Chimeric antigen receptors targeting cluster of differentiation 19 (CD19) and cluster of differentiation 22 (CD22)

Abstract

The application provides chimeric antigen receptors (CARs) that target cluster of differentiation 19 (CD 19) and / or cluster of differentiation 22 (CD22), and their uses in tumor immunotherapy. The application also provides polynucleotides and vectors that encode the CARs, as well as host cells comprising the CARs. The application also provides methods for preparing host cells comprising the CARs and methods for treating patients using the modified host cells.

Description

Attorney Docket No: 243734.000231CHIMERIC ANTIGEN RECEPTORS TARGETING CLUSTER OF DIFFERENTIATION 19 (CD19) AND CLUSTER OF DIFFERENTIATION 22 (CD22)CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U. S. Provisional Application No. 63 / 745,761, filed January 15, 2025, the contents of which is incorporated herein by reference in its entirety.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on December 15, 2025, is named 243734_000231_SL.xml and is 165,520 bytes in size.FIELD OF THE INVENTION

[0003] The application relates to chimeric antigen receptors (CARs), particularly CARs targeting cluster of differentiation 19 (CD 19) and / or cluster of differentiation 22 (CD22), and their uses in tumor immunotherapy (e.g., adoptive cell therapy). The application further relates to therapeutic cells that express such CARs and methods for treating patients using the CAR-expressing therapeutic cells.BACKGROUND

[0004] CAR-T cell therapies targeting cluster of differentiation (CD) 19 (CD 19) have demonstrated efficacy in patients with relapsed and chemotherapy refractory B cell acute lymphoblastic leukemia (B-ALL). Though CAR-T cell therapies in acute lymphoblastic leukemia show tremendous potential, not all patients respond. CD 19-negative relapse is the predominant cause of therapy failure in patients treated with anti-CD19 CAR-T as a standalone therapy. CD 19 antigen loss has been observed in this setting. Similarly, relapse has been observed in patients treated with CAR-T cell therapies targeting CD22 due to CD22 loss or diminution of surface expression.Attorney Docket No: 243734.000231SUMMARY OF THE INVENTION

[0005] As specified in the Background section above, there is a need in the art for chimeric antigen receptor (CAR) systems that target CD 19 and CD22 to treat various cancer types, including hematological cancers. The present application addresses these and other needs.

[0006] In one aspect, provided herein is a polynucleotide encoding a dual-targeting chimeric antigen receptor (CAR) system comprising two CARs, wherein each CAR comprises an extracellular domain comprising a target binding moiety; wherein optionally, one of the CARs lacks an activation domain; and wherein one target binding moiety binds to cluster of differentiation 19 (CD 19) and the other target binding moiety binds to cluster of differentiation 22 (CD22).

[0007] In one aspect, provided herein is a pair of polynucleotides encoding a dual-targeting chimeric antigen receptor (CAR) system comprising two CARs, wherein the first polynucleotide encodes a first CAR of the dual-targeting CAR system, and the second polynucleotide encodes a second CAR of the dual-targeting CAR system, wherein each CAR comprises an extracellular domain comprising a target binding moiety; wherein optionally, one of the CARs lacks an activation domain; and wherein one target binding moiety binds to cluster of differentiation 19 (CD 19) and the other target binding moiety binds to cluster of differentiation 22 (CD22).

[0008] In one aspect, provided herein is a polynucleotide encoding a chimeric antigen receptor (CAR) comprising an extracellular domain comprising a target binding moiety, wherein the CAR lacks an activation domain; and wherein the target binding moiety binds to cluster of differentiation 19 (CD 19) or cluster of differentiation 22 (CD22).

[0009] In some embodiments of the polynucleotide or the pair of polynucleotides, one of the CARs lacks an activation domain.

[0010] In some embodiments of the polynucleotide or the pair of polynucleotides, the CAR lacking the activation domain further lacks a costimulatory domain.

