RU2816370C2 - Rituximab-resistant chimeric antigen receptors and ways of use thereof - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to an isolated polynucleotide encoding a polypeptide containing a chimeric antigen receptor for CD19 (CAR), in which the polypeptide does not contain a rituximab binding site, and wherein the polynucleotide comprises a short EF1a promoter which is capable of expressing a chimeric antigen receptor for CD19 (CAR) in a mammalian T cell, as well as to a vector and a T-cell containing it. Also disclosed is a pharmaceutical composition containing said cell.

EFFECT: invention is effective for treating a disease or disorder associated with high CD19 levels in a subject.

25 cl, 13 dwg, 5 tbl, 4 ex

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[001] This application claims benefit from U.S. Provisional Patent Application No. 62/839455, filed April 26, 2019, and U.S. Provisional Patent Application No. 63/005041, filed April 3, 2020, the contents of which are incorporated herein by reference herein. in full.

TECHNICAL FIELD

[002] The present invention relates to chimeric antigen receptors (CARs) containing an antigen binding molecule that binds to CD19, polynucleotides encoding them, and methods of treating cancer in a patient using them.

LIST OF SEQUENCES

[003] This application contains a sequence listing that was submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy in question, created on April 21, 2020, is called AT-028_03WO_SL.txt and is 81460 bytes in size.

BACKGROUND OF THE ART

[004] Adoptive transfer of immune cells genetically modified to recognize antigens associated with cancer is a promising new approach to cancer treatment (see, for example, Brenner et al., Current Opinion in Immunology, 22(2): 251-257 (2010); Rosenberg et al., Nature Reviews Cancer, 8(4): 299-308 (2008)). Immune cells can be genetically modified to express chimeric antigen receptors (CARs), fusion proteins consisting of a CD19 antigen recognition fragment and T cell activation domains (see, for example, Eshhar et al., Proc. Natl. Acad. Sci USA, 90(2): 720-724 (1993) and Sadelain et al., Curr. Opin. Immunol, 21(2): 215-223 (2009)). Immune cells that contain CARs, such as CAR T cells (CAR-T), are engineered to be specific for an antigen while maintaining or enhancing their ability to recognize and kill a target cell.

[005] There is a need for treatments for cancer, and in particular malignancies, involving aberrant expression of CD19. Provided herein are methods and compositions that satisfy this need.

SHORT DESCRIPTION

[006] Provided herein are chimeric antigen receptors (CARs) that contain a CD19 antigen-binding domain that specifically binds to CD19; polynucleotides encoding such CARs; and immune cells expressing such CD19-specific CARs, such as CAR T cells. Methods for producing and using such CD19-specific CARs and immune cells containing such CD19-specific CARs are also provided.

[007] In one aspect, the present invention provides an isolated polynucleotide encoding a polypeptide comprising a CD19 chimeric antigen receptor (CAR) that is at least 70% identical to SEQ ID NO: 9, wherein the polypeptide does not contain a rituximab binding site, and wherein the polynucleotide comprises a short EF1a promoter that is capable of expressing a chimeric antigen receptor for CD19 (CAR) in a mammalian T cell.

[008] In some embodiments, the short EF1a promoter does not contain an intronic sequence within the nucleic acid sequence of SEQ ID NO: 15. In some embodiments, the intron contains the nucleic acid sequence of SEQ ID NO: 39.

[009] In some embodiments, the promoter comprises the nucleic acid sequence of SEQ ID NO: 16.

[010] In some embodiments, the promoter is a full-length EF1a promoter comprising the nucleic acid sequence of SEQ ID NO: 15.

[011] In some embodiments, the promoter comprises the nucleic acid sequence of SEQ ID NO: 15, and the polynucleotide encodes a polypeptide that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99% or 100% identical to any of SEQ ID NO: 8-14.

[012] In some embodiments, the polypeptide further comprises a safety switch.

[013] In some embodiments, the safety switch is linked to the anti-CD19 CAR using a linker peptide.

[014] In some embodiments, the safety switch is linked to the CD19 CAR using a T2A linker.

[015] In some embodiments, the safety switch comprises an antibody binding site.

[016] In some embodiments, the security switch comprises a mutated CD20 mimotope.

[017] In some embodiments, the polypeptide further comprises a CD8 hinge/transmembrane domain.

[018] In some embodiments, the polypeptide contains a CD34 epitope.

[019] In some embodiments, the CD34 epitope is a QBEND-10 epitope.

[020] In some embodiments, the isolated polynucleotide contains a nucleic acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to any of SEQ ID NO: 1-7.

[021] In some embodiments, the isolated polynucleotide encodes a polypeptide that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to any of SEQ ID NO: 8 -14.

[022] In another aspect, the present invention provides a vector containing an isolated polynucleotide described herein.

[023] In some embodiments, the vector is a retroviral vector, DNA vector, plasmid, RNA vector, adenoviral vector, adeno-associated virus vector, lentiviral vector, or any combination thereof.

[024] In one aspect, the present invention provides an engineered immune cell containing an isolated polynucleotide described herein.

[025] In some embodiments, the present invention provides an engineered immune cell comprising a polynucleotide that contains a nucleic acid sequence that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99% or 100% identical to SEQ ID NO: 3.

[026] In some embodiments, the present invention provides an engineered immune cell comprising a polynucleotide that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to a lentiviral CD19 v1.2, v1.3, v1.4, v1.5, or v1.6 CAR construct as shown in Table 1.

[027] In some embodiments, the present invention provides an engineered immune cell comprising a polynucleotide that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100% identical to a lentiviral CD19 v1.2 CAR construct as shown in Table 1.

[028] In some embodiments, the present invention provides an engineered immune cell comprising a polynucleotide that encodes a polypeptide that is at least about 80%, 85%, 90%, 95%, 96%, 98%, 99%, or 100 % identical to SEQ ID NO: 9 or SEQ ID NO: 10, with or without a signal sequence.

[029] In some embodiments, the present invention provides an engineered immune cell comprising a polynucleotide that contains the nucleic acid sequence of SEQ ID NO: 3, operating through a short EF1a promoter containing the nucleic acid of SEQ ID NO: 16.

[030] In some embodiments, the present invention provides an engineered immune cell comprising a polynucleotide that encodes the polypeptide sequence of SEQ ID NO: 9 or 10, with or without a signal sequence, operating through a short EF1a promoter comprising the nucleic acid of SEQ ID NO: 16. In some embodiments, the promoter does not contain the first intron of the EF1a gene.

[031] In some embodiments, the engineered immune cell does not contain rituximab mimotope.

[032] In some embodiments, the engineered immune cell comprises a polynucleotide comprising an anti-CD19 CAR v1.2 lentiviral construct, as illustrated in FIG. 1 (may also be called ALLO-501A).

[033] In some embodiments, the engineered immune cell comprises a vector described herein.

[034] In some embodiments, the immune cell is a T cell, tumor infiltrating lymphocyte (TIL), NK cell, TCR expressing cell, dendritic cell, or NK T cell.

[035] In some embodiments, the cell is an autologous T cell.

[036] In some embodiments, the cell is an allogeneic T cell.

[037] In one aspect, the present invention provides an engineered immune cell as described herein, wherein the cell is resistant to rituximab.

[038] In another aspect, the present invention provides a pharmaceutical composition comprising an engineered immune cell as described herein.

[039] In one aspect, the present invention provides a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject an engineered immune cell described herein or a pharmaceutical composition described herein.

[040] In some embodiments, the disease or disorder is non-Hodgkin's lymphoma (NHL).

[041] In some embodiments, the subject has received or is currently receiving treatment with rituximab.

[042] In one aspect, the present invention provides an article containing an engineered immune cell or a pharmaceutical composition comprising an engineered immune cell expressing the chimeric antigen receptors described herein.

BRIEF DESCRIPTION OF GRAPHIC MATERIALS

[043] In FIG. Figure 1 schematically shows rituximab-resistant chimeric antigen receptors CD19.

[044] In FIG. 2A and 2B are flow cytometry plots demonstrating CAR expression at day 5 from pan-T cells transduced with rituximab-resistant CAR expression vectors. In FIG. 2A shows CAR expression in cells from donors 541 and 604. FIG. 2B shows CAR and CD34 expression in cells from donor 410. A similar profile was observed in donor 2593 (data not shown).

[045] In FIG. Figure 3 shows normalized cell expansion and final CAR expression in all four donors at day 13.

[046] In FIG. 4A-4D show cell proliferation and CAR expression over time of pan-T cells from donors 541 (FIG. 4A), 604 (FIG. 4B), 410 (FIG. 4C), 2593 (FIG. 4D) transduced with resistant rituximab with CAR expression vectors.

[047] In FIG. 5A-5D show the CD4/CD8 ratios at days 5, 9 and 13 of pan-T cells from donors 541 (FIG. 5A), 604 (FIG. 5B), 410 (FIG. 5C), 2593 (FIG. 5D), transduced with rituximab-resistant CAR expression vectors.

[048] In FIG. 6A-6D show the phenotype and day 9 activation of pan-T cells from donors 541 (FIG. 6A), 604 (FIG. 6B), 410 (FIG. 6C), 2593 (FIG. 6D) transduced with rituximab-resistant expression CAR vectors.

[049] In FIG. Figure 7 shows the phenotype, % CD8+ activation, and anergy measured using the average of TIM3 and PD1 staining for all four donors at day 9.

[050] In FIG. 8A-8D show the phenotype and activation at day 13 of pan-T cells from donors 541 (FIG. 8A), 604 (FIG. 8B), 410 (FIG. 8C), 2593 (FIG. 8D) transduced with rituximab-resistant expression CAR vectors.

[051] In FIG. Figure 9 shows the phenotype, %CD8+ activation, and anergy measured using TIM3 and PD1 staining for all four donors at day 13.

[052] In FIG. Figure 10 shows the average short-term (24 h) cell death assay values using Raji cells as target cells for each CAR construct.

[053] In FIG. 11A-11D show average long-term cell death assay values using A549-CD19+ cells as target cells with E:T of 8:1 (FIG. 11A), 4:1 (FIG. 11B), 2:1 (FIG. 11C) and 1:1 (FIG. 11D) for each CAR design.

[054] In FIG. 12 shows the % level of CAR+ T cells on day 5 after transduction of pan-T cells with serial dilutions of lentiviral preparations of rituximab-resistant lentiviral constructs (ALLO-501v1.2 and v1.3) or rituximab-resistant lentiviral construct (ALLO-501v1. 0).

[055] In FIG. 13A-13B show the in vivo efficacy of pan-T cells from donors 541 (FIG. 13A) and 604 (FIG. 13B) transduced with a rituximab-resistant CAR expression vector (ALLO-501v1.2) or a rituximab-sensitive CAR expression vector (ALLO -501v1.0) when tested in a mouse model of NSG tumor bearing Raji cells.

DETAILED DESCRIPTION

[056] Chimeric antigen receptor (CAR) therapy is a promising approach in the treatment of cancer. The CAR construct, described herein as v1.0, is an exemplary CD19 CAR that expresses a synthetic RQR8 peptide that serves as a safety switch. RQR8 contains two rituximab-binding mimotopes. In the event of an adverse event, patients may be treated with rituximab to reduce circulating levels of CD19 v1.0. Rituximab is also used as standard of care for some features of non-Hodgkin's lymphoma (NHL) and is given in high doses. Due to the long half-life of rituximab v1.0 for CD19, it should not be administered to patients until circulating levels of rituximab have reached low concentrations. Therapy with a rituximab-resistant CD19 CAR would enable patients who have previously been treated with rituximab to receive CAR-T therapy directly, without having to wait for rituximab levels to decline and without having to subject patients to apheresis. Provided herein are CD19 chimeric antigen receptors (CARs) that are resistant to the CD20 binding antibody rituximab. New CAR constructs are designed to eliminate binding to rituximab while maintaining CAR expression and activity.

I. Chimeric antigen receptors

[057] As used herein, chimeric antigen receptors (CARs) are proteins that specifically recognize target antigens (eg, target antigens on cancer cells). When bound to a target antigen, a CAR can activate an immune cell to attack and destroy the cell carrying that antigen (such as a cancer cell). CARs may also include costimulatory or signaling domains to enhance their effectiveness. See Krause et al., J. Exp. Med., Volume 188, No. 4, 1998 (619-626); Finney et al., Journal of Immunology, 1998, 161: 2791-2797, Song et al., Blood 119:696-706 (2012); Kalos et al., Sci. Transl. Med. 3:95 (2011); Porter et al., N. Engl. J. Med. 365:725-33 (2011) and Gross et al., Annu. Rev. Pharmacol. Toxicol. 56:59-83 (2016); US patents Nos. 7741465 and 6319494.

[058] The chimeric antigen receptors described herein comprise an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises a CD19 antigen-binding domain that specifically binds to CD19. In some embodiments, the CD19-specific CAR comprises the following elements from the 5' to the 3' end: a signal sequence, a CD19 antigen-binding domain (e.g., a 4G7-derived scFv), a hinge and transmembrane region, and one or more from subsequent signaling domains. In some embodiments, the antibody binding domain binds to CD19 to treat hematologic malignancies associated with CD19 expression.

[059] The scFv portion of the chimeric antigen receptor (CAR) used in the allogeneic CD19 CAR v1.0 is derived from the mouse anti-human CD19 antibody clone 4G7. 4G7 is an anti-CD19 monoclonal antibody that recognizes CD19. Single chain variable fragments (scFv) derived from 4G7 contain the targeting component of certain chimeric antigen receptors (CARs) (see WO 2014184143 A1). In some embodiments, the scFv derived from the anti-CD19 monoclonal antibody 4G7 comprises a portion of the anti-CD19 monoclonal antibody 4G7 immunoglobulin gamma heavy chain 1 (GenBank: CAD88275.1; SEQ ID NO: 17), and a portion of the monoclonal antibody immunoglobulin kappa light chain to CD19 4G7 (GenBank: CAD88204.1; SEQ ID NO: 35) linked together via a flexible linker. (Peipp M., D. Saul, et al., 2004. Efficient eukaryotic expression of fluorescent scFv fusion proteins directed against CD antigens for FACS applications. J. Immunol. Methods 285: 265-280). In some embodiments, the scFv comprises variable fragments of the anti-CD19 monoclonal antibody 4G7 immunoglobulin heavy chain gamma chain 1, and variable fragments of the 4G7 anti-CD19 monoclonal antibody immunoglobulin kappa light chain, linked together by a flexible linker.

[060] Anti-CD19 monoclonal antibody immunoglobulin gamma chain 1 heavy gamma chain 4G7 (signal sequence underlined)

[062] Anti-CD19 monoclonal antibody immunoglobulin kappa light chain 4G7 (signal sequence underlined)

[064] In some embodiments, the scFv comprises a portion of the amino acid sequences of SEQ ID NO: 17 and/or SEQ ID NO: 18. In some embodiments, the scFv is characterized by at least 70%, at least 80%, at least 90% , at least 95%, at least 97%, or at least 99% sequence identity with the variable region of the amino acid sequence of SEQ ID NO: 34 and/or SEQ ID NO: 35. Disclosed herein are antigen-binding molecules, including including antibodies that specifically bind to the 4G7-derived scFv for CD19, as well as molecules containing these sequences and cells presenting such molecules. Humanized forms of antigen binding molecules are also an aspect of the present invention. Routes of application and use of these antigen-binding molecules are also disclosed.

A. Antigen binding domain

[065] As discussed above, the CD19 CARs described herein contain an antigen binding domain. As used herein, the term “antigen binding domain” means any polypeptide that binds a particular target antigen, for example, the particular target antigen may be a CD19 protein or a fragment thereof. In some embodiments, the antigen binding domain binds to a CD19 antigen on a tumor cell. In some embodiments, the antigen binding domain binds to a CD19 antigen on a cell involved in a hyperproliferative disease.

[066] In some embodiments, the antigen binding domain comprises a variable heavy chain, a variable light chain, and/or one or more CDRs. In some embodiments, the antigen binding domain is a single chain variable fragment (scFv) comprising CDR light chains CDR1, CDR2 and CDR3 and CDR heavy chains CDR1, CDR2 and CDR3. Variants of the antigen binding domains (eg, CDR variants VH and/or VL) are also within the scope of the present invention, for example, each of the variable light and/or variable heavy chains is characterized by at least 70-80%, 80-85%, 85- 90%, 90-95%, 95-97%, 97-99%, or greater than 99% amino acid sequence identity to the antigen binding domain sequences described herein. In some cases, such molecules include at least one heavy chain and one light chain, while in other cases, the variant forms contain two variable light chains and two variable heavy chains (or subparts thereof). One skilled in the art will be able to determine suitable variants of the antigen binding domains set forth herein using well known techniques. In certain embodiments, one skilled in the art can identify suitable regions of the molecule that can be altered without interfering with activity by targeting regions not believed to be important for activity.

[067] In certain embodiments, the polypeptide structure of the antigen binding domains is based on antibodies, including, but not limited to, monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies (sometimes referred to herein as “antibody mimetics”), chimeric antibodies, humanized antibodies, human antibodies, fusion antibodies (sometimes referred to herein as “antibody conjugates”) and fragments thereof, respectively. In some embodiments, the antigen binding domain contains or consists of avimers.

[068] A CD19 antigen-binding domain is considered “selective” when it binds to one target more strongly than to a second target. In some embodiments, the CD19 antigen binding domain is a scFv.

[069] In some embodiments, the present invention provides isolated polynucleotides encoding any of the CD19 chimeric antigen receptors (CARs) described herein. In some embodiments, the present invention provides isolated polynucleotides encoding the CD19 CAR described in Table 1. Also provided herein are vectors containing the polynucleotides and methods for producing them.

b. Safety switches and monoclonal antibody-specific epitopes

Security switches

[070] It should be appreciated that adverse events may be minimized by transducing immune cells (containing one or more CARs) with a suicide gene other than the rituximab-binding epitope. It is also necessary to include an inducible “on” or “speed up” switch in immune cells. Suitable techniques include the use of inducible caspase-9 (US Patent Application No. 2011/0286980) or thymidine kinase before, after, or simultaneously with transduction of cells with the CAR construct of the present invention. Additional methods for introducing suicide genes and/or switches include the use of TALENS, zinc fingers, RNAi, siRNA, shRNA, antisense technology and other techniques known in the art.

[071] In accordance with the present invention, additional on-off switches or other types of controlled switching techniques may be included herein. These techniques can implement the use of dimerization domains and optional dimerization activators of such domains. These techniques include, for example, those described by Wu et al., Science 2014 350 (6258), using FKBP/Rapalog dimerization systems in certain cells, the contents of which are incorporated herein by reference in their entirety. Additional dimerization technology is described, for example, in Fegan et al. Chem. Rev. 2010, 110, 3315-3336, as well as in US patent Nos. 5830462, 5834266, 5869337 and 6165787, the contents of which are incorporated herein by reference in their entirety. Additional dimerization pairs may include cyclosporine-A/cyclophilin receptor, estrogen/estrogen receptor (optionally using tamoxifen), glucocorticoids/glucocorticoid receptor, tetracycline/tetracycline receptor, vitamin D/vitamin D receptor. Additional examples of dimerization technology can be found in, e.g. WO 2014/127261, WO 2015/090229, US 2014/0286987, US 2015/0266973, US 2016/0046700, US Patent No. 8486693, US 2014/0171649 and US 2012/0130076, the contents of which are further included in this document via link in them in full.

