Methods for manufacturing car t cells

Abstract

The present disclosure relates generally to methods of making a population of trispecific CAR-expressing immune cells 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 No.: 063384-523001 WOMETHODS FOR MANUFACTURING CAR T CELLSCROSS-REFERENCE TO RELATED APPLICATION

[0001] The present application claims priority to U. S. Provisional Patent Application Serial No. 63 / 737,380, filed on December 20, 2024. The contents of the above-referenced application is herein expressly incorporated by reference in its entirety, including any drawings.INCORPORATION BY REFERENCE OF SEQUENCE LISTING

[0002] This application contains a Sequence Listing which has been submitted in. XML format via EFS-WEB and is hereby incorporated by reference in its entirety. Said. XML copy, created on December 19, 2025, is named 063384-523001WO.xml, and is 94,208 bytes in size.FIELD

[0003] The present disclosure relates generally to the fields of immuno-therapeutics and cell therapy and more particularly relates to improved methods of making and / or manufacturing a population of trispecific chimeric antigen receptor (CAR)-expressing immune cells. The disclosure provides populations of trispecific CAR-expressing immune cells made and / or manufactured by the improved methods, and pharmaceutical compositions comprising the same. The disclosure also describes methods for treating a health condition in a subject in need thereof.BACKGROUND

[0004] Autologous CAR-T cell (chimeric antigen receptor T cell) therapies have been used for the treatment of hematologic malignancies including leukemias and lymphomas and for patients having such diseases who have relapsed after receiving chemotherapy. Despite advances made by commercially available CAR-T cell therapies, there remain key resistance mechanisms that can limit the strength and quality of T cell response and lead to disease progression, including loss or down-regulation of target antigen expression, loss of costimulation and limited CAR-T-cell persistence and activity. Therefore, there remains an urgent medical need for improved CAR-T immunotherapies.

[0005] 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 wideAttorney Docket No.: 063384-523001 WOapplication 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.

[0006] Current manufacturing processes for CAR-T cells have various 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

[0007] The present disclosure relates generally to, inter alia, improved methods of making and / or manufacturing a population of trispecific chimeric antigen receptor (CAR)-expressing immune cells. 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.

[0008] In one aspect of the disclosure, provided herein is a method of making a population of CAR-expressing immune cells, the method comprising: obtaining a liquid sample comprising an initial population of autologous cells comprising immune cells from a human subject; processing the initial population of autologous cells to generate a washed population of cells comprising immune cells; on day 0, enriching the washed population of cells comprising immune cells for CD4+ and CD 8+ T cells to generate an enriched population of cells comprising immune cells, and culturing the enriched population of cells in a volume of a medium comprising an activation agent to generate an enriched and activated population of cells comprising immune cells; transducing the enriched and activated population of cells comprising immune cells with one or more recombinant polynucleotides encoding three CARs thereby generating an enriched, activated, and transduced population of cells comprising CAR-expressing immune cells; expanding the enriched, activated, and transduced population of cells comprising CAR-expressing immune cells in media to yield an expanded population of enriched, activated, and transduced cells comprising CAR-expressing immune cells; harvesting the expanded population of enriched, activated, and transduced cells comprising CAR-expressing immune cells on or before Day 9 after the seeding in step (c); and formulating the harvested population of enriched, activated, and transduced cells comprising CAR-expressing immune cells for cryopreservation and administration to patients.Attorney Docket No.: 063384-523001 WO

[0009] In some embodiments, the method further includes a step of cryopreserving the initial population of autologous cells prior to step (c) to generate a population of cryopreserved cells.

[0010] In some embodiments, the liquid sample including an initial population of autologous cells comprises a leukapheresis product.

[0011] In some embodiments, the processing in step (b) comprises a step of washing, concentrating and eluting or resuspending the washed population of cells in a buffer. In some embodiments, the washing comprises PBS / EDTA + 0.5% HSA.

[0012] In some embodiments, the processing in step (b) further comprises a step of reducing the number of platelets in the washed population of cells. In some embodiments, the number of platelets is reduced by spinning membrane filtration. In some embodiments, the spinning membrane filtration system is a Cue Cell Processing System.

[0013] In some embodiments, the washed or washed and concentrated population of cells has a reduced number of platelets compared to the initial population of cells is eluted or resuspended in a buffer. In some embodiments, the buffer comprises human serum albumin (HSA), Plasma-Lyte ATM, phosphate buffered saline, ethylenediamine tetraacetic acid (EDTA), 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.

[0014] In some embodiments, the washed or washed and concentrated population of cells having a reduced number of platelets compared to the initial 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 to generate a cryopreserved population of cells.

[0015] In some embodiments, the cryopreserved population of cells is thawed prior to step (c) to generate a thawed population of cells comprising immune cells.

[0016] In some embodiments, the enriching step (c) comprises mixing the washed or washed, concentrated, and thawed population of cells with magnetic beads derivatized with CD4-specific binding gents and CD8-specific binding agents, washing, and eluting to generate the enriched population of cells.

[0017] In some embodiments, the culturing of the enriched population of cells comprises seeding 3×108cells from the enriched population of cells in a vessel. In some embodiments, the vessel is a G-Rex® vessel.Attorney Docket No.: 063384-523001 WO

[0018] In some embodiments, the culturing of the enriched population of cells comprises seeding 2.5×106cells from the enriched population of cells in a vessel.

[0019] In some embodiments, the culturing of the enriched population of cells comprises seeding between at least 2.5×106cells and at least 3×108cells from the enriched population of cells in a vessel.

[0020] In some embodiments, the culturing of the enriched population of cells comprises seeding at least about 2.5×106, 3xl06, 3.5 xlO6, 4x106, 4.5x106, 5x106, 5.5 xlO6, 6xl06, 6.5xl06, 7xl06, 7.5xl06, 8xl06, 8.5xl06, 9xl06, 9.5xl06, IxlO7, 1.5xl07, 2xl07, 2.5 xlO7, 3xl07, 3.5xl07, 4xl07, 4.5xl07, 5xl07, 5.5xl07, 6xl07, 6.5xl07, 7xl07, 7.5x107, 8xl07, 8.5xl07, 9xl07, 9.5xl07, IxlO8, 1.5xl08, 2xl08, 2.5xl08, 3xl08, 3.5xl08, 4xl08, 4.5xl08, or 5 x 108cells in a vessel.

[0021] In some embodiments, the enriched population of cells is cultured in a volume of media comprising modified T Cell Culture Medium. In some embodiments, the volume of media brings the culture volume to 70 mL. In some embodiments, the modified T Cell Culture Medium comprises Prime XV CDM Medium supplemented with a 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.

[0022] In some embodiments, the modified T Cell Culture Medium further comprises an effective amount of an activation reagent comprising agonists of CD3 and CD28. In some embodiments, the activation reagent comprising CD3 and CD28 agonists comprises the T cell TransActTMreagent.

[0023] In some embodiments, the one or more recombinant polynucleotides encoding three CARs further comprises one or more lentiviral expression vectors.

[0024] In some embodiments, one or more lentiviral expression vectors comprises one lentiviral expression vector comprising one recombinant polynucleotide encoding three separate and distinct C ARs.

[0025] In some embodiments, the lentiviral expression vector(s) is / are manufactured using a suspension cell culture method.

[0026] In some embodiments, the transduction in step (e) is performed on Day 1.

[0027] In some embodiments, the transduction in step (e) is performed 22-26 hours after culturing of the enriched and activated population of cells comprising immune cells on Day 0.Attorney Docket No.: 063384-523001 WO

[0028] In some embodiments, the amount of vector used to transduce the enriched 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 culture the enriched population of cells comprising immune cells such that the transduction is performed with a multiplicity of infection (MOI) of 3.0.

[0029] In some embodiments, the transduction is performed with an MOI of 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.

[0030] In some embodiments, the one lentiviral expression vector comprising one recombinant polynucleotide encoding three CARs is thawed, diluted into modified T Cell Culture Medium, and added to the enriched population of cells comprising immune cells. In some embodiments, the modified T Cell Culture Medium comprises Prime XV CDM Medium supplemented with a 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.

[0031] In some embodiments, the final volume in which the transduction in step (e) is performed comprises 100 mL.

[0032] In some embodiments, the method further includes adding pre-warmed T Cell Culture Medium to the transduced population of cells.

[0033] In some embodiments, the method further includes removing at least 50% of the volume of the medium on or before Day 4 after the culturing in step (c).

[0034] In some embodiments, the expansion in step (e) comprises determining the number of viable CD3+ CAR-expressing T cells, continuing to expand the expanded population of cells, and exchanging the medium. In some embodiments, determining the number of viable CD3+ CAR-expressing T cells is performed on Day 4 by taking a sample of the expanded population of cells and calculating the amount of C AR+ cells in the cell culture based on the transduction efficiency.

[0035] In some embodiments, when the amount of CAR+ cells on Day 4 is greater than a minimum harvest threshold of 115×106, 177×106or 269×106CAR+ cells, the harvesting step (f) is performed on Day 5.

[0036] In some embodiments, when the amount of CAR+ cells on Day 4 is less than a minimum harvest threshold of 115×106, 177×106or 269×106CAR+ cells, the expanded population of cells is expanded for another day, a sample of the expanded population of cells is taken on Day 5, and the number of C AR+ cells is recalculated.Attorney Docket No.: 063384-523001 WO

[0037] In some embodiments, when the amount of CAR+ cells on Day 5 is greater than the minimum harvest threshold of 115×106, 177×106or 269×106CAR+ cells the harvesting step (f) is performed on Day 5.

[0038] In some embodiments, when the amount of C AR+ cells on Day 5 is less than a minimum harvest threshold of 115×106, 177×106or 269×106CAR+ cells, the expanded population of cells is expanded for another day, a sample of the expanded population of cells is taken on Day 6 and the number of CAR+ cells is recalculated.

[0039] In some embodiments, the amount of CAR+ cells on Day 6 is greater than the minimum harvest threshold of 115×106, 177×106or 269×106CAR+ cells, and the harvesting step (f) is performed on Day 7.

[0040] In some embodiments, the amount of CA R+ cells on Day 6 is less than a minimum harvest threshold of 115×106, 177×106or 269×106CAR+ cells, the expanded population of cells is expanded for another day, a sample of the expanded population of cells is taken on Day 8 and the number of CAR+ cells is recalculated.

[0041] In some embodiments, the amount of CAR+ cells on Day 8 is greater than the minimum harvest threshold of 115×106, 177×106or 269×106CAR+ cells, and the harvesting step (f) is performed on Day 9.

[0042] In some embodiments, the minimum harvest threshold is 269×106CAR+ cells.

[0043] In some embodiments, the harvested population of cells is formulated with Plasma- Lyte A® + 4% (w / v) HSA, diluted 1:1 with Cryostor® CS10 and frozen.

[0044] In some embodiments, the formulation step is automated. In some embodiments, the automated formulation step is performed using a Cue ScaleReady Cell Processing System, and the harvested population of cells is resuspended to the desired concentration in Final Formulation Medium comprising a 1:1 mixture of Plasma-Lyte A+4% (w / v) that is subsequently diluted 1:1 with Cryostor® CS10 and cryopreserved.

[0045] In some embodiments, the harvested population of cells including CAR-expressing immune cells comprises autologous CAR-expressing immune cells.

[0046] In some embodiments, the harvested population of cells including CAR-expressing immune cells comprises autologous T cells expressing a CD19-specific C AR, a CD20-specific CAR, and a CD22-specific CAR.

[0047] In some embodiments, the recombinant polynucleotide encoding three CARs encodes a CD19-specific CAR, a CD20-specific CAR, and a CD22-specific CAR.Attorney Docket No.: 063384-523001 WO

[0048] In some embodiments, the recombinant polynucleotide encoding an autologous CD19-specific CAR, CD20-specific CAR, and CD22-specific CAR further comprises a lentiviral expression vector.

[0049] In some embodiments, the lentiviral expression vector is manufactured using a suspension cell culture method.

[0050] In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the CD19-specific C AR, a polynucleotide sequence encoding the CD20-specific CAR, and a polynucleotide sequence encoding the CD22-specific CAR, wherein each CAR-encoding polynucleotide sequence is separated from the other CAR-encoding polynucleotide sequences by a sequence encoding a viral ribosome skipping peptide selected from the group consisting of a P2A peptide, a T2A peptide, an E2A peptide, and an F2A peptide.

[0051] In some embodiments, the recombinant polynucleotide comprises from 5’ to 3’, a sequence encoding the CD22-specific CAR; a sequence encoding a viral P2A ribosome skipping peptide; a sequence encoding the CD19-specific CAR; a sequence encoding a viral T2A ribosome skipping peptide; and a sequence encoding the CD20-specific CAR.

[0052] In some embodiments, the CD22-specific CAR comprises a CD22-specific binding domain, a transmembrane domain, and an intracellular domain.

[0053] In some embodiments, the CD22-specific CAR comprises a CD22-specific binding domain, a spacer, a hinge domain, a transmembrane domain, a peptide linker, and an intracellular domain.

[0054] In some embodiments, the CD22-specific binding domain comprises an antibody that binds CD22 or an antigen-binding fragment thereof.

[0055] In some embodiments, the CD22-specific binding domain comprises an antibody that binds human CD22.

[0056] In some embodiments, the CD22-specific binding domain comprises an antigenbinding fragment of an antibody that binds human CD22.

[0057] In some embodiments, the antigen-binding fragment of an antibody that binds human CD22 is a single chain variable fragment (scFv) that binds CD22.

[0058] In some embodiments, the scFv that binds CD22 has the sequence of QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRS KWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIW GQGTMVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGAttorney Docket No.: 063384-523001 WOKAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQ GTKLEIK (SEQ ID NO: 1).

[0059] In some embodiments, the scFv that binds 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.

[0060] In some embodiments, the CD22-specific CAR comprises a CD8a hinge domain comprising the sequence of TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 23) and a CD8a transmembrane domain comprising the sequence of IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 24) and optionally a peptide linker having the sequence of LYC.

[0061] In some embodiments, the CD22-specific CAR comprises a CD8a 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: 23 and a CD8a 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: 24.

[0062] In some embodiments, the CD8a transmembrane domain further comprises a spacer having the sequence of LYC.

[0063] In some embodiments, the CD22-specific CAR comprises an intracellular domain further comprising a primary T cell activating domain comprising an immunoreceptor tyrosine-based activation motif (IT AM) and a costimulatory signaling domain.

[0064] In some embodiments, the primary T cell activating domain comprising an IT AM comprises a CD3^ intracellular signaling domain.

[0065] In some embodiments, the CD3^ intracellular signaling domain comprises the sequence of RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR (SEQ ID NO: 26).

[0066] In some embodiments, the CD3C, 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: 26.

[0067] In some embodiments, the costimulatory signaling domain comprises a 4-1BB / CD137 signaling domain.Attorney Docket No.: 063384-523001 WO

[0068] In some embodiments, the 4-1BB / CD137 signaling domain comprises the sequence of KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 25).

[0069] 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: 25.

[0070] In some embodiments, the CD22-specific CAR comprises the sequence of QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIWGQGTMVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIW APLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEG GCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRR KNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALH MQALPPR (SEQ ID NO: 29).

[0071] In some embodiments, the CD22-specific 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: 29.

[0072] In some embodiments, the CD22-specific CAR comprises the sequence of MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAW NWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPED TAVYYCAREVTGDLEDAFDIWGQGTMVTVSSGGGGSDIQMTQSPSSLSASVGDRVT ITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQ AEDFATYYCQQSYSIPQTFGQGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACR PAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFM RPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREE YDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKG HDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 30).

[0073] In some embodiments, the CD22-specific 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: 30.

[0074] In some embodiments, the CD19-specific CAR comprises a CD19-specific binding domain, a transmembrane domain, and an intracellular domain.Attorney Docket No.: 063384-523001 WO

[0075] In some embodiments, the CD19-specific CAR comprises a CD19-specific binding domain, a hinge domain, a transmembrane domain, a spacer, and an intracellular domain.

[0076] In some embodiments, the CD19-specific binding domain comprises an antibody that binds CD 19 or an antigen-binding fragment thereof.

[0077] In some embodiments, the CD19-specific binding domain comprises an antibody that binds human CD 19.

[0078] In some embodiments, the CD19-specific binding domain comprises an antigenbinding fragment of an antibody that binds human CD 19.

[0079] In some embodiments, the antigen-binding fragment of an antibody that binds human CD 19 is a single chain variable fragment (scFv) that binds CD 19.

[0080] In some embodiments, the scFv capable of binding CD19 has the sequence of EVQLVQSGAEVKKPGESLKISCKASGYRFTNYWIAWVRQRPGKGLEWMGRIDPSDS YTHYSPSFQGHVTMSTDKSISTAYLQWSSLKASDTAMYYCARPGDILTGWAMDVW GQGTLVTVSSAAASGGGGSGGGGSGGGGSALQSVLTQPPSVSAAPGQKVTISCSGSS SNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQAEDE ADYYCQSYDSSLSGNYVFGTGTKVTVL (SEQ ID NO: 34).

[0081] In some embodiments, the scFv capable of binding CD 19 comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 34.

[0082] In some embodiments, the CD19-specific CAR comprises a CD28 hinge domain comprising the sequence of IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 43) and a CD28 transmembrane domain comprising the sequence of FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 44).

[0083] In some embodiments, the CD19-specific CAR comprises a CD28 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: 43 and a CD28 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: 44.

[0084] In some embodiments, the CD19-specific CAR comprises an intracellular domain further comprising a primary T cell activating domain comprising an immunoreceptor tyrosine-based activation motif (IT AM) and a costimulatory signaling domain.

[0085] In some embodiments, the primary T cell activating domain comprising an ITAM comprises a CD3C, intracellular signaling domain.Attorney Docket No.: 063384-523001 WO

[0086] In some embodiments, the CD3C, intracellular signaling domain comprises the sequence of RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR (SEQ ID NO: 26).

[0087] In some embodiments, the CD3C, 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 I D NO: 26.

[0088] In some embodiments, the costimulatory signaling domain comprises a CD28 signaling domain.

[0089] In some embodiments, the CD28 signaling domain comprises the sequence of RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 45).

[0090] In some embodiments, the CD28 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: 45.

[0091] In some embodiments, the CD19-specific CAR comprises the sequence of EVQLVQSGAEVKKPGESLKISCKASGYRFTNYWIAWVRQRPGKGLEWMGRIDPSDS YTHYSPSFQGHVTMSTDKSISTAYLQWSSLKASDTAMYYCARPGDILTGWAMDVW GQGTLVTVSSAAASGGGGSGGGGSGGGGSALQSVLTQPPSVSAAPGQKVTISCSGSS SNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQAEDE ADYYCQSYDSSLSGNYVFGTGTKVTVLIEVMYPPPYLDNEKSNGTIIHVKGKHLCPS PLFPGPSKPFWVLVWGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPG PTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY QGLSTATKDTYDALHMQALPPR (SEQ ID NO: 47).

[0092] In some embodiments, the CD19-specific 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: 47.

[0093] In some embodiments, the CD19-specific CAR comprises the sequence of MALPVTALLLPLALLLHAARPEVQLVQSGAEVKKPGESLKISCKASGYRFTNYWIA WVRQRPGKGLEWMGRIDPSDSYTHYSPSFQGHVTMSTDKSISTAYLQWSSLKASDT AMYYCARPGDILTGWAMDVWGQGTLVTVSSAAASGGGGSGGGGSGGGGSALQSV LTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDAttorney Docket No.: 063384-523001 WORFSGSKSGTSATLGITGLQAEDEADYYCQSYDSSLSGNYVFGTGTKVTVLIEVMYPPP YLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFIIFWV RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYKQ GQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAE AYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 48).

[0094] In some embodiments, the CD19-specific 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: 48.

[0095] In some embodiments, the CD20-specific CAR comprises a CD20-specific binding domain, a transmembrane domain, and an intracellular domain.

[0096] In some embodiments, the CD20-specific binding domain comprises an antibody that binds CD20 or an antigen-binding fragment thereof.

[0097] In some embodiments, the CD20-specific binding domain comprises an antibody that binds human CD20.

[0098] In some embodiments, the CD20-specific binding domain comprises an antigenbinding fragment of an antibody that binds human CD20.

[0099] In some embodiments, the antigen-binding fragment of an antibody that binds human CD20 is a single chain variable fragment (scFv) that binds CD20.

[0100] In some embodiments, the scFv capable of binding CD20 has the sequence of DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWA STRESGVPDRFSGSGSGSDFTLTISSLQAEDVAVYYCQQYYSFYQTFGQGTKVEIKGG GGSGGGGSGGGGSEVQLVESGGGWRPGGSLRLSCTASGFTFGDYGMSWVRQAPG KGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCA RKSYYGSGSPDVFDIWGQGTMVTVSS (SEQ ID NO: 50).

[0101] In some embodiments, the scFv capable of binding CD20 comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 50.

[0102] In some embodiments, the CD20-specific CAR comprises a CD28 hinge domain comprising the sequence of IEVM YPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 43) and a CD28 transmembrane domain comprising the sequence of FWVLVWGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 44).

[0103] In some embodiments, the CD20-specific CAR comprises a CD28 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: 43 and a CD28 transmembraneAttorney Docket No.: 063384-523001 WOdomain comprising a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 44.

[0104] In some embodiments, the CD20-specific CAR comprises an intracellular domain further comprising a primary T cell activating domain comprising an immunoreceptor tyrosine-based activation motif (IT AM) and a costimulatory signaling domain.

[0105] In some embodiments, the primary T cell activating domain comprising an IT AM comprises a CD3^ intracellular signaling domain.

[0106] In some embodiments, the CD3^ intracellular signaling domain comprises the sequence of RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR (SEQ ID NO: 26).

[0107] In some embodiments, the CD3C, 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: 26.

[0108] In some embodiments, the costimulatory signaling domain comprises a CD2 signaling domain.

[0109] In some embodiments, the CD2 signaling domain comprises the sequence of KRKKQRSRRNDEELETRAHRVATEERGRKPHQIPASTPQNPATSQHPPPPPGHRSQAP SHRPPPPGHRVQHQPQKRPPAPSGTQVHQQKGPPLPRPRVQPKPPHGAAENSLSPSSN(SEQ ID NO: 59).

[0110] In some embodiments, the CD2 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:59.

[0111] In some embodiments, the CD20-specific CAR comprises the sequence of MLLLVTSLLLCELPHPAFLLIPDIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKN YLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGSDFTLTISSLQAEDVAVYYC QQYYSFYQTFGQGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSC TASGFTFGDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKN SLYLQMNSLRAEDTALYYCARKSYYGSGSPDVFDIWGQGTMVTVSSIEVMYPPPYL DNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFIIFWVKR KKQRSRRNDEELETRAHRVATEERGRKPHQIPASTPQNPATSQHPPPPPGHRSQAPSH RPPPPGHRVQHQPQKRPPAPSGTQVHQQKGPPLPRPRVQPKPPHGAAENSLSPSSNRAttorney Docket No.: 063384-523001 WOVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 64).

[0112] In some embodiments, the CD20-specific 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: 64.

[0113] In some embodiments, the CD20-specific CAR comprises the sequence of DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWA STRESGVPDRFSGSGSGSDFTLTISSLQAEDVAVYYCQQYYSFYQTFGQGTKVEIKGG GGSGGGGSGGGGSEVQLVESGGGWRPGGSLRLSCTASGFTFGDYGMSWVRQAPG KGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCA RKSYYGSGSPDVFDIWGQGTMVTVSSIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPL FPGPSKPFWVLVWGGVLACYSLLVTVAFIIFWVKRKKQRSRRNDEELETRAHRVAT EERGRKPHQIPASTPQNPATSQHPPPPPGHRSQAPSHRPPPPGHRVQHQPQKRPPAPS GTQVHQQKGPPLPRPRVQPKPPHGAAENSLSPSSNRVKFSRSADAPAYKQGQNQLY NELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGM KGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 62).

[0114] In some embodiments, the CD20-specific 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: 62.

[0115] Also provided herein is a method of making a population of CAR-expressing immune cells, the method comprising: obtaining a liquid sample comprising an initial population of autologous cells comprising immune cells from a human subject; processing the initial population of autologous cells generate a washed population of cells comprising immune cells; on day 0, enriching the washed population of cells comprising immune cells for CD4+ and CD8+ T cells to generate an enriched population of cells comprising immune cells, and cul turing the enriched popul ation of cells in a volume of a medium comprising an activation agent to generate an enriched and activated population of cells comprising immune cells, wherein the enriched and activated population of cells is seeded with at least 3. Ox 108cells from the activated population of cells into a volume of at least 70 mL of a medium comprising IL-7 and IL- 15; transducing the enriched and activated population of cells comprising immune cells with one or more recombinant polynucleotides encoding a CD19-specific CAR, a CD20-specific CAR, and a CD22-specific CAR, thereby generating an enriched, activated, and transduced population of cells comprising CAR-expressing immuneAttorney Docket No.: 063384-523001 WOcells; adding a volume of first medium to the enriched, activated, and transduced population of cells on Day 2 after the culturing step in (c) to bring the total volume to at least 1000 mL; expanding the enriched, activated, and transduced population of cells in media to yield an expanded population of enriched, activated, and transduced cells comprising CAR-expressing immune cells; determining the number of viable CD3+ CAR-expressing T cells; and either continuing to expand the enriched, activated, and transduced population of cells and exchanging the medium, or harvesting the enriched, activated, and transduced population of cells comprising CAR-expressing immune cells on or before Day 9 after the seeding in step (c); and formulating the harvested population of cells for cryopreservation and administration to patients.BRIEF DESCRIPTION OF THE DRAWINGS

[0116] FIG. 1 shows the experimental workflow for comparison of process performance and drug product using fresh and cryopreserved (“cryo”) apheresis product.

[0117] FIG. 2 shows enrichment recovery, as defined by total target cells enriched divided by total target cells loaded onto CliniMACS® Prodigy (Target = CD4+ and CD8+ of CD45+).

[0118] FIG. 3 shows the percentage of cell loss as measured from culture day 0 seeding at 300 x 106target Total Viable Cells (TVC) to pre-transduction on day 1.

[0119] FIG. 4 shows the number of fold expansions between the study arms for each donor as measured by the TVC at harvest day 5 divided by the seeding density of 300*106on day 0.

[0120] FIG. 5 shows the paired-wise comparisons of tri-CAR transduction efficiency as measured from in-process day 5 cellular material.

[0121] FIG. 6 shows total CAR+ cells at day 5 harvest between the cryo and fresh conditions for the three donors investigated in the study.

[0122] FIG. 7 shows Vector Copy Number (VCN) / Tri-CAR+ cell for drug product from fresh or cryo Apheresis.

[0123] FIG. 8 shows T cell memory subsets in drug product manufactured from fresh or cryopreserved apheresis.

[0124] FIG. 9 shows T cell activation markers in drug product manufactured from fresh or cryopreserved apheresis.

[0125] FIG. 10 shows individual T cell exhaustion marker expression in drug product manufactured from fresh or cryopreserved apheresis.Attorney Docket No.: 063384-523001 WO

[0126] FIG. 11 shows LAG-3+PD-l+TIGIT+TIM-3+exhaustion marker co-expression in drug product manufactured from fresh or cryopreserved apheresis.

[0127] FIG. 12 shows cell health in drug product manufactured from fresh or cryopreserved apheresis.

[0128] FIG. 13 shows IFNy secretion in fresh and cryo drug product samples.

[0129] FIG. 14 shows Total Viable Count (TVC) from Day 5 to Day 9 (No Split).

[0130] FIG. 15 shows Viability (%) of Day 5, Day 7, and Day 9 Culture (No Split).

[0131] FIG. 16 shows cumulative doublings across harvest day (No Split).

[0132] FIG. 17 shows total viable count from Day 5 to Day 9 (Split).

[0133] FIG. 18 shows cumulative doublings across harvest day in (Split).

[0134] FIG. 19 shows CD3+ out of CD45+ of Live Cells (No Split).

[0135] FIG. 20 shows transduction efficiency (no split) and transduction efficiency (Split).

[0136] FIG. 21A shows CD4+ / CD8+ Ratio (No Split).

[0137] FIG. 21B shows CD4+ / CD8+ Ratio (Split).

[0138] FIG. 22A shows total Tri-CAR-l- Cells (No Split).

[0139] FIG. 22B shows total Tri-CAR-l- Cells (Split).

[0140] FIG. 23 shows growth curves of transduced T Cells using LV-023 65L Pilot Lot 24 P009 (OXB) and LV-023 5L Lot 23_E082 (OXB) at various MOIs.

[0141] FIG. 24 shows a small-scale LV-023 65L Pilot Lot 24 P009 (OXB) titration curve for Donors ENO 1000800 and BOO 1001987.

[0142] FIG. 25A shows small-scale titration VCN per Tri-CAR-l- Cell transduced with LV- 023 65L Pilot Lot 24 P009 (OXB).

[0143] FIG. 25B shows small-scale titration VCN per tri-CAR-l- Cell transduced with LV-023 65L Pilot Lot 24 P009 (OXB).

[0144] FIG. 26 shows CD3+CD45+ purity of CRG-023 produced using LV-023 65L Pilot Lot 24 P009 (OXB) in At-Scale Healthy Donors BOO 1002139, DOO 1006680, DOO 1006110.

[0145] FIG. 27 shows healthy donor cell fold expansion, using LV-023 65 L Pilot Lot 24 P009 (OXB), from Day 0 to Day 5.

[0146] FIG. 28 shows healthy donor cell viability, throughout the process, transduced with LV-023 65L Pilot Lot 24 P009 (OXB).

[0147] FIG. 29 shows healthy donor total viable cells (TVC) transduced with LV-023 65L Pilot Lot 24 P009 (OXB).

[0148] FIG. 30 shows transduction efficiency of healthy donor cells transduced with LV-023 65L Pilot Lot 24 P009 (OXB).Attorney Docket No.: 063384-523001 WO

[0149] FIG. 31 shows total CRG-023 Viable CD3+ Tri-CAR+ cells at harvest (Day 5) transduced with LV-023 65L Pilot Lot 24 P009 (OXB).

[0150] FIG. 32 shows CD3+CD45+ purity of CRG-023 produced using LV-023 65L Pilot Lot 24 P009 (OXB) in DLBCL Patient Donors.

[0151] FIG. 33 shows DLBCL patient-derived material total viable cells, throughout the process, transduced with LV-023 65L Pilot Lot 24 P009 (OXB).

[0152] FIG. 34 shows DLBCL patient-derived material viability, throughout the process, transduced with LV-023 65L Pilot Lot 24 P009 (OXB).

[0153] FIG. 35 shows transduction efficiency of DLBCL patient-derived material, throughout the process, transduced with LV-023 65L Pilot Lot 24 P009 (OXB).

[0154] FIG. 36 shows total CRG-023 viable CD3+ Tri-C AR+ Cells, throughout the process, transduced with LV-023 65L Pilot Lot 24 P009 (OXB).

[0155] FIG. 37 shows D001004593 titration with LV-023 Lot 2602-*-*-l (OXB) Cell Growth.

[0156] FIG. 38 shows DOO 1004593 titration with LV-023 Lot 24P0017 (YPK) Cell Growth.

[0157] FIG. 39 shows D001003932 titration with LV-023 Lot 2602-*-*-l (OXB) Cell Growth.

[0158] FIG. 40 shows DOO 1003932 LV-023 Lot 24P0017 (YPK) cell growth.

[0159] FIG. 41 shows small scale LV-023 Lot 2602-*-*- 1 (OXB) titration curve of thawed drug product.

[0160] FIG. 42 shows small scale LV-023 Lot 24P0017 (YPK) titration curve of thawed drug product.

[0161] FIG. 43A shows small scale titration VCN per Tri-CAR+ cell transduced with LV-023 Lot 2602-*-*-l (OXB).

[0162] FIG. 43B shows small scale titration VCN per Tri-CAR+ cell for healthy donor D001003932 transduced with LV-023 Lot 2602-*-*-l (OXB).

[0163] FIG. 43C shows small scale titration VCN per Tri-CAR+ cell for healthy donor D001004593 transduced with LV-023 Lot 2602-*-*-l (OXB).

[0164] FIG. 44A shows small scale titration VCN per Tri-C AR+ cell transduced with LV-023 Lot 24P0017 (YPK).

[0165] FIG. 44B shows small scale titration VCN per Tri-CAR+ cell for healthy donor DOO 1003932 transduced with LV-023 Lot 24P0017 (YPK).

[0166] FIG. 44C shows small scale titration VCN per Tri-CAR+ cell for healthy donor DOO 1004593 transduced with LV-023 Lot 24P0017 (YPK).Attorney Docket No.: 063384-523001 WO

[0167] FIG. 45 shows CD3+CD45+ purity of CRG-023 produced using LV-023 lot 2602-*-*-l (OXB) and LV-023 Lot 24P0017 (YPK) - Healthy Donors D001004593, D001006875, and D001003932.

[0168] FIG. 46 shows healthy donor total viable cells (TVC) throughout the process. Red curves represent LV-023 Lot 2602-*-*-! (OXB), Blue curves represent LV-023 Lot 24P0017 (YPK).

[0169] FIG. 47 shows healthy donor cell viability throughout the process. Red curves represent LV-023 Lot 2602-*-*-! (OXB), Blue curves represent LV-023 Lot 24P0017 (YPK).

[0170] FIG. 48 shows transduction efficiency of healthy donor runs at harvest, n=l per LV-023 Lot per Donor (total n==2 per LV-023 lot).

[0171] FIG. 49 shows Total Tri-CAR+ cells at harvest for healthy donors, n=l per LV-023 Lot per Donor.

[0172] FIG. 50 shows VCN / Tri-CAR+ cell for DP transduced with LV-023 Lot 2602-*-*-l (OXB) and LV-023 Lot 24P0017 (YPK), n=3.

[0173] FIG. 51 shows CD3+CD45+ Purity of DLBCL 2215 113-1013 patient-derived material, produced using LV-023 Lot 2602-*-*-! (OXB), throughout the process.

[0174] FIG. 52 shows DLBCL 2215 113-1013 patient-derived material total viable cells, produced using LV-023 Lot 2602-*-*-! (OXB), throughout the process.

[0175] FIG. 53 shows transduction Efficiency of DLBCL 2215 113-1013 patient-derived material, using LV-023 Lot 2602-*-*- 1 (OXB), throughout the Process.

[0176] FIG. 54 shows total tri-CAR+ Cells of DLBCL 2215 113-1013 patient-derived material, using LV-023 Lot 2602-*-*- 1 (OXB), throughout the Process.

[0177] FIG. 55 shows VCN / Tri-CAR+ cell in DLBCL 2215 113-1013 patient-derived drug product at various seeding densities and harvest days using LV-023 Lot 2602-*-*- 1 (OXB).

[0178] FIG. 56 shows CAR+CD4+ memory phenotype in DLBCL 2215 113-1013 patient-derived drug product at various seeding densities and harvest days using LV- 023 Lot 2602-*-*-1 (OXB).

[0179] FIG. 57 shows C AR+CD8+ memory phenotype in DLBCL 2215 113-1013 patient-derived drug product at various seeding densities and harvest days using LV- 023 Lot 2602- *-*-1 (OXB).

[0180] FIG. 58 shows CAR+CD4+ T Cell activation markers on DLBCL 2215_113-1013 patient- derived drug product at different seeding densities and harvest days using LV- 023 Lot 2602-*-*-! (OXB).Attorney Docket No.: 063384-523001 WO

[0181] FIG. 59 shows CAR+CD8+ t cell activation markers on DLBCL 2215 113-1013 patient- derived drug product at different seeding densities and harvest days using LV- 023 Lot 2602-*-*-l (OXB).

