Compositions and methods for preparing engineered lymphocytes for cell therapy

Abstract

The present specification provides compositions and methods for producing engineered lymphocytes. Also provided are prepared engineered lymphocytes with an increased proportion of naive lymphocytes that result in increased therapeutic efficacy. The methods in various embodiments produce lymphocytes that are accelerated compared to the prior art and have improved survival rates, transduction success rates, and in vivo anti-tumor efficacy.

Description

Technical Field

[0001] (Cross - reference to Related Applications) This application claims priority to U.S. Provisional Patent Application No. 63 / 346,709, filed May 27, 2022; U.S. Provisional Patent Application No. 63 / 485,623, filed Feb. 17, 2023; and U.S. Provisional Patent Application No. 63 / 490,162, filed Mar. 14, 2023, which are hereby incorporated by reference in their entirety.

[0002] (Field of the Invention) The present disclosure relates to the field of cell therapy, and more specifically, to compositions and methods for manufacturing engineered lymphocytes.

Background Art

[0003] Immune cells can be modified to target and kill a patient's cancer cells. To increase the ability of immune cells to target and kill specific cancer cells, methods have been developed to genetically engineer immune cells to express constructs that direct the immune cells to specific target cancer cells. Chimeric antigen receptors (CARs) containing binding domains that can interact with specific tumor antigens and engineered T cell receptors (TCRs) enable immune cells to target and kill cancer cells expressing specific tumor antigens.

[0004] Typically, very complex autologous cell manipulation and production processes that take at least a week and can take several weeks also pose significant challenges. During the process, lymphocytes collected from the patient need to be transported to a processing center, while the produced cells need to be cryopreserved and then transported to the patient for transplantation. This very complex process inevitably results in high costs and limits clinical applications.

[0005] In addition, long processes can lead to reduced cell viability and increased lymphocyte maturity, both of which are detrimental to in vivo efficacy. Thus, there is a strong unmet need to develop a process that not only takes less time but also generates immune cells with improved therapeutic efficacy.

SUMMARY OF THE INVENTION

[0006] As provided, current autologous CAR cell manufacturing processes typically take about 7 days and can be much longer. The present disclosure describes an improved process that can be completed within 6 days or even within 4 days (or within 5 days or even within 3 days after the enrichment step). In various examples, a 5-day process (i.e., 5 days after enrichment) includes a transduction preparation and execution step in which a greater number of lymphocytes contact a vector immobilized on recombinant fibronectin coated on the inner surface of a closed system. Such improved transduction procedures enable a very short post-transduction cell expansion step.

[0007] Furthermore, an even more improved process that does not require any post-transduction expansion can be completed within just 3 days after the initial enrichment step. Surprisingly, it has been discovered that both 3-day and 5-day processes produce transduced lymphocytes with a higher percentage of naive cells. At least in part due to the increased naive cell population, these cell products exhibited significantly improved in vivo anti-tumor efficacy compared to the conventional 7-day process.

[0008] Accordingly, in one embodiment of the present disclosure, there is provided a method for preparing transduced lymphocytes, comprising incubating a sample containing lymphocytes obtained from a donor subject with a polynucleotide vector to transduce the lymphocytes to produce transduced lymphocytes, and culturing a sample containing the transduced lymphocytes for less than 72 hours before the lymphocytes are collected to produce a collected sample.

[0009] In some embodiments, the transduced lymphocytes are cultured for less than 48 hours before being harvested. In some embodiments, the transduced lymphocytes are cultured for less than 36 hours before being harvested.

[0010] In some embodiments, the incubation is performed in a closed system. In some embodiments, the closed system has an inner surface area of at least 1500 cm 2 . In some embodiments, the closed system has an inner surface coated with recombinant human fibronectin, and the coating is performed with a solution containing about 1-10 μg / ml of recombinant human fibronectin. In some embodiments, the inner surface is further contacted with a second solution containing a polynucleotide vector, and the second solution has a volume of about 200 mL. In some embodiments, the coating further includes draining the second solution. In some embodiments, the sample in the closed system contains at least 1.5×10 8 lymphocytes. In some embodiments, the sample contains at least 4×10 8 lymphocytes.

[0011] In some embodiments, the lymphocytes are peripheral blood mononuclear cells (PBMCs) or T cells. In some embodiments, the harvested sample contains CD3+ cells. In some embodiments, the harvested sample contains CD4+ T cells and CD8+ T cells. In some embodiments, at least 20% of the harvested CD4+ T cells are naive T cells, and 12% or less of the harvested CD4+ T cells are effector memory T cells. In some embodiments, at least 25% of the harvested CD4+ T cells are naive T cells, and 9% or less of the harvested CD4+ T cells are effector memory T cells. In some embodiments, at least 80% of the harvested CD4+ T cells are CCR7+ cells. In some embodiments, at most 20% of the harvested CD4+ T cells are a combination of effector memory T cells and effector T cells.

[0012] In some embodiments, at least 10% of the CD8+ T cells collected are naive T cells, and 30% or less of the CD8+ T cells collected are effector memory T cells. In some embodiments, at least 20% of the CD8+ T cells collected are naive T cells, and 20% or less of the CD8+ T cells collected are effector memory T cells. In some embodiments, naive T cells are characterized as CCR7+ and CD45RA+. In some embodiments, effector memory T cells are characterized as CCR7-, CD45RO+, and CD95+. In some embodiments, at least 60% of the CD8+ T cells collected are CCR7+ cells. In some embodiments, at most 40% of the CD8+ T cells collected are a combination of effector memory T cells and effector T cells.

[0013] In some embodiments, the method further comprises obtaining lymphocytes from a donor subject. In some embodiments, the method further comprises enriching the lymphocytes. In some embodiments, the method further comprises contacting the sample with a lymphocyte stimulant to activate the lymphocytes.

[0014] In some embodiments, activating the sample is prior to incubating the sample with the polynucleotide vector. In some embodiments, the sample contains at least 1×10 9 lymphocytes. In some embodiments, activating the sample is after the sample has been incubated with the polynucleotide vector. In some embodiments, the lymphocyte stimulant comprises an anti-CD3 antibody and / or an anti-CD28 antibody.

[0015] In some embodiments, the method further comprises administering the collected lymphocytes to the subject after collection, or freezing the collected lymphocytes. In some embodiments, the subject is the same as the donor subject. In some embodiments, a total of 10,000 to 3,000,000 collected lymphocytes per kilogram of the subject are administered to the subject. In some embodiments, a total of 20,000 to 400,000 collected lymphocytes per kilogram of the subject are administered to the subject. In some embodiments, at least 15% of the collected lymphocytes are transduced with a vector.

[0016] In some embodiments, the polynucleotide vector is a viral vector. In some embodiments, the viral vector is a retroviral vector or a lentiviral vector. In some embodiments, the vector encodes a chimeric antigen receptor (CAR) or a T cell receptor (TCR). In some embodiments, the CAR comprises an intracellular co-stimulatory domain.In some embodiments, the intracellular co-stimulatory domain is the signaling domain of a protein selected from the group consisting of DAP-10, CD28, OX-40, 4-1BB (CD137), CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell co-stimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD11a / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), tumor necrosis factor superfamily member 14, TNFSF14, LIGHT, NKG2C, Ig alpha (CD79a), Fc gamma receptor, MHC class I molecule, TNF receptor protein, immunoglobulin-like protein, cytokine receptor, integrin, signaling lymphocyte activation molecule (SLAM protein), activated NK cell receptor, BTLA, Toll ligand receptor, CDS, GITR, BAFFR, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD (CD11d), ITGAE (CD103), ITGAL (CD11a), ITGAM (CD11b), ITGAX (CD11c), ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, TNFR2, TRANCE (RANKL), DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG (Cbp), CD19a, CD83, and ligands that specifically bind thereto, and combinations thereof.In some embodiments, the intracellular co-stimulatory domain is the signaling region of CD28.

[0017] In some embodiments, the CAR or TCR recognizes a tumor antigen. In some embodiments, the tumor antigen is CD19. In some embodiments, the lymphocytes comprising the CAR are axicabtagene ciloleucel or brexucabtagene autoleucel. In some embodiments, the tumor antigen is CD19 and / or CD20. In some embodiments, the tumor antigen is CLL-1.

[0018] In some embodiments, the CAR or TCR is 2B4 (CD244), 4-1BB, 5T4, A33 antigen, adenocarcinoma antigen, adrenoceptor beta 3 (ADRB3), A kinase anchor protein 4 (AKAP-4), alpha-fetoprotein (AFP), anaplastic lymphoma kinase (ALK), androgen receptor, B7H3 (CD276), β2-integrin, BAFF, B lymphoma cells, B cell maturation antigen (BCMA), bcr-abl (a cancer gene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia virus cancer gene homolog 1 (Abl)), BhCG, bone marrow stromal cell antigen 2 (BST2), CCCTC-binding factor (zinc finger protein)-like (brother of BORIS or imprint site regulator), BST2, C242 antigen, 9-O-acetyl-CA19-9 marker, CA-125, CAEX, calreticulin, carbonic anhydrase 9 (carbonic anhydrase9, CAIX, C-MET, CCR4, CCR5, CCR8, CD2, CD3, CD4, CD5, CD8, CD7, CD10, CD16, CD19, CD20, CD22, CD23 (IgE receptor), CD24, CD25, CD27, CD28, CD30 (TNFRSF8), CD33, CD34, CD38, CD40, CD40L, CD41, CD44, CD44V6, CD49f, CD51, CD52, CD56, CD63, CD70, CD72, CD74, CD79a, CD79b, CD80, CD84, CD96, CD97, CD100, CD123, CD125, CD133, CD137, CD138, CD150, CD152 (CTLA-4), CD160, CD171, CD179a, CD200, CD221, CD229, CD244, CD272 (BTLA), CD274 (PDL-1, B7H1), CD279 (PD-1), CD352, CD358, CD300 molecule-like family member f (CD300 molecule-like family member f, CD300LF), carcinoembryonic antigen (CEA), claudin 6 (CLDN6), C-type lectin-like molecule-1 (C-type lectin-like molecule-1, CLL-1 or CLECL1), C-type lectin domain family 12 member A (C-type lectin domain family 12 member A, CLEC12A), cytomegalovirus (CMV) infected cell antigen, CNT0888, CRTAM (CD355), CS-1 (also known as CD2 subset 1, CRACC, CD319, and 19A24), CTLA-4, cyclin B1, chromosome X open reading frame 61 (chromosome X open reading frame 61, CXORF61), Cytochrome P450 1B1 (Cytochrome P450 1B1, CYP1B1), DNAM-1 (CD226), desmoglein 4, DR3, DR5, E-cadherin neoepitope, epidermal growth factor receptor (epidermal growth factor receptor, EGFR), EGF1R, epidermal growth factor receptor variant III (epidermal growthfactor receptor variant III, EGFRvIII), epithelial glycoprotein-2 (epithelial glycoprotein-2, EGP-2), epithelial glycoprotein-40 (epithelial glycoprotein-40, EGP-40), EGF-like module-containing mucin-like hormone receptor-like 2 (EGF-like module-containing mucin-like hormone receptor-like 2, EMR2), elongation factor 2 mutated (elongation factor 2 mutated, ELF2M), endothelin, Epithelial cell adhesion molecule (Epithelial cell adhesion molecule, EPCAM), ephrin type-A receptor 2 (ephrin type-A receptor 2, EphA2), ephrin B2, receptor tyrosine-protein kinase erb-B2, 3, 4 (erb-B2, 3, 4), ERBB, ERBB2 (Her2 / neu), ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene), ETA, ETS translocation variant gene 6 located on chromosome 12p (ETV6-AML), Fc fragment of IgA receptor (Fc fragment of IgA receptor, FCAR or CD89), fibroblast activation protein alpha (fibroblast activation protein alpha, FAP), FBP, Fc receptor-like 5 (Fc receptor-like 5, FCRL5), fetal acetylcholine receptor (fetal acetylcholine receptor, AChR), fibronectin extra domain B, Fms-like tyrosine kinase 3 (Fms-Like Tyrosine Kinase 3, FLT3), folate-binding protein (folate-binding protein, FBP), folate receptor 1, folate receptor alpha, folate receptor beta, Fos-related antigen 1, fucosyl, fucosyl GM1; GM2, ganglioside G2 (GD2), ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer), o-acetyl-GD2 ganglioside (o-acetyl-GD2ganglioside, OAcGD2), GITR (TNFRSF18), GM1, ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer), GP100, hexasaccharide moiety of GloboH glycosphingolipid (GloboH), glycoprotein 75, Glypican-3 (GPC3), glycoprotein 100 (gp100), GPNMB, G protein-coupled receptor 20 (GPR20), G protein-coupled receptor class C group 5, member D (GPRC5D), Hepatitis A virus cellular receptor 1 (HAVCR1), human Epidermal Growth Factor Receptor 2 (HER-2), HER2 / neu, HER3, HER4, HGF, high molecular weight-melanoma-associated antigen (HMWMAA), human papilloma virus E6 (HPV E6), human papilloma virus E7 (HPV E7), heat shock protein 70-2 mutated (mut hsp70-2), human scatter factor receptor kinase, human Telomerase reverse transcriptase (hTERT), HVEM, ICOS, Insulin-like growth factor receptor 1 (IGF-1 receptor), IGF-I, IgGl, immunoglobulin lambda-like polypeptide 1 (IGLL1), IL-6, Interleukin 11 receptor alpha (IL-11Ra), IL-13, Interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2), insulin-like growth factor 1 receptor (IGF1-R), integrin α5β1, integrin ανβ3, intestinal carboxylesterase, κ light chain, KCS1, kinase insert domain receptor (KDR), KIR, KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL2, KIR-L, KG2D ligand, KIT (CD117), KLRGI, LAGE-la, LAG3, lymphocyte-specific protein tyrosine kinase (LCK), Leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), legumain, Leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), Lewis (Y) antigen, LeY, LG, L1 cell adhesionmolecule, L1-CAM), LIGHT, LMP2, lymphocyte antigen 6 complex, LTBR, locus K9 (LY6K), Ly-6, lymphocyte antigen 75 (LY75), melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2), MAGE, melanoma-associated antigen 1 (MAGE-A1), MAGE-A3 melanoma antigen 1 recognized by T cells (MelanA or MARTI), MelanA / MARTl, mesothelin, MAGE A3, melanoma inhibitor of apoptosis (ML-IAP), melanoma-specific chondroitin-sulfate proteoglycan (MCSCP), MORAb-009, MS4A1, Mucin1 (MUC1), MUC2, MUC3, MUC4, MUC5AC, MUC5b, MUC7, MUC16, mucin CanAg, Mullerian inhibitory substance (MIS) receptor type II, v-myc avian myelocytomatosis virus oncogene homolog derived from neuroblastoma (MYCN), N-glycolylneuraminic acid, N-acetylglucosaminyltransferase V (NA17), neural cell adhesion molecule (NCAM), NKG2A, NKG2C, NKG2D, NKG2E ligand, NKR-P1A, NPC-1C, NTB-A, breast differentiation antigen (NY-BR-1), NY-ESO-1, tumor fetal antigen (h5T4), olfactory receptor 51E2 (OR51E2), OX40, plasma cell antigen, poly SA, proacrosin-binding protein sp32 (OY-TES1), p53, p53 variant, pannexin 3 (PANX3), prostatic acid phosphatase (PAP), paired box protein Pax-3 (PAX3), paired box protein Pax-5 (PAX5), prostate carcinoma tumor antigen-1 (PCTA-1 or galectin 8), PD-1H, platelet-derived growth factor receptor alpha (Platelet-derived growth factorreceptor alpha, PDGFR-alpha, PDGFR-beta, PDL192, PEN-5, phosphatidylserine, placenta-specific 1 (PLAC1), polysialic acid, prostase, prostate cancer cells, prostain, protease serine 21 (testisin or PRSS21), proteinase 3 (PR1), prostate stem cell antigen, prostate-specific membrane antigen (PSMA), proteasome (Prosome, Macropain) subunit, beta type, Receptor for Advanced Glycation Endproducts (RAGE-1), RANKL, Ras variant, Ras homolog family member C (RhoC), RON, Receptor tyrosine kinase-like orphan receptor 1 (ROR1), renal ubiquitous 1 (RU1), renal ubiquitous 2 (RU2), sarcoma translocation breakpoint, Squamous Cell Carcinoma Antigen Recognized By T Cells 3 (SART3), SAS, SDC1, SLAMF7, sialyl Lewis adhesion molecule (sLe), Siglec-3, Siglec-7, Siglec-9, sonic hedgehog (SHH), sperm protein 17 (SPA17), Stage-specific embryonic antigen-4 (SSEA-4), STEAP, sTn antigen, synovial sarcoma, X breakpoint 2 (SSX2), survivin, Tumor-associated glycoprotein 72 (TAG72), TCR5y, TCRa, TCRB, TCR Gamma Alternate Reading Frame protein (TCR Gamma Alternate Reading FrameIt recognizes an antigen comprising Protein, TARP, telomerase, TIGIT, TNF-α precursor, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), tenascin C, TGF beta 2, TGF-β, transglutaminase 5 (TGS5), angiopoietin-binding cell surface receptor 2 (Tie2), TIM1, TIM2, TIM3, Tn Ag, TRAIL-R1, TRAIL-R2, tyrosinase-related protein 2 (TRP-2), thyroid stimulating hormone receptor (TSHR), tumor antigen CTAA16.88, tyrosinase, ROR1, TAG-72, uroplakin 2 (UPK2), VEGF-A, VEGFR-1, vascular endothelial growth factor receptor 2 (VEGFR2), and vimentin, Wilms tumor protein (WT1), or X antigen family, member 1A (XAGE1), or a combination thereof.

[0019] Also, in one embodiment, there is provided a population of cells prepared from a blood sample of a donor subject, comprising CD4+ T cells and CD8+ T cells, wherein at least 20% of the CD4+ T cells collected are naive T cells, no more than 12% of the CD4+ T cells collected are effector memory T cells, at least 10% of the CD8+ T cells collected are naive T cells, no more than 30% of the CD8+ T cells collected are effector memory T cells, at least 50% of the cells collected are CD3+ T cells, and at least 25% of all T cells are transduced with a polynucleotide vector encoding a CAR or TCR.

[0020] In one embodiment, a population of cells prepared from a blood sample of a donor subject is provided, which population contains CD4+ T cells and CD8+ T cells, at least 80% of the CD4+ T cells are CCR7+ cells, at least 60% of the CD8+ T cells are CCR7+ cells, at most 20% of the CD4+ T cells are a combination of effector memory T cells and effector T cells, and at most 40% of the CD8+ T cells are a combination of effector memory T cells and effector T cells that have been transduced with a polynucleotide vector encoding a CAR or TCR.

[0021] In some embodiments, at least 25% of the CD4+ T cells collected are naive T cells, and 9% or less of the CD4+ T cells collected are effector memory T cells. In some embodiments, at least 20% of the CD8+ T cells collected are naive T cells, and 20% or less of the CD8+ T cells collected are effector memory T cells. In some embodiments, naive T cells are characterized as CCR7+ and CD45RA+. In some embodiments, effector memory T cells are characterized as CCR7-, CD45RO+, and CD95+. In some embodiments, at least 65% of the cells collected are CD3+ T cells. In some embodiments, at least 15% of all T cells are transduced with a polynucleotide vector.

[0022] In some embodiments, at least 80% of the CD4+ T cells collected are CCR7+ cells. In some embodiments, at most 20% of the CD4+ T cells collected are a combination of effector memory T cells and effector T cells. In some embodiments, at least 60% of the CD8+ T cells collected are CCR7+ cells. In some embodiments, at most 40% of the CD8+ T cells collected are a combination of effector memory T cells and effector T cells.

[0023] In some embodiments, the polynucleotide vector is a viral vector. In some embodiments, the viral vector is a retroviral vector or a lentiviral vector. In some embodiments, the CAR comprises an intracellular co-stimulatory domain. In some embodiments, the intracellular co-stimulatory domain is the signaling region of CD28. In some embodiments, the CAR recognizes a tumor antigen. In some embodiments, the tumor antigen is CD19. In some embodiments, the population of cells comprising the CAR is axicabtagene ciloleucel or brexucabtagene autoleucel.

[0024] Also provided is a pharmaceutical composition comprising the population of cells described herein.

[0025] Yet another embodiment provides a method for administering T cells to a subject, the method comprising injecting into the subject a harvested sample prepared by the methods of the present disclosure, or a pharmaceutical composition. In some embodiments, the subject has cancer. In some embodiments, the cancer is a B cell malignancy. In some embodiments, the cancer is non-Hodgkin's lymphoma (NHL), diffuse large B cell lymphoma (DLBCL), small lymphocytic lymphoma (SLL / CLL), mantle cell lymphoma (MCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), extranodal (MALT lymphoma), nodal (monocytoid B cell lymphoma), splenic, diffuse large cell lymphoma, B cell chronic lymphocytic leukemia / lymphoma, Burkitt lymphoma, lymphoblastic lymphoma, acute myeloid leukemia, or multiple myeloma.

DETAILED DESCRIPTION OF THE INVENTION

[0026] Definitions

[0027] To facilitate a better understanding of the present disclosure, certain terms are first defined below. Additional definitions of the following terms and other terms are set forth throughout this specification.

[0028] As used herein, unless specifically stated or otherwise apparent from the context, the term "or" is understood to be inclusive and encompasses both "or" and "and".

[0029] As used herein, the term "and / or" should be construed as a specific disclosure of each of the two specified features or components, regardless of the presence or absence of others. Thus, as used herein, the term "and / or" in phrases such as "A and / or B" is intended to include A and B, A or B, A alone, and B alone. Similarly, the term "and / or" in phrases such as "A, B, and / or C" is intended to include each of the following aspects: A, B, and C, A, B, or C, A or C, A or B, B or C, A and C, A and B, B and C, A alone, B alone, and C alone.

