AU2023202826A1 - Central Memory T Cells For Adoptive T Cell Therapy - Google Patents

Abstract

Chimeric transmembrane immunoreceptors (CAR) which include an extracellular domain that includes IL-13 or a variant thereof that binds interleukin-13Ra2 (ILl3Ra2), a transmembrane region, a costimulatory domain and an intracellular signaling domain are described. Dy4oe 1-

Description

Central Memory T Cells for Adoptive T Cell Therapy

BACKGROUND

[001] Tumor-specific T cell based immunotherapies, including therapies employing engineered T cells, have been investigated for anti-tumor treatment. In some cases the T cells used in such therapies do not remain active in vivo for a long enough period. In some cases, the tumor-specificity of the T cells is relatively low. Therefore, there is a need in the art for tumor-specific cancer therapies with longer term anti-tumor functioning.

[002] Adoptive T cell therapy (ACT) utilizing chimeric antigen receptor (CAR) engineered T cells may provide a safe and effective way to reduce recurrence rates of MG, since CAR T cells can be engineered to specifically recognize antigenically-distinct tumor populations (Cartellieri et al. 2010 JBiomed Biotechnol 2010:956304; Ahmed et al. 2010 Clin CancerRes 16:474; Sampson et al. 2014 Clin CancerRes 20:972; Brown et al. 2013 Clin Cancer Res 2012 18:2199; Chow et al. 2013 Mol Ther 21:629), and T cells can migrate through the brain parenchyma to target and kill infiltrative malignant cells (Hong et al. 2010 Clin CancerRes 16:4892; Brown et al. 2007 JImmunol 179:3332; Hong et al. 2010 Clin CancerRes 16:4892; Yaghoubi 2009 Nat Clin PRact Oncol 6:53). Preclinical studies have demonstrated that IL13Ra2-targeting CAR+ T cells exhibit potent major histocompatibility complex (MHC)-independent, IL13Ra2-specific cytolytic activity against both stem-like and differentiated glioma cells, and induce regression of established glioma xenografts in vivo (Kahlon et al. 2004 CancerRes 64:9160; Brown et al. 2012 Clin CancerRes 18:2199).

SUMMARY

[003] Described herein are cell populations that comprise central memory T cells (Tcm). The cell populations include both CD4+ T cells and CD8+ T cells. The cells in the cell populations are useful for expressing chimeric transmembrane immunoreceptors (chimeric antigen receptors or "CARs") which comprise an extracellular domain (e.g., an scFv that binds to a selected target), a transmembrane region and an intracellular signaling domain, e.g., an intracellular domain that includes the signaling domain from the zeta chain of the human CD3 complex (CD3Q) and one or more costimulatory domains, e.g., a 4-1BB costimulatory domain. The extracellular domain enables the CAR, when expressed on the surface of a T cell, to direct T cell activity to those cells expressing a target recognized by the extracellular domain. The inclusion of a costimulatory domain, such as the 4-1BB (CD137) costimulatory domain in series with CD3Q in the intracellular region enables the T cell to receive co-stimulatory signals.

[004] The Tem cells described herein, for example, patient-specific, autologous or allogenic Tem cells can be engineered to express a desired CAR and the engineered cells can be expanded and used in ACT. The population of TCM cells are CD45RO+CD62L and include CD4+ and CD8+ cells.

[005] In various embodiments: the population of human T cells comprise a vector expressing a chimeric antigen receptor; the population of human central memory T cells (Tem cells ) comprise at least 10% CD4+ cells and at least 10% CD8+ cells (e.g., at least %, 30%, 40%, 50% 60%, 70%, 80% of the cells are Tem cells; at least 15%, 20%, %, 30%, 35% of the Tem cells are CD4+ and at least 15%, 20%, 25%, 30%, 35% of the Tem cells are CD8+ cells).

[006] Also described is a method of treating cancer in a patient comprising administering a population of autologous or allogeneic human T cells (e.g., autologous or allogenic T cells comprising Tem cells, e.g., at least 20%, 30%, 40%, 50% 60%, 70%, % of the cells are Tem cells; at least 15%, 20%, 25%, 30%, 35% of the Tem cells are CD4+ and at least 15%, 20%, 25%, 30%, 35% of the Tem cells are CD8+ cells) transduced by a vector comprising an expression cassette encoding a chimeric antigen receptor.

[007] Described herein a population of human T cells of transduced with a vector expressing a chimeric antigen receptor wherein at least 50% of the transduced human T cells are central memory T cells. In various embodiments: at least 10% of the transduced cells central memory T cells are CD4+; at least 10% of the transduced central memory T cells are CD8+; at leastl5% of the central memory T cells are CD4+ and at least 15% are CD8+; and at least 50% of the transduced human T cells are CD4+/CD8+/CD62L+.

[008] Also described herein is a population of human T cells of transduced with a vector expressing a chimeric antigen receptor wherein: at least 50% of the transduced human T cells are CD45R+, CD62L+ and CD45Ra-, at least 10% of the cells are CD4+ and at least 10% of the cells are CD4+. In various embodiments: at least 10% of the cells that are CD45R+, CD62L+ and CD45Ra- are also CD4+ and at least 10% of the cells that are CD45RO+, CD62L+ and CD45Ra- are also CD8+; and at least 15% of the cells that are CD45RO+, CD62L+ and CD45Ra- are also CD4+ and at least 15% of the cells that are CD45RO+, CD62L+ and CD45Ra- are also CD4+.

[009] Also described is a method of treating cancer comprising administering to a patient in need thereof a pharmaceutical composition comprising the central memory T cells described herein. The T cells can be autologous to the patient or are allogenic to the patient.

[0010] Also described is a method for preparing a population central memory T cells comprising: obtaining a population of T cells from a human subject; depleting the population of T cells for cells that express CD25, cells that express CD14, and cells that express CD45+ to prepared a depleted T cell population; enriching the depleted T cell population for cells expressing CD62L, thereby preparing a population of central memory T cells, wherein the method does not comprising a step of depleting a cell population for cells expressing CD4 and does not comprising a step of depleting a cell population of cells expressing CD8+. In various embodiments: at least 50% (e.g., at least 60%, 70%, %, 90% or 95%) of the cells in the population central memory T cells are CD45R+, CD62L+ and CD45Ra-, at least 10% of the cells are CD4+ and at least 10% of the cells are CD4+; at least 50% (e.g., at least 60%, 70%, 80%, 90% or 95%) of the cells in the population central memory T cells are CD45RO+, CD62L+ and CD45Ra-, at least 15% of the cells are CD4+ and at least 15% of the cells are CD4+; at least 50% of the cells in the population central memory T cells are CD45RO+, CD62L+ and CD45Ra-, at least 20% (e.g., at least 30%, 40% or 50%) of the cells are CD4+ and at least 20% (e.g., at least

%, 40% or 50%) of the cells are CD4+. The method can further comprise stimulating the population of central memory T cells (e.g., by contacting the population of central memory T cells with CD3 and/or CD28); can comprise transducing the cells with a vector expressing a recombinant protein to create a population of genetically modified central memory T cells; and can comprise expanding the population of generically modified central memory T cells (e.g., of expanding the population of generically modified central memory T cells by exposing the cells to one or both of IL-2 and IL-15.

DESCRIPTION OF DRAWINGS

[0011] Figure 1 is a schematic depiction of IL13(E13Y)-zetakine CAR (Left) composed of the IL13Ra2-specific human IL-13 variant (huIL-13(E13Y)), human IgG4 Fc spacer (huY4F), human CD4 transmembrane (huCD4 tm), and human CD3Q chain cytoplasmic (huCD3Q cyt) portions as indicated. Also depicted is a IL13(EQ)BBQ CAR which is the same as the IL13(E13Y)-zetakine with the exception of the two point mutations, L235E and N297Q indicated in red, that are located in the CH2 domain of the IgG4 spacer, and the addition of a costimulatory 4-1BB cytoplasmic domain (4-1BB cyt).

[0012] Figures 2A-C depict certain vectors an open reading frames. A is a diagram of the cDNA open reading frame of the 2670 nucleotide IL13(EQ)BBZ-T2ACD19t construct, where the IL13Ra2-specific ligand IL13(E13Y), IgG4(EQ) Fc hinge, CD4 transmembrane, 4-1BB cytoplasmic signaling, three-glycine linker, and CD3Q cytoplasmic signaling domains of the IL13(EQ)BBZ CAR, as well as the T2A ribosome skip and truncated CD19 sequences are indicated. The human GM-CSF receptor alpha and CD19 signal sequences that drive surface expression of the IL13(EQ)BBQ CAR and CD19t are also indicated. B is a diagram of the sequences flanked by long terminal repeats (indicated by 'R') that will integrate into the host genome. C is a map of the IL13(EQ)BBZ-T2A-CD19t epHIV7 plasmid.

[0013] Figure 3 depicts the construction of pHIV7.

[0014] Figure 4 depicts the elements of pHIV7.

[0015] Figure 5 depicts a production scheme for IL13(EQ)BBQ/CD19t+ TCM.

[0016] Figures 6A-C depicts the results of flow cytometric analysis of surface transgene and T cell marker expression. IL3(EQ)BBQ/CD19t+ TCM HDO06.5 and HD187.1 were co-stained with anti-IL13-PE and anti-CD8-FITC to detect CD8+ CAR+ and CD4+ (i.e., CD8 negative) CAR+ cells (A), or anti-CD19-PE and anti-CD4-FITC to detect CD4+ CD19t+ and CD8+ (i.e., CD4 negative) CAR+ cells (B). IL3(EQ)BBQ/CD19t+ TCM HD006.5 and HD187.1 stained with fluorochromeconjugatedanti-CD3, TCR, CD4, CD8, CD62L and CD28 (grey histograms) or isotype controls (black histograms) (C). In all cases the percentages based on viable lymphocytes (DAPI negative) stained above isotype.

[0017] Figures 7A-B depict the in vitro functional characterization of IL13Ra2-specific effector function of IL13(EQ)BBZ+ TCM. IL13(EQ)BBZ/CD19t+ TCM HDO06.5 and HD187.1 were used as effectors in a 6-hour "Cr release assay using a 10:1 E:T ratio based on CD19t expression. The IL13Ra2-positive tumor targets were K562 engineered to express IL13Ra2 (K562-IL13Ra2) and primary glioma line PBT030-2, and the IL13Ra2-negative tumor target control was K562 parental line (A). IL13(EQ)BBZ/CD19t+ TCMHD006.5 and HD187.1 were evaluated for antigen dependent cytokine production following overnight co-culture at a 10:1 E:T ratio with IL13Ra2-positive and negative targets. Cytokine levels were measured using the Bio Plex Pro Human Cytokine TH1/TH2 Assay kit and INF-y are reported (B).

