Allogeneic Cell Therapy Of B Cell Malignancies Using Genetically Engineered T Cells Targeting Cd19

Abstract

A population of genetically engineered immune cells (e.g., T cells), which express a chimeric antigen receptor (CAR) specific to CD19 and contain a disrupted TRAC gene, a disrupted B2M gene, or both, for use in treating a B cell malignancy.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of the filing dates of U.S. Provisional Application No. 62/840,913, filed Apr. 30, 2019, the entire contents of which are incorporated by reference herein.

BACKGROUND OF THE INVENTION

[0002] Chimeric antigen receptor (CAR) T cell therapies are adoptive T cell therapeutics used to treat human malignancies. Although CAR T cell therapy has led to tremendous clinical success, including durable remission in relapsed/refractory non-Hodgkin lymphoma (NHL) and pediatric acute lymphoblastic leukemia (ALL), the approved products are autologous and require patient-specific cell collection and manufacturing. Because of this, some patients have experienced disease progression or death while awaiting treatment. Accordingly, there remains a need for improved CAR T cell therapeutics.

SUMMARY OF THE INVENTION

[0003] The present disclosure is based, at least in part, on the development of allogeneic cell therapy for B cell malignancies such as transformed FL or DLBCL using genetically engineered T cells (e.g., CTX110 cells, a.k.a., TC1 cells) expressing an anti-CD19 chimeric antigen receptor (CAR) and having disrupted TRAC gene and B2M gene. The allogeneic CAR-T cell therapy disclosed herein showed treatment efficacies in human patients having B cell malignancies disclosed herein, including complete responses in certain patients and long durability of responses. Further, the allogeneic CAR-T cell therapy disclosed herein exhibited desired pharmacokinetic features in the human patients, including prolonged CAR-T cell expansion and persistence after infusion.

[0004] Accordingly, some aspects of the present disclosure provides a method for treating a B-cell malignancy in a human patient, the method comprising: (i) subjecting a human patient having a B-cell malignancy to a lymphodepletion treatment; and (ii) administering to the human patient a population of genetically engineered T cells after step (i). In some embodiments, step (i) can be performed about 2-7 days prior to step (ii). In some embodiments, the population of genetically engineered T cells is allogeneic.

[0005] The population of the genetically engineered T cells may comprise T cells that comprise: (a) a disrupted T cell receptor alpha constant (TRAC) gene, (b) a nucleic acid coding for a chimeric antigen receptor (CAR) that binds CD19, wherein the CAR comprises an anti-CD19 single chain variable fragment (scFv) that comprises a heavy chain variable region set forth in SEQ ID NO: 51, and a light chain variable region set forth in SEQ ID NO: 52, and wherein the nucleic acid is inserted in the disrupted TRAC gene, and (c) a disrupted beta 2-microglobulin (.beta.2M) gene. In some embodiments, the disrupted TRAC gene comprises a deletion of a fragment comprising the nucleotide sequence of SEQ ID NO: 26.

[0006] In some embodiments, the population of genetically engineered T cells is administered to the human patient at a dose of about 1.times.10.sup.7 to about 1.times.10.sup.9 CAR.sup.+ T cells. In some examples, the population of genetically engineered T cells is administered to the human patient at a dose of about 1.times.10.sup.7 CAR.sup.+ T cells. In some examples, the population of genetically engineered T cells is administered to the human patient at a dose of about 3.times.10.sup.7 CAR T cells. In some examples, the population of genetically engineered T cells is administered to the human patient at a dose of about 1.times.10.sup.8 CAR.sup.+ T cells. In some examples, the population of genetically engineered T cells is administered to the human patient at a dose of about 3.times.10.sup.8 CAR.sup.+ T cells. In some examples, the population of genetically engineered T cells is administered to the human patient at a dose of about 1.times.10.sup.9 CAR.sup.+ T cells. In any event, the population of genetically engineered T cells administered to the human patient per dose contains no more than 7.times.10.sup.4 TCR.sup.+ T cells/kg.

[0007] In some embodiments, the lymphodepletion treatment in step (i) comprises co-administration to the human patient fludarabine at about 30 mg/m.sup.2 and cyclophosphamide at about 500-750 mg/m.sup.2 per day for three days. For example, the lymphodepletion treatment in step (i) comprises co-administration to the human patient fludarabine at about 30 mg/m.sup.2 and cyclophosphamide at about 500 mg/m.sup.2 per day for three days. In other examples, the lymphodepletion treatment in step (i) comprises co-administration to the human patient fludarabine at about 30 mg/m.sup.2 and cyclophosphamide at about 750 mg/m.sup.2 per day for three days.

[0008] In some embodiments, prior to step (i), the human patient does not show one or more of the following features: (a) significant worsening of clinical status, (b) requirement for supplemental oxygen to maintain a saturation level of greater than 91%, (c) uncontrolled cardiac arrhythmia, (d) hypotension requiring vasopressor support, (e) active infection, and (f) grade .gtoreq.2 acute neurological toxicity.

[0009] In some embodiments, after step (i) and prior to step (ii), the human patient does not show one or more of the following features: (a) active uncontrolled infection; (b) worsening of clinical status compared to the clinical status prior to step (i); and (c) grade .gtoreq.2 acute neurological toxicity.

[0010] Any of the methods disclosed herein may further comprise (iii) monitoring the human patient for development of acute toxicity after step (ii); and (iv) managing the acute toxicity if occurs. In some embodiments, step (iii) can be performed for at least 28 days after administration of the population of genetically engineered T cells. Exemplary acute toxicity may comprise tumor lysis syndrome (TLS), cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), B cell aplasia, hemophagocytic lymphohistiocytosis (HLH), cytopenia, graft-versus-host disease (GvHD), hypertension, renal insufficiency, or a combination thereof.

[0011] In some embodiments, the B cell malignancy is non-Hodgkin lymphoma. Examples include, but are not limited to, diffuse large B cell lymphoma (DLBCL), high grade B cell lymphoma with MYC and BCL2 and/or BCL6 rearrangement, transformed follicular lymphoma (FL), or grade 3b FL. In some instances, DLBCL is DLBCL not otherwise specified (NOS). In some examples, the B cell malignancy is refractory and/or relapsed.

[0012] In some embodiments, the human patient may have at least one measurable lesion that is fluorodeoxyglucose positron emission tomography (PET)-positive. In some embodiments, the human patient has undergone one or more lines of prior anti-cancer therapies. In some examples, the human patient has undergone two or more lines of prior anti-cancer therapies. Exemplary prior anti-cancer therapies may comprise an anti-CD20 antibody, an anthracycline-containing regimen, or a combination thereof.

[0013] In some examples, the human patient has refractory or relapsed transformed FL and has undergone at least one line of chemotherapy for disease after transformation to DLBCL. In other examples, the B cell malignancy is refractory, and the human patient has progressive disease on last therapy, or has stable disease following at least two cycles of therapy with duration of stable disease of up to 6 months. In yet other examples, the human patient has failed prior autologous hematopoietic stem cell transplantation (HSCT) or ineligible for prior autologous HSCT. Alternatively or in addition, the human patient is subject to an additional anti-cancer therapy after treatment with the population of genetically engineered T cells.

[0014] In any of the methods disclosed herein, the human patient has one or more of the following features:

[0015] (a) has an Eastern Cooperative Oncology Group (ECOG) performance status 0 or 1;

[0016] (b) adequate renal, liver, cardiac, and/or pulmonary function;

[0017] (c) free of prior gene therapy or modified cell therapy;

[0018] (d) free of prior treatment comprising an anti-CD19 antibody;

[0019] (e) free of prior allogeneic HSCT;

[0020] (f) free of detectable malignant cells from cerebrospinal fluid;

[0021] (g) free of brain metastases;

[0022] (h) free of prior central nervous system disorders;

[0023] (i) free of unstable angina, arrhythmia, and/or myocardial infarction;

[0024] (j) free of uncontrolled infection;

[0025] (k) free of immunodeficiency disorders or autoimmune disorders that require immunosuppressive therapy; and

[0026] (l) free of infection by human immunodeficiency virus, hepatitis B virus, or hepatitis C virus.

[0027] In any of the methods disclosed herein, the anti-CD19 CAR expressed by the genetically engineered T cells may comprise an extracellular antigen binding domain, which is an anti-CD19 scFv comprising the amino acid sequence of SEQ ID NO: 47. In some embodiments, the anti-CD19 CAR may comprise the amino acid sequence of SEQ ID NO: 40.

[0028] In some embodiments, the nucleic acid encoding the anti-CD19 CAR is inserted at the site of deletion in the disrupted TRAC gene. In some examples, the disrupted TRAC gene comprises the nucleotide sequence of SEQ ID NO: 54. Alternatively or in addition, the disrupted .beta.2M gene in the population of genetically engineered T cells comprises at least one of the nucleotide sequence set forth in SEQ ID NOs: 9-14.

[0029] In some embodiments, at least 90% of the T cells in the population of genetically engineered T cells do not express a detectable level of TCR surface protein. For example, at least 70% of the T cells in the population of genetically engineered T cells do not express a detectable level of TCR surface protein; at least 50% of the T cells in the population of genetically engineered T cells do not express a detectable level of B2M surface protein; and/or at least 30% of the T cells in the population of genetically engineered T cells express a detectable level of the CAR. In some examples, at least 99.5% of the T cells in the population of genetically engineered T cells do not express a detectable level of TCR surface protein. In some examples, at least 70% of the T cells in the population of genetically engineered T cells do not express a detectable level of B2M surface protein. In specific examples, at least 85% of the T cells in the population of the genetically engineered T cells do not express a detectable level of B2M surface protein. In some examples, at least 50% of the T cells in the population of genetically engineered T cells express a detectable level of the CAR. In specific examples, at least 70% of the T cells in the population of genetically engineered T cells express a detectable level of the CAR.

[0030] In a specific example, the population of genetically engineered T cells for use in any of the methods disclosed herein are CTX110 cells.

[0031] In any of the methods disclosed herein, the population of genetically engineered T cells are administered to the human patient via intravenous infusion. In some examples, the population of genetically engineered T cells may be suspended in a cryopreservation solution.

[0032] Also within the scope of the present disclosure are pharmaceutical compositions for use in treating a B-cell malignancy, the pharmaceutical composition comprising any of the population of genetically engineered T cells disclosed herein (e.g., the CTX110 cells), as well as use of the genetically engineered T cells for manufacturing a medicament for use in treating a B-cell malignancy as disclosed herein.

[0033] The details of one or more embodiments of the invention are set forth in the description below. Other features or advantages of the present invention will be apparent from the following drawings and detailed description of several embodiments, and also from the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0034] FIG. 1 is a series of flow cytometry plots of human primary T-cells, TRAC-/B2M-CD19CAR+T cells (TC1), 8 days post-editing. The graphs show reduced surface expression of TRAC and B2M. TCR/MHC I double knockout cells express high levels of the CAR transgene (bottom panel). Negative selection of TC1 cells with purification beads leads to a reduction in TCR positive cells (right panel).

[0035] FIG. 2 is a graph depicting high editing rates achieved at the TRAC and B2M loci in TRAC-/B2M-CD19CAR+T cells (TC1). Surface expression of TCR and MHCI, which is the functional output of gene editing, was measured and plotted as editing percentage on the y-axis. High efficiency (e.g., greater than 50%) site-specific integration and expression of the CAR from the TRAC locus were detected. These data demonstrate greater than 50% efficiency for the generation of TRAC-/B2M-/anti-CD19CAR+ T cells.

[0036] FIG. 3 is a graph depicting a statistically significant decrease in tumor volume (mm.sup.3) (p=0.007) in NOG Raji mice following treatment with TRAC-/.beta.2M-/CD19 CAR+ T cells (TC1).

[0037] FIG. 4 is a survival curve graph demonstrating increased survival of NOG Raji mice treated with TC1 cells in comparison to NOG Raji mice receiving no treatment.

[0038] FIG. 5 is a survival curve graph demonstrating increased survival of NOG Raji mice treated with TC1 cells on day 4, in comparison to control mice receiving no treatment on day 1.

[0039] FIGS. 6A and 6B include diagrams showing persistence and anti-tumor activity of TC1 cells in mice. 6A: a series of flow cytometry plots demonstrating that TC1 cells persist in NOG Raji mice. 6B: a graph demonstrating that TC1 cells selectively eradicate splenic Raji cells in NOG Raji mice treated with TC1 in comparison to controls (NOG Raji mice with no treatment or NOG mice). The effect is depicted as a decreased splenic mass in NOG Raji mice treated with TC1 in comparison to controls.

[0040] FIG. 7 is a series of flow cytometry plots demonstrating that persistent splenic TC1 cells are edited in two independent NOG Raji mice with TC1 treatment.

[0041] FIG. 8 is a Kaplan-Meier survival plot demonstrating increased survival of NOG Nalm6 mice treated with TC1 cells on day 4, in comparison to control mice receiving no treatment on day 1.

[0042] FIG. 9 is a Kaplan-Meier survival plot demonstrating an increase survival of mice bearing a disseminated Nalm6 B-cell acute lymphoblastic leukemia (B-ALL) after treatment with different concentrations of TC1, in comparison to control mice receiving no treatment.

[0043] FIG. 10 is a graph depicting a statistically significant inhibition in tumor cell expansion in the disseminated Nalm6 B-cell acute lymphoblastic leukemia (B-ALL) tumor model following treatment with TC1 cells.

[0044] FIG. 11 is a Kaplan-Meier survival plot of healthy mice treated with TC1 cells or various control cells (PBMCs or electroporated (EP) T cells) after radiation, or mice that only received radiation ("RT only").

[0045] FIG. 12 is a graph showing percentage of body weight change of the mice treated in FIG. 18.

[0046] FIG. 13 is a Kaplan-Meier survival plot of healthy mice treated with a low dose (2.times.10.sup.7) or high dose (4.times.10.sup.7) of TC1 cells, or unedited T cells after radiation, or mice that only received radiation ("Vehicle-RT").

[0047] FIG. 14 is a graph showing percentage of body weight change of the mice treated in FIG. 20, in addition to mice that were not irradiated and not dosed with cells ("Vehicle--no RT").

[0048] FIG. 15 is a bar graph showing percentage of CD27+CD45RO- cells within the unedited CD8+ T cell subset of peripheral blood cells from six different donors.

[0049] FIG. 16 provides flow cytometry results of TCR.alpha..beta. and B2M expression on TC1 cells before and after depletion of TCR.alpha..beta.+ cells.

[0050] FIG. 17 is a graph the percentage loss of protein for TCR- and MHC I-(B2M) after gene editing, and percentage of cells expressing an anti-CD19 CAR in edited TC1 cells from individual lots of TC1 production.

[0051] FIG. 18 provides graphs showing the percentage of PD1+(top left), LAG3+(top right), TIM3+(bottom left) or CD57+(bottom right) in the T cell population from six different donors before and after editing.

[0052] FIG. 19 is a graph showing the percentage of cell lysis of CD19-positive cell lines (Nalm6; Raji; and K562-CD19) and CD19-negative cells (K562) when co-cultured at different ratios with TC1 cells or unedited T cells.

[0053] FIG. 20 is a graph showing the number of viable TC1 cells when cultured in the presence of T-cell media (serum+IL2+IL7; Complete Media), media containing serum but no IL2 or IL7 cytokines (5% Serum, No cytokines) or no serum or cytokines (No Serum, No Cytokines). Cells were counted on the indicated days post gene editing. Mean values from three lots shown.+-.SD.

[0054] FIG. 21 is a schematic depicting the clinical study design to evaluate CTX110 cells, (a.k.a., TC1 cells) administered after lymphodepletion to human subjects having CD19+ malignancies.

DETAILED DESCRIPTION OF THE INVENTION

[0055] Cluster of Differentiation 19 (CD19) is an antigenic determinant detectable on leukemia precursor cells. The human and murine amino acid and nucleic acid sequences can be found in a public database, such as GenBank, UniProt and Swiss-Prot. For example, the amino acid sequence of human CD19 can be found as UniProt/Swiss-Prot Accession No. P15391 and the nucleotide sequence encoding of the human CD19 can be found at Accession No. NM_001178098. CD19 is expressed on most B lineage cancers, including, e.g., acute lymphoblastic leukemia, chronic lymphocyte leukemia and non-Hodgkin's lymphoma. It is also an early marker of B cell progenitors. See, e.g., Nicholson et al. Mol. Immun. 34 (16-17): 1157-1165 (1997).

[0056] The present disclosure provides an allogeneic CAR-T cell therapy for B cell malignancies. The CAR-T cell therapy involves a population of genetically engineered T cells expressing an anti-CD19 CAR and having disrupted TRAC gene and B2M gene, the nucleic acid coding for the anti-CD19 CAR being inserted into the TRAC gene locus, thereby disrupting expression of the TRAC gene. The allogenic anti-CD19 CAR-T cells are prepared using parent T cells obtained from healthy donors. As such, the CAR-T therapy is available to a patient having the target B cell malignancy immediately after diagnosis, as opposed to at least three week gap between diagnosis and treatment in autologous CAR-T therapy required for manufacturing the CAR-T cells from the patient's own T cells. The allogeneic CAR T therapy can be stored and inventoried at the site of care to facilitate treatment immediately following diagnosis. The immediate availability of the allogeneic anti-CD19 CAR T therapy eliminates the need for bridging chemo-therapy, which may be required when autologous CAR-T cells are manufactured from the patient's own cells. The allogeneic anti-CD19 CAR-T cell therapy disclosed herein showed treatment efficacies in human patients having B cell malignancies disclosed herein, including complete responses in certain patients and long durability of responses. Further, the allogeneic CAR-T cell therapy disclosed herein exhibited desired pharmacokinetic features in the human patients, including prolonged CAR-T cell expansion and persistence after infusion.

[0057] Accordingly, provided herein are methods for treating a B-cell malignancy in a human patient using a population of genetically engineered immune cells such as T cells, which collectively comprises a disrupted TRAC gene, a disrupted B2M, and a nucleic acid encoding an anti-CD19 CAR (e.g., SEQ ID NO: 40, encoded by SEQ ID NO:39). The nucleic acid encoding the anti-CD19 CAR and optionally comprising a promoter sequence and one or more regulatory elements may be inserted in the disrupted TRAC gene locus, e.g., replacing the segment of SEQ ID NO: 26 in the TRAC gene. The human patient is subject to a lymphodepletion treatment prior to administration of the population of genetically engineered T cells.

I. Anti-CD19 CAR T Cells

[0058] Disclosed herein are anti-CD19 CAR T cells (e.g., CTX110 cells) for use in treating B cell malignancies. In some embodiments, the anti-CD19 CAR T cells are human T cells expressing an anti-CD19 CAR and having a disrupted TRAC gene, a disrupted B2M gene, or a combination thereof. In specific examples, the anti-CD19 CART cells express an anti-CD19 CAR and have endogenous TRAC and B2M genes disrupted.

[0059] (i) Anti-CD19 Chimeric Antigen Receptor (CAR)

[0060] The genetically engineered immune cells such as T cells disclosed here express a chimeric antigen receptor (CAR) that binds CD19 (an anti-CD19 CAR). A chimeric antigen receptor (CAR) refers to an artificial immune cell receptor that is engineered to recognize and bind to an antigen expressed by undesired cells, for example, disease cells such as cancer cells. A T cell that expresses a CAR polypeptide is referred to as a CAR T cell. CARs have the ability to redirect T-cell specificity and reactivity toward a selected target in a non-MHC-restricted manner. The non-MHC-restricted antigen recognition gives CAR-T cells the ability to recognize an antigen independent of antigen processing, thus bypassing a major mechanism of tumor escape. Moreover, when expressed on T-cells, CARs advantageously do not dimerize with endogenous T-cell receptor (TCR) alpha and beta chains.

[0061] There are various generations of CARs, each of which contains different components. First generation CARs join an antibody-derived scFv to the CD3zeta ((or z) intracellular signaling domain of the T-cell receptor through hinge and transmembrane domains. Second generation CARs incorporate an additional co-stimulatory domain, e.g., CD28, 4-1BB (41BB), or ICOS, to supply a costimulatory signal. Third-generation CARs contain two costimulatory domains (e.g., a combination of CD27, CD28, 4-1BB, ICOS, or OX40) fused with the TCR CD3.zeta. chain. Maude et al., Blood. 2015; 125(26):4017-4023; Kakarla and Gottschalk, Cancer J. 2014; 20(2):151-155). Any of the various generations of CAR constructs is within the scope of the present disclosure.

[0062] Generally, a CAR is a fusion polypeptide comprising an extracellular domain that recognizes a target antigen (e.g., a single chain fragment (scFv) of an antibody or other antibody fragment) and an intracellular domain comprising a signaling domain of the T-cell receptor (TCR) complex (e.g., CD3.zeta.) and, in most cases, a co-stimulatory domain. (Enblad et al., Human Gene Therapy. 2015; 26(8):498-505). A CAR construct may further comprise a hinge and transmembrane domain between the extracellular domain and the intracellular domain, as well as a signal peptide at the N-terminus for surface expression. Examples of signal peptides include MLLLVTSLLLCELPHPAFLLIP (SEQ ID NO: 30) and MALPVTALLLPLALLLHAARP (SEQ ID NO: 31). Other signal peptides may be used.

[0063] The anti-CD19 CAR may comprise an anti-CD19 single-chain variable fragment (scFv) specific for CD19, followed by hinge domain and transmembrane domain (e.g., a CD8 hinge and transmembrane domain) that is fused to an intracellular co-signaling domain (e.g., a CD28 co-stimulatory domain) and a CD3.zeta. signaling domain. Exemplary components for use in constructing the anti-CD19 CAR disclosed herein can be found in the Sequence Table provided below.

[0064] (a) Antigen Binding Extracellular Domain

[0065] The antigen-binding extracellular domain is the region of a CAR polypeptide that is exposed to the extracellular fluid when the CAR is expressed on cell surface. In some instances, a signal peptide may be located at the N-terminus to facilitate cell surface expression. In some embodiments, the antigen binding domain can be a single-chain variable fragment (scFv, which may include an antibody heavy chain variable region (V.sub.H) and an antibody light chain variable region (V.sub.L) (in either orientation). In some instances, the V.sub.H and V.sub.L fragment may be linked via a peptide linker. The linker, in some embodiments, includes hydrophilic residues with stretches of glycine and serine for flexibility as well as stretches of glutamate and lysine for added solubility. The scFv fragment retains the antigen-binding specificity of the parent antibody, from which the scFv fragment is derived. In some embodiments, the scFv may comprise humanized V.sub.H and/or V.sub.L domains. In other embodiments, the V.sub.H and/or V.sub.L domains of the scFv are fully human.

[0066] The antigen-binding extracellular domain in the CAR polypeptide disclosed herein is specific to CD19 (e.g., human CD19). In some examples, the antigen-binding extracellular domain may comprise a scFv extracellular domain capable of binding to CD19. The anti-CD19 scFv may comprise a heavy chain variable domain (V.sub.H) having the same heavy chain complementary determining regions (CDRs) as those in SEQ ID NO: 51 and a light chain variable domain (V.sub.L) having the same light chain CDRs as those in SEQ ID NO: 52. Two antibodies having the same V.sub.H and/or V.sub.L CDRs means that their CDRs are identical when determined by the same approach (e.g., the Kabat approach, the Chothia approach, the AbM approach, the Contact approach, or the IMGT approach as known in the art. See, e.g., bioinf.org.uk/abs/). In some examples, the anti-CD19 scFv comprises the V.sub.H of SEQ ID NO: 51 and/or the V.sub.L of SEQ ID NO: 52. In specific examples, the anti-CD19 scFv may comprise the amino acid sequence of SEQ ID NO: 47.

[0067] (b) Transmembrane Domain

[0068] The anti-CD19 CAR polypeptide disclosed herein may contain a transmembrane domain, which can be a hydrophobic alpha helix that spans the membrane. As used herein, a "transmembrane domain" refers to any protein structure that is thermodynamically stable in a cell membrane, preferably a eukaryotic cell membrane. The transmembrane domain can provide stability of the CAR containing such.

[0069] In some embodiments, the transmembrane domain of a CAR as provided herein can be a CD8 transmembrane domain. In other embodiments, the transmembrane domain can be a CD28 transmembrane domain. In yet other embodiments, the transmembrane domain is a chimera of a CD8 and CD28 transmembrane domain. Other transmembrane domains may be used as provided herein. In one specific example, the transmembrane domain in the anti-CD19 CAR is a CD8a transmembrane domain having the amino acid sequence of SEQ ID NO: 32.

[0070] (c) Hinge Domain

[0071] In some embodiments, a hinge domain may be located between an extracellular domain (comprising the antigen binding domain) and a transmembrane domain of a CAR, or between a cytoplasmic domain and a transmembrane domain of the CAR. A hinge domain can be any oligopeptide or polypeptide that functions to link the transmembrane domain to the extracellular domain and/or the cytoplasmic domain in the polypeptide chain. A hinge domain may function to provide flexibility to the CAR, or domains thereof, or to prevent steric hindrance of the CAR, or domains thereof.

[0072] In some embodiments, a hinge domain may comprise up to 300 amino acids (e.g., 10 to 100 amino acids, or 5 to 20 amino acids). In some embodiments, one or more hinge domain(s) may be included in other regions of a CAR. In some embodiments, the hinge domain may be a CD8 hinge domain. Other hinge domains may be used.

[0073] (d) Intracellular Signaling Domains

[0074] Any of the anti-CD19 CAR constructs disclosed herein contain one or more intracellular signaling domains (e.g., CD3.zeta., and optionally one or more co-stimulatory domains), which are the functional end of the receptor. Following antigen recognition, receptors cluster and a signal is transmitted to the cell.

[0075] CD3.zeta. is the cytoplasmic signaling domain of the T cell receptor complex. CD3.zeta. contains three (3) immunoreceptor tyrosine-based activation motif (ITAM)s, which transmit an activation signal to the T cell after the T cell is engaged with a cognate antigen. In many cases, CD3.zeta. provides a primary T cell activation signal but not a fully competent activation signal, which requires a co-stimulatory signaling. In some examples, the anti-CD19 CAR construct disclosed herein comprise a CD3.zeta. cytoplasmic signaling domain, which may have the amino acid sequence of SEQ ID NO: 38.

[0076] In some embodiments, the anti-CD19 CAR polypeptides disclosed herein may further comprise one or more co-stimulatory signaling domains. For example, the co-stimulatory domains of CD28 and/or 4-1BB may be used to transmit a full proliferative/survival signal, together with the primary signaling mediated by CD3.zeta.. In some examples, the CAR disclosed herein comprises a CD28 co-stimulatory molecule, for example, a CD28 co-stimulatory signaling domain having the amino acid sequence of SEQ ID NO:36. In other examples, the CAR disclosed herein comprises a 4-1BB co-stimulatory molecule, for example, a 4-1BB co-stimulatory signaling domain having the amino acid sequence of SEQ ID NO: 34.

[0077] In specific examples, an anti-CD19 CAR disclosed herein may include a CD3.zeta. signaling domain (e.g., SEQ ID NO: 38) and a CD28 co-stimulatory domain (e.g., SEQ ID NO: 36).

[0078] It should be understood that methods described herein encompasses more than one suitable CAR that can be used to produce genetically engineered T cells expressing the CAR, for example, those known in the art or disclosed herein. Examples can be found in, e.g., International Application Number PCT/IB2018/001619, filed May 11, 2018, which published as WO 2019/097305A2, and International Application Number PCT/IB2019/000500, filed May 10, 2019, the relevant disclosures of each of the prior applications are incorporated by reference herein for the purpose and subject matter referenced herein.

[0079] In specific examples, the anti-CD19 CAR disclosed herein may comprise the amino acid sequence of SEQ ID NO: 40, which may be encoded by the nucleotide sequence of SEQ ID NO: 39. See the sequence table provided below.

[0080] In the genetically engineered T cells disclosed herein, a nucleic acid comprising the coding sequence of the anti-CD19 CAR, and optionally regulatory sequences for expression of the anti-CD19 CAR (e.g., a promoter such as the EF1a promoter provided in the sequence Table) may be inserted into a genomic locus of interest. In some examples, the nucleic acid is inserted in the endogenous TRAC gene locus, thereby disrupting expression of the TRAC gene. In specific examples, the nucleic acid may replace a fragment in the TRAC gene, for example, a fragment comprising the nucleotide sequence of SEQ ID NO: 26.

[0081] (ii) Knock-Out of TRAC and B2M Genes

[0082] The anti-CD19 CAR-T cells disclosed herein may further have a disrupted TRAC gene, a disrupted B2M gene, or a combination thereof. The disruption of the TRAC locus results in loss of expression of the T cell receptor (TCR) and is intended to reduce the probability of Graft versus Host Disease (GvHD), while the disruption of the .beta.2M locus results in lack of expression of the major histocompatibility complex type I (MHC I) proteins and is intended to improve persistence by reducing the probability of host rejection. The addition of the anti-CD19 CAR directs the modified T cells towards CD19-expressing tumor cells.

[0083] As used herein, the term "a disrupted gene" refers to a gene containing one or more mutations (e.g., insertion, deletion, or nucleotide substitution, etc.) relative to the wild-type counterpart so as to substantially reduce or completely eliminate the activity of the encoded gene product. The one or more mutations may be located in a non-coding region, for example, a promoter region, a regulatory region that regulates transcription or translation; or an intron region. Alternatively, the one or more mutations may be located in a coding region (e.g., in an exon). In some instances, the disrupted gene does not express or expresses a substantially reduced level of the encoded protein. In other instances, the disrupted gene expresses the encoded protein in a mutated form, which is either not functional or has substantially reduced activity. In some embodiments, a disrupted gene is a gene that does not encode functional protein. In some embodiments, a cell that comprises a disrupted gene does not express (e.g., at the cell surface) a detectable level (e.g. by antibody, e.g., by flow cytometry) of the protein encoded by the gene. A cell that does not express a detectable level of the protein may be referred to as a knockout cell. For example, a cell having a .beta.2M gene edit may be considered a .beta.2M knockout cell if .beta.2M protein cannot be detected at the cell surface using an antibody that specifically binds .beta.2M protein.

[0084] In some embodiments, a disrupted gene may be described as comprising a mutated fragment relative to the wild-type counterpart. The mutated fragment may comprise a deletion, a nucleotide substitution, an addition, or a combination thereof. In other embodiments, a disrupted gene may be described as having a deletion of a fragment that is present in the wild-type counterpart. In some instances, the 5' end of the deleted fragment may be located within the gene region targeted by a designed guide RNA such as those disclosed herein (known as on-target sequence) and the 3' end of the deleted fragment may go beyond the targeted region. Alternatively, the 3' end of the deleted fragment may be located within the targeted region and the 5' end of the deleted fragment may go beyond the targeted region.

[0085] In some instances, the disrupted TRAC gene in the anti-CD19 CAR-T cells disclosed herein may comprise a deletion, for example, a deletion of a fragment in Exon 1 of the TRAC gene locus. In some examples, the disrupted TRAC gene comprises a deletion of a fragment comprising the nucleotide sequence of SEQ ID NO: 26, which is the target site of TRAC guide RNA TA-1. See sequence table below. In some examples, the fragment of SEQ ID NO: 26 may be replaced by a nucleic acid encoding the anti-CD19 CAR. Such a disrupted TRAC gene may comprise the nucleotide sequence of SEQ ID NO: 39.

[0086] The disrupted B2M gene in the anti-CD19 CAR-T cells disclosed herein may be generated using the CRISPR/Cas technology. In some examples, a B2M gRNA provided in the sequence table below can be used. The disrupted B2M gene may comprise a nucleotide sequence of any one of SEQ ID Nos: 9-14.

[0087] (iii) Exemplary Population of Anti-CD19 CAR-T Cells for Allogeneic Therapy

[0088] Also provided herein is population of genetically engineered immune cells (e.g., T cells such as human T cells) comprising the anti-CD19 CAR-T cells disclosed herein, which express any of the anti-CD19 CAR disclosed herein (e.g., the anti-CD19 CAR comprising the amino acid sequence of SEQ ID NO: 40), and a disrupted TRAC gene and/or a disrupted B2M gene as also disclosed herein. In some examples, the population of genetically engineered T cells are CTX110 cells, which are CD19-directed T cells having disrupted TRAC gene and B2M gene. The nucleic acid encoding the anti-CD19 CAR can be inserted in the disrupted TRAC gene at the site of SEQ ID NO: 26, which is replaced by the nucleic acid encoding the anti-CD19 CAR, thereby disrupting expression of the TRAC gene. The disrupted TRAC gene in the CTX110 cells may comprise the nucleotide sequence of SEQ ID NO: 39.

[0089] CTX110 cells can be produced via ex vivo genetic modification using the CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein 9) technology to disrupt targeted genes (TRAC and B2M genes), and adeno-associated virus (AAV) transduction to deliver the anti-CD19 CAR construct. CRISPR-Cas9-mediated gene editing involves two guide RNAs (sgRNAs): TA-1 sgRNA (SEQ ID NO: 18), which targets the TRAC locus, and B2M-1 sgRNA (SEQ ID NO: 20), which targets the .beta.2M locus. For any of the gRNA sequences provided herein, those that do not explicitly indicate modifications are meant to encompass both unmodified sequences and sequences having any suitable modifications.

