CD19 and CD22 chimeric antigen receptors and uses thereof

Abstract

The present disclosure provides compositions and methods for treating diseases associated with CD19 and / or CD22 expression, for example, by administering recombinant T cells or natural killer (NK) cells comprising CD22 CARs and CD19 CARs as described herein. The present disclosure also relates to CAR molecules specific for CD22 and / or CD19, methods for making cells comprising the same, and vectors encoding same.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS AND INCORPORATION OF SEQUENCE LISTINGS This application claims priority to U.S. Provisional Patent Application No. 62 / 940,600, filed November 26, 2019, which is incorporated herein by reference in its entirety. The Sequence Listing contained in file entitled "PAT058691-WO-PCT SQL_ST25," created November 24, 2020, and measuring 182,837 bytes (measured using the MS-Windows operating system), is hereby filed and incorporated herein by reference.

[0002] The present invention generally relates to the use of T cells or natural killer (NK) cells engineered to express a chimeric antigen receptor (CAR) comprising a cluster of differentiation 19 protein (CD19) binding domain and / or a cluster of differentiation 22 protein (CD22) binding domain to treat diseases associated with CD19 and / or CD22 expression. [Background technology]

[0003] Many patients with B-cell malignancies are incurable with standard therapies. In addition, conventional treatment options often have severe side effects. While attempts have been made at cancer immunotherapy, several obstacles make this an extremely difficult goal to achieve clinical efficacy. Hundreds of so-called tumor antigens have been identified, but they are generally self-derived and therefore poorly immunogenic. Furthermore, tumors use several mechanisms to render themselves unsuitable for initiating and propagating immune attack.

[0004] Recent developments using chimeric antigen receptor (CAR)-modified autologous T cell (CART) therapy, which relies on redirecting T cells to suitable cell surface molecules on cancer cells, such as B cell malignancies, have shown promising results in harnessing the power of the immune system to treat B cell malignancies and other cancers (see, e.g., Non-Patent Document 1). Clinical results with murine-derived CART19 (i.e., "CTL019") have shown promise for complete remission in patients with CLL and pediatric ALL (see, e.g., Non-Patent Document 2, Non-Patent Document 3, Non-Patent Document 4). In addition to the ability of the chimeric antigen receptor on genetically modified T cells to recognize and destroy target cells, successful therapeutic T cell therapy requires their ability to proliferate and persist long-term to monitor for leukemia relapse. Variations in T cell quality due to anergy, suppression, or exhaustion can affect the performance of CAR-transformed T cells, which those skilled in the art currently have limited control over. To be effective, CAR-transformed patient T cells must persist and maintain their ability to proliferate in response to cognate antigens. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Sadelain et al., Cancer Discovery 3:388-398(2013) [Non-patent document 2] Kalos et al., Sci Transl Med 3:95ra73(2011) [Non-patent document 3] Porter et al.,NEJM 365:725-733(2011) [Non-patent document 4] Grupp et al.,NEJM 368:1509-1518(2013) Summary of the Invention [Means for solving the problem]

[0006] The present disclosure features, inter alia, novel nucleic acid molecules encoding chimeric antigen receptor (CAR) molecules comprising a first CAR comprising a CD22 CAR and a second CAR comprising a CD19 CAR, e.g., dual CARs as described herein. In some embodiments, the CD22 CAR comprises a CD22 antigen-binding domain and a first transmembrane domain; a first costimulatory signaling domain; and / or a first primary signaling domain. In some embodiments, the CD19 CAR comprises a CD19 antigen-binding domain and a second transmembrane domain; a second costimulatory signaling domain; and / or a second primary signaling domain. In some embodiments of the CAR molecules disclosed herein, the CAR molecule comprises two identical polypeptide sequences, e.g., the first and second transmembrane domains; the first and second costimulatory domains; and / or the first and second primary signaling domains, encoded by different nucleotide sequences. Also disclosed herein are methods of using the CAR molecules. Further disclosed herein are CARs comprising a bispecific antigen-binding domain comprising a CD22 antigen-binding domain and a CD19 antigen-binding domain, e.g., a tandem CAR as described herein. Nucleic acids encoding the compositions, host cells, vectors, and methods of making and using the compositions are also disclosed.

[0007] Dual Car In one aspect, the present disclosure provides a nucleic acid molecule encoding a chimeric antigen receptor (CAR) molecule, said CAR molecule comprising: (a) a first CAR comprising: a first antigen-binding domain that binds to CD22; a first transmembrane domain; a first costimulatory signaling domain; and / or a first primary signaling domain; and (b) a second CAR comprising a second antigen-binding domain that binds to CD19; a second transmembrane domain; a second costimulatory signaling domain; and / or a second primary signaling domain. Including, (i) the first transmembrane domain and the second transmembrane domain comprise the amino acid sequence of SEQ ID NO: 65, or an amino acid sequence having at least 90% identity thereto, and optionally, the nucleotide sequence encoding the first transmembrane domain and contained in the nucleic acid molecule is different from the nucleotide sequence encoding the second transmembrane domain and contained in the nucleic acid molecule; (ii) the first costimulatory signaling domain and the second costimulatory signaling domain comprise the amino acid sequence of any one of SEQ ID NO: 70, or an amino acid sequence having at least 90% identity thereto, and optionally, the nucleotide sequence encoding the first costimulatory signaling domain and contained in the nucleic acid molecule is different from the nucleotide sequence encoding the second costimulatory signaling domain and contained in the nucleic acid molecule; and / or (iii) the first primary signaling domain and the second primary signaling domain comprise the amino acid sequence of SEQ ID NO: 75, or an amino acid sequence having at least 90% identity thereto, and optionally, the nucleotide sequence encoding the primary signaling domain and contained in the nucleic acid molecule is different from the nucleotide sequence encoding the second primary signaling domain and contained in the nucleic acid molecule.

[0008] In one embodiment, the first CAR comprises a first antigen-binding domain that binds to CD22, a first transmembrane domain, and a first costimulatory signaling domain. In one embodiment, the first CAR comprises a first antigen-binding domain that binds to CD22, a first transmembrane domain, and a first primary signaling domain. In one embodiment, the first CAR comprises a first antigen-binding domain that binds to CD22, a first transmembrane domain, a first costimulatory signaling domain, and a first primary signaling domain.

[0009] In some embodiments, the second CAR comprises a second antigen-binding domain that binds to CD19; a second transmembrane domain; and a second costimulatory signaling domain. In some embodiments, the second CAR comprises a second antigen-binding domain that binds to CD19; a second transmembrane domain; and a second primary signaling domain. In some embodiments, the second CAR comprises a second antigen-binding domain that binds to CD19; a second transmembrane domain; a second costimulatory signaling domain; and a second primary signaling domain.

[0010] In one embodiment, the CD22 antigen binding domain comprises one or more (e.g., all three) light chain complementarity determining region 1 (LC CDR1), light chain complementarity determining region 2 (LC CDR2), and light chain complementarity determining region 3 (LC CDR3) of a CD22 binding domain described herein, e.g., in Table 1A, Table 2A, or Table 3A; and / or one or more (e.g., all three) heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2), and heavy chain complementarity determining region 3 (HC CDR3) of a CD22 binding domain described herein, e.g., in Table 1A, Table 2A, or Table 3A. In one embodiment, the CD22 binding domain comprises (i) HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, LC CDR3 comprising the amino acid sequences of SEQ ID NOs: 20, 21, 22, 28, 29, and 30, respectively; (ii) SEQ ID NOs: 23, 24, 22, 31, 32, and 33, respectively; or (iii) SEQ ID NOs: 25, 26, 27, 34, 32, and 30, respectively.

[0011] In one embodiment, the CD22 antigen-binding domain comprises an scFv comprising an amino acid sequence having one, two, or three or more alterations (e.g., substitutions), but not more than 30, 20, or 10 alterations (e.g., substitutions), of a CD22 scFv sequence provided in Table 1A or Table 3A, e.g., SEQ ID NO: 50, 53, or 55. In one embodiment, the CD22 antigen-binding domain comprises an scFv comprising an amino acid sequence having at least 95% identity to a CD22 scFv sequence provided in Table 1A or Table 3A, e.g., SEQ ID NO: 50, 53, or 55. In one embodiment, the CD22 antigen-binding domain comprises an scFv comprising the amino acid sequence of a CD22 scFv sequence provided in Table 1A or Table 3A, e.g., SEQ ID NO: 50, 53, or 55.

[0012] In one embodiment, the CD22 antigen binding domain comprises an scFv encoded by a nucleotide sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity to a CD22 scFv sequence provided in Table 1A or Table 3A, e.g., SEQ ID NO: 49, 51, 52, 54, 56 or 57.

[0013] In one embodiment, the CD19 antigen binding domain comprises one or more (e.g., all three) light chain complementarity determining region 1 (LC CDR1), light chain complementarity determining region 2 (LC CDR2), and light chain complementarity determining region 3 (LC CDR3) of a CD19 antigen binding domain described herein, e.g., in Table 1A, Table 2A, Table 3A, or Table 5A; and / or one or more (e.g., all three) heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2), and heavy chain complementarity determining region 3 (HC CDR3) of a CD19 antigen binding domain described herein, e.g., in Table 1A, Table 2A, Table 3A, or Table 5A. In one embodiment, the CD19 binding domain comprises (i) HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, LC CDR3 comprising the amino acid sequences of SEQ ID NOs: 35, 36, 39, 40, 41, and 42, respectively; (ii) SEQ ID NOs: 35, 37, 39, 40, 41, and 42, respectively; or (iii) SEQ ID NOs: 35, 38, 39, 40, 41, and 42, respectively.

[0014] In one embodiment, the CD19 antigen-binding domain comprises an scFv comprising an amino acid sequence having one, two, or three or more alterations (e.g., substitutions) of a CD19 scFv sequence provided in Table 1A, Table 3A, or Table 5A, e.g., SEQ ID NO: 44 or 47, but not more than 30, 20, or 10 alterations (e.g., substitutions). In one embodiment, the CD19 antigen-binding domain comprises an scFv comprising an amino acid sequence having at least 95% identity to a CD19 scFv sequence provided in Table 1A, Table 3A, or Table 5A, e.g., SEQ ID NO: 44 or 47. In one embodiment, the CD19 antigen-binding domain comprises an scFv comprising a CD19 scFv sequence provided in Table 1A, Table 3A, or Table 5A, e.g., the amino acid sequence of SEQ ID NO: 44 or 47. In one embodiment, the CD19 antigen binding domain comprises an scFv encoded by a nucleotide sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity to a CD19 scFv sequence provided in Table 1A, Table 3A or Table 5A, e.g., SEQ ID NO: 43, 45, 46 or 48.

[0015] In certain embodiments of the nucleic acids encoding the CAR molecules disclosed herein, the nucleotide sequence encoding the first transmembrane domain differs from the nucleotide sequence encoding the second transmembrane domain by at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100%. In certain embodiments, the nucleotide sequence encoding the first transmembrane domain differs from the nucleotide sequence encoding the second transmembrane domain by at least 1 nucleotide, 10 nucleotides, 20 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 60 nucleotides, 70 nucleotides, 80 nucleotides, 90 nucleotides, 100 nucleotides, 150 nucleotides, 200 nucleotides, or all nucleotides.

[0016] In certain embodiments of the nucleic acids encoding the CAR molecules disclosed herein, the nucleotide sequence encoding the first costimulatory signaling domain differs from the nucleotide sequence encoding the second costimulatory signaling domain by at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100%. In certain embodiments, the nucleotide sequence encoding the first costimulatory signaling domain differs from the nucleotide sequence encoding the second costimulatory signaling domain by at least 1 nucleotide, 10 nucleotides, 20 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 60 nucleotides, 70 nucleotides, 80 nucleotides, 90 nucleotides, 100 nucleotides, 150 nucleotides, 200 nucleotides, or all nucleotides.

[0017] In certain embodiments of the nucleic acids encoding the CAR molecules disclosed herein, the nucleotide sequence encoding the first primary signaling domain differs from the nucleotide sequence encoding the second primary signaling domain by at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 100%. In certain embodiments, the nucleotide sequence encoding the first primary signaling domain differs from the nucleotide sequence encoding the second primary signaling domain by at least 1 nucleotide, 10 nucleotides, 20 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 60 nucleotides, 70 nucleotides, 80 nucleotides, 90 nucleotides, 100 nucleotides, 150 nucleotides, 200 nucleotides, or all nucleotides.

[0018] In some embodiments of the nucleic acid sequence encoding a CAR molecule disclosed herein, the CAR molecule is encoded by the nucleotide sequence of SEQ ID NO: 11, 15, or 19, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0019] In some embodiments, the CAR molecule disclosed herein comprises the amino acid sequence of SEQ ID NO: 12 or 16, or an amino acid sequence having at least 90% identity thereto.

[0020] In some embodiments, the present disclosure provides a cell (e.g., an immune effector cell) comprising a nucleic acid sequence encoding a chimeric antigen receptor (CAR) molecule, wherein the CAR molecule comprises: (a) a first CAR comprising a first antigen-binding domain that binds to CD22 and a first transmembrane domain; a first costimulatory signaling domain; and / or a first primary signaling domain; and (b) a second CAR comprising a second antigen-binding domain that binds to CD19 and a second transmembrane domain; a second costimulatory signaling domain; and / or a second primary signaling domain. Including, (i) the first transmembrane domain and the second transmembrane domain comprise the amino acid sequence of SEQ ID NO: 65, or an amino acid sequence having at least 90% identity thereto, and optionally, the nucleotide sequence encoding the first transmembrane domain and contained in the nucleic acid molecule is different from the nucleotide sequence encoding the second transmembrane domain and contained in the nucleic acid molecule; (ii) the first costimulatory signaling domain and the second costimulatory signaling domain comprise the amino acid sequence of SEQ ID NO: 70, or an amino acid sequence having at least 90% identity thereto, and optionally, the nucleotide sequence encoding the first costimulatory signaling domain and contained in the nucleic acid molecule is different from the nucleotide sequence encoding the second costimulatory signaling domain and contained in the nucleic acid molecule; and / or (iii) the first primary signaling domain and the second primary signaling domain comprise the amino acid sequence of SEQ ID NO: 75, or an amino acid sequence having at least 90% identity thereto, and optionally, the nucleotide sequence encoding the first primary signaling domain and contained in the nucleic acid molecule is different from the nucleotide sequence encoding the second primary signaling domain and contained in the nucleic acid molecule.

[0021] In one embodiment, the first primary signaling domain and the second primary signaling domain comprise the amino acid sequence of SEQ ID NO: 108, or an amino acid sequence having at least 90% identity thereto, and optionally, the nucleotide sequence encoding the first primary signaling domain and contained in the nucleic acid molecule differs from the nucleotide sequence encoding the second primary signaling domain and contained in the nucleic acid molecule.

[0022] In another aspect, provided herein is a cell (e.g., immune effector cell) comprising a chimeric antigen receptor (CAR) molecule, wherein the CAR molecule is (a) a first CAR comprising a first antigen-binding domain that binds to CD22 and a first transmembrane domain; a first costimulatory signaling domain; and / or a first primary signaling domain; and (b) a second CAR comprising a second antigen-binding domain that binds to CD19 and a second transmembrane domain; a second costimulatory signaling domain; and / or a second primary signaling domain. Including, (i) the first transmembrane domain and the second transmembrane domain comprise the amino acid sequence of SEQ ID NO: 65, or an amino acid sequence having at least 90% identity thereto, and optionally, the nucleotide sequence encoding the first transmembrane domain and contained in the nucleic acid molecule is different from the nucleotide sequence encoding the second transmembrane domain and contained in the nucleic acid molecule; (ii) the first costimulatory signaling domain and the second costimulatory signaling domain comprise the amino acid sequence of SEQ ID NO: 70, or an amino acid sequence having at least 90% identity thereto, and optionally, the nucleotide sequence encoding the first costimulatory signaling domain and contained in the nucleic acid molecule is different from the nucleotide sequence encoding the second costimulatory signaling domain and contained in the nucleic acid molecule; and / or (iii) the first primary signaling domain and the second primary signaling domain comprise the amino acid sequence of SEQ ID NO: 75, or an amino acid sequence having at least 90% identity thereto, and optionally, the nucleotide sequence encoding the first primary signaling domain and contained in the nucleic acid molecule is different from the nucleotide sequence encoding the second primary signaling domain and contained in the nucleic acid molecule.

[0023] In some embodiments, the cell is an immune effector cell, such as a T cell (e.g., a CD3+, CD4+, or CD8+ T cell) or an NK cell. In some embodiments, the cell is a human cell.

[0024] In an embodiment, provided herein is a method of providing anti-tumor immunity, comprising administering to a subject in need thereof an effective amount of a population of cells, e.g., immune effector cells, comprising, e.g., expressing, a CAR molecule disclosed herein, e.g., a dual CAR molecule disclosed herein.

[0025] In another aspect, the present disclosure provides a method of treating a subject having a disease associated with an antigen (e.g., CD19 and / or CD22), comprising administering to a subject in need thereof an effective amount of a population of cells, e.g., immune effector cells, comprising, e.g., expressing, a CAR molecule disclosed herein, e.g., a dual CAR molecule disclosed herein.

[0026] Tandem Car In certain embodiments, the present disclosure provides a bispecific antigen-binding domain comprising a first antigen-binding domain that binds to CD22 and a second antigen-binding domain that binds to CD19.

[0027] In another aspect, provided herein is a chimeric antigen receptor (CAR) comprising a bispecific antigen-binding domain described herein.

[0028] In yet another aspect, the present disclosure provides a nucleic acid encoding a chimeric antigen receptor (CAR) comprising a bispecific antigen-binding domain described herein.

[0029] In some embodiments of the bispecific antigen-binding domains described herein, e.g., a CAR comprising a bispecific antigen-binding domain or a nucleic acid encoding a CAR comprising a bispecific antigen-binding domain, the first antigen-binding domain can be upstream (e.g., toward the N-terminus) of the second antigen-binding domain, or the first antigen-binding domain can be downstream (e.g., toward the C-terminus) of the second antigen-binding domain.

[0030] In some embodiments, each of the first and second antigen-binding domains comprises an scFv, e.g., a light chain variable (VL) domain and a heavy chain variable (VH) domain. In some embodiments, the first antigen-binding domain comprises an scFv comprising a first VH (VH1) and a first VL (VL1). In some embodiments, the second antigen-binding domain comprises an scFv comprising a second VH (VH2) and a second VL (VL2).

[0031] In some embodiments, the bispecific antigen-binding domain has any one of the following configurations from N-terminus to C-terminus: VL1-VH1-VH2-VL2; VH1-VL1-VH2-VL2; VL1-VH1-VL2-VH2; VH1-VL1-VL2-VH2, VH2-VL2-VL1-VH1; VL2-VH2-VL1-VH1; VH2-VL2-VH1-VL1; or VL2-VH2-VH1-VL1.

[0032] In one embodiment, the CAR comprising the bispecific antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 2 and is encoded by the nucleic acid sequence of SEQ ID NO: 1. In another embodiment, the CAR comprising the bispecific antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 4 and is encoded by the nucleic acid sequence of SEQ ID NO: 3.

[0033] In another embodiment, the CAR comprising the bispecific antigen-binding domain comprises the amino acid sequence of SEQ ID NO:6 and is encoded by the nucleic acid sequence of SEQ ID NO:5.

[0034] In another embodiment, the CAR comprising the bispecific antigen-binding domain comprises the amino acid sequence of SEQ ID NO:8 and is encoded by the nucleic acid sequence of SEQ ID NO:7.

[0035] In another embodiment, the CAR comprising the bispecific antigen-binding domain comprises the amino acid sequence of SEQ ID NO: 10 and is encoded by the nucleic acid sequence of SEQ ID NO: 9.

[0036] In some aspects, the present disclosure provides a vector comprising a nucleic acid molecule encoding a CAR molecule disclosed herein, a nucleic acid encoding a bispecific antigen-binding domain disclosed herein, or a nucleic acid encoding a CAR comprising a bispecific antigen-binding domain disclosed herein.

[0037] In another aspect, provided herein is a pharmaceutical composition comprising a nucleic acid encoding a CAR molecule disclosed herein or a pharmaceutical composition comprising a CAR molecule disclosed herein. In some embodiments, the pharmaceutical composition comprises an excipient, carrier, diluent, and / or stabilizer.

[0038] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising a bispecific antigen-binding domain disclosed herein, a CAR comprising a bispecific antigen-binding domain disclosed herein, or a CAR nucleic acid encoding a bispecific antigen-binding domain disclosed herein. In some embodiments, the pharmaceutical composition comprises an excipient, carrier, diluent, and / or stabilizer.

[0039] In an embodiment, provided herein is a method of providing anti-tumor immunity, comprising administering to a subject in need thereof an effective amount of a population of cells, e.g., immune effector cells, comprising, e.g., expressing, a CAR disclosed herein, e.g., a tandem CAR disclosed herein.

[0040] In another aspect, the present disclosure provides a method of treating a subject having a disease associated with an antigen (e.g., CD19 and / or CD22), comprising administering to a subject in need thereof an effective amount of a population of cells, e.g., immune effector cells, comprising, e.g., expressing, a CAR disclosed herein, e.g., a tandem CAR disclosed herein.

[0041] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following recitation of embodiments.

[0042] Enumeration of Embodiments 1. A nucleic acid molecule encoding a chimeric antigen receptor (CAR) molecule, said CAR molecule comprising: (a) a first CAR comprising: a first antigen-binding domain that binds to CD22; a first transmembrane domain; a first costimulatory signaling domain; and / or a first primary signaling domain; and (b) a second CAR comprising a second antigen-binding domain that binds to CD19; a second transmembrane domain; a second costimulatory domain; and / or a second primary signaling domain. Including, (i) the first transmembrane domain and the second transmembrane domain comprise the amino acid sequence of SEQ ID NO: 65, or an amino acid sequence having at least 90% identity thereto, and optionally, the nucleotide sequence encoding the first transmembrane domain and contained in the nucleic acid molecule is different from the nucleotide sequence encoding the second transmembrane domain and contained in the nucleic acid molecule; (ii) the first costimulatory signaling domain and the second costimulatory signaling domain comprise the amino acid sequence of SEQ ID NO: 70, or an amino acid sequence having at least 90% identity thereto, and optionally, the nucleotide sequence encoding the first costimulatory signaling domain and contained in the nucleic acid molecule is different from the nucleotide sequence encoding the second costimulatory signaling domain and contained in the nucleic acid molecule; and / or (iii) the first primary signaling domain and the second primary signaling domain comprise the amino acid sequence of SEQ ID NO: 75, or an amino acid sequence having at least 90% identity thereto, and optionally, the nucleotide sequence encoding the primary signaling domains and contained in the nucleic acid molecule is different from the nucleotide sequence encoding the second primary signaling domain and contained in the nucleic acid molecule.

[0043] 2. The first CAR is a first antigen-binding domain that binds to CD22, a first transmembrane domain, and a first costimulatory signaling domain; a first antigen-binding domain that binds to CD22, a first transmembrane domain, and a first primary signaling domain; or a first antigen-binding domain that binds to CD22, a first transmembrane domain, a first costimulatory signaling domain, and a first primary signaling domain. 2. The nucleic acid molecule of embodiment 1, comprising:

[0044] 3. The second CAR is a second antigen-binding domain that binds to CD19; a second transmembrane domain; and a second costimulatory signaling domain; a second antigen-binding domain that binds to CD19; a second transmembrane domain; and a second primary signaling domain; or a second antigen-binding domain that binds to CD19; a second transmembrane domain; a second costimulatory signaling domain; and a second primary signaling domain. 3. The nucleic acid molecule of embodiment 1 or 2, comprising:

[0045] 4. The CD22 antigen-binding domain is one or more (e.g., all three) light chain complementarity determining region 1 (LC CDR1), light chain complementarity determining region 2 (LC CDR2), and light chain complementarity determining region 3 (LC CDR3) of a CD22 binding domain described herein, e.g., in Table 1A, Table 2A, or Table 3A; and / or One or more (e.g., all three) heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2), and heavy chain complementarity determining region 3 (HC CDR3) of a CD22 binding domain described herein, e.g., in Table 1A, Table 2A, or Table 3A 10. The nucleic acid molecule of any one of the preceding embodiments, comprising:

[0046] 5. The nucleic acid molecule of embodiment 4, wherein the CD22 antigen-binding domain comprises the LC CDR1, LC CDR2, and LC CDR3 of a CD22 binding domain described herein, e.g., in Table 1A, Table 2A, or Table 3A; and / or the HC CDR1, HC CDR2, and HC CDR3 of a CD22 binding domain described herein, e.g., in Table 1A, Table 2A, or Table 3A.

[0047] 6. The nucleic acid molecule of embodiment 4 or 5, wherein the CD22 antigen binding domain comprises a nucleotide sequence encoding an LC CDR1 of SEQ ID NO: 28, 31 or 34, an LC CDR2 of SEQ ID NO: 29 or 32; an LC CDR3 of SEQ ID NO: 30 or 33; and / or an HC CDR1, an HC CDR2 of SEQ ID NO: 20, 23 or 25, or an HC CDR3 of SEQ ID NO: 21, 24 or 26; or an HC CDR3 of SEQ ID NO: 22 or 27.

[0048] 7. The nucleic acid molecule of any one of embodiments 4-6, wherein the CD22 antigen binding domain (e.g., scFv) comprises a light chain variable (VL) region of a CD22 binding domain described herein, e.g., in Table 1A or Table 3A; and / or a heavy chain variable (VH) region of a CD22 binding domain described herein, e.g., in Table 1A or Table 3A.

[0049] 8. The CD22 antigen-binding domain is a VL region comprising an amino acid sequence having one, two, or three or more alterations (e.g., substitutions) of a CD22 VL region sequence provided in Table 1A or Table 3A, but not more than 30, 20, or 10 alterations (e.g., substitutions); a VL region comprising an amino acid sequence having at least 95% identity to a CD22 VL region sequence provided in Table 1A or Table 3A; or VL regions encoded by nucleotide sequences encoding the amino acid sequences of the CD22 VL region sequences provided in Table 1A or Table 3A 8. The nucleic acid molecule of embodiment 7, comprising:

[0050] 9. The CD22 antigen-binding domain is a VH region comprising an amino acid sequence having one, two, or three or more alterations (e.g., substitutions) of a CD22 VH region sequence provided in Table 1A or Table 3A, but not more than 30, 20, or 10 alterations (e.g., substitutions); a VH region comprising an amino acid sequence having at least 95% identity to a CD22 VH region sequence provided in Table 1A or Table 3A; or VH regions encoded by nucleotide sequences encoding the amino acid sequences of the CD22 VH region sequences provided in Table 1A or Table 3A 9. The nucleic acid molecule of embodiment 7 or 8, comprising:

[0051] 10. The nucleic acid molecule of any one of embodiments 7-9, wherein the VH and VL regions of the CD22 antigen-binding domain are linked by a linker having at least 95%, 96%, 97%, 98%, 99%, or 100% identity to a linker described herein, e.g., a linker disclosed in Table 4A.

[0052] 11. The CD22 antigen-binding domain is an scFv comprising an amino acid sequence having one, two, or three or more alterations (e.g., substitutions) of a CD22 scFv sequence provided in Table 1A or Table 3A, e.g., SEQ ID NO: 50, but not more than 30, 20, or 10 alterations (e.g., substitutions); an scFv comprising an amino acid sequence having at least 95% identity to a CD22 scFv sequence provided in Table 1A or Table 3A, e.g., SEQ ID NO: 50; a CD22 scFv sequence provided in Table 1A or Table 3A, e.g., an scFv comprising the amino acid sequence of SEQ ID NO: 50; or scFvs encoded by a nucleotide sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity to a CD22 scFv sequence provided in Table 1A or Table 3A, e.g., SEQ ID NO: 49 or 51. 10. The nucleic acid molecule of any one of the preceding embodiments, comprising:

[0053] 12. The CD19 antigen-binding domain is one or more (e.g., all three) light chain complementarity determining region 1 (LC CDR1), light chain complementarity determining region 2 (LC CDR2), and light chain complementarity determining region 3 (LC CDR3) of a CD19 binding domain described herein, e.g., in Table 1A, Table 2A, Table 3A, or Table 5A; and / or One or more (e.g., all three) heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2), and heavy chain complementarity determining region 3 (HC CDR3) of a CD19 binding domain described herein, e.g., in Table 1A, Table 2A, Table 3A, or Table 5A 10. The nucleic acid molecule of any one of the preceding embodiments, comprising:

[0054] 13. The nucleic acid molecule of embodiment 12, wherein the CD19 antigen-binding domain comprises the LC CDR1, LC CDR2 and LC CDR3 of a CD19 binding domain described herein, e.g., in Table 1A or Table 2A; and / or the HC CDR1, HC CDR2 and HC CDR3 of a CD19 binding domain described herein, e.g., in Table 1A, Table 2A or Table 3A.

[0055] 14. The nucleic acid molecule of embodiment 12 or 13, wherein the CD19 antigen-binding domain comprises an LC CDR1 of SEQ ID NO: 40, an LC CDR2 of SEQ ID NO: 41; and an LC CDR3 of SEQ ID NO: 42; and / or an HC CDR1 of SEQ ID NO: 35, an HC CDR2 of SEQ ID NOs: 36-38; and an HC CDR3 of SEQ ID NO: 39.

[0056] 15. The nucleic acid molecule of any one of embodiments 12-14, wherein the CD19 antigen binding domain (e.g., scFv) comprises a light chain variable (VL) region of a CD19 binding domain described herein, e.g., in Table 1A, Table 3A, or Table 5A; and / or a heavy chain variable (VH) region of a CD19 binding domain described herein, e.g., in Table 1A, Table 3A, or Table 5A.

[0057] 16. The CD19 antigen-binding domain is an amino acid sequence having one, two, or three or more alterations (e.g., substitutions) of a CD19 VL region sequence provided in Table 1A, Table 3A, or Table 5A, but not more than 30, 20, or 10 alterations (e.g., substitutions); an amino acid sequence having at least 95% identity to a CD19 VL region sequence provided in Table 1A, Table 3A, or Table 5A; or The amino acid sequence of the CD19 VL region sequence provided in Table 1A, Table 3A, or Table 5A 16. The nucleic acid molecule of embodiment 15, comprising a VL region comprising:

[0058] 17. The CD19 antigen-binding domain is an amino acid sequence having one, two, or three or more alterations (e.g., substitutions) of a CD19 VH region sequence provided in Table 1A, Table 3A, or Table 5A, but not more than 30, 20, or 10 alterations (e.g., substitutions); an amino acid sequence having at least 95% identity to a CD19 VH region sequence provided in Table 1A, Table 3A, or Table 5A; or The amino acid sequence of the CD19 VH region sequence provided in Table 1A, Table 3A, or Table 5A 17. The nucleic acid molecule of embodiment 15 or 16, comprising a VH region comprising:

[0059] 18. The nucleic acid molecule of any one of embodiments 15-17, wherein the VH and VL regions of the CD19 antigen-binding domain are connected by a linker having at least 95%, 96%, 97%, 98%, 99%, or 100% identity to a linker described herein, e.g., a linker disclosed in Table 4A.