[0018] Figures 8A-C depict the result of studies demonstrating the regression of established glioma tumor xenografts after adoptive transfer of IL13(EQ)BBQ/CD19t+ TCM. EGFP-ffLuc+ PBT030-2 tumor cells (1x 10) were stereotactically implanted into the right forebrain of NSG mice. On day 5, mice received either 2x10 6 IL13(EQ)BBQ/CD19t+ TCM(1.1X10 6 CAR+; n=6), 2x10 6 mock TCM (no CAR; n=6) or PBS (n=6). Representative mice from each group showing relative tumor burden using Xenogen Living Image (A). Quantification of ffLuc flux (photons/sec) shows that IL13(EQ)BBQ/CD19t+ TCM induce tumor egression as compared to mock-transduced TCM and PBS (#p<0.02, *p<0.001, repeated measures ANOVA) (B). Kaplan Meier survival curve (n=6 per group) demonstrating significantly improved survival (p=0.0008; log-rank test) for mice treated with IL13(EQ)BBQ/CD19t+ TCM (C)

[0019] Figures 9A-C depict the results of studies comparing ant-tumor efficacy of IL13(EQ)BBZ TCM and IL13-zetakine CTL clones. EGFP-ffLuc+ PBT030-2 TSs (1x10) were stereotactically implanted into the right forebrain of NSG mice. On day 8, mice received either 1.6x10 6 mock TCM (no CAR),1.x106 CAR+ IL13(EQ)BBQ TCM (1.6x106 total T cells; 63% CAR), 1.0x10 6 IL13-zetakine CD8+ CTL cl. 2D7 (clonal CAR+), or no treatment (n=6 per group). Representative mice from each group showing relative tumor burden using Xenogen Living Image (A). Linear regression lines of natural log of ffLuc flux (photons/sec) over time, P-values are for group by time interaction comparisons (B). Kaplan Meier survival analysis (n= 6 per group) demonstrate significantly improved survival (p=0.02; log-rank test) for mice treated with IL13(EQ)BBQ TCM as compared to1 IL13-zetakine CD8+ CTL cl. 2D7 (C).

[0020] Figures 10A-C depict the results of studies comparing ant-tumor efficacy of IL13(EQ)BBQ TCM and IL13-zetakine CTL clones. EGFP-ffLuc+ PBT030-2 TSs (1x10') were stereotactically implanted into the right forebrain of NSG mice. On day 8, mice received either 1.3x10 6 mock TCM (no CAR; n=6), 1.0, 0.3 or 0.1x10 6 CAR+ IL13(EQ)BBQ TCM (78% CAR+; n=6-7),1.0,0.3 or 0.1x10 6 IL13-zetakine CD8+ CTL cl. 2D7 (clonal CAR+; n=6-7), or no treatment (n=5). Xenogen imaging of representative mice from each group showing relative tumor burden (A). Linear regression lines of natural log of ffLuc flux (photons/sec) shows that IL3(EQ)BBQ TCM achieve superior tumor regression as compared to first-generation IL13-zetakine CTL cl. 2D7, mock TCM and tumor only (B). Average flux per group at day 27 post tumor injection demonstrating thatthe 0.1x10 6 IL13(EQ)BBQ TCM dose outperforms the ten-fold higher 1.0x10 6 dose of IL13-zetakine CD8+ CTL cl. 2D7 (p = 0.043; Welch two sample t- test) (C).

[0021] Figure 11 depicts the results of studies demonstrating IL13(EQ)BB1 m Te display improved persistence compared IL13-zetakine CTL clones. CD3 immunohistochemistry evaluating T cell persistence at the tumor site 7-days post T cell infusion. Significant numbers of T cells are detected for IL13(EQ)BB1 m Te (top panel). By contrast, very few viable CD3+ IL13-zetakine T cells are detected (bottom panel).

[0022] Figures 12A-D depict the results of experiments comparing route of CAR+ T cell delivery (i.e. versus i.v.) for large established tumors. EGFP-ffLuc+ PBT030-2 TSs (1x10) were implanted into the right forebrain of NSG mice. On days 19 and 26, mice were injected i.v. through the tail vein with either 5x10 6 CAR+ IL13(EQ)BBQ+ Tem

(11.8x10 6 total cells; n=4), or mock Tem (11.8x106 cells; n=4). Alternatively, on days 19, 22, 26 and 29 mice were injected i.e. with either x106 CAR+ IL13(EQ)BBQ+ Tem (2.4x106 total cells; n=4), or mock Tem (2.4x106 cells; n=5). Average ffLuc flux (photons/sec) over time shows that i.e. delivered IL13(EQ)BB1 m Te mediates tumor regression of day 19 tumors. By comparison, i.v. delivered T cells do not shown reduction in tumor burden as compared to untreated or mock Tem controls (A). Kaplan Meier survival curve demonstrates improved survival for mice treated i.e. IL13(EQ)BBZ Tem as compared to mice treated with i.v. administered CAR+ Tem (p = 0.0003 log rank test) (B). Representative H&E and CD3 IHC of mice treated i.v. (C) versus i.e. (D) with IL13(EQ)BBZ+ Tcm. CD3+ T cells were only detected in the i.e. treated group, with no CD3+ cells detected in the tumor or surrounding brain parenchyma for i.v. treated mice.

[0023] Figures 13A-B depict the results of studies showing that CAR+ T cell injected intracranially, either intratumoral (i.c.t.) or intraventricular (i.c.v.), can traffic to tumors on the opposite hemisphere. EGFP-ffLuc+ PBT030-2 TSs (1x105) were stereotactically implanted into the right and left forebrains of NSG mice. On day 6, mice were injected i.e. at the right tumor site with1.0x106 IL13(EQ)BBQ+ Tem (1.6x106 total cells; 63% CAR; n=4). Schematic of multifocal glioma experimental model (A). CD3 IHC showing T cells infiltrating both the right and left tumor sites (B).

[0024] Figure 14 depicts the amino acid sequence of IL3(EQ)BBQ/CD19t+ (SEQ ID NO:10).

[0025] Figure 15 depicts a sequence comparison of IL13(EQ)41BBJIL13{EQ}41BBQ T2A-CD19tepHIV7; pF02630] (SEQ ID NO:12) and CD19RopepHIV7 (pJ01683) (SEQ ID NO:13).

DETAILED DESCRIPTION

[0026] A TCM cell population that includes CD4+ cells and CD8+ cells ("TCM CD4+/CD8+" cell population) is described below. The cells of the TCM CD4+/CD8+ cell population can be activated with anti-CD3/CD28 and transduced with, for example, a SIN lentiviral vector that directs the expression of a CAR to create genetically modified cells. The activated/genetically modified cells can be expanded in vitro with IL-2/IL-15 and then used or cryopreserved and used later. Described is an example of the use of TCM CD4+/CD8+ cell population to express a CAR targeted to IL13Ra2.

[0027] The CAR used in the examples below is referred to as IL13(EQ)BBQ. This CAR includes a variety of important features including: a IL13U2 ligand having an amino acid change that improves specificity of biding to IL13a2; the domain of CD137 (4-1BB) in series with CD3Q to provide beneficial costimulation; and an IgG4 Fc region that is mutated at two sites within the CH2 region (L235E; N297Q) in a manner that reduces binding by Fc receptors (FcRs). This CAR and others can be produced using a vector in which the CAR open reading frame is followed by a T2A ribosome skip sequence and a truncated CD19 (CD19t), which lacks the cytoplasmic signaling tail (truncated at amino acid 323). In this arrangement, co-expression of CD19t provides an inert, non immunogenic surface marker that allows for accurate measurement of gene modified cells, and enables positive selection of gene-modified cells, as well as efficient cell tracking and/or imaging of the therapeutic T cells in vivo following adoptive transfer. Co expression of CD19t provides a marker for immunological targeting of the transduced cells in vivo using clinically available antibodies and/or immunotoxin reagents to selectively delete the therapeutic cells, and thereby functioning as a suicide switch.

[0028] Gliomas, express IL13 receptors, and in particular, high-affinity IL13 receptors. However, unlike the IL13 receptor, glioma cells overexpress a unique IL13Ra2 chain capable of binding IL13 independently of the requirement for IL4RP or yc44. Like its homolog IL4, IL13 has pleotropic immunoregulatory activity outside the CNS. Both IL13 and IL4 stimulate IgE production by B lymphocytes and suppress pro-inflammatory cytokine production by macrophages.

[0029] Detailed studies using autoradiography with radiolabeled IL13 have demonstrated abundant IL13 binding on nearly all malignant glioma tissues studied. This binding is highly homogeneous within tumor sections and in single cell analysis. However, molecular probe analysis specific for IL13Ra2 mRNA did not detect expression of the glioma-specific receptor by normal brain elements and autoradiography with radiolabeled IL13 also could not detect specific IL13 binding in the normal CNS. These studies suggest that the shared IL13Ral/IL40/yc receptor is not expressed detectably in the normal CNS. Therefore, IL13Ra2 is a very specific cell-surface target for glioma and is a suitable target for a CAR designed for treatment of a glioma.

[0030] Binding of IL13-based therapeutic molecules to the broadly expressed IL13Ral/IL40/yc receptor complex, however, has the potential of mediating undesired toxicities to normal tissues outside the CNS, and thus limits the systemic administration of these agents. An amino acid substitution in the IL13 alpha helix A at amino acid 13 of tyrosine for the native glutamic acid selectively reduces the affinity of IL13 to the IL13Ral/IL40/yc receptor. Binding of this mutant (termed IL13(E13Y)) to IL13Ra2, however, was increased relative to wild-type IL13. Thus, this minimally altered IL13 analog simultaneously increases IL13's specificity and affinity for glioma cells. Therefore, CAR described herein include an IL13 containing a mutation (E to Y or E to some other amino acid such as K or R or L or V) at amino acid 13 (according to the numbering of Debinski et al. 1999 Clin CancerRes 5:3143s). IL13 having the natural sequence also may be used, however, and can be useful, particularly in situations where the modified T cells are to be locally administered, such as by injection directly into a tumor mass.

[0031] The CAR described herein can be produced by any means known in the art, though preferably it is produced using recombinant DNA techniques. Nucleic acids encoding the several regions of the chimeric receptor can be prepared and assembled into a complete coding sequence by standard techniques of molecular cloning known in the art (genomic library screening, PCR, primer-assisted ligation, site-directed mutagenesis, etc.) as is convenient. The resulting coding region is preferably inserted into an expression vector and used to transform a suitable expression host cell line, preferably a T lymphocyte cell line, and most preferably an autologous T lymphocyte cell line.