[0090] The anti-CD19 CAR of CTX110 cells is composed of an anti-CD19 single-chain antibody fragment (scFv, which may comprise the amino acid sequence of SEQ ID NO: 47), followed by a CD8 hinge and transmembrane domain (e.g., comprising the amino acid sequence of SEQ ID NO: 32) that is fused to an intracellular co-signaling domain of CD28 (e.g., SEQ ID NO: 36) and a CD3.zeta. signaling domain (e.g., SEQ ID NO: 38). In specific examples, the anti-CD19 CAR in CTX110 cells comprises the amino acid sequence of SEQ ID NO:40.

[0091] In some embodiments, at least 30% of a population of CTX110 cells express a detectable level of the anti-CD19 CAR. For example, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the CTX110 cells express a detectable level of the anti-CD19 CAR.

[0092] In some embodiments, at least 50% of a population of CTX110 cells may not express a detectable level of .beta.2M surface protein. For example, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the CTX110 cells may not express a detectable level of .beta.2M surface protein. In some embodiments, 50%-100%, 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-100%, 60%-90%, 60%-80%, 60%-70%, 70%-100%, 70%-90%, 70%-80%, 80%-100%, 80%-90%, or 90%-100% of the engineered T cells of a population does not express a detectable level of .beta.2M surface protein.

[0093] Alternatively or in addition, at least 50% of a population of CTX110 cells may not express a detectable level of TCR surface protein. For example, at least 55%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the CTX110 cells may not express a detectable level of TCR surface protein. In some embodiments, 50%-100%, 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-100%, 60%-90%, 60%-80%, 60%-70%, 70%-100%, 70%-90%, 70%-80%, 80%-100%, 80%-90%, or 90%-100% of the engineered T cells of a population does not express a detectable level of TRAC surface protein. In specific examples, more than 90% (e.g., more than 99.5%) of the CTX110 cells do not express a detectable TCR surface protein.

[0094] In some embodiments, a substantial percentage of the population of CTX110 T cells may comprise more than one gene edit, which results in a certain percentage of cells not expressing more than one gene and/or protein.

[0095] For example, at least 50% of a population of CTX110 cells may not express a detectable level of two surface proteins, e.g., does not express a detectable level of $2M and TRAC proteins. In some embodiments, 50%-100%, 50%-90%, 50%-80%, 50%-70%, 50%-60%, 60%-100%, 60%-90%, 60%-80%, 60%-70%, 70%-100%, 70%-90%, 70%-80%, 80%-100%, 80%-90%, or 90%-100% of the CTX110 T cells do not express a detectable level of TRAC and B2M surface proteins. In another example, at least 50% of a population of the CTX110 cells do not express a detectable level of TRAC and B2M surface proteins.

[0096] In some embodiments, the population of CTX110 T cells may comprise more than one gene edit (e.g., in more than one gene), which may be an edit described herein. For example, the population of CTX110 T cells may comprise a disrupted TRAC gene via the CRISPR/Cas technology using the TA-1 TRAC gRNA. In some examples, the CTX110 cells may comprise a deletion in the TRAC gene relative to unmodified T cells. For example, the CTX110 T cells may comprise a deletion of the fragment AGAGCAACAGTGCTGTGGCC (SEQ ID NO: 26) in the TRAC gene. This fragment can be replaced by the nucleic acid encoding the anti-CD19 CAR (e.g., SEQ ID NO: 39). Alternatively or in addition, the population of CTX110 cells may comprise a disrupted .beta.2M gene via CRISPR/Cas9 technology using the gRNA of B2M-1. Such CTX110 cells may comprise Indels in the .beta.2M gene, which comprise one or more of the nucleotide sequences of SEQ ID NOs: 9-14. In specific examples, CTX110 cells comprise .gtoreq.30% CAR T cells, .ltoreq.50% B2M.sup.+ cells, and .ltoreq.30% TCR.alpha..beta..sup.+ cells. In additional specific examples, CTX110 cells comprise .gtoreq.30% CAR.sup.+ T cells, .ltoreq.30% B2M.sup.+ cells, and .ltoreq.0.5% TCR.alpha..beta..sup.+ cells.

[0097] See also WO 2019/097305A2, and WO2019215500, the relevant disclosures of each of which are incorporated by reference for the subject matter and purpose referenced herein.

[0098] (iv) Pharmaceutical Compositions

[0099] In some aspects, the present disclosure provides pharmaceutical compositions comprising any of the populations of genetically engineered anti-CD19 CAR T cells as disclosed herein, for example, CTX110 cells, and a pharmaceutically acceptable carrier. Such pharmaceutical compositions can be used in cancer treatment in human patients, which is also disclosed herein.

[0100] As used herein, the term "pharmaceutically acceptable" refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and/or bodily fluids of the subject without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio. As used herein, the term "pharmaceutically acceptable carrier" refers to solvents, dispersion media, coatings, antibacterial agents, antifungal agents, isotonic and absorption delaying agents, or the like that are physiologically compatible. The compositions can include a pharmaceutically acceptable salt, e.g., an acid addition salt or a base addition salt. See, e.g., Berge et al., (1977) J Pharm Sci 66:1-19.

[0101] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable salt. Non-limiting examples of pharmaceutically acceptable salts include acid addition salts (formed from a free amino group of a polypeptide with an inorganic acid (e.g., hydrochloric or phosphoric acids), or an organic acid such as acetic, tartaric, mandelic, or the like). In some embodiments, the salt formed with the free carboxyl groups is derived from an inorganic base (e.g., sodium, potassium, ammonium, calcium or ferric hydroxides), or an organic base such as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, or the like).

[0102] In some embodiments, the pharmaceutical composition disclosed herein comprises a population of the genetically engineered anti-CD19 CAR-T cells (e.g., CTX110 cells) suspended in a cryopreservation solution (e.g., CryoStor.RTM. C55). The cryopreservation solution for use in the present disclosure may also comprise adenosine, dextrose, dextran-40, lactobionic acid, sucrose, mannitol, a buffer agent such as N-)2-hydroxethyl) piperazine-N'-(2-ethanesulfonic acid) (HEPES), one or more salts (e.g., calcium chloride, magnesium chloride, potassium chloride, potassium bicarbonate, potassium phosphate, etc.), one or more base (e.g., sodium hydroxide, potassium hydroxide, etc.), or a combination thereof. Components of a cryopreservation solution may be dissolved in sterile water (injection quality). Any of the cryopreservation solution may be substantially free of serum (undetectable by routine methods).

[0103] In some instances, a pharmaceutical composition comprising a population of genetically engineered anti-CD19 CAR-T cells such as the CTX110 cells suspended in a cryopreservation solution (e.g., substantially free of serum) may be placed in storage vials.

[0104] Any of the pharmaceutical compositions disclosed herein, comprising a population of genetically engineered anti-CD19 CAR T cells as also disclosed herein (e.g., CTX110 cells), which optionally may be suspended in a cryopreservation solution as disclosed herein may be stored in an environment that does not substantially affect viability and bioactivity of the T cells for future use, e.g., under conditions commonly applied for storage of cells and tissues. In some examples, the pharmaceutical composition may be stored in the vapor phase of liquid nitrogen at .ltoreq.-135.degree. C. No significant changes were observed with respect to appearance, cell count, viability, % CAR.sup.+ T cells, % TCR.sup.+ T cells, and % B2M.sup.+ T cells after the cells have been stored under such conditions for a period of time.

II. Preparation of Genetically Engineered Immune Cells

[0105] Any suitable gene editing methods known in the art can be used for making the genetically engineered immune cells (e.g., T cells such as CTX110 cells) disclosed herein, for example, nuclease-dependent targeted editing using zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or RNA-guided CRISPR-Cas9 nucleases (CRISPR/Cas9; Clustered Regular Interspaced Short Palindromic Repeats Associated 9). In specific examples, the genetically engineered immune cells such as CTX110 cells are produced by the CRISPR technology in combination with homologous recombination using an adeno-associated viral vector (AAV) as a donor template.

[0106] (1) CRISPR-Cas9-Mediated Gene Editing System

[0107] The CRISPR-Cas9 system is a naturally-occurring defense mechanism in prokaryotes that has been repurposed as an RNA-guided DNA-targeting platform used for gene editing. It relies on the DNA nuclease Cas9, and two noncoding RNAs, crisprRNA (crRNA) and trans-activating RNA (tracrRNA), to target the cleavage of DNA. CRISPR is an abbreviation for Clustered Regularly Interspaced Short Palindromic Repeats, a family of DNA sequences found in the genomes of bacteria and archaea that contain fragments of DNA (spacer DNA) with similarity to foreign DNA previously exposed to the cell, for example, by viruses that have infected or attacked the prokaryote. These fragments of DNA are used by the prokaryote to detect and destroy similar foreign DNA upon re-introduction, for example, from similar viruses during subsequent attacks. Transcription of the CRISPR locus results in the formation of an RNA molecule comprising the spacer sequence, which associates with and targets Cas (CRISPR-associated) proteins able to recognize and cut the foreign, exogenous DNA. Numerous types and classes of CRISPR/Cas systems have been described (see, e.g., Koonin et al., (2017) Curr Opin Microbiol 37:67-78).

[0108] crRNA drives sequence recognition and specificity of the CRISPR-Cas9 complex through Watson-Crick base pairing typically with a 20 nucleotide (nt) sequence in the target DNA. Changing the sequence of the 5' 20 nt in the crRNA allows targeting of the CRISPR-Cas9 complex to specific loci. The CRISPR-Cas9 complex only binds DNA sequences that contain a sequence match to the first 20 nt of the crRNA, if the target sequence is followed by a specific short DNA motif (with the sequence NGG) referred to as a protospacer adjacent motif (PAM).

[0109] TracrRNA hybridizes with the 3' end of crRNA to form an RNA-duplex structure that is bound by the Cas9 endonuclease to form the catalytically active CRISPR-Cas9 complex, which can then cleave the target DNA.

[0110] Once the CRISPR-Cas9 complex is bound to DNA at a target site, two independent nuclease domains within the Cas9 enzyme each cleave one of the DNA strands upstream of the PAM site, leaving a double-strand break (DSB) where both strands of the DNA terminate in a base pair (a blunt end).

[0111] After binding of CRISPR-Cas9 complex to DNA at a specific target site and formation of the site-specific DSB, the next key step is repair of the DSB. Cells use two main DNA repair pathways to repair the DSB: non-homologous end joining (NHEJ) and homology-directed repair (HDR).

[0112] NHEJ is a robust repair mechanism that appears highly active in the majority of cell types, including non-dividing cells. NHEJ is error-prone and can often result in the removal or addition of between one and several hundred nucleotides at the site of the DSB, though such modifications are typically <20 nt. The resulting insertions and deletions (indels) can disrupt coding or noncoding regions of genes. Alternatively, HDR uses a long stretch of homologous donor DNA, provided endogenously or exogenously, to repair the DSB with high fidelity. HDR is active only in dividing cells, and occurs at a relatively low frequency in most cell types. In many embodiments of the present disclosure, NHEJ is utilized as the repair operant.

[0113] (a) Cas9

[0114] In some embodiments, the Cas9 (CRISPR associated protein 9) endonuclease is used in a CRISPR method for making the genetically engineered T cells as disclosed herein. The Cas9 enzyme may be one from Streptococcus pyogenes, although other Cas9 homologs may also be used. It should be understood, that wild-type Cas9 may be used or modified versions of Cas9 may be used (e.g., evolved versions of Cas9, or Cas9 orthologues or variants), as provided herein. In some embodiments, Cas9 comprises a Streptococcus pyogenes-derived Cas9 nuclease protein that has been engineered to include C- and N-terminal SV40 large T antigen nuclear localization sequences (NLS). The resulting Cas9 nuclease (sNLS-spCas9-sNLS) is a 162 kDa protein that is produced by recombinant E. coli fermentation and purified by chromatography. The spCas9 amino acid sequence can be found as UniProt Accession No. Q99ZW2, which is provided herein as SEQ ID NO: 55.

[0115] (b) Guide RNAs (gRNAs)

[0116] CRISPR-Cas9-mediated gene editing as described herein includes the use of a guide RNA or a gRNA. As used herein, a "gRNA" refers to a genome-targeting nucleic acid that can direct the Cas9 to a specific target sequence within a TRAC gene or a .beta.2M gene for gene editing at the specific target sequence. A guide RNA comprises at least a spacer sequence that hybridizes to a target nucleic acid sequence within a target gene for editing, and a CRISPR repeat sequence.

[0117] An exemplary gRNA targeting a TRAC gene is provided in SEQ ID NO: 18 or 22. See the sequence table below. See also WO 2019/097305A2, the relevant disclosures of which are incorporated by reference herein for the subject matter and purpose referenced herein. Other gRNA sequences may be designed using the TRAC gene sequence located on chromosome 14 (GRCh38: chromosome 14: 22,547,506-22,552,154; Ensembl; ENSG00000277734). In some embodiments, gRNAs targeting the TRAC genomic region and Cas9 create breaks in the TRAC genomic region resulting Indels in the TRAC gene disrupting expression of the mRNA or protein.

[0118] An exemplary gRNA targeting a .beta.2M gene is provided in SEQ ID NO: 20 or 24. See the sequence table below. See also WO 2019/097305A2, the relevant disclosures of which are incorporated by reference herein for the purpose and subject matter referenced herein. Other gRNA sequences may be designed using the .beta.2M gene sequence located on Chromosome 15 (GRCh38 coordinates: Chromosome 15: 44,711,477-44,718,877; Ensembl: ENSG00000166710). In some embodiments, gRNAs targeting the .beta.2M genomic region and RNA-guided nuclease create breaks in the .beta.2M genomic region resulting in Indels in the .beta.2M gene disrupting expression of the mRNA or protein.

[0119] In Type II systems, the gRNA also comprises a second RNA called the tracrRNA sequence. In the Type II gRNA, the CRISPR repeat sequence and tracrRNA sequence hybridize to each other to form a duplex. In the Type V gRNA, the crRNA forms a duplex. In both systems, the duplex binds a site-directed polypeptide, such that the guide RNA and site-direct polypeptide form a complex. In some embodiments, the genome-targeting nucleic acid provides target specificity to the complex by virtue of its association with the site-directed polypeptide. The genome-targeting nucleic acid thus directs the activity of the site-directed polypeptide.

[0120] As is understood by the person of ordinary skill in the art, each guide RNA is designed to include a spacer sequence complementary to its genomic target sequence. See Jinek et al., Science, 337, 816-821 (2012) and Deltcheva et al., Nature, 471, 602-607 (2011).

[0121] In some embodiments, the genome-targeting nucleic acid (e.g., gRNA) is a double-molecule guide RNA. In some embodiments, the genome-targeting nucleic acid (e.g., gRNA) is a single-molecule guide RNA.

[0122] A double-molecule guide RNA comprises two strands of RNA molecules. The first strand comprises in the 5' to 3' direction, an optional spacer extension sequence, a spacer sequence and a minimum CRISPR repeat sequence. The second strand comprises a minimum tracrRNA sequence (complementary to the minimum CRISPR repeat sequence), a 3' tracrRNA sequence and an optional tracrRNA extension sequence.

[0123] A single-molecule guide RNA (referred to as a "sgRNA") in a Type II system comprises, in the 5' to 3' direction, an optional spacer extension sequence, a spacer sequence, a minimum CRISPR repeat sequence, a single-molecule guide linker, a minimum tracrRNA sequence, a 3' tracrRNA sequence and an optional tracrRNA extension sequence. The optional tracrRNA extension may comprise elements that contribute additional functionality (e.g., stability) to the guide RNA. The single-molecule guide linker links the minimum CRISPR repeat and the minimum tracrRNA sequence to form a hairpin structure. The optional tracrRNA extension comprises one or more hairpins. A single-molecule guide RNA in a Type V system comprises, in the 5' to 3' direction, a minimum CRISPR repeat sequence and a spacer sequence.

[0124] The "target sequence" is in a target gene that is adjacent to a PAM sequence and is the sequence to be modified by Cas9. The "target sequence" is on the so-called PAM-strand in a "target nucleic acid," which is a double-stranded molecule containing the PAM-strand and a complementary non-PAM strand. One of skill in the art recognizes that the gRNA spacer sequence hybridizes to the complementary sequence located in the non-PAM strand of the target nucleic acid of interest. Thus, the gRNA spacer sequence is the RNA equivalent of the target sequence.

[0125] For example, if the TRAC target sequence is 5'-AGAGCAACAGTGCTGTGGCC-3' (SEQ ID NO: 26), then the gRNA spacer sequence is 5'-AGAGCAACAGUGCUGUGGCC-3' (SEQ ID NO: 19). In another example, if the .beta.2M target sequence is 5'-GCTACTCTCTCTTTCTGGCC-3' (SEQ ID NO: 27), then the gRNA spacer sequence is 5'-GCUACUCUCUCUUUCUGGCC-3' (SEQ ID NO: 21). The spacer of a gRNA interacts with a target nucleic acid of interest in a sequence-specific manner via hybridization (i.e., base pairing). The nucleotide sequence of the spacer thus varies depending on the target sequence of the target nucleic acid of interest.

[0126] In a CRISPR/Cas system herein, the spacer sequence is designed to hybridize to a region of the target nucleic acid that is located 5' of a PAM recognizable by a Cas9 enzyme used in the system. The spacer may perfectly match the target sequence or may have mismatches. Each Cas9 enzyme has a particular PAM sequence that it recognizes in a target DNA. For example, S. pyogenes recognizes in a target nucleic acid a PAM that comprises the sequence 5'-NRG-3', where R comprises either A or G, where N is any nucleotide and N is immediately 3' of the target nucleic acid sequence targeted by the spacer sequence.

[0127] In some embodiments, the target nucleic acid sequence has 20 nucleotides in length. In some embodiments, the target nucleic acid has less than 20 nucleotides in length. In some embodiments, the target nucleic acid has more than 20 nucleotides in length. In some embodiments, the target nucleic acid has at least: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides in length. In some embodiments, the target nucleic acid has at most: 5, 10, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30 or more nucleotides in length. In some embodiments, the target nucleic acid sequence has 20 bases immediately 5' of the first nucleotide of the PAM. For example, in a sequence comprising 5'-NNNNNNNNNNNNNNNNNNNNNRG-3', the target nucleic acid can be the sequence that corresponds to the Ns, wherein N can be any nucleotide, and the underlined NRG sequence is the S. pyogenes PAM. Examples are provides as SEQ ID NOs: 15-17.

[0128] The guide RNA disclosed herein may target any sequence of interest via the spacer sequence in the crRNA. In some embodiments, the degree of complementarity between the spacer sequence of the guide RNA and the target sequence in the target gene can be about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100%. In some embodiments, the spacer sequence of the guide RNA and the target sequence in the target gene is 100% complementary. In other embodiments, the spacer sequence of the guide RNA and the target sequence in the target gene may contain up to 10 mismatches, e.g., up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 mismatch.

[0129] Non-limiting examples of gRNAs that may be used as provided herein are provided in WO 2019/097305A2, and WO2019/215500, the relevant disclosures of each of which are herein incorporated by reference for the purposes and subject matter referenced herein. For any of the gRNA sequences provided herein, those that do not explicitly indicate modifications are meant to encompass both unmodified sequences and sequences having any suitable modifications.

[0130] The length of the spacer sequence in any of the gRNAs disclosed herein may depend on the CRISPR/Cas9 system and components used for editing any of the target genes also disclosed herein. For example, different Cas9 proteins from different bacterial species have varying optimal spacer sequence lengths. Accordingly, the spacer sequence may have 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, 35, 40, 45, 50, or more than 50 nucleotides in length. In some embodiments, the spacer sequence may have 18-24 nucleotides in length. In some embodiments, the targeting sequence may have 19-21 nucleotides in length. In some embodiments, the spacer sequence may comprise nucleotides in length.

[0131] In some embodiments, the gRNA can be a sgRNA, which may comprise a 20 nucleotide spacer sequence at the 5' end of the sgRNA sequence. In some embodiments, the sgRNA may comprise a less than 20 nucleotide spacer sequence at the 5' end of the sgRNA sequence. In some embodiments, the sgRNA may comprise a more than 20 nucleotide spacer sequence at the 5' end of the sgRNA sequence. In some embodiments, the sgRNA comprises a variable length spacer sequence with 17-30 nucleotides at the 5' end of the sgRNA sequence.

[0132] In some embodiments, the sgRNA comprises no uracil at the 3' end of the sgRNA sequence. In other embodiments, the sgRNA may comprise one or more uracil at the 3' end of the sgRNA sequence. For example, the sgRNA can comprise 1-8 uracil residues, at the 3' end of the sgRNA sequence, e.g., 1, 2, 3, 4, 5, 6, 7, or 8 uracil residues at the 3' end of the sgRNA sequence.

[0133] Any of the gRNAs disclosed herein, including any of the sgRNAs, may be unmodified. Alternatively, it may contain one or more modified nucleotides and/or modified backbones. For example, a modified gRNA such as a sgRNA can comprise one or more 2'-O-methyl phosphorothioate nucleotides, which may be located at either the 5' end, the 3' end, or both.

[0134] In certain embodiments, more than one guide RNAs can be used with a CRISPR/Cas nuclease system. Each guide RNA may contain a different targeting sequence, such that the CRISPR/Cas system cleaves more than one target nucleic acid. In some embodiments, one or more guide RNAs may have the same or differing properties such as activity or stability within the Cas9 RNP complex. Where more than one guide RNA is used, each guide RNA can be encoded on the same or on different vectors. The promoters used to drive expression of the more than one guide RNA is the same or different.

[0135] It should be understood that more than one suitable Cas9 and more than one suitable gRNA can be used in methods described herein, for example, those known in the art or disclosed herein. In some embodiments, methods comprise a Cas9 enzyme and/or a gRNA known in the art. Examples can be found in, e.g., WO 2019/097305A2, and WO2019/215500, the relevant disclosures of each of which are herein incorporated by reference for the purposes and subject matter referenced herein.

[0136] (ii) AAV Vectors for Delivery of CAR Constructs to T Cells

[0137] A nucleic acid encoding an anti-CD19 CAR construct as disclosed herein can be delivered to a cell using an adeno-associated virus (AAV). AAVs are small viruses which integrate site-specifically into the host genome and can therefore deliver a transgene, such as CAR. Inverted terminal repeats (ITRs) are present flanking the AAV genome and/or the transgene of interest and serve as origins of replication. Also present in the AAV genome are rep and cap proteins which, when transcribed, form capsids which encapsulate the AAV genome for delivery into target cells. Surface receptors on these capsids which confer AAV serotype, which determines which target organs the capsids will primarily bind and thus what cells the AAV will most efficiently infect. There are twelve currently known human AAV serotypes. In some embodiments, the AAV for use in delivering the CAR-coding nucleic acid is AAV serotype 6 (AAV6).

[0138] Adeno-associated viruses are among the most frequently used viruses for gene therapy for several reasons. First, AAVs do not provoke an immune response upon administration to mammals, including humans. Second, AAVs are effectively delivered to target cells, particularly when consideration is given to selecting the appropriate AAV serotype. Finally, AAVs have the ability to infect both dividing and non-dividing cells because the genome can persist in the host cell without integration. This trait makes them an ideal candidate for gene therapy.

[0139] A nucleic acid encoding an anti-CD19 CAR can be designed to insert into a genomic site of interest in the host T cells. In some embodiments, the target genomic site can be in a safe harbor locus.

[0140] In some embodiments, a nucleic acid encoding the anti-CD19 CAR (e.g., via a donor template, which can be carried by a viral vector such as an adeno-associated viral (AAV) vector) can be designed such that it can insert into a location within a TRAC gene to disrupt the TRAC gene in the genetically engineered T cells and express the CAR polypeptide. Disruption of TRAC leads to loss of function of the endogenous TCR. For example, a disruption in the TRAC gene can be created with an endonuclease such as those described herein and one or more gRNAs targeting one or more TRAC genomic regions. Any of the gRNAs specific to a TRAC gene and the target regions can be used for this purpose, e.g., those disclosed herein.

[0141] In some examples, a genomic deletion in the TRAC gene and replacement by a CAR coding segment can be created by homology directed repair or HDR (e.g., using a donor template, which may be part of a viral vector such as an adeno-associated viral (AAV) vector). In some embodiments, a disruption in the TRAC gene can be created with an endonuclease as those disclosed herein and one or more gRNAs targeting one or more TRAC genomic regions, and inserting a CAR coding segment into the TRAC gene.

[0142] A donor template as disclosed herein can contain a coding sequence for a CAR. In some examples, the CAR-coding sequence may be flanked by two regions of homology to allow for efficient HDR at a genomic location of interest, for example, at a TRAC gene using CRISPR-Cas9 gene editing technology. In this case, both strands of the DNA at the target locus can be cut by a CRISPR Cas9 enzyme guided by gRNAs specific to the target locus. HDR then occurs to repair the double-strand break (DSB) and insert the donor DNA coding for the CAR. For this to occur correctly, the donor sequence is designed with flanking residues which are complementary to the sequence surrounding the DSB site in the target gene (hereinafter "homology arms"), such as the TRAC gene. These homology arms serve as the template for DSB repair and allow HDR to be an essentially error-free mechanism. The rate of homology directed repair (HDR) is a function of the distance between the mutation and the cut site so choosing overlapping or nearby target sites is important. Templates can include extra sequences flanked by the homologous regions or can contain a sequence that differs from the genomic sequence, thus allowing sequence editing.

[0143] Alternatively, a donor template may have no regions of homology to the targeted location in the DNA and may be integrated by NHEJ-dependent end joining following cleavage at the target site.

[0144] A donor template can be DNA or RNA, single-stranded and/or double-stranded, and can be introduced into a cell in linear or circular form. If introduced in linear form, the ends of the donor sequence can be protected (e.g., from exonucleolytic degradation) by methods known to those of skill in the art. For example, one or more dideoxynucleotide residues are added to the 3' terminus of a linear molecule and/or self-complementary oligonucleotides are ligated to one or both ends. See, for example, Chang et al., (1987) Proc. Natl. Acad. Sci. USA 84:4959-4963; Nehls et al., (1996) Science 272:886-889. Additional methods for protecting exogenous polynucleotides from degradation include, but are not limited to, addition of terminal amino group(s) and the use of modified internucleotide linkages such as, for example, phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyribose residues.

[0145] A donor template can be introduced into a cell as part of a vector molecule having additional sequences such as, for example, replication origins, promoters and genes encoding antibiotic resistance. Moreover, a donor template can be introduced into a cell as naked nucleic acid, as nucleic acid complexed with an agent such as a liposome or poloxamer, or can be delivered by viruses (e.g., adenovirus, AAV, herpesvirus, retrovirus, lentivirus and integrase defective lentivirus (IDLV)).

[0146] A donor template, in some embodiments, can be inserted at a site nearby an endogenous promoter (e.g., downstream or upstream) so that its expression can be driven by the endogenous promoter. In other embodiments, the donor template may comprise an exogenous promoter and/or enhancer, for example, a constitutive promoter, an inducible promoter, or tissue-specific promoter to control the expression of the CAR gene. In some embodiments, the exogenous promoter is an EF1a promoter. Other promoters may be used.

[0147] Furthermore, exogenous sequences may also include transcriptional or translational regulatory sequences, for example, promoters, enhancers, insulators, internal ribosome entry sites, sequences encoding 2A peptides and/or polyadenylation signals.

[0148] To prepare the genetically engineered immune cells (e.g., T cells disclosed herein), immune cells such as T cells from a suitable source may be obtained, e.g., blood cells from a human donor, who may be a healthy donor or a patient need CAR-T cell therapy. The CTX110 cells can be made using blood cells from one or more healthy human donors. Manufacturing from healthy donor cells minimizes the risk of unintentionally transducing malignant lymphoma/leukemia cells and potentially may improve the functionality of the CAR T cells. The components of the CRISPR system (e.g., Cas9 protein and the gRNAs), optionally the AAV donor template, may be delivered into the host immune cells via conventional approaches. In some examples, the Cas9 and the gRNAs can form a ribonucleoprotein complex (RNP), which can be delivered to the host immune cells by electroporation. Optionally, the AAV donor template may be delivered to the immune cells concurrently with the RNP complex. Alternatively, delivery of the RNPs and the AAV donor template can be performed sequentially. In some examples, the T cells may be activated prior to delivery of the gene editing components.

[0149] After delivery of the gene editing components and optionally the donor template, the cells may be recovered and expanded in vitro. Gene editing efficiency can be evaluated using routine methods for confirm knock-in of the anti-CD19 CAR and knock-out of the target genes (e.g., TRAC, B2M, or both). In some examples, TCR.alpha..beta..sup.+ T cells may be removed. Additional information for preparation of the genetically engineered immune cells disclosed herein such as the CTX110 cells can be found in U.S. Patent Application No. 62/934,991, the relevant disclosures of which are incorporated by reference for the purpose and subject matter referenced herein.

III. Allogeneic CAR-T Cell Therapy of B Cell Malignancies

[0150] In some aspects, provided herein are methods for treating a human patient having a B cell malignancy using a population of any of the genetically engineered anti-CD19 CAR T cells such as the CTX110 T cells as disclosed herein. The allogeneic anti-CD19 CART cell therapy may comprise two stages of treatment: (i) a conditioning regimen (lymphodepleting treatment), which comprises giving one or more doses of one or more lymphodepleting agents to a suitable human patient, and (ii) a treatment regimen (allogeneic anti-CD19 CAR T cell therapy), which comprises administration of the population of allogeneic anti-CD19 CAR T cells such as the CTX110 T cells as disclosed herein to the human patient.

[0151] (i) Patient Population

[0152] A human patient may be any human subject for whom diagnosis, treatment, or therapy is desired. A human patient may be of any age. In some embodiments, the human patient is an adult (e.g., a person who is at least 18 years old). In some examples, the human patient may have a body weight of 50 kg or higher. In some embodiments, the human patient can be a child.

[0153] A human patient to be treated by the methods described herein can be a human patient having, suspected of having, or a risk for having a B cell malignancy. A subject suspected of having a B cell malignancy might show one or more symptoms of B cell malignancy, e.g., unexplained weight loss, fatigue, night sweats, shortness of breath, or swollen glands. A subject at risk for a B cell malignancy can be a subject having one or more of the risk factors for B cell malignancy, e.g., a weakened immune system, age, male, or infection (e.g., Epstein-Barr virus infection). A human patient who needs the anti-CD19 CAR T cell (e.g., CTX110 T cell) treatment may be identified by routine medical examination, e.g., physical examination, laboratory tests, biopsy (e.g., bone marrow biopsy and/or lymph node biopsy), magnetic resonance imaging (MRI) scans, or ultrasound exams.

[0154] Examples of B cell malignancies that may be treated using the methods described herein include, but are not limited to, diffuse large B cell lymphoma (DLBCL), high grade B cell lymphoma with MYC and BCL2 and/or BCL6 rearrangement, transformed follicular lymphoma (FL), grade 3b FL, or Richter's transformation of chronic lymphocytic leukemia (CLL). In some examples, the B cell malignancy is DLBCL, e.g., high grade DLBCL or DLBCL not otherwise specified (NOS). In some examples, the B cell malignancy is transformed FL or grade 3b FL. In some examples, the human patient has at least one measurable lesion that is fluorodeoxyglucose positron emission tomography (PET)-positive.

[0155] In some embodiment, the human patient to be treated has DLBCL and exhibits pararectal mass, retroperitoneal mass, diffuse lymph nodes (LN), lytic lesions, tonsillar lesion, or a combination thereof. Alternatively or in addition, the human patient may have bone marrow diffusion. In other examples, the human patient is free of bone marrow diffusion.

[0156] In some embodiments, the human patient to be treated has transformed FL. Such a human patient may exhibit diffuse LN. In some instances, the human patient may have bone marrow diffusion. In other instances, the human patient may be free of bone marrow diffusion.

[0157] A human patient to be treated by methods described herein may be a human patient that has relapsed following a treatment and/or that has been become resistant to a treatment and/or that has been non-responsive to a treatment. As used herein, "relapsed" or "relapses" refers to a B cell malignancy such as those disclosed herein that returns following a period of complete response. Progressive disease refers to an instance when a disease worsens after the last evaluation (e.g., stable disease or partial response). In some embodiments, progression occurs during the treatment. In some embodiments, relapse occurs after the treatment. A lack of response may be determined by routine medical practice. For example, the human patient to be treated by methods described herein may be a human patient that has had one or more lines of prior anti-cancer therapies. In some instances, the human patient may have undergone two or more lines of prior anti-cancer therapies, e.g., a chemotherapy, an immunotherapy, a surgery, or a combination thereof. In some examples, the prior anti-cancer therapies may comprise an anti-CD20 antibody therapy, an anthracycline-containing therapy, or a combination thereof.

[0158] In some instances, the human patient has a refractory B cell malignancy. As used herein, "refractory" refers to a B cell malignancy such as those disclosed herein that does not respond to or becomes resistant to a treatment. A human patient having a refractory B cell malignancy may have progressive disease on last therapy, or has stable disease following at least two cycles of therapy with duration of stable disease of up to 6 months (e.g., up to 5 months, up to 4 months, or up to 3 months or up to 2 months or up to 1 month). In some instances, the human patient may have undergone a prior autologous hematopoietic stem cell transplantation (HSCT) and showed no response to such (failed) or have progressed or relapsed after achieving some response. In other instances, the human patient may not be eligible for prior autologous HSCT.