[0060] 19. The CD19 antigen-binding domain is an scFv comprising an amino acid sequence having one, two, or three or more alterations (e.g., substitutions) of a CD19 scFv sequence provided in Table 1A, Table 3A, or Table 5A, e.g., SEQ ID NO: 44, but not more than 30, 20, or 10 alterations (e.g., substitutions); an scFv comprising an amino acid sequence having at least 95% identity to a CD19 scFv sequence provided in Table 1A, Table 3A, or Table 5A, e.g., SEQ ID NO: 44; a CD19 scFv sequence provided in Table 1A, Table 3A, or Table 5A, e.g., an scFv comprising the amino acid sequence of SEQ ID NO: 44; or scFvs encoded by a nucleotide sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity to a CD19 scFv sequence provided in Table 1A, Table 3A or Table 5A, e.g., SEQ ID NO: 43 or 48. 10. The nucleic acid molecule of any one of the preceding embodiments, comprising:

[0061] 20. The nucleic acid molecule of any of the preceding embodiments, wherein the first transmembrane domain and the second transmembrane domain comprise an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 65.

[0062] 21. The nucleic acid molecule of any of the preceding embodiments, wherein the first transmembrane domain and the second transmembrane domain comprise an amino acid sequence having 1, 2, 3, 4, 5, 6, or 7 alterations (e.g., substitutions) relative to the amino acid sequence of SEQ ID NO: 65.

[0063] 22. The nucleic acid molecule of any of the preceding embodiments, wherein the first transmembrane domain and the second transmembrane domain comprise the amino acid sequence of SEQ ID NO: 65.

[0064] 23. The nucleic acid molecule of any of the preceding embodiments, wherein the nucleotide sequence encoding the first transmembrane domain differs from the nucleotide sequence encoding the second transmembrane domain by at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100%.

[0065] 24. The nucleic acid molecule of any of the preceding embodiments, wherein the nucleotide sequence encoding the first transmembrane domain differs from the nucleotide sequence encoding the second transmembrane domain by at least 1 nucleotide, 10 nucleotides, 20 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 60 nucleotides, 70 nucleotides, 80 nucleotides, 90 nucleotides, 100 nucleotides, 150 nucleotides, 200 nucleotides, or all nucleotides.

[0066] 25. The nucleic acid molecule of any of the preceding embodiments, wherein the nucleotide sequence encoding the first transmembrane domain is selected from sequences having at least 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 64 or 66.

[0067] 26. The nucleic acid molecule of any of the preceding embodiments, wherein the nucleotide sequence encoding the second transmembrane domain is selected from sequences having at least 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 67 or 68.

[0068] 27. The nucleic acid molecule of any of the preceding embodiments, wherein the first costimulatory signaling domain and the second costimulatory signaling domain comprise an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 70.

[0069] 28. The nucleic acid molecule of any of the preceding embodiments, wherein the first costimulatory signaling domain and the second costimulatory signaling domain comprise amino acid sequences having 1, 2, 3, 4, or 5 alterations (e.g., substitutions) relative to the amino acid sequence of any one of SEQ ID NO: 70.

[0070] 29. The nucleic acid molecule of any of the preceding embodiments, wherein the first costimulatory signaling domain and the second costimulatory signaling domain comprise the amino acid sequence of any one of SEQ ID NO: 70.

[0071] 30. The nucleic acid molecule of any of the preceding embodiments, wherein the nucleotide sequence encoding the first costimulatory signaling domain differs from the nucleotide sequence encoding the second costimulatory signaling domain by at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100%.

[0072] 31. The nucleic acid molecule of any of the preceding embodiments, wherein the nucleotide sequence encoding the first costimulatory signaling domain differs from the nucleotide sequence encoding the second costimulatory signaling domain by at least 1 nucleotide, 10 nucleotides, 20 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 60 nucleotides, 70 nucleotides, 80 nucleotides, 90 nucleotides, 100 nucleotides, 120 nucleotides, or all nucleotides.

[0073] 32. The nucleic acid molecule of any of the preceding embodiments, wherein the nucleotide sequence encoding the first costimulatory domain is selected from sequences having at least 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 69 or 72.

[0074] 33. The nucleic acid molecule of any of the preceding embodiments, wherein the nucleotide sequence encoding the second costimulatory domain is selected from sequences having at least 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 71 or 73.

[0075] 34. The nucleic acid molecule of any of the preceding embodiments, wherein the first primary signaling domain and the second primary signaling domain comprise an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 75.

[0076] 35. The nucleic acid molecule of any of the preceding embodiments, wherein the first primary signaling domain and the second primary signaling domain comprise an amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 alterations (e.g., substitutions) relative to the amino acid sequence of SEQ ID NO: 75.

[0077] 36. The nucleic acid molecule of any of the preceding embodiments, wherein the first primary signaling domain and the second primary signaling domain comprise the amino acid sequence of SEQ ID NO: 75.

[0078] 37. The nucleic acid molecule of any of the preceding embodiments, wherein the nucleotide sequence encoding the first primary signaling domain differs from the nucleotide sequence encoding the second primary signaling domain by at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or 100%.

[0079] 38. The nucleic acid molecule of any of the preceding embodiments, wherein the nucleotide sequence encoding the first primary signaling domain differs from the nucleotide sequence encoding the second primary signaling domain by at least 1 nucleotide, 10 nucleotides, 20 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 60 nucleotides, 70 nucleotides, 80 nucleotides, 90 nucleotides, 100 nucleotides, 150 nucleotides, 200 nucleotides, 250 nucleotides, 300 nucleotides, or all nucleotides.

[0080] 39. The nucleic acid molecule of any of the preceding embodiments, wherein the nucleotide sequence encoding the first primary signaling domain is selected from a sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 74 or 77.

[0081] 40. The nucleic acid molecule of any of the preceding embodiments, wherein the nucleotide sequence encoding the second primary signaling domain is selected from a sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 76 or 78.

[0082] 41. The nucleic acid molecule of any of the preceding embodiments, wherein the first CAR and / or the second CAR comprises a signal peptide, e.g., a peptide comprising a stretch of hydrophobic amino acids, e.g., 5 to 16 residues.

[0083] 42. The nucleic acid molecule of embodiment 41, wherein the signal peptide is selected from the group consisting of CD8α signal peptide, interleukin-2 signal peptide, human albumin signal peptide, human chymotrypsinogen signal peptide, human trypsinogen-2 signal peptide, or other similar signal peptides disclosed in Stern B. et al. "Improving mammalian cell factories: The selection of signal peptide has a major impact on recombinant protein synthesis and secretion in mammalian cells" (2007).

[0084] 43. The signal peptide is Contains a signal peptide provided in Table 4A; comprising the amino acid sequence of SEQ ID NO: 59; or 43. The nucleic acid molecule of embodiment 41 or 42, encoded by a nucleic acid of any one of SEQ ID NOs: 58, 60, 61, 62 or 63, or a nucleic acid having at least 95%, 96%, 97%, 98% or 99% identity thereto.

[0085] 44. The nucleic acid molecule of any of the preceding embodiments, comprising, in the 5' to 3' direction, a first CAR followed by a second CAR.

[0086] 45. The nucleic acid molecule of any of embodiments 1-43, comprising, in the 5' to 3' direction, a second CAR followed by a first CAR.

[0087] 46. ​​The nucleic acid molecule of any of the preceding embodiments, further comprising a protease cleavage site (e.g., a T2A, P2A, E2A, or F2A cleavage site) or an internal ribosome entry site.

[0088] 47. The nucleic acid molecule of embodiment 46, wherein the protease cleavage site is a P2A site.

[0089] 48. The nucleic acid molecule of embodiment 46 or 47, wherein the P2A site comprises a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 86; or a nucleotide sequence of SEQ ID NO: 85 or 87.

[0090] 49. The nucleic acid molecule of any one of embodiments 46-48, wherein the protease cleavage site or internal ribosome entry site is located between the first CAR and the second CAR.

[0091] 50. The nucleic acid molecule of any one of embodiments 46-49, wherein the protease cleavage site is positioned such that the cell can express a fusion protein comprising the first CAR and the second CAR, and optionally, the fusion protein is processed by proteolytic cleavage into two peptides.

[0092] 51. The nucleic acid molecule of any of the preceding embodiments, wherein the CAR molecule comprises the nucleotide sequence of SEQ ID NO: 11, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0093] 52. The nucleic acid molecule of any of the preceding embodiments, wherein the CAR molecule comprises a first CAR comprising the amino acid sequence of SEQ ID NO: 13, or amino acids with at least 95%, 96%, 97%, 98%, or 99% identity thereto, and a second CAR comprising the amino acid sequence of SEQ ID NO: 14, or amino acids with at least 95%, 96%, 97%, 98%, or 99% identity thereto.

[0094] 53. The nucleic acid molecule of any of the preceding embodiments, wherein the CAR molecule comprises the amino acid sequence of SEQ ID NO: 12, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity thereto.

[0095] 54. The nucleic acid molecule of any of embodiments 1-50, wherein the CAR molecule is encoded by the nucleotide sequence of SEQ ID NO: 15 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0096] 55. The nucleic acid molecule of any one of embodiments 1 to 50 or 54, wherein the CAR molecule comprises a first CAR comprising the amino acid sequence of SEQ ID NO: 17 or amino acids having at least 95%, 96%, 97%, 98% or 99% identity thereto, and a second CAR comprising the amino acid sequence of SEQ ID NO: 18 or amino acids having at least 95%, 96%, 97%, 98% or 99% identity thereto.

[0097] 56. The nucleic acid molecule of any one of embodiments 1 to 50, or 54, or 55, wherein the CAR molecule is encoded by a nucleic acid encoding the amino acid sequence of SEQ ID NO: 16, or an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity thereto.

[0098] 57. The nucleic acid molecule of any of embodiments 1-50, wherein the CAR molecule is encoded by the nucleotide sequence of SEQ ID NO: 19 or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0099] 58. The nucleic acid molecule of any one of embodiments 1 to 50 or 57, wherein the CAR molecule comprises a first CAR comprising the amino acid sequence of SEQ ID NO: 13 or amino acids having at least 95%, 96%, 97%, 98% or 99% identity thereto, and a second CAR comprising the amino acid sequence of SEQ ID NO: 14 or amino acids having at least 95%, 96%, 97%, 98% or 99% identity thereto.

[0100] 59. The nucleic acid molecule of any one of embodiments 1 to 50 or 57 or 58, wherein the CAR molecule comprises the amino acid sequence of SEQ ID NO: 12 or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto.

[0101] 60. A nucleic acid molecule encoding a chimeric antigen receptor (CAR) molecule, wherein the CAR molecule comprises, in a 5' to 3' direction: (a) a first CAR comprising a first signal peptide; a first antigen-binding domain that binds to CD22; a first transmembrane domain; a first costimulatory signaling domain; and a first primary signaling domain; (b) P2A protease cleavage site; (c) a second CAR comprising a second signal peptide; a second antigen-binding domain that binds to CD19; a second transmembrane domain; a second costimulatory domain; and a second primary signaling domain. Including, The CAR molecule is a nucleic acid molecule encoded by a nucleic acid encoding the nucleotide sequence of SEQ ID NO: 11; or the amino acid sequence of SEQ ID NO: 12.

[0102] 61. A nucleic acid molecule encoding a chimeric antigen receptor (CAR) molecule, wherein the CAR molecule comprises, in a 5' to 3' direction: (a) a second CAR comprising a second signal peptide; a second antigen-binding domain that binds to CD19; a second transmembrane domain; a second costimulatory domain; and a second primary signaling domain; (b) P2A protease cleavage site; (c) a first CAR comprising: a first signal peptide; a first antigen-binding domain that binds to CD22; a first transmembrane domain; a first costimulatory signaling domain; and a first primary signaling domain. Including, The CAR molecule is a nucleic acid molecule encoded by a nucleic acid encoding the nucleotide sequence of SEQ ID NO: 15; or the amino acid sequence of SEQ ID NO: 16.

[0103] 62. A nucleic acid molecule encoding a chimeric antigen receptor (CAR) molecule, wherein the CAR molecule comprises, in a 5' to 3' direction: (a) a first CAR comprising a first signal peptide; a first antigen-binding domain that binds to CD22; a first transmembrane domain; a first costimulatory signaling domain; and a first primary signaling domain; (b) P2A protease cleavage site; (c) a second CAR comprising a second signal peptide; a second antigen-binding domain that binds to CD19; a second transmembrane domain; a second costimulatory domain; and a second primary signaling domain. Including, The CAR molecule is a nucleic acid molecule encoded by a nucleic acid encoding the nucleotide sequence of SEQ ID NO: 19; or the amino acid sequence of SEQ ID NO: 12.

[0104] 63. The nucleic acid molecule of any of the preceding embodiments, further comprising a promoter sequence, for example the EF1 promoter.

[0105] 64. The nucleic acid molecule of any of the preceding embodiments, wherein the nucleotide sequence encoding the first CAR and the nucleotide sequence encoding the second CAR are located in a single nucleic acid construct.

[0106] 65. The nucleic acid molecule of embodiment 64, wherein the nucleotide sequence encoding the first CAR and the nucleic acid encoding the second CAR are located in the same vector.

[0107] 66. The nucleic acid molecule of any of the preceding embodiments, wherein the nucleotide sequence encoding the first CAR and the nucleotide sequence encoding the second CAR are located on different nucleic acid constructs, e.g., the nucleotide sequence encoding the first CAR is located on the first nucleic acid construct and the nucleotide sequence encoding the second CAR is located on the second nucleic acid construct.

[0108] 67. The nucleic acid molecule of embodiment 66, wherein the nucleotide sequence encoding the first CAR is placed in a first vector.

[0109] 68. The nucleic acid molecule of embodiment 66, wherein the nucleotide sequence encoding the second CAR is placed in a second vector.

[0110] 69. The nucleic acid molecule comprises a viral element, for example, a viral packaging element.

[0111] 70. A vector comprising the nucleic acid molecule of any of embodiments 1 to 69.

[0112] 71. The vector of embodiment 70, which is selected from DNA, RNA, a plasmid, a lentiviral vector, an adenoviral vector, or a retroviral vector.

[0113] 72. A cell (e.g., an immune effector cell) comprising the vector of embodiment 70 or 71 or the nucleic acid molecule of any of embodiments 1 to 69.

[0114] 73. A cell (e.g., an immune effector cell) comprising a nucleic acid molecule encoding a chimeric antigen receptor (CAR) molecule, wherein the CAR molecule is: (a) a first CAR comprising a first antigen-binding domain that binds to CD22 and a first transmembrane domain; a first costimulatory signaling domain; and / or a first primary signaling domain; and (b) a second CAR comprising a second antigen-binding domain that binds to CD19 and a second transmembrane domain; a second costimulatory domain; and / or a second primary signaling domain. Including, (i) the first transmembrane domain and the second transmembrane domain comprise the amino acid sequence of SEQ ID NO: 65, or an amino acid sequence having at least 90% identity thereto, and optionally, the nucleotide sequence encoding the first transmembrane domain and contained in the nucleic acid molecule is different from the nucleotide sequence encoding the second transmembrane domain and contained in the nucleic acid molecule; (ii) the first costimulatory signaling domain and the second costimulatory signaling domain comprise the amino acid sequence of SEQ ID NO: 70, or an amino acid sequence having at least 90% identity thereto, and optionally, the nucleotide sequence encoding the first costimulatory signaling domain and contained in the nucleic acid molecule is different from the nucleotide sequence encoding the second costimulatory signaling domain and contained in the nucleic acid molecule; and / or (iii) the first primary signaling domain and the second primary signaling domain comprise the amino acid sequence of any one of SEQ ID NO: 75, or an amino acid sequence having at least 90% identity thereto, and optionally, the nucleotide sequence encoding the primary signaling domains and contained in the nucleic acid molecule is different from the nucleotide sequence encoding the second primary signaling domain and contained in the nucleic acid molecule.

[0115] 74. A cell (e.g., an immune effector cell) comprising a chimeric antigen receptor (CAR) molecule, wherein the CAR molecule is: (a) a first CAR comprising a first antigen-binding domain that binds to CD22 and a first transmembrane domain; a first costimulatory signaling domain; and / or a first primary signaling domain; and (b) a second CAR comprising a second antigen-binding domain that binds to CD19 and a second transmembrane domain; a second costimulatory domain; and / or a second primary signaling domain. Including, (i) the first transmembrane domain and the second transmembrane domain comprise the amino acid sequence of SEQ ID NO: 65, or an amino acid sequence having at least 90% identity thereto, and optionally, the nucleotide sequence encoding the first transmembrane domain and contained in the nucleic acid molecule is different from the nucleotide sequence encoding the second transmembrane domain and contained in the nucleic acid molecule; (ii) the first costimulatory signaling domain and the second costimulatory signaling domain comprise the amino acid sequence of SEQ ID NO: 70, or an amino acid sequence having at least 90% identity thereto, and optionally, the nucleotide sequence encoding the first costimulatory signaling domain and contained in the nucleic acid molecule is different from the nucleotide sequence encoding the second costimulatory signaling domain and contained in the nucleic acid molecule; and / or (iii) the first primary signaling domain and the second primary signaling domain comprise the amino acid sequence of SEQ ID NO: 75, or an amino acid sequence having at least 90% identity thereto, and optionally, the nucleotide sequence encoding the primary signaling domains and contained in the nucleic acid molecule is different from the nucleotide sequence encoding the second primary signaling domain and contained in the nucleic acid molecule.

[0116] 75. The cell of embodiment 74, comprising a nucleic acid encoding a CAR molecule.

[0117] 76. A cell according to embodiment 73 or 75, comprising a nucleic acid molecule according to any of embodiments 1 to 69 or a vector according to embodiment 70 or 71.

[0118] 77. (a) A first CAR comprising: a first signal peptide; a first antigen-binding domain that binds to CD22; a first transmembrane domain; a first costimulatory signaling domain; and a first primary signaling domain; (b) a second CAR comprising a second signal peptide; a second antigen-binding domain that binds to CD19; a second transmembrane domain; a second costimulatory domain; and a second primary signaling domain. A cell comprising a chimeric antigen receptor (CAR) molecule, comprising: A cell, wherein the CAR molecule is encoded by the nucleotide sequence of SEQ ID NO: 11; or comprises the amino acid sequence of SEQ ID NO: 12.

[0119] 78. (a) A second CAR comprising a second signal peptide; a second antigen-binding domain that binds to CD19; a second transmembrane domain; a second costimulatory domain; and a second primary signaling domain; (b) a first CAR comprising: a first signal peptide; a first antigen-binding domain that binds to CD22; a first transmembrane domain; a first costimulatory signaling domain; and a first primary signaling domain. A cell comprising a chimeric antigen receptor (CAR) molecule, comprising: A cell, wherein the CAR molecule is encoded by the nucleotide sequence of SEQ ID NO: 15; or comprises the amino acid sequence of SEQ ID NO: 16.

[0120] 79. (a) A first CAR comprising: a first signal peptide; a first antigen-binding domain that binds to CD22; a first transmembrane domain; a first costimulatory signaling domain; and a first primary signaling domain; (b) a second CAR comprising a second signal peptide; a second antigen-binding domain that binds to CD19; a second transmembrane domain; a second costimulatory domain; and a second primary signaling domain. A cell comprising a chimeric antigen receptor (CAR) molecule, comprising: A cell, wherein the CAR molecule is encoded by the nucleotide sequence of SEQ ID NO: 19; or comprises the amino acid sequence of SEQ ID NO: 12.

[0121] 80. The cell of any one of embodiments 72-79, which is an immune effector cell, such as a T cell (e.g., a CD3+, CD4+, or CD8+ T cell) or an NK cell.

[0122] 81. The cell of any one of embodiments 72 to 80, which is a human cell.

[0123] 82. A method for producing cells (e.g., immune effector cells), comprising transducing immune effector cells, e.g., T cells or NK cells, with the vector of embodiment 70 or 71.

[0124] 83. A method for producing a cell (e.g., an immune effector cell), comprising introducing into an immune effector cell, e.g., a T cell or an NK cell, a nucleic acid molecule of any one of embodiments 1-69.

[0125] 84. A method for generating a population of RNA-engineered cells, comprising introducing in vitro transcribed or synthetic RNA into cells, wherein the RNA comprises a nucleic acid molecule of any one of embodiments 1 to 69.

[0126] 85. A bispecific antigen-binding domain comprising a first antigen-binding domain that binds to CD22 and a second antigen-binding domain that binds to CD19.

[0127] 86. The bispecific antigen-binding domain of embodiment 85, wherein the first antigen-binding domain can be upstream of the second antigen-binding domain (e.g., towards the N-terminus), or the first antigen-binding domain can be downstream of the second antigen-binding domain (e.g., towards the C-terminus).

[0128] 87. The bispecific antigen-binding domain of embodiment 85 or 86, wherein each of the first and second antigen-binding domains is an scFv, e.g., comprising a light chain variable (VL) domain and a heavy chain variable (VH) domain.

[0129] 88. The bispecific antigen-binding domain of embodiment 87, wherein the VH can be upstream or downstream of the VL.

[0130] 89. The bispecific antigen-binding domain of embodiment 87 or 88, wherein the first antigen-binding domain comprises an scFv comprising a first VH (VH1) and a first VL (VL1).

[0131] 90. The bispecific antigen-binding domain of any one of embodiments 87-89, wherein the second antigen-binding domain comprises an scFv comprising a second VH (VH2) and a second VL (VL2).

[0132] 91. The bispecific antigen-binding domain of any one of embodiments 87-90, wherein the first antigen-binding domain is arranged comprising a VH1 upstream of a VL1.

[0133] 92. The bispecific antigen-binding domain of any one of embodiments 87-90, wherein the first antigen-binding domain is arranged comprising a VL1 upstream of a VH1.

[0134] 93. The bispecific antigen-binding domain of any one of embodiments 87-92, wherein the second antigen-binding domain is arranged comprising a VH2 upstream of a VL2.

[0135] 94. The bispecific antigen-binding domain of any one of embodiments 87-92, wherein the second antigen-binding domain is arranged comprising a VL2 upstream of a VH2.

[0136] 95. The bispecific antigen-binding domain of any one of embodiments 87-90 or 92-93, wherein the antigen-binding domain has the following configuration from N-terminus to C-terminus: VL1-VH1-VH2-VL2.

[0137] 96. The bispecific antigen-binding domain of any one of embodiments 87-90, 91 or 93, wherein the antigen-binding domain has the following configuration from N-terminus to C-terminus: VH1-VL1-VH2-VL2.

[0138] 97. The bispecific antigen-binding domain of any one of embodiments 87-90, 92, or 94, wherein the antigen-binding domain has the following configuration from N-terminus to C-terminus: VL1-VH1-VL2-VH2.

[0139] 98. The bispecific antigen-binding domain of any one of embodiments 87-90, 91 or 94, wherein the antigen-binding domain has the following configuration from N-terminus to C-terminus: VH1-VL1-VL2-VH2.

[0140] 99. The bispecific antigen-binding domain of any one of embodiments 85-98, wherein a linker is positioned between the first antigen-binding domain and the second antigen-binding domain.

[0141] 100. The bispecific antigen-binding domain of embodiment 99, wherein a linker is positioned between the scFv of the first antigen-binding domain and the scFv of the second antigen-binding domain.

[0142] 101. The linker between VH1 and VH2 when the construct has the configuration VL1-VH1-VH2-VL2; between VL1 and VH2 when the construct has the configuration VH1-VL1-VH2-VL2; between VH1 and VL2 when the construct has the configuration VL1-VH1-VL2-VH2; or When the construct has a VH1-VL1-VL2-VH2 configuration, the VL1 and VL2 101. The bispecific antigen-binding domain of embodiment 100, wherein

[0143] 102. The bispecific antigen-binding domain of any one of embodiments 99-101, wherein the linker is long enough to avoid mispairing between the domains of the two scFvs.

[0144] 103. The bispecific antigen-binding domain of any one of embodiments 99-102, wherein the linker is a linker described herein, such as a linker provided in Table 1A or Table 4A.

[0145] 104. The bispecific antigen-binding domain of any one of embodiments 99-103, wherein the linker is a (Gly4-Ser)n linker, where n is 1, 2, 3, 4, 5, or 6.

[0146] 105. The bispecific antigen-binding domain of embodiment 104, wherein n=1, and for example, the linker has the amino acid sequence Gly4-Ser.

[0147] 106. The bispecific antigen-binding domain of embodiment 104, wherein n=3, for example SEQ ID NO: 82.

[0148] 107. The bispecific antigen-binding domain of any one of embodiments 99-103, wherein the linker comprises the amino acid sequence: LAEAAAK, such as SEQ ID NO: 80.

[0149] 108. The bispecific antigen-binding domain of any one of embodiments 99-107, wherein a linker, such as a linker described herein, is positioned between the VL and VH of the scFv of the first antigen-binding domain.

[0150] 109. The bispecific antigen-binding domain of any one of embodiments 99-108, wherein a linker, such as a linker described herein, is positioned between the VL and VH of the scFv of the second antigen-binding domain.

[0151] 110. The bispecific antigen-binding domain of any one of embodiments 99-109, comprising the amino acid sequence of the antigen-binding domain as provided in Table 1A or Table 4A, such as any one of SEQ ID NOs: 2, 4, 6, 8, 10, 44, 47, 53 or 55, or an amino acid sequence with at least 95%, 96%, 97%, 98% or 99% identity thereto.

[0152] 112. The bispecific antigen-binding domain of any one of embodiments 99-109, encoded by the nucleotide sequence of the antigen-binding domain provided in Table 1A or Table 4a, such as any one of SEQ ID NOs: 1, 3, 5, 7, 9, 43, 45, 46, 52, 54, 56 or 57, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0153] 113. A bispecific chimeric antigen receptor (CAR) comprising the bispecific antigen-binding domain of any one of embodiments 85 to 112.

[0154] 114. A nucleic acid construct encoding a bispecific chimeric antigen receptor (CAR), wherein the nucleic acid construct encodes the bispecific antigen-binding domain of any one of embodiments 85 to 112.

[0155] 115. A first antigen-binding domain that binds to CD22 and a second antigen-binding domain that binds to CD19. wherein the CAR comprises a transmembrane domain, a costimulatory domain, and / or a primary signaling domain.

[0156] 116. The CAR of embodiment 115, comprising the bispecific antigen-binding domain of any one of embodiments 86 to 112.

[0157] 117. Bispecific antigen-binding domain; transmembrane domain; and costimulatory signaling domain; a bispecific antigen-binding domain; a transmembrane domain; and a primary signaling domain; or a bispecific antigen-binding domain; a transmembrane domain; a costimulatory signaling domain; and a first primary signaling domain. 117. The CAR of embodiment 115 or 116, comprising:

[0158] 118. The CAR of any one of embodiments 115 to 117, wherein the CAR comprises a transmembrane domain, which is selected from the α, β or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD123, CD134, CD137 or CD154.

[0159] 119. The CAR of embodiment 118, wherein the bispecific antigen-binding domain is linked to the transmembrane domain by a hinge region, for example a hinge region described herein.

[0160] 120. The CAR of any one of embodiments 115-119, wherein the CAR comprises a costimulatory domain, the costimulatory domain comprising a signaling domain of OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278) or 4-1BB (CD137).

[0161] 121. The CAR of any one of embodiments 115-120, wherein the costimulatory domain comprises a 4-1BB signaling domain.

[0162] 122. The CAR of any one of embodiments 115-121, wherein the CAR comprises a primary signaling domain comprising the signaling domain of CD3ζ.

[0163] 123. The CAR of any one of embodiments 115-122, wherein the CAR comprises an amino acid sequence provided in Table 4A, such as any one of SEQ ID NOs: 2, 4, 6, 8, 10, or an amino acid sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto.

[0164] 124. A chimeric antigen receptor (CAR) comprising a bispecific antigen-binding domain comprising a first antigen-binding domain that binds to CD22 and a second antigen-binding domain that binds to CD19, (i) the first and second antigen-binding domains are each scFvs; (ii) the second antigen-binding domain is oriented upstream of the first antigen-binding domain; and (iii) a linker is disposed between the first antigen-binding domain and the second antigen-binding domain; CAR comprises a transmembrane domain, a costimulatory domain, and a primary signaling domain; The CAR comprises the amino acid sequence of SEQ ID NO: 2 or a sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto.

[0165] 125. A chimeric antigen receptor (CAR) comprising a bispecific antigen-binding domain comprising a first antigen-binding domain that binds to CD22 and a second antigen-binding domain that binds to CD19, (i) the first and second antigen-binding domains are each scFvs; (ii) the first antigen-binding domain is oriented upstream of the second antigen-binding domain; and (iii) a linker is disposed between the first antigen-binding domain and the second antigen-binding domain; CAR comprises a transmembrane domain, a costimulatory domain, and a primary signaling domain; The CAR comprises the amino acid sequence of SEQ ID NO: 4 or a sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto.

[0166] 126. A chimeric antigen receptor (CAR) comprising a bispecific antigen-binding domain comprising a first antigen-binding domain that binds to CD22 and a second antigen-binding domain that binds to CD19, (i) the first and second antigen-binding domains are each scFvs; (ii) the first antigen-binding domain is oriented upstream of the second antigen-binding domain; and (iii) a linker is disposed between the first antigen-binding domain and the second antigen-binding domain; CAR comprises a transmembrane domain, a costimulatory domain, and a primary signaling domain; The CAR comprises the amino acid sequence of SEQ ID NO: 6 or a sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto.

[0167] 127. A chimeric antigen receptor (CAR) comprising a bispecific antigen-binding domain comprising a first antigen-binding domain that binds to CD22 and a second antigen-binding domain that binds to CD19, (i) the first and second antigen-binding domains are each scFvs; (ii) the first antigen-binding domain is oriented upstream of the second antigen-binding domain; and (iii) a linker is disposed between the first antigen-binding domain and the second antigen-binding domain; CAR comprises a transmembrane domain, a costimulatory domain, and a primary signaling domain; The CAR comprises the amino acid sequence of SEQ ID NO: 8 or a sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto.

[0168] 128. A chimeric antigen receptor (CAR) comprising a bispecific antigen-binding domain comprising a first antigen-binding domain that binds to CD22 and a second antigen-binding domain that binds to CD19, (i) the first and second antigen-binding domains are each scFvs; (ii) the first antigen-binding domain is oriented upstream of the second antigen-binding domain; and (iii) a linker is disposed between the first antigen-binding domain and the second antigen-binding domain; CAR comprises a transmembrane domain, a costimulatory domain, and a primary signaling domain; The CAR comprises the amino acid sequence of SEQ ID NO: 10 or a sequence having at least 95%, 96%, 97%, 98% or 99% identity thereto.

[0169] 129. A nucleic acid encoding a chimeric antigen receptor (CAR nucleic acid), wherein the CAR is: a first antigen-binding domain that binds to CD22 and a second antigen-binding domain that binds to CD19 a bispecific antigen-binding domain comprising The CAR is a CAR nucleic acid comprising a transmembrane domain, a costimulatory domain, and / or a primary signaling domain.

[0170] 130. The CAR nucleic acid of embodiment 129, comprising a nucleic acid encoding the bispecific antigen-binding domain of any one of embodiments 86 to 112.