[0011] In some embodiments of the polynucleotide or the pair of polynucleotides, each target binding moiety is independently an antibody or antigen-binding fragment thereof, or a peptide.

[0012] In some embodiments of the polynucleotide or the pair of polynucleotides, the antibody or antigen-binding fragment thereof is selected from a single chain variable fragment (scFv), Fab, Fab’, F(ab’)2, Fv fragment, disulfide-linked Fv (dsFv), diabody, VHH, VNAR, single-domain antibody (sdAb), nanobody, dAb fragment, Fd’ fragment, and Fd fragment.Attorney Docket No: 243734.000231

[0013] In some embodiments of the polynucleotide or the pair of polynucleotides, the antiCD 19 target binding moiety is an anti-CD19 antibody or antigen-binding fragment thereof.

[0014] In some embodiments of the polynucleotide or the pair of polynucleotides, the anti-CD19 antibody or antigen-binding fragment thereof is derived from FMC63, HD37, CAT19, budoprutug, coltuximab, denintuzumab, duvortuxizumab, emfizatamab, englumafusp alfa, inebilizumab, loncastuximab, obexelimab, tafasitamab, taplitumomab, or zeripatamig.

[0015] In some embodiments of the polynucleotide or the pair of polynucleotides, the anti-CD19 target binding moiety is an anti-CD19 scFv.

[0016] In some embodiments of the polynucleotide or the pair of polynucleotides, the antiCD 19 scFv is derived from FMC63.

[0017] In some embodiments of the polynucleotide or the pair of polynucleotides, the antiCD 19 scFv comprises a heavy chain complementarity determining region 1 (HCDR1), an HCDR2, and an HCDR3 contained within a heavy chain variable domain (VH) comprising the amino acid sequence EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYY NSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVT VSS (SEQ ID NO: 20), or an amino acid sequence having at least 80% identity thereto; and / or a light chain complementarity determining region 1 (LCDR1), an LCDR2, and an LCDR3 contained within a light chain variable domain (VL) comprising the amino acid sequence DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPS RFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT (SEQ ID NO: 18), or an amino acid sequence having at least 80% identity thereto.

[0018] In some embodiments of the polynucleotide or the pair of polynucleotides, the anti-CD19 scFv comprises an HCDR1 comprising the amino acid sequence DYGVS (SEQ ID NO: 127), an HCDR2 comprising the amino acid sequence VIWGSETTYYNSALKS (SEQ ID NO: 128), and an HCDR3 comprising the amino acid sequence HYYYGGSYAMDY (SEQ ID NO: 129); and / or an LCDR1 comprising the amino acid sequence RASQDISKYLN (SEQ ID NO: 130), an LCDR2 comprising the amino acid sequence HTSRLHS (SEQ ID NO: 131), and an LCDR3 comprising the amino acid sequence QQGNTLPYT (SEQ ID NO: 132).

[0019] In some embodiments of the polynucleotide or the pair of polynucleotides, the antiCD 19 scFv comprises a VH comprising the amino acid sequenceAttorney Docket No: 243734.000231EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYY NSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVT VSS (SEQ ID NO: 20), or an amino acid sequence having at least 80% identity thereto; and / or a VL comprising the amino acid sequence DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPS RFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT (SEQ ID NO: 18), or an amino acid sequence having at least 80% identity thereto.

[0020] In some embodiments of the polynucleotide or the pair of polynucleotides, the nucleotide sequence encoding the VH of the anti-CD19 scFv comprises the sequence of SEQ ID NO: 4, or a nucleotide sequence having at least 80% identity thereto; and / or the nucleotide sequence encoding the VL of the anti-CD19 scFv comprises the sequence of SEQ ID NO: 2, or a nucleotide sequence having at least 80% identity thereto.

[0021] In some embodiments of the polynucleotide or the pair of polynucleotides, the anti-CD22 target binding moiety is an anti-CD22 antibody or antigen-binding fragment thereof.