[0030] Unless specifically stated or apparent from the context, the term "about" refers to a value or composition within an acceptable error range for a particular value or composition as determined by one of ordinary skill in the art, which depends to some extent on how the value or composition is measured or determined, i.e., the limitations of the measuring system. For example, "about" or "consisting essentially of" can mean within the range of 1 or more standard deviations in accordance with the practice in the art. "About" or "consisting essentially of" can mean a range of up to 10% (i.e., ±10%). Thus, "about" can be understood to be within a range of 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001% greater or less than the stated value. For example, about 5 mg can include any amount from 4.5 mg to 5.5 mg. Further, especially with respect to biological systems or processes, the term can mean up to one order of magnitude or up to five-fold the value. When a particular value or composition is presented in the present disclosure, unless otherwise specified, it should be assumed that the meaning of "about" or "consisting essentially of" is within the acceptable error range for that particular value or composition.

[0031] "Administering" refers to the physical introduction of an agent, such as a modified T cell disclosed herein, to a subject using any of a variety of methods and delivery systems known to those of skill in the art. Exemplary routes of administration for the formulations disclosed herein include, for example, intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration by injection or infusion. The phrase "parenteral administration" means a mode of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, arterial, intrathecal, lymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subdural, intraspinal, epidural, and intrasternal injections and infusions, as well as in vivo electroporation. In some embodiments, the formulation is administered orally, for example, via a non-parenteral route. Other non-parenteral routes include topical, epidermal or mucosal routes of administration, for example, intranasal, intravaginal, rectal, sublingual, or topical. Administration can also be carried out, for example, once, multiple times, and / or over one or more extended periods of time.

[0032] The term "allogeneic" refers to any material that is derived from one individual and then introduced into another individual of the same species, for example, an allogeneic T cell transplant.

[0033] The term "antibody" (Ab) includes, but is not limited to, glycoprotein immunoglobulins that specifically bind to an antigen. Generally, an antibody can include at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or its antigen-binding molecule. Each H chain includes a heavy chain variable region (abbreviated as VH herein) and a heavy chain constant region. The heavy chain constant region includes three constant domains, CH1, CH2, and CH3. Each light chain includes a light chain variable region (abbreviated as VL herein) and a light chain constant region. The light chain constant region includes one constant domain CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) in which more conserved regions called framework regions (FRs) are interspersed. Each VH and VL includes three CDRs and four FRs arranged in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from the amino terminus toward the carboxy terminus. The variable regions of the heavy and light chains include a binding domain that interacts with the antigen. The constant region of the Ab can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. Generally, a human antibody is a ~150 kD tetrameric agent composed of two identical heavy (H) chain polypeptides (each ~50 kD) and two identical light (L) chain polypeptides (each ~25 kD) that associate with each other in what is commonly referred to as a "Y-shaped" structure. The heavy and light chains are linked or connected to each other by a single disulfide bond, and two other disulfide bonds connect the heavy chain hinge regions to each other, such that the dimers are connected to each other to form a tetramer. Naturally produced antibodies are also glycosylated, for example, on this CH2 domain.

[0034] "Antigen-binding molecule", "antigen-binding portion", "antigen-binding fragment", or "antibody fragment" refers to any molecule that includes the antigen-binding portion (e.g., CDR) of the antibody from which the molecule is derived. The antigen-binding molecule may include antigen complementarity-determining regions (CDRs). Examples of antibody fragments include, but are not limited to, Fab, Fab’, F(ab’)2, and Fv fragments, dAb, linear antibodies, scFv antibodies, and multispecific antibodies formed from antigen-binding molecules. Peptibodies (i.e., Fc fusion molecules containing a peptide-binding domain) are another example of suitable antigen-binding molecules. In some embodiments, the antigen-binding molecule binds to an antigen on a tumor cell. In some embodiments, the antigen-binding molecule binds to an antigen on a cell involved in a proliferative disorder or a viral antigen or a bacterial antigen. In certain embodiments, the antigen-binding molecule is a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR).

[0035] The terms "variable region" or "variable domain" are used interchangeably. The variable region typically refers to a part of an antibody, generally part of the light chain or the heavy chain, typically the approximately 110-120 amino acids at the amino terminus of the mature heavy chain and the approximately 90-115 amino acids of the mature light chain, which vary greatly in sequence between antibodies and are used for the binding and specificity of a particular antibody to its particular antigen. The variability of the sequence is concentrated in regions called complementarity-determining regions (CDRs), and the more highly conserved regions within the variable domain are called framework regions (FRs). Without wishing to be bound by any particular mechanism or theory, the CDRs of the light and heavy chains are thought to be mainly responsible for the antibody's interaction and specificity with the antigen. In certain embodiments, the variable region is a human variable region. In certain embodiments, the variable region includes rodent or mouse CDRs and human framework regions (FRs). In certain embodiments, the variable region is a primate (e.g., non-human primate) variable region. In certain embodiments, the variable region includes rodent or mouse CDRs and primate (e.g., non-human primate) framework regions (FRs).

[0036] The terms "VL" and "VL domain" are used interchangeably to refer to the variable light chain region of an antibody or its antigen-binding molecule.

[0037] The terms "VH" and "VH domain" are used interchangeably to refer to the variable heavy chain region of an antibody or its antigen-binding molecule.

[0038] Many definitions of CDRs are commonly used: Kabat numbering, Chothia numbering, AbM numbering, or contact numbering. The AbM definition is a compromise between two used by Oxford Molecular's AbM antibody modeling software. The contact definition is based on the analysis of available complex crystal structures.

[0039] The term "autologous" refers to any material derived from the same individual that is reintroduced later. For example, the methods of engineered autologous cell therapy (eACT™) described herein include the collection of lymphocytes from a patient, which are then engineered, for example, to express a CAR construct and then administered to the same patient.

[0040] "Chimeric antigen receptor" or "CAR" refers to a molecule engineered to include a binding motif and means of activating an immune cell (e.g., a T cell such as a naive T cell, central memory T cell, effector memory T cell, or a combination thereof) upon antigen binding. CARs are also known as artificial T cell receptors, chimeric T cell receptors, or chimeric immune receptors. In some embodiments, a CAR includes a binding motif, an extracellular domain, a transmembrane domain, one or more co-stimulatory domains, and an intracellular signaling domain. T cells genetically engineered to express a chimeric antigen receptor may be referred to as CAR T cells. The "extracellular domain" (or "ECD") refers to the portion of a polypeptide that is understood to be present outside the cell membrane, in the extracellular space, when the polypeptide is present in the cell membrane.

[0041] "T cell receptor" or "TCR" refers to the antigen recognition molecule present on the surface of T cells. During the development of normal T cells, each of the four TCR genes, α, β, γ, and δ, can rearrange to yield a very diverse TCR protein.

[0042] The term "heterologous" means derived from any source other than a naturally occurring sequence. For example, a heterologous sequence included as part of a costimulatory protein is an amino acid that does not occur naturally as a wild-type human costimulatory protein, i.e., does not align with the wild-type human costimulatory protein. For example, a heterologous nucleotide sequence refers to a nucleotide sequence other than the nucleotide sequence of the wild-type human costimulatory protein coding sequence.

[0043] The term "identity" refers to the overall relatedness between polymer molecules, such as between nucleic acid molecules (e.g., DNA molecules and / or RNA molecules), and / or between polypeptide molecules. Methods for calculating the percent identity between two provided polypeptide sequences are known. For example, the calculation of the percent identity of two nucleic acid or polypeptide sequences can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second sequences for optimal alignment, and non-identical sequences can be ignored for comparison purposes). Next, the nucleotides or amino acids at the corresponding positions are compared. If the position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is optionally a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap, which may need to be introduced for the optimal alignment of the two sequences. The comparison or alignment of sequences and the determination of the percent identity between two sequences can be achieved using a mathematical algorithm such as BLAST (Basic Local Alignment Search Tool). In some embodiments, polymer molecules are considered to be "homologous" to each other if their sequences are at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identical (e.g., 85-90%, 85-95%, 85-100%, 90-95%, 90-100%, or 95-100%).

[0044] The immune cells for immunotherapy can be derived from any source known in the art. For example, immune cells can be differentiated in vitro from a population of hematopoietic stem cells, or immune cells can be obtained from a subject. Immune cells can be obtained, for example, from peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infected site, ascites, pleural effusion, spleen tissue, and tumors. Furthermore, immune cells can be derived from one or more immune cell lines available in the art. Immune cells can also be obtained from a blood unit collected from a subject using various techniques known to those of skill in the art, such as FICOLL™ separation, OPTIPREP™ separation, and / or apheresis. Additional methods for isolating immune cells for immunotherapy are disclosed in U.S. Patent Application Publication No. 2013 / 0287748, which is hereby incorporated by reference in its entirety.

[0045] "Patient" includes any human suffering from cancer (e.g., lymphoma or leukemia). The terms "subject" and "patient" are used interchangeably herein.

[0046] The term "pharmaceutically acceptable" refers to a molecule or composition that, when administered to a recipient, is not harmful to that recipient or has a benefit to that recipient that outweighs any harmful effects. With respect to carriers, diluents, or excipients used to formulate the compositions disclosed herein, a pharmaceutically acceptable carrier, diluent, or excipient must be compatible with the other components of the composition and not be harmful to the recipient or have a benefit to the recipient that outweighs any harmful effects. The term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material involved in the transport or delivery of a drug from one part of the body to another (e.g., from one organ to another). Each carrier present in a pharmaceutical composition must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient, or having a benefit to the recipient that outweighs any harmful effects. Some examples of materials that can function as pharmaceutically acceptable carriers include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; tragacanth powder; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, soybean oil; glycols such as propylene glycol; polyols such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; pH buffering solutions; polyesters, polycarbonates, and / or polyanhydrides; and other non-toxic compatible substances used in pharmaceutical formulations.

[0047] The term "pharmaceutical composition" refers to a composition in which an active agent is formulated together with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in an amount of a unit dose suitable for administration in a treatment regimen that exhibits a statistically significant probability of achieving a predetermined therapeutic effect when administered to a relevant subject or population. In some embodiments, the pharmaceutical composition may be formulated for administration in solid or liquid form, including, but not limited to, forms adapted for the following: oral administration, such as, for example, a medicated drink (aqueous or non-aqueous solution or suspension), tablets, such as, for example, buccal, sublingual, and those targeted for systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, such as, for example, by subcutaneous, intramuscular, intravenous, or epidural injection as a sterile solution or suspension, or as a sustained release formulation; topical administration, such as, for example, as a cream, ointment, or sustained release patch or spray applied to the skin, lung, or oral cavity; intravaginal or rectal, such as, for example, as a pessary, cream, or foam; sublingual; for the eyes; transdermal; or for the nasal, lung, and other mucosal surfaces.

[0048] The terms "reduce" and "decrease" are used interchangeably herein and indicate any change to less than the original. "Reduce" and "decrease" are relative terms and require a comparison between before and after measurement. "Reduce" and "decrease" include complete depletion.

[0049] The term "reference" describes the standard or control against which a comparison is made. For example, in some embodiments, the agent, animal, individual, population, sample, sequence, or value of interest is compared to a reference or control that is an agent, animal, individual, population, sample, sequence, or value. In some embodiments, the reference or control is tested, measured, and / or determined substantially simultaneously with the test, measurement, or determination of interest. In some embodiments, the reference or control is optionally a reference or control from the past embodied in a tangible medium. Generally, the reference or control is determined or characterized under conditions or circumstances equivalent to those under evaluation. If the similarity is sufficient to justify the dependence on and / or comparison to the selected reference or control.

[0050] A "therapeutically effective amount", "effective dose", "effective amount", or "therapeutically effective dosage" of a therapeutic agent, e.g., engineered CAR T cells, is any amount that, when used alone or in combination with another therapeutic agent, protects a subject from the onset of a disease or promotes regression of the disease as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of periods without disease symptoms, or a prevention of impairment or disability resulting from the disease. The ability of a therapeutic agent to promote regression of a disease can be evaluated using various methods known to those of skill in the art, such as in human subjects during clinical trials, in animal model systems that predict efficacy in humans, or by assaying the activity of the agent in in vitro assays.

[0051] The terms "transfection" and "transfected" refer to the process by which foreign nucleic acid is introduced into a cell via a viral vector (see Jones et al., "Genetics: principles and analysis," Boston: Jones & Bartlett Publ. (1998)). In some embodiments, the vector is a retroviral vector, a DNA vector, an RNA vector, an adenoviral vector, a baculoviral vector, an Epstein-Barr viral vector, a papovaviral vector, a vaccinia viral vector, a herpes simplex viral vector, an adeno-associated vector, a lentiviral vector, or any combination thereof.

[0052] "Treatment" or "treating" of a subject refers to any type of intervention or process performed on, or administration of an active agent to, a subject for the purpose of reversing, alleviating, ameliorating, inhibiting, slowing, or preventing the onset, progression, development, severity, or recurrence of a symptom, complication, or condition, or a biochemical marker associated with a disease. In one embodiment, "treatment" or "treating" includes partial remission. In another embodiment, "treatment" or "treating" includes complete remission. In some embodiments, treatment can be of a subject who does not exhibit symptoms of a related disease, disorder, and / or condition, and / or of a subject who exhibits only early signs of a disease, disorder, and / or condition. In some embodiments, such treatment can be of a subject who exhibits one or more definitive signs of a related disease, disorder, and / or condition. In some embodiments, treatment can be of a subject diagnosed with having a related disease, disorder, and / or condition. In some embodiments, treatment can be of a subject known to have one or more susceptibility factors that are statistically correlated with an increased risk of developing a related disease, disorder, and / or condition.

[0053] The term "vector" refers to a recipient nucleic acid molecule that contains or has been modified to incorporate the provided nucleic acid sequence. One type of vector is a "plasmid", which refers to a circular double-stranded DNA molecule into which additional DNA can be ligated. Another type of vector is a viral vector, into which additional DNA segments can be ligated to the viral genome. Certain vectors are capable of autonomous replication in the host cells into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) may integrate into the genome of the host cell upon introduction into the host cell and thereby be replicated along with the host genome. Further, certain vectors contain sequences that direct the expression of an inserted gene to which they are operably linked. Such vectors may be referred to herein as "expression vectors". Standard techniques can be used to manipulate vectors, e.g., as found in Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference.

[0054] The term "7-day process" refers to a CAR cell manufacturing process that takes approximately 7 days after the initial enrichment and / or activation step. The 7-day process is at least 8 days in length from the initial enrichment and / or activation step to the harvesting step and can be a total of 8 to 11 days if it includes a concentration and / or activation step. In some embodiments, there is no initial activation step or no activation prior to transduction.

[0055] The term "5-day process" refers to a CAR cell manufacturing process that takes approximately 5 days after the first enrichment and / or activation step. The 5-day process is 6 days in length from the first enrichment and / or activation step to the harvesting step, and can be a total of 6 - 9 days if a concentration and / or activation step is included. In some embodiments, there is no first activation step, or there is no activation prior to transduction.

[0056] The term "3-day process" refers to a CAR cell manufacturing process that takes approximately 3 days from the first enrichment and / or activation step. The 3-day process is approximately 4 days in length from the first enrichment and / or activation step to the harvesting step. The 3-day process does not include a cell expansion step that includes 1 or more days after the transduction step and before the harvesting step. In some embodiments, there is no first activation step, or there is no activation prior to transduction.

[0057] In some embodiments, the 3-day process described herein is approximately 5 days in length from the first enrichment and / or activation step to the harvesting step. In some embodiments, the 3-day process is approximately 3 - 4 days in length or approximately 72 - 96 hours in length (e.g., lengths of about 72 hours, 74 hours, 76 hours, 78 hours, 80 hours, 82 hours, 84 hours, 86 hours, 88 hours, 90 hours, 92 hours, 94 hours, 96 hours) from the first enrichment and / or activation step to the harvesting step. In some embodiments, the 3-day process is approximately 4 - 5 days in length or approximately 96 - about 120 hours in length (e.g., lengths of about 96 hours, 98 hours, 100 hours, 102 hours, 104 hours, 106 hours, 108 hours, 110 hours, 112 hours, 114 hours, 116 hours, 118 hours, 120 hours) from the first enrichment and / or activation step to the harvesting step. Some embodiments are that the 3-day process is less than 5 days or 120 hours in length from the first enrichment and / or activation step to the harvesting step.

[0058] Preparation of Engineered Lymphocytes

[0059] Conventional autologous CAR cell manufacturing processes take about 7 days and can be much longer. At least, due to the limited supply of lymphocytes obtained from apheresis collections from donor subjects as the starting material, the relatively low efficiency of transduction, and the need to expand the transduced cells, a long process has been considered necessary. Non-limiting examples of CAR cell manufacturing processes are described in WO 2015 / 120096 and WO 2016 / 191755, each of which is incorporated herein by reference in its entirety.

[0060] However, as demonstrated in the attached examples, such a long process is neither necessary nor beneficial to the patient. As shown in Table 7, for example, each washing step during the expansion process significantly reduced the viability of the transduced cells. Also, while the multi-day expansion process increased the total number of transduced cells, it decreased the percentage of younger cells (e.g., naive T cells, stem memory T cells, and central memory T cells) and increased the percentage of more mature cells (e.g., effector memory T cells) (Tables 2 and 6). However, it has been shown that younger lymphocytes are more effective than more mature lymphocytes (Tables 5, 10, and 11).

[0061] The present disclosure describes an improved process that can be completed within 5 days or even 3 days after the enrichment step. In various examples, the 5-day process includes a transduction preparation and execution step in which a greater number of lymphocytes contact a vector immobilized on recombinant fibronectin coated on the inner surface of a closed system. Such improved transduction procedures enable a highly shortened post-transduction cell expansion step. As shown in Table 2, the transduced cells prepared from the 5-day process were biased towards younger cells. Results of in vivo anti-tumor efficacy are shown in (Table 5).

[0062] Furthermore, an even more improved process that requires no expansion after transduction can be completed within just 3 days after the enrichment step. Surprisingly, compared to the 5-day process, this 3-day process generates a cell population with a higher percentage of young cells (comparing Table 6 to Table 2) and a decreased percentage of more mature, differentiated, and activated cells. Thus, not only did the cell product from the 3-day process exhibit the best in vivo antitumor efficacy, but such greatly improved efficacy could be achieved even at much lower doses (Tables 10 - 11).

[0063] Yet another surprising discovery is that the accelerated lymphocyte preparation process improved the efficacy of all CAR types tested, but it was particularly beneficial for CAR molecules with a CD28 co-stimulatory domain (compared to those with a 4-1BB co-stimulatory domain) (Table 13).

[0064] Thus, according to one embodiment of the present disclosure, a method for preparing transduced (or similarly transfected) lymphocytes is provided. In some embodiments, the method involves incubating a sample of lymphocytes with a polynucleotide vector to transduce the lymphocytes and produce transduced lymphocytes, and culturing a sample containing the transduced lymphocytes to produce a sample taken before the lymphocytes were collected.

[0065] In some embodiments, the culturing step is shortened compared to a conventional process that takes about 4 days. In some embodiments, the culturing step is completed within 96 hours or within 72 hours, 60 hours, 50 hours, 48 hours, 42 hours, 36 hours, 30 hours, 29 hours, 28 hours, 27 hours, 26 hours, 25 hours, 24 hours, 23 hours, 22 hours, 21 hours, 20 hours, 19 hours, 18 hours, 17 hours, 16 hours, 15 hours, 14 hours, 13 hours, 12 hours, 11 hours, 10 hours, 9 hours, 8 hours, 7 hours, 6 hours, 5 hours, or 4 hours.

[0066] In some embodiments, the time of the culturing step is counted from the completion of the transduction step (e.g., removal of cells from a system having an immobilized vector) to the harvesting of cells for storage, transportation, or clinical use.

[0067] The culturing of the transduced lymphocytes can be performed in media and conditions known in the art. In some embodiments, the culturing of the transduced lymphocytes can be performed at a temperature and / or in the presence of CO 2 2. In certain embodiments, the temperature can be about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, or about 39°C. In certain embodiments, the temperature can be about 34 - 39°C. In certain embodiments, the predetermined temperature can be from about 35 - 37°C. In certain embodiments, the preferred predetermined temperature can be from about 36 - 38°C. In certain embodiments, the predetermined temperature can be about 36 - 37°C or more preferably about 37°C.

[0068] In some embodiments, the culturing of the transduced lymphocytes can be performed in the presence of a predetermined level of CO 2 2. In certain embodiments, the predetermined level of CO 2 2 can be 1.0 - 10% CO 2 2. In certain embodiments, the predetermined level of CO 2 2 can be about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, or about 10.0% CO 2 2. In certain embodiments, the predetermined level of CO 2 2 can be about 4.5 - 5.5% CO 2 2. In certain embodiments, the predetermined level of CO 2 2 can be about 5% CO 2 2. In certain embodiments, the predetermined level of CO 2 2 can be about 3.5%, about 4.0%, about 4.5%, about 5.0%, about 5.5%, or about 6.5% CO 2 2. In some embodiments, the step of expanding a population of transduced T cells is at a predetermined temperature and / or a predetermined level of CO2 It can be carried out in any combination in the presence of. For example, in one embodiment, the step of expanding the population of transduced T cells is carried out at a predetermined temperature of about 36-38 °C and a CO 2 at a predetermined level of CO 2 in the presence of.

[0069] Any suitable culture medium A T cell growth medium can be used to culture the cells in suspension. For example, the T cell growth medium can include, but is not limited to, a sterile low-glucose solution containing a suitable amount of buffer, magnesium, calcium, sodium pyruvate, and sodium bicarbonate. In one embodiment, the culture medium is OpTmizer™ (Life Technologies), although those skilled in the art will understand how to produce a similar medium. In one embodiment, the culture medium is EX-VIVO™ serum-free medium (Lonza Bioscience).