[0171] 131.CAR is a bispecific antigen-binding domain; a transmembrane domain; and a costimulatory signaling domain; a bispecific antigen-binding domain; a transmembrane domain; and a primary signaling domain; or a bispecific antigen-binding domain; a transmembrane domain; a costimulatory signaling domain; and a first primary signaling domain. 131. The CAR nucleic acid of embodiment 129 or 130, comprising:

[0172] 132. The CAR nucleic acid of any one of embodiments 129 to 131, wherein the CAR comprises a transmembrane domain, and the transmembrane domain is selected from the alpha, beta or zeta chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD123, CD134, CD137 or CD154.

[0173] 133. The CAR nucleic acid of embodiment 132, wherein the bispecific antigen-binding domain is linked to the transmembrane domain by a hinge region, e.g., a hinge region described herein.

[0174] 134. The CAR nucleic acid of any one of embodiments 129 to 133, wherein the CAR comprises a costimulatory domain, and the costimulatory domain comprises a signaling domain of OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278) or 4-1BB (CD137).

[0175] 135. The CAR nucleic acid of embodiment 134, wherein the costimulatory domain comprises a 4-1BB signaling domain.

[0176] 136. The CAR nucleic acid of any one of embodiments 129-135, wherein the CAR comprises a primary signaling domain comprising the signaling domain of CD3ζ.

[0177] 137. The CAR nucleic acid of any one of embodiments 129-136, comprising a nucleotide sequence provided in Table 4A, such as any one of SEQ ID NOs: 1, 3, 5, 7, or 9, or a nucleotide sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0178] 138. A nucleic acid encoding a chimeric antigen receptor (CAR nucleic acid), wherein the CAR comprises a bispecific antigen-binding domain comprising a first antigen-binding domain that binds to CD22 and a second antigen-binding domain that binds to CD19; (i) the first and second antigen-binding domains are each scFvs; (ii) the second antigen-binding domain is oriented upstream of the first antigen-binding domain; and (iii) a linker is disposed between the first antigen-binding domain and the second antigen-binding domain; CAR comprises a transmembrane domain, a costimulatory domain, and a primary signaling domain; The CAR is a CAR nucleic acid comprising the amino acid sequence of SEQ ID NO: 2 or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0179] 139. A nucleic acid encoding a chimeric antigen receptor (CAR nucleic acid), wherein the CAR comprises a bispecific antigen-binding domain comprising a first antigen-binding domain that binds to CD22 and a second antigen-binding domain that binds to CD19; (i) the first and second antigen-binding domains are each scFvs; (ii) the first antigen-binding domain is oriented upstream of the second antigen-binding domain; and (iii) a linker is disposed between the first antigen-binding domain and the second antigen-binding domain; CAR comprises a transmembrane domain, a costimulatory domain, and a primary signaling domain; The CAR is a CAR nucleic acid comprising the amino acid sequence of SEQ ID NO: 4 or a sequence having at least 80%, 85%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0180] 140. A nucleic acid encoding a chimeric antigen receptor (CAR nucleic acid), wherein the CAR comprises a bispecific antigen-binding domain comprising a first antigen-binding domain that binds to CD22 and a second antigen-binding domain that binds to CD19; (i) the first and second antigen-binding domains are each scFvs; (ii) the first antigen-binding domain is oriented upstream of the second antigen-binding domain; and (iii) a linker is disposed between the first antigen-binding domain and the second antigen-binding domain; CAR comprises a transmembrane domain, a costimulatory domain, and a primary signaling domain; The CAR is a CAR nucleic acid comprising the amino acid sequence of SEQ ID NO: 6 or a sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0181] 141. A nucleic acid encoding a chimeric antigen receptor (CAR nucleic acid), wherein the CAR comprises a bispecific antigen-binding domain comprising a first antigen-binding domain that binds to CD22 and a second antigen-binding domain that binds to CD19; (i) the first and second antigen-binding domains are each scFvs; (ii) the first antigen-binding domain is oriented upstream of the second antigen-binding domain; and (iii) a linker is disposed between the first antigen-binding domain and the second antigen-binding domain; CAR comprises a transmembrane domain, a costimulatory domain, and a primary signaling domain; The CAR is a CAR nucleic acid comprising the amino acid sequence of SEQ ID NO: 8 or a sequence having at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0182] 142. A nucleic acid encoding a chimeric antigen receptor (CAR nucleic acid), wherein the CAR comprises a bispecific antigen-binding domain comprising a first antigen-binding domain that binds to CD22 and a second antigen-binding domain that binds to CD19; (i) the first and second antigen-binding domains are each scFvs; (ii) the first antigen-binding domain is oriented upstream of the second antigen-binding domain; and (iii) a linker is disposed between the first antigen-binding domain and the second antigen-binding domain; CAR comprises a transmembrane domain, a costimulatory domain, and a primary signaling domain; The CAR is a CAR nucleic acid comprising the amino acid sequence of SEQ ID NO: 10 or a sequence having at least 95%, 96%, 97%, 98%, or 99% identity thereto.

[0183] 143. A vector comprising the bispecific antigen-binding domain of any one of embodiments 85 to 112, the CAR of any one of embodiments 113 or 115 to 128, or the CAR nucleic acid of any one of embodiments 114 or 129 to 142.

[0184] 144. A cell (e.g., an immune effector cell) comprising the bispecific antigen-binding domain of any one of embodiments 85 to 112, the CAR of any one of embodiments 113 or 115 to 128, the CAR nucleic acid of any one of embodiments 114 or 129 to 142, or the vector of embodiment 143.

[0185] 145. A cell comprising a chimeric antigen receptor (CAR), wherein the CAR is: a first antigen-binding domain that binds to CD22 and a second antigen-binding domain that binds to CD19 a bispecific antigen-binding domain comprising The CAR comprises a transmembrane domain, a costimulatory domain and / or a primary signaling domain.

[0186] 146. The cell of embodiment 145, comprising a CAR of any one of embodiments 116 to 123.

[0187] 147. A method of producing cells (e.g., immune effector cells), comprising: Transducing immune effector cells, such as T cells or NK cells, with the vector of embodiment 143; or Introducing the CAR nucleic acid molecule of any one of embodiments 129-142 into immune effector cells, such as T cells or NK cells. A method comprising:

[0188] 148. A pharmaceutical composition comprising a nucleic acid encoding a CAR molecule of any one of embodiments 1 to 69, a bispecific antigen-binding domain of any one of embodiments 85 to 112, a CAR of any one of embodiments 113 or 115 to 128, or a CAR nucleic acid of any one of embodiments 114 or 129 to 142, optionally comprising an excipient, carrier, diluent and / or stabilizer.

[0189] 149. A method for providing anti-tumor immunity, comprising administering to a subject in need thereof an effective amount of a population of cells, e.g., immune effector cells, comprising, e.g., expressing, a nucleic acid encoding the CAR molecule of any one of embodiments 1-69, the bispecific antigen-binding domain of any one of embodiments 85-112, the CAR of any one of embodiments 113 or 115-128, or the CAR nucleic acid of any one of embodiments 114 or 129-142.

[0190] 150. A population of cells, e.g., immune effector cells, comprising, e.g., expressing, a nucleic acid encoding the CAR molecule of any one of embodiments 1-69, the bispecific antigen-binding domain of any one of embodiments 85-112, the CAR of any one of embodiments 113 or 115-128, or the CAR nucleic acid of any one of embodiments 114 or 129-142, for use in a method for providing anti-tumor immunity to a subject.

[0191] 151. The method of embodiment 149 or the use of embodiment 150, wherein the cells are T cells or NK cells.

[0192] 152. The method of embodiment 149 or 151 or the use of embodiment 150 or 151, wherein the cells are autologous or allogeneic cells.

[0193] 153. The method of embodiment 149 or the use of embodiment 150, wherein the subject is a human.

[0194] 154. A method of treating a subject having a disease associated with an antigen (e.g., CD19 and / or CD22), comprising administering to a subject in need thereof an effective amount of a population of cells, e.g., immune effector cells, comprising, e.g., expressing, a nucleic acid encoding the CAR molecule of any one of embodiments 1-69, the bispecific antigen-binding domain of any one of embodiments 85-112, the CAR of any one of embodiments 113 or 115-128, or the CAR nucleic acid of any one of embodiments 114 or 129-142.

[0195] 155. A population of cells, e.g., immune effector cells, comprising, e.g., expressing, a nucleic acid encoding the CAR molecule of any one of embodiments 1-69, the bispecific antigen-binding domain of any one of embodiments 85-112, the CAR of any one of embodiments 113 or 115-128, or the CAR nucleic acid of any one of embodiments 114 or 129-142, for use in a method of treating a subject having a disease associated with an antigen (e.g., CD19 and / or CD22).

[0196] 156. The method of embodiment 154 or the use of embodiment 155, wherein the cells are T cells or NK cells.

[0197] 157. The method of embodiment 154 or 155 or the use of embodiment 154 or 155, wherein the cells are autologous or allogeneic cells.

[0198] 158. The method of embodiment 154 or the use of embodiment 155, wherein the subject is a human.

[0199] 159. The method of any one of embodiments 154 or 155-158 or the use of any one of embodiments 155-158, wherein the disease associated with CD19 and / or CD22 is selected from a proliferative disease, such as a cancer or malignant tumor, a precancerous condition, such as myelodysplasia, myelodysplastic syndrome or preleukemia, or a non-cancer-related indication associated with expression of CD19 and / or CD22.

[0200] 160. The method or use of embodiment 159, wherein the disease is cancer, for example blood cancer.

[0201] 161. The method of any one of embodiments 154 or 155-160 or the use of any one of embodiments 155-160, wherein the disease is a B-cell malignancy.

[0202] 162. Blood cancers include acute myeloid leukemia (AML), B-cell acute lymphoblastic leukemia (BALL), small lymphocytic leukemia (SLL), acute lymphoblastic leukemia (ALL), chronic myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, hairy lymphoma, and leukemia. 162. The method or use of embodiment 160 or 161, wherein the tumor is selected from: small cell leukemia, small cell lymphoma, large follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplastic or myelodysplastic syndrome, myeloproliferative neoplasm, non-Hodgkin's lymphoma, Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, preleukemia, or a combination thereof.

[0203] 163. To the subject, drugs that increase the efficacy of cells expressing CAR molecules; an agent that ameliorates one or more side effects associated with the administration of cells expressing a CAR molecule; or Agents for treating diseases associated with CD19 and / or CD22 The method of any one of embodiments 154 or 155-162 or the use of any one of embodiments 155-162, comprising administering [Brief explanation of the drawings]

[0204] [Figures 1A-1C]

[0023] Figure 1A shows a schematic diagram of the dual CAR constructs disclosed herein. These dual CAR constructs comprise a CD22 CAR and a CD19 CAR. Figure 1A shows a dual CAR construct comprising a CD22 CAR from the N-terminus to the C-terminus, followed by a CD19 CAR. Figure 1B shows a different dual CAR construct comprising a CD22 CAR from the N-terminus to the C-terminus, followed by a CD19 CAR. Figure 1C shows a dual CAR construct comprising a CD19 CAR from the N-terminus to the C-terminus, followed by a CD22 CAR. [Figure 2] Schematic diagram of the tandem CD19 / CD22 CAR constructs of the present disclosure. Each tandem CAR comprises a bispecific antigen-binding domain comprising a CD19 antigen-binding domain and a CD22 antigen-binding domain. [Figure 3] Figure 3 shows the in vitro activity of tandem and dual CAR T cells targeting CD19 and CD22. Figure 3A is a graph depicting the cytolytic activity (cell killing) of various constructs against a CD22-negative ALL cell line (CD22KO Nalm6-Luc). Figure 3B is a graph depicting the cytolytic activity (cell killing) of various constructs against a CD19-negative ALL cell line (CD19KO Nalm6-Luc). Figures 3C-3D are graphs showing IFNg cytokine production by various CAR constructs in response to target cells expressing CD22 and / or CD19. [Figure 4] Figure 4 shows the in vivo activity of tandem and dual CAR T cells targeting CD19 and CD22 in a B-cell acute lymphoblastic leukemia xenograft relapse model. Figure 4A is a graph showing total flux (mean bioluminescence) for all treatment groups. Figure 4B is a graph depicting the expansion kinetics of various CAR-T cells. [Figure 5] Figure 5 shows flow cytometry analysis of the percentage of CAR19+, CAR22+, and double-positive CAR T cells targeting CD19 and CD22 produced by the activation process. Figures 5A and 5B depict results from the small-scale manufacturing process at 72 hours and 144 hours post-harvest, respectively. Figure 5C depicts results from the large-scale manufacturing process. [Figure 6] Figure 6 shows the in vivo activity of mono- and dual-CAR T cells targeting CD19 and / or CD22 in a B-cell acute lymphoblastic leukemia xenograft model. Figure 6A is a graph showing total flux (mean bioluminescence) for all treatment groups. Figure 6B shows a head-to-head comparison of the 0.3 x 10 (0.3e6) dose groups. Figure 6C is a graph depicting the expansion kinetics of various CAR-T cells, shown as the number of CAR+ cells per 20 μl of blood (number of CAR-T cells, nCARtot). DETAILED DESCRIPTION OF THE INVENTION

[0205] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0206] The terms "a" and "an" refer to one or to more than one (i.e., to at least one) of the grammatical referent of the article. By way of example, "an element" means one element or more than one element.

[0207] The term "about," when referring to a measurable value, such as an amount, a temporal duration, or the like, means that variations of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% from the specified value are encompassed, as such variations are appropriate for the practice of the methods of the present disclosure.

[0208] As used herein, the term "pharmaceutically acceptable salt" refers to a salt that is, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject without undue toxicity, irritation, allergic reaction, etc., and that is commensurate with a reasonable benefit-risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, Berge et al. provide a detailed description of pharmaceutically acceptable salts in J. Pharmaceutical Sciences (1977) 66:1-19.

[0209] The term "chimeric antigen receptor," "CAR," or "CAR molecule" refers to a set of polypeptides, typically two in the simplest embodiment, that, when present in an immune effector cell, confers specificity for a target cell, typically a cancer cell, and intracellular signal generation to that cell. In some embodiments, a CAR comprises at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain (also referred to herein as an "intracellular signaling domain") that comprises a functional signaling domain derived from a stimulatory molecule and / or a costimulatory molecule, as defined below. In some aspects, the set of polypeptides are contiguous with each other, e.g., within the same polypeptide chain, e.g., comprising a chimeric fusion protein. In some embodiments, the set of polypeptides are not contiguous with each other, e.g., on different polypeptide chains. In some embodiments, the set of polypeptides comprises a dimerization switch that can couple the polypeptides to each other when a dimerization molecule is present, e.g., can couple the antigen-binding domain to the intracellular signaling domain. In one aspect, the stimulatory molecule is the zeta chain associated with the T cell receptor complex. In one aspect, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule, as defined below. In one embodiment, the costimulatory molecule is selected from a costimulatory molecule described herein, e.g., 4-1BB (i.e., CD137), CD27, and / or CD28. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a stimulatory molecule. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising a functional signaling domain derived from a costimulatory molecule and a functional signaling domain derived from a stimulatory molecule. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain comprising two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule.In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and an intracellular signaling domain comprising at least two functional signaling domains derived from one or more costimulatory molecules and a functional signaling domain derived from a stimulatory molecule. In one embodiment, the CAR comprises an optional leader sequence at the amino-terminus (N-ter) of the CAR fusion protein. In one embodiment, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen-binding domain, wherein the leader sequence is optionally cleaved from the antigen-binding domain (e.g., scFv) during cellular processing and cell membrane localization of the CAR.

[0210] The term "signaling domain" refers to a functional portion within a protein that functions by transmitting information within the cell to regulate cellular activity through a defined signaling pathway, either by generating second messengers or by functioning as an effector in response to such messengers.

[0211] As used herein, the term "CD19" refers to the cluster of differentiation 19 protein, an antigenic determinant detectable on leukemia progenitor cells. Human and mouse amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequence of human CD19 can be found under UniProt / Swiss-Prot accession number P15391, and the nucleic acid sequence encoding human CD19 can be found under accession number NM_001178098. CD19 is expressed in most B-cell lineage cancers, including, for example, acute lymphoblastic leukemia, chronic lymphocytic leukemia, and non-Hodgkin's lymphoma. Other cells that express CD19 are provided below in the definition of "diseases associated with CD19 expression." It is also an early marker of B-cell progenitor cells. See, for example, Nicholson et al. Mol. Immun. 34(16-17):1157-1165 (1997). In one embodiment, the antigen-binding portion of the CART recognizes and binds to an antigen within the extracellular domain of the CD19 protein. In one embodiment, the CD19 protein is expressed on cancer cells. As used herein, "CD19" includes proteins containing mutations of full-length wild-type CD19, such as point mutations, fragments, insertions, deletions, and splice variants.

[0212] As used herein, the term "CD22" refers to an antigenic determinant known to be detectable on leukemia progenitor cells. Human and mouse amino acid and nucleic acid sequences can be found in public databases such as GenBank, UniProt, and Swiss-Prot. For example, the amino acid sequences of human CD22 isoforms 1-5 can be found under accession numbers NP 001762.2, NP 001172028.1, NP 001172029.1, NP 001172030.1, and NP 001265346.1, respectively, and nucleic acid sequences encoding human CD22 variants 1-5 can be found under accession numbers NM 001771.3, NM 001185099.1, NM 001185100.1, NM 001185101.1, and NM 001278417.1, respectively. In one embodiment, the antigen-binding portion of the CAR recognizes and binds to an antigen within the extracellular domain of the CD22 protein. In one embodiment, the CD22 protein is expressed on cancer cells. As used herein, "CD22" includes proteins containing mutations of full-length wild-type CD22, such as point mutations, fragments, insertions, deletions, and splice variants.

[0213] As used herein, the term "binding domain" (e.g., a "CD20 binding domain") refers to a protein comprising at least one immunoglobulin variable domain sequence, e.g., an immunoglobulin chain or fragment thereof. The term "binding domain" (also referred to herein as "antibody molecule") or "antibody molecule" encompasses antibodies and antibody fragments. In certain embodiments, an antibody molecule is a multispecific antibody molecule, e.g., it comprises a plurality of immunoglobulin variable domain sequences, wherein a first immunoglobulin variable domain sequence of the plurality has binding specificity for a first epitope and a second immunoglobulin variable domain sequence of the plurality has binding specificity for a second epitope. In certain embodiments, a multispecific antibody molecule is a bispecific antibody molecule. A bispecific antibody has specificity for no more than two antigens. A bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence that has binding specificity for a first epitope and a second immunoglobulin variable domain sequence that has binding specificity for a second epitope.

[0214] The term "antibody fragment" refers to at least a portion of an antibody that retains the ability to specifically interact with an epitope of an antigen (e.g., by binding, steric hindrance, stabilization / destabilization, spatial distribution). Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab'), Fv fragments, scFv antibody fragments, disulfide-linked Fv (sdFv), Fd fragments consisting of VH and CH1 domains, linear antibodies, single domain antibodies (either VL or VH) such as sdAb, camelid VHH domains, multispecific antibodies formed from antibody fragments such as a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, and isolated CDRs or other epitope-binding fragments of an antibody. Antigen-binding fragments can also be incorporated into single-domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, Nature Biotechnology 23:1126-1136, 2005). Antigen-binding fragments can also be grafted onto polypeptide-based scaffolds, such as fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide minibodies). The term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light and heavy chain variable regions are closely linked, e.g., by a synthetic linker, e.g., a short flexible polypeptide linker, and can be expressed as a single polypeptide chain, and the scFv retains the specificity of the intact antibody from which it is derived. Unless otherwise specified, as used herein, an scFv can have the VL and VH variable regions in either order, e.g., relative to the N- and C-terminal ends of the polypeptide, and can comprise a VL-linker-VH or a VH-linker-VL.

[0215] The term "complementarity determining region" or "CDR" as used herein refers to a sequence of amino acids within an antibody variable region that confers antigen specificity and binding affinity. The exact amino acid sequence boundaries of a given CDR can be found in Kabat et al. (1991), Sequences of Proteins of Immunological Interest, 5 thEd. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme), Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme) and ImMunoGenTics (IMGT) numbering (Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003) ("IMGT" numbering scheme). For example, for the classical format, according to Kabat, the CDR amino acid residues of the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues of the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). According to Chothia, the CDR amino acids of the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues of the VL are numbered 26-32 (LCDR1), 52-56 (HCDR2), and 95-102 (HCDR3). Combining the CDR definitions of both Kabat and Chothia, the CDRs are numbered as amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) of human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) of human VL. Based on IMGT, the CDR amino acid residues of VH are numbered approximately 26 to 35 (CDR1), 51 to 57 (CDR2), and 93 to 102 (CDR3), and the CDR amino acid residues of VL are numbered approximately 27 to 32 (CDR1), 50 to 52 (CDR2), and 89 to 97 (CDR3) (numbering according to "IMGT"). Based on IMGT, the CDR regions of an antibody can be determined using the program IMGT / DomainGapAlign.

[0216] The portion of the CAR of the present invention comprising an antibody or antibody fragment thereof can exist in various forms, with the antigen-binding domain expressed as part of a contiguous polypeptide chain, including, for example, a single-domain antibody fragment (sdAb), a single-chain antibody (scFv), a humanized antibody, or a bispecific antibody (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In one embodiment, the antigen-binding domain of the CAR composition of the present invention comprises an antibody fragment. In a further embodiment, the CAR comprises an antibody fragment comprising an scFv.

[0217] The term "antibody heavy chain" refers to the larger of the two polypeptide chains present in antibody molecules in their naturally occurring conformation, and which usually determines the class to which the antibody belongs.

[0218] The term "antibody light chain" refers to the smaller of the two polypeptide chains present in antibody molecules in their naturally occurring conformation. Kappa (κ) and lambda (λ) light chains refer to the two major antibody light chain isotypes.

[0219] The term "recombinant antibody" refers to an antibody made using recombinant DNA techniques, such as, for example, an antibody expressed in a bacteriophage or yeast expression system. The term should also be construed to mean an antibody made by synthesis of a DNA molecule encoding the antibody and which expresses the antibody protein, or an amino acid sequence specifying that antibody, where the DNA or amino acid sequence was obtained using well-known recombinant DNA or amino acid sequence techniques available in the art.

[0220] The term "antigen" or "Ag" refers to a molecule that elicits an immune response. This immune response may include either or both antibody production or activation of specific immunologically competent cells. Those skilled in the art will understand that any macromolecule can be an antigen, including virtually any protein or peptide. Furthermore, antigens can be derived from recombinant or genomic DNA. Those skilled in the art will therefore understand that any DNA containing a nucleic acid sequence or partial nucleic acid sequence that encodes a protein that elicits an immune response encodes an "antigen" as that term is used herein. Furthermore, those skilled in the art will understand that an antigen need not be encoded solely by the full-length nucleic acid sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleic acid sequences of two or more genes, and that these nucleic acid sequences are arranged in various combinations to encode a polypeptide that elicits a desired immune response. Furthermore, those skilled in the art will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen can be synthetically produced, obtained from a biological sample, or can be a macromolecule other than a polypeptide. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells or bodily fluids, along with other biological components.

[0221] The term "anti-cancer effect" refers to a biological effect that may be manifested by various means, including, but not limited to, a reduction in tumor volume, a reduction in the number of cancer cells, a reduction in the number of metastases, an increase in life expectancy, a reduction in cancer cell proliferation, a reduction in cancer cell survival, or an improvement in various physiological symptoms associated with a cancerous condition. An "anti-cancer effect" may also be manifested by the ability of the peptides, polynucleotides, cells, and antibodies of the present invention in preventing cancer from occurring in the first place. The term "anti-tumor effect" refers to a biological effect that may be manifested by various means, including, but not limited to, a reduction in tumor volume, a reduction in tumor cell number, a reduction in tumor cell proliferation, or a reduction in tumor cell survival. The term "autologous" refers to any material derived from the same individual that is subsequently reintroduced into that individual.

[0222] The term "allogeneic" refers to any material derived from a different animal of the same species as the individual into which the material is introduced. Two or more individuals are said to be allogeneic to each other when the genes at one or more loci are not identical. In some embodiments, allogeneic materials from individuals of the same species may be genetically different enough to interact antigenically.

[0223] The term "xenogeneic" refers to a graft derived from an animal of a different species.

[0224] The term "combination" refers to either a fixed combination in a single dosage unit form, or a combination administration in which a compound of the present invention and a combination partner (e.g., another drug, as described below, also referred to as a "therapeutic agent" or "co-drug") can be independently administered separately at the same time or within a time interval, particularly where these time intervals allow the combination partners to exhibit a cooperative effect, e.g., a synergistic effect. Single components can be packaged in a kit or separately. One or both of the components (e.g., powder or liquid) can be reconstituted or diluted to the desired dose before administration. The terms "co-administration" or "co-administration," etc., as used herein, are meant to encompass the administration of selected combination partners to a single subject (e.g., patient) in need thereof, and are intended to include therapeutic regimens in which the agents are not necessarily administered by the same route of administration or at the same time. The term "pharmaceutical combination," as used herein, refers to a product obtained by mixing or combining two or more therapeutic agents, and includes both fixed and non-fixed combinations of therapeutic agents. The term "fixed combination" means that both therapeutic agents, e.g., a compound of the present invention and a combination partner, are administered to a patient simultaneously in the form of a single entity or dosage. The term "non-fixed combination" means that both therapeutic agents, e.g., a compound of the present invention and a combination partner, are administered to a patient simultaneously, in parallel, or alternatively sequentially as separate entities without any specific time limit, such administration providing therapeutically effective levels of the two compounds in the patient's body. The latter also applies to cocktail therapy, e.g., the administration of three or more therapeutic agents.

[0225] The term "cancer" refers to a disease characterized by the rapid, uncontrolled growth of abnormal cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Various examples of cancer are described herein and include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, etc. The terms "tumor" and "carcinoma" are used interchangeably herein, e.g., both terms encompass solid and liquid cancers, e.g., tumors. As used herein, the term "cancer" or "tumor" includes precancerous and malignant cancers and tumors.

[0226] As used herein, the phrase "disease associated with CD22 expression" includes, but is not limited to, a disease associated with expression of CD22 (e.g., wild-type or mutant CD22) or a condition associated with cells that express, or have at any time expressed, CD22 (e.g., wild-type or mutant CD22), including, for example, a proliferative disease such as a cancer or malignant tumor, or a precancerous condition such as myelodysplasia, myelodysplastic syndrome, or preleukemia; or a non-cancer-related indication associated with cells that express CD22 (e.g., wild-type or mutant CD22). For the avoidance of doubt, a disease associated with CD22 expression may include a condition associated with cells that once expressed CD22 but no longer express CD22, e.g., due to downregulation of CD22 expression, e.g., by treatment with a molecule that targets CD22, e.g., a CD22 CAR. In one embodiment, the cancer associated with CD22 expression is a hematological cancer. In one aspect, hematological cancers include, but are not limited to, AML, myelodysplastic syndrome, ALL, hairy cell leukemia, prolymphocytic leukemia, chronic myeloid leukemia, Hodgkin's lymphoma, blastic plasmacytoid dendritic cell neoplasm, etc. Furthermore, diseases associated with CD22 expression include, but are not limited to, atypical and / or non-classical cancers, malignancies, precancerous conditions, or proliferative disorders associated with CD22 expression. Non-cancer-related symptoms associated with CD22 expression may also be included. In some embodiments, CD22-expressing cells express or have expressed CD22 mRNA at some time. In certain embodiments, CD22-expressing cells produce CD22 protein (e.g., wild-type or mutant), and the CD22 protein may be present at normal or low levels. In certain embodiments, CD22-expressing cells produced detectable levels of CD22 protein at one time, but subsequently ceased to produce substantially detectable CD22 protein.

[0227] The phrase "disease associated with CD19 expression" includes diseases associated with expression of CD19 (e.g., wild-type or mutant CD19), or conditions associated with cells that express or have at any time expressed CD19 (e.g., wild-type or mutant CD19), or non-cancer-related indications associated with cells that express CD19, including, but not limited to, proliferative diseases such as cancer or malignant tumors, or precancerous conditions such as myelodysplasia, myelodysplastic syndrome, or preleukemia. For the avoidance of doubt, diseases associated with CD19 expression may include conditions associated with cells that once expressed CD19 but no longer express CD19, e.g., due to downregulation of CD19 expression by, for example, treatment with a CD19-targeting molecule, e.g., a CD19 CAR. In one embodiment, the cancer associated with CD19 expression is a hematological cancer. In one embodiment, the hematological cancer is leukemia or lymphoma. In one aspect, cancers associated with expression of CD19 include cancers and malignancies including, but not limited to, one or more acute leukemias, for example, but not limited to, B-cell acute lymphoblastic leukemia (BALL), T-cell acute lymphoblastic leukemia (TALL), acute lymphoblastic leukemia (ALL), one or more chronic leukemias, for example, but not limited to, chronic myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL). Additional cancers or hematological conditions associated with expression of CD19 include, but are not limited to, B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, mantle cell lymphoma (MCL), marginal zone lymphoma, multiple myeloma, myelodysplastic and myelodysplastic syndromes, non-Hodgkin's lymphoma, Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, and "preleukemias," a group of diverse hematological conditions united by ineffective production (or dysplasia) of myeloid blood cells. Additionally, diseases associated with the expression of CD19 expression include, but are not limited to, atypical and / or non-classical cancers, malignancies, precancerous conditions or proliferative disorders associated with the expression of CD19.Non-cancer-related indications associated with CD19 expression include, but are not limited to, autoimmune diseases (e.g., lupus), inflammatory disorders (allergies and asthma), and transplantation. In some embodiments, CD19-expressing cells express, or have expressed at some time, CD19 mRNA. In certain embodiments, CD19-expressing cells produce CD19 protein (e.g., wild-type or mutant), which may be present at normal or reduced levels. In certain embodiments, CD19-expressing cells transiently produce detectable levels of CD19 protein, but subsequently cease to produce substantially detectable CD19 protein.

[0228] As used herein, unless otherwise specified, the terms "prevent," "preventing," and "prevention" refer to actions taken before a subject begins to suffer from a condition or the recurrence of a condition. Prevention may not result in complete prevention of a condition; the term encompasses partial prevention or reduction of a condition or symptoms of a condition, or a reduction in the risk of developing a condition.

[0229] As used herein, "administered in combination" means that two (or more) different therapies are delivered to a subject during the course of the subject's illness with a disorder, e.g., two or more therapies are delivered after the subject is diagnosed with the disorder and before the disorder is cured or resolved, or before the treatment is discontinued for other reasons. In some embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, resulting in overlap in administration. This is sometimes referred to herein as "simultaneous" or "concurrent delivery." In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In some embodiments, either case, the treatments are more effective because of the combined administration. For example, the effectiveness of the second treatment is increased compared to what would have been observed if the second treatment had been administered without the first treatment, e.g., the second treatment could be reduced with an equivalent effect or the second treatment could reduce symptoms to a greater extent, or the same situation could be observed with the first treatment. In some embodiments, the delivery is such that the reduction in symptoms or other parameters associated with the disorder is greater than what would have been observed if one treatment had been delivered without the other. The effects of the two therapies can be partially additive, fully additive, or greater than additive. The delivery is such that the effect of the first therapy is still detectable upon delivery of the second. In one embodiment, the CAR-expressing cells are administered at a dose and / or dosing schedule described herein, and the B-cell inhibitory agent or agent that enhances the activity of the CD19 CAR-expressing cells is administered at a dose and / or dosing schedule described herein.

