume of a first media, wherein the third population of cells is a subset of the second population of cells, wherein the third population of cells is seeded into a volume of at least 250 mL of first media with at least 3.0 ^108 cells from the second population of cells; (e) transducing the third population of cells comprising immune cells with a recombinant polynucleotide encoding a CAR thereby generating a fourth population of cells, wherein the fourth population of cells comprises CAR-expressing immune cells; (f) expanding the fourth population of cells in media to yield a fifth population of cells comprising CAR-expressing immune cells; (g) removing at least 50% of the volume of the media on or before Day 4 after the seeding in step (d); (h) determining the number of viable CD3+ CAR-expressing T cells; and either (i) continuing to expand the fifth population of cells and exchanging the media, or (j) harvesting the fifth population of cells comprising CAR-expressing immune cells on or before Day 9 after the seeding in step (d), wherein at least 2.4% of cells in the fifth population of cells including CAR-expressing immune cells are CCR7+CD45RA+ immune cells. In some embodiments, the method further includes a step of (k) formulating the fifth population of cells for cryopreservation and administration to patients. [0136] In various embodiments of disclosure, the third population of cells comprising immune cells is seeded in a volume of the first buffer that comprises growth media. In these instances, the first buffer is also referred to as the first media. DEFINITIONS [0137] In the following description, certain specific details are set forth in order to provide a thorough understanding of various embodiments. However, one skilled in the art will understand that the embodiments provided may be practiced without these details. Unless the context requires otherwise, throughout the specification and claims which follow, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense, that is, as “including, but not limited to.” As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the content clearly dictates otherwise. It should also be noted that the term “or” is generally employed in its sense including “and/or” unless the content clearly dictates otherwise. Further, headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed embodiments. [0138] Certain ranges are presented herein with numerical values being preceded by the term “about” which, as used herein, has its ordinary meaning of approximate. The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number. If the degree of approximation is not otherwise clear from the context, “about” means either within plus or minus 10% of the provided value, or rounded to the nearest significant figure, in all cases inclusive of the provided value. In some embodiments, the term “about” indicates the designated value ± up to 10%, up to ± 5%, or up to ± 1%. For example, in some embodiments of the disclosure, the term “about” refers to an amount that is near the stated amount by 10%, 5%, or 1%, including increments therein. In some embodiments, the term “about” in reference to a percentage refers to an amount that is greater or less the stated percentage by 10%, 5%, or 1%, including increments therein. [0139] The term “Chimeric Antigen Receptor” or alternatively a “CAR” refers to a synthetic receptor encoded by one or more polypeptides including at least the following functional domains: an extracellular antigen-binding domain, a transmembrane domain or a hinge and transmembrane domain, and an intracellular or cytoplasmic signaling domain, which when expressed in an immune cell, provides the cell with the ability to specifically bind a target cell expressing a particular antigen, for example a cancer cell, and with the ability to generate a signal (without engaging the T cell receptor) via the intracellular signaling domain following engagement of the antigen-binding domain by its cognate antigen. In some embodiments, a CAR includes at least an extracellular antigen binding domain, a transmembrane domain and an intracellular or cytoplasmic signaling domain (also referred to herein as “an intracellular signaling domain”) including a functional signaling domain derived from one or more primary T cell activation molecules and/or one or more costimulatory molecules as defined below. [0140] The term “antibody” or “antigen-binding domain” herein is used in the broadest sense to encompass monoclonal antibodies, bispecific antibodies, intact antibodies and functional (antigen-binding) antibody fragments thereof, including fragment antigen binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single chain antibody fragments, including single chain variable fragments (sFv or scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific, e.g., bispecific, antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless otherwise stated, the term “antibody” should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full- length antibodies, including antibodies of any isotype, class or sub-class, including IgG and sub-classes thereof, IgM, IgE, IgA, and IgD. The antibody can include a human IgG1 constant region. The antibody can include a human IgG4 constant region. [0141] The term “scFv” or “single chain variable fragment” refers to a fusion protein including at least one antibody fragment including a variable region of a light chain and at least one antibody fragment including a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked, e.g., via a synthetic linker, e.g., a short flexible peptide or polypeptide linker, and capable of being expressed as a single chain polypeptide, wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, as used herein an scFv may encode the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may include VL-linker-VH or may include VH-linker-VL. [0142] The antigen-binding domain of a CAR (i.e., the portion of a CAR including an antibody or antibody fragment thereof) may exist in a variety of forms where the antigen binding domain is expressed as part of a contiguous polypeptide chain including, for example, a single domain antibody fragment (sdAb), a single chain antibody (scFv) and a humanized antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, N.Y.; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In one embodiment, the antigen binding domain of a CAR includes an antibody fragment. In a further embodiment, the CAR includes an antibody fragment that includes a scFv. [0143] As used herein, the term “binding domain”, “antigen binding domain”, or “antibody molecule” refers to a protein, e.g., an immunoglobulin polypeptide or fragment thereof, including at least one immunoglobulin variable domain sequence. The term “binding domain” or “antibody molecule” encompasses antibodies and antibody fragments. In an embodiment, an antibody molecule is a multispecific antibody molecule, e.g., it includes a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In an embodiment, a multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity for no more than two antigens. A bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence which has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope. [0144] The term “heavy chain,” refers to the longer of the two types of polypeptides present in antibody molecules in their naturally occurring conformations, and which normally determines the class to which the antibody belongs. [0145] The term “light chain,” refers to the shorter of the two types of polypeptides present in antibody molecules in their naturally occurring conformations. There are two major antibody light chain polypeptide isotypes, designated kappa (κ) and lambda (λ). [0146] The term “antigen” or “Ag” refers to a molecule capable of provoking an immune response. This immune response may be innate or adaptive, and may involve either antibody production, the activation of specific immunologically-competent cells, or both. The skilled artisan will understand that any macromolecule, including virtually all proteins, polypeptides, peptides can be antigenic. Furthermore, antigens can be derived from (e.g., expressed from) recombinant or genomic DNA. A skilled artisan will understand that any DNA which includes a nucleotide sequence or a partial nucleotide sequence encoding a protein or protein fragment can elicit an immune response and therefore encodes an “antigen” as that term is used herein. Furthermore, one skilled in the art will understand that an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the present disclosure includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to encode polypeptides that elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample or might be macromolecule besides a polypeptide. Such a biological sample can include, but is not limited to a tissue sample, a tumor sample, a cell, or a fluid including other biological components. [0147] The term “cancer” refers to a disease characterized by the presence of cells possessing several characteristics typical of cancer-causing cells, such as uncontrolled growth/proliferation of cells (e.g., aberrant cells). Cancer cells can but do not always aggregate into a mass, such as a tumor, or can exist alone within a subject. A tumor can be a solid tumor, a liquid tumor a soft tissue tumor, or a metastatic lesion. Cancers can grow locally or in distant locations following metastasis, a process by which cancer cells are shed from a tumor and spread through the bloodstream and lymphatic system to other parts of the body. As such, cancers may be solid (e.g., cancer of the lung, colon, or breast) or liquid (e.g., cancer of myeloid cells (e.g., myeloma) or B cells (e.g., diffuse large B cell lymphoma). Examples of various cancers are described herein and include but are not limited to, lymphoma and leukemia. Also of interest may be breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain cancer, lung cancer and the like. The term “cancer” encompasses solid and liquid, e.g., diffuse or circulating, tumors. As used herein, the term “cancer” includes premalignant, as well as malignant cancers and tumors. [0148] “Derived from” as that term is used herein, indicates a relationship between a first and a second molecule. It generally refers to structural similarity between the first molecule and a second molecule and does not connote or include a process or source limitation on a first molecule that is derived from a second molecule. For example, in the case of an intracellular signaling domain that is derived from a CD3 ^ molecule, the intracellular signaling domain retains sufficient CD3 ^ structure such that it has the required function, namely, the ability to generate a signal under the appropriate conditions. It does not connote or include a limitation to a particular process of producing the intracellular signaling domain, e.g., it does not mean that, to provide the intracellular signaling domain, one must start with a CD3 ^ sequence and delete unwanted sequence, or introduce mutations, to arrive at the intracellular signaling domain. [0149] The term “relapse” as used herein refers to reappearance of a disease (e.g., cancer) after an initial period of remission or response to an anti-cancer treatment, e.g., after prior treatment with a therapy, e.g., cancer therapy (e.g., complete response or partial response). The initial period of responsiveness may involve the level of cancer cells falling below a certain threshold, e.g., below 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or below the limit of detection. The reappearance may involve the level of cancer cells rising above a certain threshold, e.g., above 20%, 15%, 10%, 5%, 4%, 3%, 2%, or 1%. For example, e.g., in the context of B-ALL, the reappearance may involve, e.g., a reappearance of blasts in the blood, bone marrow (BM) (>5%), or any extramedullary site, after a complete response. A complete response, in this context, may involve <5% blasts in the bone marrow. In some embodiments, a response (e.g., complete response or partial response) can involve the absence of detectable disease (e.g., MRD, or minimal residual disease). In some embodiments, the initial period of responsiveness lasts at least 1, 2, 3, 4, 5, or 6 days; at least 1, 2, 3, or 4 weeks; at least 1, 2, 3, 4, 6, 8, 10, or 12 months; or at least 1, 2, 3, 4, or 5 years. [0150] “Refractory” as used herein refers to a disease, e.g., cancer, that does not respond to a treatment. In embodiments, a refractory cancer can be resistant to a treatment before or at the beginning of the treatment. In other embodiments, the refractory cancer can become resistant during a treatment. A refractory cancer is also called a resistant cancer. [0151] The term “stimulation,” refers to a primary response induced by binding of a stimulatory molecule (e.g., a TCR/CD3 complex or CAR) with its cognate ligand thereby mediating a signal transduction event, such as, but not limited to, signal transduction via the TCR/CD3 complex or signaling domains of the CAR. Stimulation can mediate altered expression of certain molecules. [0152] “Immune cell,” as that term is used herein, refers to cells that are involved in an adaptive immune response, e.g., in the promotion of an immune effector response. Exemplary immune cell types include T cells, e.g., alpha/beta T cells and gamma/delta T cells, regulatory T cells (Tregs) B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, macrophages, and myeloid-derived phagocytes. [0153] The term “expression” refers to the transcription and/or translation of a particular nucleotide sequence of interest. Such nucleotide sequences can be naturally occurring or non- naturally occurring (e.g., a synthetic nucleotide sequence like, for example, a nucleotide sequence encoding a chimeric antigen receptor or “CAR”). [0154] The term “expression vector” refers to a vector including a recombinant polynucleotide to be expressed and various expression control sequences operatively linked to the recombinant nucleotide sequence. An expression vector includes sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. The term expression vectors as used herein encompasses those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes) and viral vectors (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) capable of incorporating and expressing the recombinant polynucleotide. [0155] The term “lentivirus” refers to members of a genus in the Retroviridae family. Lentiviruses are unique among the retroviruses in being able to infect non-dividing cells; they can deliver a considerable amount of genetic information into the DNA of the host cell, so they are one of the most efficient methods of a gene delivery vector. HIV, SIV, and FIV are all examples of lentiviruses. [0156] The term “lentiviral vector” refers to a vector derived from at least a portion of a lentivirus genome, including especially a self-inactivating lentiviral vector as provided in Milone et al., Mol. Ther.17(8): 1453-1464 (2009). Nonclinical types of lentiviral vectors are also available and would be known to one skilled in the art. [0157] The term “isolated” used in reference to a substance means it has been removed from its natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell. [0158] As used herein, a “subject” or an “individual” includes animals, such as human (e.g., human individuals). In some embodiments, a “subject” or “individual” is a patient under the care of a physician. Thus, the subject can be a human patient or an individual who has, is at risk of having, or is suspected of having a health condition of interest (e.g., a cancer) and/or one or more symptoms of the health condition. The subject can also be an individual who is diagnosed with a risk of the health condition of interest at the time of diagnosis or later. [0159] As used herein, the terms “treat”, “treatment” and “treating” refer to the administration of a drug or therapeutic resulting in a reduction or amelioration of the progression, severity and/or duration of a disorder (e.g., a neoplastic disorder such as cancer), or the amelioration of one or more symptoms (e.g., one or more discernible symptoms) of a proliferative disorder resulting from the administration of one or more therapies (e.g., one or more therapeutic agents such as a CAR-expressing immune cell as provided herein) into a subject in need thereof, e.g., a patient. In some embodiments, the terms “treat”, “treatment” and “treating” refer to the amelioration of at least one measurable physical parameter of a neoplastic disorder, such as growth of a tumor, not necessarily discernible by the subject, e.g., patient. In some embodiments the terms “treat”, “treatment” and “treating” refer to the inhibition of the progression of a proliferative disorder, either physically by, e.g., stabilization of a discernible symptom, physiologically by, e.g., stabilization of a physical parameter of the disease or disorder, or both. In some embodiments, the terms “treat”, “treatment” and “treating” refer to the reduction or stabilization of tumor size or cancer cell count. [0160] The term “transfected” or “transformed” or “transduced” refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A “transfected” or “transformed” or “transduced” cell is one which has been transfected, transformed or transduced with exogenous nucleic acid, e.g., a lentiviral expression vector encoding a CAR. The term “transduced”, “transfected”, or “transformed” cell includes the primary subject cell and its progeny. [0161] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub- combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub- combination was individually and explicitly disclosed herein. METHODS OF MANUFACTURING A POPULATION OF CAR-EXPRESSING IMMUNE CELLS [0162] In one aspect, provided herein are methods of making a population of chimeric antigen receptor CAR-expressing immune cells. In certain embodiments, the methods described herein may include the steps shown in Figure 1. The methods may include one or more of the following steps: cell collection, cell processing, cell cryopreservation, cell thawing, cell seeding, cell transduction, cell expansion, cell washing, media exchange, cell harvesting, cell packaging, and cell storing. For purposes of the methods described herein, Day 0 is the day on which a processed, cryopreserved leukapheresis or apheresis product is thawed and the method is initiated. [0163] In one aspect, described herein is a method of making a population of chimeric antigen receptor CAR-expressing immune cells, the method including the steps of: (a) obtaining a liquid sample including a first population of cells including immune cells from a human subject; (b) processing the first population of cells to remove platelets thereby generating a second population of cells including immune cells, wherein the second population of cells includes at least 1 ^104 total cells and wherein less than 20% of the total number of cells in the second population of cells are platelets; (c) seeding a third population of cells including immune cells in a volume of a first buffer, wherein the third population of cells is a subset of the second population of cells; (d) transducing the third population of cells including immune cells with a recombinant polynucleotide encoding a CAR thereby generating a fourth population of cells, wherein the fourth population of cells includes CAR- expressing immune cells; (e) expanding the fourth population of cells to yield a fifth population of cells including CAR-expressing immune cells; and (f) harvesting the fifth population of cells on Day 5 or later after the seeding in step (c). In some embodiments, the method further includes a step of removing at least 50% of the volume of the first buffer on or before Day 4 after seeding. In some embodiments, at least 2.4% of cells in the fifth population of cells are CCR7+CD45RA+ immune cells. In some embodiments, the liquid sample including a first population of cells including immune cells from a human subject comprises a leukapheresis product or an apheresis product. [0164] In one aspect, described herein is a method of making a population of CAR- expressing immune cells, the method including the steps of: (a) obtaining a liquid sample including a first population of cells including immune cells from a human subject; (b) processing the first population of cells thereby generating a second population of cells including immune cells; (c) seeding a third population of cells including immune cells in a volume of a first buffer, wherein the third population of cells is a subset of the second population of cells; (d) transducing the third population of cells with a recombinant polynucleotide encoding a CAR thereby generating a fourth population of cells, wherein the fourth population of cells includes CAR-expressing immune cells; (e) expanding the fourth population of cells including CAR-expressing immune cells yielding a fifth population of cells including CAR-expressing immune cells; (f) removing at least 50% of the volume of the first buffer on or before Day 4 after the seeding in step (c); and (g) harvesting the fifth population of cells on Day 5 or later after the seeding in step (c). [0165] In some embodiments, at least 2.4% of cells in the fifth population of cells are CCR7+CD45RA+ immune cells. In some embodiments, the liquid sample including a first population of cells including immune cells from a human subject comprises an apheresis product. [0166] In one aspect, described herein is a method of making a population of CAR- expressing immune cells, the method including the steps of: (a) obtaining a liquid sample including a first population of cells including immune cells from a human subject; (b) processing the first population of cells thereby generating a second population of cells including immune cells; (c) seeding a third population of cells including immune cells in a volume of a first buffer, wherein the third population of cells is a subset of the second population of cells; (d) transducing the third population of cells with a recombinant polynucleotide encoding a CAR thereby generating a fourth population of cells, wherein the fourth population of cells includes CAR-expressing immune cells; (f) expanding the fourth population of cells to yield a fifth population of cells comprising CAR-expressing immune cells; and (g) harvesting the fifth population of cells on Day 5 or later after the seeding of step (c), wherein at least 2.4% of cells in the fifth population of cells are CCR7+ CD45RA+ immune cells. In some embodiments, the liquid sample including a first population of cells including immune cells from a human subject comprises a leukapheresis product or an apheresis product. [0167] In another aspect, described herein is a method of making a population of CAR- expressing immune cells, the method including: (a) obtaining a liquid sample including a first population of cells including immune cells from a human subject; (b) processing the first population of cells to remove platelets thereby generating a second population of cells including immune cells, wherein the second population of cells includes at least 1 ^104 total cells and wherein less than 20% of the total number of cells in the second population of cells are platelets; and (c) seeding a third population of cells including immune cells in a volume of a first buffer, wherein the third population of cells is a subset of the second population of cells, wherein the third population of cells includes at least 2.0 ^108 cells; (d) transducing the third population of cells with a recombinant polynucleotide encoding a CAR thereby generating a fourth population of cells, wherein the fourth population of cells includes CAR- expressing immune cells; (e) expanding the fourth population of cells yielding a fifth population of cells comprising CAR-expressing immune cells; (f) removing at least 50% of the volume of the first buffer on or before Day 4 after the seeding in step (c); and (g) harvesting the fifth population of cells on or before Day 9 after the seeding in step (c), wherein at least 2.4% of cells in the fifth population of cells are CCR7+CD45RA+ immune cells. In some embodiments, the liquid sample including a first population of cells including immune cells from a human subject comprises an apheresis product. METHODS OF MAKING CAR-EXPRESSING IMMUNE CELLS WITH REDUCED AMOUNT OF PLATELETS [0168] In another aspect, described herein is a method of making a population of chimeric antigen receptor CAR-expressing immune cells, the method including the steps of: (a) obtaining a liquid sample including a first population of cells including immune cells from a human subject; (b) processing the first population of cells to remove platelets thereby generating a second population of cells including immune cells, wherein the second population of cells includes at least 1 ^104 total cells and wherein less than 20% of the total number of cells in the