[0182] FIG. 60 shows exhaustion marker co-expression on DLBCL 2215 113-1013 patient-derived drug product at different seeding densities and harvest days using LV- 023 Lot 2602-*-*-1 (OXB).

[0183] FIG. 61 shows CAR+ CD4 / CD8 ratio in DLBCL 2215 113-1013 patient-derived drug product at different seeding densities and harvest days using LV-023 Lot 2602-*-*-l (OXB).

[0184] FIG. 62 shows IFNy production across harvest days in four different target cell lines expressing the CD 19 C AR.

[0185] FIG. 63 shows IFNy across harvest days in four different target cell lines expressing the CD20 CAR.

[0186] FIG. 64 shows IFNy across harvest days in four different target cell lines expressing the CD22 CAR.

[0187] FIG. 65 shows IFNy across harvest days in four different Triple Knock-Out (TKO) target cell lines.DETAILED DESCRIPTION OF THE DISCLOSURE

[0188] The present disclosure generally relates to, inter alia, improved methods of making and / or manufacturing a population of immune cells expressing one or more chimeric antigen receptors (CARs). 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.

[0189] As discussed in greater detail below, the present disclosure provides methods that allow an 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 successful production of drug product from apheresis starting material of varying quality. The manufactured product is highly enriched in more naive T cell memory subpopulations (central memory T cells (TCM) in particular) and exhibits significant potency as demonstrated by secretion of IFNy.

[0190] 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 obtaining aAttorney Docket No.: 063384-523001 WOliquid sample comprising an initial population of autologous cells comprising immune cells from a human subject; processing the initial population of autologous cells to generate a washed population of cells comprising immune cells; on day 0, enriching the washed population of cells comprising immune cells for CD4+ and CD8+ T cells to generate an enriched population of cells comprising immune cells, and culturing the enriched population of cells in a volume of a medium comprising an activation agent to generate an enriched and activated population of cells comprising immune cells; transducing the enriched and activated population of cells comprising immune cells with one or more recombinant polynucleotides encoding three CARs thereby generating an enriched, activated, and transduced population of cells comprising CAR-expressing immune cells; expanding the enriched, activated, and transduced population of cells comprising CAR-expressing immune cells in growth medium to yield an expanded population of enriched, activated, and transduced cells comprising CAR-expressing immune cells; harvesting the expanded population of enriched, activated, and transduced cells comprising CAR-expressing immune cells on or before Day 9 after the seeding; and formulating the harvested population of enriched, activated, and transduced cells comprising CAR-expressing immune cells for cryopreservation and administration to patients.DEFINITIONS

[0191] 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.

[0192] Certain ranges are presented herein with numerical values being preceded by the term “about” which, as used herein, has its ordinary meaning of approximate.

[0193] 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 singleAttorney Docket No.: 063384-523001 WOembodiment. 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 discl osure 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

[0194] 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 one or more of the following steps: cell collection, cell processing e.g., washing and / or concentration), cell cryopreservation, cell thawing, cell seeding, cell transduction, cell expansion, 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.

[0195] 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 comprising an initial population of autologous cells comprising immune cells from a human subject; (b) processing the initial population of autologous cells to generate a washed population of cells comprising immune cells; (c) on day 0, enriching the washed population of cells comprising immune cells for CD4+ and CD8+ T cells to generate an enriched population of cells comprising immune cells, and culturing the enriched population of cells in a volume of a medium comprising an activation agent to generate an enriched and activated population of cells comprising immune cells; (d) transducing the enriched and activated population of cells comprising immune cells with one or more recombinant polynucleotides encoding three CARs thereby generating an enriched, activated, and transduced population of cells comprising CAR-expressing immune cells; (e) expanding the enriched, activated, and transduced population of cells comprising CAR-expressing immune cells in medium to yield an expanded population of enriched, activated, and transduced cells comprising CAR-expressing immune cells; (f) harvesting the expanded population of enriched, activated, and transduced cells comprising CAR-expressing immuneAttorney Docket No.: 063384-523001 WOcells on or before Day 9 after the seeding in step (c); and (g) formulating the harvested population of enriched, activated, and transduced cells comprising CAR-expressing immune cells for cryopreservation and administration to patients.Cell Collection

[0196] The first step in the method described herein involves obtaining a liquid sample comprising an initial population of autologous cells comprising immune cells from a human subject.

[0197] In some embodiments, a human subject can be a patient under the care of a physician. Thus, the subject can be a human patient 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 a human who is diagnosed with a risk of the health condition of interest at the time of diagnosis or later.

[0198] As described above, the liquid sample comprises an initial population of autologous (i.e., patient-derived) cells comprising immune cells. In some embodiments, the liquid sample including an initial population of autologous cells comprising immune cells comprises a leukapheresis product or an apheresis product. As used herein, the terms “leukapheresis” and “apheresis” are used interchangeably to refer to a process involving the removal of certain blood components (e.g., stem cells, platelets, white blood cells, red blood cells) from subject’s bloodstream.

[0199] The term “immune cells” used in reference to the cells present in the liquid sample 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. In some instances, the T cells are CD8-positive T cells, CD4-positive T cells, regulatory T cells, cytotoxic T cells, or tumor infiltrating lymphocytes. The immune cells can also be precursor cells, e.g., cells that are capable of differentiating into immune cells.

[0200] In some embodiments, the initial population of 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 (TCTLS), helper T cells (TH) or tumor infdtrating lymphocytes (TILs). In some instances, the mammal is a primate. In some instances, the primate is a human.Attorney Docket No.: 063384-523001 WO

[0201] T cells can also 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 coll ected from an individual using any number of known techniques such as sedimentation, e.g., FICOLL™ separation or apheresis. In some embodiments, the T cells are from a blood sample. In some embodiments, the T cells are obtained by apheresis.

[0202] 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.. 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. Suitable approaches for such sorting are known and include, e.g., magnetic-activated cell sorting (MACS), where TN are CD45RO-CD45RA+ CD62L+ CD95-; TSCM are CD45RO-CD45RA+CCR7+; TCM are CD45RO+CD45RA-CCR7+; TMEM are CD45RO+, (TEFF) are CD45RO+CD45RA-CCR7-, and TEM are CD45RO+CCR7-CD45RA-. An exemplary approach for such sorting is described in Wang et al., (2016) Blood 127(24):2980- 90.

[0203] In some instances, the initial population of autologous cells is a heterogeneous mixture of cells of different cell types. In some instances, the initial population of cells comprises a heterogenous mixture of different cell types comprising at least about 1 x 104cells, about IxlO5cells, about IxlO6cells, about IxlO7cells, about IxlO8cells, or about IxlO9cells. In some instances, the initial population of cells comprises at least about IxlO9cells. In some instances, the population of cells comprises at least about 2x 109cells. In some instances, the population of cells comprises at least about 3X109cells. In some instances, the population of cells comprises at least about 4xl09cells. In some instances, the population of cells comprises at least about 5 x 109cells. In some instances, the population of cells comprises at least about 6X109cells. In some instances, the population of cells comprises at least about 7xl09cells.Cell ProcessingAttorney Docket No.: 063384-523001 WO

[0204] Following obtention of the initial population of autologous cells, the initial population of autologous cells is processed.

[0205] In some embodiments, the processing of the initial population of cells comprises the step of washing the cells to generate a washed population of cells comprising immune cells.

[0206] In some embodiments, the initial population of cells is processed by washing the cells in a buffer comprising HSA.

[0207] In some embodiments, the concentration of HSA in the buffer 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).

[0208] In some embodiments, the wash buffer may include HSA, Plasma-Lyte A®, phosphate buffered saline (PBS), ethylenediamine tetraacetic acid (EDTA), sodium chloride, sodium bicarbonate buffer, glutathione, biotin, vitamin B12, inositol, choline, L-glutamine, sodium pyruvate, glucose, or a combination thereof.

[0209] In some embodiments, the initial population of cells is processed by washing the cells in buffer comprising PBS / 1 mM EDTA + 0.5% HSA.

[0210] In some embodiments, the method then proceeds to the enrichment and activation step (c) as further described in the next section.

[0211] In some embodiments, the processing of the initial population of cells further includes a step of cryopreserving the initial population of cells to generate a population of cryopreserved cells before proceeding to the enrichment and activation step (c).

[0212] In instances where the initial population of cells is cryopreserved, in some embodiments, the processing step includes the steps of washing the initial population of cells, concentrating the initial population of cells, and eluting and / or resuspending the initial population of cells, thereby generating a washed and concentrated population of cells.

[0213] In some embodiments, the processing of the initial popula tion of cells further comprises a step of reducing the number of platelets in the initial population of cells. In someAttorney Docket No.: 063384-523001 WOembodiments, the washed population of cells is washed, concentrated, and eluted or resuspended and the number of platelets is reduced by elutriation using an automated system. In some embodiments, the automated system comprises a spinning membrane filtration system. In some embodiments, the spinning membrane filtration system is a Cue Cell Processing System.

[0214] In some embodiments, the washed and concentrated population of cells is eluted or resuspended in a buffer comprising human serum albumin (HSA), Plasma-Lyte A, phosphate buffered saline (PBS), sodium chloride, ethylenediamine tetraacetic acid (EDTA), 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.

[0215] In some embodiments, the washed and concentrated 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 can 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 can be diluted after elution / resuspension into a dilution buffer. The dilution buffer may include dimethyl sulfoxide (DMSO), sucrose, sodium hydroxide, potassium hydroxide, fructose, HSA, Plasma-Lyte A®, phosphate buffered saline (PBS), 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 lOmL, 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 lOOmL, 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 IL, 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 popula tion 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 theAttorney Docket No.: 063384-523001 WOcell population (i.e., a 1:1 dilution). In some embodiments, the cell population may be cryopreserved after transfer to a sterile container. In some embodiments, the cell population can 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.

[0216] 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 to generate a cryopreserved population of cells. In some embodiments, the cryoprotectant is CryoStor®.T Cell Enrichment and Activation

[0217] As described above, following the processing of the initial population of autologous cells to generate the washed, washed and concentrated, or cryopreserved population of cells, the method described herein next involves the step of, on day 0, enriching the washed, washed and concentrated, or cryopreserved population of cells comprising immune cells for CD4+ and CD8+ T cells to generate an enriched population of cells comprising immune cells, and culturing the enriched population of cells in a volume of a medium comprising an activation agent to generate an enriched and activated population of cells comprising immune cells.

[0218] In embodiments where a cryopreserved population of cells has been generated, the cryopreserved population of cells is thawed before it is enriched and activated in step (c) to generate a thawed population of cells. For purposes of the methods described herein, Day 0 is the day on which a freshly processed is enriched and activated or a processed and cryopreserved leukapheresis or apheresis product is thawed, enriched, and activated.

[0219] In some embodiments, the enriching for CD4+ and CD8+ T cells further 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 population of cells.

[0220] In some embodiments, the amount of cryopreserved apheresis product (i.e., the cryopreserved population of cells) to be thawed for processing to enrich for CD4+ and CD8+ T cells is determined by calculating the following: Target Cells in each aliquot of cryopreserved Apheresis Product = Viable Cell Density post-Cue Cell Processing (cells / mL)xvolume of aliquot (mL) x (CD4% + CD8%). The number of aliquots ofAttorney Docket No.: 063384-523001 WOcryopreserved Apheresis Product to be thawed for use in the enrichment step is determined to be the amount that results in at least l. OxlO9CD3+ cells and the closest in absolute value to 3.0xl09CD3+ cells.

[0221] In some embodiments, the enriching for CD4+ and CD8+ T cells comprises a step mixing the washed, washed and concentrated, or thawed population of cells with magnetic beads derivatized with CD4-specific binding agents and CD8-specific binding agents, followed by washing and eluting to generate the enriched population of cells. In some embodiments, this enrichment step takes place within a CliniMACS Prodigy® system.

[0222] Following generation of the enriched population of cells comprising CD4+ and CD8+ T cells, the enriched population of cells is cultured in a volume of a medium comprising an activation agent to generate an enriched and activated population of cells comprising CD4+ and CD8+ immune cells.

[0223] In some embodiments, the enriched population of cells is cultured by seeding 3xl08cells from the enriched population of cells in a vessel. In some embodiments, the enriched population of cells is cultured by seeding 2.5xl06cells from the enriched population of cells in a vessel. In some embodiments, the enriched population of cells is cultured by seeding between at least 2.5x106cells and at least 3×108cells from the enriched population in a vessel. In some embodiments, the enriched population of cells is cultured by seeding at least about 2.5xl06, 3xl06, 3.5xl06, 4xl06, 4.5xl06, 5xl06, 5.5xl06, 6xl06, 6.5xl06, 7xl06, 7.5xl06, 8xl06, 8.5xl06, 9xl06, 9.5xl06, IxlO7, 1.5xl07, 2xl07, 2.5 xlO7, 3xl07, 3.5xl07, 4xl07, 4.5xl07, 5xl07, 5.5xl07, 6xl07, 6.5xl07, 7xl07, 7.5xl07, 8xl07, 8.5xl07, 9xl07, 9.5xl07, IxlO8, 1.5xl08, 2xl08, 2.5X108, 3xl08, 3.5xl08, 4xl08, 4.5xl08, or 5xl08cells from the enriched population in a vessel. In some embodiments, the vessel is a G-REX® vessel.

[0224] In some embodiments the enriched population of cells is seeded in modified T Cell Culture Medium. In some embodiments the enriched population of cells is seeded in modified T Cell Culture Medium to bring the total culture volume to 70 mL. In some embodiments, the modified T Cell Culture Medium comprises Prime XV CDM Medium supplemented with a 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 (hlL-15) comprises 12.5 ng / mL hIL-7 and 12.5 ng / mL hIL-15. In some embodiments, the modified T Cell Culture 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 Trans Act™ reagent.Attorney Docket No.: 063384-523001 WOTransduction

[0225] As described for the methods herein, the enriched and activated population of cells is then transduced with one or more recombinant polynucleotides encoding three CARs thereby generating an enriched, activated, and transduced population of cells comprising CAR-expressing immune cells.

[0226] Transduction can refer to a process by which exogenous nucleic acid is transferred or introduced into the host cell. For example, a “transduced” cell is one which has been transduced with exogenous nucleic acid, e.g., a lentiviral expression vector encoding one or more CARs. The term “transduced” can include the primary subject cell and its progeny.

[0227] In some embodiments, the one or more recombinant polynucleotides encoding three C ARs further comprises one or more lentiviral expression vectors. In some embodiments, one or more lentiviral expression vectors comprises one lentiviral expression vector comprising one recombinant polynucleotide encoding three separate and distinct CARs.

[0228] A lentiviral vector as used herein can refer 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. As used herein, 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. In addition, 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 used in immune cell engineering. HIV, SIV, and FIV are all examples of lentiviruses.

[0229] A “lentiviral expression vector” can refer to a lentiviral vector including a recombinant polynucleotide to be expressed and various expression control sequences operatively linked to the recombinant nucleotide sequence. A lentiviral 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. 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 C AR construct may be incorporated into a lentiviral vector backbone, which may then be packaged using a packaging system known in the art. Vector backbones suitable for use include the pCCL-c-MNDU3-X expression vector for use in mammalian cells. In some embodiments, nucleic acid inserts encoding the subject CARs can be operably linked to a promoter, which is selected based on, for example, the cell type in which expression is sought. In some embodiments, the pCCL-c-MNDU3-XAttorney Docket No.: 063384-523001 WOexpression vector further comprises the MNDU3 promoter. In some embodiments, the MNDU3 promoter is replaced with the EFla promoter.

[0230] 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 of the lenti virus construct into T cells. In some embodiments of the disclosure, immune cells are transduced with a lentiviral expression vector comprising one recombinant polynucleotide encoding three separate and distinct CARs, and the lentiviral expression vector is 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 comprising one recombinant polynucleotide encoding three separate and distinct CARs, and the lentiviral expression vector is produced using a suspension cell culture -based LV manufacturing platform.

[0231] In some embodiments, the three CAR constructs comprise a CD19-specific CAR, a CD20-specific CAR, and a CD22-specific CAR. Such CAR constructs are described in more detail in sections herein below.

[0232] In some embodiments, the transduction in step (d) is performed on Day 1. In some embodiments, the transduction in step (d) is performed 22-26 hours after culturing of the enriched and activated population of cells comprising immune cells on Day 0.

[0233] In some embodiments, the amount of vector used to transduce the enriched and activated population of cells comprising immune cells in step (d) is determined based on the infectious titer of the l entiviral vector and the number of cells used to culture the enriched and activated population of cells comprising immune cells such that the transduction is performed with a multiplicity of infection (MOI) of 3.0. In some embodiments, the transduction is performed with a MOI of at least 1.3, at least 1.4, 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.Attorney Docket No.: 063384-523001 WO

[0234] In some embodiments, the one or more lentiviral expression vectors comprising one or more recombinant polynucleotides encoding three CARs is thawed, diluted into modified T Cell Culture Medium, and added to the enriched and activated population of cells comprising immune cells. In some embodiments, the modified T Cell Culture Medium comprises Prime XV CDM Medium supplemented with a 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 final volume in which the transduction in step (d) is performed comprises 100 mL.

[0235] In some embodiments, an additional culture feeding step is performed. In some embodiments, 22-26 hours after transduction, pre-warmed T Cell Culture Medium is added to the transduced population of cells comprising immune cells. In some embodiments, 900 mL of pre-wanned T Cell Culture Medium is added to the vessel to bring the total volum e of the culture to 1000 mL.Expansion and Harvest

[0236] As described herein, following the transduction step (d), the enriched, activated, and transduced population of cells comprising CAR-expressing immune cells is expanded in growth medium to yield an expanded population of enriched, activated, and transduced cells comprising CAR-expressing immune cells, e.g., CD4+ and CD8+ T cells comprising TN, TMEM, TSCM, TCM, TEM, and TEFF T cell subpopulations.

[0237] In some embodiments, the expansion step (e) further includes removing at least 50% of the volume of the medium on or before Day 4 after the culturing in step (c). In some embodiments, approximately 700 mL of the medium is removed from the vessel.

[0238] In some embodiments, the expansion step (e) comprises determining the number of viable CD3+ CAR-expressing T cells, continuing to expand the expanded population of cells, and exchanging the medium. In some embodiments, the number of viable CD3+ C AR-expressing T cells is determined on Day 4 by taking a sample of the expanded population of cells and calculating the amount of CAR+ cells in the cell culture based on the transduction efficiency.

[0239] For the purposes of the methods described herein, the harvesting step (I) is triggered based on meeting a minimum harvest threshold (MHT). The MHT refers to the required number of viable CAR+ T cells needed to produce a product dose, product samples for release testing and retains, product samples for extended characterization, while alsoAttorney Docket No.: 063384-523001 WOaccounting for harvest operation losses. The MHT selected depends upon the desired product dose. In some embodiments, the desired product dose is 25 x 106CAR+ cells, and the MHT is 115×106viable CAR+ cells. In some embodiments, the desired product dose is 75xl06CAR+ cells, and the the MHT is 177×106viable CAR+ cells. In some embodiments, the desired product dose is 150x106CAR+ cells, and the MHT is 269×106viable CAR+ cells.

[0240] In some embodiments, when the am ount of viable CAR+ cells on Day 4 is greater than a minimum harvest threshold of 115×106, 177×106or 269×106viable CAR+ cells, the harvesting step (I) is performed on Day 5.

[0241] In some embodiments, when the amount of viable CAR+ cells on Day 4 is less than a minimum harvest threshold of 115×106, 177×106or 269×106viable CAR+ cells, the expanded population of cells is expanded for another day, a sample of the expanded population of cells is taken on Day5, and the number of viable CAR+ cells is recalculated.

[0242] In some embodiments, when the amount of viable CAR+ cells on Day 5 is greater than the minimum harvest threshold of 115×106, 177×106or 269×106viable CAR+ cells the harvesting step (f) is performed on Day 5.

[0243] In some embodiments, when the amount of viable CAR+ cells on Day 5 is less than a minimum harvest threshold of 115×106, 177×106or 269×106viable CAR+ cells, the expanded population of cells is expanded for another day, a sample of the expanded population of cells is taken on Day 6 and the number of viable CAR+ cells is recalculated.

[0244] In some embodiments, when the am ount of viable CA R+ cells on Day 6 is greater than the minimum harvest threshold of 115 x 106, 177×106or 269×106viable CAR+ cells, and the harvesting step (!) is performed on Day 7.

[0245] In some embodiments, when the amount of viable CAR+ cells on Day 6 is less than a minimum harvest threshold of 115×106, 177×106or 269×106viable CAR+ cells, the expanded population of cells is expanded for another day, a sample of the expanded population of cells is taken on Day 8 and the number of viable CAR+ cells is recalculated.

[0246] In some embodiments, when the amount of viable CAR+ cells on Day 8 is greater than the minimum harvest threshold of 115×106, 177×106or 269×106viable CAR+ cells, and the harvesting step (I) is performed on Day 9.

[0247] In some embodiments, the expanded population of CAR+ cells is harvested on Day 5, Day 6, Day 7, Day 8, or Day 9. In some embodiments, the expanded population of CAR+ cells is harvested on Day 5, 7, and / or Day 9. In some embodiments, the expanded population of CAR+ cells is harvested on Day 5. In some embodiments, the expanded population ofAttorney Docket No.: 063384-523001 WOCAR+ cells is harvested on Day 7. In some embodiments, the expanded population of CAR+ cells is harvested on Day 9.

[0248] In some embodiments, the minimum harvest threshold is 269x106viable CAR+ cells. In some embodiments, the minimum harvest threshold is 115 x 106viable CAR+ cells. In some embodiments, the minimum harvest threshold is 177×106viable CAR+ cells.

[0249] In some embodiments, the harvesting step (I) comprises removing media from the expanded population of viable CAR+ cells and resuspending the cells (in what?).

[0250] In some embodiments, the harvested population of cells including viable CAR-expressing immune cells comprises autologous CAR-expressing immune cells.

[0251] In some embodiments, the harvested population of cells including CAR-expressing immune cells comprises autologous T cells expressing a CD19-specific C AR, a CD20-specific CAR, and a CD22-specific CAR.

[0252] In some embodiments, at least 70%, 75%, 80%, 85%, or 90% of the cells in the harvested population of cells are viable. In some embodiments, the number of viable cells is measured by flow cytometry for CAR+CD4+ and CAR+CD8+ cells. In some embodiments, the number of viabl e cells is measured by flow cytometry for CAR+CD4+ and C AR+CD8+ cells with viable cells defined as annexin V7-AAD", dying cells defined as annexin V+7-AAD", and dead cells defined as annexin V^7-AAD+. In some embodiments, the harvested population of cells is cryopreserved after harvesting.

[0253] In some instances, the harvested population of cells does not substantially express one or more of the following exhaustion-specific markers: PD-1, TIGIT, TIM3, and LAG3.

[0254] 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. 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). 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 TAttorney Docket No.: 063384-523001 WOcells (TTM), effector T cells (TEFF), or combinations 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.

[0255] In some embodiments, at least 0.1% of cells in the harvested population of cells are CCR7+CD45RA+ immune cells. In some embodiments, at least 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, or 2.0% of cells in the harvested population of cells are CCR7+CD45RA+ T stem celllike 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). 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, the CCR7+CD45RA+CD3+ immune cells are also CD4+ (i.e., CCR7+CD45RA+CD3+CD4+).

[0256] In some embodiments, at least 74.1%, 75.0%, 75.5.0%, 76.0%, 76.5%, 77.0%, 77.5%, 78.0%, 78.5%, 79%, 79.5%, 80.0%, 80.5%, 81.0%, 81.5%, 82.0%, 82.1%, 83.0%, 84.0%, 85.0%, 86.0%, 87.0%, 88.0%, 88.8%, 89.0%, 89.8% 90.0%, 91.0%, 91.9%, 92.0%, 92.2%, 92.3%, 92.9%, 93.0%, 93.1%, 93.4%, 93.6%, 93.8%, 94.0%, 94.9%, 95.0%, 95.1%, 96.0%, 96.5%, 97.0%, 97.6%, 98.0%, 98.2%, or 99.0% of the cells in the harvested population of cells are CCR7+CD45RA- T central memory (TCM) 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, the CCR7+CD45RA-CD3+ immune cells are also CD4+ (i.e., CCR7+CD45RA-CD3+CD4+).

[0257] In some embodiments, the harvested population of CAR+ immune cells secretes IFNy. In some embodiments, the harvested population of cells secretes at least about 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 g / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 21 ng / mL, 21 ng / mL, 22 ng / mL, 23 ng / mL, 24 ng / mL, 25 ng / mL, 26 ng / mL, 27 ng / mL, 28Attorney Docket No.: 063384-523001 WOng / mL, 29 ng / mL, 30 ng / mL, 31 ng / mL, 32 ng / mL, 33 ng / mL, 34 ng / mL, 35 ng / mL 36 ng / mL, 37 ng / mL, 38 ng / mL, 39 ng / mL, 40 ng / mL, 41 ng / mL. 42 ng / mL, 43 ng / mL, 44 ng / mL, 45 ng / mL, 46 ng / mL, 47 ng / mL, 48 ng / mL, 49 ng / mL, or 50 ng / mL of IFNy.Formulation

[0258] Upon completion of cell harvesting, the harvested population of enriched, activated, and transduced cells comprising CAR-expressing immune cells is formulated forcryopreservation and administration to patients.

[0259] In some embodiments, the formulation step is automated. In some embodiments, the automated formulation step is performed using a Cue ScaleReady Cell Processing System.

[0260] In some embodiments, the harvested population of enriched, activated, and transduced cells comprising CAR-expressing immune cells is resuspended to the desired concentration in Final Formulation Medium comprising a 1:1 mixture of Plasma-Lyte A®+4% HS A (w / v) that is subsequently diluted 1:1 with Cryostor® CS10 and cryopreserved. In some embodiments, the desired concentration comprises 2.5 106CAR+ cells / mL. In some embodiments, the desired concentration comprises 1.0 106CAR+ cells / mL. In some embodiments, the desired concentration comprises 1.5 106CAR+ cells / mL. In some embodiments, the desired concentration comprises 2.0 106CAR+ cells / mL. In some embodiments, the desired concentration comprises 2.5 106CAR+ cells / mL. In some embodiments, the desired concentration comprises 3.0 IO6CAR+ cells / mL. In some embodiments, the desired concentration comprises 3.5 106CAR+ cells / mL. In some embodiments, the desired concentration comprises 4.0 106C AR+ cells / mL. In some embodiments, the desired concentration comprises 4.5 106CAR+ cells / mL. In some embodiments, the desired concentration comprises 5.0 106CAR+ cells / mL. In some embodiments, the desired concentration comprises 5.5 106CAR+ cells / mL. In some embodiments, the desired concentration comprises 6.0 106CAR+ cells / mL. In some embodiments, the desired concentration comprises 6.5 106C AR+ cells / mL. In some embodiments, the desired concentration comprises 7.0 106CAR+ cells / mL. In some embodiments, the desired concentration comprises 7.5 IO6CAR+ cells / mL. In some embodiments, the desired concentration comprises 8.0 106CAR+ cells / mL. In some embodiments, the desired concentration comprises 8.5 106CAR+ cells / mL. In some embodiments, the desired concentration comprises 9.0 106CAR+ cells / mL. In some embodiments, the desired concentration comprises 9.5 106CAR+ cells / mL. In some embodiments, the desired concentration comprises 10.0x106CAR+ cells / mL.Attorney Docket No.: 063384-523001 WORECOMBINANT POLYNUCLEOTIDES ENCODING CARS

[0261] As described above, the methods described herein involve transducing a population of cells with one or more recombinant polynucleotides encoding three chimeric antigen receptors or alternatively “CARs”. As used herein, the term “CAR” refers to a class of synthetic receptors 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. When expressed in an immune cell, the CAR can provide the cell with the ability to specifically bind a target cell expressing a particular antigen, for example a cancer cell, and the ability to generate an activation signal in a non-MHC-restricted fashion ( / .<?., without engaging the T cell receptor) via the intracellular signaling domain following engagement of the antigen-binding domain by its cognate antigen.

[0262] In some embodiments, a CAR includes at least an extracellular antigen binding domain, a transmembrane domain or a hinge and 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.

[0263] In some embodiments, the one or more recombinant polynucleotides encode an autologous CD19-specific CAR, CD20-specific CAR, and CD22-specific CAR.

[0264] In another aspect, the present disclosure describes a recombinant polynucleotide comprising a sequence encoding a polypeptide of an anti-CD19 chimeric antigen receptor (CAR), a recombinant polynucleic acid molecule comprising a sequence encoding a polypeptide of an anti-CD20 chimeric antigen receptor (CAR), and a recombinant polynucleic acid molecule comprising a sequence encoding a polypeptide of an anti-CD22 chimeric antigen receptor (CAR).

[0265] In some embodiments, the recombinant polynucleotide comprises a sequence encoding a polypeptide of an anti-CD19 CAR, an anti-CD20 CAR, and an anti-CD22 CAR.

[0266] In some embodiments, the recombinant polynucleotide comprises a sequence from 5’ to 3’ encoding an anti-CD22 CAR, an anti-CD19 CAR and an anti-CD20 CAR.

[0267] In some embodiments, the three CARs are separated by a self-cleavage site. In some embodiments, the self-cleavage site is a P2A or T2A self-cleaving peptide (also referred to as “viral ribosome skipping peptide”). These viral peptide sequences cause the ribosome to skip ahead along an mRNA transcript such that it does not make a peptide bond at the skippedAttorney Docket No.: 063384-523001 WOposition, thereby terminating translation of the upstream polypeptide. As used herein, the terms “self-cleaving peptide” and “viral ribosome skipping peptide” refer to the same virus-derived peptides and the terms are used interchangeably herein. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence encoding the CD 19-specific CAR, a polynucleotide sequence encoding the CD20-specific CAR, and a polynucleotide sequence encoding the CD22-specific CAR, wherein each CAR-encoding polynucleotide sequence is separated from the other CAR-encoding polynucleotide sequences by a sequence encoding a viral ribosome skipping peptide selected from the group consisting of a P2A peptide, a T2A peptide, an E2A peptide, and an F2 A peptide.

[0268] In some embodiments, the sequence encoding an anti-CD19 CAR, the sequence encoding an anti-CD20 CAR, and the sequence encoding an anti-CD22 CAR are separated by a sequence encoding a P2A site, a T2A self-cleaving peptide, an E2A self-cleaving peptide or an F2A self-cleaving peptide.

[0269] In some embodiments, the recombinant polynucleotide comprises from 5’ to 3’, a sequence encoding the CD22-specific CAR; a sequence encoding a viral P2A ribosome skipping peptide; a sequence encoding the CD19-specific CAR; a sequence encoding a viral T2A ribosome skipping peptide; and a sequence encoding the CD20-specific CAR.

[0270] In some embodiments, the recombinant polynucleic acid molecule is a viral expression vector. In some embodiments, the viral expression vector is a lentiviral expression vector.Chimeric Antigen Receptors

[0271] As described above, each CAR contains 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. In some embodiments, the one or more recombinant polynucleotides encode an autologous CD19-specific CAR, CD20-specific CAR, and CD22- specific C AR.CD22-specific CARs

[0272] In some embodiments, the CD22-specific CAR comprises a CD22-specific binding domain, a transmembrane domain, and an intracellular domain. In some embodiments, the CD22-specific CAR comprises a CD22-specific binding domain, a spacer, a hinge domain, a transmembrane domain, a peptide linker, and an intracellular domain.Attorney Docket No.: 063384-523001 WO

[0273] The CD22-specific binding domain can be any molecule that binds to CD22 with sufficient affinity and specificity, and is often an antibody or an antibody derivative, such as an scFv, single domain antibody (sdAb), Fab' fragment, (Fab')2 fragment, nanobody, diabody, or the like. As used herein, “sufficient affinity and specificity” can refer to the property that, under designated conditions, binding domain binds preferentially to its particular target protein (i.e., CD22) and does not bind in a significant amount to other proteins in a sample or subject. In certain embodiments, the anti-CD22 binding domain will have a sufficiently high binding affinity for CD22, for example, the antibody may bind CD22 with a Kd value of between 1×10'7M to 10xl0'10M. Antibody affinities may be determined, e.g., by a surface plasmon resonance based assay (such as the BIAcore® assay as described in PCT Application Publication No. W02005 / 012359); enzyme-linked immunoabsorbent assay (ELISA); and competition assays (e.g. radioimmunoassays (RIAs)). Alternatively, 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 designed using phage display techniques (see, e.g., E. Romao et al., Curr Pharm Des (2016) 22(43):6500-18).

[0274] In some embodiments, the CD22-specific binding domain is a single chain variable fragment (scFv). In some embodiments, the anti-CD22 scFv comprises an antibody heavyAttorney Docket No.: 063384-523001 WOchain 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: 22).

[0275] In some embodiments, the CD22-specific 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 CD22-specific binding domain comprises a VH that comprises an HCDR1 sequence in Table 1. In some embodiments, the CD22-specific 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 CD22-specific binding domain includes a VH that includes an HCDR2 sequence in Table 1. In some embodiments, the CD22-specific 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 CD22-specific binding domain includes a VH that includes an HCDR3 sequence in Table 1. In some embodiments, the CD22-specific 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 CD22-specific 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 CD22-specific binding domain includes the heavy chain HCDR1, HCDR2, HCDR3 sequences as set forth in SEQ ID NOs: 16, 17, and 18, respectively.

[0276] In some embodiments, the CD22-specific 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 CD22-specific binding domain includes a VL comprising an LCDR1 sequence in Table 1. In some embodiments, the CD22-specific 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 CD22-specific bindingAttorney Docket No.: 063384-523001 WOdomain comprises a VL that comprises an LCDR2 sequence in Table 1. In some embodiments the CD22-specific 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 CD22-specific 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 CD22-specific 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.

[0277] In some embodiments, the CD22-specific 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 CD22-specific binding domain comprises a VH with the sequence QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRS KWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIW GQGTMVTVSS (SEQ ID NO: 2).

[0278] In some embodiments, the CD22-specific 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 CD22-specific binding domain comprises a VL with the sequence DIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGV PSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIK (SEQ ID NO: 3).Attorney Docket No.: 063384-523001 WO

[0279] In some embodiments, the CD22-specific 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 CD22-specific 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 CD22-specific 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.

[0280] In some embodiments, the CD22-specific binding domain is an scFv. In some embodiments, the anti-CD22 scF v comprises a peptide linker between the VH and the VL domains. In some embodiments, the peptide linker comprises the sequence GGGGS (SEQ ID NO: 22). In some embodiments, the CD22-specific 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 QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIWGQGTMVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIK (SEQ ID NO: 1). In some embodiments, the CD22-specific binding domain comprises the sequence QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIWGQGTMVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIK (SEQ ID NO: 1).