[0022] In some embodiments of the polynucleotide or the pair of polynucleotides, the anti-CD22 antibody or antigen-binding fragment thereof is derived from m971, LT22, or 16P.

[0023] In some embodiments of the polynucleotide or the pair of polynucleotides, the anti-CD22 target binding moiety is an anti-CD22 scFv.

[0024] In some embodiments of the polynucleotide or the pair of polynucleotides, the anti-CD22 scFv is derived from m971.

[0025] In some embodiments of the polynucleotide or the pair of polynucleotides, the anti-CD22 scFv comprises an HCDR1, an HCDR2, and an HCDR3 contained within a VH comprising the amino acid sequence QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSK WYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIWGQG TMVTVSS (SEQ ID NO: 26), or an amino acid sequence having at least 80% identity thereto; and / or an LCDR1, an LCDR2, and an LCDR3 contained within a VL comprising the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIKR (SEQ ID NO: 27), or an amino acid sequence having at least 80% identity thereto.Attorney Docket No: 243734.000231

[0026] In some embodiments of the polynucleotide or the pair of polynucleotides, the anti-CD22 scFv comprises an HCDR1 comprising the amino acid sequence GDSVSSNSAA (SEQ ID NO: 117), an HCDR2 comprising the amino acid sequence TYYRSKWYN (SEQ ID NO: 118), and an HCDR3 comprising the amino acid sequence AREVTGDLEDAFDI (SEQ ID NO: 119); and / or an LCDR1 comprising the amino acid sequence QTIWS (SEQ ID NO: 120), an LCDR2 comprising the amino acid sequence AAS, and an LCDR3 comprising the amino acid sequence QQSYSIPQT (SEQ ID NO: 122).

[0027] In some embodiments of the polynucleotide or the pair of polynucleotides, the anti-CD22 scFv comprises a VH comprising the amino acid sequence QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSK WYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIWGQG TMVTVSS (SEQ ID NO: 26), or an amino acid sequence having at least 80% identity thereto; and / or a VL comprising the amino acid sequenceDIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIKR (SEQ ID NO: 27), or an amino acid sequence having at least 80% identity thereto.

[0028] In some embodiments of the polynucleotide or the pair of polynucleotides, the anti-CD22 scFv comprises a nucleotide sequence encoding the VH of the anti-CD22 scFv comprises the sequence of SEQ ID NO: 10, or SEQ ID NO: 35, or a nucleotide sequence having at least 80% identity thereto; wherein the nucleotide sequence encoding the VL of the anti-CD22 scFv comprises the sequence of SEQ ID NO: 12, or SEQ ID NO: 37, or a nucleotide sequence having at least 80% identity thereto.

[0029] In some embodiments of the polynucleotide or the pair of polynucleotides, the scFv further comprises a linker between the VH and VL.

[0030] In some embodiments of the polynucleotide or the pair of polynucleotides, the linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 19), GGGGS (SEQ ID NO: 91), GGGGSGGGGS (SEQ ID NO: 92), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 93), KESGSVSSEQLAQFRSLD (SEQ ID NO: 94), EGKSSGSGSESKST (SEQ ID NO: 95), EGKSSGSGSESKSTQ (SEQ ID NO: 96), GSTSGSGKSSEGKG (SEQ ID NO: 97), SSADDAKKDDAKKDDAKKDDAKKDG (SEQ ID NO: 98), EGKSSGSGSESKVD (SEQ IDAttorney Docket No: 243734.000231NO: 99), ESGSVSSEELAFRSLD (SEQ ID NO: 100), or an amino acid sequence having at least 80% sequence identity thereto.

[0031] In some embodiments of the polynucleotide or the pair of polynucleotides, the linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 19), or an amino acid sequence having at least 80% sequence identity thereto.