[0070] The incubation (and / or transduction) step can be carried out in a closed system, although not limited thereto. In certain embodiments, the closed system is a closed bag culture system using any suitable cell culture bag (e.g., Mitenyi Biotec MACS® GMP Cell Differentiation Bags, Origen Biomedical PermaLife™ Cell Culture bags). In some embodiments, the closed system has an inner surface area of at least 500 cm 2 In some embodiments, the closed system has an inner surface area of at least 1000 cm 2 , 1200 cm 2 , 1400 cm 2 , 1500 cm 2 , 1600 cm 2 , 1800 cm 2 , 2000 cm 2 , 2200 cm 2 , 2500 cm 2 , or 3000 cm 2 of inner surface area. In some embodiments, the closed system is 1500 cm2 、 1600 cm 2 、 1800 cm 2 、 2000 cm 2 、 2200 cm 2 、 2500 cm 2 、 or 3000 cm 2 and has the following inner surface area.

[0071] In some embodiments, the cell culture bag used in a closed system is coated with a recombinant human fibronectin protein. The recombinant human fibronectin fragment can include three functional domains, namely, a central cell-binding domain, a heparin-binding domain II, and a CS1 sequence. The recombinant human fibronectin protein or a fragment thereof can be used to increase the genetic efficiency of viral transduction of immune cells by assisting the co-localization of target cells or vectors. In certain embodiments, the recombinant human fibronectin fragment is RetroNectin® (Takara Bio, Japan). In certain embodiments, the cell culture bag can be coated with a recombinant human fibronectin fragment at a concentration of about 0.1 - 60 μg / mL, preferably 0.5 - 40 μg / mL. In certain embodiments, the cell culture bag can be coated with a recombinant human fibronectin fragment at a concentration of about 0.5 - 20 μg / mL, 20 - 40 μg / mL, or 40 - 60 μg / mL. In certain embodiments, the cell culture bag can be coated with a recombinant human fibronectin fragment at about 0.5 μg / mL, 1 μg / mL, about 2 μg / mL, about 3 μg / mL, about 4 μg / mL, about 5 μg / mL, about 6 μg / mL, about 7 μg / mL, about 8 μg / mL, about 9 μg / mL, about 10 μg / mL, about 11 μg / mL, about 12 μg / mL, about 13 μg / mL, about 14 μg / mL, about 15 μg / mL, about 16 μg / mL, about 17 μg / mL, about 18 μg / mL, about 19 μg / mL, or about 20 μg / mL. In certain embodiments, the cell culture bag can be coated with a recombinant human fibronectin fragment at about 2 - 5 μg / mL, about 2 - 10 μg / mL, about 2 - 20 μg / mL, about 2 - 25 μg / mL, about 2 - 30 μg / mL, about 2 - 35 μg / mL, about 2 - 40 μg / mL, about 2 - 50 μg / mL, or about 2 - 60 μg / mL.In certain embodiments, the cell culture bag can be coated with recombinant human fibronectin fragments at least about 2 μg / mL, at least about 5 μg / mL, at least about 10 μg / mL, at least about 15 μg / mL, at least about 20 μg / mL, at least about 25 μg / mL, at least about 30 μg / mL, at least about 40 μg / mL, at least about 50 μg / mL, or at least about 60 μg / mL. In certain embodiments, the cell culture bag can be coated with recombinant human fibronectin fragments at least about 10 μg / mL. In certain embodiments, the cell culture bag may not be coated with recombinant human fibronectin fragments.

[0072] In some embodiments, the transduction enhancer is introduced into the closed system. Non-limiting examples of such transduction enhancers include Vectofusin™ transduction mixtures.

[0073] In certain embodiments, the cell culture bag used in the closed bag culture system can be blocked with human albumin serum (HSA). In alternative embodiments, the cell culture bag is not blocked with HSA.

[0074] When the closed system is coated with recombinant fibronectin, a solution containing the vector is added to the closed system such that the vector can be immobilized on the inner surface of the closed system by the recombinant fibronectin. Such immobilization can improve the transduction efficiency when cells are added.

[0075] In some embodiments, the vector can be a viral vector such as a lentiviral vector and a retroviral vector. Several recombinant viruses have been used as viral vectors to deliver genetic materials into cells. Viral vectors that can be used according to the transduction process include, but are not limited to, recombinant retroviral vectors, recombinant lentiviral vectors, recombinant adenoviral vectors, and recombinant adeno-associated viral (AAV) vectors, and can be any ecotropic or amphotropic viral vector. In one embodiment, the viral vector is an MSGV1 gamma retroviral vector. In some embodiments, the vector is a non-viral vector.

[0076] In some embodiments, a solution containing a vector with a total volume of at least 100 mL is used. In some embodiments, a solution containing a vector with a total volume of at least 110 mL, 120 mL, 130 mL, 140 mL, 150 mL, 160 mL, 170 mL, 180 mL, 190 mL, 200 mL, 210 mL, 220 mL, 230 mL, 240 mL, 250 mL, 260 mL, 270 mL, 280 mL, 290 mL, 300 mL, 350 mL, or 400 mL is used. In some embodiments, a solution containing a vector with a total volume of 150 mL, 160 mL, 170 mL, 180 mL, 190 mL, 200 mL, 210 mL, 220 mL, 230 mL, 240 mL, 250 mL, 260 mL, 270 mL, 280 mL, 290 mL, 300 mL, 350 mL, 400 mL, or 500 mL or less is used.

[0077] In some embodiments, the vector solution contains 3 ~ 12 1×10 transduction units (TU / ml) of the viral vector.

[0078] When the closed system is coated with recombinant fibronectin and the vector is immobilized, the vector solution can be removed. In some embodiments, the closed system does not contain recombinant fibronectin. In some embodiments, the removal of the vector solution is performed by gravity or syringe drainage, which helps to retain the immobilized vector on the inner surface while removing impurities. Lymphocyte transfection can be performed in a closed system coated with the immobilized vector. In some embodiments, the transfection is performed using a sample containing lymphocytes. In some embodiments, the sample contains at least 2.5×10 7 lymphocytes (e.g., T cells). In some embodiments, the sample contains at least 3×10 7 , 4×10 7 , 5×10 7 , 6×10 7 , 7×10 7 , 8×10 7 , 9×10 7 , 1×10 8 , 1.2×10 8 , 1.5×10 8 , 1.8×10 8 , 2×10 8 , 2.2×10 8 , 2.5×10 8 , 2.6×10 8 , 2.7×10 8 , 2.8×10 8 , 2.9×10 8 , 3×10 8 , 3.1×10 8 , 3.2×10 8 , 3.3×10 8 , 3.4×10 8 , 3.5×10 8 , 3.6×10 8 , 3.7×10 8 , 3.8×10 8 , 3.9×10 8 , 4×10 8 , 4.1×10 8 , 4.2×10 8 , 4.3×10 8 , 4.4×10 8 , 4.5×10 8, 4.6×10 8 , 4.7×10 8 , 4.8×10 8 , 4.9×10 8 , 5×10 8 , 5.1×10 8 , 5.2×10 8 , 5.3×10 8 , 5.4×10 8 , 5.5×10 8 , 5.6×10 8 , 5.7×10 8 , 5.8×10 8 , 5.9×10 8 , 6×10 8 , 6.1×10 8 , 6.2×10 8 , 6.3×10 8 , 6.4×10 8 , 6.5×10 8 , 6.6×10 8 , 6.7×10 8 , 6.8×10 8 , 6.9×10 8 , 7×10 8 , 7.5×10 8 , 8×10 8 , 9×10 8 , or 10×10 8 contains lymphocytes (e.g., T cells). In some embodiments, the sample is 3×10 8 , 3.1×10 8 , 3.2×10 8 , 3.3×10 8 , 3.4×10 8 , 3.5×10 8 , 3.6×10 8 , 3.7×10 8 , 3.8×10 8 , 3.9×10 8 , 4×10 8 , 4.1×10 8 , 4.2×10 8 , 4.3×10 8 , 4.4×10 8 , 4.5×10 8 , 4.6×10 8 , 4.7×10 8 , 4.8×10 8 , 4.9×10 8, 5×10 8 , 5.1×10 8 , 5.2×10 8 , 5.3×10 8 , 5.4×10 8 , 5.5×10 8 , 5.6×10 8 , 5.7×10 8 , 5.8×10 8 , 5.9×10 8 , 6×10 8 , 6.1×10 8 , 6.2×10 8 , 6.3×10 8 , 6.4×10 8 , 6.5×10 8 , 6.6×10 8 , 6.7×10 8 , 6.8×10 8 , 6.9×10 8 , 7×10 8 , 7.5×10 8 , 8×10 8 , 9×10 8 , or 10×10 8 and contain 10 or fewer lymphocytes (e.g., T cells).

[0079] In some embodiments, lymphocyte transduction is performed in a closed system that does not contain an immobilizing agent (e.g., recombinant fibronectin).

[0080] The lymphocytes used in the methods disclosed in the present invention are typically obtained from a donor subject, which can be a cancer patient (i.e., an autologous donor) to be treated with a population of cells generated by the methods described herein, or an individual (i.e., an allogeneic donor) who provides a lymphocyte sample used to treat a different individual or cancer patient at the time of generation of the cell population generated by the methods described herein. Lymphocytes can be obtained from a donor subject by any suitable method used in the art. For example, lymphocytes can be obtained by any suitable ex vivo method, venipuncture, or other blood collection method from which a sample of blood and / or lymphocytes is obtained. In one embodiment, lymphocytes are obtained by apheresis.

[0081] Optionally, in some embodiments, the methods described herein further include enriching a population of lymphocytes obtained from a donor subject prior to transduction. Lymphocyte enrichment can be achieved by any suitable separation method including, but not limited to, use of a separation medium (e.g., Ficoll-Paque™, RosetteSep™ HLA Pan Lymphocyte Enrichment Cocktail, Lymphocyte Separation Medium (LSA) (MP Biomedical catalog number 0850494X), a nonionic iodixanol-based medium such as OptiPrep™, etc.), cell size, shape, or density separation by filtration or elution, immunomagnetic separation (e.g., magnetic-activated cell sorting system, MACS), fluorescence separation (e.g., fluorescence activated cell sorting system, FACS), or bead-based column separation.

[0082] Optionally, in some embodiments, circulating lymphoma cells are removed from the sample via positive enrichment of CD4 + / CD8 + cells. In some such embodiments, after incubation with the selection reagent, the incubated cells containing the cells to which the selection reagent has bound are transferred to a system for separation based on the immunophilicity of the cells. In some embodiments, the system for separation based on immunophilicity is or includes a magnetic separation column.

[0083] In some such embodiments, the isolation method involves the separation of different cell types based on the expression or presence of one or more specific molecules, such as surface markers, e.g., surface proteins, intracellular markers, or nucleic acids, in the cells. In some embodiments, any known method for separation based on such markers can be used. In some embodiments, the separation is separation based on affinity or immunoaffinity. For example, in some embodiments, the isolation typically involves the separation of cells and cell populations based on the expression or expression level of one or more markers, which are typically cell surface markers, e.g., incubation with an antibody or binding partner that specifically binds to such markers, usually followed by a washing step and separation of the cells bound to the antibody or binding partner from the cells not bound to the antibody or binding partner. Such separation steps may be based on positive selection where the cells bound to the reagent are secured for further use, and / or negative selection where the cells not bound to the antibody or binding partner are secured. In some examples, both fractions are secured for further use.

[0084] In some such embodiments, negative selection, where an antibody that specifically identifies cell types within a heterogeneous population is not available, may be particularly useful such that the separation is performed best based on markers expressed by cells other than the desired population.

[0085] The separation does not need to result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection or enrichment of a particular type of cell, e.g., a cell expressing a marker, refers to increasing the number or percentage of such cells, but does not need to result in the complete absence of cells that do not express the marker. Similarly, negative selection, removal, or depletion of a particular type of cell, e.g., a cell expressing a marker, refers to decreasing the number or percentage of such cells, but does not need to result in the complete removal of all such cells.

[0086] In some examples, a fraction that has been positively or negatively selected in one step is subjected to another separation step, for example, multiple rounds of separation steps where the fraction is then subjected to positive or negative selection. In some examples, for instance, by incubating cells with a plurality of antibodies or binding partners each specific for a marker that is a target for negative selection, cells that simultaneously express multiple markers can be removed in a single separation step. Similarly, by incubating cells with a plurality of antibodies or binding partners expressed in various cell types, multiple cell types can be positively selected simultaneously.

[0087] For example, in some embodiments, a particular subpopulation of T cells, e.g., cells that are positive or express a high level of one or more surface markers, such as CD28+, CD62L+, CCR7+, CD27+, CD127+, CD4+, CD8+, CD45RA+, and / or CD45RO+ T cells, are isolated by positive or negative selection techniques. For example, CD3+, CD28+ T cells can be positively selected using anti-CD3 / anti-CD28 conjugate magnetic beads (e.g., DYNABEADS® M-450 CD3 / CD28 T Cell Expander). In some embodiments, a population of cells is enriched for T cells having a naive phenotype (CD45RA+CCR7+).

[0088] In some embodiments, isolation is performed by enrichment of a particular cell population by positive selection or depletion of a particular cell population by negative selection. In some embodiments, positive or negative selection is achieved by incubating the cells with one or more antibodies or other binding agents that specifically bind to one or more surface markers that are expressed or expressed at a relatively higher level (marker high) on the cells that are to be positively or negatively selected, respectively.

[0089] In certain embodiments, a biological sample, e.g., a sample of PBMC or other white blood cells, is subjected to the selection of CD4+ T cells where both negative and positive fractions are secured. In certain embodiments, CD8+ T cells are secured from the negative fraction. In some embodiments, the biological sample is subjected to the selection of CD8+ T cells where both negative and positive fractions are secured. In certain embodiments, CD4+ T cells are secured from the negative fraction.

[0090] In some embodiments, T cells are isolated from a PBMC sample by negative selection of markers expressed on non-T cells, e.g., B cells, monocytes, or other white blood cells, such as CD14. In some embodiments, CD4+ or CD8+ selection steps are used to isolate CD4+ helper and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into subpopulations by positive or negative selection for markers that are expressed on or relatively more highly expressed on one or more naive T cell, memory T cell, and / or effector T cell subpopulations.

[0091] In one example, monoclonal antibody cocktails for enriching CD4+ cells by negative selection typically include antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is attached to a solid support or solid matrix, such as magnetic beads or paramagnetic beads, to enable separation of cells in positive and / or negative selection. For example, in some embodiments, cells and cell populations are separated or isolated using immunomagnetic (or affinity magnetic) separation techniques. In some embodiments, a sample or composition of cells to be separated is incubated with small magnetizable or magnetoresponsive materials, such as magnetoresponsive particles or microparticles, such as paramagnetic beads (e.g., Dynabeads™ or MACS beads). The magnetoresponsive material, e.g., the particles, is typically directly or indirectly attached to a binding partner, e.g., a molecule, e.g., an antibody that specifically binds to a surface marker present on the cells, multiple cells, or population of cells that are desired to be separated, e.g., negatively or positively selected.

[0092] In some embodiments, the magnetic particles or beads comprise a magnetic responsive material conjugated to a specifically binding member such as an antibody or other binding partner. There are a number of well-known magnetic responsive materials used in magnetic separation methods. Incubation is typically performed under conditions such that molecules such as antibodies or binding partners conjugated to the magnetic particles or beads, or secondary antibodies or other reagents that specifically bind to such antibodies or binding partners, specifically bind to cell surface molecules (if present on the cells in the sample). In some embodiments, the sample is placed in a magnetic field and the cells conjugated with magnetic responsive particles or magnetizable particles are attracted to a magnet and separated from the unlabeled cells. In positive selection, the cells attracted to the magnet are secured, and in negative selection, the cells not attracted (unlabeled cells) are secured. In some embodiments, a combination of positive and negative selection is performed during the same selection step, where the positive and negative fractions are secured and further processed or subjected to further separation steps. In some embodiments, the magnetic responsive particles are coated with a primary antibody or other binding partner, a secondary antibody, a lectin, an enzyme, or streptavidin. In certain embodiments, the magnetic particles are conjugated to cells by coating with a primary antibody specific for one or more markers. In certain embodiments, instead of beads, the cells are labeled with a primary antibody or binding partner and then magnetic particles coated with a secondary antibody or other binding partner specific for the cell type (e.g., streptavidin) are added. In certain embodiments, magnetic particles coated with streptavidin are used with biotinylated primary or secondary antibodies. In some embodiments, the magnetic responsive particles are left conjugated to the cells to be subsequently incubated, cultured, and / or manipulated, and in some embodiments, the particles are left conjugated to the cells for administration to a patient. In some embodiments, the magnetizable particles or magnetic responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, for example, the use of competing unlabeled antibodies and magnetizable particles or antibodies conjugated with cleavable linkers.In some embodiments, the magnetizable particles are biodegradable.

[0093] In some embodiments, affinity-based selection is by magnetic-activated cell sorting (MACS) (Miltenyi Biotec, Auburn, CA). The magnetic-activated cell sorting (MACS) system enables high-purity selection of cells to which magnetized particles are bound. In certain embodiments, MACS operates in such a way that non-target and target species are eluted sequentially after the application of an external magnetic field. That is, cells bound to the magnetized particles are retained in place and unbound species are eluted. Then, after this first elution step is complete, the species that were captured within the magnetic field and had their elution blocked are released in some manner such that they can be eluted and recovered. In certain embodiments, non-target cells are labeled and depleted from a heterogeneous population of cells.

[0094] In some embodiments, isolation or separation is performed using a system, device, or apparatus that performs one or more of the isolation, cell preparation, separation, processing, incubation, culture, and / or formulation steps of the method. In some embodiments, the system is used to perform each of these steps in a closed or sterile environment, for example, to minimize errors, user handling, and / or contamination. In one example, the system is the system described in International Publication No. WO 2009 / 072003 or U.S. Patent Application Publication No. 20110003380(A1), each of which is incorporated herein by reference. In some embodiments, the system or apparatus performs one or more of the isolation, processing, manipulation, and formulation steps in an integrated or self - contained system and / or in an automated or programmable manner. In some embodiments, the system or apparatus includes a computer and / or computer program that communicates with the system or apparatus and that enables a user to program, control, evaluate the results of, and / or adjust various embodiments of the processing, isolation, manipulation, and formulation steps. In some embodiments, the separation and / or other steps are performed using a CliniMACS system (Miltenyi Biotec) for the automated separation of cells at the clinical - scale level within a closed sterile system. Components may include an embedded microcomputer, a magnetic separation unit, a peristaltic pump, and various pinch valves. In some embodiments, the embedded microcomputer controls the components of the instrument and instructs the system to perform repetitive procedures in a standardized sequence. In some embodiments, the magnetic separation unit includes a movable permanent magnet and a holder for the selection column. The peristaltic pump controls the flow rate within the tubing set and, together with the pinch valves, ensures a controlled flow of buffer and continuous suspension of cells through the system.

[0095] In some embodiments, the CliniMACS system uses magnetizable particles coupled to an antibody that are supplied in a sterile, non-pyrogenic solution. In some embodiments, after labeling of the cells with magnetic particles, the cells are washed and excess particles are removed. The cell preparation bag is then connected to a tubing set, which is then connected to a bag containing buffer and a cell collection bag. The tubing set consists of pre-assembled sterile tubing that includes a pre-column and a separation column and is intended for single use only. After initiation of the separation program, the system automatically applies the cell sample onto the separation column. The labeled cells are retained within the column while unlabeled cells are removed by a series of washing steps. In some embodiments, the cell population used in the methods described herein is unlabeled and not retained within the column. In some embodiments, the cell population used in the methods described herein is labeled and retained within the column. In some embodiments, the cell population for use in the methods described herein is eluted from the column after removal of the magnetic field and collected within the cell collection bag.

[0096] In certain embodiments, the separation and / or other procedures are performed using a CliniMACS Prodigy system (Miltenyi Biotec). In some embodiments, the CliniMACS Prodigy system comprises a cell processing unit that enables automated washing and fractionation of cells by centrifugation. The CliniMACS Prodigy system may also include an on-board camera and image recognition software that determines the optimal cell fractionation endpoint by identifying the macroscopic layer of the source cell product. For example, peripheral blood is automatically separated into red blood cells, white blood cells, and plasma layers. The CliniMACS Prodigy system may also include an integrated cell culture chamber that performs cell culture protocols such as cell differentiation and expansion, antigen loading, and long-term cell culture. The inlets may allow for sterile removal and replenishment of the medium and the cells may be monitored using an integrated microscope.

[0097] In some embodiments, the cell populations described herein are collected and enriched (or depleted) by flow cytometry in which cells stained for multiple cell surface markers are carried in a fluid stream. In some embodiments, the cell populations described herein are collected and enriched (or depleted) by sorting on a fluorescence-activated cell sorting (FACS) scale. In certain embodiments, the cell populations described herein are collected and enriched (or depleted) by use of a microelectromechanical system (MEMS) chip combined with a FACS-based detection system (see, e.g., International Publication No. WO 2010 / 033140, Cho et al. (2010) Lab Chip 10, 1567-1573, and Godin et al. (2008) J Biophoton. 1(5):355-376). In any case, the cells can be labeled with multiple markers, which allows for the isolation of well-defined T cell subsets with high purity.

[0098] In some embodiments, the antibody or binding partner is labeled with one or more detectable markers to facilitate separation by positive and / or negative selection. For example, the separation can be based on binding to a fluorescently labeled antibody. In some instances, the separation of cells based on the binding of an antibody or other binding partner specific for one or more cell surface markers is performed in a fluid stream, e.g., by fluorescence-activated cell sorting (FACS) including a fluorescence-activated cell sorter (FACS) and / or by a microelectromechanical system (MEMS) chip combined with a flow cytometry detection system. Such methods allow for simultaneous positive and negative selection based on multiple markers.

[0099] In some embodiments, at least 0.5×10 9 lymphocytes are obtained from a donor, optionally enriched, and / or subjected to stimulation. In some embodiments, at least 0.6×10 9 , 0.7×10 9 , 0.8×10 9 , 0.9×10 9 , 1×109 , 1.1×10 9 , 1.2×10 9 , 1.3×10 9 , 1.4×10 9 , 1.5×10 9 , 1.6×10 9 , 1.7×10 9 , 1.8×10 9 , 1.9×10 9 , 2×10 9 , 2.5×10 9 , or 3×10 9 lymphocytes are obtained from a donor and, optionally, enriched and / or subjected to stimulation. In some embodiments, 1×10 9 , 1.1×10 9 , 1.2×10 9 , 1.3×10 9 , 1.4×10 9 , 1.5×10 9 , 1.6×10 9 , 1.7×10 9 , 1.8×10 9 , 1.9×10 9 , 2×10 9 , 2.5×10 9 , or 3×10 9 or fewer lymphocytes are obtained from a donor and, optionally, enriched and / or subjected to stimulation.