[0281] 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 ConstructsAttorney Docket No.: 063384-523001 WOConstructs Amino Acid Sequence SEQ ID NO.Anti-CD22 scFv full length QVQLQQSGPGLVKPSQTLSLTCA1SGDSVSSNSAA 1 WNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVK SR1T1NPDTSKNQFSLQLNSVTPEDTAVYYCAREVT GDLEDAFDIWGQGTMVTVSSGGGGSD1QMTQSPSSLSAS VGDRVTF1 CRASQT1W S YLN W YQQRPGKAPN LL1YAASSLQSGVPSRFSGRGSGTDFTLT1SSLQAED FATYYCQQSYS1PQTFGQGTKLE1KAnti-CD22 VH Q VQLQQSGPGL VKPSQTLSLTC Al SGD S VS SN S AA 2WNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVK SRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVT GDLEDAFDIWGQGTMVTVSSAnti-CD22 VL DIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWY 3QQRPGKAPNLLIYAASSLQSGVPSRFSGRGSGTDFT LTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIK HCDR1 (Chothia) GDSVSSNSA 4 HCDR2 (Chothia) YYRSKWY 5 HCDR3 (Chothia) EVTGDLEDAFDI 6 LCDR1 (Chothia) RASQTIWSYLN 7 LCDR2 (Chothia) AASSLQS 8 LCDR3 (Chothia) QQSYSIPQT 9 HCDR1 (Kabat) SNSAAWN 10 HCDR2 (Kabat) RTYYRSKWYNDYAVSVKS 11 HCDR3 (Kabat) EVTGDLEDAFDI 12 LCDR1 (Kabat) RASQTIWSYLN 13 LCDR2 (Kabat) AASSLQS 14 LCDR3 (Kabat) QQSYSIPQT 15 HCDR1 (1MGT) GDSVSSNSAA 16 HCDR2 (1MGT) TYYRSKWYN 17 HCDR3 (1MGT) AREVTGDLEDAFDI 18 LCDR1 (1MGT) QTIWSY 19 LCDR2 (1MGT) AAS 20 LCDR3 (1MGT) QQSYSIPQT 21

[0282] In some embodiments, the CD22-specific 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.,Attorney Docket No.: 063384-523001 WOmore similar to the corresponding human sequences. One type of humanized anti-CD22 binding domain is a C DR-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.

[0283] In some embodiments, the recombinant polynucleotide encodes a CD22-specific CAR comprising: (i) an extracellular domain comprising an anti-CD22 binding domain, (ii) a transmembrane domain, and (iii) a cytoplasmic domain comprising an intracellular signaling domain. In some embodiments, the polypeptide is a CD22-specific CAR comprising: (i) an extracellular domain comprising an anti-CD22 binding domain and a hinge domain from CD8a or CD28, (ii) a transmembrane domain from CD8a or CD28, and (iii) a cytoplasmic domain comprising an intracellular co -stimulatory signaling domain from 4- IBB and an intracellular signaling domain from CD3Q In some embodiments, the polypeptide is an anti-CD22 CAR comprising: (i) an extracellular domain comprising an anti-CD22 binding domain and a hinge domain from CD8a, (ii) a transmembrane domain from CD8a, and (iii) a cytoplasmic domain comprising an intracellular co-stimulatory signaling domain from 4- IBB and an intracellular signaling domain from CD3^.

[0284] In some embodiments, the linker between the anti-CD22 binding domain and the hinge domain from CD8a or CD28 comprises the sequence AAA.

[0285] In some embodiments, the hinge domain of the CD22-specific CAR is derived from CD8a. In some embodiments, the CD8a hinge domain of the CD22-specific CAR comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 23). In some embodiments, the CD8a hinge domain of the CD22-specific CAR comprises the sequence TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 23).

[0286] In some embodiments, the transmembrane domain of the CD22-specific CAR is from CD8a. In some embodiments, the CD8a transmembrane domain of the CD22-specific CAR comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 24). In someAttorney Docket No.: 063384-523001 WOembodiments, the transmembrane domain of the anti-CD22 CAR comprises the sequence IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 24).

[0287] In some embodiments, the CD22-specific CAR comprises a portion of a CD8a cytoplasmic domain. In some embodiments, the portion of a CD8a cytoplasmic domain of the anti-CD22 CAR comprises the sequence LYC.

[0288] In some embodiments, the intracellular co-stimulatory domain of the CD22-specific CAR is derived from 4- IBB. In some embodiments, the 4- IBB co-stimulatory signaling domain of the CD22-specific CAR comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 25). In some embodiments, the 4- I BB co-stimulatory signaling domain of the CD22-specific CAR comprises the sequence KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 25).

[0289] In some embodiments, the CD22-specific CAR comprises an intracellular signaling domain comprising a primary T cell activating domain comprising an immunoreceptor tyrosine-based activation motif (IT AM) or ITAM-containing signaling domain. In some embodiments, the intracellular signaling domain can be an ITAM-containing domain. In some embodiments, the ITAM-containing domain is derived from CD3Q In some embodiments, the intracellular signaling domain of the CD22-specific CAR is derived from CD3Q In some embodiments, the CD3^ intracellular signaling domain of the CD22-specific CAR comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR (SEQ ID NO: 26). In some embodiments, the CD3C, intracellular signaling domain of the CD22-specific CAR comprises the sequence RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR (SEQ ID NO: 26).

[0290] In some embodiments, the CD22-specific CAR comprises a signal peptide sequence derived from CD8a or GMCSFRa. In some embodiments, the signal peptide sequence is derived from GMCSFRa and comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 27).Attorney Docket No.: 063384-523001 WO

[0291] In some embodiments, the signal peptide sequence is derived from GMCSFRa and comprises the sequence MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 27).

[0292] In some embodiments, the signal peptide sequence is derived from CD8a and comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence MALPVTALLLPLALLLHAARP (SEQ ID NO: 28).

[0293] In some embodiments, the signal peptide sequence is derived from CD8a and comprises the sequence MALPVTALLLPLALLLHAARP (SEQ ID NO: 28).

[0294] In some embodiments, the CD22-specific CAR comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIWGQGTMVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 29).

[0295] In some embodiments, the CD22-specific CAR comprises the sequence QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIWGQGTMVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 29).

[0296] In some embodiments, the CD22-specific CAR comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAW NWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPED TAVYYCAREVTGDLEDAFDIWGQGTMVTVSSGGGGSDIQMTQSPSSLSASVGDRVT ITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAttorney Docket No.: 063384-523001 WOAEDFATYYCQQSYSIPQTFGQGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACR PAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFM RPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREE YDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKG HDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 30).

[0297] In some embodiments, the CD22-specific CAR comprises the sequence MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAW NWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPED TAVYYCAREVTGDLEDAFDIWGQGTMVTVSSGGGGSDIQMTQSPSSLSASVGDRVT ITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQ AEDFATYYCQQSYSIPQTFGQGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACR PAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFM RPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREE YDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKG HDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 30).

[0298] In some embodiments, the CD22-specific CAR comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence MALPVTALLLPLALLLHAARPEVQLVESGGGWRPGGSLRLSCTASGFTFGDYGMS WVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAED TALYYCARKSYYGSGSPDVFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIVMTQSP DSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVP DRFSGSGSGSDFTLTISSLQAEDVAVYYCQQYYSFYQTFGQGTKVEIKIEVMYPPPYL DNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFIIFWVKR KKQRSRRNDEELETRAHRVATEERGRKPHQIPASTPQNPATSQHPPPPPGHRSQAPSH RPPPPGHRVQHQPQKRPPAPSGTQVHQQKGPPLPRPRVQPKPPHGAAENSLSPSSNR VKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 31).

[0299] In some embodiments, the CD22-specific CAR comprises a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGWRPGGSLRLSCTASGFTFGDYGMS WVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAED TALYYCARKSYYGSGSPDVFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIVMTQSP DSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPAttorney Docket No.: 063384-523001 WODRFSGSGSGSDFTLTISSLQAEDVAVYYCQQYYSFYQTFGQGTKVEIKIEVMYPPPYL DNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVKR KKQRSRRNDEELETRAHRVATEERGRKPHQIPASTPQNPATSQHPPPPPGHRSQAPSH RPPPPGHRVQHQPQKRPPAPSGTQVHQQKGPPLPRPRVQPKPPHGAAENSLSPSSNR VKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 32).CD 19-specific CAR

[0300] In some embodiments, the CD 19-specific CAR comprises a CD 19-specific binding domain, a transmembrane domain, and an intracellular domain. In some embodiments, the CD 19-specific CAR comprises a CD 19-specific binding domain, a spacer, a hinge domain, a transmembrane domain, and an intracellular domain.

[0301] The CD 19-specific binding domain can be any molecule that binds to CD 19 with sufficient affinity and specificity, and is often an antibody or an antibody derivative, such as an scFv, single domain antibody (sdAb), Fab' fragment, (Fab')2 fragment, nanobody, diabody, or the like. As used herein, “sufficient affinity and specificity” can refer to the property that, under designated conditions, binding domain binds preferentially to its particular target protein (z.e., CD 19) and does not bind in a significant amount to other proteins in a sample or subject. In certain embodiments, the CD 19-specific binding domain will have a sufficiently high binding affinity for CD 19, for example, the antibody may bind CD 19 with a Kd value of between 1×10'7to 10×10'10M. Antibody affinities may be determined, e.g., by a surface plasmon resonance based assay (such as the BIAcore® assay as described in PCT Application Publication No. W02005 / 012359); enzyme-linked immunoabsorbent assay (ELISA); and competition assays e.g., radioimmunoassays (RIAs)). Alternatively, the anti-CD19 binding domain can be a receptor or a receptor fragment that binds specifically to CD 19. The CD 19-specific 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.Attorney Docket No.: 063384-523001 WOSmith 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 (CD 19 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 designed using phage display techniques (see, e.g., E. Romao et al., Curr Pharm Des (2016) 22(43):6500-18).

[0302] In some embodiments, the CD19-specific binding domain is a single chain variable fragment (scFv). In some embodiments, the anti-CD19 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-CD19 scFv further comprises a peptide linker disposed between the VH and VL polypeptides. In some embodiments, the anti-CD19 scFv comprises, from N-terminus to C-terminus, a VH polypeptide, a polypeptide linker, and a VL polypeptide. In some embodiments, the anti-CD19 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 AAASGGGGSGGGGSGGGGSAL (SEQ ID NO: 33).

[0303] In some embodiments, the CD19-specific 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 2. In some embodiments, the CD19-specific binding domain comprises a VH that comprises an HCDR1 sequence in Table 2. In some embodiments, the CD19-specific 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 2. In some embodiments, the CD19-specific binding domain includes a VH that includes an HCDR2 sequence in Table 2.In some embodiments, the CD19-specific 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 2. In some embodiments, the CD19-specific binding domain includes a VH that includes an HCDR3 sequence in Table 2. In some embodiments, the CD19-specific binding domain includes theAttorney Docket No.: 063384-523001 WOheavy chain CDRs 1, 2, and 3 (HCDR1, HCDR2, HCDR3) sequences as set forth in SEQ ID NOs: 37-39, 66-68, or 72-74, respectively.

[0304] In some embodiments, the CD19-specific 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 2. In some embodiments, the CD 19-specific binding domain includes a VL comprising an LCDR1 sequence in Table 2. In some embodiments, the CD19-specific 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 2. In some embodiments, the CD19-specific binding domain comprises a VL that comprises an LCDR2 sequence in Table 2. In some embodiments, the CD19-specific 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 2. In some embodiments, the CD19-specific binding domain comprises a VL that comprises an LCDR3 sequence in Table 2. In some embodiments, the CD19-specific binding domain includes the light chain CDRs 1, 2, and 3 (LCDR1, LCDR2, LCDR3) sequences as set forth in SEQ ID NOs: 40-42, 69-71, or 75-77, respectively.

[0305] In some embodiments, the CD19-specific 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 EVQLVQSGAEVKKPGESLKISCKASGYRFTNYWIAWVRQRPGKGLEWMGRIDPSDS YTHYSPSFQGHVTMSTDKSISTAYLQWSSLKASDTAMYYCARPGDILTGWAMDVW GQGTLVTVSS (SEQ ID NO: 35). In some embodiments, the CD19-specific binding domain comprises a VH with the sequence EVQLVQSGAEVKKPGESLKISCKASGYRFTNYWIAWVRQRPGKGLEWMGRIDPSDS YTHYSPSFQGHVTMSTDKSISTAYLQWSSLKASDTAMYYCARPGDILTGWAMDVW GQGTLVTVSS (SEQ ID NO: 35).

[0306] In some embodiments, the CD19-specific 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 QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSG IPDRFSGSKSGTSATLGITGLQAEDEADYYCQSYDSSLSGNYVFGTGTKVTVL (SEQAttorney Docket No.: 063384-523001 WOID NO: 36). In some embodiments, the CD19-specific binding domain comprises a VL with the sequence QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSG IPDRFSGSKSGTSATLGITGLQAEDEADYYCQSYDSSLSGNYVFGTGTKVTVL (SEQ ID NO: 36).

[0307] In some embodiments, the CD19-specific 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: 37-42, 66-71, or 72-77, respectively.

[0308] In some embodiments, the CD19-specific binding domain is an scFv. In some embodiments, the anti-CD19 scFv comprises a peptide linker between the VH and the VL domains. In some embodiments, the peptide linker comprises the sequence AAASGGGGSGGGGSGGGGSAL (SEQ ID NO: 33). In some embodiments, the CD 19-specific 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 EVQLVQSGAEVKKPGESLKISCKASGYRFTNYWIAWVRQRPGKGLEWMGRIDPSDS YTHYSPSFQGHVTMSTDKSISTAYLQWSSLKASDTAMYYCARPGDILTGWAMDVW GQGTLVTVSSAAASGGGGSGGGGSGGGGSALQSVLTQPPSVSAAPGQKVTISCSGSS SNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQAEDE ADYYCQSYDSSLSGNYVFGTGTKVTVL (SEQ ID NO: 34). In some embodiments, the CD19-specific binding domain comprises the sequence EVQLVQSGAEVKKPGESLKISCKASGYRFTNYWIAWVRQRPGKGLEWMGRIDPSDS YTHYSPSFQGHVTMSTDKSISTAYLQWSSLKASDTAMYYCARPGDILTGWAMDVW GQGTLVTVSSAAASGGGGSGGGGSGGGGSALQSVLTQPPSVSAAPGQKVTISCSGSS SNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQAEDE ADYYCQSYDSSLSGNYVFGTGTKVTVL (SEQ ID NO: 34).

[0309] In some embodiments, the antigen binding domain is a mouse anti-human CD 19 binding domain. In some embodiments, the antigen binding domain is a chimeric mouse antihuman CD 19 binding domain. In some embodiments, the antigen binding domain is a humanized anti-human CD 19 binding domain. In some embodiments, the antigen binding domain is a fully human anti -human CD 19 binding domain.TABLE 2: Exemplary anti-CD19 scFv ConstructsAttorney Docket No.: 063384-523001 WOConstructs Amino Acid Sequence SEQ ID NO.Anti-CD19 scFv full length EVQLVQSGAEVKKPGESLKISCKASGYRFTNYWIAWVRQRPGKGLEWMGRIDPSDSYTHYSPSFQGHVTMSTDKSISTAYLQWSSLKASDTAMYYCARPGDILTGWAMDVWGQGTLVTVSSAAASGGGGSGGGGSGGGGSALQSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQAEDEADYYCQSYDSSLSGNYVFGTGTKVTVLAnti-CD19 VH EVQLVQSGAEVKKPGESLKISCKASGYRFTNYWIAWVRQRPGKGLEWMGRIDPSDSYTHYSPSFQGHVTMSTDKSISTAYLQWSSLKASDTAMYYCARPGDILTGWAMD V WGQGTL VT VS SAnti-CD19 VL QSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSW 36 YQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSA TLGITGLQAEDEADYYCQSYDSSLSGNYVFGTGTKVTVL HCDR1 (1MGT) GYRFTNYW 37 HCDR2 (1MGT) IDPSDSYT 38 HCDR3 (1MGT) ARPGDILTGWAMDV 39 LCDR1 (1MGT) SSNIGNNY 40 LCDR2 (1MGT) DNN 41 LCDR3 (1MGT) QSYDSSLSGNYV 42 HCDR1 (Chothia) GYRFTNY 66 HCDR2 (Chothia) DPSDSY 67 HCDR3 (Chothia) PGDILTGWAMDV 68 LCDR1 (Chothia) SGSSSNIGNNYVS 69 LCDR2 (Chothia) DNNKRPS 70 LCDR3 (Chothia) QSYDSSLSGNYV 71 HCDR1 (Kabat) NYWIA 72 HCDR2 (Kabat) RIDPSDSYTHYSPSFQG 73 HCDR3 (Kabat) PGDILTGWAMDV 74 LCDR1 (Kabat) SGSSSNIGNNYVS 75 LCDR2 (Kabat) DNNKRPS 76 LCDR3 (Kabat) QSYDSSLSGNYV 77

[0310] In some embodiments, the recombinant polynucleic acid comprises a sequence encoding a polypeptide encoding a CD19-specific CAR comprising: (i) an extracellular domain comprising an anti-CD19 binding domain, (ii) a transmembrane domain, and (iii) a cytoplasmic domain comprising an intracellular signaling domain. In some embodiments, theAttorney Docket No.: 063384-523001 WOpolypeptide is an anti-CD19 CAR comprising: (i) an extracellular domain comprising an anti-CD19 binding domain and a hinge domain from CD8a or CD28, (ii) a transmembrane domain from CD8a or CD28, and (iii) a cytoplasmic domain comprising an intracellular costimulatory signaling domain from CD28 and an intracellular signaling domain from CD3 In some embodiments, the polypeptide is a CD 19-specific CAR comprising: (i) an extracellular domain comprising an anti-CD19 binding domain and a hinge domain from CD28, (ii) a transmembrane domain from CD28, and (iii) a cytoplasmic domain comprising an intracellular co-stimulatory signaling domain from CD28 and an intracellular signaling domain from CD3 The anti-CD19 binding domain can be any of the anti-CD19 binding domains provided herein.

[0311] In some embodiments, the hinge domain sequence of the CD 19-specific CAR comprises a hinge domain derived from CD28. In some embodiments, the CD28 hinge domain of the anti-CD19 CAR comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 43). In some embodiments, the CD28 hinge domain of the CD 19-specific CAR comprises the sequence IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 43).

[0312] In some embodiments, the transmembrane domain sequence of the CD 19-specific CAR comprises a transmembrane domain derived from CD28. In some embodiments, the CD28 transmembrane domain of the CD 19-specific CAR comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence FWVLVWGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 44). In some embodiments, the CD28 transmembrane domain of the CD 19-specific CAR comprises the sequence FWVLVWGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 44).

[0313] In some embodiments, the intracellular co-stimulatory signaling domain of the CD19- specific CAR is a CD28 intracellular co-stimulatory signaling domain. In some embodiments, the CD28 intracellular co-stimulatory signaling domain of the CD 19-specific CAR comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 45). In some embodiments, the CD28 intracellular co-stimulatory signaling domain of the CD 19-specific C AR comprises the sequence RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 45).Attorney Docket No.: 063384-523001 WO

[0314] In some embodiments, the CD19-specific CAR comprises an intracellular signaling domain comprising an immunoreceptor tyrosine-based activation motif (ITAM) or ITAM-containing signaling domain. In some embodiments, the ITAM-containing signaling domain is a CD3C intracellular signaling domain. In some embodiments, the CD3C intracellular signaling domain of the CD 19-specific CAR comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR (SEQ ID NO: 26). In some embodiments, the CD3^ intracellular signaling domain of the CD 19-specific CAR comprises the sequence RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR (SEQ ID NO: 26).

[0315] In some embodiments, the CD19-specific CAR comprises a signal peptide sequence derived from CD8a or granulocyte -macrophage colony-stimulating factor receptor (GMCSFRa). In some embodiments, the CD 19-specific CAR comprises a signal peptide sequence derived from CD8a. In some embodiments, the CD8a signal peptide sequence comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence MALPVTALLLPLALLLHAARP (SEQ ID NO: 28). In some embodiments, the CD 8a signal peptide sequence comprises the sequence MALPVTALLLPLALLLHAARP (SEQ ID NO: 28).

[0316] In some embodiments, the CD 19-specific CAR comprises a signal peptide sequence derived from GMCSFRa. In some embodiments, the GMCSFRa signal peptide sequence comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 27). In some embodiments, the GMCSFRa signal peptide sequence comprises the sequence MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 27).

[0317] In some embodiments, the CD 19-specific CAR comprises a CD28 hinge domain, a CD28 transmembrane domain, a CD28 intracellular costimulatory signaling domain and a CD3^ intracellular signaling domain comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLWVGGVLACYSLLVT VAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSAAttorney Docket No.: 063384-523001 WODAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 46). In some embodiments, the CD19-specific CAR comprises a CD28 hinge domain, a CD28 transmembrane domain, a CD28 intracellular costimulatory signaling domain and a CD3C intracellular signaling domain comprising the sequence IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLWVGGVLACYSLLVT VAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSA DAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQ KDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 46).

[0318] In some embodiments, the CD19-specific CAR comprises an scFv derived from a human CD 19 antibody designated 19c493, a CD28 hinge domain, a CD28 transmembrane domain, a CD28 intracellular costimulatory signaling domain, and a CD3C intracellular signaling domain together having the sequence EVQLVQSGAEVKKPGESLKISCKASGYRFTNYWIAWVRQRPGKGLEWMGRIDPSDS YTHYSPSFQGHVTMSTDKSISTAYLQWSSLKASDTAMYYCARPGDILTGWAMDVW GQGTLVTVSSAAASGGGGSGGGGSGGGGSALQSVLTQPPSVSAAPGQKVTISCSGSS SNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQAEDE ADYYCQSYDSSLSGNYVFGTGTKVTVLIEVMYPPPYLDNEKSNGTIIHVKGKHLCPS PLFPGPSKPFWVLVWGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPG PTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY QGLSTATKDTYDALHMQALPPR (SEQ ID NO: 47)

[0319] In some embodiments, the CD19-specific CAR comprises a signal sequence derived from CD8a, an scFv derived from a human CD 19 antibody designated 19c493, a CD28 hinge domain, a CD28 transmembrane domain, a CD28 intracellular costimulatory signaling domain, and a CD3C intracellular signaling domain together having the sequence MALPVTALLLPLALLLHAARPEVQLVQSGAEVKKPGESLKISCKASGYRFTNYWIA WVRQRPGKGLEWMGRIDPSDSYTHYSPSFQGHVTMSTDKSISTAYLQWSSLKASDT AMYYCARPGDILTGWAMDVWGQGTLVTVSSAAASGGGGSGGGGSGGGGSALQSV LTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPD RFSGSKSGTSATLGITGLQAEDEADYYCQSYDSSLSGNYVFGTGTKVTVLIEVMYPPP YLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFIIFWVAttorney Docket No.: 063384-523001 WORSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYKQ GQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAE AYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 48).CD20-specific CAR

[0320] In some embodiments, the CD20-specific CAR comprises a CD20-specific binding domain, a transmembrane domain, and an intracellular domain.

[0321] The CD20-specific binding domain can be any molecule that binds to CD20 with sufficient affinity and specificity, and is often an antibody or an antibody derivative, such as an scFv, single domain antibody (sdAb), Fab' fragment, (Fab’)2 fragment, nanobody, diabody, or the like. As used herein, the phrase “sufficient affinity and specificity” refers to the property that, under designated conditions, binding domain binds preferentially to its particular target protein (i.e., CD20) and does not bind in a significant amount to other proteins in a sample or subject. In certain embodiments, the CD20-specific binding domain will have a sufficiently high binding affinity for CD20, for example, the antibody may bind CD20 with a Kd value of between lx IO"7to 6x1 O’10M. Antibody affinities may be determined, e.g., by a surface plasmon resonance based assay (such as the BIAcore® assay described in PCT Application Publication No. W02005 / 012359); enzyme-linked immunoabsorbent assay (ELISA); and competition assays (e.g., radioimmunoassays (RIAs)). Alternatively, the CD20-specific binding domain can be a receptor or a receptor fragment that binds specifically to CD20. The CD20-specific 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 (CD 19 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,Attorney Docket No.: 063384-523001 WOminibody, 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 designed using phage display techniques (see, e.g., E. Romao et al., Curr Pharm Des (2016) 22(43):6500-18).

[0322] In some embodiments, the CD20-specific binding domain is a single chain variable fragment (scFv). In some embodiments, the anti-CD20 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-CD20 scFv further comprises a peptide linker disposed between the VH and VL polypeptides. In some embodiments, the anti-CD20 scFv comprises, from N-terminus to C-temiinus, a VH polypeptide, a polypeptide linker, and a VL polypeptide. In some embodiments, the anti-CD20 scFv comprises, fromN-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 GGGGSGGGGSGGGGS (SEQ ID NO: 49).

[0323] In some embodiments, the CD20-specific 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 3. In some embodiments, the CD20-specific binding domain comprises a VH that comprises an HCDR1 sequence in Table 3. In some embodiments, the CD20-specific 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 3. In some embodiments, the CD20-specific binding domain includes a VH that includes an HCDR2 sequence in Table 3.In some embodiments, the CD20-specific 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 3. In some embodiments, the CD20-specific binding domain includes a VH that includes an HCDR3 sequence in Table 3. In some embodiments, the CD20-specific binding domain includes the heavy chain CDRs 1, 2, and 3 (HCDR1, HCDR2, HCDR3) sequences as set forth in SEQ ID NOs: 53-55, 78-80, or 84-86, respectively.

[0324] In some embodiments, the CD20-specific 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 3. In some embodiments, the CD20-specific binding domain includes a VL comprising an LCDR1 sequence in Table 3. In some embodiments, the CD20-specific binding domain comprises a VL that comprises a light chainAttorney Docket No.: 063384-523001 WOCDR2 (LCDR2) sequence with at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to an LCDR2 sequence in Table 3. In some embodiments, the CD20-specific binding domain comprises a VL that comprises an LCDR2 sequence in Table 3. In some embodiments, the CD20-specific 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 3. In some embodiments, the CD20-specific binding domain comprises a VL that comprises an LCDR3 sequence in Table 3. In some embodiments, the CD20-specific binding domain includes the light chain CDRs 1, 2, and 3 (LCDR1, LCDR2, LCDR3) sequences as set forth in SEQ ID NOs: 56-58, 81-83, or 87-89, respectively.

[0325] In some embodiments, the CD20-specific 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 EVQLVESGGGVVRPGGSLRLSCTASGFTFGDYGMSWVRQAPGKGLEWVSGINWNG GSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCARKSYYGSGSPDVFDI WGQGTMVTVSS (SEQ ID NO: 51). In some embodiments, the CD20-specific binding domain comprises a VH with the sequence EVQLVESGGGVVRPGGSLRLSCTASGFTFGDYGMSWVRQAPGKGLEWVSGINWNG GSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCARKSYYGSGSPDVFDI WGQGTMVTVSS (SEQ ID NO: 51).

[0326] In some embodiments, the CD20-specific 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 DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWA STRESGVPDRFSGSGSGSDFTLTISSLQAEDVAVYYCQQYYSFYQTFGQGTKVEIK(SEQ ID NO: 52). In some embodiments, the CD20-specific binding domain comprises a VL with the sequence DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWA STRESGVPDRFSGSGSGSDFTLTISSLQAEDVAVYYCQQYYSFYQTFGQGTKVEIK(SEQ ID NO: 52).

[0327] In some embodiments, the CD20-specific binding domain includes the heavy chain CDRs 1, 2, and 3 (HCDR1, HCDR2, HCDR3) sequences and the light chain CDRs 1, 2, andAttorney Docket No.: 063384-523001 WO3 (LCDR1, LCDR2, LCDR3) sequences as set forth in SEQ ID NOs: 53-58, 78-83, or 84-89, respectively.

[0328] In some embodiments, the CD20-specific binding domain is an scFv. In some embodiments, the anti-CD20 scFv comprises a peptide linker between the VH and the VL domains. In some embodiments, the peptide linker comprises the sequence GGGGSGGGGSGGGGS (SEQ ID NO: 49). In some embodiments, the CD20-specific 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 DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWA STRESGVPDRFSGSGSGSDFTLTISSLQAEDVAVYYCQQYYSFYQTFGQGTKVEIKGG GGSGGGGSGGGGSEVQLVESGGGWRPGGSLRLSCTASGFTFGDYGMSWVRQAPG KGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCA RKSYYGSGSPDVFDIWGQGTMVTVSS (SEQ ID NO: 50). In some embodiments, the CD20-specific binding domain comprises the sequence DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWA STRESGVPDRFSGSGSGSDFTLTISSLQAEDVAVYYCQQYYSFYQTFGQGTKVEIKGG GGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSCTASGFTFGDYGMSWVRQAPG KGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCA RKSYYGSGSPDVFDIWGQGTMVTVSS (SEQ ID NO: 50).

[0329] In some embodiments, the antigen binding domain is a mouse anti-human CD20 binding domain. In some embodiments, the antigen binding domain is a chimeric mouse antihuman CD20 binding domain. In some embodiments, the antigen binding domain is a humanized anti-human CD20 binding domain. In some embodiments, the antigen binding domain is a fully human anti-human CD20 binding domain.TABLE 3: Exemplary anti-CD20 scFv ConstructsConstructs Amino Acid Sequence SEQ ID NO.Anti-CD20 scFv full length DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNK 50NYLAWYQQKPGQPPKLLIYWASTRES GVPDRFSGS GSGSDFTLTISSLQAEDVAVYYCQQYYSFYQTFGQ GTKVEIKG GGGSGGGGSGGGGSEVQLVESGGGVV RPGGSLRLSCTASGFTFGDYGMSWVRQAPGKGLE WVSGINWNGGSTGYADSVKGRFTISRDNAKNSLY LQMNSLRAEDTALYYCARKSYYGSGSPDVFDIWG QGTMVTVSSAttorney Docket No.: 063384-523001 WOConstructs Amino Acid Sequence SEQ ID NO.Anti-CD20 VH EVQLVESGGGVVRPGGSLRLSCTASGFTFGDYGMS 51 WVRQAPGKGLEWVSGINWNGGSTGYADSVKGRF TISRDNAKNSLYLQMNSLRAEDTALYYCARKSYY GSGSPDVFDIWGQGTMVTVSSAnti-CD20 VL DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNK 52 NYLAWYQQKPGQPPKLLIYWASTRES GVPDRFSGS GSGSDFTLTISSLQAEDVAVYYCQQYYSFYQTFGQGTKVEIK HCDR1 (IMGT) GFTFGDYG 53 HCDR2 (IMGT) INWNGGST 54 HCDR3 (IMGT) ARKSYYGSGSPDVFDI 55 LCDR1 (IMGT) QSVLYSSNNKNY 56 LCDR2 (IMGT) WAS 57 LCDR3 (IMGT) QQYYSFYQT 58 HCDR1 (Chothia) GFTFGDY 78 HCDR2 (Chothia) NWNGGS 79 HCDR3 (Chothia) KSYYGSGSPDVFDI 80 LCDR1 (Chothia) KSSQSVLYSSNNKNYLA 81 LCDR2 (Chothia) WASTRES 82 LCDR3 (Chothia) QQYYSFYQT 83 HCDR1 (Kabat) DYGMS 84 HCDR2 (Kabat) GINWNGGSTGYADSVKG 85 HCDR3 (Kabat) KSYYGSGSPDVFDI 86 LCDR1 (Kabat) KSSQSVLYSSNNKNYLA 87 LCDR2 (Kabat) WASTRES 88 LCDR3 (Kabat) QQYYSFYQT 89

[0330] In some embodiments, the recombinant polynucleic acid comprises a sequence encoding a polypeptide encoding a CD20-specific CAR comprising: (i) an extracellular domain comprising an anti-CD20 binding domain, (ii) a transmembrane domain, and (iii) a cytoplasmic domain comprising an intracellular signaling domain. In some embodiments, the polypeptide is a CD20-specific CAR comprising: (i) an extracellular domain comprising an anti-CD20 binding domain and a hinge domain from CD28, (ii) a transmembrane domain from CD28, and (iii) a cytoplasmic domain comprising an intracellular co -stimulatory signaling domain from CD2 and an intracellular signaling domain from CD3^. The anti-CD20 binding domain can be any of the anti-CD20 binding domains provided herein.Attorney Docket No.: 063384-523001 WO

[0331] In some embodiments, the hinge domain sequence of the CD20-specific CAR comprises a hinge domain derived from CD28. In some embodiments, the CD28 hinge domain of the CD20-specific CAR comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 43). In some embodiments, the CD28 hinge domain of the CD20-specific CAR comprises the sequence IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 43).

[0332] In some embodiments, the transmembrane domain sequence of the CD20-specific CAR comprises a transmembrane domain derived from CD28. In some embodiments, the CD28 transmembrane domain of the CD20-specific CAR comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence FWVLVWGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 44). In some embodiments, the transmembrane domain of the C D20-specific CAR comprises the sequence FWVLVWGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 44).

[0333] In some embodiments, the intracellular co-stimulatory signaling domain of the CD20-specific CAR is derived from CD2. In some embodiments, the CD2 intracellular costimulatory signaling domain of the CD20-specific CAR comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence KRKKQRSRRNDEELETRAHRVATEERGRKPHQIPASTPQNPATSQHPPPPPGHRSQAP SHRPPPPGHRVQHQPQKRPPAPSGTQVHQQKGPPLPRPRVQPKPPHGAAENSLSPSSN(SEQ ID NO: 59). In some embodiments, the CD2 intracellular co-stimulatory signaling domain of the CD20-specific CAR comprises the sequence KRKKQRSRRNDEELETRAHRVATEERGRKPHQIPASTPQNPATSQHPPPPPGHRSQAP SHRPPPPGHRVQHQPQKRPPAPSGTQVHQQKGPPLPRPRVQPKPPHGAAENSLSPSSN(SEQ ID NO: 59).

[0334] In some embodiments, the CD20-specific CAR comprises an intracellular signaling domain comprising an immunoreceptor tyrosine -based activation motif (IT AM) or ITAM-containing signaling domain. In some embodiments, the intracellular signaling domain can be derived from an ITAM-containing domain. In some embodiments, the ITAM-containing domain is derived from CD3^. In some embodiments, the intracellular signaling domain of the CD20-specific CAR is derived from CD3^. In some embodiments, the CD3^ intracellular signaling domain of the CD20-specific CAR comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequenceRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQAttorney Docket No.: 063384-523001 WOEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR (SEQ ID NO: 26). In some embodiments, the CD3C, intracellular signaling domain of the CD20-specific CAR comprises the sequence RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR (SEQ ID NO: 26).

[0335] In some embodiments, the CD20-specific CAR comprises a signal peptide sequence derived from CD8a or granulocyte-macrophage colony-stimulating factor receptor (GMCSFRa). In some embodiments, the CD20-specific CAR comprises a signal peptide sequence derived from CD8a. In some embodiments, the signal peptide sequence derived from CD8a comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence MALPVTALLLPLALLLHAARP (SEQ ID NO: 28). In some embodiments, the signal peptide sequence derived from CD8a comprises the sequence MALPVTALLLPLALLLHAARP (SEQ ID NO: 28).

[0336] In some embodiments, the CD20-specific CAR comprises a signal peptide sequence derived from granulocyte-macrophage colony-stimulating factor receptor (GMCSFRa). In some embodiments, the CD20-specific CAR comprises a signal peptide sequence derived from GMCSFRa. In some embodiments, the signal peptide sequence derived from GMCSFRa comprises a sequence having at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the sequence MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 27). In some embodiments, the signal peptide sequence derived from GMSCFR a comprises the sequence MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 27).