[072] In some embodiments, a CAR immune cell (eg, a CAR T cell) of the present invention comprises a polynucleotide encoding a “suicide” polypeptide that is deficient in rituximab binding. In some embodiments, the suicide peptide comprises a mutated RQR8 sequence. See, for example, WO 2013153391 A, which is hereby incorporated by reference in its entirety. In a CAR immune cell (eg, a CAR T cell) containing a polynucleotide, the “suicide” polypeptide is expressed on the surface of the CAR immune cell (eg, a CAR T cell). In some embodiments, the “suicide” polypeptide contains the amino acid sequence shown under SEQ ID NO: 19.

[073] The "suicide" polypeptide may also contain a signal peptide at the amino terminus, for example, MGTSLLCWMALCLLGADHADA (SEQ ID NO: 20). In some embodiments, the “suicide” polypeptide comprises the amino acid sequence shown under SEQ ID NO: 21, which includes the signal sequence of SEQ ID NO: 20.

[074] In some embodiments, the suicide peptide comprises an amino acid sequence that contains one or more mutated residues, inserted residues, or deleted residues that reduce or eliminate binding to rituximab.

[075] If a suicide polypeptide is expressed on the surface of a CAR immune cell (eg, a CAR T cell), then binding of the antibody to the suicide polypeptide gene epitopes causes cell lysis. Deletion of a CD19-specific CAR immune cell (eg, a CAR T cell) can occur in vivo, for example, when a suicide drug is administered to a patient. The decision to delete the transferred cells may be driven by the identification of adverse effects associated with the transferred cells in the patient, such as the detection of unacceptable levels of toxicity. As used herein, the term “suicide agent” refers to a molecule that binds to a CAR-containing immune cell and causes lysis of the CAR-expressing immune cell.

[076] In some embodiments, the suicide polypeptide is expressed on the surface of a cell. In some embodiments, the “suicide” polypeptide is included in the CAR construct. In some embodiments, the “suicide” polypeptide is not part of the CD19 CAR construct.

[077] In some embodiments, the extracellular domain of any of the CD19-specific CARs disclosed herein may contain one or more epitopes specific to (ie, specifically recognized by) the monoclonal antibody. These epitopes are also referred to herein as mAb-specific epitopes. Exemplary mAb-specific epitopes are disclosed in International Patent Publication No. WO 2016/120216, which is incorporated herein by reference in its entirety. In such embodiments, the extracellular domain of the CAR contains antigen binding domains that specifically bind to CD19, and one or more epitopes that bind to one or more monoclonal antibodies (mAbs). CARs containing mAb-specific epitopes can be single-chain or multi-chain.

[078] Incorporation of monoclonal antibody-specific epitopes into the extracellular domain of the CAR described herein allows the sorting and depletion of engineered CAR-expressing immune cells. In some embodiments, this feature also promotes the recovery of endogenous CO19-expressing cells that have been depleted by administration of engineered CAR-expressing immune cells. In some embodiments, the depletion capability is provided by a safety switch in the event of harmful effects, such as when administered to a subject.

[079] Therefore, in some embodiments, the present invention provides a method for sorting and/or depleting engineered immune cells bearing CARs containing mAb-specific epitopes, and a method for promoting recovery of endogenous CD19-expressing cells.

[080] Multiple epitope-monoclonal antibody pairs can be used to create CARs containing monoclonal antibody-specific epitopes; in particular those already approved for medical or GMP manufacturing use, such as the CD34/QBEND-10 epitope, as a non-limiting example.

[081] The present invention also includes methods for sorting engineered immune cells having CARs specific for CD19 and expressing mAb-specific epitope(s), and therapeutic methods in which activating engineered immune cells having these CARs are modulated by cell depletion using an antibody that targets the outer ligand binding domain of said CARs. Table 2 provides exemplary mimotopic sequences that may be inserted into the extracellular domains of any of the CARs of the present invention.

[082] In some embodiments, the CAR comprises an epitope or mimotope amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95 %, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the epitope or mimotope amino acid sequences set forth herein in Table 2. B In some embodiments, the CAR comprises an epitope or mimotope amino acid sequence that is not or does not contain SEQ ID NO: 22. In some embodiments, the CAR comprises an epitope or mimotope comprising the amino acid sequence of SEQ ID NO: 30.

With. Hinge domain

[083] The extracellular domain of the CAR of the present invention may comprise a “hinge” domain (or hinge region). The term generally refers to any polypeptide that functions to link the transmembrane domain of a CAR to the extracellular antigen binding domain of a CAR. In particular, hinge domains can be used to provide greater flexibility and accessibility to the extracellular antigen binding domain.

[084] The hinge domain may contain no more than 300 amino acids, in some embodiments from 10 to 100 amino acids, or in some embodiments from 25 to 50 amino acids. The hinge domain may be derived from all or part of a naturally occurring molecule, such as all or part of the extracellular region of CD8, CD4, CD28, 4-1BB, or IgG (in particular, the hinge region of an IgG; it should be appreciated that the hinge region may contain part or all of the immunoglobulin family, such as IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, IgM or fragments thereof), or from all or part of the heavy chain constant region of an antibody. Alternatively, the hinge domain may be a synthetic sequence that corresponds to a naturally occurring hinge sequence, or may be a completely synthetic hinge sequence. In some embodiments, said hinge domain is part of the human CD8α chain (eg, NP_001139345.1). In another specific embodiment, said hinge and transmembrane domains form part of the human CD8α chain. In some embodiments, the hinge domain of the CARs described herein comprises a CD8α, IgG1, IgG4, PD-1, or FcγRIIIα subsequence, particularly the hinge region of any one of CD8α, IgG1, IgG4, PD-1, or FcγRIIIα. In some embodiments, the hinge domain includes a human CD8α hinge, a human IgG1 hinge, a human IgG4 hinge, a human PD-1 hinge, or a human FcγRIIIα hinge. In some embodiments, the CARs disclosed herein comprise scFv, human CD8α hinge and transmembrane domains, CD3ζ signaling domain, and 4-1 BB signaling domain. Table 3 provides the amino acid sequences for the exemplary hinges provided herein.

[085] In some embodiments, the hinge region comprises an amino acid sequence that is at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to the hinge domain amino acid sequences set forth herein in Table 3.

d. Transmembrane domain

[086] The CARs of the present invention are designed with a transmembrane domain that is fused to the extracellular domain of the CAR. Likewise, it can be fused to the intracellular domain of CAR. In some cases, the transmembrane domain may be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins in order to minimize levels of interaction with other members of the receptor complex. In some embodiments, short linkers may form connections between any or some of the extracellular, transmembrane, and intracellular domains of the CAR. In some embodiments, the linker contains glycine repeat sequences. In some embodiments, the linker comprises (GGGGS)n, where n is 1, 2, 3, 4, or 5 (SEQ ID NO: 41).

[087] Suitable transmembrane domains for CARs disclosed herein have the ability to (a) be expressed on the surface of an immune cell, such as, for example, but not limited to a lymphocyte such as a helper (T _h ) T cell, a cytotoxic (T _c ) cell A T cell, a regulatory (T _reg ) cell, or a natural killer (NK) cell, and/or (b) interact with an extracellular antigen-binding domain and an intracellular signaling domain to direct the immune cell's cellular response against a target cell.

[088] The transmembrane domain can be obtained from either a natural or synthetic source. If the source is natural, the domain can be derived from any membrane-associated or transmembrane protein.

[089] Transmembrane regions, particularly useful in the present invention, can be derived from (contained in or correspond to) CD28, OX-40, 4-1BB/CD137, CD2, CD7, CD27, CD30, CD40, programmed death protein-1 cells (PD-1), inducible T-cell costimulator (ICOS), lymphocyte functionally associated antigen-1 (LFA-1, CD1-1a/CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7- H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class 1 molecules, TNF receptor proteins, immunoglobulin protein, cytokine receptor, integrins, signaling lymphocyte activating molecules (SLAM proteins ), activating NK cell receptors, BTLA, Toll-like receptors, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44 , NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1 1d, ITGAE, CD103, ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE/RANKL, DNAM1 ( CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, a ligand that specifically binds to CD83, or any combination thereof.

[090] As non-limiting examples, the transmembrane region may be derived from, or may be part of, a T cell receptor such as an α, β, γ, or δ, CD3 complex polypeptide, IL-2 receptor p55 (chain), p75 ( β chain) or γ chain, subunit chain of Fc receptors, in particular Fcγ receptor III, or CD proteins. Alternatively, the transmembrane domain may be synthetic and may contain predominantly hydrophobic residues such as leucine and valine. In some embodiments, said transmembrane domain is derived from a human CD8α chain {eg, NP_001139345.1).

[091] In some embodiments, the transmembrane domain in the CAR of the present invention is a CD8α transmembrane domain. In some embodiments, the transmembrane domain in the CAR of the present invention is a CD8α transmembrane domain comprising the amino acid sequence IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 35). In some embodiments, the transmembrane domain of the CD8α contains a sequence of nucleic acid, which encodes a transmembrane amino acid sequence for Seq ID No: 35. In some embodiments, a hinge and a trans -membrane domain in the present invention are a CD8α and a transmembrane domain containing an amino acid sequence Aptiasqplslrpeacrpaaggavhtrgldfacdiywaplagtcgvllslvit (SEQ ID NO: 36).

e. Intracellular domain

[092] The intracellular (cytoplasmic) domain of the CAR of the present invention may enable activation of at least one of the normal effector functions of the CAR-containing immune cell. T cell effector function, for example, may relate to cytolytic activity or helper activity, including cytokine secretion.

In some embodiments, the activating intracellular signaling domain for use in a CAR may be cytoplasmic sequences, such as, but not limited to, the T cell receptor and co-receptors that act together to initiate signal transduction upon binding to an antigen receptor, as well as any derivative or variant of these sequences and any synthetic sequence that has the same functionality.

[093] It will be appreciated that suitable (e.g., activating) intracellular domains include, but are not limited to, signaling domains derived from (or corresponding to) CD28, OX-40, 4-1BB/CD137, CD2, CD7, CD27, CD30, CD40, programmed cell death protein-1 (PD-1), inducible T-cell costimulator (ICOS), lymphocyte function-associated antigen-1 (LFA-1, CD1-1a/CD18), CD3 gamma, CD3 delta, CD3 epsilon , CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class 1 molecules, TNF receptor proteins, immunoglobulin protein, cytokine receptor, integrins, signaling lymphocyte-activating molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll-like receptors, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7 , NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA -6, CD49f, ITGAD, CD1 1d, ITGAE, CD103, ITGAL, CD1 1a, LFA-1, ITGAM, CD1 1b, ITGAX, CD1 1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2 , TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 ( NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, a ligand that specifically binds to CD83, or any combination thereof.

[094] The intracellular domains of the CARs of the present invention may include, in addition to the activating domains described above, costimulatory signaling domains (interchangeably referred to herein as costimulatory molecules) to enhance their effectiveness. Costimulatory domains can provide a signal in addition to the primary signal provided by the activating molecule, as described herein.

[095] It will be appreciated that suitable costimulatory domains within the scope of the present invention may be derived from (or correspond to) from, for example, CD28, OX40, 4-1BB/CD137, CD2, CD3 (alpha, beta, delta, epsilon, gamma, zeta), CD4, CD5, CD7, CD9, CD16, CD22, CD27, CD30, CD33, CD37, CD40, CD45, CD64, CD80, CD86, CD 134, CD 137, CD 154, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1 (CD1 1a/CD18), CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10 , Fc gamma receptor, MHC class 1 molecules, TNFR, integrin, signal lymphocyte activating molecule, BTLA, Toll-like receptors, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR ), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, SV8alpha, CD8beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1-1d, ITGAE, CD103, ITGAL, CD1-1a, LFA-1, ITGAM, CD1-1b, ITGAX, CD1-1c, ITGB1, CD29, ITGB2, CD 18 , LFA-1, ITGB7, NKG2D, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRT AM, Ly9 (CD229), CD160 (BY55), PSGL1 , CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Lyl08), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/ Cbp, CD19a, CD83 ligand, or fragments or combinations thereof. It should be appreciated that additional costimulatory molecules or fragments thereof not listed above are within the scope of the present invention.

[096] In some embodiments, the intracellular/cytoplasmic domain of the CAR may be designed to contain the 41BB/CD137 domain by itself or in combination with any other desired intracellular domain(s). -s) applicable in the context of the CARs of the present invention. The complete native amino acid sequence of 41BB/CD137 is described in the NCBI reference sequence database: NP_001552.2. The complete native nucleic acid sequence of 41BB/CD137 is described in the NCBI reference sequence database: NM_001561.5.

[097] In some embodiments, the intracellular/cytoplasmic domain of the CAR may be designed to contain the CD28 domain by itself or in combination with any other desired intracellular domain(s). ami) applicable in the context of the CAR of the present invention. The complete native amino acid sequence of CD28 is described in the NCBI reference sequence database: NP_006130.1. The complete native CD28 nucleic acid sequence is described in the NCBI reference sequence database: NM_006139.1.

[098] In some embodiments, the intracellular/cytoplasmic domain of the CAR may be designed to contain a CD3 zeta domain by itself or in combination with any other desired intracellular domain(s). -s) applicable in the context of the CARs of the present invention. In some embodiments, the CAR intracellular signaling domain may comprise a CD3ξ signaling domain that has an amino acid sequence with at least about 70%, at least 80%, at least 90%, 95%, 97%, or 99% sequence identity with the amino acid the sequence set forth at SEQ ID NO: 38. For example, the intracellular domain of a CAR may contain a portion of the CD3 zeta chain and a portion of a costimulatory signaling molecule. The intracellular signal sequences in the intracellular signal portion of the CAR of the present invention may be linked to each other in a random or specific order. In some embodiments, the intracellular domain is designed to contain a CD3 zeta activating domain and a CD28 signaling domain.

[099] In some embodiments, the intracellular domain is designed to contain a CD3 zeta activating domain and a 4-1BB signaling domain. In some embodiments, 4-1BB (intracellular domain) comprises the amino acid sequence

[0100] The amino acid sequence of CD3 zeta may comprise SEQ ID NO: 38.

[0101] In some embodiments, the intracellular signaling domain of the CAR of the present invention comprises a costimulatory molecule domain. In some embodiments, the intracellular signaling domain of the CAR of the present invention comprises a portion of a co-stimulatory molecule selected from the group consisting of fragments 41BB (GenBank: AAA53133.) and CD28 (NP_006130.1). In some embodiments, the intracellular signaling domain of a CAR of the present invention comprises an amino acid sequence that has at least 70%, at least 80%, at least 90%, 95%, 97%, or 99% sequence identity with the amino acid sequence represented by under SEQ ID NO: 37 and SEQ ID NO: 38. In some embodiments, the intracellular signaling domain of the CAR of the present invention contains an amino acid sequence that is characterized by at least 70%, at least 80%, at least 90%, 95% , 97% or 99% sequence identity with the amino acid sequence presented under SEQ ID NO: 37, and/or at least 70%, at least 80%, at least 90%, 95%, 97% or 99% identity sequence with the amino acid sequence shown under SEQ ID NO: 38.

[0102] In illustrative embodiments, the CAR of the present invention comprises, from N-terminus to C-terminus: a CD8α signal sequence, a CD19 scFv, a CD8α hinge and transmembrane region, a 41BB cytoplasmic signaling domain, and a CD3ξ cytoplasmic signaling domain.

III. Immune cells containing CAR

A. Immune cells

[0103] Provided herein are engineered immune cells expressing the CARs of the present invention (eg, CAR T cells).

[0104] In some embodiments, the engineered immune cell contains a population of CARs, with each CAR containing different extracellular antigen-binding domains. In some embodiments, the immune cell contains a population of CARs, wherein each CAR contains extracellular antigen-binding domains.

[0105] Engineered immune cells can be allogeneic or autologous.

[0106] In some embodiments, the engineered immune cell is a T cell (e.g., inflammatory T lymphocyte, cytotoxic T lymphocyte, regulatory T lymphocyte, helper T lymphocyte, tumor infiltrating lymphocyte (TIL)), NK cell , NK T cell, TCR-expressing cell, dendritic cell, killer dendritic cell, mast cell or B cell. In some embodiments, the cell may be derived from a group consisting of CD4+ T lymphocytes and CD8+ T lymphocytes. In some illustrative embodiments, the engineered immune cell is a T cell. In some illustrative embodiments, the engineered immune cell is a gamma delta T cell. In some illustrative embodiments, the engineered immune cell is a macrophage.

[0107] In some embodiments, the engineered immune cell may be derived from, for example, but not limited to, a stem cell. The stem cells may be mature stem cells that are not human embryonic stem cells, more specifically non-human stem cells, umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells, or hematopoietic stem cells.

[0108] In some embodiments, the cell is obtained or isolated from peripheral blood. In some embodiments, the cell is derived from or isolated from peripheral blood mononuclear cells (PBMC). In some embodiments, the cell is obtained or isolated from bone marrow. In some embodiments, the cell is obtained or isolated from umbilical cord blood. In some embodiments, the cell is a human cell. In some embodiments, the cell is transfected or transduced with a nucleic acid vector using a method selected from the group consisting of electroporation, sonoporation, biolistics (e.g., gene gun), lipid transfection, polymer transfection, nanoparticles, viral transfection (e.g., using a retrovirus, lentivirus, AAV) or polyplexes.

[0109] In some embodiments, the engineered immune cells expressing on the surface of their cell membrane the CD19-specific CAR of the present invention have a percentage of memory stem cells and central memory cells of greater than 10%, 20%, 30 %, 40%, 50%, 60%, 70%, 80%, 90% or 100%. In some embodiments, the engineered immune cells expressing on the surface of their cell membrane the CD19-specific CAR of the present invention have a percentage of memory stem cells and central memory cells of from about 10% to about 100%, from about 10 % to about 90%, from about 10% to about 80%, from about 10% to about 70%, from about 10% to about 60%, from about 10% to about 50%, from about 10% to about 40% , from about 10% to about 30%, from about 10% to about 20%, from about 15% to about 100%, from about 15% to about 90%, from about 15% to about 80%, from about 15% to about 70%, from about 15% to about 60%, from about 15% to about 50%, from about 15% to about 40%, from about 15% to about 30%, from about 20% to about 100%, from about 20% to about 90%, from about 20% to about 80%, from about 20% to about 70%, from about 20% to about 60%, from about 20% to about 50%, from about 20% to about 40%, from about 20% to about 30%, from about 30% to about 100%, from about 30% to about 90%, from about 30% to about 80%, from about 30% to about 70%, from about 30% to about 60%, about 30% to about 50%, about 30% to about 40%, about 40% to about 100%, about 40% to about 90%, about 40% to about 80%, from about 40% to about 70%, from about 40% to about 60%, from about 40% to about 50%, from about 50% to about 100%, from about 50% to about 90%, from about 50% to about 80%, from about 50% to about 70%, from about 50% to about 60%, from about 60% to about 100%, from about 60% to about 90%, from about 60% to about 80 %, from about 60% to about 70%, from about 70% to about 90%, from about 70% to about 80%, from about 80% to about 100%, from about 80% to about 90%, from about 90 % to about 100%, from about 25% to about 50%, from about 75% to about 100%, or from about 50% to about 75%.

[0110] In some embodiments, the immune cell is an inflammatory T lymphocyte that expresses any of the CARs described herein. In some embodiments, the immune cell is a cytotoxic T lymphocyte that expresses any of the CARs described herein. In some embodiments, the immune cell is a regulatory T lymphocyte that expresses any of the CARs described herein. In some embodiments, the immune cell is a helper T lymphocyte that expresses any of the CARs described herein.