[0159] A human patient may be screened to determine whether the patient is eligible to undergo a conditioning regimen (lymphodepleting treatment) and/or an allogeneic anti-CD19 CAR-T cell therapy as disclosed herein. For example, a human patient who is eligible for lymphodepletion treatment does not show one or more of the following features: (a) significant worsening of clinical status, (b) requirement for supplemental oxygen to maintain a saturation level of greater than 90%, (c) uncontrolled cardiac arrhythmia, (d) hypotension requiring vasopressor support, (e) active infection, and (f) grade .gtoreq.2 acute neurological toxicity. In another example, a human patient who is eligible for a treatment regimen does not show one or more of the following features: (a) active uncontrolled infection, (b) worsening of clinical status compared to the clinical status prior to lymphodepletion treatment, and (c) grade .gtoreq.2 acute neurological toxicity.

[0160] A human patient may be screened and excluded from the conditioning regimen and/or treatment regimen based on such screening results. For example, a human patient may be excluded from a conditioning regimen and/or the allogeneic anti-CD19 CAR-T cell therapy, if the patient meets one or more of the following exclusion criteria: (a) has an Eastern Cooperative Oncology Group (ECOG) performance status 0 or 1; (b) adequate renal, liver, cardiac, and/or pulmonary function; (c) free of prior gene therapy or modified cell therapy; (d) free of prior treatment comprising an anti-CD19 antibody; (e) free of prior allogeneic HSCT; (f) free of detectable malignant cells from cerebrospinal fluid; (g) free of brain metastases; (h) free of prior central nervous system disorders; (i) free of unstable angina, arrhythmia, and/or myocardial infarction; (j) free of uncontrolled infection; (k) free of immunodeficiency disorders or autoimmune disorders that require immunosuppressive therapy; and (1) free of infection by human immunodeficiency virus, hepatitis B virus, or hepatitis C virus.

[0161] (ii) Conditioning Regimen (Lymphodepleting Therapy)

[0162] Any human patients suitable for the treatment methods disclosed herein may receive a lymphodepleting therapy to reduce or deplete the endogenous lymphocyte of the subject.

[0163] Lymphodepletion refers to the destruction of endogenous lymphocytes and/or T cells, which is commonly used prior to immunotransplantation and immunotherapy. Lymphodepletion can be achieved by irradiation and/or chemotherapy. A "lymphodepleting agent" can be any molecule capable of reducing, depleting, or eliminating endogenous lymphocytes and/or T cells when administered to a subject. In some embodiments, the lymphodepleting agents are administered in an amount effective in reducing the number of lymphocytes by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 96%, 97%, 98%, or at least 99% as compared to the number of lymphocytes prior to administration of the agents. In some embodiments, the lymphodepleting agents are administered in an amount effective in reducing the number of lymphocytes such that the number of lymphocytes in the subject is below the limits of detection. In some embodiments, the subject is administered at least one (e.g., 2, 3, 4, 5 or more) lymphodepleting agents.

[0164] In some embodiments, the lymphodepleting agents are cytotoxic agents that specifically kill lymphocytes. Examples of lymphodepleting agents include, without limitation, fludarabine, cyclophosphamide, bendamustin, 5-fluorouracil, gemcitabine, methotrexate, dacarbazine, melphalan, doxorubicin, vinblastine, cisplatin, oxaliplatin, paclitaxel, docetaxel, irinotecan, etopside phosphate, mitoxantrone, cladribine, denileukin diftitox, or DAB-IL2. In some instances, the lymphodepleting agent may be accompanied with low-dose irradiation. The lymphodepletion effect of the conditioning regimen can be monitored via routine practice.

[0165] In some embodiments, the method described herein involves a conditioning regimen that comprises one or more lymphodepleting agents, for example, fludarabine and cyclophosphamide. A human patient to be treated by the method described herein may receive multiple doses of the one or more lymphodepleting agents for a suitable period (e.g., 1-5 days) in the conditioning stage. The patient may receive one or more of the lymphodepleting agents once per day during the lymphodepleting period. In one example, the human patient receives fludarabine at about 20-50 mg/m.sup.2 (e.g., 30 mg/m.sup.2) per day for 2-4 days (e.g., 3 days) and cyclophosphamide at about 500-750 mg/m.sup.2 (e.g., 500 or 750 mg/m.sup.2) per day for 2-4 days (e.g., 3 days). In specific examples, the human patient may receive fludarabine at about 30 mg/m.sup.2 and cyclophosphamide at about 500 mg/m.sup.2 per day for three days. In other specific examples, the human patient may receive fludarabine at about 30 mg/m.sup.2 and cyclophosphamide at about 750 mg/m.sup.2 per day for three days.

[0166] The human patient may then be administered any of the anti-CD19 CAR T cells such as CTX110 cells within a suitable period after the lymphodepleting therapy as disclosed herein. For example, a human patient may be subject to one or more lymphodepleting agent about 2-7 days (e.g., for example, 2, 3, 4, 5, 6, 7 days) before administration of the anti-CD19 CAR+ T cells (e.g., CTX110 cells). In some instances, a human patient is administered the anti-CD19 CAR+ T cells (e.g., CTX110 cells) within about 4-5 days after the lymphodepleting therapy.

[0167] Since the allogeneic anti-CD19 CAR-T cells such as CTX110 cells can be prepared in advance and may be stored at the treatment site, the lymphodepleting therapy as disclosed herein may be applied to a human patient having a B cell malignancy within a short time window (e.g., within 2 weeks) after the human patient is identified as suitable for the allogeneic anti-CD19 CAR-T cell therapy disclosed herein. For example, the first dose of the lymphodepleting therapy (e.g., fludarabine at about 30 mg/m.sup.2 and cyclophosphamide at about 500 mg/m.sup.2 or 750 mg/m.sup.2) may be administered to the human patient within two weeks (e.g., within 10 days, within 9 days, within 8 days, within 7 days, within 6 days, within 5 days, within 4 days, within 3 days, within two days, or less) after the human patient is identified as suitable for the allogeneic anti-CD19 CAR-T cell therapy. In some examples, the lymphodepleting therapy may be performed to the human patient within 24-72 hours (e.g., within 24 hours) after the human patient is identified as suitable for the treatment. The patient can then be administered the CAR-T cells within 2-7 days (e.g., for example, 2, 3, 4, 5, 6, or 7 days) after the lymphodepleting treatment. This allows for timely treatment of the human patient with the allogeneic anti-CD19 CAR-T cells disclosed herein such as CTX110 cells after disease diagnosis and/or patient identification without delay (e.g., delay due to preparation of the therapeutic cells). In certain instances, a patient may receive the treatment during inpatient hospital care. In certain instances, a patient may receive the treatment in outpatient care.

[0168] Prior to any of the lymphodepletion steps, a human patient may be screened for one or more features to determine whether the patient is eligible for lymphodepletion treatment. For example, prior to lymphodepletion, a human patient eligible for lymphodepletion treatment does not show one or more of the following features: (a) significant worsening of clinical status, (b) requirement for supplemental oxygen to maintain a saturation level of greater than 90%, (c) uncontrolled cardiac arrhythmia, (d) hypotension requiring vasopressor support, (e) active infection, and (f) grade .gtoreq.2 acute neurological toxicity.

[0169] Following lymphodepletion, a human patient may be screened for one or more features to determine whether the patient is eligible for treatment with anti-CD19 CAR T cells such as the CTX110 cells. For example, prior to anti-CD19 CART cell treatment and after lymphodepletion treatment, a human patient eligible for anti-CD19 CAR T cells treatment does not show one or more of the following features: (a) active uncontrolled infection, (b) worsening of clinical status compared to the clinical status prior to lymphodepletion treatment, and (c) grade .gtoreq.2 acute neurological toxicity.

[0170] (iii) Administration of Anti-CD19 CAR T Cells

[0171] Administering anti-CD19 CAR T cells may include placement (e.g., transplantation) of a genetically engineered T cell population as disclosed herein (e.g., the CTX110 cells) into a human patient as also disclosed herein by a method or route that results in at least partial localization of the genetically engineered T cell population at a desired site, such as a tumor site, such that a desired effect(s) can be produced. The genetically engineered T cell population can be administered by any appropriate route that results in delivery to a desired location in the subject where at least a portion of the implanted cells or components of the cells remain viable. The period of viability of the cells after administration to a subject can be as short as a few hours, e.g., twenty-four hours, to a few days, to several weeks or months, to as long as several years, or even the life time of the subject, i.e., long-term engraftment. In certain instances, a patient may receive the genetically engineered T cell population (e.g., CTX110 cells) during inpatient hospital care. In certain instances, a patient may receive genetically engineered T cell population (e.g., CTX110 cells) in outpatient care.

[0172] For example, in some aspects described herein, an effective amount of the genetically engineered T cell population can be administered via a systemic route of administration, such as an intraperitoneal or intravenous route.

[0173] In some embodiments, the genetically engineered T cell population is administered systemically, which refers to the administration of a population of cells other than directly into a target site, tissue, or organ, such that it enters, instead, the subject's circulatory system and, thus, is subject to metabolism and other like processes. Suitable modes of administration include injection, infusion, instillation, or ingestion. Injection includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal, intracerebro spinal, and intrasternal injection and infusion. In some embodiments, the route is intravenous.

[0174] An effective amount refers to the amount of a genetically engineered T cell population needed to prevent or alleviate at least one or more signs or symptoms of a medical condition (e.g., a B cell malignancy), and relates to a sufficient amount of a genetically engineered T cell population to provide the desired effect, e.g., to treat a subject having a medical condition. An effective amount also includes an amount sufficient to prevent or delay the development of a symptom of the disease, alter the course of a symptom of the disease (for example but not limited to, slow the progression of a symptom of the disease), or reverse a symptom of the disease. It is understood that for any given case, an appropriate effective amount can be determined by one of ordinary skill in the art using routine experimentation.

[0175] An effective amount of a genetically engineered T cell population may comprise about 1.times.10.sup.7 CAR+ cells to about 1.times.10.sup.9 CAR+ cells, e.g., about 1.times.10.sup.7 cells to about 1.times.10.sup.9 cells that express a CAR that binds CD19 (CAR+ cells). In some embodiments, an effective amount of a genetically engineered T cell population may comprise at least 1.times.10.sup.7 CAR.sup.+ CTX110 cells, at least 3.times.10.sup.7 CAR.sup.+ CTX110 cells, at least 1.times.10.sup.8 CAR.sup.+ CTX110 cells, at least 3.times.10.sup.8 CAR.sup.+ CTX110 cells, or at least 1.times.10.sup.9 CAR.sup.+ CTX110 cells. In some embodiments, an effective amount of a genetically engineered T cell population may comprise a dose of the genetically engineered T cell population, e.g., a dose comprising about 1.times.10.sup.7 CTX110 cells to about 1.times.10.sup.9 CTX110 cells.

[0176] The efficacy of anti-CD19 CAR T cell therapy described herein can be determined by the skilled clinician. An anti-CD19 CART cell therapy (e.g., involving CTX110 cells) is considered "effective", if any one or all of the signs or symptoms of, as but one example, levels of CD19 are altered in a beneficial manner (e.g., decreased by at least 10%), or other clinically accepted symptoms or markers of a B cell malignancy are improved or ameliorated. Efficacy can also be measured by failure of a subject to worsen as assessed by hospitalization or need for medical interventions (e.g., progression of the B cell malignancy is halted or at least slowed). Methods of measuring these indicators are known to those of skill in the art and/or described herein. Treatment includes any treatment of a B cell malignancy in a human patient and includes: (1) inhibiting the disease, e.g., arresting, or slowing the progression of symptoms; or (2) relieving the disease, e.g., causing regression of symptoms; and (3) preventing or reducing the likelihood of the development of symptoms.

[0177] Following each dosing of anti-CD110 CAR T cells, a human patient may be monitored for acute toxicities such as tumor lysis syndrome (TLS), cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), B cell aplasia, hemophagocytic lymphohistiocytosis (HLH), cytopenia, graft-versus-host disease (GvHD), hypertension, renal insufficiency, or a combination thereof.

[0178] When a human patient exhibits one or more symptoms of acute toxicity, the human patient may be subjected to toxicity management. Treatments for patients exhibiting one or more symptoms of acute toxicity are known in the art. For example, a human patient exhibiting a symptom of CRS (e.g., cardiac, respiratory, and/or neurological abnormalities) may be administered an anti-cytokine therapy. In addition, a human patient that does not exhibit a symptom of CRS may be administered an anti-cytokine therapy to promote proliferation of anti-CTX110 CAR T cells.

[0179] Alternatively, or in addition to, when a human patient exhibits one or more symptoms of acute toxicity, treatment of the human patient may be terminated. Patient treatment may also be terminated if the patient exhibits one or more signs of an adverse event (AE), e.g., the patient has an abnormal laboratory finding and/or the patient shows signs of disease progression.

[0180] The allogeneic anti-CD19 CAR T cell therapy (e.g., involving the CTX110 cells) described herein may also be used in combination therapies. For example, anti-CD19 CAR T cells treatment methods described herein may be co-used with other therapeutic agents, for treating a B cell malignancy, or for enhancing efficacy of the genetically engineered T cell population and/or reducing side effects of the genetically engineered T cell population.

IV. Kit for Allogeneic CAR-T Cell Therapy of B Cell Malignancies

[0181] The present disclosure also provides kits for use of a population of anti-CD19 CAR T cells such as CTX110 cells as described herein in methods for treating a B cell malignancy. Such kits may include one or more containers comprising a first pharmaceutical composition that comprises one or more lymphodepleting agents, and a second pharmaceutical composition that comprises any nucleic acid or population of genetically engineered T cells (e.g., those described herein), and a pharmaceutically acceptable carrier. Kits comprising the genetically engineered CAR-T cells as disclosed herein, such at the CTX110 cells, may be stored and inventoried at the site of care, allowing for rapid treatment of human patients following diagnosis.

[0182] In some embodiments, the kit can comprise instructions for use in any of the methods described herein. The included instructions can comprise a description of administration of the first and/or second pharmaceutical compositions to a subject to achieve the intended activity in a human patient. The kit may further comprise a description of selecting a human patient suitable for treatment based on identifying whether the human patient is in need of the treatment. In some embodiments, the instructions comprise a description of administering the first and second pharmaceutical compositions to a human patient who is in need of the treatment.

[0183] The instructions relating to the use of a population of anti-CD19 CAR T cells such as CTX110 T cells described herein generally include information as to dosage, dosing schedule, and route of administration for the intended treatment. The containers may be unit doses, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions supplied in the kits of the disclosure are typically written instructions on a label or package insert. The label or package insert indicates that the population of genetically engineered T cells is used for treating, delaying the onset, and/or alleviating a T cell or B cell malignancy in a subject.

[0184] The kits provided herein are in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging, and the like. Also contemplated are packages for use in combination with a specific device, such as an inhaler, nasal administration device, or an infusion device. A kit may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access port. At least one active agent in the pharmaceutical composition is a population of the anti-CD19 CAR-T cells such as the CTX110 T cells as disclosed herein.

[0185] Kits optionally may provide additional components such as buffers and interpretive information. Normally, the kit comprises a container and a label or package insert(s) on or associated with the container. In some embodiment, the disclosure provides articles of manufacture comprising contents of the kits described above.

General Techniques

[0186] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature, such as Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed. 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1989) Academic Press; Animal Cell Culture (R. I. Freshney, ed. 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds. 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (D. M. Weir and C. C. Blackwell, eds.): Gene Transfer Vectors for Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); Current Protocols in Molecular Biology (F. M. Ausubel, et al. eds. 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., eds. 1994); Current Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (C. A. Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practice approach (D. Catty, ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and J. D. Capra, eds. Harwood Academic Publishers, 1995); DNA Cloning: A practical Approach, Volumes I and II (D. N. Glover ed. 1985); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. (1985; Transcription and Translation (B. D. Hames & S. J. Higgins, eds. (1984; Animal Cell Culture (R. I. Freshney, ed. (1986; Immobilized Cells and Enzymes (IRL Press, (1986; and B. Perbal, A practical Guide To Molecular Cloning (1984); F. M. Ausubel et al. (eds.).

[0187] Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for the purposes or subject matter referenced herein.

Example 1: Preparation of CD19 Targeting Allogeneic CAR-T Cells

[0188] Allogeneic T cells expressing a chimeric antigen receptor (CAR) specific for CD19 were prepared from healthy donor peripheral blood mononuclear cells as described in US Publication No. US 2018-0325955, incorporated herein by reference. Briefly, primary human T cells were first electroporated with Cas9 or Cas9:sgRNA ribonucleoprotein (RNP) complexes targeting TRAC (AGAGCAACAGTGCTGTGGCC (SEQ ID NO: 26)) and B2M (GCTACTCTCTCTTTCTGGCC (SEQ ID NO: 27)). The DNA double stranded break at the TRAC locus was repaired by homology directed repair with an AAV6-delivered DNA template (SEQ ID NO: 56) containing right and left homology arms to the TRAC locus flanking a chimeric antigen receptor (CAR) cassette. The CAR comprised a single-chain variable fragment (scFv) derived from a murine antibody specific for CD19, a CD8 hinge region and transmembrane domain and a signaling domain comprising CD3z and CD28 signaling domains. The amino acid sequence of the CAR, and nucleotide sequence encoding the same, is set forth in SEQ ID NOs: 40 and 39, respectively. The gRNAs used in this Example comprise the following spacer sequences: TRAC gRNA spacer (AGAGCAACAGUGCUGUGGCC (SEQ ID NO: 19)); and B2M gRNA spacer (GCUACUCUCUCUUUCUGGCC (SEQ ID NO: 21)). A population of cells comprising TRAC.sup.-/.beta.2M.sup.-/anti-CD19 CAR.sup.+ T cells are referred to herein as "TC1 cells" or "CTX110 cells".

[0189] With CRISPR/Cas9 editing technology, high frequency knockout of the constant region of the TCR.alpha. gene (TRAC) with .about.98% reduction of TCR surface expression in human primary T-cells from healthy donors, which aims to significantly impair graft-versus-host disease (GVHD), was achieved. High frequency knockout of the .beta.-2-microglobulin (B2M) gene could also be obtained, which aims to increase persistence in patients, potentially leading to increased potency overall. TRAC/B2M double knockout frequencies have been obtained in .about.80% of T cells without any subsequent antibody-based purification or enrichment. Human T cells expressing a CD19-specific CAR from within a disrupted TRAC locus, produced by homology-directed repair using an AAV6-delivered donor template, along with knockout of the B2M gene have been consistently produced at a high efficiency. This site-specific integration of the CAR protects against the potential outgrowth of CD3+CAR+ cells, further reducing the risk of GVHD, while also reducing the risk of insertional mutagenesis associated with retroviral or lentiviral delivery mechanisms. These engineered allogeneic CAR-T cells show CD19-dependent T-cell cytokine secretion and potent CD19-specific cancer cell lysis.

[0190] The production of allogeneic anti-CD19 CAR-T product (FIG. 1) exhibited efficiency editing (e.g., greater than 50% TRAC-/B2M-/anti-CD19 CAR+T cells efficiency) (FIG. 2).

Example 2: Dose Escalation Study to Determine the Efficacy of CAR-T Cells in the Subcutaneous Raji Human Burkitt's Lymphoma Tumor Xenograft Model in NOG Mice

[0191] The efficacy of CD19 targeting CAR-T cells against the subcutaneous Raji Human Burkitt's Lymphoma tumor xenograft model in NOG mice was evaluated using methods employed by Translational Drug Development, LLC (Scottsdale, Ariz.). In brief, 12, 5-8 week old female, CIEA NOG (NOD.Cg-Prkd.sup.scidIl2rg.sup.tm1Sug/JicTac) mice were individually housed in ventilated microisolator cages, maintained under pathogen-free conditions, 5-7 days prior to the start of the study. On Day 1 mice received a subcutaneous inoculation of 5.times.10.sup.6 Raji cells/mouse. The mice were further divided into 3 treatment groups as shown in Table 1. On Day 8 (7 days post inoculation with the Raji cells), treatment group 2 and group 3 received a single 200 .mu.l intravenous dose of TRAC.sup.-/B2M.sup.-/anti-CD19 CAR+ cells (TC1) according to Table 1.

TABLE-US-00001 TABLE 1 Treatment groups. Group Raji Cells (s.c.) TC1 Treatment (i.v.) N 1 5 .times. 10.sup.6 cells/mouse None 4 2 5 .times. 10.sup.6 cells/mouse 5 .times. 10.sup.6 cells/mouse 4 3 5 .times. 10.sup.6 cells/mouse 1 .times. 10.sup.7 cells/mouse 4

[0192] Tumor volume and body weight was measured and individual mice were euthanized when tumor volume was .gtoreq.500 mm.sup.3.

[0193] By Day 18, the data show a statistically significant decrease in the tumor volume in response to TC1 cells as compared to untreated mice (FIG. 3). The effect on tumor volume was dose-dependent (Table 2); mice receiving higher doses of TC1 cells showed significantly reduced tumor volume when compared to mice receiving either a lower dose of TC1 cells or no treatment. An increase in survival was also observed in the treated group (Table 2).

TABLE-US-00002 TABLE 2 Tumor response and survival. Tumor Tumor Group volume (Day 18) volume (Day 20) Survival (Days) N 1 379.6 .+-. 67.10 482 .+-. 47.37 20-22 4 2 214.0 .+-. 20.73 372.2 .+-. 78.21 25 4 3 107.5 .+-. 7.33* 157.1 .+-. 10.62** 27 (end of study) 4 p = 0.007 compared to control (Group 1) **p = 0.0005 compared to control (Group 1)

Example 3: Assessment of CD19 Targeting CAR-T Cells Efficacy in Intravenous Disseminated Models in NOG Mice

[0194] To further assess the efficacy of TRAC.sup.-/B2M.sup.-/anti-CD19 CAR+ cells (TC1), disseminated mouse models were utilized.

[0195] Intravenous Disseminated Raji Human Burkitt's Lymphoma Tumor Xenograft Model

[0196] The Intravenous Disseminated Model (Disseminated Model) using the Raji Human Burkitt's Lymphoma tumor cell line in NOG mice was used to further demonstrate the efficacy of TC1. Efficacy of TC1 was evaluated in the Disseminated Model using methods employed by Translations Drug Development, LLC (Scottsdale, Ariz.) and described herein. In brief, 24, 5-8 week old female CIEA NOG (NOD.Cg-Prkde.sup.scidI12rg.sup.t1Sug/JicTac) mice were individually housed in ventilated microisolator cages, maintained under pathogen-free conditions, 5-7 days prior to the start of the study. At the start of the study, the mice were divided into 5 treatment groups as shown in Table 9. On Day 1 mice in Groups 2-5 received an intravenous injection of 0.5.times.10.sup.6 Raji cells/mouse. The mice were inoculated intravenously to model disseminated disease. On Day 8 (7 days post injection with the Raji cells), treatment Groups 3-5 received a single 200 .mu.l intravenous dose of TC1 cells (Table 3).

TABLE-US-00003 TABLE 3 Treatment groups. Group Raji Cells (i.v.) TC1 Treatment (i.v.) N 1 None None 8 2 0.5 .times. 10.sup.6 cells/mouse None 4 3 0.5 .times. 10.sup.6 cells/mouse 1 .times. 10.sup.6 cells/mouse 4 (~0.5 .times. 10.sup.6 CAR-T+ cells) 4 0.5 .times. 10.sup.6 cells/mouse 2 .times. 10.sup.6 cells/mouse 4 (~1.0 .times. 10.sup.6 CAR-T+ cells) 5 0.5 .times. 10.sup.6 cells/mouse 4 .times. 10.sup.6 cells/mouse 4 (~2.0 .times. 10.sup.6 CAR-T+ cells)

[0197] During the course of the study mice were monitored daily and body weight was measured two times weekly. A significant endpoint was the time to peri-morbidity and the effect of T-cell engraftment was also assessed. The percentage of animal mortality and time to death were recorded for every group in the study. Mice were euthanized prior to reaching a moribund state. Mice may be defined as moribund and sacrificed if one or more of the following criteria were met:

[0198] Loss of body weight of 20% or greater sustained for a period of greater than 1 week;

[0199] Tumors that inhibit normal physiological function such as eating, drinking, mobility and ability to urinate and or defecate;

[0200] Prolonged, excessive diarrhea leading to excessive weight loss (>20%); or

[0201] Persistent wheezing and respiratory distress.

[0202] Animals were also considered moribund if there was prolonged or excessive pain or distress as defined by clinical observations such as: prostration, hunched posture, paralysis/paresis, distended abdomen, ulcerations, abscesses, seizures and/or hemorrhages.

[0203] Similar to the subcutaneous xenograph model (Example 2), the Disseminated Model revealed a statistically significant survival advantage in mice treated with TRAC.sup.-/B2M.sup.-/anti-CD19 CAR+ cells (TC1) as shown in FIG. 4, p<0.0001. The effect of TC1 treatment on survival in the disseminated model was also dose dependent (Table 4).

TABLE-US-00004 TABLE 4 Animal survival. Raji TC1 Max Median Group Cells (i.v.) Treatment (i.v.) survival (days) survival (days) 1 No No Max Max 2 Yes No 20 20 3 Yes 1 .times. 10.sup.6 cells/mouse 21 21 4 Yes 2 .times. 10.sup.6 cells/mouse 25 25 5 Yes 4 .times. 10.sup.6 cells/mouse 32 26

[0204] A second experiment was run using the Intravenous Disseminated model described above.

[0205] On Day 1 mice in Groups 2-4 received an intravenous injection of 0.5.times.10.sup.6 Raji cells/mouse. The mice were inoculated intravenously to model disseminated disease. On Day 4 (3 days post injection with the Raji cells), treatment Groups 2-4 received a single 200 .mu.l intravenous dose of TC1 cells per Table 5.

TABLE-US-00005 TABLE 5 Treatment groups. Group Raji Cells (i.v.) TC1 Treatment (i.v.) N 1 0.5 .times. 10.sup.6 cells/mouse None 6 2 0.5 .times. 10.sup.6 cells/mouse 0.6 .times. 10.sup.6 CAR.sup.+ cells/mouse 7 3 0.5 .times. 10.sup.6 cells/mouse 1.2 .times. 10.sup.6 CAR.sup.+ cells/mouse 5 4 0.5 .times. 10.sup.6 cells/mouse 2.4 .times. 10.sup.6 CAR.sup.+ cells/mouse 5

[0206] Again, the Disseminated Model revealed a statistically significant survival advantage in mice treated with TRAC.sup.-/B2M.sup.-/anti-CD19 CAR+ cells (TC1) as shown in FIG. 5, p=0.0016. The effect of TC1 treatment on survival in the disseminated model was also dose dependent (Table 6).

TABLE-US-00006 TABLE 6 Animal survival. Raji Max Median Cells TC1 survival survival Group (i.v.) Treatment (i.v.) (days) (days) Significance 1 Yes No 20 20 2 Yes 0.6 .times. 10.sup.6 CAR.sup.+ 35 27 p = 0.005 cells/mouse 3 Yes 1.2 .times. 10.sup.6 CAR.sup.+ 39 37 p = 0.016 cells/mouse 4 Yes 2.4 .times. 10.sup.6 CAR.sup.+ 49 46 p = 0.016 cells/mouse

[0207] Evaluation of Splenic Response to TC1 Treatment

[0208] The spleen was collected from mice 2-3 weeks following Raji injection and the tissue was evaluated by flow cytometry for the persistence of TC1 cells and eradication of Raji cells in the spleen.

[0209] The spleen was transferred to 3 mL of 1.times.DPBS CMF in a C tube and dissociated using the MACS Octo Dissociator. The sample was transferred through a 100 micron screen into a 15 mL conical tube, centrifuged (1700 rpm, 5 minutes, ART with brake) and resuspended in 1 mL of 1.times.DPBS CMF for counting using the Guava PCA. Bone marrow was centrifuged and resuspended in 1 mL of 1.times.DPBS CMF for counting using the Guava PCA. Cells were resuspended at a concentration of 10.times.10.sup.6 cells/mL in 1.times.DPBS CMF for flow cytometry staining.

[0210] Specimens (50 .mu.L) were added to 1 mL 1.times. Pharm Lyse and incubated for 10-12 minutes at room temperature (RT). Samples were centrifuged and then washed once with 1.times.DPBS CMF. Samples were resuspended in 50 .mu.L of 1.times.DPBS and incubated with Human and Mouse TruStain for 10-15 minutes at RT. The samples were washed once with 1 mL 1.times.DPBS CMF and resuspend in 50 .mu.L of 1.times.DPBS CMF for staining. Surface antibodies were added and the cells incubated for 15-20 minutes in the dark at RT and then washed with 1 mL 1.times.DPBS CMF. Then samples were resuspended in 125 .mu.L of 1.times.DPBS CMF for acquisition on the flow cytometer. Cells were stained with the following surface antibody panel:

TABLE-US-00007 TABLE 7 Antibody panel. FITC PE APC C3 APCCy7 V421 V510 huCD3 huCD45 huCD19 7AAD CD8 CD4 mCD45 (UCHT1) (HI30) (HIB19) (SK1) (RPA-T4) (30-F11)

[0211] Cell populations were determined by electronic gating (Pl=total leukocytes) on the basis of forward versus side scatter. Compensation to address spill over from one channel to another was performed upon initial instrument set up using Ultra Comp Beads from Thermo Fisher. The flow cytometer was set to collect 10,000 CD45+ events in each tube. Flow cytometric data acquisition was performed using the FACSCantoll.TM. flow cytometer. Data was acquired using BO FACSDiva.TM. software (version 6.1.3 or 8.0.1). Flow cytometry data analysis was in the form of Flow Cytograms, which are graphical representations generated to measure relative percentages for each cell type.

[0212] This example demonstrates that following TC1 cell treatment, the therapeutically beneficial TRAC.sup.-/B2M.sup.-/anti-CD19 CAR+ cells persist in the spleen and selectively eradicate Raji cells from the tissue (FIG. 6A). In addition, treatment with TC1 cells do not exhibit Raji induced increase in cell mass (FIG. 6A). Further, FIG. 7 shows that the remaining human cells in spleens of mice treated with TRAC.sup.-/B2M.sup.-/anti-CD19 CAR+ cells are CD8+. These CD8+ T cells are also CD3 negative proving that persistent T cells in this model remain TCR/CD3 negative and are thus edited.

[0213] Intravenous Disseminated Nalm-6 Human Acute Lymphoblastic Leukemia Tumor Xenograft Model

[0214] The Intravenous Disseminated Model (Disseminated Model) using the Nalm-6 Human Acute Lymphoblastic Leukemia tumor cell line in NOG mice was used in to further demonstrate the efficacy of TC1. Efficacy of TC1 was evaluated in the Disseminated Model using methods employed by Translations Drug Development, LLC (Scottsdale, Ariz.) and described herein. In brief, 24, 5-8 week old female CIEA NOG (NOD.Cg-Prkd.sup.scidIl2rg.sup.tm1Sug/JicTac) mice were individually housed in ventilated microisolator cages, maintained under pathogen-free conditions, 5-7 days prior to the start of the study. At the start of the study, the mice were divided into 5 treatment groups as shown in Table 14. On Day 1 mice in Groups 2-4 received an intravenous injection of 0.5.times.10.sup.6 Nalm6 cells/mouse. The mice were inoculated intravenously to model disseminated disease. On Day 4 (3 days post injection with the Nalm6 cells), treatment Groups 2-4 received a single 200 .mu.l intravenous dose of TC1 cells per Table 8.

TABLE-US-00008 TABLE 8 Treatment groups. Group Nalm6 Cells (i.v.) TC1 Treatment (i.v.) N 1 0.5 .times. 10.sup.6 cells/mouse None 6 2 0.5 .times. 10.sup.6 cells/mouse 1 .times. 10.sup.6 CAR.sup.+ cells/mouse 6 3 0.5 .times. 10.sup.6 cells/mouse 2 .times. 10.sup.6 CAR.sup.+ cells/mouse 6 4 0.5 .times. 10.sup.6 cells/mouse 4 .times. 10.sup.6 CAR.sup.+ cells/mouse 6

[0215] During the course of the study mice were monitored daily and body weight was measured two times weekly as described above.

[0216] Similar to the Raji intravenous disseminated model (above), the Nalm6 Model also showed a statistically significant survival advantage in mice treated with TRAC.sup.-/B2M.sup.-/anti-CD19 CAR+ cells (TC1) as shown in FIG. 8, p=0.0004. The effect of TC1 treatment on survival in the Nalm6 disseminated model was also dose dependent (Table 9).

TABLE-US-00009 TABLE 9 Animal survival. Nalm6 TC1 Max Median Cells Treatment survival Survival Group (i.v.) (i.v.) (days) (days) Significance 1 Yes No 31 25.5 2 Yes 1 .times. 10.sup.6 CAR.sup.+ 32 31 p = 0.03 cells/mouse 3 Yes 2 .times. 10.sup.6 CAR.sup.+ 38 36 p = 0.0004 cells/mouse 4 Yes 4 .times. 10.sup.6 CAR.sup.+ 52 46 p = 0.0004 cells/mouse

Example 4: Further Assessment of CD19 Targeting CAR-T Cells Efficacy in Intravenous Disseminated Models in NOG Mice

[0217] The purpose of this study was to evaluate the anti-tumor activity of anti-CD19 CAR+ T cells at multiple dose levels against the Nalm6-Fluc-GFP acute lymphoblastic leukemia tumor cell line in NOG mice. The mice were inoculated intravenously to model disseminated disease. Significant endpoint was time to pen-morbidity. Bioluminescent imaging was performed to monitor progression of disseminated disease.

