Compositions and methods for preparing engineered lymphocytes for cell therapy

Abstract

Provided herein are compositions and methods for making engineered lymphocytes. Also provided are engineered lymphocytes prepared having an increased proportion of juvenile / immature lymphocytes, resulting in an increased therapeutic efficacy. The methods in various embodiments are accelerated compared to conventional techniques and produce lymphocytes with increased viability, transduction success rate, and in vivo anti-tumor efficacy.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 346,709 filed on May 27, 2022, U.S. Provisional Application No. 63 / 485,623 filed on February 17, 2023, and U.S. Provisional Application No. 63 / 490,162 filed on March 14, 2023, which are hereby incorporated by reference in their entirety. Technical Field

[0003] The present disclosure relates to the field of cell therapy, and more particularly to compositions and methods for making engineered lymphocytes. Background Art

[0004] Immune cells can be modified to target and kill cancer cells in a patient. To increase the ability of immune cells to target and kill specific cancer cells, methods have been developed to engineer immune cells to express constructs that direct immune cells to specific target cancer cells. Chimeric antigen receptors (CARs) and engineered T cell receptors (TCRs) that include binding domains capable of interacting with specific tumor antigens allow immune cells to target and kill cancer cells that express specific tumor antigens.

[0005] A significant challenge comes from the highly complex autologous cell engineering and production process, which usually takes at least a week and may even take several weeks. In this process, lymphocytes collected from patients must be transported to a processing center, and the resulting cells must be cryopreserved and then transported back to the patient for transplantation. This highly complex process inevitably leads to high costs and limited clinical applications.

[0006] Additionally, lengthy processes may result in reduced cell viability and increased lymphocyte maturation, both of which are detrimental to in vivo efficacy. Therefore, there is a strong need to develop processes that are not only shorter in duration, but also produce immune cells with improved therapeutic efficacy. Summary of the invention

[0007] As provided, the current autologous CAR cell manufacturing process typically takes about 7 days, and may be longer. The present disclosure describes an improved process that can be completed within 6 days or even 4 days (or within 5 days or even 3 days after the enrichment step). In various examples, the 5-day process (ie, 5 days after enrichment) includes transduction preparation and specific implementation steps, in which a greater number of lymphocytes are contacted with a vector that is fixed to a recombinant fibronectin coated on the inner surface of a closed system. This improved transduction procedure allows the post-transduction cell expansion step to be greatly shortened.

[0008] Yet another improved process that does not require post-transduction expansion at all can be completed in only 3 days after the initial enrichment step. It was surprisingly found that both the 3-day and 5-day processes produced transduced lymphocytes with a higher percentage of naive cells. Due at least in part to the increased naive cell population, these cell products exhibited greatly improved in vivo anti-tumor efficacy compared to the conventional 7-day process.

[0009] Therefore, according to one embodiment of the present disclosure, a method for preparing transduced lymphocytes is provided, the method comprising: incubating a sample comprising lymphocytes obtained from a donor subject with a polynucleotide vector to transduce the lymphocytes, thereby producing transduced lymphocytes; and culturing the sample comprising the transduced lymphocytes for less than 72 hours, followed by harvesting the lymphocytes to produce a harvested sample.

[0010] 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.

[0011] In some embodiments, the incubation is performed in a closed system. In some embodiments, the closed system has at least 1500 cm 2 In some embodiments, the closed system has an inner surface coated with recombinant human fibronectin, wherein the coating is performed using a solution comprising about 1 µg / ml to 10 µg / ml of the recombinant human fibronectin. In some embodiments, the inner surface is also contacted with a second solution comprising the polynucleotide carrier, wherein the second solution has a volume of about 200 mL. In some embodiments, the coating further comprises draining the second solution. In some embodiments, the sample in the closed system comprises at least 1.5×10 8 In some embodiments, the sample includes at least 4×10 8 Lymphocytes.

[0012] In some embodiments, the lymphocyte is a peripheral blood mononuclear cell (PBMC) or a T cell. In some embodiments, the harvested sample includes CD3+ cells. In some embodiments, the harvested sample includes CD4+ T cells and CD8+ T cells. In some embodiments, at least 20% of the harvested CD4+ T cells are naive T cells, and no more than 12% 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 no more than 9% 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, up to 20% of the harvested CD4+ T cells are a combination of effector memory T cells and effector T cells.

[0013] In some embodiments, at least 10% of the harvested CD8+ T cells are naive T cells, and no more than 30% of the harvested CD8+ T cells are effector memory T cells. In some embodiments, at least 20% of the harvested CD8+ T cells are naive T cells, and no more than 20% of the harvested CD8+ T cells are effector memory T cells. In some embodiments, the naive T cells are characterized as CCR7+ and CD45RA+. In some embodiments, the effector memory T cells are characterized as CCR7-, CD45RO+ and CD95+. In some embodiments, at least 60% of the harvested CD8+ T cells are CCR7+ cells. In some embodiments, up to 40% of the harvested CD8+ T cells are a combination of effector memory T cells and effector T cells.

[0014] In some embodiments, the method further comprises: obtaining the lymphocyte from the donor subject. In some embodiments, the method further comprises: enriching the lymphocyte. In some embodiments, the method further comprises: contacting the sample with a lymphocyte stimulator to activate the lymphocyte.

[0015] In some embodiments, the activating the sample is prior to incubating the sample with the polynucleotide carrier. In some embodiments, the sample comprises at least 1×10 9 In some embodiments, the activating the sample is after incubating the sample with the polynucleotide carrier. In some embodiments, the lymphocyte stimulator comprises an anti-CD3 antibody and / or an anti-CD28 antibody.

[0016] In some embodiments, the method further comprises: after the harvest, administering the harvested lymphocytes to the subject or freezing the harvested 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 harvested lymphocytes per kilogram of the subject are administered to the subject. In some embodiments, a total of 20,000 to 400,000 harvested lymphocytes per kilogram of the subject are administered to the subject. In some embodiments, at least 15% of the harvested lymphocytes are transduced using the vector.

[0017] 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 includes an intracellular co-stimulatory domain. In some embodiments, the intracellular co-stimulatory domain is a signaling 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), CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), tumor necrosis factor superfamily member 14, TNFSF14, LIGHT), NKG2C, Ig α (CD79a), Fc γ receptor, MHC Class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activated NK cell receptors, BTLA, Toll ligand receptors, CDS, GITR, BAFFR, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 α, CD8 β, IL2R β, IL2R γ, IL7R α, ITGA4, VLA1, CD49a, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD (CD11d), ITGAE (CD103), ITGAL (CD11a), ITGAM (CD11b), IT GAX (CD11c), ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, TNFR2, TRANCE (RANKL), DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, C D96 (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, a ligand that specifically binds to CD83, and a combination thereof. In some embodiments, the intracellular co-stimulatory domain is a signaling region of CD28.

[0018] In some embodiments, the CAR or TCR recognizes a tumor antigen. In some embodiments, the tumor antigen is CD19. In some embodiments, the lymphocyte comprising the CAR is axicabtageneciloleucel or brexucabtagene autoleucel. In some embodiments, the tumor antigen is CD19 and / or CD20. In some embodiments, the tumor antigen is CLL-1.

[0019] In some embodiments, the CAR or TCR recognizes an antigen that includes 2B4 (CD244), 4-1BB, 5T4, A33 antigen, adenocarcinoma antigen, adrenergic receptor beta 3 (ADRB3), A kinase anchoring protein 4 (AKAP-4), alpha-fetoprotein (AFP), anaplastic lymphoma kinase (ALK), androgen receptor, B7H3 (CD276), beta 2-integrin, BAFF, B lymphoma cells, B cell maturation antigen (BCMA), bcr-abl (oncogene fusion protein composed of breakpoint cluster region (BCR) and Aberson murine leukemia virus oncogene homolog 1 (Abl)), BhCG, bone marrow stromal cell antigen 2 (BST2), CCCTC-binding factor (zinc finger protein)-like (BORIS or imprinting site regulator brother brother), BST2, C242 antigen, 9-0-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, C D44, CD44V6, CD49f, CD51, CD52, CD56, CD63, CD70, CD72, CD74, CD79a, CD79b, CD80, CD84, CD96, CD97, CD100, CD123, CD125, CD1 33. CD137, CD138, CD150, CD152 (CTLA-4), CD160, CD171, CD179a, CD200, CD221, CD229, CD244, CD272 (BTLA), CD274 (PDL-1, B7H 1), CD279 (PD-1), CD352, CD358, CD300 molecule-like family member f (CD300LF), carcinoembryonic antigen (CEA), occludin 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 subclass 1, CRACC, CD319 and 19A24), CTLA-4, cyclin B l, chromosome X open reading frame 61 (CXORF61), cytochrome P450 1B1 (CYP1B1), DNAM-1 (CD226), desmoglein 4, DR3, DR5, E-cadherin neo-epitope, epidermal growth factor receptor (EGFR), EGF1R, epidermal growth factor receptor variant III (EGFRvIII), epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), mucin-like hormone receptor-like 2 containing an EGF-like module (EMR2), elongation factor 2 mutant (ELF2M), endosialin, epithelial cell adhesion molecule (EPCAM), 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 (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(l-4)bDGlcp(ll)Cer), o-acetyl-GD2 ganglioside (OAcGD2), GITR (TNFRSF 18), GM1, ganglioside GM3 (aNeu5Ac(2-3)bDGalp(l-4)bDGlcp(ll)Cer), GP 100, hexose portion of globoH glycoceramide (GloboH), glycoprotein 75, phosphatidylinositol proteoglycan 3 (GPC3), glycoprotein 100 (gp1OO), 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 mutant (muthsp70-2), 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, kappa-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), legumin, 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 K 9 (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 recognized by T cells 1 (MelanA or MARTI), MelanA / MARTl, mesothelin, MAGEA3, melanoma cell apoptosis inhibitor (ML-IAP), melanoma specific chondroitin sulfate proteoglycan (MCSCP), MOR Ab-009, MS4A1, mucin 1 (MUCl), MUC2, MUC3, MUC4, MUC5AC, MUC5b, MUC7, MUC16, mucin CanAg, Müllerian inhibitory substance (MIS) type II receptor, v-myc avian myelocytic tumor viral oncogene neuroblastoma-derived homolog (MYCN), N-glycolylneuraminic acid, N-acetylglucosaminyltransferase V (NA17), neural cell adhesion molecule (NCAM), NKG2A, NKG2C, NKG2D, NKG2E ligand, NKR-PIA, NPC-1C, NTB-A, mammary differentiation antigen (NY-BR-1), NY-ESO-1, carcinoembryonic antigen (h5T4), olfactory receptor 51E2 (OR51E2), OX40, plasma cell antigen, poly SA, acrosomal protease binding protein sp32 (OY-TESl), p53, p53 mutant, pan-linked protein 3 (PANX3), prostatic 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-α), PDGFR-β, PDL192, PEN-5, phosphatidylserine, placenta-specific 1 (PLAC1), polysialic acid, prostate enzyme, prostate cancer cell, prostaglandin, protease serine 21 (testosterone or PRSS21), protease 3 (PR1), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), proteasome (precursor, megalin factor) subunit beta type, advanced glycation end product receptor RAGE-1, RANKL, Ras mutant, Ras homolog family member C (RhoC), RON, receptor tyrosine kinase-like orphan receptor 1 (ROR1), renal ubiquitin 1 (RU1), renal ubiquitin 2 (RU2), sarcoma translocation breakpoints, 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 (TARP), telomerase, TIGIT, TNF-α precursor, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), tenascin C, TGFβ2, TGF-β, 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, urokinase 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.

[0020] In one embodiment, a cell population prepared from a blood sample of a donor subject is also provided, the cell population comprising CD4+ T cells and CD8+ T cells, wherein: at least 20% of the harvested CD4+ T cells are naive T cells, and no more than 12% of the harvested CD4+ T cells are effector memory T cells; at least 10% of the harvested CD8+ T cells are naive T cells, and no more than 30% of the harvested CD8+ T cells are effector memory T cells; at least 50% of the harvested cells are CD3+ T cells; and at least 25% of all T cells are transduced with a polynucleotide vector encoding a CAR or TCR.

[0021] In one embodiment, a cell population prepared from a blood sample of a donor subject is also provided, the cell population 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, which are transduced using a polynucleotide vector encoding CAR or TCR.

[0022] In some embodiments, at least 25% of the harvested CD4+ T cells are naive T cells, and no more than 9% of the harvested CD4+ T cells are effector memory T cells. In some embodiments, at least 20% of the harvested CD8+ T cells are naive T cells, and no more than 20% of the harvested CD8+ T cells are effector memory T cells. In some embodiments, the naive T cells are characterized as CCR7+ and CD45RA+. In some embodiments, the effector memory T cells are characterized as CCR7-, CD45RO+ and CD95+. In some embodiments, at least 65% of the harvested cells are CD3+ T cells. In some embodiments, at least 15% of all T cells are transduced using the polynucleotide vector.

[0023] In some embodiments, at least 80% of the harvested CD4+ T cells are CCR7+ cells. In some embodiments, up to 20% of the harvested CD4+ T cells are a combination of effector memory T cells and effector T cells. In some embodiments, at least 60% of the harvested CD8+ T cells are CCR7+ cells. In some embodiments, up to 40% of the harvested CD8+ T cells are a combination of effector memory T cells and effector T cells.

[0024] 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 includes an intracellular co-stimulatory domain. In some embodiments, the intracellular co-stimulatory domain is a 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 cell population including the CAR is Akilensai or brexucabtagene autoleucel.

[0025] Also provided is a pharmaceutical composition comprising a cell population as described herein.

[0026] Another embodiment provides a method for administering T cells to a subject, the method comprising: injecting the subject with a sample of the harvest prepared by the method of the present disclosure or the pharmaceutical composition. In some embodiments, the subject suffers from cancer. In some embodiments, the cancer is a B cell malignancy. In some embodiments, 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 (monocytic B cell lymphoma), splenic diffuse large cell lymphoma, B cell chronic lymphocytic leukemia / lymphoma, Burkitt's lymphoma, lymphoblastic lymphoma, acute myeloid leukemia or multiple myeloma. DETAILED DESCRIPTION

[0027] definition

[0028] In order to more easily understand the present disclosure, certain terms are first defined below. Additional definitions of the following terms and other terms are set forth throughout the specification.

[0029] Unless otherwise stated or obvious from the context, as used herein, the term "or" is to be construed as inclusive and encompasses both "or" and "and."

[0030] As used herein, the term "and / or" should be considered as a specific disclosure of each of the two specified features or components, with or without the other. Thus, the term "and / or" as used in phrases such as "A and / or B" herein is intended to include A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following: 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).

[0031] Unless otherwise specified or apparent from the context, the term "about" refers to a value or composition within an acceptable error range of a particular value or composition as determined by a person of ordinary skill in the art, which will depend in part on how to measure or determine the value or composition, i.e., the limitations of the measurement system. For example, according to the practice of the art, "about" or "consisting essentially of" may mean within one or more than one standard deviation. "About" or "consisting essentially of" may mean a range of up to 10% (i.e., ±10%). Therefore, "about" may be understood as being within 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 value. For example, about 5 mg may include any amount between 4.5 mg and 5.5 mg. In addition, particularly with respect to a biological system or process, these terms may mean up to an order of magnitude or up to 5 times of a certain value. When a specific value or composition is provided in the present disclosure, unless otherwise stated, the meaning of "about" or "consisting essentially of should be assumed to be within an acceptable error range for the specific value or composition.

[0032] "Administering" refers to the physical introduction of an agent into a subject using any of the various methods and delivery systems known to those skilled in the art, such as the modified T cells disclosed herein. Exemplary routes of administration for the preparations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration (e.g., by injection or infusion). The phrase "parenteral administration" means a mode of administration (usually by injection) other than enteral and topical administration, and includes but is not limited to intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcutaneous, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, and in vivo electroporation. In some embodiments, the preparation is administered via a non-parenteral route (e.g., oral). Other non-parenteral routes include topical, epidermal or mucosal administration routes, such as intranasal, vaginal, rectal, sublingual or topical. Administration can also be performed, for example, once, multiple times, and / or over one or more extended periods of time.

[0033] The term "allogeneic" refers to any material derived from one individual and subsequently introduced into another individual of the same species, such as an allogeneic T cell transplant.

[0034] The term "antibody" (Ab) includes, but is not limited to, glycoprotein immunoglobulins that specifically bind to antigens. In general, an antibody may comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding molecule thereof. Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region comprises three constant domains CH1, CH2, and CH3. Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region comprises one constant domain CL. The VH region and the VL region can be further subdivided into hypervariable regions (called complementarity determining regions (CDRs)), with more conserved regions (called framework regions (FRs)) interspersed therebetween. Each VH and VL comprises three CDRs and four FRs, which are arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains comprise binding domains that interact with antigens. The constant region of Ab can mediate the binding of immunoglobulins 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 speaking, human antibodies are tetrameric agents of approximately 150 kD, consisting of two identical heavy (H) chain polypeptides (each approximately 50 kD) and two identical light (L) chain polypeptides (each approximately 25 kD), which associate with each other into a structure generally referred to as a "Y-shaped" structure. The heavy and light chains are linked or connected to each other by a single disulfide bond; two additional disulfide bonds connect the heavy chain hinge regions to each other, so that the dimers are connected to each other and form a tetramer. Naturally produced antibodies are also glycosylated, for example, on the CH2 domain.

[0035] "Antigen binding molecule", "antigen binding portion", "antigen binding fragment", "antibody fragment" refers to any molecule that comprises an antigen binding portion (e.g., CDR) of an antibody from which the molecule is derived. An antigen binding molecule may comprise an antigen complementarity determining region (CDR). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, dAbs, linear antibodies, scFv antibodies, and multispecific antibodies formed by antigen binding molecules. Peptibodies (i.e., Fc fusion molecules comprising 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 hyperproliferative disease or a viral or bacterial antigen. In certain embodiments, the antigen binding molecule is a chimeric antigen receptor (CAR) or an engineered T cell receptor (TCR).