Example 1: Construction and Structure of an IL13Ra2-specific CAR

[0032] The structure of a useful IL13Ra2-specific CAR is described below. The codon optimized CAR sequence contains a membrane-tethered IL-13 ligand mutated at a single site (E13Y) to reduce potential binding to IL13Ral, an IgG4 Fc spacer containing two mutations (L235E; N297Q) that greatly reduce Fc receptor-mediated recognition models, a CD4 transmembrane domain, a costimulatory 4-1BB cytoplasmic signaling domain, and a CD3Q cytoplasmic signaling domain. A T2A ribosome skip sequence separates this IL13(EQ)BBQ CAR sequence from CD19t, an inert, non-immunogenic cell surface detection/selection marker. This T2A linkage results in the coordinate expression of both IL13(EQ)BB and CD19t from a single transcript. Figure 1A is a schematic drawing of the 2670 nucleotide open reading frame encoding the IL13(EQ)BBZ-T2ACD19t construct. In this drawing, the IL3Ra2-specific ligand IL13(E13Y), IgG4(EQ) Fc, CD4 transmembrane, 4-1BB cytoplasmic signaling, three-glycine linker, and CD3Q cytoplasmic signaling domains of the IL13(EQ)BBZ CAR, as well as the T2A ribosome skip and truncated CD19 sequences are all indicated. The human GM-CSF receptor alpha and CD19 signal sequences that drive surface expression of the IL13(EQ)BBZ CAR and CD19t are also indicated. Thus, the IL13(EQ)BBZ-T2ACD19t construct includes a IL13Ra2-specific, hinge-optimized, costimulatory chimeric immunoreceptor sequence (designated IL13(EQ)BBZ), a ribosome-skip T2A sequence, and a CD19t sequence.

[0033] The IL13(EQ)BBZ sequence was generated by fusion of the human GM-CSF receptor alpha leader peptide with IL13(E13Y) ligand 5 L235E/N297Q-modified IgG4 Fc hinge (where the double mutation interferes with FcR recognition), CD4 transmembrane, 4-1BB cytoplasmic signaling domain, and CD3Q cytoplasmic signaling domain sequences. This sequence was synthesized de novo after codon optimization. The T2A sequence was obtained from digestion of a T2A-containing plasmid. The CD19t sequence was obtained from that spanning the leader peptide sequence to the transmembrane components (i.e., basepairs 1-972) of a CD19-containing plasmid. All three fragments, 1)

IL13(EQ)BBZ, 2) T2A, and 3) CD19t, were cloned into the multiple cloning site of the epHIV7 lentiviral vector. When transfected into appropriate cells, the vector integrates the sequence depicted schematically in Figure 1B into the host cells genome. Figure 1C provides a schematic drawing of the 9515 basepair IL13(EQ)BBZ-T2A-CD19t _epHIV7 plasmid itself.

[0034] As shown schematically in Figure 2, IL13(EQ)BBZ CAR differs in several important respects from a previously described IL13Ra2-specific CAR referred to as IL13(E13Y)-zetakine (Brown et al. 2012 ClinicalCancer Research 18:2199). The IL13(E13Y)-zetakine is composed of the IL13Ra2-specific human IL-13 mutein (huIL 13(E13Y)), human IgG4 Fc spacer (huy4Fc), human CD4 transmembrane (huCD4 tm), and human CD3Q chain cytoplasmic (huCD3Q cyt) portions as indicated. In contrast, the IL13(EQ)BB ) has two point mutations, L235E and N297Q that are located in the CH2 domain of the IgG4 spacer, and a costimulatory 4-1BB cytoplasmic domain (4-1BB cyt).

Example 2: Construction and Structure of epHIV7 used for Expression of an IL13Ra2-specific CAR

[0035] The pHIV7 plasmid is the parent plasmid from which the clinical vector IL13(EQ)BBZ-T2A-CD19t epHIV7 was derived in the T cell Therapeutics Research Laboratory (TCTRL) at City of Hope (COH). The epHIV7 vector used for expression of the CAR was produced from pHIV7 vector. Importantly, this vector uses the human EF promoter to drive expression of the CAR. Both the 5' and 3' sequences of the vector were derived from pv653RSN as previously derived from the HXBc2 provirus. The polypurine tract DNA flap sequences (cPPT) were derived from HIV-1 strain pNL4-3 from the NIH AIDS Reagent Repository. The woodchuck post-transcriptional regulatory element (WPRE) sequence was previously described.

[0036] Construction of pHIV7 is schematically depicted in Figure 3. Briefly, pv653RSN, containing 653 bp from gag-pol plus 5' and 3' long-terminal repeats (LTRs) with an intervening SL3-neomycin phosphotransferase gene (Neo), was subcloned into pBluescript, as follows: In Step 1, the sequences from 5' LTR to rev-responsive element

(RRE) made p5'HIV-151, and then the 5' LTR was modified by removing sequences upstream of the TATA box, and ligated first to a CMV enhancer and then to the SV40 origin of replication (p5'HIV-2). In Step 2, after cloning the 3'LTR into pBluescript to make p3'HIV-1, a 400-bp deletion in the 3' LTR enhancer/promoter was made to remove cis-regulatory elements in HIV U3 and form p3'HIV-2. In Step 3, fragments isolated from the p5'HIV-3 and p3'HIV-2 were ligated to make pHIV-3. In Step 4, the p3'HIV-2 was further modified by removing extra upstream HIV sequences to generate p3'HIV-3 and a 600-bp BamHI-SalI fragment containing WPRE was added to p3'HIV-3 to make the p3'HIV-4. In Step 5, the pHIV-3 RRE was reduced in size by PCR and ligated to a 5' fragment from pHIV-3 (not shown) and to the p3'HIV-4, to make pHIV-6. In Step 6, a 190-bp BglII-BamHI fragment containing the cPPT DNA flap sequence from HIV-1 pNL4-3 (55) was amplified from pNL4-3 and placed between the RRE and the WPRE sequences in pHIV6 to make pHIV-7. This parent plasmid pHIV7-GFP (GFP, green fluorescent protein) was used to package the parent vector using a four-plasmid system.

[0037] A packaging signal, psi V, is required for efficient packaging of viral genome into the vector. The RRE and WPRE enhance the RNA transcript transport and expression of the transgene. The flap sequence, in combination with WPRE, has been demonstrated to enhance the transduction efficiency of lentiviral vector in mammalian cells.

[0038] The helper functions, required for production of the viral vector), are divided into three separate plasmids to reduce the probability of generation of replication competent lentivirus via recombination: 1) pCgp encodes the gag/pol protein required for viral vector assembly; 2) pCMV-Rev2 encodes the Rev protein, which acts on the RRE sequence to assist in the transportation of the viral genome for efficient packaging; and 3) pCMV-G encodes the glycoprotein of the vesiculo-stomatitis virus (VSV), which is required for infectivity of the viral vector.

[0039] There is minimal DNA sequence homology between the pHIV7 encoded vector genome and the helper plasmids. The regions of homology include a packaging signal region of approximately 600 nucleotides, located in the gag/pol sequence of the pCgp helper plasmid; a CMV promoter sequence in all three helper plasmids; and a RRE sequence in the helper plasmid pCgp. It is highly improbable that replication competent recombinant virus could be generated due to the homology in these regions, as it would require multiple recombination events. Additionally, any resulting recombinants would be missing the functional LTR and tat sequences required for lentiviral replication.

[0040] The CMV promoter was replaced by the EFla-HTLV promoter (EFIp), and the new plasmid was named epHIV7 (Figure 4). The EFIp has 563 bp and was introduced into epHIV7 using NruI and NheI, after the CMV promoter was excised.

[0041] The lentiviral genome, excluding gag/pol and rev that are necessary for the pathogenicity of the wild-type virus and are required for productive infection of target cells, has been removed from this system. In addition, the IL13(EQ)BBZ T2ACD19tepHIV7 vector construct does not contain an intact 3'LTR promoter, so the resulting expressed and reverse transcribed DNA proviral genome in targeted cells will have inactive LTRs. As a result of this design, no HIV-I derived sequences will be transcribed from the provirus and only the therapeutic sequences will be expressed from their respective promoters. The removal of the LTR promoter activity in the SIN vector is expected to significantly reduce the possibility of unintentional activation of host genes (56). Table 4 summarizes the various regulator elements present in IL13(EQ)BBZ T2ACD19tepHIV7.

Table 4 Functional elements of I L13(EQ)4 IBBZ-T2A-CDI9tepH IV7 Regulatory Elements Location Comments and Genes (Nucleotide Numbers) U5 87-171 5' Unique sequence psi 233-345 Packaging signal RRE 957-1289 Rev-responsive element Contains polypurine track flap 1290-1466 sequence and central termination sequence to facilitate nuclear import of pre-integration complex EF1-alpha Eukaryotic Promoter EFIp Promoter 1524-2067 sequence driving expression of CD19Rop

II3B-Z ta-T2- 2084-4753 Therapeutic insert

Table 4 Functional elements of IL13(EQ)4LBBZ-T2A-CDI9tepH IV7 Regulatory Elements Location Comments and Genes (Nucleotide Numbers) CD19t Woodchuck hepatitis virus derived WPRE 4790-5390 regulatory element to enhance viral RNA transportation delU3 5405-5509 3' U3 with deletion to generate SIN vector R 5510-5590 Repeat sequence within LTR U5 5591-5704 3' U5 sequence in LTR AmpR 6540-7398 Ampicillin-resistance gene CoEl ori 7461-8342 Replication origin of plasmid SV40 ori 8639-8838 Replication origin of SV40 CMV promoter 8852-9451 CMV promoter to generate viral genome RNA R 9507-86 Repeat sequence within LTR

Example 3: Production of Vectors for Transduction of Patient T Cells

[0042] For each plasmid (IL13(EQ)BBZ-T2A-CD19t epHIV7; pCgp; pCMV-G; and pCMV-Rev2), a seed bank is generated, which is used to inoculate the fermenter to produce sufficient quantities of plasmid DNA. The plasmid DNA is tested for identity, sterility and endotoxin prior to its use in producing lentiviral vector.

[0043] Briefly, cells were expanded from the 293T working cell (WCB), which has been tested to confirm sterility and the absence of viral contamination. A vial of 293T cells from the 293T WCB was thawed. Cells were grown and expanded until sufficient numbers of cells existed to plate an appropriate number of 10 layer cell factories (CFs) for vector production and cell train maintenance. A single train of cells can be used for production.