[0111] Prior to expansion and genetic modification, the source of cells can be obtained from a subject using a variety of non-limiting methods. Cells can be obtained from a number of non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, T cells or NK cells derived from stem cells or iPSCs, tissue from a site of infection, ascites, pleural effusion, splenic tissue and tumors. In some embodiments, any number of T cell lines available and known to those skilled in the art may be used. In some embodiments, the cells may be obtained from a healthy donor, from a patient diagnosed with cancer, or from a patient diagnosed with an infection. In some embodiments, the cells may be part of a mixed population of cells that have different phenotypic characteristics.

[0112] Also provided herein are cell lines derived from a transformed immune cell (eg, T cell) according to any of the methods described above. Also presented herein are modified cells that are resistant to immunosuppressive treatment. In some embodiments, an isolated cell in accordance with the present invention contains a polynucleotide encoding a CAR.

[0113] The immune cells of the present invention can be activated and expanded before or after genetic modification of the immune cells using generally known methods. Typically, the engineered immune cells of the present invention can be expanded, for example, by contacting an agent that stimulates the CD3 TCR complex and a costimulatory molecule on the surface of the T cells to create an activation signal for the T cell. For example, chemicals such as the calcium ionophore A23187, phorbol 12-myristate 13-acetate (PMA), or mitogenic lectins such as phytohemagglutinin (PHA) can be used to create an activation signal for the T cell.

[0114] In some embodiments, T cell populations can also be stimulated in vitro by contacting, for example, an anti-CD3 antibody or antigen-binding fragment thereof, or a surface-immobilized anti-CD28 antibody, or by contacting a protein kinase C activator (for example, bryostatin) in combination with a calcium ionophore. To co-stimulate an accessory molecule on the surface of T cells, a ligand is used that binds the accessory molecule. For example, a population of T cells may be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions suitable to stimulate T cell proliferation. The anti-CD3 antibody and the anti-CD28 antibody may be located on a bead, plate, or other substrate. Conditions suitable for culture of T cells include a suitable medium {e.g., minimal essential medium, or RPMI 1640, or X-vivo 15 (Lonza)), which may contain factors necessary for proliferation and viability, including blood serum {e.g. , fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-2, IL-15, TGF beta and TNF, or any other cell culture additives known to one skilled in the art. Other cell culture additives include, but are not limited to, surfactant, plasmanate, and reducing agents such as N-acetylcysteine and 2-mercaptoethanol. Media may include RPMI 1640, A1M-V, DMEM, MEM, a-MEM, F-12, X-Vivo 15 and X-Vivo 20, Optimizer with added amino acids, sodium pyruvate and vitamins, either without blood serum or supplemented with appropriate the amount of blood serum (or plasma), or a certain set of hormones, and/or the amount of cytokine(s) sufficient for the growth and proliferation of T cells (for example, IL-7 and/or IL-15). Antibiotics, such as penicillin and streptomycin, are included only in the experimental cultures and not in the cell cultures that are to be administered to the subject. The target cells are maintained under conditions necessary to support growth, for example, at an appropriate temperature (eg, 37° C.) and atmosphere (eg, air plus 5% CO ₂ ). T cells exposed to varying amounts of time stimulation may exhibit different characteristics.

In some embodiments, the cells of the present invention can be propagated by co-culture with tissue or cells. The cells can also be expanded in vivo, for example, in the blood of a subject after the cell is administered to the subject.

[0115] In some embodiments, an engineered immune cell in accordance with the present invention may contain one or more disrupted or inactivated genes. In some embodiments, an engineered immune cell in accordance with the present invention contains one disrupted or inactivated gene selected from the group consisting of CD52, CD19, GR, PD-1, CTLA-4, LAG3, TIM3, BTLA, BY55, TIGIT, B7H5 , LAIR1, SIGLEC10, 2B4, HLA, TCRα and TCRβ, and/or expresses a CAR, multichain CAR and/or pTa transgene. In some embodiments, the isolated cell contains polynucleotides encoding polypeptides containing a multi-chain CAR. In some embodiments, an isolated cell in accordance with the present invention contains two disrupted or inactivated genes selected from the group consisting of CD52 and GR, CD52 and TCRα, CDR52 and TCRβ, CD19 and CD52, CD19 and TCRα, CD19 and TCRβ, GR and TCRα, GR and TCRβ, TCRα and TCRβ, PD-1 and TCRα, PD-1 and TCRβ, CTLA-4 and TCRα, CTLA-4 and TCRβ, LAG3 and TCRα, LAG3 and TCRβ, TIM3 and TCRα, Tim3 and TCRβ, BTLA and TCRα, BTLA and TCRβ, BY55 and TCRα, BY55 and TCRβ, TIGIT and TCRα, TIGIT and TCRβ, B7H5 and TCRα, B7H5 and TCRβ, LAIR1 and TCRα, LAIR1 and TCRβ, SIGLEC10 and TCRα, SIGLEC10 and TCRβ, 2B4 and TCRα, 2B4 and TCRβ, and/or expresses CAR, multichain CAR and pTa transgene. In some embodiments, the method involves disrupting or inactivating one or more genes by introducing into cells an endonuclease capable of selectively inactivating the gene by selectively cleaving DNA. In some embodiments, the endonuclease may be, for example, a zinc finger nuclease (ZFN), a megaTAL nuclease, a meganuclease, a transcription activator-like effector nuclease (TALE nuclease), or a CRIPR (eg, Cas9) endonuclease.

[0116] In some embodiments, the TCR becomes nonfunctional in cells in accordance with the present invention due to disruption or inactivation of the TCRα gene and/or TCRβ gene(s). In some embodiments, a method is provided for producing modified cells obtained from an individual, wherein the cells can proliferate independently of the major histocompatibility complex (MHC) signaling pathway. Modified cells that can proliferate independently of the MHC signaling pathway that can be produced by this method are within the scope of the present invention. The modified cells disclosed herein can be used in treating patients in need thereof for host versus graft (HvG) rejection and graft versus host disease (GvHD); therefore, a method of treating patients in need thereof against host-versus-graft (HvG) rejection and graft-versus-host disease (GvHD), comprising treating said patient by administering to said patient an effective amount of modified cells containing disrupted or inactivated TCRα genes and /or TCRβ is within the scope of the present invention.

[0117] In some embodiments, immune cells are engineered to be resistant to one or more chemotherapy drugs. The chemotherapeutic drug may be, for example, a purine nucleotide analogue (PNA), thereby making the immune cell suitable for cancer treatment combining adoptive immunotherapy and chemotherapy. Exemplary PNAs include, for example, clofarabine, fludarabine, cyclophosphamide, and cytarabine, alone or in combination. PNAs are metabolized by deoxycytidine kinase (dCK) into mono-, di-, and triphosphate PNAs. Their triphosphate forms compete with ATP for DNA synthesis, act as proapoptotic agents, and are potent inhibitors of ribonucleotide reductase (RNR), which is involved in the production of trinucleotides. Provided herein are CD19-specific CAR T cells containing a disrupted or inactivated dCK gene. In some embodiments, dCK knockout cells are generated by transfecting T cells using polynucleotides encoding a specific TAL nuclease targeting dCK genes, using, for example, mRNA electroporation. CD19-specific dCK knockout CAR T cells are resistant to PNAs, including, for example, chloropharabine and/or fludarabine, and maintain T cell cytotoxic activity toward CD19-expressing cells.

[0118] In some embodiments, isolated cells or cell lines of the present invention may contain pTα or a functional variant thereof. In some embodiments, the isolated cell or cell line may be further genetically modified by disrupting or inactivating the TCRα gene.

[0119] The present invention also provides engineered immune cells containing any of the CAR polynucleotides described herein.

With. Methods of obtaining

[0120] Provided herein are methods for producing CARs and CAR-containing immune cells of the present invention. In the production of polynucleotides, polypeptides, vectors, antigen binding domains, immune cells, compositions, etc. A number of known techniques can be used in accordance with the present invention.

Polynucleotides and vectors

[0121] In some embodiments, the CAR can be introduced into an immune cell as a transgene using a plasmid vector. In some embodiments, the plasmid vector may also contain, for example, a selection marker that allows for the identification and/or selection of cells that have received the vector.

[0122] CAR polypeptides can be synthesized in situ in a cell after polynucleotides encoding the CAR polypeptides are introduced into the cell. Alternatively, CAR polypeptides can be produced outside of cells and then introduced into cells. Methods for introducing a polynucleotide construct into cells are known in the art. In some embodiments, stable transformation methods (eg, using a lentiviral vector) can be used to integrate a polynucleotide construct into the genome of a cell. In other embodiments, transient transformation methods may be used to transiently express a polynucleotide construct, and the polynucleotide construct is not integrated into the genome of the cell. In other embodiments, methods using virus-mediated effects may be used. Polynucleotides can be introduced into a cell by any suitable means, such as, for example, recombinant viral vectors (eg, retroviral, adenoviral), liposomes, and the like. Temporary transformation methods include, for example, but are not limited to, microinjection, electroporation, or particle bombardment. The polynucleotides can be included in vectors, such as, for example, plasmid vectors or viral vectors.

[0123] In some embodiments, isolated nucleic acids are provided comprising a promoter operably linked to a first polynucleotide encoding a CD19 antigen binding domain, at least one costimulatory molecule, and an activating domain. In some embodiments, the nucleic acid construct is contained in a viral vector. In some embodiments, the viral vector is selected from the group consisting of retroviral vectors, murine leukemia virus vectors, SFG vectors, adenoviral vectors, lentiviral vectors, adeno-associated virus (AAV) vectors, herpes virus vectors, and vaccinia virus vectors. In some embodiments, the nucleic acid is contained in a plasmid.

[0124] In one aspect, the present invention provides a polynucleotide sequence comprising a promoter that is capable of expressing a CAR transgene in a mammalian T cell. In some embodiments, the promoter is an EF1a promoter. The native EF1a promoter initiates expression of the alpha subunit of the elongation factor-1 complex, which is responsible for the enzymatic delivery of aminoacyl-tRNA to the ribosome. The EF1a promoter is widely used in mammalian expression plasmids and has been shown to be effective in driving CAR expression from transgenes cloned into a lentiviral vector. See, for example, Milone et al., Mol. Ther. 17(8): 1453-1464 (2009). In some embodiments, the EF1a promoter comprises the sequence shown under SEQ ID NO: 15.

[0125] The EF1a promoter sequence shown above contains the first exon (in bold) and the first intron (underlined, SEQ ID NO: 39) followed by the N-terminal portion of the second exon of the EF1a gene. In some embodiments, the polynucleotide provided herein comprises a short EF1a promoter. In some embodiments, the polynucleotide provided herein contains an EF1a promoter that is shorter than the nucleic acid sequence of SEQ ID NO: 15. In some embodiments, the polynucleotide provided herein contains an EF1a promoter that does not contain the first intron of the EF1a gene. In some embodiments, the polynucleotide provided herein comprises an EF1a promoter that does not contain the nucleic acid sequence of SEQ ID NO: 39.

[0126] In some embodiments, the promoter comprises the sequence shown under ID NO: 16.

[0127] Prior to in vitro manipulation or genetic modification of immune cells described herein, the cells may be obtained from a subject. Cells expressing CAR for CD19 can be obtained using allogeneic or autologous methods.

Raw material

[0128] In some embodiments, immune cells include T cells. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells (PBMC), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, splenic tissue, and tumors. In certain embodiments, T cells can be obtained from a unit of blood taken from a subject using any number of techniques known to one skilled in the art, such as FICOLL™ separation.

[0129] Cells can be obtained from the circulating blood of an individual through apheresis. The product obtained by apheresis typically contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells and platelets. In certain embodiments, cells collected by apheresis can be washed to remove the blood plasma fraction and placed in appropriate buffer or media for subsequent processing.

[0130] In certain embodiments, T cells are isolated from PBMCs by lysing erythrocytes and depleting monocytes, for example, by PERCOLL™ gradient centrifugation. Specific subsets of T cells (e.g. CD28+, CD4+, CD45RA- and CD45RO+ T cells or CD28+, CD4+, CDS+, CD45RA-, CD45RO+ and CD62L+ T cells) can be further isolated using positive or negative techniques known in the art. selection. For example, enrichment of a T cell population by negative selection can be achieved using a combination of antibodies directed to surface markers unique to the negatively selected cells. One application herein is to sort and/or select cells by negative magnetic immunoadhesion or flow cytometry, which uses a cocktail of monoclonal antibodies directed to cell surface markers present on negatively selected cells. For example, to enrich for CD4+ cells by negative selection, the monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CDllb, CD16, HLA-DR, and CD8. Flow cytometry and cell sorting can also be used to isolate cell populations of interest for use in the present invention.

[0131] PBMC can be used directly for genetic modification by immune cells (such as CAR or TCR) using the methods described herein. In certain embodiments, once PBMCs are isolated, T lymphocytes may be further isolated, and both cytotoxic and helper T lymphocytes may be sorted into subpopulations of naive, memory T cells, and effector T cells, either before or after genetic modification and/or reproduction.

[0132] In some embodiments, CD8+ cells are further sorted into intact, memory stem cells, central memory cells, and effector cells by identifying cell surface antigens that are associated with each of these CD8+ cell types. In some embodiments, expression of the phenotypic markers of central memory T cells includes CD45RO, CD62L, CCR7, CD28, CD3, and CD127 and is negative for granzyme B. In some embodiments, the stem memory T cells are CD45RO-, CD62L+, CD8+ T cells. In some embodiments, the central memory T cells are CD45RO+, CD62L+, CD8+ T cells. In some embodiments, the effector T cells are negative for CD62L, CCR7, CD28, and CD127 and positive for granzyme B and perforin. In certain embodiments, the CD4+ T cells are further sorted into subpopulations. For example, CD4+ T helper cells can be sorted into intact, central memory and effector cells by identifying cell populations that have cell surface antigens.

Stem cell-derived immune cells

[0133] In some embodiments, immune cells can be derived from embryonic stem (ES) or induced pluripotent stem (iPS) cells. Suitable HSCs, ES cells, iPS cells and other types of stem cells can be cultured immortalized cell lines or can be isolated directly from the patient. Various methods for isolating, developing and/or culturing stem cells are known in the art and can be used to practice the present invention.

[0134] In some embodiments, the immune cell is an induced pluripotent stem cell (iPSC) derived from a reprogrammed T cell. In some embodiments, the starting material may be an induced pluripotent stem cell (iPSC) derived from a T cell or a cell other than a T cell. The starting material may be an embryonic stem cell. The starting material may be a B cell or any other cell from peripheral blood mononuclear cell isolates, a hematopoietic progenitor cell, a hematopoietic stem cell, a mesenchymal stem cell, an adipose stem cell, or any other type of somatic cell.

Genetic modification of isolated cells

[0135] Immune cells, such as T cells, can be genetically modified after isolation using known methods, or immune cells can be activated and expanded (or differentiated in the case of progenitors) in vitro before genetic modification. In some embodiments, the isolated immune cells are genetically modified to reduce or eliminate the expression of endogenous TCRα and/or CD52. In some embodiments, cells are genetically modified using gene editing technology (e.g., CRISPR/Cas9, zinc finger nuclease (ZFN), TALEN, MegaTAL, meganuclease) to reduce or eliminate the expression of endogenous proteins (e.g., TCRα and/or CD52 ). In another embodiment, immune cells, such as T cells, are genetically modified with chimeric antigen receptors described herein (e.g., transduced with a viral vector containing one or more nucleotide sequences encoding a CAR) and then activated and/or expanded in vitro . Methods for activating and propagating T cells are known in the art and are described, for example, in US Patent No. 6905874, US Patent No. 6867041, US Patent No. 6797514 and PCT WO 2012/079000, the contents of which are incorporated herein by reference in their entirety. volume. Typically, such methods involve contacting PBMCs or isolated T cells with an additional stimulatory molecule and a co-stimulatory molecule, such as anti-CD3 and anti-CD28 antibodies, typically attached to a bead or other surface, in a culture medium with appropriate cytokines such as IL -2. Anti-CD3 and anti-CD28 antibodies attached to the same bead serve as a “mock” of an antigen presenting cell (APC). One example is the Dynabeads® system, a CD3/CD28 activator/stimulator system for physiological activation of human T cells. In other embodiments, T cells can be activated and stimulated to proliferate by feeder cells and appropriate antibodies and cytokines using methods such as those described in US Pat. No. 6,040,177, US Pat. No. 5,827,642, and WO 2012129514, the contents of which are incorporated herein by references in their entirety.

[0136] Certain methods for producing the constructs and engineered immune cells of the present invention are described in PCT application PCT/US15/14520, the contents of which are incorporated herein by reference in their entirety.

[0137] It should be appreciated that PBMCs may additionally include other cytotoxic lymphocytes, such as NK cells or NKT cells. An expression vector carrying a chimeric receptor coding sequence as disclosed herein can be introduced into a population of donor human T cells, NK cells, or NKT cells. Successfully transduced T cells carrying an expression vector can be sorted using flow cytometry to isolate CO3-positive T cells and then expanded to increase the number of these CAR-expressing T cells in addition to activating the cells using anti-CD3 antibodies and IL-2 or other methods known in the art, as described elsewhere herein. Standard procedures are used to cryopreserve CAR-expressing T cells for storage and/or production for use in a human subject. In one embodiment, transduction, culture and/or expansion of T cells in vitro is carried out in the absence of products derived from non-human animals, such as fetal bovine serum and fetal bovine serum.

[0138] To clone polynucleotides, a vector can be introduced into a host cell (an isolated host cell) to allow replication of the vector itself and thereby amplify copies of the polynucleotide contained therein. Cloning vectors may contain sequence components, typically including, but not limited to, origin of replication, promoter sequences, transcription initiation sequences, enhancer sequences and selectable markers. These elements can be selected accordingly by one of ordinary skill in the art. For example, the origin of replication may be selected to facilitate autonomous replication of the vector in the host cell.

[0139] In certain embodiments, the present invention provides isolated host cells containing a vector provided herein. Host cells containing the vector can be used to express or clone the polynucleotide contained in the vector. Suitable host cells may include, but are not limited to, prokaryotic cells, fungal cells, yeast cells, or higher eukaryotic cells such as mammalian cells. Suitable prokaryotic cells for this purpose include, but are not limited to, eubacteria such as gram-negative or gram-positive organisms, e.g. Enterobacteriaceae, such as Escherichia, e.g. E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g. Salmonella typhimurium, Serratia, e.g. Serratia marcescens and Shigella, as well as bacilli such as B. subtilis and B. licheniformis, Pseudomonas such as P. aeruginosa and Streptomyces.

[0140] The vector can be introduced into a host cell using any suitable methods known in the art, including, but not limited to, DEAE-dextran mediated delivery, calcium phosphate precipitation method, cationic lipid mediated delivery, liposome mediated transfection, electroporation , microparticle bombardment, receptor-mediated gene delivery, polylysine-mediated delivery of histone, chitosan and peptides. Standard methods for transfecting and transforming cells to express a vector of interest are well known in the art. In a further embodiment, a mixture of different expression vectors can be used to genetically modify a donor population of immune effector cells, where each vector encodes a different CAR, as disclosed herein. The resulting transduced immune effector cells form a mixed population of engineered cells, with a portion of the engineered cells expressing more than one different CAR.