second population of cells are platelets. In some embodiments, less than 18%, less than 15%, less than 12%, less than 10%, less than 8%, or less than 5% of the total number of cells in the second population of cells are platelets. [0169] In some embodiments, the processing of the first population of cells in step (b) includes diluting the liquid sample including the first population of cells with a first buffer, thereby generating a diluted liquid sample including the first population of cells. In some embodiments, the liquid sample is diluted prior to removing platelets. In some embodiments, the liquid sample has a total volume of at least 50 mL, e.g., about 50 mL to 10 L, about 50 mL to 1 L, about 50 mL to 500 mL, about 100 mL to 600 mL, about 200 mL to 700 mL, about 300 mL to 800 mL, about 400 mL to 900 mL, about 500 mL to 1 L, about 600 mL to 1.5 L, or about 600 mL to 2 L. In some embodiments, the liquid sample has a total volume of at least 50 mL, at least 100 mL, at least 200 mL, at least 250 mL, at least 300 mL, at least 400 mL, at least 500 mL, at least 600 mL, at least 700 mL, at least 800 mL, at least 900 mL, at least 1 L, or at least 2 L. [0170] In some embodiments, the step of diluting the liquid sample including the first population of cells includes adding a volume of the first buffer to the liquid sample, wherein the volume of the first buffer added to the liquid sample is at least the same as the total volume of liquid sample prior to diluting (i.e., the dilution is at least 1:1). The dilution buffer may include human serum albumin (HSA), Plasma-Lyte A, phosphate buffered saline, sodium chloride, sodium bicarbonate buffer, glutathione, biotin, vitamin B12, inositol, choline, L-glutamine, sodium pyruvate, glucose, or a combination thereof. In some embodiments, the first buffer includes human serum albumin (HSA). In some embodiments, the first buffer includes Plasma-Lyte A. In some embodiments, the first buffer includes equal volumes of Plasma-Lyte A and 4% (w/v) human serum albumin. [0171] In some embodiments, the concentration of HSA may be between about 0.25-2% (w/v), about 1-4% (w/v), about 2-6% (w/v), about 4-8% (w/v), about 5-10% (w/v), or about 0.25% (w/v), about 0.5% (w/v), about 0.75% (w/v), about 1% (w/v), about 1.25% (w/v), about 1.5% (w/v), about 1.75% (w/v), about 2% (w/v), about 2.25% (w/v), about 2.5% (w/v), about 2.75% (w/v), about 3% (w/v), about 3.25% (w/v), about 3.5% (w/v), about 3.75% (w/v), about 4% (w/v), about 4.25% (w/v), about 4.5% (w/v), about 4.75% (w/v), about 5% (w/v), about 5.25% (w/v), about 5.5% (w/v), about 5.75% (w/v), about 6% (w/v), about 6.25% (w/v), about 6.5% (w/v), about 6.75% (w/v), about 7% (w/v), about 7.25% (w/v), about 7.5% (w/v), about 7.75% (w/v), about 8% (w/v), about 8.25% (w/v), about 8.5% (w/v), about 8.75% (w/v), about 9% (w/v), about 9.25% (w/v), about 9.5% (w/v), about 9.75% (w/v), or about 10% (w/v). [0172] In some embodiments, the volume of the first buffer added to the liquid sample may be at least about 5mL, at least about 10mL, at least about 15mL, at least about 20mL, at least about 25mL, at least about 30mL, at least about 40mL, at least about 50mL, at least about 75mL, at least about 100mL, at least about 150mL, at least about 200mL, at least about 250mL, at least about 300mL, at least about 400mL, at least about 500mL, at least about 600mL, at least about 700mL, at least about 800mL, at least about 900mL, at least about 1L, at least about 1.5L, at least about 2L, at least about 2.5L, at least about 3L, at least about 3.5L, at least about 4L, at least about 4.5L, or at least about 5L. [0173] In some embodiments, the diluted liquid sample has a total volume that is at least twice (2X) the total volume of the liquid sample prior to diluting, e.g., at least 2X to 10X, at least 2X to 5X, at least 2X to 4X, at least 2X to 3X, at least 3X to 7X, at least 4X to 8X, at least 5X to 9X, at least 6X to 10X, at least 3X to 5X, or at least 4X to 6X. In some embodiments, the diluted liquid sample has a total volume that is at least two times (2X), at least three times (3X), or at least five times (5X) the total volume of the liquid sample prior to diluting. In some embodiments, the diluted liquid sample has a total volume that is at least three times (3X) the total volume of the liquid sample prior to diluting. In some embodiments, the diluted liquid sample has a total volume that is at least four (4X) times the total volume of the liquid sample prior to diluting. In some embodiments, the diluted liquid sample has a total volume that is at least five (5X) times the total volume of the liquid sample prior to diluting. [0174] In some embodiments, the processing of the first population of cells includes the steps of washing the first population of cells, concentrating the first population of cells, and eluting and/or resuspending the first population of cells, thereby generating the second population of cells. In some embodiments, concentrating the first population of cells is accomplished using an automated centrifugation system. In some embodiments, the automated centrifugation system concentrates the first population of cells by elutriation. In some embodiments, the particle size filter has a size cutoff sufficient to retain immune cells on the filter. In some embodiments, concentrating the first population of cells is accomplished using elutriation to keep larger immune cells in the centrifuge chamber and elute out smaller particles. [0175] In some embodiments, the wash buffer may include HSA, Plasma-Lyte A, phosphate buffered saline, sodium chloride, sodium bicarbonate buffer, glutathione, biotin, vitamin B12, inositol, choline, L-glutamine, sodium pyruvate, glucose, or a combination thereof. The cell population may be concentrated after washing. The washing step may result in a higher concentration of cells than the concentration of cells on the automated instrument. The cell population may be eluted and/or resuspended from the automated cell processing instrument after concentration. [0176] In some embodiments, the cell population is transferred to a sterile container after elution/resuspension. In some embodiments, the transfer is performed manually. In some embodiments, the transfer is automated. The sterile container may include a bag, a bottle, a tube, a glass jar, or a plastic container. In some embodiments, before transfer to a sterile container, the cell population may be diluted after elution/resuspension into a dilution buffer. The dilution buffer may include dimethyl sulfoxide, sucrose, sodium hydroxide, potassium hydroxide, fructose, HSA, Plasma-Lyte A, phosphate buffered saline, sodium chloride, sodium bicarbonate buffer, glutathione, biotin, vitamin B12, inositol, choline, L-glutamine, sodium pyruvate, glucose, or a combination thereof. The volume of dilution buffer added to the cell population after elution and/or resuspension may be at least about 5mL, at least about 10mL, at least about 15mL, at least about 20mL, at least about 25mL, at least about 30mL, at least about 40mL, at least about 50mL, at least about 75mL, at least about 100mL, at least about 150mL, at least about 200mL, at least about 250mL, at least about 300mL, at least about 400mL, at least about 500mL, at least about 600mL, at least about 700mL, at least about 800mL, at least about 900mL, at least about 1L, at least about 1.5L, at least about 2L, at least about 2.5L, at least about 3L, at least about 3.5L, at least about 4L, at least about 4.5L, or at least about 5L. In some embodiments, the volume of dilution buffer added to the cell population after elution and/or resuspension is more than the volume of the cell population. In some embodiments, the volume of dilution buffer added to the cell population is less than the volume of the cell population. In some embodiments, the volume of the dilution buffer added to the cell population is the same as the volume of the cell population. In some embodiments, the cell population may be cryopreserved after transfer to a sterile container. In some embodiments, the cell population may be cryopreserved after dilution and transfer to a sterile container. In some embodiments, the sterile packaged cell population is cryopreserved using a controlled rate freezer. In some embodiments, the sterile packaged cell population is cryopreserved by immersion in liquid nitrogen. [0177] In some embodiments, the processing of the first population of cells in step (b) further includes a step of adding a second buffer to the second population of cells and cryopreserving the second population of cells. In some embodiments, the second buffer includes one or more of the following: phosphate-buffered saline, dimethyl sulfoxide, sodium hydroxide, potassium hydroxide, and sucrose. In some embodiments, the second buffer includes phosphate buffered saline, dimethyl sulfoxide, sodium hydroxide, potassium hydroxide, and sucrose. [0178] In some embodiments, the cryopreserved second population of cells is thawed before it is transduced with a recombinant polynucleotide encoding a CAR in step (d). For purposes of the methods described herein, Day 0 is the day on which a processed, cryopreserved leukapheresis or apheresis product is thawed and the method is initiated. In some embodiments, the recombinant polynucleotide comprises a lentiviral expression vector encoding a chimeric antigen receptor. In some embodiments, the lentiviral expression vector is manufactured in an adherent cell culture. In some embodiments, the lentiviral expression vector is manufactured in a suspension cell culture. [0179] In some embodiments, the second population of cells after processing may include fewer platelets compared to the cell sample before processing. In some embodiments, the amount of platelets in the second cell population after processing may comprise less than 60%, less than 55%, less than 50%, less than 45%, less than, 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, less than 2% of the amount of platelets present in the sample before processing. [0180] In some embodiments, the amount of viable cells left in the sample after processing may be at least about 65%, at least about 68%, at least about 70%, at least about 72%, at least about 74%, at least about 76%, at least about 78%, at least about 80%, at least about 82%, at least about 84%, at least about 86%, at least about 88%, at least about 90%, at least about 92%, at least about 94%, at least about 96%, at least about 98%, or at least about 100% of the amount of viable cells in the sample before processing. In some embodiments, the cell recovery after processing may be at least about 65%, at least about 68%, at least about 70%, at least about 72%, at least about 74%, at least about 76%, at least about 78%, at least about 80%, at least about 82%, at least about 84%, at least about 86%, at least about 88%, at least about 90%, at least about 92%, at least about 94%, at least about 96%, at least about 98%, or at least about 100% of the cells in the sample before processing. METHODS OF MAKING A POPULATION OF CAR EXPRESSING IMMUNE CELLS COMPRISING A STEP OF HARVESTING THE CELLS ON DAY 5 OR LATER AFTER SEEDING [0181] As discussed above, one aspect of the disclosure relates to a method of making a population of CAR-expressing immune cells, the method including the steps of: (a) obtaining a liquid sample including a first population of cells including immune cells from a human subject; (b) processing the first population of cells thereby generating a second population of cells including immune cells; (c) seeding a third population of cells in a volume of a first buffer, wherein the third population of cells is a subset of the second population of cells; (d) transducing the third population of cells with a recombinant polynucleotide encoding a CAR thereby generating a fourth population of cells, wherein the fourth population of cells includes CAR-expressing immune cells; (e) expanding the fourth population of cells to yield a fifth population of cells comprising CAR-expressing immune cells; (f) removing at least 50% of the volume of the first buffer on or before Day 4 after the seeding in step (c); and (g) harvesting the fifth population of cells on Day 5 or later after the seeding in step (c). [0182] Non-limiting exemplary embodiments of the methods in accordance with this aspect and other aspects of the disclosure may include one or more of the following features. In some embodiments, the processing of the first population of cells in step (b) includes a step of adding a second buffer to the second population of cells and cryopreserving the second population of cells. In some embodiments, at least 2.4% of cells in the fifth population of cells are CCR7+CD45RA+ immune cells. In some embodiments, the liquid sample including a first population of cells including immune cells from a human subject comprises a leukapheresis product or an apheresis product. [0183] Another aspect of the disclosure relates to a method of making a population of CAR- expressing immune cells, the method including the steps of: (a) obtaining a liquid sample including a first population of cells including immune cells from a human subject; (b) processing the first population of cells thereby generating a second population of cells including immune cells and optionally cryopreserving the second population of cells; (c) seeding a third population of cells in a volume of a first buffer, wherein the third population of cells is a subset of the second population of cells; (d) transducing the third population of cells with a recombinant polynucleotide encoding a CAR thereby generating a fourth population of cells, wherein the fourth population of cells includes CAR-expressing immune cells; (f) expanding the fourth population of cells yielding a fifth population of cells; and (g) harvesting the fifth population of cells on Day 5 or later after the seeding in step (c), wherein at least 2.4% of cells in the fifth population of cells are CCR7+ CD45RA+ immune cells. In some embodiments, the processing of the first population of cells in step (b) includes a step of adding a second buffer to the second population of cells and cryopreserving the second population of cells. In some embodiments, the liquid sample including a first population of cells including immune cells from a human subject comprises an apheresis product. [0184] Another aspect of the disclosure relates a method of making a population of CAR- expressing immune cells, the method including: (a) obtaining a liquid sample including a first population of cells including immune cells from a human subject; (b) processing the first population of cells to remove platelets thereby generating a second population of cells including immune cells, wherein the second population of cells includes at least 1 ^104 total cells, wherein less than 20% of the total number of cells in the second population of cells are platelets and optionally cryopreserving the second population of cells; and (c) seeding a third population of cells with a portion of the second population of cells in a volume of a first media or thawing the cryopreserved second population of cells on Day 0 and seeding a third population of cells with a portion of the thawed cryopreserved second population of cells in a volume of a first media, wherein the third population of cells is a subset of the second population of cells, wherein the third population of cells includes at least 2.0 ^108 cells; (d) transducing the third population of cells with a recombinant polynucleotide encoding a CAR thereby generating a fourth population of cells, wherein the fourth population of cells includes CAR-expressing immune cells; (e) expanding the fourth population of cells to yield a fifth population of cells comprising CAR-expressing immune cells; (f) removing at least 50% of the volume of the first media on or before Day 4 after the seeding in step (c); and (g) harvesting the fifth population of cells on or before Day 9 after the seeding in step (c), wherein at least 2.4% of cells in the fifth population of cells are CCR7+CD45RA+ immune cells. In some embodiments, the processing of the first population of cells in step (b) includes a step of adding a second buffer to the second population of cells and cryopreserving the second population of cells. In some embodiments, the liquid sample including a first population of cells including immune cells from a human subject comprises an apheresis product. [0185] In some embodiments, at least 2.4% of cells in the fifth population of cells are CCR7+CD45RA+ immune cells. In some embodiments, at least 2.5%, 2.8%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, or 5.5% of cells in the fifth population of cells are CCR7+CD45RA+ immune cells. In some embodiments, at least 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.9%, 5.0%, 5.1%, 5.2%, 5.3%, 5.4%, or 5.5% of cells in the fifth population of cells are CCR7+ CD45RA+ immune cells. In some embodiments, the CCR7+ CD45RA+ immune cells are also CD3+. In some embodiments, the CCR7+CD45RA+CD3+ immune cells are also CD8+ (i.e., CCR7+CD45RA+CD3+CD8+). In some embodiments, at least 78.3%, 78.5%, 79.0%, 79.5%, 80%, 80.5%, 81.0%, 81.4%, 81.5%, 82.0%, 83.0%, 84.0%, 85.0%, 86.0%, or 87.0% of the cells in the fifth population of cells are CCR7+CD45RA- immune cells. In some embodiments, the CCR7+CD45RA+CD3+ immune cells are also CD4+ (i.e., CCR7+CD45RA+CD3+CD4+). In some embodiments, at least 78.0%, 78.1%, 78.2%, 78.3%, 78.4%, 78.5%, 78.6%, 78.7%, 78.8%, 78.9%, 79.0%, 79.1%, 79.2%, 79.3%, 79.4%, 79.5%, 79.6%, 79.7%, 79.8%, 79.9%, 80.0%, 80.1%, 80.2%, 80.3%, 80.4%, 80.5%, 80.6%, 80.7%, 80.8%, 80.9%, or 81.0% of cells in the fifth population of cells are CCR7+ CD45RA- immune cells. In some embodiments, at least 81.0%, 81.1%, 81.2%, 81.3%, 81.4%, 81.5%, 81.6%, 81.7%, 81.8%, 81.9%, 82.0%, 82.0%, 82.1%, 82.2%, 82.3%, 82.4%, 82.5%, 82.6%, 82.7%, 82.8%, 82.9%, 83.0%, 83.1%, 83.2%, 83.3%, 83.4%, 83.5%, 83.6%, 83.7%, 83.8%, 83.9%, or 84.0% of cells in the fifth population of cells are CCR7+ CD45RA- immune cells. In some embodiments, at least 84.0%, 84.1%, 84.2%, 84.3%, 84.4%, 84.5%, 84.6%, 84.7%, 84.8%, 84.9%, 85.0%, 85.0%, 85.1%, 85.2%, 85.3%, 85.4%, 85.5%, 85.6%, 85.7%, 85.8%, 85.9%, 86.0%, 86.1%, 86.2%, 86.3%, 86.4%, 86.5%, 86.6%, 86.7%, 86.8%, 86.9%, or 87.0% of cells in the fifth population of cells are CCR7+ CD45RA- immune cells. [0186] In some embodiments, at most 16.1%, 16.0%, 15.0%, 14.0%, 13.0%, 12.0%, 11.0%, 10.0%, 9.0%, 8.0%, 7.5%, 7%, 6.5%, 6.0%, 5.5%, 5.0%, 4.5%, 4.0%, 3.5%, or 3.4% of the cells in the fifth population of cells are CCR7-CD45RA- immune cells. In some embodiments, at most 16.5%, 16.0%, 15.5%, 15.0%, 14.5%, 14.0%, 13.5%, 13.0%, 12.5%, 12.0%, 11.5%, 11.0%, 10.5%, 10.0%, 9.5%, 9.0%, 8.5%, 8.0%, 7.5%, 7.0%, 6.5%, 6.0%, 5.5%, 5.0%, 4.5%, 4.0%, 3.5%, or 3.5% of cells in the fifth population of cells are CCR7- CD45RA- immune cells. [0187] In some embodiments, the harvested immune cells in the fifth population in (e) include at least 2.3%, at least 2.4%, at least 2.5%, at least 2.6%, at least 2.7%, at least 2.8%, at least 2.9%, at least 3%, at least 3.1%, at least 3.2%, at least 3.3%, at least 3.4%, at least 3.5%, at least 3.6%, at least 3.7%, at least 3.8%, at least 3.9%, at least 4%, at least 4.1%, at least 4.2%, at least 4.3%, at least 4.4%, at least 4.5%, at least 4.6%, at least 4.7%, at least 4.8%, at least 4.9%, at least 5%, at least 5.1%, at least 5.2%, at least 5.3%, at least 5.4%, at least 5.5%, at least 5.6%, at least 5.7%, at least 5.8%, at least 5.9%, at least 6%, at least 6.1%, at least 6.2%, at least 6.3%, at least 6.4%, at least 6.5%, at least 6.6%, at least 6.7%, at least 6.8%, at least 6.9%, at least 7%, at least 7.1%, at least 7.2%, at least 7.3%, at least 7.4%, at least 7.5%, at least 7.6%, at least 7.7%, at least 7.8%, at least 7.9%, at least 8%, at least 8.1%, at least 8.2%, at least 8.3%, at least 8.4%, at least 8.5%, at least 8.6%, at least 8.7%, at least 8.8%, at least 8.9%, at least 9%, at least 9.1%, at least 9.2%, at least 9.3%, at least 9.4%, at least 9.5%, at least 9.6%, at least 9.7%, at least 9.8%, at least 9.9%, or at least 10% of CCR7+CD45RA+ T stem cell-like memory (TN/TSCM) cells. See, e.g., Y. Tian et al., “Unique phenotypes and clonal expansions of human CD4 effector memory T cells re-expressing CD45RA”, Nat. Commun.8:1473 (2017). [0188] In some embodiments, the harvested immune cells in the fifth population in (e) include at least 78.3%, at least 78.5%, at least 79.0%, at least 79.5%, at least 80.0%, at least 80.5%, at least 81.0%, at least 81.4%, at least 81.8%, at least 82.4%, at least 82.8%, at least 83.4%, at least 83.8%, at least 84.4%, at least 84.8%, at least 85.4%, at least 85.8%, at least 86.4%, at least 86.8%, at least 87.4%, at least 87.8%, at least 88.4%, at least 88.8%, at least 89.4%, at least 89.8%, at least 90.4%, at least 90.8%, at least 91.4%, at least 91.8%, at least 92.4%, at least 92.8%, at least 93.4%, at least 93.8%, at least 94.4%, at least 94.8%, at least 95.4%, at least 95.8%, at least 96.4%, at least 96.8%, at least 97.4%, at least 97.8%, at least 98.4%, at least 98.8%, or at least 99.4% of CCR7+CD45RA- T central memory (TCM) cells. [0189] In some embodiments, the harvested immune cell population in (e) includes fewer than 16%, fewer than 15.8%, fewer than 15.6%, fewer than 15.4%, fewer than 15.2%, fewer than 15%, fewer than 14.8%, fewer than 14.6%, fewer than 14.4%, fewer than 14.2%, fewer than 14%, fewer than 13.8%, fewer than 13.6%, fewer than 13.4%, fewer than 13.2%, fewer than 13%, fewer than 12.8%, fewer than 12.6%, fewer than 12.4%, fewer than 12.2%, fewer than 12%, fewer than 11.8%, fewer than 11.6%, fewer than 11.4%, fewer than 11.2%, fewer than 11%, fewer than 10.8%, fewer than 10.6%, fewer than 10.4%, fewer than 10.2%, fewer than 10%, fewer than 9.8%, fewer than 9.6%, fewer than 9.4%, fewer than 9.2%, fewer than 9%, fewer than 8.8%, fewer than 8.6%, fewer than 8.4%, fewer than 8.2%, fewer than 8%, fewer than 7.8%, fewer than 7.6%, fewer than 7.4%, fewer than 7.2%, fewer than 7%, fewer than 6.8%, fewer than 6.6%, fewer than 6.4%, fewer than 6.2%, fewer than 6%, fewer than 5.8%, fewer than 5.6%, fewer than 5.4%, fewer than 5.2%, fewer than 5%, fewer than 4.8%, fewer than 4.6%, fewer than 4.4%, fewer than 4.2%, fewer than 4%, fewer than 3.8%, fewer than 3.6%, fewer than 3.4%, or fewer than 3.2% of CCR7-CD45RA- T effector memory (TEM) cells. [0190] In some embodiments, less than 60%, less than 55%, less than 50%, less than 45%, less than, 45%, less than 40%, less than 35%, less than 30%, less than 25%, less than 20%, less than 18%, less than 15%, less than 12%, less than 10%, less than 8%, less than 5%, or less than 2% of the total number of cells in the second population of cells are platelets. [0191] In some embodiments, the efficiency of transduction of the third population of cells is at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%. In some embodiments, the efficiency of transduction of the third population of cells is at most 60%, at most 55%, at most 50%, at most 45%, at most 40%, at most 35%, at most 30%, at most 25%, at most 20%, at most 15%, at most 10%. [0192] In some embodiments, the third population of cells is transduced at a multiplicity of infection (MOI) of at least 0.5, at least 1, at least 1.5, at least 2, at least 2.5, at least 3, at least 3.5, at least 4.0, at least 4.5, at least 5.0, or at least 5.5. In some embodiments, the third population of cells is transduced at an MOI of at most 0.5, at most 1, at most 1.5, at most 2, at most 2.5, at most 3, at most 3.5, at most 4.0, at most 4.5, at most 5.0, or at most 5.5. [0193] In some embodiments, the third population of cells is enriched for CD3+CD4+ T cells before seeding. In some embodiments, the third population of cells is enriched for CD3+CD8+ T cells before seeding. In some embodiments, the third population of cells is enriched for CD4+CD8+ T cells before seeding. [0194] In some embodiments, the third population of cells is activated by at least one cytokine. In some embodiments, the at least one cytokine includes IL-2, IL-4, IL-7, IL-9, IL- 15, IL-21, or a combination thereof. In some embodiments, the at least one cytokine includes a combination of IL-7 and IL-15. [0195] In some embodiments, the third population of cells is seeded in a first buffer. In some embodiments, the first buffer includes 12.5ng/mL IL-7, 12.5ng/mL IL-15, Plasma-Lyte A, and 4% human serum albumin. [0196] In some embodiments, the third population of cells includes at least 1.1×108 cells from the second population of cells. In some embodiments, the third population of cells includes at least at least 2.5 ^106, at least 5 ^106, at least 7.5 ^106, at least 1 ^107, at least 2.5 ^107, at least 5 ^107, at least 7.5 ^107, at least 1 ^108, 1.2×108, at least 1.4×108, at least 1.6×108, at least 1.8×108, at least 2.0×108, at least 2.2 ^108, at least 2.4 ^108, at least 2.6 ^108, at least 2.8 ^108, or at least 