[0100] Optionally, the methods described herein further comprise stimulating the lymphocytes with one or more lymphocyte stimulants. In some embodiments, the stimulation is performed before the transduction step. In some embodiments, the stimulation is performed after the transduction step. The stimulation step is also referred to herein as the activation step.

[0101] Any combination of one or more suitable lymphocyte stimulants can be used to stimulate (activate) lymphocytes. Non-limiting examples include antibodies or functional fragments thereof that target T cell stimulatory or costimulatory molecules (e.g., anti-CD2 antibody, anti-CD3 antibody, anti-CD28 antibody, or functional fragments thereof), T cell cytokines (e.g., interleukin 1 (IL-1), interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 5 (IL-5), interleukin 7 (IL-7), interleukin 15 (IL-15), tumor necrosis factor α (TNFα), etc., any isolated, wild-type, or recombinant cytokine), or any other suitable mitogen for T cell stimulatory or costimulatory molecules (e.g., tetradecanoyl phorbol acetate (TPA), phytohemagglutinin (PHA), concanavalin A (ConA), lipopolysaccharide (LPS), pokeweed mitogen (PWM)) or natural ligands. In some embodiments, the stimulant is an anti-CD3 antibody and / or an anti-CD28 antibody.

[0102] In some embodiments, the lymphocyte stimulant can be a bead-based activator such as T-cell TransAct™ (Miltenyi Biotec), Dynabeads® (Thermo Fisher Scientific), or Cloudz™ T Cell Activation Kit (R&D Systems).

[0103] In some embodiments, the step of stimulating the lymphocytes described herein is performed at a predetermined temperature, for a predetermined time, and / or at a predetermined level of CO 2In the presence of, it may involve stimulating lymphocytes with one or more stimulants. In certain embodiments, the predetermined temperature for stimulation can be about 34°C, about 35°C, about 36°C, about 37°C, about 38°C, or about 39°C. In certain embodiments, the predetermined temperature for stimulation can be about 34 - 39°C. In certain embodiments, the step of stimulating lymphocytes includes stimulating lymphocytes with one or more stimulants for a predetermined time. In certain embodiments, the predetermined time for stimulation can be about 24 - 72 hours. In certain embodiments, the predetermined time for stimulation can be about 24 - 36 hours. In certain embodiments, the step of stimulating lymphocytes is at a predetermined level of CO 2 In the presence of, it may include stimulating lymphocytes with one or more stimulants. In certain embodiments, the predetermined level of CO 2 for stimulation can be about 1.0 - 10% CO 2 In certain embodiments, the predetermined level of CO 2 for stimulation can be about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, or about 10.0% CO 2 In certain embodiments, the predetermined level of CO

[0104] In some embodiments, an anti-CD3 antibody (or a functional fragment thereof), an anti-CD28 antibody (or a functional fragment thereof), or a combination of an anti-CD3 antibody and an anti-CD28 antibody can be used according to the step of stimulating a population of lymphocytes. Any soluble or immobilized anti-CD3 and / or anti-CD28 antibody or a functional fragment thereof can be used (e.g., clone OKT3 (anti-CD3), clone 145-2C11 (anti-CD3), clone UCHT1 (anti-CD3), clone L293 (anti-CD28), clone 15E8 (anti-CD28)). In some aspects, the antibodies can be commercially purchased from vendors known in the art including, but not limited to, Miltenyi Biotec, BD Biosciences (e.g., MACS GMP CD3 Pure 1 mg / mL, Part No. 170-076-116), and eBioscience, Inc. Further, those skilled in the art will understand methods for producing anti-CD3 antibodies and / or anti-CD28 antibodies by standard methods. Any antibody used in the methods described herein needs to be produced under Good Manufacturing Practices (GMP) in order to comply with the relevant institutional guidelines for biological products.

[0105] In certain embodiments, the T cell stimulant can contain an anti-CD3 or anti-CD28 antibody at a concentration from about 20 ng / mL to 100 ng / mL. In certain embodiments, the concentration of the anti-CD3 or anti-CD28 antibody can be about 20 ng / mL, about 30 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, or about 100 ng / mL.

[0106] In some embodiments, the three-day process of the present disclosure may include the following steps: collection of apheresis material, a first washing step performed on day 0, a second washing step performed on day 0, an enrichment step performed on day 0, a third washing step performed on day 0, an activation step performed from day 0 up to a maximum of day 1, a viral transduction step performed from day 1 up to a maximum of day 4, and a fourth washing and concentration step performed from day 3 up to a maximum of day 4 (the collection day). The apheresis material can be fresh apheresis material, cryopreserved apheresis material, or cryopreserved T cells.

[0107] In some embodiments, the three-day process may include an activation step on day 0, day 1, or day 2. In some embodiments, the three-day process may include a viral vector transduction step on day 1, day 2, or day 3.

[0108] In some embodiments, the three-day process may optionally include one, two, three, four, five, six, seven, eight, or more washing steps. Each washing step may include the same washing procedure or a different washing procedure. In some embodiments, one or more washing steps may occur before the enrichment step. In some embodiments, the first washing may occur after the enrichment step. In some embodiments, the first washing may occur after the enrichment step and before the activation step. In some embodiments, the first washing may occur after both the enrichment step and the activation step. In some embodiments, the washing step may be performed on the collection day.

[0109] Prepared and manipulated lymphocytes

[0110] The prepared lymphocytes contained a higher proportion of young lymphocytes (e.g., naive T cells), as demonstrated in the attached experimental examples. Thus, one embodiment of the present disclosure provides a population of lymphocytes prepared by the method and containing CD4+ T cells and CD8+ T cells.

[0111] In some embodiments, at least 20% of the CD4+ T cells are naive T cells. In some embodiments, at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% of the CD4+ T cells are naive T cells.

[0112] In some embodiments, 25% or less of the CD4+ T cells are effector memory T cells. In some embodiments, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, or 5% or less of the CD4+ T cells are effector memory T cells.

[0113] In some embodiments, 44% or less of the CD4+ T cells are central memory T cells. In some embodiments, 43%, 42%, 41%, or 40% or less of the CD4+ T cells are central memory T cells.

[0114] In some embodiments, 1.5% or less of the CD4+ T cells are effector T cells. In some embodiments, 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, or 0.5% or less of the CD4+ T cells are effector T cells.

[0115] In some embodiments, at least 80% of the harvested CD4+ T cells are CCR7+ cells. In some embodiments, at most 20% of the harvested CD4+ T cells are a combination of effector memory T cells and effector T cells.

[0116] In some embodiments, at least 5% of the CD8+ T cells are naive T cells. In some embodiments, at least 10%, 15%, 20%, 25%, 30%, or 35% of the CD8+ T cells are naive T cells.

[0117] In some embodiments, 30% or less of the CD8+ T cells are effector memory T cells. In some embodiments, 28%, 27%, 25%, 22%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, or 10% or less of the CD8+ T cells are effector memory T cells.

[0118] In some embodiments, 60% or less of the CD8+ T cells are central memory T cells. In some embodiments, 58%, 56%, 55%, 54%, 52%, or 50% or less of the CD8+ T cells are central memory T cells.

[0119] In some embodiments, at least 60% of the harvested CD8+ T cells are CCR7+ cells. In some embodiments, at most 40% of the harvested CD8+ T cells are a combination of effector memory T cells and effector T cells.

[0120] Each type of T cell can be characterized by cell surface markers well known in the art. For example, naive T cells can be characterized as CCR7+, CD45RO-, and CD95-. Additional markers for naive T cells include CD45RA+, CD62L+, CD27+, CD28+, CD127+, CD132+, CD25-, CD44-, and HLA-DR-.

[0121] Surface markers for stem memory T cells (Tscm) include, but are not limited to, CD45RO-, CCR7+, CD45RA+, CD62L+ (L-selectin), CD27+, CD28+, IL-7Ra+, CD95+, IL-2RP+, CXCR3+, and LFA-.

[0122] Surface markers of effector memory T cells (Tem) include, but are not limited to, CCR7−, CD45RO+, and CD95+. A further marker of effector memory T cells is IL-2Rβ+. For central memory T cells (Tcm), suitable markers include CD45RO+, CD95+, IL-2Rβ+, CCR7+, and CD62L+. For effector T cells (Teff), suitable markers include, but are not limited to, CD45RA+, CD95+, IL-2Rβ+, CCR7−, and CD62L−.

[0123] The term "naive cells" as referred to throughout includes one or more of naive T cells, stem memory T cells (Tscm), and central memory T cells (Tcm). These cells are characterized, in part, by the expression of CCR7+.

[0124] The collected lymphocytes preferably contain CD3+ T cells in a favorable proportion. In some embodiments, at least 25%, 35%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the collected lymphocytes are CD3+ T cells.

[0125] The lymphocytes taken are preferably those that have been transduced at a good rate. In some embodiments, at least 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 42%, 44%, 45%, 46%, 47%, 48%, 49%, or 50% of the lymphocytes taken are transduced with the vector. In some embodiments, each transduced lymphocyte contains at least 1 copy of the vector (or the coding sequence contained therein) integrated into the host genome. In some embodiments, each transduced lymphocyte contains at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 copies of the vector integrated into the host genome.

[0126] In some embodiments, the vector contains a transgene encoding a polypeptide. The polypeptide can be, but is not limited to, a CAR or a TCR. In some embodiments, the CAR or TCR contains an antigen-binding molecule. The antigen-binding molecule, in some embodiments, has binding specificity for an antigenic moiety. In some embodiments, the antigenic moiety is a tumor antigen (e.g., a protein or other molecule produced by cancer cells).

[0127] In some embodiments, the vector contains two or more transgenes encoding two or more CAR molecules or TCR molecules that contain antigen-binding molecules with specificities for different antigenic moieties. In some embodiments, the vector contains two or more transgenes encoding two or more CAR molecules or TCR molecules that contain antigen-binding molecules with specificities for two different tumor antigens.

[0128] In some embodiments, the antigenic portion is an antigen associated with cancer or cancer cells. Such antigens include 707-AP (707 alanine proline), AFP (alpha(a)-fetoprotein), ART-4 (an adenocarcinoma antigen recognized by T4 cells), BAGE (B antigen, b-catenin / m, b-catenin / variant), BCMA (B cell maturation antigen), Bcr-abl (breakpoint cluster region-Abelson), CAIX (carbonic anhydrase IX), CD19 (cluster of differentiation 19), CD20 (cluster of differentiation 20), CD22 (cluster of differentiation 22), CD30 (cluster of differentiation 30), CD33 (cluster of differentiation 33), CD44v7 / 8 (cluster of differentiation 44, exon 7 / 8), CAMEL (a CTL-recognized antigen on melanoma), CAP-1 (cancer fetal antigen peptide-1), CASP-8 (caspase-8), CDC27m (cell division cycle 27, variant), CDK4 / m (cyclin-dependent kinase 4, variant), CEA (carcinoembryonic antigen), CT (cancer / testis (antigen)), Cyp-B (cyclophilin B), DAM (differentiation antigen, melanoma), EGFR (epidermal growth factor receptor), EGFRvIII (epidermal growth factor receptor, variant III), EGP-2 (epithelial glycoprotein 2), EGP-40 (epithelial glycoprotein 40), Erbb2, 3, 4 (erythroblastic leukemia viral oncogene homolog-2, -3, 4), ELF2M (elongation factor 2, variant), ETV6-AML1 (Ets variant gene 6 / acute myeloid leukemia 1 gene ETS), FBP (folate binding protein), fAchR (fetal acetylcholine receptor), G250 (glycoprotein 250), GAGE (G antigen), GD2 (disialoganglioside 2), GD3 (disialoganglioside 3), GnT-V (N-acetylglucosaminyltransferase V), Gp100 (glycoprotein 100kD), HAGE (helicos antigen), HER-2 / neu (human epidermal receptor-2 / neurological, also known as EGFR2), HLA-A (human leukocyte antigen-A), HPV (human papillomavirus), HSP70-2M (heat shock protein 70-2 variant), HST-2 (human ring finger tumor-2), hTERT or hTRT (human telomerase reverse transcriptase), iCE (intestinal carboxylesterase), IL-13R-a2 (interleukin-13 receptor subunit alpha-2),KIAA0205, KDR (Kinase Insert Domain Receptor), κ-light chain, LAGE (L antigen), LDLR / FUT (Low Density Lipoprotein Receptor / GDP-L-fucose:β-D-galactosidase 2-α-L-fucosyltransferase), LeY (Lewis-Y antibody), L1CAM (L1 cell adhesion molecule), MAGE (Melanoma Antigen), MAGE-A1 (Melanoma-associated Antigen 1), Mesothelin, Mouse CMV-infected cells, MART-1 / Melan-A (Melanoma Antigen recognized by T cells-1 / Melanoma Antigen A), MC1R (Melanocortin 1 Receptor), Myosin / m (Myosin variant), MUC1 (Mucin 1), MUM-1, -2, -3 (Melanoma ubiquitous, variant 1, 2, 3), NA88-A (NA cDNA clone of patient M88), NKG2D (Natural Killer group 2, member D) ligand, NY-BR-1 (New York Breast Differentiation Antigen 1), NY-ESO-1 (New York Esophageal Squamous Cell Carcinoma-1), Tumor fetal antigen (h5T4), P15 (Protein 15), p190 minor bcr-abl (190KD bcr-abl protein), Pml / RARa (Promyelocytic leukemia / Retinoic acid receptor a), PRAME (Preferentially expressed antigen in melanoma), PSA (Prostate-specific antigen), PSCA (Prostate stem cell antigen), PSMA (Prostate-specific membrane antigen), RAGE (Renal antigen), RU1 or RU2 (Renal ubiquitous 1 or 2), SAGE (Sarcoma antigen), SART-1 or SART-3 (Flat antigen that rejects tumors 1 or 3), SSX1, -2, -3, 4 (Synovial sarcoma X1, -2, -3, -4), TAA (Tumor-associated antigen), TAG-72 (Tumor-associated glycoprotein 72), TEL / AML1 (Translocated Ets-family leukemia / Acute Myeloid Leukemia 1), TPI / m (Triose Phosphate Isomerase variant), TRP-1 (Tyrosinase-related protein 1, or gp75), TRP-2 (Tyrosinase-related protein 2), TRP-2 / INT2 (TRP-2 / Intron 2), VEGF-R2 (Vascular Endothelial Growth Factor Receptor 2), or WT1 (Wilms tumor gene), or any combination thereof may be included, but is not limited thereto.,

[0129] Additional examples of tumor antigens include 2B4 (CD244), 4-1BB, 5T4, A33 antigen, adenocarcinoma antigen, adrenergic receptor beta 3 (ADRB3), A kinase anchor protein 4 (AKAP-4), alpha-fetoprotein (AFP), anaplastic lymphoma kinase (ALK), androgen receptor, B7H3 (CD276), beta2-integrin, BAFF, B lymphoma cells, B cell maturation antigen (BCMA), bcr-abl (a cancer gene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia virus cancer gene homolog 1 (Abl)), BhCG, bone marrow stromal cell antigen 2 (BST2), CCCTC-binding factor (zinc finger protein)-like (BORIS or brother of the imprinting site regulator), BST2, C242 antigen, 9-O-acetyl-CA19-9 marker, CA-125, CAEX, calreticulin, carbonic anhydrase 9 (CAIX), C-MET, CCR4, CCR5, CCR8, CD2, CD3, CD4, CD5, CD8, CD7, CD10, CD16, CD19, CD20, CD22, CD23 (IgE receptor), CD24, CD25, CD27, CD28, CD30 (TNFRSF8), CD33, CD34, CD38, CD40, CD40L, CD41, CD44, CD44V6, CD49f, CD51, CD52, CD56, CD63, CD70, CD72, CD74, CD79a, CD79b, CD80, CD84, CD96, CD97, CD100, CD123, CD125, CD133, CD137, CD138, CD150, CD152 (CTLA-4), CD160, CD171, CD179a, CD200, CD221, CD229, CD244, CD272 (BTLA), CD274 (PDL-1, B7H1), CD279 (PD-1), CD352, CD358, CD300 molecule-like family member f (CD300LF), carcinoembryonic antigen (CEA), claudin 6 (CLDN6), C-type lectin-like molecule-1 (CLL-1 or CLECL1), C-type lectin domain family 12 member A (CLEC12A), cytomegalovirus (CMV) infected cell antigen, CNT0888, CRTAM (CD355), CS-1 (also known as CD2 subset 1, CRACC, CD319, and 19A24), CTLA-4, cyclin B1,Chromosome X open reading frame 61 (CXORF61), cytochrome P450 1B1 (CYP1B1), DNAM-1 (CD226), desmoglein 4, DR3, DR5, E-cadherin neoepitope, epidermal growth factor receptor (EGFR), EGF1R, epidermal growth factor receptor variant III (EGFRvIII), epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), EGF-like module-containing mucin-like hormone receptor-like 2 (EMR2), elongation factor 2 variant (ELF2M), endosialin, epithelial cell adhesion molecule (EPCAM), ephrin type-A receptor 2 (EphA2), ephrin B2, receptor tyrosine-protein kinases erb-B2, 3, 4 (erb-B2, 3, 4), ERBB, ERBB2 (Her2 / neu), ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene), ETA, ETS translocation variant gene 6 located on chromosome 12p (ETV6-AML), Fc fragment of IgA receptor (FCAR or CD89), fibroblast activation protein alpha (FAP), FBP, Fc receptor-like 5 (FCRL5), fetal acetylcholine receptor (AChR), fibronectin extra domain B, Fms-like tyrosine kinase 3 (FLT3), folate-binding protein (FBP), folate receptor 1, folate receptor alpha, folate receptor beta, Fos-related antigen 1, fucosyl, fucosyl GM1, GM2, ganglioside G2 (GD2), ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer), o-acetyl-GD2 ganglioside (OAcGD2), GITR (TNFRSF18), GM1, ganglioside GM3 (aNeu5Ac(2-3)bDGalp(l-4)bDGlcp(l-l)Cer), GP100, hexasaccharide moiety of GloboH glycosphingolipid (GloboH), glycoprotein 75, glypican-3 (GPC3), glycoprotein 100 (gp100), GPNMB, G protein-coupled receptor 20 (GPR20), G protein-coupled receptor class C group 5, member D (GPRC5D), hepatitis A virus cellular receptor 1 (HAVCR1), human epidermal growth factor receptor 2 (HER-2), HER2 / neu, HER3, HER4, HGF, high-molecular weight melanoma-associated antigen (HMWMAA),Human papillomavirus E6 (HPV E6), Human papillomavirus E7 (HPV E7), heat shock protein 70-2 variant (mut hsp70-2), human scatter factor receptor kinase, human telomerase reverse transcriptase (hTERT), HVEM, ICOS, insulin-like growth factor receptor 1 (IGF-1 receptor), IGF-I, IgGl, immunoglobulin lambda-like polypeptide 1 (IGLL1), IL-6, interleukin 11 receptor alpha (IL-11Ra), IL-13, interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2), insulin-like growth factor I receptor (IGF1-R), integrin α5β1, integrin ανβ3, intestinal carboxylesterase, κ light chain, KCS1, kinase insert domain receptor (KDR), KIR, KIR2DL1, KIR2DL2, KIR2DL3, KIR3DL2, KIR-L, KG2D ligand, KIT (CD117), KLRGI, LAGE-la, LAG3, lymphocyte-specific protein tyrosine kinase (LCK), leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2), legumain, leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), Lewis (Y) antigen, LeY, LG, LI cell adhesion molecule (LI-CAM), LIGHT, LMP2, lymphocyte antigen 6 complex, LTBR, locus K9 (LY6K), Ly-6, lymphocyte antigen 75 (LY75), melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2), MAGE, melanoma-associated antigen 1 (MAGE-A1), MAGE-A3 melanoma antigen 1 recognized by T cells (MelanA or MARTI), MelanA / MARTl, mesothelin, MAGE A3, melanoma inhibitor of apoptosis (ML-IAP), melanoma-specific chondroitin sulfate proteoglycan (MCSCP), MORAb-009, MS4A1, Mucin1 (MUC1), MUC2, MUC3, MUC4, MUC5AC, MUC5b, MUC7, MUC16, mucin CanAg, Mullerian-inhibiting substance (MIS) receptor type II, v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN), N-glycolylneuraminic acid,N-acetylglucosaminyltransferase V (NA17), neural cell adhesion molecule (NCAM), NKG2A, NKG2C, NKG2D, NKG2E ligand, NKR-P IA, NPC-1C, NTB-A, mammary differentiation antigen (NY-BR-1), NY-ESO-1, tumor fetal antigen (h5T4), olfactory receptor 51E2 (OR51E2), OX40, plasma cell antigen, poly SA, proacrosin-binding protein sp32 (OY-TES 1), p53, p53 variant, pannexin 3 (PANX3), prostate acid phosphatase (PAP), paired box protein Pax-3 (PAX3), paired box protein Pax-5 (PAX5), prostate cancer tumor antigen-1 (PCTA-1 or galectin 8), PD-1H, platelet-derived growth factor receptor alpha (PDGFR-alpha), PDGFR-beta, PDL192, PEN-5, phosphatidylserine, placenta-specific 1 (PLAC1), polysialic acid, prostase, prostate cancer cell, prostain, protease serine 21 (testisin or PRSS21), proteinase 3 (PR1), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), proteasome (Prosome, Macropain) subunit, beta type, receptor for advanced glycation end products (RAGE-1), RANKL, Ras variant, Ras homolog family member C (RhoC), RON, receptor tyrosine kinase-like orphan receptor 1 (ROR1), renal ubiquitous 1 (RU1), renal ubiquitous 2 (RU2), sarcoma translocation breakpoint, squamous cell carcinoma antigen recognized by T cells 3 (SART3), SAS, SDC1, SLAMF7, sialyl Lewis adhesion molecule (sLe), Siglec-3, Siglec-7, Siglec-9, Sonic hedgehog (SHH), sperm protein 17 (SPA17), stage-specific embryonic antigen 4 (SSEA-4), STEAP, sTn antigen, synovial sarcoma, X breakpoint 2 (SSX2), survivin, tumor-associated glycoprotein 72 (TAG72), TCR5y, TCRa, TCRB, TCR gamma alternative reading frame protein (TARP), telomerase, TIGIT TNF-alpha precursor, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), tenascin C, TGF beta2, TGF-beta,Transglutaminase 5 (TGS5), angiopoietin-binding cell surface receptor 2 (Tie 2), TIM1, TIM2, TIM3, Tn Ag, TRAIL-R1, TRAIL-R2, tyrosinase-related protein 2 (TRP-2), thyroid-stimulating hormone receptor (TSHR), tumor antigen CTAA16.88, tyrosinase, ROR1, TAG-72, uroplakin 2 (UPK2), VEGF-A, VEGFR-1, vascular endothelial growth factor receptor 2 (VEGFR2), and vimentin, Wilms tumor protein (WT1), or X antigen family, member 1A (XAGE1) are mentioned.