[0141]

[0142] In some embodiments, the vector comprises a lentiviral vector. A lentiviral vector containing a CAR coding sequence can be introduced into a lentiviral packaging cell line, and the lentivirus produced by the packaging cell line can be used to transduce T cells to create CAR T cells. To produce CAR-encoding lentivirus, HEK-293T cells can be seeded at 0.4 million cells per ml in 2 ml DMEM (Gibco) supplemented with 10% FBS (Hyclone or JR Scientific) per well of a 6-well plate on day 0 On day 1, lentivirus can be produced by mixing together the lentiviral packaging vectors 1.5 μg psPAX2, 0.5 μg pMD2G, and 0.5 μg of the corresponding CAR transfer vector in 250 μl Opti-MEM (Gibco) per well of a 6-well plate (“mixture of DNA”). 10 µl Lipofectamine 2000 (Invitrogen) in 250 µl Opti-MEM can be incubated at room temperature for 5 minutes and then added to the DNA mixture. The mixture can be incubated at room temperature for 20 minutes and a total volume of 500 µl slowly added to the sides of the wells containing HEK-293T. General methods for the production and transduction of CAR-containing lentiviruses are widely known in the art, for example, see Milone et al., Leukemia, 2018, 32:1529-1541; Sanber et al., Construction of stable packaging cell lines for clinical lentiviral vector production, Nature 2015, DOI: 10.1038; Roddie et al., Cytotherapy 2019, 21:327-340, all of which are incorporated herein by reference in their entirety. In one embodiment, the present invention provides a method for storing genetically engineered cells expressing a CAR or TCR that target the CD19 protein. It involves cryopreserving immune cells so that the cells remain viable after thawing. The fraction of immune cells expressing CAR can be cryopreserved by methods known in the art to provide a constant source of such cells for future treatment of patients affected by malignancies. If necessary, cryopreserved transformed immune cells can be thawed, grown and expanded to produce more such cells.

[0143] In some embodiments, cells are constituted by first collecting them from the culture medium and then washing and concentrating the cells in a medium and container system suitable for administration (a "pharmaceutically acceptable" carrier) in a treatment-effective amount. Suitable infusion media can be any formulation of isotonic media, regular normal saline, Normosol™ R (Abbott), or Plasma-Lyte™ A (Baxter), but 5% dextrose in water or lactated Ringer's may also be used. The infusion medium may be supplemented with human serum albumin.

Allogeneic CAR T cells

[0144] The process for obtaining allogeneic T cell therapies with CARs or AlloCARs™ involves the collection of healthy, selected, screened and tested T cells from healthy donors. T cells are then engineered to express CARs that recognize specific cell surface proteins (eg, CD19) expressed in hematologic or solid tumors. Allogeneic T cells undergo gene editing to reduce the risk of graft-versus-host disease (GvHD) and prevent allogeneic rejection. T cell receptor gene (eg, TCRα, TCRβ) is knocked out to avoid GvHD. The CD52 gene can be knocked out to make the CAR T cell product resistant to treatment with the kCD52 antibody. Thus, kCD52 antibody treatment can be used to suppress the host immune system and enable the engraftment of CAR T cells to achieve full therapeutic effect. The engineered T cells then undergo a purification step and are ultimately cryopreserved in vials for delivery to patients.

Autologous CAR T cells

[0145] Autologous chimeric antigen receptor (CAR) T cell therapy involves collecting the patient's own cells (e.g., white blood cells, including T cells) and genetically engineering the T cells to express CARs that recognize the target, expressed on the cell surface of one or more specific cancer cells, and destroy the cancer cells. The engineered cells are then cryopreserved and subsequently administered to the patient.

IV. Treatment methods

[0146] The present invention provides methods for treating or preventing a condition associated with undesirable and/or elevated levels of CD19 in a patient, comprising administering to a patient in need thereof an effective amount of at least one CAR or immune cells containing a CAR disclosed herein .

[0147] Methods are provided for treating diseases or disorders, including cancer. In some embodiments, the present invention relates to generating a T cell-mediated immune response in a subject, comprising administering to the subject an effective amount of engineered immune cells according to the present application. In some embodiments, the T cell-mediated immune response is directed against a target cell or cells. In some embodiments, the engineered immune cell comprises a chimeric antigen receptor (CAR). In some embodiments, the target cell is a tumor cell. In some aspects, the present invention provides a method of treating or preventing cancer, the method comprising administering to a subject in need thereof an effective amount of at least one isolated antigen binding domain described herein. In some aspects, the present invention provides a method of treating or preventing cancer, wherein the method comprises administering to a subject in need thereof an effective amount of at least one immune cell, wherein the immune cell contains at least one chimeric antigen receptor, T cell receptor and/or dedicated antigen binding domain described herein.

[0148] The CAR-containing immune cells of the present invention can be used to treat cancers associated with aberrant expression of CD19. In some embodiments, the CAR-containing immune cells of the present invention can be used to treat cancer. As used herein, the term “cancer” includes, without limitation, solid tumors and tumors derived from blood cells. The term "cancer" refers to a disease of the tissues of the skin, organs, blood and blood vessels, including, but not limited to, forms of cancer of the bladder, bone or blood, brain, breast, cervix, breast, colon, endometrium, esophagus, eye, head, kidney, liver, lymph nodes, lung, mouth, neck, ovaries, pancreas, prostate, rectum, stomach, testicle, larynx and uterus. Specific forms of cancer include, but are not limited to, advanced tumor, amyloidosis, neuroblastoma, meningioma, hemangiopericytoma, multiple brain metastases, glioblastoma multiforme, glioblastoma, brain stem glioma, malignant brain tumor with poor prognosis, malignant glioma, recurrent malignant glioma, anaplastic astrocytoma, anaplastic oligodendroglioma, neuroendocrine tumor, rectal adenocarcinoma, colorectal cancer stages C and D according to the Duke staging system, unresectable colorectal carcinoma, metastatic hepatocellular carcinoma, Kaposi's sarcoma, acute myeloid leukemia with its karyotype, Hodgkin's lymphoma, non-Hodgkin's lymphoma (N HL) , leather T-cell lymphoma, leather B-cell lymphoma, diffuse B-circuit lymphoma, low-deferenced follicular lymphoma, malignant melanoma, malignant mesothelioma, malignant pleural spille of the mesothelioma, peritoneal carcine, papallar carcinomal, papallar carcin , gynecological sarcoma, sarcoma of soft tissue, scleroderma, cutaneous vasculitis, Langerhans cell histiocytosis, leiomyosarcoma, fibrodysplasia ossificans progressiva, hormone-resistant prostate cancer, high-risk resected soft tissue sarcoma, unresectable hepatocellular carcinoma, Waldenström's macroglobulinemia, flaccid myeloma, unexpressed myeloma, cancer fallopian tubes, androgen-independent prostate cancer, androgen-dependent non-metastatic stage IV prostate cancer, hormone therapy-insensitive prostate cancer, chemotherapy-insensitive prostate cancer, papillary thyroid carcinoma, follicular thyroid carcinoma, medullary thyroid carcinoma and leiomyoma. In a specific embodiment, the cancer is metastatic. In another embodiment, the cancer is difficult to treat or resistant to chemotherapy or radiation.

[0149] In illustrative embodiments, CAR-containing immune cells, such as the CAR T cells of the present invention, are used to treat NHL.

[0150] Methods of reducing the size of a tumor in a subject are also provided, comprising administering to the subject an engineered cell of the present invention to the subject, wherein the cell contains a chimeric antigen receptor containing a CD19 antigen binding domain and binds to a CD19 antigen in the tumor.

[0151] In some embodiments, the subject has a solid tumor or blood malignancy, such as lymphoma or leukemia. In some embodiments, the engineered cell is delivered to the tumor bed. In some embodiments, the cancer is present in the bone marrow of the subject. In some embodiments, the engineered cells are autologous immune cells, such as autologous T cells. In some embodiments, the engineered cells are allogeneic immune cells, such as allogeneic T cells. In some embodiments, the engineered cells are heterologous immune cells, such as heterologous T cells. In some embodiments, the engineered cells of this application are transfected or transduced in vivo. In other embodiments, the engineered cells are transfected or transduced ex vivo. As used herein, the term “in vitro cell” refers to any cell that is cultured ex vivo.

[0152] The terms "therapeutically effective amount", "effective dose", "effective amount" or "therapeutically effective dosage" of a therapeutic agent, for example, engineered CAR T cells, mean any amount that, when used alone or in combination with another The therapeutic agent protects the subject from the manifestation of the disease or promotes regression of the disease, as evidenced by a decrease in the severity of symptoms of the disease, an increase in the frequency and duration of asymptomatic periods of the disease, or prevents deterioration or disability due to the disease. The ability of a therapeutic agent to provide disease regression can be assessed using various methods known to the skilled practitioner, for example, in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by analyzing the activity of the agent in in vitro assays.

[0153] The terms "patient" and "subject" are used interchangeably and include human and non-human animal subjects, as well as subjects with recognized disorders, subjects without officially recognized disorders, subjects receiving medical care, subjects at risk of developing violations, etc.

[0154] The terms “treat” and “treatment” include therapeutic treatments, prophylactic treatments, and uses that reduce the risk that a subject will develop a disorder or other risk factor. Treatment does not require complete cure of the disorder and includes embodiments in which symptoms or underlying risk factors are reduced. The term “warn” does not require 100% exclusion of the probability of an event. Rather, it means that the likelihood of the event occurring has been reduced by the presence of the compound or by the use of the method.

[0155] The required therapeutic amounts of cells in the composition are typically at least 2 cells (e.g., a subset of at least 1 CD8+ central memory T cell and at least 1 CD4+ helper T cell), or more often more than 10 ² cells and not more than 10 ⁶ , not more than 10 ⁸ or 10 ⁹ cells inclusive and may be more than 1010 cells. The number of cells will depend on the desired route of application for which the composition is intended and the type of cells included therein. The required cell density typically exceeds 10 ⁶ cells/ml and is typically greater than 10 ⁷ cells/ml, typically 10 8 cells/ml or greater. A clinically significant number of immune cells can be divided into multiple infusions that collectively equal or exceed 10 ⁵ , 10 ⁶ , 10 ⁷ , 10 ⁸ , 10 ⁹ , 10 ¹⁰ , 10 ¹¹ or 10 ¹² cells. In some aspects of the present invention, particularly since all cells to be infused will be redirected to a specific target antigen (CD19), smaller numbers of cells in the range of 10 ⁶ /kg (10 ⁶ -10 ¹¹ per patient) can be administered. CAR-based treatments can be administered multiple times at dosages within these ranges. The cells may be autologous, allogeneic, or heterologous to the patient undergoing therapy.

[0156] In some embodiments, the therapeutically effective number of CAR T cells is about 1 x 10 ⁵ cells/kg, about 2 x 10 ⁵ cells/kg, about 3 x 10 ⁵ cells/kg, about 4 x 10 ⁵ cells/ kg, approximately 5×10 ⁵ cells/kg, approximately 6×10 ⁵ cells/kg, approximately 7×10 ⁵ cells/kg, approximately 8×10 ⁵ cells/kg, approximately 9×10 ⁵ cells/kg, 2×10 ⁶ cells/kg, approximately 3×10 ⁶ cells/kg, approximately 4×10 ⁶ cells/kg, approximately 5×10 ⁶ cells/kg, approximately 6×10 ⁶ cells/kg, approximately 7×10 ⁶ cells/kg, approximately 8×10 ⁶ cells/kg, approximately 9×10 ⁶ cells/kg, approximately 1×10 ⁷ cells/kg, approximately 2×10 ⁷ cells/kg, approximately 3×10 ⁷ cells/kg, approximately 4×10 ⁷ cells/kg, approximately 5×10 ⁷ cells/kg, approximately 6×10 ⁷ cells/kg, approximately 7×10 ⁷ cells/kg, approximately 8×10 ⁷ cells/kg, or approximately 9×10 ⁷ cells/kg.

[0157] In some embodiments, target doses for CAR+/CAR-T+/TCR+ cells range from 1x10 ⁶ to 2x10 ⁸ cells/kg, for example, 2x10 ⁶ cells/kg. It should be understood that dosages above and below this range may be appropriate for certain subjects, and appropriate dosage levels may be determined by the healthcare provider, as appropriate. In addition, multiple doses of cells can be provided according to the present invention.

[0158] In some aspects, the present invention provides a pharmaceutical composition comprising at least one antigen binding domain described herein and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition further comprises an additional active agent.

[0159] The CAR-expressing cell populations of the present invention can be administered either alone or as a pharmaceutical composition in combination with diluents and/or other components, such as IL-2 or other cytokines or cell populations. The pharmaceutical compositions of the present invention may contain a population of CAR or TCR expressing cells, for example, T cells described herein, in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may contain buffers such as neutral buffered saline, phosphate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (eg aluminum hydroxide) and preservatives. The compositions of the present invention are preferably formulated for intravenous administration.

[0160] Pharmaceutical compositions (solutions, suspensions, etc.) may include one or more of the following: sterile diluents such as water for injection, saline, preferably saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono- or diglycerides, which can serve as a solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates; and tonicity agents such as sodium chloride or dextrose. The parenteral drug may be contained in ampoules, single-use syringes, or multiple-dose vials made of glass or plastic. The injectable pharmaceutical composition is preferably sterile.

[0161] In some embodiments, when administered to a patient, engineered immune cells expressing on their cell surface any of the CD19-specific CARs described herein can reduce, kill, or lyse the patient's endogenous CD19-expressing cells. In one embodiment, the percentage of reduction or lysis of CD19-expressing endogenous cells or cells from a CD19-expressing cell line by engineered immune cells expressing any of the CD19-specific CARs described herein is at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more. In one embodiment, the percentage of reduction or lysis of CD19-expressing endogenous cells or cells from a CD19-expressing cell line by engineered immune cells expressing a CD19-specific CAR is from about 5% to about 95%, from about 10% to about 95%, from about 10% to about 90%, from about 10% to about 80%, from about 10% to about 70%, from about 10% to about 60%, from about 10% to about 50%, from about 10% to about 40%, about 20% to about 90%, about 20% to about 80%, about 20% to about 70%, about 20% to about 60%, about 20% to about 50%, from about 25% to about 75%, or from about 25% to about 60%. In one embodiment, the endogenous CD19-expressing cells are endogenous CD19-expressing bone marrow cells.

[0162] In one embodiment, the percentage of reduction or lysis of target cells, for example, a CD19-expressing cell line, by engineered immune cells expressing on their cell surface membrane a CD19-specific CAR of the present invention can be measured using an assay disclosed in this document.

[0163] The methods may further include administering one or more chemotherapeutic agents. In certain embodiments, the chemotherapeutic agent is a lymphodepletion (preconditioning) chemotherapeutic agent. For example, methods of improving the physical condition of a patient in need of T-cell therapy include administering to the patient specific effective doses of cyclophosphamide (200 mg/m ² /day to 2000 mg/m ² /day, from about 100 mg/m ² /day to approximately 2000 mg/ ^m2 /day; for example, approximately 100 mg/ ^m2 /day, approximately 200 mg/ ^m2 /day, approximately 300 mg/ ^m2 /day, approximately 400 mg/ ^m2 /day, approximately 500 mg /m ² /day, approximately 600 mg/m ² /day, approximately 700 mg/m ² /day, approximately 800 mg/m ² /day, approximately 900 mg/m ² /day, approximately 1000 mg/m ² /day , approximately 1500 mg/m ² /day or approximately 2000 mg/m ² /day) and certain doses of fludarabine (from 20 mg/m ² /day to 900 mg/m ² /day, from approximately 10 mg/m ² /day up to about 900 mg/m ² /day; for example, about 10 mg/m ² /day, about 20 mg/m ² /day, about 30 mg/m ² /day, about 40 mg/m ² /day, about 40 mg/ ^m2 /day, approximately 50 mg/ ^m2 /day, approximately 60 mg/ ^m2 /day, approximately 70 mg/ ^m2 /day, approximately 80 mg/ ^m2 /day, approximately 90 mg/ ^m2 / day, approximately 100 mg/m ² /day, approximately 500 mg/m ² /day or approximately 900 mg/m ² /day). A preferred dosing regimen involves treating the patient by administering approximately 300 mg/m ² /day of cyclophosphamide and approximately 30 mg/m ² /day of fludarabine to the patient daily for three days before administering a therapeutically effective amount of the engineered T cells to the patient.

[0164] In some embodiments, lymphodepletion further involves administering an anti-CD52 antibody. In some embodiments, the anti-CD52 antibody is administered at a dose of approximately 13 mg/day IV.

[0165] In other embodiments, each of the antigen binding domain, transduced (or otherwise engineered) cells, and a chemotherapeutic agent is administered in an amount effective to treat a disease or condition in a subject.

[0166] In certain embodiments, compositions containing CAR-expressing immune effector cells disclosed herein may be administered in combination with any number of chemotherapeutic agents. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN™); alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa and uredopa; ethylenimines and methylmelamines, including altretamine, triethylene amine, triethylene phosphoramide, triethylene thiophosphoramide and trimethyl olomelamine resin; nitrogen mustards such as chlorambucil, chlornaphasine, chlorphosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembiquin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorosotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomycin, actinomycin, anthramycin, azaserin, bleomycin, cactinomycin, calichemycin, carabicin, carminomycin, carcinophilin, chromomycin, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-b-norleucine, doxorubicin, epirubicin, ezorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rhodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogues such as fludarabine, 6-mercaptopurine, thiamiprin, thioguanine; pyrimidine analogues such as ancesitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocytabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epithiostanol, mepithiostane, testolactone; drugs that inhibit the functions of the adrenal cortex, such as aminoglutethimide, mitotane, trilostane; a folate supplement such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatrexate; defofamine; demecolcine; diaziquon; elformitine; elliptinium acetate; ethoglucide; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamole; nitracrine; pentostatin; phenomet; pirarubicin; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; sisofiran; spirogermanium; tenuazonic acid; triaziquon; 2,2',2''-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustin; mitobronitol; mitolactol; pipobromance; gacytosine; arabinoside (“Ara-C”); cyclophosphamide; thiotepu; taxoids, for example, paclitaxel (TAXOL®, Bristol-Myers Squibb) and doxetaxel (TAXOTERE®, Rhne-Poulenc Rorer); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; Novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; SRT-11; topoisomerase inhibitor RF S2000; difluoromethylomithine (DMFO); retinoic acid derivatives such as Targretin™ (bexarotene), Panretin™ (aliretinoin); ONTAK™ (denileukin diftitox); esperamycins; capecitabine; as well as pharmaceutically acceptable salts, acids and derivatives of any of the foregoing. Also included in this definition are antihormonal agents that regulate or suppress the action of hormones on tumors, such as antiestrogens, including, for example, tamoxifen, raloxifene, 4(5)-imidazoles, aromatase inhibitors, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018 , onapristone and toremifene (Fareston); and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; as well as pharmaceutically acceptable salts, acids and derivatives of any of the foregoing. Also, if necessary, combinations of chemotherapeutic agents are administered, including without limitation CHOP, i.e. cyclophosphamide (Cytoxan®), doxorubicin (hydroxydoxorubicin), vincristine (Oncovin®), and prednisone.

[0167] In some embodiments, the chemotherapeutic agents are administered at the same time or one week after administration of the engineered cell, polypeptide, or nucleic acid. In other embodiments, the chemotherapy agent is administered over 1 to 4 weeks, or 1 week to 1 month, 1 week to 2 months, 1 week to 3 months, 1 week to 6 months, 1 week to 9 months, or 1 week to 12 months after administration of the engineered cell, polypeptide, or nucleic acid. In other embodiments, the chemotherapeutic agent is administered at least 1 month prior to administration of the cell, polypeptide, or nucleic acid. In some embodiments, the methods further include administering two or more chemotherapeutic agents.