[0036] The terms "variable region" or "variable domain" are used interchangeably. The variable region generally refers to a part of an antibody, generally a part of a light chain or a heavy chain, usually about 110 to 120 amino acids at the amino terminal in a mature heavy chain and about 90 to 115 amino acids in a mature light chain, which are widely different in sequence between antibodies and are used for the binding and specificity of a specific antibody to its specific antigen. The variability of the sequence is concentrated in those regions called complementary determining regions (CDRs), while the more highly conserved regions in the variable domain are called framework regions (FRs). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody 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 specific 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).

[0037] The terms "VL" and "VL domain" are used interchangeably to refer to the light chain variable region of an antibody or antigen binding molecule thereof.

[0038] The terms "VH" and "VH domain" are used interchangeably to refer to the heavy chain variable region of an antibody or antigen binding molecule thereof.

[0039] Many definitions of CDRs are commonly used: Kabat numbering, Chothia numbering, AbM numbering, or Contact numbering. The AbM definition is a compromise between the two definitions used by Oxford Molecular's AbM antibody modeling software. The Contact definition is based on analysis of available complex crystal structures.

[0040] The term "autologous" refers to any material that originates from the same individual and is later reintroduced into that individual. For example, the engineered autologous cell therapy (eACT) described herein ™ ) method involves collecting lymphocytes from a patient, then engineering them to express, for example, a CAR construct, and subsequently administering them back to the same patient.

[0041] "Chimeric antigen receptor" or "CAR" refers to a molecule that is engineered to contain a binding motif and a means of activating an immune cell (e.g., a T cell, such as a naive T cell, a central memory T cell, an effector memory T cell, or a combination thereof) after antigen binding. CAR is also referred to as an artificial T cell receptor, a chimeric T cell receptor, or a chimeric immunoreceptor. In some embodiments, CAR comprises a binding motif, an extracellular domain, a transmembrane domain, one or more co-stimulatory domains, and an intracellular signaling domain. T cells that have been genetically engineered to express a chimeric antigen receptor can be referred to as CAR T cells. "Extracellular domain" (or "ECD") refers to a portion of a polypeptide that, when the polypeptide is present in a cell membrane, is understood to be located outside the cell membrane, in the extracellular space.

[0042] "T cell receptor" or "TCR" refers to an antigen recognition molecule present on the surface of a T cell. During normal T cell development, each of the four TCR genes, α, β, γ, and δ, can rearrange, resulting in a highly diverse set of TCR proteins.

[0043] The term "heterologous" refers to 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, i.e., is not identical to a wild-type human costimulatory protein. For example, a heterologous nucleotide sequence refers to a nucleotide sequence that is different from a wild-type human costimulatory protein coding sequence.

[0044] The term "identity" refers to the overall correlation between polymeric molecules, such as nucleic acid molecules (e.g., DNA molecules and / or RNA molecules) and / or polypeptide molecules. Methods for calculating the percentage identity between two provided polypeptide sequences are known. For example, the percentage identity of the two sequences can be calculated by comparing two nucleic acid or polypeptide sequences for the purpose of optimal comparison (e.g., spaces can be introduced in one or both of the first sequence and the second sequence for optimal comparison, and non-identical sequences can be ignored for comparison purposes). The nucleotides or amino acids at the corresponding positions are then compared. When 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 percentage identity between the two sequences is a function of the number of identical positions shared by the sequences, optionally taking into account the number of spaces and the length of each space, and it may be necessary to introduce spaces for optimal comparison of the two sequences. The comparison or comparison of sequences and the determination of the percentage identity between the two sequences can be achieved using mathematical algorithms such as BLAST (Basic Local Alignment Search Tool). In some embodiments, polymer molecules are considered "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% identical).

[0045] The immune cells of immunotherapy can be from any source known in the art. For example, immune cells can be differentiated from a hematopoietic stem cell population in vitro, or can be obtained from a subject. Immune cells can be obtained from, for example, peripheral blood mononuclear cells (PBMC), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In addition, immune cells can be derived from one or more immune cell lines available in the art. A variety of techniques known to technicians (such as FICOLL ™ Separation, OPTIPREP ™ Immune cells are obtained from a unit of blood collected from a subject by separation and / or apheresis. Additional methods for isolating immune cells for use in immune cell therapy are disclosed in U.S. Patent Publication No. 2013 / 0287748, which is incorporated herein by reference in its entirety.

[0046] A "patient" includes any person suffering from cancer (eg, lymphoma or leukemia). The terms "subject" and "patient" are used interchangeably herein.

[0047] The term "pharmaceutically acceptable" refers to a molecule or composition that is harmless to the recipient when applied to the recipient, or any harmful effects are outweighed by the beneficial effects to the recipient. In terms of the carrier, diluent or excipient used to prepare the composition as disclosed herein, the pharmaceutically acceptable carrier, diluent or excipient must be compatible with the other components of the composition and harmless to the recipient, or any harmful effects must be outweighed by the beneficial effects to the recipient. The term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition or vehicle that involves carrying or transporting an agent from one part of the body to another part (e.g., from one organ to another), such as a liquid or solid filler, diluent, excipient or solvent encapsulating material. Each carrier present in the pharmaceutical composition must be "acceptable" in the following sense: compatible with the other ingredients of the preparation and harmless to the patient, or any harmful effects must be outweighed by the beneficial effects to the recipient. Some examples of materials that can serve 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 carboxymethylcellulose, ethylcellulose, and cellulose acetate; powdered tragacanth; 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, and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; pH buffered solutions; polyesters, polycarbonates, and / or polyanhydrides; and other nontoxic compatible substances employed in pharmaceutical formulations.

[0048] The term "pharmaceutical composition" refers to a composition in which an active agent is formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose suitable for administration in a treatment regimen that, when administered to a relevant subject or population, shows a statistically significant probability of achieving a predetermined therapeutic effect. In some embodiments, the pharmaceutical composition can be formulated for administration in solid or liquid form, including but not limited to forms suitable for the following: oral administration, such as drenches (aqueous or non-aqueous solutions or suspensions), tablets (e.g., those targeted for buccal, sublingual, and systemic absorption), boluses, powders, granules, pastes for application to the tongue; parenteral administration, such as by subcutaneous, intramuscular, intravenous, or epidural injection, as, for example, sterile solutions or suspensions, or sustained-release formulations; topical application, such as as a cream, ointment, or controlled-release patch, or a spray applied to the skin, lungs, or mouth; intravaginal or rectal administration, for example, as a vaginal suppository, cream, or foam; sublingual administration; ophthalmic administration; transdermal administration; or nasal, pulmonary, and other mucosal surfaces.

[0049] The terms "reduce" and "reduction" are used interchangeably herein and indicate any change that is less than the original value. "Reduce" and "reduction" are relative terms requiring a comparison between before and after measurement. "Reduce" and "reduction" include complete depletion.

[0050] The term "reference" describes a standard or control relative to which a comparison is made. For example, in some embodiments, an agent of interest, an animal, an individual, a colony, a sample, a sequence or a value is compared with a reference or control as an agent, an animal, an individual, a colony, a sample, a sequence or a value. In some embodiments, a reference or control is tested, measured and / or measured substantially simultaneously with a test, measurement or determination of interest. In some embodiments, a reference or control is a historical reference or control, which is optionally embodied in a tangible medium. Generally speaking, a reference or control is measured or characterized under conditions or circumstances comparable to the conditions or circumstances of the assessment. When there are enough similarities to prove that dependence and / or comparison to the selected reference or control is correct.

[0051] A "therapeutically effective amount," "effective dose," "effective amount," or "therapeutically effective dose" 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 disease or promotes disease regression (as evidenced by a reduction in the severity of disease symptoms, an increase in the frequency and duration of asymptomatic disease periods, or the prevention of impairment or disability resulting from the disease). The ability of a therapeutic agent to promote disease regression can be assessed using a variety of methods known to the skilled artisan, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by measuring the activity of the agent in an in vitro assay.

[0052] The terms "transduction" and "transduced" refer to the process of introducing exogenous nucleic acid into cells 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 virus vector, a papillomavirus vector, a vaccinia virus vector, a herpes simplex virus vector, an adenovirus-associated vector, a lentiviral vector, or any combination thereof.

[0053] "Treatment" or "treatment" of a subject refers to any type of intervention or process performed on a subject, or the administration of an active agent to a subject, with the purpose of reversing, alleviating, improving, inhibiting, slowing down or preventing the onset, progression, development, severity or recurrence of symptoms, complications or conditions or biochemical indicators associated with a disease. In one embodiment, "treatment" includes partial relief. In another embodiment, "treatment" or "treatment" includes complete relief. In some embodiments, treatment may be treatment of subjects who do not show signs of the relevant disease, disorder and / or condition and / or subjects who only show early signs of the disease, disorder and / or condition. In some embodiments, such treatment may be treatment of subjects who show one or more established signs of the relevant disease, disorder and / or condition. In some embodiments, treatment may be treatment of subjects who have been diagnosed with the relevant disease, disorder and / or condition. In some embodiments, treatment may be treatment of subjects who are known to have one or more susceptibility factors that are statistically associated with an increased risk of the development of the relevant disease, disorder and / or condition.

[0054] The term "vector" refers to a receptor nucleic acid molecule modified to include or incorporate a provided nucleic acid sequence. One type of vector is a "plasmid", which refers to a circular double-stranded DNA molecule to which additional DNA can be connected. Another type of vector is a viral vector, in which additional DNA segments can be connected to a viral genome. Some vectors can replicate autonomously in the host cells they introduce (e.g., bacterial vectors with bacterial replication origins, and additional mammalian vectors). Other vectors (e.g., non-additional mammalian vectors) can be integrated into the genome of the host cell when introduced into the host cell, and thus replicated with the host genome. In addition, some vectors include sequences that guide the expression of inserted genes operably connected to them. Such vectors may be referred to as "expression vectors" in this article. Standard techniques may be used for vector engineering, for example, as present in Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)), the document is incorporated herein by reference.

[0055] The term "7-day process" refers to a CAR cell manufacturing process that requires about 7 days after the initial enrichment and / or activation step. From the initial enrichment and / or activation step to the harvesting step, the length of the 7-day process is at least 8 days, and when the enrichment and / or activation step is included, it can be 8 to 11 days in total. In some embodiments, there is no initial activation step, or there is no activation before transduction.

[0056] The term "5-day process" refers to a CAR cell manufacturing process that requires about 5 days after the initial enrichment and / or activation step. From the initial enrichment and / or activation step to the harvesting step, the length of the 5-day process is 6 days, and when the enrichment and / or activation step is included, it can be 6 to 9 days in total. In some embodiments, there is no initial activation step, or there is no activation before transduction.

[0057] The term "3-day process" refers to a CAR cell manufacturing process that requires about 3 days from the initial enrichment and / or activation step. The length of the 3-day process is about 4 days from the initial enrichment and / or activation step to the harvesting step. The 3-day process does not include a cell expansion step, which includes one or more days after the transduction step and before the harvesting step. In some embodiments, there is no initial activation step, or there is no activation before transduction.

[0058] In some embodiments, the length of the 3-day process described herein is about 5 days from the initial enrichment and / or activation step to the harvesting step. In some embodiments, the length of the 3-day process is about 3 to 4 days from the initial enrichment and / or activation step to the harvesting step or about 72 hours to 96 hours (e.g., 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 in length). In some embodiments, the length of the 3-day process is about 4 to 5 days from the initial enrichment and / or activation step to the harvesting step or about 96 hours to about 120 hours (e.g., a length 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). In some embodiments, a 3 day process is less than 5 days or 120 hours in length from the initial enrichment and / or activation step to the harvesting step.

[0059] Preparation of engineered lymphocytes

[0060] Conventional autologous CAR cell manufacturing processes take about 7 days, and may be longer. It is believed that a lengthy process is required, at least because of the limited supply of starting material (i.e., lymphocytes obtained from apheresis collection of donor subjects), relatively low transduction efficiency, and the need to expand transduced cells. Non-limiting examples of CAR cell manufacturing processes are described in WO 2015120096 and WO 2016191755, each of which is incorporated herein in its entirety.

[0061] However, as demonstrated in the accompanying examples, such a lengthy 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. In addition, the expansion process of several days, while increasing the total number of transduced cells, reduced the percentage of younger cells (e.g., naive T cells, stem cell 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, younger lymphocytes were shown to be more effective than more mature lymphocytes (Tables 5, 10, and 11).

[0062] 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 transduction preparation and specific implementation steps, in which a larger number of lymphocytes are contacted with a carrier that is fixed on a recombinant fibronectin coated on the inner surface of a closed system. This improved transduction procedure allows for a greatly shortened post-transduction cell expansion step. As shown in Table 2, the transduced cells prepared by the 5-day process are biased towards younger cells. In vivo anti-tumor efficacy results are shown in (Table 5).

[0063] Yet another improved process that does not require post-transduction expansion at all can be completed in just 3 days after the enrichment step. Surprisingly, the 3-day process produced a cell population with a higher percentage of naive cells and a reduced percentage of more mature, differentiated and activated cells (compared to Tables 6 to 2) compared to the 5-day process. Thus, not only did the cell product from the 3-day process exhibit the best in vivo anti-tumor efficacy, this greatly improved efficacy was even achieved at much lower doses (Tables 10 to 11).

[0064] In yet another surprising finding, while the accelerated lymphocyte preparation process improved the efficacy of all CAR types tested, it was particularly beneficial for CAR molecules with a CD28 co-stimulatory domain (compared to CAR molecules with a 4-1BB co-stimulatory domain) (Table 13).

[0065] Therefore, according to one embodiment of the present disclosure, a method for preparing transduced (or similarly transfected) lymphocytes is provided. In some embodiments, the method requires incubating a lymphocyte sample with a polynucleotide vector to transduce the lymphocytes so as to produce transduced lymphocytes, and culturing a sample containing the transduced lymphocytes, followed by harvesting the lymphocytes, thereby producing a harvested sample.

[0066] In some embodiments, the culturing step is shortened compared to a conventional process that requires 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.

[0067] In some embodiments, the time of the culturing step is counted from the completion of the transduction step (eg, removal of cells from a system with immobilized vectors) to harvesting of cells for storage, transport, or clinical use.

[0068] The culture of transduced lymphocytes can be carried out under culture medium and conditions known in the art. In some embodiments, the culture of transduced lymphocytes can be carried out at a certain temperature and / or in the presence of CO2. 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°C to 39°C. In certain embodiments, the predetermined temperature can be about 35°C to 37°C. In certain embodiments, the preferred predetermined temperature can be about 36°C to 38°C. In certain embodiments, the preferred predetermined temperature can be about 36°C to 37°C, or more preferably about 37°C.

[0069] In some embodiments, the culture of transduced lymphocytes can be carried out in the presence of a predetermined level of CO2. In certain embodiments, the predetermined level of CO2 can be 1.0% to 10% CO2. In certain embodiments, the predetermined level of CO2 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% CO2. In certain embodiments, the predetermined level of CO2 can be about 4.5% to 5.5% CO2. In certain embodiments, the predetermined level of CO2 can be about 5% CO2. In certain embodiments, the predetermined level of CO2 can be about 3.5%, about 4.0%, about 4.5%, about 5.0%, about 5.5% or about 6.5% CO2. In some embodiments, the step of amplifying the transduced T cell colony can be performed in any combination at a predetermined temperature and / or in the presence of a predetermined level of CO2. For example, in one embodiment, the step of expanding the transduced T cell population may comprise a predetermined temperature of about 36°C to 38°C and in the presence of a predetermined level of CO2 of about 4.5% to 5.5% CO2.

[0070] Any suitable medium T cell growth medium can be used to suspend the cells. For example, the T cell growth medium can include, but is not limited to, a sterile low glucose solution comprising appropriate amounts of a buffer, magnesium pyruvate, calcium pyruvate, sodium pyruvate, and sodium bicarbonate. In one embodiment, the medium is OpTmizer ™ (Life Technologies), but those skilled in the art will understand how to generate similar culture media. In one embodiment, the culture medium is EX-VIVO ™ Serum-free medium (Lonza Bioscience).

[0071] The incubation (and / or transduction) step can be performed without limitation in a closed system. In certain embodiments, the closed system is a cell culture bag using any suitable cell culture bag (e.g., Mitenyi Biotec MACS GMP Cell Differentiation Bags, Origen Biomedical PermaLife ™ In some embodiments, the closed system has at least 500 cm 2 In some embodiments, the closed system has an internal surface area of ​​at least 1000 cm 2 、1200cm 2 、1400cm 2 、1500cm 2 、1600cm 2 、1800cm 2, 2000cm 2 、2200cm 2 、2500cm 2 or 3000cm 2 In some embodiments, the closed system has an internal surface area of ​​no greater than 1500 cm 2 、1600cm 2 、1800cm 2 , 2000cm 2 、2200cm 2 、2500cm 2 or 3000cm 2 The internal surface area.