[0044] The lentiviral vector was produced in sub-batches of up to 10 CFs. Two sub batches can be produced in the same week leading to the production of approximately 20 L of lentiviral supernatant/week. The material produced from all sub-batches were pooled during the downstream processing phase, in order to produce one lot of product. 293T cells were plated in CFs in 293T medium (DMEM with 10% FBS). Factories were placed in a 37°C incubator and horizontally leveled in order to get an even distribution of the cells on all the layers of the CF. Two days later, cells were transfected with the four lentiviral plasmids described above using the CaPO4 method, which involves a mixture of Tris:EDTA, 2M CaCl2, 2X HBS, and the four DNA plasmids. Day 3 after transfection, the supernatant containing secreted lentiviral vectors was collected, purified and concentrated. After the supernatant was removed from the CFs, End-of-Production Cells were collected from each CF. Cells were trypsinized from each factory and collected by centrifugation. Cells were resuspended in freezing medium and cryopreserved. These cells were later used for replication-competent lentivirus (RCL) testing.

[0045] To purify and formulate vectors crude supernatant was clarified by membrane filtration to remove the cell debris. The host cell DNA and residual plasmid DNA were degraded by endonuclease digestion (Benzonase@). The viral supernatant was clarified of cellular debris using a 0.45 pm filter. The clarified supernatant was collected into a pre weighed container into which the Benzonase@ is added (final concentration 50 U/mL). The endonuclease digestion for residual plasmid DNA and host genomic DNA as performed at 370 C for 6 h. The initial tangential flow ultrafiltration (TFF) concentration of the endonuclease-treated supernatant was used to remove residual low molecular weight components from the crude supernatant, while concentrating the virus ~20 fold. The clarified endonuclease-treated viral supernatant was circulated through a hollow fiber cartridge with a NMWCO of 500 kD at a flow rate designed to maintain the shear rate at ~4,000 sec-i or less, while maximizing the flux rate. Diafiltration of the nuclease-treated supernatant was initiated during the concentration process to sustain the cartridge performance. An 80% permeate replacement rate was established, using 4% lactose in PBS as the diafiltration buffer. The viral supernatant was brought to the target volume, representing a 20-fold concentration of the crude supernatant, and the diafiltration was continued for 4 additional exchange volumes, with the permeate replacement rate at 100%.

[0046] Further concentration of the viral product was accomplished by using a high speed centrifugation technique. Each sub-batch of the lentivirus was pelleted using a

Sorvall RC-26 plus centrifuge at 6000 RPM (6,088 RCF) at 6oC for 16-20 h. The viral pellet from each sub-batch was then reconstituted in a 50 mL volume with 4% lactose in PBS. The reconstituted pellet in this buffer represents the final formulation for the virus preparation. The entire vector concentration process resulted in a 200-fold volume reduction, approximately. Following the completion of all of the sub-batches, the material was then placed at -8OoC, while samples from each sub-batch were tested for sterility. Following confirmation of sample sterility, the sub-batches were rapidly thawed at 37oC with frequent agitation. The material was then pooled and manually aliquoted in the Class II Type A/B3 biosafety cabinet in the viral vector suite. A fill configuration of 1 mL of the concentrated lentivirus in sterile USP class 6, externally threaded O-ring cryovials was used. Center for Applied Technology Development (CATD)'s Quality Systems (QS) at COH released all materials according to the Policies and Standard Operating Procedures for the CBG and in compliance with current Good Manufacturing Practices (cGMPs).

[0047] To ensure the purity of the lentiviral vector preparation, it was tested for residual host DNA contaminants, and the transfer of residual host and plasmid DNA. Among other tests, vector identity was evaluated by RT-PCR to ensure that the correct vector is present. All release criteria were met for the vector intended for use in this study.

Example 4: Preparation of a TCM CD4+/CD8+ Cell Population Suitable for Use in ACT

[0048] An outline of the manufacturing strategy for a TCM CD4+/CD8+ cell population-is depicted in Figure 8 (Manufacturing schema for IL13(EQ)BBQ/CD19t+ TCM). Specifically, apheresis products obtained from consented research participants are ficolled, washed and incubated overnight. Cells are then depleted of monocyte, regulatory T cell and naYve T cell populations using GMP grade anti-CD14, anti-CD25 and anti CD45RA reagents (Miltenyi Biotec) and the CliniMACSTM separation device. Following depletion, negative fraction cells are enriched for CD62L+ TCM cells using DREG56 biotin (COH clinical grade) and anti-biotin microbeads (Miltenyi Biotec) on the

CliniMACSTM separation device. The cells are not depleted for CD4+ cells or for CD8+ cells.

[0049] Following enrichment, TCM CD4+/CD8+ cells are formulated in complete X Vivo15 plus 50 IU/mL IL-2 and 0.5 ng/mL IL-15 and transferred to a Teflon cell culture bag, where they are stimulated with Dynal ClinEx TM Vivo CD3/CD28 beads. Up to five days after stimulation, cells are transduced with a desired vector expressing a CAR (e.g., IL13(EQ)BBZ-T2A-CD19t epHIV7 lentiviral vector) at a multiplicity of infection (MOI) of, for example, 1.0 to 0.3. Cultures are maintained for up to 42 days with addition of complete X-Vivo15 and IL-2 and IL-15 cytokine as required for cell expansion (keeping cell density between 3x10 5 and 2x10 6 viable cells/mL, and cytokine supplementation every Monday, Wednesday and Friday of culture). Cells typically expand to approximately 10 9 cells under these conditions within 21 days. At the end of the culture period cells are harvested, washed twice and formulated in clinical grade cryopreservation medium (Cryostore CS5, BioLife Solutions).

[0050] On the day(s) of T cell infusion, the cryopreserved and released product is thawed, washed and formulated for re-infusion. The cryopreserved vials containing the released cell product are removed from liquid nitrogen storage, thawed, cooled and washed with a PBS/2% human serum albumin (HSA) Wash Buffer. After centrifugation, the supernatant is removed and the cells resuspended in a Preservative-Free Normal Saline (PFNS)/ 2% HSA infusion diluent. Samples are removed for quality control testing.

[0051] Two qualification runs on cells procured from healthy donors were performed using the manufacturing platform described above. Each preclinical qualification run product was assigned a human donor (HD) number - HD006.5 and HD187.1. Importantly, as shown in Table 5, these qualification runs expanded >80 fold within 28 days and the expanded cells expressed the IL13(EQ)BBy/CD19t transgenes.

Table 5: Summary of Expression Data from Pre-clinical Qualification Run Product

Cell CAR CD19 CD4+ CD8+ Fold Expansion Product

HDO06.5 20% 22% 24% 76% 84-fold (28 days)

Hd187.1 18% 25% 37% 63% 259-fold (28 days)

Example 5: Flow cytometric analysis of surface transgene and T cell marker expression in IL 13(EQ)BBy/CD1 9 t+TCM

[0052] The two preclinical qualification run products described in Example 4 were used in pre-clinical studies to as described below. Figures 6A-C depict the results of flow cytometric analysis of surface transgene and T cell marker expression. IL13(EQ)BBy/CD19t+ TCM HD006.5 and HD187.1 were co-stained with anti-IL13-PE and anti-CD8-FITC to detect CD8+ CAR+ and CD4+ (i.e., CD8 negative) CAR+ cells (Figure 6A), or anti-CD19-PE and anti-CD4-FITC to detect CD4+ CD19t+ and CD8+ (i.e., CD4 negative) CAR+ cells (Figure 6B). IL13(EQ)BBy/CD19t+ TCM HD06.5 and HD187.1 were stained with fluorochrome-conjugated anti-CD3, TCR, CD4, CD8, CD62L and CD28 (grey histograms) or isotype controls (black histograms). (Figure 6C). In each of Figures 6A-C, the percentages indicated are based on viable lymphocytes (DAPI negative) stained above isotype.

Example 6: Effector Activity of IL13(EQ)BBy/CD19t+ TCM

[0053] The effector activity of IL13(EQ)BBQ/CD19t+ TCM was assessed and the results of this analysis are depicted in Figures 7A-B. Briefly, IL13(EQ)BBy/CD19t+ TCM HD006.5 and HD187.1 were used as effectors in a 6-hour 51Cr-release assay using a 1OE:1T ratio based on CD19t expression. The IL13Ra2-positive tumor targets were K562 engineered to express IL13Ra2 (K562-IL13Ra2) and primary glioma line PBT030-2, and the ILI 3Ra2-negative tumor target control was the K562 parental line (Figure 7A). IL13(EQ)BBy/CD19t+ HD006.5 and HD187.1 were evaluated for antigen-dependent cytokine production following overnight co-culture at a 10E:1T ratio with the same IL13Ra2-positive and negative targets as described in above. Cytokine levels were measured using the Bio-Plex Pro Human Cytokine TH1/TH2 Assay kit and INF-y levels are depicted (Figure 7B).

Example 7: In vivo Anti-tumor Activity of IL 3(EQ)BBy/CD19t+ TCM

[0054] The studies described below demonstrate that IL13(EQ)BBy/CD19t+ TCM exhibit anti-tumor efficacy in in vivo mouse models. Specifically, we have evaluated the anti tumor potency of IL13(EQ)BBy/CD19t+ TCM against the IL13Ra2+ primary low-passage glioblastoma tumor sphere line PBT030-2, which has been engineered to express both EGFP and firefly luciferase (ffLuc) reporter genes (PBT030-2 EGFP:ffLuc) (k). A panel of primary lines (PBT) from patient glioblastoma specimens grown as tumor spheres (TSs) in serum-free media. These expanded TS lines exhibit stem cell-like characteristics, including expression of stem cell markers, multilineage differentiation and capacity to initiate orthotopic tumors in immunocompromised mice (NSG) at low cell numbers. The PBT030-2 EGFP:ffLuc TS-initiated xenograft model (0.1x10 6 cells; 5 day engraftment) has been previously used to evaluate in vivo anti-tumor activity in NSG mice of IL13Ra2-specific CAR expressing T cells, whereby three injections of 2x10 6cytolytic T lymphocytes (CTLs) over a course of 2 weeks were shown to reduce tumor growth. However, in those experiments the majority of the PBT030-2 tumors eventually recurred. By comparison, a single injection of IL13(EQ)BBy/CD19t+ TCM (1.1X106 CAR+ TCM; 2x10 6 total TCM) exhibited robust anti-tumor activity against PBT030-2 EGFP:ffLuc TS initiated tumors (0.1x10 6 cells; 5 day engraftment) as shown in Figures 8A-C. As compared to NSG mice treated with either PBS or mock transduced TCM (no CAR), IL13(EQ)BBy/CD19t+ TCMsignificantly reduce ffLuc flux (p < 0.001 at >18-days) and significantly improve survival (p = 0.0008).