[0230] As used herein, the term "derived from" refers to a relationship between a first molecule and a second molecule. This generally refers to the structural similarity between the first and second molecules and does not imply or include a limitation on the process or source from which the first molecule is derived from the second molecule. For example, in the case of an intracellular signaling domain derived from the CD3ζ molecule, the intracellular signaling domain retains sufficient CD3ζ structure so that it has the required function, i.e., the ability to generate a signal under appropriate conditions. This does not imply or include a limitation to a particular process for generating the intracellular signaling domain; for example, it does not mean that one must start with the CD3ζ sequence and delete or mutate undesired sequences to provide the intracellular signaling domain.

[0231] The term "conservative sequence modifications" refers to amino acid modifications that do not significantly affect or alter the binding characteristics of an antibody or antibody fragment containing that amino acid sequence. Such conservative modifications include amino acid substitutions, additions, and deletions. Modifications can be introduced into an antibody or antibody fragment of the present invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are those in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within a CAR of the invention can be replaced with other amino acid residues from the same side chain family, and the altered CAR can be tested using the functional assays described herein.

[0232] The term "stimulation" refers to a primary response resulting from binding of a stimulatory molecule (e.g., a TCR / CD3 complex or a CAR) to its cognate ligand (or tumor antigen in the case of a CAR), thereby mediating a signal transduction event, such as, but not limited to, signaling through the TCR / CD3 complex or signaling through the signaling domain of an appropriate NK receptor or CAR. Stimulation can mediate changes in the expression of certain molecules.

[0233] The term "stimulatory molecule" refers to a molecule expressed by an immune cell, e.g., a T cell, NK cell, or B cell, that provides one or more cytoplasmic signaling sequences that regulate immune cell activation in a stimulatory manner for at least some aspect of an immune cell signaling pathway. In one embodiment, the signal is a primary signal, e.g., initiated by binding of the TCR / CD3 complex to a peptide-loaded MHC molecule, which leads to mediation of a T cell response, including, but not limited to, proliferation, activation, differentiation, etc. Primary cytoplasmic signaling sequences (also referred to as "primary signaling domains") that function in a stimulatory manner can contain signaling motifs known as immunoreceptor tyrosine-based activation motifs, or ITAMs. Examples of ITAM-containing cytoplasmic signaling sequences that are particularly useful in the present invention include, but are not limited to, those derived from CD3ζ, common FcRγ (FCER1G), FcγRIIa, FcRβ (FcεR1b), CD3γ, CD3δ, CD3ε, CD79a, CD79b, DAP10, and DAP12. In specific CARs of the invention, the intracellular signaling domain of any one or more CARs of the invention comprises an intracellular signaling sequence, e.g., the primary signaling sequence of CD3-zeta. In specific CARs of the invention, the primary signaling sequence of CD3-zeta is the sequence provided as SEQ ID NO: 96, or equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc.

[0234] The term "antigen-presenting cell" or "APC" refers to a cell of the immune system, such as an accessory cell (e.g., a B cell, a dendritic cell, etc.), that presents foreign antigens complexed with major histocompatibility complexes (MHC) on its surface. T cells can recognize such complexes using their T cell receptors (TCRs). APCs process antigens and present them to T cells.

[0235] "Immune effector cells," as the term is used herein, refer to cells that are involved in an immune response, e.g., promoting an immune effector response. Examples of immune effector cells include T cells, e.g., α / β T cells and γ / δ T cells, B cells, natural killer (NK) cells, natural killer T (NK-T) cells, mast cells, and myeloid-derived phagocytes.

[0236] "Immune effector function or immune effector response," as the term is used herein, refers to a function or response, e.g., of an immune effector cell, that enhances or promotes an immune attack of a target cell. For example, an immune effector function or response refers to a property of a T cell or NK cell that promotes the killing or growth or proliferation inhibition of a target cell. In the case of T cells, primary stimulation and costimulation are examples of immune effector functions or responses.

[0237] "Intracellular signaling domain," as that term is used herein, refers to the intracellular portion of a molecule. The intracellular signaling domain generates a signal that promotes immune effector function of a CAR-containing cell, such as a CART cell or a CAR-expressing NK cell. For example, examples of immune effector function in a CART cell or a CAR-expressing NK cell include helper activity, including cytolytic activity and cytokine secretion.

[0238] In certain embodiments, the intracellular signaling domain can comprise a primary intracellular signaling domain. Exemplary primary intracellular signaling domains include those derived from molecules involved in primary stimulation or antigen-dependent stimulation. In certain embodiments, the intracellular signaling domain can comprise a costimulatory intracellular domain. Exemplary costimulatory intracellular signaling domains include those derived from molecules involved in costimulatory signals or antigen-independent stimulation. For example, in the case of CART, the primary intracellular signaling domain can comprise a cytoplasmic sequence of a T cell receptor, and the costimulatory intracellular signaling domain can comprise a cytoplasmic sequence from a co-receptor or costimulatory molecule.

[0239] The primary intracellular signaling domain can contain a signaling motif known as an immunoreceptor tyrosine-based activation motif, or ITAM. Examples of primary cytoplasmic signaling sequences containing ITAMs include, but are not limited to, those derived from CD3ζ, common FcRγ (FCER1G), FcRβ (FcεR1b), CD3γ, CD3δ, CD3ε, CD79a, CD79b, DAP10, and DAP12.

[0240] The terms "ζ" or alternatively "ζ chain," "CD3-ζ," or "TCR-ζ" are defined as the protein provided under GenBank Accession No. BAG36664.1, or equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc., and "ζ stimulatory domain" or alternatively "CD3-ζ stimulatory domain" or "TCR-ζ stimulatory domain" are defined as the amino acid residues from the cytoplasmic domain of the ζ chain or a functional derivative thereof sufficient to functionally transmit an initial signal necessary for T cell activation. In one embodiment, the cytoplasmic domain of ζ comprises residues 52-164 of GenBank Accession No. BAG36664.1, or equivalent residues from a non-human species that is a functional ortholog thereof, e.g., mouse, rodent, monkey, ape, etc. In one embodiment, the "ζ stimulatory domain" or "CD3-ζ stimulatory domain" is the sequence provided as SEQ ID NO:96.

[0241] The term "costimulatory molecule" refers to a cognate binding partner on a T cell that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the T cell, such as, but not limited to, proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that contribute to an efficient immune response. Costimulatory molecules include, but are not limited to, MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD5, ICAM-1, LFA-1 (CD11a / CD18), 4-1BB ( CD137), B7-H3, CDS, ICAM-1, ICOS5 (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NK p44, NKp30, NKp46, CD19, CD4, CD8α, CD8β, IL2Rβ, IL2Rγ, IL7Rα, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA- 6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD84, CD96(Tactile), CEA These include ligands that specifically bind to CAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, CD19a, and CD83.

[0242] The costimulatory intracellular signaling domain can be the intracellular portion of a costimulatory molecule. Costimulatory molecules can be representative of the following protein families: TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), and activating NK cell receptors. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CSD, CD7, CD287, LIGHT, NKG2C, NKG2D, SLAMF7, NKp80, NKp30, NKp44, NKp46, CD160, B7-H3, and ligands that specifically bind to CD83.

[0243] The intracellular signaling domain can comprise the entire intracellular portion of the molecule from which it is derived, or the entire naturally occurring intracellular signaling domain, or a functional fragment or derivative thereof.

[0244] The term "4-1BB" refers to a member of the TNFR superfamily having the amino acid sequence provided as GenBank Accession No. AAA62478.2, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc., and a "4-1BB costimulatory domain" is defined as amino acid residues 214-255 of GenBank Accession No. AAA62478.2, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc. In one embodiment, the "4-1BB costimulatory domain" is the sequence provided as SEQ ID NO: 70, or the equivalent residues from a non-human species, e.g., mouse, rodent, monkey, ape, etc.

[0245] The term "encoding" refers to the inherent property of a specific nucleotide sequence within a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes that have either a defined nucleotide sequence (e.g., rRNA, tRNA, and mRNA) or a defined amino acid sequence. Thus, a gene, cDNA, or RNA encodes a protein when the protein is produced in a cell or other biological system by transcription and translation of the mRNA corresponding to that gene. Both the coding strand, whose nucleic acid sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, which is used as a transcription template for a gene or cDNA, can be said to encode the protein or other product of that gene's cDNA.

[0246] Unless otherwise specified, a "nucleic acid sequence encoding an amino acid sequence" includes all nucleic acid sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleic acid sequence encoding a protein or RNA can also include introns, to the extent that the nucleic acid sequence encoding the protein may, in some versions, contain one or more introns.

[0247] The terms "effective amount" or "therapeutically effective amount" are used interchangeably herein and refer to an amount of a compound, formulation, material, or composition as described herein that is effective to achieve a particular biological result.

[0248] The term "endogenous" refers to any material that is derived from or produced within an organism, cell, tissue, or system.

[0249] The term "exogenous" refers to any material introduced from or produced outside an organism, cell, tissue or system.

[0250] The term "expression" refers to the transcription and / or translation of a particular nucleic acid sequence driven by a promoter.

[0251] The term "transfer vector" refers to a composition containing an isolated nucleic acid that can be used to deliver the isolated nucleic acid into a cell. Many vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "transfer vector" includes self-replicating plasmids or viruses. This term should be construed to further include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds and liposomes. Examples of viral transfer vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, lentiviral vectors, and the like.

[0252] The term "expression vector" refers to a vector containing a recombinant polynucleotide comprising expression control sequences operably linked to a nucleic acid sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Expression vectors include all those known in the art, including cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0253] The term "lentivirus" refers to a genus of the Retroviridae family. Lentiviruses are unique among retroviruses in that they can infect non-dividing cells, and they can deliver large amounts of genetic information into the DNA of host cells, making them one of the most efficient gene delivery vectors. HIV, SIV, and FIV are all examples of lentiviruses.

[0254] The term "lentiviral vector" refers to a vector derived from at least a portion of a lentiviral genome, including, inter alia, self-inactivating lentiviral vectors as provided in Milone et al., Mol. Ther. 17(8):1453-1464 (2009). Other examples of lentiviral vectors that may be used clinically include, but are not limited to, the LENTIVECTOR® gene delivery technology from Oxford BioMedica and the LENTIMAX™ vector system from Lentigen. Non-clinical types of lentiviral vectors are also available and will be known to those skilled in the art.

[0255] The term "homologous" or "identity" refers to the subunit sequence identity between two polymer molecules, e.g., between two nucleic acid molecules, such as two DNA molecules or two RNA molecules, or between two polypeptide molecules. When a subunit position in both of the two molecules is occupied by the same monomer subunit, e.g., if each position in two DNA molecules is occupied by adenine, they are homologous or identical at that position. The homology between two sequences is a direct function of the number of matching or homologous positions. For example, if half of the positions in two sequences are homologous (e.g., 5 positions in a polymer 10 subunits long), the two sequences are 50% homologous. If 90% of the positions (e.g., 9 out of 10) are matched or homologous, the two sequences are 90% homologous.

[0256] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies and antibody fragments thereof are those in which residues from a recipient complementarity-determining region (CDR) of a human immunoglobulin (recipient antibody or antibody fragment) are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies / antibody fragments can comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. These modifications can further refine and optimize antibody or antibody fragment performance. Generally, a humanized antibody or antibody fragment thereof will comprise substantially all of at least one, and typically two, variable domains, with all or substantially all of the CDR regions corresponding to those of a non-human immunoglobulin and all or most of the FR regions being those of a human immunoglobulin sequence. The humanized antibody or antibody fragment can also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321:522-525, 1986; Reichmann et al., Nature, 332:323-329, 1988; Presta, Curr. Op. Struct. Biol., 2:593-596, 1992.

[0257] "Fully human" refers to an immunoglobulin, such as an antibody or antibody fragment, whose entire molecule is of human origin or consists of an amino acid sequence identical to a human form of an antibody or immunoglobulin.

[0258] "Murine" refers to a mouse or rat. For example, a murine antibody or fragment thereof comprises the sequence of an antibody or fragment thereof isolated from a murine, e.g., a mouse or rat.

[0259] The term "isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide that is naturally present in a living animal is not "isolated," but the same nucleic acid or peptide that has been partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in a substantially purified form, or can exist in a non-native environment, such as, for example, a host cell.

[0260] In the context of the present invention, the following abbreviations are used for commonly occurring nucleobases: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0261] The term "operably linked" or "transcriptional control" refers to a functional linkage between a regulatory sequence and a heterologous nucleic acid sequence that results in expression of the latter. For example, a first nucleic acid sequence is operably linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter is operably linked to a coding sequence if it affects the transcription or expression of the coding sequence. Operably linked DNA sequences can be contiguous with each other and, for example, in the same reading frame, as necessary to join two protein-coding regions.

[0262] The term "parenteral" administration of an immunogenic composition includes, for example, subcutaneous (sc), intravenous (iv), intramuscular (im), or intrasternal injection, intratumoral, or infusion techniques.

[0263] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof, in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise indicated, a particular nucleic acid sequence implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions can be achieved by creating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0264] The terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids, with no limit on the maximum number of amino acids that can comprise a protein or peptide sequence. A polypeptide includes any peptide or protein containing two or more amino acids joined to each other by peptide bonds. As used herein, the term refers to both short chains, commonly referred to in the art as peptides, oligopeptides, and oligomers, and to the numerous types of longer chains generally referred to in the art as proteins. "Polypeptide" specifically includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins. A polypeptide includes natural peptides, recombinant peptides, or combinations thereof.

[0265] The term "promoter" refers to a DNA sequence recognized by the synthetic machinery of a cell or introduced synthetic machinery necessary to initiate the specific transcription of a polynucleotide sequence.

[0266] The term "promoter / regulatory sequence" refers to a nucleic acid sequence required for expression of a gene product operably linked to that promoter / regulatory sequence. In some instances, this sequence may be a core promoter sequence, and in other instances, this sequence may also include an enhancer sequence and other regulatory elements required for expression of the gene product. The promoter / regulatory sequence may, for example, be one that confers tissue-specific expression of the gene product.

[0267] The term "constitutive" promoter refers to a nucleic acid sequence that, when operably linked to a polynucleotide that encodes or specifies a gene product, causes the production of the gene product in a cell under most or all physiological conditions of the cell.

[0268] The term "inducible" promoter refers to a nucleic acid sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes production of the gene product in a cell substantially only when an inducer corresponding to the promoter is present in the cell.

[0269] The term "tissue-specific" promoter refers to a nucleic acid sequence that, when operably linked to a polynucleotide that encodes or specifies a gene product, causes the production of the gene product in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[0270] The term "flexible polypeptide linker" or "linker," when used in the context of an scFv, refers to a peptide linker composed of amino acids such as glycine and / or serine residues used alone or in combination to link the variable heavy and variable light chain regions together. In one embodiment, the flexible polypeptide linker is a Gly / Ser linker and comprises the amino acid sequence (Gly-Gly-Gly-Ser) (SEQ ID NO: 89) repeated n times, where n is a positive integer equal to or greater than 1, e.g., n=1, n=2, n=3, N=4, n=5, and n=6, n=7, n=8, n=9, and n=10. In one embodiment, the flexible polypeptide linker is (Gly4Ser)3 (SEQ ID NO: 82). In another embodiment, the linker comprises multiple repeats of (Gly2Ser) and (GlySer). In another embodiment, the polypeptide does not comprise a linker, e.g., (n=0). Also included within the scope of the present invention are the linkers described in WO 2012 / 138475, which is incorporated herein by reference.

[0271] As used herein, a 5' cap (RNA cap, RNA 7-methylguanosine cap or RNA m 7 A 5' cap (also called a G-cap) is a modified guanine nucleotide added to the "pre" or 5' end of eukaryotic messenger RNA immediately after transcription initiation. The 5' cap consists of a terminal group attached to the first transcribed nucleotide. Its presence is important for ribosome recognition and protection from RNases. Capping is coupled to transcription and occurs cotranscriptionally, with each cap affecting the other. Shortly after transcription initiation, a cap-synthesizing complex associated with RNA polymerase binds to the 5' end of the mRNA being synthesized. This enzyme complex catalyzes the chemical reactions required for mRNA capping. Synthesis proceeds as a multistep biochemical reaction. The capping moiety can be modified to adjust mRNA functions such as its stability or translation efficiency.

[0272] As used herein, "in vitro transcribed RNA" refers to in vitro synthesized RNA, preferably mRNA. Generally, in vitro transcribed RNA is generated from an in vitro transcription vector. The in vitro transcription vector contains a template used to generate the in vitro transcribed RNA.

[0273] As used herein, "poly(A)" refers to a series of adenosines added to mRNA by polyadenylation. In preferred embodiments of constructs for transient expression, the poly(A) is 50 to 5,000, preferably greater than 64, more preferably greater than 100, and most preferably greater than 300 or 400. The poly(A) sequence can be chemically or enzymatically modified to adjust mRNA function, such as localization, stability, or translation efficiency.

[0274] As used herein, "polyadenylation" refers to the covalent attachment of a polyadenylyl moiety, or modified variants thereof, to a messenger RNA molecule. In eukaryotes, most messenger RNA (mRNA) molecules are polyadenylated at the 3' end. The 3' poly(A) tail is a long sequence (often several hundred) of adenine nucleotides added to pre-mRNA by the action of the enzyme polyadenylate polymerase. In higher eukaryotes, the poly(A) tail is added to transcripts containing a specific sequence, the polyadenylation signal. The poly(A) tail and its associated proteins help protect the mRNA from exonuclease degradation. Polyadenylation is also important for transcription termination, mRNA nuclear export, and translation. Polyadenylation occurs in the nucleus immediately after DNA-to-RNA transcription, but can also occur later in the cytoplasm. After transcription is terminated, the mRNA strand is cleaved by the action of an endonuclease complex associated with RNA polymerase. The cleavage site is usually characterized by the presence of the base sequence AAUAAA near the cleavage site. After the mRNA is cleaved, an adenosine residue is added to the free 3' end of the cleavage site.

[0275] As used herein, "transient" refers to expression of a non-integrated transgene for a period of hours, days, or weeks, which is shorter than the period of expression of the gene when integrated into the genome or contained in a stable plasmid replicon in the host cell.

[0276] As used herein, the terms "treat," "treatment," and "treating" refer to a reduction or amelioration of the progression, severity, and / or duration of a proliferative disorder, or an amelioration of one or more symptoms (preferably one or more discernible symptoms) of a proliferative disorder, brought about by the administration of one or more therapies (e.g., one or more therapeutic agents, such as a CAR of the invention). In specific embodiments, the terms "treat," "treatment," and "treating" refer to an improvement in at least one measurable physical parameter of a proliferative disorder, such as tumor growth, which may not necessarily be discernible to the patient. In some embodiments, the terms "treat," "treatment," and "treating" refer to either or both of a physical inhibition of the progression of a proliferative disorder, e.g., by stabilization of a discernible symptom, or a physiological inhibition, e.g., by stabilization of a physical parameter. In some embodiments, the terms "treat," "treatment," and "treating" refer to a reduction or stabilization of tumor size or cancerous cell number.

[0277] The term "signal transduction pathway" refers to the biochemical relationships between various signaling molecules that play a role in transmitting a signal from one part of a cell to another part of the cell. The phrase "cell surface receptor" includes molecules and molecular complexes that have the ability to receive a signal and transmit the signal across the membrane of a cell.

[0278] The term "subject" is intended to include living organisms (eg, mammals, humans) in which an immune response can be generated.

[0279] The term "substantially purified" cells refers to cells that are essentially free of other cell types. Substantially purified cells also refer to cells that have been separated from other cell types with which they are normally associated in their naturally occurring state. In some instances, a population of substantially purified cells refers to a homogenous population of cells. In other instances, the term simply refers to cells that have been separated from the cells with which they are naturally associated in their natural state. In some embodiments, the cells are cultured in vitro. In other embodiments, the cells are not cultured in vitro.

[0280] The term "therapeutic" as used herein means treatment. A therapeutic effect is achieved by the reduction, suppression, amelioration, or eradication of a disease state.

[0281] The term "prevention," as used herein, means the prevention of or prophylactic treatment for a disease or disease state.

[0282] In the context of the present invention, a "tumor antigen," or a "hyperproliferative disorder antigen," or an "antigen associated with a hyperproliferative disorder" refers to an antigen common to a specific hyperproliferative disorder. In certain embodiments, the hyperproliferative disorder antigen of the present invention is derived from a cancer, including, but not limited to, primary or metastatic melanoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin's lymphoma, leukemia, uterine cancer, cervical cancer, bladder cancer, renal cancer, and adenocarcinoma, such as breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, etc.

[0283] The terms "transfected" or "transformed" or "transduced" refer to the process of transferring or introducing exogenous nucleic acid into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed or transduced with exogenous nucleic acid. The cell includes the primary subject cell and its progeny.

[0284] The term "specifically binds" refers to an antibody or ligand that recognizes and binds to a binding partner (e.g., a stimulatory tumor antigen) protein present in a sample, but does not substantially recognize or bind to other molecules in the sample.

[0285] "Refractory," as used herein, refers to a disease, e.g., cancer, that does not respond to treatment. In embodiments, a refractory cancer may be resistant to treatment before or at the time of initiation of treatment. In some embodiments, a refractory cancer may become resistant during treatment. A refractory cancer is also referred to as a resistant cancer.

[0286] A subject "responds" to a treatment if a parameter of the cancer (e.g., a hematological cancer, e.g., cancer cell growth, proliferation, and / or survival) in the subject is slowed or reduced by a detectable amount, e.g., about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, as determined by any appropriate measure, e.g., mass, cell count, or volume. In one example, a subject responds to a treatment if the subject's life expectancy is extended by about 5%, 10%, 20%, 30%, 40%, 50% or more over the life expectancy expected if the treatment is not administered. In another example, a subject responds to a treatment if the subject has an increase in disease-free survival, overall survival, or an increase in time to progression. Whether a patient responds to a treatment can be determined using several methods, including, for example, the criteria provided by the NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®). For example, in the context of B-ALL, a complete response or complete responder may be associated with one or more of: <5% BM blasts, >1000 neutrophils / ANC ( / μL), >100,000 platelets ( / μL) without circulating blasts or extramedullary disease (no lymphadenopathy, splenomegaly, skin / gingival involvement / testicular mass / CNS lesions), trilineage hematopoiesis, and 4-week relapse-free survival. A partial responder may be associated with one or more of: a ≥50% reduction in BM blasts, >1000 neutrophils / ANC ( / μL), >100,000 platelets ( / μL). Non-responders may exhibit disease progression, e.g., >25% BM blasts. In one embodiment, a complete responder is defined as having 7% or more CD27+ CD45RO- cells in the CD8+ population. In one embodiment, the percent of CAR+ cells at pre-harvest levels distinguishes responders (e.g., complete and partial responders) from non-responders (NR).

[0287] The term "relapse," as used herein, refers to the reappearance of cancer after an initial period of response (e.g., complete or partial response). The initial period of response may involve levels of cancer cells below a certain threshold, e.g., below 20%, 1%, 10%, 5%, 4%, 3%, 2%, or 1%. Reappearance may include levels of cancer cells above a certain threshold, e.g., above 20%, 1%, 10%, 5%, 4%, 3%, 2%, or 1%. For example, in the context of B-ALL, reappearance may include the reappearance of blasts in the blood, bone marrow (>5%), or any extramedullary site after a complete response. In this context, a complete response may include <5% BM blasts. More generally, in certain embodiments, a response (e.g., complete or partial response) may include the absence of detectable MRD (minimal residual disease). In certain embodiments, the initial response period lasts for at least 1, 2, 3, 4, 5, or 6 days, at least 1, 2, 3, or 4 weeks, at least 1, 2, 3, 4, 6, 8, 10, or 12 months, or at least 1, 2, 3, 4, or 5 years.

[0288] As that term is used herein, a "regulatable chimeric antigen receptor (RCAR)" refers to a set of polypeptides, typically two polypeptides in the simplest embodiment, that, when present in an RCARX cell, can confer specificity for a target cell, typically a cancer cell, and regulatable intracellular signal generation or proliferation to the RCARX cell, thereby optimizing the immune effector properties of the RCARX cell. RCARX cells rely, at least in part, on an antigen-binding domain to confer specificity for target cells that contain the antigen to which the antigen-binding domain binds. In one embodiment, the RCAR contains a dimerization switch that can couple the intracellular signaling domain to the antigen-binding domain when a dimerizing molecule is present.

[0289] "Membrane anchor" or "membrane tethering domain," as that term is used herein, refers to a polypeptide or moiety, e.g., a myristoyl group, sufficient to anchor an extracellular or intracellular domain to a cell membrane.

[0290] A "switch domain," as the term is used herein, e.g., in reference to an RCAR, refers to an entity, typically a polypeptide-based entity, that associates with another switch domain in the presence of a dimerization molecule. This association results in functional coupling between a first entity linked to, e.g., fused to, a first switch domain and a second entity linked to, e.g., fused to, a second switch domain. The first and second switch domains are collectively referred to as a dimerization switch. In embodiments, the first and second switch domains are the same as each other, e.g., polypeptides having the same primary amino acid sequence, and are collectively referred to as a homodimerization switch. In embodiments, the first and second switch domains are different from each other, e.g., polypeptides having different primary amino acid sequences, and are collectively referred to as a heterodimerization switch. In embodiments, the switch is intracellular. In embodiments, the switch is extracellular. In embodiments, the switch domain is a polypeptide-based entity, e.g., FKBP- or FRB-based, and the dimerization molecule is a small molecule, e.g., a rapalog. In embodiments, the switch domain is a polypeptide-based entity, e.g., an scFv that binds a myc peptide, and the dimerization molecule is a polypeptide, fragment thereof, or multimer of a polypeptide, e.g., a myc ligand or multimer of a myc ligand that binds one or more myc scFvs. In embodiments, the switch domain is a polypeptide-based entity, e.g., a myc receptor, and the dimerization molecule is an antibody or fragment thereof, e.g., a myc antibody.

[0291] "Dimerization molecule," as the term is used herein, e.g., in reference to RCAR, refers to a molecule that promotes association of a first switch domain with a second switch domain. In embodiments, the dimerization molecule is not naturally present in a subject or is not present at concentrations that would result in significant dimerization. In embodiments, the dimerization molecule is a small molecule, e.g., rapamycin or a rapalog, e.g., RAD001.

[0292] The term "bioequivalent" refers to the amount of an agent other than a reference compound (e.g., RAD001) required to produce an effect equivalent to that produced by a reference dose or amount of a reference compound (e.g., RAD001). In some embodiments, the effect is the level of mTOR inhibition, e.g., as measured by P70 S6 kinase inhibition, e.g., as assessed in an in vivo or in vitro assay, e.g., an assay described herein, e.g., the Boulay assay, or by measuring phosphorylated S6 levels by Western blot. In some embodiments, the effect is a change in the ratio of PD-1-positive / PD-1-negative T cells, as measured by cell sorting. In some embodiments, a bioequivalent amount or dose of an mTOR inhibitor is an amount or dose that achieves the same level of P70 S6 kinase inhibition as a reference dose or reference amount of a reference compound. In some embodiments, a bioequivalent amount or dose of an mTOR inhibitor is an amount or dose that achieves the same level of change in the ratio of PD-1-positive / PD-1-negative T cells as a reference dose or reference amount of a reference compound.

[0293] The term "immune-enhancing low-dose," when used in conjunction with an mTOR inhibitor, e.g., an allosteric mTOR inhibitor, e.g., RAD001 or rapamycin, or a catalytic mTOR inhibitor, refers to a dose of the mTOR inhibitor that partially, but not completely, inhibits mTOR activity, e.g., as measured by inhibition of P70 S6 kinase activity. Methods for assessing mTOR activity, e.g., by inhibition of P70 S6 kinase, are discussed herein. The dose is insufficient to result in complete immunosuppression, but sufficient to enhance the immune response. In some embodiments, the immune-enhancing low-dose of an mTOR inhibitor results in a decrease in the number of PD-1-positive T cells and / or an increase in the number of PD-1-negative T cells, or an increase in the ratio of PD-1-negative T cells to PD-1-positive T cells. In some embodiments, the immune-enhancing low-dose of an mTOR inhibitor results in an increase in the number of naive T cells. In some embodiments, the immune-enhancing low-dose of an mTOR inhibitor results in one or more of the following: For example, the following markers on memory T cells, e.g., on memory T cell precursors: CD62L high , CD127high , CD27 + , and increased expression of one or more of BCL2; Decreased expression of KLRG1, e.g., on memory T cells, e.g., on memory T cell precursors; and Memory T cell precursors, e.g., those with the following characteristics: CD62L high Increased CD127 high Increased CD27 + an increase in the number of cells with any one or combination of an increase in KLRG1, a decrease in KLRG1, and an increase in BCL2 wherein any of the above-described changes occur, e.g., at least transiently, e.g., when compared to an untreated subject.

[0294] Ranges: Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be construed to include all the possible subranges specifically disclosed as well as individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be construed to include specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numerical values ​​within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. As another example, a range such as 95-99% identity includes those with 95%, 96%, 97%, 98%, or 99% identity, and includes subranges such as 96-99%, 96-98%, 96-97%, 97-99%, 97-98%, and 98-99% identity. This applies regardless of the width of the range.

[0295] Dual Car The present disclosure features, at least in part, novel nucleic acid molecules encoding chimeric antigen receptor (CAR) molecules, e.g., dual CARs as described herein, comprising a first CAR comprising a CD22 CAR and a second CAR comprising a CD19 CAR. In some embodiments, the CD22 CAR comprises a CD22 antigen-binding domain and a first transmembrane domain; a first costimulatory signaling domain; and / or a first primary signaling domain. In some embodiments, the CD19 CAR comprises a CD19 antigen-binding domain and a second transmembrane domain; a second costimulatory signaling domain; and / or a second primary signaling domain. In some embodiments of the CAR molecules disclosed herein, the CAR molecule comprises two identical polypeptide sequences, e.g., a first and a second transmembrane domain; a first and a second costimulatory domain; and / or a first and a second primary signaling domain, wherein these polypeptide sequences are encoded by different nucleotide sequences. Also disclosed herein are methods of using the CAR molecules.

[0296] Without wishing to be bound by theory, it is believed that in some embodiments, the nucleic acid molecule encoding the CAR molecule, e.g., the dual CAR molecule, is optimized, e.g., codon-optimized, to prevent recombination, e.g., homologous recombination. In some embodiments, the CAR molecule, e.g., the dual CAR molecule, comprises two domains, e.g., a first transmembrane domain and a second transmembrane domain, each of which comprises a similar amino acid sequence but is encoded by a different nucleotide sequence.

[0297] In some embodiments, a CAR molecule disclosed herein comprises a first CAR comprising a first antigen binding domain that binds to CD22; a first transmembrane domain; a first costimulatory signaling domain; and / or a first primary signaling domain.

[0298] In one embodiment, the CD22 antigen binding domain comprises one or more (e.g., all three) light chain complementarity determining region 1 (LC CDR1), light chain complementarity determining region 2 (LC CDR2), and light chain complementarity determining region 3 (LC CDR3) of a CD22 binding domain described herein, e.g., in Table 1A, Table 2A, or Table 3A; and / or one or more (e.g., all three) heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2), and heavy chain complementarity determining region 3 (HC CDR3) of a CD22 binding domain described herein, e.g., in Table 1A, Table 2A, or Table 3A. In one embodiment, the CD22 antigen binding domain comprises the LC CDR1, LC CDR2, and LC CDR3 of a CD22 binding domain described herein, e.g., in Table 1A, Table 2A, or Table 3A; and / or the HC CDR1, HC CDR2, and HC CDR3 of a CD22 binding domain described herein, e.g., in Table 1A, Table 2A, or Table 3A.