[0072] In some embodiments, the cell culture bag used in the closed system is coated with recombinant human fibronectin. The recombinant human fibronectin fragment may include three functional domains: a central cell binding domain, a heparin binding domain II, and a CS1 sequence. Recombinant human fibronectin or its fragments can increase the gene 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 (TakaraBio, Japan). In certain embodiments, the cell culture bag may be coated with a recombinant human fibronectin fragment at a concentration of about 0.1µg / mL to 60µg / mL, preferably 0.5µg / mL to 40µg / mL. In certain embodiments, the cell culture bag may be coated with a recombinant human fibronectin fragment at a concentration of about 0.5µg / mL to 20µg / mL, 20µg / mL to 40µg / mL, or 40µg / mL to 60µg / mL. In certain embodiments, the cell culture bag may be coated with about 0.5µg / mL, 1µg / mL, about 2µg / mL, about 3µg / mL, about 4µg / mL, about 5µg / mL, about 6µg / mL, about 7µg / mL, about 8µg / mL, about 9µg / mL, about 10µg / mL, about 11µg / mL, about 12µg / mL, about 13µg / mL, about 14µg / mL, about 15µg / mL, about 16µg / mL, about 17µg / mL, about 18µg / mL, about 19µg / mL, or about 20µg / mL of a recombinant human fibronectin fragment. In certain embodiments, the cell culture bag may be coated with about 2µg / mL to 5µg / mL, about 2µg / mL to 10µg / mL, about 2µg / mL to 20µg / mL, about 2µg / mL to 25µg / mL, about 2µg / mL to 30µg / mL, about 2µg / mL to 35µg / mL, about 2µg / mL to 40µg / mL, about 2µg / mL to 50µg / mL, or about 2µg / mL to 60µg / mL of recombinant human fibronectin fragment. In certain embodiments, the cell culture bag may be coated with at least about 2µg / mL, at least about 5µg / mL, at least about 10µg / mL, at least about 15µg / mL, at least about 20µg / mL, at least about 25µg / mL, at least about 30µg / mL, at least about 40µg / mL, at least about 50µg / mL, or at least about 60µg / mL of recombinant human fibronectin fragment. In certain embodiments, the cell culture bag may be coated with at least about 10 μg / mL of a recombinant human fibronectin fragment. In certain embodiments, the cell culture bag may not be coated with a recombinant human fibronectin fragment.

[0073] In some embodiments, a transduction enhancer is introduced into the closed system. Non-limiting examples of such transduction enhancers include Vectofusin ™ Transduction mixture.

[0074] In certain embodiments, the cell culture bags used in the closed bag culture system can be blocked with human albumin serum (HSA). In alternative embodiments, the cell culture bags are not blocked with HSA.

[0075] Once the closed system is coated with recombinant fibronectin, a solution containing the vector is added to the closed system so that the vector can be fixed to the inner surface of the closed system by the recombinant fibronectin. Once cells are added, this fixation can improve the transduction efficiency.

[0076] 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 material to cells. The viral vector that can be used according to the transduction step can be any affine or bi-directional viral vector, including but not limited to recombinant retroviral vectors, recombinant lentiviral vectors, recombinant adenoviral vectors, and recombinant adeno-associated virus (AAV) vectors. In one embodiment, the viral vector is a MSGV1 γ retroviral vector. In some embodiments, the vector is a non-viral vector.

[0077] In some embodiments, a total volume of at least 100 mL of a solution containing the carrier is used. In some embodiments, 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 of a solution containing the carrier is used. In some embodiments, a total volume of no more than 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 of a solution containing the vector is used.

[0078] In some embodiments, the carrier solution includes 1×10 3 Transduction units per ml (TU / ml) up to 1 × 10 12 Transducing units of viral vector per milliliter (TU / ml).

[0079] Once 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 include recombinant fibronectin. In some embodiments, the removal of the vector solution is performed by gravity or syringe discharge, which helps to retain the immobilized vector on the inner surface while removing impurities. Lymphocyte transduction can be performed in a coated closed system with an immobilized vector. In some embodiments, transduction is performed using a sample containing lymphocytes. In some embodiments, the sample includes at least 2.5×10 7In some embodiments, the sample includes 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 In some embodiments, the sample includes no more than 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 lymphocytes (e.g., T cells).

[0080] In some embodiments, lymphocyte transduction is performed in a closed system that does not include a fixative (eg, recombinant fibronectin).

[0081] The lymphocytes used in the currently disclosed methods are generally obtained from a donor subject, which may be a cancer patient (i.e., an autologous donor) who will be treated with a cell population produced by the methods described herein, or may be an individual (i.e., an allogeneic donor) who donates a lymphocyte sample that will be used to treat a different individual or cancer patient after generating a cell population produced by the methods described herein. Lymphocytes may be obtained from a donor subject by any suitable method used in the art. For example, lymphocytes may be obtained by any suitable in vitro method, venipuncture, or other blood collection method, by which a blood sample and / or lymphocytes are obtained. In one embodiment, lymphocytes are obtained by a single collection.

[0082] Optionally, in some embodiments, the methods described herein further comprise the step of enriching the lymphocyte population obtained from the donor subject prior to transduction. Enriching lymphocytes can be accomplished by any suitable separation method, including but not limited to the use of a separation medium (e.g., Ficoll-Paque ™ 、RosetteSep ™ HLA Total Lymphocyte Enrichment Cocktail, Lymphocyte Separation Medium (LSA) (MP Biomedical Cat. No. 0850494X), etc.), non-ionic iodixanol-based media such as OptiPrep ™ etc.), cell size, shape or density separation by filtration or panning, immunomagnetic separation (e.g., magnetic-activated cell sorting system, MACS), fluorescent separation (e.g., fluorescence-activated cell sorting system, FACS), or bead-based column separation.

[0083] Optionally, in some embodiments, by using a selection agent to select CD4 + / CD8 +The cells are positively enriched to remove circulating lymphoma cells from the sample. In some such embodiments, after incubation with the selection agent, the incubated cells (including cells to which the selection agent has been bound) are transferred to a system for immunoaffinity-based cell separation. In some embodiments, the system for immunoaffinity-based separation is or comprises a magnetic separation column.

[0084] In some such embodiments, the separation method includes separating different cell types based on the expression or presence of one or more specific molecules in the cell, such as surface markers, for example, surface proteins, intracellular markers or nucleic acids. In some embodiments, any known separation method based on such markers can be used. In some embodiments, separation is separation based on affinity or immunoaffinity. For example, separation in some embodiments includes separating cells and cell colonies based on cell expression or expression levels of one or more markers (usually cell surface markers), such as by incubating with antibodies or binding partners that specifically bind to these markers, followed by washing steps and separating cells bound to antibodies or binding partners from cells not bound to antibodies or binding partners. Such separation steps can be based on positive selection and / or negative selection, retaining cells bound to reagents in positive selection for further use, and retaining cells not bound to antibodies or binding partners in negative selection. In some examples, two parts are retained for further use.

[0085] In some such embodiments, negative selection may be particularly useful when antibodies that specifically identify cell types in a heterogeneous population are not available, such that separation is best based on markers expressed by cells other than the desired population.

[0086] Separation need not 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 (such as cells expressing a marker) refers to increasing the number or percentage of such cells, but does not necessarily result in the complete absence of cells that do not express the marker. Similarly, positive selection, removal or depletion of a particular type of cell (such as cells expressing a marker) refers to reducing the number or percentage of such cells, but does not necessarily result in the complete removal of all such cells.

[0087] In some examples, multiple rounds of separation steps are performed, wherein the positive or negatively selected fraction from one step is subjected to another separation step, such as a subsequent positive or negative selection. In some examples, a single separation step can deplete cells that simultaneously express multiple markers, such as by incubating the cells with multiple antibodies or binding partners, each of which is specific for the marker targeted by the negative selection. Similarly, multiple cell types can be positively selected simultaneously by incubating the cells with multiple antibodies or binding partners expressed on multiple cell types.

[0088] For example, in some embodiments, specific subpopulations of T cells, such as cells that are positive or express high levels 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, anti-CD3 / anti-CD28 conjugated magnetic beads (e.g., DYNABEADS M-450 CD3 / CD28 T cell expander) to positively select CD3+, CD28+ T cells. In some embodiments, the cell population is enriched for T cells with a naive phenotype (CD45RA+ CCR7+).

[0089] In some embodiments, separation is performed by enriching a particular cell population by positive selection, or depleting a particular cell population by negative selection. In some embodiments, positive or negative selection is accomplished by incubating cells with one or more antibodies or other binding agents that specifically bind to one or more surface markers expressed (marker+) or expressed at relatively high levels (markerhigh) on the positively or negatively selected cells, respectively.

[0090] In specific embodiments, a biological sample (e.g., a sample of PBMCs or other leukocytes) is subjected to selection for CD4+ T cells, wherein both negative and positive fractions are retained. In certain embodiments, CD8+ T cells are selected from the negative fraction. In some embodiments, a biological sample is subjected to selection for CD8+ T cells, wherein both negative and positive fractions are retained. In certain embodiments, CD4+ T cells are selected from the negative fraction.

[0091] In some embodiments, T cells are isolated from PBMC samples by negative selection for markers expressed on non-T cells such as B cells, monocytes or other leukocytes such as CD14. In some embodiments, CD4+ or CD8+ selection steps are used to separate CD4+ helper T cells and CD8+ cytotoxic T cells. Such CD4+ and CD8+ populations can be further sorted into subpopulations by positive or negative selection for markers expressed or expressed at a relatively high degree on one or more naive, memory and / or effector T cell subsets.

[0092] In one example, to enrich CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibodies or binding partners are bound to a solid support or matrix, such as magnetic beads or paramagnetic beads, to allow separation of cells for positive and / or negative selection. For example, in some embodiments, cells and cell populations are separated or separated using immunomagnetic (or affinity magnetic) separation techniques. In some embodiments, a sample or composition of cells to be separated is incubated with a small, magnetizable or magnetically responsive material, such as magnetically responsive particles or microparticles, such as paramagnetic beads (e.g., such as Dynabeads ™ or MACS beads). The magnetically responsive material (e.g., particle) is typically attached directly or indirectly to a binding partner (e.g., antibody) that specifically binds to a molecule (e.g., surface marker) present on one or more cells or cell populations for which separation (e.g., negative or positive selection is desired) is desired.

[0093] In some embodiments, the magnetic particles or beads contain magnetically responsive materials bound to specific binding members (such as antibodies or other binding partners). Many well-known magnetically responsive materials are used in magnetic separation methods. Incubation is usually carried out under conditions where an antibody or binding partner or a molecule (such as a secondary antibody or other reagent) that specifically binds to such an antibody or binding partner attached to a magnetic particle or bead specifically binds to a cell surface molecule (if present on a cell within the sample). In some embodiments, the sample is placed in a magnetic field, and those cells attached with magnetically responsive or magnetizable particles will be attracted to the magnet and separated from unlabeled cells. For positive selection, cells attracted to the magnet are retained; for negative selection, cells that are not attracted (unlabeled cells) are retained. In some embodiments, a combination of positive and negative selections is performed during the same selection step, wherein the positive and negative portions are retained and further processed or undergo further separation steps. In some embodiments, the magnetically responsive particles are coated in a first antibody or other binding partner, a second antibody, a lectin, an enzyme, or streptavidin. In certain embodiments, the magnetic particles are attached to the cells via a coating of a primary antibody specific for one or more markers. In some embodiments, cells (rather than beads) are labeled with a primary antibody or binding partner, and magnetic particles coated with a cell type-specific secondary antibody or other binding partner (e.g., streptavidin) are then added. In some embodiments, streptavidin-coated magnetic particles are used in combination with biotinylated primary or secondary antibodies. In some embodiments, the magnetically responsive particles are attached to cells that will then be incubated, cultured, and / or engineered; in some embodiments, the particles remain attached to the cells for administration to a patient. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, for example, the use of competitive non-labeled antibodies, and magnetizable particles or antibodies conjugated to cleavable linkers. In some embodiments, the magnetizable particles are biodegradable.

[0094] In some embodiments, affinity-based selection is performed via magnetic activated cell sorting (MACS) (Miltenyi Biotec, Auburn, CA). The magnetic activated cell sorting (MACS) system is capable of selecting cells attached to magnetized particles with high purity. In certain embodiments, MACS operates in a mode in which non-target and target materials are eluted sequentially after application of an external magnetic field. That is, cells attached to the magnetized particles are held in place, while unattached materials are eluted. Then, after this first elution step is complete, the materials that were trapped in the magnetic field and prevented from eluting are released in a manner so that they can be eluted and recovered. In certain embodiments, non-target cells are labeled and removed from the heterogeneous cell population.

[0095] In some embodiments, use the system, device or equipment of one or more steps in the separation, cell preparation, separation, processing, incubation, cultivation and / or preparation step of carrying out these methods to separate or separate.In some embodiments, this system is used for carrying out each step in these steps in a closed or sterile environment, for example, to minimize error, user processing and / or pollution.In one example, system is the system described in International Patent Application Publication No. WO 2009 / 072003 or US20110003380 A1, and this International Patent Application is incorporated herein by reference separately.In some embodiments, system or device are with integrated or independent system and / or in automatic or programmable mode to carry out one or more steps in separation, processing, engineering and preparation step.In some embodiments, system or equipment comprise the computer and / or computer program communicated with this system or equipment, and it allows the user to program, control the various embodiments of processing, separation, engineering and preparation step, assess their result and / or adjust the various embodiments of processing, separation, engineering and preparation step. In some embodiments, separation and / or other steps are performed using a CliniMACS system (Miltenyi Biotec), for example, for automatically separating cells at a clinical scale in a closed and sterile system. Components may include an integrated microcomputer, a magnetic separation unit, a peristaltic pump, and various pinch valves. In some embodiments, the integrated computer controls the components of the apparatus and directs 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 selecting a column. The peristaltic pump controls the flow rate of the entire tubing set and, together with the pinch valve, ensures the controlled flow of buffer through the system and the continuous suspension of cells.

[0096] In some embodiments, the CliniMACS system uses magnetizable particles coupled with antibodies provided in a sterile, pyrogen-free solution. In some embodiments, after labeling cells with magnetic particles, the cells are washed to remove excess particles. The cell preparation bag is then connected to a tubing set, which is in turn connected to a bag containing a buffer and a cell collection bag. The tubing set consists of preassembled sterile tubing, including a pre-column and a separation column, and is only for single use. After starting the separation procedure, the system automatically applies the cell sample to the separation column. The labeled cells are retained in the column, while the unlabeled cells are removed by a series of washing steps. In some embodiments, the cell colony used for the method described herein is not labeled and is not retained in the column. In some embodiments, the cell colony used for the method described herein is labeled and retained in the column. In some embodiments, the cell colony used for the method described herein is eluted from the column after removing the magnetic field and collected in a cell collection bag.

[0097] In certain embodiments, separation and / or other steps are performed using the CliniMACS Prodigy system (Miltenyi Biotec). In some embodiments, the CliniMACS Prodigy system is equipped with a cell processing unit that allows automatic washing and fractionation of cells by centrifugation. The CliniMACS Prodigy system may also include an onboard camera and image recognition software to determine the optimal cell fractionation endpoint by identifying the macroscopic layers 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 completes cell culture protocols such as cell differentiation and expansion, antigen loading, and long-term cell culture. The input port may allow for aseptic removal and replenishment of culture medium, and cells may be monitored using an integrated microscope.

[0098] In some embodiments, the cell populations described herein are collected and enriched (or consumed) by flow cytometry, wherein cells stained for a variety of cell surface markers are carried in a fluid stream. In some embodiments, the cell populations described herein are collected and enriched (or consumed) by preparative scale (FACS) sorting. In certain embodiments, the cell populations described herein are collected and enriched (or consumed) by using a combination of a microelectromechanical system (MEMS) chip and a FACS-based detection system (see, e.g., WO 2010 / 033140, Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al. (2008) J Biophoton. 1 (5): 355-376). In both cases, cells can be labeled with a variety of markers, thereby allowing well-defined T cell subsets to be separated with high purity.

[0099] In some embodiments, the antibody or binding partner is labeled with one or more detectable markers to facilitate separation of positive and / or negative selection. For example, separation can be based on binding to a fluorescently labeled antibody. In some examples, cell separation based on binding of antibodies or other binding partners specific to one or more cell surface markers is performed in a fluid stream, such as by fluorescence activated cell sorting (FACS), including preparative (FACS) and / or microelectromechanical systems (MEMS) chips, for example, in combination with a flow cytometry detection system. Such methods allow positive and negative selection based on multiple markers simultaneously.

[0100] In some embodiments, at least 0.5×10 9 lymphocytes, and optionally enrich and / or stimulate them. In some embodiments, at least 0.6×10 9 , 0.7×109 , 0.8×10 9 , 0.9×10 9 , 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 lymphocytes, and optionally enrich and / or stimulate them. In some embodiments, no more than 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 lymphocytes, and optionally enrich and / or stimulate them.

[0101] Optionally, the methods described herein also include the step of stimulating lymphocytes using one or more lymphocyte stimulators. In some embodiments, stimulation is performed before the transduction step. In some embodiments, stimulation is performed after the transduction step. The stimulation step is also referred to as the activation step in this article.

[0102] Any combination of one or more suitable lymphocyte stimulators can be used to stimulate (activate) lymphocytes. Non-limiting examples include antibodies or functional fragments thereof targeting T cell stimulatory or costimulatory molecules (e.g., anti-CD2 antibodies, anti-CD3 antibodies, anti-CD28 antibodies or their functional fragments), T cell cytokines (e.g., any isolated, wild-type or recombinant cytokines, such as: 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)) or any other suitable mitogen (e.g., tetradecanoylphorbol acetate (TPA), phytohemagglutinin (PHA), concanavalin A (conA), lipopolysaccharide (LPS), pokeweed mitogen (PWM)) or natural ligands of T cell stimulatory or costimulatory molecules. In some embodiments, the stimulator is an anti-CD3 antibody and / or an anti-CD28 antibody.

[0103] In some embodiments, the lymphocyte stimulator 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).

[0104] In some embodiments, the step of stimulating lymphocytes described herein may require stimulating lymphocytes at a predetermined temperature, within a predetermined amount of time, and / or in the presence of a predetermined level of CO2 using one or more stimulants. In certain embodiments, the predetermined temperature for stimulation may 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 may be about 34°C to 39°C. In certain embodiments, the step of stimulating lymphocytes includes stimulating lymphocytes for a predetermined time using one or more stimulants. In certain embodiments, the predetermined time for stimulation may be about 24 hours to 72 hours. In certain embodiments, the predetermined time for stimulation may be about 24 hours to 36 hours. In certain embodiments, the step of stimulating lymphocytes may include stimulating lymphocytes using one or more stimulants in the presence of a predetermined level of CO2. In certain embodiments, the predetermined level of CO2 may be about 1.0% to about 10% CO2. In certain embodiments, the predetermined level of CO2 for stimulation may 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% CO2.