[0130] In other embodiments, the antigenic portion is associated with a cell infected with a virus (i.e., a viral antigenic portion). Such antigenic portions include, but are not limited to, Epstein-Barr virus (EBV) antigens (e.g., EBNA-1, EBNA-2, EBNA-3, LMP-1, LMP-2), hepatitis A virus antigens (e.g., VP1, VP2, VP3), hepatitis B virus antigens (e.g., HBsAg, HBcAg, HBeAg), hepatitis C virus antigens (e.g., envelope glycoproteins E1 and E2), herpes simplex virus type 1, 2, or 8 (HSV1, HSV2, or HSV8) virus antigens (e.g., glycoproteins gB, gC, gC, gE, gG, gH, gI, gJ, gK, gL, gM, UL20, UL32, US43, UL45, UL49A), cytomegalovirus (CMV) virus antigens (e.g., glycoproteins gB, gC, gC, gE, gG, gH, gI, gJ, gK, gL, gM, or other envelope proteins), human immunodeficiency virus (HIV) virus antigens (glycoproteins gp120, gp41, or p24), influenza virus antigens (e.g., hemagglutinin (HA) or neuraminidase (NA)), measles or mumps virus antigens, human papillomavirus (HPV) virus antigens (e.g., L1, L2), parainfluenza virus virus antigens, rubella virus virus antigens, respiratory syncytial virus (RSV) virus antigens, or varicella zoster virus virus antigens. In such embodiments, the cell surface receptor can be any TCR or any CAR that recognizes any of the aforementioned viral antigens on a cell infected with the target virus.

[0131] In other embodiments, the antigenic moiety is associated with cells having immune or inflammatory dysfunction. Such antigenic moieties include, but are not limited to, myelin basic protein (MBP), proteolipid protein (PLP), myelin oligodendrocyte glycoprotein (MOG), carcinoembryonic antigen (CEA), proinsulin, glutamic acid decarboxylase (GAD65, GAD67), heat shock protein (HSP), or any other tissue-specific antigen involved in or associated with a pathogenic autoimmune process.

[0132] In some embodiments, the TCR has specificity for an antigenic moiety on a cancer cell. Non-limiting examples of TCRs include anti-707-AP TCR, anti-AFP TCR, anti-ART-4 TCR, anti-BAGE TCR, anti-Bcr-abl TCR, anti-CAMEL TCR, anti-CAP-1 TCR, anti-CASP-8 TCR, anti-CDC27m TCR, anti-CDK4 / m TCR, anti-CEA TCR, anti-CT TCR, anti-Cyp-B TCR, anti-DAM TCR, anti-TCR, anti-EGFRvIII TCR, anti-ELF2M TCR, anti-ETV6-AML1 TCR, anti-G250 TCR, GAGE TCR, anti-GnT-V TCR, anti-Gp100 TCR, anti-HAGE TCR, anti-HER-2 / neu TCR, anti-HLA-A TCR, anti-HPV TCR, anti-HSP70-2M TCR, anti-HST-2 TCR, anti-hTERT TCR or anti-hTRT TCR, anti-iCE TCR, anti-KIAA0205, anti-LAGE (L antigen), anti-LDLR / FUT TCR, anti-MAGE TCR, anti-MART-1 / Melan-A TCR, anti-MC1R TCR, anti-Myosin / m TCR, anti-MUC1 TCR, anti-MUM-1, -2, -3 TCR, anti-NA88-A TCR, anti-NY-ESO-1 TCR, anti-P15 TCR, anti-p190 minor bcr-abl TCR, anti-Pml / RARa TCR, anti-PRAME TCR, anti-PSA TCR, anti-PSMA TCR, anti-RAGE TCR, anti-RU1 TCR or anti-RU2 TCR, anti-SAGE TCR, anti-SART-1 TCR or anti-SART-3 TCR, anti-SSX1, -2, -3, 4 TCR, anti-TEL / AML1 TCR, anti-TPI / m TCR, anti-TRP-1 TCR, anti-TRP-2 TCR, anti-TRP-2 / INT2 TCR, or anti-WT1 TCR.

[0133] The CARs of the present disclosure may include a hinge, a transmembrane domain, and / or an intracellular domain in addition to the antigen-binding molecule. In some embodiments, the intracellular domain may include a co-stimulatory domain and an activation domain.

[0134] The hinge can be the extracellular domain of the antigen-binding system located between the binding motif and the transmembrane domain. The hinge can also be referred to as the extracellular domain or "spacer". The hinge can contribute to the expression, activity, and / or stability of the receptor. The hinge can also provide flexibility for accessing the target antigen. In some embodiments, the hinge domain is located between the binding motif and the transmembrane domain.

[0135] In some embodiments, the hinge is an immunoglobulin-like hinge domain, from an immunoglobulin-like hinge domain, or derived from an immunoglobulin-like hinge domain (e.g., including all or a fragment of the immunoglobulin-like hinge domain). In some embodiments, the hinge domain is from an immunoglobulin or derived from an immunoglobulin. In some embodiments, the hinge domain is selected from the hinge of IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, or IgM, or a fragment thereof.

[0136] In some embodiments, the hinge is CD2, CD3 delta, CD3 epsilon, CD3 gamma, CD4, CD7, CD8 alpha, CD8 beta, CD11a (ITGAL), CD11b (ITGAM), CD11c (ITGAX), CD11d (ITGAD), CD18 (ITGB2), CD19 (B4), CD27 (TNFRSF7), CD28, CD28T, CD29 (ITGB1), CD30 (TNFRSF8), CD40 (TNFRSF5), CD48 (SLAMF2), CD49a (ITGA1), CD49d (ITGA4), CD49f (ITGA6), CD66a (CEACAM1), CD66b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), CD79A (B cell antigen receptor complex associated alpha chain), CD79B (B cell antigen receptor complex associated beta chain), CD84 (SLAMF5), CD96 (Tactile), CD100 (SEMA4D), CD103 (ITGAE), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD158A (KIR2DL1), CD158B1 (KIR2DL2), CD158B2 (KIR2DL3), CD158C (KIR3DP1), CD158D (KIRDL4), CD158F1 (KIR2DL5A), CD158F2 (KIR2DL5B), CD158K (KIR3DL2), CD160 (BY55), CD162 (SELPLG), CD226 (DNAM1), CD229 (SLAMF3), CD244 (SLAMF4), CD247 (CD3-zeta), CD258 (LIGHT), CD268 (BAFFR), CD270 (TNFSF14), CD272 (BTLA), CD276 (B7-H3), CD279 (PD-1), CD314 (NKG2D), CD319 (SLAMF7), CD335 (NK-p46), CD336 (NK-p44), CD337 (NK-p30), CD352 (SLAMF6), CD353 (SLAMF8), CD355 (CRTAM), CD357 (TNFRSF18), inducible T cell co-stimulatory factor (ICOS), LFA-1 (CD11a / CD18), NKG2C,DAP-10, ICAM-1, NKp80 (KLRF1), IL-2R beta, IL-2R gamma, IL-7R alpha, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, CD83 ligand, Fc gamma receptor, MHC class 1 molecule, MHC class 2 molecule, TNF receptor protein, immunoglobulin protein, cytokine receptor, integrin, activated NK cell receptor, or Toll ligand receptor, or fragments or combinations thereof, or from them, or derived from them (e.g., including all or fragments thereof).

[0137] In some embodiments, the hinge is the hinge of CD8 alpha, from the hinge of CD8 alpha, or derived from the hinge of CD8 alpha (e.g., including all or fragments of the hinge of CD8 alpha). In some embodiments, the hinge is the hinge of CD28, from the hinge of CD28, or derived from the hinge of CD28. In some embodiments, the hinge is a fragment of the hinge of CD8 alpha or a fragment of the hinge of CD28, from a fragment of the hinge of CD8 alpha or a fragment of the hinge of CD28, or derived from a fragment of the hinge of CD8 alpha or a fragment of the hinge of CD28, and the fragment is smaller than the whole. In some embodiments, the fragment of the CD8 alpha hinge or the CD28 hinge comprises an amino acid sequence that excludes at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 amino acids at the N-terminus and / or C-terminus of the CD8 alpha hinge or the CD28 hinge.

[0138] "Transmembrane domain" refers to a domain that has the attribute of being present within the membrane when present in a molecule on the cell surface or cell membrane (e.g., spanning a portion or all of the cell membrane). It is not necessary for all amino acids in the transmembrane domain to be present within the membrane. For example, in some embodiments, a transmembrane domain is characterized in that a designated stretch or portion of the protein is substantially located within the membrane. Amino acid sequences or nucleic acid sequences can be analyzed using various algorithms for predicting the intracellular localization of a protein (e.g., transmembrane localization). Programs such as psort (PSORT.org) and Prosite (prosite.expasy.org) are examples of such programs.

[0139] Transmembrane domains are those of the alpha, beta, or zeta chains of the T cell receptor, CD28, CD3 epsilon, CD3 delta, CD3 gamma, CD45, CD4, CD5, CD7, CD8, CD8 alpha, CD8 beta, CD9, CD11a, CD11b, CD11c, CD11d, CD16, CD22, CD27, CD33, CD37, CD64, CD80, CD86, CD134, CD137, TNFSFR25, CD154, 4-1BB / CD137, activated NK cell receptor, immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD276 (B7-H3), CD29, CD30, CD40, CD49a, CD49D, CD49f, CD69, CD84, CD96 (Tactile), CD5, CEACAM1, CRTAM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell co-stimulator (ICOS), integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LFA-1, ligand that binds to CD83, LIGHT, LIGHT, LTBR, Ly9 (CD229), lymphocyte function-associated antigen-1 (LFA-1; CD1-1a / CD18), MHC class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1; CD150; IPO-3), SLAMF4 (CD244; 2B4), SLAMF6 (NTB-A;It may be derived from any membrane-bound protein or transmembrane protein such as Ly108), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or fragments, cleavages, or combinations thereof.;

[0140] The intracellular domain (or cytoplasmic domain) contains one or more signaling domains that, when the target antigen binds to the binding motif, for example, cause and / or mediate an intracellular signal that activates one or more immune cell effector functions (such as innate immune cell effector functions). In some embodiments, the signaling domain of the intracellular domain mediates the activation of at least one of the normal effector functions of immune cells. The effector functions of T cells can be, for example, cytolytic activity or helper activity including the secretion of cytokines. In some embodiments, the signaling domain of the intracellular domain mediates the activation, proliferation, survival, and / or other T cell functions. The intracellular domain may contain a signaling domain that is an activation domain. The intracellular domain may contain a signaling domain that is a co-stimulatory signaling domain.

[0141] Intracellular signaling domains that can transmit signals upon binding of an antigen to an immune cell are known. For example, the cytoplasmic sequence of the T cell receptor (TCR) is known to initiate signal transduction after binding of the TCR to an antigen (e.g., Brownlie et al., Nature Rev. Immunol. 13:257-269 (2013)).

[0142] In certain embodiments, suitable signaling domains include, but are not limited to, 4-1BB / CD137, activating NK cell receptor, immunoglobulin protein, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 delta, CD3 epsilon, CD3 gamma, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8 alpha, CD8 beta, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CD5, CEACAM1, CRT AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc gamma receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, Ig alpha (CD79a), IL-2R beta, IL-2R gamma, IL-7R alpha, inducible T cell co-stimulator (ICOS), integrin, ITGA4, ITGA4, ITGA6, ITGAD, ITGAE, ITGAL, ITGAM, ITGAX, ITGB2, ITGB7, ITGB1, KIRDS2, LAT, LFA-1, LFA-1, ligand binding to CD83, LIGHT, LIGHT, LTBR, Ly9 (CD229), Ly108), lymphocyte function-associated antigen-1 (LFA-1, CD1-1a / CD18), MHC class 1 molecule, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecule (SLAM protein), SLAM (SLAMF1, CD150, IPO-3), SLAMF4 (CD244, 2B4), SLAMF6 (NTB-A, SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1, or VLA-6, or fragments, truncations, or combinations thereof.

[0143] The CAR may also include a co-stimulatory signaling domain, for example, to increase signaling efficacy. See U.S. Patent Nos. 7,741,465, 6,319,494, and Krause et al and Finney et al. (supra), Song et al., Blood 119:696-706 (2012), Kalos et al., Sci Transl. Med. 3:95 (2011), Porter et al., N. Engl. J. Med. 365:725-33 (2011), and Gross et al., Annu. Rev. Pharmacol. Toxicol. 56:59-83 (2016). Signals generated only through the TCR may be insufficient for complete activation of T cells, and secondary or co-stimulatory signals can increase activation. Thus, in some embodiments, the signaling domain further includes one or more additional signaling domains (e.g., co-stimulatory signaling domains) that activate one or more immune cell effector functions (e.g., the innate immune cell effector functions described herein). In some embodiments, a portion of such a co-stimulatory signaling domain can be used as long as that portion transduces an effector function signal. In some embodiments, the cytoplasmic domains described herein include one or more cytoplasmic sequences of a T cell co-receptor (or fragment thereof). Non-limiting examples of such T cell co-receptors include ligands that bind to CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), MYD88, CD2, CD7, LIGHT, NKG2C, B7-H3, and CD83. Exemplary co-stimulatory proteins have the amino acid sequences of co-stimulatory proteins naturally found on T cells, and the complete native amino acid sequences of such co-stimulatory proteins are described in NCBI reference sequence: NP_0.1. In certain examples, the CAR includes a 4-1BB co-stimulatory domain. In certain examples, the CAR includes a CD28 co-stimulatory domain. In certain examples, the CAR includes a DAP-10 co-stimulatory domain.

[0144] In some embodiments, the co-stimulatory signaling domain is the signaling domain of CD28. As shown in the experimental examples, CAR molecules having the CD28 co-stimulatory signaling domain can particularly benefit from a newly developed rapid manufacturing process. In some embodiments, the CAR is encoded by a nucleic acid molecule incorporated into a viral vector. In some embodiments, the CAR is encoded by a nucleic acid molecule incorporated into a lentiviral vector.

[0145] In some embodiments, the CAR further comprises an ITAM. Examples of ITAM-containing primary cytoplasmic signaling sequences that are particularly useful in the present disclosure include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66d. In some embodiments, the ITAM comprises CD3 zeta.

[0146] In some embodiments, the CAR molecule can be any anti-CD19 CAR molecule. In one aspect, the anti-CD19 CAR comprises an extracellular scFv domain, an intracellular and / or transmembrane portion of the CD28 molecule, an optional extracellular portion of the CD28 molecule, and an intracellular CD3 zeta domain as described in International Publication No. WO 2015 / 120096 or International Publication No. WO 2016 / 191755, each of which is incorporated herein by reference in its entirety.

[0147] In certain embodiments, the anti-CD19 CAR may also include additional domains such as a CD8 extracellular and / or transmembrane region, an extracellular immunoglobulin Fc domain (e.g., IgG1, IgG2, IgG3, IgG4), or one or more additional signaling domains such as 4-1BB, OX40, CD2, CD16, CD27, CD30, CD40, PD-1, ICOS, LFA-1, IL-2 receptor, Fc gamma receptor, or any other co-stimulatory domain having an immunoreceptor tyrosine-based activation motif.

[0148] In certain embodiments, the cell surface receptor is an anti-CD19 CAR such as the FMC63-28Z CAR or the FMC63-CD828BBZ CAR described in Kochenderfer et al., J Immunother. 2009 September;32(7):689-702, "Construction and Preclinical Evaluation of an Anti-CD19 Chimeric Antigen Receptor," which is incorporated herein by reference for the purpose of providing methods of constructing vectors for use in producing T cells that express the FMC63-28Z CAR or the FMC63-CD828BBZ CAR.

[0149] In some embodiments, the T cells comprising the CAR molecule are Yescarta® (axicabtagene ciloleucel). In some embodiments, the T cells comprising the CAR molecule are Tecartus® (brexucabtagene autoleucel).

[0150] In some embodiments, the engineered lymphocytes comprise a dual-targeted antigen-binding system. The dual-targeted antigen-binding system can comprise a bispecific CAR or TCR and / or a bicistronic CAR or TCR. The bispecific and bicistronic CARs can comprise two binding motifs (in a single CAR molecule or two CAR molecules, respectively). In some embodiments, the vectors of the disclosure encode a bicistronic and / or bispecific CAR (e.g., a bicistronic and / or bispecific CAR that binds to CD20 and CD19).

[0151] In some embodiments, there is provided a pharmaceutical composition comprising a population of engineered lymphocytes produced by the methods described herein. In certain embodiments, the pharmaceutical composition may also include a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier can be a pharmaceutically acceptable material, composition, or vehicle involved in the transport or delivery of the desired cells from one tissue, organ, or part of the body to another tissue, organ, or part of the body. For example, the carrier can be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or some combination thereof. Each component of the carrier needs to be "pharmaceutically acceptable" in that it is compatible with the other ingredients of the formulation. It also needs to be suitable for contact with any tissue, organ, or part of the body it may encounter, meaning that it should not have a risk of toxicity, irritation, allergic reaction, immunogenicity, or any other complication that unduly outweighs its therapeutic benefit.

[0152] Treatment and Use, and Optional Storage

[0153] The lymphocytes prepared by the methods, or the lymphocyte populations disclosed herein, can be used to treat a variety of diseases and conditions.

[0154] In some embodiments, if lymphocytes are not used immediately, they can be cryopreserved so that they can be used at a later date. Such methods can include the steps of washing and concentrating the population of engineered lymphocytes with a diluent. In some embodiments, the diluent is normal saline, 0.9% saline, PlasmaLyte A (PlasmaLyte, PL), 5% dextrose / 0.45% NaCl saline solution (D5), human serum albumin (HSA), or a combination thereof. In some embodiments, HSA can be added to the washed and concentrated cells to improve cell viability and cell recovery after thawing. In another embodiment, the wash solution is saline and the washed and concentrated cells are supplemented with HSA (5%). This method may also include the step of generating a cryopreservation mixture, where the cryopreservation mixture includes a population of diluted cells in a diluent and a suitable cryopreservation solution. In some embodiments, the cryopreservation solution can be any suitable cryopreservation solution including, but not limited to, CryoStor 10 (BioLife Solution), and is mixed with the diluent of the engineered immune cells in a ratio of 1:1 or 2:1.

[0155] In certain embodiments, HSA can be added to provide a final concentration of HSA in the cryopreserved mixture of about 1.0 to 10%. In certain embodiments, HSA can be added to provide a final concentration of HSA in the cryopreserved mixture of about 1.0%, about 2.0%, about 3.0%, about 4.0%, about 5.0%, about 6.0%, about 7.0%, about 8.0%, about 9.0%, or about 10.0%. In certain embodiments, HSA can be added to provide a final concentration of about 1 to 3% HSA, about 1 to 4% HSA, about 1 to 5% HSA, about 1 to 7% HSA, about 2 to 4% HSA, about 2 to 5% HSA, about 2 to 6% HSA, or about 2 to 7% HSA in the cryopreserved mixture. In certain embodiments, HSA can be added to provide a final concentration of HSA of about 2.5% in the cryopreserved mixture. For example, in certain embodiments, cryopreservation of the engineered T cell population can include washing the cells with 0.9% saline, adding HSA to the washed cells at a final concentration of 5%, and diluting the cells 1:1 with CryoStor™ CS10 (2.5% final concentration of HSA in the final cryopreserved mixture). In some embodiments, the method also includes the step of freezing the cryopreserved mixture. In one aspect, the cryopreserved mixture is frozen in a controlled rate freezer using a defined freezing cycle at a cell concentration of about 1e6 to about 1.5e7 cells per mL of cryopreserved mixture. The method can also include the step of storing the cryopreserved mixture in vapor phase liquid nitrogen.

[0156] Methods and uses for treating a disease or pathological condition in a subject having the disease or pathological condition are also provided. In some embodiments, the method involves administering to the subject a therapeutically effective amount or dose of engineered lymphocytes. Pathological conditions that can be treated with the engineered T cells produced by the methods described herein include, but are not limited to, cancer, viral infection, acute or chronic inflammation, autoimmune disease, or any other immune deficiency.