3.0 ^108 cells from the second population of cells. In some embodiments, the third population of cells includes at least about 2×108 cells from the second population of cells. In some embodiments, the third population of cells includes at least about 3×108 cells from the second population of cells. In some embodiments, the third population of cells includes at least about 1×109 cells, at least about 1.05×109 cells, at least about 1.1×109 cells, at least about 1.15×109 cells, at least about 1.2×109 cells, at least about 1.25×109 cells, at least about 1.3×109 cells, at least about 1.35×109 cells, at least about 1.4×109 cells, at least about 1.45×109 cells, at least about 1.5×109 cells, at least about 1.55×109 cells, at least about 1.6×109 cells, at least about 1.65×109 cells, at least about 1.7×109 cells, at least about 1.75×109 cells, at least about 1.8×109 cells, at least about 1.85×109 cells, at least about 1.9×109 cells, at least about 1.95×109 cells, at least about 2×109 cells, at least about 2.05×109 cells, at least about 2.1×109 cells, at least about 2.15×109 cells, at least about 2.2×109 cells, at least about 2.25×109 cells, at least about 2.3×109 cells, at least about 2.35×109 cells, at least about 2.4×109 cells, at least about 2.45×109 cells, at least about 2.5×109 cells, at least about 2.55×109 cells, at least about 2.6×109 cells, at least about 2.65×109 cells, at least about 2.7×109 cells, at least about 2.75×109 cells, at least about 2.8×109 cells, at least about 2.85×109 cells, at least about 2.9×109 cells, at least about 2.95×109 cells, at least about 3×109 cells, at least about 3.05×109 cells, at least about 3.1×109 cells, at least about 3.15×109 cells, at least about 3.2×109 cells, at least about 3.25×109 cells, at least about 3.3×109 cells, at least about 3.35×109 cells, at least about 3.4×109 cells, at least about 3.45×109 cells, at least about 3.5× 109 cells, at least about 3.55×109 cells, at least about 3.6×109 cells, at least about 3.65×109 cells, at least about 3.7×109 cells, at least about 3.75×109 cells, at least about 3.8×109 cells, at least about 3.85×109 cells, at least about 3.9×109 cells, at least about 3.95×109 cells, or at least about 4×109 cells from the second population of cells. [0197] In some embodiments, the fifth population of cells is harvested on Day 5, Day 6, Day 7, Day 8, Day 9, Day 10, Day 11, Day 12, Day 13, Day 14, or Day 15. In some embodiments, the fifth population of cells is harvested on Day 5, Day 6, Day 7, Day 8, or Day 9. In some embodiments, the fifth population of cells is harvested on Day 5, 7, and/or Day 9. In some embodiments, the fifth population of cells is harvested on Day 5. In some embodiments, the fifth population of cells is harvested on Day 6. In some embodiments, the fifth population of cells is harvested on Day 7. In some embodiments, the fifth population of cells is harvested on Day 8. In some embodiments, the fifth population of cells is harvested on Day 9. [0198] In some embodiments, the harvested fifth population of cells includes at least 1×103, at least 1×104, at least 1×105, at least 1×106, at least 1×107, at least 1×108, at least 2.2 ^108, at least 2.4 ^108, at least 2.6 ^108, at least 2.8 ^108, or at least 3.0 ^108 CAR-expressing immune cells. [0199] In some embodiments, at least 70%, 75%, 80%, 85%, or 90% of the cells in the fifth population of cells are viable. In some embodiments, the fifth population of cells is cryopreserved after harvesting. [0200] In some embodiments, the number of CAR+ cells harvested in step (f) or step (g) may be 1.0×108 cells, 1.5×108 cells, at least about 2×108 cells, at least about 2.5×108 cells, at least about 3×108 cells, at least about 3.5×108 cells, at least about 4×108 cells, at least about 4.5×108 cells, at least about 5×108 cells, at least about 5.5×108 cells, at least about 6×108 cells, at least about 6.5×108 cells, at least about 7×108 cells, at least about 7.5×108 cells, at least about 8×108 cells, at least about 8.5×108 cells, at least about 9×108 cells, at least about 9.5×108 cells, at least about 1×109 cells, at least about 1.05×109 cells, at least about 1.1×109 cells, at least about 1.15×109 cells, at least about 1.2×109 cells, at least about 1.25×109 cells, at least about 1.3×109 cells, at least about 1.35×109 cells, at least about 1.4×109 cells, at least about 1.45×109 cells, at least about 1.5×109 cells, at least about 1.55×109 cells, at least about 1.6×109 cells, at least about 1.65×109 cells, at least about 1.7×109 cells, at least about 1.75×109 cells, at least about 1.8×109 cells, at least about 1.85×109 cells, at least about 1.9×109 cells, at least about 1.95×109 cells, at least about 2×109 cells, at least about 2.05×109 cells, at least about 2.1×109 cells, at least about 2.15×109 cells, at least about 2.2×109 cells, at least about 2.25×109 cells, at least about 2.3×109 cells, at least about 2.35×109 cells, at least about 2.4×109 cells, at least about 2.45×109 cells, at least about 2.5×109 cells, at least about 2.55×109 cells, at least about 2.6×109 cells, at least about 2.65×109 cells, at least about 2.7×109 cells, at least about 2.75×109 cells, at least about 2.8×109 cells, at least about 2.85×109 cells, at least about 2.9×109 cells, at least about 2.95×109 cells, at least about 3×109 cells, at least about 3.05×109 cells, at least about 3.1×109 cells, at least about 3.15×109 cells, at least about 3.2×109 cells, at least about 3.25×109 cells, at least about 3.3×109 cells, at least about 3.35×109 cells, at least about 3.4×109 cells, at least about 3.45×109 cells, at least about 3.5×109 cells, at least about 3.55×109 cells, at least about 3.6×109 cells, at least about 3.65×109 cells, at least about 3.7×109 cells, at least about 3.75×109 cells, at least about 3.8×109 cells, at least about 3.85×109 cells, at least about 3.9×109 cells, at least about 3.95×109 cells, or at least about 4×109 cells. In some embodiments, the percentage of CAR+ cells within the total harvested cell population may be at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, or at least 80%. [0201] The harvested CAR-T cells may include different cell subsets. In some embodiments, multiple T-cell subsets may be present within the harvested CAR-T cell sample. In some embodiments, the T cell subsets may be CD4+, CD8+, CD3+, CD5+, CD2+, CD7+, or a combination thereof. In some embodiments, the T cell subsets may be CD45RA+, CCR7+, CD45RO+, or a combination thereof. In some embodiments, the T cell subsets may be CD45RA-, CCR7-, CD45RO-, or a combination thereof. In some embodiments, the T cell subsets may include stem cell-like memory T cells (TSCM), central memory T cells (TCM), transitional memory T cells (TTM), effector T cells (TEFF), or a combination thereof. In some embodiments, TSCM cells may include CD45RA+, CD45RO- and CCR7+ cells. In some embodiments, TCM cells may include CD45RA-, CD45RO+ and CCR7+ cells. In some embodiments, TTM cells may include CD45RA+, CD45RO- and CCR7- cells. In some embodiments, TEFF cells may include CD45RA-, CD45RO+ and CCR7- cells. In some embodiments, marker expression is analyzed using flow cytometry. In some embodiments, a determination of positive or negative expression for a given marker may be determined by a gating analysis of flow cytometry data. In some embodiments, the gating analysis is performed manually. In some embodiments, the gating analysis is performed automatically. METHODS OF MANUFACTURING CAR T CELLS, INCLUDING CD22 CAR T CELLS [0202] In another aspect, provided herein are methods of making a population of CAR- expressing immune cells. In some embodiments, the method comprises the steps of: (a) obtaining a liquid sample comprising a first population of cells including immune cells from a human subject; (b) processing the first population of cells thereby generating a second population of cells including immune cells, wherein the second population of cells includes at least 1 ^104 total cells, wherein less than 20% of the total number of cells in the second population of cells are platelets; (c) cryopreserving the second population of cells; (d) on Day 0, thawing the cryopreserved second population of cells including immune cells, processing the thawed second population of cells including immune cells, and seeding a third population of cells including immune cells with a portion of the processed second population of cells including immune cells in a volume of a first media, wherein the third population of cells is a subset of the second population of cells, wherein the third population of cells is seeded into a volume of at least 250 mL of media with at least 2.0 ^108 cells from the second population of cells; (e) transducing the third population of cells including immune cells with a recombinant polynucleotide encoding a CAR thereby generating a fourth population of cells, wherein the fourth population of cells includes CAR-expressing immune cells; (f) expanding the fourth population of cells in media to yield a fifth population of cells including CAR-expressing immune cells; (g) removing at least 50% of the volume of the first media on or before Day 4 after the seeding in step (d); (h) determining the number of viable CD3+ CAR-expressing T cells; and either (i) continuing to expand the fifth population of cells and exchanging the media or (j) harvesting the fifth population of cells including CAR-expressing immune cells on or before Day 9 after the seeding in step (d), wherein at least 2.4% of cells in the fifth population of cells including CAR-expressing immune cells are CCR7+CD45RA+ immune cells; and (k) formulating the fifth population of cells for cryopreservation and administration to patients. In some embodiments, the third population of cells is a subset of the second population of cells and include at least 3.0 ^108 cells from the second population of cells. In some embodiments, the third population of cells is a subset of the second population of cells, wherein the third population of cells is seeded into a volume of at least 250 mL of media with at least 3.0 ^108 cells from the second population of cells. [0203] In some embodiments, the liquid sample including a first population of cells including immune cells from a human subject comprises a leukapheresis product or an apheresis product. [0204] In some embodiments, at least 2.4% of cells in the fifth population of cells are CCR7+CD45RA+ immune cells. In some embodiments, at least 2.5%, 2.8%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, or 5.5% of cells in the fifth population of cells are CCR7+CD45RA+ immune cells. In some embodiments, at least 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.9%, 5.0%, 5.1%, 5.2%, 5.3%, 5.4%, or 5.5% of cells in the fifth population of cells are CCR7+CD45RA+ immune cells. In some embodiments, the CCR7+CD45RA+ immune cells are also CD3+. In some embodiments, the CCR7+CD45RA+CD3+ immune cells are also CD8+ (i.e., CCR7+CD45RA+CD3+CD8+). In some embodiments, at least 78.3%, 78.5%, 79.0%, 79.5%, 80%, 80.5%, 81.0%, 81.4%, 81.5%, 82.0%, 83.0%, 84.0%, 85.0%, 86.0%, or 87.0% of the cells in the fifth population of cells are CCR7+CD45RA- immune cells. In some embodiments, the CCR7+CD45RA+CD3+ immune cells are also CD4+ (i.e., CCR7+CD45RA+CD3+CD4+). In some embodiments, at least 78.0%, 78.1%, 78.2%, 78.3%, 78.4%, 78.5%, 78.6%, 78.7%, 78.8%, 78.9%, 79.0%, 79.1%, 79.2%, 79.3%, 79.4%, 79.5%, 79.6%, 79.7%, 79.8%, 79.9%, 80.0%, 80.1%, 80.2%, 80.3%, 80.4%, 80.5%, 80.6%, 80.7%, 80.8%, 80.9%, or 81.0% of cells in the fifth population of cells are CCR7+CD45RA- immune cells. In some embodiments, at least 81.0%, 81.1%, 81.2%, 81.3%, 81.4%, 81.5%, 81.6%, 81.7%, 81.8%, 81.9%, 82.0%, 82.0%, 82.1%, 82.2%, 82.3%, 82.4%, 82.5%, 82.6%, 82.7%, 82.8%, 82.9%, 83.0%, 83.1%, 83.2%, 83.3%, 83.4%, 83.5%, 83.6%, 83.7%, 83.8%, 83.9%, or 84.0% of cells in the fifth population of cells are CCR7+CD45RA- immune cells. In some embodiments, at least 84.0%, 84.1%, 84.2%, 84.3%, 84.4%, 84.5%, 84.6%, 84.7%, 84.8%, 84.9%, 85.0%, 85.0%, 85.1%, 85.2%, 85.3%, 85.4%, 85.5%, 85.6%, 85.7%, 85.8%, 85.9%, 86.0%, 86.1%, 86.2%, 86.3%, 86.4%, 86.5%, 86.6%, 86.7%, 86.8%, 86.9%, or 87.0% of cells in the fifth population of cells are CCR7+CD45RA- immune cells. [0205] In some embodiments, at most 16.1%, 16.0%, 15.0%, 14.0%, 13.0%, 12.0%, 11.0%, 10.0%, 9.0%, 8.0%, 7.5%, 7%, 6.5%, 6.0%, 5.5%, 5.0%, 4.5%, 4.0%, 3.5%, or 3.4% of the cells in the fifth population of cells are CCR7-CD45RA- immune cells. In some embodiments, at most 16.5%, 16.0%, 15.5%, 15.0%, 14.5%, 14.0%, 13.5%, 13.0%, 12.5%, 12.0%, 11.5%, 11.0%, 10.5%, 10.0%, 9.5%, 9.0%, 8.5%, 8.0%, 7.5%, 7.0%, 6.5%, 6.0%, 5.5%, 5.0%, 4.5%, 4.0%, 3.5%, or 3.5% of cells in the fifth population of cells are CCR7- CD45RA- immune cells. [0206] In some embodiments, the harvested immune cells in the fifth population in (j) include at least 2.3%, at least 2.4%, at least 2.5%, at least 2.6%, at least 2.7%, at least 2.8%, at least 2.9%, at least 3%, at least 3.1%, at least 3.2%, at least 3.3%, at least 3.4%, at least 3.5%, at least 3.6%, at least 3.7%, at least 3.8%, at least 3.9%, at least 4%, at least 4.1%, at least 4.2%, at least 4.3%, at least 4.4%, at least 4.5%, at least 4.6%, at least 4.7%, at least 4.8%, at least 4.9%, at least 5%, at least 5.1%, at least 5.2%, at least 5.3%, at least 5.4%, at least 5.5%, at least 5.6%, at least 5.7%, at least 5.8%, at least 5.9%, at least 6%, at least 6.1%, at least 6.2%, at least 6.3%, at least 6.4%, at least 6.5%, at least 6.6%, at least 6.7%, at least 6.8%, at least 6.9%, at least 7%, at least 7.1%, at least 7.2%, at least 7.3%, at least 7.4%, at least 7.5%, at least 7.6%, at least 7.7%, at least 7.8%, at least 7.9%, at least 8%, at least 8.1%, at least 8.2%, at least 8.3%, at least 8.4%, at least 8.5%, at least 8.6%, at least 8.7%, at least 8.8%, at least 8.9%, at least 9%, at least 9.1%, at least 9.2%, at least 9.3%, at least 9.4%, at least 9.5%, at least 9.6%, at least 9.7%, at least 9.8%, at least 9.9%, or at least 10% of CCR7+CD45RA+ T stem cell-like memory (TN/TSCM) cells. See, e.g., Y. Tian et al., “Unique phenotypes and clonal expansions of human CD4 effector memory T cells re-expressing CD45RA”, Nat. Commun.8:1473 (2017). [0207] In some embodiments, the harvested immune cells in the fifth population in (j) include at least 78.3%, at least 78.5%, at least 79.0%, at least 79.5%, at least 80.0%, at least 80.5%, at least 81.0%, at least 81.4%, at least 81.8%, at least 82.4%, at least 82.8%, at least 83.4%, at least 83.8%, at least 84.4%, at least 84.8%, at least 85.4%, at least 85.8%, at least 86.4%, at least 86.8%, at least 87.4%, at least 87.8%, at least 88.4%, at least 88.8%, at least 89.4%, at least 89.8%, at least 90.4%, at least 90.8%, at least 91.4%, at least 91.8%, at least 92.4%, at least 92.8%, at least 93.4%, at least 93.8%, at least 94.4%, at least 94.8%, at least 95.4%, at least 95.8%, at least 96.4%, at least 96.8%, at least 97.4%, at least 97.8%, at least 98.4%, at least 98.8%, or at least 99.4% of CCR7+CD45RA- T central memory (TCM) cells. [0208] In some embodiments, the harvested immune cell population in (j) includes fewer than 16%, fewer than 15.8%, fewer than 15.6%, fewer than 15.4%, fewer than 15.2%, fewer than 15%, fewer than 14.8%, fewer than 14.6%, fewer than 14.4%, fewer than 14.2%, fewer than 14%, fewer than 13.8%, fewer than 13.6%, fewer than 13.4%, fewer than 13.2%, fewer than 13%, fewer than 12.8%, fewer than 12.6%, fewer than 12.4%, fewer than 12.2%, fewer than 12%, fewer than 11.8%, fewer than 11.6%, fewer than 11.4%, fewer than 11.2%, fewer than 11%, fewer than 10.8%, fewer than 10.6%, fewer than 10.4%, fewer than 10.2%, fewer than 10%, fewer than 9.8%, fewer than 9.6%, fewer than 9.4%, fewer than 9.2%, fewer than 9%, fewer than 8.8%, fewer than 8.6%, fewer than 8.4%, fewer than 8.2%, fewer than 8%, fewer than 7.8%, fewer than 7.6%, fewer than 7.4%, fewer than 7.2%, fewer than 7%, fewer than 6.8%, fewer than 6.6%, fewer than 6.4%, fewer than 6.2%, fewer than 6%, fewer than 5.8%, fewer than 5.6%, fewer than 5.4%, fewer than 5.2%, fewer than 5%, fewer than 4.8%, fewer than 4.6%, fewer than 4.4%, fewer than 4.2%, fewer than 4%, fewer than 3.8%, fewer than 3.6%, fewer than 3.4%, or fewer than 3.2% of CCR7-CD45RA- T effector memory (TEM) cells. [0209] In some embodiments, the processing of the first population of cells in step (b) comprises a step of washing, concentrating and eluting or resuspending the second population of cells in a buffer. In some embodiments, the processing of the first population of cells further comprises a step of reducing the number of platelets in the first population of cells. In some embodiments, the second population of cells is washed, concentrated, and eluted or resuspended and the number of platelets is reduced by elutriation using a centrifugation system having a molecular weight cutoff sufficient to retain the immune cells. In some embodiments, the centrifugal filtration system is a CTSTM RoteaTM Counterflow Centrifugation System. In some embodiments, the concentrated second population of cells is resuspended in a buffer comprising human serum albumin (HSA), Plasma-Lyte A, phosphate buffered saline, sodium chloride, sodium bicarbonate buffer, glutathione, biotin, vitamin B12, inositol, choline, L-glutamine, sodium pyruvate, glucose, or any combination thereof. In some embodiments, the buffer comprises human serum albumin (HSA). In some embodiments, the buffer comprises Plasma-Lyte A. In some embodiments, the buffer comprises equal volumes of Plasma-Lyte A and 4% (w/v) human serum albumin. In some embodiments, the first population of cells is resuspended in a buffer comprising equal volumes of Plasma-Lyte A and 4% (w/v) HSA and further diluted 1:1 with a cryoprotectant. In some embodiments, the cryoprotectant is CryoStorR. [0210] In some embodiments, the processing of the thawed second population of cells in step (d) comprises a step of enriching for CD4+ and CD8+ T cells. In some embodiments, the enriching for CD4+ and CD8+ T cells comprises a step of measuring the total viable cells and determining the percentage (%) of cells that are CD3+ (%CD3+), CD4+CD8- (%CD4+CD8- ), CD8+CD4- (%CD8+CD4-), and CD4+CD8+ (%CD4+CD8+) in the thawed second population of cells. In some embodiments, the enriching for CD4+ and CD8+ T cells further comprises a step mixing the thawed second population of cells with magnetic beads derivatized with CD4-specific binding agents and CD8-specific binding agents, washing, and eluting the thawed second population of cells enriched for CD4+ and CD8+ cells. In some embodiments, the amount of cryopreserved apheresis product (i.e., the second population of cells) to be thawed for processing to enrich for CD4+ and CD8+ is determined by calculating the following: (1) Target Cells in each aliquot of cryopreserved Apheresis Product = (%CD4+CD8+ cells + %CD8+CD4- cells +%CD4+CD8+ cells)×Viable Cell Density post- RoteaTM (cells/mL)×volume of aliquot (mL) and (2) Total CD3+ cells in each aliquot of cryopreserved Apheresis Product = (%CD3+)×Viable Cell Density post-RoteaTM (cells/mL)×volume of aliquot (mL). The number of aliquots of cryopreserved Apheresis Product to be thawed for use in the enrichment step is determined to be the amount that results in at least 1.0×109 CD3+ cells and the closest in absolute value to 3.0×109 CD3+ cells. In some embodiments, the step of enriching the thawed second population of cells for CD4+ and CD8+ T cells is performed on a CliniMACS Prodigy using CliniMACS buffer + 2% (w/v) or 0.5% (w/v) HSA. [0211] In some embodiments, the third population of cells is seeded with 300×106 cells from the processed second population of cells. In some embodiments, the third population of cells is seeded with at least 2.5×106 cells from the processed second population of cells. In some embodiments, the third population of cells is seeded with between at least 2.5×106 cells and at least 300×106 cells from the processed second population of cells. In some embodiments, the third population of cells is seeded with at least about 2.5×106, 3×106, 3.5×106, 4×106, 4.5×106, 5×106, 5.5×106, 6×106, 6.5×106, 7×106, 7.5×106, 8×106, 8.5×106, 9×106, 9.5×106, 1×107, 1.5×107, 2×107, 2.5 ×107, 3×107, 3.5×107, 4×107, 4.5×107, 5×107, 5.5×107, 6×107, 6.5×107, 7×107, 7.5×107, 8×107, 8.5×107, 9×107, 9.5×107, 1×108, 1.5×108, 2×108, 2.5×108, 3×108, 3.5×108, 4×108, 4.5×108, or 5×108 cells from the processed second population of cells. In some embodiments the third population of cells is seeded in modified TexMACSTM medium (MTM). In some embodiments, the MTM comprises TexMACSTM medium supplemented with 3% human AB plasma (HABS) and 1:1 mixture of recombinant human cytokines IL-7 (hIL-7) and IL-15 (hIL-15). In some embodiments, the modified TexMACSTM medium further comprises an effective amount of a reagent comprising agonists of CD3 and CD28. In some embodiments, the reagent comprising CD3 and CD28 agonists comprises the T cell TransActTM reagent. [0212] In some embodiments, the recombinant polynucleotide encoding a CAR further comprises a lentiviral expression vector (i.e., it comprises a lentiviral expression vector comprising a recombinant polynucleotide encoding a CAR). In some embodiments, the lentiviral expression vector is manufactured using a suspension cell culture method. In some embodiments, the lentiviral expression vector is manufactured using an adherent cell culture method. In some embodiments, the transduction in step (e) is performed on Day 1. In some embodiments, the transduction in step (e) is performed 22-26 hours after seeding of the third population of cells comprising immune cells on Day 0. In some embodiments, the amount of vector used to transduce the third population of cells comprising immune cells in step (e) is determined based on the infectious titer of the lentiviral vector and the number of cells used to seed the third population of cells comprising immune cells such that the transduction is performed with a multiplicity of infection (MOI) of 2.0. In some embodiments, the transduction is performed with a MOI of at least 1.5, at least 1.6, at least 1.7, at least 1.8, at least 1.9, at least 2.0, at least 2.1, at least 2.2, at least 2.3, at least 2.4, at least 2.5, at least 2.6, at least 2.7, at least 2.8, at least 2.9, at least 3.0, at least 3.5, at least 4.0, at least 4.5, or at least 5.0. In some embodiments, the recombinant polynucleotide encoding a CAR further comprising a lentiviral expression vector is thawed, diluted into modified TexMACSTM medium (MTM), and added to the third population of cells comprising immune cells. In some embodiments, the MTM comprises TexMACSTM medium supplemented with 3% human AB plasma (HABS) and 1:1 mixture of recombinant human cytokines IL-7 (hIL-7) and IL-15 (hIL-15). In some embodiments, the modified TexMACSTM medium further comprises an effective amount of a reagent comprising agonists of CD3 and CD28. In some embodiments, the reagent comprising CD3 and CD28 agonists comprises the T cell TransActTM reagent. In some embodiments, the final volume in which the transduction in step (e) is performed comprises 100 mL. [0213] In some embodiments, the fifth population of cells in step (f) is expanded in MTM in a volume of 200 mL. In some embodiments, the fifth population of cells is washed to remove T cell TransActTM and residual lentiviral vector. In some embodiments the washing step is performed on Day 4. In some embodiments, the washing step is performed using the automated CliniMACS Prodigy program. In some embodiments, the washing step is performed on Day 4 using the CliniMACS Prodigy program. In some embodiments, the washing step comprises removal of cell culture supernatant and resuspension of the fifth population of cells in MTM without T cell TransActTM reagent. In some embodiments, the washing step comprises removal of 65% of the culture volume and resuspension of the fifth population in the desired volume of MTM without T cell TransActTM. [0214] In some embodiments, the step (h) of determining the number of viable CD3+ CAR- expressing T cells is performed on Day 5 by taking a sample of the fifth population of cells and calculating the Dose Factor = Total Viable Cells (cell number)×Day 4 Transduction Efficiency (%) / Dose Target (1.0×106 viable CD3+CAR+ cells/kg)×Patient weight (kg). In some embodiments, there are enough viable CD3+CAR+ cells to meet the dose requirements on Day 5 and the harvesting step (j) is performed. In some embodiments, there are not enough viable CD3+CAR+ cells on Day 5, the fifth population of cells is expanded for another day, a sample of the fifth population of cells is taken on Day 6 and the Dose Factor is recalculated assuming a 7% increase in Day 4 Transduction Efficiency. In some embodiments, there are enough viable CD3+CAR+ cells to meet the dose requirements on Day 6 and the harvesting step (j) is performed. In some embodiments, there are not enough viable CD3+CAR+ cells on Day 6, the fifth population of cells is expanded for another