[0105] In some embodiments, anti-CD3 antibodies (or functional fragments thereof), anti-CD28 antibodies (or functional fragments thereof), or a combination of anti-CD3 and anti-CD28 antibodies may be used according to the step of stimulating a lymphocyte population. Any soluble or fixed anti-CD3 antibody and / or anti-CD28 antibody or their functional fragments may 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, antibodies may be purchased commercially from suppliers known in the art, including but not limited to Germany Tianmei Biotech, BD Biosciences (e.g., 1 mg / mL pure MACS GMP CD3, part number 170-076-116) and eBioscience. In addition, those skilled in the art will understand how to produce anti-CD3 antibodies and / or anti-CD28 antibodies by standard methods. Any antibodies used in the methods described herein should be produced under good manufacturing practices (GMP) in compliance with the relevant agency's guidelines for biological products.

[0106] In certain embodiments, the T cell stimulator may include an anti-CD3 antibody or anti-CD28 antibody at a concentration of about 20 ng / mL to 100 ng / mL. In certain embodiments, the concentration of the anti-CD3 antibody or anti-CD28 antibody may 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.

[0107] In some embodiments, the 3-day process of the present disclosure may include the following steps: apheresis material collection, 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 on day 0 to day 1, a viral transduction step performed on day 1 to day 4, and a fourth washing and concentration step performed on day 3 to day 4 (harvest day). The apheresis material may be fresh apheresis material, cryopreserved apheresis material, or cryopreserved T cells.

[0108] In some embodiments, the 3-day process may include an activation step on day 0, day 1, or day 2. In some embodiments, the 3-day process may include a viral vector transduction step on day 1, day 2, or day 3.

[0109] In some embodiments, the process of 3 days 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 different washing procedures.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 harvest day.

[0110] Engineered lymphocytes

[0111] As demonstrated in the attached experimental examples, the prepared lymphocytes include a higher proportion of immature lymphocytes (eg, naive T cells). Therefore, one embodiment of the present disclosure provides a lymphocyte population including CD4+ T and CD8+ T cells prepared by the present method.

[0112] 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.

[0113] In some embodiments, no more than 25% of CD4+ T cells are effector memory T cells. In some embodiments, no more than 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6% or 5% of CD4+ T cells are effector memory T cells.

[0114] In some embodiments, no more than 44% of CD4+ T cells are central memory T cells. In some embodiments, no more than 43%, 42%, 41% or 40% of CD4+ T cells are central memory T cells.

[0115] In some embodiments, no more than 1.5% of CD4+ T cells are effector T cells. In some embodiments, no more than 1.4%, 1.3%, 1.2%, 1.1%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, or 0.5% of CD4+ T cells are effector T cells.

[0116] 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.

[0117] 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.

[0118] In some embodiments, no more than 30% of CD8+ T cells are effector memory T cells. In some embodiments, no more than 28%, 27%, 25%, 22%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11% or 10% of CD8+ T cells are effector memory T cells.

[0119] In some embodiments, no more than 60% of CD8+ T cells are central memory T cells. In some embodiments, no more than 58%, 56%, 55%, 54%, 52% or 50% of CD8+ T cells are central memory T cells.

[0120] In some embodiments, at least 60% of the harvested CD8+ T cells are CCR7+ cells. In some embodiments, up to 40% of the harvested CD8+ T cells are a combination of effector memory T cells and effector T cells.

[0121] As is well known in the art, each type of T cell can be characterized by cell surface markers. 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-.

[0122] Surface markers of stem cell 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-.

[0123] Surface markers for effector memory T cells (Tem) include, but are not limited to, CCR7-, CD45RO+, and CD95+. An additional marker for 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-.

[0124] The term "naive cells" referred to throughout the text includes one or more of naive T cells, stem cell memory T cells (Tscm) and central memory T cells (Tcm). These cells are characterized in part by the expression of CCR7+.

[0125] The harvested lymphocytes preferably include a large proportion of CD3+ T cells. In some embodiments, at least 25%, 35%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% or 90% of the harvested lymphocytes are CD3+ T cells.

[0126] The harvested lymphocytes preferably include a large proportion that have been transduced. 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 harvested lymphocytes are transduced with the vector. In some embodiments, each transduced lymphocyte includes at least one copy of the vector (or the coding sequence included) integrated into the host genome. In some embodiments, each transduced lymphocyte includes at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of the vector integrated into the host genome.

[0127] In some embodiments, the vector includes a transgene encoding a polypeptide. The polypeptide can be a CAR or a TCR, but is not limited thereto. In some embodiments, the CAR or TCR includes an antigen binding molecule. In some embodiments, the antigen binding molecule has binding specificity to an antigenic portion. In some embodiments, the antigenic portion is a tumor antigen (e.g., a protein or other molecule produced by a cancer cell).

[0128] In some embodiments, the vector includes more than one transgene encoding more than one CAR or TCR molecule, including antigen binding molecules with specificity for different antigen portions. In some embodiments, the vector includes more than one transgene encoding more than one CAR or TCR molecule, including antigen binding molecules with specificity for two different tumor antigens.

[0129] In some aspects, the antigenic portion is an antigen associated with a cancer or cancer cell. Such antigens may include, but are not limited to, 707-AP (707 alanine proline), AFP (alpha (a)-fetoprotein), ART-4 (adenocarcinoma antigen recognized by T4 cells), BAGE (B antigen; b-catenin / m, b-catenin / mutated), BCMA (B cell maturation antigen), Bcr-abl (cleft punctate 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 (antigen recognized by CTL on melanoma), CAP-1 (carcinoembryonic antigen peptide 1), CASP-8 (caspase 8), CDC27m (mutated cell division cyclin 27), CDK4 / m (mutated cyclin-dependent kinase 4), CEA (carcinoembryonic antigen), CT (carcinoma / testis). granules (antigens), Cyp-B (cyclophilin B), DAM (differentiation antigen on melanoma), EGFR (epidermal growth factor receptor), EGFRvIII (epidermal growth factor receptor variant III), EGP-2 (epidermal glycoprotein 2), EGP-40 (epidermal glycoprotein 40), Erbb2, 3, 4 (erythroblastic leukemia viral oncogene homolog-2, -3, 4), ELF2M (mutated elongation factor 2), ETV6-AML1 (Ets mutant somatic 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-acetylglucosamine transferase V), Gp100 (glycoprotein 100kD), HAGE (helicase antigen), HER-2 / neu (human epidermal receptor 2 / neural;also known as EGFR2), HLA-A (human leukocyte antigen A), HPV (human papillomavirus), HSP70-2M (mutated heat shock protein 70-2), HST-2 (human cyclic tumor factor 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), kappa light chain, LAGE (L antigen), LDLR / FUT (low-density lipid receptor / GDP-L-fucose: bD-galactosidase 2-a L-fucosyltransferase), LeY (Lewis-Y antibody), L1CAM (L1 cell adhesion molecule), MAGE (melanoma antigen), MAGE-A1 (melanoma associated antigen 1), mesothelin, murine CMV-infected cells, MART-1 / Melan-A (melanoma antigen recognized by T cells 1 / melanoma antigen A), MC1R (melanocortin 1 receptor), myosin / m (mutated myosin), MUC1 (mucin 1), MUM-1, -2, -3 (melanoma ubiquitously mutated proteins 1, 2, 3), NA88-A (NA from patient M88) cDNA clone), 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), carcinoembryonic antigen (h5T4), P15 (protein 15), p190 smaller bcr-abl (190KD protein bcr-abl), 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 ubiquitin 1 or 2), SAGE (meat ; SART-1 or SART-3 (squamous antigen for tumor rejection 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 (translocation Ets family leukemia / acute myeloid leukemia 1), TPI / m (mutated triosephosphate isomerase), 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), WT1 (Wilms tumor gene), or any combination thereof. ;

[0130] Additional examples of tumor antigens include 2B4 (CD244), 4-1BB, 5T4, A33 antigen, adenocarcinoma antigen, adrenergic receptor beta 3 (ADRB3), A kinase anchoring protein 4 (AKAP-4), alpha-fetoprotein (AFP), anaplastic lymphoma kinase (ALK), androgen receptor, B7H3 (CD276), beta 2-integrin, BAFF, B lymphoma cells, B cell maturation antigen (BCMA), bcr-abl (oncogene fusion protein composed of breakpoint cluster region (BCR) and Aberson murine leukemia virus oncogene homolog 1 (Abl)), BhCG, bone marrow stromal cell antigen 2 (BST2), CCCTC-binding factor (zinc finger protein)-like (BORIS or imprinting site regulator) brothers), BST2, C242 antigen, 9-0-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, CD 133. CD137, CD138, CD150, CD152 (CTLA-4), CD160, CD171, CD179a, CD200, CD221, CD229, CD244, CD272 (BTLA), CD274 (PDL-1, B7 H1), CD279 (PD-1), CD352, CD358, CD300 molecule-like family member f (CD300LF), carcinoembryonic antigen (CEA), occludin 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 subclass 1, CRACC, CD319, and 19A24), CTLA-4, cyclin B l, chromosome X open reading frame 61 (CXORF61), cytochrome P450 1B1 (CYP1B1), DNAM-1 (CD226), desmoglein 4, DR3, DR5, E-cadherin neo-epitope, epidermal growth factor receptor (EGFR), EGF1R, epidermal growth factor receptor variant III (EGFRvIII), epithelial glycoprotein-2 (EGP-2), epithelial glycoprotein-40 (EGP-40), mucin-like hormone receptor-like 2 containing an EGF-like module (EMR2), elongation factor 2 mutant (ELF2M), endosialin, epithelial cell adhesion molecule (EPCAM), 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 (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(l-4)bDGlcp(ll)Cer), o-acetyl-GD2 ganglioside (OAcGD2), GITR (TNFRSF 18), GM1, ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer), GP 100, hexose portion of globoH glycoceramide (GloboH), glycoprotein 75, phosphatidylinositol proteoglycan 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 (HPVE7), heat shock protein 70-2 mutation (muthsp70-2), 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, kappa-light chain, KCS1, kinase insert 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), legumin, 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 K 9 (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 recognized by T cells 1 (MelanA or MARTI), MelanA / MARTl, mesothelin, MAGE A3, melanoma cell inhibitor of apoptosis (ML-IAP), melanoma-specific chondroitin sulfate proteoglycan (MCSCP), MORAb-009, MS4A1, mucin 1 (MUCl), MUC2, MUC3, MUC4, MUC5AC, MUC5b, MUC7, MUC16, mucin CanAg, Müllerian inhibitory substance (MIS) type II receptor, v-myc avian myelocytic tumor 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, breast differentiation antigen (NY-BR-1), NY-ESO-1, carcinoembryonic antigen (h5T4), olfactory receptor 51E2 (OR51E2), OX40, plasma cell antigen, poly SA, acrosome protease binding protein sp32 (OY-TESl), p53, p53 mutant, pan-linked protein 3 (PANX3), prostatic 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-α), PDGFR-β, PDL192, PEN-5, phosphatidylserine, placenta-specific 1 (PLAC1), polysialic acid, prostate enzyme, prostate cancer cell, prostaglandin, protease serine 21 (testosterone or PRSS21), protease 3 (PR1), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), proteasome (precursor, megalin factor) subunit beta type, advanced glycation end product receptor RAGE-1, RANKL, Ras mutant, Ras homolog family member C (RhoC), RON, receptor tyrosine kinase-like orphan receptor 1 (ROR1), renal ubiquitin 1 (RU1), renal ubiquitin 2 (RU2), sarcoma translocation breakpoints, 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 (TARP), telomerase, TIGIT, TNF-α precursor, tumor endothelial marker 1 (TEM1 / CD248), tumor endothelial marker 7-related (TEM7R), tenascin C, TGFβ2, TGF-β, 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, urokinase 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).

[0131] In other embodiments, the antigenic portion is associated with a virus-infected cell (i.e., a viral antigenic portion). Such antigenic portions can 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, type 2, or type 8 (HSV1, HSV2, or HSV8) viral antigens (e.g., glycoproteins gB, gC, gC, gE, gG, gH, gI, gJ, gK, gL, gM, UL20, UL32, US43, UL45, UL49A), cytomegalovirus (CMV) viral antigens (e.g., glycoprotein gB, gC, gC, gE, gG, gH, gI, gJ, gK, gL, gM or other envelope proteins), human immunodeficiency virus (HIV) viral antigens (glycoprotein gp120, gp41 or p24), influenza virus antigens (e.g., hemagglutinin (HA) or neuraminidase (NA)), measles or mumps virus antigens, human papillomavirus (HPV) viral antigens (e.g., L1, L2), viral antigens of parainfluenza virus, viral antigens of rubella virus, viral antigens of respiratory syncytial virus (RSV) virus, or viral antigens of varicella-zoster virus. In this embodiment, the cell surface receptor can be any TCR, or any CAR that recognizes any of the aforementioned viral antigens on a virus-infected target cell.

[0132] In other embodiments, the antigenic portion is associated with cells with immune or inflammatory dysfunction. Such antigenic portions may include, but are not limited to, myelin basic protein (MBP), myelin proteolipid protein (PLP), myelin oligodendrocyte glycoprotein (MOG), carcinoembryonic antigen (CEA), proinsulin, glutamine decarboxylase (GAD65, GAD67), heat shock protein (HSP), or any other tissue-specific antigen involved in or associated with a pathogenic autoimmune process.

[0133] In some embodiments, the TCR is specific for an antigenic portion 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-human papillomavirus TCR, anti-HSP70-2M TCR, anti-HST-2 TCR, anti-hTERT TCR, or anti-hTRT TCR, anti-iCETCR, 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-PSATCR, 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.

[0134] In addition to the antigen binding molecules, the CAR of the present disclosure may include a hinge, a transmembrane domain and / or an intracellular domain. In some embodiments, the lymphocyte domain may include a costimulatory domain and an activation domain.

[0135] The hinge can be an extracellular domain of an antigen binding system located between the binding motif and the transmembrane domain. The hinge can also be referred to as an extracellular domain or a "spacer". The hinge can contribute to receptor expression, activity and / or stability. The hinge can also provide flexibility in accessing the target antigen. In some embodiments, the hinge domain is located between the binding motif and the transmembrane domain.

[0136] In some embodiments, the hinge is, is from, or is derived from an immunoglobulin-like hinge domain (e.g., including all or fragments thereof). In some embodiments, the hinge domain is from or is 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.

[0137] In some embodiments, the hinge is, is from or is derived from (e.g., comprises all or a fragment of) CD2, CD3 δ, CD3 ε, CD3 γ, CD4, CD7, CD8.α., CD8.β., 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), CD66a (CEACAM2), CD66a (CEACAM3), CD66a (CEACAM4), CD66a (CEACAM5), CD66a (CEACAM6), CD66a (CEACAM1), CD66a (CEACAM1), CD66a (CEACAM2), CD66a (CEACAM1), CD66a (CEACAM2), CD66a (CEACAM3), CD66a (CEACAM1), CD66a (CEACAM2 ... 6b (CEACAM8), CD66c (CEACAM6), CD66d (CEACAM3), CD66e (CEACAM5), CD69 (CLEC2), CD79A (B cell antigen receptor complex associated α chain), CD79B (B cell antigen receptor complex associated β chain), CD84 (SLAMF5), CD96 (Tactile), CD100 (SEMA4D), CD103 (ITGAE), CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD158A (KIR2DL1), CD158B1 (K IR2DL2), CD158B2 (KIR2DL3), CD158C (KIR3DP1), CD158D (KIRDL4), CD158F1 (KIR2DL5A), CD158F2 (KIR2DL5B), CD158K (KIR3DL2), CD160 (BY 55), CD162 (SELPLG), CD226 (DNAM1), CD229 (SLAMF3), CD244 (SLAMF4), CD247 (CD3-ζ), CD258 (LIGHT), CD268 (BAFFR), CD270 (TNFSF14), CD27 2 (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-stimulator (ICOS), LFA-1 (CD11a / CD18), NKG2C, DAP-10, ICAM-1, NKp80 (KLRF1), IL-2Rβ, IL-2R γ, IL-7R α, LFA-1, SLAMF9, LAT, GADS (GrpL), SLP-76 (LCP2), PAG1 / CBP, CD83 ligand, Fc γ receptor, MHC class 1 molecule, MHC class 2 molecule, TNF receptor protein, immunoglobulin, cytokine receptor, integrin, activated NK cell receptor, or Toll ligand receptor or fragments or combinations thereof.

[0138] In some embodiments, the hinge is, is or is derived from a hinge of CD8 alpha (e.g., including all or fragments thereof). In some embodiments, the hinge is, is or is derived from a hinge of CD28. In some embodiments, the hinge is, is or is derived from a fragment of a CD8 alpha hinge or a fragment of a CD28 hinge, wherein the fragment is any fragment that is less than the whole. In some embodiments, a fragment of a CD8 alpha hinge or a fragment of a 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 or C-terminus or both of a CD8 alpha hinge or a CD28 hinge.

[0139] A "transmembrane domain" refers to a domain that has the property of being present in a membrane when present in a molecule on the cell surface or cell membrane (e.g., spanning a portion or all of a cell membrane). It is not necessary for every amino acid in a transmembrane domain to be present in the membrane. For example, in some embodiments, a transmembrane domain is characterized in that a specified fragment or portion of a protein is substantially located in the membrane. A variety of algorithms can be used to analyze amino acid or nucleic acid sequences to predict protein subcellular localization (e.g., transmembrane localization). The programs psort (PSORT.org) and Prosite (prosite.expasy.org) are examples of such programs.

[0140] The transmembrane domain may be derived from any membrane-bound or transmembrane protein, such as the α, β or ζ chains of the T cell receptor, CD28, CD3 ε, CD3 δ, CD3 γ, CD45, CD4, CD5, CD7, CD8, CD8 α, CD8 β, 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, 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, CRT AM, cytokine receptor, DAP-10, DNAM1 (CD226), Fc γ receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, Ig α (CD79a), IL-2R β, IL-2R γ, IL-7R α, 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), lymphocyte function-associated antigen-1 (LFA-1; CD1-1a / CD18), MHC Class 1 molecules, 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; Ly108), SLAMF7, SLP-76, TNF receptor protein, TNFR2, TNFSF14, Toll ligand receptor, TRANCE / RANKL, VLA1 or VLA-6, or fragments, truncated forms or combinations thereof.