[0299] In one embodiment, the CD22 binding domain comprises an LC CDR1 of SEQ ID NO: 28, an LC CDR2 of SEQ ID NO: 29, and an LC CDR3 of SEQ ID NO: 30. In one embodiment, the CD22 binding domain comprises an LC CDR1 of SEQ ID NO: 31, an LC CDR2 of SEQ ID NO: 32, and an LC CDR3 of SEQ ID NO: 33. In one embodiment, the CD22 binding domain comprises an LC CDR1 of SEQ ID NO: 34, an LC CDR2 of SEQ ID NO: 32, and an LC CDR3 of SEQ ID NO: 30.

[0300] In one embodiment, the CD22 binding domain comprises an HC CDR1 of SEQ ID NO: 20, an HC CDR2 of SEQ ID NO: 21, and an HC CDR3 of SEQ ID NO: 22. In one embodiment, the CD22 binding domain comprises an HC CDR1 of SEQ ID NO: 23, an HC CDR2 of SEQ ID NO: 24, and an HC CDR3 of SEQ ID NO: 22. In one embodiment, the CD22 binding domain comprises an HC CDR1 of SEQ ID NO: 25, an HC CDR2 of SEQ ID NO: 26, and an HC CDR3 of SEQ ID NO: 27.

[0301] In one embodiment, the CD22 binding domain comprises a LC CDR1 of SEQ ID NO:28, a LC CDR2 of SEQ ID NO:29, and a LC CDR3 of SEQ ID NO:30; and a HC CDR1 of SEQ ID NO:20, a HC CDR2 of SEQ ID NO:21, and a HC CDR3 of SEQ ID NO:22.

[0302] In one embodiment, the CD22 binding domain comprises a LC CDR1 of SEQ ID NO: 31, a LC CDR2 of SEQ ID NO: 32, and a LC CDR3 of SEQ ID NO: 33; and a HC CDR1 of SEQ ID NO: 23, a HC CDR2 of SEQ ID NO: 24, and a HC CDR3 of SEQ ID NO: 22.

[0303] In one embodiment, the CD22 binding domain comprises a LC CDR1 of SEQ ID NO: 34, a LC CDR2 of SEQ ID NO: 32, and a LC CDR3 of SEQ ID NO: 30; and a HC CDR1 of SEQ ID NO: 25, a HC CDR2 of SEQ ID NO: 26, and a HC CDR3 of SEQ ID NO: 27.

[0304] In some embodiments, the CD22 antigen-binding domain (e.g., scFv) comprises a light chain variable (VL) region of a CD22 binding domain described herein, e.g., in Table 1A or Table 3A; and / or a heavy chain variable (VH) region of a CD22 binding domain described herein, e.g., in Table 1A or Table 3A. In some embodiments, the CD22 antigen-binding domain comprises a VL region comprising an amino acid sequence having one, two, or three or more alterations (e.g., substitutions) of a CD22 VL-region sequence provided in Table 1A or Table 3A, but not more than 30, 20, or 10 alterations (e.g., substitutions). In some embodiments, the CD22 antigen-binding domain comprises a VL region comprising an amino acid sequence having at least 95% identity to a CD22 VL-region sequence provided in Table 1A or Table 3A. In some embodiments, the CD22 antigen-binding domain comprises a VL region comprising the amino acid sequence of a CD22 VL-region sequence provided in Table 1A or Table 3A. In one embodiment, a CD22 antigen-binding domain comprises a VH region comprising an amino acid sequence having one, two, or three or more alterations (e.g., substitutions) of a CD22 VH region sequence provided in Table 1A or Table 3A, but not more than 30, 20, or 10 alterations (e.g., substitutions). In one embodiment, a CD22 antigen-binding domain comprises a VH region comprising an amino acid sequence having at least 95% identity to a CD22 VH region sequence provided in Table 1A or Table 3A. In one embodiment, a CD22 antigen-binding domain comprises a VH region comprising the amino acid sequence of a CD22 VH region sequence provided in Table 1A or Table 3A.

[0305] In some embodiments, the CD22 antigen binding comprises an scFv comprising an amino acid sequence having one, two, or three or more alterations (e.g., substitutions) of a CD22 scFv sequence provided in Table 1A or Table 3A, e.g., SEQ ID NO: 50, but not more than 30, 20, or 10 alterations (e.g., substitutions). In some embodiments, the CD22 antigen binding comprises an scFv comprising an amino acid sequence having at least 95% identity to a CD22 scFv sequence provided in Table 1A or Table 3A, e.g., SEQ ID NO: 50. In some embodiments, the CD22 antigen binding comprises an scFv comprising a CD22 scFv sequence provided in Table 1A or Table 3A, e.g., the amino acid sequence of SEQ ID NO: 50. In some embodiments, the CD22 antigen binding comprises an scFv encoded by a nucleotide sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% identity to a CD22 scFv sequence provided in Table 1A or Table 3A, e.g., SEQ ID NO: 49 or 51.

[0306] In some embodiments, a CAR molecule disclosed herein comprises a second CAR comprising a second antigen binding domain that binds to CD19 and a second transmembrane domain; a second costimulatory domain; and / or a second primary signaling domain.

[0307] In some embodiments, the CD19 antigen binding domain comprises one or more (e.g., all three) light chain complementarity determining region 1 (LC CDR1), light chain complementarity determining region 2 (LC CDR2), and light chain complementarity determining region 3 (LC CDR3) of a CD19 binding domain described herein, e.g., in Table 1A, Table 2A, Table 3A, or Table 5A; and / or one or more (e.g., all three) heavy chain complementarity determining region 1 (HC CDR1), heavy chain complementarity determining region 2 (HC CDR2), and heavy chain complementarity determining region 3 (HC CDR3) of a CD19 binding domain described herein, e.g., in Table 1A, Table 2A, Table 3A, or Table 5A. In some embodiments, the CD19 antigen-binding domain comprises an LC CDR1, an LC CDR2, and an LC CDR3 of a CD19 binding domain described herein, e.g., in Table 1A or Table 2A; and / or an HC CDR1, an HC CDR2, and an HC CDR3 of a CD19 binding domain described herein, e.g., in Table 1A, Table 2A, or Table 3A. In some embodiments, the CD19 antigen-binding domain comprises an LC CDR1 of SEQ ID NO: 40, an LC CDR2 of SEQ ID NO: 41, and an LC CDR3 of SEQ ID NO: 42; and / or an HC CDR1 of SEQ ID NO: 35, an HC CDR2 of SEQ ID NOs: 36-38, and an HC CDR3 of SEQ ID NO: 39.

[0308] In some embodiments, the CD19 antigen-binding domain (e.g., scFv) comprises a light chain variable (VL) region of a CD19 binding domain described herein, e.g., in Table 1A, Table 3A, or Table 5A; and / or a heavy chain variable (VH) region of a CD19 binding domain described herein, e.g., in Table 1A, Table 3A, or Table 5A. In some embodiments, the CD19 antigen-binding domain comprises a VL region comprising an amino acid sequence having one, two, or three or more alterations (e.g., substitutions) of a CD19 VL-region sequence provided in Table 1A, Table 3A, or Table 5A, but not more than 30, 20, or 10 alterations (e.g., substitutions). In some embodiments, the CD19 antigen-binding domain comprises a VL region comprising an amino acid sequence having at least 95% identity to a CD19 VL-region sequence provided in Table 1A, Table 3A, or Table 5A. In some embodiments, the CD19 antigen-binding domain comprises a VL region comprising the amino acid sequence of a CD19 VL region sequence provided in Table 1A, Table 3A, or Table 5A. In some embodiments, the CD19 antigen-binding domain comprises a VH region comprising an amino acid sequence having one, two, or three or more alterations (e.g., substitutions) of a CD19 VH region sequence provided in Table 1A, Table 3A, or Table 5A, but not more than 30, 20, or 10 alterations (e.g., substitutions). In some embodiments, the CD19 antigen-binding domain comprises a VH region comprising an amino acid sequence having at least 95% identity to a CD19 VH region sequence provided in Table 1A, Table 3A, or Table 5A. In some embodiments, the CD19 antigen-binding domain comprises a VH region comprising the amino acid sequence of a CD19 VH region sequence provided in Table 1A, Table 3A, or Table 5A.

[0309] In another embodiment, the CD19 antigen-binding domain comprises an scFv comprising an amino acid sequence having one, two, or three or more alterations (e.g., substitutions), but not more than 30, 20, or 10 alterations (e.g., substitutions), of a CD19 scFv sequence provided in Table 1A, Table 3A, or Table 5A, e.g., SEQ ID NO: 44. In another embodiment, the CD19 antigen-binding domain comprises an scFv comprising an amino acid sequence having at least 95% identity to a CD19 scFv sequence provided in Table 1A, Table 3A, or Table 5A, e.g., SEQ ID NO: 44. In another embodiment, the CD19 antigen-binding domain comprises an scFv comprising a CD19 scFv sequence provided in Table 1A, Table 3A, or Table 5A, e.g., the amino acid sequence of SEQ ID NO: 44. In other embodiments, the CD19 antigen binding domain comprises an scFv encoded by a nucleotide sequence having at least 95%, 96%, 97%, 98%, 99% or 100% identity to a CD19 scFv sequence provided in Table 1A, Table 3A or Table 5A, e.g., SEQ ID NO: 43 or 48.

[0310] In some aspects, the CAR molecule disclosed herein comprises a first CAR comprising a first transmembrane domain and a second CAR comprising a second transmembrane domain. In some embodiments, the first transmembrane domain and the second transmembrane domain comprise the same amino acid sequence, for example, as disclosed herein. In some embodiments, the first transmembrane domain and the second transmembrane domain are encoded by a first nucleotide sequence and a second nucleotide sequence, respectively. In some embodiments, the first nucleotide sequence and the second nucleotide sequence have at least one nucleotide difference.

[0311] In some embodiments, the first transmembrane domain and the second transmembrane domain are the same transmembrane domain, e.g., selected from the α, β, or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD123, CD134, CD137, or CD154.

[0312] In some embodiments, the first transmembrane domain and the second transmembrane domain are different transmembrane domains, e.g., selected from the α, β, or ζ chain of the T cell receptor, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD123, CD134, CD137, or CD154.

[0313] In some embodiments, a nucleic acid molecule encoding a CAR molecule described herein comprises a first CAR comprising a first transmembrane domain and a second CAR comprising a second transmembrane domain. In some embodiments, the first transmembrane domain and the second transmembrane domain comprise a CD8α transmembrane domain. In some embodiments, the first transmembrane domain and the second transmembrane domain comprise the amino acid sequence of SEQ ID NO: 65, or an amino acid sequence having at least 90% identity thereto.

[0314] In some embodiments, the nucleotide sequence encoding the first transmembrane domain and contained in the nucleic acid molecule is different from the nucleotide sequence encoding the second transmembrane domain and contained in the nucleic acid molecule.

[0315] In some aspects, the CAR molecule disclosed herein comprises a first CAR comprising a first costimulatory domain and a second CAR comprising a second costimulatory domain. In some embodiments, the first costimulatory domain and the second costimulatory domain comprise the same amino acid sequence, for example, as disclosed herein. In some embodiments, the first costimulatory domain and the second costimulatory domain are encoded by a first nucleotide sequence and a second nucleotide sequence, respectively. In some embodiments, the first nucleotide sequence and the second nucleotide sequence have at least one nucleotide difference.

[0316] In some embodiments, the first costimulatory domain and the second costimulatory domain are the same costimulatory domain, e.g., selected from the signaling domains of OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), or 4-1BB (CD137).

[0317] In some embodiments, the first costimulatory domain and the second costimulatory domain are different costimulatory domains, e.g., selected from the signaling domains of OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), or 4-1BB (CD137).

[0318] In some embodiments, a nucleic acid molecule encoding a CAR molecule described herein comprises a first CAR comprising a first costimulatory domain and a second CAR comprising a second costimulatory domain. In some embodiments, the first costimulatory domain and the second costimulatory domain comprise a 4-1BB costimulatory domain. In some embodiments, the first costimulatory domain and the second costimulatory domain comprise the amino acid sequence of SEQ ID NO: 65, or an amino acid sequence having at least 90% identity thereto.

[0319] In some embodiments, the nucleotide sequence encoding the first costimulatory domain and contained in the nucleic acid molecule is different from the nucleotide sequence encoding the second costimulatory domain and contained in the nucleic acid molecule.

[0320] In some aspects, the present disclosure provides nucleic acid molecules encoding CAR molecules, for example, including (i) a first CAR comprising a CD22 antigen-binding domain and (ii) a second CAR comprising a CD19 antigen-binding domain. In embodiments, the nucleic acid comprises RNA or DNA. In embodiments, the nucleic acid sequences encoding (i) and (ii) are oriented in the same direction, e.g., transcription of the nucleic acid sequences encoding (i) and (ii) proceeds in the same direction. In embodiments, the nucleic acid sequences encoding (i) and (ii) are oriented in opposite directions. In embodiments, a single promoter controls the expression of the nucleic acid sequences encoding (i) and (ii). In embodiments, a nucleic acid encoding a protease cleavage site (such as a T2A, P2A, E2A, or F2A cleavage site) is located between the nucleic acid sequences encoding (i) and (ii). In embodiments, the protease cleavage site is positioned to allow the cell to express a fusion protein comprising (i) and (ii), which is subsequently processed by proteolytic cleavage into two peptides. In some embodiments, the nucleic acid sequence encoding (i) is upstream of the nucleic acid sequence encoding (ii), or the nucleic acid sequence encoding (ii) is upstream of the nucleic acid sequence encoding (i). In embodiments, a first promoter controls expression of the nucleic acid sequence encoding (i) and a second promoter controls expression of the nucleic acid sequence encoding (ii). In embodiments, the nucleic acid is a plasmid. In embodiments, the nucleic acid comprises a viral packaging element. In some aspects, the present disclosure provides a cell, e.g., an immune effector cell, comprising a nucleic acid described herein, e.g., a nucleic acid comprising (i) and (ii) as described above. The cell may comprise a protease (e.g., endogenous or exogenous) that cleaves the T2A, P2A, E2A, or F2A cleavage site.

[0321] Exemplary nucleotide and amino acid sequences of CAR molecules, e.g., dual CAR molecules, disclosed herein are provided in Table 1A.

[0322] [Table 1]

[0323] Table 2

[0324] Table 3

[0325] Table 4

[0326] Table 5

[0327] Table 6

[0328] Table 7

[0329] Table 8

[0330] Table 9

[0331] Table 10

[0332] Table 11

[0333] [Table 12]

[0334] [Table 13]

[0335] [Table 14]

[0336] The CD22 and CD19 CDRs of the dual or tandem CARs of the present disclosure are provided in Table 2A.

[0337] [Table 15]

[0338] [Table 16]

[0339] Table 3A provides the nucleotide and amino acid sequences of the CD19 and CD22 binding domains of the dual or tandem CARs disclosed herein.

[0340] [Table 17]

[0341] [Table 18]

[0342] [Table 19]

[0343] [Table 20]

[0344] [Table 21]

[0345] Table 4A provides the nucleotide and amino acid sequences of additional CAR components, such as the signal peptide, linker, and P2A site.

[0346] [Table 22]

[0347] [Table 23]

[0348] [Table 24]

[0349] [Table 25]

[0350] [Table 26]

[0351] [Table 27]

[0352] [Table 28]

[0353] [Table 29]

[0354] Tandem Car In some embodiments, the present specification discloses a CAR that comprises a bispecific antigen binding domain, for example, a tandem CAR. In some embodiments, the bispecific antigen binding domain comprises two antigen binding domains, for example, a first antigen binding domain and a second antigen binding domain. In some embodiments, the tandem CAR comprises a bispecific antigen binding domain that comprises a CD22 antigen binding domain and a CD19 antigen binding domain.

[0355] In some embodiments of the bispecific antigen-binding domain, the first antigen-binding domain is an antibody molecule, e.g., an antibody binding domain (e.g., an scFv). In some embodiments of the bispecific antigen-binding domain, the second antigen-binding domain is an antibody molecule, e.g., an antibody binding domain (e.g., an scFv). Within each antibody molecule, e.g., an scFv, of the bispecific antigen-binding domain, the VH can be upstream or downstream of the VL.

[0356] In some embodiments, the upstream antibody or antibody fragment (e.g., scFv) is arranged such that its VH (VH1) is upstream of its VL (VL1), and the downstream antibody or antibody fragment (e.g., scFv) is arranged such that its VL (VL2) is upstream of its VH (VH2), such that the bispecific antibody molecule has the overall N-terminal to C-terminal arrangement VH1-VL1-VL2-VH2.

[0357] In some embodiments, the upstream antibody or antibody fragment (e.g., scFv) is arranged such that its VL (VL1) is upstream of its VH (VH1), and the downstream antibody or antibody fragment (e.g., scFv) is arranged such that its VH (VH2) is upstream of its VL (VL2), such that the bispecific antibody molecule has the overall N-terminal to C-terminal arrangement VL1-VH1-VH2-VL2.

[0358] In some embodiments, the upstream antibody or antibody fragment (e.g., scFv) is arranged such that its VL (VL1) is upstream of its VH (VH1), and the downstream antibody or antibody fragment (e.g., scFv) is arranged such that its VL (VL2) is upstream of its VH (VH2), such that the bispecific antibody molecule has the overall N-terminal to C-terminal arrangement VL1-VH1-VL2-VH2.

[0359] Furthermore, in some embodiments, the upstream antibody or antibody fragment (e.g., scFv) is arranged such that its VH (VH1) is upstream of its VL (VL1), and the downstream antibody or antibody fragment (e.g., scFv) is arranged such that its VH (VH2) is upstream of its VL (VL2), such that the bispecific antibody molecule has the overall N-terminal to C-terminal arrangement VH1-VL1-VH2-VL2.

[0360] In any of the above configurations, a linker is optionally placed between the two antibodies or antibody fragments (e.g., scFvs), for example, between VL1 and VL2 when the construct is configured as VH1-VL1-VL2-VH2; between VH1 and VH2 when the construct is configured as VL1-VH1-VH2-VL2; between VH1 and VL2 when the construct is configured as VL1-VH1-VL2-VH2; or between VL1 and VH2 when the construct is configured as VH1-VL1-VH2-VL2. Generally, the linker between the two scFvs should be long enough to avoid mispairing between the domains of the two scFvs. The linker can be as described herein. In some embodiments, the linker is a (Gly4-Ser)n linker (where n is 1, 2, 3, 4, 5, or 6). In some embodiments, the linker is (Gly4-Ser)n, where n=1, e.g., the linker has the amino acid sequence Gly4-Ser. In some embodiments, the linker is (Gly4-Ser)n, where n=4 (SEQ ID NO: 82). In some embodiments, the linker comprises, e.g., consists of, the amino acid sequence: LAEAAAK (e.g., SEQ ID NO: 80).

[0361] In any of the above configurations, optionally, a linker is placed between the VL and VH of the first scFv. Optionally, a linker is placed between the VL and VH of the second scFv. In constructs with multiple linkers, any two or more of the linkers can be the same or different. Thus, in some embodiments, a bispecific CAR comprises a VL, a VH, and optionally one or more linkers, arranged as described herein.

[0362] In some embodiments, each antibody molecule, e.g., each antigen-binding domain (e.g., each scFv), comprises a linker between the VH region and the VL region. In some embodiments, the linker between the VH region and the VL region is a (Gly4-Ser)n linker, where n is 1, 2, 3, 4, 5, or 6. In some embodiments, the linker is (Gly4-Ser)n, where n=1, e.g., the linker has the amino acid sequence Gly4-Ser. In some embodiments, the linker is (Gly4-Ser)n, where n=4 (SEQ ID NO: 82). In some embodiments, the VH region and the VL region are joined without a linker.

[0363] Split CAR In some embodiments, the CAR-expressing cell uses a split CAR. The split CAR approach is described in further detail in International Publication Nos. 2014 / 055442 and 2014 / 055657 (incorporated herein by reference). Briefly, the split CAR system comprises a cell expressing a first CAR having a first antigen-binding domain and a costimulatory domain (e.g., 4-1BB), and the cell also expresses a second CAR having a second antigen-binding domain and an intracellular signaling domain (e.g., CD3ζ). When the cell encounters the first antigen, the costimulatory domain is activated, causing the cell to proliferate. When the cell encounters the second antigen, the intracellular signaling domain is activated, initiating cell-killing activity. Thus, the CAR-expressing cell is only fully activated in the presence of both antigens.

[0364] RNA transfection Disclosed herein are methods for producing in vitro transcribed RNA CARs. The present invention also includes CAR-encoding RNA constructs that can be directly transfected into cells. Methods for producing mRNA for use in transfection include in vitro transcription (IVT) of a template using specifically designed primers, followed by poly(A) addition, to produce constructs, typically 50-2000 bases long, containing 3' and 5' untranslated sequences ("UTRs"), a 5' cap and / or internal ribosome entry site (IRES), the nucleic acid to be expressed, and a poly(A) tail. RNAs produced in this manner can be efficiently translated in cells of different species. In one embodiment, the template contains the sequence of the CAR.

[0365] In one embodiment, the CAR, e.g., a dual CAR or a tandem CAR, is encoded by messenger RNA (mRNA). In one embodiment, the mRNA encoding the CAR, e.g., a dual CAR or a tandem CAR, is introduced into an immune effector cell, e.g., a T cell or an NK cell, to generate a CAR-expressing cell, e.g., a CART cell or a CAR NK cell.

[0366] In one embodiment, in vitro transcribed RNA of a CAR can be introduced into cells as a form of transient transfection. The RNA is produced by in vitro transcription using a polymerase chain reaction (PCR)-generated template. Using appropriate primers and RNA polymerase, DNA of interest from any source can be directly converted into a template for in vitro mRNA synthesis by PCR. The source of DNA can be, for example, genomic DNA, plasmid DNA, phage DNA, cDNA, synthetic DNA sequence, or any other suitable DNA source. A desirable template for in vitro transcription is the CAR of the present invention. For example, the template for an RNA CAR includes an extracellular region including a single-chain variable domain of an anti-tumor antibody; a hinge region, a transmembrane domain (e.g., the transmembrane domain of CD8a); and a cytoplasmic region including an intracellular signaling domain, for example, including the signaling domain of CD3-zeta and the signaling domain of 4-1BB.

[0367] In one embodiment, the DNA used for PCR comprises an open reading frame. The DNA can be from a naturally occurring DNA sequence from the genome of an organism. In one embodiment, the nucleic acid can include some or all of the 5' and / or 3' untranslated regions (UTRs). The nucleic acid can include exons and introns. In one embodiment, the DNA used for PCR is a human nucleic acid sequence. In another embodiment, the DNA used for PCR is a human nucleic acid sequence including the 5' and 3' UTRs. The DNA can alternatively be an artificial DNA sequence that is not normally expressed in a naturally occurring organism. An exemplary artificial DNA sequence is one that includes portions of genes that are ligated together to form an open reading frame encoding a fusion protein. The portions of DNA ligated together can be from a single organism or from two or more organisms.

[0368] Templates for in vitro transcription of mRNA for transfection are generated using PCR. Methods for performing PCR are well known in the art. Primers used in PCR are designed to have a region substantially complementary to a region of DNA used as a PCR template. "Substantially complementary," as used herein, refers to a sequence of nucleotides in which most or all of the bases in the primer sequence are complementary, or in which one or more bases are non-complementary or mismatched. A substantially complementary sequence is capable of annealing or hybridizing with the intended DNA target under the annealing conditions used in PCR. Primers can be designed to be substantially complementary to any portion of the DNA template. For example, primers can be designed to amplify a portion of a nucleic acid normally transcribed in cells (open reading frame), including the 5' and 3' UTRs. Primers can also be designed to amplify a portion of a nucleic acid encoding a specific domain of interest. In one embodiment, primers are designed to amplify the coding region of a human cDNA, including all or a portion of the 5' and 3' UTRs. Primers useful for PCR can be generated by synthetic methods well known in the art. A "forward primer" is a primer that contains a nucleotide region that is substantially complementary to nucleotides on a DNA template that is upstream of the DNA sequence to be amplified. "Upstream" is used herein to refer to a position 5' of the DNA sequence to be amplified relative to the coding strand. A "reverse primer" is a primer that contains a nucleotide region that is substantially complementary to a double-stranded DNA template that is downstream of the DNA sequence to be amplified. "Downstream" is used herein to refer to a position 3' of the DNA sequence to be amplified relative to the coding strand.

[0369] Any DNA polymerase useful in PCR can be used in the methods disclosed herein. Reagents and polymerases are commercially available from several sources.

[0370] Chemical structures capable of promoting stability and / or translation efficiency may also be used. The RNA preferably has 5' and 3' UTRs. In one embodiment, the 5' UTR is 1 to 3,000 nucleotides in length. The length of the 5' and 3' UTR sequences added to the coding region can be varied by various methods, including, but not limited to, designing PCR primers that anneal to different regions of the UTR. Using this approach, one skilled in the art can modify the 5' and 3' UTR lengths necessary to achieve optimal translation efficiency after transfection of the transcribed RNA.

[0371] The 5' and 3' UTRs may be naturally occurring endogenous 5' and 3' UTRs for the nucleic acid of interest. Alternatively, a UTR sequence that is not endogenous to the nucleic acid of interest can be added by introducing the UTR sequence into the forward and reverse primers or by any other modification of the template. The use of a UTR sequence that is not endogenous to the nucleic acid of interest can be useful for modifying RNA stability and / or translation efficiency. For example, it is known that AU-rich elements in the 3' UTR sequence can reduce mRNA stability. Therefore, the 3' UTR can be selected or designed to increase the stability of the transcribed RNA based on the properties of UTRs known in the art.

[0372] In one embodiment, the 5' UTR can contain the Kozak sequence of the endogenous nucleic acid. Alternatively, if a 5' UTR that is not endogenous to the nucleic acid of interest is added by PCR as described above, the consensus Kozak sequence can be redesigned by adding a 5' UTR sequence. While the Kozak sequence can increase the translation efficiency of some RNA transcripts, it does not appear to be necessary for all RNAs to enable efficient translation. The requirement for a Kozak sequence for many mRNAs is known in the art. In some embodiments, the 5' UTR can be the 5' UTR of an RNA virus whose RNA genome is stable in cells. In some embodiments, various nucleotide analogs can be used in the 3' or 5' UTR to prevent exonuclease degradation of the mRNA.

[0373] To enable RNA synthesis from a DNA template without the need for gene cloning, a transcription promoter must be added to the DNA template upstream of the sequence to be transcribed. When a sequence that functions as a promoter for RNA polymerase is added to the 5' end of the forward primer, the RNA polymerase promoter is incorporated upstream of the open reading frame to be transcribed in the PCR product. In a preferred embodiment, the promoter is a T7 polymerase promoter, as described elsewhere herein. Other useful promoters include, but are not limited to, T3 and SP6 RNA polymerase promoters. Consensus nucleic acid sequences for T7, T3, and SP6 promoters are known in the art.

[0374] In a preferred embodiment, the mRNA has both a 5'-end cap and a 3' poly(A) tail, which determine ribosome binding, translation initiation, and mRNA stability in cells. On circular DNA templates, such as plasmid DNA, RNA polymerase produces long concatemeric products that are not suitable for expression in eukaryotic cells. Transcription of plasmid DNA linearized at the end of the 3' UTR, even if polyadenylated after transcription, results in mRNA of a normal size that is not effective in eukaryotic transfection.

[0375] On a linear DNA template, phage T7 RNA polymerase can extend the 3' end of the transcript beyond the last base of the template (Schenborn and Mierendorf, Nucleic Acids Res., 13:6223-36 (1985); Nacheva and Berzal-Herranz, Eur. J. Biochem., 270:1485-65 (2003)).

[0376] The traditional method for incorporating polyA / T stretches into DNA templates is molecular cloning. However, polyA / T sequences incorporated into plasmid DNA can also cause plasmid instability, which is why plasmid DNA templates obtained from bacterial cells are often highly contaminated with deletions and other abnormalities. This makes the cloning procedure not only laborious and time-consuming but also unreliable. Therefore, a method that allows for the construction of DNA templates with polyA / T 3' stretches without cloning is highly desirable.

[0377] The poly(A) / T segment of the transcription DNA template can be generated during PCR using a reverse primer containing a poly(T) tail, such as a 100T tail (which can range in size from 50 to 5000T), or can be generated after PCR by any other method, including but not limited to, DNA ligation or in vitro recombination. The poly(A) tail also provides stability to the RNA, reducing its degradation. Generally, the length of the poly(A) tail positively correlates with the stability of the transcribed RNA. In one embodiment, the poly(A) tail is 100 to 5000 adenosines.

[0378] The poly(A) tail of an RNA can be further extended after in vitro transcription using a poly(A) polymerase, such as Escherichia coli (E. coli) poly(A) polymerase (E-PAP). In one embodiment, increasing the length of the poly(A) tail from 100 nucleotides to 300-400 nucleotides approximately doubles the translation efficiency of the RNA. In addition, adding different chemical groups to the 3' end can enhance mRNA stability. Such additions can include modified / artificial nucleotides, aptamers, and other compounds. For example, poly(A) polymerase can be used to incorporate ATP analogs into the poly(A) tail. ATP analogs can further enhance RNA stability.

[0379] A 5' cap also provides stability to an RNA molecule. In a preferred embodiment, the RNA produced by the methods disclosed herein includes a 5' cap. The 5' cap can be provided using techniques known in the art and described herein (Cougot, et al., Trends in Biochem. Sci., 29:436-444 (2001); Stepinski, et al., RNA, 7:1468-95 (2001); Elango, et al., Biochim. Biophys. Res. Commun., 330:958-966 (2005)).

[0380] The RNA produced by the methods disclosed herein may also contain an internal ribosome entry site (IRES) sequence. The IRES sequence may be any viral, chromosomal, or artificially designed sequence that initiates cap-independent ribosome binding to mRNA to facilitate translation initiation. Any solute suitable for cell electroporation may be included, including factors that promote cell penetration and viability, such as sugars, peptides, lipids, proteins, antioxidants, and detergents.

[0381] RNA can be introduced into target cells using any of several different methods, including, but not limited to, electroporation (Amaxa Nucleofector-II (Amaxa Biosystems, Cologne, Germany)), (ECM 830(BTX) (Harvard Instruments, Boston, Mass.) or Gene Pulser II (BioRad, Denver, Colo.), Multiporator (Eppendort, Hamburg, Germany), cationic liposome-mediated transfection using lipofection, polymer encapsulation, peptide-mediated transfection, or biolistic particle delivery systems such as "gene guns" (see, e.g., Nishikawa, et al., Hum Gene Ther., 12(8):861-70 (2001)).

[0382] Non-viral delivery methods In some embodiments, non-viral methods can be used to deliver a nucleic acid encoding a CAR described herein to a cell, tissue, or subject.