day, a sample of the fifth population of cells is taken on Day 7 and the Dose Factor is recalculated assuming a 7% increase in Day 4 Transduction Efficiency. In some embodiments, a further media exchange is performed on Day 7. In some embodiments, the further media exchange on Day 7 comprises removing 60% of the culture volume and replacing it with fresh MTM. In some embodiments, there are enough viable CD3+CAR+ cells to meet the dose requirements on Day 7 and the harvesting step (j) is performed. In some embodiments, there are not enough viable CD3+CAR+ cells on Day 7, the fifth population of cells is expanded for another day, a sample of the fifth population of cells is taken on Day 8 and the Dose Factor is recalculated assuming a 7% increase in Day 4 Transduction Efficiency. In some embodiments, a further media exchange is performed on Day 8. In some embodiments, the further media exchange on Day 8 comprises removing 60% of the culture volume and replacing it with fresh MTM. In some embodiments, there are enough viable CD3+CAR+ cells to meet the dose requirements on Day 8 and the harvesting step (j) is performed. In some embodiments, there are not enough viable CD3+CAR+ cells on Day 8, the fifth population of cells is expanded for another day, and the harvesting step (j) is performed. In some embodiments, there are not enough viable CD3+CAR+ cells on Day 8, the fifth population of cells is expanded for another day, and the harvesting step (j) is performed on Day 9. [0215] In some embodiments, the harvesting step (j) further comprises a step of calculating viable cell density and determining whether the post-harvest viable cell density is ≥ the minimum transduced viable cell density for the formulation step. In some embodiments, the post-harvest viable cell density is ≥ the minimum transduced viable cell density and the harvested fifth population of cells is formulated for cryopreservation and administration to patients. In some embodiments, the post-harvest viable cell density is < the minimum transduced viable cell density and the harvested fifth population of cells is concentrated using a RoteaTM so that the post-harvest viable cell density is ≥ the minimum transduced viable cell density and the concentrated harvested fifth population of cells is formulated for cryopreservation and administration to patients. In some embodiments, the minimum transduced viable cell density is 1×106 CD3+CAR+ cells/kg. In some embodiments, the formulation step is performed manually, and the harvested fifth population of cells or the concentrated harvested fifth population of cells is resuspended to the desired concentration in Final Formulation Medium comprising Plasma-Lyte A + 4% (w/v) HSA, diluted 1:1 with CryostorR CS10 and frozen. In some embodiments, the formulation step is automated. In some embodiments, the automated formulation step is performed using a FINIAR Fill and Finish System, and the harvested fifth population of cells or the concentrated harvested fifth population of cells is resuspended to the desired concentration in Final Formulation Medium comprising Plasma-Lyte A+4% (w/v) HSA, diluted 1:1 with CryostorR CS10 and frozen. In some embodiments, the automated formulation step is performed using a Cue ScaleReady Cell Processing System, and the harvested fifth population of cells or the concentrated harvested fifth population of cells is resuspended to the desired concentration in Final Formulation Medium comprising Plasma-Lyte A+4% (w/v) HSA, diluted 1:1 with CryostorR CS10 and frozen. [0216] In some embodiments, the fifth population of cells including CAR-expressing immune cells comprises autologous CAR-expressing immune cells. In some embodiments, the fifth population of cells including CAR-expressing immune cells comprises autologous T cells expressing a CD22 CAR (i.e., a CD22-specific CAR). In some embodiments, the recombinant polynucleotide encoding an autologous CD22 CAR further comprises a lentiviral expression vector. In some embodiments, the lentiviral expression vector is manufactured using a suspension cell culture platform. In some embodiments, the lentiviral expression vector is manufactured using an adherent cell culture platform. [0217] In some embodiments, the recombinant polynucleotide encoding a CAR encodes a CD22-specific CAR. In some embodiments, the CD22 CAR comprises a CD22-specific binding domain, a transmembrane domain, and an intracellular domain. In some embodiments, the CD22 CAR comprises a CD22-specific binding domain, a hinge domain, a transmembrane domain, a spacer, and an intracellular domain, In some embodiments, the CD22-specific binding domain comprises an antibody capable of binding CD22 or an antigen-binding fragment thereof. In some embodiments, the CD22-specific binding domain comprises an antibody capable of binding human CD22. In some embodiments, the CD22- specific binding domain comprises an antigen-binding fragment of an antibody capable of binding human CD22. [0218] In some embodiments, the anti-CD22 binding domain includes a VH comprising a heavy chain CDR1 (HCDR1) sequence with at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an HCDR1 sequence in Table 1. In some embodiments, the anti- CD22 binding domain comprises a VH that comprises an HCDR1 sequence in Table 1. In some embodiments, the anti-CD22 binding domain includes a VH that includes a heavy chain CDR2 (HCDR2) sequence with at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an HCDR2 sequence in Table 1. In some embodiments, the anti-CD22 binding domain includes a VH that includes an HCDR2 sequence in Table 1. In some embodiments, the anti-CD22 binding domain includes a heavy chain variable region (VH) comprising a heavy chain CDR3 (HCDR3) sequence with at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an HCDR3 sequence in Table 1. In some embodiments, the anti- CD22 binding domain includes a VH that includes an HCDR3 sequence in Table 1. In some embodiments, the anti-CD22 binding domain includes the heavy chain CDRs 1, 2, and 3 (HCDR1, HCDR2, HCDR3) sequences as set forth in SEQ ID NOs: 4, 5, and 6, respectively. In some embodiments, the anti-CD22 binding domain includes the heavy chain HCDR1, HCDR2, HCDR3 sequences as set forth in SEQ ID NOs: 10, 11, and 12, respectively. In some embodiments, the anti-CD22 binding domain includes the heavy chain HCDR1, HCDR2, HCDR3 sequences as set forth in SEQ ID NOs: 16, 17, and 18, respectively. [0219] In some embodiments, the anti-CD22 binding domain includes a VL comprising a light chain CDR1 (LCDR1) sequence with at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an LCDR1 sequence in Table 1. In some embodiments, the anti-CD22 binding domain includes a VL comprising an LCDR1 sequence in Table 1. In some embodiments, the anti-CD22 binding domain comprises a VL that comprises a light chain CDR2 (LCDR2) sequence with at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an LCDR2 sequence in Table 1. In some embodiments, the anti-CD22 binding domain comprises a VL that comprises an LCDR2 sequence in Table 1. In some embodiments, the anti-CD22 binding domain comprises a VL that comprises a light chain CDR3 (LCDR3) sequence with at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an LCDR3 sequence in Table 1. In some embodiments, the anti-CD22 binding domain comprises a VL that comprises an LCDR3 sequence in Table 1. In some embodiments, the anti-CD22 binding domain includes the light chain CDRs 1, 2, and 3 (LCDR1, LCDR2, LCDR3) sequences as set forth in SEQ ID NOs: 7, 8, and 9, respectively. In some embodiments, the anti-CD22 binding domain includes the light chain LCDR1, LCDR2, LCDR3 sequences as set forth in SEQ ID NOs: 13, 14, and 15, respectively. In some embodiments, the anti-CD22 binding domain includes the light chain LCDR1, LCDR2, LCDR3 sequences as set forth in SEQ ID NOs: 19, 20, and 21, respectively. [0220] In some embodiments, the anti-CD22 binding domain includes the heavy chain CDRs 1, 2, and 3 (HCDR1, HCDR2, HCDR3) sequences and the light chain CDRs 1, 2, and 3 (LCDR1, LCDR2, LCDR3) sequences as set forth in SEQ ID NOs: 4, 5, 6, 7, 8, and 9, respectively. In some embodiments, the anti-CD22 binding domain includes the heavy chain HCDR1, HCDR2, HCDR3 sequences and the light chain LCDR1, LCDR2, LCDR3 sequences as set forth in SEQ ID NOs: 10, 11, 12, 13, 14, and 15, respectively. In some embodiments, the anti-CD22 binding domain includes the heavy chain HCDR1, HCDR2, HCDR3 sequences and the light chain LCDR1, LCDR2, LCDR3 sequences as set forth in SEQ ID NOs: 16, 17, 18, 19, 20, and 21, respectively. [0221] In some embodiments, the antigen-binding fragment of an antibody capable of binding human CD22 is a single chain variable fragment (scFv) capable of binding CD22. In some embodiments, the scFv capable of binding CD22 has the sequence of SEQ ID NO: 1. In some embodiments, the scFv capable of binding CD22 comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 1. In some embodiments, the CD22 CAR comprises a CD8 ^ hinge domain comprising the sequence of SEQ ID NO: 24 and a CD8 ^ transmembrane domain comprising the sequence of SEQ ID NO: 25. In some embodiments, the CD22 CAR comprises a CD8 ^ hinge domain comprising the sequence of SEQ ID NO:24, a CD8 ^ transmembrane domain comprising the sequence of SEQ ID NO:25, and a peptide linker having the sequence of SEQ ID NO:31. In some embodiments, the CD22 CAR comprises a CD8 ^ hinge domain comprising a sequence comprising 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 24 and a CD8 ^ transmembrane domain comprising a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 25. In some embodiments, the CD8 ^ transmembrane domain further comprises a spacer having the sequence of LYC. In some embodiments, the CD8 ^ transmembrane domain further comprises a spacer having the sequence of SEQ ID NO:31. In some embodiments, the CD22 CAR comprises an intracellular domain further comprising a primary T cell activating domain comprising an immunoreceptor tyrosine-based activation motif (ITAM) and a costimulatory signaling domain. In some embodiments, the primary T cell activating domain comprising an ITAM comprises a CD3 ^ intracellular signaling domain. In some embodiments, the CD3 ^ intracellular signaling domain comprises the sequence of SEQ ID NO: 27. In some embodiments, the CD3 ^ intracellular signaling domain comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 27. In some embodiments, the CD3 ^ intracellular signaling domain comprises the sequence of SEQ ID NO: 30. In some embodiments, the CD3 ^ intracellular signaling domain comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 30. In some embodiments the costimulatory signaling domain comprises a 4-1BB/CD137 signaling domain. In some embodiments, the 4-1BB/CD137 signaling domain comprises the sequence of SEQ ID NO: 26. In some embodiments, the 4-1BB/CD137 costimulatory signaling domain comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 26. In some embodiments, the CD22 CAR comprises the sequence of SEQ ID NO: 22. In some embodiments, the CD22 CAR comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 22. In some embodiments, the CD22 CAR comprises the sequence of SEQ ID NO: 23. In some embodiments, the CD22 CAR comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 23. See, e.g., Table 2 and paragraph [0198] above. [0222] In some embodiments, the anti-CD22 binding domain includes a VH comprising a heavy chain CDR1 (HCDR1) sequence with at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an HCDR1 sequence in Table 1. In some embodiments, the anti- CD22 binding domain comprises a VH that comprises an HCDR1 sequence in Table 1. In some embodiments, the anti-CD22 binding domain includes a VH that includes a heavy chain CDR2 (HCDR2) sequence with at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an HCDR2 sequence in Table 1. In some embodiments, the anti-CD22 binding domain includes a VH that includes an HCDR2 sequence in Table 1. In some embodiments, the anti-CD22 binding domain includes a heavy chain variable region (VH) comprising a heavy chain CDR3 (HCDR3) sequence with at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an HCDR3 sequence in Table 1. In some embodiments, the anti- CD22 binding domain includes a VH that includes an HCDR3 sequence in Table 1. In some embodiments, the anti-CD22 binding domain includes the heavy chain CDRs 1, 2, and 3 (HCDR1, HCDR2, HCDR3) sequences as set forth in SEQ ID NOs: 4, 5, and 6, respectively. In some embodiments, the anti-CD22 binding domain includes the heavy chain HCDR1, HCDR2, HCDR3 sequences as set forth in SEQ ID NOs: 10, 11, and 12, respectively. In some embodiments, the anti-CD22 binding domain includes the heavy chain HCDR1, HCDR2, HCDR3 sequences as set forth in SEQ ID NOs: 16, 17, and 18, respectively. [0223] In some embodiments, the anti-CD22 binding domain includes a VL comprising a light chain CDR1 (LCDR1) sequence with at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an LCDR1 sequence in Table 1. In some embodiments, the anti-CD22 binding domain includes a VL comprising an LCDR1 sequence in Table 1. In some embodiments, the anti-CD22 binding domain comprises a VL that comprises a light chain CDR2 (LCDR2) sequence with at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an LCDR2 sequence in Table 1. In some embodiments, the anti-CD22 binding domain comprises a VL that comprises an LCDR2 sequence in Table 1. In some embodiments, the anti-CD22 binding domain comprises a VL that comprises a light chain CDR3 (LCDR3) sequence with at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an LCDR3 sequence in Table 1. In some embodiments, the anti-CD22 binding domain comprises a VL that comprises an LCDR3 sequence in Table 1. In some embodiments, the anti-CD22 binding domain includes the light chain CDRs 1, 2, and 3 (LCDR1, LCDR2, LCDR3) sequences as set forth in SEQ ID NOs: 7, 8, and 9, respectively. In some embodiments, the anti-CD22 binding domain includes the light chain LCDR1, LCDR2, LCDR3 sequences as set forth in SEQ ID NOs: 13, 14, and 15, respectively. In some embodiments, the anti-CD22 binding domain includes the light chain LCDR1, LCDR2, LCDR3 sequences as set forth in SEQ ID NOs: 19, 20, and 21, respectively. [0224] In some embodiments, the anti-CD22 binding domain includes the heavy chain CDRs 1, 2, and 3 (HCDR1, HCDR2, HCDR3) sequences and the light chain CDRs 1, 2, and 3 (LCDR1, LCDR2, LCDR3) sequences as set forth in SEQ ID NOs: 4, 5, 6, 7, 8, and 9, respectively. In some embodiments, the anti-CD22 binding domain includes the heavy chain HCDR1, HCDR2, HCDR3 sequences and the light chain LCDR1, LCDR2, LCDR3 sequences as set forth in SEQ ID NOs: 10, 11, 12, 13, 14, and 15, respectively. In some embodiments, the anti-CD22 binding domain includes the heavy chain HCDR1, HCDR2, HCDR3 sequences and the light chain LCDR1, LCDR2, LCDR3 sequences as set forth in SEQ ID NOs: 16, 17, 18, 19, 20, and 21, respectively. CHIMERIC ANTIGEN RECEPTOR
Figure imgf000060_0001
the CAR-expressing immune cells of the disclosure express a CAR. In some embodiments, the CAR comprises an antigen-binding domain, optionally a hinge domain, a transmembrane domain, optionally a peptide linker, and an intracellular signaling domain. In some embodiments, the CAR includes an antigen-binding domain having binding affinity for one or more antigens. In some embodiments, the one or more antigens are selected from the group consisting of CD22, CD20, and CD19. In some embodiments, the CAR includes an anti-CD22 antigen-binding domain. In some embodiments, the CAR includes an anti-CD20 antigen-binding domain. In some embodiments, the CAR includes an anti-CD19 antigen-binding domain. In some embodiments, the CAR includes a bispecific binding domain having binding affinity for two different antigens. In some embodiments, the CAR includes an anti-CD19 antigen-binding domain and an anti-CD20 antigen-binding domain. In some embodiments, the CAR includes an anti-CD19 antigen-binding domain and an anti-CD22 antigen binding domain. In some embodiments, the CAR includes an anti-CD20 antigen-binding domain and an anti-CD22 antigen-binding domain. Anti-CD22 binding domain [0226] The anti-CD22 binding domain can be any molecule having a binding affinity and/or specificity to CD22. In some embodiments, the anti-CD22 binding domain is an antibody or an antibody derivative, such as an scFv, single domain antibody (sdAb), Fab' fragment, (Fab')2 fragment, nanobody, diabody, or the like. In some embodiments, the anti-CD22 binding domain can be a receptor or a receptor fragment that binds specifically to CD22. The anti-CD22 binding domain can be attached to the rest of the receptor directly (covalently) or indirectly (for example, through the noncovalent binding of two or more binding partners). Antibody derivatives are molecules that resemble antibodies in their mechanism of ligand binding, and include, for example, nanobodies, duobodies, diabodies, triabodies, minibodies, F(ab')2 fragments, Fab fragments, single chain variable fragments (scFv), single domain antibodies (sdAb), and functional fragments thereof. See, for example, D.L. Porter et al., N Engl J Med ( 2011) 365(8):725-33 (scFv); E.L. Smith et al., Mol Ther (2018)26(6): 1447-56 (scFv); S.R. Banihashemi et al., Iran J Basic Med Sci (2018) 21(5):455-64 (CD19 nanobody); F. Rahbarizadeh et al., Adv Drug Deliv Rev (2019) 141:41-46 (sdAb); S.M. Kipriyanov et al., Int J Cancer (1998) 77(5):763-72 (diabody); F. Le Gall et al., FEBS Lett (1999) 453(1-2): 164-68 (triabody); M.A. Ghetie et al., Blood (1994) 83(5): 1329-36 (F(ab')2); and M.A. Ghetie et al., Clin Cancer Res (1999) 5(12):3920-27 (F(ab')2 and Fab'). Antibody derivatives can also be prepared from therapeutic antibodies, for example without limitation, by preparing a nanobody, duobody, diabody, triabody, minibody, F(ab')2 fragment, Fab fragment, single chain variable fragment (scFv), or single domain antibody (sdAb) based on a therapeutic antibody. Antibody derivatives can also be identified using phage display techniques (see, e.g., E. Romao et al., Curr Pharm Des (2016) 22(43):6500-18). [0227] In some embodiments, the anti-CD22 binding domain is a single chain variable fragment (scFv). In some embodiments, the anti-CD22 scFv comprises an antibody heavy chain variable domain polypeptide (VH) covalently linked to an antibody light chain variable domain polypeptide (VL). In some embodiments, the anti-CD22 scFv further comprises a peptide linker disposed between the VH and VL polypeptides. In some embodiments, the anti-CD22 scFv comprises, from N-terminus to C-terminus, a VH polypeptide, a polypeptide linker, and a VL polypeptide. In some embodiments, the anti-CD22 scFv comprises, from N- terminus to C-terminus, a VL polypeptide, a polypeptide linker, and a VH polypeptide. In some embodiments, the polypeptide linker comprises a polypeptide having the sequence GGGGS (SEQ ID NO: 29). [0228] In some embodiments, the anti-CD22 binding domain includes a VH comprising a heavy chain CDR1 (HCDR1) sequence with at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an HCDR1 sequence in Table 1. In some embodiments, the anti- CD22 binding domain comprises a VH that comprises an HCDR1 sequence in Table 1. In some embodiments, the anti-CD22 binding domain includes a VH that includes a heavy chain CDR2 (HCDR2) sequence with at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an HCDR2 sequence in Table 1. In some embodiments, the anti-CD22 binding domain includes a VH that includes an HCDR2 sequence in Table 1. In some embodiments, the anti-CD22 binding domain includes a heavy chain variable region (VH) comprising a heavy chain CDR3 (HCDR3) sequence with at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an HCDR3 sequence in Table 1. In some embodiments, the anti- CD22 binding domain includes a VH that includes an HCDR3 sequence in Table 1. In some embodiments, the anti-CD22 binding domain includes the heavy chain CDRs 1, 2, and 3 (HCDR1, HCDR2, HCDR3) sequences as set forth in SEQ ID NOs: 4, 5, and 6, respectively. In some embodiments, the anti-CD22 binding domain includes the heavy chain HCDR1, HCDR2, HCDR3 sequences as set forth in SEQ ID NOs: 10, 11, and 12, respectively. In some embodiments, the anti-CD22 binding domain includes the heavy chain HCDR1, HCDR2, HCDR3 sequences as set forth in SEQ ID NOs: 16, 17, and 18, respectively. [0229] In some embodiments, the anti-CD22 binding domain includes a VL comprising a light chain CDR1 (LCDR1) sequence with at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an LCDR1 sequence in Table 1. In some embodiments, the anti-CD22 binding domain includes a VL comprising an LCDR1 sequence in Table 1. In some embodiments, the anti-CD22 binding domain comprises a VL that comprises a light chain CDR2 (LCDR2) sequence with at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an LCDR2 sequence in Table 1. In some embodiments, the anti-CD22 binding domain comprises a VL that comprises an LCDR2 sequence in Table 1. In some embodiments, the anti-CD22 binding domain comprises a VL that comprises a light chain CDR3 (LCDR3) sequence with at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an LCDR3 sequence in Table 1. In some embodiments, the anti-CD22 binding domain comprises a VL that comprises an LCDR3 sequence in Table 1. In some embodiments, the anti-CD22 binding domain includes the light chain CDRs 1, 2, and 3 (LCDR1, LCDR2, LCDR3) sequences as set forth in SEQ ID NOs: 7, 8, and 9, respectively. In some embodiments, the anti-CD22 binding domain includes the light chain LCDR1, LCDR2, LCDR3 sequences as set forth in SEQ ID NOs: 13, 14, and 15, respectively. In some embodiments, the anti-CD22 binding domain includes the light chain LCDR1, LCDR2, LCDR3 sequences as set forth in SEQ ID NOs: 19, 20, and 21, respectively. [0230] In some embodiments, the anti-CD22 binding domain comprises a VH with a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRS KWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIW GQGTMVTVSS (SEQ ID NO: 2). In some embodiments, the anti-CD22 binding domain comprises a VH with the sequence QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRS KWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIW GQGTMVTVSS (SEQ ID NO: 2). [0231] In some embodiments, the anti-CD22 binding domain comprises a VL with a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence DIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGV PSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIK (SEQ ID NO: 3). In some embodiments, the anti-CD20 binding domain comprises a VL with the sequence DIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGV PSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIK (SEQ ID NO: 3). [0232] In some embodiments, the anti-CD22 binding domain includes the heavy chain CDRs 1, 2, and 3 (HCDR1, HCDR2, HCDR3) sequences and the light chain CDRs 1, 2, and 3 (LCDR1, LCDR2, LCDR3) sequences as set forth in SEQ ID NOs: 4, 5, 6, 7, 8, and 9, respectively. In some embodiments, the anti-CD22 binding domain includes the heavy chain HCDR1, HCDR2, HCDR3 sequences and the light chain LCDR1, LCDR2, LCDR3 sequences as set forth in SEQ ID NOs: 10, 11, 12, 13, 14, and 15, respectively. In some embodiments, the anti-CD22 binding domain includes the heavy chain HCDR1, HCDR2, HCDR3 sequences and the light chain LCDR1, LCDR2, LCDR3 sequences as set forth in SEQ ID NOs: 16, 17, 18, 19, 20, and 21, respectively. [0233] In some embodiments, the anti-CD22 binding domain is an scFv. In some embodiments, the anti-CD22 scFv comprises a peptide linker between the VH and the VL domains. In some embodiments, the peptide linker comprises the sequence GGGGS (SEQ ID NO: 29). In some embodiments, the anti-CD22 binding domain comprises a sequence having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRS KWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIW GQGTMVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPG KAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQ GTKLEIK (SEQ ID NO: 1). In some embodiments, the anti-CD22 binding domain comprises the sequence QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRS KWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIW GQGTMVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPG KAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQ GTKLEIK (SEQ ID NO: 1). [0234] In some embodiments, the antigen binding domain is a mouse anti-human CD22 binding domain. In some embodiments, the antigen binding domain is a chimeric mouse anti- human CD22 binding domain. In some embodiments, the antigen binding domain is a humanized anti-human CD22 binding domain. In some embodiments, the antigen binding domain is a fully human anti-human CD22 binding domain. TABLE 1: Exemplary anti-CD22 scFv Constructs Constructs Amino Acid Sequence SEQ ID NO.
Figure imgf000064_0001
Constructs Amino Acid Sequence SEQ ID NO.
Figure imgf000065_0001
Constructs Amino Acid Sequence SEQ ID NO.