[0337] In some embodiments, the CD20-specific CAR comprises a CD28 hinge domain, a CD28 transmembrane domain, a CD2 intracellular costimulatory signaling domain, and a CD3^ intracellular signaling domain comprising a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the sequence IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLWVGGVLACYSLLVT VAFIIFWVKRKKQRSRRNDEELETRAHRVATEERGRKPHQIPASTPQNPATSQHPPPP PGHRSQAPSHRPPPPGHRVQHQPQKRPPAPSGTQVHQQKGPPLPRPRVQPKPPHGAA ENSLSPSSNRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPR (SEQ ID NO: 60).

[0338] In some embodiments, the CD20-specific CAR comprises a CD28 hinge domain, a CD28 transmembrane domain, a CD2 intracellular costimulatory signaling domain, and aAttorney Docket No.: 063384-523001 WOCD3^ intracellular signaling domain comprising the sequence IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVT VAFIIFWVKRKKQRSRRNDEELETRAHRVATEERGRKPHQIPASTPQNPATSQHPPPP PGHRSQAPSHRPPPPGHRVQHQPQKRPPAPSGTQVHQQKGPPLPRPRVQPKPPHGAA ENSLSPSSNRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPR (SEQ ID NO: 60).

[0339] In some embodiments, the CD20-specific CAR comprises a Flag tag, an scFv derived from a human CD20 antibody designated 20c27, a CD28 hinge domain, a CD28 transmembrane domain, a CD2 intracellular costimulatory signaling domain, and a CD3^ intracellular signaling domain together having the sequence DYKDDDDKDIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQ PPKLLIYWASTRESGVPDRFSGSGSGSDFTLTISSLQAEDVAVYYCQQYYSFYQTFGQ GTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGWRPGGSLRLSCTASGFTFGDYGM SWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAE DTALYYCARKSYYGSGSPDVFDIWGQGTMVTVSSIEVMYPPPYLDNEKSNGTIIHVK GKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFIIFWVKRKKQRSRRNDEEL ETRAHRVATEERGRKPHQIPASTPQNPATSQHPPPPPGHRSQAPSHRPPPPGHRVQHQ PQKRPPAPSGTQVHQQKGPPLPRPRVQPKPPHGAAENSLSPSSNRVKFSRSADAPAY KQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKM AEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPR (SEQ ID NO: 61).

[0340] In some embodiments, the CD20-specific CAR comprises an scFv derived from a human CD20 antibody designated 20c27, a CD28 hinge domain, a CD28 transmembrane domain, a CD2 intracellular costimulatory signaling domain, and a CD3^ intracellular signaling domain together having the sequence DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLLIYWA STRESGVPDRFSGSGSGSDFTLTISSLQAEDVAVYYCQQYYSFYQTFGQGTKVEIKGG GGSGGGGSGGGGSEVQLVESGGGWRPGGSLRLSCTASGFTFGDYGMSWVRQAPG KGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCA RKSYYGSGSPDVFDIWGQGTMVTVSSIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPL FPGPSKPFWVLWVGGVLACYSLLVTVAFIIFWVKRKKQRSRRNDEELETRAHRVAT EERGRKPHQIPASTPQNPATSQHPPPPPGHRSQAPSHRPPPPGHRVQHQPQKRPPAPS GTQVHQQKGPPLPRPRVQPKPPHGAAENSLSPSSNRVKFSRSADAPAYKQGQNQLYAttorney Docket No.: 063384-523001 WONELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGM KGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 62).

[0341] In some embodiments, the CD20-specific CAR comprises a signal sequence derived from GMCSFRa, a FLAG tag, an scFv derived from a human CD20 antibody designated 20c27, a CD28 hinge domain, a CD28 transmembrane domain, a CD2 intracellular costimulatory signaling domain, and a CD3^ intracellular signaling domain together having the sequence MLLLVTSLLLCELPHPAFLLIPDYKDDDDKDIVMTQSPDSLAVSLGERATINCKSSQS VLYSSNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGSDFTLTISSLQ AEDVAVYYCQQYYSFYQTFGQGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGW RPGGSLRLSCTASGFTFGDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVK. GR FTISRDNAKNSLYLQMNSLRAEDTALYYCARKSYYGSGSPDVFDIWGQGTMVTVSSI EVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVT VAFIIFWVKRKKQRSRRNDEELETRAHRVATEERGRKPHQIPASTPQNPATSQHPPPP PGHRSQAPSHRPPPPGHRVQHQPQKRPPAPSGTQVHQQKGPPLPRPRVQPKPPHGAA ENSLSPSSNRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYD ALHMQALPPR (SEQ ID NO: 63).

[0342] In some embodiments, the CD20-specific CAR comprises a signal sequence derived from GMCSFRa, an scFv derived from a human CD20 antibody designated 20c27, a CD28 hinge domain, a CD28 transmembrane domain, a CD2 intracellular costimulatory signaling domain, and a CD3^ intracellular signaling domain together having the sequence MLLLVTSLLLCELPHPAFLLIPDIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKN YLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGSDFTLTISSLQAEDVAVYYC QQYYSFYQTFGQGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGVVRPGGSLRLSC TASGFTFGDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKN SLYLQMNSLRAEDTALYYCARKSYYGSGSPDVFDIWGQGTMVTVSSIEVMYPPPYL DNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFIIFWVKR KKQRSRRNDEELETRAHRVATEERGRKPHQIPASTPQNPATSQHPPPPPGHRSQAPSH RPPPPGHRVQHQPQKRPPAPSGTQVHQQKGPPLPRPRVQPKPPHGAAENSLSPSSNR VKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQE GLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 64).Attorney Docket No.: 063384-523001 WOExemplary Recombinant Polynucleotides

[0343] In some embodiments, the recombinant polynucleotide described herein comprises a single polynucleic acid comprising a sequence encoding a CD22 CAR, a P2A ribosome skipping peptide, a CD 19 CAR, a T2A ribosome skipping peptide, and a CD20 CAR together having the sequence:MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAW NWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPED TAVYYCAREVTGDLEDAFDIWGQGTMVTVSSGGGGSDIQMTQSPSSLSASVGDRVT ITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQ AEDFATYYCQQSYSIPQTFGQGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEACR PAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFM RPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREE YDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKG HDGLYQGLSTATKDTYDALHMQALPPRATNFSLLKQAGDVEENPGPMALPVTALLL PLALLLHAARPEVQLVQSGAEVKKPGESLKISCKASGYRFTNYWIAWVRQRPGKGL EWMGRIDPSDSYTHYSPSFQGHVTMSTDKSISTAYLQWSSLKASDTAMYYCARPGDI LTGWAMDVWGQGTLVTVSSAAASGGGGSGGGGSGGGGSALQSVLTQPPSVSAAPG QKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFSGSKSGTSATL GITGLQAEDEADYYCQSYDSSLSGNYVFGTGTKVTVLIEVMYPPPYLDNEKSNGTIIH VKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDY MNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYKQGQNQLYNELNL GRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERR RGKGHDGLYQGLSTATKDTYDALHMQALPPREGRGSLLTCGDVEENPGPMLLLVTS LLLCELPHPAFLLIPDIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQ QKPGQPPKLLIYWASTRESGVPDRFSGSGSGSDFTLTISSLQAEDVAVYYCQQYYSFY QTFGQGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGWRPGGSLRLSCTASGFTF GDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSLYLQM NSLRAEDTALYYCARKSYYGSGSPDVFDIWGQGTMVTVSSIEVMYPPPYLDNEKSN GTIIHVKGKHLCPSPLFPGPSKPFWVLWVGGVLACYSLLVTVAFIIFWVKRKKQRSR RNDEELETRAHRVATEERGRKPHQIPASTPQNPATSQHPPPPPGHRSQAPSHRPPPPG HRVQHQPQKRPPAPSGTQVHQQKGPPLPRPRVQPKPPHGAAENSLSPSSNRVKFSRS ADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNEL QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 90).Attorney Docket No.: 063384-523001 WO

[0344] In some embodiments, the recombinant polynucleotide described herein comprises a single polynucleic acid comprising the sequence atgcttctgctcgtgacaagcctgctgctgtgcgagctgccccaccctgcctttctgctgatccctCAAGTCCAGCTGCAG CAATCTGGCCCTGGCCTGGTGAAGCCTAGCCAGACCCTGAGCCTGACATGTGCCA TCAGCGGAGATTCTGTTAGCAGCAACAGCGCGGCCTGGAACTGGATCAGACAGA GCCCTAGCAGAGGACTGGAGTGGCTGGGCCGGACCTACTACAGAAGCAAGTGGT ACAACGACTACGCCGTGTCCGTGAAAAGCAGAATCACCATCAACCCCGACACCA GCAAGAACCAGTTCTCCCTGCAACTGAATAGCGTCACACCCGAGGATACAGCCG TGTACTACTGCGCCAGAGAGGTTACAGGCGACCTGGAAGATGCCTTTGACATCTG GGGCCAGGGCACGATGGTGACCGTCAGTAGCGGCGGCGGAGGAAGCGACATCC AGATGACCCAATCTCCTAGCAGCCTGTCTGCCTCTGTGGGCGACAGAGTGACCAT CACCTGCCGCGCCTCTCAAACAATCTGGTCCTACCTGAACTGGTATCAGCAAAGA CCTGGCAAAGCCCCAAACCTGCTGATCTACGCCGCCAGCTCCCTGCAAAGCGGA GTGCCCAGCAGATTCAGCGGCCGGGGCAGCGGCACTGATTTTACCCTGACTATTT CTTCCCTGCAAGCCGAGGACTTCGCCACATACTACTGTCAGCAGAGCTACAGCAT CCCTCAGACCTTCGGCCAGGGCACCAAGCTGGAAATCAAGGCGGCCGCCACAAC CACCCCTGCTCCCCGCCCACCTACCCCTGCTCCTACAATCGCCTCGCAACCTCTG AGCCTGAGACCTGAGGCCTGTAGACCCGCCGCTGGCGGCGCCGTGCACACCAGA GGCCTGGACTTCGCCTGCGACATCTACATCTGGGCCCCACTGGCCGGCACATGCG GCGTGCTGCTGCTGTCCCTGGTGATCACCCTGTATTGCAAGAGAGGCAGAAAGA AACTGCTGTACATCTTCAAGCAGCCTTTCATGCGGCCCGTGCAGACCACCCAGGA GGAAGATGGTTGCTCATGCCGGTTCCCCGAGGAAGAGGAAGGCGGATGTGAACT GAGAGTGAAGTTCAGCCGCTCCGCTGACGCACCCGCCTATAAACAGGGGCAGAA TCAACTTTATAACGAACTGAACCTTGGAAGAAGGGAGGAATATGACGTGCTTGA TAAGAGAAGAGGCAGAGATCCTGAAATGGGAGGGAAACCTCGGAGAAAAAATC CACAAGAGGGGCTCTACAATGAACTCCAGAAAGACAAGATGGCTGAAGCTTACT CTGAGATTGGAATGAAAGGGGAGCGGCGCCGGGGCAAAGGGCACGACGGATTG TACCAAGGACTCAGTACAGCTACTAAAGACACATATGACGCCCTGCATATGCAG GCACTGCCCCCACGGGCCACCAACTTCAGCCTGCTGAAGCAGGCCGGCGACGTG GAAGAGAACCCCGGCCCTATGGCCTTGCCCGTGACTGCTCTGCTCCTGCCCCTCG CACTGCTGCTGCACGCCGCGAGGCCCGAAGTGCAGCTGGTCCAATCTGGCGCCG AGGTGAAGAAGCCCGGCGAGAGCCTGAAGATCAGCTGTAAAGCCAGCGGATAT AGATTCACCAACTACTGGATCGCCTGGGTGCGGCAGCGGCCTGGCAAGGGCCTG GAATGGATGGGCAGAATCGACCCATCTGATAGCTACACCCACTACAGCCCTAGCAttorney Docket No.: 063384-523001 WOTTCCAAGGCCACGTGACCATGAGCACAGATAAGAGCATCAGCACCGCCTACCTG CAGTGGTCCTCCCTGAAAGCTAGCGACACCGCTATGTACTACTGCGCCAGACCTG GCGACATCCTGACCGGCTGGGCTATGGATGTGTGGGGCCAGGGAACACTGGTGA CAGTGTCCAGCGCTGCAGCAAGTGGAGGCGGAGGAAGTGGAGGCGGTGGTTCAG GAGGAGGAGGATCTGCTCTGCAATCTGTGCTCACACAGCCCCCCAGCGTGTCTGC CGCTCCTGGACAGAAAGTGACCATCAGCTGCAGCGGATCTAGCTCCAACATCGG CAACAACTACGTGTCCTGGTATCAGCAGCTGCCTGGCACCGCCCCTAAGCTGCTG ATCTACGACAACAACAAGCGGCCATCTGGCATCCCTGATCGCTTCAGCGGCAGC AAGTCCGGCACCAGCGCCACCCTGGGAATCACCGGCCTGCAGGCCGAGGACGAA GCCGACTACTACTGTCAGAGCTACGATAGCAGCCTGAGCGGCAATTACGTGTTCG GCACAGGCACAAAGGTCACCGTGCTGATCGAGGTGATGTACCCTCCTCCTTACCT GGATAATGAGAAGTCCAACGGAACAATCATCCACGTGAAGGGCAAGCACCTGTG CCCCTCTCCTCTGTTTCCGGGCCCTTCTAAGCCCTTCTGGGTGCTGGTCGTGGTGG GCGGAGTGCTCGCTTGTTACTCTCTGCTTGTGACCGTGGCCTTCATTATCTTCTGG GTCCGCAGCAAGCGGTCCAGACTGCTGCACAGCGACTACATGAACATGACCCCT CGGAGACCTGGACCCACCAGAAAGCACTACCAGCCTTACGCCCCTCCACGCGAC TTCGCCGCTTATCGCAGCCGCGTTAAATTTTCACGCAGCGCGGACGCCCCTGCGT ACAAGCAGGGACAGAACCAGCTGTACAACGAGCTCAACCTGGGCCGCCGGGAG GAGTACGACGTGCTGGACAAGCGCCGGGGGCGTGATCCAGAGATGGGCGGCAA ACCCCGCCGCAAGAATCCTCAGGAGGGCTTATACAACGAGCTGCAGAAGGACAA AATGGCCGAGGCCTACAGCGAGATCGGCATGAAGGGAGAGCGCCGGCGTGGAA AGGGCCATGACGGCCTCTATCAGGGTCTGTCCACCGCCACCAAGGACACCTACG ACGCTCTACACATGCAGGCCTTGCCTCCGCGCGAGGGCAGAGGCAGCCTGCTGA CCTGCGGCGATGTGGAGGAGAACCCCGGACCTATGCTGTTGCTTGTTACAAGCCT CCTCCTGTGCGAATTGCCTCACCCCGCATTTCTCCTGATACCCGACATCGTGATG ACCCAGTCCCCAGACAGCCTGGCTGTGTCCCTGGGCGAACGGGCCACCATCAAC TGCAAGAGCAGCCAGAGCGTGCTGTACAGCAGCAACAACAAGAATTACCTGGCC TGGTACCAGCAGAAACCTGGCCAGCCTCCCAAGCTGCTGATCTACTGGGCCAGC ACCAGAGAGAGCGGAGTGCCTGATAGATTCAGCGGCAGCGGATCTGGCTCTGAT TTTACCCTGACAATCAGCAGCCTCCAGGCCGAGGACGTCGCCGTGTACTATTGTC AGCAATACTACTCCTTCTACCAAACATTCGGCCAGGGCACCAAAGTTGAAATCA AGGGAGGAGGAGGGTCTGGAGGTGGAGGAAGTGGCGGAGGGGGAAGTGAGGTG CAGCTGGTCGAGAGCGGCGGAGGCGTGGTGCGGCCTGGAGGATCTCTCAGACTG AGCTGTACCGCCAGCGGATTTACCTTCGGCGACTACGGCATGAGCTGGGTGCGCCAttorney Docket No.: 063384-523001 WOAGGCCCCTGGCAAAGGCCTGGAATGGGTTTCCGGCATCAACTGGAACGGCGGCT CTACAGGCTACGCCGACAGCGTGAAGGGCAGATTCACCATCAGCCGGGACAACG CCAAGAATAGCCTGTACCTGCAAATGAACAGCCTGAGAGCTGAAGATACAGCTC TGTATTACTGCGCCAGAAAGTCCTACTACGGCTCTGGCAGCCCCGATGTGTTCGA CATCTGGGGCCAGGGCACAATGGTGACCGTGTCCAGCATAGAAGTAATGTACCC TCCGCCTTATCTCGATAATGAAAAAAGCAACGGGACAATTATTCATGTGAAGGG CAAGCATCTTTGTCCCTCACCGCTTTTCCCTGGACCATCAAAACCATTTTGGGTGC TCGTCGTCGTGGGAGGGGTCCTCGCCTGCTATTCTCTGTTGGTGACTGTAGCCTTT ATCATTTTTTGGGTTAAACGGAAGAAGCAGAGATCTAGACGGAACGACGAGGAA CTGGAAACCCGCGCTCACAGGGTGGCCACCGAGGAAAGAGGCAGAAAGCCACAC CAGATCCCCGCCTCAACACCTCAGAATCCCGCCACAAGCCAGCACCCCCCACCTC CTCCTGGCCATCGCAGCCAGGCCCCTAGCCACCGGCCTCCACCTCCCGGTCACAG AGTGCAGCACCAACCTCAAAAACGGCCCCCCGCTCCAAGCGGCACCCAAGTTCA CCAGCAGAAGGGCCCTCCTCTGCCTAGACCTAGAGTCCAGCCTAAGCCTCCTCAC GGCGCTGCTGAGAACAGCCTGAGCCCTTCTTCGAATCGGGTCAAATTCAGTCGCT CTGCCGATGCTCCAGCCTACAAACAAGGCCAAAACCAATTATACAATGAACTTA ATCTCGGGCGCAGGGAAGAGTACGATGTTCTCGATAAAAGGCGGGGACGCGACC CCGAAATGGGTGGAAAGCCAAGAAGGAAGAACCCGCAGGAAGGACTGTATAAT GAGTTACAAAAGGATAAGATGGCAGAGGCATATTCAGAAATCGGGATGAAGGG CGAAAGAAGAAGAGGCAAGGGACACGATGGGCTTTATCAAGGCTTAAGCACTGC AACAAAGGATACTTATGATGCACTCCATATGCAAGCTCTCCCACCTAGA (SEQ ID NO: 65).PHARMACEUTICAL COMPOSITIONS

[0345] 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.

[0346] 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)Attorney Docket No.: 063384-523001 WOor 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).

[0347] 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.

[0348] 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.

[0349] 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.

[0350] 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,Attorney Docket No.: 063384-523001 WOalanine, 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).

[0351] 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.

[0352] 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.

[0353] In some embodiments, a surfactant may also be added to the formulation to reduce aggregation and / or minimize the forma tion of particula tes 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.Attorney Docket No.: 063384-523001 WO

[0354] 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 Cryostor® CS10 and frozen.METHODS OF TREATMENT

[0355] Administration of 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).

[0356] 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 neopl astic 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.

[0357] 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. InAttorney Docket No.: 063384-523001 WOsome 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.

[0358] 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 anticancer 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.

[0359] In some instances, the disease, disorder, or condition is cancer. The term “cancer” can refer 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. Cancers for treatment with the manufactured drug products of the present disclosure include liquid cancers (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. The term “cancer”Attorney Docket No.: 063384-523001 WOencompasses liquid, e.g., diffuse or circulating, tumors. As used herein, the term “cancer” includes premalignant, as well as malignant cancers and tumors.

[0360] 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).

[0361] 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.

[0362] 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.

[0363] In some embodiments, the cancer is a relapsed cancer. The term “relapse” as used herein refers to reappearance of a disease (e.g., cancer) after an initial period of remission orAttorney Docket No.: 063384-523001 WOresponse 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.

[0364] In some embodiments, the cancer is a refractory cancer. “Refractory” as used herein refers to a 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.Administration of pharmaceutical compositions comprising CAR-expressing immune cells to a subject

[0365] Methods for administering pharmaceutical compositions comprising CAR-expressing immune cells for the treatment of cancer, e.g., hematological 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.

[0366] 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.

[0367] 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 anyAttorney Docket No.: 063384-523001 WOappropriate 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.

[0368] 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.

[0369] 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 25x106cells, at least 30x106cells, at least 35x106cells at least 40x106cells, at least 50x106cells, at least 55x106cells, at least 60x106cells, at least 65x106cells, at least 70x106cells, at least 75x106cells, at least 80x106cells, at least 85x106cells, at least 90x106cells, at least 95 x 106cells, at least 100x106cells, at least 105x106cells, at least 110x106cells, at least 115×106cells, at least 120x106cells, at least 125x106cells, at least 130x106cells, at least 135x106cells, at least 140x106cells, at least 145x106cells, or at least 150x106cells.

[0370] In some embodiments, the pharmaceutical compositions include a therapeutically effective amount of CD22 / CD20 / CD19 CAR T cells as disclosed herein, which can be at least 25x106cells, at least 30x106cells, at least 35x106cells at least 40x106cells, at least 50x106cells, at least 55 x 106cells, at least 60x106cells, at least 65x106cells, at least 70x106cells, at least 75 x 106cells, at least 80x106cells, at least 85 x 106cells, at least 90x106cells, at least 95x106cells, at least 100x106cells, at least 105 x 106cells, at least 110x106cells, at least 115X106cells, at least 120x106cells, at least 125x106cells, at least 130x106cells, at least 135x106cells, at least 140x106cells, at least 145x106cells, or at least 150x106cells.

[0371] 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 x 105cells, at least 25x106cells, at least 30x106cells, at least 35x106cells at least 40x106cells, at least 50x106cells, at least 55 x 106cells, at least 60x106cells, at least 65x106cells, at least 70x106cells, at least 75 x 106cells, at least 80x106cells, at least 85 x 106cells, at least 90x106cells, at least 95x106cells, at least 100x106cells, at least 105x106cells, at least 110x106cells, at least 115×106cells, at least 120x106cells, at least 125x106cells, at least 130x106cells, at leastAttorney Docket No.: 063384-523001 WO135x106cells, at least 140x106cells, at least 145x106cells, or at least 150x106cells, 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.

[0372] In some embodiments, the CAR-expressing immune cells are administered systemically, e.g., via intravenous infusion or injection.

[0373] 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.

[0374] 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 hematological cancer, suitable parameters can include number of months of progression-free survival, overall survival, stage or grade of the disease, the rate of disease progression, 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.

[0375] 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, anAttorney Docket No.: 063384-523001 WOeffective 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

[0376] 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.

[0377] 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.EXAMPLE 1Comparison of Process Performance and Drug Product Using Fresh and Cryopreserved Apheresis

[0378] This example describes studies aimed to evaluate the CRG-023 manufacturing process using either cryopreserved apheresis or fresh apheresis.

[0379] Apheresis from two healthy donors was collected and volumetrically split in half for each donor as seen in the process flow diagram from FIG. 1.

[0380] For each donor, the following steps were executed on half of the split apheresis to represent the cryopreservation arm of the study. On Day-X, the apheresis was loaded on to the Cue® Cell Processing System, an automated and functionally closed cell processing system, for an automated dilution and platelet reduction wash step followed by elution in Plasma-Lyte A + 4% (w / v) HS A utilizing the CryoApheresis CARGO protocol. Finally, CryoStor® CS10 was loaded on the Cue® to perform a 1:1 volumetric dilution for formulation of three product bags at 50.0 mL and 10.0 mL left in the bulk bag for retain samples. Allocated volume for retain samples were aliquoted into 1.0 mL / vial for further characterization and product retains.

[0381] The cryopreservation of formulated apheresis product bags and retain sample vials was performed in the CryoMed™ controlled rate freezer (CRF; Thermo Fisher). The cryopreserved apheresis bags were then stored in a temperature monitored LN2 freezer at < -Attorney Docket No.: 063384-523001 WO130°C until further manufacturing for Day 0 operations. The recommended shelflife of cryopreserved apheresis for subsequent Day 0 operations is up to one year.

[0382] Cryopreserved apheresis bag(s) were retrieved from LN2 freezer storage and transferred into the manufacturing space via temperature-controlled storage container. The cryo-apheresis bag(s) were placed into a secondary bag then thawed using the Plasmatherm (Barkey) at 37°C for a target of 3 minutes, up to 5 minutes, if needed. Cold TCM from a 2-8°C refrigerator was added to the thawed apheresis. The diluted, thawed apheresis bag(s) were placed on cold gel pack(s) and combined if more than one apheresis bag was thawed. Then a sample was taken from the diluted apheresis for cell count and viability using the NucleoCounter® NC-200™ (Chemometec). If the post-thaw resulted in a quantity of target cells greater than 3 x 109, then the excess apheresis material was removed prior to loading onto the CliniMACS Prodigy®.

[0383] From this point, each arm of the fresh versus cryopreserved treatments was carried through the CRG-023 process described herein.1. Performance Indicators

[0384] There are five main performance indicators by which the differences between cryopreserved apheresis and fresh apheresis were evaluated for in-process performance (Table 4).Table 4: Process Parameter Performance IndicatorsResponseProcess Parameter Response Category Rationale CategoryNot anticipated to affect the likelihood Prodigy Enrichment Recovery Secondaryof a conforming batchNot anticipated to the likelihood of a Day 0 to Day 1 Contraction Secondaryconforming batchAnticipated to affect the likelihood of a Fold Expansions Primaryconforming batchAnticipated to affect the likelihood of a Transduction Efficiency Primaryconforming batchAnticipated to affect the likelihood of a Total CAR+ Cell Yield Primaryconforming batchSome responses of performance were of greater importance to ensure generating a conforming dose by the end of the harvest window. For the sake of clarity, these categories have been delineated by chronology in FIG. 1, but it is important to discuss which of these parameters are most critical when choosing whether to process from fresh or cryopreserved material. Responses that are determined to be of higher importance have been labeled with a primary demarcation, while less important responses have a secondary demarcation.Attorney Docket No.: 063384-523001 WOi. Prodigy Enrichment Recovery

[0385] Differences in data with respect to apheresis starting material between each patient for purposes of Prodigy enrichment recovery are shown in Table 5.Table 5: Apheresis Starting Material (Prodigy Input) _ _ _Fresh Cryo Fresh Cryo Donor, Vector D001006110 (LV- D001006765 (LV- 0235 L) 02365 L) Cell Lot Number Unit PS. CL051 PS. CL052 PS. CL062 PS. CL063 Fresh or Frozen NA Fresh Cryo Fresh Cryo Volume mL 106.5 190 35 96 Viable Cell Cone. Cells / mL 6.57E+07 1.98E+07 1.32E+08 5.30E+07 Viability % 96.4% 72.4% 98.0% 75.6% Total TVC Input cells 7.00E+09 3.76E+09 4.62E+09 5.09E+09 CD45+CD3+ % of CD45 491%(1) 68%(1) CD45+CD3+CD4+CD8- % of CD45 31%(1 ) 40%(1) CD45+CD3+CD4-CD8+ % of CD45 21%(1 ) 23%(1) Frequency CD4+ and CD8+ of % of CD45 53%(1 ) 63%(1) CD45+Total Target Input cells 3.69E+09 1.98E+09 2.91E+09 3.21E+09CD4: CD8 Ratio Ratio 1.44 1.721All values which are merged across fresh and cryo columns indicate that the corresponding data is shared between the arms from sampling the apheresis before the volumetric split.

[0386] Differences in data with respect to post enrichment cells between each patient for purposes of Prodigy output are shown in Table 6.Table 6: Post Enrichment Cells Data (Prodigy Output)Fresh Cryo Fresh Cryo Apheresis Donor, Vector Lot D001006110 (LV- D001006765 (LV- 0235 L) 02365 L) Cell Lot Number Unit PS. CL051 PS. CL180 PS. CL187 PS. CL251 Enrichment Method Prodigy TCT Prodigy TCT Number of Stages stages 3 3 3 3 Viable Cell Cone. Cells / mL 1.06E+07 5.99E+06 1.20E+07 1.38E+07 Viability % 93.1% 76.4% 98.1% 87.5% Volume mL 120 120 120 120 Total Viable Cells cells 1.27E+09 7.19E+08 1.44E+09 1.66E+09 CD45+CD3+ % of CD45 97% 95% 97% 98% CD45+CD3+CD4+CD8- % of CD45 65% 67% 60% 61% CD45+CD3+CD4-CD8+ % of CD45 37% 35% 41% 39%CD4: CD8 Enr Ratio ratio 1.74 1.92 1.45 1.56Attorney Docket No.: 063384-523001 WOFrequency CD4+ and CD8+ of % of 99% 98% 98% 98%CD45+CD3+Actual Enrichment Recovery % 34% 36% 49% 52%

[0387] Next, enrichment recovery from the Prodigy at Day 0 was evaluated. Enrichment recovery was defined as total target cells enriched divided by total target cells loaded onto prodigy (Target = CD4+ and CD8+ of CD45+).

[0388] The data in FIG. 2 demonstrates that there was no clear trend in enrichment recovery between Fresh and Cyro apheresis study arms. Additionally, this enrichment yield was determined to be a secondary response because it is generally not anticipated to affect the likelihood of a conforming batch. For this reason, while important to observe differences between fresh and cryopreserved apheresis in enrichment behavior, it should not have a large weighting in determining the decision of which option (fresh or cryo) to prioritize for manufacturing.ii. Day 0 to Day 1 Contraction

[0389] The second Process Parameter Performance Indicator, Day 0 to Day 1 Contraction, was measured next. The percentage of cell loss as measured from culture day 0 seeding at 300× 106target TVC to pre-transduction on day 1 is shown in FIG. 3. It was concluded that cryopreserved apheresis resulted in a slightly higher contraction from day 0 to day 1 across the donors examined. Additionally, as an overall response, the contraction percentage has been labelled to be of secondary importance due to its upstream nature. This parameter hence is useful for understanding process behavior but again should not be the highest weighted factor in prioritizing the manufacturing option.Hi. Fold Expansions

[0390] The day 0 to day 5 fold expansion was then measured. Table 7 shows the paired values which determine the primary process responses to be considered between fresh and cryopreserved apheresis manufacturing.Attorney Docket No.: 063384-523001 WOTable 7: In-Process Drug Product PerformanceFresh Cryo Fresh Cryo Apheresis Donor, Vector Lot D001006110 (LV- D001006765 (LV- 0235 L) 02365 L) Cell Lot Number Unit PS. CL051 PS. CL180 PS. CL187 PS. CL251 Day of Query / Harvest Day 5 5 5 5 Pre-H Viable Cell Cone. Cells / mL 5.55E+06 1.53E+07 6.55E+06 6.04E+06 Pre-H Viability % 90.3% 89.3% 89.2% 92.7% Pre-H Volume mL 277 139.9 270 269 Pre-H Total Viable Cells Cells 1.54E+09 2.14E+09 1.77E+09 1.62E+09 Fold Expansion (Total Viable Cells Day 0 to fold 5.12 7.13 5.89 5.41 Day 5)Post-H Viable Cell Cone. cells / mL 3.40E+06 1.14E+07 4.18E+06 4.24E+06 Post-H Viability % 94.5% 90.7% 92.7% 92.8% Post-H Volume mL 469 163 432 468 Post-H Total Viable Cells cells 1.59E+09 1.85E+09 1.80E+09 1.98E+09 CD45+CD3+ % of CD45 100% 98% 97% 99% CD3+CD19CAR+CD20CAR+CD22CAR+ % of CD45 33% 65% 58% 63% (Tri-CAR+)CD3+ CD19CAR+CD20CAR+CD22CAR+ % of CD45 47% 59% 44% 45% CD4+CD3+ CD19CAR+CD20CAR+CD22CAR+ % of CD45 52% 36% 55% 54% CD8+CD4: CD8 Ratio ratio 0.9 1.6 0.8 0.8 Frequency CD4+ and CD8+ of CD45+ % of 99% 95% 99% 98% CD45Pre-H Total CAR+ Cells Cells 5.04E+08 1.39E+09 1.03E+09 1.02E+09 Post-Harvest Total CAR+ Cells Cells 5.23E+08 1.21E+09 1.05E+09 1.25E+09Post-Harvest Total Cell Recovery % 104% 86% 102% 122%

[0391] The number of fold expansions between the study arms for each donor as measured by the TVC at harvest day 5 divided by the seeding density of 300× 106on day 0 was also determined as shown in FIG. 4. This graph demonstrates the observed increase in fold expansions within the cryopreserved apheresis group at harvest time. Each paired-wise cryo arm was larger than the respective fresh arm in fold expansions. Both the fresh arm and cryo arms were cultured for similar amounts of time and harvested on day 5 of culturing to ensure comparable growth periods between conditions. This is the first in-process factor which should be considered of primary importance due to its direct effect on the ability to produce a conforming batch. Generating enough fold expansions to manufacture enough cells earlier on in the harvest window is critical to the CRG-023 program’s ability to make a dose and therefore this difference should be weighted heavily.Attorney Docket No.: 063384-523001 WOiv. Transduction Efficiency

[0392] To analyze transduction efficiency, paired-wise comparisons of tri-CAR transduction efficiency as measured from in-process D5 cellular material were made. As shown in FIG. 5, it is possible to see a marked difference in transduction efficiency (%TE Tr-CAR) between the cryo arms and the fresh arms, where cryo arm exhibits higher %TE than fresh. Similarly to the Day 0 to Day 5 fold expansion parameter that was inspected across the fresh and ciyo conditions, transduction efficiency is of primary importance as well because it directly implicates the CRG-023 process performance in its ability to generate enough CAR+ cells for a dose. Maximizing the ability to generate harvests with higher %TE will improve manufacturing success rate, especially for the slower growing patients during clinical trial. It is yet to be determined, however, the difference in %TE between fresh and cry apheresis starting material derived from patients, as the data presented in Figure 5 is derived from a healthy donor. For this comparison, all MOIs used were the same within each donor and the cells were transduced on day 1 of the process as denoted in the process description RPT- 0102.v. Total CAR+ Yield

[0393] Total CAR+ yield (Viable CD3+ Tri-CAR+ cells) was then compared by examining total CAR+ cells at day 5 harvest between the cryo and fresh conditions for the three donors investigated in the study. From FIG. 6, there is a clear increase in total CAR+ yield at harvest on day 5 in the cryopreserved donors. Similarly to the trends observed between fresh and cryo apheresis starting material for the previous parameters of TVC and %TE, total CAR+ yield is the primary factor in determining process performance differences between fresh and cryo apheresis due to its immediate and direct impact on the ability to formulate to a dose. Generating more CAR+ cells at harvest would improve the likelihood of avoiding any nonconforming batches that do not meet dose due to slower growing batches.