[0055] Briefly, EGFP-ffLuc+ PBT030-2 tumor cells (1x10') were stereotactically implanted into the right forebrain of NSG mice. On day 5, mice received either 2x10 6 IL13(EQ)BBy/CD19t+ TCM(1.1x106 CAR+; n=6), 2x10 6 mock TCM (no CAR; n=6) or PBS (n=6). Figure 8A depicts representative mice from each group showing relative tumor burden using Xenogen Living Image. Quantification of ffLuc flux (photons/sec) shows that IL13(EQ)BBC/CD19t+ TCMinduce tumoregression as compared to mock transduced TCM and PBS (#p<0.02, *p<0.001, repeated measures ANOVA) (Figure 8B). As shown in Figure 8C, a Kaplan Meier survival curve (n=6 per group) demonstrates significantly improved survival (p=0.0008; log-rank test) for mice treated with IL13(EQ)BBy/CD19t+ TCM.

Example 8: Comparison of IL 3(EQ)BBC+ Tcm and Non-Tcm IL 13-zetakine CD8+ CTL Clones in Antitumor Efficacy and T cell Persistence

[0056] The studies described below compare IL13(EQ)BBQ+ Tem and a previously created IL13Ra2-specific human CD8+ CTLs (IL13-zetakine CD8+ CTL (described in Brown et al. 2012 Clin CancerRes 18:2199 and Kahlon et al. 2004 CancerRes 64:9160). The IL13-zetakine uses a CD3Q stimulatory domain, lacks a co-stimulatory domain and uses the same IL13 variant as IL3(EQ)BBQ+.

[0057] A panel of primary lines (PBT) from patient glioblastoma specimens grown as tumor spheres (TSs) in serum-free media was generated (Brown et al. 2012 Clin Cancer Res 18:2199; Brown et al. 2009 Cancer Res 69:8886). These expanded TS lines exhibit stem cell-like characteristics, including expression of stem cell markers, multi-lineage differentiation and capacity to initiate orthotopic tumors in immunocompromised mice (NSG) at low cell numbers. The IL13Ra2+ primary low-passage glioblastoma TS line PBT030-2, which has been engineered to express both EGFP and firefly luciferase (ffLuc) reporter genes (PBT030-2 EGFP:ffLuc) (Brown et al. 2012 Clin Cancer Res 18:2199) was used for the experiments outlined below.

[0058] First, a single dose (1x10 6 CAR T cells) of IL3(EQ)BBQ+ Tem product was compared to IL13-zetakine CD8+ CTL clones evaluated against day 8 PBT030-2 EGFP:ffuc TS-initiated xenografts (0.1x10 6 cells). While both IL13Ra2-specific CAR T cells (IL13-zetakine CTL and IL13(EQ)BBQ Tcm) demonstrated antitumor activity against established PBT030-2 tumors as compared to untreated and mock Tem (CAR negative) controls (Figures 9A and 9B), IL13(EQ)BBZ+ Tem mediated significantly improved survival and durable tumor remission with mice living >150 days as compared to our first-generation IL13-zetakine CD8+ CTL clones (Figure 9C).

[0059] To further compare the therapeutic effectiveness of these two IL13Ra2-CAR T cell products, a dose titration of 1.0, 0.3 andO.1x10 6 CAR T cells against day 8 PBT030 2 EGFP:ffuc TS-initiated tumors was performed (Figures 10A-C). The highest dose

(1x10 6) of IL13-zetakine CD8+ CTL cl. 2D7 mediated antitumor responses as measured by Xenogen flux in 3 of 6 animals (Figure 10C), but no significant antitumor responses were observed at lower CAR T cell doses. By comparison, injection of IL13(EQ)BBQ+ Tcm product mediated complete tumor regression in the majority of mice at all dose levels, including treatment with as few as 0.1x106 CAR T cells. These data demonstrate that IL13(EQ)BBQ+ Tcm is at least 10-fold more potent than IL13-zetakine CD8+ CTL clones in antitumor efficacy. The improved anti-tumor efficacy of is due to improved T cell persistence in the tumor microenvironment. Evaluation of CD3+ T cells 7-days post i.e. injection revealed significant numbers of IL13(EQ)BBQ+ Tcm in the tumor microenvironment, whereas very few first-generation IL13-zeta CTLs were present (Figure 11).

Example 9: Comparison of CAR T cell delivery route for treatment of large TS initiated PBT tumors

[0060] Described below are studies that compare the route of delivery, intraveneous (i.v.) or intracranial (i.c.), on antitumor activity against invasive primary PBT lines. In pilot studies (data not shown), it was unexpectedly observed that i.v. administered IL13(EQ)BBQ+ Tcm provided no therapeutic benefit as compared to PBS for the treatment of small (day 5) PBT030-2 EGFP:ffLuc tumors. This is in contrast to the robust therapeutic efficacy observed with i.e. administered CAR+ T cells. Reasoning that day 5 PBT030-2 tumors may have been too small to recruit therapeutic T cells from the periphery, a comparison was made of i.v. versus i.e. delivery against larger day 19 PBT030-2 EGFP:ffLuc tumors. For these studies, PBT030-2 engrafted mice were treated with either two i.v. infusions (5 x 106 CAR+ Tem; days 19 and 26) or four i.e. infusions

(1 x 106 CAR+ Tem; days 19,22,26 and 29) of IL13(EQ)BBZ+ Tcm, or mock Tcm (no CAR). Here too no therapeutic benefit as monitored by Xenogen imaging or Kaplan Meier survival analysis for i.v. administered CAR+ T cells (Figures 12A and 12B). In contrast, potent antitumor activity was observed for i.c. administered IL13(EQ)BBQ+

Tern (Figures 12A-B). Next, brains from a cohort of mice 7 days post T cell injection were harvested and evaluated for CD3+ human T cells by IHC. Surprisingly, for mice treated i.v. with either mock Tem or IL13(EQ)BB1 m Te there were no detectable CD3+ human T cells in the tumor or in others mouse brain regions where human T cells typically reside (i.e. the leptomeninges) (Figure 12C), suggesting a deficit in tumor tropism. This is in contrast to the significant number of T cells detected in the i.e. treated mice (Figure 12D).

[0061] Tumor derived cytokines, particularly MCP-1/CCL2, are important in recruiting T cells to the tumor. Thus, PBT030-2 tumor cells were evaluated and it was found that this line produces high levels of MCP-1/CCL2 comparable to U25IT cells (data not shown), a glioma line previously shown to attract i.v. administered effector CD8+ T cells to i.e. engrafted tumors. Malignant gliomas are highly invasive tumors and are often multi focal in presentation. The studies described above establish that IL13BBZ TCM can eliminate infiltrated tumors such as PBT030-2, and mediate long-term durable antitumor activity. The capacity of intracranially delivered CAR T cells to traffic to multifocal disease was also examined. For this study PBT030-2 EGFP:ffLuc TSs were implanted in both the left and right hemispheres (Figure 13A) and CAR+ T cells were injected only at the right tumor site. Encouragingly, for all mice evaluated (n=3) we detected T cells by CD3 IHC 7-days post T cell infusion both at the site of injection (i.e. right tumor), as well within the tumor on the left hemisphere (Figure 13B). These findings provide evidence that CAR+ T cells are able to traffic to and infiltrate tumor foci at distant sites. Similar findings were also observed in a second tumor model using the U25IT glioma cell line (data not shown).

Example 10: IL13(EQ)BBC/CD19t Sequences

[0062] The complete amino acid sequence of IL13(EQ)BBQ/CD19t is depicted in Figure 17. The entire sequence (SEQ ID NO:1) includes: a 22 amino acid GMCSF signal peptide (SEQ ID NO:2), a 112 amino acid IL-13 sequence (SEQ ID NO:3; amino acid substitution E13Y shown in bold); a 229 amino acid IgG4 sequence (SEQ ID NO:4; with amino acid substitutions L235E and N297Q shown in bold); a 22 amino acid CD4 transmembrane sequence (SEQ ID NO:5); a 42 amino acid 4-1BB sequence (SEQ ID NO:6); a 3 amino acid Gly linker; a 112 amino acid CD3Q sequence (SEQ ID NO:7); a 24 amino acid T2A sequence (SEQ ID NO:8); and a 323 amino acid CD19t sequence (SEQ ID NO:9).

[0063] The mature chimeric antigen receptor sequence (SEQ ID NO:10) includes: a 112 amino acid IL-13 sequence (SEQ ID NO:3; amino acid substitution E13Y shown in bold); a 229 amino acid IgG4 sequence (SEQ ID NO:4; with amino acid substitutions L235E and N297Q shown in bold); at 22 amino acid CD4 sequence (SEQ ID NO:5); a 42 amino acid 4-1BB sequence (SEQ ID NO:6); a 3 amino acid Gly linker; and a 112 amino acid CD3Q sequence (SEQ IDNO:7). Within this CAR sequence (SEQ ID NO:10) is the IL-13/IgG4/CD4t/41-BB sequence (SEQ ID NO:11), which includes: a 112 amino acid IL-13 sequence (SEQ ID NO:3; amino acid substitution E13Y shown in bold); a 229 amino acid IgG4 sequence (SEQ ID NO:4; with amino acid substitutions L235E and N297Q shown in bold); at 22 amino acid CD4 sequence (SEQ ID NO:5); and a 42 amino acid 4-1BB sequence (SEQ ID NO:6). The IL13/IgG4/CD4t/4-1BB sequence (SEQ ID NO:11) can be joined to the 112 amino acid CD3Q sequence (SEQ ID NO:7) by a linker such as a Gly Gly Gly linker. The CAR sequence (SEQ ID NO:10) can be preceded by a 22 amino acid GMCSF signal peptide (SEQ ID NO:2). Figure 18 depicts a comparison of the sequences of IL13(EQ)41BBJIL13{EQ}41BBQ T2A-CD19t epHIV7; pF02630] (SEQ ID NO:12) and CD19RopepHIV7 (pJO1683) (SEQ ID NO:13).