Figure imgf000066_0001
[0235] In some embodiments, the anti-CD22 binding domain is a humanized binding domain. For example, a humanized anti-CD22 binding domain may include heavy and light chain variable region sequences from a non-human species (e.g., a mouse) but in which at least a portion of the VH and/or VL sequence has been altered to be more “human-like”, i.e., more similar to the corresponding human sequences. One type of humanized anti-CD22 binding domain is a CDR-grafted binding domain in which human CDR sequences are introduced into non-human VH and VL sequences to replace the corresponding non-human CDR sequences. Another type of humanized antibody is a framework region (FWR)-grafted antibody in which human FWR sequences are introduced into non-human VH and VL sequences to replace corresponding non-human FWR sequences. In some embodiments, the anti-CD22 binding domain of the CD22 CARs disclosed herein is or comprises a scFv derived from a fully human anti-CD22 antibody. [0236] In some embodiments, the anti-CD22 CAR includes a hinge domain. Non-limiting examples of hinge domains suitable for the methods disclosed herein include hinge domains from LFA-1 (CD11a/CD18), LFA-2 (CD2), CD4, CD5, CD8 ^, CD8 ^, CD27 (TNFRSF7), CD28, CD70, 4-1BB (CD137), OX40 (CD134), CD152 (CTLA4), ICOS (CD278), and the IgG1 Fc region, IgG4 Fc region. In some embodiments, the hinge domain is from CD8 ^ or CD28. In some embodiments, the hinge domain is from CD28. In some embodiments, the hinge domain is from CD8 ^. In some embodiments, the anti-CD22 CAR further comprises a linker positioned between the anti-CD22 binding domain and the hinge domain. In some embodiments, the linker between the anti-CD22 binding domain and the hinge domain comprises the sequence AAA. In some embodiments, the hinge domain is from CD8 ^ and the linker includes the sequence AAA. In some embodiments, the CD8 ^ hinge domain of the anti-CD22 CAR comprises the sequence TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 24). In some embodiments, the anti-CD22 CAR further includes a transmembrane domain. In some embodiments, the transmembrane domain is from CD8 ^. In some embodiments, the CD8 ^ transmembrane domain of the anti-CD22 CAR comprises the sequence IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 25). [0237] In some embodiments, the anti-CD22 CAR comprises a peptide linker (e.g., spacer) disposed between the transmembrane domain and the intracellular signaling domain. In some embodiments, the peptide linker (e.g., spacer) comprises a portion of a CD8 ^ cytoplasmic domain. In some embodiments, the peptide linker (e.g., spacer) comprising a portion of a CD8 ^ cytoplasmic domain of the anti-CD22 CAR comprises the sequence LYC (SEQ ID NO: 31). [0238] In some embodiments, the anti-CD22 CAR includes an intracellular signaling domain. In some embodiments, the intracellular signaling domain comprises a signaling domain from an immunoreceptor tyrosine-based activation motif (“ITAM”)-containing protein and a signaling domain from a co-stimulatory protein. Non-limiting exemplary ITAM-containing proteins having ITAM-containing signaling domains suitable for use in the intracellular signaling domain of the anti-CD22 CAR used in the methods of the disclosure include the T cell receptor-associated proteins CD3 ^, CD3 ^, CD3 ^, and CD3 ^, the B cell receptor- associated proteins Ig ^ and Ig ^, the Fc ^RI ^ polypeptide, the Fc ^RI-, Fc ^RII-, and Fc ^RIII ^ polypeptide, DAP12, Dectin-1, CLEC-1, CD28, and CD27. In some embodiments, the signaling domain from an ITAM-containing protein is an ITAM-containing signaling domain from a CD3 ^ polypeptide. In some embodiments, the CD3 ^ intracellular signaling domain comprises the sequence RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR (SEQ ID NO: 27). In some embodiments, the CD3 ^ intracellular signaling domain comprises the sequence RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR (SEQ ID NO: 30) [0239] Non-limiting exemplary co-stimulatory proteins having co-stimulatory signaling domains suitable for use in the intracellular signaling domain of the anti-CD22 CAR used in the methods of the disclosure include co-stimulatory domains from 4-1BB (CD137), CD27 (TNFRSF7), CD28, OX40 (CD134), CD70, LFA-2 (CD2), CD5, ICAM-1 (CD54), LFA-1 (CD11a/CD18), DAP10, and DAP12. In some embodiments, the signaling domain from a co- stimulatory protein is a co-stimulatory signaling domain from 4-1BB (CD137). In some embodiments, the 4-1BB co-stimulatory domain includes the sequence KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 26). [0240] In some embodiments, the anti-CD22 CAR further includes a signal peptide sequence. In some embodiments, the signal peptide sequence of the anti-CD22 CAR comprises the sequence MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 28). [0241] In some embodiments of the disclosure, the anti-CD22 CAR includes (i) an anti- CD22 scFv, (ii) a CD8 ^ hinge domain, (iii) CD8 ^ transmembrane (TM) domain; (iv) a 4- 1BB co-stimulatory domain, and (v) a CD3 ^ intracellular signaling domain. In some embodiments of the disclosure, the anti-CD22 CAR includes (i) anti-CD22 scFv of SEQ ID NO: 1, (ii) a CD8 ^ hinge domain of SEQ ID NO: 24, (iii) CD8 ^ transmembrane (TM) domain of SEQ ID NO: 25; (iv) a 4-1BB co-stimulatory domain of SEQ ID NO: 26, and (v) a CD3 ^ intracellular signaling domain of SEQ ID NO: 27 or CD3 ^ intracellular signaling domain of SEQ ID NO: 30. In some embodiments of the disclosure, the anti-CD22 CAR includes (i) a signal peptide sequence, (ii) an anti-CD22 scFv, (iii) a CD8 ^ hinge domain, (iv) CD8 ^ transmembrane (TM) domain; (v) a 4-1BB co-stimulatory domain, and (vi) a CD3 ^ intracellular signaling domain. In some embodiments of the disclosure, the anti-CD22 CAR includes (i) a signal peptide sequence of SEQ ID NO: 28, (ii) an anti-CD22 scFv of SEQ ID NO: 1, (iii) a CD8 ^ hinge domain of SEQ ID NO: 24, (iv) CD8 ^ transmembrane (TM) domain of SEQ ID NO: 25; (v) a 4-1BB co-stimulatory domain of SEQ ID NO: 26, and (vi) a CD3 ^ intracellular signaling domain of SEQ ID NO: 27 or SEQ ID NO: 30. [0242] In some embodiments, anti-CD22 scFv includes an anti-CD22 VH and an anti-CD22 VL linked to one another by a linker. In some embodiments, anti-CD22 scFv includes an anti-CD22 VH of SEQ ID NO: 2 and an anti-CD22 VL of SEQ ID NO: 3 linked to one another by a linker of SEQ ID NO: 29. IMMUNE CELLS [0243] In another aspect, the methods disclosed herein are used to manufacture CAR- expressing immune cells for use as human therapeutics. In some embodiments, the immune cell is an immune system cell, e.g., a lymphocyte (for example without limitation, a T cell, natural killer cell or NK cell, natural killer T cell or NKT cell, a B cell, a plasma cell, tumor- infiltrating lymphocyte (TIL)), a monocyte or macrophage, or a dendritic cell. In some instances, the immune system cell is selected from the group consisting of B cells, T cells, monocytes, dendritic cells, and epithelial cells. In some instances, the immune system cell is a T lymphocyte. In some instances, the T cell is a CD8-positive T cell, a CD4-positive T cell, a regulatory T cell, a cytotoxic T cell, or a tumor infiltrating lymphocyte. The immune cell can also be a precursor cell, e.g., a cell that is capable of differentiating into an immune cell. In some embodiments, the immune cell is from a blood sample. In some embodiments, the immune cell is obtained by apheresis. [0244] Techniques for transducing or transforming a wide variety of the above-mentioned host cells and species with an expression vector, e.g., a lentiviral expression vector, comprising a recombinant polynucleotide acid encoding a CAR are known in the art and described in the technical and scientific literature. In some instances, the nucleic acid molecule is introduced into a host cell by transduction, or electroporation. Accordingly, in some embodiments, a sample comprising a population of immune cells is transformed with an expression vector encoding a CAR construct to produce a population of cells for use in any of the methods described herein. In some embodiments, the expression vector is a lentiviral expression vector. Lentiviral vectors and various lentiviral components useful for the production of a lentiviral vector are known in the art. For example, an expression cassette encoding a CAR construct may be incorporated into a lentiviral vector backbone, which may then be packaged using a packaging system known in the art. A number of techniques suitable for lentiviral manufacturing are also generally known in the art and described in the technical and scientific literature. In some embodiments, the lentiviral expression vector comprising an expression cassette encoding a CAR construct is manufactured using an adherent cell-based process. In some embodiments, the lentiviral expression vector comprising an expression cassette encoding a CAR construct is manufactured using a suspension cell-based process, which may help ease lentiviral manufacturing at scale with the use of stirred tank bioreactors and, in some instances, provides significantly better transduction efficiency. As described in greater detail below, in some embodiments of the disclosure, immune cells are transduced with a lentiviral expression vector encoding a CAR construct produced using an adherent cell-based lentiviral vector manufacturing platform. In some other embodiments of the disclosure, immune cells are transduced with a lentiviral expression vector encoding a CAR construct produced using a suspension cell culture-based LV manufacturing platform. [0245] In some embodiments, the CAR construct comprises a CD22 CAR. Methods and systems suitable for generating and maintaining cell cultures are known in the art. [0246] In some embodiments, the population of CAR-expressing immune cells described herein may be autologous (e.g., obtained from the same subject who will be treated with them) or allogeneic (e.g., obtained from a healthy donor and administered to multiple subjects, e.g., patients). In some embodiments, the cells are T cells obtained from a mammal. In some embodiments, the T cells obtained from a mammal are CD8-positive T cells, CD4- positive T cells, regulatory T cells (Tregs), cytotoxic T cells (CTLs), or tumor infiltrating lymphocytes (TILs). In some instances, the mammal is a primate. In some instances, the primate is a human. [0247] T cells can be obtained from a number of sources including, but not limited to, peripheral blood, peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some instances, T cells are obtained from a unit of blood collected from an individual using any number of known techniques such as sedimentation, e.g., FICOLL™ separation or apheresis. [0248] In some instances, an isolated or purified population of T cells is used. In some instances, TCTL and TH lymphocytes are purified from PBMCs. In some embodiments, the T cells are obtained directly from the patient by leukapheresis and/or apheresis. In some instances, the TCTL and TH lymphocytes are sorted into naive (TN), memory (TMEM), stem cell memory (TSCM), central memory (TCM), effector memory (TEM), and effector (TEFF) T cell subpopulations either before or after activation, expansion, and/or genetic modification. Suitable approaches for such sorting are known and include, e.g., magnetic-activated cell sorting (MACS), where TN are CD45RA+ CD62L+ CD95-; TSCM are CD45RA+CCR7+; TCM are CD45RA-CCR7+; and TEM are CCR7-CD45RA-. An exemplary approach for such sorting is described in Wang et al. (2016) Blood 127(24):2980- 90. [0249] A specific subpopulation of T cells expressing one or more of the following markers: CD3, CD4, CD8, CD28, CD45RA, CD45RO, CD62, CD127, and HLA-DR can be further isolated by positive or negative selection techniques. In some instances, a specific subpopulation of T cells, expressing one or more of the markers selected from the group consisting of CD62L, CCR7, CD28, CD27, CD122, CD127, CD197; or CD38 or CD62L, CD127, CD197, and CD38, is further isolated by positive or negative selection techniques. In some instances, the manufactured T cell compositions do not express one or more of the following markers: CD57, CD244, CD 160, PD-1, CTLA4, TIM3, and LAG3. In some instances, the manufactured T cell compositions do not substantially express one or more of the following markers: CD57, CD244, CD 160, PD-1, CTLA4, TIM3, and LAG3. [0250] In some embodiments, the cell is a mammalian cell. In some instances, the mammalian cell is a primate cell or a human cell. In some embodiments, the mammalian cell is a human cell. In some instances, the human cell is a blood cell. The cell can be a human cell. The cell can be a blood cell. In some instances, the blood cell is a lymphocyte. In some instances, the lymphocyte is a T cell. In some instances, the T cell obtained from a mammal is a CD8-positive T cell, a CD4-positive T cell, a regulatory T cell (Treg), a cytotoxic T cells (CTL), or a tumor infiltrating lymphocyte (TIL). In some instances, the cell is a population of cells. In some instances, the population of cells is a population of blood cells. The blood cells can be lymphocytes. The lymphocytes can be T cells. In some instances, the T cells are CD8- positive T cells, CD4-positive T cells, regulatory T cells (Tregs), cytotoxic T cells (CTLs), or tumor infiltrating lymphocytes (TILs). In some instances, the population of cells is a homogeneous mixture of cells of the same cell type. In some instances, the population of cells is a heterogeneous mixture of cells of different cell types. In some instances, the population of cells comprises at least about 1×103 cells. In some instances, the population of cells comprises at least about 1×104 cells. In some instances, the population of cells comprises at least about 1×105 cells. In some instances, the population of cells comprises at least about 1×106 cells. In some instances, the population of cells comprises at least about 1×107 cells. In some instances, the population of cells comprises at least about 1×108 cells. In some instances, the population of cells comprises at least about 1×109 cells. In some embodiments, the population of cells comprises from about 1×103 cells to about 1×109 cells. In some embodiments, the population of cells comprises from about 1×103 cells to about 1×108 cells. In some embodiments, the population of cells comprises from about 1×103 cells to about 1×107 cells. In some embodiments, the population of cells comprises from about 1×103 cells to about 1×106 cells. In some embodiments, the population of cells comprises from about 1×103 cells to about 1×105 cells. In some embodiments, the population of cells comprises from about 1×103 cells to about 1×104 cells. In some embodiments, the population of cells comprises from about 1×104 cells to about 1×109 cells. In some embodiments, the population of cells comprises from about 1×104 cells to about 1×108 cells. In some embodiments, the population of cells comprises from about 1×104 cells to about 1×107 cells. In some embodiments, the population of cells comprises from about 1×104 cells to about 1×106 cells. In some embodiments, the population of cells comprises from about 1×104 cells to about 1×105 cells. In some instances, the population of cells comprises from about 1×105 cells to about 1×109 cells. In some instances, the population of cells comprises from about 1×105 cells to about 1×108 cells. In some instances, the population of cells comprises from about 1×105 cells to about 1×107 cells. In some instances, the population of cells comprises from about 1×105 cells to about 1×106 cells. PHARMACEUTICAL COMPOSITIONS [0251] In some embodiments, the CAR-expressing immune cells of the disclosure can be formulated as compositions, including pharmaceutical compositions. Such compositions generally include the CAR-expressing immune cells as described herein and a pharmaceutically acceptable carrier. Accordingly, one aspect of the present disclosure is related to pharmaceutical compositions comprising a population of CAR-expressing immune cells made according to the methods described herein. [0252] The pharmaceutical compositions generally include a therapeutically effective amount of cells. By “therapeutically effective amount” is meant a number of cells sufficient to produce a desired result, e.g., an amount sufficient to effect beneficial or desired therapeutic (including preventative) results, such as a reduction in a symptom of a disease (e.g., cancer) or disorder associated, e.g., with the target cell or a population thereof (e.g., cancer cells), as compared to a control. An effective amount can be administered in one or more administrations. The amount of CAR-expressing immune cells that comprises a “therapeutically effective amount” may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the cells to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the cells are outweighed by the therapeutically beneficial effects. The term “therapeutically effective amount” includes an amount that is effective to “treat” an individual, e.g., a patient. When a therapeutic amount is indicated, the precise amount of the compositions contemplated in particular embodiments to be administered, can be determined by a physician in view of the specification and with consideration of individual differences in age, weight, tumor size, extent of infection or metastasis, and condition of the patient (individual). In some embodiments, a pharmaceutical composition of the present disclosure includes from 1×103 to 5×1010 of the cells of the present disclosure. [0253] The CAR-expressing immune cells of the present disclosure can be incorporated into a variety of formulations for therapeutic administration. More particularly, the CAR- expressing immune cells of the present disclosure can be formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable excipients or diluents. [0254] Formulations of the cells suitable for administration to a patient (e.g., suitable for human administration) are generally sterile and may further be free of detectable pyrogens or other contaminants contraindicated for administration to a patient according to a selected route of administration. [0255] The cells may be formulated for parenteral (e.g., intravenous, intra-arterial, intraosseous, intramuscular, intracerebral, intracerebroventricular, intrathecal, subcutaneous, etc.) administration, or any other suitable route of administration. [0256] Pharmaceutical compositions that include the cells of the present disclosure may be prepared by mixing the cells having the desired degree of purity with optional physiologically acceptable carriers, excipients, stabilizers, surfactants, buffers and/or tonicity agents. Acceptable carriers, excipients and/or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid, glutathione, cysteine, methionine and citric acid; preservatives (such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, or combinations thereof); amino acids such as arginine, glycine, ornithine, lysine, histidine, glutamic acid, aspartic acid, isoleucine, leucine, alanine, phenylalanine, tyrosine, tryptophan, methionine, serine, proline and combinations thereof; monosaccharides, disaccharides and other carbohydrates; low molecular weight (less than about 10 residues) polypeptides; proteins, such as gelatin or serum albumin; chelating agents such as EDTA; sugars such as trehalose, sucrose, lactose, glucose, mannose, maltose, galactose, fructose, sorbose, raffinose, glucosamine, N-methylglucosamine, galactosamine, and neuraminic acid; and/or non-ionic surfactants such as Tween, Brij Pluronics, Triton-X, or polyethylene glycol (PEG). [0257] An aqueous formulation of the recombinant polypeptides, proteases, nucleic acids, expression vectors, and/or cells may be prepared in a pH-buffered solution, e.g., at pH ranging from about 7.0 to 8.0, 4.0 to about 7.0, or from about 5.0 to about 6.0, or alternatively about 5.5. Examples of buffers that are suitable for a pH within this range include phosphate-, histidine-, citrate-, succinate-, acetate-buffers and other organic acid buffers. The buffer concentration can be from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, depending, e.g., on the buffer and the desired tonicity of the formulation. [0258] A tonicity agent may be included in the formulation to modulate the tonicity of the formulation. Exemplary tonicity agents include sodium chloride, potassium chloride, glycerin and any component from the group of amino acids, sugars as well as combinations thereof. In some embodiments, the aqueous formulation is isotonic, although hypertonic or hypotonic solutions may be suitable. The term “isotonic” denotes a solution having the same tonicity as some other solution with which it is compared, such as physiological salt solution or serum. Tonicity agents may be used in an amount of about 5 mM to about 350 mM, e.g., in an amount of 100 mM to 350 mM. [0259] In some embodiments, a surfactant may also be added to the formulation to reduce aggregation and/or minimize the formation of particulates in the formulation and/or reduce adsorption. Example surfactants include polyoxyethylensorbitan fatty acid esters (Tween), polyoxyethylene alkyl ethers (Brij), alkylphenylpolyoxyethylene ethers (Triton-X), polyoxyethylene- polyoxypropylene copolymer (Poloxamer, Pluronic), and sodium dodecyl sulfate (SDS). Examples of suitable polyoxyethylenesorbitan-fatty acid esters are polysorbate 20, (sold under the trademark Tween 20™) and polysorbate 80 (sold under the trademark Tween 80™). Examples of suitable polyethylene-polypropylene copolymers are those sold under the names Pluronic® F68 or Poloxamer 188™. Examples of suitable Polyoxyethylene alkyl ethers are those sold under the trademark Brij™. Exemplary concentrations of surfactant may range from about 0.001% to about 1% w/v. [0260] In some instances, the pharmaceutical composition includes CAR-expressing immune cells of the present disclosure, and one or more of the above-identified agents (e.g., a surfactant, a buffer, a stabilizer, a tonicity agent) and is essentially free of one or more preservatives, such as ethanol, benzyl alcohol, phenol, m-cresol, p-chlor-m-cresol, methyl or propyl parabens, benzalkonium chloride, and combinations thereof. In other embodiments, a preservative is included in the formulation, e.g., at concentrations ranging from about 0.001 to about 2% (w/v). In some embodiments, the pharmaceutical composition comprises the harvested fifth population of cells comprising CAR-expressing immune cells resuspended to the desired concentration in Final Formulation Medium comprising Plasma-Lyte A+4% (w/v) HSA, diluted 1:1 with CryostorR CS10 and frozen. METHODS OF TREATMENT
Figure imgf000075_0001
any one of the therapeutic compositions described herein, e.g., a pharmaceutical composition comprising a therapeutically effective amount of CAR- expressing immune cells, can be used in the prevention and/or treatment of relevant health conditions, such as proliferative diseases (e.g., cancer). Further disclosed here include methods for preventing and/or treating a disease, disorder, or health condition in a subject in need thereof, comprising administering a therapeutically effective amount of a pharmaceutical composition comprising a therapeutically effective amount of CAR- expressing immune cells as disclosed herein, wherein the pharmaceutical composition comprising a therapeutically effective amount of CAR-expressing immune cells is administered to the subject individually as a single therapy (monotherapy) or as a first therapy in combination with at least one additional therapies (e.g., second therapy). In some embodiments, the health condition is a cancer, e.g., a hematologic cancer. In some embodiments, the administered CAR-expressing immune cells or the pharmaceutical composition is administered to or provide anti-tumor immunity to the subject. In some embodiments, the first therapy and the second therapy are administered concomitantly. In some embodiments, the first therapy is administered at the same time as the second therapy. In some embodiments, the first therapy and the second therapy are administered sequentially. In some embodiments, the first therapy is administered before the second therapy. In some embodiments, the first therapy is administered after the second therapy. In some embodiments, the first therapy is administered before and/or after the second therapy. In some embodiments, the first therapy and the second therapy are administered in rotation. In some embodiments, the first therapy and the second therapy are administered together in a single formulation. [0262] The CAR-expressing immune cells and pharmaceutical compositions of the disclosure can be administered alone or in combination with other agents (e.g., an antibody or an antigen binding fragment thereof, or a molecule). In some embodiments, a vaccine, an oncoloytic virus, a checkpoint inhibitor, a T cell agonist antibody, chemotherapy, and/or a bispecific antibody can be combined with the pharmaceutical composition disclosed herein. In some instances, the pharmaceutical composition is administered with other cells (e.g., CAR T cells or other adoptively transferred T cells). Administration “in combination with” one or more additional therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order. In some embodiments, the one or more additional therapeutic agents, chemotherapeutics, anti-cancer agents, or anti-cancer therapies is selected from the group consisting of chemotherapy, radiotherapy, immunotherapy, hormonal therapy, toxin therapy, and surgery. “Chemotherapy” and “anti-cancer agent” are used interchangeably herein. Various classes of anti-cancer agents can be used. Non-limiting examples of anti- cancer agents include: alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, podophyllotoxin, antibodies (e.g., monoclonal or polyclonal), checkpoint inhibitors, immunomodulators, cytokines, nanoparticles, radiation therapy, tyrosine kinase inhibitors (for example, imatinib mesylate), hormone treatments, soluble receptors and other antineoplastics. [0263] In some instances, the disease, disorder, or condition is a cancer, an inflammatory disease, a neuronal disorder, HIV/AIDS, diabetes, a cardiovascular disease, an infectious disease, or an autoimmune disease. In some instances, the disease, disorder, or condition is cancer. In some instances, the cancer is lymphoma or leukemia. In some instances, the disease, disorder, or condition is a hyperproliferative disorder. Hyperproliferative disorders include cancers and hyperplasia characterized by the unregulated overgrowth of cells. Hyperproliferative disorders frequently display loss of genetic regulatory mechanisms and may express native proteins inappropriately (including expression of proteins from other cell types or developmental stages, expression of mutated proteins, and expression of proteins at levels higher or lower than normal). [0264] B-cell hyperproliferative disorders include B-cell leukemias and lymphomas such as, but not limited to, acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), B-cell prolymphocytic leukemia, precursor B lymphoblastic leukemia, hairy cell leukemia, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, marginal zone lymphoma, mantle cell lymphoma, Burkitt’s lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, Waldenstrom’s macroglobulinemia, and/or other disorders characterized by the overgrowth of B-lineage cells. [0265] In some embodiments, the B-cell hyperproliferative disorder is a lymphoma. In some embodiments, the lymphoma is selected from a group consisting of diffuse large B cell lymphoma (DLBCL), large B cell lymphoma (LBCL), mantle cell lymphoma (MCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), Burkitt’s lymphoma, anaplastic large-cell lymphoma, angioimmunoblastic T cell lymphoma, and Hodgkin lymphoma. In some embodiments, the lymphoma is large B cell lymphoma. In some embodiments, the B- cell hyperproliferative disorder is a leukemia. In some embodiments, the leukemia is selected from a group consisting of acute lymphocytic leukemia (ALL), acute lymphoblastic leukemia (ALL), B cell acute lymphocytic leukemia (B-ALL), B cell acute lymphoblastic leukemia (B- ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), B cell prolymphocytic leukemia (B-PLL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), chronic myelomonocytic leukemia (CMML), hairy cell leukemia (HCL), juvenile myelomonocytic leukemia (JMML), large granular lymphocytic leukemia (LGLL), and T cell prolymphocytic leukemia (T-PLL). In some embodiments, the leukemia is ALL or B-ALL. In some embodiments, the leukemia is pediatric ALL or B- ALL. [0266] Hyperproliferative disorders include diseases such as, but not limited to, bladder cancer, including upper tract tumors and urothelial carcinoma of the prostate; bone cancer, including chondrosarcoma, Ewing's sarcoma, and osteosarcoma; breast cancer, including noninvasive, invasive, phyllodes tumor, Paget's disease, and breast cancer during pregnancy; central nervous system cancers, adult low-grade