[0218] In brief, 6 week old female, CIEA NOG (NOD.Cg-Prkdc.sup.scidIl2rg.sup.tm1Sug/JicTac) mice were housed in ventilated microisolator cages, maintained under pathogen-free conditions, 5-7 days prior to the start of the study. On Day 1 mice received an intravenous inoculation of 5.times.10.sup.4 Nalm6-Fluc-GFP (Nalm6-Fluc-Neo/eGFP--Puro; Imanis Life Sciences (Rochester, Minn.)) cells/mouse. Three (3) days post inoculation with Nalm6-Fluc-GFP cells, the mice were divided into treatment groups and dosed with T cell populations comprising TRAC-/B2M-/anti-CD19 CAR+ T cells, as indicated in Table 10. Region of Interest values (ROI) values were captured and reported. Body weight was measured twice daily and bioluminescence was measured twice weekly starting on Day 4 (3 Days Post inoculation of Nalm6-Fluc-GFP cells) through Day 67, once weekly starting Day 74 to study end. To measure bioluminescence mice were injected intraperitoneally with 200 .mu.l of D-Luciferin 150 mg/kg. Kinetics images were taken at the beginning of the study and as needed throughout to determine optimal post D-Luciferin dose and exposure time to image the mice. Mice were imaged by capturing luminescence signal (open emission) using an AMI 1000 imaging unit with software version 1.2.0 (Spectral Instruments Imaging Inc.; Tucson, Ariz.).

TABLE-US-00010 TABLE 10 Treatment groups. # of T Cells Anti-CD19 Group Anti-CD19 CAR T Cell injected (iv) CAR+ T cells N 1 N/A N/A N/A 5 2 TRAC-/.beta.2M-/anti-CD19 3 .times. 10.sup.6 ~1.8 .times. 10.sup.6 5 cells/mouse 3 TRAC-/.beta.2M-/anti-CD19 6 .times. 10.sup.6 ~3.6 .times. 10.sup.6 5 cells/mouse 4 TRAC-/.beta.2M-/anti-CD19 12 .times. 10.sup.6 7.2 .times. 10.sup.6 4 cells/mouse

[0219] Individual mice were euthanized at pen-morbidity (clinical signs suggesting high tumor burden (e.g., lack of motility, hunch back, hypoactivity) or 20% or greater body weight loss sustained for a period of greater than 1-week). Mice were euthanized prior to reaching a moribund state. The study was ended on Day 99 when the final mouse was euthanized as a long-term survivor.

[0220] FIG. 9 shows prolonged survival of mice that received different doses of TC1 cells relative to untreated mice. FIG. 10 shows low to undetectable levels of bioluminescence in mice that received the highest dose of TC1 cells (12.times.10.sup.6 cells/mouse) and which resulted in the longest survival as shown in FIG. 9. At day 74 bioluminescence was detected in all 4 mice, indicative of tumor cell expansion in the treatment group.

[0221] Overall, these results show a single injection of TC1 cells can prolong survival of mice that were administered a lethal dose of Nalm6 B-ALL cells. This prolonged survival is dose dependent with a graded survival response observed between low, middle and high doses of TC1 cells.

Example 5: Analysis of Graft Versus Host Disease in Mice Administered Allogeneic CD19 Targeting CAR T Cells

[0222] A study in mice was conducted to evaluate the potential for both unedited human T cells and TC1 cells to cause graft versus host disease (GvHD). After total body irradiation with 200 cGy, NOG female mice were administered a single intravenous slow bolus injection of unedited human T cells or TC1 cells. Animals were followed for up to 119 days after radiation only (Group 1) or radiation plus a single dose administration of PBMCs (Group 2), electroporated T cells (Group 3) or TC1 cells (Group 4). Cells were administered approximately 6 hours post radiation on Day 1. Table 11 summarizes the groups and study design.

TABLE-US-00011 TABLE 11 Treatment groups. Total Number of Group Dose Level Irradiation Animals Number Test Article (cells/mouse) Dose (Female) 1 Radiation Only 0 200 cGy 12 2 Radiation + PBMCs 6 .times. 10P.sup.6 6 3 Radiation + EP T cells 3 .times. 10P.sup.7 6 4 Radiation + TC1 cells 3 .times. 10P.sup.7 6

[0223] The endpoints of the study were survival, kinetics of appearance of GvHD symptoms, and body weight measurements.

[0224] Mortality was observed in Group 1 (3 of 12 animals), Group 2 (6 of 6 animals) and Group 3 (2 of 6 animals) during the first 30 days post-treatment (FIG. 11). All animals in Group 4 (TC1 cells) survived until scheduled necropsy (FIG. 11). Moribund animals in Groups 1, 2 and 3 experienced weight loss and/or clinical observations consistent with the development of GvHD (slight to severe cold to touch, slight to moderate emaciation, slight to marked hunched posture, severe weight loss, mild to severe alopecia, severe hypoactivity, moderate labored respiration, and marked tachypnea). Animals in Groups 1 and 4, and non-moribund animals in Group 3, experienced mild weight loss following radiation which improved over the course of the study (FIG. 12). No notable clinical observations were recorded.

[0225] This study demonstrated that unedited human PBMCs induce fatal GvHD in irradiated NOG mice in all animals (Group 2), with onset 2 to 3 weeks after administration of cells. In contrast, no mice that received TC1 cells (Group 4) developed GvHD during the study (119 days), despite the higher number of cells that were administered to these animals (3.times.10.sup.7 TC1 cells per mouse compared to 6.times.10.sup.6 PBMCs per mouse). The irradiation procedure induced transient weight loss in all groups and recovered in all groups that did not receive unedited PBMCs.

[0226] A second study was conducted to further evaluate the potential for both unedited human T cells and TC1 cells to cause GvHD. Specifically, NOD/SCID/IL2R.gamma.null (NSG) female mice were administered a single intravenous slow bolus injection of unedited human T cells or TC1 cells after a total body irradiation (total irradiation dose of 200 cGy, 160 cGy/min; targeted LDR.sub.0/140R). The endpoints of this study were survival, kinetics of appearance of symptoms of GvHD and body weight measurements. Histopathology was also performed on all collected tissues. Exposure was assessed in mouse blood and tissues by flow cytometry and immunohistochemistry (IHC), where appropriate.

[0227] The cells were administered as a single dose via intravenous slow bolus as described in Table 12.

TABLE-US-00012 TABLE 12 Study Design. Concen- Total Number Dose tration Ir- of Group (Cells/ (Cells/ radiation Animals Number Test Article Mouse) mL) Dose M F 1 Vehicle - no RT.sup.a 0 0 0 cGy 5 5 2 Vehicle - RT.sup.a 0 0 200 cGy 15 15 3 Unedited T cells 1 .times. 10.sup.7 4 .times. 10.sup.7 15 15 4 TC1 - low dose 2 .times. 10.sup.7 8 .times. 10.sup.7 15 15 5 TC1 - high dose 4 .times. 10.sup.7 16 .times. 10.sup.7 15 15 .sup.aGroup 1 animals were not irradiated and were not dosed with cells (animals were administered with vehicle, PBS 1X). Group 2 animals were irradiated but were not dosed with cells (animals were administered with vehicle, phosphate-buffered saline [PBS]).

[0228] Animals were randomized into treatment groups by body weight using a validated preclinical software system (Provantis). Due to the large size of this study, dosing and necropsy activities were staggered over nine days. To minimize bias, animals from the control and TC1 groups (Groups 4 and 5) were dosed and necropsied on the same day. Necropsy occurred on Study Day 85 for all groups.

[0229] Mortality was observed for all animals that received unedited human T cells (Group 3), with onset at Day 14 (FIG. 13). All mice that received unedited human T cells (Group 3), were either found dead or sent to unscheduled euthanasia by Day 29. Clinical signs in these animals were consistent with the development of GvHD and included dull fur, slight to severe decreased activity, hunched back posture, slight to moderate thinness, and increased respiratory rate. Marked changes in hematology parameters were observed at euthanasia in mice that received unedited human T cells (Group 3), including decreases in red blood cells, hemoglobin, platelets, white blood cells and reticulocyte counts. Minimal to moderate inflammation was observed in the liver, lung, kidney, spleen, and thymus of Group 3 animals. Necrosis often accompanied inflammation in these tissues. These findings were consistent with the development of GvHD. Additionally, mild to severe hypocellularity in the femoral and sternal bone marrow was also present in the majority of Group 3 animals, which was likely attributable to the effects of total body irradiation. This was likely only observed in this group due to the early necropsy dates (2-4 weeks post-radiation), compared to 12 weeks for all other groups. Consistent with the presence of GvHD, immunohistochemical analysis of Group 3 animals revealed the presence of human CD45P.sup.+P cells in all tissues examined (kidney, liver, spleen, lung, skin, and the digestive tract). All animals in the other Groups survived until the scheduled necropsy.

[0230] Further, no significant weight loss was observed in Groups 1, 2, 4, or 5 (FIG. 14). No notable clinical observations that were consistent with GvHD, characterized by observations of at least two symptoms considered likely to denote GvHD, were recorded in these groups. Several animals from Groups 4 and 5 exhibited symptoms such as dull fur, slight to moderate decreased activity, and/or slight thinness throughout the study. Although these symptoms are often associated with GvHD, they did not appear to be TC1-related as they were infrequently observed, transient and of short duration, and were also seen in some irradiated control animals (Group 2).

[0231] Overall the results from these two studies confirmed TC1 cells do not induce graft versus host disease.

Example 6: Preparation and Characterization of Developmental Lots of Allogeneic CD19 Targeting CAR T Cells

[0232] TC1 cells for the purposes of the clinical study were prepared from healthy donor peripheral blood mononuclear cells obtained via a standard leukopheresis procedure. The mononuclear cells were enriched for T cells and activated with anti-CD3/CD28 antibody-coated beads, then electroporated with CRISPR-Cas9 ribonucleoprotein complexes and transduced with a CAR gene-containing recombinant adeno-associated virus (AAV) vector. The modified T cells were expanded in cell culture, purified, formulated into a suspension, and cryopreserved.

[0233] Prior to modifying the cells, T cells from six different healthy donors were evaluated for expression of various cell surface markers. CD27+CD45RO- T cells within the CD8+ subset were previously shown to correlate with complete responses in chronic lymphocytic leukemia (CLL) when treated with anti-CD19 CAR T cell therapy (Fraietta et al., Nat Med, Vol. 24(5): 563-571, 2018). Accordingly, the percent of CD27+CD45O- T cells within the CD8+ subset of six different donors was evaluated by flow cytometry. In brief, 1.times.10.sup.6 cells were incubated with Fab-Biotin or IgG-Biotin antibodies as a negative control. Cells were washed with staining buffer and incubated with mouse anti-IgG to capture excess primary antibodies. Cells were washed again and incubated with the full panel of secondary antibodies (CD8, Biolegend: Catalog #300924, CD45RO, Biolegend: Catalog #304230, CD27, Biolegend: Catalog #560612) and viability dye. Cells were washed a final time with staining buffer and run on the flow cytometer to capture various stained populations. FIG. shows the levels of CD27+CD45RO- T cells within their CD8+ subsets. Allogeneic CAR-T manufacturing allows for the selection of donor input material with favorable characteristics, such as high CD27+CD45RO- cells in the CD8+ fraction of a donor of interest.

[0234] More specifically, leukopaks from 18 to 40 year-old male donors were used to isolate CD4+ and CD8+ T cells. After isolation, enrichment and activation of CD4+ and CD8+ T cells, cells were electroporated with ribonucleoprotein complexes comprising Cas9 nuclease protein, TRAC sgRNA (SEQ ID NO: 26) or B2M sgRNA (SEQ ID NO: 27). The TRAC and B2M ribonucleoprotein complexes were combined prior to electroporation. After electroporation, freshly thawed rAAV comprising a donor template (SEQ ID NO: 54) encoding the anti-CD19 CAR (SEQ ID NO: 40) was added to the cells, and cells were incubated. Cells were then expanded in culture and supplemented with rhIL-2 and rhIL-7 every three to four days. Cells set up for monitoring were tested for T cell identity and gene editing with a TCR panel (CD5, CD4, CD8, TCR.alpha..beta., B2M and CD45). Upon confirmation of T cell identity, TCR.alpha..beta. depletion was performed by incubating the cells with a biotin-conjugated anti-TCR.alpha..beta. antibody and anti-biotin beads. The depleted cells were recovered and formulated for administration. The resulting population of cells had less than 0.5% TCR.alpha..beta.+ cells. FIG. 16 shows the analysis of TCR.alpha..beta.+ cells before and after purification.

[0235] Eight development lots of TC1 cells were tested for T cell identity. Average results from eight tested lots showed 84.58% knock-out of B2M (i.e., 15.42% B2M.sup.+ cells) and 99.98% of cells were TCR- (i.e., 0.2% TCR+), and .about.50% knock-in of anti-CD19 CAR (FIG. 17).

[0236] In addition, exhaustion and senescent markers were evaluated in donors before and after T cell editing. Specifically, the percentage of PD1+, LAG3+, TIM3+ and CD57+ cells were determined from total T cell populations. Expression of the markers was assessed by flow cytometry, as described above, using the following secondary antibodies: Mouse Anti-PD1 PeCy7, Biolegend, Catalog #329918; Mouse Anti-TIM3BV421, Biolegend, Catalog #345008; Mouse Anti-CD57 PerCp Cy5.5, Biolegend, Catalog #359622; and Mouse Anti-LAG3 PE, Biolegend, Catalog #369306. FIG. 18 shows that exhaustion or senescent markers never increased over 15% of the total T cell population after genome editing.

[0237] In addition, selective killing by three different lots of TC1 cells was evaluated in vitro. Specifically, TC1 cells were incubated with CD19-positive cell lines (K562-CD19; Raji; and Nalm6), or a CD19-negative cell line (K562). Killing was measured using a flow cytometry-based cytotoxicity assay after .about.24 hours. Specifically, target cells were labeled with 5 .mu.M efluor670 (Thermo Fisher Scientific, Waltham, Mass.), washed and incubated overnight (50,000 target cells/well; 96-well U-bottom plate [Corning, Tewksbury, Mass.]) in co-cultures with TC1 or control T cells at varying ratios (from 0.1:1 up to 4:1 T cells to target cells). The next day, wells were washed and media was replaced with 200 .mu.L of fresh media containing a 1:500 dilution of 5 mg/mL 4',6-diamidino-2-phenylindole (DAPI) (Thermo Fisher Scientific, Waltham, Mass.) to enumerate dead/dying cells. Finally, 25 .mu.L of CountBright beads (Thermo Fisher Scientific) was added to each well, and cells were then analyzed by flow cytometry using a Novocyte flow cytometer (ACEA Biosciences, San Diego, Calif.). Flowjo software (v10, Flowjo, Ashland, Oreg.) was used to analyze flow cytometry data files (fcs files). TCR.alpha..beta.+ T cells (unedited cells) were used as controls. TC1 cells efficiently killed CD19-positive cells at higher rates than unedited T cells, and CD19-negative cells showed low levels of cell lysis in the presence of TC1 cells that were no more than when co-cultured with unedited T cells (FIG. 19).

[0238] TC1 cells produced from three unique donors were also used to assess growth in the absence of cytokine and/or serum. Specifically, TC1 cells were grown in full T cell media for 14 days. On Day 0, cells from culture were grown either in complete T-cell media (containing X-VIVO 15 (Lonza, Basel, Switzerland), 5% human AB serum (Valley Biomedical, Winchester, Va.), IL-2 (Miltenyi, Bergisch Gladbach, Germany) and IL-7 (Cellgenix, Frieburg, Germany)) (Complete Media), media containing serum but no IL-2 or IL-7 cytokines (5% serum, no cytokines), or no serum or cytokines (No serum, No Cytokines). Cells were enumerated as above for up to 35 days after removal of cytokines and/or serum. No outgrowth of TC1 cells was observed in the absence of cytokine and/or serum (FIG. 20).

[0239] For administration, TC1 cells are resuspended in cryopreservative solution (CryoStor CS-5) and supplied in a 6 mL infusion vial. The total dose is contained in one or more vials. The infusion of each vial occurs within 20 minutes of thawing.

Example 7: A Phase I, Open-Label, Multicenter, Dose Escalation and Cohort Expansion Study of the Safety and Efficacy of Allogeneic CRISPR-Cas9 Engineered T Cells (CTX110) in Subjects with Relapsed or Refractory B Cell Malignancies

[0240] CTX110 is a CD19-directed chimeric antigen receptor (CAR) T cell immunotherapy comprised of allogeneic T cells that are genetically modified ex vivo using CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) gene editing components (single guide RNA and Cas9 nuclease). The modifications include targeted disruption of the T cell receptor (TCR) alpha constant (TRAC) and beta-2 microglobulin (B2M) loci, and the insertion of an anti-CD19 CAR transgene into the TRAC locus via an adeno-associated virus expression cassette. The anti-CD19 CAR (SEQ ID NO: 40) is composed of an anti-CD19 single-chain variable fragment comprising the SEQ ID NO: 47, the CD8 transmembrane domain of SEQ ID NO: 32, a CD28 co-stimulatory domain of SEQ ID NO: 36, and a CD3.zeta. signaling domain of SEQ ID NO: 38.

[0241] In this study, eligible human patients received an intravenous (IV) infusion of CTX110 following lymphodepleting (LD) chemotherapy.

Study Population

[0242] Dose escalation and cohort expansion include adult subjects with B cell malignancies. Subjects are assigned to independent dose escalation groups based on disease histology. Enrolled adult subjects include those with select subtypes of non-Hodgkin lymphoma (NHL), including diffuse large B cell lymphoma (DLBCL) not otherwise specified (NOS), high grade B cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements, transformed follicular lymphoma (FL), grade 3b FL or Richter's transformation of CLL.

Study Purpose and Rationale

[0243] The purpose of the Phase 1 dose escalation study is to evaluate the safety and efficacy of anti-CD19 allogeneic CRISPR-Cas9 engineered T cells (CTX110 cells) in subjects with relapsed or refractory B cell malignancies.

[0244] Outcomes for patients with relapsed/refractory B cell malignancies are historically poor. However, the use of autologous CAR T cell therapy in this setting has produced complete and durable responses where previous treatment options were palliative (June et al., (2018) Science, 359, 1361-1365; Maus and June, (2016) Clin Cancer Res, 22, 1875-1884; Neelapu et al., (2017) N Engl J Med, 377,2531-2544; Schuster et al., (2019) N Engl J Med, 380, 45-56; Schuster et al., (2017) N Engl J Med, 377, 2545-2554). Autologous CAR T cell therapies require patient-specific cell collection and manufacturing. Unfortunately, some patients are not candidates to undergo leukapheresis, or they experience disease progression or death while awaiting treatment. An allogeneic off-the-shelf CAR T cell product such as CTX110 could provide the benefit of immediate availability, reduce manufacturing variability, and prevent individual subject manufacturing failures.

[0245] Further, patients treated with multiple rounds of chemotherapy may have T cells with exhausted or senescent phenotypes. The low response rates in patients with chronic lymphocytic leukemia (CLL) treated with autologous CAR T cell therapy have been partially attributed to the exhausted T cell phenotype (Fraietta et al., (2018) Nat Med, 24, 563-571; Riches et al., (2013) Blood, 121, 1612-1621). By starting with chemotherapy-naive T cells from a healthy donor, allogeneic approaches could increase the consistency and potency of CAR T therapy as compared to autologous products.

[0246] The main barrier to the use of allogeneic CAR T cells has been the risk of graft versus host disease (GvHD). CRISPR Cas9 gene-editing technology allows for reliable multiplex cellular editing. The CTX110 manufacturing process couples the introduction of the CAR construct to the disruption of the TRAC locus through homologous recombination. The delivery and precise insertion of the CAR at the TRAC genomic locus using an AAV-delivered DNA donor template and HDR contrasts with the random insertion of genetic material using lentiviral and retroviral transduction methods. CAR gene insertion at the TRAC locus results in elimination of TCR in nearly all cells expressing the CAR, which minimizes risk of GvHD. Furthermore, manufacturing from healthy donor cells removes the risk of unintentionally transducing malignant B cells (Ruella et al., (2018) Nat Med, 24, 1499-1503). This first-in-human trial in subjects with relapsed/refractory B cell malignancies aims to evaluate the safety as well as efficacy of CTX110 with this CRISPR-Cas9-modified allogeneic CAR T cell approach.

[0247] CTX110, a CD19-directed genetically modified allogeneic T-cell immunotherapy, is manufactured from the cells of healthy donors; therefore, the resultant manufactured cells are intended to provide each subject with a consistent, final product of reliable quality. Furthermore, the manufacturing of CTX110, through precise delivery and insertion of the CAR at the TRAC site using AAV and homology-directed repair (HDR), does not present the risks associated with random insertion of lentiviral and retroviral vectors.

Objectives

[0248] Primary objective, Part A (Dose escalation): To assess the safety of escalating doses of CTX110 in combination with various lymphodepletion agents in subjects with relapsed or refractory B cell malignancies to determine the recommended Part B dose.

[0249] Primary objective, Part B (Cohort expansion): To assess the efficacy of CTX110 in subjects with relapsed or refractory B cell malignancies, as measured by objective response rate (ORR).

[0250] Secondary objectives (Parts A and B): To further characterize the efficacy, safety, and pharmacokinetics of CTX110.

[0251] Exploratory objectives (Parts A and B): To identify genomic, metabolic, and/or proteomic biomarkers associated with CTX110 that may indicate or predict clinical response, resistance, safety, or pharmacodynamic activity.

Endpoints

Primary Endpoints

[0252] Part A: The incidence of adverse events, defined as dose-limiting toxicities. [0253] Part B: The objective response rate (ORR) defined as complete response (CR)+partial response (PR) per the Lugano Response Criteria for Malignant Lymphoma (Cheson et al., (2014) J Clin Oncol, 32, 3059-3068), as determined by independent central radiology review.

[0254] The Lugano Classification provides a standardized way to assess imaging in lymphoma subjects. It is comprised of radiologic assessments of tumor burden on diagnostic CT, and metabolic assessments on F.sup.18 FDG-PET for FDG-avid histologies (see Tables 13-14).

TABLE-US-00013 TABLE 13 Lugano Classification Assessment Components. Diagnostic CT/MRI F.sup.18 FDC-PET Target Lymph Nodes and Extra Nodal 5 Point Scale (Deauville) PET Score Lesions (Lymph Nodes and Extra Lymphatic Sites) * Up to 6 of the largest target nodes, nodal masses, The 5-point scale scores the site of the most or other lymphomatous lesions that are intense FDG uptake for the time point, as follows: measurable in two diameters (longest diameter Score Criteria [LDi] and shortest diameter) should be identified 1 No uptake from different body regions representative of the 2 Uptake .ltoreq. mediastinum subject's overall disease burden and include 3 Uptake > mediastinum but .ltoreq. liver mediastinal and retroperitoneal disease, if 4 Uptake moderately higher than liver involved. (moderately indicates uptake greater Nodal disease: Must have an LDi > 1.5 cm than normal liver) Extranodal disease: Must have an 5 Uptake markedly higher than liver LDi > 1.0 cm (markedly indicates much higher than Non-Measured Lesions normal liver) All other lesions (including nodal, extranodal, and/or and assessable disease) should be followed as New lesions nonmeasured disease (e.g., cutaneous, GI, bone, X New areas of uptake unlikely to be spleen, liver, kidneys, pleural or pericardial related to lymphoma effusions, ascites). Bone Marrow: FDG uptake assessed as Organ Enlargement (Spleen) No FDG uptake consistent with lymphoma The spleen is considered enlarged Focal FDG uptake consistent with (splenomegaly) when >13 cm in the cranial to lymphoma caudal dimension. Diffuse FDG uptake consistent with New Lesions lymphoma Nodal disease: Must have an LDi > 1.5 cm Extranodal disease: Any size CT: computed tomography; F.sup.18 FDG: fluorodeoxyglucose F18; LDi: longest diameter; MRI: magnetic resonance imaging; PET: positron emission tomography. * See (Barrington et al., (2014) J Clin Oncol, 32, 3048-3058).

TABLE-US-00014 TABLE 14 Lugano Criteria for Response Assessment. At each follow-up time point, a PET-based response and a CT-based response is made per the definitions below. Response and Site PET-based Response CT-based Response COMPLETE Complete Metabolic Response* Complete Radiologic Response ALL of the following ALL of the following Lymph nodes, Score of 1, 2, or 3* Lymph nodes: All <1.5 cm in longest extranodal lesions diameter. Extralymphatic disease absent. Nonmeasured lesion N/A Absent Organ enlargement N/A Spleen: normal size New lesions No new metabolically active lesions None (new lesions drive score 5) Bone marrow No FDG-avid disease in marrow Normal by morphology; if indeterminate, IHC negative. PARTIAL Partial Remission Partial Metabolic Response ALL of the following Lymph nodes, Score of 4, or 5 with reduced uptake .gtoreq.50% decrease in SPD of all extranodal lesions from baseline and residual masses of target lesions from baseline any size Nonmeasured lesion N/A Absent, normal, or regressed, but no increase Organ enlargement N/A Spleen: .gtoreq.50% decrease from baseline in enlarged portion New lesions None None Bone marrow Residual uptake higher than uptake in N/A normal marrow but reduced compared with baseline (e.g., persistent focal changes in the marrow with nodal response) NO RESPONSE/STABLE DISEASE No Metabolic Response Stable Disease Lymph nodes, Score of 4, or 5 with no significant <50% decrease in SPD of all target extranodal lesions change in FDG uptake from baseline lesion from baseline No progression Nonmeasured lesion N/A No increase consistent with progression Organ enlargement N/A Spleen: No increase consistent with progression New lesions None None Bone marrow No change from baseline N/A PROGRESSION Progressive Disease Progressive Metabolic Response ANY of the following Lymph nodes, Lymph nodes/nodal masses: PPD Progression extranodal lesions Score of 4 or 5 with increased An individual node/extranodal lesion must uptake compared to baseline. be abnormal (nodal disease with LDi > 1.5 Extranodal lesions: New FDG cm, extranodal disease with and LDi > 1.0 avid foci consistent with cm) with: lymphoma. Increase of .gtoreq.50% from the product of the perpendicular diameters (PPD) from nadir AND Increase in LDi or SDi from nadir .gtoreq.0.5 cm for lesions .ltoreq.2 cm .gtoreq.1.0 cm for lesions >2 cm Nonmeasured lesion None Unequivocal progression Organ enlargement None Progression of pre-existing splenomegaly: Splenic length must increase by 50% of the extent of its prior increase beyond baseline (e.g., a 15-cm spleen must increase to 16 cm). New splenomegaly: Spleen must increase by at least 2 cm from baseline Or Recurrent splenomegaly New lesions New FDG-avid foci consistent with Regrowth of previously resolved lesions lymphoma rather than another etiology New node > 1.5 cm in any axis New extranodal site > 1.0 cm in any axis New extranodal site < 1.0 cm in any axis or unequivocal/attributable to lymphoma New assessable disease unequivocal/ attributable to lymphoma of any size Bone marrow New/recurrent FDG-avid foci New or recurrent involvement FDG: fluorodeoxyglucose; NC: immunohistochemistry; LDi: longest diameter; N/A: not applicable; PPD: perpendicular diameters; SDi: shortest diameter; SPD: sum of the products of diameters. *Deauville score of 3 represent a complete metabolic response (Barrington et al., (2014) J Clin Oncol, 32, 3048-3058). Note: It is recognized that in Waldeyer's ring or extranodal sites with high physiologic uptake or with activation within spleen or marrow (e.g., with chemotherapy or myeloid colony-stimulating factors), uptake may be greater than normal mediastinum and/or liver. In this circumstance, complete metabolic response may be inferred if uptake at sites of initial involvement is no greater than surrounding normal tissue even if the tissue has high physiologic uptake.

[0255] Secondary Endpoints (Dose Escalation and Cohort Expansion)

[0256] Efficacy [0257] Duration of response/remission (central read/assessment). Duration of response/remission is reported only for subjects who have had objective response events. This is calculated as the time between first objective response and date of disease progression or death due to any cause. [0258] Progression-free/event-free survival (central read/assessment). Progression-free survival (PFS) and event-free survival is calculated as the difference between date of CTX110 infusion and date of disease progression or death due to any cause. Subjects who have not progressed and are still on study at the data cutoff date are censored at their last assessment date. [0259] Overall survival. Overall survival is calculated as the time between date of first dose of CTX110 and death due to any cause. Subjects who are alive at the data cutoff date are censored at their last date known to be alive.

[0260] Safety

[0261] Frequency and severity of AEs and clinically significant laboratory abnormalities.

[0262] Pharmacokinetic [0263] Levels of CTX110 in blood over time.

[0264] Exploratory Endpoints (Dose Escalation and Cohort Expansion) [0265] Levels of CTX110 in tissues (e.g., trafficking of CTX110 in bone marrow, CSF, and/or tumor tissue may be evaluated in any samples collected per protocol-specific sampling). [0266] Levels of cytokines in blood and other tissues. [0267] Incidence of anti-CTX110 antibodies. [0268] Levels of B cells and immunoglobulins overtime. [0269] Impact of anti-cytokine therapy on CTX110 proliferation, CRS, and response. [0270] Incidence of autologous or allogeneic HSCT following CTX110 therapy. [0271] Incidence and type of subsequent anticancer therapy. [0272] Time to complete response/remission. [0273] First subsequent therapy-free survival. [0274] Other genomic, protein, metabolic, or pharmacodynamic endpoints.

Study Design

[0275] This is an open-label, multicenter, Phase 1 study evaluating the safety and efficacy of CTX110 in subjects with relapsed or refractory B cell malignancies. The study is divided into 2 parts: dose escalation (Part A) followed by cohort expansion (Part B).

[0276] Part A investigates escalating doses of CTX110 in Cohort A in adult subjects with 1 of the following NHL subtypes: DLBCL NOS, high grade B cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements, grade 3b FL, or transformed FL.

[0277] In the dose escalation part of the study, 1 additional cohort (Cohort B) with an NHL population similar to Cohort A has been added to explore an increased dose of cyclophosphamide (750 mg/m.sup.2) relative to Cohort A (500 mg/m.sup.2). Subjects in Cohort B are treated with an increased dose of cyclophosphamide to explore the effects of a longer suppression of lymphocytes on CAR T cell expansion following CTX110 infusion (see Table 15).

TABLE-US-00015 TABLE 15 Cohort A and Cohort B. Cohort Disease Subset Treatment A Adult subjects with DLBCL NOS, LD chemotherapy: Co-administration of high grade B cell lymphoma with fludarabine MYC and BCL2 and/or BCL6 30 mg/m.sup.2 + cyclophosphamide 500 mg/m.sup.2 IV rearrangements, grade 3b FL, and daily for 3 days transformed FL CTX110 starting at DL1 B Same as Cohort A LD chemotherapy: Co-administration of fludarabine 30 mg/m.sup.2 + cyclophosphamide 750 mg/m.sup.2 IV daily for 3 days CTX110 starting at DL2 DL1/2: Dose Level 1 or 2; DLBCL: diffuse large B cell lymphoma; FL: follicular lymphoma; IV: intravenously; LD: lymphodepleting.

[0278] The study is divided into 2 parts: dose escalation (Part A) followed by cohort expansion (Part B). Both parts of the study will consist of 3 main stages: screening, treatment, and follow-up. A schematic depiction of the study schema is shown in FIG. 21.

[0279] A schedule of assessments is provided in Table 16 and Table 17.

Stage 1--Screening to determine eligibility for treatment (up to 14 days). Stage 2--Lymphodepleting (LD) chemotherapy and infusion of CTX110 (1-2 weeks). Prior to both the initiation of LD chemotherapy and infusion of CTX110, the clinical eligibility of subjects must be reconfirmed.

[0280] Stage 2A--LD chemotherapy: [0281] Cohort A: Co-administration of fludarabine 30 mg/m.sup.2 and cyclophosphamide 500 mg/m.sup.2 intravenously (IV) daily for 3 days. [0282] Cohort B: Co-administration of fludarabine 30 mg/m.sup.2 and cyclophosphamide 750 mg/m.sup.2 intravenously (IV) daily for 3 days.

[0283] Stage 2B--CTX110 infusion: [0284] Cohort A (NHL subsets): Lymphodepleting (LD) chemotherapy (fludarabine 30 mg/m.sup.2 and cyclophosphamide 500 mg/m.sup.2 intravenously [IV] daily for 3 days) completed at least 48 hours (but no more than 7 days) prior to CTX110 infusion (dose escalation from Dose Level [DL] 1). [0285] Cohort B (higher LD chemotherapy dose): LD chemotherapy (fludarabine 30 mg/m.sup.2 and cyclophosphamide 750 mg/m.sup.2 IV daily for 3 days) completed at least 48 hours (but no more than 7 days) prior to CTX110 infusion (dose escalation from Dose Level [DL] 2).

[0286] Stage 3--Follow up (5 years after the last CTX110 infusion).

[0287] For both dose escalation and cohort expansion, subjects must remain within proximity of the investigative site (i.e., 1-hour transit time) for 28 days after CTX110 infusion. During this acute toxicity monitoring period, subjects will be routinely assessed for adverse events (AEs), including cytokine release syndrome (CRS), neurotoxicity, and GvHD. Toxicity management guidelines are provided in the study protocol. During dose escalation, all subjects will be hospitalized for the first 7 days following CTX110 infusion, or longer if required by local regulation or site practice.

[0288] After the acute toxicity monitoring period, subjects will be subsequently followed for up to 5 years after CTX110 infusion with physical exams, regular laboratory and imaging assessments, and AE evaluations. After completion of this study, subjects will be required to participate in a separate long-term follow-up study for an additional 10 years to assess long-term safety and survival.