Sequence Listing 1 Sequence Listing Information May 2023

1-1 File Name 40056-0002AU6_SL_ST26.xml 1-2 DTD Version V1_3 1-3 Software Name WIPO Sequence 1-4 Software Version 2.3.0 1-5 Production Date 2023-05-09 1-6 Original free text language en code 1-7 Non English free text language code 2 General Information 2-1 Current application: IP AU Office 2023202826

2-2 Current application: Application number 2-3 Current application: Filing date 2-4 Current application: 40056-0002AU6 Applicant file reference 2-5 Earliest priority application: US IP Office 2-6 Earliest priority application: 62/053,068 Application number 2-7 Earliest priority application: 2014-09-19 Filing date 2-8en Applicant name CITY OF HOPE 2-8 Applicant name: Name Latin 2-9en Inventor name 2-9 Inventor name: Name Latin 2-10en Invention title CENTRAL MEMORY T CELLS FOR ADOPTIVE T CELL THERAPY 2-11 Sequence Total Quantity 13

3-1 Sequences 3-1-1 Sequence Number [ID] 1 3-1-2 Molecule Type AA 3-1-3 Length 889 May 2023

3-1-4 Features source 1..889 Location/Qualifiers mol_type=protein organism=synthetic construct NonEnglishQualifier Value 3-1-5 Residues MLLLVTSLLL CELPHPAFLL IPGPVPPSTA LRYLIEELVN ITQNQKAPLC NGSMVWSINL 60 TAGMYCAALE SLINVSGCSA IEKTQRMLSG FCPHKVSAGQ FSSLHVRDTK IEVAQFVKDL 120 LLHLKKLFRE GRFNESKYGP PCPPCPAPEF EGGPSVFLFP PKPKDTLMIS RTPEVTCVVV 180 DVSQEDPEVQ FNWYVDGVEV HNAKTKPREE QFQSTYRVVS VLTVLHQDWL NGKEYKCKVS 240 NKGLPSSIEK TISKAKGQPR EPQVYTLPPS QEEMTKNQVS LTCLVKGFYP SDIAVEWESN 300 GQPENNYKTT PPVLDSDGSF FLYSRLTVDK SRWQEGNVFS CSVMHEALHN HYTQKSLSLS 360 LGKMALIVLG GVAGLLLFIG LGIFFKRGRK KLLYIFKQPF MRPVQTTQEE DGCSCRFPEE 420 EEGGCELGGG RVKFSRSADA PAYQQGQNQL YNELNLGRRE EYDVLDKRRG RDPEMGGKPR 480 RKNPQEGLYN ELQKDKMAEA YSEIGMKGER RRGKGHDGLY QGLSTATKDT YDALHMQALP 540 2023202826

PRLEGGGEGR GSLLTCGDVE ENPGPRMPPP RLLFFLLFLT PMEVRPEEPL VVKVEEGDNA 600 VLQCLKGTSD GPTQQLTWSR ESPLKPFLKL SLGLPGLGIH MRPLAIWLFI FNVSQQMGGF 660 YLCQPGPPSE KAWQPGWTVN VEGSGELFRW NVSDLGGLGC GLKNRSSEGP SSPSGKLMSP 720 KLYVWAKDRP EIWEGEPPCV PPRDSLNQSL SQDLTMAPGS TLWLSCGVPP DSVSRGPLSW 780 THVHPKGPKS LLSLELKDDR PARDMWVMET GLLLPRATAQ DAGKYYCHRG NLTMSFHLEI 840 TARPVLWHWL LRTGGWKVSA VTLAYLIFCL CSLVGILHLQ RALVLRRKR 889 3-2 Sequences 3-2-1 Sequence Number [ID] 2 3-2-2 Molecule Type AA 3-2-3 Length 22 3-2-4 Features source 1..22 Location/Qualifiers mol_type=protein organism=synthetic construct NonEnglishQualifier Value 3-2-5 Residues MLLLVTSLLL CELPHPAFLL IP 22 3-3 Sequences 3-3-1 Sequence Number [ID] 3 3-3-2 Molecule Type AA 3-3-3 Length 112 3-3-4 Features source 1..112 Location/Qualifiers mol_type=protein organism=synthetic construct NonEnglishQualifier Value 3-3-5 Residues GPVPPSTALR YLIEELVNIT QNQKAPLCNG SMVWSINLTA GMYCAALESL INVSGCSAIE 60 KTQRMLSGFC PHKVSAGQFS SLHVRDTKIE VAQFVKDLLL HLKKLFREGR FN 112 3-4 Sequences 3-4-1 Sequence Number [ID] 4 3-4-2 Molecule Type AA 3-4-3 Length 229 3-4-4 Features source 1..229 Location/Qualifiers mol_type=protein organism=synthetic construct NonEnglishQualifier Value 3-4-5 Residues ESKYGPPCPP CPAPEFEGGP SVFLFPPKPK DTLMISRTPE VTCVVVDVSQ EDPEVQFNWY 60 VDGVEVHNAK TKPREEQFQS TYRVVSVLTV LHQDWLNGKE YKCKVSNKGL PSSIEKTISK 120 AKGQPREPQV YTLPPSQEEM TKNQVSLTCL VKGFYPSDIA VEWESNGQPE NNYKTTPPVL 180 DSDGSFFLYS RLTVDKSRWQ EGNVFSCSVM HEALHNHYTQ KSLSLSLGK 229 3-5 Sequences 3-5-1 Sequence Number [ID] 5 3-5-2 Molecule Type AA 3-5-3 Length 22 3-5-4 Features source 1..22 Location/Qualifiers mol_type=protein organism=synthetic construct NonEnglishQualifier Value 3-5-5 Residues MALIVLGGVA GLLLFIGLGI FF 22 3-6 Sequences 3-6-1 Sequence Number [ID] 6 3-6-2 Molecule Type AA 3-6-3 Length 42 3-6-4 Features source 1..42 Location/Qualifiers mol_type=protein organism=synthetic construct

NonEnglishQualifier Value 3-6-5 Residues KRGRKKLLYI FKQPFMRPVQ TTQEEDGCSC RFPEEEEGGC EL 42 3-7 Sequences 3-7-1 Sequence Number [ID] 7 May 2023

3-7-2 Molecule Type AA 3-7-3 Length 112 3-7-4 Features source 1..112 Location/Qualifiers mol_type=protein organism=synthetic construct NonEnglishQualifier Value 3-7-5 Residues RVKFSRSADA PAYQQGQNQL YNELNLGRRE EYDVLDKRRG RDPEMGGKPR RKNPQEGLYN 60 ELQKDKMAEA YSEIGMKGER RRGKGHDGLY QGLSTATKDT YDALHMQALP PR 112 3-8 Sequences 3-8-1 Sequence Number [ID] 8 3-8-2 Molecule Type AA 3-8-3 Length 24 2023202826

3-8-4 Features source 1..24 Location/Qualifiers mol_type=protein organism=synthetic construct NonEnglishQualifier Value 3-8-5 Residues LEGGGEGRGS LLTCGDVEEN PGPR 24 3-9 Sequences 3-9-1 Sequence Number [ID] 9 3-9-2 Molecule Type AA 3-9-3 Length 323 3-9-4 Features source 1..323 Location/Qualifiers mol_type=protein organism=synthetic construct NonEnglishQualifier Value 3-9-5 Residues MPPPRLLFFL LFLTPMEVRP EEPLVVKVEE GDNAVLQCLK GTSDGPTQQL TWSRESPLKP 60 FLKLSLGLPG LGIHMRPLAI WLFIFNVSQQ MGGFYLCQPG PPSEKAWQPG WTVNVEGSGE 120 LFRWNVSDLG GLGCGLKNRS SEGPSSPSGK LMSPKLYVWA KDRPEIWEGE PPCVPPRDSL 180 NQSLSQDLTM APGSTLWLSC GVPPDSVSRG PLSWTHVHPK GPKSLLSLEL KDDRPARDMW 240 VMETGLLLPR ATAQDAGKYY CHRGNLTMSF HLEITARPVL WHWLLRTGGW KVSAVTLAYL 300 IFCLCSLVGI LHLQRALVLR RKR 323 3-10 Sequences 3-10-1 Sequence Number [ID] 10 3-10-2 Molecule Type AA 3-10-3 Length 867 3-10-4 Features source 1..867 Location/Qualifiers mol_type=protein organism=synthetic construct NonEnglishQualifier Value 3-10-5 Residues GPVPPSTALR YLIEELVNIT QNQKAPLCNG SMVWSINLTA GMYCAALESL INVSGCSAIE 60 KTQRMLSGFC PHKVSAGQFS SLHVRDTKIE VAQFVKDLLL HLKKLFREGR FNESKYGPPC 120 PPCPAPEFEG GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SQEDPEVQFN WYVDGVEVHN 180 AKTKPREEQF QSTYRVVSVL TVLHQDWLNG KEYKCKVSNK GLPSSIEKTI SKAKGQPREP 240 QVYTLPPSQE EMTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFL 300 YSRLTVDKSR WQEGNVFSCS VMHEALHNHY TQKSLSLSLG KMALIVLGGV AGLLLFIGLG 360 IFFKRGRKKL LYIFKQPFMR PVQTTQEEDG CSCRFPEEEE GGCELGGGRV KFSRSADAPA 420 YQQGQNQLYN ELNLGRREEY DVLDKRRGRD PEMGGKPRRK NPQEGLYNEL QKDKMAEAYS 480 EIGMKGERRR GKGHDGLYQG LSTATKDTYD ALHMQALPPR LEGGGEGRGS LLTCGDVEEN 540 PGPRMPPPRL LFFLLFLTPM EVRPEEPLVV KVEEGDNAVL QCLKGTSDGP TQQLTWSRES 600 PLKPFLKLSL GLPGLGIHMR PLAIWLFIFN VSQQMGGFYL CQPGPPSEKA WQPGWTVNVE 660 GSGELFRWNV SDLGGLGCGL KNRSSEGPSS PSGKLMSPKL YVWAKDRPEI WEGEPPCVPP 720 RDSLNQSLSQ DLTMAPGSTL WLSCGVPPDS VSRGPLSWTH VHPKGPKSLL SLELKDDRPA 780 RDMWVMETGL LLPRATAQDA GKYYCHRGNL TMSFHLEITA RPVLWHWLLR TGGWKVSAVT 840 LAYLIFCLCS LVGILHLQRA LVLRRKR 867 3-11 Sequences 3-11-1 Sequence Number [ID] 11 3-11-2 Molecule Type AA 3-11-3 Length 405 3-11-4 Features source 1..405 Location/Qualifiers mol_type=protein organism=synthetic construct NonEnglishQualifier Value 3-11-5 Residues GPVPPSTALR YLIEELVNIT QNQKAPLCNG SMVWSINLTA GMYCAALESL INVSGCSAIE 60 KTQRMLSGFC PHKVSAGQFS SLHVRDTKIE VAQFVKDLLL HLKKLFREGR FNESKYGPPC 120 PPCPAPEFEG GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SQEDPEVQFN WYVDGVEVHN 180 AKTKPREEQF QSTYRVVSVL TVLHQDWLNG KEYKCKVSNK GLPSSIEKTI SKAKGQPREP 240 QVYTLPPSQE EMTKNQVSLT CLVKGFYPSD IAVEWESNGQ PENNYKTTPP VLDSDGSFFL 300