infiltrative supratentorial astrocytoma/oligodendroglioma, adult intracranial ependymoma, anaplastic astrocytoma/anaplastic oligodendroglioma/glioblastoma multiforme, carcinomatous lymphomatous meningitis, non-immunosuppressed primary CNS lymphoma, and metastatic spine tumors; cervical cancer; colon cancer, rectal cancer, anal carcinoma; esophageal cancer; gastric (stomach) cancer; head and neck cancers, including ethmoid sinus tumors, maxillary sinus tumors, salivary gland tumors, cancer of the lip, cancer of the oral cavity, cancer of the oropharynx, cancer of the hypopharynx, occult primary, cancer of the glottic larynx, cancer of the supraglottic larynx, cancer of the nasopharynx, and advanced head and neck cancer; hepatobiliary cancers, including hepatocellular carcinoma, gallbladder cancer, intrahepatic cholangiocarcinoma, and extrahepatic cholangiocarcinoma; Hodgkin disease/lymphoma; kidney cancer; melanoma; multiple myeloma, systemic light chain amyloidosis, Waldenstrom's macroglobulinemia; myelodysplastic syndromes; neuroendocrine tumors, including multiple endocrine neoplasia, type 1, multiple endocrine neoplasia, type 2, carcinoid tumors, islet cell tumors, pheochromocytoma, poorly differentiated/small cell/atypical lung carcinoids; Non-Hodgkin's Lymphomas, including chronic lymphocytic leukemia/small lymphocytic lymphoma, follicular lymphoma, marginal zone lymphoma, mantle cell lymphoma, diffuse large B-Cell lymphoma, Burkitt's lymphoma, lymphoblastic lymphoma, AIDS-Related B-Cell lymphoma, peripheral T Cell lymphoma, and mycosis fungoides/Sëzary Syndrome; non-melanoma skin cancers, including basal and squamous cell skin cancers, dermatofibrosarcoma protuberans, Merkel cell carcinoma; non-small cell lung cancer (NSCLC), including thymic malignancies; occult primary; ovarian cancer, including epithelial ovarian cancer, borderline epithelial ovarian cancer (Low Malignant Potential), and less common ovarian histologies; pancreatic adenocarcinoma; prostate cancer; small cell lung cancer and lung neuroendocrine tumors; soft tissue sarcoma, including soft-tissue extremity, retroperitoneal, intra-abdominal sarcoma, and desmoid; testicular cancer; thymic malignancies, including thyroid carcinoma, nodule evaluation, papillary carcinoma, follicular carcinoma, Hürthle cell neoplasm, medullary carcinoma, and anaplastic carcinoma; uterine neoplasms, including endometrial cancer and/or uterine sarcoma. Administration of pharmaceutical compositions comprising CAR-expressing immune cells to a subject [0267] Methods for administering pharmaceutical compositions comprising CAR-expressing immune cells for the treatment of cancer, e.g., hematologica cancers, are known and may be used in connection with the provided methods and compositions. For example, adoptive T cell therapy methods are described in US 2003/0170238; US 4690915; S.A. Rosenberg, Nat Rev Clin Oncol (2011) 8(10):577-85. See also M. Themeli et al., Nat Biotechnol (2013) 31(10):928-33; and T. Tsukahara et al., Biochem Biophys Res Commun (2013) 438(l):84-89. [0268] In some embodiments, this administering step can be accomplished using any method of delivery known in the art. For example, the CAR-expressing immune cells can be infused intravenously directly into the subject’s bloodstream or otherwise administered to the subject. [0269] The step of administering, which term is used interchangeably with the terms “introducing,” implanting,” and “transplanting,” CAR-expressing immune cells into an individual, by a method or route such that a desired effect(s) is/are produced. The CAR- expressing immune cells, or their differentiated progeny can be administered by any appropriate route for the disease being treated that results in at least a portion of the administered cells or components of the cells remaining viable. The period of viability of the cells after administration to a subject can be as short as a few hours, e.g., twenty-four hours, to a few days, to as long as several years, or even the lifetime of the individual, e.g., long-term engraftment. [0270] When provided prophylactically, the CAR-expressing immune cells described herein can be administered to a subject in advance of any symptom of a disease or health condition to be treated. Accordingly, in some embodiments the prophylactic administration of a pharmaceutical composition comprising a population of CAR-expressing immune cells prevents the occurrence of symptoms of the disease or health condition. [0271] When provided therapeutically in some embodiments, CAR-expressing immune cells are provided at (or after) the onset of a symptom or indication of a disease or health condition, e.g., upon the onset of disease or health condition. [0272] For use in the various embodiments, e.g., the pharmaceutical compositions, described herein, a therapeutically effective amount of CAR-expressing immune cells, e.g., T cells, as disclosed herein, can be at least 102 cells, at least 5 × 102 cells, at least 103 cells, at least 5 × 103 cells, at least 104 cells, at least 5 × 104 cells, at least 105 cells, at least 2 × 105 cells, at least 3 × 105 cells, at least 4 × 105 cells, at least 5 × 105 cells, at least 6 × 105 cells, at least 7 × 105 cells, at least 8 × 105 cells, at least 9 × 105 cells, at least 1 × 106 cells, at least 2 × 106 cells, at least 3 × 106 cells, at least 4 × 106 cells, at least 5 × 106 cells, at least 6 × 106 cells, at least 7 × 106 cells, at least 8 × 106 cells, at least 9 × 106 cells, or multiples thereof. [0273] In some embodiments, the pharmaceutical compositions include a therapeutically effective amount of CD22 CAR T cells as disclosed herein, which can be at least 102 cells, at least 5 × 102 cells, at least 103 cells, at least 5 × 103 cells, at least 104 cells, at least 5 × 104 cells, at least 105 cells, at least 2 × 105 cells, at least 3 × 105 cells, at least 4 × 105 cells, at least 5 × 105 cells, at least 6 × 105 cells, at least 7 × 105 cells, at least 8 × 105 cells, at least 9 × 105 cells, at least 1 × 106 cells, at least 2 × 106 cells, at least 3 × 106 cells, at least 4 × 106 cells, at least 5 × 106 cells, at least 6 × 106 cells, at least 7 × 106 cells, at least 8 × 106 cells, at least 9 × 106 cells, or multiples thereof. [0274] In some embodiments, the pharmaceutical compositions include a therapeutically effective amount of CD22 CAR T cells as disclosed herein, which may be about 1 × 106 cells/kg or 1 × 106 cells/kg. The maximum dose will be fixed at the dose for a subject of 100 kg for any subjects weighing >100 kg at time of treatment. [0275] A pharmaceutical composition comprising a therapeutically effective amount of CAR-expressing immune cells can be administered by any appropriate route that results in effective treatment in the subject, e.g., administration results in delivery to a desired location in the subject where at least a portion of the composition delivered, e.g., at least 1 × 105 cells, at least 105 cells, at least 2 × 105 cells, at least 3 × 105 cells, at least 4 × 105 cells, at least 5 × 105 cells, at least 6 × 105 cells, at least 7 × 105 cells, at least 8 × 105 cells, at least 9 × 105 cells, at least 1 × 106 cells, at least 2 × 106 cells, at least 3 × 106 cells, at least 4 × 106 cells, at least 5 × 106 cells, at least 6 × 106 cells, at least 7 × 106 cells, at least 8 × 106 cells, or at least 9 × 106 cells, is delivered to the desired site for a period of time. For the delivery of cells, delivery by injection or intravenous infusion is often considered a standard mode of administration. [0276] In some embodiments, the CAR-expressing immune cells are administered systemically, e.g., via intravenous infusion or injection. [0277] The efficacy of a treatment including any of the compositions provided herein for the prevention or treatment of a disease or health condition can be determined by a skilled clinician. However, one skilled in the art will appreciate that a prevention or treatment is considered effective if any one or all of the signs or symptoms or markers of disease are improved or ameliorated. Efficacy can also be measured by failure of a subject to worsen as assessed by decreased hospitalization or need for medical interventions (e.g., progression of the disease is halted or at least slowed). Methods of measuring these indicators are known to those of skill in the art and/or described herein. Treatment includes any treatment of a disease in a subject or an animal (some non-limiting examples include a human, or a mammal) and includes: (1) inhibiting the disease, e.g., arresting, or slowing the progression of symptoms; or (2) relieving the disease, e.g., causing regression of symptoms, slowing tumor growth or otherwise reducing tumor burden; and (3) preventing or reducing the likelihood of the development of symptoms. [0278] Measurement of the degree of efficacy is based on parameters selected with regard to the disease being treated and the symptoms experienced. In general, a parameter is selected that is known or accepted as correlating with the degree or severity of the disease, such as a parameter accepted or used in the medical community. For example, in the treatment of a solid cancer, suitable parameters can include reduction in the number and/or size of metastases, number of months of progression-free survival, overall survival, stage or grade of the disease, the rate of disease progression, the reduction in diagnostic biomarkers (for example without limitation, a reduction in circulating tumor DNA or RNA, a reduction in circulating cell-free tumor DNA or RNA, and the like), and combinations thereof. It will be understood that the effective dose and the degree of efficacy will generally be determined with relation to a single subject and/or a group or population of subjects. Therapeutic methods of the disclosure reduce symptoms and/or disease severity and/or disease biomarkers by at least about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100% as compared to a reference subject that has not been administered with the CAR-expressing immune cells of the disclosure. [0279] As discussed above, a therapeutically effective amount of a pharmaceutical composition can be an amount of the pharmaceutical composition that is sufficient to promote a particular beneficial effect when administered to a subject, such as one who has, is suspected of having, or is at risk for a disease or health condition. In some embodiments, an effective amount includes an amount sufficient to prevent or delay the development of a symptom of the disease or health condition, alter the course of a symptom of the disease or health condition (for example but not limited to, slow the progression of a symptom of the disease), or reverse a symptom of the disease or health condition. EXAMPLES [0280] The practice of the present disclosure will employ, unless otherwise indicated, techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are known to those skilled in the art. Such techniques are explained fully in the literature cited herein. [0281] Additional embodiments are disclosed in further detail in the following examples, which are provided by way of illustration and are not in any way intended to limit the scope of this disclosure or the claims. [0282] Examples 1-18 below describe, inter alia, two different protocols of manufacturing T cells expressing a CD22 CAR (i.e., a CD22-specific CAR) in accordance with some non- limiting embodiments of the disclosure. In Process 1 (Protocol v1), the lentiviral vector encoding CD22 CAR was generated in an adherent cell-based lentiviral vector manufacturing process. In Protocol v1, the step of processing and cryopreserving the samples comprising immune cells obtained from human subject is optional (see, e.g., Example 1). In Process 2 (Protocol v2), the lentiviral vector encoding CD22 CAR was generated in an suspension cell- based-based lentiviral vector manufacturing process, which produces a much higher concentration of vector as compared to the adherent cell-based manufacturing process in Process 1. In Protocol v2, the step of processing and cryopreserving the samples comprising immune cells obtained from human subject is mandatory. Protocol 2 also includes an expansion step where the cell media undergoes one or more media exchange steps, wherein a portion of the culture volume was removed and replaced with new media between Day 4 and Day 6 after seeding (see, e.g., Example 2 and 5). EXAMPLE 1 Manufacturing CD22 CAR T cells (protocol v1) [0283] In this Example, a population of CD22 CAR-T cells was generated from a blood sample collected from a patient using a protocol for manufacturing CD22 CAR T cells, termed the v1 protocol, as illustrated in Figure 1A. [0284] The patient leukapheresis (also referred to interchangeably as “apheresis”) sample was collected by standard protocol, and CD4+ and CD8+ T-cell enrichment was performed using beads that enrich for CD4+ and CD8+ cells. Post-enrichment cells were washed and resuspended in a buffer in a process shown in Figure 2A. A subset of the cells (1×108 cells) was seeded. On Day 1 after seeding, transduction was performed by adding a solution of lentiviral vector encoding CRG-022, a CD22-specific CAR, to the cell sample to produce CAR-expressing cells. After transduction, the culture was expanded. The media used in the seeding step was the same media for expansion, e.g., TexMACSTM culture medium supplemented with appropriate cytokines (IL-7 and IL-15). In these studies, the lentiviral vector was generated using an adherent cell-based lentiviral vector manufacturing process. As shown in Figure 12, cells were subsequently washed and actively shaken on Day 3. On Day 6, 125mL of buffer was removed and 125mL of new buffer was added. The transduction efficiency on Day 6 was analyzed. Cells were harvested on Day 7 or later; harvesting consists of washing, concentration, and manual fill and finish steps. The cells were filled in CryoMACS 50 bags and QC vials. The packaged cells were stored at -150° Celsius or colder. EXAMPLE 2 Manufacturing CD22 CAR T cells (protocol v2) [0285] In this Example, a population of CD22 CAR-T cells was generated from a blood sample collected from a patient using a protocol for manufacturing CD22 CAR T cells, termed the v2 protocol as illustrated in Figure 1B. [0286] A patient apheresis sample was collected by standard protocol and used to manufacture a CD22 CAR-T cell population. The cells were filtered (e.g., concentrated the sample into a smaller volume), washed and cryopreserved prior to enrichment using beads. Briefly, the washing and cryopreservation steps were performed on an automated cell processing instrument. The details of this process are shown in Figure 2B. Apheresis samples were diluted in Plasma-Lyte A and 4% (w/v) human serum albumin (HSA). The diluted samples were then washed, concentrated, eluted/resuspended, and filled in a FINIA 250 tubing set with Cryostor 10 buffer. Cells were cryopreserved in a controlled rate freezer until use. Cells were then thawed using a dry heating device at 37°C, after which cells were enriched for CD4+ T cells and CD8+ T cells using beads that bind to these corresponding targets. A subset of the enriched cells (2×108 cells) were activated by cytokines IL-7 and IL- 15 and seeded on Day 0. On Day 1 after seeding, transduction was performed by adding a solution of lentiviral vector encoding CRG-022, a CD22-specific CAR, was added to the cell sample to produce CAR-expressing cells. After transduction, the culture was expanded. The media used in the seeding step was the same media for expansion, e.g., TexMACSTM culture medium supplemented with appropriate cytokines (IL-7 and IL-15). In these studies, the lentiviral vector encoding CRG-022 was generated in a suspension cell-based lentiviral vector manufacturing process. As shown in Figure 11, cells were subsequently washed and actively shaken on Day 3. On Day 4, 130mL of buffer was removed and 180mL of new buffer was added. Cells were harvested between Day 5 and Day 9 after seeding, at which point the cells were washed, concentrated, and automatically filled and finished in CryoMACS 50 bags and QC vials. The packaged cells were stored at -150° Celsius or colder. EXAMPLE 3 Optimization of platelet removal prior to cryopreservation [0287] Three different methods for processing apheresis product for cryopreservation were compared (Fig.2A-2C). The first method, used in the v1 cell manufacturing protocol described in Example 1, comprised a workflow using a Prodigy concentration instrument, wherein cells were loaded onto the instrument, washed, reformulated, harvested and subsequently frozen (Fig.2A). The second method, used in the v2 cell manufacturing protocol described in Example 2, comprised a workflow using a Rotea instrument, wherein cells were diluted prior to loading on an instrument, cells were loaded onto the instrument, washed, concentrated, eluted/resuspended and subsequently frozen (Fig.2B). The third method comprised a centrifugation step, wherein cells were loaded into conical tubes, centrifuged at 1936 ^g for 15 minutes, and the platelet layer manually removed after centrifugation. Subsequently, the sample was diluted with buffer, and frozen (Fig.2C). For each of the three methods, healthy donor derived leukapheresis samples were used. [0288] After processing, cell viability, cell recovery and residual platelet levels were measured using methods described herein. Residual platelet levels were measured by the Sysmex instrument. The results for each of these metrics for each method are shown in Figure 3. The number of input cells is listed in Figures 3A-3C. As shown in Figures 3D and 3E, white blood cell recovery and viability post-processing were similar across the three methods. In contrast, however, a substantial difference was observed in the level of residual platelet levels detected in each of samples analyzed after processing. As shown in Figures 3F and 3G, the residual platelet percentage in the second method (labeled as Rotea v2) was below 10%, whereas the third method resulted in a residual platelet level of approximately 80% and the first method (labeled Prodigy) resulted in a residual platelet level of over 40%. Lower platelet levels improve efficiency of subsequent T cell enrichment steps, because the presence of platelets can lead to aggregation of cells, reducing the efficiency of the enrichment process. [0289] Blood samples collected from two donors were processed and frozen according to the methods described in Examples 1 and 2. The frozen samples were thawed, and cell viability was measured according to the methods described in Example 12. As shown in Figure 4, the total cell counts and percent cell viability before processing (post-thaw) and after processing (pre-freeze) were similar across each of the methods. Recovery levels after processing were also calculated. For each of the donor samples, the level of recovery was highest with the v2 method, with a recovery of 95% for the second donor sample. Figure 5A showed that after centrifugation residual platelet levels was down to 6%, while cell viability and cell recovery remained above 95%. Figure 5B showed that post-thaw the cell viability and cell recovery were above 70%. [0290] Cells were analyzed before (thawed cryoapheresis) and after CD4+/CD8+ enrichment (enriched fraction) by flow cytometry. The sample analyzed before enrichment was designated the “thawed cryoapheresis” sample. The sample analyzed after enrichment was designated the “enriched fraction” sample. As shown in Figure 6A, the enriched fraction contained 94% CD3+CD4+ and CD3+CD8+ cells, compared to just 50% in the thawed cyroapheresis samples. The enriched fraction also had a higher percentage of viable cells (93.3%) compared to the thawed cryoapheresis samples (83.2%). [0291] Overall, the second method (the Rotea method) used in the v2 manufacturing protocol generated a cell population with substantially lower residual platelets compared to the first method used in the v1 manufacturing protocol (the Prodigy method). The cryopreservation apheresis preparation using v2 method also yielded similar or higher levels of viability and recovery compared to the other two methods. EXAMPLE 4 Cell seeding evaluation [0292] Blood samples from donors were processed according to the methods described in Example 3 and transduced with lentiviral vectors. Thereafter, a population of the transfected cells were used to seed growth to generate a larger population of CAR-expressing cells. Two different seed quantities were tested: 100×106 and 200×106. Cells were expanded and generated at two different manufacturing sites using the manufacturing protocols described in Example 1 and Example 2. Cells were harvested on Day 7 and analyzed to compare cell characteristics between samples initially seeded with different CAR T quantities. As shown in Figure 7, the overall number of viable cells was comparable between the two seeding conditions, but the yield of CAR+ cells was slightly higher in the 200×106 condition compared to the 100×106 condition. Both the percentage of cell viability on Day 7 and the percentage contraction on Day 1 were similar between the two seed conditions (Figure 8). The post-harvest transduction efficiency on D7 was slightly higher in the 200×106 condition compared to the 100×106 condition, as measured by the % CD3+/CAR+ cells. [0293] These results indicated that seeding with 200×106 cells increased CAR+ yield. EXAMPLE 5 Media exchange protocol optimization [0294] The CD22 CAR-T manufacturing processes described herein comprised an expansion step, where activated, transfected CAR-T cells were cultured in growth medium for multiple days after seeding. The cells were grown in suspension. During this growth period, the cell media might be supplemented with additional volume and/or exchanged, wherein volume was removed and replaced with new media between Day 4 and Day 6 after seeding. Six different media exchange protocols (G-Rex 1, G-Rex 2, G-Rex 3, G-Rex 4, G-Rex 5, G-Rex 6) were tested to determine the optimal media exchange protocol. The details of each of the conditions are listed in Figure 9. For each condition, cells were seeded on Day 0 and harvested for analysis on both Day 5 and Day 7 after seeding. As shown in Figure 10A, the total viable cell yield on Day 7 was slightly higher for conditions that incorporated media exchange steps on Day 4. Figure 10B showed that the cell viability and transduction efficiencies were similar for all conditions on Day 5 and Day 7. Figure 10C showed that yield of CAR expressing (CAR+) cells was higher for samples that incorporated Day 4 washes. Figure 10D shows that the cell phenotype, as assessed by flow cytometry measuring CD3+CD4+ cells and CD3+C8+ cells was similar for all conditions. [0295] The data from the six tested conditions was used to inform the protocol for a scaled media exchange screen. Two different protocols were tested: the media exchange protocol used in the v2 cell manufacturing protocol (Figure 11) and the media exchange protocol used in the v1 cell manufacturing protocol (Figure 12). CAR-expression products were prepared according to the v1 or v2 protocol described in examples 1 and 2. Cells were harvested on Days 3-11 and analyzed for population doubling, viability and CAR expression (CAR+). As shown in Figure 13A, the cumulative population doubling was higher for the v2 protocol on Days 5-11 compared with the v1 protocol. As shown in Figure 13B, the v2 protocol showed cell viability percentages between the two samples were similar and the v2 protocol had slightly higher cell viability on Day 6 and 7 compared with the v1 protocol. Figure 13C showed that transduction efficiency between samples collected on different days for the two different protocols were similar. [0296] Overall, the data suggested that media exchange on Day 4 increased yield and resulted in comparable cell viability and transduction efficiency. EXAMPLE 6 T cell harvesting time optimization [0297] CD22-specific CAR T cells were prepared according to the v1 or v2 protocol described in Examples 1 and 2. Cells were harvested between Day 5 and 9. Population doubling time and transduction efficiency were measured. Samples with the highest population doublings and highest transduction efficiencies enable the harvesting of cells earlier in the process. As shown in Figure 14, v2 product harvested on Day 5 achieved similar number of population doublings and transduction efficiency as the v1.0 product harvested on Day 6 and v2 product harvested on Day 7. The earlier harvesting date was likely due to the optimized seeding density and media exchange strategy. [0298] CD22 CAR-T products were generated for two healthy donors (4224BW and 4267BW) and a patient sample (CCT-5029-047) according to the v2 method described in Example 2. CD22 CAR-T products were harvested on Day 4, 5, 7, or 9. Total CAR+ yield, cumulative population doublings, viability, and transduction efficiencies were measured. Figure 15 and Figure 16A-C show that CAR+ T cell yield in the cells harvested on Day 5 met the final dose fill requirement for both healthy donor and patient. EXAMPLE 7 Characterization of T cell subtypes [0299] Six CAR-T products were prepared according to the v1 or v2 cell manufacturing protocol described in Examples 1 and 2. T cell memory phenotypes were analyzed using flow cytometry analysis based on the gating scheme described in Figure 38. An equivalence test with matched-pairs Two One-sided Test (TOST) was used to compare the results from the v1 and v2 protocol with respect to the production of certain T cell memory phenotypes. As shown in Figure 18, a more juvenile, less differentiated phenotype (with increased TSCM and TCM, and decreased TEM) for the v2 protocol was produced compared to the v1 protocol. EXAMPLE 8 Characterization of T-cell exhaustion and activation [0300] Six preparations of CAR-T products were prepared according to the v1 or v2 protocol described in Examples 1 and 2. T-cell exhaustion and activation phenotypes were analyzed using a flow cytometry analysis measuring 4-1BB, CD69 and PD-1 expression (Figure 39), to assess the quality of the starting apheresis product. Since apheresis are collected taken from sick patients that have already been treated with other B cell-specific immunotherapies, their T cells are often poor quality and already close to exhausted. Therefore, measuring 4- 1BB, CD69 and PD-1 expression is a well-accepted methodology to evaluate the starting material for manufacturing. [0301] An equivalence test with matched-pairs TOST was used to compare the results from the v1 and v2 protocol related to expression of markers of T cell activation/exhaustion. Figure 19 showed higher levels of T cell activation/exhaustion markers were observed in cells generated by the v2 protocol, where levels of 4-1BB, CD69 and PD-1 were all increased in the drug product made with the v2 protocol as compared to the v1 protocol. EXAMPLE 9 In vivo tumor clearance study [0302] A cohort of 17 NSG (NOD.Cg-Prkdc scid Il2rgamma tm1Wjl/SzJ) mice were used to analyze in vivo tumor clearance using the CD22 CAR-T cell drug product described herein. All mice were infused with 1×106 Nalm6 tumor cells. Nalm6 is a B cell precursor leukemia cell line used as a xenograft model of acute lymphoblastic leukemia. Four days later, the mice were infused with CD22 CAR+ cells, un-transduced T cell controls or no cells according to the workflow scheme shown in Figure 20. Ten mice in each group received CD22 CAR-T cells generated using the v2 protocol and harvested on Day 5, Day 7 or Day 9, respectively, and ten mice received CAR-T cells generated using the v1 protocol and harvested on Day 7. For each protocol and time point, two different dosing schemes were used: five mice were infused with a low dose (1×106 cells) and five mice were infused with a high dose (5×106 cells). Total flux readings were obtained by bioluminescence imaging as a measure of tumor growth. As shown in Figure 21, mice treated with T cells generated by harvesting on D5 using the v2 cell manufacturing protocol continued to limit tumor burden out to Day 53 post injection. In contrast, tumor progression was much faster in other groups. Tumors in both the mice infused with un-transduced cells and the mice with tumor only reached termination size on Day 12, and tumors in mice treated with CD22 CAR+ T cells generated using the v1 protocol harvested on D7 and using the v2 protocol but harvested on D7 or D9 reached termination size around Day 53. Figure 22 shows that more than 50% survival was observed at the end of study in mice treated with the low dose of CD22 CAR-T cells generated by harvesting on D7 using the v1 cell manufacturing protocol and CD22 CAR-T cells generated by harvesting on D5 using the v2 cell manufacturing protocol. More than 50% survival was observed at the end of study in mice treated with the high dose of CAR-T cells generated by harvesting on D5, D7 and D9 using the v2 cell manufacturing protocol, but less than 50% survival was observed at the end of study in mice treated with the high dose of CAR-T cells generated by harvesting on D7 