[0032] In some embodiments of the polynucleotide or the pair of polynucleotides, the nucleotide sequence encoding the linker comprises the nucleotide sequence GGCGGCGGAGGAAGCGGGGGCGGAGGATCTGGTGGTGGTGGATCT (SEQ ID NO: 3), GGTGGCGGAGGTAGTGGTGGTGGCGGAAGTGGCGGAGGTGGAAGC (SEQ ID NO: 11), GGCGGCGGAGGAAGCGGAGGCGGAGGATCTGGTGGTGGTGGATCT (SEQ ID NO: 32), or GGAGGCGGAGGTTCTGGTGGCGGAGGAAGTGGTGGCGGCGGATCT (SEQ ID NO: 36), or a nucleotide sequence having at least 80% sequence identity thereto.

[0033] In some embodiments of the polynucleotide or the pair of polynucleotides, the antiCD 19 scFv comprises the amino acid sequence DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSG VPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGG5GGG G5GGGG5EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIROPPRKGLEWLGVIW GSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYW GQGTSVTVSS (SEQ ID NO: 61) or EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQK PDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYT FGGGTKLEIT (SEQ ID NO: 133), or an amino acid sequence having at least 80% sequence identity thereto.

[0034] In some embodiments of the polynucleotide or the pair of polynucleotides, the nucleotide sequence encoding the anti-CD19 scFv comprises the sequence SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 134, or SEQ ID NO: 135, or a nucleotide sequence having at least 80% identity thereto.

[0035] In some embodiments of the polynucleotide or the pair of polynucleotides, the anti-CD22 scFv comprises the amino acid sequenceAttorney Docket No: 243734.000231QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNW YQQRPGKAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSY SIPQTFGQGTKLEIKR (SEQ ID NO 63) or DIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSG VPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIKRGGGGSGG GG5GGGGSQVQLQQSGPGLVKPSOTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLG RTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDA FDIWGQGTMVTVSS (SEQ ID NO: 136), or an amino acid sequence having at least 80% sequence identity thereto.

[0036] In some embodiments of the polynucleotide or the pair of polynucleotides, the nucleotide sequence encoding the anti-CD22 scFv comprises the sequence SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 137, or SEQ ID NO: 138, or a nucleotide sequence having at least 80% identity thereto.

[0037] In some embodiments of the polynucleotide or the pair of polynucleotides, each CAR comprises a cytoplasmic domain comprising a costimulatory domain.

[0038] In some embodiments of the polynucleotide or the pair of polynucleotides, the costimulatory domain present in one CAR is different from the costimulatory domain present in the other CAR.

[0039] In some embodiments of the polynucleotide or the pair of polynucleotides, each costimulatory domain is independently derived from CD28, 4-1BB (CD137), CD27, CD40, CD134 (0X40), CD226, CD79A, ICOS, BTLA, CD30, GITR, HVEM, IL-2Rβ, a STAT3-binding YXXQ in which X is any amino acid, or MyD88, or any combination thereof.

[0040] In some embodiments of the polynucleotide or the pair of polynucleotides, each costimulatory domain is independently derived from CD28 or 4-1BB.

[0041] In some embodiments of the polynucleotide or the pair of polynucleotides, the costimulatory domain of the anti-CD22 CAR is derived from CD28; and / or the costimulatory domain of the anti-CD19 CAR is derived from 4-1BB.Attorney Docket No: 243734.000231

[0042] In some embodiments of the polynucleotide or the pair of polynucleotides, the costimulatory domain of the anti-CD19 CAR is derived from CD28; and / or the costimulatory domain of the anti-CD22 CAR is derived from 4- IBB.

[0043] In some embodiments of the polynucleotide or the pair of polynucleotides, the CD28 costimulatory domain comprises the amino acid sequence RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 30), or an amino acid sequence having at least 80% sequence identity thereto.