[0157] As used herein, "cancer" refers to acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adenoid cystic cancer, adrenocortical cancer, AIDS-related cancer, anal cancer, appendiceal cancer, astrocytoma, atypical teratoid / rhabdoid tumor, central nervous system, B-cell leukemia, lymphoma or other B-cell malignancies, basal cell cancer, bile duct cancer, bladder cancer, bone cancer, osteosarcoma and malignant fibrous histiocytoma, brainstem glioma, brain tumor, breast cancer, bronchial tumor, Burkitt lymphoma, carcinoid tumor, central nervous system cancer, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myelogenous leukemia (CML), chronic myeloproliferative disorder, colon cancer, colorectal cancer, craniopharyngioma, cutaneous T-cell lymphoma, germinoma, central nervous system, endometrial cancer, epithelioblastoma, epithelioma, esophageal cancer,esthesioneuroblastoma, Ewing sarcoma family of tumors, extracranial primitive neuroectodermal tumor, extragonadal primitive neuroectodermal tumor, extrahepatic bile duct cancer, eye cancer, malignant fibrous histiocytoma of bone and osteosarcoma, gallbladder cancer, gastric (stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), soft tissue sarcoma, germ cell tumor, gestational trophoblastic tumor, glioma, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, islet cell tumor (pancreas), Kaposi sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral cavity cancer, liver cancer (primary), lobular carcinoma in situ (LCIS), lung cancer, lymphoma, macroglobulinemia, male breast cancer, malignant fibrous histiocytoma and osteosarcoma of bone, medulloblastoma, medulloepithelioma, melanoma, Merkel cell cancer, mesothelioma, metastatic squamous cell carcinoma with potential primary midline cancer associated with the NUT gene, oral cancer, multiple endocrine neoplasia syndrome, multiple myeloma / plasma cell tumor, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasm, myeloid leukemia, chronic (myelogenousLeukemia, chronic (CML), myeloid leukemia, acute (AML), multiple myeloma, myeloproliferative disorders, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, hypopharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma of bone, ovarian cancer, pancreatic cancer, papilloma, paraganglioma, paranasal sinus and nasal cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, intermediate pineal parenchymal tumor, pineoblastoma and supratentorial primitive neuroectodermal tumor (PNET), pituitary tumor, plasma cell tumor / multiple myeloma, pleuropulmonary blastoma, pregnancy and breast cancer, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, renal pelvis and ureter, transitional cell carcinoma, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, Sézary syndrome, small cell lung cancer, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, squamous cell carcinoma of the neck, gastric (stomach) cancer, supratentorial primitive neuroectodermal tumor (PNET), T-cell lymphoma, skin, testicular cancer, pharyngeal cancer, thymoma and thymic cancer, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, choriocarcinoma, ureter and renal pelvis cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, Wilms tumor, including but not limited to, any cancer associated with a surface antigen or cancer marker.

[0158] In some embodiments, the cancer is a B cell malignancy. Examples of B cell malignancies include, but are not limited to, non-Hodgkin lymphoma (NHL), diffuse large B cell lymphoma (DLBCL), small lymphocytic lymphoma (SLL / CLL), mantle cell lymphoma (MCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), extranodal (MALT lymphoma), nodal (monocytoid B cell lymphoma), splenic, diffuse large cell lymphoma, B cell chronic lymphocytic leukemia / lymphoma, Burkitt lymphoma, and lymphoblastic lymphoma. As used herein, "viral infection" can be an infection caused by any virus that causes a disease or pathological condition in a host. Examples of viral infections that can be treated with engineered T cells produced by the methods described herein include viral infections caused by Epstein-Barr virus (EBV); viral infections caused by hepatitis A virus, hepatitis B virus, or hepatitis C virus; viral infections caused by herpes simplex type 1 virus, herpes simplex type 2 virus, or herpes simplex type 8 virus, viral infections caused by cytomegalovirus (CMV), viral infections caused by human immunodeficiency virus (HIV), viral infections caused by influenza virus, viral infections caused by measles or mumps virus, viral infections caused by human papillomavirus (HPV), viral infections caused by parainfluenza virus, viral infections caused by rubella virus, viral infections caused by respiratory syncytial virus (RSV), or viral infections caused by varicella zoster virus, but are not limited thereto. In some embodiments, the viral infection can lead to or result in the development of cancer in a subject having the viral infection (e.g., HPV infection can cause or be associated with the development of several cancers including cervical cancer, vulvar cancer, vaginal cancer, penile cancer, anal cancer, and oropharyngeal cancer, and HIV infection can cause the development of Kaposi sarcoma).Examples of chronic inflammatory diseases, autoimmune diseases, or any other immunodeficiencies that can be treated with engineered T cells produced by the methods described herein include, but are not limited to, multiple sclerosis, lupus, and psoriasis.

[0159] Further examples of chronic inflammatory diseases, autoimmune diseases, or any other immunodeficiencies that can be treated with engineered T cells produced by the methods described herein include rheumatoid arthritis, allergies, asthma, Crohn's disease, IBD, IBS, fibromyalgia, mastocytosis, and celiac disease.

[0160] As used herein, the terms "treat," "treating," or "treatment" with respect to a condition or disease can refer to preventing the condition or disease, slowing the rate of onset or development of the condition or disease, reducing the risk of developing the condition or disease, preventing or delaying the onset of symptoms associated with the condition or disease, reducing or eliminating the symptoms associated with the condition or disease, causing complete or partial regression of the condition or disease, or some combination thereof.

[0161] "Therapeutically effective amount" or "therapeutically effective dosage" is the amount of engineered lymphocytes that, by killing target cells, produces a desired therapeutic effect in a subject, such as preventing or treating a target condition or alleviating symptoms associated with the condition. The most effective results regarding the effectiveness of treatment in a given subject will vary depending on various factors including the characteristics of the engineered lymphocytes (including lifespan, activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological state of the subject (including age, sex, type and stage of disease, general physical condition, responsiveness to a given dosage, and type of drug), the nature of any pharmaceutically acceptable carrier(s) in any composition used, and the route of administration, among others. The therapeutically effective dosage of engineered lymphocytes may also depend on the cell surface receptors expressed by the lymphocytes (e.g., the affinity and density of cell surface receptors expressed on the cells), the type of target cells, the nature of the disease or disorder being treated, or a combination of both.

[0162] As shown in the examples, the engineered lymphocytes prepared by this process have a much increased in vivo effectiveness compared to the prior art and, thus, require a much lower dosage.

[0163] Thus, in some embodiments, the therapeutically effective dose of engineered lymphocytes is less than about 2,000,000 engineered lymphocytes per kilogram of body weight of the subject in need of treatment (cells / kg). Thus, in some embodiments, the therapeutically effective dose of engineered lymphocytes is from about 10,000 to about 2,500,000 engineered lymphocytes / kg. In certain embodiments, the therapeutically effective dose of engineered lymphocytes is from about 10,000 to about 1,500,000 engineered lymphocytes / kg. In certain embodiments, the therapeutically effective dose is from about 20,000 to about 1,200,000 engineered lymphocytes / kg. In certain embodiments, the therapeutically effective dose is from about 20,000 to about 1,000,000 engineered lymphocytes / kg. In certain embodiments, the therapeutically effective dose is from about 20,000 to about 500,000 engineered lymphocytes / kg. In certain embodiments, the therapeutically effective dose is from about 20,000 to about 400,000 engineered lymphocytes / kg. In certain embodiments, the therapeutically effective dose is from about 40,000 to about 400,000 engineered lymphocytes / kg. In certain embodiments, the therapeutically effective dose is from about 50,000 to about 200,000 engineered lymphocytes / kg. In certain embodiments, the therapeutically effective dose is from about 50,000 to about 100,000 engineered lymphocytes / kg.

[0164] In some embodiments, the therapeutically effective dose of engineered lymphocytes is from about 1,600,000 to about 2,500,000 engineered lymphocytes per kilogram of body weight of the subject in need of treatment (cells / kg). In some embodiments, the therapeutically effective dose of engineered lymphocytes is from about 2,000,000 to about 2,400,000 engineered lymphocytes per kilogram of body weight of the subject in need of treatment (cells / kg).

[0165] In some embodiments, the administered T cells are Yescarta® (axicabtagene ciloleucel). In some embodiments, the administered T cells are Tecartus® (brexucabtagene autoleucel).

[0166] The following examples are intended to illustrate various embodiments of the present invention. Accordingly, the specific embodiments considered should not be construed as limitations on the scope of the present invention. For example, the following examples are directed to T cells transduced with an anti-CD19 chimeric antigen receptor (CAR), but those skilled in the art will understand that the methods described herein can be applied to T cells transduced with any CAR. It is clear that various equivalents, changes, and modifications can be made without departing from the scope of the invention, and it is understood that such equivalent embodiments are included herein. Further, all references cited in this disclosure are hereby incorporated by reference in their entirety as if fully set forth herein.

[0167] Example 1: 7-Day Lymphocyte Production Process

[0168] This example describes a process for preparing lymphocytes transduced with a polynucleotide vector such as a viral vector encoding a therapeutic protein. The prepared lymphocytes can be useful for treating various diseases such as cancer, particularly when the therapeutic protein is a chimeric antigen receptor (CAR) or a T cell receptor (TCR) designed to target cancer cells.

[0169] When used throughout, the terms "7-day process" and "7-day lymphocyte production process" are used interchangeably and refer to a CAR cell production process that takes approximately 7 days after the initial enrichment and activation steps. The 7-day process has a length of at least 8 days from the initial enrichment and activation steps to the harvesting step, and can be 8 - 11 days in total when including the enrichment and activation steps.

[0170] Apheresis collection. White blood cells were collected (leukapheresis) using a standard apheresis device such as Cobe® Spectra, Spectra Optia®, Fenwal™ Amicus®, or equivalent. Typically, about 200 - 400 mL of apheresis product was obtained from the patient by the leukapheresis process. The apheresis product can be subjected to the manufacturing process on-site or, optionally, transported to the facility at 1 - 10 °C and undergo the manufacturing process at a different location. Further process steps can be carried out in an ISO 7 cell culture process suite (or a similar cleanroom-type environment).

[0171] Volume reduction. If necessary, the volume reduction step was performed using a cell processing device such as Sepax® 2 laboratory equipment (Biosafe SA, Houston, TX) or equivalent, and executed using a standard sterile tube kit. Considering the variability in the number of cells from each subject and the volume of the incoming source material (about 200 - 400 mL), the volume reduction step was designed to standardize the cell volume to about 120 mL. If the apheresis volume is less than 120 mL, there is no need to perform the volume reduction step, and the cells are directly carried to the lymphocyte enrichment step. The volume reduction step is designed to standardize the volume of cells received from each subject, retain mononuclear cells, achieve consistent cell yields and high cell viability, and maintain a closed system to minimize the risk of contamination.

[0172] Lymphocyte enrichment. After the volume reduction step, the cells were subjected to ficoll-based separation using a cell processing device such as Sepax® 2 or equivalent, using a standard sterile tube kit and a separation protocol developed and recommended by the equipment manufacturer (NeatCell Program). The lymphocyte enrichment step reduces product-related impurities such as RBC and granulocytes, enriches and concentrates mononuclear cells, washes and reduces process-related residues such as ficoll, formulates the cells in a growth medium for cell activation preparation, and achieves consistent cell yields and high cell viability. The closed system minimizes environmental contamination.

[0173] The process can be carried out within an ISO 7 area at ambient temperature, and all connections can be made using a sterile tube welder or within an ISO 5 laminar flow hood.

[0174] Lymphocyte activation. The lymphocyte activation step can be carried out using either newly processed cells from lymphocyte enrichment or pre-frozen and stored cells. If frozen and stored cells are used, the cells can be thawed using a protocol developed prior to use.

[0175] The lymphocyte activation step selectively activates lymphocytes to make them receptive to retroviral vector transduction, reduces the population of all other cell types that survive, achieves consistent cell yields and high lymphocyte viability, and maintains a closed system to minimize the risk of contamination. Lymphocyte activation can be achieved using lymphocyte stimulants such as anti-CD3 antibody and IL-2.

[0176] Washing 1. After the lymphocyte activation step, the cells were washed using a cell processing device such as Sepax® 2 or equivalent, using a protocol developed by the manufacturer, in a standard sterile kit with fresh culture medium. For preparation for retroviral vector transduction, the cells were optionally concentrated to a final volume of approximately 100 mL. The washing 1 step reduces process-related residues such as anti-CD3 antibody, used growth medium, and cell debris, achieves consistent cell yields and high T cell viability, maintains a closed system to minimize the risk of contamination, and concentrates and delivers a sufficient number of viable T cells in a small volume suitable for the start of transduction.

[0177] Transfection. Activated cells from the washing step 1 in fresh cell growth medium were transferred to a cell culture bag (Origen Biomedical PL240 or equivalent) pre-prepared by first coating with a recombinant fibronectin such as RetroNectin® (Takara Bio, Japan) or a fragment thereof, and then incubated with a retroviral vector according to a defined procedure prior to the introduction of the activated cells. RetroNectin® coating (10 μg / mL) was performed at a temperature of 2 - 8 °C for 20 ± 4 hours, washed with dilution buffer, and then incubated at 37 ± 1 °C and 5 ± 0.5% CO 2 for about 180 - 210 minutes with the thawed retroviral vector. After addition of the cells to the bag, transfection was performed at 37 ± 1 °C and 5 ± 0.5% CO 2 for 20 ± 4 hours. The retroviral transfection process cultures activated T cells in the presence of a retroviral vector under controlled conditions to enable efficient transfection to occur, achieve consistent cell yields and high cell viability, and maintain a closed system to minimize the risk of contamination.

[0178] Washing step 2. After the retroviral transfection process, the cells were washed with fresh growth medium using a cell processing device such as Sepax® 2 or equivalent in a standard sterile kit using a protocol developed by the manufacturer, and the cells were concentrated to a final volume of about 100 mL in preparation for the expansion process. The washing step 2 is designed to reduce process-related residues such as retroviral vector particles, vector production process residues, used growth medium, and cell debris, achieve consistent cell yields and high cell viability, maintain a closed system to minimize the risk of contamination, and replace the used growth medium with fresh medium having a target cell number in a specific volume suitable for the start of the expansion process.

[0179] Lymphocyte expansion. Cells from the two washing steps are aseptically transferred to a culture bag (Origen Biomedical PL325 or equivalent), diluted with fresh cell growth medium, and cultured at 37 ± 1 °C and 5 ± 0.5% CO 2 for approximately 72 hours. Cell density was measured daily starting on day 5. Since the doubling time of T cells may vary slightly for each subject, additional growth time beyond 72 hours (i.e., 3 - 6 days) may be required if the total cell number is insufficient to deliver the target dose of CAR-positive T cells / kg of subject body weight. The lymphocyte expansion process is designed to culture cells under controlled conditions to produce a sufficient number of transduced cells to deliver an effective dose, maintain a closed system to minimize the risk of contamination, and achieve consistent cell yields and high cell viability. One such effective or target dose is 2 × 10 6 FMC63-28Z CAR-positive or FMC63-CD828BBZ CAR-positive T cells / kg of subject body weight (±20%), both of which are produced via transduction with either the MSGV-FMC63-28Z retroviral vector or the MSGV-FMC63-CD828BBZ retroviral vector, as described in detail in Kochenderfer et al., J Immunother. 2009 September;32(7):689 - 702.

[0180] Washing 3 and concentration. After the lymphocyte expansion process, the cells are washed with 0.9% saline using cell processing equipment such as Sepax® 2 or equivalent in a standard sterile kit using a protocol developed by the manufacturer, and concentrated to a final volume of approximately 35 mL for formulation and cryopreservation. The washing 3 process is designed to reduce process-related residues such as retrovirus production process residues, used growth medium, and cell debris, maintain a closed system to achieve consistent cell yields and high cell viability, and minimize the risk of contamination.

[0181] When the cells are concentrated and washed in 0.9% saline, an appropriate cell dose can be formulated for the preparation of the final cryopreserved product.

[0182] The embodiments described herein provide for the efficient production of lymphocyte therapy engineered within 7 days.

[0183] Example 2: 5-Day Lymphocyte Manufacturing Process

[0184] This example developed an accelerated process based on the 7-day process described in Example 1.

[0185] When used throughout, the terms "5-day process" and "5-day lymphocyte manufacturing process" are used interchangeably and refer to a CAR cell manufacturing process that takes approximately 5 days after the initial enrichment and activation steps. The 5-day process is 6 days in length from the initial enrichment and activation steps to the harvest step and can be 6 - 9 days in total when including the enrichment and activation steps.

[0186] During the 7-day process, lymphocytes are enriched and activated on day 0, the transduction bag is coated with recombinant fibronectin on day 1, viral transduction is performed on day 2, the transduced lymphocytes are washed, then expanded on days 3 and 4, the expansion is continued by changing the medium daily on days 5 and 6, and the final cell product is harvested on day 7. From approximately 1.2×10 9 lymphocytes obtained from apheresis, approximately 2.4×10 8 lymphocytes are incubated with a viral vector for transduction.

[0187] In the newly developed 5-day process, no changes were made to the process on day 0. However, on days 1 and 2, larger bags were used. Instead of the Origen Biomedical PL240 bags used for transduction, Origen Biomedical PL325 bags, or more preferably PL750 bags, were used. The larger bags allowed for the use of a larger volume of vector (200 mL instead of 100 mL) and more lymphocytes (3.2×10 8 cells instead of 2.4×10 8 ~6×10 8 cells) in the transduction step.

[0188] Interestingly, along with more vector starting lymphocytes, the increase in transduction volume did not result in an unacceptable reduction in transduction efficiency (54% - 35.15%) or cell viability (92% - 92.4%) (see Table 1). Thus, the cell expansion step, which required 4 days in the 7-day process, was reduced to 2 days, enabling the collection of the final cell product on day 5. TIFF2025518111000001.tif33128

[0189] However, a slight reduction in transduction rate was not correlated with clinical efficacy or patient safety. Also importantly, cell products from the 5-day process were found to contain an increase in the percentage of naive cells in both the CD4+ T cell and CD8+ T cell populations (Table 2), which is thought to be related to improved therapeutic efficacy. Note that these variations are within the range of donor performance history collected from the 7-day process. In Table 2 below, the historical average from the 7-day process runs is reflected in the column labeled "Historical Average", and the data from the 5-day and 7-day runs conducted in this study are shown in the columns labeled "5-day" and "7-day", respectively. TIFF2025518111000002.tif46129CM: Central memory T cells, Teff: T effector cells, EM: Effector memory T cells.

[0190] Therefore, this example shows that the shortened 5-day process met the specification requirements for transduction efficiency, effectiveness, and safety. On the other hand, the 5-day product showed a younger phenotype within the historical range.

[0191] The in vivo effectiveness of the cell product from the 5-day process was tested in a mouse model transplanted iv with 5×10 5 cells of the Nalm6 cell line (human acute lymphoblastic leukemia (ALL) strain) in 100 μL solution. The CAR used targeted CD19 and was injected on day 6 at a dose of 5×10 6 cells, 1×10 6 cells, or 0.2×10 6 cells / kg body weight (Table 3). TIFF2025518111000003.tif55128

[0192] The body weight changes (Table 4) correlated with the increase in tumor burden, indicating that all of these treatments were safe for the animals. TIFF2025518111000004.tif80170

[0193] The results of tumor inhibition are shown in Table 5 (measured as photons / second). TIFF2025518111000005.tif84170

[0194] 5×10 6 When dosed at cells / kg, the cell product from the 5-day process showed complete tumor growth inhibition. At all doses, the cell product from the 5-day process performed better than the cell product from the 7-day process. The cells from the 5-day process were also more effective than the cells from the 7-day process even when the percentage of CAR+ cells matched (i.e., there were fewer CAR-T cells from the 5-day process).

[0195] Example 3: 3-day lymphocyte manufacturing process

[0196] Based on the 7-day process described in Example 1 and the 5-day process described in Example 2, this example developed a further accelerated process.

[0197] When used throughout, the terms "3-day process" and "3-day lymphocyte manufacturing process" are used interchangeably and refer to a CAR cell manufacturing process that takes approximately 3 days from the initial enrichment and activation steps. The 3-day process is approximately 4 days in length from the initial enrichment and activation steps to the collection step. The 3-day process does not include a cell expansion step that includes more than 1 day after the transduction step and before the collection step.

[0198] In this new process, the procedures on days 0-1 are the same as the 5-day process, including fibronectin coating of a larger bag (e.g., Origen Biomedical PL325 or preferably PL750) for subsequent transduction on day 2. However, on day 2, only about 4.8×10 8 cells (instead of 6×10 8 cells) of lymphocytes were used in the transduction step together with the same amount of viral vector (200 mL). Another important difference is that, unlike the 5-day and 7-day processes, no specific step of T cell expansion was performed. Instead, the transduced lymphocytes were collected on day 3, and the entire process was completed within 3 days from the initial enrichment and activation steps.

[0199] Considering the lack of a specific T cell expansion step in this newly developed 3-day process, the collected cell product contained a slightly lower percentage of T cells (CD3+). However, importantly, the product on day 3 contained a higher percentage of naive T cells (Table 6). Note that in Table 6 below, the historical average from running the 7-day process is reflected in the column labeled "Historical Average", and the data from the 3-day and 7-day runs performed in this study are shown in the columns labeled "3-day" and "7-day", respectively. TIFF2025518111000006.tif46129CM: Central memory T cells, Teff: T effector cells, EM: Effector memory T cells.

[0200] Even if lymphocytes from the same donor were not used for testing, the comparison between Tables 2 and 6 shows that the percentage of naive T cells from the 3-day process was also significantly higher than that from the 5-day process. Within CD4+ T cells, the 3-day process generated approximately 55.75% naive T cells, while the 5-day process generated approximately 40.65%. Within CD8+ T cells, the 3-day process generated approximately 37.35% naive T cells, while the 5-day process generated approximately 3.93%.

[0201] In other words, the 3-day process results in an approximately 1.4-fold increase in the percentage of CD4+ naive T cells compared to the percentage of such cells observed from the 5-day process, and an approximately 9.5-fold increase in the percentage of CD8+ naive T cells compared to the percentage of such cells observed from the 5-day process. Also, the 3-day process results in an approximately 3.0-fold increase in the percentage of CD4+ naive T cells compared to the historical average of such cells observed from the 7-day process, and an approximately 18.0-fold increase in the percentage of CD8+ naive T cells compared to the historical average of such cells observed from the 7-day process.