[0289] LD chemotherapy it to be delayed if any of the following signs or symptoms are present: [0290] Significant worsening of clinical status that, according to the investigator, increases the potential risk of AEs associated with LD chemotherapy. [0291] Requirement for supplemental oxygen to maintain a saturation level of >91%. [0292] New uncontrolled cardiac arrhythmia. [0293] Hypotension requiring vasopressor support. [0294] Active infection: Positive blood cultures for bacteria, fungus, or virus not responding to treatment. [0295] Grade .gtoreq.2 acute neurological toxicity.

TABLE-US-00016 [0295] TABLE 16 Schedule of Assessments (Screening to Month 24). Treat- ment Screen- (Stage 2) Study ing .sup.1 D-5 Follow-up (Stage 3) Stage (Stage to D2 .+-. D3 .+-. D5 .+-. D8 .+-. D10 .+-. D14 .+-. D21 .+-. Day 1) D-3 D1.sup.2 2 d 2 d 2 d 2 d 2 d 2 d 2 d Informed X consent Medical X history.sup.3 Physical X X X X X X X X X X exam Vital X X X X X X X X X X signs .sup.4 Height, X X X X X weight .sup.5 Preg- X X nancy test .sup.6 ECOG X X status Echo- X cardio- gram 12-lead X X X ECG .sup.7 Brain X MRI Lumbar X punc- ture .sup.8 ICE X X X X X X assess- ment .sup.9 Patient- X reported outcome Con- Continuous comitant meds .sup.10 Adverse Continuous events .sup.11 Hospital Continuous util- ization Treat- ment LD X chemo- ther- apy .sup.12 CTX110 X infu- sion .sup.13 NHL Disease Re- sponse/ Assess- ment (Central and Local) PET/CT X scan .sup.14 BM X biopsy .sup.15 Tumor biopsy .sup.16 Tumor X path- ology.sup.17 Adult B Cell ALL Disease Re- sponse/ Assess- ment BM X biopsy and aspirate (central and lo- cal) .sup.14, 15 Peri- X pheral blood chim- erism (local) .sup.19 Lab - oratory Assess- ments (Local) CBC w/ X X X X X X X X X X differ- ential Serum X X X X X X X X X X chemistry Coagu- X X X X X X X X X lation para- meters Viral X serol- ogy .sup.20 Immuno- X X globulins Ferritin, X X X X X X X X X CRP Lympho- X X X X X X X X cyte subsets .sup.21 B cells X X X (CD19, CD20) Blood type, Ab screen .sup.22 Bio- markers (Blood, Central) CTX110 X X25 X X X X X X X PK .sup.23, 24 pre/ post Cyto- X X X X X X X X X kines .sup.26 Anti- X Cas9 Ab .sup.24 Anti- X CTX110 Ab .sup.24 Immuno- X X25 X X X X X pheno- pre/ type post DNA X Cell-free X DNA PBMCs X Explor- X X.sup.28 X X X X X X X X atory bio- mar- kers .sup.27 Study Follow-up (Stage 3) Stage D28 .+-. M2 .+-. M3 .+-. M6 .+-. M9 .+-. M12 .+-. M15 .+-. M18 .+-. M24 .+-. Day 4 d 7 d 7 d 14 d 14 d 14 d 14 d 14 d 21 d Informed consent Medical history.sup.3 Physical X X X X X X X X X exam Vital X X X X X X X X X signs .sup.4 Height, X X X X X X X X X weight .sup.5 Preg- nancy test .sup.6 ECOG X X status Echo- cardio- gram 12-lead X ECG .sup.7 Brain MRI Lumbar punc- ture .sup.8 ICE X assess- ment .sup.9 Patient- X X X X reported outcome Con- Continuous comitant meds .sup.10 Adverse Continuous events .sup.11 Hospital Continuous util- ization Treat- ment LD chemo- ther- apy .sup.12 CTX110 infu- sion .sup.13 NHL Disease Re- sponse/ Assess- ment (Central and Local) PET/CT X X X X X X X scan .sup.14 BM X biopsy .sup.15 Tumor X biopsy .sup.16 Tumor path- ology.sup.17 Adult B Cell ALL Disease Re- sponse/ Assess- ment BM X X.sup.18 X.sup.18 biopsy and aspirate (central and lo- cal) .sup.14, 15 Peri- pheral blood

chim- erism (local) .sup.19 Lab - oratory Assess- ments (Local) CBC w/ X X X X X X X X X differ- ential Serum X X X X X X X X X chemistry Coagu- X lation para- meters Viral serol- ogy .sup.20 Immuno- X X X X X X X X X globulins Ferritin, X CRP Lympho- X X X X X X cyte subsets .sup.21 B cells X X X X X X X X X (CD19, CD20) Blood type, Ab screen .sup.22 Bio- markers (Blood, Central) CTX110 X X X X X X X X X PK .sup.23, 24 Cyto- X X kines .sup.26 Anti- X X X X Cas9 Ab .sup.24 Anti- X X X X CTX110 Ab .sup.24 Immuno- X X X X X X X X X pheno- type DNA Cell-free X X X X X X X DNA PBMCs X X X X X X X Explor- X X X X X X X X X atory bio- mar- kers .sup.27 Ab: antibody; AE: adverse event; BM: bone marrow; Cas9: CRISPR-associated protein 9; CBC: complete blood count; CNS: central nervous system; CRISPR: clustered regularly interspaced short palindromic repeats; CRP: C-reactive protein; CT: computed tomography; D or d: day; EC.sub.90: 90% effectiveconcentration; ECG: electrocardiogram; ECOG: Eastern Cooperative Oncology Group; HBV: hepatitis B virus; HCV: hepatitis C virus; HIV-1/-2: human immunodeficiency virus type 1 or 2; HSCT: hematopoietic stem cell transplant; ICE: immune effector cell-associated encephalopathy; ICF: informed consent form; IPI: International Prognostic Index; LD: lymphodepleting; LP: lumbar puncture; M: month; MRI: magnetic resonance imaging; PBMC: peripheral blood mononuclear cell; PCR: polymerase chain reaction; PET: positron emission tomography; PK: pharmacokinetic(s); Q: every; TBNK: T-, B-, natural killer (cells). .sup.1 Screening assessments completed within 14 days of informed consent. Subjects allowed 1-time rescreening within 3 months of initial consent. .sup.2 All baseline assessments on Day 1 are to be performed prior to CTX110 infusion unless otherwise specified. .sup.3 Includes complete surgical and cardiac history. .sup.4 Includes sitting blood pressure, heart rate, respiratory rate, pulse oximetry, and temperature. .sup.5 Height at screening only. .sup.6 For female subjects of childbearing potential. Serum pregnancy test at screening. Serum or urine pregnancy test within 72 hours before start of LD chemotherapy. .sup.7 Prior to LD chemotherapy, and prior to CTX110 infusion. .sup.8 LP at screening on subjects with high risk for CNS involvement (e.g., high-grade B cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements, subjects with testicular involvement of lymphoma, or subjects with high-risk CNS IPI score). For LPs performed during neurotoxicity, samples should be sent to central laboratory for CTX110 PK and exploratory biomarkers whenever possible. .sup.9 On Day 1 prior to CTX110 administration. If CNS symptoms persist after Day 28, ICE assessment should continue to be performed approximately every 2 days until symptom resolution to grade 1 or baseline. .sup.10 All concomitant medications will be collected .ltoreq. 3 months post-CTX110, after which only select concomitant medications will be collected. 11 Collect all AEs from informed consent to Month 3 visit, collect all SAEs and AESIs after Month 3 visit to Month 60 visit. Only SAEs and AESIs should be reported for .ltoreq. 3 months post-CTX110 if subject begins new anticancer therapy before Month 3 visit. Only AESIs will be reported if subject begins new anticancer therapy after Month 3 visit. .sup.12 Start LD chemotherapy within 7 days of study enrollment. After completion of LD chemotherapy, ensure washout period of .gtoreq. 48 hours (but .ltoreq. 7 days) before CTX110 infusion. Physical exam, weight, and coagulation laboratories performed prior to first dose of LD chemotherapy. Vital signs, CBC, clinical chemistry, and AEs/concomitant medications assessed and recorded daily (i.e., 3 times) during LD chemotherapy. .sup.13 CTX110 administered 48 hours to 7 days after completion of LD chemotherapy. .sup.14 Baseline disease assessment (PET/CT for subjects with NHL) to be performed within 28 days prior to CTX110 infusion. MRI with contrast allowed if CT clinically contraindicated, or as required by local regulation. .sup.15 BM biopsy to confirm complete response as part of disease evaluation. BM biopsy may also be performed at time of disease relapse. Samples from BM aspirate after CTX110 infusion should be sent for CTX110 PK and exploratory biomarkers. To be performed .+-. 5 days of visit date. .sup.16 Optional: For subjects who have disease amenable to biopsy and who provide separate consent. To be performed .+-. 5 days of visit date. .sup.17It is preferred that subjects undergo tumor biopsy during screening. However, if a biopsy of relapsed/refractory disease was performed within 3 months prior to enrollment and after the most recent line of therapy, archival tissue may be used. If relapse occurs on study, every attempt should be made to obtain biopsy of relapsed tumor and send to central pathology. Tumor biopsy refers to tissue other than bone marrow. .sup.18 Assessments at Months 2 and 3 to confirm CR if not achieved at Month 1. .sup.19 To be performed only in subjects who have received prior allogeneic HSCT. .sup.20 Infectious disease testing (HIV-1, HIV-2, HCV antibody/PCR, HBV surface antigen, HBV surface antibody, HBV core antibody) .ltoreq. 30 days of signing ICF may be considered for subject eligibility. .sup.21 Lymphocyte subset assessment at screening, before start of first day of LD chemotherapy, before CTX110 infusion, then all listed time points. To include 6-color TBNK panel, or equivalent for T, B, and natural killer cells. .sup.22 Blood type and antibody screen. .sup.23 Samples for CTX110 PK should be sent from any LP, BM biopsy, or tissue biopsy performed following CTX110 infusion. If CRS occurs, samples for assessment of CTX110 levels will be collected every 48 hours between scheduled visits until CRS resolves. .sup.24 Sponsor may request discontinuation of sample collection if consecutive tests are negative. Continue sample collection for all listed time points until otherwise instructed by sponsor. .sup.25 Two samples collected on Day 1: One pre-CTX110 infusion and one 20 (.+-. 5) minutes after the end of CTX110 infusion. .sup.26 Additional cytokine samples should be collected daily for the duration of CRS. Day 1 samples to be collected prior to CTX110 infusion. .sup.27 Samples for exploratory biomarkers should be sent from any LP or BM biopsy performed following CTX110 infusion. If CRS occurs, samples for assessment of exploratory biomarkers will be collected every 48 hours between scheduled visits until CRS resolves. .sup.28 Prior to first day of LD chemotherapy only.

TABLE-US-00017 TABLE 17 Schedule of Assessments (Months 30-60). M30 (.+-. M36 (.+-. M42 (.+-. M48(.+-. M54(.+-. M60 (.+-. Progressive Secondary Assessments 21 days) 21 days) 21 days) 21 days) 21 days) 21 days) Disease Follow-Up .sup.1 Vital signs .sup.2 X X X X X X X X Physical exam X X X X X X X X Concomitant medications .sup.3 X X X X X X X X Disease assessment .sup.4 X X X X X X X CBC with differential .sup.5 X X X X X X X X Serum chemistry .sup.5 X X X X X X X X Immunoglobulins .sup.5, 6 X X X X X X X Lymphocyte subsets .sup.5, 6 X X X X X X X CTX110 persistence X X X X X X X X (blood, central) .sup.6, 7 Exploratory biomarkers X X X X X X X X (blood, central) Anti-Cas9 Ab X X X X (blood, central) .sup.6 Anti-CTX110 , X X X X anti-daratumumab Ab (blood, central) .sup.6 Patient-reported outcome X X X X Adverse events .sup.8 X X X X X X X X Ab: antibody; AESI: adverse event of special interest; BM: bone marrow; Cas9: CRISPR-associated protein 9; CBC: complete blood count; CRISPR: clustered regularly interspaced short palindromic repeats; CT: computed tomography; NHL: non-Hodgkin lymphoma; PET: positron emission tomography; PK: pharmacokinetic; SAE: serious adverse event; TBNK: T-, B-, natural killer (cells). .sup.1 Subjects with progressive disease or who undergo stem cell transplant will discontinue the normal schedule of assessments and attend annual study visits. Visits will occur at 12-month intervals. Subjects who partially withdraw consent will undergo these procedures at minimum. .sup.2 Includes temperature, blood pressure, pulse rate, and respiratory rate. .sup.3 Only select concomitant medications will be collected. .sup.4 Disease assessment will consist of investigator review of physical exam, CBC, clinical chemistry, and lactate dehydrogenase for NHL subjects, and of physical exam, CBC with differential, and clinical chemistry for B cell ALL. NHL subjects with suspected malignancy will undergo PET/CT imaging and/or a BM biopsy to confirm relapse. Every attempt should be made to obtain a biopsy of the relapsed tumor in subjects who progress. Assessed at local laboratory. To include 6-color TBNK panel, or equivalent for T, B, and natural killer cells. .sup.6 Sponsor may request discontinuation of sample collection. Continue sample collection for all listed time points until otherwise instructed by sponsor. .sup.7 Samples for CTX110 PK analysis should be sent to the central laboratory from any lumbar puncture, BM biopsy, or tissue biopsy performed following CTX110 infusion. .sup.8 SAEs and AESIs should be reported for up to 3 months after CTX110 infusion if a subject begins new anticancer therapy before Month 3 study visit. Only AESIs will be reported if a subject begins new anticancer therapy after Month 3 study visit.

[0296] The goal of lymphodepletion is to allow for significant CAR T cell expansion following infusion. LD chemotherapy consisting of fludarabine and cyclophosphamide across different doses has been successfully utilized in several autologous CAR T cell trials. The rationale for the use of LD chemotherapy is to eliminate regulatory T cells and other competing elements of the immune system that act as `cytokine sinks,` enhancing the availability of cytokines such as interleukin 7 (IL-7) and interleukin 15 (IL-15) (Dummer et al., (2002) J Clin Invest, 110, 185-192; Gattinoni et al., (2005) J Exp Med, 202, 907-912). Additionally, it is postulated that naive T cells begin to proliferate and differentiate into memory-like T cells when total numbers of naive T cells are reduced below a certain threshold (Dummer et al., (2002) J Clin Invest, 110, 185-192). Cohort A will use cyclophosphamide (500 mg/m.sup.2) and fludarabine (30 mg/m.sup.2) at doses that are consistent with doses used in registrational clinical trials of axicabtagene ciloleucel. Cohort B will use a higher dose of cyclophosphamide (750 mg/m.sup.2) to evaluate whether increased intensity of lymphodepletion may facilitate expansion of an allogeneic CAR T cell product. Doses of cyclophosphamide within this range (total of >120 mg/kg or 3 g/m.sup.2) have been used in prior CAR T cell therapy studies in hematological malignancies (Brentjens et al., (2011) Blood, 118, 4817-4828; Kochenderfer et al., (2015) J Clin Oncol, 33, 540-549; Turtle et al., (2016) Sci Transl Med, 8, 355ra116). When used as a part of higher intensity LD chemotherapy, increased doses of cyclophosphamide are associated with improved efficacy (Hirayama et al., (2019) Blood, 133, 1876-1887).

[0297] CTX110 infusion is to be delayed if any of the following signs or symptoms are present: [0298] New active uncontrolled infection. [0299] Worsening of clinical status compared to prior to start of LD chemotherapy that, in the opinion of the investigator, places the subject at increased risk of toxicity. [0300] Grade .gtoreq.2 acute neurological toxicity.

CTX110 Dose

[0301] CTX110 cells are administered IV using a flat dosing schema based on the number of CAR+ T cells. The starting dose is 3.times.10.sup.7 CAR+ T cells, which is approximately 1 log lower than the doses of autologous CAR T cells currently approved for NHL including KYMRIAH.RTM. (5.times.10.sup.8 total CAR T cells) and YESCARTA.RTM. (2.times.10.sup.6 kg, maximum 2.times.10.sup.8 CAR T cells).

Dose Escalation

[0302] Dose escalation will be performed using a standard 3+3 design. The following doses of CTX110, based on CAR.sup.+ T cells, may be evaluated in this study beginning with DL1 for Cohort A. Only after assessment and confirmation of safety of DL2 in Cohort A by the Safety Review Committee (SRC) may subsequent Cohort B be opened/enrolled and begin dose escalation from DL2. Due to the study's dose limit of 7.times.10.sup.4 TCR+ cells/kg, the study may proceed with DL4 in Cohort A and/or Cohort B if a subject weighs .gtoreq.60 kg (see Table 18).

TABLE-US-00018 TABLE 18 Dose Levels. Dose Level Total CAR+ T Cell Dose -1 1 .times. 10.sup.7 1 3 .times. 10.sup.7 2 1 .times. 10.sup.8 3 3 .times. 10.sup.8 4 1 .times. 10.sup.9 CAR: chimeric antigen receptor.

[0303] The DLT evaluation period begins with CTX110 infusion and last for 28 days. The first 3 subjects in each cohort will be treated in a staggered manner, such that the 2.sup.nd and 3.sup.rd subjects will only receive CTX110 after the previous subject has completed the DLT evaluation period. In subsequent dose levels or expansion of the same dose level, cohorts of up to 3 subjects may be enrolled and dosed concurrently.

[0304] Subjects must receive CTX110 to be evaluated for DLT. If a subject discontinues the study any time prior to CTX110 infusion, the subject will not be evaluated for DLT and a replacement subject will be enrolled at the same dose level as the discontinued subject. If a DLT-evaluable subject has signs or symptoms of a potential DLT, the DLT evaluation period will be extended according to the protocol-defined window to allow for improvement or resolution before a DLT is declared.

[0305] Toxicities are graded and documented according to National Cancer Institute Common Terminology Criteria for Adverse Events (CTCAE) version 5, except for CRS (Lee criteria), neurotoxicity (ICANS, immune effector cell-associated neurotoxicity syndrome criteria and CTCAE v5.0), and GvHD (Mount Sinai Acute GVHD International Consortium [MAGIC] criteria).

[0306] A DLT will be defined as any of the following events occurring during the DLT evaluation period that persists beyond the specified duration (relative to the time of onset): [0307] Grade .gtoreq.2 GvHD that is steroid-refractory (e.g., progressive disease after 3 days of steroid treatment [e.g., 1 mg/kg/day], stable disease after 7 days, or partial response after 14 days of treatment). [0308] Death during the DLT period (except due to disease progression). [0309] Any grade 3 or 4 toxicity that is clinically significant according to the investigator's judgement and does not improve within 72 hours. [0310] The following will NOT be considered as DLTs: [0311] Grade 3 or 4 CRS that improves to grade .ltoreq.2 within 72 hours. [0312] Grade 3 or 4 neurotoxicity (e.g., encephalopathy, confusion) that improves to grade .ltoreq.2 within 14 days. [0313] Grade 3 or 4 fever. [0314] Bleeding in the setting of thrombocytopenia (platelet count <50.times.10.sup.9/L); documented bacterial infections or fever in the setting of neutropenia (absolute neutrophil count <1000/mm.sup.3). [0315] Grade 3 or 4 hypogammaglobulinemia. [0316] Grade 3 or 4 pulmonary toxicity that resolves to grade .ltoreq.2 within 7 days. For subjects intubated due to fluid overload from supportive care, this may be extended to 14 days. [0317] Grade 3 or 4 liver function studies that improve to grade .ltoreq.2 within 14 days. [0318] Grade 3 or 4 renal insufficiency that improves to grade .ltoreq.2 within 21 days. [0319] Grade 3 or 4 thrombocytopenia or neutropenia will be assessed retrospectively. After at least 6 subjects are infused, if .gtoreq.50% of subjects have prolonged cytopenias (i.e., lasting more than 28 days post-infusion), dose escalation will be suspended pending SRC assessment.

[0320] AEs that have no plausible causal relationship with CTX110 will not be considered DLTs.

Toxicity Management

[0321] Subjects must be closely monitored for at least 28 days after CTX110 infusion. Significant toxicities have been reported with autologous CAR T cell therapies and investigators are required to proactively monitor and treat all adverse events in accordance with protocol guidance.

[0322] The following general recommendations are provided based on prior experience with CD19-directed autologous CAR T cell therapies: [0323] Fever is the most common early manifestation of cytokine release syndrome (CRS); however, subjects may also experience weakness, hypotension, or confusion as first presentation. [0324] Diagnosis of CRS should be based on clinical symptoms and NOT laboratory values. [0325] In subjects who do not respond to CRS-specific management, always consider sepsis and resistant infections. Subjects should be continually evaluated for resistant or emergent bacterial infections, as well as fungal or viral infections. [0326] CRS, hemophagocytic lymphohistiocytosis (HLH), and tumor lysis syndrome (TLS) may occur at the same time following CAR T cell infusion. Subjects should be consistently monitored for signs and symptoms of all the conditions and managed appropriately. [0327] Neurotoxicity may occur at the time of CRS, during CRS resolution, or following resolution of CRS. Grading and management of neurotoxicity will be performed separately from CRS. [0328] Tocilizumab must be administered within 2 hours from the time of order.

[0329] The safety profile of CTX110 will be continually assessed throughout the study, and investigators will be updated on a regular basis with new information regarding the identification and management of potential CTX110-related toxicity.

Infusion Reactions

[0330] Infusion reactions have been reported in autologous CD19-directed CAR T cell trials, including transient fever, chills, and/or nausea. Acetaminophen (paracetamol) and diphenhydramine hydrochloride (or another H1-antihistamine) may be repeated every 6 hours after CTX110 infusion, as needed, if an infusion reaction occurs. Nonsteroidal anti-inflammatory medications may be prescribed, as needed, if the subject continues to have fever not relieved by acetaminophen. Systemic steroids should not be administered except in cases of life-threatening emergency, as this intervention may have a deleterious effect on CAR T cells.

Febrile Reaction and Infection Prophylaxis

[0331] Infection prophylaxis should occur according to the institutional standard of care for patients with B cell malignancies in an immunocompromised setting. In the event of febrile reaction, an evaluation for infection should be initiated and the subject managed appropriately with antibiotics, fluids, and other supportive care as medically indicated and determined by the treating physician. Viral and fungal infections should be considered throughout a subject's medical management if fever persists. If a subject develops sepsis or systemic bacteremia following CTX110 infusion, appropriate cultures and medical management should be initiated. Additionally, consideration of CRS should be given in any instances of fever following CTX110 infusion within 30 days post-infusion.

Tumor Lysis Syndrome (TLS)

[0332] Subjects receiving CAR T cell therapy are at increased risk of TLS. Subjects should be closely monitored for TLS via laboratory assessments and symptoms from the start of LD chemotherapy until 28 days following CTX110 infusion. All subjects should receive prophylactic allopurinol (or a non-allopurinol alternative, such as febuxostat) and increased oral/IV hydration during screening and before initiation of LD chemotherapy. Prophylaxis can be stopped after 28 days following CTX110 infusion or once the risk of TLS passes.

[0333] Sites should monitor and treat TLS as per their institutional standard of care, or according to published guidelines (Cairo and Bishop, (2004) Br J Haematol, 127, 3-11). TLS management, including administration of rasburicase, should be instituted promptly when clinically indicated.

Cytokine Release Syndrome (CRS)

[0334] CRS is a major toxicity reported with autologous CD19-directed CAR T cell therapy. CRS is due to hyperactivation of the immune system in response to CAR engagement of the target antigen, resulting in multi-cytokine elevation from rapid T cell stimulation and proliferation (Frey et al., (2014) Blood, 124, 2296; Maude et al., (2014) Cancer J, 20, 119-122). When cytokines are released, a variety of clinical signs and symptoms associated with CRS may occur, including cardiac, gastrointestinal (GI), neurological, respiratory (dyspnea, hypoxia), skin, cardiovascular (hypotension, tachycardia), and constitutional (fever, rigors, sweating, anorexia, headaches, malaise, fatigue, arthralgia, nausea, and vomiting) symptoms, and laboratory (coagulation, renal, and hepatic) abnormalities.

[0335] The goal of CRS management is to prevent life-threatening sequelae while preserving the potential for the antitumor effects of CTX110. Symptoms usually occur 1 to 14 days after autologous CAR T cell therapy, but the timing of symptom onset has not been fully defined for allogeneic CAR T cells.

[0336] CRS should be identified and treated based on clinical presentation and not laboratory cytokine measurements. If CRS is suspected, grading and management should be performed according to the recommendations in Table 19, which are adapted from published guidelines (Lee et al., (2014) Blood, 124, 188-195). Since the development of the original Lee CRS grading criteria, physicians using CAR T cell therapies have gained further understanding of the presentation and time course of CRS. The recent American Society for Blood and Marrow Transplantation (ASBMT) consensus criteria (Lee et al., (2018) Biol Blood Marrow Transplant) recommend that grading should be based on the presence of fever with hypotension and/or hypoxia, and that other end organ toxicities should be managed separately with supportive care. Accordingly, in this protocol neurotoxicity will be graded and managed using a different scale (see section entitled "Immune Effector Cell-Associated Neurotoxicity Syndrome (ICANS)"), and end organ toxicity in the context of CRS management refers only to hepatic and renal systems (as in the Penn Grading criteria; (Porter et al., (2018) J Hematol Oncol, 11, 35). The sponsor may elect to revise the CRS grading criteria and toxicity management algorithms to reflect the ASBMT consensus proposal based on clinical experience with CTX110 and other CAR T cell therapies.

TABLE-US-00019 TABLE 19 Cytokine Release Syndrome Grading and Management Guidance. CRS Severity .sup.1 Tocilizumab Corticosteroids Grade 1 N/A N/A Symptoms require symptomatic treatment only (e.g., fever, fatigue, headache, myalgia, malaise). Grade 2 Administer tocilizumab.sup.3 8 mg/kg Manage per grade 3 if no Symptoms require and respond to IV over 1 hour (not to exceed improvement within 24 hours moderate intervention. Oxygen 800 mg). after starting tocilizumab. requirement < 40% FiO.sub.2 or Repeat tocilizumab every 8 hypotension responsive to fluids hours as needed if not responsive or low dose of 1 vasopressor or to IV fluids or increasing grade 2 organ toxicity..sup.2 supplemental oxygen. Limit to .ltoreq.3 doses in a 24-hour period; maximum total of 4 doses. Grade 3 Per grade 2. If no improvement within 24 Symptoms require and respond to hours after starting tocilizumab, aggressive intervention. Oxygen administer methylprednisolone requirement .gtoreq.40% FiO.sub.2 or 1 mg/kg IV twice daily. hypotension requiring high-dose Continue corticosteroid use until or multiple vasopressors.sup.4 or grade the event is grade .ltoreq.1, then taper 3 organ toxicity or grade 4 over 3 days. transaminitis Grade 4 Per grade 2. Per grade 3. Life-threatening symptoms. If no response to multiple doses Requirements for ventilator of tocilizumab and steroids, support, continuous veno-venous consider using other anti- hemodialysis or grade 4 organ cytokine therapies (e.g., toxicity (excluding transaminitis) siltuximab). CRS: cytokine release syndrome; FiO.sub.2: fraction of inspired oxygen; IV: intravenously; N/A: not applicable. .sup.1 See (Lee et al., (2014) Blood, 124, 188-195). .sup.2Refer to entitled "Immune Effector Cell-Associated Neurotoxicity Syndrome (ICANS)" for management of neurologic toxicity. Organ toxicity refers to hepatic and renal systems only. .sup.3Refer to tocilizumab prescribing information. .sup.4 See Table 20 for information on high-dose vasopressors.

TABLE-US-00020 TABLE 20 High-dose Vasopressors. Pressor Dose* Norepinephrine monotherapy .gtoreq.20 .mu.g/min Dopamine monotherapy .gtoreq.10 .mu.g/kg/min Phenylephrine monotherapy .gtoreq.200 .mu.g/min Epinephrine monotherapy .gtoreq.10 .mu.g/min If on vasopressin Vasopressin + norepinephrine equivalent of .gtoreq.10 .mu.g/min** If on combination vasopressors Norepinephrine equivalent (not vasopressin) of .gtoreq.20 .mu.g/min** *All doses are required for .gtoreq.3 hours. **VASST Trial vasopressor equivalent equation: norepinephrine equivalent dose = [norepinephrine (.mu.g/min) + (.mu.g/min)/2] + [epinephrine (.mu.g/min)] + [phenylephrine (.mu.g/min)/10]

[0337] Throughout the duration of CRS, subjects should be provided with supportive care consisting of antipyretics, IV fluids, and oxygen. Subjects who experience grade .gtoreq.2 CRS (e.g., hypotension, not responsive to fluids, or hypoxia requiring supplemental oxygenation) should be monitored with continuous cardiac telemetry and pulse oximetry. For subjects experiencing grade 3 CRS, consider performing an echocardiogram to assess cardiac function. For grade 3 or 4 CRS, consider intensive care supportive therapy. Intubation for airway protection due to neurotoxicity (e.g., seizure) and not due to hypoxia should not be captured as grade 4 CRS. Similarly, prolonged intubation due to neurotoxicity without other signs of CRS (e.g., hypoxia) is not considered grade 4 CRS. Investigators should always consider the potential of an underlying infection in cases of severe CRS, as the presentation (fever, hypotension, hypoxia) is similar. Resolution of CRS is defined as resolution of fever (temperature .gtoreq.38.degree. C.), hypoxia, and hypotension (Lee et al., (2018) Biol Blood Marrow Transplant).

[0338] Immune Effector Cell-associated Neurotoxicity Syndrome (ICANS) Neurotoxicity has been observed with autologous CD19-directed CAR T cell therapies. It may occur at the time of CRS, during the resolution of CRS, or following resolution of CRS, and its pathophysiology is unclear. The recent ASBMT consensus further defined neurotoxicity associated with CRS as immune effector cell-associated neurotoxicity syndrome (ICANS), a disorder characterized by a pathologic process involving the CNS following any immune therapy that results in activation or engagement of endogenous or infused T cells and/or other immune effector cells (Lee et al., (2018) Biol Blood Marrow Transplant). Signs and symptoms can be progressive and may include aphasia, altered level of consciousness, impairment of cognitive skills, motor weakness, seizures, and cerebral edema. ICANS grading was developed based on CAR T cell-therapy-associated TOXicity (CARTOX) working group criteria used previously in autologous CAR T cell trials (Neelapu et al., (2018) N Engl J Med, 377, 2531-2544). ICANS incorporates assessment of level of consciousness, presence/absence of seizures, motor findings, presence/absence of cerebral edema, and overall assessment of neurologic domains by using a modified assessment tool called the ICE (immune effector cell-associated encephalopathy) assessment tool (see Table 21).

[0339] Evaluation of any new onset neurotoxicity should include a neurological examination (including ICE assessment tool, Table 22), brain MRI, and examination of the CSF (via lumbar puncture) as clinically indicated. If a brain MRI is not possible, all subjects should receive a non-contrast CT to rule out intracerebral hemorrhage. Electroencephalogram should also be considered as clinically indicated. Endotracheal intubation may be needed for airway protection in severe cases.

[0340] Non-sedating, anti-seizure prophylaxis (e.g., levetiracetam) should be considered in all subjects for at least 21 days following CTX110 infusion or upon resolution of neurological symptoms (unless the investigator considers the antiseizure medication to be contributing to the detrimental symptoms). Subjects who experience ICANS grade .gtoreq.2 should be monitored with continuous cardiac telemetry and pulse oximetry. For severe or life-threatening neurologic toxicities, intensive care supportive therapy should be provided. Neurology consultation should always be considered. Monitor platelets and for signs of coagulopathy, and transfuse blood products appropriately to diminish risk of intracerebral hemorrhage. Table 21 provides neurotoxicity grading and Table 23 provides management guidance.

[0341] For subjects who receive active steroid management for more than 3 days, antifungal and antiviral prophylaxis is recommended to mitigate a risk of severe infection with prolonged steroid use. Consideration for antimicrobial prophylaxis should also be given.

TABLE-US-00021 TABLE 21 ICANS Grading. Neuro- toxicity Grade Grade Grade Grade Domain 1 2 3 4 ICE 7-9 3-6 0-2 0 (subject is score .sup.1 unarousable and unable to undergo ICE assessment) Depressed Awakens Awakens Awakens only to Subject is level of spon- to voice tactile stimulus unarousable or conscious- taneously requires vigorous ness .sup.2 or repetitive tactile stimuli to arise; stupor or coma Seizure N/A N/A Any clinical Life-threatening seizure, focal or prolonged seizure generalized, that (>5 min) or resolves rapidly, repetitive or nonconvulsive clinical or electrical seizures on seizures without EEG that resolve return to baseline with intervention in between Motor N/A N/A N/A Deep focal motor findings .sup.3 weakness such as hemiparesis or paraparesis Elevated N/A N/A Focal/local Diffuse cerebral ICP/ edema on edema on cerebral neuroimaging .sup.4 neuroimaging, edema decerebrate or decorticate posturing, cranial nerve VI palsy, papilladema, or Cushing's triad CTCAE: Common Terminology Criteria for Adverse Events; EEG: electroencephalogram; ICANS: immune effector cell-associated neurotoxicity syndrome; ICE: immune effector cell-associated encephalopathy (assessment tool); ICP: intracranial pressure; N/A: not applicable. ICANS grade is determined by the most severe event (ICE score, level of consciousness, seizure, motor findings, raised ICP/cerebral edema) not attributable to any other cause. .sup.1 A subject with an ICE score of 0 may be classified as grade 3 ICANS if awake with global aphasia, but a subject with an ICE score of 0 may be classified as grade 4 ICANS if unarousable. .sup.2 Depressed level of consciousness should be attributable to no other cause (e.g., sedating medication). .sup.3 Tremors and myoclonus associated with immune effector therapies should be graded according to CTCAE v5.0 but do not influence ICANS grading.