[0141] The intracellular domain (or cytoplasmic domain) comprises one or more signaling domains that cause and / or mediate intracellular signals after the target antigen binds to the binding motif, such as intracellular signals that activate one or more immune cell effector functions (e.g., natural immune cell effector functions). In some embodiments, the signaling domain of the intracellular domain mediates the activation of at least one normal effector function of an immune cell. The effector function of a T cell can be, for example, cytolytic activity or auxiliary activity, including the secretion of cytokines. In some embodiments, the signaling domain of the intracellular domain mediates T cell activation, proliferation, survival, and / or other T cell functions. The intracellular domain may comprise a signaling domain that is an activation domain. The intracellular domain may comprise a signaling domain that is a co-stimulatory signaling domain.

[0142] Intracellular signaling domains are known to transduce signals when an antigen binds to an immune cell. For example, cytoplasmic sequences of the T cell receptor (TCR) are known to initiate signal transduction after TCR binds to an antigen (see, e.g., Brownlie et al., Nature Rev. Immunol. 13:257-269 (2013)).

[0143] In certain embodiments, suitable signaling domains include, but are not limited to, 4-1BB / CD137, activating NK cell receptors, immunoglobulins, B7-H3, BAFFR, BLAME (SLAMF8), BTLA, CD100 (SEMA4D), CD103, CD160 (BY55), CD18, CD19, CD19a, CD2, CD247, CD27, CD276 (B7-H3), CD28, CD29, CD3 δ, CD3 ε, CD3 γ, CD30, CD4, CD40, CD49a, CD49D, CD49f, CD69, CD7, CD84, CD8α, CD8β, CD96 (Tactile), CD11a, CD11b, CD11c, CD11d, CD5, CEACAM1, CRT AM, cytokine receptors, DAP-10, DNAM1 (CD226), Fc γ receptor, GADS, GITR, HVEM (LIGHTR), IA4, ICAM-1, ICAM-1, Ig α (CD79a), IL-2R β, IL-2Rγ, IL-7R α, 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 molecules, NKG2C, NKG2D, NKp30, NKp44, NKp46, NKp80 (KLRF1), OX-40, PAG / Cbp, programmed death-1 (PD-1), PSGL1, SELPLG (CD162), signaling lymphocyte activation molecules (SLAM proteins), 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, truncated forms or combinations thereof.

[0144] CARs may also include a co-stimulatory signaling domain, for example, to increase signaling efficacy. See U.S. Pat. Nos. 7,741,465 and 6,319,494, as well as 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). The signal generated by TCR alone may not be sufficient to fully activate T cells, and secondary or co-stimulatory signals can increase activation. Therefore, in some embodiments, the signaling domain also includes one or more additional signaling domains (e.g., co-stimulatory signaling domains) that activate one or more immune cell effector functions (e.g., 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 the portion transduces an effector function signal. In some embodiments, the cytoplasmic domain described herein comprises one or more cytoplasmic sequences of a T cell co-receptor (or a fragment thereof). Non-limiting examples of such T cell co-receptors include 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 a ligand that binds to CD83. An exemplary co-stimulatory protein has an amino acid sequence of a co-stimulatory protein naturally found on T cells, and the complete native amino acid sequence of the co-stimulatory protein is described in NCBI Reference Sequence: NP 0.1. In some cases, CAR includes a 4-1BB co-stimulatory domain. In some cases, CAR includes a CD28 co-stimulatory domain. In some cases, CAR includes a DAP-10 co-stimulatory domain.

[0145] In some embodiments, the co-stimulatory signaling domain is a signaling domain of CD28. As shown in the experimental examples, CAR molecules with CD28 co-stimulatory signaling domains can benefit particularly from the newly developed accelerated manufacturing process. In some embodiments, CAR is encoded by a nucleic acid molecule embedded in a viral vector. In some embodiments, CAR is encoded by a nucleic acid molecule embedded in a lentiviral vector.

[0146] In some embodiments, CAR also includes ITAM. Examples of primary cytoplasmic signaling sequences containing ITAM that are particularly useful in the present disclosure include those derived from TCR ζ, FcR γ, FcR β, CD3 γ, CD3 δ, CD3 ε, CD5, CD22, CD79a, CD79b and CD66d. In some embodiments, ITAM includes CD3 ζ.

[0147] In some embodiments, the CAR molecule can be any anti-CD19 CAR molecule. On the one hand, as described in WO2015120096 or WO 2016191755, anti-CD19 CAR includes an extracellular scFv domain, an intracellular portion and / or a transmembrane portion of a CD28 molecule, an optional extracellular portion of a CD28 molecule and an intracellular cd3ζ domain, each of which is incorporated herein in its entirety.

[0148] In certain embodiments, the anti-CD19 CAR may further include additional domains, such as CD8 extracellular and / or transmembrane regions, extracellular immunoglobulin Fc domains (e.g., IgG1, IgG2, IgG3, IgG4), or one or more additional signaling domains, such as 41BB, OX40, CD2 CD16, CD27, CD30 CD40, PD-1, ICOS, LFA-1, IL-2 receptor, Fcγ receptor, or any other co-stimulatory domain with an immunoreceptor tyrosine-based activation motif.

[0149] In certain embodiments, the cell surface receptor is an anti-CD19 CAR, such as the FMC63-28Z CAR or FMC63-CD828BBZ CAR described in Kochenderfer et al., J Immunother. 2009 Sep;32(7):689-702, “Construction and Preclinical Evaluation of an Anti-CD19 Chimeric Antigen Receptor”, the subject matter of which is hereby incorporated by reference for the purpose of providing methods for constructing vectors for generating T cells expressing FMC63-28Z CAR or FMC63-CD828BBZ CAR.

[0150] In some embodiments, the T cell comprising the CAR molecule is Yescarta (Akiramu). In some embodiments, the T cell comprising the CAR molecule is Tecartus (brexucabtagene autoleucel).

[0151] In some embodiments, the engineered lymphocytes include a dual-targeted antigen binding system. The dual-targeted antigen binding system may include a bispecific CAR or TCR and / or a bicistronic CAR or TCR. Bispecific and bicistronic CARs may include two binding motifs (in a single CAR molecule or two CAR molecules, respectively). In some embodiments, the vectors of the present disclosure encode bicistronic and / or bispecific CARs (e.g., bicistronic and / or bispecific CARs that bind CD20 and CD19).

[0152] In some embodiments, a pharmaceutical composition is provided, the pharmaceutical composition comprising an engineered lymphocyte colony produced by the method described herein. In certain embodiments, the pharmaceutical composition may also include a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier may be a pharmaceutically acceptable material, composition or vehicle that participates in carrying or transporting the cell of interest from a tissue, organ or part of a body to another tissue, organ or part of a body. For example, the carrier may be a liquid or solid filler, a diluent, an excipient, a solvent or an encapsulating material or a certain combination thereof. Each component of the carrier must be "pharmaceutically acceptable" because it must be compatible with the other ingredients of the formulation. It must also be suitable for contact with any body tissue, organ or part that it may encounter, which means that it may not carry the risk of toxicity, irritation, allergic reaction, immunogenicity or any other complication that far exceeds its therapeutic benefit.

[0153] Treatment and use and optional storage

[0154] Lymphocytes prepared by the present method or lymphocyte populations as disclosed herein can be used to treat a variety of diseases and disorders.

[0155] In some embodiments, if the lymphocytes are not used immediately, they can be cryopreserved for later use. This method may include the step of washing and concentrating the engineered lymphocyte population using a diluent solution. In some aspects, the diluent solution is saline, 0.9% saline, PlasmaLyte A (PL), 5% glucose / 0.45% NaCl saline solution (D5), human serum albumin (HSA), or a combination thereof. In some aspects, HSA can be added to the washed and concentrated cells to improve cell viability and cell recovery after thawing. In another aspect, the washing solution is saline, and HSA (5%) is supplemented to the washed and concentrated cells. The method may also include the step of producing a cryopreservation mixture, wherein the cryopreservation mixture includes a diluted cell population in a diluent solution and a suitable cryopreservation solution. In some aspects, the cryopreserved solution can be any suitable cryopreservation solution, including but not limited to CryoStor10 (BioLifeSolution), which is mixed with a diluent solution of engineered lymphocytes at a ratio of 1:1 or 2:1.

[0156] In certain embodiments, HSA may be added to provide a final concentration of about 1.0% to 10% HSA in a mixture of cryopreservation. In certain embodiments, HSA may be added to provide a final concentration 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% HSA in a mixture of cryopreservation. In certain embodiments, HSA may 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 a mixture of cryopreservation. In certain embodiments, HSA may be added to provide a final concentration of about 2.5% HSA in a mixture of cryopreservation. For example, in certain embodiments, cryopreservation of an engineered T cell population may include washing the cells with 0.9% saline, adding HSA to a final concentration of 5% to the washed cells, and cryopreserving the cells with a CryoStor. ™ CS10 dilutes the cells 1:1 (for a final concentration of 2.5% HSA in the final cryopreservation mixture). In some embodiments, the method further comprises the step of freezing the cryopreservation mixture. In one aspect, the cryopreservation mixture is frozen in a controlled rate freezer using a defined freezing cycle at a cell concentration between about 1e6 and about 1.5e7 cells / mL of cryopreservation mixture. The method may further comprise the step of storing the cryopreservation mixture in vapor phase liquid nitrogen.

[0157] Also provided are methods and uses for treating a disease or pathological condition in a subject suffering from the disease or pathological condition. In some embodiments, the method requires administering to the subject a therapeutically effective amount or therapeutically effective dose of engineered lymphocytes. Pathological conditions that can be treated with 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 dysfunction.

[0158] As referred to herein, "cancer" may be a cancer associated with a surface antigen or a cancer marker, including but not limited to acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), adenoid cystic carcinoma, adrenocortical carcinoma, cancer, AIDS-related cancer, anal cancer, appendix cancer, astrocytoma, atypical teratoid / rhabdoma, central nervous system cancer, B-cell leukemia, lymphoma or other B-cell malignancies, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, osteosarcoma and malignant fibrous histiocytoma, brain stem glioma, brain tumor, breast cancer, bronchial tumor, Burkitt's lymphoma, carcinoid tumor, central nervous system cancer, cervical cancer, chordoma, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML ), chronic myeloid proliferative disorder, colon cancer, colorectal cancer, craniopharyngioma, cutaneous t-cell lymphoma, embryonal tumor, central nervous system cancer, endometrial cancer, ependymoblastoma, ependymoma, esophageal cancer, esthesia neuroblastoma, Ewing sarcoma family of tumors, extracranial germ cell tumor, extracranial germ cell tumor, extrahepatic bile duct cancer, eye cancer, bone fibrous histiocytoma, malignant tumor, osteosarcoma, gallbladder cancer, gastric (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 (endocrine pancreas), Kaposi's sarcoma, kidney cancer, Langerhans cell histiocytosis, laryngeal cancer, leukemia, lip and oral 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 carcinoma, mesothelioma, metastatic squamous neck cancer with occult primary midline tract cancer (involving the NUT gene), oral cancer, multiple endocrine neoplasms syndrome, multiple myeloma / plasma cell neoplasms, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative neoplasms, chronic myeloid leukemia (CML), acute myeloid leukemia (AML) , multiple myeloma, myeloproliferative disorders, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oral cancer, oropharyngeal cancer, osteosarcoma and malignant fibrous histiocytoma of bone, ovarian cancer, pancreatic cancer, papillomatosis, paraganglioma, sinus and nasal cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, intermediate differentiation pinealoma, pineoblastoma and supratentorial primitive neuroectodermal tumor, pituitary tumor, plasma cell neoplasms / multiple myeloma, pleuropulmonary blastoma, pregnancy and breast cancer, primary central nervous system (CNS) lymphoma, prostate cancer, rectal cancer, renal cell (kidney) cancer, transitional cell carcinoma of the renal pelvis and ureter, retinoblastoma, rhabdomyosarcoma, salivary gland cancer,Sarcoma, Sezary syndrome, small cell lung cancer, small intestinal cancer, soft tissue sarcoma, squamous cell carcinoma, squamous neck cancer, gastric (gastric / stomach) cancer, supratentorial primitive neuroectodermal tumor, T-cell lymphoma, skin cancer, testicular cancer, laryngeal cancer, thymoma and thymic cancer, thyroid cancer, transitional cell carcinoma of the renal pelvis and ureter, trophoblastic tumor, ureter and renal pelvis cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, Wilms' tumor.

[0159] In some aspects, 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), 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's lymphoma and lymphoblastic lymphoma. As mentioned 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, but are not limited to, 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 virus type 1, herpes simplex virus type 2, or herpes simplex virus type 8, 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. In some aspects, viral infection can cause or contribute to the development of cancer in a subject with the viral infection (e.g., HPV infection can lead to the development of or be associated with the development of several cancers, including cervical cancer, vulvar cancer, vaginal cancer, penile cancer, anal cancer, oropharyngeal cancer, and HIV infection can lead to the development of Kaposi's sarcoma). Examples of chronic inflammatory diseases, autoimmune diseases, or any other immune dysfunction 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.

[0160] Other examples of chronic inflammatory diseases, autoimmune diseases, or any other immune dysfunction that can be treated using the engineered T cells produced by the methods described herein include rheumatoid arthritis, allergies, asthma, Crohn's disease, IBD, IBS, fibromyalgia, mastocytosis, and celiac disease.

[0161] The term "treating," "treating," "treating," or "treatment" as used herein with respect to a disorder or disease may refer to preventing the disorder or disease, slowing the rate of onset or development of a disorder or disease, reducing the risk of developing a disorder or condition, preventing or delaying the development of symptoms associated with the disorder or disease, alleviating or ending symptoms associated with the disorder or disease, producing complete or partial regression of the disorder or disease, or some combination thereof.

[0162] A "therapeutically effective amount" or "therapeutically effective dose" is the amount of engineered lymphocytes that produces the desired therapeutic effect in a subject, such as preventing or treating a target disorder or alleviating symptoms associated with the disorder by killing target cells. Depending on the efficacy of the treatment for a given subject, the most effective result will depend on various factors, including, but not limited to, the characteristics of the engineered lymphocytes (including lifespan, activity, pharmacokinetics, pharmacodynamics, and bioavailability), the physiological condition of the subject (including age, sex, disease type and stage, general physical condition, responsiveness to a given dose, and type of drug), the nature of any one or more pharmaceutically acceptable carriers in any composition used, and the route of administration. The therapeutically effective dose of engineered lymphocytes also depends on the cell surface receptors expressed by the lymphocytes (e.g., the affinity and density of the cell surface receptors expressed on the cells), the type of target cells, the nature of the disease or pathological condition being treated, or a combination of both.

[0163] As shown in the examples, the in vivo efficacy of engineered lymphocytes prepared by the present process is greatly increased compared to conventional techniques, thereby requiring much lower doses.

[0164] Therefore, in some aspects, the therapeutically effective dose of engineered lymphocytes is less than about 2 million engineered lymphocytes per kilogram of subject body weight (cells / kg) that needs to be treated. Therefore, in some aspects, the therapeutically effective dose of engineered lymphocytes is about 10,000 to about 2,500,000 engineered lymphocytes / kg. In certain embodiments, the therapeutically effective dose of engineered lymphocytes is about 10,000 to about 1,500,000 engineered lymphocytes / kg. In certain embodiments, the therapeutically effective dose is about 20,000 to about 1,200,000 engineered lymphocytes / kg. In certain embodiments, the therapeutically effective dose is about 20,000 to about 1,000,000 engineered lymphocytes / kg. In certain embodiments, the therapeutically effective dose is about 20,000 to about 500,000 engineered lymphocytes / kg. In certain embodiments, the therapeutically effective dose is about 20,000 to about 400,000 engineered lymphocytes / kg. In certain embodiments, the therapeutically effective dose is about 40,000 to about 400,000 engineered lymphocytes / kg. In certain embodiments, the therapeutically effective dose is about 50,000 to about 200,000 engineered lymphocytes / kg. In certain embodiments, the therapeutically effective dose is about 50,000 to about 100,000 engineered lymphocytes / kg.

[0165] In some aspects, the therapeutically effective dose of engineered lymphocytes is about 1,600,000 to about 2,500,000 engineered lymphocytes per kilogram of body weight of the subject to be treated (cells / kg). In some embodiments, the therapeutically effective dose of engineered lymphocytes is about 2,000,000 to about 2,400,000 engineered lymphocytes per kilogram of body weight of the subject to be treated (cells / kg).

[0166] In some embodiments, the T cells administered are Yescarta (Akiramucirumab). In some embodiments, the T cells administered are Tecartus (brexucabtagene autoleucel).

[0167] The following examples are intended to illustrate various embodiments of the present invention. Therefore, the specific embodiments discussed should not be interpreted as limiting the scope of the present invention. For example, although the following examples are directed to T cells transduced with anti-CD19 chimeric antigen receptors (CARs), those skilled in the art will understand that the methods described herein are applicable to T cells transduced with any CAR. It will be apparent to those skilled in the art that various equivalents, changes and modifications may be made without departing from the scope of the present invention, and it should be understood that such equivalent embodiments will be included herein. In addition, all references cited in this disclosure are hereby incorporated by reference in their entirety, as if fully set forth herein.

[0168] Example 1: Seven-day lymphocyte production process

[0169] This example describes a process for preparing lymphocytes transduced with a polynucleotide vector encoding a therapeutic protein, such as a viral vector. The prepared lymphocytes can be used to treat various diseases such as cancer, especially when the therapeutic protein is a chimeric antigen receptor (CAR) or T cell receptor (TCR) designed to target cancer cells.

[0170] As used throughout the text, the terms "7-day process" and "7-day lymphocyte manufacturing process" are used interchangeably and refer to a CAR cell manufacturing process that requires about 7 days after the initial enrichment and activation steps. From the initial enrichment and activation steps to the harvesting step, the 7-day process is at least 8 days in length, and when including the enrichment and activation steps, it can be 8 to 11 days in total.