[0202] Conversely, within CD4+ T cells, the 3-day process generated only approximately 4.35% effector memory T cells, while the 5-day process generated approximately 8.55%. Within CD8+ T cells, the 3-day process generated only approximately 9.85% effector memory T cells, while the 5-day process generated approximately 15.85%.

[0203] In other words, the 3-day process results in a decrease of approximately 2.0-fold in the percentage of CD4+ effector memory T cells compared to the percentage of such cells observed in the 5-day process, and a decrease of approximately 1.6-fold in the percentage of CD8+ effector memory T cells compared to the percentage of such cells observed in the 5-day process. Also, the 3-day process produces a decrease of approximately 6.0-fold in the percentage of CD4+ effector memory T cells compared to the historical average of such cells observed in the 7-day process, and a decrease of approximately 3.5-fold in the percentage of CD8+ effector memory T cells compared to the historical average of such cells observed in the 7-day process.

[0204] Also, as can be understood from Table 6, the percentage of CD4+CCR7+ cells (i.e., CM and naive cells) in the population finally collected from the 3-day process is at least 95% of the collected CD4+ T cell population. In other words, the maximum percentage of Teff / TEMRA and EM CD4+ cells in the population finally collected from the 3-day process is approximately 5%. Thus, there are at least 19 times more CD4+CCR7+ cells than Teff / TEMRA and EM CD4+ cells in the collected population. In comparison, the percentage of CD4+CCR7+ cells (i.e., CM and naive cells) in the population finally collected from the 7-day process is approximately 71% of the collected CD4+ T cell population. In other words, the maximum percentage of Teff / TEMRA and EM CD4+ cells in the population finally collected from the 7-day process is approximately 29%. Thus, the 3-day process generates a CD4+ T cell population in which the percentage of CD4+CCR7+ cells is increased by approximately 1.3-fold compared to the 7-day process, and the percentage of Teff / TEMRA and EM CD4+ cells is decreased by approximately 5-fold.

[0205] As can be seen in Table 6, the percentage of CD8+CCR7+ cells (i.e., CM and naive cells) in the population finally harvested from the 3-day process is at least 84% of the harvested CD8+ T cell population. In other words, the maximum percentage of Teff / TEMRA and EM CD4+ cells in the population finally harvested from the 3-day process is approximately 16%. Thus, there are at least about five times more CD8+CCR7+ cells than Teff / TEMRA and EM CD4+ cells in the harvested population. In comparison, the percentage of CD8+CCR7+ cells (i.e., CM and naive cells) in the population finally harvested from the 7-day process is approximately 66% of the harvested CD8+ T cell population. In other words, the maximum percentage of Teff / TEMRA and EM CD8+ cells in the population finally harvested from the 7-day process is approximately 34%. Thus, the 3-day process generates a CD8+ T cell population with a percentage of CD8+CCR7+ cells increased by about 1.3 times that of the 7-day process and a percentage of Teff / TEMRA and EM CD8+ cells decreased by about 2 times.

[0206] Cell viability measurements showed that cell viability remained high (>90%) throughout the 3-day process, but the washing step on day 2 caused the greatest reduction in cell viability. In contrast, the 5-day and 7-day processes included additional washing steps, each of which contributed to an additional decrease in cell viability (Table 7). TIFF2025518111000007.tif64128

[0207] Example 4: In Vivo Efficacy of Cells Prepared by the 3-Day Process

[0208] This example compared the in vivo efficacy of cells prepared by the 3-day process described in Example 3 with that from the 7-day process using the same animal model and procedure described in Example 2.

[0209] The experimental groups are shown in Table 8, and the body weight changes are shown in Table 9. TIFF2025518111000008.tif59149TIFF2025518111000009.tif45170

[0210] The results of tumor inhibition are shown in Table 10 (measured in photons / second). TIFF2025518111000010.tif50170

[0211] At all three doses (0.5×, 1×, and 3×10 6 cells / kg), the cell products from the 3-day process were able to completely inhibit tumor growth throughout the observation period without significant difference. In contrast, the cells from the 7-day process were able to inhibit tumor growth only through 15 days (0.5 and 1×10 6 cells / kg), and 23 days (3×10 6 cells / kg).

[0212] Therefore, this example shows that the 3-day process can prepare cell products with significantly improved anti-tumor efficacy compared to the 7-day process. Furthermore, considering the significantly improved efficacy, the dose required to achieve a much more complete anti-tumor response is much lower.

[0213] In further experiments, even lower doses of cells from the 3-day process (0.2×10 6 cells / kg, 3 different donors) were compared to high doses of cells from the 7-day process (1×10 6 cells / kg). The results (Table 11) show that even with a 5-fold difference, the results of the 3-day process were far superior to those of the 7-day process. TIFF2025518111000011.tif88170

[0214] Example 5: Influence of Early Collection on CAR

[0215] This example found that when the CAR construct contains the CD28 co-stimulatory domain, the improvement in the performance of the shortened (earlier harvested) process is more significant compared to the 4-1BB co-stimulatory domain.

[0216] Both types of CARs contain a CD19-binding antigen-binding fragment. One, CAR number 1, contains the CD28 co-stimulatory domain, and the other, CAR number 2, contains the 4-1BB co-stimulatory domain. Both were delivered by a lentiviral vector. Cells were harvested on days 4, 7, and 14, and the doses for the in vivo animal study included 0.2×10 6 cells / kg and 1×10 6 cells / kg (Table 12). The results are shown in Table 13. TIFF2025518111000012.tif55128TIFF2025518111000013.tif86170

[0217] As shown in Table 13, at the later harvest (day 7 or 14), the difference in performance between the two constructs was limited. However, at the early harvest, which benefited both constructs, CAR number 1 (G3) showed significantly higher in vivo efficacy than CAR number 2 (G8).

[0218] Furthermore, a CAR targeting CD19 and containing the CD28 co-stimulatory domain was tested in vivo using a Nalm6 mouse model of leukemia. The results showed that the early harvest (day 4) resulted in better in vivo antitumor efficacy than the normal harvest (day 7), which in turn was more effective than the late harvest (day 14). In fact, T cells harvested at a low dose (2×10 5 cells) on day 4 were five times more effective than T cells harvested at a high dose (10 6 cells) on day 7.

[0219] Example 6 In vivo efficacy of cells prepared by a 3-day process

[0220] This example complements Example 4 and presents additional data comparing the in vivo efficacy of anti-CD19 CAR T cells prepared by the 3-day process described in Example 3 with those prepared by a 7-day process.

[0221] Anti-CD19 CAR T cells prepared by a 3-day process and anti-CD19 CAR T cells prepared by a 7-day process were manufactured from healthy human donors, along with their respective non-transduced (NTD) day 3 and NTD day 7 cells. NALM6-luc cells (5×10 5 cells) were IV transplanted via the lateral tail vein into 8-week-old female NSG mice (5 mice per group). On day 6 post-tumor transplantation, the mice received either a control (NTD day 3 or NTD day 7) or one of the anti-CD19 CAR T cell products as a single-dose cohort or a multiple-dose cohort. A total of five dose cohorts (1×10 6 , 5×10 5 , 3×10 5 , 2×10 5 , and 1×10 5 CAR + T cells) were tested for cells derived from the 3-day process, and four dose cohorts (1×10 7 , 5×10 6 , 3×10 6 , and 1×10 6 CAR + T cells) were tested for cells derived from the 7-day process. Anti-tumor activity was evaluated by determining the effect of the anti-CD19 CAR T cell product on tumor burden and animal survival.

[0222] Antitumor activity was assessed by evaluating the change in tumor burden, as measured by bioluminescence imaging (BLI), from day 5 (one day before CAR T cell treatment) to day 22 (16 days after CAR T cell treatment, the last time point when all groups were intact). Tumor growth inhibition was observed in all four dose cohorts of mice treated with anti-CD19 CAR T cells derived from the 7-day process and in all five dose cohorts of anti-CD19 CAR T cells derived from the 3-day process (Tables 14, 15, and 16). For anti-CD19 CAR T cells derived from the 3-day process, tumor growth inhibition compared to the no treatment group was 1×10 6 , 5×10 5 , 3×10 5 , 2×10 5 , and 1 × 10 5 CARs + In animals treated with T cells, the tumor growth inhibition was 128.1%, 126.7%, 117.7%, 113.4%, and 65.7%, respectively. For anti-CD19 CAR T cells derived from the 7-day process, tumor growth inhibition ranged from 109.0% to 115.1% for the dose cohorts tested (Table 16). Tumor burden was 1×10 6 of anti-CD19 CAR T cells prepared by the 3-day process, which resulted in delayed tumor control by day 26. 5 CARs + All groups and all CARs tested, except for the T cell dose cohort. + At doses of T cells, regression occurred at day 22. Antitumor efficacy at all doses of anti-CD19 CAR T cells prepared by the 3-day process and anti-CD19 CAR T cells prepared by the 7-day process reached statistical significance at day 22 when compared to untreated mice or NTD controls (Tukey's post-hoc test, p<0.05 considered significant). TIFF2025518111000014.tif64128TIFF2025518111000015.tif113170TIFF2025518111000016.tif28170

[0223] In Table 16, the tumor burden (measured by BLI) was log 10 normalized and the means for each cohort on Day 5 and Day 20 of the study were tabulated. The change in tumor BLI from Day 5 to Day 20 was calculated, and the percent change in tumor burden relative to the untreated group was calculated as ΔT / ΔC. Tumor growth inhibition was defined as (1 - [ΔT / ΔC]×100) and represented the percentage change in tumor volume in the treatment group relative to the control (i.e., mice that did not receive treatment). 0% tumor growth inhibition (TGI) indicated that the mean tumor growth in the group was equivalent to the mean tumor growth observed in the untreated group, while 100% TGI indicated that no tumor growth was observed from Day 5 to Day 20. TGI > 100% indicated that the mean tumor burden regressed from Day 5 to Day 20, while TGI < 0 indicated that the increase in tumor burden was greater than that seen in the untreated group (G1).

[0224] Treatment of mice with anti-CD19 CAR T cells prepared by a 3-day process or anti-CD19 CAR T cells prepared by a 7-day process resulted in an extension of survival compared to control mice that received NTD or no treatment. NTD controls and untreated mice were euthanized by Day 8 based on a survival endpoint defined by bioluminescence of <5×10 8 photons / second. In contrast, the group treated with anti-CD19 CAR T cells prepared by a 3-day process experienced significantly greater survival, with 100% survival achieved at the study endpoint on Day 64 in mice from all dose cohorts. Mice treated with anti-CD19 CAR T cells prepared by a 7-day process had lower survival compared to the group treated with anti-CD19 CAR T cells prepared by a 3-day process, with 80% survival in the 1×10 7 individual CAR + T cell dose cohort, 20% survival in the 5×10 6 individual CAR + T cell dose cohort, and 3×10 6 and 1×106 Individual CAR + It was in the range of 0% survival of the cohort of T cell doses.

[0225] Mice that received anti-CD19 CAR T cells prepared by the 3-day process at all doses had 3×10 6 and 1×10 6 Individual CAR + For mice that received anti-CD19 CAR T cells prepared by the 7-day process at the T cell dose, a statistically significant improvement in response was achieved, but it was not statistically different from the 1×10 7 Individual CAR + T cell dose. All doses of anti-CD19 CAR T cells prepared by the 3-day process resulted in complete antitumor responses and 100% survival by the end of the study (day 64), but the highest dose (1×10 7 Individual CAR + T cells) of anti-CD19 CAR T cells prepared by the 7-day process resulted in 80% survival by the end of the study.

[0226] Finally, the increase in tumor burden over time in the untreated group resulted in an average weight loss of >18% and / or adverse clinical signs by day 26, after which all animals were excluded from the study. Animals that received NTD T cells showed a similar pattern of weight loss due to uncontrolled tumor burden by day 29. In contrast, all doses of anti-CD19 CAR T cells prepared by the 3-day process of 1×10 6 ~1×10 5 Individual cells had good tolerance, and the animals maintained a consistent body weight during the study period. For the groups treated with anti-CD19 CAR T cells prepared by the 7-day process, all mice at all doses tested (1×10 7 , 5×10 6 , 3×10 6 , and 1×10 6 Individual CAR + T cells) maintained a consistent body weight during the study period.

[0227] Example 7 Characterization of Cells Prepared by a 3-Day Process

[0228] This example describes the functional and phenotypic characterization of anti-CD19 / CD20 CAR T cells collected on day 3 compared to anti-CD19 / CD20 CAR T cells collected on day 6.

[0229] Briefly, positively selected CD4 + and CD8 + T cells from healthy human donors were activated and transduced using a lentiviral vector encoding a bispecific anti-CD19 CAR and anti-CD20 CAR. Subsequently, the transduced cells were collected on day 3. Similarly, another set of anti-CD19 / CD20 CAR T cell products was produced from T cells collected on day 6 from the same donor (referred to as "anti-CD19 / CD20 CAR day 6" in Examples 7 and 8). Subsequently, both CAR T cell products were functionally characterized and compared to their respective NTD control T cells (NTD day 3 and NTD day 6) produced in parallel from the same donor material.

[0230] Anti-CD19 / CD20 CAR T cells collected on day 3 were produced by the following 3-day process starting from apheresis material collection. The starting apheresis material can optionally be fresh or cryopreserved apheresis or cryopreserved T cells. The steps of Wash 1, Wash 2, T cell enrichment, and Wash 3 were performed on day 0. T cell activation was performed from day 0 to a maximum of day 1. Lentiviral transduction was performed from day 1 to a maximum of day 4. Collection, washing, and concentration were performed from day 3 to a maximum of day 4.

[0231] The anti-CD19 / CD20 CAR T cells collected on the 6th day were produced by the following process starting from apheresis material collection. The steps of Wash 1, Wash 2, T cell enrichment, and Wash 3 were performed on the 0th day. T cell activation was performed from the 0th day to a maximum of the 2nd day. Lentiviral transduction was performed from the 2nd day to a maximum of the 5th day. Wash 4 was performed from the 4th day to a maximum of the 5th day. T cell expansion was performed from the 4th day to a maximum of the 15th day. Collection wash and concentration were performed from the 6th day to a maximum of the 15th day.

[0232] The experimental groups for the study described in this example are shown in Table 17. TIFF2025518111000017.tif29128

[0233] The anti-CD19 / CD20 CAR T cells produced by the 3-day process and the anti-CD19 / CD20 CAR 6-day T cell product were evaluated for CAR cell surface expression to establish the CAR transduction efficiency. The cell surface expression of both anti-CD19 CAR and anti-CD20 CAR was individually detected by flow cytometry using a fluorescently labeled anti-idiotype antibody. The individual expression of anti-CD19 CAR was 68% for the anti-CD19 / CD20 CAR T cells produced by the 3-day process and 35% for the anti-CD19 / CD20 CAR 6-day T cell product, and the expression of anti-CD20 CAR was 65% and 37% respectively in the same samples. The transduction efficiency measured by the total of the total anti-CD19 and anti-CD20 CAR expression (i.e., the percentage of anti-CD19 CAR antibody + cells + anti-CD29CAR antibody + cells percentage) was 72% for the anti-CD19 / CD20 CAR T cells produced by the 3-day process and 40% for the anti-CD19 / CD20 CAR 6-day T cell product.

[0234] CD4 + and CD8 +To evaluate the presence of cells and the relative composition of the mostly undifferentiated T cell subsets, the phenotype of the CAR T cell product was characterized by flow cytometry after incubation with fluorescently labeled antibodies. The CD45RA and CCR7 markers can be used to define various T cell populations, including naive and stem memory T cells, also known as young T cells (CD45RA + CCR7 + ). The anti-CD19 / CD20 CAR T cells produced by the 3-day process showed a slightly higher CD4 / CD8 ratio than the anti-CD19 / CD20 CAR 6-day T cell product (Table 18). TIFF2025518111000018.tif31128

[0235] Both the CD4 + and CD8 + populations of the anti-CD19 / CD20 CAR T cells produced by the 3-day process showed an increase in the frequency of young cells compared to the anti-CD19 / CD20 CAR 6-day T cell product (Tables 19 and 20). TIFF2025518111000019.tif17128TIFF2025518111000020.tif17128

[0236] The functionality of the CAR T cell product was evaluated in co - culture assays with antigen - positive target cells and antigen - negative target cells. The NTD T cell product was included in the experiment as a control to assess the level of alloreactivity. Cytotoxicity was measured 1 day and 4 days after the start of co - culture of the T cell product with luciferase - expressing target cells. Cytotoxicity was measured as the reduction of the luciferase signal released in the test wells upon addition of luciferin, compared to wells seeded with target cells alone. Multiple E:T ratios were used in these assays in the range of 1:1 to 1:243. Four major cell lines expressing various levels of CD19 and CD20 antigens were used: Raji, a B - cell lymphoma cell line expressing high levels of CD19 and CD20 antigens; Nalm6, a B - cell leukemia cell line expressing high levels of CD19 but low levels of CD20; ST486, a B - cell lymphoma cell line expressing low levels of CD19 but high levels of CD20; and K - 562, a chronic myelogenous leukemia cell line that does not express CD19 or CD20 antigens. In addition, the controls consisted of Raji CD19 knockout (KO) cells and Raji CD20 KO cells. The Raji cell line was engineered to express either antigen: Raji CD19 KO cells express only CD20 and not CD19, and Raji CD20 KO cells express only CD19 and not CD20. Antigen KO was confirmed at the DNA level by tracking insertions or deletions (indels) by decomposition (TIDE) analysis and at the cell - surface protein level by flow cytometry.

[0237] On day 4 of co-culture, the anti-CD19 / CD20 CAR T cells and anti-CD19 / CD20 CAR D6 T cell products produced by the 3-day process showed equivalent dose-dependent specific cytotoxicity against antigen-positive target cells, regardless of the expression of either or both antigens (Tables 21A-21F). The basal level of antigen-independent cytotoxicity mediated by alloreactivity against target cells was observed at the highest E:T ratio by the cytotoxicity of NTD D3 control T cells against ST486 cells (low CD19 / high CD20 expression). (Table 21A). Both the anti-CD19 / CD20 CAR T cells produced by the 3-day process and the anti-CD19 / CD20 CAR D6 T cell products derived from healthy donors had equivalent cytotoxic activity across the various target cell lines tested and lacked killing activity against antigen-negative K-562 cells (Table 21F). TIFF2025518111000021.tif40160TIFF2025518111000022.tif40149TIFF2025518111000023.tif40164TIFF2025518111000024.tif40164TIFF2025518111000025.tif34170TIFF2025518111000026.tif40139

[0238] Specific activity was further evaluated by measuring cytokine levels in the culture supernatant after an overnight co-culture with Nalm6, ST486, Raji, Raji CD19KO, Raji CD20KO, and K-562 target cells at an E:T ratio of 1:1. Quantification of IFN-γ, IL-2, tumor necrosis factor-α (TNF-α) pro-inflammatory cytokine production was performed.

[0239] Anti-CD19 / CD20 CAR T cells and anti-CD19 / CD20 CAR T cell products produced by the 3-day process demonstrated robust antigen-dependent IFN-γ, IL-2, and TNF-α pro-inflammatory cytokine production from co-culture with antigen-positive target cells, Nalm6, ST486, Raji, Raji CD19KO, and Raji CD20KO (Tables 22A - 22C). Both anti-CD19 / CD20 CAR T cells and anti-CD19 / CD20 CAR T cell products produced by the 3-day process showed equivalent cytokine levels of IL-2 and TNF-α across the various target cell lines tested. The functionality of each individual CAR was demonstrated by cytokine production when the T cell product was co-cultured with target cells expressing a single antigen (Raji CD19KO or Raji CD20KO), but was at lower levels than those induced by co-culture with the Raji parental cells expressing both antigens. In contrast, in the absence of target cells (i.e., T cells alone), cytokine production was not detected, and as shown, no cytokine production was observed from CAR T cells co-cultured with antigen-negative K-562 (CD19 - CD20 - ) cells (Tables 22A - 22C). TIFF2025518111000027.tif71170TIFF2025518111000028.tif72170TIFF2025518111000029.tif71170

[0240] To evaluate antigen-specific proliferation of the CAR T cell products upon binding to the target antigen, T cell products co-cultured with Raji, Nalm6, Raji CD19KO, Raji CD20KO, and K-562 target cells were harvested 4 days after the start of co-culture, centrifuged to pellet, and prepared for flow cytometry evaluation. The T cells were pre-labeled with a fluorescent dye used to track multiple generations of cells by analysis of dye dilution during cell division. A 1:1 E:T ratio from the co-cultures was used, and T cell products cultured in the absence of target cells (T cells alone) were used as controls. Antigen-negative K-562 (CD19 - CD20- ) T cells co-cultured with cells were used to evaluate the specificity and level of antigen-independent proliferation, while T cells alone were used to evaluate the basal level of proliferation in the absence of stimulation. At the time of collection, the cells were stained with fluorescently labeled antibodies against CD3, CD4, CD8, CD19 CAR, CD20 CAR, CD25 and a viability dye.

[0241] Anti-CD19 / CD20 CAR T cells produced by a 3-day process and anti-CD19 / CD20 CAR T cell products on day 6 showed equivalent proliferation in co-culture with Nalm6, Raji, Raji CD19KO, and Raji CD20KO target cells, but did not show equivalent proliferation in co-culture with antigen-negative K-562 cells, indicating that both anti-CD19 CAR and anti-CD20 CAR in the T cell products are functional. NTD T cell controls showed varying levels of non-specific basal proliferation when co-cultured with different target cell lines, but overall, day 3 NTD cells showed more basal proliferation than day 6 NTD cells.

[0242] Example 8 In vivo efficacy of cells prepared by a 3-day process

[0243] In vivo studies were performed in a disseminated xenograft mouse model of human B-ALL consisting of severely immunodeficient non-obese diabetic (NOD), severe combined immunodeficient (scid) interleukin-2 receptor gamma chain null (NSG) mice injected intravenously with Nalm6-luc cells expressing high levels of CD19 and CD20. Antitumor efficacy was evaluated for CAR T cell products generated from T cells derived from one healthy donor and previously characterized by the in vitro studies described in Example 7.