[0044] In some embodiments of the polynucleotide or the pair of polynucleotides, the nucleotide sequence encoding the CD28 costimulatory domain comprises the sequence CGAAGCAAGCGGAGCCGGCTGCTGCACTCCGACTACATGAACATGACCCCTAGACG GCCTGGACCTACCAGAAAGCACTACCAGCCTTACGCTCCTCCTAGAGACTTCGCCGC CTACCGGTCC (SEQ ID NO: 15), or CGAAGCAAGCGGAGCCGGCTGCTGCACAGCGACTACATGAACATGACCCCTAGACG GCCCGGACCAACCAGAAAGCACTACCAGCCTTACGCTCCTCCTAGAGATTTCGCCGC CTACCGGTCC (SEQ ID NO: 40), or a nucleotide sequence having at least 80% sequence identity thereto.

[0045] In some embodiments of the polynucleotide or the pair of polynucleotides, the 4- IBB costimulatory domain comprises the amino acid sequence KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 23), or an amino acid sequence having at least 80% sequence identity thereto.

[0046] In some embodiments of the polynucleotide or the pair of polynucleotides, the nucleotide sequence encoding the 4- IBB costimulatory domain comprises the sequence AAGCGGGGCAGAAAGAAACTGCTCTACATCTTCAAGCAGCCCTTCATGCGGCCCGT GCAGACCACACAAGAGGAAGATGGCTGCTCCTGCAGATTCCCCGAGGAAGAAGAA GGCGGCTGCGAGCTG (SEQ ID NO: 7), or a nucleotide sequence having at least 80% sequence identity thereto.

[0047] In some embodiments of the polynucleotide or the pair of polynucleotides, the anti-CD22 CAR comprises an activation domain.

[0048] In some embodiments of the polynucleotide or the pair of polynucleotides, the activation domain is derived from CD3 zeta (ζ), CD3 delta (δ), CD3 epsilon (ε), CD3 gamma (γ), zeta-chain-associated protein kinase (ZAP) 70 (ZAP70), DNAX-activating protein (DAP) 10Attorney Docket No: 243734.000231(DAP10), DAP12, Fc epsilon receptor I γ chain (FCER1G), CD226, NKG2C, NKG2D, NKG2E, FcR beta (β), CD5, CD22, CD66d, CD79B, or CD79A.

[0049] In some embodiments of the polynucleotide or the pair of polynucleotides, the activation domain is derived from CD3ζ.

[0050] In some embodiments of the polynucleotide or the pair of polynucleotides, the CD3^ activation domain comprises the amino acid sequence RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEG LYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 24), or an amino acid sequence having at least 80% sequence identity thereto.

[0051] In some embodiments of the polynucleotide or the pair of polynucleotides, the nucleotide encoding the CD3ζ activation domain comprises the sequence AGAGTGAAGTTCAGCAGATCCGCCGACGCTCCTGCCTATCAGCAGGGCCAAAACCA GCTGTACAACGAGCTGAACCTGGGGAGAAGAGAAGAGTACGACGTGCTGGACAAG CGGAGAGGCAGAGATCCTGAAATGGGCGGCAAGCCCAGACGGAAGAATCCTCAAG AGGGCCTGTATAATGAGCTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATC GGAATGAAGGGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTACCAGGGCC TGAGCACCGCCACCAAGGATACCTATGATGCCCTGCACATGCAGGCCCTGCCTCCAA GA (SEQ ID NO: 8), AGAGTGAAATTCTCCAGAAGCGCTGACGCCCCAGCTTACCAGCAGGGACAGAATCA GCTCTATAACGAACTGAATCTCGGCAGGCGCGAGGAATATGATGTGCTGGATAAGA GGCGCGGCAGGGACCCAGAGATGGGAGGAAAGCCTCGGCGGAAGAACCCACAAGA AGGACTTTACAACGAACTGCAAAAGGATAAGATGGCAGAAGCTTACTCCGAGATTG GCATGAAGGGCGAACGTCGGAGAGGAAAAGGCCACGACGGACTCTATCAGGGACT GTCCACAGCCACAAAGGACACATACGACGCACTCCATATGCAGGCTCTCCCACCTA GA (SEQ ID NO: 16), or AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACC AGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAG AGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGG AAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATT GGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCTAttorney Docket No: 243734.000231CAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC (SEQ ID NO: 41), or a nucleotide sequence having at least 80% sequence identity thereto.