[0383] In one embodiment, the non-viral method involves the use of transposons (also called transposable elements). In one embodiment, a transposon is a segment of DNA that can insert itself into a location in the genome, e.g., a segment of DNA that is self-replicating and can insert its copy into a genome, or a segment of DNA that can be spliced ​​out of a longer nucleic acid and inserted elsewhere in the genome. For example, a transposon comprises a DNA sequence made up of inverted repeats flanking a transposable gene.

[0384] Exemplary methods of nucleic acid delivery using transposons include the Sleeping Beauty transposon system (SBTS) and the piggyBac (PB) transposon system. For example, Aronovich et al.Hum.Mol.Genet.20.R1(2011):R14-20;Singh et al.Cancer Res.15(2008):2961-2971;Huang et al.Mol.Ther.16(2008):580-589;Grabundzija et al. al.Mol.Ther.18(2010):1200-1209;Kebriaei et al.Blood.122.21(2013):166;Williams.Molecular Therapy 16.9(2008):1515-16;Bell et al.Nat.Protoc.2.12(2007):3153-65; and Ding et al. al. Cell. 122.3(2005):473-83, all of which are incorporated herein by reference.

[0385] SBTS contains two components: 1) a transposon containing a transgene, and 2) a source of transposase enzyme. The transposase can transfer the transposon from a carrier plasmid (or other donor DNA) to target DNA, such as a host cell chromosome / genome. For example, the transposase binds to the carrier plasmid / donor DNA, excises the transposon (containing the transgene) from the plasmid, and inserts it into the genome of the host cell. See, e.g., Aronovich et al.

[0386] Exemplary transposons include pT2-based transposons. See, e.g., Grabundzija et al. Nucleic Acids Res. 41.3 (2013): 1829-47; and Singh et al. Cancer Res. 68.8 (2008): 2961-2971, all of which are incorporated herein by reference. Exemplary transposases include Tc1 / mariner-type transposases, such as SB10 transposase or SB11 transposase (e.g., hyperactive transposases that can be expressed from a cytomegalovirus promoter). See, e.g., Aronovich et al.; Kebriai et al.; and Grabundzija et al., all of which are incorporated herein by reference.

[0387] The use of SBTS allows for efficient integration and expression of a transgene, e.g., a nucleic acid encoding a CAR described herein. Provided herein are methods for generating cells, e.g., T cells or NK cells, that stably express a CAR described herein using a transposon system such as SBTS.

[0388] According to the methods described herein, in some embodiments, one or more nucleic acids, e.g., plasmids, comprising SBTS components are delivered to cells (e.g., T cells or NK cells). For example, the nucleic acids are delivered by standard methods for nucleic acid (e.g., plasmid DNA) delivery, such as those described herein, e.g., electroporation, transfection, or lipofection. In some embodiments, the nucleic acid comprises a transposon comprising a transgene, e.g., a nucleic acid encoding a CAR described herein. In some embodiments, the nucleic acid comprises a transposon comprising a transgene (e.g., a nucleic acid encoding a CAR described herein) and a nucleic acid sequence encoding a transposase enzyme. In some embodiments, a system comprising two nucleic acids, e.g., a dual plasmid system, is provided, e.g., wherein a first plasmid comprises a transposon comprising a transgene and a second plasmid comprises a nucleic acid sequence encoding a transposase enzyme. For example, the first and second nucleic acids are co-delivered to a host cell.

[0389] In some embodiments, cells, e.g., T cells or NK cells, that express a CAR described herein are generated using a combination of gene insertion using SBTS and gene editing using nucleases (e.g., zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR / Cas systems, or engineered meganucleases or re-engineered homing endonucleases).

[0390] In some embodiments, non-viral delivery methods allow for the reprogramming of cells, such as T cells or NK cells, and the direct infusion of the cells into a subject. Advantages of non-viral vectors include, but are not limited to, ease and relatively low cost of production in sufficient quantities to satisfy a patient population, stability upon storage, and lack of immunogenicity.

[0391] Nucleic acid construct encoding CAR The present invention also provides nucleic acid molecules encoding one or more CAR constructs described herein. In one embodiment, the nucleic acid molecule is provided as a messenger RNA transcript. In one embodiment, the nucleic acid molecule is provided as a DNA construct.

[0392] Nucleic acid sequences encoding the desired molecules can be obtained using standard techniques, for example, by screening libraries from cells which express the gene, by derivatizing the gene from a vector known to contain it, or by isolating it directly from cells and tissues known to contain it, using recombinant methods known in the art. Alternatively, the gene of interest can be produced synthetically rather than cloned.

[0393] The present invention also provides a vector into which the DNA of the present invention is inserted. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow long-term stable integration of transgenes and their transmission to daughter cells. Lentiviral vectors have the added advantage over vectors derived from onco-retroviruses, such as murine leukemia viruses, of being able to transduce non-proliferating cells, such as hepatocytes. Furthermore, they have the additional advantage of being less immunogenic.

[0394] In another embodiment, the vector containing the nucleic acid encoding the desired CAR of the present invention is an adenoviral vector (A5 / 35). In another embodiment, expression of the nucleic acid encoding the CAR can be achieved using transposons such as sleeping beauty, crisper, CAS9, and zinc finger nucleases. See June et al. 2009 Nature Reviews Immunology 9.10:704-716, infra, which is incorporated herein by reference.

[0395] In general, expression of a natural or synthetic nucleic acid encoding a CAR is typically achieved by operably linking a nucleic acid encoding a CAR polypeptide, or a portion thereof, to a promoter and incorporating the construct into an expression vector. The vector may be suitable for replication and integration in eukaryotes. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for controlling the expression of the desired nucleic acid sequence.

[0396] Expression of the constructs of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods for gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entireties. In another embodiment, the present invention provides a gene therapy vector.

[0397] Nucleic acids can be cloned into numerous types of vectors, including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0398] Furthermore, the expression vector can be provided to cells in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY, and other virology and molecular biology manuals. Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, suitable vectors contain an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (e.g., WO 01 / 96584; WO 01 / 29058; and U.S. Pat. No. 6,326,193).

[0399]

[0003] A number of viral-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. A gene of choice can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject in vivo or ex vivo. Many retroviral systems are known in the art. In one embodiment, an adenoviral vector is used. Many adenoviral vectors are known in the art. In one embodiment, a lentiviral vector is used.

[0400] Additional promoter elements, such as enhancers, control the frequency of transcription initiation. Typically, these are located in the region 30–110 bp upstream of the start site, although many promoters have been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements is often flexible, such that promoter function is maintained when elements are inverted or moved relative to one another. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, individual elements appear to be able to function cooperatively or independently to activate transcription. Exemplary promoters include the CMV IE gene, EF-1α, ubiquitin C, or phosphoglycerokinase (PGK) promoters.

[0401] An example of a promoter capable of expressing a CAR transgene in mammalian T cells is the EF-1 alpha (EF1a) promoter. The native EF1a promoter drives the expression of the alpha subunit of the elongation factor-1 complex, which is responsible for the enzymatic delivery of aminoacyl-tRNA to ribosomes. 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 lentiviral vectors. See, for example, Milone et al., Mol. Ther. 17(8):1453-1464 (2009). In one embodiment, the EF1a promoter comprises a sequence known in the art.

[0402] Another example of a promoter is the immediate-early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked to it. However, other constitutive promoter sequences, including but not limited to the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate-early promoter, and Rous sarcoma virus promoter, as well as human gene promoters, such as but not limited to the actin promoter, myosin promoter, elongation factor-1α promoter, hemoglobin promoter, and creatine kinase promoter, may also be used. Furthermore, the present invention should not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of an inducible promoter provides a molecular switch that can activate expression of an operably linked polynucleotide sequence when expression is desired and shut it off when expression is not desired. Examples of inducible promoters include, but are not limited to, a metallothionein promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0403] To assess the expression of a CAR polypeptide or a portion thereof, the expression vector introduced into cells may contain a selectable marker gene or a reporter gene, or both, to facilitate identification and selection of expressing cells from a population of cells being sought for gene transfection or infection with a viral vector. In other embodiments, the selectable marker is carried on a separate piece of DNA and used in a co-transfection method. Both the selectable marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes, such as neo.

[0404] Reporter genes are used to identify potentially transfected cells and evaluate the functionality of regulatory sequences. Generally, reporter genes are genes that encode polypeptides that are not present or expressed in the recipient organism or tissue, and whose expression is manifested by an easily detectable property, such as enzymatic activity. Expression of the reporter gene is assayed a suitable time after DNA is introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well known and can be prepared by known techniques or commercially available. Generally, constructs with minimal 5' flanking regions that exhibit the highest level of reporter gene expression are identified as promoters. Such promoter regions can be linked to reporter genes and used to evaluate drugs for their ability to modulate promoter-driven transcription.

[0405] Methods for introducing and expressing genes in cells are known in the art. With respect to expression vectors, the vectors can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method known in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0406] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art (see, e.g., Sambrook et al., 2012, MOLECULAR CLONING: A LABORATORY MANUAL, volumes 1-4, Cold Spring Harbor Press, NY). A preferred method for introducing polynucleotides into host cells is calcium phosphate transfection.

[0407] Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human, cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, etc. See, e.g., U.S. Patent Nos. 5,350,674 and 5,585,362.

[0408] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. A representative colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., artificial membrane vesicle). Other state-of-the-art methods for targeted delivery of nucleic acids are available, such as delivery of polynucleotides using targeted nanoparticles or other suitable submicron-sized delivery systems.

[0409] When a non-viral delivery system is utilized, a typical delivery vehicle is a liposome. The use of lipid formulations is intended for the introduction of nucleic acids into host cells (in vitro, ex vivo, or in vivo). In another embodiment, the nucleic acid can be bound to a lipid. The lipid-bound nucleic acid can be encapsulated in the aqueous interior of a liposome, dispersed within the lipid bilayer of a liposome, bound to a liposome via a linking molecule that binds both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a lipid-containing solution, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed within a micelle, or otherwise bound to a lipid. Lipid, lipid / DNA, or lipid / expression vector bound compositions are not limited to any particular structure in solution. For example, they can exist in a bilayer structure, as micelles, or have a "collapsed" structure. They can also simply be dispersed in a solution and form aggregates, possibly not uniform in size or shape. Lipids are fatty substances that can be naturally occurring or synthetic lipids. For example, lipids include the lipid droplets that occur naturally in the cytoplasm as well as a class of compounds containing long-chain aliphatic hydrocarbons such as fatty acids, alcohols, amines, amino alcohols, and aldehydes, and their derivatives.

[0410] Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") can be obtained from Sigma, St. Louis, MO; dicetyl phosphate ("DCP") can be obtained from K&K Laboratories (Plainview, NY); cholesterol ("Choi") can be obtained from Calbiochem-Behring; and dimyristyl phosphatidylglycerol ("DMPG") and other lipids can be obtained from Avanti Polar Lipids, Inc. (Birmingham, AL). Stock solutions of lipids in chloroform or chloroform / methanol can be stored at approximately -20°C. Chloroform is used as the sole solvent because it volatilizes more readily than methanol. "Liposome" is a general term that encompasses a variety of mono- and multilamellar lipid vesicles formed by the production of enclosed lipid bilayers or aggregates. Liposomes can be characterized by having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components self-aggregate before forming a closed structure, encapsulating water and dissolved solutes between the lipid bilayers (Ghosh et al., 1991 Glycobiology 5:505-10). However, compositions with structures in solution that differ from the typical vesicle structure are also encompassed. For example, lipids may be considered to be in a micellar structure or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also contemplated.

[0411] Regardless of the method by which exogenous nucleic acid is introduced into a host cell or otherwise exposes the cell to an inhibitor of the present invention, a variety of assays can be performed to confirm the presence of the recombinant DNA sequence in the host cell. Such assays include, for example, "molecular biological" assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR, and "biochemical" assays, such as detecting the presence or absence of specific peptides by, for example, immunological means (ELISA and Western blot), for use in drug identification or the assays described herein that fall within the scope of the present invention.

[0412] The present invention further provides a vector comprising a CAR-encoding nucleic acid molecule. In one embodiment, the CAR vector can be directly transduced into cells, such as T cells or NK cells. In one embodiment, the vector is a cloning or expression vector, such as, but not limited to, one or more plasmids (e.g., expression plasmids, cloning vectors, minicircles, minivectors, double minute chromosomes), retroviral and lentiviral vector constructs. In one embodiment, the vector is capable of expressing the CAR construct in mammalian T cells or NK cells. In one embodiment, the mammalian T cells are human T cells.

[0413] Manufacturing / Production Method The present invention also provides methods of producing the cells disclosed herein, e.g., methods of engineering T cells or NK cells to express nucleic acid molecules encoding one or more CAR constructs described herein. In some embodiments, the manufacturing methods disclosed herein are used to produce cells comprising nucleic acid molecules encoding two CARs disclosed herein (e.g., a CD19 / CD22 tandem and / or dual CAR disclosed herein). In some embodiments, the manufacturing methods disclosed herein are used to produce cells comprising nucleic acid molecules encoding a diabody CAR disclosed herein, e.g., an anti-CD22 / anti-CD19 diabody CAR disclosed herein. In some embodiments, the manufacturing methods disclosed herein are used to produce cells comprising two nucleic acid molecules each encoding a CAR disclosed herein (e.g., one nucleic acid molecule encoding an anti-CD22 CAR and one nucleic acid molecule encoding an anti-CD19 CAR). In some embodiments, provided herein are populations of cells (e.g., immune effector cells, e.g., T cells or NK cells) produced by any of the manufacturing processes described herein.

[0414] Activation Process In some embodiments, the methods disclosed herein can produce immune effector cells engineered to express one or more CARs in less than 24 hours. Without wishing to be bound by theory, the methods provided herein maintain T cells of an undifferentiated phenotype, such as naive T cells, during the manufacturing process. These CAR-expressing cells with an undifferentiated phenotype persist longer and / or expand better in vivo after infusion. In some embodiments, CART cells produced by the manufacturing methods provided herein contain a higher percentage of stem cell memory T cells compared to CART cells produced by conventional manufacturing processes, as measured, for example, using single-cell or bulk RNA-seq or flow cytometry using markers known in the art. In some embodiments, CART cells produced by the manufacturing methods provided herein contain a lower percentage of effector T cells compared to CART cells produced by conventional manufacturing processes, as measured, for example, using single-cell RNA-seq. In some embodiments, CART cells produced by the manufacturing methods provided herein better maintain T cell stemness compared to CART cells produced by conventional manufacturing processes, e.g., as measured using scRNA-seq. In some embodiments, CART cells produced by the manufacturing methods provided herein exhibit lower levels of hypoxia compared to CART cells produced by conventional manufacturing processes, e.g., as measured using scRNA-seq. In some embodiments, CART cells produced by the manufacturing methods provided herein exhibit lower levels of autophagy compared to CART cells produced by conventional manufacturing processes, e.g., as measured using scRNA-seq. In some embodiments, immune effector cells are engineered to contain nucleic acid molecules encoding a tandem or dual CAR disclosed herein (e.g., a CD19 / CD22 tandem or dual CAR disclosed herein).In some embodiments, immune effector cells are engineered to contain nucleic acid molecules encoding a tandem or dual CAR disclosed herein, such as an anti-CD22 / anti-CD19 tandem or dual CAR disclosed herein. In some embodiments, immune effector cells are engineered to contain two nucleic acid molecules, each encoding a CAR disclosed herein (e.g., one nucleic acid molecule encoding an anti-CD22 CAR and one nucleic acid molecule encoding an anti-CD19 CAR). In other embodiments, immune effector cells are engineered to contain one nucleic acid molecule encoding one or two CARs disclosed herein (e.g., one nucleic acid molecule encoding an anti-CD22 / anti-CD19 tandem or anti-CD22 / anti-CD19 CAR).

[0415] In some embodiments, the methods disclosed herein do not include the use of beads such as Dynabeads® (e.g., CD3 / CD28 Dynabeads®) and do not include a de-beading step. In some embodiments, CART cells produced by the methods disclosed herein can be administered to a subject with minimal, e.g., less than 2 days, less than 1 day, less than 12 hours, less than 8 hours, less than 6 hours, less than 4 hours, less than 3 hours, less than 2 hours, less than 1 hour, or no ex vivo expansion. Thus, the methods described herein provide a high-throughput manufacturing process for generating improved CAR-expressing cell products for use in treating a disease in a subject.

[0416] In some embodiments, the disclosure provides a method of generating a population of cells (e.g., T cells) expressing a chimeric antigen receptor (CAR) (e.g., one or more CARs, e.g., two CARs), comprising: (i) contacting a population of cells (e.g., T cells, e.g., T cells isolated from frozen or fresh leukapheresis products) with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the cells; (ii) contacting the population of cells (e.g., T cells) with one or more CARs encoding one or more CARs; with a plurality of nucleic acid molecules (e.g., DNA or RNA molecules), thereby providing a population of cells (e.g., T cells) comprising the nucleic acid molecules; and (iii) recovering the population of cells (e.g., T cells) for storage (e.g., reformulating the population of cells in a cryopreservation medium) or for administration, wherein (a) step (ii) is performed together with step (i), or within 20 hours after the initiation of step (i), e.g., 12, 13, 14, 15, 16, 18, 20, 22, 24, 26, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 110, 111, 112, 113, 114, (b) step (ii) is carried out together with step (i) or within 20 hours after the start of step (i), for example within 12, 13, 14, 15, 16, 17 or 18 hours after the start of step (i), for example within 18 hours after the start of step (i), and step (iii) is carried out within 26 hours after the start of step (i), for example within 22, 23 or 24 hours after the start of step (i), for example within 24 hours after the start of step (i); and (c) the population of cells from step (iii) is not expanded, or is expanded by no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40%, e.g., no more than 10%, as assessed by viable cell count compared to the population of cells at the start of step (i);or d) the population of cells from step (iii) is reduced or decreased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%, e.g., as assessed by viable cell count compared to the population of cells at the start of step (i). In some embodiments, the nucleic acid molecule of step (ii) is a DNA molecule. In some embodiments, the nucleic acid molecule of step (ii) is an RNA molecule. In some embodiments, the nucleic acid molecule of step (ii) is on a viral vector, e.g., a viral vector selected from a lentiviral vector, an adenoviral vector, or a retroviral vector. In some embodiments, the nucleic acid molecule of step (ii) is on a non-viral vector. In some embodiments, the nucleic acid molecule of step (ii) is on a plasmid. In some embodiments, the nucleic acid molecule of step (ii) is not on any vector. In some embodiments, step (ii) comprises transducing a population of cells (e.g., T cells) with one or more viral vectors comprising nucleic acid molecules encoding one or more CARs;

[0417] In some embodiments of the aforementioned methods, the method further comprises adding a supplement or transduction-enhancing reagent to the cell culture medium to enhance transduction efficiency. In some embodiments, the supplement or transduction-enhancing reagent comprises a cationic polymer. In some embodiments, the supplement or transduction-enhancing reagent is selected from LentiBOOST™ (Sirion Biotech), vectofusin-1, F108 (Poloxamer 338 or Pluronic® F-38), hexadimethrine bromide (polybrene), PEA, Pluronic F68, Pluronic F127, protamine sulfate, Synperonic, or LentiTrans™. In some embodiments, the supplement is LentiBOOST™ (Sirion Biotech). In other embodiments, the supplement is F108 (Poloxamer 338 or Pluronic® F-38).

[0418] In some embodiments, the population of cells (eg, T cells) is obtained from an apheresis sample (eg, a leukapheresis sample) from a subject.

[0419] In some embodiments, an apheresis sample (e.g., a leukapheresis sample) is collected from a subject and shipped as a frozen sample (e.g., a cryopreserved sample) to a cell manufacturing facility. The frozen apheresis sample is then thawed, and T cells (e.g., CD4+ T cells and / or CD8+ T cells) are selected from the apheresis sample, for example, using a cell sorting machine (e.g., a CliniMACS® Prodigy® device). The selected T cells (e.g., CD4+ T cells and / or CD8+ T cells) are then seeded and subjected to CART manufacturing using the activation process described herein. In some embodiments, the selected T cells (e.g., CD4+ T cells and / or CD8+ T cells) undergo one or more rounds of freeze-thawing before seeding for CART manufacturing.

[0420] In some embodiments, an apheresis sample (e.g., a leukapheresis sample) is collected from a subject and shipped as a fresh product (e.g., an unfrozen product) to a cell manufacturing facility. For example, T cells (e.g., CD4+ T cells and / or CD8+ T cells) are selected from the apheresis sample using a cell sorting machine (e.g., a CliniMACS® Prodigy® device). The selected T cells (e.g., CD4+ T cells and / or CD8+ T cells) are then seeded and subjected to CART manufacturing using the activation process described herein. In some embodiments, the selected T cells (e.g., CD4+ T cells and / or CD8+ T cells) undergo one or more rounds of freeze-thawing before seeding for CART manufacturing.

[0421] In some embodiments, an apheresis sample (e.g., a leukapheresis sample) is collected from a subject. T cells (e.g., CD4+ T cells and / or CD8+ T cells) are selected from the apheresis sample, for example, using a cell sorting machine (e.g., a CliniMACS® Prodigy® device). The selected T cells (e.g., CD4+ T cells and / or CD8+ T cells) are then shipped as a frozen sample (e.g., a cryopreserved sample) to a cell manufacturing facility. The selected T cells (e.g., CD4+ T cells and / or CD8+ T cells) are later thawed, seeded, and subjected to CART manufacturing using the activation process described herein.

[0422] In some embodiments, cells (e.g., T cells) are contacted with anti-CD3 and anti-CD28 antibodies, e.g., immediately thereafter, transduced with a vector (e.g., a lentiviral vector) (e.g., one or more vectors) encoding a CAR (e.g., one or more CARs). After 24 hours of culture, the cells are washed and formulated for storage or administration.

[0423] Without wishing to be bound by theory, brief stimulation of CD3 and CD28 may promote efficient transduction of self-renewing T cells. Compared to conventional CART production techniques, the activation process provided herein does not involve prolonged ex vivo expansion. Similar to the cytokine process, the activation process provided herein also maintains undifferentiated T cells during CART production.

[0424] In some embodiments, cells (e.g., T cells) are contacted with anti-CD3 and anti-CD28 antibodies for, e.g., 12 hours, and then transduced with a vector (e.g., a lentiviral vector) (e.g., one or more vectors) encoding a CAR (e.g., one or more CARs). After 24 hours of culture, the cells are washed and formulated for storage or administration.

[0425] Without wishing to be bound by theory, brief stimulation of CD3 and CD28 may promote efficient transduction of self-renewing T cells. Compared to conventional CART production techniques, the activation process provided herein does not involve prolonged ex vivo expansion. Similar to the cytokine process, the activation process provided herein also maintains undifferentiated T cells during CART production.

[0426] In some embodiments, the population of cells is contacted with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the cells.

[0427] In some embodiments, T cells are selected using anti-CD4 and anti-CD8 beads, for example, by positive selection using a cell sorting machine (e.g., a CliniMACS® Prodigy® device).

[0428] In some embodiments, T cells are selected using anti-CD45RA and anti-CCR7 beads, for example, by positive selection using a cell sorting machine (e.g., a CliniMACS® Prodigy® instrument).

[0429] In some embodiments, T cells are selected using anti-CD45RA and anti-CD27 beads, for example, by positive selection using a cell sorting machine (e.g., a CliniMACS® Prodigy® device).

[0430] In some embodiments, T cells are selected using anti-CD3 and anti-CD28 beads, for example, by positive selection using a cell sorting machine (e.g., a CliniMACS® Prodigy® device).

[0431] In some embodiments, T cells are selected using anti-lineage beads (excluding T cells), for example, by negative selection using a cell sorting machine (e.g., a CliniMACS® Prodigy® instrument).

[0432] In some embodiments, the agent that stimulates the CD3 / TCR complex is an agent that stimulates CD3. In some embodiments, the agent that stimulates a costimulatory molecule is an agent that stimulates CD28, ICOS, CD27, HVEM, LIGHT, CD40, 4-1BB, OX40, DR3, GITR, CD30, TIM1, CD2, CD226, or any combination thereof. In some embodiments, the agent that stimulates a costimulatory molecule is an agent that stimulates CD28. In some embodiments, the agent that stimulates the CD3 / TCR complex is selected from an antibody (e.g., a single domain antibody (e.g., a heavy chain variable domain antibody), a peptibody, a Fab fragment, or an scFv), a small molecule, or a ligand (e.g., a naturally occurring ligand, a recombinant ligand, or a chimeric ligand). In some embodiments, the agent that stimulates the CD3 / TCR complex is an antibody. In some embodiments, the agent that stimulates the CD3 / TCR complex is an anti-CD3 antibody. In some embodiments, the agent that stimulates a costimulatory molecule is selected from an antibody (e.g., a single domain antibody (e.g., a heavy chain variable domain antibody), a peptibody, a Fab fragment, or an scFv), a small molecule, or a ligand (e.g., a naturally occurring ligand, a recombinant ligand, or a chimeric ligand). In some embodiments, the agent that stimulates a costimulatory molecule is an antibody. In some embodiments, the agent that stimulates a costimulatory molecule is an anti-CD28 antibody. In some embodiments, the agent that stimulates the CD3 / TCR complex or the agent that stimulates a costimulatory molecule does not comprise beads. In some embodiments, the agent that stimulates the CD3 / TCR complex comprises an anti-CD3 antibody covalently attached to a colloidal polymer nanomatrix. In some embodiments, the agent that stimulates a costimulatory molecule comprises an anti-CD28 antibody covalently attached to a colloidal polymer nanomatrix. In some embodiments, the agent that stimulates the CD3 / TCR complex and the agent that stimulates a costimulatory molecule comprise T Cell TransAct™.

[0433] In some embodiments, the matrix comprises or consists of a polymeric, e.g., biodegradable or biocompatible, inert material that is non-toxic to cells. In some embodiments, the matrix is ​​composed of hydrophilic polymer chains, which achieve maximum mobility in aqueous solution due to hydration of the chains. In some embodiments, the mobile matrix can be collagen, purified proteins, purified peptides, polysaccharides, glycosaminoglycans, or extracellular matrix compositions. Polysaccharides can include, for example, cellulose ethers, starch, gum arabic, agarose, dextran, chitosan, hyaluronic acid, pectin, xanthan, guar gum, or alginic acid. Other polymers can include polyesters, polyethers, polyacrylates, polyacrylamides, polyamines, polyethyleneimines, polyquaternium polymers, polyphosphazenes, polyvinyl alcohols, polyvinyl acetates, polyvinylpyrrolidones, block copolymers, or polyurethanes. In some embodiments, the mobile matrix is ​​a dextran polymer.

[0434] In some embodiments, the population of cells is contacted with a nucleic acid molecule (e.g., one or more nucleic acid molecules) encoding a CAR (e.g., one or more CARs). In some embodiments, the population of cells is transduced with a DNA molecule (e.g., one or more DNA molecules) encoding a CAR (e.g., one or more CARs).

[0435] In some embodiments, contacting the population of cells with one or more nucleic acid molecules encoding one or more CARs is performed simultaneously with contacting the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the cells, as described above. In some embodiments, contacting the population of cells with one or more nucleic acid molecules encoding one or more CARs is performed within 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0.5 hours after the start of contacting the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the cells, as described above. In some embodiments, contacting the population of cells with one or more nucleic acid molecules encoding one or more CARs occurs within 20 hours after initiating contacting the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the cells, as described above. In some embodiments, contacting the population of cells with one or more nucleic acid molecules encoding one or more CARs occurs within 19 hours after initiating contacting the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the cells, as described above. In some embodiments, contacting the population of cells with one or more nucleic acid molecules encoding one or more CARs occurs within 18 hours after initiating contacting the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the cells, as described above. In some embodiments, contacting the population of cells with one or more nucleic acid molecules encoding one or more CARs occurs within 17 hours of initiating contacting the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the cells, as described above.In some embodiments, contacting the population of cells with one or more nucleic acid molecules encoding one or more CARs occurs within 16 hours after initiating contacting the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the cells, as described above. In some embodiments, contacting the population of cells with one or more nucleic acid molecules encoding one or more CARs occurs within 15 hours after initiating contacting the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the cells, as described above. In some embodiments, contacting the population of cells with one or more nucleic acid molecules encoding one or more CARs occurs within 14 hours after initiating contacting the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the cells, as described above. In some embodiments, contacting the population of cells with one or more nucleic acid molecules encoding one or more CARs occurs within 14 hours after initiating contacting the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the cells, as described above. In some embodiments, contacting the population of cells with one or more nucleic acid molecules encoding one or more CARs occurs within 13 hours after initiating contacting the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the cells, as described above. In some embodiments, contacting the population of cells with one or more nucleic acid molecules encoding one or more CARs occurs within 12 hours after initiating contacting the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the cells, as described above. In some embodiments, contacting the population of cells with one or more nucleic acid molecules encoding one or more CARs occurs within 11 hours of initiating contacting the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the cells, as described above.In some embodiments, contacting the population of cells with one or more nucleic acid molecules encoding one or more CARs occurs within 10 hours after initiating contacting the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with one or more nucleic acid molecules encoding one or more CARs occurs within 9 hours after initiating contacting the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with one or more nucleic acid molecules encoding one or more CARs occurs within 8 hours after initiating contacting the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with one or more nucleic acid molecules encoding one or more CARs occurs within 7 hours after initiating contacting the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with one or more nucleic acid molecules encoding one or more CARs occurs within 6 hours after initiating contacting the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the cells, as described above. In some embodiments, contacting the population of cells with one or more nucleic acid molecules encoding one or more CARs occurs within 5 hours after initiating contacting the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the cells, as described above. In some embodiments, contacting the population of cells with one or more nucleic acid molecules encoding one or more CARs occurs within 4 hours after initiating contacting the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the cells, as described above.In some embodiments, contacting the population of cells with one or more nucleic acid molecules encoding one or more CARs occurs within 3 hours of initiating contact with the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with one or more nucleic acid molecules encoding one or more CARs occurs within 2 hours of initiating contact with the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with one or more nucleic acid molecules encoding one or more CARs occurs within 1 hour of initiating contact with the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, contacting the population of cells with one or more nucleic acid molecules encoding one or more CARs occurs within 30 minutes of initiating contact with the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the cells described above.

[0436] In some embodiments, the population of cells is collected for storage or administration.

[0437] In some embodiments, the population of cells is harvested for storage or administration within 72, 60, 48, 36, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 hours after initiating contact of the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the cells, as described above. In some embodiments, the population of cells is harvested for storage or administration within 26 hours after initiating contact of the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the cells, as described above. In some embodiments, the population of cells is harvested for storage or administration within 25 hours after initiating contact of the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the cells, as described above. In some embodiments, the population of cells is collected for storage or administration within 24 hours after initiating contact of the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the cells, as described above. In some embodiments, the population of cells is collected for storage or administration within 23 hours after initiating contact of the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the cells, as described above. In some embodiments, the population of cells is collected for storage or administration within 22 hours after initiating contact of the population of cells with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the cells, as described above.

[0438] In some embodiments, the population of cells is not expanded ex vivo.