[0171] Apheresis is performed using standard apheresis equipment (such as Cobe Spectra, Spectra Optia 、Fenwal ™ Amicus The leukocyte apheresis process typically produces approximately 200 mL to 400 mL of apheresis product from the patient. The apheresis product can be processed on-site or optionally shipped at 1°C to 10°C to a facility for manufacturing at another location. Additional process steps can be performed in an ISO 7 cell culture process suite (or similar cleanroom type environment).

[0172] Where appropriate, use cell processing devices (such as Sepax The volume reduction step was performed using 2 laboratory instruments (Biosafe SA; Houston, TX) or equivalent instruments and standard sterile tubing kits. Given the variability in the number of cells from each subject and the volume of input source material (approximately 200 mL to 400 mL), the volume reduction step was designed to standardize the cell volume to approximately 120 mL. In cases where the apheresis volume was less than 120 mL, the volume reduction step was not required and the cells proceeded directly to the lymphocyte enrichment step. The volume reduction step was designed to standardize the volume of cells received from each subject, retain mononuclear cells, achieve consistent cell yield and high cell viability, and maintain a closed system to minimize contamination risk.

[0173] Lymphocyte enrichment. After the volume reduction step, the cells are isolated using a separation protocol developed and recommended by the device manufacturer (NeatCell procedure) and using standard sterile tubing kits on a cell processing device (such as Sepax 2 or equivalent). The lymphocyte enrichment step reduces product-related impurities (such as RBCs and granulocytes), enriches and concentrates mononuclear cells, washes and reduces process-related residues (such as Ficoll), and formulates cells in growth medium to prepare for cell activation and achieve consistent cell yields and high cell viability. The closed system minimizes environmental contamination.

[0174] The process can be performed in an ISO 7 zone at ambient temperature, and all connections can be made using a sterile tubing welder, or in an ISO 5 laminar flow hood.

[0175] Lymphocyte Activation. The lymphocyte activation step can be performed with freshly processed cells from lymphocyte enrichment or with previously cryopreserved cells. In the case of using cryopreserved cells, the cells can be thawed prior to use using a developed protocol.

[0176] The lymphocyte activation step selectively activates lymphocytes, making them receptive to retroviral vector transduction, depletes the viable cell population of all other cell types, achieves consistent cell yield and high lymphocyte viability, and maintains a closed system to minimize contamination risk. Lymphocyte activation can be achieved with lymphocyte stimulators such as anti-CD3 antibodies and IL-2.

[0177] Wash 1. After the lymphocyte activation step, use a cell processing device (such as Sepax 2 or equivalent), using fresh medium from a standard sterile kit, using a protocol developed by the manufacturer to wash the cells. Optionally concentrate the cells to a final volume of approximately 100 mL in preparation for retroviral vector transduction. The Wash 1 step reduces process-related carryover (such as anti-CD3 antibodies), spent growth medium, and cell debris; achieves consistent cell yields and high T cell viability, maintaining a closed system to minimize contamination risk; and concentrates and delivers sufficient numbers of viable T cells with a small volume suitable for starting transduction.

[0178] Transduction. Transfer the activated cells from the wash 1 step in fresh cell growth medium to a cell culture bag (Origen Biomedical PL240 or similar) that has been previously transduced by first incubating with recombinant fibronectin or a fragment thereof (such as RetroNectin The bag was coated with a 50% lipoprotein (Takara Bio, Japan) and then incubated with the retroviral vector according to a defined procedure before introduction into activated cells. RetroNectin was performed at a temperature of 2°C to 8°C. Coating (10µg / mL) lasts for 20±4 hours and is subsequently incubated with thawed retroviral vectors for approximately 180 to 210 minutes at 37±1°C and 5±0.5% CO2. After adding cells to the bag, transduction is performed for 20±4 hours at 37±1°C and 5±0.5% CO2. Retroviral transduction step Activated T cells are cultured in the presence of retroviral vectors under controlled conditions to allow efficient transduction to occur, achieve consistent cell yields and high cell viability, and maintain a closed system to minimize the risk of contamination.

[0179] Wash 2. Following the retroviral transduction step, wash the cells using a cell handling device (such as Sepax) in a standard sterile kit using the manufacturer's developed protocol. 2 or equivalent), wash the cells with fresh growth medium and concentrate the cells to a final volume of approximately 100 mL in preparation for the expansion step. The Wash 2 step is designed to reduce process-related carryover, such as retroviral vector particles, vector production process carryover, spent growth medium, and cell debris, achieve consistent cell yields and high cell viability; maintain a closed system to minimize contamination risk; and exchange spent growth medium for a target number of cells with a specified volume suitable for starting the expansion step.

[0180] Lymphocyte Expansion. Cells from the Wash 2 step are aseptically transferred to a culture bag (Origen Biomedical PL325 or equivalent) and diluted with fresh cell growth medium and cultured at 37±1°C and 5±0.5% CO2 for approximately 72 hours. Beginning on Day 5, cell density is measured daily. Because the doubling time of T cells can vary slightly from subject to subject, additional growth time beyond 72 hours (i.e., 3 to 6 days) may be required in cases where the total cell number is insufficient to deliver the target dose of CAR-positive T cells / kg subject body weight. The lymphocyte expansion step is designed to culture cells under controlled conditions in order to produce sufficient numbers of transduced cells for delivery of an efficacious dose, maintain a closed system to minimize the risk of contamination, and achieve consistent cell yield and high cell viability. One such efficacious dose or target dose includes 2×10 6 FMC63-28Z CAR-positive or FMC63-CD828BBZ CAR-positive T cells / kg (±20%) of subject body weight generated by transduction with MSGV-FMC63-28Z retroviral vector or MSGV-FMC63-CD828BBZ retroviral vector, respectively, both of which are described in detail in the following references: Kochenderfer et al., J Immunother. 2009 Sep;32(7):689–702.

[0181] Wash 3 and concentrate. Following the lymphocyte expansion step, the cells are concentrated using a cell processing instrument (such as Sepax ELISA) in a standard sterile kit using the manufacturer's developed protocol. 2 or equivalent), wash the cells with 0.9% saline, and concentrate the cells to a final volume of approximately 35 mL in preparation for formulation and cryopreservation. The Wash 3 step is designed to reduce process-related carryover (such as carryover from retroviral production processes), spent growth medium, and cell debris; achieve consistent cell yields and high cell viability; and maintain a closed system to minimize contamination risk.

[0182] Once the cells are concentrated and washed into 0.9% saline, the appropriate cell dose can be formulated to prepare the final cryopreserved product.

[0183] The embodiments described herein efficiently produced engineered lymphocyte therapy within 7 days.

[0184] Example 2: Five-day lymphocyte production process

[0185] This example develops an accelerated process based on the 7 day process described in Example 1.

[0186] As used throughout the text, the terms "5-day process" and "5-day lymphocyte manufacturing process" are used interchangeably and refer to a CAR cell manufacturing process that requires about 5 days after the initial enrichment and activation steps. From the initial enrichment and activation steps to the harvesting step, the length of the 5-day process is 6 days, and when the enrichment and activation steps are included, the total can be 6 to 9 days.

[0187] During the 7-day process, lymphocytes were enriched and activated on day 0; the transduction bag was coated with recombinant fibronectin on day 1; viral transduction was performed on day 2; the transduced lymphocytes were washed and then expanded on days 3 and 4; expansion was continued on days 5 and 6 with daily media changes; and the final cell product was harvested on day 7. 9 lymphocytes, about 2.4×10 8 Lymphocytes were incubated with the viral vectors used for transduction.

[0188] 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 using an Origen Biomedical PL240 bag for transduction, an Origen Biomedical PL325 bag, or more preferably a PL750 bag, was used. The larger bag allowed for the use of a larger volume of vector (200 mL instead of 100 mL) and a larger number of lymphocytes (at 3.2 × 10 8 and 6×10 8 Instead of 2.4×10 8 ).

[0189] Interestingly, the increase in transduction volume and the addition of more vector and starting lymphocytes did not result in an unacceptable decrease in transduction efficiency (54% to 35.15%) or cell viability (92% to 92.4%) (see Table 1). Thus, the cell expansion step that required 4 days in a 7-day process was reduced to 2 days, allowing the final cell product to be harvested on day 5.

[0190] Table 1. Evaluation of 5-day process and 7-day process

[0191]

[0192] However, the modest reduction in transduction rate was not associated with clinical efficacy or patient safety. Equally important, the cellular product from the 5-day process was found to include an increased percentage of naive cells in both the CD4+ T and CD8+ T cell populations (Table 2), which is believed to be associated with improved therapeutic efficacy. It is important to note that these changes are within the historical range of donor runs collected from the 7-day process. It is important to note that in Table 2 below, the historical averages from the 7-day process run are reflected in the column labeled "Historical Averages", and the data from the 5-day and 7-day runs performed in the current study are depicted in the columns labeled "5 Days" and "7 Days", respectively.

[0193] Table 2. Young cells within the 5-day bias history range

[0194]

[0195] CM: central memory T cells; Teff: T effector cells; EM: effector memory T cells.

[0196] Thus, this example demonstrates that the shortened 5-day process meets regulatory requirements for transduction efficiency, efficacy, and safety. At the same time, the 5-day product exhibits a more juvenile phenotype within the historical range.

[0197] The in vivo efficacy of the cell products from the 5-day process was tested in a mouse model in which 5 × 10 cells in 100 µL of solution were implanted iv. 5 Nalm6 cells (human acute lymphoblastic leukemia (ALL) line). The CAR used was injected on day 6 and targeted CD19 at a dose of 5 × 10 6 cells, 1×10 6 cells or 0.2×10 6 cells / body weight (Table 3).

[0198] Table 3. Experimental groups

[0199]

[0200] The changes in body weight associated with the increase in tumor burden (Table 4) indicated that all these treatments were safe for the animals.

[0201] Table 4. Body weight changes in the days after tumor implantation

[0202]

[0203] The tumor inhibition results are shown in Table 5 (measured in photons / second).

[0204] Table 5. Tumor mass several days after tumor implantation

[0205]

[0206] When 5×10 6 The cell product from the 5-day process showed complete tumor growth inhibition when dosed at 10 cells / kg. The cell product from the 5-day process was superior to the cell product from the 7-day process at all doses. The cells from the 5-day process were also more potent than the cells from the 7-day process, even when the percentage of CAR+ cells was matched (i.e., there were fewer CAR-T cells from the 5-day process).

[0207] Example 3: Three-day lymphocyte production process

[0208] This example develops a further accelerated process based on the 7 day process as described in Example 1 and the 5 day process as described in Example 2.

[0209] As used throughout the text, the terms "3-day process" and "3-day lymphocyte manufacturing process" are used interchangeably and refer to a CAR cell manufacturing process that takes about 3 days starting from the initial enrichment and activation steps. The length of the 3-day process is about 4 days from the initial enrichment and activation steps to the harvesting step. The 3-day process does not include the cell expansion step, which includes one or more days after the transduction step and before the harvesting step.

[0210] In this new process, the Day 0 to Day 1 procedure is similar to the 5-day process, including fibronectin coating of larger bags (e.g., Origen Biomedical PL325 or preferably PL750) for subsequent transduction on Day 2. However, on Day 2, only approximately 4.8 × 10 8 (Instead of 6×10 8 ) lymphocytes, the same amount of viral vector (200 mL) was used. Another important difference is that, unlike the 5-day process and the 7-day process, no specific steps are performed for T cell expansion. Instead, transduced lymphocytes are harvested on day 3, allowing the entire process to be completed in 3 days starting from the initial enrichment and activation steps.

[0211] Given the lack of a specific T cell expansion step in this newly developed 3-day process, the harvested cell product included a slightly smaller percentage of T cells (CD3+). Importantly, however, the 3-day product included an even greater percentage of naive (immature) T cells (Table 6). Note that in Table 6 below, the historical averages from a 7-day process run are reflected in the column labeled "Historical Averages," and data from the 3-day and 7-day runs performed in the current study are depicted in the columns labeled "3 Days" and "7 Days," respectively.

[0212] Table 6. Phenotype of cell products

[0213]

[0214] CM: central memory T cells; Teff: T effector cells; EM: effector memory T cells.

[0215] Although not tested with lymphocytes from the same donor, comparison between Table 2 and Table 6 indicates that the percentage of naive T cells from the 3-day process was also significantly higher than that from the 5-day process. In CD4+ T cells, the 3-day process produced about 55.75% naive T cells, while the 5-day process produced about 40.65%; in CD8+ T cells, the 3-day process produced about 37.35% naive T cells, while the 5-day process produced about 3.93%.

[0216] In other words, the 3-day process produced an approximately 1.4-fold increase in the percentage of CD4+ naive T cells observed over the 5-day process, and an approximately 9.5-fold increase in the percentage of CD8+ naive T cells observed over the 5-day process. In addition, the 3-day process produced an approximately 3.0-fold increase in the percentage of CD4+ naive T cells observed over the 7-day process, and an approximately 18.0-fold increase in the percentage of CD8+ naive T cells observed over the 7-day process.

[0217] In contrast, among CD4+ T cells, the 3-day process produced only about 4.35% of effector memory T cells, while the 5-day process produced about 8.55%; among CD8+ T cells, the 3-day process produced only about 9.85% of effector memory T cells, while the 5-day process produced about 15.85%.

[0218] In other words, the 3-day process produced an approximately 2.0-fold decrease in the percentage of CD4+ effector memory T cells observed over a 5-day process, and an approximately 1.6-fold decrease in the percentage of CD8+ effector memory T cells observed over a 5-day process. In addition, the 3-day process produced an approximately 6.0-fold decrease in the percentage of CD4+ effector memory T cells observed over a 7-day process, and an approximately 3.5-fold decrease in the percentage of CD8+ effector memory T cells observed over a 7-day process.

[0219] In addition, as can be seen from Table 6, in the final harvest population from the 3-day process, the percentage of CD4+ CCR7+ cells (i.e., CM cells and naive cells) is at least 95% of the harvested CD4+ T cell population. In other words, in the final harvest population from the 3-day process, the maximum percentage of Teff / TEMRA and EM CD4+ cells is about 5%. Therefore, the CD4+ CCR7+ cells in the harvested population are at least 19 times of Teff / TEMRA and EM CD4+ cells. In contrast, in the final harvest population from the 7-day process, the percentage of CD4+ CCR7+ cells (i.e., CM cells and naive cells) is about 71% of the harvested CD4+ T cell population. In other words, in the final harvest population from the 7-day process, the maximum percentage of Teff / TEMRA and EMCD4+ cells is about 29%. Thus, a 3-day process produced a CD4+ T cell population with an approximately 1.3-fold increase in the percentage of CD4+ CCR7+ cells and an approximately 5-fold decrease in the percentage of Teff / TEMRA and EM CD4+ cells compared to a 7-day process.

[0220] As also seen in Table 6, in the final harvest population from the 3-day process, the percentage of CD8+ CCR7+ cells (i.e., CM cells and naive cells) was at least 84% of the harvested CD8+ T cell population. In other words, in the final harvest population from the 3-day process, the maximum percentage of Teff / TEMRA and EM CD4+ cells was about 16%. Therefore, the CD8+ CCR7+ cells in the harvested population were at least about 5 times more Teff / TEMRA and EM CD4+ cells. In contrast, in the final harvest population from the 7-day process, the percentage of CD8+ CCR7+ cells (i.e., CM cells and naive cells) was about 66% of the harvested CD8+ T cell population. In other words, in the final harvest population from the 7-day process, the maximum percentage of Teff / TEMRA and EM CD8+ cells was about 34%. Thus, a 3-day process produced a CD8+ T cell population with an approximately 1.3-fold increase in the percentage of CD8+CCR7+ cells and an approximately 2-fold decrease in the percentage of Teff / TEMRA and EM CD8+ cells compared to a 7-day process.

[0221] Cell viability measurements showed that cell viability remained high (>90%) throughout the 3-day process, with the wash step causing the greatest decrease in cell viability on day 2. In contrast, the 5-day process and the 7-day process included additional wash steps, each of which resulted in additional decreases in cell viability (Table 7).

[0222] Table 7. Changes in cell viability during the process

[0223]

[0224] Example 4: In vivo efficacy of cells prepared by a three-day process

[0225] This example compares the in vivo efficacy of cells prepared by a 3-day process as described in Example 3 with cells prepared by a 7-day process using the same animal model and procedures as described in Example 2.

[0226] The experimental groups are shown in Table 8 and the body weight changes are shown in Table 9.

[0227] Table 8. Experimental groups

[0228]

[0229] Table 9. Body weight changes in the days after tumor implantation

[0230]

[0231] The tumor inhibition results are shown in Table 10 (measured in photons / second).

[0232] Table 10. Tumor mass several days after tumor implantation

[0233]

[0234] At all three doses (0.5×, 1×, and 3×10 6 The cell products from the 3-day process were able to completely inhibit tumor growth throughout the observation period with no significant differences at 0.5 and 1 × 10 cells / kg. In contrast, cells from the 7-day process were only able to inhibit tumor growth by 15 days (0.5 and 1 × 10 6 cells / kg) and 23 days (3×10 6 cells / kg) to inhibit tumor growth.

[0235] Thus, this example demonstrates that a 3-day process can produce a cell product with greatly improved anti-tumor efficacy compared to a 7-day process. Furthermore, given the greatly improved efficacy, a much lower dose is required to achieve a more complete anti-tumor response.

[0236] In another experiment, a lower dose of cells (0.2 × 10 6 cells / kg; three different donors) versus a high dose (1 × 10 6 The results (Table 11) show that even with a 5-fold difference, the results of the 3-day process are far superior to those of the 7-day process.

[0237] Table 11. Tumor mass several days after tumor implantation

[0238]

[0239] Example 5: Effect of earlier harvest on CAR

[0240] This example found that the performance improvement of the shortened (early harvest) process was more significant when the CAR construct included a CD28 co-stimulatory domain compared to a 4-1BB co-stimulatory domain.