TABLE-US-00022 TABLE 22 ICE Assessment. Maximum Domain Assessment Score Orientation Orientation to year, month, city, hospital 4 points Naming Name 3 objects (e.g., point to clock, pen, 3 points button) Following Ability to follow commands (e.g., "Show 1 point command me 2 fingers" or "Close your eyes and stick out your tongue") Writing Ability to write a standard sentence 1 point (includes a noun and verb) Attention Ability to count backward from 100 by 10 1 point ICE score will be reported as the total number of points (0-10) across all assessments.

[0342] The ICE assessment will be performed at screening, before administration of CTX110 on Day 1, and on Days 2, 3, 5, 8, and 28. If a subject experiences CNS symptoms, the ICE assessment should continue to be performed approximately every 2 days until resolution of symptoms. To minimize variability, whenever possible the assessment should be performed by the same research staff member who is familiar with or trained in administration of the ICE assessment.

TABLE-US-00023 TABLE 23 ICANS Management Guidance. Severity Management Grade 2 Consider administering dexamethasone 10 mg IV every 6 hours (or equivalent methylprednisolone) unless subject already on equivalent dose of steroids for CRS. Continue dexamethasone use until event is grade Grade 3 Administer dexamethasone 10 mg IV every 6 hours, unless subject already on equivalent dose of steroids for CRS. Continue dexamethasone use until event is grade Grade 4 Administer methylprednisolone 1000 mg IV per day for 3 days; if improves, manage as above. CRS: cytokine release syndrome; ICANS: immune effector cell-associated neurotoxicity syndrome; IV: intravenously.

[0343] Headache, which may occur in a setting of fever or after chemotherapy, is a nonspecific symptom. Headache alone may not necessarily be a manifestation of ICANS and further evaluation should be performed. Weakness or balance problem resulting from deconditioning and muscle loss are excluded from definition of ICANS. Similarly, is intracranial hemorrhage with or without associated edema may occur due to coagulopathies in these subjects and are also excluded from definition of ICANS. These and other neurotoxicities should be captured in accordance with CTCAE v5.0.

B Cell Aplasia

[0344] B cell aplasia may occur and will be monitored by following immunoglobulin G blood levels. IV gammaglobulin will be administered for clinically significant hypogammaglobulinemia (systemic infections) according to institutional standard of care.

Hemophagocytic Lymphohistiocytosis (HLH)

[0345] HLH has been reported after treatment with autologous CD19-directed CAR T cells (Barrett et al., (2014) Curr Opin Pediatr, 26, 43-49; Maude et al., (2014) N Engl J Med, 371, 1507-1517; Maude et al., (2015) Blood, 125, 4017-4023; Porter et al., (2015) Sci Transl Med, 7, 303ra139; Teachey et al., (2013) Blood, 121, 5154-5157. HLH is a clinical syndrome that is a result of an inflammatory response following infusion of CAR T cells in which cytokine production from activated T cells leads to excessive macrophage activation. Signs and symptoms of HLH may include fevers, cytopenias, hepatosplenomegaly, hepatic dysfunction with hyperbilirubinemia, coagulopathy with significantly decreased fibrinogen, and marked elevations in ferritin and C-reactive protein (CRP). Neurologic findings have also been observed (Jordan et al., (2011) Blood, 118, 4041-4052; La Rosde, (2015) Hematology Am Soc Hematol Educ Program, 190-196.

[0346] CRS and HLH may possess similar clinical syndromes with overlapping clinical features and pathophysiology. HLH will likely occur at the time of CRS or as CRS is resolving. HLH should be considered if there are unexplained elevated liver function tests or cytopenias with or without other evidence of CRS. Monitoring of CRP and ferritin may assist with diagnosis and define the clinical course.

[0347] If HLH is suspected: [0348] Frequently monitor coagulation parameters, including fibrinogen. These tests may be done more frequently than indicated in the schedule of assessments, and frequency should be driven based on laboratory findings. [0349] Fibrinogen should be maintained .gtoreq.100 mg/dL to decrease risk of bleeding. [0350] Coagulopathy should be corrected with blood products. [0351] Given the overlap with CRS, subjects should also be managed per CRS treatment guidance in Table 19.

Cytopenias

[0352] Grade 3 neutropenia and thrombocytopenia, at times lasting more than 28 days post-infusion, have been reported in subjects treated with autologous CD19-directed CAR T cell products (Kymriah USPI, 2017; Yescarta USPI, 2017). Therefore, subjects receiving CTX110 should be monitored for such toxicities and appropriately supported. Consideration should be given to antimicrobial and antifungal prophylaxis for any subject with prolonged neutropenia.

[0353] G-CSF may be considered in cases of grade 4 neutropenia 21 days post-CTX110 infusion, when the risk of CRS has passed.

Graft Versus Host Disease

[0354] GvHD is seen in the setting of allogeneic HSCT and is the result of immunocompetent donor T cells (the graft) recognizing the recipient (the host) as foreign. The subsequent immune response activates donor T cells to attack the recipient to eliminate foreign antigen-bearing cells. GvHD is divided into acute, chronic, and overlap syndromes based on both the time from allogeneic HSCT and clinical manifestations. Signs of acute GvHD may include a maculopapular rash; hyperbilirubinemia with jaundice due to damage to the small bile ducts, leading to cholestasis; nausea, vomiting, and anorexia; and watery or bloody diarrhea and cramping abdominal pain (Zeiser and Blazar, (2017) N Engl J Med, 377, 2167-2179.

[0355] To support the proposed clinical study, a nonclinical Good Laboratory Practice (GLP)-compliant GvHD and tolerability study was performed in immunocompromised mice at 2 doses that exceed all proposed clinical dose levels by at least 10-fold. Further, due to the specificity of CAR insertion at the TRAC locus, it is highly unlikely for a T cell to be both CAR+ and TCR+. Remaining TCR+ cells are removed during the manufacturing process by immunoaffinity chromatography on an anti-TCR antibody column to achieve <0.5% TCR+ cells in the final product. A dose limit of 7.times.10.sup.4 TCR+ cells/kg will be imposed for all dose levels. This limit is lower than the limit of 1.times.10.sup.5 TCR+ cells/kg based on published reports on the number of allogeneic cells capable of causing severe GvHD during SCT with haploidentical donors (Bertaina et al., (2014) Blood, 124, 822-826. Through this specific editing, purification, and strict product release criteria, the risk of GvHD following CTX110 should be low, although the true incidence is unknown. Subjects should be monitored closely for signs of acute GvHD following infusion of CTX110. The timing of potential symptoms is unknown. However, given that CAR T cell expansion is antigen-driven and will likely occur only in TCR- cells, it is unlikely that the number of TCR+ cells will appreciably increase above the number infused.

[0356] Diagnosis and grading of GvHD should be based on published criteria (Harris et al., (2016) Biol Blood Marrow Transplant, 22, 4-10), as outlined in Table 24.

TABLE-US-00024 TABLE 24 Criteria for Grading Acute GvHD Skin Liver Lower GI (active (bilirubin Upper (stool Stage erythema only) mg/dL) GI output/day) 0 No active <2 No or <500 ml/day or (erythematous) intermittent <3 episodes/day GvHD rash nausea, vomiting, or anorexia 1 Maculopapular 2-3 Persistent 500-999 ml/day or rash <25% nausea, 3-4 episodes/day BSA vomiting, or anorexia 2 Maculopapular 3.1-6 1000-1500 ml/day or rash 25-50% 5-7 episodes/day BSA 3 Maculopapular 6.1-15 >1500 ml/day or rash >50% >7 episodes/day BSA 4 Generalized >15 Severe abdominal erythroderma pain with or (>50% BSA) without ileus, or plus bullous grossly bloody formation and stool (regardless desquamation of stool >5% BSA volume) BSA: body surface area; GI: gastrointestinal; GvHD: graft versus host disease.

[0357] Overall GvHD grade will be determined based on most severe target organ involvement. [0358] Grade 0: No stage 1-4 of any organ. [0359] Grade 1: Stage 1-2 skin without liver, upper GI, or lower GI involvement. [0360] Grade 2: Stage 3 rash and/or stage 1 liver and/or stage 1 upper GI and/or stage 1 lower GI. [0361] Grade 3: Stage 2-3 liver and/or stage 2-3 lower GI, with stage 0-3 skin and/or stage 0-1 upper GI. [0362] Grade 4: Stage 4 skin, liver, or lower GI involvement, with stage 0-1 upper GI.

[0363] Potential confounding factors that may mimic GvHD such as infections and reactions to medications should be ruled out. Skin and/or GI biopsy should be obtained for confirmation before or soon after treatment has been initiated. In instance of liver involvement, liver biopsy should be attempted if clinically feasible. Sample(s) of all biopsies will also be sent to a central laboratory for pathology assessment. Details of sample preparation and shipment are contained in the Laboratory Manual.

[0364] Recommendations for management of acute GvHD are outlined in Table 25. To allow for intersubject comparability at the end of the trial, investigators should follow these recommendations except in specific clinical scenarios in which following them could put the subject at risk.

TABLE-US-00025 TABLE 25 Acute GvHD Management Grade Management 1 Skin: Topical steroids or immunosuppressants; if stage 2: prednisone 1 mg/kg (or equivalent dose). 2-4 Initiate prednisone 2 mg/kg daily (or equivalent dose). IV form of steroid such as methylprednisolone should be considered if there are concerns with malabsorption. Steroid taper may begin after improvement is seen after .gtoreq.3 days of steroids. Taper should be 50% decrease of total daily steroid dose every 5 days. GI: In addition to steroids, start anti-diarrheal agents per standard practice. GI: gastrointestinal; IV: intravenous.

[0365] Decisions to initiate second-line therapy should be made sooner for subjects with more severe GvHD. For example, secondary therapy may be indicated after 3 days with progressive manifestations of GvHD, after 1 week with persistent grade 3 GvHD, or after 2 weeks with persistent grade 2 GvHD. Second-line systemic therapy may be indicated earlier in subjects who cannot tolerate high-dose glucocorticoid treatment (Martin et al., (2012) Biol Blood Marrow Transplant, 18, 1150-1163). Choice of secondary therapy and when to initiate will be based on the treating investigator's clinical judgement and local practice.

[0366] Management of refractory acute GvHD or chronic GvHD will be per institutional guidelines. Anti-infective prophylaxis measures should be instituted per local guidelines when treating subjects with immunosuppressive agents (including steroids).

Hypotension and Renal Insufficiency

[0367] Hypotension and renal insufficiency have been reported with CAR T cell therapy and should be treated with IV administration of normal saline boluses according to institutional practice guidelines. Dialysis should be considered when appropriate.

Study Eligibility

Inclusion Criteria

[0368] To be considered eligible to participate in this study, a subject must meet the inclusion criteria listed below (unless indicated as optional):

[0369] 1. .gtoreq.18 years of age and weight >50 kg (optional).

[0370] 2. Able to understand and comply with protocol-required study procedures and voluntarily sign a written informed consent document.

[0371] 3. Diagnosed with 1 of the following B cell malignancies: Histologically confirmed B cell NHLs: DLBCL NOS, high grade B cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements, transformed FL, or grade 3b FL. [0372] Confirmation of tumor histology from local pathology lab (archival tissue from last relapse/progression [within 3 months of enrollment] or biopsy during screening). [0373] At least 1 measurable lesion that is fluorodeoxyglucose positron emission tomography (PET)-positive, as defined by Lugano criteria (score of 4 or 5 on Lugano criteria 5-point scale). Previously irradiated lesions will be considered measurable only if progression is documented following completion of radiation therapy.

[0374] 4. Refractory or relapsed disease, as evidenced by the following cohort-specific criteria:

[0375] Two or more lines of prior therapy, including an anti-CD20 monoclonal antibody and an anthracycline-containing regimen, and have failed prior autologous hematopoietic stem cell transplantation (HSCT) or ineligible for or refused prior autologous HSCT. Subjects who have received autologous HSCT must have recovered from HSCT-related toxicities. [0376] For refractory disease, subjects must have progressive disease on last therapy, or have stable disease following at least 2 cycles of therapy with duration of stable disease of up to 6 months. [0377] For subjects with transformed FL, subjects must have received at least 1 line of chemotherapy for disease after transformation to DLBCL.

[0378] 5. Eastern Cooperative Oncology Group (ECOG) performance status 0 or 1.

[0379] 6. Meets criteria to undergo LD chemotherapy and CAR T cell infusion.

[0380] 7. Adequate organ function: [0381] Renal: Estimated glomerular filtration rate >50 mL/min/1.73 m.sup.2. [0382] Liver: Aspartate transaminase or alanine transaminase <3.times. upper limit of normal (ULN); total bilirubin <1.5.times.ULN (for subjects with Gilbert's syndrome, total bilirubin <2 mg/dL). [0383] Cardiac: Hemodynamically stable and left ventricle ejection fraction .gtoreq.45% by echocardiogram. [0384] Pulmonary: Oxygen saturation level on room air >91% per pulse oximetry.

[0385] 8. Female subjects of childbearing potential (postmenarcheal with an intact uterus and at least 1 ovary, who are less than 1 year postmenopausal) must agree to use acceptable method(s) of contraception from enrollment through at least 12 months after CTX110 infusion.

[0386] 9. Male subjects must agree to use effective contraception from enrollment through at least 12 months after CTX110 infusion.

[0387] 10. Agree to participate in an additional long-term follow-up study after completion of this study.

Exclusion Criteria

[0388] To be eligible for entry into the study, the subject must not meet any of the exclusion criteria listed below:

[0389] 1. Eligible for and agrees to autologous HSCT.

[0390] 2. Treatment with the following therapies as described below: [0391] Prior treatment with any gene therapy or genetically modified cell therapy, including CAR T cells. [0392] Prior treatment with a CD19-directed antibody, bispecific T cell engager, or antibody-drug conjugate, unless there is confirmed CD19 expression (by immunohistochemistry or flow cytometry) after progression or relapse following most recent CD19-directed treatment.

[0393] 3. Prior allogeneic HSCT.

[0394] 4. Known contraindication to cyclophosphamide, fludarabine, or any of the excipients of CTX110 product.

[0395] 5. Detectable malignant cells from cerebrospinal fluid (CSF) or magnetic resonance imaging (MRI) indicating brain metastases during screening, or a history of central nervous system (CNS) involvement by malignancy (CSF or imaging).

[0396] 6. History of a seizure disorder, cerebrovascular ischemia/hemorrhage, dementia, cerebellar disease, or any autoimmune disease with CNS involvement.

[0397] 7. Unstable angina, clinically significant arrhythmia, or myocardial infarction within 6 months prior to screening.

[0398] 8. Uncontrolled, acute life-threatening bacterial, viral, or fungal infection.

[0399] 9. Positive for presence of human immunodeficiency virus (HIV) type 1 or 2, or active hepatitis B virus (HBV) or hepatitis C virus (HCV) infection. Subjects with prior history of HBV or HBC infection who have documented undetectable viral load (by quantitative polymerase chain reaction [PCR] or nucleic acid testing) are permitted. Infectious disease testing (HIV-1, HIV-2, HCV antibody and PCR, HBV surface antigen, HBV surface antibody, HBV core antibody) performed within 30 days of signing the informed consent form may be considered for subject eligibility.

[0400] 10. Previous or concurrent malignancy, except basal cell or squamous cell skin carcinoma, adequately resected and in situ carcinoma of cervix, or a previous malignancy that was completely resected and has been in remission for .gtoreq.5 years.

[0401] 11. Radiation therapy within 14 days of enrollment.

[0402] 12. Use of systemic antitumor therapy or investigational agent within 14 days or 5 half-lives, whichever is longer, of enrollment. Exceptions are made for 1) prior inhibitory/stimulatory immune checkpoint molecule therapy, which is prohibited within 3 half-lives of enrollment, and 2) rituximab use within 30 days prior to screening is prohibited.

[0403] 13. Primary immunodeficiency disorder or active autoimmune disease requiring steroids and/or other immunosuppressive therapy.

[0404] 14. Diagnosis of significant psychiatric disorder or other medical condition that could impede the subject's ability to participate in the study.

[0405] 15. Women who are pregnant or breastfeeding.

Statistical Methods

Sample Size

[0406] The sample size in the dose escalation part of the study will be approximately 6 to 54 subjects, depending on the number of dose levels and cohorts evaluated, and the occurrence of DLTs.

[0407] If the study proceeds to cohort expansion, an optimal Simon 2-stage design will be employed. The sample size for each cohort will depend on the assumption of effect size for the specific indication.

[0408] For expansion of Cohort A, in the first stage, up to 30 subjects will be enrolled. If 10 or more of the first 30 subjects in the full analysis set achieve an objective response, the study will expand enrollment to include an additional 47 subjects (77 total) in the second stage. A final sample size of 77 subjects will have 90% power (.alpha.=0.05, 2-sided test) to test for a difference between a ORR of 45% with CTX110 and an ORR of 26%, the estimated ORR to standard salvage therapy in patients with relapsed/refractory DLBCL.

[0409] As in Cohort A, upon completion of the dose escalation part of the study, Cohort B may go on to cohort expansion after a protocol amendment.

[0410] To date, all subjects that participated in this study have completed Stage 1 (eligibility screening) within 14 days. One subject completed Stage 1 within 2 days. A subject who met the eligibility criteria started lymphodepleting therapy within 24 hours of completing Stage 1. All eligible subjects have completed the screening period (stage 1) and received LD chemotherapy in less than 15 days, with one patient completing screening and starting an LD chemo dose within 72 hrs. Some of the eligible subjects have DLBCL (e.g., NOS, high grade); others have transformed FL and Richter's transformation.

[0411] All subjects receiving LD chemotherapy have progressed to receiving the DL1 or DL2 dose of CTX110 within 2-7 days following completion of the LD chemotherapy. Results obtained from these patients to date are summarized below.

[0412] Subjects in both DL1 and DL2 doses experienced decreased tumor metabolic activity (FDG uptake on PET scan) and/or decrease in tumor size. A dose dependent response has been observed, including a complete and durable response for >60 days at DL2. None of the treated patients exhibited any DLTs so far. Further, the allogeneic CAR-T cell therapy exhibited desired pharmacokinetic features in the treated human subjects, including CAR-T cell expansion and persistence after infusion. A dose dependent effect has also been observed in both CTX110 expansion and persistence. All subjects in DL2 have exhibited CTX110 expansion and persistence Up to 90-fold expansion of CTX110 in peripheral blood has been observed in one subject. Further, persistence of CTX110 cells can be detected in DL2 subjects at least 8-10 days following treatment and has been detected up to 28 days post-infusion.

TABLE-US-00026 SEQUENCE TABLE SEQ ID NO Description Sequence 1 TRAC gene-edit AAGAGCAACAAATCTGACT 2 TRAC gene-edit AAGAGCAACAGTGCTGTGCCTGGAGCAACAAATCTGACT AAGAGCAACAAATCTGACT 3 TRAC gene-edit AAGAGCAACAGTGCTGGAGCAACAAATCTGACT AAGAGCAACAAATCTGACT 4 TRAC gene-edit AAGAGCAACAGTGCCTGGAGCAACAAATCTGACT AAGAGCAACAAATCTGACT 5 TRAC gene-edit AAGAGCAACAGTGCTGACTAAGAGCAACAAATCTGACT 6 TRAC gene-edit AAGAGCAACAGTGCTGTGGGCCTGGAGCAACAAATCTGA CT AAGAGCAACAAATCTGACT 7 TRAC gene-edit AAGAGCAACAGTGCTGGCCTGGAGCAACAAATCTGACT AAGAGCAACAAATCTGACT 8 TRAC gene-edit AAGAGCAACAGTGCTGTGTGCCTGGAGCAACAAATCTGA CT AAGAGCAACAAATCTGACT 9 B2M gene-edit CGTGGCCTTAGCTGTGCTCGCGCTACTCTCTCTTTCTGCCT GGAGGCTATCCAGCGTGAGTCTCTCCTACCCTCCCGCT 10 B2M gene-edit CGTGGCCTTAGCTGTGCTCGCGCTACTCTCTCTTTCGCCTG GAGGCTATCCAGCGTGAGTCTCTCCTACCCTCCCGCT 11 B2M gene-edit CGTGGCCTTAGCTGTGCTCGCGCTACTCTCTCTTTCTGGA GGCTATCCAGCGTGAGTCTCTCCTACCCTCCCGCT 12 B2M gene-edit CGTGGCCTTAGCTGTGCTCGCGCTACTCTCTCTTTCTGGAT AGCCTGGAGGCTATCCAGCGTGAGTCTCTCCTACCCTCCC GCT 13 B2M gene-edit CGTGGCCTTAGCTGTGCTCGCGCTATCCAGCGTGAGTCTC TCCTACCCTCCCGCT 14 B2M gene-edit CGTGGCCTTAGCTGTGCTCGCGCTACTCTCTCTTTCTGTGG CCTGGAGGCTATCCAGCGTGAGTCTCTCCTACCCTCCCGC T 15 sgRNA nnnnnnnnnnnnnnnnnnnnguuuuagagcuagaaauagcaaguuaaaauaaggcu aguccguuaucaacuugaaaaaguggcaccgagucggugcuuuu 16 sgRNA nnnnnnnnnnnnnnnnnnnnguuuuagagcuagaaauagcaaguuaaaauaaggcu aguccguuaucaacuugaaaaaguggcaccgagucggugc 17 sgRNA n.sub.(17-30)guuuuagagcuagaaauagcaaguuaaaauaaggcuaguccguuauc aacuugaaaaaguggcaccgagucggugcu.sub.(1-8) 18 TRAC sgRNA (TA-1) AGAGCAACAGUGCUGUGGCCguuuuagagcuagaaauagcaaguuaa unmodified aauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcUUUU 19 TRAC sgRNA spacer AGAGCAACAGUGCUGUGGCC unmodified 20 B2M sgRNA GCUACUCUCUCUUUCUGGCCguuuuagagcuagaaauagcaaguuaaa unmodified auaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcUUUU 21 B2M sgRNA spacer GCUACUCUCUCUUUCUGGCC unmodified 22 TRAC sgRNA (TA-1) A*G*A*GCAACAGUGCUGUGGCCguuuuagagcuagaaauagcaagu modified uaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcU*U* *: 2'-O-methyl U*U phosphorothioate residue 23 TRAC sgRNA spacer A*G*A*GCAACAGUGCUGUGGCC modified *: 2'-O-methyl phosphorothioate residue 24 B2M sgRNA G*C*U*ACUCUCUCUUUCUGGCCguuuuagagcuagaaauagcaagu modified uaaaauaaggcuaguccguuaucaacuugaaaaaguggcaccgagucggugcU*U* *: 2'-O-methyl U*U phosphorothioate residue 25 B2M sgRNA spacer G*C*U*ACUCUCUCUUUCUGGCC modified *: 2'-O-methyl phosphorothioate residue 26 TRAC target AGAGCAACAGTGCTGTGGCC sequence 27 B2M target sequence GCTACTCTCTCTTTCTGGCC 28 TRAC target AGAGCAACAGTGCTGTGGCC (TGG) sequence with (PAM) 29 B2M target sequence GCTACTCTCTCTTTCTGGCC (TGG) with (PAM) 30 signal peptide MLLLVTSLLLCELPHPAFLLIP 31 signal peptide MALPVTALLLPLALLLHAARP 32 CD8a transmembrane IYIWAPLAGTCGVLLLSLVITLY domain 33 4-1BB nucleotide AAACGGGGCAGAAAGAAACTCCTGTATATATTCAAACAA sequence CCATTTATGAGACCAGTACAAACTACTCAAGAGGAAGAT GGCTGTAGCTGCCGATTTCCAGAAGAAGAAGAAGGAGGA TGTGAACTG 34 4-1BB amino acid KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL sequence 35 CD28 nucleotide TCAAAGCGGAGTAGGTTGTTGCATTCCGATTACATGAATA sequence TGACTCCTCGCCGGCCTGGGCCGACAAGAAAACATTACC AACCCTATGCCCCCCCACGAGACTTCGCTGCGTACAGGTC C 36 CD28 amino acid SKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS sequence 37 CD3-zeta nucleotide CGAGTGAAGTTTTCCCGAAGCGCAGACGCTCCGGCATAT sequence CAGCAAGGACAGAATCAGCTGTATAACGAACTGAATTTG GGACGCCGCGAGGAGTATGACGTGCTTGATAAACGCCGG GGGAGAGACCCGGAAATGGGGGGTAAACCCCGAAGAAA GAATCCCCAAGAAGGACTCTACAATGAACTCCAGAAGGA TAAGATGGCGGAGGCCTACTCAGAAATAGGTATGAAGGG CGAACGACGACGGGGAAAAGGTCACGATGGCCTCTACCA AGGGTTGAGTACGGCAACCAAAGATACGTACGATGCACT GCATATGCAGGCCCTGCCTCCCAGA 38 CD3-zeta amino acid RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRG sequence RDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGER RRGKGHDGLYQGLSTATKDTYDALHMQALPPR 39 FMC63-28Z ATGCTTCTTTTGGTTACGTCTCTGTTGCTTTGCGAACTTCC (FMC63-CD8[tm]- TCATCCAGCGTTCTTGCTGATCCCCGATATTCAGATGACT CD28[co-stimulatory CAGACCACCAGTAGCTTGTCTGCCTCACTGGGAGACCGA domain]-CD3z) GTAACAATCTCCTGCAGGGCAAGTCAAGACATTAGCAAA TACCTCAATTGGTACCAGCAGAAGCCCGACGGAACGGTA AAACTCCTCATCTATCATACGTCAAGGTTGCATTCCGGAG TACCGTCACGATTTTCAGGTTCTGGGAGCGGAACTGACTA TTCCTTGACTATTTCAAACCTCGAGCAGGAGGACATTGCG ACATATTTTTGTCAACAAGGTAATACCCTCCCTTACACTT TCGGAGGAGGAACCAAACTCGAAATTACCGGGTCCACCA GTGGCTCTGGGAAGCCTGGCAGTGGAGAAGGTTCCACTA AAGGCGAGGTGAAGCTCCAGGAGAGCGGCCCCGGTCTCG TTGCCCCCAGTCAAAGCCTCTCTGTAACGTGCACAGTGAG TGGTGTATCATTGCCTGATTATGGCGTCTCCTGGATAAGG CAGCCCCCGCGAAAGGGTCTTGAATGGCTTGGGGTAATA TGGGGCTCAGAGACAACGTATTATAACTCCGCTCTCAAA AGTCGCTTGACGATAATAAAAGATAACTCCAAGAGTCAA GTTTTCCTTAAAATGAACAGTTTGCAGACTGACGATACCG CTATATATTATTGTGCTAAACATTATTACTACGGCGGTAG TTACGCGATGGATTATTGGGGGCAGGGGACTTCTGTCAC AGTCAGTAGTGCTGCTGCCTTTGTCCCGGTATTTCTCCCA GCCAAACCGACCACGACTCCCGCCCCGCGCCCTCCGACA CCCGCTCCCACCATCGCCTCTCAACCTCTTAGTCTTCGCC CCGAGGCATGCCGACCCGCCGCCGGGGGTGCTGTTCATA CGAGGGGCTTGGACTTCGCTTGTGATATTTACATTTGGGC TCCGTTGGCGGGTACGTGCGGCGTCCTTTTGTTGTCACTC GTTATTACTTTGTATTGTAATCACAGGAATCGCTCAAAGC GGAGTAGGTTGTTGCATTCCGATTACATGAATATGACTCC TCGCCGGCCTGGGCCGACAAGAAAACATTACCAACCCTA TGCCCCCCCACGAGACTTCGCTGCGTACAGGTCCCGAGTG AAGTTTTCCCGAAGCGCAGACGCTCCGGCATATCAGCAA GGACAGAATCAGCTGTATAACGAACTGAATTTGGGACGC CGCGAGGAGTATGACGTGCTTGATAAACGCCGGGGGAGA GACCCGGAAATGGGGGGTAAACCCCGAAGAAAGAATCC CCAAGAAGGACTCTACAATGAACTCCAGAAGGATAAGAT GGCGGAGGCCTACTCAGAAATAGGTATGAAGGGCGAACG ACGACGGGGAAAAGGTCACGATGGCCTCTACCAAGGGTT GAGTACGGCAACCAAAGATACGTACGATGCACTGCATAT GCAGGCCCTGCCTCCCAGA 40 FMC63-28Z MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTI (FMC63-CD8[tm]- SCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFS CD28[co-stimulatory GSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEI domain]-CD3z) TGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCT Amino Acid VSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALK SRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSY AMDYWGQGTSVTVSSAAAFVPVFLPAKPTTTPAPRPPTPAP TIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGT CGVLLLSLVITLYCNHRNRSKRSRLLHSDYMNMTPRRPGPT RKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYN ELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNEL QKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTY DALHMQALPPR 41 TRAC-LHA (800 bp) GAGATGTAAGGAGCTGCTGTGACTTGCTCAAGGCCTTAT ATCGAGTAAACGGTAGTGCTGGGGCTTAGACGCAGGTGT TCTGATTTATAGTTCAAAACCTCTATCAATGAGAGAGCAA TCTCCTGGTAATGTGATAGATTTCCCAACTTAATGCCAAC ATACCATAAACCTCCCATTCTGCTAATGCCCAGCCTAAGT TGGGGAGACCACTCCAGATTCCAAGATGTACAGTTTGCTT TGCTGGGCCTTTTTCCCATGCCTGCCTTTACTCTGCCAGA GTTATATTGCTGGGGTTTTGAAGAAGATCCTATTAAATAA AAGAATAAGCAGTATTATTAAGTAGCCCTGCATTTCAGGT TTCCTTGAGTGGCAGGCCAGGCCTGGCCGTGAACGTTCAC TGAAATCATGGCCTCTTGGCCAAGATTGATAGCTTGTGCC TGTCCCTGAGTCCCAGTCCATCACGAGCAGCTGGTTTCTA AGATGCTATTTCCCGTATAAAGCATGAGACCGTGACTTGC CAGCCCCACAGAGCCCCGCCCTTGTCCATCACTGGCATCT GGACTCCAGCCTGGGTTGGGGCAAAGAGGGAAATGAGAT CATGTCCTAACCCTGATCCTCTTGTCCCACAGATATCCAG AACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAA TCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATT CTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGT ATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGG ACTTCA 42 TRAC-RHA (800 bp) TGGAGCAACAAATCTGACTTTGCATGTGCAAACGCCTTCA ACAACAGCATTATTCCAGAAGACACCTTCTTCCCCAGCCC AGGTAAGGGCAGCTTTGGTGCCTTCGCAGGCTGTTTCCTT GCTTCAGGAATGGCCAGGTTCTGCCCAGAGCTCTGGTCA ATGATGTCTAAAACTCCTCTGATTGGTGGTCTCGGCCTTA TCCATTGCCACCAAAACCCTCTTTTTACTAAGAAACAGTG AGCCTTGTTCTGGCAGTCCAGAGAATGACACGGGAAAAA AGCAGATGAAGAGAAGGTGGCAGGAGAGGGCACGTGGC CCAGCCTCAGTCTCTCCAACTGAGTTCCTGCCTGCCTGCC TTTGCTCAGACTGTTTGCCCCTTACTGCTCTTCTAGGCCTC ATTCTAAGCCCCTTCTCCAAGTTGCCTCTCCTTATTTCTCC CTGTCTGCCAAAAAATCTTTCCCAGCTCACTAAGTCAGTC TCACGCAGTCACTCATTAACCCACCAATCACTGATTGTGC CGGCACATGAATGCACCAGGTGTTGAAGTGGAGGAATTA AAAAGTCAGATGAGGGGTGTGCCCAGAGGAAGCACCATT CTAGTTGGGGGAGCCCATCTGTCAGCTGGGAAAAGTCCA AATAACTTCAGATTGGAATGTGTTTTAACTCAGGGTTGAG AAAACAGCTACCTTCAGGACAAAAGTCAGGGAAGGGCTC TCTGAAGAAATGCTACTTGAAGATACCAGCCCTACCAAG GGCAGGGAGAGGACCCTATAGAGGCCTGGGACAGGAGC TCAATGAGAAAGG 43 EF1a GGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCC ACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGA ACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAA AGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTG GGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACG TTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAG TGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGT TATGGCCCTTGCGTGCCTTGAATTACTTCCACTGGCTGCA GTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGG TGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCG CCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCC GCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGC

TGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGA CCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAA ATGCGGGCCAAGATCTGCACACTGGTATTTCGGTTTTTGG GGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCAC ATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAG AATCGGACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCT GGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGG GCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCG GAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCA AAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGA GTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGC CGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCC AGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGT CTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCC CCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTG GCACTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGT TTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCA AAGTTTTTTTCTTCCATTTCAGGTGTCGTGA 44 GM-CSF signal ATGCTTCTTTTGGTTACGTCTCTGTTGCTTTGCGAACTTCC peptide TCATCCAGCGTTCTTGCTGATCCCC 45 GM-CSF signal MLLLVTSLLLCELPHPAFLLIP peptide 46 Anti-CD19 scFv GATATTCAGATGACTCAGACCACCAGTAGCTTGTCTGCCT CACTGGGAGACCGAGTAACAATCTCCTGCAGGGCAAGTC AAGACATTAGCAAATACCTCAATTGGTACCAGCAGAAGC CCGACGGAACGGTAAAACTCCTCATCTATCATACGTCAA GGTTGCATTCCGGAGTACCGTCACGATTTTCAGGTTCTGG GAGCGGAACTGACTATTCCTTGACTATTTCAAACCTCGAG CAGGAGGACATTGCGACATATTTTTGTCAACAAGGTAAT ACCCTCCCTTACACTTTCGGAGGAGGAACCAAACTCGAA ATTACCGGGTCCACCAGTGGCTCTGGGAAGCCTGGCAGT GGAGAAGGTTCCACTAAAGGCGAGGTGAAGCTCCAGGAG AGCGGCCCCGGTCTCGTTGCCCCCAGTCAAAGCCTCTCTG TAACGTGCACAGTGAGTGGTGTATCATTGCCTGATTATGG CGTCTCCTGGATAAGGCAGCCCCCGCGAAAGGGTCTTGA ATGGCTTGGGGTAATATGGGGCTCAGAGACAACGTATTA TAACTCCGCTCTCAAAAGTCGCTTGACGATAATAAAAGA TAACTCCAAGAGTCAAGTTTTCCTTAAAATGAACAGTTTG CAGACTGACGATACCGCTATATATTATTGTGCTAAACATT ATTACTACGGCGGTAGTTACGCGATGGATTATTGGGGGC AGGGGACTTCTGTCACAGTCAGTAGT 47 CD19 scFv amino DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPD acid sequence GTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIA Linker underlined TYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGE VKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRK GLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNS LQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS 48 CD8a extracellular + GCTGCTGCCTTTGTCCCGGTATTTCTCCCAGCCAAACCGA CD8a transmembrane + CCACGACTCCCGCCCCGCGCCCTCCGACACCCGCTCCCAC 5' Linker CATCGCCTCTCAACCTCTTAGTCTTCGCCCCGAGGCATGC (underlined) CGACCCGCCGCCGGGGGTGCTGTTCATACGAGGGGCTTG GACTTCGCTTGTGATATTTACATTTGGGCTCCGTTGGCGG GTACGTGCGGCGTCCTTTTGTTGTCACTCGTTATTACTTTG TATTGTAATCACAGGAATCGC 49 CD8a extracellular + TTTGTCCCGGTATTTCTCCCAGCCAAACCGACCACGACTC CD8a transmembrane CCGCCCCGCGCCCTCCGACACCCGCTCCCACCATCGCCTC (without linker) TCAACCTCTTAGTCTTCGCCCCGAGGCATGCCGACCCGCC GCCGGGGGTGCTGTTCATACGAGGGGCTTGGACTTCGCTT GTGATATTTACATTTGGGCTCCGTTGGCGGGTACGTGCGG CGTCCTTTTGTTGTCACTCGTTATTACTTTGTATTGTAATC ACAGGAATCGC 50 CD8a extracellular + FVPVFLPAKPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGG CD8a transmembrane AVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNHRNR 51 CD19 VH EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPR KGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMN SLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS 52 CD19 VL DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPD GTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIA TYFCQQGNTLPYTFGGGTKLEIT 53 CD19 linker GSTSGSGKPGSGEGSTKG 54 LHA to RHA GAGATGTAAGGAGCTGCTGTGACTTGCTCAAGGCCTTAT ATCGAGTAAACGGTAGTGCTGGGGCTTAGACGCAGGTGT TCTGATTTATAGTTCAAAACCTCTATCAATGAGAGAGCAA TCTCCTGGTAATGTGATAGATTTCCCAACTTAATGCCAAC ATACCATAAACCTCCCATTCTGCTAATGCCCAGCCTAAGT TGGGGAGACCACTCCAGATTCCAAGATGTACAGTTTGCTT TGCTGGGCCTTTTTCCCATGCCTGCCTTTACTCTGCCAGA GTTATATTGCTGGGGTTTTGAAGAAGATCCTATTAAATAA AAGAATAAGCAGTATTATTAAGTAGCCCTGCATTTCAGGT TTCCTTGAGTGGCAGGCCAGGCCTGGCCGTGAACGTTCAC TGAAATCATGGCCTCTTGGCCAAGATTGATAGCTTGTGCC TGTCCCTGAGTCCCAGTCCATCACGAGCAGCTGGTTTCTA AGATGCTATTTCCCGTATAAAGCATGAGACCGTGACTTGC CAGCCCCACAGAGCCCCGCCCTTGTCCATCACTGGCATCT GGACTCCAGCCTGGGTTGGGGCAAAGAGGGAAATGAGAT CATGTCCTAACCCTGATCCTCTTGTCCCACAGATATCCAG AACCCTGACCCTGCCGTGTACCAGCTGAGAGACTCTAAA TCCAGTGACAAGTCTGTCTGCCTATTCACCGATTTTGATT CTCAAACAAATGTGTCACAAAGTAAGGATTCTGATGTGT ATATCACAGACAAAACTGTGCTAGACATGAGGTCTATGG ACTTCAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACA TCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGC AATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAAC TGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCG AGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCG TGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACA GGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTT ACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACTG GCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGA AGTGGGTGGGAGAGTTCGAGGCCTTGCGCTTAAGGAGCC CCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCT GGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCT GTCTCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTT TGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTC TTGTAAATGCGGGCCAAGATCTGCACACTGGTATTTCGGT TTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCA GCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCC ACCGAGAATCGGACGGGGGTAGTCTCAAGCTGGCCGGCC TGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCG CCCTGGGCGGCAAGGCTGGCCCGGTCGGCACCAGTTGCG TGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGCAGGG AGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGC GGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTC CTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCG CCGTCCAGGCACCTCGATTAGTTCTCGAGCTTTTGGAGTA CGTCGTCTTTAGGTTGGGGGGAGGGGTTTTATGCGATGGA GTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCC AGCTTGGCACTTGATGTAATTCTCCTTGGAATTTGCCCTTT TTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGT GGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGACCAC CATGCTTCTTTTGGTTACGTCTCTGTTGCTTTGCGAACTTC CTCATCCAGCGTTCTTGCTGATCCCCGATATTCAGATGAC TCAGACCACCAGTAGCTTGTCTGCCTCACTGGGAGACCG AGTAACAATCTCCTGCAGGGCAAGTCAAGACATTAGCAA ATACCTCAATTGGTACCAGCAGAAGCCCGACGGAACGGT AAAACTCCTCATCTATCATACGTCAAGGTTGCATTCCGGA GTACCGTCACGATTTTCAGGTTCTGGGAGCGGAACTGACT ATTCCTTGACTATTTCAAACCTCGAGCAGGAGGACATTGC GACATATTTTTGTCAACAAGGTAATACCCTCCCTTACACT TTCGGAGGAGGAACCAAACTCGAAATTACCGGGTCCACC AGTGGCTCTGGGAAGCCTGGCAGTGGAGAAGGTTCCACT AAAGGCGAGGTGAAGCTCCAGGAGAGCGGCCCCGGTCTC GTTGCCCCCAGTCAAAGCCTCTCTGTAACGTGCACAGTGA GTGGTGTATCATTGCCTGATTATGGCGTCTCCTGGATAAG GCAGCCCCCGCGAAAGGGTCTTGAATGGCTTGGGGTAAT ATGGGGCTCAGAGACAACGTATTATAACTCCGCTCTCAA AAGTCGCTTGACGATAATAAAAGATAACTCCAAGAGTCA AGTTTTCCTTAAAATGAACAGTTTGCAGACTGACGATACC GCTATATATTATTGTGCTAAACATTATTACTACGGCGGTA GTTACGCGATGGATTATTGGGGGCAGGGGACTTCTGTCA CAGTCAGTAGTGCTGCTGCCTTTGTCCCGGTATTTCTCCC AGCCAAACCGACCACGACTCCCGCCCCGCGCCCTCCGAC ACCCGCTCCCACCATCGCCTCTCAACCTCTTAGTCTTCGC CCCGAGGCATGCCGACCCGCCGCCGGGGGTGCTGTTCAT ACGAGGGGCTTGGACTTCGCTTGTGATATTTACATTTGGG CTCCGTTGGCGGGTACGTGCGGCGTCCTTTTGTTGTCACT CGTTATTACTTTGTATTGTAATCACAGGAATCGCTCAAAG CGGAGTAGGTTGTTGCATTCCGATTACATGAATATGACTC CTCGCCGGCCTGGGCCGACAAGAAAACATTACCAACCCT ATGCCCCCCCACGAGACTTCGCTGCGTACAGGTCCCGAGT GAAGTTTTCCCGAAGCGCAGACGCTCCGGCATATCAGCA AGGACAGAATCAGCTGTATAACGAACTGAATTTGGGACG CCGCGAGGAGTATGACGTGCTTGATAAACGCCGGGGGAG AGACCCGGAAATGGGGGGTAAACCCCGAAGAAAGAATC CCCAAGAAGGACTCTACAATGAACTCCAGAAGGATAAGA TGGCGGAGGCCTACTCAGAAATAGGTATGAAGGGCGAAC GACGACGGGGAAAAGGTCACGATGGCCTCTACCAAGGGT TGAGTACGGCAACCAAAGATACGTACGATGCACTGCATA TGCAGGCCCTGCCTCCCAGATAATAATAAAATCGCTATCC ATCGAAGATGGATGTGTGTTGGTTTTTTGTGTGTGGAGCA ACAAATCTGACTTTGCATGTGCAAACGCCTTCAACAACA GCATTATTCCAGAAGACACCTTCTTCCCCAGCCCAGGTAA GGGCAGCTTTGGTGCCTTCGCAGGCTGTTTCCTTGCTTCA GGAATGGCCAGGTTCTGCCCAGAGCTCTGGTCAATGATG TCTAAAACTCCTCTGATTGGTGGTCTCGGCCTTATCCATT GCCACCAAAACCCTCTTTTTACTAAGAAACAGTGAGCCTT GTTCTGGCAGTCCAGAGAATGACACGGGAAAAAAGCAGA TGAAGAGAAGGTGGCAGGAGAGGGCACGTGGCCCAGCC TCAGTCTCTCCAACTGAGTTCCTGCCTGCCTGCCTTTGCTC AGACTGTTTGCCCCTTACTGCTCTTCTAGGCCTCATTCTAA GCCCCTTCTCCAAGTTGCCTCTCCTTATTTCTCCCTGTCTG CCAAAAAATCTTTCCCAGCTCACTAAGTCAGTCTCACGCA GTCACTCATTAACCCACCAATCACTGATTGTGCCGGCACA TGAATGCACCAGGTGTTGAAGTGGAGGAATTAAAAAGTC AGATGAGGGGTGTGCCCAGAGGAAGCACCATTCTAGTTG GGGGAGCCCATCTGTCAGCTGGGAAAAGTCCAAATAACT TCAGATTGGAATGTGTTTTAACTCAGGGTTGAGAAAACA GCTACCTTCAGGACAAAAGTCAGGGAAGGGCTCTCTGAA GAAATGCTACTTGAAGATACCAGCCCTACCAAGGGCAGG GAGAGGACCCTATAGAGGCCTGGGACAGGAGCTCAATGA GAAAGG 55 spCas9 MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDR HSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYL QEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVD EVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFR GHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVD AKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTP NFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLF LAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDL TLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFY KFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHL GELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDK NLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLS GEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGV EDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLF EDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKL INGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQ KAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKV MGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKEL GSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDIN RLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELD KAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREV KVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVV GTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATA KYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKG RDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDK LIARKKDWDPKKYGGFD SPTVAYSVLVVAKVEKGKSKKLK SVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKY SLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHY EKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYF DTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD 56 rAAV CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCG CCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTG AGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATC ACTAGGGGTTCCTGCGGCCGCACGCGTGAGATGTAAGGA GCTGCTGTGACTTGCTCAAGGCCTTATATCGAGTAAACGG TAGTGCTGGGGCTTAGACGCAGGTGTTCTGATTTATAGTT CAAAACCTCTATCAATGAGAGAGCAATCTCCTGGTAATG TGATAGATTTCCCAACTTAATGCCAACATACCATAAACCT CCCATTCTGCTAATGCCCAGCCTAAGTTGGGGAGACCACT CCAGATTCCAAGATGTACAGTTTGCTTTGCTGGGCCTTTT TCCCATGCCTGCCTTTACTCTGCCAGAGTTATATTGCTGG GGTTTTGAAGAAGATCCTATTAAATAAAAGAATAAGCAG TATTATTAAGTAGCCCTGCATTTCAGGTTTCCTTGAGTGG CAGGCCAGGCCTGGCCGTGAACGTTCACTGAAATCATGG CCTCTTGGCCAAGATTGATAGCTTGTGCCTGTCCCTGAGT CCCAGTCCATCACGAGCAGCTGGTTTCTAAGATGCTATTT CCCGTATAAAGCATGAGACCGTGACTTGCCAGCCCCACA GAGCCCCGCCCTTGTCCATCACTGGCATCTGGACTCCAGC CTGGGTTGGGGCAAAGAGGGAAATGAGATCATGTCCTAA CCCTGATCCTCTTGTCCCACAGATATCCAGAACCCTGACC CTGCCGTGTACCAGCTGAGAGACTCTAAATCCAGTGACA AGTCTGTCTGCCTATTCACCGATTTTGATTCTCAAACAAA TGTGTCACAAAGTAAGGATTCTGATGTGTATATCACAGAC AAAACTGTGCTAGACATGAGGTCTATGGACTTCAGGCTC

CGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGT CCCCGAGAAGTTGGGGGGAGGGGTCGGCAATTGAACCGG TGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGA TGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGA GAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTT TTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCG TGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGG CCCTTGCGTGCCTTGAATTACTTCCACTGGCTGCAGTACG TGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGA GAGTTCGAGGCCTTGCGCTTAAGGAGCCCCTTCGCCTCGT GCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGC GTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTT TCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGC TGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCG GGCCAAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCG CGGGCGGCGACGGGGCCCGTGCGTCCCAGCGCACATGTT CGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCG GACGGGGGTAGTCTCAAGCTGGCCGGCCTGCTCTGGTGC CTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGC AAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAG ATGGCCGCTTCCCGGCCCTGCTGCAGGGAGCTCAAAATG GAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCAC CCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCG CTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCAGGC ACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTT AGGTTGGGGGGAGGGGTTTTATGCGATGGAGTTTCCCCA CACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCA CTTGATGTAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTG GATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAG TTTTTTTCTTCCATTTCAGGTGTCGTGACCACCATGCTTCT TTTGGTTACGTCTCTGTTGCTTTGCGAACTTCCTCATCCAG CGTTCTTGCTGATCCCCGATATTCAGATGACTCAGACCAC CAGTAGCTTGTCTGCCTCACTGGGAGACCGAGTAACAAT CTCCTGCAGGGCAAGTCAAGACATTAGCAAATACCTCAA TTGGTACCAGCAGAAGCCCGACGGAACGGTAAAACTCCT CATCTATCATACGTCAAGGTTGCATTCCGGAGTACCGTCA CGATTTTCAGGTTCTGGGAGCGGAACTGACTATTCCTTGA CTATTTCAAACCTCGAGCAGGAGGACATTGCGACATATTT TTGTCAACAAGGTAATACCCTCCCTTACACTTTCGGAGGA GGAACCAAACTCGAAATTACCGGGTCCACCAGTGGCTCT GGGAAGCCTGGCAGTGGAGAAGGTTCCACTAAAGGCGAG GTGAAGCTCCAGGAGAGCGGCCCCGGTCTCGTTGCCCCC AGTCAAAGCCTCTCTGTAACGTGCACAGTGAGTGGTGTAT CATTGCCTGATTATGGCGTCTCCTGGATAAGGCAGCCCCC GCGAAAGGGTCTTGAATGGCTTGGGGTAATATGGGGCTC AGAGACAACGTATTATAACTCCGCTCTCAAAAGTCGCTTG ACGATAATAAAAGATAACTCCAAGAGTCAAGTTTTCCTT AAAATGAACAGTTTGCAGACTGACGATACCGCTATATAT TATTGTGCTAAACATTATTACTACGGCGGTAGTTACGCGA TGGATTATTGGGGGCAGGGGACTTCTGTCACAGTCAGTA GTGCTGCTGCCTTTGTCCCGGTATTTCTCCCAGCCAAACC GACCACGACTCCCGCCCCGCGCCCTCCGACACCCGCTCCC ACCATCGCCTCTCAACCTCTTAGTCTTCGCCCCGAGGCAT GCCGACCCGCCGCCGGGGGTGCTGTTCATACGAGGGGCT TGGACTTCGCTTGTGATATTTACATTTGGGCTCCGTTGGC GGGTACGTGCGGCGTCCTTTTGTTGTCACTCGTTATTACTT TGTATTGTAATCACAGGAATCGCTCAAAGCGGAGTAGGT TGTTGCATTCCGATTACATGAATATGACTCCTCGCCGGCC TGGGCCGACAAGAAAACATTACCAACCCTATGCCCCCCC ACGAGACTTCGCTGCGTACAGGTCCCGAGTGAAGTTTTCC CGAAGCGCAGACGCTCCGGCATATCAGCAAGGACAGAAT CAGCTGTATAACGAACTGAATTTGGGACGCCGCGAGGAG TATGACGTGCTTGATAAACGCCGGGGGAGAGACCCGGAA ATGGGGGGTAAACCCCGAAGAAAGAATCCCCAAGAAGG ACTCTACAATGAACTCCAGAAGGATAAGATGGCGGAGGC CTACTCAGAAATAGGTATGAAGGGCGAACGACGACGGGG AAAAGGTCACGATGGCCTCTACCAAGGGTTGAGTACGGC AACCAAAGATACGTACGATGCACTGCATATGCAGGCCCT GCCTCCCAGATAATAATAAAATCGCTATCCATCGAAGAT GGATGTGTGTTGGTTTTTTGTGTGTGGAGCAACAAATCTG ACTTTGCATGTGCAAACGCCTTCAACAACAGCATTATTCC AGAAGACACCTTCTTCCCCAGCCCAGGTAAGGGCAGCTT TGGTGCCTTCGCAGGCTGTTTCCTTGCTTCAGGAATGGCC AGGTTCTGCCCAGAGCTCTGGTCAATGATGTCTAAAACTC CTCTGATTGGTGGTCTCGGCCTTATCCATTGCCACCAAAA CCCTCTTTTTACTAAGAAACAGTGAGCCTTGTTCTGGCAG TCCAGAGAATGACACGGGAAAAAAGCAGATGAAGAGAA GGTGGCAGGAGAGGGCACGTGGCCCAGCCTCAGTCTCTC CAACTGAGTTCCTGCCTGCCTGCCTTTGCTCAGACTGTTT GCCCCTTACTGCTCTTCTAGGCCTCATTCTAAGCCCCTTCT CCAAGTTGCCTCTCCTTATTTCTCCCTGTCTGCCAAAAAA TCTTTCCCAGCTCACTAAGTCAGTCTCACGCAGTCACTCA TTAACCCACCAATCACTGATTGTGCCGGCACATGAATGCA CCAGGTGTTGAAGTGGAGGAATTAAAAAGTCAGATGAGG GGTGTGCCCAGAGGAAGCACCATTCTAGTTGGGGGAGCC CATCTGTCAGCTGGGAAAAGTCCAAATAACTTCAGATTG GAATGTGTTTTAACTCAGGGTTGAGAAAACAGCTACCTTC AGGACAAAAGTCAGGGAAGGGCTCTCTGAAGAAATGCTA CTTGAAGATACCAGCCCTACCAAGGGCAGGGAGAGGACC CTATAGAGGCCTGGGACAGGAGCTCAATGAGAAAGGTAA CCACGTGCGGACCGAGGCTGCAGCGTCGTCCTCCCTAGG AACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGC TCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCG ACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCG AGCGCGCAGCTGCCTGCAGG *indicates a nucleotide with a 2'-O-methyl phosphorothioate modification. "n" refers to the spacer sequence at the 5' end.

OTHER EMBODIMENTS

[0413] All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

[0414] From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the claims.

EQUIVALENTS

[0415] While several inventive embodiments have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the function and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the inventive embodiments described herein. More generally, those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the inventive teachings is/are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific inventive embodiments described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, inventive embodiments may be practiced otherwise than as specifically described and claimed. Inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and/or methods, if such features, systems, articles, materials, kits, and/or methods are not mutually inconsistent, is included within the inventive scope of the present disclosure.

[0416] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.

[0417] All references, patents and patent applications disclosed herein are incorporated by reference with respect to the subject matter for which each is cited, which in some cases may encompass the entirety of the document.

[0418] The indefinite articles "a" and "an," as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

[0419] The phrase "and/or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and/or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and/or" clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to "A and/or B", when used in conjunction with open-ended language such as "comprising" can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0420] As used herein in the specification and in the claims, "or" should be understood to have the same meaning as "and/or" as defined above. For example, when separating items in a list, "or" or "and/or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of" or "exactly one of," or, when used in the claims, "consisting of," will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e. "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of" "Consisting essentially of," when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0421] As used herein in the specification and in the claims, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B," or, equivalently "at least one of A and/or B") can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0422] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited.

Sequence CWU 1

1

56119DNAArtificial SequenceSynthetic Polynucleotide 1aagagcaaca aatctgact 19258DNAArtificial SequenceSynthetic Polynucleotide 2aagagcaaca gtgctgtgcc tggagcaaca aatctgacta agagcaacaa atctgact 58352DNAArtificial SequenceSynthetic Polynucleotide 3aagagcaaca gtgctggagc aacaaatctg actaagagca acaaatctga ct 52453DNAArtificial SequenceSynthetic Polynucleotide 4aagagcaaca gtgcctggag caacaaatct gactaagagc aacaaatctg act 53538DNAArtificial SequenceSynthetic Polynucleotide 5aagagcaaca gtgctgacta agagcaacaa atctgact 38660DNAArtificial SequenceSynthetic Polynucleotide 6aagagcaaca gtgctgtggg cctggagcaa caaatctgac taagagcaac aaatctgact 60757DNAArtificial SequenceSynthetic Polynucleotide 7aagagcaaca gtgctggcct ggagcaacaa atctgactaa gagcaacaaa tctgact 57860DNAArtificial SequenceSynthetic Polynucleotide 8aagagcaaca gtgctgtgtg cctggagcaa caaatctgac taagagcaac aaatctgact 60979DNAArtificial SequenceSynthetic Polynucleotide 9cgtggcctta gctgtgctcg cgctactctc tctttctgcc tggaggctat ccagcgtgag 60tctctcctac cctcccgct 791078DNAArtificial SequenceSynthetic Polynucleotide 10cgtggcctta gctgtgctcg cgctactctc tctttcgcct ggaggctatc cagcgtgagt 60ctctcctacc ctcccgct 781175DNAArtificial SequenceSynthetic Polynucleotide 11cgtggcctta gctgtgctcg cgctactctc tctttctgga ggctatccag cgtgagtctc 60tcctaccctc ccgct 751284DNAArtificial SequenceSynthetic Polynucleotide 12cgtggcctta gctgtgctcg cgctactctc tctttctgga tagcctggag gctatccagc 60gtgagtctct cctaccctcc cgct 841355DNAArtificial SequenceSynthetic Polynucleotide 13cgtggcctta gctgtgctcg cgctatccag cgtgagtctc tcctaccctc ccgct 551482DNAArtificial SequenceSynthetic Polynucleotide 14cgtggcctta gctgtgctcg cgctactctc tctttctgtg gcctggaggc tatccagcgt 60gagtctctcc taccctcccg ct 8215100RNAArtificial SequenceSynthetic Polynucleotidemisc_feature(1)..(20)n is a, c, g, or u 15nnnnnnnnnn nnnnnnnnnn guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 1001696RNAArtificial SequenceSynthetic Polynucleotidemisc_feature(1)..(20)n is a, c, g, or u 16nnnnnnnnnn nnnnnnnnnn guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugc 9617114RNAArtificial SequenceSynthetic Polynucleotidemisc_feature(1)..(30)n is a, c, g, or umisc_feature(18)..(30)n at positions 18 to 30 is optionalmisc_feature(107)..(114)u at positions 2 to 8 is optional 17nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn guuuuagagc uagaaauagc aaguuaaaau 60aaggcuaguc cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu uuuu 11418100RNAArtificial SequenceSynthetic Polynucleotide 18agagcaacag ugcuguggcc guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 1001920RNAArtificial SequenceSynthetic Polynucleotide 19agagcaacag ugcuguggcc 2020100RNAArtificial SequenceSynthetic Polynucleotide 20gcuacucucu cuuucuggcc guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 1002120RNAArtificial SequenceSynthetic Polynucleotide 21gcuacucucu cuuucuggcc 2022100RNAArtificial SequenceSynthetic Polynucleotidemisc_feature(1)..(3)nucleotide with a 2'-O-methyl phosphorothioate modificationmisc_feature(97)..(99)nucleotide with a 2'-O-methyl phosphorothioate modification 22agagcaacag ugcuguggcc guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 1002320RNAArtificial SequenceSynthetic Polynucleotidemisc_feature(1)..(3)nucleotide with a 2'-O-methyl phosphorothioate modification 23agagcaacag ugcuguggcc 2024100RNAArtificial SequenceSynthetic Polynucleotidemisc_feature(1)..(3)nucleotide with a 2'-O-methyl phosphorothioate modificationmisc_feature(97)..(99)nucleotide with a 2'-O-methyl phosphorothioate modification 24gcuacucucu cuuucuggcc guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 1002520RNAArtificial SequenceSynthetic Polynucleotidemisc_feature(1)..(3)nucleotide with a 2'-O-methyl phosphorothioate modification 25gcuacucucu cuuucuggcc 202620DNAArtificial SequenceSynthetic Polynucleotide 26agagcaacag tgctgtggcc 202720DNAArtificial SequenceSynthetic Polynucleotide 27gctactctct ctttctggcc 202823DNAArtificial SequenceSynthetic Polynucleotide 28agagcaacag tgctgtggcc tgg 232923DNAArtificial SequenceSynthetic Polynucleotide 29gctactctct ctttctggcc tgg 233022PRTArtificial SequenceSynthetic Polypeptide 30Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro1 5 10 15Ala Phe Leu Leu Ile Pro 203121PRTArtificial SequenceSynthetic Polypeptide 31Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu1 5 10 15His Ala Ala Arg Pro 203223PRTArtificial SequenceSynthetic Polypeptide 32Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu1 5 10 15Ser Leu Val Ile Thr Leu Tyr 2033126DNAArtificial SequenceSynthetic Polynucleotide 33aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120gaactg 1263442PRTArtificial SequenceSynthetic Polypeptide 34Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met1 5 10 15Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe 20 25 30Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 35 4035120DNAArtificial SequenceSynthetic Polynucleotide 35tcaaagcgga gtaggttgtt gcattccgat tacatgaata tgactcctcg ccggcctggg 60ccgacaagaa aacattacca accctatgcc cccccacgag acttcgctgc gtacaggtcc 1203640PRTArtificial SequenceSynthetic Polypeptide 36Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro1 5 10 15Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro 20 25 30Arg Asp Phe Ala Ala Tyr Arg Ser 35 4037336DNAArtificial SequenceSynthetic Polynucleotide 37cgagtgaagt tttcccgaag cgcagacgct ccggcatatc agcaaggaca gaatcagctg 60tataacgaac tgaatttggg acgccgcgag gagtatgacg tgcttgataa acgccggggg 120agagacccgg aaatgggggg taaaccccga agaaagaatc cccaagaagg actctacaat 180gaactccaga aggataagat ggcggaggcc tactcagaaa taggtatgaa gggcgaacga 240cgacggggaa aaggtcacga tggcctctac caagggttga gtacggcaac caaagatacg 300tacgatgcac tgcatatgca ggccctgcct cccaga 33638112PRTArtificial SequenceSynthetic Polypeptide 38Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly1 5 10 15Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25 30Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys 35 40 45Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys 50 55 60Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg65 70 75 80Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala 85 90 95Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110391518DNAArtificial SequenceSynthetic Polynucleotide 39atgcttcttt tggttacgtc tctgttgctt tgcgaacttc ctcatccagc gttcttgctg 60atccccgata ttcagatgac tcagaccacc agtagcttgt ctgcctcact gggagaccga 120gtaacaatct cctgcagggc aagtcaagac attagcaaat acctcaattg gtaccagcag 180aagcccgacg gaacggtaaa actcctcatc tatcatacgt caaggttgca ttccggagta 240ccgtcacgat tttcaggttc tgggagcgga actgactatt ccttgactat ttcaaacctc 300gagcaggagg acattgcgac atatttttgt caacaaggta ataccctccc ttacactttc 360ggaggaggaa ccaaactcga aattaccggg tccaccagtg gctctgggaa gcctggcagt 420ggagaaggtt ccactaaagg cgaggtgaag ctccaggaga gcggccccgg tctcgttgcc 480cccagtcaaa gcctctctgt aacgtgcaca gtgagtggtg tatcattgcc tgattatggc 540gtctcctgga taaggcagcc cccgcgaaag ggtcttgaat ggcttggggt aatatggggc 600tcagagacaa cgtattataa ctccgctctc aaaagtcgct tgacgataat aaaagataac 660tccaagagtc aagttttcct taaaatgaac agtttgcaga ctgacgatac cgctatatat 720tattgtgcta aacattatta ctacggcggt agttacgcga tggattattg ggggcagggg 780acttctgtca cagtcagtag tgctgctgcc tttgtcccgg tatttctccc agccaaaccg 840accacgactc ccgccccgcg ccctccgaca cccgctccca ccatcgcctc tcaacctctt 900agtcttcgcc ccgaggcatg ccgacccgcc gccgggggtg ctgttcatac gaggggcttg 960gacttcgctt gtgatattta catttgggct ccgttggcgg gtacgtgcgg cgtccttttg 1020ttgtcactcg ttattacttt gtattgtaat cacaggaatc gctcaaagcg gagtaggttg 1080ttgcattccg attacatgaa tatgactcct cgccggcctg ggccgacaag aaaacattac 1140caaccctatg cccccccacg agacttcgct gcgtacaggt cccgagtgaa gttttcccga 1200agcgcagacg ctccggcata tcagcaagga cagaatcagc tgtataacga actgaatttg 1260ggacgccgcg aggagtatga cgtgcttgat aaacgccggg ggagagaccc ggaaatgggg 1320ggtaaacccc gaagaaagaa tccccaagaa ggactctaca atgaactcca gaaggataag 1380atggcggagg cctactcaga aataggtatg aagggcgaac gacgacgggg aaaaggtcac 1440gatggcctct accaagggtt gagtacggca accaaagata cgtacgatgc actgcatatg 1500caggccctgc ctcccaga 151840506PRTArtificial SequenceSynthetic Polypeptide 40Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro1 5 10 15Ala Phe Leu Leu Ile Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser 20 25 30Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser 35 40 45Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly 50 55 60Thr Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val65 70 75 80Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr 85 90 95Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln 100 105 110Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 115 120 125Thr Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser 130 135 140Thr Lys Gly Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala145 150 155 160Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu 165 170 175Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu 180 185 190Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser 195 200 205Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln 210 215 220Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr225 230 235 240Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr 245 250 255Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser Ala Ala Ala Phe Val 260 265 270Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro 275 280 285Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro 290 295 300Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu305 310 315 320Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys 325 330 335Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn His Arg 340 345 350Asn Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met 355 360 365Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala 370 375 380Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg385 390 395 400Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn 405 410 415Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg 420 425 430Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro 435 440 445Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala 450 455 460Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His465 470 475 480Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp 485 490 495Ala Leu His Met Gln Ala Leu Pro Pro Arg 500 50541800DNAArtificial SequenceSynthetic Polynucleotide 41gagatgtaag gagctgctgt gacttgctca aggccttata tcgagtaaac ggtagtgctg 60gggcttagac gcaggtgttc tgatttatag ttcaaaacct ctatcaatga gagagcaatc 120tcctggtaat gtgatagatt tcccaactta atgccaacat accataaacc tcccattctg 180ctaatgccca gcctaagttg gggagaccac tccagattcc aagatgtaca gtttgctttg 240ctgggccttt ttcccatgcc tgcctttact ctgccagagt tatattgctg gggttttgaa 300gaagatccta ttaaataaaa gaataagcag tattattaag tagccctgca tttcaggttt 360ccttgagtgg caggccaggc ctggccgtga acgttcactg aaatcatggc ctcttggcca 420agattgatag cttgtgcctg tccctgagtc ccagtccatc acgagcagct ggtttctaag 480atgctatttc ccgtataaag catgagaccg tgacttgcca gccccacaga gccccgccct 540tgtccatcac tggcatctgg actccagcct gggttggggc aaagagggaa atgagatcat 600gtcctaaccc tgatcctctt gtcccacaga tatccagaac cctgaccctg ccgtgtacca 660gctgagagac tctaaatcca gtgacaagtc tgtctgccta ttcaccgatt ttgattctca 720aacaaatgtg tcacaaagta aggattctga tgtgtatatc acagacaaaa ctgtgctaga 780catgaggtct atggacttca 80042804DNAArtificial SequenceSynthetic Polynucleotide 42tggagcaaca aatctgactt tgcatgtgca aacgccttca acaacagcat tattccagaa 60gacaccttct tccccagccc aggtaagggc agctttggtg ccttcgcagg ctgtttcctt 120gcttcaggaa tggccaggtt ctgcccagag ctctggtcaa tgatgtctaa aactcctctg 180attggtggtc tcggccttat ccattgccac caaaaccctc tttttactaa gaaacagtga 240gccttgttct ggcagtccag agaatgacac gggaaaaaag cagatgaaga gaaggtggca 300ggagagggca cgtggcccag cctcagtctc tccaactgag ttcctgcctg cctgcctttg 360ctcagactgt ttgcccctta ctgctcttct aggcctcatt ctaagcccct tctccaagtt 420gcctctcctt atttctccct gtctgccaaa aaatctttcc cagctcacta agtcagtctc 480acgcagtcac tcattaaccc accaatcact gattgtgccg gcacatgaat gcaccaggtg 540ttgaagtgga ggaattaaaa agtcagatga ggggtgtgcc cagaggaagc accattctag 600ttgggggagc ccatctgtca gctgggaaaa gtccaaataa cttcagattg gaatgtgttt 660taactcaggg ttgagaaaac agctaccttc aggacaaaag tcagggaagg gctctctgaa 720gaaatgctac ttgaagatac cagccctacc aagggcaggg agaggaccct atagaggcct 780gggacaggag ctcaatgaga aagg 804431178DNAArtificial SequenceSynthetic Polynucleotide 43ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg 60ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt 120gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca 180gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc 240gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt 300acttccactg gctgcagtac gtgattcttg atcccgagct tcgggttgga agtgggtggg 360agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt gaggcctggc 420ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt 480tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt tttttctggc 540aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt tttggggccg 600cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag 660cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg 720gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg tcggcaccag 780ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca aaatggagga 840cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg gcctttccgt 900cctcagccgt