[0168] A number of additional therapeutic agents can be used in combination with the compositions described herein. For example, potentially useful additional therapeutics include PD-1 inhibitors such as nivolumab (Opdivo®), pembrolizumab (Keytruda®), pembrolizumab, pidilizumab, and atezolizumab.

[0169] Additional therapeutic agents suitable for use in combination according to the present invention include, but are not limited to, ibrutinib (Imbruvica®), ofatumumab (Arzerra®, rituximab (Rituxan®), bevacizumab (Avastin®), trastuzumab (Herceptin®), trastuzumab emtansine (KADCYLA®, imatinib (Gleevec®), cetuximab (Erbitux®, panitumumab) (Vectibix®), catumaxomab, ibritumomab, ofatumumab, tositumomab, brentuximab, alemtuzumab, gemtuzumab, erlotinib, gefitinib, vandetanib, afatinib, lapatinib, neratinib, axitinib, mazitinib, pazopanib, sunitinib, sorafenib, toceranib, lestaurtinib, axitinib, cediranib, lenvatinib, nintedanib, pazopanib, regorafenib, semaxanib, sorafenib, sunitinib, tivozanib, toceranib, vandetanib, entrectinib, sabozantinib, and matinib, dasatinib, nilotinib, ponatinib, radotinib, bosutinib, lestaurtinib, ruxolitinib, pacritinib, cobimetinib, selumetinib, trametinib, binimetinib, alectinib, ceritinib, crizotinib, aflibercept, adipotide, denileukin diftitox, mTOR inhibitors such as everolimus and temsirolimus, hedgehog inhibitors such as sonidegib and vismodegib , CDK inhibitors, such as a CDK inhibitor (palbociclib).

[0170] In some embodiments, a composition comprising CAR-containing immune cells may be administered in a therapeutic regimen to prevent cytokine release syndrome (CRS) or neurotoxicity. Therapeutic regimen to prevent cytokine release syndrome (CRS) or neurotoxicity may include administration of lenzilumab, tocilizumab, atrial natriuretic peptide (ANP), anakinra, iNOS inhibitors (eg, L-NIL or 1400W). In additional embodiments, a composition comprising CAR-containing immune cells may be administered with an anti-inflammatory agent. Anti-inflammatory drugs or drugs include, but are not limited to, steroids and glucocorticoids (including betamethasone, budesonide, dexamethasone, hydrocortisone acetate, hydrocortisone, hydrocortisone, methylprednisolone, prednisolone, prednisone, triamcinolone), non-steroidal anti-inflammatory drugs (NSAIDs), including aspirin, ibuprofen, naproxen, methotrexate, sulfasalazine, leflunomide, anti-TNF drugs, cyclophosphamide and mycophenolate. Illustrative NSAIDs include ibuprofen, naproxen, naproxen sodium, Cox-2 inhibitors and sialylates. Exemplary analgesics include acetaminophen, oxycodone, tramadol, and propoxyphene hydrochloride. Exemplary glucocorticoids include cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, or prednisone. Exemplary biological response modifiers include molecules directed against cell surface markers (eg, CD4, CD5, etc.), cytokine inhibitors such as TNF antagonists (eg, etanercept (ENBREL®), adalimumab (HUMIRA®), and infliximab (REMICADE ®), chemokine inhibitors, and adhesion molecule inhibitors. Biological response modifiers include monoclonal antibodies as well as recombinant forms of the molecules. Illustrative DMARDs include azathioprine, cyclophosphamide, cyclosporine, methotrexate, penicillamine, leflunomide, sulfasalazine, hydroxychloroquine, Gold (oral (auranofine) and intramuscular ) and minocycline.

[0171] In certain embodiments, the compositions described herein are administered in combination with a cytokine. Examples of cytokines are lymphokines, monokines and traditional polypeptide hormones. Cytokines include growth hormones such as human growth hormone, N-methionyl human growth hormone, and bovine growth hormone; parathyroid hormone; thyroxine; insulin; proinsulin; relaxin; prorelaxin; glycoprotein hormones such as follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH) and luteinizing hormone (LH); liver growth factor (HGF); fibroblast growth factor (FGF); prolactin; placental lactogen; Mullerian inhibitory substance; mouse gonadotropin-associated peptide; inhibin; activin; vascular endothelial growth factors; integrin; thrombopoietin (TPO); nerve growth factors (NGFs), such as NGF-beta; platelet-derived growth factor; transforming growth factors (TGFs), such as TGF-alpha and TGF-beta; insulin-like growth factor I and II; erythropoietin (EPO); osteoinductive factors; interferons such as interferon-alpha, -beta and -gamma, colony-stimulating factors (CSF) such as macrophage CSF (M-CSF); granulocyte and macrophage CSF (GM-CSF) and granulocyte CSF (G-CSF); interleukins (IL) such as IL-1, 1b-1alpha, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10 , IL-11, IL-12, IL-15, IL-21; tumor necrosis factor such as TNF-alpha or TNF-beta; and other polypeptide factors, including LIF and KIT ligand (KL). As used herein, the term “cytokine” includes proteins from natural sources or from recombinant cell culture and the biologically active equivalents of native sequence cytokines.

V. Methods of sorting and depletion

[0172] In some embodiments, methods are provided for sorting an immune cell population in vitro, wherein a subset of the immune cell population comprises engineered immune cells expressing any of CD19-specific CARs containing monoclonal antibody-specific epitopes (e.g., illustrative mimotope sequences). The method involves contacting a population of immune cells with a capitope-specific monoclonal antibody and selecting immune cells that bind to the monoclonal antibody to produce a cell population enriched in engineered immune cells expressing a CD19-specific CAR.

[0173] In some embodiments, said monoclonal antibody specific for said epitope is optionally conjugated to a fluorophore. In this embodiment, the step of selecting cells that bind to the monoclonal antibody can be performed by fluorescence-activated cell sorting (FACS).

[0174] In some embodiments, said monoclonal antibody specific for said epitope is optionally conjugated to a magnetic particle. In this embodiment, the step of selecting cells that bind to the monoclonal antibody may be performed by magnetically activated cell sorting (MACS).

[0175] In some embodiments, the mAb used in the method of sorting immune cells expressing CAR is selected from alemtuzumab, ibritumomab tiuxetan, muromonab-POP, tositumomab, abciximab, basiliximab, brentuximab vedotin, cetuximab, infliximab, rituximab, bevacizumab, certolizumab pegola, daclizumab, eculizumab, efalizumab, gemtuzumab, natalizumab, omalizumab, palivizumab, ranibizumab, tocilizumab, trastuzumab, vedolizumab, adalimumab, belimumab, canakinumab, denosumab, golimumab, ipilimumab, ofatumumab, panitumumab, QBEND-10 and /or ustekinumab. In some embodiments, said mAb is rituximab. In another embodiment, said mAb is QBEND-10.

[0176] In some embodiments, the population of CAR-expressing immune cells obtained using the in vitro sorting method for CAR-expressing immune cells described above contains at least 70%, 75%, 80%, 85%, 90%, 95% expressing CAR immune cells. In some embodiments, the population of CAR-expressing immune cells obtained using an in vitro sorting method for CAR-expressing immune cells contains at least 85% CAR-expressing immune cells.

[0177] In some embodiments, a population of CAR-expressing immune cells obtained using the in vitro sorting method for CAR-expressing immune cells described above exhibits increased in vitro cytotoxic activity compared to the original (unsorted) cell population. In some embodiments, said in vitro cytotoxic activity is increased by 10%, 20%, 30%, or 50%. In some embodiments, the immune cells are T cells.

[0178] In some embodiments, the mAb is pre-bound to a support or surface. Non-limiting examples of a solid support may include a bead, an agarose bead, a magnetic bead, a plastic well plate, a glass well plate, a ceramic well plate, a column, or a cell culture bag.

[0179] CAR-expressing immune cells to be administered to a recipient can be enriched in vitro from the original population. Methods for propagating source populations may involve selecting cells that express an antigen, such as CD34 antigen, using combinations of density gradient centrifugation, immunomagnetic bead purification, affinity chromatography, and fluorescence-activated cell sorting.

[0180] Flow cytometry can be used to quantify specific cell types within a population of cells. In general, flow cytometry is a method of quantifying components or structural features of cells, primarily through optical means. Because different cell types can be distinguished by quantifying structural features, flow cytometry and cell sorting can be used to count and sort cells of different phenotypes in a mixture.

[0181] Flow cytometry analysis involves two main steps: 1) labeling selected cell types with one or more labeled markers and T) determining the number of labeled cells relative to the total number of cells in the population. In some embodiments, the method of labeling cell types involves linking the labeled antibodies to markers expressed by a particular cell type. Antibodies can either be directly labeled with a fluorescent compound or indirectly labeled using, for example, a fluorescently labeled second antibody that recognizes the first antibody.

[0182] In some embodiments, the method used to sort CAR-expressing T cells is magnetic activated cell sorting (MACS). Magnetic activated cell sorting (MACS) is a method of separating different populations of cells based on their surface antigens (CD molecules) using superparamagnetic nanoparticles and columns. MACS can be used to obtain a pure population of cells. Cells in a single-cell suspension can be magnetically labeled using microbeads. The sample is applied to a column consisting of ferromagnetic spheres coated with a cell-friendly coating, allowing for rapid and gentle cell separation. Unlabeled cells pass through, but magnetically labeled cells remain inside the column. The flow through can be collected as a fraction of unlabeled cells. After the washing step, the column is removed from the separator and the magnetically labeled cells are eluted from the column.

[0183] A detailed protocol for purifying a specific cell population, such as T cells, can be found in Basil S et al. (2010). (Basu S, Campbell HM, Dittel BN, Ray A. Purification of specific cell populations by fluorescence activated cell sorting (FACS). J Vis Exp. (41): 1546).

[0184] In some aspects, the present invention provides a method of depleting CD19-specific CAR-expressing immune cells by in vivo depletion. In vivo depletion may involve administering a treatment (eg, a molecule that binds an epitope on a CAR) to a mammal to stop the proliferation of CAR-expressing immune cells by inhibition or elimination.

[0185] In one aspect, the present invention provides a method for in vivo depletion of an engineered immune cell expressing a CD19 CAR containing a mAb-specific epitope, comprising contacting said engineered immune cell or said CAR-expressing immune cell with at least one epitope-specific mAb. In another aspect, the present invention provides a method for in vivo depletion of a CAR-expressing immune cell that contains a chimeric scFv (eg, formed by insertion of an mAb-specific epitope) by contacting said engineered immune cell with epitope-specific antibodies. In some embodiments, the immune cells are T cells and/or the antibodies are monoclonal.

[0186] In one embodiment, in vivo depletion of engineered immune cells is performed on engineered immune cells that have been presorted using the in vitro method of the present invention. In this case, the same infused mAb can be used. In some embodiments, the mAb-specific antigen is CD20 antigen and the mAb-specific antigen is rituximab. In some embodiments, the present invention provides a method for in vivo depletion of an engineered immune cell expressing a CAR containing a mAb-specific epitope (CAR-expressing immune cell) from a patient, comprising contacting said CAR-expressing immune cell with at least one epitope-specific mAb.

[0187] In some embodiments, the step of contacting said engineered immune cell or said CAR-expressing immune cell with at least one epitope-specific mAb involves infusing the patient with an epitope-specific mAb (eg, rituximab). In some embodiments, the amount of the epitope-specific mAb administered to the patient is sufficient to kill at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the CAR-expressing immune cells in the patient .

[0188] In some embodiments, the step of contacting said engineered immune cell or said CAR-expressing immune cell with at least one epitope-specific mAb involves infusing the patient with 375 mg/m ² rituximab one or more times. In some embodiments, the mAb (eg, rituximab) is administered once per week.

[0189] In some embodiments, by depleting immune cells expressing a CAR containing an mAb-specific epitope (CAR-expressing immune cells) in a complement dependent cytotoxicity (CDC) assay using the epitope-specific mAb, the number of viable CAR-expressing immune cells is reduced . In some embodiments, the number of viable CAR-expressing immune cells is reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%. In some embodiments, the mAb-specific epitope is a CD20 epitope or mimotope, and/or the epitope-specific mAb is rituximab.

[0190] In certain embodiments, in vivo depletion of CAR-engineered immune cells is accomplished through infusions of bispecific antibodies. By definition, a bispecific monoclonal antibody (BsAb) is an artificial protein that is composed of fragments of two different monoclonal antibodies and therefore binds to two different types of antigens. These BsAbs and their use in immunotherapy have been reviewed in Muller D and Kontermann R.E. (2010) Bispecific Antibodies for Cancer Immunotherapy, BioDrugs 24 (2): 89-98.

[0191] In another specific embodiment, the infused bispecific mAb is capable of binding both an mAb-specific epitope carried on engineered immune cells expressing the chimeric scFv and a surface antigen on an effector and cytotoxic cell (e.g., immune cells such such as lymphocytes, macrophages, dendritic cells, natural killer cells (NK cells), cytotoxic T lymphocytes (CTL)). Thus, BsAb-induced depletion of engineered immune cells may occur due to antibody-dependent cellular cytotoxicity (ADCC). (Deo Y M, Sundarapandiyan K, Keler T, Wallace PK, and Graziano RF, (2000), Journal of Immunology, 165 (10):5954-5961).

[0192] In some embodiments, the cytotoxic drug is coupled to an epitope-specific mAb that can be used to deplete CAR-expressing immune cells. By combining the targeting abilities of monoclonal antibodies with the ability of cytotoxic drugs to kill cancer cells, an antibody-drug conjugate (ADC) can clearly distinguish between healthy and diseased tissue compared to using the drug alone. Marketing approvals have been received for several ADCs; the technology for their preparation, especially on linkers, is described in (Payne, G. (2003) Cancer Cell 3:207-212; Trail et al (2003) Cancer Immunol. Immunother. 52:328-337; Syrigos and Epenetos (1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drug Del. Rev. 26:151-172; US Patent No. 4975278).

[0193] In some embodiments, the epitope-specific mAb to be infused is preconjugated to a molecule capable of promoting complement-dependent cytotoxicity (CDC). Therefore, the complement system enhances or complements the ability of antibodies to clear pathogens from the body. Upon stimulation, an activation cascade is triggered as a massive increase in response and activation of the membrane attack complex, which destroys cells. Various molecules, such as glycans, can be used for conjugation with mAbs (Courtois, A., Gac-Breton, S., Berthou, C., Guezermec, J., Bordron, A. and Boisset, C. (2012), Complement dependent cytotoxicity activity of therapeutic antibody fragments may be acquired by immunogenic glycan coupling, Electronic Journal of Biotechnology ISSN: 0717-3458; http://www.ejbiotechnology.info DOI: 10.2225/voll5-issue5).

VI. Sets and products

[0194] The present application provides kits comprising any of the CD19-containing CARs or CD19 CARs containing immune cells described herein and pharmaceutical compositions thereof. In some illustrative embodiments, the kit of the present invention contains allogeneic T cells containing a CD19 CAR and a CD52 antibody for administration to a subject in accordance with a lymphodepletion and CAR-T regimen.

[0195] This application also provides for articles containing any of the therapeutic compositions or kits described herein. Examples of articles include vials (eg, stoppered vials).

EXAMPLES

Example 1: Generation of Rituximab-Resistant Immune Cells with CAR for CD19

[0196] Rituximab-resistant chimeric antigen receptor CD19 constructs were created that do not express the rituximab binding site as shown in FIG. 1 and Table 4. Lentiviral vector constructs were introduced into a viral packaging cell line and lentiviruses containing the CD19 CAR were produced in Allogene.

[0197] Pan T cells from four human donors (541, 604, 410 and 2593) were thawed and activated at a concentration of 1.5 x 10 ⁶ cells/ml using large-scale T Cell TransAct™ (ratio 1:15) at presence of IL-2 (100 IU/ml). After 2 days, 1.5×10 ⁶ cells (in 3 ml) were transduced with 2 ml of freshly obtained lentivirus containing the vectors described in Table 4. Schematic representations of the modified vectors are presented in Figure 1. IL-2 (100 IU/ml) was added on days 0, 2, 5, 7, 9 and 12. A total of 6 x 10 ⁶ cells were transferred to a 6-well G-Rex plate on day 5 and media exchanged on days 9 and 12. Cells were frozen on day 13.

[0198] Flow cytometry experiments were performed on transduced cells, displaying data for lymphocytes, live CD3+, CAR+, CD4/CD8, and downstream markers. Human cell transduction validation and analysis using the CD34 panel was performed on day 5 and day 13 using the CD3, CD4, CD8, viability, CD34 and anti-CAR antibody panel for CD19 (anti-Id 4G7). In FIG. 2A and 2B are flow cytometry plots showing CAR expression at day 5 obtained from pan-T cells transduced with the CAR expression vectors shown in Table 4 using anti-CAR antibody anti-Id for CD19.

[0199] Flow cytometry using human phenotype and activation panel was performed on days 9 and 13. The panel included CD3, CD4, CD8, viability, CD45RO, CD62L, CD25, 4-1 BB, PD-1, anti-CAR antibody anti-idiotype for CD19 and TIM3. Cells were normalized for cell expansion and final CAR expression for all four donors at day 13 (FIG. 3). Data in FIG. 3 show that although v1.2 showed a higher degree of transduction (%CAR+), cells transduced with v1.2 showed lower levels of CAR expression (CAR MFI) compared to, for example, v1.0 and vl.l. Cell expansion and CAR expression for pan T cells from donor 541 (FIG. 4A), 604 (FIG. 4B), 410 (FIG. 4C), 2593 (FIG. 4D) transduced with rituximab-resistant CAR expression vectors, monitored over time.

[0200] CD4/CD8 ratio was measured on days 5, 9 and 13 for pan-T cells from donor 541 (FIG. 5A), 604 (FIG. 5B), 410 (FIG. 5C), 2593 (FIG. 5D) , transduced with rituximab-resistant CAR expression vectors. In FIG. 6A-6D show the phenotype and day 9 activation of pan-T cells from donors 541 (FIG. 6A), 604 (FIG. 6B), 410 (FIG. 6C), 2593 (FIG. 6D) transduced with rituximab-resistant expression CAR vectors. In FIG. Figure 7 shows phenotype, % CD8+ activation, and T cell anergy measured using the average of TIM3 and PD1 staining for all four donors on day 9. Phenotype and activation were measured on day 13 for pan T cells from donor 541 (FIG. 8A ), 604 (FIG. 8B), 410 (FIG. 8C), 2593 (FIG. 8D), transduced with rituximab-resistant CAR expression vectors. In FIG. Figure 9 shows the phenotype, % CD8+ activation, and T cell anergy measured using TIM3 and PD1 staining values for all four donors at day 13.

Example 2 Short- and Long-Term In Vitro Cell Death Assays

[0201] The transduced CAR cells from Example 1 were tested to determine short- and long-term cell death characteristics. Co-cultures of CAR T cells with Raji cells (2:1 effector-to-target ratio) were prepared for subsequent Luminex analysis. Mean values of short-term (24 h) cell death assays using Raji cells as target cells were determined for each CAR construct (FIG. 10). In FIG. 11A-11D show average long-term cell death assay values using A549-CD19+ cells as target cells with E:T of 8:1 (FIG. 11A), 4:1 (FIG. 11B), 2:1 (FIG. 11C) and 1:1 (FIG. 11D) for each CAR design.