[0052] In some embodiments of the polynucleotide or the pair of polynucleotides, each CAR further comprises a transmembrane domain.

[0053] In some embodiments of the polynucleotide or the pair of polynucleotides, each transmembrane domain is independently derived from α, β or ζ chain of the T-cell receptor, CD8α, CD28, CD8, CD3 epsilon, CD45, CD4, CD3ζ, CD5, CD9, CD16, CD22, CD33, CD37, CD40, CD64, CD80, CD86, CD 134 (0X40), CD 137, or CD 154, or CD7, or a combination thereof.

[0054] In some embodiments of the polynucleotide or the pair of polynucleotides, each transmembrane domain is independently derived from CD8a or CD28.

[0055] In some embodiments of the polynucleotide or the pair of polynucleotides, the transmembrane domain of the anti-CD22 CAR is derived from CD28; and / or the transmembrane domain of the anti-CD19 CAR is derived from CD28.

[0056] In some embodiments of the polynucleotide or the pair of polynucleotides, the transmembrane domain of the anti-CD22 CAR is derived from CD28; and / or the transmembrane domain of the anti-CD19 CAR is derived from CD8a.

[0057] In some embodiments of the polynucleotide or the pair of polynucleotides, the CD8a transmembrane domain comprises the amino acid sequence IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 22), IYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLH (SEQ ID NO: 46), or IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 124), or an amino acid sequence having at least 80% sequence identity thereto.

[0058] In some embodiments of the polynucleotide or the pair of polynucleotides, the nucleotide sequence encoding the CD8a transmembrane domain comprises the sequence ATCTACATCTGGGCCCCTCTGGCTGGAACATGTGGCGTGCTGCTGCTGAGCCTGGTC ATCACCCTGTATTGC (SEQ ID NO: 6), ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTA TCACCCTTTACTGC (SEQ ID NO: 39), ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTA TCACCCTTTACTGCCGAAGCAAGCGGAGCCGGCTGCTGCACTAA (SEQ ID NO: 45), ATCTACATCTGGGCCCCTCTGGCTGGAACATGTGGCGTGCTGCTGCTGAGCCTGGTC ATCACC (SEQ ID NO: 123), orAttorney Docket No: 243734.000231ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTA TCACC (SEQ ID NO: 126), or a nucleotide sequence having at least 80% sequence identity thereto.

[0059] In some embodiments of the polynucleotide or the pair of polynucleotides, the CD28 transmembrane domain comprises the amino acid sequence FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 29), or FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLH (SEQ ID NO: 48), or an amino acid sequence having at least 80% sequence identity thereto.

[0060] In some embodiments of the polynucleotide or the pair of polynucleotides, the nucleotide encoding the CD28 transmembrane domain comprises the sequence TTCTGGGTGCTCGTTGTTGTTGGCGGCGTGCTGGCCTGTTATAGCCTGCTGGTTACCG TGGCCTTCATCATCTTTTGGGTC (SEQ ID NO: 14), TTTTGGGTCTTGGTGGTTGTGGGCGGCGTACTGGCCTGCTACTCCCTGCTAGTCACCG TCGCGTTCATCATCTTCTGGGTC (SEQ ID NO: 34), TTCTGGGTGCTCGTTGTTGTTGGCGGCGTGCTGGCCTGTTACAGCCTGCTGGTTACCG TGGCCTTCATCATCTTTTGGGTC (SEQ ID NO: 43), or TTCTGGGTGCTCGTTGTTGTTGGCGGCGTGCTGGCCTGTTACAGCCTGCTGGTTACCG TGGCCTTCATCATCTTTTGGGTCCGAAGCAAGCGGAGCCGGCTGCTGCAC (SEQ ID NO: 47), or a nucleotide sequence having at least 80% sequence identity thereto.