YSRLTVDKSR WQEGNVFSCS VMHEALHNHY TQKSLSLSLG KMALIVLGGV AGLLLFIGLG 360 IFFKRGRKKL LYIFKQPFMR PVQTTQEEDG CSCRFPEEEE GGCEL 405 3-12 Sequences 3-12-1 Sequence Number [ID] 12 3-12-2 Molecule Type DNA May 2023

3-12-3 Length 7754 3-12-4 Features source 1..7754 Location/Qualifiers mol_type=other DNA organism=synthetic construct NonEnglishQualifier Value 3-12-5 Residues gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc 60 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg 120 taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg 180 aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt 240 gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg 300 actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga 360 attagatcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat ataaattaaa 420 2023202826

acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg gcctgttaga 480 aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc agacaggatc 540 agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc atcaaaggat 600 agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa acaaaagtaa 660 gaaaaaagca cagcaagcag cagctgacac aggacacagc aatcaggtca gccaaaatta 720 ccctatagtg cagaacatcc aggggcaaat ggtacatcag gccatatcac ctagaacttt 780 aaatgcatgg gtaaaagtag tagaagagaa ggctttcagc ccagaagtga tacccatgtt 840 ttcagcatta tcagaaggag ccaccccaca agatttaaac accatgctaa acacagtggg 900 gggacatcaa gcagccatgc aaatgttaaa agagaccatc aatgaggaag ctgcaggcaa 960 agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg ttccttgggt 1020 tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg gtacaggcca 1080 gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct attgaggcgc 1140 aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca agaatcctgg 1200 ctgtggaaag atacctaaag gatcaacagc tcctggggat ttggggttgc tctggaaaac 1260 tcatttgcac cactgctgtg ccttggatct acaaatggca gtattcatcc acaattttaa 1320 aagaaaaggg gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac 1380 agacatacaa actaaagaat tacaaaaaca aattacaaaa attcaaaatt ttcgggttta 1440 ttacagggac agcagagatc cagtttgggg atcaattgca tgaagaatct gcttagggtt 1500 aggcgttttg cgctgcttcg cgaggatctg cgatcgctcc ggtgcccgtc agtgggcaga 1560 gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt gaaccggtgc 1620 ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt 1680 tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg 1740 caacgggttt gccgccagaa cacagctgaa gcttcgaggg gctcgcatct ctccttcacg 1800 cgcccgccgc cctacctgag gccgccatcc acgccggttg agtcgcgttc tgccgcctcc 1860 cgcctgtggt gcctcctgaa ctgcgtccgc cgtctaggta agtttaaagc tcaggtcgag 1920 accgggcctt tgtccggcgc tcccttggag cctacctaga ctcagccggc tctccacgct 1980 ttgcctgacc ctgcttgctc aactctacgt ctttgtttcg ttttctgttc tgcgccgtta 2040 cagatccaag ctgtgaccgg cgcctacggc tagcgccgcc accatgctgc tgctggtgac 2100 cagcctgctg ctgtgcgagc tgccccaccc cgcctttctg ctgatccctg gccccgtgcc 2160 ccctagcacc gccctgcgct acctgatcga ggaactggtg aacatcaccc agaaccagaa 2220 agcccccctg tgcaacggca gcatggtgtg gagcatcaac ctgaccgccg gcatgtactg 2280 tgccgccctg gaaagcctga tcaacgtgag cggctgcagc gccatcgaga aaacccagcg 2340 gatgctgtcc ggcttctgcc cccacaaggt gtccgccgga cagttcagca gcctgcacgt 2400 gcgggacacc aagatcgagg tggcccagtt cgtgaaggac ctgctgctgc acctgaagaa 2460 gctgttccgg gagggccggt tcaactacaa gaccaccccc cctgtgctgg acagcgacgg 2520 cagcttcttc ctgtacagca ggctgaccgt ggacaagagc cggtggcagg aaggcaacgt 2580 ctttagctgc agcgtgatgc acgaggccct gcacaaccac tacacccaga agagcctgtc 2640 cctgagcctg ggcaagcggg tgaagttcag ccggtccgcc gacgcccctg cctaccagca 2700 gggccagaac cagctgtaca acgagctgaa cctgggcagg cgggaggaat acgacgtgct 2760 ggacaagcgg agaggccggg accctgagat gggcggcaag cctcggcgga agaaccccca 2820 ggaaggcctg tataacgaac tgcagaaaga caagatggcc gaggcctaca gcgagatcgg 2880 catgaagggc gagcggaggc ggggcaaggg ccacgacggc ctgtatcagg gcctgtccac 2940 cgccaccaag gatacctacg acgccctgca catgcaggcc ctgcccccaa ggtctagacc 3000 cgggctgcag gaattcgata tcaagcttat cgataatcaa cctctggatt acaaaatttg 3060 tgaaagattg actggtattc ttaactatgt tgctcctttt acgctatgtg gatacgctgc 3120 tttaatgcct ttgtatcatg ctattgcttc ccgtatggct ttcattttct cctccttgta 3180 taaatcctgg ttgctgtctc tttatgagga gttgtggccc gttgtcaggc aacgtggcgt 3240 ggtgtgcact gtgtttgctg acgcaacccc cactggttgg ggcattgcca ccacctgtca 3300 gctcctttcc gggactttcg ctttccccct ccctattgcc acggcggaac tcatcgccgc 3360 ctgccttgcc cgctgctgga caggggctcg gctgttgggc actgacaatt ccgtggtgtt 3420 gtcggggaaa tcatcgtcct ttccttggct gctcgcctgt gttgccacct ggattctgcg 3480 cgggacgtcc ttctgctacg tcccttcggc cctcaatcca gcggaccttc cttcccgcgg 3540 cctgctgccg gctctgcggc ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat 3600 ctccctttgg gccgcctccc cgcatcgata ccgtcgacta gccgtacctt taagaccaat 3660 gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg 3720 gctaattcac tcccaaagaa gacaagatct gctttttgcc tgtactgggt ctctctggtt 3780 agaccagatc tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca 3840 ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa 3900 ctagagatcc ctcagaccct tttagtcagt gtggaaaatc tctagcagaa ttcgatatca 3960 agcttatcga taccgtcgac ctcgaggggg ggcccggtac ccaattcgcc ctatagtgag 4020 tcgtattaca attcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt 4080 acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag 4140 gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg gaaattgtaa 4200 May 2023 gcgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc 4260 aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag atagggttga 4320 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag 4380 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc taatcaagtt 4440 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta 4500 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag 4560 cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg 4620 cgcttaatgc gccgctacag ggcgcgtcag gtggcacttt tcggggaaat gtgcgcggaa 4680 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac 4740 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg 4800 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc 4860 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg 4920 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga 4980 2023202826 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc 5040 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag 5100 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga 5160 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg 5220 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga 5280 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt 5340 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact 5400 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt 5460 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg 5520 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta 5580 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac 5640 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta 5700 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt 5760 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt 5820 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt 5880 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc 5940 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg 6000 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg 6060 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt 6120 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac 6180 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg 6240 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg 6300 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat 6360 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt 6420 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg 6480 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa 6540 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc 6600 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga 6660 aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg 6720 ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc 6780 acacaggaaa cagctatgac catgattacg ccaagctcga aattaaccct cactaaaggg 6840 aacaaaagct ggagctccac cgcggtggcg gcctcgaggt cgagatccgg tcgaccagca 6900 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat 6960 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc 7020 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag 7080 cttcgacggt atcgattggc tcatgtccaa cattaccgcc atgttgacat tgattattga 7140 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc 7200 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat 7260 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc 7320 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc 7380 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt 7440 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta 7500 ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg 7560 gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac 7620 gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg 7680 tacggaattc ggagtggcga gccctcagat cctgcatata agcagctgct ttttgcctgt 7740 actgggtctc tctg 7754 3-13 Sequences 3-13-1 Sequence Number [ID] 13 3-13-2 Molecule Type DNA 3-13-3 Length 8732 3-13-4 Features source 1..8732 Location/Qualifiers mol_type=other DNA organism=synthetic construct NonEnglishQualifier Value 3-13-5 Residues gttagaccag atctgagcct gggagctctc tggctaacta gggaacccac tgcttaagcc 60 tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg 120 taactagaga tccctcagac ccttttagtc agtgtggaaa atctctagca gtggcgcccg 180 aacagggact tgaaagcgaa agggaaacca gaggagctct ctcgacgcag gactcggctt 240 gctgaagcgc gcacggcaag aggcgagggg cggcgactgg tgagtacgcc aaaaattttg 300 actagcggag gctagaagga gagagatggg tgcgagagcg tcagtattaa gcgggggaga 360 attagatcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat ataaattaaa 420 May 2023 acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg gcctgttaga 480 aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc agacaggatc 540 agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc atcaaaggat 600 agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa acaaaagtaa 660 gaaaaaagca cagcaagcag cagctgacac aggacacagc aatcaggtca gccaaaatta 720 ccctatagtg cagaacatcc aggggcaaat ggtacatcag gccatatcac ctagaacttt 780 aaatgcatgg gtaaaagtag tagaagagaa ggctttcagc ccagaagtga tacccatgtt 840 ttcagcatta tcagaaggag ccaccccaca agatttaaac accatgctaa acacagtggg 900 gggacatcaa gcagccatgc aaatgttaaa agagaccatc aatgaggaag ctgcaggcaa 960 agagaagagt ggtgcagaga gaaaaaagag cagtgggaat aggagctttg ttccttgggt 1020 tcttgggagc agcaggaagc actatgggcg cagcgtcaat gacgctgacg gtacaggcca 1080 gacaattatt gtctggtata gtgcagcagc agaacaattt gctgagggct attgaggcgc 1140 aacagcatct gttgcaactc acagtctggg gcatcaagca gctccaggca agaatcctgg 1200 2023202826 ctgtggaaag atacctaaag gatcaacagc tcctggggat ttggggttgc tctggaaaac 1260 tcatttgcac cactgctgtg ccttggatct acaaatggca gtattcatcc acaattttaa 1320 aagaaaaggg gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac 1380 agacatacaa actaaagaat tacaaaaaca aattacaaaa attcaaaatt ttcgggttta 1440 ttacagggac agcagagatc cagtttgggg atcaattgca tgaagaatct gcttagggtt 1500 aggcgttttg cgctgcttcg cgaggatctg cgatcgctcc ggtgcccgtc agtgggcaga 1560 gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt gaaccggtgc 1620 ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt 1680 tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg 1740 caacgggttt gccgccagaa cacagctgaa gcttcgaggg gctcgcatct ctccttcacg 1800 cgcccgccgc cctacctgag gccgccatcc acgccggttg agtcgcgttc tgccgcctcc 1860 cgcctgtggt gcctcctgaa ctgcgtccgc cgtctaggta agtttaaagc tcaggtcgag 1920 accgggcctt tgtccggcgc tcccttggag cctacctaga ctcagccggc tctccacgct 1980 ttgcctgacc ctgcttgctc aactctacgt ctttgtttcg ttttctgttc tgcgccgtta 2040 cagatccaag ctgtgaccgg cgcctacggc tagcgccgcc accatgctgc tgctggtgac 2100 cagcctgctg ctgtgcgagc tgccccaccc cgcctttctg ctgatccccg acatccagat 2160 gacccagacc acctccagcc tgagcgccag cctgggcgac cgggtgacca tcagctgccg 2220 ggccagccag gacatcagca agtacctgaa ctggtatcag cagaagcccg acggcaccgt 2280 caagctgctg atctaccaca ccagccggct gcacagcggc gtgcccagcc ggtttagcgg 2340 cagcggctcc ggcaccgact acagcctgac catctccaac ctggaacagg aagatatcgc 2400 cacctacttt tgccagcagg gcaacacact gccctacacc tttggcggcg gaacaaagct 2460 ggaaatcacc ggcagcacct ccggcagcgg caagcctggc agcggcgagg gcagcaccaa 2520 gggcgaggtg aagctgcagg aaagcggccc tggcctggtg gcccccagcc agagcctgag 2580 cgtgacctgc accgtgagcg gcgtgagcct gcccgactac ggcgtgagct ggatccggca 2640 gccccccagg aagggcctgg aatggctggg cgtgatctgg ggcagcgaga ccacctacta 2700 caacagcgcc ctgaagagcc ggctgaccat catcaaggac aacagcaaga gccaggtgtt 2760 cctgaagatg aacagcctgc agaccgacga caccgccatc tactactgcg ccaagcacta 2820 ctactacggc ggcagctacg ccatggacta ctggggccag ggcaccagcg tgaccgtgag 2880 cagcgagagc aagtacggcc ctccctgccc cccttgccct gcccccgagt tcctgggcgg 2940 acccagcgtg ttcctgttcc cccccaagcc caaggacacc ctgatgatca gccggacccc 3000 cgaggtgacc tgcgtggtgg tggacgtgag ccaggaagat cccgaggtcc agttcaattg 3060 gtacgtggac ggcgtggagg tgcacaacgc caagaccaag cccagggaag agcagttcaa 3120 cagcacctac cgggtggtgt ccgtgctgac cgtgctgcac caggactggc tgaacggcaa 3180 agaatacaag tgcaaggtgt ccaacaaggg cctgcccagc agcatcgaga aaaccatcag 3240 caaggccaag ggccagcctc gggagcccca ggtgtacacc ctgccccctt cccaggaaga 3300 gatgaccaag aatcaggtgt ccctgacctg cctggtgaag ggcttctacc ccagcgacat 3360 cgccgtggag tgggagagca acggccagcc cgagaacaac tacaagacca ccccccctgt 3420 gctggacagc gacggcagct tcttcctgta cagcaggctg accgtggaca agagccggtg 3480 gcaggaaggc aacgtcttta gctgcagcgt gatgcacgag gccctgcaca accactacac 3540 ccagaagagc ctgtccctga gcctgggcaa gatggccctg atcgtgctgg gcggcgtggc 3600 cgggctgctg ctgttcatcg gcctgggcat ctttttccgg gtgaagttca gccggtccgc 3660 cgacgcccct gcctaccagc agggccagaa ccagctgtac aacgagctga acctgggcag 3720 gcgggaggaa tacgacgtgc tggacaagcg gagaggccgg gaccctgaga tgggcggcaa 3780 gcccaggcgg aagaaccctc aggaaggcct gtataacgaa ctgcagaaag acaagatggc 3840 cgaggcctac agcgagatcg gcatgaaggg cgagcggcgg aggggcaagg gccacgacgg 3900 cctgtaccag ggcctgagca ccgccaccaa ggatacctac gacgccctgc acatgcaggc 3960 cctgcccccc aggtgacccg ggctgcagga attcgatatc aagcttatcg ataatcaacc 4020 tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac 4080 gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt 4140 cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt 4200 tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg 4260 cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac 4320 ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac 4380 tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt 4440 tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc 4500 ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg 4560 ccctcagacg agtcggatct ccctttgggc cgcctccccg catcgatacc gtcgactagc 4620 cgtaccttta agaccaatga cttacaaggc agctgtagat cttagccact ttttaaaaga 4680 aaagggggga ctggaagggc taattcactc ccaaagaaga caagatctgc tttttgcctg 4740 tactgggtct ctctggttag accagatctg agcctgggag ctctctggct aactagggaa 4800 cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct 4860 gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc 4920 tagcagaatt cgatatcaag cttatcgata ccgtcgacct cgaggggggg cccggtaccc 4980 May 2023 aattcgccct atagtgagtc gtattacaat tcactggccg tcgttttaca acgtcgtgac 5040 tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc 5100 tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat 5160 ggcgaatgga aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa 5220 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat 5280 agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg 5340 tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac 5400 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta 5460 aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag 5520 ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg 5580 taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcaggt ggcacttttc 5640 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc 5700 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga 5760 2023202826 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt 5820 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag 5880 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag 5940 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta 6000 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg 6060 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca 6120 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag 6180 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc 6240 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg 6300 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc 6360 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg 6420 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg 6480 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga 6540 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac 6600 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa 6660 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca 6720 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag 6780 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac 6840 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa 6900 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc 6960 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag 7020 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac 7080 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc 7140 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc 7200 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca 7260 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc 7320 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg 7380 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct 7440 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata 7500 ccgctcgccg cagccgaacg accgagcgca gcgagtcagt gagcgaggaa gcggaagagc 7560 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg 7620 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca 7680 ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg 7740 tgagcggata acaatttcac acaggaaaca gctatgacca tgattacgcc aagctcgaaa 7800 ttaaccctca ctaaagggaa caaaagctgg agctccaccg cggtggcggc ctcgaggtcg 7860 agatccggtc gaccagcaac catagtcccg cccctaactc cgcccatccc gcccctaact 7920 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag 7980 gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc 8040 ctaggctttt gcaaaaagct tcgacggtat cgattggctc atgtccaaca ttaccgccat 8100 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata 8160 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc 8220 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag 8280 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac 8340 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg 8400 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 8460 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat 8520 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt 8580 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc 8640 aaatgggcgg taggcgtgta cggaattcgg agtggcgagc cctcagatcc tgcatataag 8700 cagctgcttt ttgcctgtac tgggtctctc tg 8732