using the v1 cell manufacturing protocol. Body weight was maintained throughout the study because, in these studies, tumor growth did not appear to significantly change body weight. [0303] Peripheral mouse blood was sampled at various timepoints during the course of the experiment from each animal. Blood cells were analyzed by flow cytometry to detect CD3+/CD19- cells, CD3+/CD19-/CD22CAR+ cells, CD3+/CD19-/CD22CAR+CD4+ cells, and CD3+/CD19-/CD22CAR+CD8+ cells. Additionally, VCN per CD22 CAR+ per cell was analyzed by measuring the number of UC462 copies. As shown in Figure 23, the mice treated with the CAR-T cells generated by the v2 cell manufacturing protocol and harvested on Day 5 resulted in the highest levels of detected CD3+ T cells, CD22 CAR+ T cells, CD3+/CD19-/CD22CAR+ cells, and CD3+/CD19-/CD22CAR+/CD4+ cells in the blood compared to the other CAR-T cell drug products. Additionally, the mice treated with the high dose of the CAR-T cells generated by the v2 protocol and harvested on Day 5 resulted in a much higher VCN on Day 28 compared to any other analyzed data point. [0304] CD22 drug products were also manufactured from excess patient apheresis for two patients (CCT-5029-050 and CCT-5029-047) using the v1 and v2 cell manufacturing protocols described herein. Cell viability %, number of viable cells/mL and population doublings over time were measured (Figure 24). Both patient-derived CAR-T drug products yielded high % viability when harvested on Day 5 and onward. Additionally, the generated cell density was similar for both samples. Cell samples were harvested on Day 5, Day 7 and Day 9 for the v2 protocol and Day 7 for the v1 protocol. Cells were analyzed using the IFNgamma (IFN ^) ELISpot assay described previously (Figure 25). CAR-T cells generated from the CCT-5029-050 sample showed similar IFNgamma (IFN ^) secretion, whereas cells generated from the CCT-5029-047 sample showed higher IFNgamma (IFN ^) secretion by CAR-T cells generated using v2 cell manufacturing protocol. CAR-T cells for each time point and protocol were also analyzed using flow cytometry to assess the frequency of different T cell activation and exhaustion markers using the flow cytometry protocol previously described. Figure 26 showed that CAR-T cells generated using the v2 protocol and harvested on Day 5 had higher levels of CD3+CAR+PD1+ and CD3+CAR+TIGIT+ cells and therefore showed higher T-cell activation in the population compared to CAR-T cells generated using the v1 protocol and CAR-T cells generated using the v2 protocol and harvested on D7 or D9. [0305] Transduction efficiencies and total CAR+ yield were analyzed for each of the process Days 5, 7 and 9. Figure 27 showed that transduction efficiency was similar in CAR-T cells harvested at different time points, and the total CAR+ yield increased as cells were harvested later. EXAMPLE 10 v1 and v2 products equivalency [0306] A comparability experiment was performed between products generated by the v1 and v2 protocols. The experiment used a prospective side-by-side (matched pairs) evaluation of the drug product from 6 split batches of healthy donor apheresis collections. This number of split paired healthy donor runs was determined to ensure the study was powered sufficiently based on equivalence acceptance criteria. Apheresis was collected from normal peripheral blood of healthy donors and was split approximately equally into six samples. CAR-T products were generated using the v1 or v2 cell manufacturing protocol. Cells were harvested and then evaluated for cell viability, CD3 expression, transduction efficiency and vector copy number using the protocols described herein. An equivalence test with matched- pairs TOST was used to compare the results from the v1 and v2 protocol related to each of these features. As shown in Figure 28 and Figure 29, CAR-T products generated using the v1 and v2 cell manufacturing protocol presented similar % viability, % transduction efficiency, % CD3+, and VCN numbers. Therefore, the products generated using the v1 and v2 cell manufacturing protocol passed the Tier 1 equivalence test. EXAMPLE 11 Characterization of product potency [0307] IFNgamma (IFN ^) secretion [0308] IFNgamma (IFN ^) secretion is a proxy for CAR-T potency. IFNgamma (IFN ^) secretion was measured by the ELISpot cytokine release assay according to the protocol described herein. CAR-T cells were co-cultured with CD22-expressing target cells at a range of E:T ratios between 0:1 to 2:1 on an ELISpot IFNg plate. The plates were washed and substrate was added. IFNgamma (IFN ^) positive spots were scored by CTL Immunospot. Figure 30 shows that CAR-T cells harvested on Day 5 released similar amount of IFNgamma (IFN ^) as the CAR-T cells harvested on Day 6 and Day 7. [0309] CAR-T cells were prepared by v2 process with media exchange according to methods described herein, and cells were harvested on Days 5, 7, 9 or 11. IFNgamma (IFN ^) secretion was measured by the ELISpot cytokine release assay according to the protocol described herein. CAR-T cells were co-cultured with CD22-expressing target cells at a range of E:T ratios between 0:1 to 2:1 on an ELISpot IFNgamma plate. The plates were washed and substrate was added. IFNgamma (IFN ^) positive spots were scored by CTL Immunospot. Figure 31 shows that CAR-T cells prepared with media exchange and harvested on Day 5 induced the highest amount of IFNgamma (IFN ^) release. These results suggested that incorporation of media exchange in v2 process helped to generate a greater amount of IFNgamma (IFN ^) secreting cells. [0310] CD22 CAR-T products were prepared from two donors (donor 1 and donor 2) according to the v2 method described in Example 2. CAR-T cells were harvested on Days 5, 6, 7, 10, or 12 after seeding. CAR-T cells were co-cultured with CD22-expressing target cells at a range of E:T ratios between 0:1 to 2:1 on an ELISpot IFNgamma plate. The plates were washed and substrate was added. IFNgamma (IFN ^) positive spots were scored by CTL Immunospot. Total viable cells were measured at different time points. Figure 32A showed that the number of total viable cells increased as the cells expanded in culture. Figures 32B and 32C demonstrated that CAR-T cells harvested between Days 5 and 7 released more IFNgamma (IFN ^) than cells harvested on Day 10 and Day 12. [0311] Cytotoxicity [0312] CAR-T products were prepared according to the v2 protocol described in Example 2. Cells were harvested on Days 5, 7 and 9. CAR-T cells were co-cultured with CD22 expressing target cells or CD22 knocked out target cells at a range of E:T ratios between 1:0.03 to 2:1. Cell toxicity was measured by cytotoxicity assay as described in Figure 49. The % specific lysis = (experimental RLU – target only RLU) / (Full lysis RLU – target only RLU) *100%. (RLU = relative luminescence units). Figures 33A and 33B showed that cytotoxicity assay was specific to CD22 antigen stimulation and harvesting later resulted in lower E:T EC50. EXAMPLE 12 Quantification of cell count and viability of CD22 CAR-T products [0313] Total viable cell concentration and viability of CAR-T products were determined using the NucleoCounter NC-200 (Chemometec), an automated cell counter capable of high precision cell counting. Cells were loaded into Via2 cassettes containing Acridine Orange and DAPI dyes per the Quantification of Cell Count and Viability of CD22 CAR-T Products protocol. Acridine Orange stains all cells and DAPI stains dead cells, allowing for the quantification of viable cell concentration as well as the total percentage of viable cells. TexMACSTM culture medium was used as a negative control. System suitability criteria for this assay were: 1) Cell count readings will have < 3% aggregates, and 2) % CV of measurements will be < 10%. Flow cytometry is described in the following section. EXAMPLE 13 Measurement of CD22CAR-T transduction efficiency and CD3+ T cells by flow cytometry [0314] A single flow cytometry method, designated “Transduction Efficiency and T Cell Phenotype for CD22-CAR”, was used to measure several different product quality attributes for drug product release as follows. [0315] Cryopreserved CD22 CAR-T cells were thawed, counted by NC-200, and stained with anti-human CD3 PE-Cy7, recombinant human CD22 Fc Chimera Protein AF647, and 7AAD viability dye. Cells that are negative for the 7AAD dye are viable. Stained cells were then analyzed using the Miltenyi MACSQuant 16 flow cytometer to determine the percentage of viable CD3+ T cells (%7AAD-/CD3+) and viable CD3+/CD22 CAR+ cells (%7AAD- /CD3+/CD22 CAR+). The cytometer was calibrated with MACSQuant 16 calibration beads at time of acquisition and fluorescent minus one (FMOs) controls were used for gate placement of CD3+ T Cells and CD3+CD22 CAR+ cells. [0316] Results for %7AAD-/CD3+ cells were used to assess purity and %7AAD- /CD3+/CD22-CAR+ to assess identity for lot release. System suitability criteria for this assay were: Live CD3P T Cell percentages are ≥ 80% for the positive control and live CD3PCD22 CARP cell percentages were within acceptance range of the positive control. EXAMPLE 14 Protocol for measuring IFNgamma potency by ELISpot cytokine release assay [0317] CD22 CAR-T drug product potency was evaluated by measuring the production of interferon gamma (IFNγ or IFNgamma) produced by CD22 CAR-T drug product upon stimulation with a Nalm6 cell line engineered to express CD22. [0318] The following effector cells: CD22 CAR-T drug product, untransduced CD3P T cells (CRG‑022‑NEG), and CD22 CAR-T drug product system suitability (previously characterized) CD22-CAR T-Cells (CD22 CAR-T-SS) were thawed, counted, and cultured in flasks for 18-24 hours prior to initiating the ELISpot assay to allow effector cells to recover from the effects of cryopreservation. On Day 1 of the assay, two target cell lines, Nalm6‑CD22MED/CD19KO and Nalm6-CD22KO/CD19KO (knock-out), were harvested from culture flasks, counted, and plated (in quadruplicate per effector cell sample tested) on a 96-well ELISpot plate pre-coated with anti-human IFN-γ antibody. Additionally, each target cell line was plated in quadruplicate without effector cells as target cell line negative control samples. Cultured CD22 CAR-T drug product, CD22 CAR-T-NEG, and CD22 CAR-T-SS cells (system suitability control from a v2 development run at scale) were then harvested and counted. For CD22 CAR-T drug product and CD22 CAR-T-PC cells, the concentration of CD22 CAR-TP cells in suspension were calculated by normalizing the viable cell concentration to their respective %7AAD-/CD3P/CD22 CAR P value. All three sample effector cells were then separately plated in quadruplicate and serially diluted across a range of effector:target ratios. Additionally, effector cell control samples such as effector cells alone and effector cells alone stimulated with a pre-mixed cocktail containing phorbol 12- myristate-13-acetate (PMA) and ionomycin were also plated in quadruplicate. All ELISpot plates were then incubated for 18-24 hours. After the incubation period (Day 2), ELISpot plates were decanted, washed, and stained with anti-human-IFN-γ (biotin) detection antibody. A biotin-conjugated secondary antibody was used for detection using alkaline phosphatase linked to streptavidin and a colorimetric substrate (BCIP; 5-bromo-4-chloro-3’- indolylphosphate p-toluidine). Stained plates were then imaged and analyzed using the CTL Immunospot® S6 Universal Analyzer and the Immunospot® Single‑Color Enzymatic Suite Analysis Software. Cytotoxic T-cells secreting IFN-γ are visualized as individual spots (spot- forming units, SFU) with each spot representing a single CAR-T cell. EXAMPLE 15 Measuring vector copy number in transduced cells [0319] In this Example, a method for measuring vector copy number per cell using droplet digital PCR (ddPCR) is described. [0320] The vector copy number assay, Vector Copy Number Determination, targets the product-specific sequence presented in the transduced cells to determine the number of gene copies integrated into the T cell genome in the CD22 CAR-T drug product. In this assay, DNA extracted from the drug product was used in a Droplet DigitalTM PCR (Bio-Rad QX200TM) reaction to detect a portion of the CD22 CAR DNA sequence and multiplexed with an assay for the human albumin sequence to determine the cell number. Non-transduced DNA serves as a negative control, and linearized CD22 gBlock plasmid (Integrated DNA Technologies) diluted in human gDNA served as a positive control for the assay. The number of CD22 CAR DNA copies per transduced cell is reported. System suitability criteria for this assay were: wells with < 10,000 accepted droplets were not used for analysis, 3 out of 4 replicates were required for a dilution to be valid, no template control (NTC) wells resulted in average background of ≤ 2 positive droplets and copy number percent recovery of the positive control gBlock plasmid was 75%-125% of the expected value in copies/µL. EXAMPLE 16 Quantification of vector copy number in CD22 CAR-T cells [0321] The number of vector copies per CAR-T cell can be a factor in the efficacy of treatment using CAR-T therapy. Various parameters can contribute to transduction efficiency and the resulting vector copy numbers per cell. One such parameter is the MOI ratio. In this experiment, multiple MOI ratios were used to transfect lymphocytes after apheresis cryopreservation treatment and the vector copy number (VCN) was determined thereafter using the ddPCR methods described herein and according to the formulas below. − ln( ^^ ^^ ^^ ^^ ^^ ) Concentration of target or reference (copies/µL) = ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ [0322] wherein: [0323] N = total number of droplets (target or reference). [0324] Nneg = number of negative droplets (target or reference). [0325] Vdroplet = volume of droplet (8.5 × 10-4 µL). Vector copy number = ^^ ^^ × ^^ ^^ [0326] wherein:
Figure imgf000093_0001
[0327] A = concentration of target species (copies/µL). [0328] B = concentration of reference species (copies/µL). [0329] NB = number of copies of reference loci in the genome. [0330] Samples from two donors in the Stanford phase I trial were prepared. Vector copy number and MOI were calculated. As shown in Figures 34A and 34B, VCN increases with increasing MOI. [0331] CD22 CAR-T cells were processed at two different manufacturing sites. The vector copy numbers per cell and per CAR+ cell were measured for samples prepared at both sites. As shown in Figure 35, CD22 vector copy numbers for samples prepared at the two sites were essentially the same. [0332] CD22 CAR-T cells were prepared according to v2 method described in Example 2 and harvested on Days 5, 7, and 10 post seeding. The vector copy number per cell, per CAR+ cells, and transduction efficiency were measured. As shown in Figure 36, VCN per transduced cell was well below 5 copies/CAR+ cells (generally accepted limit for VCN in autologous CAR-T products). There was a small trend observed of decreasing VCN with increased day of harvest. EXAMPLE 17 Confirmation of CD22 CAR potency using Jurkat cells [0333] Jurkat cells were transduced using a lentiviral vector encoding a CD22 CAR. The Jurkat cells were expanded, isolated and confirmed to express the CD22 CAR using a flow cytometry analysis. CD22 CAR-expressing Jurkat cells were then co-cultured in the presence of Nalm6 cells expressing CD22. An ELISpot analysis was performed to measure IL-2 release as a function of CAR-Jurkat cell function. As shown in Figure 47, the level of IL-2 released by the CAR-expressing Jurkat cells was proportional to the ratio of Jurkat cell:Nalm6 cell and also highest for the Nalm6 expressing high levels of CD22 and lowest for the Nalm6 without CD22 expression. This result demonstrates the ability of the CD22 CAR to recognize CD22 and initiate the relevant immune signaling pathways that lead to potency in a clinical context. EXAMPLE 18 Manufacturing CD22 CAR T cells (protocol v2) [0334] This Example describes the manufacturing of a population of CD22 CAR-T cells from a cryopreserved apheresis sample in accordance with some non-limiting embodiments of the disclosure. [0335] In this Example, the cryopreserved apheresis sample was thawed and the manufacturing process proceeded continuously for approximately 5 - 9 days. The patient’s T cells were isolated from the cryopreserved apheresis sample, transduced with a lentiviral vector expressing an anti-CD22 CAR comprising a leader sequence (SEQ ID NO: 28), an anti-CD22 scFv (SEQ ID NO: 1), a peptide linker (SEQ ID NO: 29), a CD8 ^ hinge domain (SEQ ID NO: 24), a CD8 ^ transmembrane domain (SEQ ID NO: 25), a peptide linker (e.g., spacer) LYC (SEQ ID NO: 31) comprising a portion of the CD8 ^ cytoplasmic domain, a 4- 1BB(CD137) co-stimulatory signaling domain (SEQ ID NO: 26) , and a CD3 ^ primary T cell activation domain (SEQ ID NO: 27) expressed under the control of an EF1 ^ promoter, and expanded to produce a dose of 1×106 viable CD3+CAR+ cells/kg, with weight capped at 100 kg per the clinical protocol. The formulated drug product was cryopreserved in a controlled- rate freezer and stored at ≤ -130°C. A detailed process flow is summarized in Table 2. TABLE 2: Key process description for the manufacturing process Manufacturing process unit Ops Unit Ops description Analytical testing
Figure imgf000095_0001
Albumin (2% w/v) + 50% CAR19
Figure imgf000096_0001
CliniMACS Prodigy® • Viability (%)
Figure imgf000097_0001
A. Media preparation [0336] Prior to the start of the manufacturing process, preparation of T cell growth medium was formulated according to the concentrations in Table 3. TexMACS™ medium was supplemented with human AB serum (HABS) to a final concentration of 3% HABS. An aliquot of TexMACS™ medium was added to a vial of lyophilized IL-7 and a vial of lyophilized IL-15, respectively. Once the cytokines were resuspended in solution, the entire amount was removed from each vial and added to the mixing assembly along with TexMACS™ and 3% HABS. This medium was referred to as modified TexMACS™ Medium (MTM). The supplemented medium was then terminally filtered using a customized XLM Media Filtration Assembly that consisted of 0.2/0.1 μm Polysulfone (PES) double layer filter membranes. Aliquots were filled as shown in Table 4 and were stored at 2 - 8°C with light protective cover until use. Medium could be formulated for a manufacturing lot up to 3 days before Day 0. [0337] Once all medium aliquots were filled, a filter integrity test was performed using a bubble point test. The filter was flushed with water for injection (WFI) and a set point of 58 psi was used. No max diffusional flow was set for the test. TABLE 3: MTM formulation concentrations Components Final Concentration Components Final Concentration
Figure imgf000098_0001
TABLE 4: Media preparation process parameters Parameter Target Values Parameter Target Values
Figure imgf000098_0002
B. Apheresis sample receipt and cryopreservation [0338] Apheresis sample was collected at an approved blood collection center and transported to the manufacturing site in a temperature-controlled shipping container at 2 - 8°C for manufacturing within 48 hours from the end of collection to spiking of the apheresis bag. At the manufacturing site, the apheresis material was initially quarantined and assessed for label integrity, bag integrity, and verification of chain of identity and chain of custody. [0339] Upon release of the fresh apheresis sample from quarantine at the manufacturing site, the apheresis sample was sampled for cell count and viability, T cell phenotype via flow cytometry (CD3/4/8) and retain is taken for sterility and VCN for CD19 CAR+. In order to prepare for the subsequent wash and concentration steps, calculations were performed based on the cell count to determine the Rotea input parameters: number of loops, load volume per loop, and harvest volume per loop. The entire apheresis sample was loaded on a CTS™ Rotea™ Counterflow Centrifugation System for an automated dilution and platelet reduction wash step before elution/resuspension in Plasma-Lyte A + 4% human serum albumin (HSA). The final collection bag containing the washed and processed apheresis sample was sampled for cell count and viability. Depending on the cell concentration and volume of the cells at formulation, additional dilution with formulation buffer may be required on a FINIA Fill and Finish System (“FINIA”). [0340] Finally, the processed apheresis sample was loaded on a FINIA, an automated formulation and fill system, where final formulation involved a 1:1 volume dilution with the cryoprotectant CryoStor® 10. The total viable cells post-Rotea wash determines the volume and number of CS250 bags that were formulated. In instances where there were >12.59×109 WBCs post-Rotea wash, three bags were formulated at a volume of 54 mL per bag. In instances where ≤12.59×109 WBCs post-Rotea wash, two bags were formulated at 70 mL per bag. A 12 mL QC bag was also filled with cell suspension on the FINIA and aliquoted in the biosafety cabinet (BSC) at 1 mL/vial for further characterization and retains. The FINIA inputs for 2 or 3 bags are summarized in Table 22 and Table 23, respectively. TABLE 22: FINIA inputs for 2 apheresis bags Parameter Value Units
Figure imgf000099_0001
QC Bag Fill Volume (mL) 12
Figure imgf000100_0003
TABLE 23: FINIA inputs for 3 apheresis bags Parameter Value Units
Figure imgf000100_0001
[0341] The cryopreservation of formulated cell suspension and sample vials was performed in a controlled-rate freezer (CRF) using a defined program as detailed in Table 25. Generally, the formulated cell suspension was placed inside the CRF before the sample vials to ensure cells experience shorter DMSO exposure (e.g., addition of DMSO to placement the inside CRF) hold time than the sample vials. TABLE 24: Key process setpoints on Day -X Parameter Target Values
Figure imgf000100_0002
Apheresis dilution by Rotea 1:1 (v/v) in Wash/Resuspension Buffer
Figure imgf000101_0001
. b Applicable when transporting cryopreserved apheresis and samples on dry ice. TABLE 25: CRF profile for apheresis Step Rate of Cooling/Warming Temperature
Figure imgf000101_0002
[0342] Identification of the storage cryogenic freezer and rack were generally performed prior to the completion of the cryopreservation. , Upon completion of the cryopreservation, cryopreserved cells were removed from the CRF before the sample vials to minimize CRF to cryogenic freezer transfer hold time. The cells were then placed on dry ice or inside a CryoPodTM and transferred to be stored in a temperature-monitored cryogenic freezer at ≤ - 130°C until ready for manufacturing. C. T cell enrichment and activation (Day 0) [0343] On Day 0, prior to the thaw of the cryopreserved apheresis sample, the CliniMACS buffer with 2% v/v or 0.5% w/v HSA was prepared in the BSC. A transfer set was spiked into the CliniMACS buffer bag and HSA was transferred into the buffer bag. [0344] Next, the Prodigy was turned on and integrity tests were performed with the pre- sterilized TS520 tubing set. The CliniMACS buffer and MTM bags were connected, and the tubing set priming was begun. [0345] To determine the number of bags of apheresis sample to thaw on Day 0, the total viable cells and flow cytometry results (CD3+%, CD4+CD8-%, CD8+CD4-%, and CD4+CD8+%) from Day -X were used. [0346] The formulas used are shown below: ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ℎ ^^ ^^ℎ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ = ( ^^ ^^4+ ^^ ^^8−% + ^^ ^^8+ ^^ ^^4−% + ^^ ^^4+ ^^ ^^8+%) × ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ( ^^ ^^ ^^ ^^ ^^⁄ ^^ ^^) × ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ( ^^ ^^) ^^ ^^ ^^ ^^ ^^ ^^ ^^3+ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ℎ ^^ ^^ℎ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ = ( ^^ ^^3+%) × ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ( ^^ ^^ ^^ ^^ ^^⁄ ^^ ^^) × ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ( ^^ ^^) [0347] The number of bags to thaw was based on the amount that results in a minimum of 1.0×109 CD3+ cells and the closest in absolute value to 3.0×109 target cells. For example, if each bag contained 1.2×109 CD3+ cells and 2.5×109 target cells, only one bag would be thawed. [0348] During priming, the cryopreserved apheresis sample was retrieved from vapor phase of liquid nitrogen storage and transported on dry ice or in a CryoPodTM. Prior to thawing, the cryopreserved apheresis sample was placed in a secondary bag. The bag(s) were thawed using the “plasma” protocol at 37±1°C with mixing via the Plasmatherm for target thaw duration of 4 minutes ± 30 seconds. [0349] The post-thaw apheresis bag(s) were placed on cold gel pack(s) and transferred into a BSC. In instances where more than one bag was needed, the bags were combined into a transfer pack prior to dilution with 2-8°C MTM to minimize the DMSO toxicity during process hold time. A sample was taken from the diluted apheresis sample for cell count and viability. This sample, in combination with the apheresis flow phenotype results on Day -X (CD3/4/8), were used to generate the number of input target cells for the subsequent step. [0350] Up to 3.0×109 CD4+/CD8+ cells were loaded on the CliniMACS Prodigy for co- enrichment of CD4+/CD8+ cells. Within the closed CliniMACS Prodigy system following a defined protocol, the cells were washed with CliniMACS buffer + 2% v/v or 0.5% w/v HSA, co-labeled with CliniMACS CD4/CD8 selection beads, and co-enriched via an integrated magnetic cell selection column. After T cell enrichment, the volume of the post-selection positive fraction containing the enriched CD4+/CD8+ cells was determined. A sample was taken from the positive fraction bag through the integrated closed-system sample pouch. The sample pouch was heat sealed and removed. An aliquot of the sample was used to measure cell count and cell viability. Samples were also aliquoted in the BSC into 5 cryovials for phenotype at 1×107 cells/vial and retained at 3×106 cells/mL for use as negative controls for VCN. The phenotype vials were cryopreserved and stored in LN2 while VCN retains were stored at -80°C. The negative fraction (non-target cells) was also aliquoted into 4 ^ 1 mL cryovials and stored in the vapor phase of LN2 for future characterization testing or investigational purposes if needed to troubleshoot the enrichment step. [0351] Next, up to 300×106 co-enriched CD4+/CD8+ cells were seeded in culture on the Prodigy in MTM and activated with 4 mL of TransAct™. The cells were cultured in the Prodigy CCU chamber at 37°C with 5% CO2 for 22 - 26 hours. [0352] After culture initiation, the bag containing the remaining CD4+/CD8+ co-enriched cells was sealed off from the Prodigy tubing set and an additional sample was removed for dry pellet, flow (cryopreserved cells) and T cell retain samples. TABLE 26: Prodigy T Cell Transduction (TCT) activity matrix P D i f Total
Figure imgf000103_0001
a Value is entered based on Day 0 activation time TABLE 27: Key process setpoint on Day 10 Parameter Target Values
Figure imgf000104_0002
D. Transduction (Day 1) [0353] On Day 1, approximately 22 - 26 hours after Day 0 culture initiation, activated T cells were transduced with lentiviral vector (CD22.BB.Z) produced by Oxford Biomedica. Vector volume consumption was calculated based on the official infectious titer of the vector batch and number of cells seeded on Day 0 (up to 200×106 co-enriched CD4+/CD8+ cells) to achieve a multiplicity of infection (MOI) of 2.0 TU/cell. [0354] Based on the vector volume needed for transduction, the lentiviral vector vials were removed from the storage freezer and thawed at room temperature for ≤ 60 minutes. The required vector volume was diluted into MTM using appropriately
Figure imgf000104_0001
syringe(s) that can measure volume to the hundredth of a unit. The medium served as a carrier for the vector formulation during the Prodigy’s automated vector addition step. After vector addition, the Prodigy rinsed the bag with additional MTM to ensure all vector volume is added, which brought the total volume to 100 mL and cell culture continued at 37°C with 5% CO2 until Day 3. TABLE 28: Key process setpoint on Day 1 Parameter Target Values
Figure imgf000104_0003
Incubation temperature 37 ± 0.5°C
Figure imgf000105_0003
E. Culture wash (Day 3) [0355] On Day 3, T cell TransActTM and residual lentiviral vector was washed out with the automated CliniMACS Prodigy Program. The wash step involved removal of supernatant, followed by addition of MTM to bring the total culture volume to 200 mL. TABLE 29: Key process setpoint on Day 3 Parameter Target Values
Figure imgf000105_0004
F. Expansion (Day 4) [0356] On Day 4, a media exchange was performed by the Prodigy and a sample was taken to determine the cell count and transduction efficiency (TE). Based on the number of viable CD3+CAR+ cells present and the patient’s weight (capped at 100kg), a projection was made whether dose would be attained on Day 5, or additional culture was required to obtain target cell number. In order to proceed with harvest, the dose factor must be ≥ 4.1 using the equation below.