[0241] Both types of CARs include a CD19 binding antigen binding fragment, while CAR No. 1 includes a CD28 co-stimulatory domain and CAR No. 2 includes a 4-1BB co-stimulatory domain, both of which are delivered by lentiviral vectors. Cells were harvested on days 4, 7, and 14, and doses used for in vivo animal studies included 0.2×10 6 cells / kg and 1×10 6 The results are shown in Table 13.

[0242] Table 12. Experimental groups

[0243]

[0244] Table 13. Tumor mass several days after tumor implantation

[0245]

[0246] As shown in Table 13, in the case of late harvest (day 7 or day 14), there was limited performance difference between the two constructs. However, in the case of early harvest, which benefited both constructs, CAR No. 1 (G3) showed significantly higher in vivo efficacy than CAR No. 2 (G8).

[0247] Additionally, a CAR targeting CD19 and including a CD28 co-stimulatory domain was tested in vivo using a Nalm6 leukemia mouse model. The results showed that early harvest (day 4) resulted in better in vivo antitumor efficacy than conventional harvest (day 7), which in turn was more effective than late harvest (day 14). In fact, a low dose (2 × 10 5 The efficacy of T cells harvested at day 7 (10 6 cells) by 5 times the amount of T cells.

[0248] Example 6 In vivo efficacy of cells prepared by a three-day process

[0249] This example supplements Example 4 and provides 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 the 7-day process.

[0250] Anti-CD19 CAR T cells prepared by a 3-day process and anti-CD19 CAR T cells prepared by a 7-day process were made from healthy human donors, as well as their corresponding non-transduced (NTD) day 3 and NTD day 7 cells. NALM6-luc cells (5 × 10 5 8-week-old female NSG mice (5 mice per group) were implanted IV via the lateral tail vein. On day 6 after tumor implantation, mice received either a single-dose cohort of control (NTD day 3 or NTD day 7) or a multi-dose cohort of anti-CD19CAR T cell products. A total of 5 dose cohorts (1×10 6 , 5×10 5 , 3 × 10 5 , 2×10 5 and 1×10 5 CAR + cells) were tested on cells derived from a 3-day process and in 4 dose cohorts (1 × 10 7 , 5×10 6 , 3×10 6 and 1×10 6 CAR + Antitumor activity was assessed by measuring the effect of anti-CD19 CAR T cell products on tumor burden and animal survival.

[0251] Antitumor activity was assessed by evaluating changes in tumor burden measured by bioluminescence imaging (BLI) from day 5 (1 day before CAR T cell treatment) to day 22 (16 days after CAR T cell treatment, and the last time point for all groups complete). Inhibition of tumor growth was observed in all 4 dose cohorts of mice treated with anti-CD19 CAR T cells derived from the 7-day course and in all 5 dose cohorts of anti-CD19 CAR T cells derived from the 3-day course (Tables 14, 15, and 16). For anti-CD19 CAR T cells derived from the 3-day course, inhibition of tumor growth was observed in all 4 dose cohorts of mice treated with anti-CD19 CAR T cells derived from the 7-day course. 6 , 5×10 5 , 3×10 5 , 2×10 5 and 1×10 5 CAR +In animals treated with T cells, tumor growth inhibition rates were 128.1%, 126.7%, 117.7%, 113.4%, and 65.7%, respectively, compared to the untreated group. For anti-CD19 CAR T cells derived from a 7-day course, tumor growth inhibition rates ranged from 109.0% to 115.1% for the dose cohorts tested (Table 16). + Tumor burden decreased at all T cell doses, but 1×10 anti-CD19 CAR T cells prepared over the 3-day process 5 CAR + The exception was the T cell dose cohort, which experienced delayed tumor control at day 26. When compared to mice that received no treatment or NTD controls, the anti-tumor efficacy of anti-CD19 CAR T cells prepared over a 3-day course for all doses and over a 7-day course reached statistical significance at day 22 (Tukey post hoc analysis, p < 0.05 was considered significant).

[0252] Table 14. Experimental groups whose results are presented in Tables 15 and 16

[0253]

[0254] Table 15. Tumor mass several days after tumor implantation (presented as the mean observed for each group at each time point; Unit = photon / second)

[0255]

[0256] Table 16. Logarithmic transformation analysis of tumor burden

[0257]

[0258] In Table 16, tumor burden (measured by BLI) is expressed as log 10 Normalized, the mean values ​​for each cohort on days 5 and 20 of the study are represented in this table. 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 represents the percent tumor volume change of the treated group relative to the control (i.e., mice that did not receive treatment). A tumor growth inhibition (TGI) of 0% indicates that the mean tumor growth of the group is comparable to that observed in the untreated group, while a TGI of 100% indicates that no tumor growth was observed between days 5 and 20. A TGI>100% indicates that the mean tumor burden decreased from day 5 to day 20, while a TGI<0 indicates that the increase in tumor burden is greater than that observed in the untreated group (G1).

[0259] Treatment of mice with anti-CD19 CAR T cells prepared over a 3-day process or anti-CD19 CAR T cells prepared over a 7-day process resulted in prolonged survival compared to control mice that received NTD or did not receive treatment. 8 The NTD control group and untreated mice were exited from the study on day 8, with a survival set point defined by 1×10 photons / second bioluminescence. In contrast, the group treated with anti-CD19 CAR T cells prepared using cells from a 3-day process experienced significantly higher survival, with mice from all dose cohorts achieving 100% survival at the study endpoint on day 64. Mice treated with anti-CD19 CAR T cells prepared using a 7-day process had lower survival rates, ranging from 1×10 7 CAR + 80% survival rate in T cell dose cohorts up to 5×10 6 CAR + The survival rate of the T cell dose cohort was 20%, and the 3×10 6 and 1×10 6 CAR + 0% survival rate for the T cell dose cohort.

[0260] With acceptance through 3×10 6 and 1×10 6 CAR + Mice that received all doses of anti-CD19 CAR T cells prepared over a 3-day course achieved statistically significant improvements in responses compared to mice that received anti-CD19 CAR T cells prepared over a 7-day course of T cell doses, but not compared to mice that received 1×10 anti-CD19 CAR T cells prepared over a 7-day course of T cells. 7 CAR + There were no statistical differences in T cell doses. All doses of anti-CD19 CAR T cells prepared over a 3-day process resulted in complete anti-tumor responses and 100% survival at the end of the study (day 64), whereas the highest dose of anti-CD19 CAR T cells (1 × 10 7 CAR + T cells) resulted in a survival rate of 80% at the end of the study.

[0261] Finally, the increase in tumor burden over time in the untreated group resulted in a mean weight loss of >18% and / or adverse clinical signs by day 26, after which all animals were removed from the study. Animals receiving NTD T cells showed a similar pattern of weight loss by day 29 due to uncontrolled tumor burden. In contrast, animals receiving NTD T cells from 1 × 10 6 to 1×10 5 All doses of anti-CD19 CAR T cells prepared using 1×10 cells were well tolerated, and animals maintained consistent weights during the study. 7 , 5×10 6 , 3×10 6 and 1×10 6 CAR + All mice under the control of T cells were kept at a consistent body weight during the study.

[0262] Example 7 Characterization of cells prepared by a three-day process

[0263] This example describes the functional and phenotypic characteristics of anti-CD19 / CD20 CAR T cells harvested on day 3 compared to anti-CD19 / CD20 CAR T cells harvested on day 6.

[0264] Briefly, positively selected CD4 T cells from healthy human donors were activated and transduced with lentiviral vectors encoding dual-targeting anti-CD19 CAR and anti-CD20 CAR. + and CD8 + T cells. The transduced cells were subsequently harvested on day 3. Similarly, another set of anti-CD19 / CD20 CAR T cell products were made from T cells from the same donor, but harvested on day 6 (referred to as "anti-CD19 / CD20 CAR on day 6" in Examples 7 and 8). Subsequently, both CAR T cell products were functionally characterized and compared to their corresponding NTD control T cells (NTD day 3 and NTD day 6), which were made in parallel from the same donor material.

[0265] The anti-CD19 / CD20 CAR T cells harvested on day 3 were generated by the following 3-day process, which starts with the collection of apheresis material. The starting apheresis material can optionally be fresh or cryopreserved apheresis T cells or cryopreserved T cells. Wash 1, Wash 2, T cell enrichment and Wash 3 steps are performed on day 0. T cell activation is performed on day 0 to day 1. Lentiviral transduction is performed on day 1 to day 4. Harvest wash and concentration are performed on day 3 to day 4.

[0266] The anti-CD19 / CD20 CAR T cells harvested on day 6 were generated by the following process, which started with the apheresis material collection. Wash 1, Wash 2, T cell enrichment, and Wash 3 steps were performed on day 0. T cell activation was performed on days 0 to 2. Lentiviral transduction was performed on days 2 to 5. Wash 4 was performed on days 4 to 5. T cell expansion was performed on days 4 to 15. Harvest wash and enrichment were performed on days 6 to 15.

[0267] The experimental groups for the study described in this example are shown in Table 17.

[0268] Table 17. Experimental groups

[0269]

[0270] Anti-CD19 / CD20 CAR T cells and anti-CD19 / CD20 CAR day 6 T cell products produced by the 3-day process were evaluated for CAR cell surface expression to establish CAR transduction efficiency. Cell surface expression of both anti-CD19 CAR and anti-CD20 CAR was detected individually by flow cytometry using fluorescently labeled anti-idiotypic antibodies. In the same sample, the expression of anti-CD19 CAR alone was 68% for anti-CD19 / CD20 CAR T cells produced by the 3-day process and 35% for anti-CD19 / CD20 CAR day 6 T cell products, while the expression rates of anti-CD20 CAR were 65% and 37%, respectively. The total expression of anti-CD19 and anti-CD20 CAR (i.e., anti-CD19 CAR antibody) was expressed by the sum of total anti-CD19 and anti-CD20 CAR. + Percentage of cells plus anti-CD29 CAR antibody + The transduction efficiency, measured as percentage of transduced cells, was 72% for the anti-CD19 / CD20 CAR T cells generated over the course of 3 days and 40% for the anti-CD19 / CD20 CAR day 6 T cell product.

[0271] Phenotypic characterization of CAR T cell products was performed by flow cytometry after incubation with fluorescently labeled antibodies to assess CD4 + and CD8 + The presence of T cells and the relative composition of less differentiated T cell subsets. The CD45RA and CCR7 markers can be used to define various populations of T cells, including naive and stem cell memory T cells (CD45RA + CCR7 + ), also known as naive T cells. The anti-CD19 / CD20 CAR T cells generated through the 3-day process showed a slightly higher CD4 / CD8 ratio than the anti-CD19 / CD20 CAR day 6 T cell product (Table 18).

[0272] Table 18. CD4 / CD8 ratio (% of CD3)

[0273]

[0274] Compared with the anti-CD19 / CD20 CAR day 6 T cell product, the CD4 + and CD8 + The populations showed an increase in the frequency of juvenile cells (Tables 19 and 20).

[0275] Table 19. CD45RA + CCR7 + (% of CD4)

[0276]

[0277] Table 20. CD45RA + CCR7 + (% of CD8)

[0278]

[0279] The functionality of the CAR T cell products was evaluated in co-culture assays with antigen-positive and antigen-negative target cells. NTD T cell products were included in the experiments as controls to assess the level of alloreactivity. Cytotoxicity was measured on days 1 and 4 after co-culture of T cell products with luciferase-expressing target cells. Cytotoxicity was measured as the reduction in luciferase signal emitted in the test wells after the addition of luciferin compared to wells seeded with target cells alone. Multiple E:T ratios ranging from 1:1 to 1:243 were used in these assays. Four major cell lines expressing different 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 myeloid leukemia cell line that does not express either CD19 or CD20 antigens. In addition, controls consisted of Raji CD19 knockout (KO) cells and Raji CD20KO cells. The Raji cell line was engineered to express either antigen: Raji CD19KO cells expressed only CD20 but not CD19, and Raji CD20KO expressed only CD19 but 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.

[0280] Anti-CD19 / CD20 CAR T cells and anti-CD19 / CD20 CAR day 6 T cell products produced by a 3-day process showed comparable dose-dependent specific cytotoxicity against antigen-positive target cells on day 4 of co-culture, regardless of the expression of both antigens or any antigen (Tables 21A to 21F). A basal level of antigen-independent cytotoxicity mediated by alloreactivity against target cells was observed by cytotoxicity of NTD D3 control T cells against ST486 cells (low CD19 / high CD20 expression) at the highest E:T ratio. (Table 21A). Both anti-CD19 / CD20 CAR T cells produced by a 3-day process and anti-CD19 / CD20 CAR D6 T cell products derived from healthy donors had comparable cytotoxic activity in various target cell lines tested and lacked killing activity against antigen-negative K-562 cells (Table 21F).

[0281] Table 21A. ST488 cells (CD19+CD20+) (% cytotoxicity)

[0282]

[0283] Table 21B. Nalm6 cells (CD19+CD20+) (% cytotoxicity)

[0284]

[0285] Table 21C. Raji cells (CD19+CD20+) (% cytotoxicity)

[0286]

[0287] Table 21D. Raji CD19KO cells (CD19-CD20+) (% cytotoxicity)

[0288]

[0289] Table 21E. Raji CD20KO cells (CD19+CD20-) (% cytotoxicity)

[0290]

[0291] Table 21F. K-562 cells (CD19-CD20-) (% cytotoxicity)

[0292]

[0293] Specific activity was further assessed by measuring cytokine levels in culture supernatants after overnight co-culture of T cell products with Nalm6, ST486, Raji, Raji CD19KO, Raji CD20KO, and K-562 target cells at a 1:1 E:T ratio. Production of proinflammatory cytokines such as IFN-γ, IL-2, and tumor necrosis factor-α (TNF-α) was quantified.

[0294] Anti-CD19 / CD20 CAR T cells and anti-CD19 / CD20 CAR day 6 T cell products produced by the three-day process showed the production of large amounts of antigen-dependent IFN-γ, IL-2, and TNF-α proinflammatory cytokines by co-culture with antigen-positive target cells Nalm6, ST486, Raji, Raji CD19KO, and Raji CD20KO (Tables 22A to 22C). Anti-CD19 / CD20 CAR T cells and anti-CD19 / CD20 CAR day 6 T cell products produced by the three-day process showed comparable cytokine levels of IL-2 and TNF-α in the various target cell lines tested. When the T cell products were co-cultured with target cells expressing a single antigen (RajiCD19KO or Raji CD20KO), the functionality of each individual CAR was demonstrated by the production of cytokines, although the levels were lower than those induced by co-culture with Raji parental cells expressing both antigens. In contrast, no cytokine production was detected in the absence of target cells (i.e., T cells alone) and with antigen-negative K-562 (CD19 – CD20 – ) cells co-cultured with CAR T cells also did not produce cytokines, as shown (Table 22A to Table 22C)

[0295] Table 22A. IFNγ Secretion (pg / mL)

[0296]

[0297] Table 22B. IL-2 secretion (pg / mL)

[0298]

[0299] Table 22C. TNFα secretion (pg / mL)

[0300]

[0301] To evaluate antigen-specific proliferation of CAR T cell products upon binding to target antigen, T cell products that had been co-cultured with Raji, Nalm6, Raji CD19KO, Raji CD20KO, and K-562 target cells were harvested 4 days after the start of co-culture, pelleted by centrifugation, and prepared for flow cytometry evaluation. T cells had been pre-labeled with a fluorescent dye that was used to track multiple generations of cells by analyzing dye dilution as cells divided. A 1:1 E:T ratio from co-cultures was used, and T cell products cultured in the absence of target cells (T cells alone) were used as controls. − CD20 −T cells co-cultured with 100 μM CARs were used to assess specific and antigen-independent proliferation levels, while T cells alone were used to assess basal levels of proliferation in the absence of stimulation. After harvest, cells were stained with fluorescently labeled antibodies against CD3, CD4, CD8, CD19 CAR, CD20 CAR, CD25, and viability dyes.

[0302] Anti-CD19 / CD20 CAR T cells and anti-CD19 / CD20 CAR day 6 T cell products generated over the three-day process showed comparable proliferation in co-culture with Nalm6, Raji, Raji CD19KO, and Raji CD20KO target cells, but not with antigen-negative K-562 cells, demonstrating that both the anti-CD19 CAR and anti-CD20 CAR in the T cell products were functional. NTD T cell controls showed various levels of nonspecific basal proliferation when co-cultured with different target cell lines, but overall, NTD day 3 cells demonstrated greater basal proliferation than NTD day 6 cells.

[0303] Example 8 In vivo efficacy of cells prepared by a three-day process

[0304] 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) IL-2 receptor-γ chain null (NSG) mice injected intravenously with Nalm6-luc cells expressing high levels of CD19 and CD20. The anti-tumor efficacy of the CAR T cell product, generated by T cells from a healthy donor and characterized by previous in vitro studies as described in Example 7, was evaluated.

[0305] Nalm6luc cells (5.0×10 5 ) were implanted into 7-week-old female NSG mice (5 mice per group) via the lateral tail vein. On day 6 after tumor implantation, mice received control (vehicle [phosphate-buffered saline] or NTD day 3 cells) or anti-CD19 / CD20 CAR T cells as follows: 3 dose levels (2.0 × 10 5 4.0×10 4 and 8.0×10 3 CAR + T cells) and anti-CD19 / CD20 CAR T cells generated at 1 dose level; 5 CAR +All animals were treated with a single IV administration of a fixed volume of 100 μL, and mice from the vehicle group received an IV injection of 100 μL PBS. Table 23 shows the experimental groups whose results are shown in Tables 24 to 26.

[0306] Table 23. Experimental groups whose results are presented in Tables 24 to 26

[0307]

[0308] Tumor burden (by bioluminescence and log quantification) was assessed from day 5 (1 day before CAR T-cell infusion) to day 22 (16 days after CAR T-cell infusion and the last time point for all groups). 10 Anti-tumor activity was assessed by changes in tumor burden (normalized to measured values). Treatment with anti-CD19 / CD20 CAR T cells generated over the course of three days resulted in a significant reduction in tumor burden when compared to mice receiving vehicle (PBS) or NTD day 3 T cells (Table 24).