cgcttcatgt gactccacgg agtaccgggc gccgtccagg cacctcgatt 960agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt tatgcgatgg 1020agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac ttgatgtaat 1080tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag cctcagacag 1140tggttcaaag tttttttctt ccatttcagg tgtcgtga 11784466DNAArtificial SequenceSynthetic Polynucleotide 44atgcttcttt tggttacgtc tctgttgctt tgcgaacttc ctcatccagc gttcttgctg 60atcccc 664522PRTArtificial SequenceSynthetic Polypeptide 45Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro1 5 10 15Ala Phe Leu Leu Ile Pro 2046735DNAArtificial SequenceSynthetic Polynucleotide 46gatattcaga tgactcagac caccagtagc ttgtctgcct cactgggaga ccgagtaaca 60atctcctgca gggcaagtca agacattagc aaatacctca attggtacca gcagaagccc 120gacggaacgg taaaactcct catctatcat acgtcaaggt tgcattccgg agtaccgtca 180cgattttcag gttctgggag cggaactgac tattccttga ctatttcaaa cctcgagcag 240gaggacattg cgacatattt ttgtcaacaa ggtaataccc tcccttacac tttcggagga 300ggaaccaaac tcgaaattac cgggtccacc agtggctctg ggaagcctgg cagtggagaa 360ggttccacta aaggcgaggt gaagctccag gagagcggcc ccggtctcgt tgcccccagt 420caaagcctct ctgtaacgtg cacagtgagt ggtgtatcat tgcctgatta tggcgtctcc 480tggataaggc agcccccgcg aaagggtctt gaatggcttg gggtaatatg gggctcagag 540acaacgtatt ataactccgc tctcaaaagt cgcttgacga taataaaaga taactccaag 600agtcaagttt tccttaaaat gaacagtttg cagactgacg ataccgctat atattattgt 660gctaaacatt attactacgg cggtagttac gcgatggatt attgggggca ggggacttct 720gtcacagtca gtagt 73547245PRTArtificial SequenceSynthetic Polypeptide 47Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly1 5 10 15Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln65 70 75 80Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Ser Thr Ser Gly 100 105 110Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Lys 115 120 125Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser 130 135 140Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser145 150 155 160Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile 165 170 175Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu 180 185 190Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn 195 200 205Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr 210 215 220Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser225 230 235 240Val Thr Val Ser Ser 24548261DNAArtificial SequenceSynthetic Polynucleotide 48gctgctgcct ttgtcccggt atttctccca gccaaaccga ccacgactcc cgccccgcgc 60cctccgacac ccgctcccac catcgcctct caacctctta gtcttcgccc cgaggcatgc 120cgacccgccg ccgggggtgc tgttcatacg aggggcttgg acttcgcttg tgatatttac 180atttgggctc cgttggcggg tacgtgcggc gtccttttgt tgtcactcgt tattactttg 240tattgtaatc acaggaatcg c 26149252DNAArtificial SequenceSynthetic Polynucleotide 49tttgtcccgg tatttctccc agccaaaccg accacgactc ccgccccgcg ccctccgaca 60cccgctccca ccatcgcctc tcaacctctt agtcttcgcc ccgaggcatg ccgacccgcc 120gccgggggtg ctgttcatac gaggggcttg gacttcgctt gtgatattta catttgggct 180ccgttggcgg gtacgtgcgg cgtccttttg ttgtcactcg ttattacttt gtattgtaat 240cacaggaatc gc 2525084PRTArtificial SequenceSynthetic Polypeptide 50Phe Val Pro Val Phe Leu Pro Ala Lys Pro Thr Thr Thr Pro Ala Pro1 5 10 15Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu 20 25 30Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg 35 40 45Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly 50 55 60Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn65 70 75 80His Arg Asn Arg51120PRTArtificial SequenceSynthetic Polypeptide 51Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln1 5 10 15Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr 20 25 30Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu 35 40 45Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys 50 55 60Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu65 70 75 80Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala 85 90 95Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110Gly Thr Ser Val Thr Val Ser Ser 115 12052107PRTArtificial SequenceSynthetic Polypeptide 52Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly1 5 10 15Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr 20 25 30Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln65 70 75 80Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr 85 90 95Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr 100 1055318PRTArtificial SequenceSynthetic Polypeptide 53Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr1 5 10 15Lys Gly544358DNAArtificial SequenceSynthetic Polynucleotide 54gagatgtaag gagctgctgt gacttgctca aggccttata tcgagtaaac ggtagtgctg 60gggcttagac gcaggtgttc tgatttatag ttcaaaacct ctatcaatga gagagcaatc 120tcctggtaat gtgatagatt tcccaactta atgccaacat accataaacc tcccattctg 180ctaatgccca gcctaagttg gggagaccac tccagattcc aagatgtaca gtttgctttg 240ctgggccttt ttcccatgcc tgcctttact ctgccagagt tatattgctg gggttttgaa 300gaagatccta ttaaataaaa gaataagcag tattattaag tagccctgca tttcaggttt 360ccttgagtgg caggccaggc ctggccgtga acgttcactg aaatcatggc ctcttggcca 420agattgatag cttgtgcctg tccctgagtc ccagtccatc acgagcagct ggtttctaag 480atgctatttc ccgtataaag catgagaccg tgacttgcca gccccacaga gccccgccct 540tgtccatcac tggcatctgg actccagcct gggttggggc aaagagggaa atgagatcat 600gtcctaaccc tgatcctctt gtcccacaga tatccagaac cctgaccctg ccgtgtacca 660gctgagagac tctaaatcca gtgacaagtc tgtctgccta ttcaccgatt ttgattctca 720aacaaatgtg tcacaaagta aggattctga tgtgtatatc acagacaaaa ctgtgctaga 780catgaggtct atggacttca ggctccggtg cccgtcagtg ggcagagcgc acatcgccca 840cagtccccga gaagttgggg ggaggggtcg gcaattgaac cggtgcctag agaaggtggc 900gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg cctttttccc gagggtgggg 960gagaaccgta tataagtgca gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg 1020ccagaacaca ggtaagtgcc gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg 1080gcccttgcgt gccttgaatt acttccactg gctgcagtac gtgattcttg atcccgagct 1140tcgggttgga agtgggtggg agagttcgag gccttgcgct taaggagccc cttcgcctcg 1200tgcttgagtt gaggcctggc ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct 1260tcgcgcctgt ctcgctgctt tcgataagtc tctagccatt taaaattttt gatgacctgc 1320tgcgacgctt tttttctggc aagatagtct tgtaaatgcg ggccaagatc tgcacactgg 1380tatttcggtt tttggggccg cgggcggcga cggggcccgt gcgtcccagc gcacatgttc 1440ggcgaggcgg ggcctgcgag cgcggccacc gagaatcgga cgggggtagt ctcaagctgg 1500ccggcctgct ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag 1560gctggcccgg tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc 1620agggagctca aaatggagga cgcggcgctc gggagagcgg gcgggtgagt cacccacaca 1680aaggaaaagg gcctttccgt cctcagccgt cgcttcatgt gactccacgg agtaccgggc 1740gccgtccagg cacctcgatt agttctcgag cttttggagt acgtcgtctt taggttgggg 1800ggaggggttt tatgcgatgg agtttcccca cactgagtgg gtggagactg aagttaggcc 1860agcttggcac ttgatgtaat tctccttgga atttgccctt tttgagtttg gatcttggtt 1920cattctcaag cctcagacag tggttcaaag tttttttctt ccatttcagg tgtcgtgacc 1980accatgcttc ttttggttac gtctctgttg ctttgcgaac ttcctcatcc agcgttcttg 2040ctgatccccg atattcagat gactcagacc accagtagct tgtctgcctc actgggagac 2100cgagtaacaa tctcctgcag ggcaagtcaa gacattagca aatacctcaa ttggtaccag 2160cagaagcccg acggaacggt aaaactcctc atctatcata cgtcaaggtt gcattccgga 2220gtaccgtcac gattttcagg ttctgggagc ggaactgact attccttgac tatttcaaac 2280ctcgagcagg aggacattgc gacatatttt tgtcaacaag gtaataccct cccttacact 2340ttcggaggag gaaccaaact cgaaattacc gggtccacca gtggctctgg gaagcctggc 2400agtggagaag gttccactaa aggcgaggtg aagctccagg agagcggccc cggtctcgtt 2460gcccccagtc aaagcctctc tgtaacgtgc acagtgagtg gtgtatcatt gcctgattat 2520ggcgtctcct ggataaggca gcccccgcga aagggtcttg aatggcttgg ggtaatatgg 2580ggctcagaga caacgtatta taactccgct ctcaaaagtc gcttgacgat aataaaagat 2640aactccaaga gtcaagtttt ccttaaaatg aacagtttgc agactgacga taccgctata 2700tattattgtg ctaaacatta ttactacggc ggtagttacg cgatggatta ttgggggcag 2760gggacttctg tcacagtcag tagtgctgct gcctttgtcc cggtatttct cccagccaaa 2820ccgaccacga ctcccgcccc gcgccctccg acacccgctc ccaccatcgc ctctcaacct 2880cttagtcttc gccccgaggc atgccgaccc gccgccgggg gtgctgttca tacgaggggc 2940ttggacttcg cttgtgatat ttacatttgg gctccgttgg cgggtacgtg cggcgtcctt 3000ttgttgtcac tcgttattac tttgtattgt aatcacagga atcgctcaaa gcggagtagg 3060ttgttgcatt ccgattacat gaatatgact cctcgccggc ctgggccgac aagaaaacat 3120taccaaccct atgccccccc acgagacttc gctgcgtaca ggtcccgagt gaagttttcc 3180cgaagcgcag acgctccggc atatcagcaa ggacagaatc agctgtataa cgaactgaat 3240ttgggacgcc gcgaggagta tgacgtgctt gataaacgcc gggggagaga cccggaaatg 3300gggggtaaac cccgaagaaa gaatccccaa gaaggactct acaatgaact ccagaaggat 3360aagatggcgg aggcctactc agaaataggt atgaagggcg aacgacgacg gggaaaaggt 3420cacgatggcc tctaccaagg gttgagtacg gcaaccaaag atacgtacga tgcactgcat 3480atgcaggccc tgcctcccag ataataataa aatcgctatc catcgaagat ggatgtgtgt 3540tggttttttg tgtgtggagc aacaaatctg actttgcatg tgcaaacgcc ttcaacaaca 3600gcattattcc agaagacacc ttcttcccca gcccaggtaa gggcagcttt ggtgccttcg 3660caggctgttt ccttgcttca ggaatggcca ggttctgccc agagctctgg tcaatgatgt 3720ctaaaactcc tctgattggt ggtctcggcc ttatccattg ccaccaaaac cctcttttta 3780ctaagaaaca gtgagccttg ttctggcagt ccagagaatg acacgggaaa aaagcagatg 3840aagagaaggt ggcaggagag ggcacgtggc ccagcctcag tctctccaac tgagttcctg 3900cctgcctgcc tttgctcaga ctgtttgccc cttactgctc ttctaggcct cattctaagc 3960cccttctcca agttgcctct ccttatttct ccctgtctgc caaaaaatct ttcccagctc 4020actaagtcag tctcacgcag tcactcatta acccaccaat cactgattgt gccggcacat 4080gaatgcacca ggtgttgaag tggaggaatt aaaaagtcag atgaggggtg tgcccagagg 4140aagcaccatt ctagttgggg gagcccatct gtcagctggg aaaagtccaa ataacttcag 4200attggaatgt gttttaactc agggttgaga aaacagctac cttcaggaca aaagtcaggg 4260aagggctctc tgaagaaatg ctacttgaag ataccagccc taccaagggc agggagagga 4320ccctatagag gcctgggaca ggagctcaat gagaaagg 4358551368PRTArtificial SequenceSynthetic Polypeptide 55Met Asp Lys Lys Tyr Ser Ile Gly Leu Asp Ile Gly Thr Asn Ser Val1 5 10 15Gly Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe 20 25 30Lys Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile 35 40 45Gly Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu 50 55 60Lys Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys65 70 75 80Tyr Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser 85 90 95Phe Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys 100 105 110His Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr 115 120 125His Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp 130 135 140Ser Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His145 150 155 160Met Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro 165 170 175Asp Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr 180 185 190Asn Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala 195 200 205Lys Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn 210 215 220Leu Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn225 230 235 240Leu Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe 245 250 255Asp Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp 260 265 270Asp Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp 275 280 285Leu Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp 290 295 300Ile Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser305 310 315 320Met Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys 325 330 335Ala Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe 340 345 350Asp Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser 355 360 365Gln Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp 370 375 380Gly Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg385 390 395 400Lys Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu 405 410 415Gly Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe 420 425 430Leu Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile 435 440 445Pro Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp 450 455 460Met Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu465 470 475 480Val Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr 485 490 495Asn Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser 500 505 510Leu Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys 515 520 525Tyr Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln 530 535 540Lys Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr545 550 555 560Val Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp 565 570 575Ser Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly 580 585 590Thr Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp 595 600 605Asn Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr 610 615 620Leu Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala625 630 635 640His Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr 645 650 655Thr Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp 660 665 670Lys Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe 675 680 685Ala Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe 690 695 700Lys Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu705 710 715 720His Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly 725 730 735Ile Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly 740 745 750Arg His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln 755 760 765Thr Thr Gln Lys Gly Gln Lys

Asn Ser Arg Glu Arg Met Lys Arg Ile 770 775 780Glu Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro785 790 795 800Val Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu 805 810 815Gln Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg 820 825 830Leu Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys 835 840 845Asp Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg 850 855 860Gly Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys865 870 875 880Asn Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys 885 890 895Phe Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp 900 905 910Lys Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr 915 920 925Lys His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp 930 935 940Glu Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser945 950 955 960Lys Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg 965 970 975Glu Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val 980 985 990Val Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe 995 1000 1005Val Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala 1010 1015 1020Lys Ser Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe 1025 1030 1035Tyr Ser Asn Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala 1040 1045 1050Asn Gly Glu Ile Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu 1055 1060 1065Thr Gly Glu Ile Val Trp Asp Lys Gly Arg Asp Phe Ala Thr Val 1070 1075 1080Arg Lys Val Leu Ser Met Pro Gln Val Asn Ile Val Lys Lys Thr 1085 1090 1095Glu Val Gln Thr Gly Gly Phe Ser Lys Glu Ser Ile Leu Pro Lys 1100 1105 1110Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys Asp Trp Asp Pro 1115 1120 1125Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala Tyr Ser Val 1130 1135 1140Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys Leu Lys 1145 1150 1155Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser Ser 1160 1165 1170Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys 1175 1180 1185Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu 1190 1195 1200Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly 1205 1210 1215Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225 1230Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser 1235 1240 1245Pro Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys 1250 1255 1260His Tyr Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys 1265 1270 1275Arg Val Ile Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala 1280 1285 1290Tyr Asn Lys His Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn 1295 1300 1305Ile Ile His Leu Phe Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala 1310 1315 1320Phe Lys Tyr Phe Asp Thr Thr Ile Asp Arg Lys Arg Tyr Thr Ser 1325 1330 1335Thr Lys Glu Val Leu Asp Ala Thr Leu Ile His Gln Ser Ile Thr 1340 1345 1350Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln Leu Gly Gly Asp 1355 1360 1365564682DNAArtificial SequenceSynthetic Polynucleotide 56cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcgtcg ggcgaccttt 60ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact 120aggggttcct gcggccgcac gcgtgagatg taaggagctg ctgtgacttg ctcaaggcct 180tatatcgagt aaacggtagt gctggggctt agacgcaggt gttctgattt atagttcaaa 240acctctatca atgagagagc aatctcctgg taatgtgata gatttcccaa cttaatgcca 300acataccata aacctcccat tctgctaatg cccagcctaa gttggggaga ccactccaga 360ttccaagatg tacagtttgc tttgctgggc ctttttccca tgcctgcctt tactctgcca 420gagttatatt gctggggttt tgaagaagat cctattaaat aaaagaataa gcagtattat 480taagtagccc tgcatttcag gtttccttga gtggcaggcc aggcctggcc gtgaacgttc 540actgaaatca tggcctcttg gccaagattg atagcttgtg cctgtccctg agtcccagtc 600catcacgagc agctggtttc taagatgcta tttcccgtat aaagcatgag accgtgactt 660gccagcccca cagagccccg cccttgtcca tcactggcat ctggactcca gcctgggttg 720gggcaaagag ggaaatgaga tcatgtccta accctgatcc tcttgtccca cagatatcca 780gaaccctgac cctgccgtgt accagctgag agactctaaa tccagtgaca agtctgtctg 840cctattcacc gattttgatt ctcaaacaaa tgtgtcacaa agtaaggatt ctgatgtgta 900tatcacagac aaaactgtgc tagacatgag gtctatggac ttcaggctcc ggtgcccgtc 960agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt 1020gaaccggtgc ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc 1080tccgcctttt tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg 1140ttctttttcg caacgggttt gccgccagaa cacaggtaag tgccgtgtgt ggttcccgcg 1200ggcctggcct ctttacgggt tatggccctt gcgtgccttg aattacttcc actggctgca 1260gtacgtgatt cttgatcccg agcttcgggt tggaagtggg tgggagagtt cgaggccttg 1320cgcttaagga gccccttcgc ctcgtgcttg agttgaggcc tggcctgggc gctggggccg 1380ccgcgtgcga atctggtggc accttcgcgc ctgtctcgct gctttcgata agtctctagc 1440catttaaaat ttttgatgac ctgctgcgac gctttttttc tggcaagata gtcttgtaaa 1500tgcgggccaa gatctgcaca ctggtatttc ggtttttggg gccgcgggcg gcgacggggc 1560ccgtgcgtcc cagcgcacat gttcggcgag gcggggcctg cgagcgcggc caccgagaat 1620cggacggggg tagtctcaag ctggccggcc tgctctggtg cctggcctcg cgccgccgtg 1680tatcgccccg ccctgggcgg caaggctggc ccggtcggca ccagttgcgt gagcggaaag 1740atggccgctt cccggccctg ctgcagggag ctcaaaatgg aggacgcggc gctcgggaga 1800gcgggcgggt gagtcaccca cacaaaggaa aagggccttt ccgtcctcag ccgtcgcttc 1860atgtgactcc acggagtacc gggcgccgtc caggcacctc gattagttct cgagcttttg 1920gagtacgtcg tctttaggtt ggggggaggg gttttatgcg atggagtttc cccacactga 1980gtgggtggag actgaagtta ggccagcttg gcacttgatg taattctcct tggaatttgc 2040cctttttgag tttggatctt ggttcattct caagcctcag acagtggttc aaagtttttt 2100tcttccattt caggtgtcgt gaccaccatg cttcttttgg ttacgtctct gttgctttgc 2160gaacttcctc atccagcgtt cttgctgatc cccgatattc agatgactca gaccaccagt 2220agcttgtctg cctcactggg agaccgagta acaatctcct gcagggcaag tcaagacatt 2280agcaaatacc tcaattggta ccagcagaag cccgacggaa cggtaaaact cctcatctat 2340catacgtcaa ggttgcattc cggagtaccg tcacgatttt caggttctgg gagcggaact 2400gactattcct tgactatttc aaacctcgag caggaggaca ttgcgacata tttttgtcaa 2460caaggtaata ccctccctta cactttcgga ggaggaacca aactcgaaat taccgggtcc 2520accagtggct ctgggaagcc tggcagtgga gaaggttcca ctaaaggcga ggtgaagctc 2580caggagagcg gccccggtct cgttgccccc agtcaaagcc tctctgtaac gtgcacagtg 2640agtggtgtat cattgcctga ttatggcgtc tcctggataa ggcagccccc gcgaaagggt 2700cttgaatggc ttggggtaat atggggctca gagacaacgt attataactc cgctctcaaa 2760agtcgcttga cgataataaa agataactcc aagagtcaag ttttccttaa aatgaacagt 2820ttgcagactg acgataccgc tatatattat tgtgctaaac attattacta cggcggtagt 2880tacgcgatgg attattgggg gcaggggact tctgtcacag tcagtagtgc tgctgccttt 2940gtcccggtat ttctcccagc caaaccgacc acgactcccg ccccgcgccc tccgacaccc 3000gctcccacca tcgcctctca acctcttagt cttcgccccg aggcatgccg acccgccgcc 3060gggggtgctg ttcatacgag gggcttggac ttcgcttgtg atatttacat ttgggctccg 3120ttggcgggta cgtgcggcgt ccttttgttg tcactcgtta ttactttgta ttgtaatcac 3180aggaatcgct caaagcggag taggttgttg cattccgatt acatgaatat gactcctcgc 3240cggcctgggc cgacaagaaa acattaccaa ccctatgccc ccccacgaga cttcgctgcg 3300tacaggtccc gagtgaagtt ttcccgaagc gcagacgctc cggcatatca gcaaggacag 3360aatcagctgt ataacgaact gaatttggga cgccgcgagg agtatgacgt gcttgataaa 3420cgccggggga gagacccgga aatggggggt aaaccccgaa gaaagaatcc ccaagaagga 3480ctctacaatg aactccagaa ggataagatg gcggaggcct actcagaaat aggtatgaag 3540ggcgaacgac gacggggaaa aggtcacgat ggcctctacc aagggttgag tacggcaacc 3600aaagatacgt acgatgcact gcatatgcag gccctgcctc ccagataata ataaaatcgc 3660tatccatcga agatggatgt gtgttggttt tttgtgtgtg gagcaacaaa tctgactttg 3720catgtgcaaa cgccttcaac aacagcatta ttccagaaga caccttcttc cccagcccag 3780gtaagggcag ctttggtgcc ttcgcaggct gtttccttgc ttcaggaatg gccaggttct 3840gcccagagct ctggtcaatg atgtctaaaa ctcctctgat tggtggtctc ggccttatcc 3900attgccacca aaaccctctt tttactaaga aacagtgagc cttgttctgg cagtccagag 3960aatgacacgg gaaaaaagca gatgaagaga aggtggcagg agagggcacg tggcccagcc 4020tcagtctctc caactgagtt cctgcctgcc tgcctttgct cagactgttt gccccttact 4080gctcttctag gcctcattct aagccccttc tccaagttgc ctctccttat ttctccctgt 4140ctgccaaaaa atctttccca gctcactaag tcagtctcac gcagtcactc attaacccac 4200caatcactga ttgtgccggc acatgaatgc accaggtgtt gaagtggagg aattaaaaag 4260tcagatgagg ggtgtgccca gaggaagcac cattctagtt gggggagccc atctgtcagc 4320tgggaaaagt ccaaataact tcagattgga atgtgtttta actcagggtt gagaaaacag 4380ctaccttcag gacaaaagtc agggaagggc tctctgaaga aatgctactt gaagatacca 4440gccctaccaa gggcagggag aggaccctat agaggcctgg gacaggagct caatgagaaa 4500ggtaaccacg tgcggaccga ggctgcagcg tcgtcctccc taggaacccc tagtgatgga 4560gttggccact ccctctctgc gcgctcgctc gctcactgag gccgggcgac caaaggtcgc 4620ccgacgcccg ggctttgccc gggcggcctc agtgagcgag cgagcgcgca gctgcctgca 4680gg 4682

Claims

1. A method for treating a B-cell malignancy in a human patient, the method comprising: (i) subjecting a human patient having a B-cell malignancy to a lymphodepletion treatment; and (ii) administering to the human patient a population of genetically engineered T cells after step (i), wherein the population of genetically engineered T cells comprising T cells that comprise: (a) a disrupted T cell receptor alpha constant (TRAC) gene, (b) a nucleic acid coding for a chimeric antigen receptor (CAR) that binds CD19, wherein the CAR comprises an anti-CD19 single chain variable fragment (scFv) that comprises a heavy chain variable region set forth in SEQ ID NO: 51, and a light chain variable region set forth in SEQ ID NO: 52, and wherein the nucleic acid is inserted in the disrupted TRAC gene, and (c) a disrupted beta 2-microglobulin (.beta.2M) gene; wherein the population of genetically engineered T cells is administered to the human patient at a dose of about 1.times.10.sup.7 to about 1.times.10.sup.9 CAR.sup.+ T cells.

2. The method of claim 1, wherein the disrupted TRAC gene comprises a deletion of a fragment comprising the nucleotide sequence of SEQ ID NO: 26,

3. The method of claim 1, wherein the population of genetically engineered T cells administered to the human patient per dose contains no more than 7.times.10.sup.4 TCR.sup.+ T cells/kg.

4. The method of claim 1, wherein the lymphodepletion treatment in step (i) comprises co-administration to the human patient fludarabine at about 30 mg/m.sup.2 and cyclophosphamide at about 500-750 mg/m.sup.2 per day for three days.

5. The method of claim 4, wherein the lymphodepletion treatment in step (i) comprises co-administration to the human patient fludarabine at about 30 mg/m.sup.2 and cyclophosphamide at about 500 mg/m.sup.2 per day for three days, or fludarabine at about 30 mg/m.sup.2 and cyclophosphamide at about 750 mg/m.sup.2 per day for three days.

6. The method of claim 1, wherein the population of genetically engineered T cells is administered to the human patient at a dose of about 1.times.10.sup.7, about 3.times.10.sup.7, about 1.times.10.sup.8, about 3.times.10.sup.8, or about 1.times.10.sup.9 CAR' T cells.

7. The method of claim 1, wherein prior to step (i), the human patient does not show one or more of the following features: (a) significant worsening of clinical status, (b) requirement for supplemental oxygen to maintain a saturation level of greater than 91%, (c) uncontrolled cardiac arrhythmia, (d) hypotension requiring vasopressor support, (e) active infection, and (f) grade .gtoreq.2 acute neurological toxicity.

8. The method of claim 1, wherein step (i) is performed about 2-7 days prior to step (ii).

9. The method of claim 1, wherein after step (i) and prior to step (ii), the human patient does not show one or more of the following features: (a) active uncontrolled infection; (b) worsening of clinical status compared to the clinical status prior to step (i); and (c) grade .gtoreq.2 acute neurological toxicity.

10. The method of claim 1, further comprising (iii) monitoring the human patient for development of acute toxicity after step (ii); and (iv) managing the acute toxicity if occurs.

11. The method of claim 10, wherein step (iii) is performed for at least 28 days after administration of the population of genetically engineered T cells.

12. The method of claim 10, wherein the acute toxicity comprises tumor lysis syndrome (TLS), cytokine release syndrome (CRS), immune effector cell-associated neurotoxicity syndrome (ICANS), B cell aplasia, hemophagocytic lymphohistiocytosis (HLH), cytopenia, graft-versus-host disease (GvHD), hypertension, renal insufficiency, or a combination thereof.

13. The method of claim 1, wherein the B cell malignancy is non-Hodgkin lymphoma, which optionally is selected from the group consisting of diffuse large B cell lymphoma (DLBCL), high grade B cell lymphoma with MYC and BCL2 and/or BCL6 rearrangement, transformed follicular lymphoma (FL), and grade 3b FL.

14. The method of claim 13, wherein DLBCL is DLBCL not otherwise specified (NOS).

15. The method of claim 1, wherein the human patient has at least one measurable lesion that is fluorodeoxyglucose positron emission tomography (PET)-positive.

16. The method of claim 1, wherein the B cell malignancy is refractory and/or relapsed.

17. The method of claim 1, wherein the human patient has undergone one or more lines of prior anti-cancer therapies.

18. The method of claim 17, wherein the human patient has undergone two or more lines of prior anti-cancer therapies.

19. The method of claim 17, wherein the prior anti-cancer therapies comprise an anti-CD20 antibody, an anthracycline-containing regimen, or a combination thereof.

20. The method of claim 17, wherein the human patient has refractory or relapsed transformed FL and has undergone at least one line of chemotherapy for disease after transformation to DLBCL.

21. The method of claim 16, wherein the B cell malignancy is refractory, and the human patient has progressive disease on last therapy, or has stable disease following at least two cycles of therapy with duration of stable disease of up to 6 months.

22. The method of claim 1, wherein the human patient has failed prior autologous hematopoietic stem cell transplantation (HSCT) or ineligible for prior autologous HSCT.

23. The method of claim 1, wherein the human patient is subject to an additional anti-cancer therapy after treatment with the population of genetically engineered T cells.

24. The method of claim 1, wherein the human patient has one or more of the following features: (a) has an Eastern Cooperative Oncology Group (ECOG) performance status 0 or 1; (b) adequate renal, liver, cardiac, and/or pulmonary function; (c) free of prior gene therapy or modified cell therapy; (d) free of prior treatment comprising an anti-CD19 antibody; (e) free of prior allogeneic HSCT; (f) free of detectable malignant cells from cerebrospinal fluid; (g) free of brain metastases; (h) free of prior central nervous system disorders; (i) free of unstable angina, arrhythmia, and/or myocardial infarction; (j) free of uncontrolled infection; (k) free of immunodeficiency disorders or autoimmune disorders that require immunosuppressive therapy; and (l) free of infection by human immunodeficiency virus, hepatitis B virus, or hepatitis C virus.

25. The method of claim 1, wherein the anti-CD19 scFv comprises the amino acid sequence of SEQ ID NO: 47.

26. The method of claim 25, wherein the CAR that binds CD19 comprises the amino acid sequence of SEQ ID NO: 40.

27. The method of claim 1, wherein the nucleic acid encoding the anti-CD19 CAR is inserted at the site of deletion in the disrupted TRAC gene.

28. The method of claim 1, wherein the disrupted TRAC gene comprises the nucleotide sequence of SEQ ID NO: 54.

29. The method of claim 1, wherein the disrupted 32M gene in the population of genetically engineered T cells comprises at least one of the nucleotide sequence set forth in SEQ ID NOs: 9-14.

30. The method of claim 1, wherein the population of genetically engineered T cells is allogeneic.

31. The method of claim 1, wherein at least 90% of the T cells in the population of genetically engineered T cells do not express a detectable level of TCR surface protein.

32. The method of claim 1, wherein at least 70% of the T cells in the population of genetically engineered T cells do not express a detectable level of TCR surface protein, wherein at least 50% of the T cells in the population of genetically engineered T cells do not express a detectable level of B2M surface protein; and/or wherein at least 30% of the T cells in the population of genetically engineered T cells express a detectable level of the CAR.

33. The method of claim 32, wherein at least 99.5% of the T cells in the population of genetically engineered T cells do not express a detectable level of TCR surface protein.

34. The method of claim 1, wherein at least 70% of the T cells in the population of genetically engineered T cells do not express a detectable level of B2M surface protein.

35. The method of claim 34, wherein at least 85% of the T cells in the population of the genetically engineered T cells do not express a detectable level of B2M surface protein.

36. The method of claim 1, wherein at least 50% of the T cells in the population of genetically engineered T cells express a detectable level of the CAR.

37. The method of claim 36, wherein at least 70% of the T cells in the population of genetically engineered T cells express a detectable level of the CAR.

38. The method of claim 1, wherein the population of genetically engineered T cells are administered to the human patient via intravenous infusion.

39. The method of claim 1, wherein the population of genetically engineered T cells are suspended in a cryopreservation solution.

40. A pharmaceutical composition for use in treating a B-cell malignancy, the pharmaceutical composition comprising a population of genetically engineered T cells that comprises: (a) a disrupted T cell receptor alpha constant (TRAC) gene, which optionally comprises a deletion of a fragment comprising the nucleotide sequence of SEQ ID NO: 26, (b) a nucleic acid coding for a chimeric antigen receptor (CAR) that binds CD19, wherein the CAR comprises an anti-CD19 single chain variable fragment (scFv) that comprises a heavy chain variable region set forth in SEQ ID NO: 51 and a light chain variable region set forth in SEQ ID NO: 52, and wherein the nucleic acid is inserted in the disrupted TRAC gene, and (c) a disrupted beta 2-microglobulin (32M) gene; wherein the composition comprises about 1.times.10.sup.7 to about 1.times.10.sup.9 CAR.sup.+ T cells.

41-42. (canceled)