[0202] The results of the cell death assay were analyzed to determine correlation with phenotypic characteristics. The percentage of cell death at day 7 at a 1:1 effector-to-target ratio was negatively correlated with CAR+CD4+41BB+, CAR+CD4+Tim3+ (p=0.0352), CAR+CD4+T _EM + (p =0.0328), CAR+CD8+PD-1+ (p=0.0269) and % CAR expression on day 13 (p=0.0245). The percentage of cell death at day 7 at an effector-to-target ratio of 1:1 was positively correlated with CAR+CD8+T _SCM +. The percentage of cell death at day 9 at a 1:1 effector-to-target ratio was negatively correlated with CAR+CD4+Tim3+, CAR+CD4+T _EM + (p=0.0031), CAR+CD8+T _cm + (p=0.0182) and % CAR expression on day 13 (p=0.0469). The percentage of cell death at day 9 at a 1:1 effector-to-target ratio was positively correlated with CAR+CD8+T _SCM +, CAR+CD8+Tim3-PD-1- (p=0.0318) and CAR+ CD4+T _SCM + (p=0.0289).

Example 3. Analysis of lentivirus titers containing different lentiviral constructs

[0203] In this experiment, lentiviral vector constructs were introduced into a cell line packaging viruses, and CD19 CARs containing lentiviruses were prepared and titers determined using Lentigen (Gaithersburg, MD, USA) according to a protocol similar to that of Example 1.

[0204] Lentivirus titers were assessed either by measuring physical titers of viral p24 protein levels or by measuring transducing titers. Surprisingly, it was found that if the RQR8 safety switch was removed from the v1.0 lentiviral construct, the viral titer was significantly reduced (comparing v1.0 with v1.1b Table 5). The titer was improved when the EF1aB v1.1 promoter was replaced with a short or truncated EF1a promoter as in v1.2 (“EE1a(short)promoter”).

[0205] To analyze the persistence of CAR v1.0 lentiviral preparations for CD19, v1.2 and vl.3, a virus titration assay was performed. Volumetric serial dilutions of lentiviral preparations v1.0, v1.2, and v1.3 were analyzed to determine % CAR+ T cells at day 5 after pan-T cell transduction. The results showed that at low dilution (eg, 10% v/v), all three constructs showed similar acceptable transduction efficiencies. However, at increasing dilutions (eg, 3.3%, 1.1% v/v), the transduction efficiency of the rituximab-resistant CAR v1.3 construct for CD19 dropped significantly compared to the other rituximab-resistant CAR v1.2 construct. for CD19. See FIG. 12. Design v1.2 was selected for in vivo analysis.

Example 4: In Vivo Assay for Biological Activity Quantification

[0206] In this in vivo experiment, the antitumor biological activity of ALLO-501v1.2 was analyzed in comparison with ALLO-501v1.0 in a mouse tumor model. CD19-positive Raji cells, which carry a luciferase reporter gene, were injected into NSG mice. Lentivirus containing lentiviral construct v1.0 or v1.2 was transduced into pan T cells from donors 541 and 604. 100,000 Raji cells with luciferase were administered to NSG mice via tail vein injection. On day 4 post-administration, Raji cell-bearing NSG mice were administered the CAR construct at the indicated doses. Raji cell engraftment and progression were assessed by intraperitoneal administration of luciferase substrate followed by measurement of cumulative luciferase signals. The results are shown in FIG. 13A (donor 541) and FIG. 13V (donor 604).

[0207] Although the disclosed teachings have been described with reference to various uses, methods, kits, and compositions, it should be understood that various changes and modifications may be made without departing from the teachings of this document and the claims set forth below. The above examples are provided to better illustrate the ideas disclosed and are not intended to limit the scope of the ideas presented herein. Although the present teachings have been described in the definitions of these illustrative embodiments, one skilled in the art will readily appreciate that numerous variations and modifications of these illustrative embodiments are possible without undue experimentation. All such variations and modifications are within the framework of the current ideas.

--->

LIST OF SEQUENCES

<110> ALLOGEN THERAPEUTICS, INC.

<120> RITUXIMAB-RESISTANT CHIMERIC ANTIGENE RECEPTORS AND PATHWAYS

THEIR APPLICATIONS

<130>AT-028/03WO

<140>

<141>

<150> 63/005,041

<151> 2020-04-03

<150> 62/839,455

<151> 2019-04-26

<160> 41

<170> PatentIn version 3.5

<210> 1

<211> 2019

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<220>

<221> source

<223> /note="chimeric antigen receptor"

<400> 1

atgctgacca gcctgctgtg ctggatggcc ctgtgcctgc tgggcgccga ccacgccgat

60

gcctgcccct acagcaaccc cagcctgtgc agcggaggcg gcggcagcga gctgcccacc

120

cagggcacct tctccaacgt gtccaccaac gtgagcccag ccaagcccac caccaccgcc

180

tgtccttatt ccaatccttc cctgtgtagc ggagggggag gcagcccagc ccccagacct

240

cccaccccag cccccaccat cgccagccag cctctgagcc tgagacccga ggcctgccgc

300

ccagccgccg gcggcgccgt gcacaccaga ggcctggatt tcgcctgcga tatctacatc

360

tgggccccac tggccggcac ctgtggcgtg ctgctgctga gcctggtgat caccctgtac

420

tgcaaccacc gcaaccgcag gcgcgtgtgc aagtgcccca ggcccgtggt gagagccgag

480

ggcagaggca gcctgctgac ctgcggcgac gtggaggaga acccaggccc catggagacc

540

gacaccctgc tgctgtgggt gctgctgctg tgggtgccag gcagcaccgg cgaggtgcag

600

ctgcagcaga gcggacccga gctgatcaag ccaggcgcca gcgtgaagat gagctgcaag

660

gccagcggct acaccttcac cagctacgtg atgcactggg tgaagcagaa gccaggccag

720

ggcctggagt ggatcggcta catcaacccc tacaacgacg gcaccaagta caacgagaag

780

ttcaagggca aggccaccct gaccagcgac aagagcagca gcaccgccta catggagctg

840

agcagcctga ccagcgagga cagcgccgtg tactactgcg ccagaggcac ctactactac

900

ggcagccggg tgttcgacta ctggggccag ggcaccaccc tgaccgtgag ctctggcgga

960

ggcggctctg gcggaggcgg ctctggcgga ggcggcagcg acatcgtgat gacccaggct

1020

gcccccagca tccccgtgac cccaggcgag agcgtgagca tcagctgccg gagcagcaag

1080

agcctgctga acagcaacgg caacacctac ctgtactggt tcctgcagcg gccaggccag

1140

agcccccagc tgctgatcta ccggatgagc aacctggcca gcggcgtgcc cgaccggttc

1200

agcggcagcg gcagcggcac cgccttcacc ctgcggatca gccgggtgga ggccgaggac

1260

gtgggcgtgt actactgcat gcagcacctg gagtacccct tcaccttcgg agccggcacc

1320

aagctggagc tgaagcggtc ggatcccacc accaccccag cccacggcc acctacccct

1380

gccccaacca tcgccagcca gcccctgagc ctgcggcctg aagcctgcag gcctgccgcc

1440

ggagggagccg tgcacacaag gggcctggac ttcgcctgcg acatctatat ctgggccccc

1500

ctggccggga catgcggggt gctgctgctg tccctggtga ttacactgta ttgcaaacgg

1560

ggccggaaga agctgctgta catcttcaag cagcccttca tgcggcccgt gcagaccacc

1620

caggaggagg acggctgcag ctgccggttc cccgaggaag aggaaggcgg ctgcgagctg

1680

cgggtgaagt tcagccggag cgccgacgcc ccagcctacc agcagggcca gaaccagctg

1740

tacaacgagc tgaacctggg acggcgggag gagtacgacg tgctggacaa gcggcgggga

1800

cgggaccccg agatgggcgg caagcctcgc cggaagaatc cccaggaggg cctgtacaac

1860

gagctgcaga aggacaagat ggccgaggcc tacagcgaga tcggcatgaa gggcgagcgg

1920

cgccggggca agggccacga cggcctgtac cagggcctga gcaccgccac caaggacacc

1980

tacgacgccc tgcacatgca ggccctgcca ccccggtga

2019

<210> 2

<211> 1488

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<220>

<221> source

<223> /note="chimeric antigen receptor"

<400> 2

atggagaccg acaccctgct gctgtgggtg ctgctgctgt gggtgccagg cagcaccggc

60

gaggtgcagc tgcagcagag cggacccgag ctgatcaagc caggcgccag cgtgaagatg

120

agctgcaagg ccagcggcta caccttcacc agctacgtga tgcactgggt gaagcagaag

180

ccaggccagg gcctggagtg gatcggctac atcaacccct acaacgacgg caccaagtac

240

aacgagaagt tcaagggcaa ggccaccctg accagcgaca agagcagcag caccgcctac

300

atggagctga gcagcctgac cagcgaggac agcgccgtgt actactgcgc cagaggcacc

360

tactactacg gcagccgggt gttcgactac tggggccagg gcaccaccct gaccgtgagc

420

tctggcggag gcggctctgg cggaggcggc tctggcggag gcggcagcga catcgtgatg

480

acccaggctg cccccagcat ccccgtgacc ccaggcgaga gcgtgagcat cagctgccgg

540

agcagcaaga gcctgctgaa cagcaacggc aacacctacc tgtactggtt cctgcagcgg

600

ccaggccaga gcccccagct gctgatctac cggatgagca acctggccag cggcgtgccc

660

gaccggttca gcggcagcgg cagcggcacc gccttcaccc tgcggatcag ccgggtggag

720

gccgaggacg tgggcgtgta ctactgcatg cagcacctgg agtacccctt caccttcgga

780

gccggcacca agctggagct gaagcggtcg gatcccacca ccaccccagc cccacggcca

840

cctacccctg ccccaaccat cgccagccag cccctgagcc tgcggcctga agcctgcagg

900

cctgccgccg gaggagccgt gcacacaagg ggcctggact tcgcctgcga catctatatc

960

tgggcccccc tggccgggac atgcggggtg ctgctgctgt ccctggtgat tacactgtat

1020

tgcaaacggg gccggaagaa gctgctgtac atcttcaagc agcccttcat gcggcccgtg

1080

cagaccaccc aggaggagga cggctgcagc tgccggttcc ccgaggaaga ggaaggcggc

1140

tgcgagctgc gggtgaagtt cagccggagc gccgacgccc cagcctacca gcagggccag

1200

aaccagctgt acaacgagct gaacctggga cggcgggagg agtacgacgt gctggacaag

1260

cggcggggac gggaccccga gatgggcggc aagcctcgcc ggaagaatcc ccaggagggc

1320

ctgtacaacg agctgcagaa ggacaagatg gccgaggcct acagcgagat cggcatgaag

1380

ggcgagcggc gccggggcaa gggccacgac ggcctgtacc agggcctgag caccgccacc

1440

aaggacacct acgacgccct gcacatgcag gccctgccac cccggtga

1488

<210> 3

<211> 1488

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<220>

<221> source

<223> /note="chimeric antigen receptor"

<400> 3

atggagacag ataccctgct gctgtgggtg ctgctgctgt gggtgcctgg ctccacagga

60

gaggtgcagc tgcagcagtc tggaccagag ctgatcaagc ctggagcatc cgtgaagatg

120

tcttgcaagg ccagcggcta tacattcacc agctacgtga tgcactgggt gaagcagaag

180

cctggccagg gcctggagtg gatcggctat atcaatccat acaacgacgg caccaagtat

240

aatgagaagt ttaagggcaa ggccacactg acctctgata agagctcctc tacagcctac

300

atggagctga gctccctgac ctctgaggac agcgccgtgt actattgcgc cagaggcaca

360

tactattacg gcagcagggt gttcgattac tggggccagg gcaccacact gaccgtgtct

420

agcggaggag gaggctccgg aggaggaggc tctggcggcg gcggcagcga catcgtgatg

480

acacaggcag caccaagcat cccagtgacc cctggcgaga gcgtgtccat ctcttgtcgg

540

tcctctaagt ccctgctgaa ctctaatggc aacacctatc tgtactggtt tctgcagcgg

600

cccggacagt ccccacagct gctgatctat aggatgagca acctggcatc cggagtgcct

660

gatcgcttca gcggctccgg ctctggaaca gcctttaccc tgaggatctc tcgggtggag

720

gcagaggacg tgggcgtgta ttactgcatg cagcacctgg agtacccctt cacatttggc

780

gcaggaacca agctggagct gaagcggagc gaccccacca caacccctgc accacggccc

840

cctacaccag cacctaccat cgcatctcag ccactgagcc tgcggcccga ggcctgtagg

900

cctgcagcag gagggagcagt gcacaccagg ggcctggact tcgcctgcga tatctatatc

960

tgggcaccac tggcaggaac atgtggcgtg ctgctgctga gcctggtcat caccctgtat

1020

tgcaagagag gcaggaagaa gctgctgtac atcttcaagc agccttttat gcggccagtg

1080

cagacaaccc aggaggagga tggctgctcc tgtagattcc cagaggagga ggagggagga

1140

tgtgagctgc gcgtgaagtt tagccggtcc gccgacgcac cagcatatca gcagggccag

1200

aatcagctgt acaatgagct gaacctgggc cggagagagg agtacgacgt gctggataag

1260

aggaggggaa gggaccccga gatgggaggc aagccacgga gaaagaatcc ccaggagggc

1320

ctgtataacg agctgcagaa ggataagatg gccgaggcct acagcgagat cggcatgaag

1380

ggagagaggc gccggggcaa gggacacgac ggcctgtatc agggcctgtc cacagccacc

1440

aaggacacct acgatgccct gcacatgcag gccctgccac caaggtga

1488

<210> 4

<211> 1875

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<220>

<221> source

<223> /note="chimeric antigen receptor"

<400> 4

atgggaacaa gcctgctgtg ctggatggct ctgtgcctgc tgggggccga ccacgctgac

60

gcctccgggg gggggggctc tcctgcccct aggcccccta cacctgcacc aaccatcgca

120

tcccagccac tgtctctgcg ccctgaggcc tgccggccag cagcaggagg agcagtgcac

180

acccgcggcc tggacttcgc ctgcgatatc tatatctggg caccactggc aggcacatgt

240

ggcgtgctgc tgctgagcct ggtcatcacc ctgtactgca atcacaggaa ccggagaagg

300

gtgtgcaagt gtccccggcc tgtggtgaga gcagagggaa ggggcagcct gctgacatgt

360

ggcgacgtgg aggagaatcc aggccctatg gagacagata ccctgctgct gtgggtgctg

420

ctgctgtggg tgcccggcag caccggagag gtgcagctgc agcagtccgg accagagctg

480

atcaagcctg gagccagcgt gaagatgtcc tgtaaggcct ctggctatac attcaccagc

540

tacgtgatgc actgggtgaa gcagaagcct ggccagggcc tggagtggat cggctatatc

600

aatccataca acgacggcac aaagtataac gagaagttta agggcaaggc cacactgacc

660

tccgataaga gctcctctac agcctacatg gagctgagct ccctgacctc tgaggacagc

720

gccgtgtact attgcgccag aggcacatac tattacggct ctagggtgtt cgattactgg

780

ggccagggca ccacactgac cgtgtctagc ggaggaggag gcagcggagg aggaggctcc

840

ggcggcggcg gctctgacat cgtgatgaca caggcagcac catccatccc agtgacccct

900

ggcgagagcg tgtccatctc ttgtcggtcc tctaagagcc tgctgaactc caatggcaac

960

acctatctgt actggtttct gcagcggccc ggacagagcc cacagctgct gatctatagg

1020

atgtctaatc tggcaagcgg cgtgcccgat cgcttcagcg gctccggctc tggcacagcc

1080

tttaccctga ggatctcccg cgtggaggca gagacgtgg gcgtgtatta ctgcatgcag

1140

cacctggagt accccttcac atttggcgca ggcaccaagc tggagctgaa gcggagcgac

1200

cccaccacaa cccctgcacc acggccaccc acaccagcac ctactattgc atcccagcca

1260

ctgagcctgc ggcccgaggc ctgtaggcct gccgccggcg gcgcagtgca cacccggggc

1320

ctggactttg cctgcgatat ctacatctgg gcacctctgg ccggcacatg cggcgtgctg

1380

ttactgagcc tggtcatcac cctgtattgc aagcggggca gaaagaagct gctgtacatc

1440

ttcaagcagc cttttatgcg gccagtgcag acaacccagg aggaggatgg ctgctcctgt

1500

agattcccag aggagggagga gggaggatgt gagctgcgcg tgaagtttag ccggtccgcc

1560

gacgcaccag catatcagca gggccagaac cagctgtaca atgagctgaa cctgggccgg

1620

agagaggagt atgacgtgct ggataagaga cggggccggg accccgagat gggaggcaag

1680

ccacgccgga agaatcccca ggagggcctg tataacgagc tgcagaagga taagatggcc

1740

gaggcctaca gcgagatcgg catgaaggga gagagaaggc gcggcaaggg acacgacggc

1800

ctgtaccagg gcctgagcac agcaacaaaa gacacctacg acgcactgca catgcaggct

1860

ctgccccctc ggtaa

1875

<210> 5

<211> 1941

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<220>

<221> source

<223> /note="chimeric antigen receptor"

<400> 5

atgggaacct ctctgctgtg ctggatggct ctgtgcctgc tggggggccga tcacgctgac

60

gcaagtggcg ggggggggtc cgaactgccc acacagggca ccttctccaa cgtgagcacc

120

aacgtgagct ccggcggagg aggcagccct gcaccaaggc cccctacacc agcacctacc

180

atcgcatctc agccactgag cctgcgcccc gaggcctgcc ggcctgcagc aggcggcgcc

240

gtgcacaccc gcggcctgga ctttgcctgc gatatctata tctgggcacc tctggcaggc

300

acatgtggcg tgctgctgct gagcctggtc atcaccctgt actgcaatca caggaaccgg

360

agaagggtgt gcaagtgtcc acggcccgtg gtgagagcag agggaagggg ctccctgctg

420

acatgtggcg acgtggagga gaatcctggc ccaatggaga cagataccct gctgctgtgg

480

gtgctgctgc tgtgggtgcc cggctccacc ggagaggtgc agctgcagca gtctggacca

540

gagctgatca agccaggagc atccgtgaag atgtcttgta aggccagcgg ctatacattc

600

accagctacg tgatgcactg ggtgaagcag aagccaggac agggcctgga gtggatcggc

660

tatatcaatc cttacaacga cggcaccaag tataacgaga agtttaaggg caaggccaca

720

ctgacctctg ataagtctag ctccacagcc tacatggagc tgtctagcct gaccagcgag

780

gactccgccg tgtactattg cgccagaggc acatactatt acggcagcag ggtgttcgat

840

tactggggcc agggcaccac actgaccgtg tcctctggag gaggaggctc cggagggagga

900

ggctctggcg gcggcggcag cgacatcgtg atgacacagg cagcaccttc catcccagtg

960

accccaggcg agtctgtgag catctcctgt cggagctcca agtccctgct gaactctaat

1020

ggcaacacct atctgtactg gtttctgcag cggcccggac agtccccaca gctgctgatc

1080

tataggatga gcaatctggc ctccggcgtg ccagatcgct tctctggcag cggctccggc

1140

acagccttta ccctgaggat ctctcgcgtg gaggcagagg acgtgggcgt gtattactgc

1200

atgcagcacc tggagtaccc attcacattt ggcgcaggca ccaagctgga gctgaagcgg

1260

agcgacccca ccacaacccc agcacctcgg ccacccacac cagcacccac catcgcatct

1320

cagcctctga gcctgcggcc cgaggcctgt aggcccgcag caggagggagc agtgcacacc

1380

cggggcctgg acttcgcctg cgatatctac atctgggcac cactggccgg cacatgcggc

1440

gtgctgttac tgagcctggt catcaccctg tattgcaagc ggggcagaaa gaagctgctg

1500

tacatcttca agcagccctt tatgcggcct gtgcagacaa cccaggagga ggatggctgc

1560

tcctgtagat tccctgagga ggaggaggga ggatgtgagc tgcgcgtgaa gttttctcgg

1620

agcgccgacg caccagcata tcagcaggga cagaaccagc tgtacaatga gctgaacctg

1680

ggccggagag aggagtatga cgtgctggat aagagacggg gccgggaccc cgagatggga

1740

ggcaagcctc gccggaagaa tccacaggag ggcctgtata acgagctgca gaaggataag

1800

atggccgagg cctacagcga gatcggcatg aagggagaga gaaggcgcgg caagggacac

1860

gacggcctgt accagggcct gagcacagca acaaaagaca cctacgacgc actgcacatg

1920

caggctctgc caccaagatg a

1941

<210> 6

<211> 2019

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<220>

<221> source

<223> /note="chimeric antigen receptor"