[0439] In some embodiments, the population of cells is expanded by no more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, or 60%, as assessed, for example, by viable cell count, compared to the population of cells prior to contact with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, the population of cells is expanded by no more than 5%, as assessed, for example, by viable cell count, compared to the population of cells prior to contact with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, the population of cells is expanded by no more than 10%, as assessed, for example, by viable cell count, compared to the population of cells prior to contact with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the cells described above. In some embodiments, the population of cells is expanded by 15% or less, as assessed, for example, by viable cell count, compared to the population of cells prior to contact with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the cells, as described above. In some embodiments, the population of cells is expanded by 20% or less, as assessed, for example, by viable cell count, compared to the population of cells prior to contact with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the cells, as described above. In some embodiments, the population of cells is expanded by 25% or less, as assessed, for example, by viable cell count, compared to the population of cells prior to contact with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the cells, as described above. In some embodiments, the population of cells is expanded by 30% or less, as assessed, for example, by viable cell count, compared to the population of cells prior to contact with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the cells, as described above.In some embodiments, the population of cells is expanded by no more than 35%, e.g., as assessed by viable cell count, compared to the population of cells prior to contact with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the cells, as described above. In some embodiments, the population of cells is expanded by no more than 40%, e.g., as assessed by viable cell count, compared to the population of cells prior to contact with an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the cells, as described above.

[0440] In some embodiments, the population of cells is expanded by no more than 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 16, 20, 24, 36, or 48 hours compared to the population of cells prior to contact with one or more cytokines described above, e.g., as assessed by viable cell count.

[0441] In some embodiments, the activation process is carried out in a serum-free cell culture medium. In some embodiments, the activation process is carried out in a cell culture medium containing one or more cytokines selected from IL-2, IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)), or IL-6 (e.g., IL-6 / sIL-6Ra). In some embodiments, hetIL-15 is [ka] In some embodiments, hetIL-15 comprises an amino acid sequence having at least about 70, 75, 80, 85, 90, 95, or 99% identity to SEQ ID NO: 109. In some embodiments, the activation process is carried out in a cell culture medium comprising an LSD1 inhibitor. In some embodiments, the activation process is carried out in a cell culture medium comprising a MALT1 inhibitor. In some embodiments, the serum-free cell culture medium comprises a serum replacement. In some embodiments, the serum replacement is CTS™ Immune Cell Serum Replacement (ICSR). In some embodiments, the level of ICSR can be, for example, up to 5%, e.g., about 1%, 2%, 3%, 4%, or 5%. Without wishing to be bound by theory, the use of a cell culture medium, such as Rapid Media, comprising ICSR, e.g., 2% ICSR, can improve cell viability during the manufacturing processes described herein.

[0442] In some embodiments, the present disclosure provides a method of generating a population of cells (e.g., T cells) expressing a chimeric antigen receptor (CAR), comprising: (a) providing an apheresis sample (e.g., a fresh or cryopreserved leukapheresis sample) collected from a subject; (b) selecting T cells from the apheresis sample (e.g., using negative selection, positive selection, or beadless selection); (c) selecting the isolated T cells from the apheresis sample (e.g., using negative selection, positive selection, or beadless selection); and (d) selecting the isolated T cells from the apheresis sample (e.g., using negative selection, positive selection, or beadless selection). 6 ~1×10 7(d) contacting the T cells with an agent that stimulates the T cells, e.g., an agent that stimulates the CD3 / TCR complex and / or an agent that stimulates a costimulatory molecule on the surface of the cell (e.g., contacting the T cells with an anti-CD3 and / or anti-CD28 antibody, e.g., contacting the T cells with TransAct); (e) contacting the T cells with one or more nucleic acid molecules (e.g., DNA or RNA molecules) encoding one or more CARs (e.g., contacting the T cells with a virus comprising one or more nucleic acid molecules encoding one or more CARs), e.g., for 6 to 48 hours, e.g., 20 to 28 hours; and (f) washing and collecting the T cells for storage (e.g., reformulating the T cells into cryopreservation medium) or administration. In some embodiments, step (f) is performed within 30 hours after the initiation of step (d) or (e), such as within 22, 23, 24, 25, 26, 27, 28, 29 or 30 hours after the initiation of step (d) or (e).

[0443] In some embodiments, provided herein are populations of cells (e.g., immune effector cells, e.g., T cells or NK cells) produced by any of the manufacturing processes described herein (e.g., the activation processes described herein).

[0444] In some embodiments, the proportion of naive cells, e.g., naive T cells, e.g., CD45RA+ CD45RO- CCR7+ T cells, in a population of cells at the end of a manufacturing process (e.g., at the end of a cytokine process or activation process described herein) is (1) the same, (2) differs by no more than, e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15%, or (3) increased by, e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25%, when compared to the proportion of naive cells, e.g., naive T cells, e.g., CD45RA+ CD45RO- CCR7+ cells, in a population of cells at the start of a manufacturing process (e.g., at the start of a cytokine process or activation process described herein). In some embodiments, the population of cells at the end of the manufacturing process (e.g., at the end of the cytokine process or activation process described herein) exhibits a higher percentage of naive cells, e.g., naive T cells, e.g., CD45RA+ CD45RO- CCR7+ T cells (e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50% higher), compared to cells produced by an otherwise similar method, e.g., lasting more than 26 hours (e.g., lasting more than 5, 6, 7, 8, 9, 10, 11, or 12 days), or a method that involves expanding the population of cells in vitro for, e.g., more than 3 days (e.g., expanding the population of cells in vitro for 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days).

[0445] In some embodiments, the percentage of naive cells, e.g., naive T cells, e.g., CD45RA+ CD45RO- CCR7+ T cells, in the population of cells at the end of the manufacturing process (e.g., at the end of the cytokine process or activation process described herein) is 20, 25, 30, 35, 40, 45, 50, 55, or 60% or more.

[0446] In some embodiments, the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, CD45RO+ central memory T cells, and / or CCR7+ central memory T cells, in the population of cells at the end of a manufacturing process (e.g., at the end of a cytokine process or activation process described herein), compared to the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the population of cells at the start of a manufacturing process (e.g., at the start of a cytokine process or activation process described herein), is (1) the same, (2) differs by no more than, e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15%, or (3) is reduced, e.g., by at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25%. In some embodiments, the population of cells at the end of the manufacturing process (e.g., at the end of a cytokine process or an activation process described herein) exhibits a lower percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells (e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50% lower) compared to cells otherwise similarly produced by, e.g., a method lasting more than 26 hours (e.g., lasting more than 5, 6, 7, 8, 9, 10, 11, or 12 days), or a method involving expanding the population of cells in vitro for, e.g., more than 3 days (e.g., expanding the population of cells in vitro for 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days).

[0447] In some embodiments, the percentage of central memory cells, e.g., central memory T cells, e.g., CD95+ central memory T cells, in the population of cells at the end of the manufacturing process (e.g., at the end of the cytokine process or activation process described herein) is 40, 45, 50, 55, 60, 65, 70, 75, or 80% or less.

[0448] In some embodiments, the population of cells at the end of the manufacturing process (e.g., at the end of a cytokine process or activation process described herein) survives longer or expands to a greater extent (e.g., at least 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90% greater) after in vivo administration compared to cells produced by an otherwise similar method, e.g., lasting longer than 26 hours (e.g., lasting longer than 5, 6, 7, 8, 9, 10, 11, or 12 days), or a method involving expanding the population of cells in vitro for, e.g., longer than 3 days (e.g., expanding the population of cells in vitro for 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days).

[0449] In some embodiments, the population of cells is enriched for IL6R-expressing cells (e.g., IL6Rα and / or IL6Rβ-positive cells) prior to the start of the manufacturing process (e.g., prior to the start of a cytokine process or activation process described herein). In some embodiments, the population of cells is enriched for IL6R-expressing cells (e.g., IL6Rα and / or IL6Rβ-positive cells) at the start of the manufacturing process (e.g., at the start of a cytokine process or activation process described herein), for example, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80% or more.

[0450] Cytokine Processes In some embodiments, the disclosure provides a method of producing a population of cells (e.g., T cells) expressing a chimeric antigen receptor (CAR) (e.g., one or more CARs, e.g., two CARs), comprising: (1) contacting the population of cells with a cytokine selected from IL-2, IL-7, IL-15, IL-21, IL-6, or a combination thereof; (2) contacting the population of cells (e.g., T cells) with one or more nucleic acid molecules (e.g., DNA or RNA molecules) encoding the one or more CARs, thereby providing a population of cells (e.g., T cells) comprising the nucleic acid molecules; and (3) formulating the population of cells (e.g., T cells) for storage (e.g., reformulating the population of cells in a cryopreservation medium) or for administration. and recovering a population of T cells (T cells), wherein (a) step (2) is performed simultaneously with step (1), or within 5 hours after the initiation of step (1), e.g., within 1, 2, 3, 4, or 5 hours after the initiation of step (1), and step (3) is performed within 26 hours after the initiation of step (1), e.g., within 22, 23, or 24 hours after the initiation of step (1), e.g., within 24 hours after the initiation of step (1); or (b) the population of cells from step (3) is not expanded, or is expanded by no more than 5, 10, 15, 20, 25, 30, 35, or 40%, e.g., no more than 10%, compared to the population of cells at the initiation of step (1), e.g., as assessed by viable cell count. In some embodiments, the nucleic acid molecule in step (2) is a DNA molecule. In some embodiments, the nucleic acid molecule in step (2) is an RNA molecule. In some embodiments, the nucleic acid molecule of step (2) is on a viral vector, e.g., a viral vector selected from a lentiviral vector, an adenoviral vector, or a retroviral vector. In some embodiments, the nucleic acid molecule of step (2) is on a non-viral vector. In some embodiments, the nucleic acid molecule of step (2) is on a plasmid. In some embodiments, the nucleic acid molecule of step (2) is not on any vector. In some embodiments, step (2) comprises transducing a population of cells (e.g., T cells) with a viral vector comprising one or more nucleic acid molecules encoding one or more CARs.In some embodiments, the cells are engineered to contain a nucleic acid molecule encoding a tandem or dual CAR disclosed herein (e.g., a CD19 / CD22 tandem or dual CAR disclosed herein). In some embodiments, the cells are engineered to contain a nucleic acid molecule encoding a diabody CAR disclosed herein, such as an anti-CD22 / anti-CD19 diabody CAR disclosed herein. In some embodiments, the cells are engineered to contain two nucleic acid molecules, each encoding a CAR disclosed herein (e.g., one nucleic acid molecule encoding an anti-CD22 CAR and one nucleic acid molecule encoding an anti-CD19 CAR).

[0451] In some embodiments, the population of cells (eg, T cells) is obtained from an apheresis sample (eg, a leukapheresis sample) from a subject.

[0452] In some embodiments, an apheresis sample (e.g., a leukapheresis sample) is collected from a subject and shipped as a frozen sample (e.g., a cryopreserved sample) to a cell manufacturing facility. The frozen apheresis sample is then thawed, and T cells (e.g., CD4+ T cells and / or CD8+ T cells) are selected from the apheresis sample, for example, using a cell sorting machine (e.g., a CliniMACS® Prodigy® device). The selected T cells (e.g., CD4+ T cells and / or CD8+ T cells) are then seeded and subjected to CAR T manufacturing using the cytokine process described herein. In some embodiments, at the end of the cytokine process, the CAR T cells are cryopreserved and later thawed and administered to a subject. In some embodiments, the selected T cells (e.g., CD4+ T cells and / or CD8+ T cells) undergo one or more rounds of freeze-thawing before seeding for CAR T manufacturing.

[0453] In some embodiments, an apheresis sample (e.g., a leukapheresis sample) is collected from a subject and shipped as a fresh product (e.g., an unfrozen product) to a cell manufacturing facility. For example, T cells (e.g., CD4+ T cells and / or CD8+ T cells) are selected from the apheresis sample using a cell sorting machine (e.g., a CliniMACS® Prodigy® device). The selected T cells (e.g., CD4+ T cells and / or CD8+ T cells) are then seeded and subjected to CART manufacturing using the cytokine process described herein. In some embodiments, the selected T cells (e.g., CD4+ T cells and / or CD8+ T cells) undergo one or more rounds of freeze-thawing before seeding for CART manufacturing.

[0454] In some embodiments, an apheresis sample (e.g., a leukapheresis sample) is collected from a subject. T cells (e.g., CD4+ T cells and / or CD8+ T cells) are selected from the apheresis sample, for example, using a cell sorting machine (e.g., a CliniMACS® Prodigy® device). The selected T cells (e.g., CD4+ T cells and / or CD8+ T cells) are then shipped as a frozen sample (e.g., a cryopreserved sample) to a cell manufacturing facility. The selected T cells (e.g., CD4+ T cells and / or CD8+ T cells) are later thawed, seeded, and subjected to CART manufacturing using the cytokine process described herein.

[0455] In some embodiments, after the cells (e.g., T cells) are seeded, one or more cytokines (e.g., IL-2, IL-7, IL-15 (e.g., one or more cytokines selected from hetIL-15 (IL15 / sIL-15Ra)), IL-21, or IL-6 (e.g., IL-6 / sIL-6R)) and a vector (e.g., a lentiviral vector) (e.g., one or more vectors) encoding a CAR (e.g., one or more CARs) are added to the cells. After a 20-24 hour incubation, the cells are washed and formulated for storage or administration.

[0456] Unlike conventional CART manufacturing techniques, the cytokine process provided herein does not involve CD3 and / or CD28 stimulation or ex vivo T cell expansion. T cells that are contacted with anti-CD3 and anti-CD28 antibodies and expanded extensively ex vivo tend to exhibit differentiation toward a central memory phenotype. Without wishing to be bound by theory, the cytokine process provided herein maintains or expands T cells of an undifferentiated phenotype during CART manufacturing, resulting in a CART formulation that may persist longer after infusion into a subject.

[0457] In some embodiments, the population of cells is contacted with one or more cytokines (e.g., one or more cytokines selected from IL-2, IL-7, IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)), IL-21, or IL-6 (e.g., IL-6 / sIL-6Ra).

[0458] In some embodiments, the population of cells is contacted with IL-2. In some embodiments, the population of cells is contacted with IL-7. In some embodiments, the population of cells is contacted with IL-15 (e.g., hetIL-15(IL15 / sIL-15Ra)). In some embodiments, the population of cells is contacted with IL-21. In some embodiments, the population of cells is contacted with IL-6 (e.g., IL-6 / sIL-6Ra). In some embodiments, the population of cells is contacted with IL-2 and IL-7. In some embodiments, the population of cells is contacted with IL-2 and IL-15 (e.g., hetIL-15(IL15 / sIL-15Ra)). In some embodiments, the population of cells is contacted with IL-2 and IL-21. In some embodiments, the population of cells is contacted with IL-2 and IL-6 (e.g., IL-6 / sIL-6Ra). In some embodiments, the population of cells is contacted with IL-7 and IL-15 (e.g., hetIL-15(IL15 / sIL-15Ra)). In some embodiments, the population of cells is contacted with IL-7 and IL-21. In some embodiments, the population of cells is contacted with IL-7 and IL-6 (e.g., IL-6 / sIL-6Ra). In some embodiments, the population of cells is contacted with IL-15 (e.g., hetIL-15(IL15 / sIL-15Ra)) and IL-21. In some embodiments, the population of cells is contacted with IL-15 (e.g., hetIL-15(IL15 / sIL-15Ra)) and IL-6 (e.g., IL-6 / sIL-6Ra). In some embodiments, the population of cells is contacted with IL-21 and IL-6 (e.g., IL-6 / sIL-6Ra). In some embodiments, the population of cells is contacted with IL-7, IL-15 (e.g., hetIL-15 (IL15 / sIL-15Ra)), and IL-21. In some embodiments, the population of cells is further contacted with an LSD1 inhibitor. In some embodiments, the population of cells is further contacted with a MALT1 inhibitor.

[0459] In some embodiments, the population of cells is contacted with 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, or 300 U / ml of IL-2. In some embodiments, the population of cells is contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 ng / ml of IL-7. In some embodiments, the population of cells is contacted with 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 ng / ml of IL-15.

[0460] In some embodiments, the population of cells is contacted with a nucleic acid molecule (e.g., one or more nucleic acid molecules) encoding a CAR (e.g., one or more CARs). In some embodiments, the population of cells is transduced with a DNA molecule (e.g., one or more DNA molecules) encoding a CAR (e.g., one or more CARs).

[0461] In some embodiments, contacting the population of cells with one or more CAR-encoding nucleic acid molecules is performed simultaneously with contacting the population of cells with one or more cytokines described above. In some embodiments, contacting the population of cells with one or more CAR-encoding nucleic acid molecules is performed within 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 hours after initiating contacting the population of cells with one or more cytokines described above. In some embodiments, contacting the population of cells with one or more CAR-encoding nucleic acid molecules is performed within 5 hours after initiating contacting the population of cells with one or more cytokines described above. In some embodiments, contacting the population of cells with one or more CAR-encoding nucleic acid molecules is performed within 4 hours after initiating contacting the population of cells with one or more cytokines described above. In some embodiments, contacting the population of cells with one or more CAR-encoding nucleic acid molecules is performed within 3 hours after initiating contacting the population of cells with one or more cytokines described above. In some embodiments, contacting the population of cells with one or more CAR-encoding nucleic acid molecules occurs within 2 hours after initiating contacting the population of cells with one or more cytokines described above. In some embodiments, contacting the population of cells with one or more CAR-encoding nucleic acid molecules occurs within 1 hour after initiating contacting the population of cells with one or more cytokines described above.

[0462] In some embodiments, the population of cells is collected for storage or administration.

[0463] In some embodiments, the population of cells is recovered for storage or administration within 72, 60, 48, 36, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 hours after initiating contacting the population of cells with one or more cytokines described above. In some embodiments, the population of cells is recovered for storage or administration within 26 hours after initiating contacting the population of cells with one or more cytokines described above. In some embodiments, the population of cells is recovered for storage or administration within 25 hours after initiating contacting the population of cells with one or more cytokines described above. In some embodiments, the population of cells is recovered for storage or administration within 24 hours after initiating contacting the population of cells with one or more cytokines described above. In some embodiments, the population of cells is recovered for storage or administration within 23 hours after initiating contacting the population of cells with one or more cytokines described above. In some embodiments, the population of cells is harvested for storage or administration within 22 hours after initiating contacting the population of cells with one or more cytokines described above.

[0464] In some embodiments, the population of cells is not expanded ex vivo.

[0465] In some embodiments, the population of cells is expanded by no more than 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, or 60% compared to the population of cells prior to contact with one or more cytokines described above, e.g., as assessed by viable cell count. In some embodiments, the population of cells is expanded by no more than 5% compared to the population of cells prior to contact with one or more cytokines described above, e.g., as assessed by viable cell count. In some embodiments, the population of cells is expanded by no more than 10% compared to the population of cells prior to contact with one or more cytokines described above, e.g., as assessed by viable cell count. In some embodiments, the population of cells is expanded by no more than 15% compared to the population of cells prior to contact with one or more cytokines described above, e.g., as assessed by viable cell count. In some embodiments, the population of cells is expanded by 20% or less, e.g., as assessed by viable cell count, compared to the population of cells prior to contact with one or more cytokines described above. In some embodiments, the population of cells is expanded by 25% or less, e.g., as assessed by viable cell count, compared to the population of cells prior to contact with one or more cytokines described above. In some embodiments, the population of cells is expanded by 30% or less, e.g., as assessed by viable cell count, compared to the population of cells prior to contact with one or more cytokines described above. In some embodiments, the population of cells is expanded by 35% or less, e.g., as assessed by viable cell count, compared to the population of cells prior to contact with one or more cytokines described above. In some embodiments, the population of cells is expanded by 40% or less, e.g., as assessed by viable cell count, compared to the population of cells prior to contact with one or more cytokines described above.

[0466] In some embodiments, the population of cells is expanded by no more than 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9, 10, 11, 12, 16, 20, 24, 36, or 48 hours compared to the population of cells prior to contact with one or more cytokines described above, e.g., as assessed by viable cell count.

[0467] In some embodiments, the population of cells is not contacted in vitro with an agent that stimulates the CD3 / TCR complex (e.g., an anti-CD3 antibody) and / or an agent that stimulates a costimulatory molecule on the surface of the cell (e.g., an anti-CD28 antibody), or if contacted, the contacting step is for less than 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 hours.

[0468] In some embodiments, the population of cells is contacted in vitro with an agent that stimulates the CD3 / TCR complex (e.g., an anti-CD3 antibody) and / or an agent that stimulates a costimulatory molecule on the surface of the cell (e.g., an anti-CD28 antibody) for 20, 21, 22, 23, 24, 25, 26, 27, or 28 hours.

[0469] In some embodiments, a population of cells produced using the cytokine processes provided herein exhibits a higher percentage of naive cells among CAR-expressing cells (e.g., at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, or 60% higher) when compared to otherwise similar cells produced by a method that further includes contacting the population of cells with, for example, an agent that binds the CD3 / TCR complex (e.g., an anti-CD3 antibody) and / or an agent that binds a costimulatory molecule on the surface of the cells (e.g., an anti-CD28 antibody).

[0470] In some embodiments, the cytokine processes provided herein are carried out in a cell culture medium comprising no more than 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or 8% serum. In some embodiments, the cytokine processes provided herein are carried out in a cell culture medium comprising an LSD1 inhibitor, a MALT1 inhibitor, or a combination thereof.

[0471] Additional Exemplary Manufacturing Methods In some embodiments, cells, e.g., T cells or NK cells, are activated, e.g., using anti-CD3 / anti-CD28 antibody-coated Dynabeads®, contacted with one or more nucleic acid molecules encoding a CAR (e.g., one or more CARs), and then expanded in vitro for, e.g., 7, 8, 9, 10, or 11 days. In some embodiments, cells, e.g., T cells or NK cells, are selected from fresh or cryopreserved leukapheresis samples, e.g., using positive or negative selection. In some embodiments, cells are contacted with a nucleic acid molecule (e.g., one or more nucleic acid molecules) encoding a CAR (e.g., one or more CARs). In some embodiments, cells are contacted with a nucleic acid molecule encoding a tandem or dual CAR (e.g., a CD19 / CD22 tandem or dual CAR) disclosed herein. In some embodiments, cells are contacted with two nucleic acid molecules, one expressing a first CAR (e.g., an anti-CD22 CAR) and the other expressing a second CAR (e.g., an anti-CD19 CAR). In some embodiments, the cells are contacted with a nucleic acid molecule encoding a diabody CAR (e.g., an anti-CD22 / anti-CD19 diabody CAR disclosed herein).

[0472] Elutriation In some embodiments, the methods described herein feature an elutriation method that removes undesirable cells, such as monocytes and blasts, thereby obtaining improved enrichment of desired immune effector cells suitable for CAR expression. In some embodiments, the elutriation methods described herein are optimized for enrichment of desired immune effector cells suitable for CAR expression from previously frozen samples, e.g., thawed samples. In some embodiments, the elutriation methods described herein provide preparations of cells with improved purity compared to preparations of cells collected from elutriation protocols known in the art. In some embodiments, the elutriation methods described herein include using a starting sample, e.g., a cell sample, e.g., a thawed cell sample, of optimized viscosity by dilution with certain isotonic solutions (e.g., PBS), and using an optimized combination of flow rate and collection volume for each fraction collected by the elutriation device. Exemplary elutriation methods that may be applied in the present invention are described on pages 48-51 of International Publication No. WO 2017 / 117112 (herein incorporated by reference in its entirety).

[0473] Density gradient centrifugation The production of adoptive cell therapy products requires the separation of desired cells, such as immune effector cells, from the complex mixture of blood cells and blood components present in the peripheral blood apheresis starting material. The separation of lymphocyte samples derived from peripheral blood has been successfully achieved using density gradient centrifugation with Ficoll solution. However, Ficoll is not clinically acceptable, making it a less desirable reagent for the separation of cells for therapeutic use. Additionally, Ficoll contains glycols, which can be toxic to cells. Furthermore, Ficoll density gradient centrifugation of thawed apheresis products after cryopreservation can result in suboptimal T cell production, as described in the Examples herein. For example, cell preparations separated by density gradient centrifugation with Ficoll solution have been observed to result in a loss of T cells in the final product, accompanied by a relative increase in non-T cells, particularly undesirable B cells, blasts, and monocytes.

[0474] Without wishing to be bound by theory, it is believed that immune effector cells, e.g., T cells, dehydrate during cryopreservation, becoming denser than fresh cells. Without wishing to be bound by theory, it is also believed that immune effector cells, e.g., T cells, remain denser for longer periods of time than other blood cells and are therefore more likely to be lost during Ficoll density gradient separation than other cells. Therefore, without wishing to be bound by theory, it is believed that media denser than Ficoll provide improved separation of desired immune effector cells compared to Ficoll or other media of the same density as Ficoll, e.g., 1.077 g / mL.

[0475] In some embodiments, the density gradient centrifugation methods described herein include the use of a density gradient medium comprising iodixanol, hi some embodiments, the density gradient medium comprises about 60% iodixanol in water.

[0476] In some embodiments, the density gradient centrifugation method described herein comprises the use of a density gradient medium with a density higher than Ficoll. In some embodiments, the density gradient centrifugation method described herein comprises the use of a density gradient medium with a density higher than 1.077 g / mL, for example, higher than 1.077 g / mL, higher than 1.1 g / mL, higher than 1.15 g / mL, higher than 1.2 g / mL, higher than 1.25 g / mL, higher than 1.3 g / mL, or higher than 1.31 g / mL. In some embodiments, the density gradient medium has a density of about 1.32 g / mL.

[0477] Further embodiments of density gradient centrifugation are described on pages 51-53 of WO 2017 / 117112, which is incorporated by reference in its entirety.

[0478] Enrichment by selection Provided herein are methods for selecting specific cells to improve enrichment of desired immune effector cells suitable for CAR expression. In some embodiments, the selection comprises positive selection, e.g., selection for desired immune effector cells. In some embodiments, the selection comprises negative selection, e.g., selection for undesired cells, e.g., removal of undesired cells. In embodiments, the positive or negative selection methods described herein are performed under flow conditions, e.g., by using a flow-through device, e.g., a flow-through device described herein. Exemplary positive and negative selection methods are described on pages 53-57 of WO 2017 / 117112 (incorporated herein by reference in its entirety). Selection methods can be performed under flow conditions, e.g., by using a flow-through device, also referred to as a cell processing system, to further enrich a cell preparation for desired immune effector cells, e.g., T cells, suitable for CAR expression. Exemplary flow-through devices are described on pages 57-70 of WO 2017 / 117112 (incorporated herein by reference in its entirety). Exemplary cell separation and de-beading methods are described on pages 70-78 of WO 2017 / 117112, which is incorporated by reference in its entirety.

[0479] Selection procedures are not limited to those described on pages 57-70 of WO 2017 / 117112. Negative T cell selection via removal of unwanted cells with CD19, CD14, and CD26 Miltenyi beads combined with column technology (CliniMACS® Plus or CliniMACS® Prodigy®) or positive T cell selection using CD4 and CD8 Miltenyi beads combined with column technology (CliniMACS® Plus or CliniMACS® Prodigy®) can be used. Alternatively, column-free technology using releasable CD3 beads (GE Healthcare) can also be used.

[0480] Additionally, bead-free technologies such as ThermoGenesis X-series instruments can be utilized as well.

[0481] Methods for producing the cells disclosed herein include those described in, for example, Chinese Patent No. 108103105, Chinese Patent No. 108085342, Chinese Patent No. 108018312, Chinese Patent No. 107287164, WO 18052947, WO 17123956, WO 17114497, WO 17103596, WO 17068421, WO 17023803, WO 17015427, WO 16196388, WO 16168595, WO 14186469, WO 17186469, WO 17186470, WO 17186471, WO 17186472, WO 17186473, WO 17186474, WO 17186475, WO 17186476, WO 171864776, WO 17186478, WO 17186479, WO 171864796, WO 171864797, WO 171864798, WO 17186479 ...

[0023] Examples of suitable fluoropolymers include those known in the art, as described in WO 17165245, WO 18106732, WO 17015490, WO 18075813, WO 18102761, WO 17127755, WO 17214333, WO 18059549, WO 17190100, WO 16180778, WO 18057823 and / or Chinese Patent No. 106957822, each of which is hereby incorporated by reference in its entirety.

[0482] Cell Source Prior to expansion and genetic or other modification, a source of cells, such as T cells or natural killer (NK) cells, can be obtained from a subject. The term "subject" is intended to include a living organism (e.g., a mammal) in which an immune response can be elicited. Examples of subjects include humans, monkeys, chimpanzees, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from a number of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In one embodiment of the present disclosure, immune effector cells, such as T cells, can be obtained from a unit of blood drawn from a subject using any technique known to those skilled in the art, such as Ficoll™ separation. In a preferred embodiment, cells from an individual's circulating blood are obtained by apheresis. The apheresis product typically includes T cells, monocytes, granulocytes, B cells, lymphocytes, including other nucleated white blood cells, red blood cells, and platelets. In one embodiment, cells collected by apheresis are washed to remove the plasma fraction, and optionally place the cells in an appropriate buffer or medium for subsequent processing. In one embodiment of the present invention, cells are washed with phosphate-buffered saline (PBS). In another embodiment, the wash solution lacks calcium, may lack magnesium, or may lack many, if not all, divalent cations. An initial activation process in the absence of calcium may lead to enhanced activation. As those skilled in the art will readily recognize, the washing step can be accomplished by semi-automated "flow-through" centrifugation (e.g., Cobe 2991 cell processor, Baxter CytoMate, or Haemonetics Cell Saver 5) following the manufacturer's instructions. After washing, cells can be resuspended in a variety of biocompatible buffers, such as Ca-free, Mg-free PBS, PlasmaLyte A, or saline solution with or without other buffering agents. Alternatively, undesirable components of the apheresis sample can be removed and the cells resuspended directly in culture medium.

[0483] In one embodiment, T cells are isolated from peripheral blood lymphocytes by lysing red blood cells and depleting monocytes, for example, by centrifugation on a PERCOLL™ gradient or by counterflow centrifugal elutriation.

[0484] The methods described herein can include selection of a specific subpopulation of immune effector cells, e.g., T cells, e.g., a T regulatory cell-depleted population, CD25+ depleted cells, e.g., using negative selection techniques, e.g., as described herein. Preferably, the population of T regulatory depleted cells comprises less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1% CD25+ cells.

[0485] In one embodiment, regulatory T cells, e.g., CD25+ T cells, are removed from the population using an anti-CD25 antibody, or fragment thereof, or a CD25-binding ligand, IL-2. In one embodiment, the anti-CD25 antibody, or fragment thereof, or CD25-binding ligand is conjugated to a substrate, e.g., a bead, or otherwise coated onto a substrate, e.g., a bead. In one embodiment, the anti-CD25 antibody, or fragment thereof, is conjugated to a substrate as described herein.

[0486] In one embodiment, regulatory T cells, e.g., CD25+ T cells, are removed from the population using CD25 depletion reagent from Militenyi™. In one embodiment, the ratio of CD25 depletion reagent to cells is 20 uL to 1e7 cells, or 15 uL to 1e7 cells, or 10 uL to 1e7 cells, or 5 uL to 1e7 cells, or 2.5 uL to 1e7 cells, or 1.25 uL to 1e7 cells.

[0487] In one embodiment, the population of immune effector cells to be depleted is about 6×10 9 In other embodiments, the population of immune effector cells to be depleted comprises about 1 x 10 9 ~1×10 10(and any integer value therebetween) CD25+ T cells. In one embodiment, the resulting population of T regulatory depleted cells comprises 2×10 9 T regulatory cells, e.g., CD25+ cells or less (e.g., 1 x 10 9 , 5×10 8 , 1×10 8 , 5×10 7 , 1×10 7 or fewer CD25+ cells).