[0244] Nalm6luc cells (5.0×10 5(number) were intravenously transplanted into 7-week-old female NSG mice (5 mice per group) via the lateral tail vein. On the 6th day after tumor transplantation, the mice received either a control (vehicle [phosphate-buffered saline] or NTD day 3 cells) or anti-CD19 / CD20 CAR T cells as follows: anti-CD19 / CD20 CAR T cells produced by a 3-day process at one of 3 dose levels (2.0×10 5 4.0×10 4 and 8.0×10 3 CAR + T cells), and anti-CD19 / CD20 CAR day 6 T cell product at 1 dose level (2.0×10 5 CAR + T cells). All animals were dosed with a single IV administration of the applicable treatment at a fixed volume of 100 μL, and mice from the vehicle group received an IV injection of 100 μL of PBS. Table 23 shows the experimental groups for the results presented in Tables 24 - 26. TIFF2025518111000030.tif38128

[0245] Antitumor activity was evaluated by assessing the change in tumor burden (measured by bioluminescence and log 10 normalized) from day 5 (1 day before CAR T cell injection) to day 22 (i.e., 16 days after CAR T cell injection and the last time point when all groups were intact). Treatment with anti-CD19 / CD20 CAR T cells produced by a 3-day process resulted in a significant reduction in tumor burden compared to mice that received either vehicle (PBS) or NTD day 3 cells (Table 24). TIFF2025518111000031.tif237170TIFF2025518111000032.tif106150

[0246] Inhibition and regression of tumor growth were observed with 2.0×10 5 (124.3%) and 4.0×10 4 CAR +Observed in the cohort of T cell doses, at the lowest dose of 8.0×10 3 CAR + T cells, the mice showed slight tumor growth inhibition (5.5%). Tumor growth was 5 CAR + controlled in animals receiving the anti-CD19 / CD20 CAR at the dose of 2.0×10 TIFF2025518111000033.tif33170

[0247] Tumor burden (measured by BLI) was log 10 normalized and the mean for each cohort at day 5 and day 22 of the study is presented in Table 25. The change in tumor BLI from day 5 to day 22 was calculated and the percent change in tumor burden relative to the vehicle group was calculated as ΔT / ΔC. Tumor growth inhibition was defined as (1 - [ΔT / ΔC] × 100) and represented the percent change in tumor volume in the study group relative to the control (i.e., mice receiving vehicle [PBS]). A TGI of 0% indicated that the mean tumor growth in the group was equivalent to the mean tumor growth observed in vehicle-treated mice, while a TGI of 100% indicated that no tumor growth was observed from day 5 to day 22. A TGI > 100% indicated that the mean tumor burden regressed from day 5 to day 22, while a TGI < 0 indicated that the increase in tumor burden was greater than the increase seen in vehicle-administered mice.

[0248] Antitumor efficacy determined by analysis of tumor burden was statistically significant at day 22 for mice treated with anti-CD19 / CD20 CAR T cells produced by the 3-day process at all doses tested (2.0×10 5 , 4.0×10 4 and 8.0×10 3 CAR + T cells) and for mice treated with the anti-CD19 / CD20 CAR day 6 product at the dose of 2.0×10 5 CAR + T cells (Table 26). TIFF2025518111000034.tif38146

[0249] In Table 26, Log on the 22nd day 10 The normalized tumor BLI data were evaluated for statistical significance across all groups using analysis of variance with Tukey's post hoc test. Significance between comparisons is shown as follows: ns, P > 0.05, ** , p < 0.01, *** , p < 0.001, **** , p < 0.0001.

[0250] Treatment of mice with anti-CD19 / CD20 CAR T cells produced by a 3-day process or anti-CD19 / CD20 CAR T cells on day 6 of the product resulted in an increase in overall survival (OS) compared to mice receiving vehicle or NTD controls. All mice receiving either vehicle or NTD control ended the study by day 15 or day 12, respectively, based on a survival set point defined by bioluminescence of tumor load < 1 × 10 10 photons / second. The groups treated with anti-CD19 / CD20 CAR T cells produced by a 3-day process at the highest doses of 2.0 × 10 5 and 4.0 × 10 4 individual CAR + T cells achieved 80% and 100% survival, respectively, at the study endpoint. The mice treated with the lowest dose (8.0 × 10 3 individual CAR + T cells) of anti-CD19 / CD20 CAR T cells produced by a 3-day process reached a median survival of 12 days, similar to the mice from the control groups (vehicle and NTD on day 3). Mice receiving the anti-CD19 / CD20 CAR T cell product on day 6 with the same dose of 2.0 × 10 5 individual CAR + T cells had a reduced median survival (36 days) compared to mice treated with anti-CD19 / CD20 CAR T cells produced by a 3-day process (the median survival point was not achieved).

[0251] In summary, treatment with anti-CD19 / CD20 CAR T cells produced by a 3-day process significantly delayed tumor growth and increased survival in the Nalm6-luc human B-ALL xenograft NSG mouse model.

[0252] Example 9: Further phenotypic characterization of cells manufactured using a 3-day process

[0253] This example complements Example 3 and presents additional data related to the phenotypic characteristics of harvested cells manufactured using a 3-day process.

[0254] Tables 27 and 28 show the final product data for cells prepared using a 3-day process. The cells are from healthy donor material (fresh apheresis or cryopreserved PBMC, N1 - N9) and patient material (cryopreserved PBMC, P1 - P4). Execution of the final product showed a higher frequency of central memory (CM) and naïve cells compared to the Teff / TEMRA and effector memory (EM) cell populations, indicating a more naïve product (Tables 27 and 28).

[0255] TIFF2025518111000035.tif72167

[0256] TIFF2025518111000036.tif72128

[0257] In Tables 27 and 28, the abbreviations are as follows: CM, central memory; EM, effector memory; HD, healthy donor; TEMRA, terminally differentiated effector memory T cells. CD45RA and CCR7 were used to define T cell phenotypic subsets including naïve T cells (CD45RA+CCR7+), CM (CD45RA-CCR7+), EM (CD45RA-CCR7-), and Teff / TEMRA (CD45RA+CCR7-).

[0258] As shown in Table 27, the percentage of CD4+CCR7+ cells (i.e., CM and naïve cells) in the ultimately harvested product is at least 80% of the harvested CD4+ T cell population. In other words, the maximum percentage of Teff / TEMRA and EM CD4+ cells in the ultimately harvested population is about 20%. Thus, there are at least four times as many CD4+CCR7+ cells as Teff / TEMRA and EM CD4+ cells in the harvested population.

[0259] As shown in Table 28, the percentage of CD8+CCR7+ cells (i.e., CM and naïve cells) in the ultimately harvested product is at least about 60% of the harvested CD8+ T cell population. In other words, the maximum percentage of Teff / TEMRA and EM CD8+ cells in the ultimately harvested population is about 40%. Thus, there are at least 1.5 times as many CD8+CCR7+ cells as Teff / TEMRA and EM CD8+ cells in the harvested population.

[0260] Example 10: Characterization of Cells Prepared by a 3-Day Process

[0261] This example describes the functional and phenotypic characterization of anti-CLL-1 CAR T cells harvested on day 3 compared to anti-CLL-1 CAR T cells harvested on day 8. The anti-CLL-1 CAR T cells described in this example were produced by a method similar to that described in Example 7.

[0262] Briefly, T cells selected from healthy human donors were activated on day 0 and transduced on day 1 using a lentiviral vector encoding anti-CLL-1 CAR. Thereafter, the transduced cells were harvested on day 3. Another set of anti-CLL-1 CAR T cells was produced from T cells derived from the same donor but harvested on day 8. Thereafter, both CAR T cell products were functionally characterized and compared to their respective NTD control T cells (NTD day 3 and NTD day 8).

[0263] The experimental groups for the research described in this example are shown in Table 29. TIFF2025518111000037.tif29128

[0264] Anti-CLL-1 CAR T cells collected on the 3rd and 8th days were evaluated for CAR cell surface expression to establish the CAR transduction efficiency. CAR expression was equivalent between those collected on the 3rd and 8th days. The expression of anti-CLL-1 CAR was 60.4% for anti-CLL-1 CAR T cells collected on the 3rd day and 78.9% for anti-CLL-1 CAR T cells collected on the 8th day.

[0265] Phenotypic characterization of the CAR T cell product was performed by flow cytometry. Anti-CLL-1 CAR T cells collected on the 3rd day had an increased naïve T cell profile of 83.7% CD45RA + CCR7 + compared to 23% CD45RA + CCR7 + cells at the 8th day collection.

[0266] Anti-CLL-1 CAR T cells collected on the 3rd day showed an increase in CD4+ cells compared to those collected on the 8th day, as shown in Table 30.

[0267] TIFF2025518111000038.tif29128

[0268] The functionality of the CAR T cell product was evaluated in a co-culture assay with antigen-positive target cells. The NTD T cell product was included in the experiment as a control. Cytotoxicity was measured 1 day and 4 days after the start of co-culture of the T cell product. An E:T ratio of 1:1 to 1:3 was used. The MV4-11 (moderate CLL-1 expression) and Kasumi-1 (very low CLL-1 expression) cell lines were used.

[0269] The anti-CLL-1 CAR T cells collected on day 3 showed an increase in in vitro cytotoxicity against Kasumi-1 cells compared to the anti-CLL-1 CAR T cells collected on day 8 (Tables 30A - 30B). The anti-CLL-1 CAR T cells collected on day 3 and day 8 showed equivalent in vitro cytotoxicity against MV4-11 cells (Tables 30C - 30D). TIFF2025518111000039.tif27170TIFF2025518111000040.tif27170TIFF2025518111000041.tif27170TIFF2025518111000042.tif27170

[0270] Example 11: Further Characterization of Cells Prepared by a 3-Day Process

[0271] In this example, the phenotypic characteristics of cells produced using a 3-day process similar to that described in Example 3 above are reported. In this example, a lentiviral vector was used to deliver the CAR.

[0272] First, as shown in Table 31, the collected cells generated from the 3-day process had a larger CD4 / CD8 ratio compared to the cells collected from the 7-day process. Table 31 discloses values from four individual samples per identified group.

[0273] TIFF2025518111000043.tif94170

[0274] Next, as shown in Table 32, the collected cells generated from the 3-day process had a larger percentage of CD45RA+CCR7+ cells and CD45RA-CCR7- cells compared to the cells collected from the 7-day process. Table 32 discloses values from four individual samples per identified group. (NTD = non-transduced control.)

[0275] TIFF2025518111000044.tif91170

[0276] Next, as shown in Table 33, the harvested cells generated from the 3-day process had a greater percentage of Tscm cells (CD27+CD28+CD45RA+CCR7+) compared to the cells harvested from the 7-day process. Table 33 discloses values from four individual samples per identified group.

[0277] TIFF2025518111000045.tif92169

[0278] Finally, as shown in Table 34, the harvested cells generated from the 3-day process had a lower percentage of finally differentiated T cells (CD45RA+CCR7-CD27-CD28-) compared to the cells harvested from the 7-day process. Table 34 discloses values from four individual samples per identified group.

[0279] TIFF2025518111000046.tif91170

[0280] The phenotypic and functional characteristics of anti-CD19 / CD20 CAR T cells produced by a process having a lentiviral transduction step on day 1 or day 2 were compared. In both processes, T cell selection and activation occurred on day 0. CAR expression was measured by detecting the anti-CD19 CAR antibody. Table 35 shows CAR expression of T cells transduced on day 1 and day 2 in cells harvested from day 3 to day 8. TIFF2025518111000047.tif29128

[0281] The T cell subset of CD27+CD28+CD45RA+CCR7+CD62L+ was measured for anti-CD19 / CD20 CAR T cells transduced on day 1 and day 2. Table 36 shows the percentage of naïve CAR+ T cells produced by each method. TIFF2025518111000048.tif40170

[0282] The sample on day 0 in Table 36 was total T cells.

[0283] Cytotoxicity functional assays were compared in anti-CD19 / CD20 CAR T cells transduced on day 1 (T-D1) or day 2 (T-D2). Table 37 shows the results of 24-hour cytotoxicity assays with a 1:3 E:T in three target cell lines (Nalm6 WT, Raji WT, and Nalm6 CD19KO) at days 3, 4, 6, and 8 of collection (H-D3, H-D4, H-D6, and H-D8, respectively). TIFF2025518111000049.tif34167

[0284] Although many embodiments have been described, it will be apparent that the present disclosure and examples may provide other embodiments that utilize the compositions and methods described herein, or other embodiments subsumed by the compositions and methods. Accordingly, it will be understood that the scope of the invention should be defined not by the embodiments presented as examples, but by what can be understood from the present disclosure and the appended claims.

Claims

1. A method for preparing transduced lymphocytes, comprising: incubating a sample containing lymphocytes obtained from a donor subject with a polynucleotide vector to transduce the lymphocytes and produce transduced lymphocytes; culturing the sample containing the transduced lymphocytes for less than 72 hours before the lymphocytes are collected to produce a collected sample.

2. The method according to claim 1, wherein the transduced lymphocytes are cultured for less than 48 hours before being collected.

3. The method according to claim 1, wherein the transduced lymphocytes are cultured for less than 36 hours before being collected.

4. The method according to any one of claims 1 to 3, wherein the incubation is performed in a closed system.

5. The method according to claim 4, wherein the closing system has an inner surface area of at least 1500 cm ２ .

6. The method according to claim 4 or 5, wherein the closed system has an inner surface coated with recombinant human fibronectin, and the coating is performed with a solution containing about 1 to 10 μg / ml of the recombinant human fibronectin.

7. The method according to claim 6, wherein the inner surface is further contacted with a second solution containing the polynucleotide vector, and the second solution has a volume of about 200 mL.

8. The method according to claim 7, wherein the coating further includes draining the second solution.

9. The sample in the closing system contains at least 1.5×10 ８ lymphocytes, and the method according to any one of claims 4 to 8.

10. The method according to claim 8, wherein the sample contains at least 4×10 ８ lymphocytes.

11. The method according to any one of claims 1 to 10, wherein the lymphocytes are peripheral blood mononuclear cells (PBMCs) or T cells.

12. The method according to any one of claims 1 to 11, wherein the collected sample contains CD3+ cells.

13. The method according to claim 12, wherein the collected sample contains CD4+ T cells and CD8+ T cells.

14. The method according to claim 13, wherein at least 20% of the CD4+ T cells are naive T cells, and 12% or less of the CD4+ T cells are effector memory T cells.

15. The method according to claim 14, wherein at least 25% of the CD4+ T cells are naive T cells, and 9% or less of the CD4+ T cells are effector memory T cells.

16. The method according to any one of claims 13 to 15, wherein at least 10% of the CD8+ T cells are naive T cells and at most 30% of the CD8+ T cells are effector memory T cells.

17. The method according to claim 16, wherein at least 20% of the CD8+ T cells are naive T cells and at most 20% of the CD8+ T cells are effector memory T cells.

18. The method according to any one of claims 13 to 17, wherein the naive T cells are characterized as CCR7+ and CD45RA+.

19. The method according to any one of claims 13 to 18, wherein the effector memory T cells are characterized as CCR7-, CD45RO+, and CD95+.

20. The method according to claim 13, wherein at least 80% of the CD4+ T cells are CCR7+ cells.

21. The method according to claim 13, wherein at least 60% of the CD8+ T cells are CCR7+ cells.

22. The method according to claim 13, wherein at most 20% of the CD4+ T cells are a combination of effector memory T cells and effector T cells.

23. The method according to claim 13, wherein at most 40% of the CD8+ T cells are a combination of effector memory T cells and effector T cells.

24. The method according to any one of claims 1 to 23, further comprising obtaining the lymphocytes from the donor subject or enriching the lymphocytes.

25. The method according to any one of claims 1 to 24, further comprising contacting the sample with a lymphocyte stimulant to activate the lymphocytes.

26. The method according to claim 25, wherein activating the sample is before incubating the sample with the polynucleotide vector.

27. The method according to claim 26, wherein the sample contains at least 1×10 ９ lymphocytes.

28. The method according to claim 25, wherein activating the sample is after the sample is incubated with the polynucleotide vector.

29. The method according to any one of claims 25 to 28, wherein the lymphocyte stimulant comprises an anti-CD3 antibody and / or an anti-CD28 antibody.

30. The method according to any one of claims 1 to 29, further comprising administering the collected lymphocytes to a subject or freezing the collected lymphocytes after collection.

31. The method according to claim 30, wherein the subject is the same as the donor subject.

32. The method according to claim 30 or 31, wherein a total of 10,000 to 1,000,000 collected lymphocytes per kilogram of the subject are administered to the subject.

33. The method according to claim 28, wherein a total of 20,000 to 400,000 collected lymphocytes per kilogram of the subject are administered to the subject.

34. The method according to claim 28 or 29, wherein at least 15% of the collected lymphocytes are transduced with the vector.

35. The method according to any one of claims 1 to 34, wherein the polynucleotide vector is a viral vector.

36. The method according to claim 31, wherein the viral vector is a retroviral vector or a lentiviral vector.

37. The method according to any one of claims 1 to 36, wherein the vector encodes a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

38. The method according to claim 34, wherein the CAR comprises an intracellular co-stimulatory domain.

39. The method according to claim 35, wherein the intracellular co-stimulatory domain is a signal transduction region of a protein selected from the group consisting of DAP-10, CD28, OX-40, 4-1BB (CD137), CD2, CD7, CD27, CD30, CD40, programmed death-1 (PD-1), inducible T cell co-stimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD11a / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), tumor necrosis factor superfamily member 14, TNFSF14, LIGHT), NKG2C, Ig alpha (CD79a), Fc gamma receptor, MHC class I molecule, TNF receptor protein, immunoglobulin-like protein, cytokine receptor, integrin, signal transduction lymphocyte activation molecule (SLAM protein), activated NK cell receptor, BTLA, Toll ligand receptor, CDS, GITR, BAFFR, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD (CD11d), ITGAE (CD103), ITGAL (CD11a), ITGAM (CD11b), ITGAX (CD11c), ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, TNFR2, TRANCE (RANKL), DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG (Cbp), CD19a, CD83, and combinations thereof, and a ligand specifically binding thereto.

40. The method according to claim 39, wherein the intracellular co-stimulatory domain is a signal transduction region of CD28.

41. The method according to any one of claims 37 to 39, wherein the CAR recognizes a tumor antigen. **Claim 42** The method according to claim 41, wherein the tumor antigen is CD19, CD20, and / or CLL-1. **Claim 43** The method according to claim 40, wherein the lymphocyte comprising the CAR is axicabtagene ciloleucel or brexucabtagene autoleucel. **Claim 44** A population of cells prepared from a blood sample of a donor subject, comprising CD4+ T cells and CD8+ T cells, wherein at least 20% of the CD4+ T cells are naive T cells, and 12% or less of the CD4+ T cells are effector memory T cells, wherein at least 10% of the CD8+ T cells are naive T cells, and 30% or less of the CD8+ T cells are effector memory T cells, wherein at least 50% of the cells are CD3+ T cells, and a population in which at least 25% of all T cells are transduced with a polynucleotide vector encoding a CAR or a TCR. **Claim 45** The population according to claim 44, wherein at least 25% of the CD4+ T cells are naive T cells, and 9% or less of the CD4+ T cells are effector memory T cells. **Claim 46** The population according to claim 44 or 45, wherein at least 20% of the CD8+ T cells are naive T cells, and 20% or less of the CD8+ T cells are effector memory T cells. **Claim 47** The population according to any one of claims 44 to 46, wherein the naive T cells are characterized as CCR7+ and CD45RA+. **Claim 48** The population according to any one of claims 44 to 47, wherein the effector memory T cells are characterized as CCR7-, CD45RO+, and CD95+. **Claim 49** The population according to any one of claims 44 to 48, wherein at least 65% of the cells are CD3+ T cells. **Claim 50** The population according to any one of claims 44 to 49, wherein at least 15% of all T cells are transduced with the polynucleotide vector. **Claim 51** The population according to any one of claims 44 to 49, wherein the polynucleotide vector is a viral vector. **Claim 52** The population according to claim 49, wherein the viral vector is a retroviral vector or a lentiviral vector. **Claim 53** The population according to any one of claims 44 to 52, wherein the CAR comprises an intracellular co-stimulatory domain. **Claim 54** The population according to claim 53, wherein the intracellular co-stimulatory domain is the signaling region of CD28. **Claim 55** The population according to any one of claims 44 to 54, wherein the CAR recognizes a tumor antigen. **Claim 56** The population according to claim 55, wherein the tumor antigen is CD19 or CD20. **Claim 57** The population according to claim 54, wherein the population of cells comprising the CAR is axicabtagene ciloleucel or brexucabtagene autoleucel. **Claim 58** A population of cells prepared from a blood sample of a donor subject, comprising CD4+ T cells and CD8+ T cells, wherein at least 80% of the CD4+ T cells are CCR7+ cells, at least 60% of the CD8+ T cells are CCR7+ cells, at most 20% of the CD4+ T cells are a combination of effector memory T cells and effector T cells, and at most 40% of the CD8+ T cells are a combination of effector memory T cells and effector T cells. **Claim 59** A pharmaceutical composition comprising the population of cells according to any one of claims 44 to 58. **Claim 60** A method for administering T cells to a subject, comprising injecting into the subject a harvested sample prepared by the method according to any one of claims 1 to 43 or the pharmaceutical composition according to claim 59. **Claim 61** The method according to claim 60, wherein the subject has cancer. **Claim 62** The method according to claim 60, wherein the cancer is a B cell malignancy. **Claim 63** The method according to claim 61, wherein the cancer is non-Hodgkin lymphoma (NHL), diffuse large B cell lymphoma (DLBCL), small lymphocytic lymphoma (SLL / CLL), mantle cell lymphoma (MCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), extranodal (MALT lymphoma), nodal (monocytoid B cell lymphoma), splenic, diffuse large cell lymphoma, B cell chronic lymphocytic leukemia / lymphoma, Burkitt lymphoma, lymphoblastic lymphoma, acute myeloid leukemia, or multiple myeloma.

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