[0394] Additional data related to the donor apheresis and process steps at Day -X is shown in Tables 8-12. Day -X refers to the day that donor apheresis is cryopreserved or used fresh. Table 8: NC-200 Count on Fresh Aph (Day-X)Fresh Cryo Fresh Cryo Apheresis Donor, Vector D001006110 (LV- D001006765 (LV-023 Lot 0235L) 65L)Cell Lot Number Unit PS. CL051 PS. CL180 PS. CL187 PS. CL251 Volume mL 213 98Viable Cell Cone. Cells / mL 6.57E+07 1.32E+08 Viability % 96.4% 98.0%Total Viable Cells cells 1.40E+10 1.29E+10Attorney Docket No.: 063384-523001 WOTable 9: Sysmex Readout on Fresh Aph (Day-X)Fresh Cryo Fresh Cryo Donor, Vector D001006110 (LV- D001006765 (LV-0230235L) 65L)Cell Lot Number Unit PS. CL051 PS. CL180 PS. CL187 PS. CL251 WBC e6 / mL 62.2 149.6RBC e9 / mL 0.26 0.435PLT e6 / mL 1918 3367HCT % 1.65% 2.90% Total WBC e6 WBC 13248.6 14660.8 Total RBC e9 RBC 55.38 42.63Total PLT e6 PLT 4.09E+05 3.30E+05 Table 10: Cue Input Values (Day-X)Cryo Cryo Apheresis Donor, Vector D001006110 (LV- D001006765 (LV-023 Lot 0235L) 65L)Cell Lot Number Unit PS. CL180 PS. CL251 Source Volume mL 108 59Total Cell Input -NC200 cells 7.10E+09 7.79E+09 Total WBC Input - Sysmex e6 cells 6720 8830Viability % 96.4% 98.0%Table 11: Cue Output Values - Post-Formulation (Bulk Bag) (Day-X)Cryo Cryo Donor, Vector D001006110 (LV- D001006765 (LV-0230235L) 65L)Cell Lot Number Unit PS. CL180 PS. CL251 Volume mL 106 52Viable Cell Cone. - NC-200 cells / mL 5.40E+07 1.13E+08 Total Cell Output - NC-200 cells 5.73E+09 5.88E+09 Viability % 94.1% 83.8%Recovery - NC-200 % 81% 75%Table 12: Flow Cytometry Analysis of Fresh Apheresis (Day-X)Fresh Cryo Fresh Cryo Donor, Vector D001006110 (LV- D001006765 (LV-0230235L) 65L)Marker Unit PS. CL051 PS. CL180 PS. CL187 PS. CL251 CD45+CD3+ % of CD45 49% 68% CD45+CD3+CD4+CD8- % of CD45 31% 40% CD45+CD3+CD4-CD8+ % of CD45 22% 23% Frequency CD4+ and CD8+ % of CD45 53% 63%of CD45+Attorney Docket No.: 063384-523001 WOThe tables related to Cue calculations and recoveries are only applicable to the cryo arms of the experiment since Cue is used for apheresis cryopreservation per the process description, but the subsequent CCV, Sysmex readouts, and fresh apheresis flow that was performed is relevant to both halves of the split apheresis. Fresh arms are not processed using the Cue since there is no need for formul ation of the apheresis with CS10 when proceeding straight to the isolation using Prodigy.2. Drug Product Release Attributes

[0395] The %TE Tri-CAR from each arm of both paired fresh and cryo runs in thawed drug product was next compared. As shown in Table 13, notably, each of the cryo arms are higher in %TE Tri-CAR than their fresh counterparts, which contribute to a larger amount of Tri-CAR+ cells generated at the harvest day 5. This data is in line with the trends seen from the in-process %TE Tri-CAR seen in FIG. 7.Table 13: %TE Tri-CAR of healthy donor derived Drug Product from fresh or frozen apheresis starting material measured with TM-0019DonorMaterial Starting Material %TE Tri-CAR ID, Vector LotDrug product Fresh 40% D001006110,(LV-0235L) Drug Product Cryo 67% D001006765, Drug Product Fresh 58%(LV-02365L) Drug Product Cryo 63%

[0396] VCN was also evaluated in two healthy donor pairs. As shown in FIG. 7 and Table 14, VCN / Tri-CAR+ cell values per TM-0028 trended higher in the cryo arm as compared to the fresh arm in both donors.Table 14: VCN / Tri-CAR+ cell for Drug Product from Fresh or Cryopreserved ApheresisSample ID PS. CL Apheresis MOI Harvest VCN / Tri- State Material Vector Day %TE CAR+ Cell D001006110 PS. CL051 Fresh LV-023 3 40 8 Healthy 5LD001006110 PS. CL180 Cryo Donor- LV-023 3 67 10 Derived 5L Day 5D001006765 PS. CL187 Fresh Drug LV-023 1.7 58 6ProductD001006765 PS. CL251 Cryo 65L 1.7 63 7Attorney Docket No.: 063384-523001 WOFor donor DOO 1006110, the difference in VCN / Tri-CAR+ between the fresh and cryo samples was 2 copies, with the cryo sample having a higher VCN of 10 copies / Tri-CAR+ cell. Transduction efficiency was also higher in the cryo sample than in the fresh sample. For the second donor, D001006765, the difference in VCN between the fresh and cryo arms was 1 copy. Though cryo VCN did trend higher than fresh VCN, the differences seen are minimal and well within the variability of the assay, suggesting that either apheresis handling method would be suitable for use in the respective manufacturing process. Respective MOIs were selected for each pilot scale 5L LV- 023 and 65L LV-023 batch by titer according to CRT-0177 and RPT-0251.3. Drug Product Extended Characterization Attributes and Analytics

[0397] Drug product samples were then assessed by extended characterization assays.

[0398] First, T Cell Memory Subsets by Flow Cytometry was performed to characterize the distribution of T cell memory subsets from C AR+CD4+ (defined by the CD3+CAR+CD4+CD8- cell population) and CAR+CD8+ (defined by the CD3+CAR+CD4-CD8+ cell population) cells in each sample. The CAR+ CD4 / CD8 ratios were also measured using this assay to track the distribution of CD4+ and CD8+ cells; reflected by CD22 CAR+ as the assay does not include CD 19 CAR or CD20 CAR detection. This is considered an acceptable surrogate for measurement of Tri-CAR+ cells as the drug product is expected to be homogeneous for the three CARs. The results are shown in Table 15 and FIG. 8.Attorney Docket No.: 063384-523001WOTable 15: T Cell Memory Phenotype of Drug Product Manufactured from Fresh or Cryopreserved AphereslsDonor ID PS. CL Material Starting TN1TSCM1TCM1TEM1TEMRAI MaterialCAR+CAR+CAR CAR CAR+CAR CAR+CAR CAR CAR+CD4+2CD8+2 +CD4+2+CD8+2CD4+2+CD8+2CD4+2+CD8+2 +CD4+2CD8+2PS. CL051 Healthy Donor- Derived Drug Fresh 0.0% 0.0% 0.4% 1.8% 91.9% 92.2% 7.7% 6.0% 0.0% 0.1% D001006110 ProductPS. CL180 Healthy Donor- Derived Drug Cryo 0.0% 0.0% 0.1% 1.0% 93.8% 93.1% 6.1% 5.7% 0.0% 0.2% ProductPS. CL187 Healthy Donor- Derived Drug Fresh 0.0% 0.0% 0.4% 0.4% 88.8% 95.1% 10.8% 4.5% 0.0% 0.0% D001006765 ProductPS. CL251 Healthy Donor- Derived Drug Cryo 0.0% 0.0% 0.2% 0.1% 93.6% 98.2% 6.3% 1.7% 0.0% 0.0%Product1TN (Naive T cell), TSCM (Stem Cell Memory T cell), TCM (Central Memory T cell), TEM (Effector Memory T cell), TEMRA (Effector Memory T cell Re-expressing CD45RA).2CAR+ reflects CD22 CAR+ cells; CD4+ reflects CAR+CD4+CD8- and CD8+ reflects CAR+CD4-CD8+. T cell memory subset analysis was performed using the TM-0002 method.Attorney Docket No.: 063384-523001 WOT cell memory phenotype was quantified by flow cytometry based on the expression of CD45RA and CCR7 cell surface markers on CAR+CD4+ (defined by the CD3+CAR+CD4+CD8- cell population) and CAR+CD8+ (defined by the CD3+CAR+CD4-CD8+ cell population) for CRG-023 drug product manufactured from forward-processed (fresh) or cryopreserved (frozen) healthy donor apheresis. The predominant distribution of memory subsets was primarily observed in central memory (TCM) and effector memory (TEM) populations, with no definitive trends in differentiation between fresh and cryo matched-pair arms.

[0399] T Cell Activation and Exhaustion Marker Analysis by Flow Cytometry was performed to characterize CAR+CD4+ and CAR+CD8+ activation and exhaustion marker expression, which can be upregulated in response to TransAct™ during the manufacturing process. As shown in Table 16 and FIG. 9, the activation marker profile showed high levels of CD25 compared to lower frequencies of CD69 and 4- IBB, indicating consistent drug product profile regardless of being manufactured from fresh or frozen healthy donor apheresis.Table 16: T Cell Activation Markers in Drug Product Manufactured from Fresh or Cryopreserved ApheresisDonor PS. CL Material Starting CD25+CD69+4-1 BB+ID Material CAR* CAR* CAR* CAR* CAR* CAR*CD4+1CD8+1CD4+1CD8+1CD4+1CD8+1D001006110 PS. CL051 Healthy Donor- Fresh 83.6% 62.0% 4.7% 3.2% 3.5% 4.7%Derived DrugProductPS. CL180 Healthy Donor- Cryo 78.8% 46.1% 2.7% 3.2% 3.9% 6.7%Derived DrugProductD001006765 PS. CL187 Healthy Donor- Fresh 82.7% 74.9% 5.1% 3.2% 2.6% 5.0%Derived DrugProductPS. CL251 Healthy Donor- Cryo 89.6% 78.4% 7.5% 3.6% 3.1% 6.1%Derived DrugProduct1CAR+ reflects CD22 CAR+ cells; CD4+ reflects CAR+CD4+CD8- and CD8+ reflects CAR+CD4-CD8-Further, individual assessment of LAG-3, PD-1, TIGIT, and TIM-3 revealed varying frequencies of expression, possibly reflecting TransAct ™-mediated activation, but generally did not show major differences between drug products manufactured from fresh and cryo apheresis (Table 17 and FIG. 10).Attorney Docket No.: 063384-523001 WOTable 17: T Cell Exhaustion Phenotype in Drug Product Manufactured from Fresh or Cryopreserved ApheresisPS. CL LAG-3* DonorI Material Startin LAG-3* PD-1* TIGIT* TIM-3* PD-1* D g TIGIT* Materia TIM-3* I CAR CAR CAR CAR CAR CAR CAR CAR CAR CAR+ + + + + + + + + + CD4+CD8+CD4+CD8+CD4+CD8+CD4+CD8+CD4+CD8+1 1 1 1 1 1 1 1 1 1 PS. CL Healty051 Donor- Derived Fresh 8.0% 12.5% 46.5% 7.8% 5.8% 5.9% 85.4% 85.4% 0.5% 1.1% DrugDOO 100611 product0 PS. CL Healty1 80 Donor- Derived Cryo 7.4% 9.8% 33.0% 8.2% 5.3% 3.8% 95.6% 96.9% 0.6% 1.1% DrugproductPS. CL Healty1 87 Donor- Derive Fresh 10.6% 14.3% 45.4% 33.7% 8.3% 2.8% 84.1% 90.2% 1.5% 1.6% d DrugProductD001006765 PS. CL Healty251 Donor- Derived Cryo 19.3% 25.0% 65.4% 21.8% 6.0% 3.3% 93.5% 93.5% 1.0% 0.7% DrugProduct1CAR+ reflects CD22 CAR+ cells; CD4+ reflects CAR+CD4+CD8- and CD8+ reflects CAR+CD4-CD8+Exhaustion marker co-expression showed minimal frequencies across drug products manufactured from either fresh or cryo apheresis (FIG. 11). These data indicate that CRG-023 drug products are not in a state of exhaustion.

[0400] The single and double CAR populations (CD 19 CAR+ CD20 CAR- CD22 CAR-, CD 19 CAR+ CD20 CAR+ CD22 CAR-, CD 19 CAR+ CD20 CAR- CD22 CAR+, CD 19 CAR- CD20 CAR+ CD22 CAR-, CD 19 CAR- CD20 CAR+ CD22 CAR+, and CD 19 CAR- CD20 CAR- CD22 CAR+) were also analyzed, which reflect the portion of surface transgene expressing-cells that are not the Tri- CAR+ subset. Frequencies were measured to be below the LOQ of 2% for all runs, indicating that the transduced drug product manufactured from fresh and cryopreserved apheresis is generally homogenous for the expression of the three CARs. Results are shown in Table 18.Table 18: Single and Double CAR+ Frequencies in Drug Product Manufactured from Fresh or Cryopreserved Apheresis Starting MaterialAttorney Docket No.: 063384-523001 WO%CD19 %CD19 %CD19 %CD19 %CD19 %CD19 CAR* CAR* CAR*Donor ID PS. CL Material Starting CAR CAR CAR Material CD20 CD20 CD20 CD20 CD20 CD20CAR CAR* CAR CAR* CAR* CAR CD22 CD22 CD22 CD22 CD22 CD22 CAR CAR CAR* CAR CAR* CAR* PS. CL051 HealthyDonor- Derived Fresh < LOQ1< LOQ1< LOQ1< LOQ1< LOQ1< LOQ1DrugproductD001006110PS. CL180 HealthyDonor- Derived Cryo < LOQ1< LOQ1< LOQ1< LOQ1< LOQ1< LOQ1DrugProductPS. CL187 HealthyDonor- Derived Fresh < LOQ1< LOQ1< LOQ1< LOQ1< LOQ1< LOQ1DrugProductD001006765PS. CL251 HealthyDonor- Derived Cryo < LOQ1< LOQ1< LOQ1< LOQ1< LOQ1< LOQ1DrugProduct

[0401] Next, a development panel utilizing annexin V and the 7-AAD viability dye was performed to evaluate cell health on post-thaw drug product samples. Populations were reported out of either CAR+CD4+ or CAR+CD8+ subsets, with live cells defined as annexin V7-AAD", dying cells defined as annexin V“7-AAD‘, and dead cells defined as annexin V+7-AAD+.

[0402] As shown in Table 19 and FIG. 12, comparison between drug products showed no obvious differences between post-thaw drug product cell health manufactured from fresh and frozen starting apheresis when comparing individual CAR+CD4+ or CAR+CD8+ subsets or when analyzing the overall cell health population out of CAR+CD3+ cells (excluding CAR+CD4+CD8+ and CAR+CD4-CD8- subsets).Attorney Docket No.: 063384-523001 WOTable 19: Cell Health frequencies of healthy donor derived DP from Fresh or Frozen Apheresis Starting MaterialLive Dying Dead Donor ID Material StartingPS. CL Material CAR+CAR+CAR+CAR+CAR+CAR+CD4+1CD8+1CD4+1CD8+1CD4+1CD8+1PS. CL051 Drug Fresh 87.5% 73.2% 3.7% 20.4% 8.4% 6.4% D001006110 ProductPS. CL180 Drug Cryo 75.4% 77.6% 1.0% 1.5% 23.0% 19.9%ProductPS. CL187 Drug Fresh 77.2% 83.5% 1.3% 1.0% 20.4% 13.9% D001006765 ProductPS. CL251 Drug Cryo 71.2% 78.6% 0.8% 1.7% 25.5% 17.2%ProductOverall Overall Overall Live2Dying2Dead2CAR+CAR+CAR+CAR+CAR+CAR+CD4+1CD8+1CD4+1CD8+1CD4+1CD8+1PS. CL051 Drug Fresh 39.6% 37.8% 1.7% 10.5% 3.8% 3.3% D001006110 ProductPS. CL180 Drug Cryo 48.8% 24.9% 0.6% 0.5% 14.9% 6.4%ProductPS. CL187 Drug Fresh 33.4% 45.4% 0.6% 0.6% 8.8% 7.6% D001006765 ProductPS. CL251 Drug Cryo 31.9% 42.1% 0.4% 0.9% 11.4% 9.2%Product'CAR+ reflects CD22 CAR+ cells; CD4+ reflects CAR+CD4+CD8- and CD8+ reflects CAR+CD4-CD8+Overall populations are calculated by multiplying the CAR+CD4+ results by the %CD4+ out of CD3+ or the CAR+CD8+ results by the %CD8+ out of CD3+ value.

[0403] Next, analysi s of the CD4 / CD8 ratio in the CAR+compartment showed no major trends between drug product manufactured from fresh or cryopreserved apheresis (Table 20). Table 20: CAR+ CD4 / CD8 Ratio in Drug Product Manufactured from Fresh or Cryopreserved ApheresisPS. CL CAR+CD4 / CD8 Donor ID Material Starting Ratio1MaterialPS. CL051 Drug product Fresh 0.8 D001006110PS. CL180 Drug Product Cryo 1.9 PS. CL187 Drug Product Fresh 0.7 D001006765PS. CL251 Drug Product Cryo 0.8'CAR+ reflects CD22 CAR; CAR+ CD4 / CD8 ratio reflects CAR+CD4+CD8- / CAR+CD4-CD8+

[0404] Finally, potency was assessed via Ella™ automated ELISA. Briefly, drug product lots that were produced from either the fresh apheresis arm or the cryopreserved apheresis arm were co-cultured with four Raji target cells that individually express CD22, CD 19, and CD20, in addition to a triple-knockout Raji cell line serving as the negative control.Attorney Docket No.: 063384-523001 WOFollowing a 24 hour co-culture, supernatants were collected and analyzed by Ella™ to measure IFN-y concentrations at different effector to target (E: T) ratios. IFNy was determined in the 1:1 Effector: Target (E: T) ratio for the CD19, CD20 and CD22 cell lines, and in the 1:1 E: T ratio for the Triple Knock-Out (TKO) cell line. These DP Potency by Ella™ runs passed all acceptance criteria outlined in TM-0027. IFNy secretion was highest in the CD20 co-culture condition across both donors tested, followed by CD 19, CD22, and TKO co-culture conditions, regardless of starting apheresis state (FIG. 13 and Table 21).Table 21: IFNy Secretion in Fresh and Cryo Drug Product samplesDonor ID Material Vector Harvest Apheresis CD19 CD20 CD22 TKO Day State (ng / mL) (ng / mL) (ng / mL) (ng / mL) Healthy Donor- D001006110 Derived Drug 5L 5 Fresh 10.55 20.01 8.35 6.52ProductHealthyD001006110 Donor-Derived 5L 5 Cryo 6.11 11.72 3.79 3.13Drug ProductHealthy Donor- D001006765 Derived 65L 5 Fresh 24.46 50.74 22.04 16.35Drug ProductHealthy Donor- D001006765 Derived Drug 65L 5 Cryo 7.22 15.65 6.98 5.41ProductMoreover, higher IFNy secretion was observed in the fresh apheresis arm compared to the cryo apheresis arm across both donors and all co-culture conditions. However, as only two donors were assessed in this dataset, no definitive conclusions can be made on whether fresh apheresis will consistently yield a more potent drug product than cryo apheresis. This data set indicates that both conditions yield a drug product that is potent, and therefore, optionality between fresh apheresis and cryo apheresis can be preserved.4. Conclusion

[0405] After evaluation of paired runs of the CRG-023 process from both cryopreserved and fresh apheresis, there are no conclusive distinctions to be made between the options with respect to in-process performance indicators. Both options for manufacturing were able to generate enough tri-CAR+ cells by harvest day to formulate to the highest proposed dose. Both cryopreserved arms from the paired runs generated a greater amount of CAR+ cells at harvest than their fresh counterparts.

[0406] Upon examination of the drug product attributes of the fresh and cryopreserved arms of the study, there are no major differences in post-thaw drug product cell health manufactured from fresh and cryopreserved starting apheresis. Additionally, the differences seen betweenAttorney Docket No.: 063384-523001 WOcryo VCN and fresh VCN are minimal. From the potency testing on the drug product, the data indicates that both conditions yield a drug product capable of secreting IFNy, and therefore, either fresh apheresis or cryo apheresis may be employed for CRG-023 manufacturing.

[0407] Overall, there is feasibility to generate an acceptable batch of CRG-023 Drug product using either fresh or cryopreserved apheresis. For the phase 1 trial, due to the increased ease in manufacturing from cryo from both a scheduling and supply chain versatility perspective, the cryo option will be used for forward processing from apheresis.EXAMPLE 2Process Performance and Drug Product Characterization from Day 5 to Day 9

[0408] Longitudinal assessments are essential for a comprehensive understanding of characteristics of CAR-T therapy. Evaluating culture days and harvest days across multiple donors increases process and product understanding in CAR-T cell production. Furthermore, longitudinal data can reveal how different donor characteristics may influence the therapy’s efficacy and safety, thereby improving patient outcomes. Understanding these long-term effects and variations helps optimize culture conditions, ultimately enhancing the overall success of CAR-T therapies.

[0409] This study aims to evaluate the l ongitudinal effects of extending the CRG-023 cell culture duration through a Day 9 harvest, while collecting retains on potential harvest Days 5 and 7. By sampling across multiple days, cellular expansion, differentiation, and potency properties of the drug product can be assessed as a function of time in culture. This study was conducted in Process Development (PD) at CARGO Therapeutics over the span of 11 days (Day -X to Day 9 harvest). The CRG-023 drug product was generated from healthy donor-derived apheresis, following a process representative of the CRG-023 clinical manufacturing process. The culture has been transduced using LV-023, a lentivirus produced by Oxford Biomedica from a tri-cistronic construct (CD 19, CD20, and CD20 CAR+). Sample retains were vialed, cryopreserved, and stored at < -130°C until the day of analytical testing.Multiple harvest days are investigated to look at the potential effects associated with prolonged culture duration.

[0410] Notably, since the minimum harvest threshold would generally be achieved earlier than the Day 9 harvest, it was not used to trigger any culturing decisions in this study.Additionally, since the culture was at risk of over-growing the G-Rex by the later days of theAttorney Docket No.: 063384-523001 WOharvest window, a split condition was implemented to prevent the G-Rex from reaching maximum cellular capacity before Day 9, which is further described in the methods section.1. Methods

[0411] For this study, drug product bags were generated from apheresis derived from two healthy donors using a process highly representative of the CRG-023 manufacturing process. The drug product was formulated and filled into 2 mL cryovials, with a fixed concentration of IxlO6viable CAR+ T cells / mL.

[0412] The apheresis material from one healthy donor (BOO 1002139) was processed using the firi-cel methodology, which included washing, formulation, and filling, while the apheresis material from the second healthy donor (DOO 1006680) was processed according to the formulation procedures outlined herein.

[0413] CRG-023 drug product was generated in the CARGO Therapeutics development lab foll owing the CRG-023 drug product process description up until Day 5 of culturing.However, since the minimum harvest threshold was not used in this study to determine harvest date, the culture was at risk of over-growing the G-Rex by the later days of the harvest window. Hence, a split condition was implemented to prevent the G-Rex from reaching maximum cellular capacity before Day 9. Each of the two healthy donor materials followed two-armed strategy: one G-Rex which was not split and carried through Day 9, and one G-Rex which was split and then also carried through Day 9 before harvest.

[0414] The splitting of the cell culture was executed by first proceeding with the media reduction on Day 5 in an identical manner to what occurs for the Day 5 pre-harvest sample outlined herein. However, at this point, the G-Rex was ingressed into the BSC where a serological pipette was used to remove 75% of the cellular material as measured volumetrically. The removed cell culture was then spun via centrifuge, at which point the supernatant was added back to the G-Rex. Finally, the initially removed media from the culture was added back via gravity drain. In this manner, the entirety of the initial volume of the split condition was able to be restored, while effectively removing 75% of the cells from the G-Rex.

[0415] On Day 5 and Day 7, retains were taken from the cultures. To collect samples from the G-Rex CS 100M vessel using the GatheRex, the process started by connecting a transfer bag to the vessel's reduction line. The GatheRex pump was then set up and media was removed until roughly 300 mL remains in the vessel as per RPT-0102. This remaining volume was swirled to resuspend the cells on the gas-permeable membrane. The transfer bag was weighed to calculate the remaining culture volume for total viable cell counts.Attorney Docket No.: 063384-523001 WO

[0416] A sample pouch was sterile welded onto the harvest line, the suspended cells were drawn into the line, and then returned to ensure thorough mixing. The cell density was assessed from the sample, and based on this density, multiple pouches were daisy-chained to obtain a sufficient volume containing 100 × 106cells. This volume was then used to aliquot 10 samples of 1×106cells / mL for further analysis and retains. These retains were formulated with PLA + 4% HSA alongside and CS10 to replicate drug product material for release testing.

[0417] To harvest, the previous sampling described above was repeated. Both split and no split arms from each donor were then harvested following the process description herein.

[0418] A luer is sterile welded onto the bag, and a 3 mL syringe is used to obtain a sample. The cell count is determined using the CRG-023 method on the NucleoCounter NC-200. Based on this count, the required number of cells for formulation is accurately partitioned. The necessary cell volume is then extracted from the bag with a syringe and transferred to a conical tube. The harvested culture is centrifuged at 300 x g for 10 minutes to pellet the cells. The supernatant is aspirated, and the cell pellet is resuspended in a 1:1 mixture of PLA + HSA and CryoStorl 0. Cryopreservation was carried out using CoolCells, and the samples were stored in LN2 at < -130°C.

[0419] To test the banked cellular material, experiments were executed on in- process retains and drug product samples for tri-CAR+ transduction efficiency. Drug product potency was evaluated by measuring the secretion of IFNy by the drug product after co-culture with single antigen expressing Raji cell lines engineered to express CD19, CD20, or CD22. The VCN assay for the drug product was performed using digital droplet PCR (ddPCR) to quantify the number of transgene copies integrated into the genome of the engineered Tri-C AR cells.2. Resultsi. Expansion Parameters

[0420] Both donor cultures, B001002139 and D001006680, were seeded at 300× 106cells for the No Split arms. By Day 5, these cultures reached total cell numbers of 1.97 billion and 1.00 billion. By Day 7, the cultures had expanded further, achieving total cell counts of approximately 3.66 billion and 2.77 billion. On Day 9, fold expansions continued, but slowed, resulting in final cell counts of 4.61 billion and 4.79 billion for BOO 1002139 and DOO 1006680, respectively (FIG. 14).

[0421] Wilson Wolfs specifications for the G-Rex® platform suggest a normalized maximum of 10xl06-40xl06cells / cm2, translating to a working maximum of approximately 4Attorney Docket No.: 063384-523001 WObillion cells per total 100 cm2of surface area in the G-Rex® 100M. By Day 9, this recommended maximum cell density was reached, potentially constraining further fold expansion. This saturation effect is particularly evident in the observed viability trends (FIG.15), with donor DOO 1006680 showing a decline in viability to 84.4% over time. In the case of healthy donor cells, which generally have superior expansion potential compared to patient- derived cells, the approach to the G-Rex® maximum is associated with reduced expansion rates, likely due to overcrowding effects within the culture environment. If the patient apheresis used in the clinic shows such high rates of growth which would approach this maximum at a later day of the harvest window, then the culture would have already been harvested earlier due to MHT being achieved on an earlier day. For this reason, the culture vessel is not a limiting factor for meeting dose.

[0422] Since the culture began to exit exponential phase of growth in the No Split arm of the study toward the end of the harvest window (FIG. 16), it became important to investigate the Split arm as well. Contrary to the No Split arm, which exited exponential phase early in the culture due to healthy donor high-performing growth, the Split arm was able to continue to grow exponentially through the entire harvest window due to culture reduction (FIG. 17 and FIG. 18). By comparing the drug product and in- process attributes of both the Split and No Split arm, the temporal behavior of CRG-023 in-process material can be further characterized.ii. Transduction Efficiency

[0423] The calculation of transduction efficiency (TE) is derived from the CD45+CD3+ population within the live cell fraction. Specifically, TE was determined by evaluating the percentage of Tri-CAR- expressing cells (CD19+CD20+CD22+) within the CD3+ population. The CD3+ cells are further gated from the CD45+ population to ensure accurate identification of T cell populations. This gating strategy assesses the purity of the cell population and allows for an accurate calculation of TE within the targeted CD3+ subset. The CD3+ purity for both donors remains consistently high, at approximately 99%, across all potential harvest days (FIG. 19). Refining the analysis of these specific biomarkers allows for reliable quantification of the populations of interest, facilitating accurate dosing configurations.Attorney Docket No.: 063384-523001 WOTable 22Donor, Batch ID Material Condition Split %TE B001002139,Drug Product Day 5 Yes 51% PS. CL161B001002139,Drug Product Day 7 Yes 55% PS. CL162B001002139,Drug Product Day 9 Yes 54% PS. CL162B001002139,Drug Product Day 5 No 53% PS. CL164B001002139,Drug Product Day 7 No 57% PS. CL164B001002139,Drug Product Day 9 No 55% PS. CL164D001006680,Drug Product Day 5 Yes 48% PS. CL158D001006680,Drug Product Day 7 Yes 54% PS. CL158D001006680,Drug Product Day 9 Yes 53% PS. CL158D001006680,Drug Product Day 5 No 50% PS. CL160D001006880,Drug Product Day 7 No 56% PS. CL160D001006880,Drug Product Day 9 No 52%PS. CL160

[0424] Donors BOO 1002139 and DOO 1006680 exhibited an increase in transduction efficiency (TE) from Day 5 to Day 7, followed by a decline from Day 7 to Day 9 in both the no split and split arms (FIG. 20). This pattern likely reflects the expansion limitations imposed by the culturing vessel, mirroring the growth kinetics observed in total viable cell counts and population doublings. The cultures likely reached a capacity threshold within the G-Rex® system by Day 7 to Day 9, leading to a growth plateau that contributed to the reduction in TE on Day 9. Cell overcrowding can negatively impact viral transduction efficiency and overall cell health, limiting the optimal expansion phase.Hi. CD4+ / CD8+ Ratio

[0425] Over the course of each harvest window, the CD4 / CD8 ratio decreased. Over longer harvest windows, this ratio typically decreased due to faster proliferation of CD8+T cells, which are more responsive to stimulation and growth factors, compared to CD4+cells.Additionally, prolonged culture can lead to differentiation and exhaustion, with CD8+cells favoring effector phenotypes, while CD4+cells may experience reduced viability or slower expansion. In the no split condition, the CD4+ / CD8+ratio exhibits a modest decline over time, most attributable to the limited expansion potential within the culture, which constrainsAttorney Docket No.: 063384-523001 WOoverall cell growth and preserves a more balanced proliferation between CD4+and CD8+T cells (FIG. 21A). Conversely, in the split condition, the ratio declined more sharply, driven by the increased space and resources available following the split, which facilitates unrestrained cell expansion (FIG. 21B). This favors more rapid proliferation of CD8+T cells. As the culture reached the vessel’s maximum capacity, the growth of CD4+cells was slower, leading to a more pronounced decline in the CD4+ / CD8+ratio as CD8 cells outcompeted CD4+cells in the expanded environment.iv. Total CAR+ Cells

[0426] By Day 5, the no split donor cultures (B001002139 and D001006680) had expanded to 1.04xl09and 4.92*108CAR+ cells, respectively. Continued expansion was observed by Day 7, with B001002139 reaching 2.06*109CAR+ cells and D001006680 reaching 1.53*109CAR+ cells. By Day 9, both cultures approached similar levels, with BOO 1002139 reaching 2.54xl09CAR+ cells and D001006680 achieving 2.47* 109CAR+ cells (FIGs. 22A-22B).

[0427] Across all time points, each required dose level was consistently achieved with healthy donor material. Analysis of the expansion kinetics revealed distinct differences between the two donors. Donor DOO 1006680 exhibited rapid cell expansion, reaching the logarithmic growth plateau by Day 9. In contrast, donor BOO 1002139 displayed similar growth kinetics, with the total cell count continuing to increase more gradually, even beyond Day 7. This suggests donor-specific variability in the time required to reach the maximum cell density.V. Extended Characterization

[0428] CRG-023 drug product samples were assessed by extended characterization assays.

[0429] T Cell Memory Subsets by Flow Cytometry assay was performed to assess the distribution of memory subpopulations within each drug product. The CAR+ CD4 / CD8 ratios were also measured from this assay to track the distribution of CD4+ and CD8+ cells in the transduced compartment. CAR+ cells by this method are defined by staining using (rh)CD22 protein and reflects CD22 CAR+ cells. This classification serves as a fit-for-purpose surrogate for the Tri-CAR+ population as the transduced portion of drug product is generally homogenous for all three CARs.

[0430] T Cell Activation and Exhaustion Marker Analysis by Flow Cytometry assay was performed to characterize drug product activation and exhaustion marker expression, which can be upregulated following exposure to TransAct™ in the CRG-023 manufacturing process. CAR+ cells by this method are defined by staining using (rh)CD22 protein and reflects CD22 CAR+ cells. This classification serves as a fit-for-purpose surrogate for theAttorney Docket No.: 063384-523001 WOTri-CAR+ population as the transduced portion of drug product is generally homogenous for all three CARs.

[0431] CRG-023 Tri-CAR Transduction Efficiency Characterization by Flow Cytometry assay reports out the single and double CAR populations in drug product (CD 19 CAR+ CD20 CAR-CD22 CAR-, CD 19 CAR+CD20 CAR+CD22 CAR-, CD 19 CAR+ CD20 CAR-CD22 CAR+, CD 19 CAR- CD20 CAR+ CD22 CAR-, CD 19 CAR- CD20 CAR+ CD22 CAR+, and CD 19 CAR- CD20 CAR- CD22 CAR+ cell populations), which inform on the homogeneity of transduced cells for expression of all three CARs.

[0432] The predominant populations in drug product derived from two independent healthy donors were found to be compartmentalized to central memory (TCM) and effector memory (TEM) T cell subsets (Table 23), with differentiation observed across harvest days, reflected by decreasing TCM frequencies and increasing TEM populations (Table 23). This data indicates that the drug product profile and directionality of differentiation of memory subsets is consistent when manufactured from healthy donor materialAttorney Docket No.: 063384-523001WOTable 23. T Cell Memory Phenotypes in Healthy Donor-Derived Drug Product Across Harvest DaysDonor HarvestID Batch Material DayTN TSCM TCM TEM TEMRA CAR+ CAR+ CAR+ CAR+ CAR+ CAR+ CAR+ CAR+ CAR+ CAR+ CD4+2CD8+2CD4+2 CD8+2 CD4+2 CD8+2 CD4+2 CD8+2 CD4+2 CD8+2 PS. CL1642Day 5 0.0% 0.0% 0.2% 0.1% 93.4% 96.5% 6.4% 3.4% 0.0% 0.0% B001002139 PS. CL164 Day 7 0.0% 0.0% 0.7% 0.2% 82.1% 92.9% 17.2% 6.9% 0.0% 0.0%HealthyDonor- PS. CL164 derived Day 9 0.0% 0.0% 0.1% 0.1% 74.1% 92.3% 25.8% 7.6% 0.0% 0.0%DrugPS. CL1603Product Day 5 0.0% 0.0% 0.1% 0.2% 97.6% 98.2% 2.3% 1.7% 0.0% 0.0% DO01006680 PS. CL160 Day 7 0.0% 0.0% 0.2% 0.0% 94.9% 97.0% 4.9% 2.9% 0.0% 0.0%PS. CL160 Day 9 0.0% 0.0% 0.5% 0.2% 89.8% 95.1% 9.8% 4.7% 0.0% 0.0%1TN (Naive T cell), TSCM (Stem Cell Memory T cell), TCM (Central Memory T cell), TEM (Effector Memory T cell), TEMRA (Effector Memory T cell Reexpressing CD45RA).2PS. CL161 was used for EC testing as a backup replicate sample when D5 material was limited.3PS. CL158 was used for EC testing as a backup replicate sample when D5 material was limitedAttorney Docket No.: 063384-523001 WO

[0433] Evaluation of activation markers in healthy donor-derived drug product revealed the predominant marker to be CD25, with lower levels of 4-1BB and CD69 (Table 24). This profile likely refl ects the dynamic nature of individual targets following Day 0 stimulation with the polyclonal T cell activator, TransAct™. Longitudinal expression across the harvest days showed a general reduction to activation state that was most prevalent for CD25 likely resulting from the higher initial levels on Day 5 (Table 24, which suggests that the directionality of markers is impacted by proximity to initial TransAct™ exposure. These data collectively show consistency in the trend of activation responses following TransAct™-mediated stimulation, which is essential for the efficient lentiviral transduction and proliferation of T cells.Table 24. T cell Activation Marker Assessment from Healthy Donor-derived Drug Product Across Harvest DaysDonor Harvest CD25+CD69+4-1 BB+ID Batch Material Day CAR CAR CAR CAR CA CAR*+ + R+CD8+1CD4 CD8 CD4 CD8 CD+1 +1 +1 +1 4+1PS. CL164 Day 5 92.2% 76.9% 2.6% 2.0% 1.6% 4.7% 2 HealthyBOO 1002139 PS. CL164 Donor- Day 7 40.8% 33.4% 3.2% 1.3% 1.2% 1.6% derivedPS. CL164 Drug Day 9 19.7% 22.5% 3.3% 1.6% 1.1% 2.5% PS. CL160 Product Day 5 96.6% 89.5% 2.8% 3.7% 2.9% 7.5% 3DOO 1006680 PS. CL160 Day 7 65.0% 54.2% 2.8% 1.6% 1.1% 1.6%PS. CL160 Day 9 35.8% 27.8% 2.7% 1.0% 0.6% 2.0% 'CAR+ reflects CD22 CAR+ cells; CAR+CD4+ reflects CAR+CD4+CD8- cells and CAR+CD8+ reflects CAR+CD4-CD8 cells.2PS. CL161 was used for EC testing as a backup replicate sample when D5 material was limited.3PS. CL158 was used for EC testing as a backup replicate sample when D5 material was limited.