[0488] In one embodiment, T regulatory cells, e.g., CD25+ cells, are removed from the population using the CliniMAC system with a depletion tubing set, e.g., tubing 162-01. In one embodiment, the CliniMAC system is run in a depletion setting, e.g., DEPLETION2.1.

[0489] The methods described herein can include two or more selection steps, e.g., two or more depletion steps. Enrichment of a T cell population by negative selection can be achieved, for example, by a combination of antibodies directed against surface markers unique to the cells to be negatively selected. One method is cell sorting and / or selection by negative magnetic immunoadhesion or flow cytometry using a cocktail of monoclonal antibodies directed against cell surface markers present on the cells to be negatively selected. For example, to enrich for CD4+ cells by negative selection, the monoclonal antibody cocktail can include antibodies against CD14, CD20, CD11b, CD16, HLA-DR, and CD8.

[0490] Also provided are methods that include removing from a population one or more cells expressing a checkpoint inhibitor, e.g., a checkpoint inhibitor described herein, e.g., PD1+ cells, LAG3+ cells, and TIM3+ cells, thereby providing a population of T regulatory-depleted, e.g., CD25+-depleted cells, and checkpoint inhibitor-depleted cells, e.g., PD1+, LAG3+, and / or TIM3+-depleted cells. Exemplary checkpoint inhibitors include B7-H1, B&-1, CD160, P1H, 2B4, PD1, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and / or CEACAM-5), LAG3, TIGIT, CTLA-4, BTLA, and LAIR1. In one embodiment, checkpoint inhibitor-expressing cells are removed simultaneously with T regulatory, e.g., CD25+ cells. For example, an anti-CD25 antibody or fragment thereof and an anti-check point inhibitor antibody or fragment thereof can be attached to the same bead, which can be used to remove cells, or an anti-CD25 antibody or fragment thereof and an anti-check point inhibitor antibody or fragment thereof can be attached to separate beads, which mixture can be used to remove cells. In some embodiments, removal of T regulatory cells, e.g., CD25+ cells, and removal of check point inhibitor-expressing cells are sequential, e.g., can occur in either order.

[0491] The methods described herein may include a positive selection step. For example, T cells may be isolated by incubating with anti-CD3 / anti-CD28 (e.g., 3x28) conjugated beads, such as DYNABEADS® M-450 CD3 / CD28 T, for a time sufficient for positive selection of the desired T cells. In one embodiment, this time period is approximately 30 minutes. In a further embodiment, the time period ranges from 30 minutes to 36 hours or longer, and all integer values ​​therebetween. In a further embodiment, the time period is at least 1, 2, 3, 4, 5, or 6 hours. In yet another preferred embodiment, the time period is 10 to 24 hours. In one embodiment, the incubation time is 24 hours. In any situation where there are only a few T cells compared to other cell types, such as isolating tumor-infiltrating lymphocytes (TILs) from tumor tissue or from immunocompromised individuals, longer incubation times may be used to isolate T cells. Furthermore, longer incubation times may increase the efficiency of CD8+ T cell capture. Thus, by simply increasing or decreasing the time that T cells are allowed to bind to the CD3 / CD28 beads, and / or by increasing or decreasing the ratio of T cells to beads (as further described herein), one can preferentially select or deselect certain populations of T cells at the beginning of the culture or at other times during the process. Additionally, by increasing or decreasing the ratio of anti-CD3 and / or anti-CD28 antibodies on the beads or other surface, one can preferentially select or deselect certain populations of T cells at the beginning of the culture or at other desired times.

[0492] In one embodiment, a T cell population can be selected that expresses one or more of T cell IFN-γ, TNFα, IL-17A, IL-2, IL-3, IL-4, GM-CSF, IL-10, IL-13, granzyme B, and perforin, or other suitable molecules, e.g., other cytokines. Methods for screening for cell expression can be determined, for example, by the methods described in WO 2013 / 126712.

[0493] To isolate a desired population of cells by positive or negative selection, the concentration of cells and surfaces (e.g., particles such as beads) can be varied. In one embodiment, it may be desirable to significantly reduce the volume in which the beads and cells are mixed together (e.g., increase the cell concentration) to ensure maximum contact between the cells and beads. For example, in one embodiment, a concentration of about 10 billion cells / ml, 9 billion cells / ml, 8 billion cells / ml, 7 billion cells / ml, 6 billion cells / ml, or 5 billion cells / ml is used. In one embodiment, a concentration of 1 billion cells / ml is used. In one embodiment, a concentration of 75, 80, 85, 90, 95, or 100 million cells / ml of cells is used. In a further embodiment, a concentration of 125 or 150 million cells / ml can be used.

[0494] High concentrations can be used to increase cell yield, cell activation, and cell proliferation. Furthermore, the use of high cell concentrations allows for more efficient capture of cells that may weakly express a target antigen of interest, such as CD28-negative T cells, or from samples in which many tumor cells are present (e.g., leukemic blood, tumor tissue, etc.). Such populations of cells may have therapeutic value and are desirable to obtain. For example, the use of high cell concentrations allows for more efficient selection of CD8+ T cells, which normally have weak CD28 expression.

[0495] In a related embodiment, it may be desirable to use a low concentration of cells. By significantly diluting the mixture of T cells and a surface (e.g., particles such as beads), particle-cell interactions are minimized. This selects for cells that express high amounts of the desired antigen that binds to the particles. For example, CD4+ T cells express high levels of CD28 and are captured more efficiently than CD8+ T cells at dilute concentrations. In one embodiment, the concentration of cells used is 5×10 6 In other embodiments, the concentration used is about 1 x 10 5 / ml ~ 1 × 10 6 / ml and any integer value therebetween.

[0496] In other embodiments, cells can be incubated on a rotator for various lengths of time at various speeds at 2-10° C. or room temperature.

[0497] T cells for stimulation may also be frozen after a washing step. Without wishing to be bound by theory, the freezing and subsequent thawing steps provide a more homogenous product by removing granulocytes and, to some extent, monocytes from the cell population. After a washing step that removes plasma and platelets, the cells may be suspended in a freezing solution. While many freezing solutions and parameters are known in the art and useful in this context, one method uses PBS containing 20% ​​DMSO and 8% human serum albumin, or culture medium containing 10% Dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO, or 31.25% Plasmalyte-A, 31.25% dextrose 5%, 0.45% NaCl, 10% Dextran 40 and 5% dextrose, 20% human serum albumin and 7.5% DMSO, or other suitable cell freezing media containing, for example, Hespan and PlasmaLyte A, and then freezes the cells to -80°C at a rate of 1° / min and stores them in a liquid nitrogen storage tank in the vapor phase. Other methods of controlled freezing, as well as uncontrolled freezing directly to -20°C or liquid nitrogen, can also be used.

[0498] In one embodiment, cryopreserved cells are thawed as described herein, washed, and allowed to rest at room temperature for 1 hour before activation using the methods of the invention.

[0499] In the context of the present invention, collection of a blood sample or apheresis product from a subject at a time prior to the time when the expanded cells described herein may be needed is also contemplated. Thus, a source of expanded cells can be harvested at any time needed, and desired cells, such as T cells, can be isolated and frozen for subsequent use in immune effector cell therapy for any number of diseases and conditions that would benefit from immune effector cell therapy, such as those described herein. In one embodiment, a blood sample or apheresis is generally collected from a healthy subject. In one embodiment, a blood sample or apheresis is generally collected from a healthy subject who is at risk of developing a disease but has not yet developed the disease, and the desired cells are isolated and frozen for subsequent use. In one embodiment, T cells can be expanded, frozen, and used at a later time. In one embodiment, a sample is collected from a patient shortly after diagnosis of a particular disease described herein, but prior to any treatment. In further embodiments, cells are isolated from a blood sample or apheresis from a subject prior to any number of relevant treatment modalities, including, but not limited to, treatment with antibodies or other immunoablative agents such as natalizumab, efalizumab, antivirals, chemotherapeutics, radiation, immunosuppressants such as cyclosporine, azathioprine, methotrexate, mycophenolate and FK506, CAMPATH, anti-CD3 antibodies, cytoxan, fludarabine, cyclosporine, FK506, rapamycin, mycophenolic acid, steroids, FR901228, and irradiation.

[0500] In a further embodiment of the present invention, T cells are obtained directly from a patient after a treatment that leaves the subject with functional T cells. It has been observed that following certain cancer treatments, particularly those with drugs that damage the immune system, the quality of the T cells obtained may be optimal or improved for their ability to expand ex vivo immediately after treatment, while the patient typically recovers from the treatment. Similarly, following ex vivo manipulation using the methods described herein, these cells may be in a favorable state for enhanced engraftment and in vivo expansion. Therefore, in the context of the present invention, it is contemplated to collect blood cells, including T cells, dendritic cells, or other cells of the hematopoietic lineage, during this recovery period. Furthermore, in one embodiment, mobilization (e.g., mobilization with GM-CSF) and conditioning regimens can be used to create conditions in the subject that favor the repopulation, recirculation, regeneration, and / or expansion of specific cell types, particularly during a defined time frame following treatment. Examples of cell types include T cells, B cells, dendritic cells, and other cells of the immune system.

[0501] In one embodiment, cells expressing an immune effector CAR molecule, e.g., a CAR molecule described herein, are obtained from a subject who has received a low, immune-enhancing dose of an mTOR inhibitor. In one embodiment, a population of immune effector cells, e.g., T cells or NK cells, engineered to express a CAR are harvested after a sufficient time, or after a sufficient dose, of the low, immune-enhancing dose of an mTOR inhibitor such that the level of PD1-negative immune effector cells, e.g., T cells or NK cells, or the ratio of PD1-negative immune effector cells, e.g., T cells / NK cells / PD1-positive immune effector cells, e.g., T cells or NK cells, in the subject or harvested from the subject is at least transiently increased.

[0502] In some embodiments, a population of immune effector cells, e.g., T cells or NK cells, that have been or will be engineered to express a CAR can be treated ex vivo by contacting them with an amount of an mTOR inhibitor that expands the number of PD1 negative immune effector cells, e.g., T cells, or increases the ratio of PD1 negative immune effector cells, e.g., T cells / NK cells / PD1 positive immune effector cells, e.g., T cells or NK cells.

[0503] In one embodiment, the T cell population is diacylglycerol kinase (DGK) deficient. DGK-deficient cells are cells that do not express DGK RNA or protein, or that have reduced or inhibited DGK activity. DGK-deficient cells can be generated by genetic methods to reduce or block DGK expression, such as administering RNA interference agents, such as siRNA, shRNA, or miRNA. Alternatively, DGK-deficient cells can be generated by treatment with a DGK inhibitor as described herein.

[0504] In one embodiment, the T cell population is Ikaros-deficient. Ikaros-deficient cells include cells that do not express Ikaros RNA or protein or that have reduced or inhibited Ikaros activity, and Ikaros-deficient cells can be generated by genetic methods to reduce or prevent Ikaros expression, such as administering RNA interference agents, e.g., siRNA, shRNA, miRNA. Alternatively, Ikaros-deficient cells can be generated by treatment with an Ikaros inhibitor, e.g., lenalidomide.

[0505] In embodiments, the T cell population is DGK- and Ikaros-deficient, e.g., does not express DGK and Ikaros, or has reduced or inhibited DGK and Ikaros activity. Such DGK- and Ikaros-deficient cells can be produced by any of the methods described herein.

[0506] In one embodiment, the NK cells are obtained from a subject. In another embodiment, the NK cells are an NK cell line, such as the NK-92 cell line (Conkwest).

[0507] Homogeneous CART In embodiments described herein, the immune effector cell can be an allogeneic immune effector cell, e.g., a T cell or an NK cell. For example, the cell is an allogeneic T cell, e.g., an allogeneic T cell that lacks expression of a functional T cell receptor (TCR) and / or a human leukocyte antigen (HLA), e.g., HLA class I and / or HLA class II.

[0508] T cells lacking a functional TCR can be engineered, for example, to not express any functional TCR on their surface, to not express one or more subunits comprising a functional TCR, or to produce minimal functional TCR on their surface. Alternatively, T cells can express a substantially impaired TCR, for example, by expression of mutated or truncated forms of one or more subunits of the TCR. The term "substantially impaired TCR" means that the TCR does not elicit a harmful immune response in the host. Such cells can be generated using one or more gene editing systems as described herein. In embodiments, the gene editing system targets sequences encoding components of the TCR, such as sequences in the TCR alpha constant chain gene (TRAC) or its regulatory elements. In embodiments, the gene editing system targets sequences encoding components of the TCR, such as sequences in the TCR beta constant chain gene (TRBC) or its regulatory elements.

[0509] The T cells described herein can be engineered, for example, to not express functional HLA on their surface. For example, the T cells described herein can be engineered to downregulate cell surface-expressed HLA, e.g., HLA class 1 and / or HLA class II. Such cells can be generated using one or more gene editing systems as described herein. In embodiments, the gene editing system targets a sequence encoding a component of one or more HLA molecules. In embodiments, the gene editing system targets a sequence encoding a factor that affects the expression of one or more HLA molecules. In embodiments, the gene editing system targets a sequence encoding a regulator of MHC class I expression, e.g., beta-2 microglobulin (B2M). In embodiments, the gene editing system targets a sequence encoding a regulator of MHC class II molecule expression, e.g., CIITA. In embodiments, a gene editing system that targets both a regulator of MHC class I expression (e.g., B2M) and a regulator of MHC class II molecule expression (e.g., CIITA) is introduced into a cell such that expression of at least one MHC class I molecule and at least one MHC class II molecule is downregulated.

[0510] In one embodiment, the T cells may lack a functional TCR and a functional HLA, for example, HLA class I and / or HLA class II.

[0511] Modified T cells lacking expression of a functional TCR and / or HLA can be obtained by any suitable means, including knockout or knockdown of one or more subunits of the TCR or HLA. For example, the T cells can include knockdown of the TCR and / or HLA using siRNA, shRNA, clustered regularly interspaced short palindromic repeats (CRISPR), transcription activator-like effector nuclease (TALEN), or zinc finger endonuclease (ZFN).

[0512] In one embodiment, the allogeneic cells can be cells that do not express or express low levels of an inhibitory molecule, e.g., by any of the methods described herein. For example, the cells can be cells that do not express or express low levels of an inhibitory molecule, which can, e.g., reduce the ability of a CAR-expressing cell to mount an immune effector response. Examples of inhibitory molecules include PD1, PD-L1, CTLA4, TIM3, CEACAM (e.g., CEACAM-1, CEACAM-3, and / or CEACAM-5), LAG3, VISTA, BTLA, TIGIT, LAIR1, CD160, 2B4, and TGFβ. Inhibition of inhibitory molecules by inhibition at the DNA, RNA, or protein level can optimize CAR-expressing cell performance. In embodiments, an inhibitory nucleic acid, e.g., an inhibitory nucleic acid, e.g., an inhibitory nucleic acid, e.g., a dsRNA, e.g., an siRNA or shRNA, a clustered regularly interspaced short palindromic repeats (CRISPR), a transcription activator-like effector nuclease (TALEN), or a zinc finger endonuclease (ZFN), can be used, e.g., as described herein.

[0513] siRNA and shRNA for inhibiting e.g. TCR or HLA In one embodiment, TCR expression and / or HLA expression can be inhibited using siRNA or shRNA targeting nucleic acids encoding TCR and / or HLA in T cells.

[0514] Expression of siRNA and shRNA in T cells can be achieved using any conventional expression system, such as, for example, a lentiviral expression system.

[0515] Exemplary shRNAs that downregulate expression of TCR components are described, for example, in U.S. Patent Application Publication No. 2012 / 0321667. Exemplary siRNAs and shRNAs that downregulate expression of HLA class I and / or HLA class II genes are disclosed, for example, in U.S. Patent Application Publication No. 2007 / 0036773.

[0516] CRISPR for example to inhibit TCR or HLA As used herein, "CRISPR," or "CRISPR to TCR and / or HLA," or "CRISPR for inhibiting TCR and / or HLA" refers to a series of clustered regularly interspaced short palindromic repeats or a system comprising such a series of repeats. As used herein, "Cas" refers to a CRISPR-associated protein. A "CRISPR / Cas" system refers to a CRISPR- and Cas-derived system that can be used to silence or mutate TCR and / or HLA genes.

[0517] Naturally occurring CRISPR / Cas systems are found in approximately 40% of sequenced eubacterial genomes and 90% of sequenced archaeal genomes. Grissa et al. (2007) BMC Bioinformatics 8:172. This system is a type of prokaryotic immune system that confers resistance to foreign genetic elements such as plasmids and phages, providing a form of acquired immunity. Barrangou et al. (2007) Science 315:1709-1712; Marragini et al. (2008) Science 322:1843-1845.

[0518] The CRISPR / Cas system has been modified for use in gene editing (silencing, enhancing, or altering specific genes) in eukaryotic organisms such as mice or primates (Wiedenheft et al. (2012) Nature 482:331-8). This is achieved by introducing into eukaryotic cells a plasmid containing a specifically designed CRISPR and one or more appropriate Cass.

[0519] CRISPR sequences, sometimes referred to as CRISPR loci, comprise alternating repeats and spacers. In naturally occurring CRISPRs, the spacers typically comprise sequences foreign to the bacterium, such as plasmid or phage sequences; in TCR and / or HLA CRISPR / Cas systems, the spacers are derived from TCR or HLA gene sequences.

[0520] RNA from CRISPR loci is constitutively expressed and processed by Cas proteins into small RNAs. These contain spacers flanked by repeat sequences. The RNA guides other Cas proteins to silence exogenous genetic elements at the RNA or DNA level. Horvath et al. (2010) Science 327:167-170; Makarova et al. (2006) Biology Direct 1:7. The spacer thus serves as a template for the RNA molecule, similar to siRNA. Pennisi (2013) Science 341:833-836.

[0521] As they occur naturally in many different types of bacteria, the exact location and structure of CRISPR, the function and number of Cas genes, and their products vary somewhat from species to species (Haft et al. (2005) PLoS Comput. Biol. 1:e60; Kunin et al. (2007) Genome Biol. 8:R61; Mojica et al. (2005) J. Mol. Evol. 60:174-182; Bolotin et al. (2005) Microbiol. 151:2551-2561; Pourcel et al. (2005) Microbiol. 151:653-663; and Stern et al. (2010) Trends. Genet. 28:335-340). For example, Cse (Cas subtype, E. coli) proteins (e.g., CasA) form a functional complex, Cascade, which processes CRISP...

Claims

1. 1. A nucleic acid molecule encoding a chimeric antigen receptor (CAR) molecule, the CAR molecule comprising: (a) a first CAR comprising a first antigen-binding domain that binds to CD22 and a first transmembrane domain; a first costimulatory signaling domain; and / or a first primary signaling domain; and (b) a second CAR comprising a second antigen-binding domain that binds to CD19, and a second transmembrane domain; a second costimulatory signaling domain; and / or a second primary signaling domain. Including, (i) the first CAR and the second CAR comprise heavy chain complementarity determining region (HCDR) 1, HCDR2, HCDR3, light chain complementarity determining region (LCDR) 1, LCDR2, and LCDR3 in SEQ ID NO: 12, respectively; or (ii) the first CAR and the second CAR each comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 16; the CAR molecule comprises the amino acid sequence of SEQ ID NO: 12 or 16, or an amino acid sequence having at least 95% identity thereto; and (i) the nucleotide sequence encoding the first transmembrane domain and contained in the nucleic acid molecule is different from the nucleotide sequence encoding the second transmembrane domain and contained in the nucleic acid molecule; (ii) the nucleotide sequence encoding the first costimulatory signaling domain and contained in the nucleic acid molecule is different from the nucleotide sequence encoding the second costimulatory signaling domain and contained in the nucleic acid molecule; and / or (iii) the nucleotide sequence encoding the first primary signaling domain and contained in the nucleic acid molecule is different from the nucleotide sequence encoding the second primary signaling domain and contained in the nucleic acid molecule; Nucleic acid molecule.

2. The first CAR comprises: a first antigen-binding domain that binds to CD22, a first transmembrane domain, and a first costimulatory signaling domain; a first antigen-binding domain that binds to CD22, a first transmembrane domain, and a first primary signaling domain; or a first antigen-binding domain that binds to CD22, a first transmembrane domain, a first costimulatory signaling domain, and a first primary signaling domain. and The second CAR comprises: a second antigen-binding domain that binds to CD19; a second transmembrane domain; and a second costimulatory signaling domain; a second antigen-binding domain that binds to CD19; a second transmembrane domain; and a second primary signaling domain; or a second antigen-binding domain that binds to CD19; a second transmembrane domain; a second costimulatory signaling domain; and a second primary signaling domain. The nucleic acid molecule of claim 1, comprising:

3. 1. A nucleic acid molecule encoding a chimeric antigen receptor (CAR) molecule, the CAR molecule comprising: (a) a first CAR comprising: a first antigen-binding domain that binds to CD22; a first transmembrane domain; a first costimulatory signaling domain; and / or a first primary signaling domain; (b) a second CAR comprising a second antigen-binding domain that binds to CD19; a second transmembrane domain; a second costimulatory signaling domain; and / or a second primary signaling domain. Including, (i) the first CAR and the second CAR each comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 12; or (ii) the first CAR and the second CAR each comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 16; The CAR molecule comprises the amino acid sequence of SEQ ID NO: 12 or 16, or an amino acid sequence having at least 95% identity thereto. Nucleic acid molecule.

4. The nucleic acid molecule of any one of claims 1 to 3, wherein the CAR molecule comprises the amino acid sequence of SEQ ID NO: 12 or 16.

5. The nucleic acid molecule of any one of claims 1 to 4, wherein the CAR molecule is encoded by the nucleotide sequence of SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 19, or a nucleotide sequence having at least 80% identity thereto.

6. A CAR molecule, the CAR molecule comprising: (a) a first CAR comprising a first antigen-binding domain that binds to CD22 and a first transmembrane domain; a first costimulatory signaling domain; and / or a first primary signaling domain; and (b) a second CAR comprising a second antigen-binding domain that binds to CD19, and a second transmembrane domain; a second costimulatory signaling domain; and / or a second primary signaling domain. Including, (i) the first CAR and the second CAR each comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 12; or (ii) the first CAR and the second CAR each comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 16; the CAR molecule comprises the amino acid sequence of SEQ ID NO: 12 or 16, or an amino acid sequence having at least 95% identity thereto; and the CAR molecule is encoded by a nucleic acid molecule; (i) the nucleotide sequence encoding the first transmembrane domain and contained in the nucleic acid molecule is different from the nucleotide sequence encoding the second transmembrane domain and contained in the nucleic acid molecule; (ii) the nucleotide sequence encoding the first costimulatory signaling domain and contained in the nucleic acid molecule is different from the nucleotide sequence encoding the second costimulatory signaling domain and contained in the nucleic acid molecule; and / or (iii) the nucleotide sequence encoding the first primary signaling domain and contained in the nucleic acid molecule is different from the nucleotide sequence encoding the second primary signaling domain and contained in the nucleic acid molecule; CAR molecule.

7. A CAR molecule, the CAR molecule comprising: (a) a first CAR comprising a first antigen-binding domain that binds to CD22, a first transmembrane domain; a first costimulatory signaling domain; and / or a first primary signaling domain; and (b) a second CAR comprising a second antigen-binding domain that binds to CD19, a second transmembrane domain; a second costimulatory signaling domain; and / or a second primary signaling domain. Including, (i) the first CAR and the second CAR each comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 12; or (ii) the first CAR and the second CAR each comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 16; The CAR molecule comprises the amino acid sequence of SEQ ID NO: 12 or 16, or an amino acid sequence having at least 95% identity thereto. CAR molecule.

8. The CAR molecule of claim 6 or 7, wherein the CAR molecule comprises the amino acid sequence of SEQ ID NO: 12 or 16.

9. 1. A cell comprising a nucleic acid molecule encoding a chimeric antigen receptor (CAR) molecule, the CAR molecule comprising: (a) a first CAR comprising: a first antigen-binding domain that binds to CD22; a first transmembrane domain; a first costimulatory signaling domain; and / or a first primary signaling domain; and (b) a second CAR comprising a second antigen-binding domain that binds to CD19; a second transmembrane domain; a second costimulatory signaling domain; and / or a second primary signaling domain. Including, (i) the first CAR and the second CAR each comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 12; or (ii) the first CAR and the second CAR each comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 16; the CAR molecule comprises the amino acid sequence of SEQ ID NO: 12 or 16, or an amino acid sequence having at least 95% identity thereto; and (i) the nucleotide sequence encoding the first transmembrane domain and contained in the nucleic acid molecule is different from the nucleotide sequence encoding the second transmembrane domain and contained in the nucleic acid molecule; (ii) the nucleotide sequence encoding the first costimulatory signaling domain and contained in the nucleic acid molecule is different from the nucleotide sequence encoding the second costimulatory signaling domain and contained in the nucleic acid molecule; and / or (iii) the nucleotide sequence encoding the first primary signaling domain and contained in the nucleic acid molecule is different from the nucleotide sequence encoding the second primary signaling domain and contained in the nucleic acid molecule; cell.

10. 1. A cell comprising a nucleic acid molecule encoding a chimeric antigen receptor (CAR) molecule, the CAR molecule comprising: (a) a first CAR comprising a first antigen-binding domain that binds to CD22, a first transmembrane domain; a first costimulatory signaling domain; and / or a first primary signaling domain; and (b) a second CAR comprising a second antigen-binding domain that binds to CD19, a second transmembrane domain; a second costimulatory signaling domain; and / or a second primary signaling domain. Including, (i) the first CAR and the second CAR each comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 12; or (ii) the first CAR and the second CAR each comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 16; The CAR molecule comprises the amino acid sequence of SEQ ID NO: 12 or 16, or an amino acid sequence having at least 95% identity thereto. cell.

11. A cell comprising a chimeric antigen receptor (CAR) molecule, the CAR molecule comprising: (a) a first CAR comprising a first antigen-binding domain that binds to CD22 and a first transmembrane domain; a first costimulatory signaling domain; and / or a first primary signaling domain; and (b) a second CAR comprising a second antigen-binding domain that binds to CD19, and a second transmembrane domain; a second costimulatory signaling domain; and / or a second primary signaling domain. Including, (i) the first CAR and the second CAR each comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 12; or (ii) the first CAR and the second CAR each comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 16; the CAR molecule comprises the amino acid sequence of SEQ ID NO: 12 or 16, or an amino acid sequence having at least 95% identity thereto; and the CAR molecule is encoded by a nucleic acid molecule; (i) the nucleotide sequence encoding the first transmembrane domain and contained in the nucleic acid molecule is different from the nucleotide sequence encoding the second transmembrane domain and contained in the nucleic acid molecule; (ii) the nucleotide sequence encoding the first costimulatory signaling domain and contained in the nucleic acid molecule is different from the nucleotide sequence encoding the second costimulatory signaling domain and contained in the nucleic acid molecule; and / or (iii) the nucleotide sequence encoding the first primary signaling domain and contained in the nucleic acid molecule is different from the nucleotide sequence encoding the second primary signaling domain and contained in the nucleic acid molecule; cell.

12. A cell comprising a CAR molecule, the CAR molecule comprising: (a) a first CAR comprising: a first antigen-binding domain that binds to CD22; a first transmembrane domain; a first costimulatory signaling domain; and / or a first primary signaling domain; and (b) a second CAR comprising a second antigen-binding domain that binds to CD19; a second transmembrane domain; a second costimulatory signaling domain; and / or a second primary signaling domain; (i) the first CAR and the second CAR each comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 12; or (ii) the first CAR and the second CAR each comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 in SEQ ID NO: 16; The CAR molecule comprises the amino acid sequence of SEQ ID NO: 12 or 16, or an amino acid sequence having at least 95% identity thereto. cell.

13. The cell of any one of claims 9 to 12, wherein the CAR molecule comprises the amino acid sequence of SEQ ID NO: 12 or 16.

14. 11. The cell of claim 9 or 10, wherein the CAR molecule is encoded by the nucleotide sequence of SEQ ID NO: 11, SEQ ID NO: 15, SEQ ID NO: 19, or a nucleotide sequence having at least 80% identity thereto.

15. A pharmaceutical composition comprising the nucleic acid molecule of any one of claims 1 to 5, the CAR molecule of any one of claims 6 to 8, or the cell of any one of claims 9 to 14, wherein the pharmaceutical composition further comprises an excipient, carrier, diluent, and / or stabilizer.

16. 15. A pharmaceutical composition comprising an effective amount of the cell of any one of claims 9 to 14, the cell comprising the nucleic acid molecule of any one of claims 1 to 5, or the CAR molecule of any one of claims 6 to 8, for use in providing anti-tumor immunity to a subject in need thereof.

17. A pharmaceutical composition comprising an effective amount of the cell of any one of claims 9 to 14, the cell comprising the nucleic acid molecule of any one of claims 1 to 5, or the CAR molecule of any one of claims 6 to 8, for use in treating an antigen-associated disease.

18. 18. The pharmaceutical composition of claim 17, wherein the cell is a T cell or an NK cell.

19. 18. The pharmaceutical composition of claim 17, wherein the disease is a blood cancer.

20. The blood cancers include acute myeloid leukemia (AML), B-cell acute lymphoblastic leukemia (BALL), small lymphocytic lymphoma (SLL), acute lymphoblastic leukemia (ALL), chronic myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, hairy cell leukemia, and the like.

20. The pharmaceutical composition of claim 19, wherein the tumor is selected from the group consisting of hematologic malignancies, small cell lymphoma, large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplastic or myelodysplastic syndromes, myeloproliferative neoplasms, non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, preleukemia, and combinations thereof.

21. 20. The pharmaceutical composition of claim 19, wherein the hematological cancer is pediatric BALL or adult BALL.

22. Use of an effective amount of the cell of any one of claims 9 to 14, the cell comprising the nucleic acid molecule of any one of claims 1 to 5, the CAR molecule of any one of claims 6 to 8, or the pharmaceutical composition of claim 15 in the manufacture of a medicament for providing anti-tumor immunity to a subject in need thereof.

23. Use of an effective amount of the cell according to any one of claims 9 to 14, the cell comprising the nucleic acid molecule according to any one of claims 1 to 5, the CAR molecule according to any one of claims 6 to 8, or the pharmaceutical composition according to claim 15 in the manufacture of a medicament for treating an antigen-associated disease.

24. 24. The use according to claim 23, wherein the cell is a T cell or an NK cell.

25. 24. The use according to claim 23, wherein the disease is a blood cancer.

26. The blood cancers include acute myeloid leukemia (AML), B-cell acute lymphoblastic leukemia (BALL), small lymphocytic lymphoma (SLL), acute lymphoblastic leukemia (ALL), chronic myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL), mantle cell lymphoma (MCL), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt's lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, hairy cell lymphoma, and the like.

26. The use of claim 25, wherein the tumor is selected from the group consisting of follicular leukemia, small cell lymphoma, large cell follicular lymphoma, malignant lymphoproliferative conditions, MALT lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplastic or myelodysplastic syndromes, myeloproliferative neoplasms, non-Hodgkin's lymphoma (NHL), Hodgkin's lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom's macroglobulinemia, preleukemia, and combinations thereof.

27. 26. The use of claim 25, wherein the hematological cancer is pediatric BALL or adult BALL.

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