Figure imgf000105_0002
Figure imgf000105_0001
[0357] If there were sufficient viable CD3+CAR+ cells to meet the dose requirements during Day 5 sampling, harvest would proceed. If total viable CD3+CAR+ cell numbers were not sufficient, cell counts were taken on Day 6, and the viable CD3+CAR+ cell numbers were calculated with Day 4 transduction efficiency (TE) added with a projected 7% increase based on historical patient runs to determine if cells could be harvested on Day 7. If viable CD3+CAR+ cell numbers were still not sufficient on Day 7 using the Day 4 TE with projected 7% increase, the culture could be extended up to Day 9. The following equation was used on Day 6 and 7 to calculate the dose factor using the projected 7% increase to Day 4 TE to determine if the batch would harvest on Day 7. [0358] Media exchange for later culture days was performed as 60% exchange on Days 7 and 8, which entailed removal of 150 mL spent media and replacement with 150 mL supplemented media, to a final total volume of 250 mL. Day 9 was the final available harvest day, and the culture was terminated regardless of viable CD3+CAR+ cell number. If the culture does not meet the intended dose of viable CD3+CAR+ cells by Day 9, harvest will proceed with a partial dose harvest and with a reduced sampling as needed or be terminated depending on management decision. G. Harvest (Day 5, 7, or 9) [0359] Harvest determination: [0360] On Day 5, a pre-harvest cell count was performed to confirm target viable CD3+CAR+ cell number was achieved based on Day 5 cell count and Day 4 TE before harvest was initiated. If target viable CD3+CAR+ cell number for harvest was not achieved based on the pre-harvest cell count and additional culture was needed, a 50% media exchange was performed on both Day 5 and 6. A portion of spent media was removed (125 mL) via centrifugation of the CentriCult Unit (CCU) and replaced with supplemented media (125 mL). Similar to Day 4, a sample was taken on Day 6 for cell count and was used (along with the reported TE from Day 4 with projected 7% increase) to assess if target viable CD3+CAR+ cell numbers were achieved for a Day 7 harvest. If harvest was initiated, a sample was also taken for mycoplasma assessment. Per the Prodigy T Cell Transduction (TCT) program, transduced cells were harvested out of the CCU via centrifugation of the CCU, followed by media exchange and elution/resuspension into Plasma-Lyte A + 4% HSA (w/v). Post-harvest samples were taken for cell count, VCN, and VSV-G testing. The harvested cells were stored at 2 - 8°C until the pre-harvest TE result was available. [0361] When the pre-harvest TE was reported by QC, calculations were performed with the post-harvest cell count to determine the volume of final formulation required to fill up to 2 Final Product bags and 1 QC bag as shown in Table 30. Based on the total transduced cells available, the cells were either further concentrated in the Rotea Counterflow System, directly formulated into drug products using the FINIA Fill and Finish System or diluted prior to formulation on the FINIA (Table 31). TABLE 30: Pots-prodigy calculation Parameter Value Units
Figure imgf000107_0001
TABLE 31: Post-prodigy harvest pathway Condition Pathway
Figure imgf000107_0002
[0362] Concentration of Rotea: [0363] If the post-Prodigy transduced viable cell density was less than the minimum transduced viable cell density for FINIA, the Rotea was used to concentrate the cells. Table 32 describes the calculations used to determine the Loop Volume, Established Bed Volume, and Harvest Volume on the Rotea pathway. TABLE 32: Rotea input calculations Parameter Value Units
Figure imgf000107_0003
Target Number of
Figure imgf000108_0001
[0364] Post-Harvest optional Rotea processing, a sample was taken for cell count and next steps were determined by completing Table 33 and Table 34. Depending on the cell concentration and volume of the cells pre-formulation, additional dilution with Final Formulation Medium (Plasma-Lyte A + 4% HSA (w/v)) may be required to achieve the target CD3+CAR+ concentration required to meet the patient specific dose for final formulation using 1:1 dilution with Cryostor® CS10. The diluent was either manually added via gravity transfer using a scale if less than 5 mL was needed, or automatically added by FINIA if more than 5 mL was needed. TABLE 33: Post-Rotea calculations Parameter Value Units
Figure imgf000109_0001
TABLE 34: Pathways Post-Rotea Condition Pathway
Figure imgf000109_0002
[0365] Dilution: [0366] If the post-Prodigy or post-Rotea viable cell density was more concentrated than the maximum viable cell density for FINIA fill, calculations were conducted using Table 54 to determine the dilution pathways. TABLE 35: Cell suspension dilution calculations Parameter Value Units
Figure imgf000109_0003
Cell Suspension TDN ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^ ^^
Figure imgf000110_0001
[0367] FINIA: [0368] Once the appropriate pre-formulation target concentration was achieved, the cell solution was diluted 1:1 with Cryostor® CS10 in the FINIA mixing bag to generate the final formulated cell suspension used to fill drug product. Drug product and QC bag labels were added to the bags and cryo-cassettes prior to the start of FINIA fill and finish steps. The cell suspension was filled into one to two of the 50 mL size product bag(s) at a target fill volume of 20 mL per bag, and one QC bag was filled at a volume of 24 mL. If 2 final bags were filled, Table 36 was used to complete a 3 Material Fill on the FINIA. If only 1 bag was filled, Table 37 and Table 38 were used to complete a 2 Material Fill on the FINIA and adjust the inputs accordingly. If there were any unexpected cell recovery losses, the QC bag fill could be reduced to as low as 20 mL without sacrificing volume needed for QC testing and retains. TABLE 36: Material fill calculations Parameter Value Units
Figure imgf000110_0002
FINIA Material 1 Target 3.5 × ^^
Figure imgf000111_0001
TABLE 37: Material fill calculations Parameter Value Units
Figure imgf000111_0002
TABLE 38: FINIA inputs for 2 material fills Condition Parameter
Figure imgf000111_0003
QC Bag Volume 2 × ^^ − 26 24 24
Figure imgf000112_0001
[0369] Upon completion of FINIA fill and finish steps, the final product bag(s) were placed in pre-cooled labelled cryo-cassettes, visually inspected, and transferred to the pre-cooled CRF at 4°C while the QC bag was aliquoted in the BSC. The cell suspension in the QC bag was immediately spiked and aliquoted into cryovials at 1 mL per vial as per the sampling plan. TABLE 39: Key process setpoints on harvest day Parameter Target Values
Figure imgf000112_0002
DMSO addition to placement in CRF hold time < 90 minutes
Figure imgf000113_0001
[0370] Prior to placing the final product cassettes inside the CRF, foam inserts were placed inside the cassettes to hold the final product bags in place and minimize movement. The cryopreservation of the final product bags and QC vials were performed in a controlled-rate freezer (CRF), and frozen using a defined program as detailed in Table 40. Generally, final product (FP) bags were placed inside the CRF before the QC vials to ensure FP cells experienced shorter DMSO exposure (e.g., addition of DMSO to placement inside the CRF) hold time than the QC vials. TABLE 40: Controlled-rate freezer profile for cryopreservation of final product Step Rate of Cooling/Warming Temperature
Figure imgf000113_0002
[0371] Generally, identification of the storage cryogenic freezer and rack was performed prior to the completion of the CRF. Upon completion of the cryopreservation, FP bags should be removed from the CRF before the QC vials to minimize CRF to cryogenic freezer transfer hold time. The FP bags were then placed on dry ice or inside a CryoPodTM and transferred to be stored in a temperature monitored cryogenic freezer at ≤ -130°C until ready for release. H. Final product attributes: [0372] The release specifications for the CD22 CAR T cell product were summarized in a Final Product Specification. The extended characterization plan for the CD22 CAR T cell product is summarized in Table 41. TABLE 41: Final product characterization Assay Sample Point
Figure imgf000113_0003
T cell Exhaustion Final Product
Figure imgf000114_0001
I. Final product packout and shipment: [0373] Upon batch release of the final product, CRG-022 FP bag(s) were removed from temperature monitored cryogenic freezer at ≤ -130°C and placed on dry ice or inside a CryoPodTM. FP labels, bag integrity and COC/COI documentation were verified on the FP cassettes that were placed on dry ice or inside a CryoPodTM. FP bag(s) were then transferred to a BioLife Solutions ModPAK and placed into a QuickSTAT SAVSU DV10 LN2 shipper. [0374] The FP must be placed inside the LN2 shipper within 10 minutes from the removal time from the storage cryogenic freezer. TABLE 42: Key process setpoint for FP packout and shipment Parameter Target Values
Figure imgf000114_0002
[0375] While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS 1. A method of making a population of CAR-expressing immune cells, the method comprising: (a) obtaining a liquid sample comprising a first population of cells comprising immune cells from a human subject; (b) processing the first population of cells thereby generating a second population of cells comprising immune cells, wherein the second population of cells includes at least 1 ^104 total cells, wherein less than 20% of the total number of cells in the second population of cells are platelets; (c) cryopreserving the second population of cells; (d) on Day 0, thawing the cryopreserved second population of cells comprising immune cells, processing the thawed second population of cells comprising immune cells, and seeding a third population of cells comprising immune cells with a portion of the processed second population of cells comprising immune cells in a volume of a first media, wherein the third population of cells is a subset of the second population of cells, wherein the third population of cells is seeded into a volume of at least 250 mL of first media with at least 3.0 ^108 cells from the second population of cells; (e) transducing the third population of cells comprising immune cells with a recombinant polynucleotide encoding a CAR thereby generating a fourth population of cells, wherein the fourth population of cells comprises CAR- expressing immune cells; (f) expanding the fourth population of cells in media to yield a fifth population of cells comprising CAR-expressing immune cells; (g) removing at least 50% of the volume of the media on or before Day 4 after the seeding in step (d); (h) determining the number of viable CD3+ CAR-expressing T cells; and either (i) continuing to expand the fifth population of cells and exchanging the media, or (j) harvesting the fifth population of cells comprising CAR-expressing immune cells on or before Day 9 after the seeding in step (d), wherein at least 2.4% of cells in the fifth population of cells including CAR-expressing immune cells are CCR7+CD45RA+ immune cells.
2. The method of claim 1, further comprising a step of (k) formulating the fifth population of cells for cryopreservation and administration to patients.
3. The method of claim 1 or claim 2, wherein the liquid sample including a first population of cells comprises a leukapheresis product.
4. A method of making a population of CAR-expressing immune cells, the method comprising: (a) obtaining a liquid sample comprising a first population of cells comprising immune cells from a human subject; (b) processing the first population of cells thereby generating a second population of cells comprising immune cells; (c) seeding a third population of cells comprising immune cells in a volume of a first media, wherein the third population of cells is a subset of the second population of cells; (d) transducing the third population of cells with a recombinant polynucleotide encoding a CAR thereby generating a fourth population of cells, wherein the fourth population of cells comprises CAR-expressing immune cells; (e) expanding the fourth population of cells to yield a fifth population of cells; and (f) harvesting the fifth population of cells on Day 5 or later after the seeding in step (c).
5. A method of making a population of chimeric antigen receptor (CAR)-expressing immune cells, the method comprising the steps of: (a) obtaining a liquid sample comprising a first population of cells comprising immune cells from a human subject; (b) processing the first population of cells to remove platelets thereby generating a second population of cells comprising immune cells, wherein the second population of cells comprises at least 1 ^104 total cells and wherein less than 20% of the total number of cells in the second population of cells are platelets; (c) seeding a third population of cells comprising immune cells in a volume of a first media, wherein the third population of cells is a subset of the second population of cells; (d) transducing the third population of cells with a recombinant polynucleotide encoding a CAR thereby generating a fourth population of cells, wherein the fourth population of cells comprises CAR-expressing immune cells; (e) expanding the fourth population of cells to yield a fifth population of cells comprising CAR-expressing immune cells; and (f) harvesting the fifth population of cells on Day 5 or later after the seeding in step (c).
6. The method of any one of claims 1-5, further comprising removing at least 50% of the volume of the first media on or before Day 4 after seeding.
7. The method of any one of claims 1-6, wherein at least 2.4% of cells in the fifth population of cells are CCR7+CD45RA+ immune cells.
8. A method of making a population of CAR-expressing immune cells, the method comprising the steps of: (a) obtaining a liquid sample comprising a first population of cells comprising immune cells from a human subject; (b) processing the first population of cells thereby generating a second population of cells comprising immune cells; (c) seeding a third population of cells comprising immune cells in a volume of a first media, wherein the third population of cells is a subset of the second population of cells; (d) transducing the third population of cells with a recombinant polynucleotide encoding a CAR thereby generating a fourth population of cells, wherein the fourth population of cells comprises CAR-expressing immune cells; (e) expanding the fourth population of cells to yield a fifth population of cells comprising CAR-expressing immune cells; (f) removing at least 50% of the volume of the first media on or before Day 4 after the seeding; and (g) harvesting the fifth population of cells on Day 5 or later after the seeding in step (c).
9. The method of claim 8, wherein at least 2.4% of cells in the fifth population of cells are CCR7+CD45RA+ immune cells.
10. A method of making a population of CAR-expressing immune cells, the method comprising the steps of: (a) obtaining a liquid sample comprising a first population of cells comprising immune cells from a human subject; (b) processing the first population of cells thereby generating a second population of cells comprising immune cells; (c) seeding a third population of cells comprising immune cells in a volume of a first media, wherein the third population of cells is a subset of the second population of cells; (d) transducing the third population of cells with a recombinant polynucleotide encoding a CAR thereby generating a fourth population of cells, wherein the fourth population of cells comprises CAR-expressing immune cells; (e) expanding the fourth population of cells yielding a fifth population of cells comprising CAR-expressing immune cells; and (f) harvesting the fifth population of cells on Day 5 or later after the seeding in step (c), wherein at least 2.4% of cells in the fifth population of cells are CCR7+ CD45RA+ immune cells.
11. A method of making a population of CAR-expressing immune cells, the method comprising: (a) obtaining a liquid sample comprising a first population of cells comprising immune cells from a human subject; (b) processing the first population of cells to remove platelets thereby generating a second population of cells, wherein the second population of cells comprises at least 1 ^104 total cells and wherein less than 20% of the total number of cells in the second population of cells are platelets; and (c) seeding a third population of cells in a volume of a first media, wherein the third population of cells is a subset of the second population of cells, wherein the third population of cells comprise at least 2.0 ^108 cells; (d) transducing the third population of cells with a recombinant polynucleotide encoding a CAR thereby generating a fourth population of cells, wherein the fourth population of cells comprises CAR-expressing immune cells; (e) expanding the fourth population of cells yielding a fifth population of cells; (f) removing at least 50% of the volume of the first media on or before Day 4 after the seeding; and (g) harvesting the fifth population of cells on or before Day 9 after the seeding, wherein at least 2.4% of cells in the fifth population of cells are CCR7+CD45RA+ immune cells.
12. The method of any one of claims 1-11, wherein less than 18%, less than 15%, less than 12%, less than 10%, less than 8%, or less than 5% of the total number of cells in the second population of cells are platelets.
13. The method of any one of claims 1-12, wherein the processing of the first population of cells comprises diluting the liquid sample comprising the first population of cells with a second buffer, thereby generating a diluted liquid sample comprising the first population of cells.
14. The method of claim 13, wherein the liquid sample comprising the first population of cells is diluted prior to removing platelets.
15. The method of any one of claims 1-14, wherein the liquid sample comprising the first population of cells has a total volume of at least 50 mL, at least 100 mL, at least 200 mL, at least 250 mL, at least 300 mL, at least 400 mL, at least 500 mL, at least 600 mL, at least 700 mL, at least 800 mL, at least 900 mL, at least 1 L, or at least 2 L.
16. The method of any one of claims 1-15, wherein the liquid sample comprising the first population of cells is a leukapheresis or apheresis sample.
17. The method of any one of claim 13-16, wherein the diluting comprises adding an equal volume of the second buffer to the liquid sample.
18. The method of any one of claims 13-17, wherein the second buffer comprises human serum albumin (HSA).
19. The method of claim 18, wherein the second buffer comprising HSA is a solution comprising between 1 and 10% (w/v) HSA or a 4% (w/v) solution of HSA.
20. The method of any one of claims 13-17, wherein the second buffer comprises Plasma- Lyte A and 4% (w/v) human serum albumin in equal volume.
21. The method of any one of claims 13-20, wherein the diluted liquid sample has a total volume that is at least two times (2X), at least three times (3X), or at least five times (5X) the total volume of the liquid sample prior to diluting.
22. The method of any one of claims 1-21, wherein the processing of the first population of cells comprises washing the first population of cells, concentrating the first population of cells, and eluting and/or resuspending the first population of cells, thereby generating the second population of cells.
23. The method of any one of claims 22, wherein the concentrating of the first population of cells comprises using an automated centrifugation system.
24. The method of claim 23, wherein the automated centrifugation system concentrates the first population of cells by elutriation.
25. The method of any one of claims 1-24, wherein the processing of the first population of cells in step (b) further comprises adding a third solution to the second population of cells and cryopreserving the second population of cells.
26. The method of claim 25, wherein the third solution comprises phosphate-buffered saline (PBS), dimethyl sulfoxide (DMSO), sodium hydroxide, potassium hydroxide, and sucrose.
27. The method of any one of claims 25-26, wherein the cryopreserved second population of cells is thawed before transducing with a recombinant polynucleotide encoding a CAR in step (d).
28. The method of any one of claims 1-27, wherein at least 2.5%, 2.8%, 3.0%, 3.5%, 4.0%, 4.5%, 5.0%, or 5.5% of cells in the fifth population of cells are CCR7+ CD45RA+ immune cells.
29. The method of any one of claims 7 or 9-28, wherein the CCR7+ CD45RA+ immune cells are also CD3+.
30. The method of claim 29, wherein the CCR7+ CD45RA+CD3+ immune cells are also CD4+.
31. The method of claim 29, wherein the CCR7+ CD45RA+CD3+ immune cells are also CD8+.
32. The method of any one of claims 1-31, where at least 78.3%, 78.5%, 79.0%, 79.5%, 80%, 80.5%, 81.0%, 81.4%, 81.5%, 82.0%, 83.0%, 84.0%, 85.0%, 86.0%, or 87.0% of cells in the fifth population of cells are CCR7+ CD45RA- immune cells.
33. The method of any one of claims 1-32, wherein at most 16.1%, 16.0%, 15.0%, 14.0%, 13.0%, 12.0%, 11.0%, 10.0%, 9.0%, 8.0%, 7.5%, 7%, 6.5%, 6.0%, 5.5%, 5.0%, 4.5%, 4.0%, 3.5%, or 3.4% of cells in the fifth population of cells are CCR7- CD45RA- immune cells.
34. The method of any one of claims 1-33, wherein the efficiency of transducing the third population of cells with a recombinant polynucleotide encoding a CAR is at least 10%, at least 20%, at least 30%, at least 35%, at least 40%, at least 45% or at least 50%.
35. The method of any one of claims 1-33, wherein the multiplicity of infection (MOI) used when transducing the third population of cells with a recombinant polynucleotide encoding a CAR is at least 1, at least 1.5, at least 2, at least 2.5, at least 3, at least 3.5, at least 4, at least 4.5, or at least 5.
36. The method of any one of claims 1-35, wherein the fifth population of cells is harvested on Day 5, Day 6, Day 7, Day 8, Day 9, Day 10, Day 11, Day 12, Day 13, Day 14, or Day 15.
37. The method of any one of claims 1-10 or 12-35, wherein the fifth population of cells is harvested on Day 5, Day 6, Day 7, Day 8, or Day 9.
38. The method of any one of claims 1-37, wherein the third population of cells is activated by at least one cytokine.
39. The method of claim 38, wherein the at least one cytokine comprises IL-2, IL-4, IL-7, IL-9, IL-15, IL-21, or a combination thereof.
40. The method of claim 38, wherein the at least one cytokine comprises a combination of IL-7 and IL-15.
41. The method of any one of claims 1-40, wherein the third population of cells is seeded in the first media.
42. The method of claim 41, wherein the first media comprises 12.5 ng/mL IL-7, 12.5 ng/mL IL-15, and 3% (w/v) human serum albumin.
43. The method of any one of claims 1-10 or 12-42, wherein the third population of cells comprises at least 1.1 ^108, at least 1.2 ^108, at least 1.4 ^108, at least 1.6 ^108, at least 1.8 ^108, at least 2.0 ^108, at least 2.2 ^108, at least 2.4 ^108, at least 2.6 ^108, at least 2.8 ^108, or at least 3.0 ^108 cells comprising a subset cells of the second population of cells.
44. The method of any one of claims 1-43, wherein the third population of cells is enriched for CD4+CD8+ cells prior to seeding.
45. The method of any one of claims 1-44, wherein the CAR-expressing fourth population of cells produced by transducing the third population of cells with a recombinant polynucleotide encoding a CAR is then expanded to produce a fifth population of cells.
46. The method of any one of claims 1-45, wherein the fifth population of cells comprises at least 1 ^103, at least 1 ^104, at least 1 ^105, at least 1 ^106, at least 1 ^107, at least 1 ^108, at least 2.2 ^108, at least 2.4 ^108, at least 2.6 ^108, at least 2.8 ^108, or at least 3.0 ^108 cells comprising CAR-expressing immune cells.
47. The method of any one of claims 1-46, wherein at least 70%, 75%, 80%, 85%, or 90% of the cells in the fifth population of cells are viable.
48. The method of any one of claims 1-47, wherein the fifth population of cells is cryopreserved after harvesting.
49. The method of any one of claims 1-48, wherein the human subject from which the liquid sample comprising a first population of cells comprising immune cells was obtained has a disease or health condition.
50. The method of claim 49, wherein the disease or health condition is cancer or a relapsed/refractory cancer.
51. The method of claim 50, wherein the cancer is a leukemia or lymphoma.
52. The method of any one of claims 1-51, wherein the recombinant polynucleotide is a viral vector.
53. The method of claim 52, wherein the viral vector is a lentiviral vector.
54. The method of any one of claims 1-2, wherein the CAR-expressing immune cells are T cells.
55. The method of any one of claims 1-53, wherein the CAR-expressing immune cells are NK cells.
56. The method of any one of claims 1-55, wherein the CAR specifically binds a B cell- specific antigen.
57. The method of claim 56, wherein the CAR comprises an anti-CD22 binding domain.
58. The method of claim 57, wherein the anti-CD22 binding domain is an antibody, an antibody fragment, or an antigen binding domain of thereof.
59. The method of claim 58, wherein the anti-CD22 binding domain is an antibody fragment.
60. The method of claim 59, wherein the antibody fragment is an anti-CD22 single chain variable fragment (scFv).
61. The method of any one of claims 57-60, wherein the anti-CD22 binding domain comprises a heavy chain variable region (VH) with a sequence with at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence of SEQ ID NO: 2.
62. The method of any one of claims 57-60, wherein the anti-CD22 binding domain comprises a VH with the sequence of SEQ ID NO: 2.
63. The method of any one of claims 57-60, wherein the anti-CD22 binding domain comprises a light chain variable region (VL) with a sequence with at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence of SEQ ID NO: 3.
64. The method of any one of claims 57-62, wherein the anti-CD22 binding domain comprises a VL with the sequence of SEQ ID NO: 3.
65. The method of any one of claims 57-62, wherein the anti-CD22 binding domain comprises a sequence at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence of SEQ ID NO: 1.
66. The method of any one of claims 57-62, wherein the anti-CD22 binding domain comprises the sequence of SEQ ID NO: 1.
67. The method of any one of claims 57-62, wherein the CD22 CAR comprises a sequence with at least 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence of SEQ ID NO: 22.
68. The method of any one of claims 57-62, wherein the CD22 CAR comprises the sequence of SEQ ID NO: 22.
69. The method of any one of claims 57-62, wherein the CD22 CAR comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence of SEQ ID: 23.
70. The method of any one of claims 57-62, wherein the anti-CD22 CAR comprises the sequence of SEQ ID NO: 23.
71. The method of claim 56, wherein the CAR is a CD19 CAR comprising an anti-CD19 binding domain.
72. The method of claim 56, wherein the CAR is a CD20 CAR comprising an anti-CD20 binding domain.
73. A method of manufacturing a population of CAR-expressing immune cells, wherein the method comprises any one of the methods of claims 1-72.
74. A method of treating a health condition in a subject, the method comprising: administering to the subject a population of CAR-expressing immune cells made according to the method of any one of claims 1-72, alone or in combination with an additional therapy, thereby preventing or treating the health condition.
75. The method of claim 74, wherein the health condition is a cancer and wherein the administered CAR-expressing immune cells treat or provide anti-tumor immunity to the subject.
76. A population of CAR-expressing immune cells made according to the method of any one of claims 1-72.
77. A pharmaceutical composition comprising the population of CAR-expressing immune cells of claim 76 and a pharmaceutically acceptable carrier.
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