[0434] Evaluation of the exhaustion phenotype in healthy donor-derived drug product showed variable levels of individual markers but minimal co- expression of LAG-3, PD-1, TIGIT, and TIM-3 immune checkpoint receptors (Table 25). As phenotypic characterization of T cell exhaustion status is typically reflected by sustained expression of multiple targets on the same cell, these data suggest that CRG-023 drug product is not active. Notably, there was no longitudinal increase in exhaustion marker co-expression (Table 25). These data demonstrate that the levels of drug product cells that co-express exhaustion markers are generally low and furthermore highlight that there is no expansion of this population throughout the CRG-023 process when initiating manufacturing with healthy donor material.Attorney Docket No.: 063384-523001WOTable 25. Exhaustion Marker Expression in Healthy Donor-derived Drug Product Across Harvest DaysLAG-3+PD-1+Donor Harvest LAG-3+PD-1+TIGIT+TIM-3+Batch Material TIGIT+TIM-3+ID DayCAR CAR CAR CAR CAR CAR CAR CAR CAR CAR CD4+1CD8+1CD4+1CD8+1CD4+1CD8+1CD4+1CD8+1CD4+1CD8+1PS. CL1642Day 5 8.9% 15.9% 72.4% 19.7% 2.6% 2.6% 67.9% 77.4% 0.3% 0.5% B001002139 PS. CL164 Day 7 5.3% 8.5% 38.0% 13.8% 3.6% 2.8% 66.6% 90.5% 0.2% 0.5%Healthy Donor- PS. CL164 derived Day 9 6.0% 6.0% 24.7% 8.6% 4.5% 3.4% 60.7% 90.9% 0.6% 0.4%Drug ProductPS. CL1603Day 5 11.5% 23.1% 64.3% 27.9% 10.9% 4.5% 70.5% 79.4% 1.2% 1.6% D001006680 PS. CL160 Day 7 9.2% 8.8% 26.6% 6.5% 11.2% 3.7% 51.9% 85.2% 0.6% 0.6%PS. CL160 Day 9 6.2% 5.6% 23.9% 5.3% 14.1% 5.5% 42.4% 84.3% 0.6% 0.6%1CAR+ reflects CD22 CAR+ cells; CAR+CD4+ reflects CAR+CD4+CD8- cells and CAR+CD8+ reflects CAR+CD4-CD8 cells.2PS. CL161 was used for EC testing as a backup replicate sample when D5 material was limited.3PS. CL158 was used for EC testing as a backup replicate sample when D5 material was limited.Attorney Docket No.: 063384-523001 WO

[0435] The CAR+ CD4 / CD8 ratios in drug product manufactured from two independent healthy donors showed a general trend of decreasing ratios across harvest days (Table 26), highlighting that the CD4 and CD8 distribution is dynamic and may be influenced by heterogeneity of the starting material.Table 26. CAR+ CAR+ CD4 / CD8 Ratios in Healthy Donor-derived Drug Product Across Harvest DaysCAR+ CD4 / Donor ID Batch Material Condition CD8 Ratio1PS. CL1642Day 5 1.6 B001002139 PS. CL164 Day 7 1.1PS. CL164 Day 9 1.0 PS. CL1603Healthy Donor- Day 5 1.6derived DrugDOO 1006680 PS. CL160 Day 7 0.8ProductPS. CL160 Day 9 0.8 'CAR+ reflects CD22 CAR; CAR+ CD4 / CD8 ratio reflects CAR+CD4+CD8- / CAR+CD4-CD8+.2PS. CL161 was used for EC testing as a backup replicate sample when D5 material was limited.3PS. CL158 was used for EC testing as a backup replicate sample when D5 material was limited

[0436] The portion of transgene expressing-cells in healthy donor drug product that were positive for single (CD 19 CAR+CD20 CAR" CD22 CAR", CD 19 CAR" CD20 CAR" CD22 CAR’, CD 19 CAR' CD20 CAR’ CD22 CAR") and double CAR" (CD 19 CAR+CD20 CAR4' CD22 CAR’, CD 19 CAR+CD20 CAR’ CD22 CAR+, CD 19 CAR’ CD20 CART CD22 CAR+) species were observed to be below the LOQ of 2% (Table 27). Importantly, longitudinal tracking showed no outgrowth of these populations across harvest days. These data collectively show that CRG-023 drug product derived from healthy donor material is primarily homogenous for the Tri-CAR+ population, with low levels of single or double CAR-expressing species and is therefore expected to maintain the capacity to recognize CD 19, CD20, and CD22 antigens.Attorney Docket No.: 063384-523001WOTable 27. Expression of Single and Double Positive CAR Populations in Healthy Donor-derived Drug Product Across Harvest Days %CD19 %CD19 %CD19 %CD19 %CD19 %CD19 Donor ID CAR+CAR+CAR+CAR' CAR' CAR'Batch Material Condition Split CD20 CD20 CD20 CD20 CD20 CD20 CAR' CD CAR+CD CAR' CD CAR+CD CAR+CD CAR' CD 22 CAR- 22 CAR' 22 CAR+22 CAR' 22 CAR+22 CAR+PS. CL162 Day 5 Yes < LOQ1< LOQ1< LOQ1< LOQ1< LOQ1< LOQ1PS. ULI 62 Day 7 Yes < LUQ' < LUQ' < LUQ' < LUQ' < LUQ' < LUQ' PS. ULI 62 HealthyDay 9 Yes < LUQ' < LUQ' < LUQ' < LUQ' < LUQ' < LUQ' B001002139 PS. ULI 64 Donor- Day 5 NO < LUQ' < LUQ' < LUQ' < LUQ' < LUQ' < LUQ' PS. ULI 64 DerivedDay 7 NO < LUQ' < LUQ' < LUQ' < LUQ' < LUQ' < LUQ' PS. ULI 64 Drug NO < LUQ' < LUQ' < LUQ' < LUQ' < LUQ' < LUQ'Product Day 9PS. CL158 Day 5 Yes < LOQ1< LOQ1< LOQ1< LOQ1< LOQ1< LOQ1Hb. UL'l 66 Day 7 Yes < LUQ' < LUQ' < LUQ' < LUQ' < LUQ' < LUQ' Hb. UL'l 66 Day 9 Yes < LUQ' < LUQ' < LUQ' < LUQ' < LUQ' < LUQ' D001006680 PS. ULI 60 Day 5 NO cLUQ1cLUQ1cLUQ1cLUQ1cLUQ1cLUQ1PS. ULI 60 Day 7 NO < LUQ' < LUQ' < LUQ' < LUQ' < LUQ' < LUQ' PS. ULI 60 NO < LUQ' < LUQ' < LUQ' < LUQ' < LUQ' < LUQ'Day 9'LOQ is 2%.Attorney Docket No.: 063384-523001 WO

[0437] Potency for healthy donor-derived drug product was determined using IFNy secretion (via Ella™ method, n=2). These Potency data show similar trend across the harvest days. All harvest days assessed, regardless of harvest or split harvest arms, had the highest level of IFNy secretion in the CD20 co-culture condition (Table 28). CD 19 and CD22 co-culture conditions had similar IFNy secretion levels within the same donor lots and were within the assay variability range of 30%. High background was observed in the TKO co-culture condition. A more suitable negative control cell line will be selected for this assay and will be implemented before the pivotal trial. While the split harvest arm yielded similar levels of IFNy secretion across all harvest days, the harvest arm yielded higher levels of IFNy secretion on Day 5 and Day 9 compared to the split harvest arm. Additionally, higher IFNy secretion was observed in the Day 7 split harvest arm when compared to the harvest arm in all co-culture conditions in both donors assessed. However, IFNy secretion trended similarly for both donors and was lowest on Day 7 harvest samples across all co-culture conditions compared to Day 5 and Day 9.

[0438] Nonetheless, this dataset suggests that DP Potency as determined by IFNy secretion follows similar trends across harvest days, regardless of harvest methods.Attorney Docket No.: 063384-523001WOTable 28. IFNy Secretion of n=2 in Healthy Donor-derived Drug Product Across Harvest DaysSeeding IFNy IFNy IFNy IFNy Donor ID Batch Material Vector Split Harvest Density (e6) secretion secretion secretion secretion Day (ng / mL), (ng / mL), (ng / mL), (ng / mL),CD19 CD20 CD22 TKO PS. CL160 No 5 300 17.39 27.66 18.81 12.13 PS. CL160 No 7 300 3.98 6.44 5.16 4.12 PS. CL160 Healthy No 9 300 8.68 13.56 11.14 8.50 D001006680 PS. CL158 Donor- 65 L Yes 5 300 11.86 23.08 15.44 11.87PS. CL158 derived Yes 7 300 8.53 16.07 11.11 8.91 PS. CL158 Drug Yes 9 300 9.94 18.49 13.05 10.52ProductPS. CL164 No 5 300 14.30 23.69 15.79 11.80 PS. CL164 No 7 300 5.41 8.10 6.73 5.96 PS. CL164 Healthy No 9 300 7.11 11.02 8.59 6.76 B001002139 PS. CL1621Donor- 65 L Yes 5 300 11.61 19.62 12.85 8.56PS. CL162 derived Yes 7 300 8.10 12.87 10.10 10.72 PS. CL162 Drug Yes 9 300 8.59 14.21 11.34 9.20Product'PS. CL161 was used for EC testing (PS. CL161 = PS. CL162).Attorney Docket No.: 063384-523001 WO

[0439] Vector Copy Number (VCN) was evaluated across two healthy donor derived drug product lots: Both lots were manufactured using LV-023 vector 65 L at- scale material. VCN results were nearly identical across DP lots, with the minor exception of donor DOO 1006680, which had a VCN / Tri-CAR+ cell of 6 copies at day 5 before stabilizing at 5 copies for days 7 and 9 like the other material (Table 29). Overall, VCN results remained consistent between Day 5 and Day 9 for both healthy donor-derived drug products evaluated.Table 29. Longitudinal VCN / Tri-CAR+ Cell Values Across DonorsHarvest VCN / Tri- Sample ID Batch Material Vector %TEDay CAR+ cell PS. CL1641Day 5 51 5 B001002139 PS. CL164 Day 7 57 5PS. CL164 Healthy Day 9 55 5 PS. CL1602At-ScaleDonor- Day 5 49 6(65 L)D001006680 PS. CL160 derived Day 7 56 5PS. CL160 Drug Day 9 52 5ProductPS. CL 161 was used for EC testing as a backup replicate sample when D5 material was limited.2PS. CL158 was used for EC testing as a backup replicate sample when D5 material was limited.3. Conclusions

[0440] This study evaluated both the drug product release and extended characterization attributes of CRG-023 drug product derived from two healthy donors in a 100M G-Rex vessel, comparing split and non-split cul ture conditions. The objective of this study was to evaluate all in-process and drug product performance parameters temporally across the entire harvest window. However, due to the limitations of development using healthy donor apheresis and the associated high-performing kinetics, the expected lag in growth with patient material in the trial is challenging to model. When the minimum harvest threshold with respect to CAR+ cells is met, the process description requires that the culture is harvested. When harvest is instead delayed and the culture is carried through Day 9 past the point of achieving MHT, cell growth plateaus and indeterminate behavior is observed with respect to attributes such as TE% or viability. Implementing this split and no split armed study allowed the isolation of longitudinal effects on CRG-023 drug product material without running into the issue of indeterminate culture behavior.

[0441] Both donors produced sufficient Tri-CAR+ cell counts for all dose levels. When controlling G-Rex overgrowth using the split arm of the study, high expansion rate and viability was maintained. Drug product exhibited similar T cell phenotypes dominated byAttorney Docket No.: 063384-523001 WOcentral memory (TCM) and effector memory (TEM) subsets across arms, with generally decreasing CD4 / CD8 ratios. Minimal exhaustion marker co-expression was observed, indicating a healthy, non- exhausted T cell population. CAR expression remained highly stable, with no quantifiable single or double CAR+ cells observed. Potency, measured via IFNy secretion, was robust among all longitudinal samples tested. Additionally, vector copy number (VCN) remained consistent across both the split and no split arms. Overall, this dataset demonstrates the feasibility of generating a homogeneous, functional CRG-023 drug product at all longitudinal timepoints of the harvest window, with strong expansion potential and transduction efficiency under clinical-scale conditions.EXAMPLE 3Vector Volume Setpoint for CRG-023 Transduction: LV-023 65L Pilot Lot 24 P009

[0442] The purpose of this study was twofold, first was to generate LV-023 65L Pilot Lot 24 P009 lentiviral vector titration data to set a recommendation for the lot-specific vector volume / MOI setpoint for the CRG-023 CAR T drug product manufacturing process in support of pre-clinical studies. Second was to generate process performance data using the recommended MOI setpoint and generate and CRG-023 test material for extended characterization which encompasses T cell memory, activation, and exhaustion phenotypes by flow, CAR, CD4 / CD8 ratios, and Potency.

[0443] The vector titration in this study was performed on LV-023 65L Pilot Lot 24 P009 from the contract development and manufacturing organization (CDMO), Oxford Biomedica (OXB).

[0444] The vector titration was performed by trained personnel in the development laboratories at CARGO Therapeutics. The cell material used to perform the titration in this study was enriched CD4 and CD8 cells produced by the CliniMACS Prodigy® TCT selection, which are representative of the CRG-023 CAR T manufacturing process.

[0445] This report contains all the information and data pertaining to the small-scale vector titration using LV-023 65L Pilot Lot 24 P009 (OXB). From this data and analysis, a lotspecific MOI setpoint was determined to move forward to at-scale execute healthy donor verification studies as well as at-scale patient-derived material verification study. This report includes all the verification study data to be used as support for the recommended MOI for LV-023 Lot 24__P009 in the pre-clinical studies.Attorney Docket No.: 063384-523001 WO

[0446] This report contains all the analytics used to evaluate the process performance of the recommended MOI setpoint used in the CRG-023 process. The process performance analytics include Purity, Viability, Growth Kinetics, Transduction Efficiency, Vector Copy Number, and Calculation of Total Tri-CAR+ cells. Extended characterization analytics were also performed to evaluate T cell memory, activation, and exhaustion phenotypes, CD4 / CD8 ratios, single and double CAR populations, and IFNy potency. The analytics are used in conjunction with the Extended Characterization testing to evaluate the efficacy of the recommended setpoint used in the CRG-023 process.1. Methodsi. Cryopreservation of Cell Starting Material and Media Formulation

[0447] Prior to the execution of the protocol, the Cue® Cell Process System (Fresenius Kabi) was used to wash and elute the cell material with Plasma-Lyte A (Baxter) + 4% (w / v) human albumin serum (FISA, Octapharma; RPT-0102). The Cue® was used to formulate the cells by the addition of CryoStor® 10 (BioLife Solutions) at a 1:1 ratio (v / v) and aliquoted into CryoStore™ 250 bags (OriGen; RPT-0102). The cells were Cryopreserved using a controlled-rate freezer (CryoMed™; Thermo Fisher) using a predefined protocol as outlined in RPT-0102.

[0448] The cryopreserved apheresis was stored in the LN2 freezer at <-l 30°C until ready for use.

[0449] One day prior to the execution of the protocol, the CliniMACS® PBS / EDTA + HSA buffer was formulated by the addition of HSA to the CliniMACS® PBS / EDTA buffer, such that the final concentration of HSA in the CliniMACS® PBS / EDTA buffer was 0.5% v / v.

[0450] One day prior to the execution of the protocol, the Complete PRIME-XV T CELL CDM media was formulated by the addition of IL-7 and IL- 15 to the PRIME-XV Media, such that the final concentration was 12.5 ng / mL for each IL-7 and IL-15.

[0451] Both the CliniMACS® PBS / EDTA + HSA buffer and the Complete PRIME-XV T CELL CDM media were stored in the fridge at 4°C until ready for the start of execution.ii. Day 0: Cell Enrichment, Cell Seeding, and Activation

[0452] The cryopreserved apheresis bags from the healthy donors were identified and loaded on to the CliniMACS Prodigy® instruments for CD4 and CD8 cell enrichment using the TCT protocol per the CRG-023 process description.

[0453] Post cell enrichment for each donor, 480 x 106cells were enumerated, from the freshly enriched CD4 / CD8 positive fraction from the CliniMACS® Prodigy TCT program, using the NC-200™ per TM-0024.Attorney Docket No.: 063384-523001 WO

[0454] 240x106enriched cells from each donor were seeded into 100M G-Rex vessels.

[0455] Each of the G-Rex vessels were QS to 70 mL in volume using pre- warmed complete PRIME-XV T CELL CDM Media. Each of the vessels were bulk activated using 4 mL of GMP TransAct™ (Miltenyi).

[0456] After activation, the G-Rex vessels were moved to the CO2 incubator at 37°C for 24 hours.Hi. Day 1: Split Seeding and Transduction

[0457] After 24 hours, a sample was taken from each G-Rex for cell count on the NucleoCounter NC-200™ using the CRG-023 counting method.

[0458] The 100M G-Rex vessels were QS to 80 mL using pre- warmed complete PRIME-XV T CELL CDM media. At this step, the small-scale process diverges from the at-scale runs. The scale down factor is 10-fold.

[0459] For small-scale titration, from one 1 OOM G-Rex vessel, the total volume was plated on a 6M G-Rex by splitting the volume evenly between 7 wells of a 6M G-Rex, one for each titration condition. This seeding technique was repeated for all 100M G-Rex vessels.

[0460] The required number of lentiviral vector vials needed to transduce all the conditions were determined and thawed. The contents of the vials were pooled, and the required amount of viral vector was added to each of the wells based off the small-scale titration scheme. This process was repeated for all donors. The small-scale titration scheme is found in Table 30. Table 30. Small-Scale Titration SchemePlate Number Well Number Volume of Virus (pL)1 6002 3003 15014 755 37.56 18.752 1 0

[0461] Post viral vector addition, all the small-scale 6M G-Rex Vessels were placed in an incubator at 37°C for 24 hours.iv. Day 2: Bulk Media Feed

[0462] After 24 hours from the viral vector addition, each of the wells of a 6M G-Rex were QS to 100 mL using pre-warmed complete PRIME-XV T CELL CDM media. All the plates were returned to the incubator at 37°C.Attorney Docket No.: 063384-523001 WOv. Day 5: Harvest

[0463] From each well of a 6M G-Rex, 60 mL of supernatant was removed and discarded without disturbing the cell bed. The remaining volume was resuspended, and 1 mL was taken for a cell count, and for flow cytometry analysis.

[0464] The remaining volume of cell suspension was prepared for cryopreservation and stored in the LN 2 freezer.vi. Cryopreservation of Samples and Evaluation of Transduction Efficiencies

[0465] For the small-scale titration, at harvest on Day 5, cells were collected and centrifuged and formulated in Plasma-Lyte A (Baxter) + 4% (w / v) human albumin serum then cryopreserved in cryogenic vials using CoolCell® (Corning) alcohol- free freezing containers placed in -80°C freezer for at least overnight. These vials were thawed and evaluated for transduction efficiency.vii. Description of At-Scale Healthy Donor and DLBCL Confirmation Runs Healthy Donor and DLBCL Confirmation Runs

[0466] The at-scale runs that were performed using healthy donors and the diffuse large B cell lymphoma (DLBCL) patient-derived material followed the procedure outlined in the CRG-023 Process Description. Three healthy donors (DOO 1006680, DOO 1006110, and B001002139) and starting apheresis material from two DLBCL patient donors, 107-1005 and 108-1007, were used to perform at-scale runs. The summarized experimental ovendew for the at-scale runs is documented in Table 31.Table 31. Summary of At-Scale Runs Experimental OverviewStarting Donor ID / SeedingCell LV-023Batch Material Patient at Day 0 MOI Day Starting BatchType Batch (cells) PS. CL163 B001002139 CryopreservedHea LV-023 3e8 5 lthyPS. CL174 D001006680 Cryopreserved 65L Pilot 3e8 5DonorPS. CL179 D001006110 Cryopreserved Lot 3e8 1.7 5 PS. CL257 DLBCL 107-1005 Cryopreserved 24_P009 3e8 9PS. CL269 Patient 108-1007 Cryopreserved (OXB) 3e8 9viii. Harvest Criteria for At-Scale Runs

[0467] For the three respective healthy donor at-scale runs, harvest day was chosen based on the pre-harvest Cell Culture Validation (CCV) which was taken on Day 5. If the culture kinetics were such that the highest dose was achieved by combination of %TE and CD3+ Purity, and Cell Culture Validation (CCV), then the batch proceeded to harvesting operations on that day. If the culture had not yet met the MHT (minimum harvest threshold), then the culture was continued until the next available harvest window. Consequently, healthy donorAttorney Docket No.: 063384-523001 WOruns BOO 1002139, DOO 1006680, D001006110 were harvested on Day 5 because the culture had met the minimum harvest threshold. By similar rationale and to elucidate the kinetics the DLBCL patient starting material, donors 107-1005 and 108-1007 were run throughout the entire harvest window (i.e., all the way until Day 9).ix. Cryopreservation of At-Scale Healthy Donor and DLBCL-patient Samples and Evaluation o f Transduction Efficiencies

[0468] To harvest the healthy donor BOO 1002139 run, the cells were processed on Day 5 using the Cue cell processing instrument as described in the CRG-023 process description. The target Tri-CAR+ dose used from the Cue for these arms of the healthy donor material was 2.5><106cells / mL in the final drug product bags. These were thawed and evaluated for transduction efficiency as per TM- 0019.

[0469] Healthy donors DOO 1006680 and D001006110 were manually formulated targeting a final concentration of 106total viable cells / mL at 1 mL per vial. These processed and formulated cells were cryopreserved using the CryoMed™ controlled-rate freezer (Thermo Fisher Scientific) using the CRG-023 profile as described in the process description. These vials were thawed and evaluated for transduction efficiency.

[0470] To harvest the DLBCL 107-1005 and 108-1007 patient run, the cells were processed on Day 9 using the Cue cell processing instrument as described in the CRG-023 process description. The target Tri-CAR-l- dose used from the Cue for these arms of the patient material was 2.5 x 106cells / mL in the final drug product bags. These were thawed and evaluated for transduction efficiency.

[0471] In process DLBCL 107-1005 and 108-1007 patient samples, that is those samples not generated at harvest (PS. CL25 and PS. CL269; Day 5 and Day 7), were not formulated on the Cue but were manually centrifuged, resuspended, formulated using our final formulation media, and frozen. These vials were thawed and evaluated for transduction efficiency.2. Resultsi. Small Scale Growth Kinetics

[0472] The growth kinetics of the transduced cultures show consistent results across all tested MOIs (FIG. 23). By Day 5, all cultures, irrespective of MOI, exhibited similar total viable cell counts. Statistical analysis using the Kruskall- Wallis Test (p=0.1511) indicated no significant differences between the groups, suggesting that transduction at all tested MOIs had no adverse effect on cell fitness or proliferation. Additionally, the viability of all cultures at harvest was > 91% (EXP24000402). This confirms that the transduction process does not negatively impact the cells' ability to grow, regardless of the MOI applied.Attorney Docket No.: 063384-523001 WOii. Transduction Efficiency

[0473] Transduction Efficiency was measured in accordance with TM-0019. The volumetric titration of the LV-023 65L Pilot Lot 24 P009 (OXB) indicates that the maximum transduction efficiency achievable is approximately 60% based on an average flow cytometry analysis of transduced primary T cells from two health donors (FIG. 24, Table 32).Table 32. %Transduction Efficiency using LV-023 65L Pilot Lot 24 P009 (OXB) at Various MOIsLVV Vol. %TE Donor %TE Donor(ML) EN01000800 B001001987600.0 57% 62%300.0 59% 57%150.0 53% 44%75.0 34% 22%37.5 16% 11%18.3 9% 5%0.0 0% 0%

[0474] The volumetric titration curve fit indicates that the curve's plateau area begins around 300 pL of LW (FIG. 24). The volume vector setpoint was selected at approximately 300 pL at a small scale, which scales to 3.0 mL at-scale (FIG. 24). The vector volume setpoint was selected in the plateau region where the maximum transduction efficiency was achieved using the least amount of vector. This approach reduces variability in transduction efficiency and minimizes the vector volume and vector copy number necessary to achieve maximum transduction efficiency.

[0475] The viral titer of the LV-023 65L Pilot Lot 24 P009 (OXB) is 1.7xl08TU / mL. The contract manufacturing organization Oxford Biomedica determined this titer via PRDSOP-047, which is documented in the Certificate of Testing for the LV-023 65L Pilot Lot 24 P009 (OXB) (CRT-0177). Since 30x106cells were seeded on Day 0, 300 pL of viral suspension results in an MOI (Multiplicity of Infection) of 1.7 as per Table 33, FIG. 24, and Equation 1.Equation 1. MOI DeterminationMultiplicity of Infection (MOI) = Vector Volume (mL)*Vector Titer (TU / mL)Total Viable Cells Seeded on Day 0 (cells)Attorney Docket No.: 063384-523001 WOTable 33. LV-02365L Pilot Lot 24 P009 (OXB) Titration MOI - Conversion of Volumetric Titration Points to MOICellsLWVol.Total TU Seeded MOI(PL) Day 0600.0 1.02E+08 3.00E+07 3.4300.0 5.10E+07 3.00E+07 1.7150.0 2.55E+07 3.00E+07 0.975.0 1.28E+07 3.00E+07 0.437.5 6.38E+06 3.00E+07 0.218.3 3.10E+06 3.00E+07 0.10.0 0.00E+00 3.00E+07 0.0Hi. Vector Copy Number (VCN) Determination by ddPCR

[0476] The samples for VCN analysis were run in accordance with TM-0028. The results show that at the chosen MOI of 1.7, the vector copy number (VCN) per transduced cell is 6. The VCN per transduced cell at the highest MOI is 7 (Table 34, FIG. 25). These levels of VCN indicate effective transduction while maintaining a balance that minimizes potential risks associated with higher vector integration.Table 34. VCN Analysis of Primary T Cells Transduced with LV-023 65L Pilot Lot 24 P009 (OXB) at Various MOIs, Thawed Drug ProductVCN / Tri- Donor Cell Lot MOI %TECAR+ cell PS. CL146 3.4 62 7 PS. CL147 1.7 57 6 PS. CL148 0.9 44 4 B001001987PS. CL149 0.4 22 4 PS. CL150 0.2 11 3 PS. CL151 0.1 5 3 PS. CL138 3.4 57 7 PS. CL139 1.7 59 6 PS. CL140 0.9 53 5EN01000800PS. CL141 0.4 34 4 PS. CL142 0.2 16 3PS. CL143 0.1 9 3

[0477] Based on these findings, the vector volume setpoint was set at 300 pL to transduce 30x106cells seeded on Day 0. When scaled up 10-fold, it is recommended to use 3.0 mL, orAttorney Docket No.: 063384-523001 WOthree vials of 1.0 mL filled LV-023 vector, to transduce 300* 106cells seeded in a GRex 100M on Day 0 (an MOI of 1.7).iv. Healthy Donor Verification At-Scale Runs Using LV-02365L Pilot Lot 24_P009 (OXB) MOI Setpoint

[0478] After the completion of the small-scale titration, three at-scale runs were performed with healthy donors to confirm the recommended MOI can effectively produce CAR T cells using the CRG-023 process. A summary of the at-scale runs performed can be seen in Table 35.Table 35. Summary of Healthy Donor At-Scale Runs using L V-02365L Pilot Lot 24 P009 (OXB)ApheresisCell LV-023 Cue At-Scale Donor ID Processing MOIStarting Batch Fill / Formulation Run Lot MethodMaterialPS. CL163 B001002139 Rotea Cryopreserved LV-023 Yes 65L1.7PS. CL174 D001006680 Cue Cryopreserved Pilot No LotPS. CL179 D001006110 Cue Cryopreserved 24_P00 No9(OXB)

[0479] The frequency of CD3+CD45+ of CRG-023 cells produced using the LV-023 65L Pilot Lot 24 P009 (OXB) batch demonstrates high purity levels. The mean purity across three healthy donor-derived samples is 99.21%, with a standard deviation of 0.54% (FIG.26).

[0480] The fold expansion values of CRG-023 CAR T cells from Day 0 to Day 5 have a mean of 6.12 and a standard deviation of 0.35 (FIG. 27). These results indicate a consistent expansion rate across different production lots and healthy donors, demonstrating reliable cell growth and scalability in the CRG-023 manufacturing process using the LV-023 65L Pilot Lot 24 P009 (OXB).

[0481] The percent viability of harvested (fresh) CRG-023 CAR T cells on Day 5 has a mean of 92.68% and a standard deviation of 1.55% (FIG. 28, Healthy Donor Cell), indicating consistently high viability across different runs and healthy donors. This high viability at harvest suggests acceptable cell fitness of CAR T cell produced using the vector volume setpoint selected for the LV-023 65L Pilot Lot 24 P009 (OXB) batch and the clinical-scale process.Attorney Docket No.: 063384-523001 WO

[0482] The growth kinetics data from an average of three healthy donors show a consistent expansion pattern of CRG-023 CAR T cells. Starting with 300x106viable cells on Day 0, the total viable cell count decreased slightly on Day 1 to an average of approximately 219x106, with a standard deviation of 30x 106(FIG. 29), which is expected during this posttransduction period. By day 5, the cells expanded significantly to an average of roughly 2.22x109viable cells, with a standard deviation of 223x106(FIG. 29). The relatively low standard deviation on Day 5 suggests high consistency in cell growth across different donors. This indicates that the process is robust and reproducible when using healthy donor-derived T cells. It demonstrates the ability of the CRG- 023 manufacturing process and the vector volume setpoint to reliably produce a substantial number of viable CAR T cells within the expected timeframe.

[0483] The transduction efficiencies (TE) of CRG-023 CAR T cells, which were transduced with the LV-023 65L Pilot Lot 24 P009 (OXB) at an MOI of 1.7, had a mean of 61.50% and a standard deviation of 0.90% (FIG. 30). This consistent TE across four unique healthy donor runs demonstrates the reliability of the CRG-023 vector volume setpoint of LV-023 65L Pilot Lot 24 P009 (OXB) and the scalability of the CRG- 023 titration process. Thus, the CRG- 023 process can consistently achieve satisfactory levels of T cell editing across multiple batches.

[0484] The TE obtained from the pre-thawed material show similar trends as the TE obtained from post-thawed material when comparing against the values in FIG. 30 (pre-thaw) and Table 33 (post -thaw). The consistency of the TE obtained from pre -thaw and post-thaw material demonstrates that cryopreservation of the final drug product does not impact the material. This reinforces the reliability of the CRG-023 process, and the stability of the drag product pre- and post-thaw.Table 36. %TE of At-Scale Healthy Donor Cells Transduced with LV-02365L Pilot Lot 24 P009 (OXB)%TE %TE Formulation Donor Material Condition(Pre-Thaw) (Post-Thaw) Method D001006680 Drug Product Day 5 62 59% Manual B001002139 Drug Product Day 5 60 60% CueD001006110 Drug Product Day 5 62 62% Manual

[0485] The total Tri-CAR+ cell counts, with a mean of approximately 1,36x109and a standard deviation of 1.31 x 108(FIG. 31), indicate that the manufacturing process for CRG- 023 consistently produces suitable numbers of CAR T cells by day 5 from each healthy donorAttorney Docket No.: 063384-523001 WOrun. The consistent values across different samples highlight the robustness of the process, ensuring that sufficient Tri-CAR+ cells are generated within the expected timeframe.

[0486] In summary, the CRG-023 manufacturing process using the vector volume setpoint of the LV-023 65L Pilot Lot 24 P009 (OXB) using the titration demonstrates robustness and consistency in producing high- purity, viable, and suitably expanded CAR T cells with reliable transduction efficiency and sufficient cell yields by Day 5 across multiple healthy donor samples without exceeding the harvest timeframe. These results demonstrate cell growth in the harvest window across all the healthy donors. This shows that the CRG-023 process and the chosen vector MOI setpoint do not negatively impact the in-process cell health or inhibit cell growth and are capable of meeting harvest requirements. The total number of Tri-C AR+ T cells and viability (fresh) met the minimum harvest threshold as described in the CRG-023 process description (RPT-0102), and dose as in CRG-023 engineering runs acceptance criteria (DOC-0244, DOC-0298).v. VCN / T -CAR+ cell is highly consistent in DP transduced with pilot LV-023

[0487] Vector copy number was assessed in three healthy donors after transduction with pilot scale LV-023 at an MOI of 1.7. The 3 donors tested exhibited a range of 5 to 7 copies / Tri-CAR+ cell (Table 37). The observed variability between donors is minimal and is well within the variability of the method.Table 37. VCN / Tri-CAR+ Cell Value Across Donors - At-Scale Healthy Donors, Thawed Drug ProductVCN / Tri- HarvestDonor ID Batch Material Vector %TE CAR+ DayCell D001006680 PS. CL174 Healthy LV-023 59 6 D001002139 Donor- 65L PilotPS. CL163 60 5 derived Lot Day 5D001006110 PS. CL179 Drug 24_P009 62 7Product (OXB)Attorney Docket No.: 063384-523001 WOTable 38. Summary of Results for At-Scale Healthy Donors using LV-02365L Pilot Lot 24 P009 (OXB) - Thawed Drug Product _ _ _ _D00100 D00100 B00100 Attribute Test Method / Test 6680 6110 2139 Category Attribute Method Number Acceptance PS. CL1 PS. CL1 PS. CL1Criteria 74 79 63 Transduction > 85% viableEfficiency and T Cell CD3+ cellsPurity Viable CD3+ 99.47% 99.T cel Phenotype for 13% 99.54% lsTri-CAR, TM-0019Transduction Tri- Tri- Tri- Identity / Efficiency and T Cell CAR CAR CAR Identity Phenotype for Tri- CARStrength Confirmed Confirmed Confirmed Tri-CAR, TM-0019 ConfirmedTransduction > 5% viableIdentity / Transduction Efficiency and T Cell CD3+ Tri- 59% 62% 60% Strength Efficiency Phenotype for CAR+Tri-CAR, TM-0019Quantification ofTri-CAR Vector Copy NumberVector Copy (VCN) in Transduced < 15Safety Number per Cells by ddPCR for copies / Tri- 6 7 5 Transduced Tri-CART cells, CAR+ cellCell TM-0024vi. Extended Characterization Assessment of Healthy Donor Verification Runs

[0488] T Cell Memory Subsets by Flow Cytometry was performed to assess the distribution of memory subpopulations within each drag product. The CART CD4 / CD8 ratios were also measured from this assay to track the distribution of CD4+ and CD8+ cells in the transduced compartment. C ART cells by this method are defined by staining using (rh)CD22 protein and reflects CD22 CART cells. This classification serves as a fit-for-purpose surrogate for the Tri-CART population as the transduced portion of drug product is generally homogenous for all three CARs.