Claims (20)

WHAT IS CLAIMED IS:

1. The population of human T cells of transduced with a vector expressing a chimeric antigen receptor wherein at least 50% of the transduced human T cells are central memory T cells.

2. The population of human T cells of claim 1 wherein at least 10% of the transduced cells central memory T cells are CD4+.

3. The population of human T cells of claim 1 wherein at least 10% of the transduced central memory T cells are CD8+.

4. The population of human T cells of claim 1 wherein at least15%of the central memory T cells are CD4+ and at least 15% are CD8+.

5. The population of human T cells of claim 1 wherein at least 50% of the transduced human T cells are CD4+/CD8+/CD62L+.

6. A population of human T cells of transduced with a vector expressing a chimeric antigen receptor wherein: at least 50% of the transduced human T cells are CD45R+, CD62L+ and CD45Ra-, at least 10% of the cells are CD4+ and at least 10% of the cells are CD4+.

7. The population of human T cells of claim 6 wherein at least 10% of the cells that are CD45R+, CD62L+ and CD45Ra- are also CD4+ and at least 10% of the cells that are CD45RO+, CD62L+ and CD45Ra- are also CD8+.

8. The population of human T cells of claim 6 wherein at least 15% of the cells that are CD45RO+, CD62L+ and CD45Ra- are also CD4+ and at least 15% of the cells that are CD45RO+, CD62L+ and CD45Ra- are also CD4+.

9. A method of treating cancer comprising administering to a patient in need thereof a pharmaceutical composition comprising the human T cells of any of claims 1-8.

10. The method of claim 9 wherein the population of human T cells are autologous to the patient.

11. The method of claim 9 wherein the population of human T cells are allogenic to the patient.

12. A method for preparing a population central memory T cells comprising: obtaining a population of T cells from a human subject; depleting the population of T cells for cells that express CD25, cells that express CD14, and cells that express CD45+ to prepared a depleted T cell population; enriching the depleted T cell population for cells expressing CD62L, thereby preparing a population of central memory T cells, wherein the method does not comprising a step of depleting a cell population for cells expressing CD4 and does not comprising a step of depleting a cell population of cells expressing CD8+.

13. The method of claim 12 wherein at least 50% of the cells in the population central memory T cells are CD45RO+, CD62L+ and CD45Ra-, at least 10% of the cells are CD4+ and at least 10% of the cells are CD4+.

14. The method of claim 12 wherein at least 50% of the cells in the population central memory T cells are CD45RO+, CD62L+ and CD45Ra-, at least 15% of the cells are CD4+ and at least 15% of the cells are CD4+.

15. The method of claim 12 wherein at least 50% of the cells in the population central memory T cells are CD45RO+, CD62L+ and CD45Ra-, at least 20% of the cells are CD4+ and at least 20% of the cells are CD4+.

16. The method of claim 12 further comprising stimulating the population of central memory T cells

17. The method of claim 16 comprising contacting the population of central memory T cells with CD3 and/or CD28.

18. The method of claim 16 further comprising transducing the cells with a vector expressing a recombinant protein to create a population of genetically modified central memory T cells.

19. The method of claim 19 further comprising expanding the population of generically modified central memory T cells.

20. The method of claim 19 wherein the step of expanding the population of generically modified central memory T cells comprising exposing the cells to one or both of IL-2 and IL-15.