<400> 6

atggggacct cactgctgtg ctggatggct ctgtgcctgc tggggggccga ccacgctgac

60

gcctgctctg gggggggggg gggctcatgc tccggagggag gaggctctga gctgccaacc

120

cagggcacat tctccaacgt gagcaccaac gtgtctcctg ccaagccaac cacaaccgca

180

tgcagcggcg gaggaggagg cagctgttcc ggcggcggcg gcagccctgc cccaaggccc

240

cctaccccag cacctacaat cgcatctcag cctctgagcc tgcgcccaga ggcctgtcgg

300

cccgcagcag gaggagcagt gcacacccgc ggcctggact ttgcctgcga tatctatatc

360

tgggcaccac tggcaggcac ctgtggcgtg ctgctgctga gcctggtcat caccctgtac

420

tgcaatcaca ggaaccggag aagggtgtgc aagtgtccac ggcccgtggt gagagcagag

480

ggaaggggct ctctgctgac ctgtggcgac gtggaggaga atcctggccc tatggagaca

540

gatacactgc tgctgtgggt gctgctgctg tgggtgcccg gcagcacagg agaggtgcag

600

ctgcagcagt ccggacctga gctgatcaag ccaggcgcct ccgtgaagat gtcttgcaag

660

gccagcggct ataccttcac aagctacgtg atgcactggg tgaagcagaa gccaggccag

720

ggcctggagt ggatcggcta tatcaatccc tacaacgacg gcaccaagta taacgagaag

780

tttaagggca aggccaccct gacaagcgat aagagctcct ctaccgccta catggagctg

840

agctccctga caagcgagga ctccgccgtg tactattgcg ccagaggcac ctactattac

900

ggctccaggg tgttcgatta ctggggccag ggcacaaccc tgacagtgtc tagcggagga

960

ggaggcagcg gaggaggagg ctccggcggc ggcggctctg acatcgtgat gacccaggca

1020

gcaccatcca tccctgtgac accaggcgag tctgtgagca tctcctgtcg gtcctctaag

1080

tccctgctga actctaatgg caacacctat ctgtactggt ttctgcagcg gcccggacag

1140

tctcctcagc tgctgatcta taggatgagc aatctggcct ccggcgtgcc tgatcgcttc

1200

tctggcagcg gctccggcac cgcctttaca ctgaggatca gccgcgtgga ggcagaggac

1260

gtgggcgtgt attactgcat gcagcacctg gagtaccctt tcacctttgg cgccggcaca

1320

aagctggagc tgaagcggag cgaccccaca accacaccag cacctcggcc acccacccca

1380

gcaccaacaa tcgcatctca gccactgagc ctgcggcccg aggcctgtag gccagccgcc

1440

ggcggcgcag tgcacacccg gggcctggac ttcgcctgcg atatctacat ctgggcccct

1500

ctggccggca cctgcggcgt gctgttactg agcctggtca tcaccctgta ttgcaagcgg

1560

ggcagaaaga agctgctgta catcttcaag cagcccttca tgcggcccgt gcagaccaca

1620

caggaggagg atggctgctc ctgtagattc cccaggagg aggagggagg atgtgagctg

1680

cgcgtgaagt tttctcggag cgccgacgca cctgcatatc agcagggaca gaaccagctg

1740

tacaatgagc tgaacctggg ccggagagag gagtatgacg tgctggataa gagacggggc

1800

cgggaccccg agatgggagg caagccccgc cggaagaatc ctcaggaggg cctgtataac

1860

gagctgcaga aggataagat ggccgaggcc tacagcgaga tcggcatgaa gggagagaga

1920

aggcgcggca agggccacga cggcctgtac cagggcctgt ccacagcaac aaaggatact

1980

tatgacgctc tgcacatgca ggctctgccc cctcggtga

2019

<210> 7

<211> 2061

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<220>

<221> source

<223> /note="chimeric antigen receptor"

<400> 7

atgggaacca gcctgctgtg ctggatggca ctgtgcctgc tgggagcaga ccacgccgat

60

gccgaactgc ctactcaggg gacattctct aatgtgagca ccaacgtgag ctctggagga

120

ggaggctccg agctgccaac cccaggcaca ttctctaatg tgagcacaaa cgtgtctccc

180

gccaagccta ccacaaccgc cgaactgcct acccagggca cattttccaa cgtgtctacc

240

aacgtgtcta gcggaggagg aggctccccc gcacctaggc cccctacccc agcaccaaca

300

atcgcaagcc agcctctgtc cctgcgccca gaggcatgca ggccagcagc aggaggagca

360

gtgcacaccc gcggcctgga ctttgcctgc gatatctata tctgggcacc actggcagga

420

acctgtggcg tgctgctgct gtctctggtc atcaccctgt actgcaatca cagaaaccgg

480

agaagggtgt gcaagtgtcc tcggccagtg gtgagagcag agggaagggg cagcctgctg

540

acctgtggcg acgtggagga gaatcccggc cctatggaga cagatacact gctgctgtgg

600

gtgctgctgc tgtgggtgcc aggctctaca ggagaggtgc agctgcagca gagcggacct

660

gagctgatca agccaggcgc ctctgtgaag atgagctgca aggcctccgg ctataccttc

720

acaagctacg tgatgcactg ggtgaagcag aagccaggcc agggcctgga gtggatcggc

780

tatatcaatc cctacaacga cggcaccaag tataacgaga agtttaaggg caaggccacc

840

ctgacatccg ataagagctc ctctaccgcc tacatggagc tgagctccct gacatccgag

900

gactctgccg tgtactattg cgccagaggc acctactatt acggctctag ggtgttcgat

960

tactggggcc agggcacaac cctgacagtg tctagcggag gaggaggctc tggagggagga

1020

ggcagcggcg gcggaggctc cgacatcgtg atgacccagg cagcaccatc catcccagtg

1080

acacctggcg agagcgtgtc catctcttgt aggtcctcta agtctctgct gaacagcaat

1140

ggcaacacct atctgtactg gtttctgcag cggcccggac agagccctca gctgctgatc

1200

tataggatgt ccaatctggc ctctggagtg cctgatcgct tcagcggctc cggctctgga

1260

accgccttta cactgaggat ctcccgcgtg gaggcagagg acgtgggcgt gtattactgc

1320

atgcagcacc tggagtaccc tttcaccttt ggcgccggca caaagctgga gctgaagcgg

1380

agcgacccca caaccacacc agcaccccgg ccaccaaccc ctgccctac aatcgcaagc

1440

cagccactgt ccctgcggcc cgaggcctgt agacctgccg ccggcggcgc cgtccatacc

1500

cgcggcctgg atttcgcctg cgatatctac atttgggccc ctctggccgg cacttgcggc

1560

gtgctgctgc tgagcctggt catcaccctg tattgcaagc ggggcagaaa gaagctgctg

1620

tacatcttca agcagccctt catgcggccc gtgcagacca cacaggagga ggatggctgc

1680

tcctgtagat tcccagagga ggaggaggga ggatgtgagc tgcgcgtgaa gtttagccgg

1740

tccgccgacg cacctgcata tcagcagggc cagaaccagc tgtacaatga gctgaacctg

1800

ggccggagag aggagtacga cgtgctggat aagagaaggg gacgggaccc cgagatggga

1860

ggcaagcccc gccggaagaa tcctcaggag ggcctgtata acgagctgca gaaggataag

1920

atggccgagg cctacagcga gatcggcatg aagggagaga gaaggcgcgg caagggacac

1980

gacggcctgt atcagggcct gtccaccgcc acaaaggaca cctacgatgc cctgcacatg

2040

caggccctgc ctccaagatg a

2061

<210> 8

<211> 672

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<220>

<221> source

<223> /note="chimeric antigen receptor"

<400> 8

Met Leu Thr Ser Leu Leu Cys Trp Met Ala Leu Cys Leu Leu Gly Ala

1 5 10 15

Asp His Ala Asp Ala Cys Pro Tyr Ser Asn Pro Ser Leu Cys Ser Gly

20 25 30

Gly Gly Gly Ser Glu Leu Pro Thr Gln Gly Thr Phe Ser Asn Val Ser

35 40 45

Thr Asn Val Ser Pro Ala Lys Pro Thr Thr Thr Ala Cys Pro Tyr Ser

50 55 60

Asn Pro Ser Leu Cys Ser Gly Gly Gly Gly Ser Pro Ala Pro Arg Pro

65 70 75 80

Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro

85 90 95

Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu

100 105 110

Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys

115 120 125

Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn His Arg

130 135 140

Asn Arg Arg Arg Val Cys Lys Cys Pro Arg Pro Val Val Arg Ala Glu

145 150 155 160

Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly

165 170 175

Pro Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val

180 185 190

Pro Gly Ser Thr Gly Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu

195 200 205

Ile Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr

210 215 220

Thr Phe Thr Ser Tyr Val Met His Trp Val Lys Gln Lys Pro Gly Gln

225 230 235 240

Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys

245 250 255

Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser

260 265 270

Ser Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser

275 280 285

Ala Val Tyr Tyr Cys Ala Arg Gly Thr Tyr Tyr Tyr Gly Ser Arg Val

290 295 300

Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly

305 310 315 320

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val

325 330 335

Met Thr Gln Ala Ala Pro Ser Ile Pro Val Thr Pro Gly Glu Ser Val

340 345 350

Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu Asn Ser Asn Gly Asn

355 360 365

Thr Tyr Leu Tyr Trp Phe Leu Gln Arg Pro Gly Gln Ser Pro Gln Leu

370 375 380

Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro Asp Arg Phe

385 390 395 400

Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Ile Ser Arg Val

405 410 415

Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His Leu Glu Tyr

420 425 430

Pro Phe Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Ser Asp

435 440 445

Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile

450 455 460

Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala

465 470 475 480

Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr

485 490 495

Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu

500 505 510

Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile

515 520 525

Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp

530 535 540

Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

545 550 555 560

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

565 570 575

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

580 585 590

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

595 600 605

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

610 615 620

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

625 630 635 640

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

645 650 655

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

660 665 670

<210> 9

<211> 495

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<220>

<221> source

<223> /note="chimeric antigen receptor"

<400> 9

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Ile

20 25 30

Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr

35 40 45

Phe Thr Ser Tyr Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly

50 55 60

Leu Glu Trp Ile Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr

65 70 75 80

Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser

85 90 95

Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala

100 105 110

Val Tyr Tyr Cys Ala Arg Gly Thr Tyr Tyr Tyr Gly Ser Arg Val Phe

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met

145 150 155 160

Thr Gln Ala Ala Pro Ser Ile Pro Val Thr Pro Gly Glu Ser Val Ser

165 170 175

Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu Asn Ser Asn Gly Asn Thr

180 185 190

Tyr Leu Tyr Trp Phe Leu Gln Arg Pro Gly Gln Ser Pro Gln Leu Leu

195 200 205

Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro Asp Arg Phe Ser

210 215 220

Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Ile Ser Arg Val Glu

225 230 235 240

Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His Leu Glu Tyr Pro

245 250 255

Phe Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Ser Asp Pro

260 265 270

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

275 280 285

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

290 295 300

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile

305 310 315 320

Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val

325 330 335

Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe

340 345 350

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

355 360 365

Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg

370 375 380

Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln

385 390 395 400

Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp

405 410 415

Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro

420 425 430

Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp

435 440 445

Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg

450 455 460

Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr

465 470 475 480

Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490 495

<210> 10

<211> 495

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<220>

<221> source

<223> /note="chimeric antigen receptor"

<400> 10

Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Ile

20 25 30

Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr

35 40 45

Phe Thr Ser Tyr Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly

50 55 60

Leu Glu Trp Ile Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr

65 70 75 80

Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser

85 90 95

Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala

100 105 110

Val Tyr Tyr Cys Ala Arg Gly Thr Tyr Tyr Tyr Gly Ser Arg Val Phe

115 120 125

Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly

130 135 140

Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met

145 150 155 160

Thr Gln Ala Ala Pro Ser Ile Pro Val Thr Pro Gly Glu Ser Val Ser

165 170 175

Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu Asn Ser Asn Gly Asn Thr

180 185 190

Tyr Leu Tyr Trp Phe Leu Gln Arg Pro Gly Gln Ser Pro Gln Leu Leu

195 200 205

Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro Asp Arg Phe Ser

210 215 220

Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Ile Ser Arg Val Glu

225 230 235 240

Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His Leu Glu Tyr Pro

245 250 255

Phe Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Ser Asp Pro

260 265 270

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

275 280 285

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

290 295 300

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile

305 310 315 320

Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val

325 330 335

Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe

340 345 350

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

355 360 365

Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg

370 375 380

Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln

385 390 395 400

Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp

405 410 415

Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro

420 425 430

Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp

435 440 445

Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg

450 455 460

Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr

465 470 475 480

Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490 495

<210> 11

<211> 624

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<220>

<221> source

<223> /note="chimeric antigen receptor"

<400> 11

Met Gly Thr Ser Leu Leu Cys Trp Met Ala Leu Cys Leu Leu Gly Ala

1 5 10 15

Asp His Ala Asp Ala Ser Gly Gly Gly Gly Ser Pro Ala Pro Arg Pro

20 25 30

Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro

35 40 45

Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu

50 55 60

Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys

65 70 75 80

Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn His Arg

85 90 95

Asn Arg Arg Arg Val Cys Lys Cys Pro Arg Pro Val Val Arg Ala Glu

100 105 110

Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly

115 120 125

Pro Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val

130 135 140

Pro Gly Ser Thr Gly Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu

145 150 155 160

Ile Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr

165 170 175

Thr Phe Thr Ser Tyr Val Met His Trp Val Lys Gln Lys Pro Gly Gln

180 185 190

Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys

195 200 205

Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser

210 215 220

Ser Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser

225 230 235 240

Ala Val Tyr Tyr Cys Ala Arg Gly Thr Tyr Tyr Tyr Gly Ser Arg Val

245 250 255

Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly

260 265 270

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val

275 280 285

Met Thr Gln Ala Ala Pro Ser Ile Pro Val Thr Pro Gly Glu Ser Val

290 295 300

Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu Asn Ser Asn Gly Asn

305 310 315 320

Thr Tyr Leu Tyr Trp Phe Leu Gln Arg Pro Gly Gln Ser Pro Gln Leu

325 330 335

Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro Asp Arg Phe

340 345 350

Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Ile Ser Arg Val

355 360 365

Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His Leu Glu Tyr

370 375 380

Pro Phe Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Ser Asp

385 390 395 400

Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile

405 410 415

Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala

420 425 430

Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr

435 440 445

Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu

450 455 460

Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile

465 470 475 480

Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp

485 490 495

Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

500 505 510

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

515 520 525

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

530 535 540

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

545 550 555 560

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

565 570 575

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

580 585 590

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

595 600 605

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

610 615 620

<210> 12

<211> 646

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<220>

<221> source

<223> /note="chimeric antigen receptor"

<400> 12

Met Gly Thr Ser Leu Leu Cys Trp Met Ala Leu Cys Leu Leu Gly Ala

1 5 10 15

Asp His Ala Asp Ala Ser Gly Gly Gly Gly Ser Glu Leu Pro Thr Gln

20 25 30

Gly Thr Phe Ser Asn Val Ser Thr Asn Val Ser Ser Gly Gly Gly Gly

35 40 45

Ser Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln

50 55 60

Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala

65 70 75 80

Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala

85 90 95

Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr

100 105 110

Leu Tyr Cys Asn His Arg Asn Arg Arg Arg Val Cys Lys Cys Pro Arg

115 120 125

Pro Val Val Arg Ala Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp

130 135 140

Val Glu Glu Asn Pro Gly Pro Met Glu Thr Asp Thr Leu Leu Leu Trp

145 150 155 160

Val Leu Leu Leu Trp Val Pro Gly Ser Thr Gly Glu Val Gln Leu Gln

165 170 175

Gln Ser Gly Pro Glu Leu Ile Lys Pro Gly Ala Ser Val Lys Met Ser

180 185 190

Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr Val Met His Trp Val

195 200 205

Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro

210 215 220

Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr

225 230 235 240

Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr Met Glu Leu Ser Ser

245 250 255

Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala Arg Gly Thr Tyr

260 265 270

Tyr Tyr Gly Ser Arg Val Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu

275 280 285

Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

290 295 300

Gly Gly Ser Asp Ile Val Met Thr Gln Ala Ala Pro Ser Ile Pro Val

305 310 315 320

Thr Pro Gly Glu Ser Val Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu

325 330 335

Leu Asn Ser Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gln Arg Pro

340 345 350

Gly Gln Ser Pro Gln Leu Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser

355 360 365

Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr

370 375 380

Leu Arg Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys

385 390 395 400

Met Gln His Leu Glu Tyr Pro Phe Thr Phe Gly Ala Gly Thr Lys Leu

405 410 415

Glu Leu Lys Arg Ser Asp Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro

420 425 430

Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu

435 440 445

Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp

450 455 460

Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly

465 470 475 480

Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg

485 490 495

Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln

500 505 510

Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu

515 520 525

Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala

530 535 540

Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu

545 550 555 560

Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp

565 570 575

Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu

580 585 590

Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile

595 600 605

Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr

610 615 620

Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met

625 630 635 640

Gln Ala Leu Pro Pro Arg

645

<210> 13

<211> 672

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<220>

<221> source

<223> /note="chimeric antigen receptor"