[0489] The predominant populations in drug products manufactured from three independent healthy donors were found to be compartmentalized to central memory (TCM) and effector memory (TEM) subsets (Table 39). These results show consistency in the memory subset distribution across all tested batches.Attorney Docket No.: 063384-523001 WOTable 39. T Cell Memory Phenotype of At Scale Healthy Donor-derived Drug Product using LV-02365L Pilot Lot 24 P009 (OXB)Don Harve TN 1 1 1or PS. C Materia1TEMRA1st TSCM TCM TEMID L 1 Day CAR CAR CAR CAR CAR CAR CAR CAR CAR CAR + + + + + + + + + + CD4 CD8+CD4 CD8 CD4+CD8 CD4+CD8 CD4+CD8 +22 +2+22 +22 +22 +2D0010066 PS. CL17 Healt 0.0% 0.0% 0.2% 0.1% 87.4 95.6 12.4 4.5% 0.0% 0.0% 80 4 hy Day % % %Donor- 5BOO 10021 PS. CL16 derived 0.0% 0.0% 0.0% 0.1% 97.3 97.1 2.6% 2.8% 0.0% 0.0%Drug39 3 % %ProductDOO 10061 PS. CL17 0.0% 0.0% 0.2% 0.1% 95.6 96.1 4.3% 3.7% 0.0% 0.0% 10 9 % %1TN (Naive T cell), TSCM (Stem Cell Memory T cell), TCM (Central Memory T cell),TEM (Effector Memory T cell), TEMRA (Effector Memory T cell Re-expressing CD45RA).2CAR+ reflects CD22 CAR+ cells; CAR+CD4+ reflects CAR+CD4+CD8- cells and CAR+CD8+ reflects CAR+CD4-CD8+ cells.

[0490] The activation marker profile of drug product manufactured from three independent healthy donors showed lower frequencies of CD69 and 4-1BB relative to higher expression for CD25 (Table 40). These results demonstrate consistency in the T cell expression profile in response to activation across the tested batches.Table 40. T Cell Activation Phenotype of Healthy Donor-derived Drug Product using LV- 02365L Pilot Lot 24 P009 (OXB)CD25+CD69+4-1 BB+Donor ndi veaiPS. CL Material CAR+CAR+CAR+CAR+CARID Day+CAR+CD4+1CD8+1CD4+1CD8+1CD4+1CD8+1D001006680 PS. CL174 88.8% 71.8% 2.5% 2.7% 1.6% 4.5%HealthyB001002139 PS. CL163 Donor- 83.8% 68.8% 2.8% 3.1% 1.8% 5.0% D00100611 derived Day 50 PS. CL179 93.0% 76.7% 2.3% 3.7% 2.6% 6.1%DrugProductCART reflects CD22 CART cef s; CAR+CD4+ ref ects CAR+CD4+CD8- cells and CAR+CD8+ reflects CAR+CD4- CD8+ cells.

[0491] Variable levels of individual LAG-3, PD-1, TIGIT, and TIM-3 markers were observed in drug products manufactured from three independent healthy donors, with lowAttorney Docket No.: 063384-523001 WOfrequencies of co-expressing cells across the tested batches (Table 41). As exhaustion is typically characterized by the sustained expression of multiple inhibitory receptors on the same cell, these data suggest that the drug product displays a minimally exhausted phenotype which is expected to maintain T cell functionality.Table 41. T Cell Exhaustion Phenotype of Healthy Donor-derived Drug Product using LV- 02365L Pilot Lot 24 P009 (OXB)LAG- Donor Harve LAG-3+PD-1+TIGIT+TIM-3+PS. CL Materi 3+PD-1+ID stal TIGIT+TIM Day -3+CAR CAR CAR CAR CAR CAR CAR CAR CAR CAR+ + + + + + + + + + CD4+CD8+CD4+CD8+CD4+CD8+CD4+CD8+CD4 CD8 1 1 1 i 1 1 1 1+i +iD0010066 PS. CL17 8.3% 14.6 56.3 20.0 11.2 4.1% 74.4 88.4 0.6% 0.7% 80 4 Healthy Day 5 % % % % % %B0010021 PS. CL16 Donor- 6.3% 10.2 60.7 10.2 2.8% 2.5% 85.6 80.4 0.4% 0.4% 39 3 derived % % % % %DrugD0010061 PS. CL17 Product 8.0% 6.1% 33.8 10.9 7.4% 4.0% 87.0 64.8 0.5% 0.3%10 9 % % % %1CAR+ reflects CD22 CAR+ cells; CAR+CD4+ reflects CAR+CD4+CD8- cells and CAR+CD8+ cells reflects CAR+CD4-CD8+ cells

[0492] Tri-CAR+ CD4 / CD8 ratios in the three drug product batches showed variable composition (Table 42), likely attributed to donor heterogeneity.Table 42. Tri-CAR+ CD4 / CD8 Ratio of Healthy Donor-derived Drug Product using LV- 02365L Pilot Lot 24 P009 (OXB)Donor Tri-CAR+ CD4 / PS. CL Material Harvest DayID CD8 Ratio1D001006680 PS. CL174 Day 5 0.9 B001002139 PS. CL163 Healthy Day 5 1.7Donor-derivedD001006110 PS. CL179 Drug Product Day 5 1.9

[0493] Single and double CAR+ frequencies in drug products manufactured from healthy donors were below the LOQ of 2% for all potential combinations (Table 43, which indicates that the drug product is largely homogenous for Tri-CAR+ cells.Attorney Docket No.: 063384-523001WOTable 43. Single and Double CAR+ Frequencies in Healthy Donor-Derived Drug Product using LV-02365L Pilot Lot 24 P009 (OXB)%CD19 %CD19 %CD19 CAR+%CD19 CAR- %CD19 CAR- %CD19 CAR- Harvest CAR+CD20 CAR+CD20 CD20 CAR' CD20 CAR+CD20 CAR+CD20 CAR' Donor ID PS. CL Material Day CAR' CD22 CAR+CD2 CD22 CD2 CD2 CD22CAR' 2 CAR' CAR+2 CAR' 2 CAR+CAR+D001006680 PS. C174 Healthy < LOQ1< LOQ1< LOQ1< LOQ1< LOQ1< LOQ1B001002139 PS. CL163 Donor-derived Day 5 < LOQ1< LOQ1< LOQ1< LOQ1< LOQ1< LOQ1D001006110 PS. CL179 Drug Product < LOQ1< LOQ1< LOQ1< LOQ1< LOQ1< LOQ1Table 44. IFNy Secretion Across Healthy Donors using LV-02365L Pilot Lot 24 P009 (OXB)IFNy IFNy IFNy IFNy Batch ID Harvest Day secretion secretion secretion secretion Donor ID Material Vector (ng / mL), CD19 (ng / mL), CD20 (ng / mL), CD22 (ng / mL), TKOB001002139 PS. CL163 65L Day 5 7.46 16.98 6.95 8.41Healthy Donor- D001006110 PS. CL179 derived Drug 65L Day 5 4.14 10.3 3.4 4.21D001006680 PS. CL174 Product 65L Day 5 7.5 19.37 8.2 8.01Attorney Docket No.: 063384-523001 WO

[0494] CRG-023 IFNy potency shows similar trends in DP generated using 65L pilot scale CRG-023 LV batches. IFNy secretion by Ella™ was measured for three different donors generated using the 65L pilot scale LV-023 at MOI 1.7. Varying levels of IFNy secretion were observed in the four cell lines tested, which can be attributed to donor heterogeneity (Table 44). All donors assessed in these studies showed similar trends in IFNy secretion wherein DP co- cultured with CD20 single-expressing target cells yielded the highest level of IFNy concentration, and CD 19, CD22, and TKO co-cultures yielded similar levels of IFNy secretion. High background was observed in the TKO co-culture condition and is a known issue. Target only co- culture conditions were assessed and were all below LLOQ, demonstrating the absence of nonspecific IFNy secretion and passing the test method specified system suitability criteria (data not shown).

[0495] Patient-derived material was also tested for verification of the MOI setpoint of 1.70 established using LV-023 65L Pilot Lot 24 P009 (OXB) from the small-scale titration data. A summary of the patient-derived material transduction information can be seen in Table 45. Table 45. Summary of DLBCL Patient At-Scale Runs using LV-02365L Pilot Lot 24 P009 (OXB)At-Scale Cell DP Cue Run Patient ID Starting LV-023 Batch MOI Fill / Formulation Lot MaterialPS. CL257 107-1005 Cryopreserved YesLV-02365L Pilot Lot24 P009 (OXB) 1.7PS. CL269 108-1007 Cryopreserved Yes

[0496] The frequency of CD3+CD45+ of CRG-023 cells produced using the L V-023 65L Pilot Lot 24 P009 (OXB) demonstrates high purity levels in runs using DLBCL starting material (FIG. 32). The mean purity, at Day 9, across two DLBCL run samples is 93.7%, with a standard deviation of 4.3%. These values indicate the robustness of the CRG-023 process in producing CD3+CD45+ CAR T cells at acceptable purity levels.

[0497] The total number of viable cells from each patient donor batch was able to grow considerably, which can be seen in FIG. 33. The growth kinetics data show a robust expansion using the CRG-023 process on DLBCL patient-derived material. Starting on Day 0 with 300e6 cells, the cells were able to significantly expand by Day 9 to produce approximately 1.49xl09cells for patient donor 107- 1005 (PS. CL257) and 2.98xl09for patient donor 108-1007 (PS. CL269) as seen in Table 29. It is seen that there is someAttorney Docket No.: 063384-523001 WOvariability between the patient donors which is a testament to the biological state of the patient. Despite the variance in the cell growth kinetics, each of the DLBCL patient-derived material batches were able to meet the minimum harvest requirement which is a testament to the effectiveness of the CRG-023 manufacturing process and optimized vector MOI setpoint.

[0498] The DLBCL patient-derived material demonstrated high viability throughout the process (fresh) as seen in FIG. 34. The percent viability on Day 9 harvest (fresh) averaged to be 93.0% with a standard deviation of 1.7%, as seen in Table 29. The high viability demonstrates the cell fitness does not get impacted by the CRG-023 process at the chosen MOI setpoint.

[0499] The average transduction efficiencies for the DLBCL patient-derived material at harvest and using an MOI of 1.70 was 31.52% with a standard deviation of 5.96% as per Table 29. The transduction efficiency throughout the process can be seen in FIG. 35. These results show that the selected MOI used in the CRG-023 process can effectively transduce cells without impacting the growth. Achieving these results also shows the robustness of the CRG-023 process, and its capability to effectively transduce DLBCL patient-derived material at the chosen MOI setpoint.

[0500] The total number of Tri-CAR+ cell count for the DLBCL patient material at Day 9 harvest was 5.13 x 108Tri-CAR+ T cells for patient donor 107-1005 (PS. CL257) and 9.35 x 108for patient donor 108-1007 (PS. CL269), which can be seen in FIG. 36 and Table 29. These results show that, using LV-023 65L Pilot Lot 24 P009 (OXB), the chosen MOI setpoint did not inhibit the growth of CAR+ cells, and sufficient Tri-CAR+ cells from the DLBCL patient-derived material are generated within the CRG-023 harvest window.Notably, the minimum harvest threshold for the highest dose level (DL3) is 2.69x10sTri-CAR+ cells, further emphasizing the robustness of the CRG-023 manufacturing process and the chosen vector MOI setpoint in meeting production requirements.

[0501] The maximum transduction efficiency of the DLBCL patient material is lower than that of the healthy donor, which suggests that the diseased state of the cells can reduce the editing efficiency. Although the transduction efficiency is lower, the data shows that the DLBCL patient material is still capable of producing sufficient Tri-CAR+ T cells by Day 9 of the process. This highlights the effectiveness of the CRG-023 manufacturing process with the chosen MOL

[0502] Overall, the in-process purity, viability, TVC, Transduction efficiency and Tri- CAR+ cell data all reflect the efficacy and robustness of the CRG-023 manufacturing process at the chosen MOI setpoint of 1.70 for LV-023 65L Pilot Lot 24 P009 (OXB). The DLBCLAttorney Docket No.: 063384-523001 WOpatient-derived material was able to meet the minimum harvest requirements without exceeding the harvest timeframe. This shows that the CRG-023 process and the chosen vector MOI setpoint do not negatively impact the cell health or inhibit cell growth and is capable of meeting harvest requirements. The total number of Tri-CAR+ T cells and viability (fresh) met the minimum harvest threshold as described in the CRG-023 process description, and in CRG-023 engineering runs acceptance criteria. This confirmed the ability of the process to generate sufficient patient-derived Tri-CAR+ T cells to harvest all dose levels.

[0503] Vector Copy Number (VCN) was evaluated across two DLBCL patient-derived drug product samples. All lots were manufactured using LV-023 65L Pilot Lot 24 P009 (OXB). For patient donor 107-1005 (PS. CL257), VCN / Tri-CAR+ cell was 18 on Day 5 and decreased to 6 copies / Tri-CAR+ on Day 9 (Table 46). The elevated copy number on Day 5, as well as the high variability between harvest days, can potentially be attributed to the low %TE observed on Day 5 and potential transient vector integration early in the culture process. For the other two patient donors, VCN results remained consistent between Day 5 and Day 9 ranging between 5 and 6 VCN / Tri-CAR+ cell.Table 46. VCN / Tri-CAR+ Cell Value of DLBCL Patient-Derived Material, At Scale DLBCL Patient Donors, Thawed Drug ProductHarvest VCN / Tri-CAR+ Patient ID Batch Material Vector %TEDay Cell Day 5 10 181107-1005 PS. CL257 LV-023Patient 65L Pilot Day 9 27 6 -derived Lot Day 5 30 5 108-1007 PS. CL269 Drug Product 24_P009 Day 7 35 6(OXB)Day 9 36 5 ^Elevated copy number may be attributed to low %TE observed on Day 5 and potentially transient vector integration early in the culture process.Attorney Docket No.: 063384-523001 WOTable 47. Summary of Results for At-Scale DLBCL Patient-Derived Material using LV- 02365L Pilot Lot 24 P009 (OXB) - Thawed Drug ProductTestAttribute Method / Test Acceptance 107-1005 108-1007AttributeCategory Method Criteria PS. CL257 PS. CL269NumberTransductionEfficiency and> 85%Viable CD3+ T CellPurity viable CD3+ 98% 99% T cells Phenotype forcellsTri-CAR,TM-0019TransductionEfficiency andIdentity / T Cell Tri-CAR Tri-CAR Tri-CAR IdentityStrength Phenotype for Confirmed Confirmed Confirmed Tri-CAR,TM-0019TransductionEfficiency and> 5% viableIdentity / Transduction T CellCD3+ 27% 36% Strength efficiency Phenotype forTri-CAR+Tri-CAR,TM-0019Quantificationof VectorTri-CAR Copy NumberVector Copy (VCN) in < 15 copies / Safety Number per Transduced Tri-CAR+ 6 5 transduced Cells by cellcell ddPCRforTri- CART cells,TM-0024vii. Extended Characterization Assessment of Patient Starting Material At- Scale Runs

[0504] Extended characterization attributes were measured as previously described, with the addition of Flow Cytometry Method for Detection of Anti-CD19 CAR T Cells in CRG-022 Patient Material, which uses recombinant human CD 19 protein to measure surface CAR expression. This approach is agnostic of specific CD 19- directed scFvs but can only be used to evaluate apheresis and CD4+ / CD8+ enriched material, as the method cannot differentiate between residual anti-CD19 CAR on incoming samples and surface CD 19 CAR transgene expression from CRG-023 drug product.

[0505] Residual levels of surface anti-CD19 CAR were near or below the LOQ on T cells from patient apheresis material (Table 48) with no increase following target cell enrichmentAttorney Docket No.: 063384-523001 WO(Table 48), suggesting that there is low probability for expansion of residual anti-CD19 CAR in the manufacturing process. The drug product was not tested using this method, as rhCD19 protein is unable to differentiate between incoming residual anti-CD19 CAR and the CD 19 CAR component of CRG-023. Nevertheless, low frequencies of incoming residual anti-CD19 CAR in apheresis and enrichment material, infer that the efficacy of drug product would largely be driven through CRG-023 transgene components and thereby minimally impacted by incoming levels of anti-CD19 CAR. Notably, CRG-023 drug product was successfully produced from patient material, indicating that low frequencies of residual anti-CD19 CAR do not appear to influence manufacturability.Table 48. Residual %CD19 CAR+ of Patient Excess Apheresis Starting Material %CD19CAR+Patient ID Material(of CD3+) Apheresis 0.4%107-1005CD4+and CD8+Enriched 0.2% Apheresis < LOQ1108-1007CD4+and CD8+Enriched < LOQ^OQ: 0.2%.

[0506] The predominant populations in drug product derived from patient material was found to be compartmentalized to central memory (TCM) and effector memory (TEM) T cell subsets (Table 46). Differentiation of T cell phenotypes was observed across harvest days, reflected by decreasing TCM frequencies and increasing TEM populations (Table 46). Differences in frequencies between harvest day-matched batches were likely attributed to starting material heterogeneity. Nevertheless, these data indicate that the drug product T cell phenotype profile is consistent when manufactured from patient material.Attorney Docket No.: 063384-523001WOTable 49. T Cell Memory Phenotype of DLBCL Patient Material-Derived In-Process Samples and Drug Product using LV-02365L Pilot Lot 24 P009 (OXB) Across Harvest DaysTN1TSCM1TCM1TEM1TEMRA1Patient Batch Material Harvest CAR+ CAR+ CAR+ CAR+ CAR+ CAR+ CAR+ CAR+ CAR+,,2 CAR+CD8+ID Day CD4+2CD8+2CD4+2CD8+2CD4+2CD8+2CD4+2CD8+2CD4+2107- PS. CL Patient- Day 9 0.0% 0.0% 0.3% 0.2% 22.5 34.1 77.0 65.6% 0.2% 0.1% 1005 257 derived In- % % %Process Day 5 0.0% 0.0% 0.2% 1.0% 93.5 89.7 6.4% 9.3% 0.0% 0.0% Sample / % %108- PS. CL Drug1007 68.1 75.6 31.7269 Product3Day 7 0.0% 0.0% 0.1% 2.6% 21.6% 0.0% 0.2%% % %Day 9 0.0% 0.0% 0.2% 0.2% 45.6 74% 54.1 25.8% 0.1% 0.1%% %1TN (Naive T cell), TSCM (Stem Cell Memory T cell), TCM (Central Memory T cell), TEM (Effector Memory T cell), TEMRA (Effector Memory T cell Re-expressing CD45RA).2CAR+ reflects CD22 CAR+ cells; CAR+CD4+ reflects CAR+CD4+CD8- cells and CAR+CD8+ reflects CAR+CD4-CD8+ cells.3Day 5 and Day 7 are in-process samples; Day 9 is the drug product.Attorney Docket No.: 063384-523001 WO

[0507] Evaluation of activation markers in patient material-derived drug product revealed the predominant marker to be CD25, with lower levels of 4-1BB and CD69 (Table 50). This profile possibly reflects the dynamic nature of individual targets foll owing Day 0 stimulation with the polyclonal T cell activator, TransAct™, with differences in frequencies across batches potentially attributed to donor heterogeneity. Longitudinal expression showed a general reduction to activation state that was most prevalent for CD25, likely resulting from the higher levels on Day 5 (Table 50) and suggests that the activation marker expression is impacted by proximity to initial TransAct™ exposure. These data show consistency in the profile of activation responses following Trans Act™-mediated stimulation, which is essential for the efficient lentiviral transduction and proliferation of T cells.Table 50. T Cell Activation Expression in DLBCL Patient Material-Derived In-Process Samples and Drug Product using LV-023 65L Pilot Lot 24 P009 (OXB) Across Harvest DaysPatient Harvest CD25+CD69+4-1 BB+Batch Material CAR* CAR* CAR* CAR* CAR* CAR* ID DayCD4+1CD8+1CD4+1CD8+1CD4+1CD8+1107-1005 PS. CL25 Day 9 44.3% 66.8% 3.3% 6.7% 1.3% 11.4%7Datient- derived In- Day 5 94.6% 80.5% 5.5% 9.3% 16.1% 43.3%Drocess108-1007 PS. CL26 Sample / Day 7 62.9% 49.3% 2.8% 5.2% 2.7% 11.8%9 DrugDay 9 20.1% 21.1% 3.7% 2.1% 0.8% 5.9%Product1CAR+ reflects CD22 CAR+ cells; CAR+CD4+ reflects CAR+CD4+CD8- cells and CAR+CD8+ reflects CAR+CD4- CD8+ cells.2Day 5 and Day 7 are in-process samples; Day 9 is the drug product.

[0508] Evaluation of the exhaustion phenotype in patient material-derived drug products showed varying levels of individual LAG-3, PD-1, TIGIT, and TIM-3 markers and minimal frequencies of co-expression (Table 51). As phenotypic characterization of T cell exhaustion status is typically reflected by sustained expression of multiple targets on the same cell, these data suggest that CRG-023 drug product is active. Notably, the Day 5 sample with appreciable levels of co-expressed targets showed a longitudinal decrease in frequencies by Day 9 (Table 51). Given that exhaustion markers can also be upregulated as a cell-intrinsic mechanism to curtail aberrant T cell activation, any reduction across harvest day may reflect a stimulated rather than exhausted state. These data demonstrate that the percentage of drug product cells that co-express exhaustion markers are generally low and furthermoreAttorney Docket No.: 063384-523001 WOhighlights that there is no expansion of this population throughout the CRG-023 process when initiating manufacturing with patient material.Attorney Docket No.: 063384-523001WOTable 51. T Cell Exhaustion Marker Expression in DLBCL Patient Material-Derived In- Process Samples and Drug Product using LV- 02365L Pilot Lot 24 P009 (OXB), Across Harvest DaysLAG-3+PD-1+Patient ID Harvest LAG-3+PD-1+TIGIT+TIM-3+Batch Material TIGIT+TIM-3+Day CAR+CAR+CAR+CAR+CAR+CAR+CAR+CAR+CAR+CAR+CD4+1CD8+1CD4+1CD8+1CD4+1CD8+1CD4+1CD8+1CD4+1CD8+1107-1005 PS. CL257 Patient- 11.5% 43.0% 27.3% 18.9% 53.1% 82.9% 89.0% 83.8% 2.8% 10.9%Derived In- Day 9Process Day 5 13.9% 48.1% 71.7% 32.5% 31.7% 17.7% 72.4% 95.5% 3.1% 7.6% 108-1007 PS. CL269 Sample / Day 7 6.6% 18.0% 44.0% 11.5% 27.0% 17.6% 78.9% 92.6% 1.3% 3.0%Drug Product2Day 9 3.4% 9.5% 32.2% 7.3% 26.8% 17.6% 53.4% 83.8% 0.7% 1.3%1CAR+ reflects CD22 CAR+ cells; CAR+CD4+ reflects CAR+CD4+CD8- cells and CAR+CD8+ reflects CAR+CD4- CD8+ cells.2Day 5 and Day 7 are in-process samples; Day 9 is the drug product.Table 52. Single and Double CAR+ Frequencies in DLBCL Patient Material-Derived In- Process Samples and Drug Product using LV-02365L Pilot Lot 24 P009 (OXB) Across Harvest Days _ _ _ _ _ _%CD19 %CD19 %CD19 %CD19 CAR %CD19 CAR %CD19 Patient Harvest CAR+CD20 CAR+CD20 CAR+CD20 CD20 CAR+CD20 CAR+CAR CD20Batch MaterialID Day CAR CD22 CAR+CD22 CAR CD22 CD2 CD2 CAR CD22CAR CAR CAR+2 CAR 2 CAR+CAR+Day 5 < LOQ1< LOQ1< LOQ13% < LOQ13% 107-1005 PS. CL257 Patient- derived In- Day 9 < LOQ1< LOQ1< LOQ1< LOQ1< LOQ1< LOQ1Process Day 5 < LOQ1< LOQ1< LOQ1< LOQ1< LOQ1< LOQ1108-1007 PS. CL269 Sample / Day 7 < LOQ1< LOQ1< LOQ1< LOQ1< LOQ1< LOQ1DrugDay 9 < LOQ1< LOQ1< LOQ1< LOQ1< LOQ1< LOQ1Product21CAR+_ reflects CD22 CAR; CAR+ CD4 / CD8 ratio reflects CAR+CD4+CD8- / CAR+CD4-CD8+.2Day 5 and Day 7 are in-process samples; Day 9 is the drug product.Attorney Docket No.: 063384-523001 WO

[0509] The majority of transgene expressing-cel Is in patient-derived drag product positive for single (CD 19 CAR+ CD20 CAR- CD22 CAR-, CD 19 CAR- CD20 CAR+ CD22 CAR-, CD 19 CAR- CD20 CAR- CD22 CAR+) and double CAR+ (CD 19 CAR+ CD20 CAR+ CD22 CAR-, CD 19 CAR+ CD20 CAR- CD22 CAR+, CD 19 CAR- CD20 CAR+ CD22 C AR+) species were below the LOQ of 2%, with a single batch on Day 5 showing levels at 3% for single CD20 CAR and single CD22 CAR populations (Table 53). Longitudinal tracking of single and double CAR+ cells across all batches demonstrated no outgrowth of these populations across harvest days (Table 53). These data show that CRG-023 drug product derived from patient material is primarily homogenous for the Tri-CAR+ population, with low levels of single or double CAR-expressing species and is therefore expected to maintain the capacity to respond to CD 19, CD20, and CD22 antigens.Table 53. Tri-CAR+ CD4 / CD8 Ratio in DLBCL Patient Material-Derived In-Process Samples and Drug Product using LV-02365L Pilot Lot 24 P009 (OXB) Across Harvest DaysTri-CAR+ Patient ID Batch Material Harvest Day CD4 / CD8 Ratio1107-1005 PS. CL257 Day 9 1.2Patient-derived Day 5 5.1 In-Process Sample108-1007 PS. CL269 Day 7 5.4 / Drug product2Day 9 5.1 'CAR+ reflects CD22 CAR; CAR+ CD4 / CD8 ratio reflects CAR+CD4+CD8- / CAR+CD4-CD8+.2Day 5 and Day 7 are in-process samples; Day 9 is the drug product.

[0510] DP Potency determined via secretion of IFNy was performed on n=2 patient donor- derived material at up to 3 harvest timepoints. Both DP lots demonstrated highest IFNy secretion in the CD20 co-culture condition (Table 54). Similar levels of IFNy secretion were observed in the CD 19 and CD22 co-culture conditions within the same donor lot, and within each harvest day evaluated for donor 108- 1007. High background was observed in the TKO co-culture condition, which may be due to the inherent characteristics of the cell line itself. A more suitable negative control cell line will be selected for this assay and will be implemented before the pivotal trial. Taken all together, this dataset suggests that DP potency as determined by IFNy secretion follows similar trends in both patient donor- derived DP lots assessed.Attorney Docket No.: 063384-523001 WOTable 54. IFNy Secretion by DP Generated by DLBCL Patient Material using LV-023 65L Pilot Lot 24 P009 (OXB) Across Harvest DaysIFNy IFNy IFNy IFNy Patien Batc Harvest secretion secretion secretion secretion h Material Vectort ID Day (ng / mL), (ng / mL), (ng / mL), (ng / mL),CD19 CD20 CD22 TKO 07-1005 PS. CL257 Patient 65L 9 3.94 8.77 4.22 3.48 08-1007 PS. CL269 Donor- 65L 5 8.36 16.65 4.36 3.13 08-1007 PS. CL269 derived 65L 7 6.23 12.33 5.15 4.29 08-1007 PS. CL269 In- 65L 9 10.89 16.67 9.57 9.05ProcessSample / DrugProduct3. Conclusion

[0511] A small-scale titration was performed to determine the vector MOI setpoint for the LV-023 65L Pilot Lot 24 P009 (OXB) to be used in the CRG-023 manufacturing process. The growth kinetics across all the MOI conditions were consistent with the cultures maintaining a viability of > 91%, which highlights that the transduction process did not negatively impact the cell growth and cell health. The maximum transduction efficiency obtained from this experiment was approximately 59.8% ± 2.8% as determined by flow cytometry. The selected vector volume set point was determined to be 300 pL at the small scale, which scales to 3.0 mL for the clinical-scale and translates to an MOI of 1.7. This volume was chosen near the plateau of the curve to achieve the maximum transduction efficiency. VCN analysis determined that a MOI of 1.7 resulted in a VCN of 6 copies / Tri- CAR+ cell. The transduction efficiency analysis, cell culture viability, and cell growth kinetics all demonstrate the selected vector MOI setpoint for the CRG-023 process is optimal for achieving adequate transduction efficiency while maintaining cell fitness, which provides a basis for the clinical-scale production of CRG-023.

[0512] At scale runs were performed using three healthy donors to verify the vector MOI setpoint to be used in the CRG-023 process for clinical-scale production. The CD4+ and CD8+ purity levels had a mean of 97.85% ± 1.53%. The cell growth kinetics showed that the cells were able to expand from 300×106viable cells on Day 0 to approximately 2.12×109±2.77×108cells with an average viability of 92.69% ± 1.26%. The healthy donors were able to achieve a transduction efficiency of 60.63% ± 1.90% with a total Tri-CART cell count of 1.29×109± 1.92 x 108cells. VCN for the three healthy donor-derived DP lots ranged from 5 to 7 copies / Tri-CAR+ cell and is highly consistent with the VCN determined in the vector MOIAttorney Docket No.: 063384-523001 WOsetpoint titration study. These results show that the CRG-023 process does not negatively impact the cell fitness or growth and can successfully achieve the maximum transduction efficiency to meet the harvest requirements at an MOI of 1.7.

[0513] Following the healthy donor verification runs, at scale runs were performed using two DLBCL patient-derived cells to verify the vector MOI setpoint to be used in the CRG-023 process for clinical-scale production. The CD4+ and CD8+ purity levels had a mean of 93.68% ± 4.29%. The cell growth kinetics showed that the cells were able to expand from 300×106viable cells on Day 0 to approximately 1.49×109PS. CL257 and 2.98×109for PS. CL269 cells with an average viability of 93.0% ± 1.7%. The patient-derived material was able to achieve a transduction efficiency of 31.52% ± 5.96% with a total Tri-CAR+ cell count of 5.13×108cells for PS. CL257 and 9.35 x 108cells for PS. CL269. VCN / Tri-CAR+ cell values were between 5-6 copies / Tri-CAR+ cell for PS. CL269. While VCN for PS. CL257 had stabilized to 6 copies / Tri-CAR+ cell, VCN for Day 5 was 18 copies / Tri-CAR+ and was largely due to the low transduction efficiency observed. These results show that while the CRG-023 process does not negatively impact the cell fitness or growth, the harvest requirements are still be met regardless of achieving a lower transduction efficiency achieved due to the diseased state of the patient material.

[0514] Extended characterization (EC) assessment of heal thy donor- and patient material-derived drug product memory subsets, activation target frequencies, exhaustion marker co¬ expression, CAR+ CD4 / CD8 ratio, and single and double CA+ populations showed differences in readouts across batches, which may be linked to starting material heterogeneity. Despite differences in magnitude, EC attribute profiles and directionality of trends were consistent.

[0515] Extended characterization of the DP to assess potency by measuring IFNy secretion demonstrated similar trends across all three healthy donors assessed. Similarly, DP generated using patient derived material yielded similar trends in IFNy secretion to the DP lots generated using healthy donor material.

[0516] Based on all the analytics, a vector MOI of 1.70 is recommended for the CRG-023 C AR T drug product manufacturing process in support of pre-clinical studies.Attorney Docket No.: 063384-523001 WOEXAMPLE 4Vector Volume Setpoint for CRG-023 Transduction: LV-023 GMP Batches

[0517] The purpose of this study was to generate LV-023 lentivirus titration data to set a recommendation for lot-specific vector volume / MOI setpoint for the CRG-023 CAR T drug product manufacturing process in support of a Phase 1 clinical trial.

[0518] The vector titration used in this study was performed on GMP LV-023 batches produced at the 200 L scale from both lenti virus’ contract development and manufacturing organizations (CDMO), Oxford Biomedica (OXB) and Yposkesi (YPK).

[0519] The vector titration was performed by trained personnel in the development laboratories at CARGO Therapeutics. The cell material used to perform the titration in this study was enriched CD4 and CD8 cells produced by the CliniMACS® Prodigy TCT selection, which are representative of the CRG-023 CAR T manufacturing process.

[0520] This report contains all the information and data pertaining to the small-scale vector titration using GMP LV-023 from OXB and YPK. From this data and analysis an MOI setpoint was determined to move forward to execute three at-scale healthy donor verification studies as well as one patient-derived material verification study. This report includes all the verification study data to be used to determine Vector Volume Setpoints for LV-023 lot 2602-*-*-! (OXB) and LV-023 Lot 24P0017 (YPK).1. Methods

[0521] Methods were performed as in Example 5 with some minor modifications.2. Resultsi. Small Scale Growth Kinetics

[0522] The growth kinetics of the transduced cultures show cell growth from Day 0 through to Day 5. The titration negative control condition 0 pL Viral Vector for CRG-023 derived from Donor D001004593 shows a lower cell count than the rest of the titration points (refer to Figure 2) The donors show a variance in growth across the different MOIs, with Donor D001004593 showing a higher cell expansion than Donor DOO 1003932.

[0523] Aside from the donor growth variability, statistical analysis using the Kruskall-Wallis Test indicated no significant total viable cell differences between the groups (p=0.99 for Donor DOO 1004593 titration with LV-023 Lot 2602-*-*-! (OXB), p=0.99 for Donor DOO 1004593 titration with LV-023 Lot 24P0017 (YPK), p=0.99 for Donor DOO 1003932 titration with LV-023 Lot 2602-*-*- 1 (OXB), and p=0.99 for Donor DOO 1003932 titration with LV-023 Lot 24P0017 (YPK)), suggesting that transduction at all tested MOIs / transduction volumes had no significant adverse effects on cell viability orAttorney Docket No.: 063384-523001 WOproliferation. The growth kinetics are found in FIG. 37, FIG. 38, FIG. 39, and FIG. 40. The MOIs are calculated from the transduction volumes as per Table 55 and Table 56.