[0061] In some embodiments of the polynucleotide or the pair of polynucleotides, each extracellular domain further comprises a hinge domain between the target-binding moiety and the transmembrane domain.

[0062] In some embodiments of the polynucleotide or the pair of polynucleotides, each hinge domain is independently derived from CD8a stalk; CD28; CD4; or IgGl, IgG2, IgG3, IgG4, IgMl, IgM2, IgAl, IgA2, IgD, or IgE, or a chimera thereof; or any combination thereof.

[0063] In some embodiments of the polynucleotide or the pair of polynucleotides, each hinge domain is independently derived from CD8a stalk or CD28.

[0064] In some embodiments of the polynucleotide or the pair of polynucleotides, the hinge domain of the anti-CD22 CAR is derived from CD28, and / or the hinge domain of the anti-CD19 CAR is derived from CD28.Attorney Docket No: 243734.000231

[0065] In some embodiments of the polynucleotide or the pair of polynucleotides, the hinge domain of the anti-CD22 CAR is derived from CD28, and / or the hinge domain of the anti-CD19 CAR is derived from CD8a stalk.

[0066] In some embodiments of the polynucleotide or the pair of polynucleotides, the CD8a stalk hinge domain comprises the amino acid sequence TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 21), or an amino acid sequence having at least 80% sequence identity thereto.

[0067] In some embodiments of the polynucleotide or the pair of polynucleotides, the nucleotide sequence encoding the CD8a stalk hinge domain comprises the sequence ACAACAACCCCTGCTCCTCGGCCTCCTACACCAGCTCCTACAATTGCCAGCCAGCCA CTGTCTCTGAGGCCCGAAGCTTGTAGACCTGCTGCTGGCGGAGCCGTGCATACAAGA GGACTGGATTTCGCCTGCGAC (SEQ ID NO: 5), or ACCACGACGCCAGCGCCGCGACCACCAACGCCGGCGCCCACCATCGCGTCGCAGCC CCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGA GGGGGCTGGACTTCGCCTGTGAT (SEQ ID NO: 38), or a nucleotide sequence having at least 80% sequence identity thereto.

[0068] In some embodiments of the polynucleotide or the pair of polynucleotides, the CD28 hinge domain comprises the amino acid sequence IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 28), or an amino acid sequence having at least 80% sequence identity thereto.

[0069] In some embodiments of the polynucleotide or the pair of polynucleotides, the nucleotide sequence encoding the CD28 hinge domain comprises the sequence ATCGAAGTGATGTACCCTCCTCCTTACCTGGACAACGAGAAGTCCAACGGCACCATC ATCCACGTGAAGGGCAAGCACCTGTGTCCTTCTCCACTGTTCCCCGGACCAAGCAAG CCT (SEQ ID NO: 13), ATCGAGGTGATGTACCCACCGCCTTACCTGGACAACGAGAAAAGCAACGGCACCAT CATTCACGTGAAGGGCAAACACCTGTGCCCATCTCCTCTGTTCCCTGGACCATCCAA GCCC (SEQ ID NO: 33), or ATCGAAGTGATGTACCCGCCTCCTTACCTGGACAACGAGAAGTCCAACGGCACCAT CATCCACGTGAAGGGAAAGCACCTGTGTCCTTCTCCACTGTTCCCCGGACCTAGCAA GCCT (SEQ ID NO: 42), or a nucleotide sequence having at least 80% sequence identity thereto.Attorney Docket No: 243734.000231

[0070] In some embodiments of the polynucleotide or the pair of polynucleotides, the IgG4 hinge domain comprises the amino acid sequence ESKYGPPCPSCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWY VDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTIS KAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP VLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 60), or an amino acid sequence having at least 80% sequence identity thereto.