[0309] Table 24. Antitumor activity of T cell products against tumor burden in NSG mice bearing Nalm6-luc (photons / second)

[0310]

[0311]

[0312] 2.0 × 10 of anti-CD19 / CD20 CAR T cells were generated through a three-day process 5 (124.3%) and 4.0×10 4 (121.1%) CAR + Inhibition of tumor growth and regression were observed in all T cell dose cohorts; and at the lowest dose of 8.0 × 10 3 CAR + In the presence of T cells, mice showed a slight tumor growth inhibition rate (5.5%). 5 CAR + Tumor growth was controlled in animals with T cell doses of anti-CD19 / CD20 CAR, with a tumor growth inhibition rate of 112.5% ​​when compared to the vehicle group (Table 24, Table 25).

[0313] Table 25. Logarithmic transformation analysis of tumor burden

[0314]

[0315] Tumor burden (measured by BLI) was log 10Normalized, the mean values ​​for each cohort on days 5 and 22 of the study are shown in Table 25. Changes in tumor BLI from day 5 to day 22 were calculated, and the percentage 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 represents the percentage tumor volume change of the study group relative to the control (i.e., mice receiving vehicle [PBS]). A TGI of 0% indicates that the average tumor growth of the group is comparable to that observed in mice receiving the vehicle, while a TGI of 100% indicates that no tumor growth was observed between days 5 and 22. TGI>100% indicates that the average tumor burden decreased from day 5 to day 22, while TGI<0 indicates that the increase in tumor burden is greater than the increase seen in mice receiving the vehicle.

[0316] Antitumor efficacy as determined by analysis of tumor burden was observed at day 22 for all tested doses (2.0 × 10 5 4.0×10 4 and 8.0×10 3 CAR + T cells) treated mice and mice treated with anti-CD19 / CD20 CAR. The product was expressed at 2.0 × 10 5 CAR + The difference reached statistical significance in mice treated with T cells (Table 26).

[0317] Table 26. Statistical analysis of tumor burden on day 22

[0318]

[0319] In Table 26, the Log 22 scores were evaluated in all groups using analysis of variance and Tukey post hoc test. 10 Statistical significance of normalized tumor BLI data. The significance of comparisons is noted as follows: ns, P>0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001.

[0320] Treatment of mice with anti-CD19 / CD20 CART cells generated over a three-day process or anti-CD19 / CD20 CAR day 6 T cell products resulted in increased overall survival (OS) compared to mice receiving vehicle or NTD controls. 10 All mice receiving vehicle or NTD control were withdrawn from the study on day 15 or day 12, respectively, at the highest dose of 2.0 × 10 5 and 4.0×104 CAR + The groups treated with anti-CD19 / CD20 CAR T cells produced through a three-day process achieved 80% and 100% survival rates, respectively. 3 CAR + T cells)-treated mice achieved a median survival of 12 days, which was similar to that of mice from the control group (vehicle and NTD day 3). 5 CAR + At the same dose of T cells, mice receiving the anti-CD19 / CD20 CAR day 6 T cell product had a decreased median survival (36 days) when compared to mice treated with anti-CD19 / CD20 CAR T cells generated over the course of three days (the median survival point was not reached).

[0321] In conclusion, treatment with anti-CD19 / CD20 CAR T cells generated over the course of three days significantly delayed tumor growth and increased survival in the Nalm6-luc human B-ALL xenograft NSG mouse model.

[0322] Example 9: Additional Phenotypic Characterization of Cells Produced Using a 3-Day Process

[0323] This example supplements Example 3 and presents additional data related to the phenotypic characteristics of harvested cells produced using the 3-day process.

[0324] Tables 27 and 28 show the final product data for cells prepared using a 3-day process. Cells were derived from healthy donor material (fresh apheresis or frozen PBMC; N1-N9) as well as from patient material (frozen PBMC; P1-P4). The final product runs showed higher frequencies of central memory (CM) and naive cells compared to Teff / TEMRA and effector memory (EM) cell populations, indicating a younger product (Tables 27 and 28).

[0325] Table 27

[0326]

[0327] Table 28

[0328]

[0329] 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 are used to define T cell phenotypic subsets, which include naive T cells (naive, CD45RA+CCR7+), CM (CD45RA-CCR7+), EM (CD45RA-CCR7-), and Teff / TEMRA (CD45RA+CCR7-)

[0330] As can be seen in Table 27, the percentage of CD4+ CCR7+ cells (i.e., CM and naive cells) in the final harvested product was at least 80% of the harvested CD4+ T cell population. In other words, the maximum percentage of Teff / TEMRA and EMCD4+ cells in the final harvest population was about 20%. Therefore, there were at least 4 times more CD4+ CCR7+ cells in the harvested population than Teff / TEMRA and EMCD4+ cells.

[0331] As can be seen in Table 28, the percentage of CD8+ CCR7+ cells (i.e., CM and naive cells) in the final harvested product was 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 final harvest population was about 40%. Thus, the CD8+ CCR7+ cells in the harvested population were at least 1.5 times more than the Teff / TEMRA and EM CD8+ cells.

[0332] Example 10: Characterization of cells prepared by a three-day process

[0333] This example describes the functional and phenotypic characteristics 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 generated by methods similar to those described in Example 7.

[0334] Briefly, T cells selected from healthy human donors were activated on day 0 and transduced with a lentiviral vector encoding an anti-CLL-1 CAR on day 1. The transduced cells were subsequently harvested on day 3. Another set of anti-CLL-1 CAR T cells was made from T cells from the same donor but harvested on day 8. Subsequently, both CAR T cell products were functionally characterized and compared with their corresponding NTD control T cells (NTD day 3 and NTD day 8).

[0335] The experimental groups for the study described in this example are shown in Table 29.

[0336] Table 29. Experimental groups

[0337]

[0338] Anti-CLL-1 CAR T cells harvested on day 3 and day 8 were evaluated for CAR cell surface expression to establish CAR transduction efficiency. CAR expression was comparable between those cells harvested on day 3 and day 8. For anti-CLL-1 CAR T cells harvested on day 3, the expression of anti-CLL-1 CAR was 60.4%, while for anti-CLL-1 CAR T cells harvested on day 8, the expression was 78.9%.

[0339] Phenotypic characterization of CAR T cell products was performed by flow cytometry. + CCR7 + In contrast, anti-CLL-1 CAR T cells harvested on day 3 showed an increased distribution of naive T cells, and CD45RA + CCR7 + The cell percentage was 83.7%.

[0340] As shown in Table 30, anti-CLL-1 CAR T cells harvested on day 3 showed an increase in CD4+ cells compared to cells harvested on day 8.

[0341] Table 30: Percentages of CD4+ and CD8+

[0342]

[0343] Functionality of the CAR T-cell products was assessed 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 on days 1 and 4 after the start of co-culture of the T-cell products. E:T ratios ranging from 1:1 to 1:3 were used. MV4-11 (moderate expression of CLL-1) and Kasumi-1 (very low expression of CLL-1) cell lines were used.

[0344] Anti-CLL-1 CAR T cells harvested on day 3 showed increased in vitro cytotoxicity against Kasumi-1 cells compared to anti-CLL-1 CAR T cells harvested on day 8 (Tables 30A-30B). Anti-CLL-1 CAR T cells harvested on days 3 and 8 showed comparable cytotoxicity against MV4-11 cells in vitro (Tables 30C-30D).

[0345] Table 30A. Kasumi-1 24-hour cytotoxicity (cytotoxicity%; n=3)

[0346]

[0347] Table 30B. Kasumi-1 96-hour cytotoxicity (cytotoxicity%; n=3)

[0348]

[0349] Table 30C. MV4-11 24-hour cytotoxicity (cytotoxicity%; n=3)

[0350]

[0351] Table 30D. MV4-11 96-hour cytotoxicity (cytotoxicity%; n=3)

[0352]

[0353] Example 11: Further characterization of cells prepared by a 3-day process

[0354] In this example, the phenotypic characterization of cells generated using a 3-day process similar to that described above in Example 3 is reported. In this example, a lentiviral vector was used to deliver the CAR.

[0355] First, the harvested cells resulting from the 3-day process had a higher CD4 / CD8 ratio compared to the cells harvested from the 7-day process, as shown in Table 31. Table 31 discloses the values ​​for 4 individual samples for each identified group.

[0356] Table 31. Cells generated from a 3-day process have higher CD4 / mRNA than cells harvested from a 7-day process. CD8 ratio. (NTD = non-transduced control.)

[0357]

[0358] Next, as shown in Table 32, the harvested cells generated from the 3-day process had a higher percentage of CD45RA+CCR7+ cells and CD45RA-CCR7- cells compared to the cells harvested from the 7-day process. Table 32 discloses the values ​​of 4 individual samples for each identification group. (NTD = non-transduced control.)

[0359] Table 32. CD45RA+CCR7+ cells and CD45RA-CCR7- cells were more abundant in cells harvested from a 3-day process. rich

[0360]

[0361] Next, as shown in Table 33, the harvested cells resulting from the 3-day process had a higher percentage of Tscm cells (CD27+CD28+CD45RA+CCR7+) compared to the cells harvested from the 7-day process. Table 33 discloses the values ​​for 4 individual samples of each identification group.

[0362] Table 33. Cells from a 3-day process had a higher percentage of Tscm cells

[0363]

[0364] Finally, as shown in Table 34, the harvested cells resulting from the 3-day process had a lower percentage of terminally differentiated T cells (CD45RA+CCR7-CD27-CD28-) compared to the cells harvested from the 7-day process. Table 34 discloses the values ​​for 4 individual samples of each identification group.

[0365] Table 34. Cells from a 3-day process had a lower percentage of Terminally differentiated T cells .

[0366]

[0367] The phenotypic and functional characteristics of anti-CD19 / CD20 CAR T cells generated by methods with 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 anti-CD19 CAR antibodies. Table 35 shows CAR expression in cells harvested on days 3 to 8 for T cells transduced on day 1 and on day 2.

[0368] Table 35: Percentage of CD3 T cells with CAR+ expression

[0369]

[0370] T cell subsets of CD27+CD28+CD45RA+CCR7+CD62L+ were measured for anti-CD19 / CD20 CAR T cells transduced on days 1 and 2. Table 36 shows the percentage of naive CAR+ T cells generated by each method.

[0371] Table 36: Percentage of CD27+CD28+CD45RA+CCR7+CD62L+ in CAR+ cells

[0372]

[0373] The day 0 samples in Table 36 are total T cells.

[0374] Functional assays for cytotoxicity 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 a 24-hour cytotoxicity assay at an E:T of 1:3 in three target cell lines (Nalm 6 WT, Raji WT, and Nalm 6 CD19 KO) at harvest days 3, 4, 6, and 8 (H-D3, H-D4, H-D6, and H-D8, respectively).

[0375] Table 37: 24 hour cytotoxicity assay (% killing), E:T = 1:3

[0376]

[0377] ***

[0378] Although multiple embodiments have been described, it is apparent that the present disclosure and embodiments may provide other embodiments utilizing or being encompassed by the compositions and methods described herein. Therefore, it should be understood that the scope of the present disclosure will be defined by what can be understood from the present disclosure and the appended claims, rather than by the embodiments that have been represented by way of example.

Claims

1. A method for preparing transduced lymphocytes, the method comprising incubating a sample including lymphocytes obtained from a donor subject with a polynucleotide vector to transduce the lymphocytes, thereby producing transduced lymphocytes; and The sample including the transduced lymphocytes is cultured for less than 72 hours, after which the lymphocytes are harvested to produce a harvested sample.

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

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

4. The method according to any preceding claim, wherein the incubation is performed in a closed system.

5. The method according to claim 4, wherein the closed system has a volume of at least 1500 cm 2 The internal surface area.

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

7. The method of claim 6, wherein the inner surface is further contacted with a second solution comprising the polynucleotide carrier, wherein the second solution has a volume of about 200 mL. The method of claim 7 , wherein the coating further comprises draining the second solution.

9. The method according to any one of claims 4 to 8, wherein the sample in the closed system comprises at least 1.5×10 8 Lymphocytes.

10. The method according to claim 8, wherein the sample comprises 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 (PBMC) or T cells.

12. The method according to any preceding claim, wherein the harvested sample comprises CD3+ cells.

13. The method of claim 12, wherein the harvested sample comprises CD4+ T cells and CD8+ T cells.

14. The method of claim 13, wherein at least 20% of the CD4+ T cells are naive T cells and no more than 12% of the CD4+ T cells are effector memory T cells.

15. The method of claim 14, wherein at least 25% of the CD4+ T cells are naive T cells and no more than 9% 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 no more than 30% of the CD8+ T cells are effector memory T cells.

17. The method of claim 16, wherein at least 20% of the CD8+ T cells are naive T cells and no more than 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 of any one of claims 13 to 18, wherein the effector memory T cells are characterized as CCR7-, CD45RO+, and CD95+.

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

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

22. The method of 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 of claim 13, wherein up to 40% of the CD8+ T cells are a combination of effector memory T cells and effector T cells.

24. The method according to any preceding claim, further comprising: The lymphocytes are obtained from the donor subject or the lymphocytes are enriched.

25. The method according to any preceding claim, further comprising: The sample is contacted with a lymphocyte stimulator to activate the lymphocytes.

26. The method of claim 25, wherein said activating said sample is prior to incubating said sample with said polynucleotide carrier.

27. The method of claim 26, wherein the sample comprises at least 1×10 9 Lymphocytes.

28. The method of claim 25, wherein activating the sample is subsequent to incubating the sample with the polynucleotide carrier.

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

30. The method of any preceding claim, further comprising: Following said harvesting, said harvested lymphocytes are administered to a subject or said harvested lymphocytes are frozen.

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

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

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

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

35. The method of any preceding claim, wherein the polynucleotide vector is a viral vector.

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

37. The method of any preceding claim, wherein the vector encodes a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

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

39. The method of claim 35, wherein the intracellular co-stimulatory domain is a signaling 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), CD3γ, CD3δ, CD3ε, CD247, CD276 (B7-H3), tumor necrosis factor superfamily member 14, TNFSF14, LIGHT), NKG2C, Ig α (CD79a), Fc γ receptor, MHC Class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activated NK cell receptors, BTLA, Toll ligand receptors, CDS, GITR, BAFFR, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 α, CD8 β, IL2R β, IL2R γ, IL7R α, ITGA4, VLA1, CD49a, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD (CD11d), ITGAE (CD103), ITGAL (CD11a), ITGAM (CD11b), IT GAX (CD11c), ITGB1, CD29, ITGB2, CD18, ITGB7, NKG2D, TNFR2, TRANCE (RANKL), DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, C D96 (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, ligands that specifically bind to CD83, and combinations thereof.

40. The method of claim 39, wherein the intracellular co-stimulatory domain is a signaling region of CD28.

41. The method of any one of claims 37 to 39, wherein the CAR recognizes a tumor antigen.

42. The method of claim 41, wherein the tumor antigen is CD19, CD20 and / or CLL-1.

43. The method of claim 40, wherein the lymphocyte comprising the CAR is akilenbine or brexucabtagene autoleucel.

44. A cell population prepared from a blood sample of a donor subject, the cell population comprising CD4+ T cells and CD8+ T cells, wherein: At least 20% of the CD4+ T cells are naive T cells, and no more than 12% of the CD4+ T cells are effector memory T cells; At least 10% of the CD8+ T cells are naive T cells, and no more than 30% of the CD8+ T cells are effector memory T cells; At least 50% of said cells are CD3+ T cells; and At least 25% of all T cells are transduced with a polynucleotide vector encoding a CAR or TCR.

45. The population of claim 44, wherein at least 25% of the CD4+ T cells are naive T cells and no more than 9% of the CD4+ T cells are effector memory T cells.

46. ​​A population according to claim 44 or 45, wherein at least 20% of the CD8+ T cells are naive T cells and no more than 20% of the CD8+ T cells are effector memory T cells.

47. A population according to any one of claims 44 to 46, wherein the naive T cells are characterized as CCR7+ and CD45RA+.

48. A population according to any one of claims 44 to 47, wherein the effector memory T cells are characterized as CCR7-, CD45RO+ and CD95+.

49. A population according to any one of claims 44 to 48, wherein at least 65% of the cells are CD3+ T cells.

50. A population according to any one of claims 44 to 49, wherein at least 15% of all T cells are transduced with the polynucleotide vector.

51. A population according to any one of claims 44 to 49, wherein the polynucleotide vector is a viral vector.

52. The population of claim 49, wherein the viral vector is a retroviral vector or a lentiviral vector.

53. A population according to any one of claims 44 to 52, wherein the CAR comprises an intracellular co-stimulatory domain.

54. A population according to claim 53, wherein the intracellular co-stimulatory domain is the signaling region of CD28.

55. A population according to any one of claims 44 to 54, wherein the CAR recognizes a tumor antigen.

56. A population according to claim 55, wherein the tumor antigen is CD19 or CD20.

57. A population according to claim 54, wherein the cell population comprising the CAR is akirafenib or brexucabtagene autoleucel.

58. A cell population prepared from a blood sample of a donor subject, the cell population 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; Up to 20% of the CD4+ T cells are a combination of effector memory T cells and effector T cells; and Up to 40% of the CD8+ T cells are a combination of effector memory T cells and effector T cells.

59. A pharmaceutical composition comprising the cell population according to any one of claims 44 to 58.

60. A method for administering T cells to a subject, the method comprising: The subject is injected with a harvested sample prepared by the method of any one of claims 1 to 43 or a pharmaceutical composition according to claim 59.

61. The method of claim 60, wherein the subject has cancer.

62. The method of claim 60, wherein the cancer is a B cell malignancy.

63. The method of 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's lymphoma, lymphoblastic lymphoma, acute myeloid leukemia, or multiple myeloma.

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