[0524] The viability of all cultures was maintained at > 88% (EXP24000963) at day of harvest. High viability of cell culture shows the cells are robust post transduction and there are no negative impacts on the cell viability. This confirms that the transduction process does not negatively affect the cell cultures’ ability to grow, regardless of the transduction volume / MOI.Table 55. LV-023 Lot 2602-*-*-! (OXB) Titration MOILVV Vol. (pL) Total TU1Cells Seeded Day 0 MOI 600 1.56E +08 3.00E+07 5.20 300 7.80E+07 3.00E+07 2.60 225 5.85E+07 3.00E+07 1.95150 3.90E+07 3.00E+07 1.3075 1.95E+07 3.00E+07 0.65 37.5 9.75E+06 3.00E+07 0.32 18.3 4.76E+06 3.00E+07 0.160 0.00E+00 3.00E+07 0.001The viral titer of LV-023 Lot 2602-*-*-! (OXB) is 2.6e8 TU / mL.Table 56. LV-023 Lot 24P0017 (YPK) Titration MOILVV Vol. (pL) Total TU1Cells Seeded Day 0 MOI 600 3.66E+08 3.00E+07 12.20 300 1.83E+08 3.00E+07 6.10 225 1.37E+08 3.00E+07 4.58 20021.22E+08 3.00E+07 4.07150 9.15E+07 3.00E+07 3.0575 4.58E+07 3.00E+07 1.53 37.5 2.29E+07 3.00E+07 0.76 18.3 1.12E+07 3.00E+07 0.370 0.00E+00 3.00E+07 0.001The viral titer of LV-023 Lot 24P0017 (YPK) is 6.1e8 TU / mL.ii. Transduction Efficiency

[0525] The volumetric titration of the LV-023 Lot 2602-*-*- 1 (OXB) indicates that the maximum achievable transduction efficiency is 69.65% for Donor DOO 1004593 and is 49.14% for Donor D001003932 based on the flow cytometry analysis of transduced primary T cells from two healthy donors. This can be seen in FIG. 41.

[0526] The volumetric titration of the LV-023 Lot 24P0017 (YPK) indicates that the maximum achievable transduction efficiency is 36.55% for Donor DOO 1004593 and is 49.37% for Donor DOO 1003932 based on the flow cytometry analysis of transduced primary T cells from two healthy donors. This can be seen FIG. 42.Attorney Docket No.: 063384-523001 WO

[0527] The volumetric titration curve of LV-023 Lot 2602-*-*-l (OXB) starts to plateau around 300 pL of vector volume added based on FIG. 41. The vector volume setpoint was selected at approximately 300 pL for the LV-023 Lot 2602-*-*-l (OXB) at a small scale, which correlates to 3.0 mL at-scale. The vector volume set point was selected in the plateau region where the transduction efficiency is highest while using minimal vector. This approach reduces variability in transduction efficiency and minimizes the vector volume and vector copy number necessary to achieve maximum transduction efficiency.

[0528] The viral titer of the LV-023 Lot 2602-*-*-l (OXB) is 2.6x108TU / mL. The contract manufacturing organization, Oxford Biomedica, determined this titer via OXB SOP-417 which is documented in the Certificate of Testing for the 200 L GMP Batch (CRT-0200). The number of cells seeded on Day 0 was 30x106; this equates 300 pL of viral suspension or an MOI of 2.60 as per Table 55 and Equation 1.Equation 1. Calculation to determine MOIMultiplicity of Infection (MOI) = Vector Volume (mD'xVector Titer (TU / mL)Total Viable Cells Seeded on Day 0 (cells)

[0529] The volumetric titration curve indicates the LV-023 Lot 24P0017 (YPK) starts to plateau around 200 pL of vector volume added based on FIG. 42. The vector volume setpoint was selected at approximately 200 pL for the LV-023 Lot 24P0017 (YPK) at a small scale, which scales to 2.0 mL at-scale. The vector volume set point was selected in the plateau region where the transduction efficiency is highest while using minimal vector. This approach reduces variability in transduction efficiency and minimizes the vector volume and vector copy number necessary to achieve maximum transduction efficiency.

[0530] The viral titer of the LV-023 Lot 24P0017 (YPK) is 6.1xl08TU / mL. The contract manufacturing organization, Yposkesi, determined this titer via YPK Q-SOP1250 which is documented in the Certificate of Testing for the 200 L GM P Batch (CRT-0205). The number of cells seeded on Day 0 was 30x106, this equates 200 pL of viral suspension or an MOI of 4.07 as per Table 56 and Equation 1.Hi. Vector Copy Number (VCN) Determination by ddPCR

[0531] The vector copy number (VCN) per Tri-CAR-l- cell obtained from the small-scale titration of two healthy donors (Donor D001004593 and Donor DOO 1003932) were averaged based on vector lot. The VCN per Tri-CAR-l- cell was measured via TM-0028.

[0532] The results FIG. 43A-43C and Table 57 show that at the chosen MOI of 2.60, for LV-023 Lot 2602-*-*-! (OXB), the vector copy number (VCN) per Tri- CAR+ cell is 5.48Attorney Docket No.: 063384-523001 WOfor D001003932, and 5.77 for D001004593. The VCN per Tri-CAR+ cell at the highest MOI (5.20) is 6.15 and 6.94, for DOO 1003932 and DOO 1004593 respectively. These levels of VCN meet the < 15 copies / Tri- CAR+ cell target per DOC-0244, indicating effective transduction while maintaining a balance that minimizes potential risks associated with higher vector integration.

[0533] The volumetric titration of the LV-023 Lot 24P0017 (YPK) starts to plateau between 5 and 6 VCN per Tri-CAR+ cell as shown in FIG. 44A-44C and Table 57. The VCN per Tri-CAR+ cell at the highest MOI (12.20) is 4.70 and 6.01, for D001003932 andDOO 1004593, respectively. These levels of VCN are acceptable, indicating effective transduction while maintaining a balance that minimizes potential risks associated with higher vector integration.Table 57. VCN Analysis of Primary T Cells Transduced with LV-023 Batch 2602-*-*-l (OXB) or Batch 24P0017 (YPK) at Various MOIsDonor Vector Lot PS. CL# MOI %TE VCN / Tri-CAR+ cell PS. CL307 0 0 0 PS. CL306 0.16 6.35 3.15 PS. CL305 0.33 15.74 3.37 PS. CL304 0.65 30.84 4 D001003932PS. CL303 1.3 41.61 4.49 PS. CL302 1.95 45.15 4.98 PS. CL301 2.6 50.08 5.48 200 L GMP OXB PS. CL300 5.2 50.18 6.15 (Batch 2602-*-*-1) PS. CL291 0 0 0PS. CL290 0.16 6.15 3.9 PS. CL289 0.33 9.16 7.17 PS. CL288 0.65 19.54 4.71 DOO 1004593PS. CL287 1.3 33.32 4.82 PS. CL286 1.95 29.48 6.63 PS. CL285 2.6 49.92 5.77 PS. CL284 5.2 64.9 6.94 Donor Vector Lot PS. CL# MOI %TE VCN / Tri-CAR+ cell PS. CL315 0 0 0 PS. CL314 0.37 20.18 3.12 PS. CL313 0.76 30.1 3.51 PS. CL312 1.53 38.63 4.05 D001003932PS. CL311 3.05 39.11 4.07 PS. CL310 4.58 41.23 4.62 PS. CL309 6.1 42.43 4.67 200 L GMP YPOPS. CL308 12.2 43.04 4.7 (Batch 24P0017)PS. CL299 0 0 0PS. CL297 0.76 23.06 6.17Attorney Docket No.: 063384-523001 WOPS. CL296 1.53 28.01 6.77 PS. CL295 3.05 29.19 7.26 PS. CL294 4.58 34.74 6.32 PS. CL293 6.1 37.15 5.92PS. CL292 12.2 37.24 6.01

[0534] Based on these small-scale findings, the vector volume setpoint, for LV- 023 Lot 2602-*-*-l (OXB), was set at 300 pL to transduce 30><106cells seeded on Day 0. When scaled up 10-fold (to at-scale), it is recommended to use 3.0 mL of LV-023 Lot 2602-*-*-l (OXB) to transduce 300×106cells seeded in GRex 100M on Day 0 (an MOI of 2.60).

[0535] The vector volume setpoint, for LV-023 Lot 24P0017 (YPK), was set at 200 pL to transduce 30x106cells seeded on Day 0. When scaled up 10- fold (to at-scale), it is recommended to use 2.0 mL of LV-023 Lot 24P0017 (YPK) to transduce 300×106cells seeded in GRex 100M on Day 0 (an MOI of 4.07).Hi. LV-023 Lot 2602-*-*-! and LV-023 Lot 24P0017 (YPK) Healthy Donor At-Scale Runs

[0536] Using the MOI setpoint recommended from the small-scale volumetric titration (2.60 for LV-023 lot 2602-*-*-! (OXB) and 4.07 for LV-023 Lot 24P0017 (YPK)), three healthy donor material batches were run at scale, with each of the two vector lots....

Claims

Attorney Docket No.: 063384-523001 WOCLAIMS1. A method of making a population of CAR-expressing immune cells, the method comprising:(a) obtaining a liquid sample comprising an initial population of autologous cells comprising immune cells from a human subject;(b) processing the initial population of autologous cells to generate a washed population of cells comprising immune cells;(c) on day 0, enriching the washed population of cells comprising immune cells for CD4+ and CD 8+ T cells to generate an enriched population of cells comprising immune cells, and culturing the enriched population of cells in a volume of a medium comprising an activation agent to generate an enriched and activated population of cells comprising immune cells;(d) transducing the enriched and activated population of cells comprising immune cells with one or more recombinant polynucleotides encoding three CARs thereby generating an enriched, activated, and transduced population of cells comprising CAR-expressing immune cells;(e) expanding the enriched, activated, and transduced population of cells comprising CAR-expressing immune cells in media to yield an expanded population of enriched, activated, and transduced cells comprising CAR- expressing immune cells;(f) harvesting the expanded population of enriched, activated, and transduced cells comprising CAR-expressing immune cells on or before Day 9 after the seeding in step (c); and(g) formulating the harvested population of enriched, activated, and transduced cells comprising CAR-expressing immune cells for cryopreservation and administration to patients.

2. The method of claim 1, further comprising a step of cryopreserving the initial population of autologous cells prior to step (c) to generate a population of cryopreserved cells.Attorney Docket No.: 063384-523001 WO3. The method of claim 1, wherein the liquid sample including an initial population of autologous cells comprises a leukapheresis product.

4. The method of any of claims 1-3, wherein the processing in step (b) comprises a step of washing, concentrating and eluting or resuspending the washed population of cells in a buffer.

5. The method of claim 4, wherein the washing comprises PBS / EDTA + 0.5% HSA.

6. The method of any of claims 1-5, wherein the processing in step (b) further comprises a step of reducing the number of platelets in the washed population of cells.

7. The method of claim 6, wherein the number of platelets is reduced by spinning membrane filtration.

8. The method of claim 7, wherein the spinning membrane filtration system is a Cue Cell Processing System.

9. The method of any of claims 1-8, wherein the washed or washed and concentrated population of cells has a reduced number of platelets compared to the initial population of cells is eluted or resuspended in a buffer.

10. The method of any of claims 1-9, wherein the buffer comprises human serum albumin (HSA), Plasma-Lyte A™, phosphate buffered saline, ethylenediamine tetraacetic acid (EDTA), sodium chloride, sodium bicarbonate buffer, glutathione, biotin, vitamin B12, inositol, choline, L-glutamine, sodium pyruvate, glucose, or any combination thereof.

11. The method of any of claims 1-10, wherein the buffer comprises human serum albumin (HSA).

12. The method of any of claims 1-11, wherein the buffer comprises Plasma-Lyte A™.

13. The method of any of claims 1-12, wherein the buffer comprises equal volumes of Plasma-Lyte A™ and 4% (w / v) human serum albumin.

14. The method of any of claims 1-13, wherein the washed or washed and concentrated population of cells having a reduced number of platelets compared to the initial 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 to generate a cryopreserved population of cells.Attorney Docket No.: 063384-523001 WO15. The method of claim 2-14, wherein the cryopreserved population of cells is thawed prior to step (c) to generate a thawed population of cells comprising immune cells.

16. The method of any of claims 1-15, wherein the enriching step (c) comprises mixing the washed or washed, concentrated, and thawed population of cells with magnetic beads derivatized with CD4-specific binding gents and CD8-specific binding agents, washing, and eluting to generate the enriched population of cells.

17. The method of any of claims 1-16, wherein the culturing of the enriched population of cells comprises seeding 3×108cells from the enriched population of cells in a vessel.

18. The method of claim 17, wherein the vessel is a G-Rex® vessel.

19. The method of any of claims 1-18, wherein the culturing of the enriched population of cells comprises seeding 2.5 x 106cells from the enriched population of cells in a vessel.

20. The method of any of claims 1-19, wherein the culturing of the enriched population of cells comprises seeding between at least 2.5x106cells and at least 3×108cells from the enriched population of cells in a vessel.

21. The method of any of claims 1 -20, wherein the culturing of the enriched population of cells comprises seeding at least about 2.5x106, 3X106, 3.5x106, 4xl06, 4.5x106, 5X106, 5.5xl06, 6xl06, 6.5xl06, 7xl06, 7.5xl06, 8x]06, 8.5xl06, 9xl06, 9.5xl06, lx107, 1.5xl07, 2xl07, 2.5 xlO7, 3xl07, 3.5xl07, 4xl07, 4.5xl07, 5X107, 5.5xl07, 6X107, 6.5xl07, 7xl07, 7.5xl07, 8xl07, 8.5xl07, 9x 107, 9.5xl07, IxlO8, 1.5xl08, 2xl08, 2.5 xlO8, 3xl08, 3.5 xlO8, 4xl08, 4.5 xlO8, or 5X108cells in a vessel.

22. The method of any of claims 1-21, wherein the enriched population of cells is cultured in a volume of media comprising modified T Cell Culture Medium.

23. The method of claim 22, wherein the volume of media brings the culture volume to 70 niL.

24. The method of any of claims 1-23, wherein the modified T Cell Culture Medium comprises Prime XV CDM Medium supplemented with a 1: 1 mixture of recombinant human cytokines IL-7 (hIL-7) and IL-15 (hIL-15).

25. The method of any of claims 1-24, wherein 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.Attorney Docket No.: 063384-523001 WO26. The method of any of claims 1-25, wherein the modified T Cell Culture Medium further comprises an effective amount of an activation reagent comprising agonists of CD3 and CD28.

27. The method of any of claims 1-26, wherein the activation reagent comprising CD3 and CD28 agonists comprises the T cell TransActTMreagent.

28. The method of any of claims 1-27, wherein the one or more recombinant polynucleotides encoding three CARs further comprises one or more lentiviral expression vectors.

29. The method of any of claims 1-28, wherein one or more lentiviral expression vectors comprises one lentiviral expression vector comprising one recombinant polynucleotide encoding three separate and distinct CARs.

30. The method of any of claims 1-29, wherein the lentiviral expression vector(s) is / are manufactured using a suspension cell culture method.

31. The method of any of claims 1 -30, wherein the transduction in step (e) is performed on Day 1.

32. The method of any of claims 1-31, wherein the transduction in step (e) is performed 22-26 hours after culturing of the enriched and activated population of cells comprising immune cells on Day 0.

33. The method of any of claims 1-32, wherein the amount of vector used to transduce the enriched 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 culture the enriched population of cells comprising immune cells such that the transduction is performed with a multiplicity of infection (MOI) of 3.0.

34. The method of any of claims 1-33, wherein the transduction is performed with an MOI of 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.

35. The method of any of claims 1-34, wherein the one lentiviral expression vector comprising one recombinant polynucleotide encoding three CARs is thawed, diluted into modified T Cell Culture Medium, and added to the enriched population of cells comprising immune cells.Attorney Docket No.: 063384-523001 WO36. The method of any of claims 1-35, wherein the modified T Cell Culture Medium comprises Prime XV CDM M edium supplemented with a 1:1 mixture of recombinant human cytokines IL-7 (hIL-7) and IL-15 (hIL-15).

37. The method of any of claims 1 -36, wherein 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.

38. The method of any of claims 1-37, wherein the final volume in which the transduction in step (e) is performed comprises 100 mL.

39. The method of any of claims 1-38, further comprising adding pre-warmed T Cell Culture Medium to the transduced population of cells.

40. The method of any of claims 1-39, further comprising removing at least 50% of the volume of the medium on or before Day 4 after the culturing in step (c).

41. The method of any of claims 1-40, wherein the expansion in step (e) comprises determining the number of viable C D3+ CAR-expressing T cells, continuing to expand the expanded population of cells, and exchanging the medium.

42. The method of claim 41, wherein determining the number of viable CD3+ CAR- expressing T cells is performed on Day 4 by taking a sample of the expanded population of cells and calculating the amount of CAR+ cells in the cell culture based on the transduction efficiency.

43. The method of claim 42, wherein when the amount of CAR+ cells on Day 4 is greater than a minimum harvest threshold of 115×106, 177×106or 269×106CAR+ cells, the harvesting step (f) is performed on Day 5.

44. The method of claim 42, wherein when the amount of CAR+ cells on Day 4 is less than a minimum harvest threshold of 115×106, 177×106or 269x106CAR+ cells, the expanded population of cells is expanded for another day, a sample of the expanded population of cells is taken on Day 5, and the number of CAR+ cells is recalculated.

45. The method of claim 44, wherein when the amount of CAR+ cells on Day 5 is greater than the minimum harvest threshold of 115 x 106, 177×106or 269×106CAR+ cells the harvesting step (f) is performed on Day 5.

46. The method of claim 44, when the amount of CAR+ cells on Day 5 is less than a minimum harvest threshold of 115×106, 177×106or 269×106CAR+ cells, theAttorney Docket No.: 063384-523001 WOexpanded population of cells is expanded for another day, a sample of the expanded population of cells is taken on Day 6 and the number of CAR+ cells is recalculated.

47. The method of claim 46, wherein the amount of CAR+ cells on Day 6 is greater than the minimum harvest threshold of 115×106, 177×106or 269×106CAR+ cells, and the harvesting step (f) is performed on Day 7.

48. The method of claim 46, wherein the amount of CAR+ cells on Day 6 is less than a minimum harvest threshold of 115 x 106, 177×106or 269×106CAR+ cells, the expanded population of cells is expanded for another day, a sample of the expanded population of cells is taken on Day 8 and the number of CAR+ cells is recalculated.

49. The method of claim 46, wherein the amount of CAR+ cells on Day 8 is greater than the minimum harvest threshold of 115×106, 177×106or 269×106CAR+ cells, and the harvesting step (f) is performed on Day 9.

50. The method of any of claims 1 -49, wherein the minimum harvest threshold is 269×106CAR+ cells.

51. The method of any of claims 1 -50, wherein the harvested population of cells is formulated with Plasma-Lyte A® + 4% (w / v) HSA, diluted 1:1 with Cryostor® CS10 and frozen.

52. The method of any of claims 1-51, wherein the formulation step is automated.

53. The method of any of claims 1-52, wherein the automated formulation step is performed using a Cue ScaleReady Cell Processing System, and the harvested population of cells is resuspended to the desired concentration in Final Formulation Medium comprising a 1:1 mixture of Plasma-Lyte A+4% (w / v) that is subsequently diluted 1:1 with Cryostor® CS10 and cryopreserved.

54. The method of any of claims 1-53, wherein the harvested population of cells including CAR-expressing immune cells comprises autologous CAR-expressing immune cells.

55. The method of any of claims 1-54, wherein the harvested population of cells including CAR-expressing immune cells comprises autologous T cells expressing a CD19- specific CAR, a CD20-specific CAR, and a CD22-specific CAR.

56. The method of any of claims 1-55, wherein the recombinant polynucleotide encoding three CARs encodes a CD19-specific CAR, a CD20-specific CAR, and a CD22- specific CAR.Attorney Docket No.: 063384-523001 WO57. The method of any of claims 1-56, wherein the recombinant polynucleotide encoding an autologous CD19-specific CAR, CD20-specific CAR, and CD22-specific CAR further comprises a lentiviral expression vector.

58. The method of any of claims 1-57, wherein the lentiviral expression vector is manufactured using a suspension cell culture method.

59. The method of any of claims 1-58, wherein the recombinant polynucleotide comprises a polynucleotide sequence encoding the CD19-specific CAR, a polynucleotide sequence encoding the CD20-specific CAR, and a polynucleotide sequence encoding the CD22-specific CAR, wherein each CAR-encoding polynucleotide sequence is separated from the other CAR-encoding polynucleotide sequences by a sequence encoding a viral ribosome skipping peptide selected from the group consisting of a P2A peptide, a T2A peptide, an E2A peptide, and an F2A peptide.

60. The method of any of claims 1 -59, wherein the recombinant polynucleotide comprises from 5’ to 3’, a sequence encoding the CD22-specific CAR; a sequence encoding a viral P2A ribosome skipping peptide; a sequence encoding the CD19-specific CAR; a sequence encoding a viral T2A ribosome skipping peptide; and a sequence encoding the CD20-specific CAR.

61. The method of any of claims 1-60, wherein the CD22-specific CAR comprises a CD22-specific binding domain, a transmembrane domain, and an intracellular domain.

62. The method of any of claims 1-61, wherein the CD22-specific CAR comprises a CD22-specific binding domain, a spacer, a hinge domain, a transmembrane domain, a peptide linker, and an intracellular domain.

63. The method of any of claims 1-62, wherein the CD22-specific binding domain comprises an antibody that binds CD22 or an antigen-binding fragment thereof.

64. The method of any of claims 1-63, wherein the CD22-specific binding domain comprises an antibody that binds human CD22.

65. The method of any of claims 1-64, wherein the CD22-specific binding domain comprises an antigen-binding fragment of an antibody that binds human CD22.Attorney Docket No.: 063384-523001 WO66. The method of any of claims 1-65, wherein the antigen-binding fragment of an antibody that binds human CD22 is a single chain variable fragment (scFv) that binds CD22.

67. The method of any of claims 1-66, wherein the scFv that binds CD22 has the sequence of QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGR TYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVT GDLEDAFDIWGQGTMVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQ TIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAE DFATYYCQQSYSIPQTFGQGTKLEIK (SEQ ID NO: 1).

68. The method of any of claims 1-67, wherein the scFv that binds 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.

69. The method of any of claims 1-68, wherein the CD22-specific CAR comprises a CD8a hinge domain comprising the sequence of TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 23) and a CD8a transmembrane domain comprising the sequence of IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 24) and optionally a peptide linker having the sequence of LYC.

70. The method of any of claims 1-69, wherein the CD22-specific CAR comprises a CD8a 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: 23 and a CD8a 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: 24.

71. The method of any of claims 1-70, wherein the CD8a transmembrane domain further comprises a spacer having the sequence of LYC.

72. The method of any of claims 1-71, wherein the CD22-specific CAR comprises an intracellular domain comprising a primary T cell activating domain comprising an immunoreceptor tyrosine-based activation motif (ITAM) and a costimulatory signaling domain.Attorney Docket No.: 063384-523001 WO73. The method of any of claims 1-72, wherein the primary T cell activating domain comprising an ITAM comprises a CD3^ intracellular signaling domain.

74. The method of any of claims 1-73, wherein the CD3C, intracellular signaling domain comprises the sequence of RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR (SEQ ID NO: 26).

75. The method of any of claims 1-74, wherein the CD3C, 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: 26.

76. The method of any of claims 1-75, wherein the costimulatory signaling domain comprises a 4-1BB / CD137 signaling domain.

77. The method of any of claims 1-76, wherein the 4-1BB / CD137 signaling domain comprises the sequence of KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 25).

78. The method of any of claims 1-77, wherein 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: 25.

79. The method of any of claims 1-78, wherein the CD22-specific CAR comprises the sequence of QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGR TYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVT GDLEDAFDIWGQGTMVTVSSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQ TIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAE DFATYYCQQSYSIPQTFGQGTKLEIKAAATTTPAPRPPTPAPTIASQPLSLRPEA CRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYI FKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQL YNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEA YSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:Attorney Docket No.: 063384-523001 WO80. The method of any of claims 1-79, wherein the CD22-specific 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: 29.

81. The method of any of claims 1 -80, wherein the CD22-specific CAR comprises the sequence of MLLLVTSLLLCELPHPAFLLIPQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSN SAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSL QLNSVTPEDTAVYYCAREVTGDLEDAFDIWGQGTMVTVSSGGGGSDIQMTQ SPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGVP SRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIKAAATTT PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCG VLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE LRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR (SEQ ID NO: 30).

82. The method of any of claims 1-81, wherein the CD22-specific 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: 30.

83. The method of any of claims 1-82, wherein the CD19-specific CAR comprises a CD19-specific binding domain, a transmembrane domain, and an intracellular domain.

84. The method of any of claims 1-83, wherein the C D19-specific CAR comprises a CD19-specific binding domain, a hinge domain, a transmembrane domain, a spacer, and an intracellular domain.

85. The method of any of claims 1-84, wherein the CD19-specific binding domain comprises an antibody that binds CD 19 or an antigen-binding fragment thereof.

86. The method of any of claims 1-85, wherein the CD19-specific binding domain comprises an antibody that binds human CD 19.

87. The method of any of claims 1-86, wherein the CD19-specific binding domain comprises an antigen-binding fragment of an antibody that binds human CD 19.Attorney Docket No.: 063384-523001 WO88. The method of any of claims 1-87, wherein the antigen-binding fragment of an antibody that binds human CD 19 is a single chain variable fragment (scFv) that binds CD19.

89. The method of any of claims 1-88, wherein the scFv capable of binding CD19 has the sequence of EVQLVQSGAEVKKPGESLKISCKASGYRFTNYWIAWVRQRPGKGLEWMGRI DPSDSYTHYSPSFQGHVTMSTDKSISTAYLQWSSLKASDTAMYYCARPGDIL TGWAMDVWGQGTLVTVSSAAASGGGGSGGGGSGGGGSALQSVLTQPPSVS AAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFS GSKSGTSATLGITGLQAEDEADYYCQSYDSSLSGNYVFGTGTKVTVL (SEQ ID NO: 34).

90. The method of any of claims 1-89, wherein the scFv capable of binding CD19 comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 34.

91. The method of any of claims 1-90, wherein the CD19-specific CA R comprises a CD28 hinge domain comprising the sequence of IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 43) and a CD28 transmembrane domain comprising the sequence of FWVLVWGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 44).

92. The method of any of claims 1-91, wherein the CD19-specific CA R comprises a CD28 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: 43 and a CD28 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: 44.

93. The method of any of claims 1-92, wherein the CD19-specific CAR comprises an intracellular domain further comprising a primary T cell activating domain comprising an immunoreceptor tyrosine -based activation motif (IT AM) and a costimulatory signaling domain.

94. The method of any of claims 1-93, wherein the primary T cell activating domain comprising an ITAM comprises a CD3^ intracellular signaling domain.Attorney Docket No.: 063384-523001 WO95. The method of any of claims 1-94, wherein the CD3^ intracellular signaling domain comprises the sequence of RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR (SEQ ID NO: 26).

96. The method of any of claims 1-95, wherein 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: 26.

97. The method of any of claims 1-96, wherein the costimulatory signaling domain comprises a CD28 signaling domain.

98. The method of any of claims 1-97, wherein the CD28 signaling domain comprises the sequence of RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 45).

99. The method of any of claims 1-98, wherein the CD28 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: 45.

100. The method of any of claims 1-99, wherein the CD19-specific CAR comprises the sequence of EVQLVQSGAEVKKPGESLKISCKASGYRFTNYWIAWVRQRPGKGLEWMGRI DPSDSYTHYSPSFQGHVTMSTDKSISTAYLQWSSLKASDTAMYYCARPGDIL TGWAMDVWGQGTLVTVSSAAASGGGGSGGGGSGGGGSALQSVLTQPPSVS AAPGQKVTISCSGSSSNIGNNYVSWYQQLPGTAPKLLIYDNNKRPSGIPDRFS GSKSGTSATLGITGLQAEDEADYYCQSYDSSLSGNYVFGTGTKVTVLIEVMY PPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVT VAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVK FSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNP QEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDAL HMQALPPR (SEQ ID NO: 47).

101. The method of any of claims 1-100, wherein the CD19-specific 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: 47.Attorney Docket No.: 063384-523001 WO102. The method of any of claims 1-99, wherein the CD19-specific CAR comprises the sequence of MALPVTALLLPLALLLHAARPEVQLVQSGAEVKKPGESLKISCKASGYRFTN YWIAWVRQRPGKGLEWMGRIDPSDSYTHYSPSFQGHVTMSTDKSISTAYLQ WSSLKASDTAMYYCARPGDILTGWAMDVWGQGTLVTVSSAAASGGGGSGG GGSGGGGSALQSVLTQPPSVSAAPGQKVTISCSGSSSNIGNNYVSWYQQLPGT APKLLIYDNNKRPSGIPDRFSGSKSGTSATLGITGLQAEDEADYYCQSYDSSLS GNYVFGTGTKVTVLIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPF WVLVWGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRK HYQPYAPPRDFAAYRSRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVL DKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKG HDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 48).

103. The method of any of claims 1-102, wherein the CD19-specific 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: 48.

104. The method of any of claims 1-103, wherein the CD20-specific CAR comprises a CD20-specific binding domain, a transmembrane domain, and an intracellular domain.

105. The method of any of claims 1-104, wherein the CD20-specific binding domain comprises an antibody that binds CD20 or an antigen-binding fragment thereof.

106. The method of any of claims 1-105, wherein the CD20-specific binding domain comprises an antibody that binds human CD20.

107. The method of any of claims 1-106, wherein the CD20-specific binding domain comprises an antigen-binding fragment of an antibody that binds human CD20.

108. The method of any of claims 1-107, wherein the antigen-binding fragment of an antibody that binds human CD20 is a single chain variable fragment (scFv) that binds CD20.

109. The method of any of claims 1-108, wherein the scFv capable of binding CD20 has the sequence of DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKL LIYWASTRESGVPDRFSGSGSGSDFTLTISSLQAEDVAVYYCQQYYSFYQTFG QGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGWRPGGSLRLSCTASGFTFAttorney Docket No.: 063384-523001 WOGDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSL YLQMNSLRAEDTALYYCARKSYYGSGSPDVFDIWGQGTMVTVSS (SEQ ID NO: 50).

110. The method of any of claims 1-109, wherein the scFv capable of binding CD20 comprises a sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity to SEQ ID NO: 50.

111. The method of any of claims 1-110, wherein the CD20-specific CAR comprises a CD28 hinge domain comprising the sequence of IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 43) and a CD28 transmembrane domain comprising the sequence of FWVLVWGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 44).

112. The method of any of claims 1-111, wherein the CD20-specific CAR comprises a CD28 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: 43 and a CD28 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: 44.

113. The method of any of claims 1-112, wherein the CD20-specific CAR comprises an intracellular domain further comprising a primary T cell activating domain comprising an immunoreceptor tyrosine -based activation motif (IT AM) and a costimulatory signaling domain.

114. The method of any of claims 1-113, wherein the primary T cell activating domain comprising an ITAM comprises a CD3^ intracellular signaling domain.

115. The method of any of claims 1-114, wherein the CD3^ intracellular signaling domain comprises the sequence of RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPR RKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDT YDALHMQALPPR (SEQ ID NO: 26).

116. The method of any of claims 1-115, wherein 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: 26.Attorney Docket No.: 063384-523001 WO117. The method of any of claims 1-116, wherein the costimulatory signaling domain comprises a CD2 signaling domain.

118. The method of any of claims 1-117, wherein the CD2 signaling domain comprises the sequence of KRKKQRSRRNDEELETRAHRVATEERGRKPHQIPASTPQNPATSQHPPPPPGH RSQAPSHRPPPPGHRVQHQPQKRPPAPSGTQVHQQKGPPLPRPRVQPKPPHGA AENSLSPSSN (SEQ ID NO: 59).

119. The method of any of claims 1-118, wherein the CD2 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:59.

120. The method of any of claims 1-119, wherein the CD20-specific CAR comprises the sequence of MLLLVTSLLLCELPHPAFLLIPDIVMTQSPDSLAVSLGERATINCKSSQSVLYSS NNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGSDFTLTISSLQA EDVAVYYCQQYYSFYQTFGQGTKVEIKGGGGSGGGGSGGGGSEVQLVESGG GWRPGGSLRLSCTASGFTFGDYGMSWVRQAPGKGLEWVSGINWNGGSTG YADSVKGRFTISRDNAKNSLYLQMNSLRAEDTALYYCARKSYYGSGSPDVF DIWGQGTMVTVSSIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPF WVLVWGGVLACYSLLVTVAFIIFWVKRKKQRSRRNDEELETRAHRVATEE RGRKPHQIPASTPQNPATSQHPPPPPGHRSQAPSHRPPPPGHRVQHQPQKRPPA PSGTQVHQQKGPPLPRPRVQPKPPHGAAENSLSPSSNRVKFSRSADAPAYKQ GQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKD KMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 64).

121. The method of any of claims 1-120, wherein the CD20-specific 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: 64.

122. The method of any of claims 1-119, wherein the CD20-specific CAR comprises the sequence of DIVMTQSPDSLAVSLGERATINCKSSQSVLYSSNNKNYLAWYQQKPGQPPKL LIYWASTRESGVPDRFSGSGSGSDFTLTISSLQAEDVAVYYCQQYYSFYQTFG QGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGWRPGGSLRLSCTASGFTFAttorney Docket No.: 063384-523001 WOGDYGMSWVRQAPGKGLEWVSGINWNGGSTGYADSVKGRFTISRDNAKNSL YLQMNSLRAEDTALYYCARKSYYGSGSPDVFDIWGQGTMVTVSSIEVMYPP PYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVWGGVLACYSLLVTVA FIIFWVKRKKQRSRRNDEELETRAHRVATEERGRKPHQIPASTPQNPATSQHP PPPPGHRSQAPSHRPPPPGHRVQHQPQKRPPAPSGTQVHQQKGPPLPRPRVQP KPPHGAAENSLSPSSNRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLD KRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGH DGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 62).

123. The method of any of claims 1-122, wherein the CD20-specific 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: 62.

124. A method of making a population of CAR-expressing immune cells, the method comprising:(a) obtaining a liquid sample comprising an initial population of autologous cells comprising immune cells from a human subject;(b) processing the initial population of autologous cells generate a washed population of cells comprising immune cells;(c) on day 0, enriching the washed population of cells comprising immune cells for CD4+ and CD 8+ T cells to generate an enriched population of cells comprising immune cells, and culturing the enriched population of cells in a volume of a medium comprising an activation agent to generate an enriched and activated population of cells comprising immune cells, wherein the enriched and activated population of cells is seeded with at least 3.0x108cells from the activated population of cells into a volume of at least 70 mL of a medium comprising IL-7 and IL-15;(d) transducing the enriched and activated population of cells comprising immune cells with one or more recombinant polynucleotides encoding a CD19-specific CAR, a CD20-specific CAR, and a CD22-specific CAR, thereby generating an enriched, activated, and transduced population of cells comprising CAR- expressing immune cells;Attorney Docket No.: 063384-523001 WO(e) adding a volume of first medium to the enriched, activated, and transduced population of cells on Day 2 after the culturing step in (c) to bring the total volume to at least 1000 mL;(f) expanding the enriched, activated, and transduced population of cells in media to yield an expanded population of enriched, activated, and transduced cells comprising CAR-expressing immune cells;(g) determining the number of viable CD3+ CAR-expressing T cells; and either (h) continuing to expand the enriched, activated, and transduced population of cells and exchanging the medium, or(i) harvesting the enriched, activated, and transduced population of cells comprising CAR-expressing immune cells on or before Day 9 after the seeding in step (c); and(j) formulating the harvested population of cells for cryopreservation and administration to patients.

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