<400> 13

Met Gly Thr Ser Leu Leu Cys Trp Met Ala Leu Cys Leu Leu Gly Ala

1 5 10 15

Asp His Ala Asp Ala Cys Ser Gly Gly Gly Gly Gly Ser Cys Ser Gly

20 25 30

Gly Gly Gly Ser Glu Leu Pro Thr Gln Gly Thr Phe Ser Asn Val Ser

35 40 45

Thr Asn Val Ser Pro Ala Lys Pro Thr Thr Thr Ala Cys Ser Gly Gly

50 55 60

Gly Gly Gly Ser Cys Ser Gly Gly Gly Gly Ser Pro Ala Pro Arg Pro

65 70 75 80

Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro

85 90 95

Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu

100 105 110

Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys

115 120 125

Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn His Arg

130 135 140

Asn Arg Arg Arg Val Cys Lys Cys Pro Arg Pro Val Val Arg Ala Glu

145 150 155 160

Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly

165 170 175

Pro Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val

180 185 190

Pro Gly Ser Thr Gly Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu

195 200 205

Ile Lys Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr

210 215 220

Thr Phe Thr Ser Tyr Val Met His Trp Val Lys Gln Lys Pro Gly Gln

225 230 235 240

Gly Leu Glu Trp Ile Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys

245 250 255

Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser

260 265 270

Ser Ser Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser

275 280 285

Ala Val Tyr Tyr Cys Ala Arg Gly Thr Tyr Tyr Tyr Gly Ser Arg Val

290 295 300

Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly

305 310 315 320

Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val

325 330 335

Met Thr Gln Ala Ala Pro Ser Ile Pro Val Thr Pro Gly Glu Ser Val

340 345 350

Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Leu Asn Ser Asn Gly Asn

355 360 365

Thr Tyr Leu Tyr Trp Phe Leu Gln Arg Pro Gly Gln Ser Pro Gln Leu

370 375 380

Leu Ile Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro Asp Arg Phe

385 390 395 400

Ser Gly Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Ile Ser Arg Val

405 410 415

Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His Leu Glu Tyr

420 425 430

Pro Phe Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Ser Asp

435 440 445

Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile

450 455 460

Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala

465 470 475 480

Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr

485 490 495

Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu

500 505 510

Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile

515 520 525

Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp

530 535 540

Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

545 550 555 560

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

565 570 575

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

580 585 590

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

595 600 605

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

610 615 620

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

625 630 635 640

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

645 650 655

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

660 665 670

<210> 14

<211> 686

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<220>

<221> source

<223> /note="chimeric antigen receptor"

<400> 14

Met Gly Thr Ser Leu Leu Cys Trp Met Ala Leu Cys Leu Leu Gly Ala

1 5 10 15

Asp His Ala Asp Ala Glu Leu Pro Thr Gln Gly Thr Phe Ser Asn Val

20 25 30

Ser Thr Asn Val Ser Ser Gly Gly Gly Gly Ser Glu Leu Pro Thr Gln

35 40 45

Gly Thr Phe Ser Asn Val Ser Thr Asn Val Ser Pro Ala Lys Pro Thr

50 55 60

Thr Thr Ala Glu Leu Pro Thr Gln Gly Thr Phe Ser Asn Val Ser Thr

65 70 75 80

Asn Val Ser Ser Gly Gly Gly Gly Ser Pro Ala Pro Arg Pro Pro Thr

85 90 95

Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala

100 105 110

Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe

115 120 125

Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val

130 135 140

Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn His Arg Asn Arg

145 150 155 160

Arg Arg Val Cys Lys Cys Pro Arg Pro Val Val Arg Ala Glu Gly Arg

165 170 175

Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met

180 185 190

Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro Gly

195 200 205

Ser Thr Gly Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Ile Lys

210 215 220

Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe

225 230 235 240

Thr Ser Tyr Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu

245 250 255

Glu Trp Ile Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn

260 265 270

Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser

275 280 285

Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val

290 295 300

Tyr Tyr Cys Ala Arg Gly Thr Tyr Tyr Tyr Gly Ser Arg Val Phe Asp

305 310 315 320

Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly

325 330 335

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr

340 345 350

Gln Ala Ala Pro Ser Ile Pro Val Thr Pro Gly Glu Ser Val Ser Ile

355 360 365

Ser Cys Arg Ser Ser Lys Ser Leu Leu Asn Ser Asn Gly Asn Thr Tyr

370 375 380

Leu Tyr Trp Phe Leu Gln Arg Pro Gly Gln Ser Pro Gln Leu Leu Ile

385 390 395 400

Tyr Arg Met Ser Asn Leu Ala Ser Gly Val Pro Asp Arg Phe Ser Gly

405 410 415

Ser Gly Ser Gly Thr Ala Phe Thr Leu Arg Ile Ser Arg Val Glu Ala

420 425 430

Glu Asp Val Gly Val Tyr Tyr Cys Met Gln His Leu Glu Tyr Pro Phe

435 440 445

Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys Arg Ser Asp Pro Thr

450 455 460

Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser

465 470 475 480

Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly

485 490 495

Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp

500 505 510

Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile

515 520 525

Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys

530 535 540

Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys

545 550 555 560

Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val

565 570 575

Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn

580 585 590

Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val

595 600 605

Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg

610 615 620

Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys

625 630 635 640

Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg

645 650 655

Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys

660 665 670

Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

675 680 685

<210> 15

<211> 1189

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<220>

<221> source

<223> /note="artificial promoter"

<400> 15

gcgtgaggct ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag

60

ttggggggag gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg

120

gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata

180

agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacaggta

240

agtgccgtgt gtggttcccg cgggcctggc ctctttacgg gttatggccc ttgcgtgcct

300

tgaattactt ccacgcccct ggctgcagta cgtgattctt gatcccgagc ttcgggttgg

360

aagtgggtgg gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt

420

tgaggcctgg cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg

480

tctcgctgct ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct

540

ttttttctgg caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt

600

ttttggggcc gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg

660

gggcctgcga gcgcggccac cgagaatcgg acggggggtag tctcaagctg gccggcctgc

720

tctggtgcct ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg

780

gtcggcacca gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc

840

aaaatggagg acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag

900

ggcctttccg tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag

960

gcacctcgat tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt

1020

ttatgcgatg gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca

1080

cttgatgtaa ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa

1140

gcctcagaca gtggttcaaa gtttttttct tccatttcag gtgtcgtga

1189

<210> 16

<211> 237

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<220>

<221> source

<223> /note="promoter"

<400> 16

gcgtgaggct ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag

60

ttggggggag gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg

120

gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata

180

agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacag

237

<210> 17

<211> 464

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<220>

<221> source

<223> /note="immunoglobulin gamma heavy chain 1"

<400> 17

Met Glu Trp Ser Trp Ile Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly

1 5 10 15

Val His Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Ile Lys

20 25 30

Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

Thr Ser Tyr Val Met His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn

65 70 75 80

Glu Lys Phe Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser

85 90 95

Thr Ala Tyr Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Gly Thr Tyr Tyr Tyr Gly Ser Arg Val Phe Asp

115 120 125

Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Ala Lys Thr Thr

130 135 140

Pro Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn

145 150 155 160

Ser Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro

165 170 175

Val Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr

180 185 190

Phe Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val

195 200 205

Thr Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val

210 215 220

Ala His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg

225 230 235 240

Asp Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser

245 250 255

Val Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu

260 265 270

Thr Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro

275 280 285

Glu Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala

290 295 300

Gln Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val

305 310 315 320

Ser Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe

325 330 335

Lys Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr

340 345 350

Ile Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile

355 360 365

Pro Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys

370 375 380

Met Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp

385 390 395 400

Asn Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp

405 410 415

Thr Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser

420 425 430

Asn Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly

435 440 445

Leu His Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys

450 455 460

<210> 18

<211> 239

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<220>

<221> source

<223> /note="immunoglobulin kappa light chain"

<400> 18

Met Arg Cys Leu Ala Glu Phe Leu Gly Leu Leu Val Leu Trp Ile Pro

1 5 10 15

Gly Ala Ile Gly Asp Ile Val Met Thr Gln Ala Ala Pro Ser Ile Pro

20 25 30

Val Thr Pro Gly Glu Ser Val Ser Ile Ser Cys Arg Ser Ser Lys Ser

35 40 45

Leu Leu Asn Ser Asn Gly Asn Thr Tyr Leu Tyr Trp Phe Leu Gln Arg

50 55 60

Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Arg Met Ser Asn Leu Ala

65 70 75 80

Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Ala Phe

85 90 95

Thr Leu Arg Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr

100 105 110

Cys Met Gln His Leu Glu Tyr Pro Phe Thr Phe Gly Ala Gly Thr Lys

115 120 125

Leu Glu Leu Lys Arg Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro

130 135 140

Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe

145 150 155 160

Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp

165 170 175

Gly Ser Glu Arg Gln Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp

180 185 190

Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys

195 200 205

Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys

210 215 220

Thr Ser Thr Ser Pro Ile Val Lys Ser Phe Asn Arg Asn Glu Cys

225 230 235

<210> 19

<211> 136

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<220>

<221> source

<223> /note="rituximab binding sequence"

<400> 19

Cys Pro Tyr Ser Asn Pro Ser Leu Cys Ser Gly Gly Gly Gly Ser Glu

1 5 10 15

Leu Pro Thr Gln Gly Thr Phe Ser Asn Val Ser Thr Asn Val Ser Pro

20 25 30

Ala Lys Pro Thr Thr Thr Ala Cys Pro Tyr Ser Asn Pro Ser Leu Cys

35 40 45

Ser Gly Gly Gly Gly Ser Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro

50 55 60

Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro

65 70 75 80

Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp

85 90 95

Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu

100 105 110

Ser Leu Val Ile Thr Leu Tyr Cys Asn His Arg Asn Arg Arg Arg Val

115 120 125

Cys Lys Cys Pro Arg Pro Val Val

130 135

<210> 20

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<220>

<221> source

<223> /note="signal peptide"

<400> 20

Met Gly Thr Ser Leu Leu Cys Trp Met Ala Leu Cys Leu Leu Gly Ala

1 5 10 15

Asp His Ala Asp Ala

20

<210> 21

<211> 157

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 21

Met Gly Thr Ser Leu Leu Cys Trp Met Ala Leu Cys Leu Leu Gly Ala

1 5 10 15

Asp His Ala Asp Ala Cys Pro Tyr Ser Asn Pro Ser Leu Cys Ser Gly

20 25 30

Gly Gly Gly Ser Glu Leu Pro Thr Gln Gly Thr Phe Ser Asn Val Ser

35 40 45

Thr Asn Val Ser Pro Ala Lys Pro Thr Thr Thr Ala Cys Pro Tyr Ser

50 55 60

Asn Pro Ser Leu Cys Ser Gly Gly Gly Gly Ser Pro Ala Pro Arg Pro

65 70 75 80

Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro

85 90 95

Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu

100 105 110

Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys

115 120 125

Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Asn His Arg

130 135 140

Asn Arg Arg Arg Val Cys Lys Cys Pro Arg Pro Val Val

145 150 155

<210> 22

<211> 9

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 22

Cys Pro Tyr Ser Asn Pro Ser Leu Cys

15

<210> 23

<211> 24

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 23

Asn Ser Glu Leu Leu Ser Leu Ile Asn Asp Met Pro Ile Thr Asn Asp

1 5 10 15

Gln Lys Lys Leu Met Ser Asn Asn

20

<210> 24

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 24

Cys Gln Phe Asp Leu Ser Thr Arg Arg Leu Lys Cys

1 5 10

<210> 25

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 25

Cys Gln Tyr Asn Leu Ser Ser Arg Ala Leu Lys Cys

1 5 10

<210> 26

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 26

Cys Val Trp Gln Arg Trp Gln Lys Ser Tyr Val Cys

1 5 10

<210> 27

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 27

Cys Met Trp Asp Arg Phe Ser Arg Trp Tyr Lys Cys

1 5 10

<210> 28

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 28

Ser Phe Val Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp

1 5 10 15

Lys Leu Ala Ala Phe Pro Glu Asp Arg

20 25

<210> 29

<211> 19

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 29

Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala Gln

1 5 10 15

Ile Lys Glu

<210> 30

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 30

Glu Leu Pro Thr Gln Gly Thr Phe Ser Asn Val Ser Thr Asn Val Ser

1 5 10 15

<210> 31

<211> 12

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 31

Gly Gln Asn Asp Thr Ser Gln Thr Ser Ser Pro Ser

1 5 10

<210> 32

<211> 16

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 32

Gly Leu Ala Val Ser Thr Ile Ser Ser Phe Phe Pro Pro Gly Tyr Gln

1 5 10 15

<210> 33

<211> 45

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 33

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp

35 40 45

<210> 34

<211> 231

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 34

Glu Pro Lys Ser Pro Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala

1 5 10 15

Pro Pro Val Ala Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

20 25 30

Asp Thr Leu Met Ile Ala Arg Thr Pro Glu Val Thr Cys Val Val Val

35 40 45

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

50 55 60

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

65 70 75 80

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

85 90 95

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

100 105 110

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

115 120 125

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

130 135 140

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

145 150 155 160

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

165 170 175

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

180 185 190

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

195 200 205

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

210 215 220

Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 35

<211> 21

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<400> 35

Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu

1 5 10 15

Ser Leu Val Ile Thr

20

<210> 36

<211> 66

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 36

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile

35 40 45

Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val

50 55 60

Ile Thr

65

<210> 37

<211> 42

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 37

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 38

<211> 113

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<400> 38

Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln

1 5 10 15

Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu

20 25 30

Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly

35 40 45

Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln

50 55 60

Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu

65 70 75 80

Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr

85 90 95

Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro

100 105 110

Arg

<210> 39

<211> 943

<212> DNA

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polynucleotide"

<220>

<221> source

<223> /note="intron"

<400> 39

gtaagtgccg tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg

60

ccttgaatta cttccacgcc cctggctgca gtacgtgatt cttgatcccg agcttcgggt

120

tggaagtggg tgggagagtt cgaggccttg cgcttaagga gccccttcgc ctcgtgcttg

180

agttgaggcc tggcctgggc gctggggccg ccgcgtgcga atctggtggc accttcgcgc

240

ctgtctcgct gctttcgata agtctctagc catttaaaat ttttgatgac ctgctgcgac

300

gctttttttc tggcaagata gtcttgtaaa tgcgggccaa gatctgcaca ctggtatttc

360

ggtttttggg gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag

420

gcggggcctg cgagcgcggc caccgagaat cggacggggg tagtctcaag ctggccggcc

480

tgctctggtg cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg caaggctggc

540

ccggtcggca ccagttgcgt gagcggaaag atggccgctt cccggccctg ctgcagggag

600

ctcaaaatgg aggacgcggc gctcgggaga gcgggcgggt gagtcaccca cacaaaggaa

660

aagggccttt ccgtcctcag ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc

720

caggcacctc gattagttct cgagcttttg gagtacgtcg tctttaggtt ggggggaggg

780

gttttatgcg atggagtttc cccacactga gtgggtggag actgaagtta ggccagcttg

840

gcacttgatg taattctcct tggaatttgc cctttttgag tttggatctt ggttcattct

900

caagcctcag acagtggttc aaagtttttt tcttccatt cag

943

<210> 40

<211> 136

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic polypeptide"

<220>

<221> source

<223> /note="mutated sequence binding

rituximab molecules"

<400> 40

Cys Ser Gly Gly Gly Gly Gly Ser Cys Ser Gly Gly Gly Gly Ser Glu

1 5 10 15

Leu Pro Thr Gln Gly Thr Phe Ser Asn Val Ser Thr Asn Val Ser Pro

20 25 30

Ala Lys Pro Thr Thr Thr Ala Cys Ser Gly Gly Gly Gly Gly Ser Cys

35 40 45

Ser Gly Gly Gly Gly Ser Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro

50 55 60

Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro

65 70 75 80

Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp

85 90 95

Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu

100 105 110

Ser Leu Val Ile Thr Leu Tyr Cys Asn His Arg Asn Arg Arg Arg Val

115 120 125

Cys Lys Cys Pro Arg Pro Val Val

130 135

<210> 41

<211> 25

<212> PROTEIN

<213> Artificial sequence

<220>

<221> source

<223> /note="Description of the artificial sequence:

synthetic peptide"

<220>

<221> AREA

<222> (1)..(25)

<223> /note="This sequence may span 1-5 'Gly'

Gly Gly Gly Ser'

repeating links"

<400> 41

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Gly Gly Gly Ser

20 25

<---

Claims (26)

1. An isolated polynucleotide encoding a polypeptide containing a chimeric antigen receptor for CD19 (CAR) that is at least 90% identical to SEQ ID NO: 9, where the polypeptide does not contain a rituximab binding site, and where the polynucleotide contains a short EF1a promoter that capable of expressing chimeric antigen receptor for CD19 (CAR) in a mammalian T cell, wherein the short EF1a promoter does not contain the nucleic acid sequence under SEQ ID NO: 39 and at the same time contains the nucleic acid sequence under SEQ ID NO: 16. 2. The isolated polynucleotide according to claim 1, wherein the short EF1a promoter is the nucleic acid sequence of SEQ ID NO: 16. 3. An isolated polynucleotide according to any of the preceding claims, wherein the polypeptide contains a safety switch. 4. The isolated polynucleotide of claim 3, wherein the safety switch is linked to the CD19 CAR using a linker peptide. 5. The isolated polynucleotide of claim 3, wherein the safety switch is linked to the anti-CD19 CAR using a T2A linker. 6. An isolated polynucleotide according to any one of paragraphs. 3-5, where the safety switch contains an antibody binding site. 7. An isolated polynucleotide according to any one of paragraphs. 3-5, where the safety switch contains a mutated CD20 mimotope. 8. An isolated polynucleotide according to any one of paragraphs. 3-7, where the polypeptide contains the CD34 epitope. 9. The isolated polynucleotide according to claim 8, wherein the CD34 epitope is the QBEND-10 epitope. 10. An isolated polynucleotide according to any one of paragraphs. 3-9, wherein the polypeptide contains the CD8 hinge/transmembrane domain. 11. An isolated polynucleotide as defined in any one of the preceding claims, comprising a nucleic acid sequence that is at least about 95%, 96%, 98%, 99%, or 100% identical to any of the nucleic acid sequences of SEQ ID NO: 1-7 . 12. An isolated polynucleotide as defined in any one of the preceding claims, wherein the polynucleotide encodes a polypeptide that is at least about 95%, 96%, 98%, 99%, or 100% identical to any of the amino acid sequences of SEQ ID NO: 8-14. 13. An expression vector containing an isolated polynucleotide according to any of the previous paragraphs. 14. The vector of claim 13, wherein the vector is a retroviral vector, a DNA vector, a plasmid, an RNA vector, an adenoviral vector, an adeno-associated virus vector, a lentiviral vector, or any combination thereof. 15. An engineered T cell that is engineered to express an anti-CD19 CAR, comprising an isolated polynucleotide according to any one of claims. 1-12, where the engineered T cell does not express the rituximab binding site. 16. An engineered T cell that is engineered to express an anti-CD19 CAR, comprising the vector of claim 13 or 14, wherein the engineered T cell does not express a rituximab binding site. 17. The engineered T cell of claim 15 or 16, wherein the immune cell is a T cell, tumor infiltrating lymphocyte (TIL), NK cell, TCR-expressing cell, dendritic cell, or NK T cell. 18. The engineered T cell of claim 17, wherein the cell is an autologous T cell. 19. The engineered T cell of claim 17, wherein the cell is an allogeneic T cell. 20. Engineered T cell according to any one of paragraphs. 15-19, wherein the cell contains a polynucleotide that is at least 90%, 95%, 96%, 98%, 99%, or 100% identical to the nucleic acid sequence of SEQ ID NO: 3. 21. The engineered T cell according to any one of paragraphs. 15-20, where the cell is resistant to rituximab. 22. A pharmaceutical composition for treating a disease or disorder associated with elevated levels of CD19 in a subject in need thereof, comprising an engineered T cell according to any one of claims. 15-21. 23. Use of a constructed T cell according to any one of paragraphs. 15-21 or the pharmaceutical composition of claim 22 for treating a disease or disorder associated with elevated CD19 levels in a subject in need thereof. 24. Use according to claim 23, wherein the disease or disorder is non-Hodgkin's lymphoma (NHL). 25. The use of claim 23 or 24, wherein the subject has been treated or is currently being treated with rituximab.