[0071] In some embodiments of the polynucleotide or the pair of polynucleotides, the nucleotide sequence encoding the IgG4 hinge domain comprises the sequence GAGTCTAAGTACGGCCCTCCTTGTCCTAGCTGCCCCGCTCCTGAATTTGAAGGCGGC CCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCAGCAGAACC CCTGAAGTGACCTGCGTGGTGGTGGACGTGTCCCAAGAGGATCCTGAGGTGCAGTTC AATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCCTAGAGAGGA ACAGTTCCAGAGCACCTACAGAGTGGTGTCCGTGCTGACAGTGCTGCACCAGGATTG GCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGGGCCTGCCTAGCAGCA TCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCAAGAGAACCCCAGGTGTACAC ACTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCAGGTGTCCCTGACCTGCCTGG TCAAGGGCTTCTACCCTTCCGATATCGCCGTGGAATGGGAGAGCAATGGCCAGCCTG AGAACAACTACAAGACCACACCTCCTGTGCTGGACAGCGACGGCTCATTCTTCCTGT ACAGCAGACTGACCGTGGACAAGAGCAGATGGCAAGAGGGCAACGTGTTCAGCTGC AGCGTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGTCTCTGAGCCTGAG CCTCGGCAAG (SEQ ID NO: 54), or a nucleotide sequence having at least 80% sequence identity thereto.

[0072] In one aspect, provided herein is a polynucleotide encoding a dual-targeting chimeric antigen receptor (CAR) system comprising two CARs, wherein the two CARs comprise:(i) an anti-cluster of differentiation 22 (CD22) CAR comprising:an extracellular domain comprising an anti-CD22 target binding moiety,a hinge domain and a transmembrane domain derived from CD28,a cytoplasmic domain comprising a CD28 costimulatory domain and a CD3ζ activation domain; and(ii) an anti-cluster of differentiation 19 (CD 19) CAR comprisingAttorney Docket No: 243734.000231an extracellular domain comprising an anti-CD19 target binding moiety,a hinge domain and a transmembrane domain derived from CD28 or CD8a,a cytoplasmic domain comprising a 4- IBB costimulatory domain and lacking an activation domain.

[0073] In one aspect, provided herein is a pair of polynucleotides encoding a dual-targeting chimeric antigen receptor (CAR) system comprising two CARs, wherein(i) the first polynucleotide encodes an anti-cluster of differentiation 22 (CD22) CAR comprising:an extracellular domain comprising an anti-CD22 target binding moiety,a hinge domain and a transmembrane domain derived from CD28,a cytoplasmic domain comprising a CD28 costimulatory domain and a CD3ζ activation domain; and(ii) the second polynucleotide encodes an anti-cluster of differentiation 19 (CD 19) CAR comprising:an extracellular domain comprising an anti-CD19 target binding moiety,a hinge domain and a transmembrane domain derived from CD28 or CD8a,a cytoplasmic domain comprising a 4-1BB costimulatory domain and lacking an activation domain.

[0074] In one aspect, provided herein is a polynucleotide encoding a dual-targeting chimeric antigen receptor (CAR) system comprising two CARs, wherein the two CARs comprise:(i) an anti-cluster of differentiation 22 (CD22) CAR comprising:an extracellular domain comprising an anti-CD22 target binding moiety,a hinge domain and a transmembrane domain derived from CD28,a cytoplasmic domain comprising a CD28 costimulatory domain and a CD3ζ activation domain; and(ii) an anti-cluster of differentiation 19 (CD 19) CAR comprisingan extracellular domain comprising an anti-CD19 target binding moiety,a hinge domain and a transmembrane domain derived from CD28 or CD8a,a cytoplasmic domain comprising a 4-1BB costimulatory domain and a CD3(^ activation domain.Attorney Docket No: 243734.000231

[0075] In one aspect, provided herein is a pair of polynucleotides encoding a dual -targeting chimeric antigen receptor (CAR) system comprising two CARs, wherein(i) the first polynucleotide encodes an anti-cluster of differentiation 22 (CD22) CAR comprising:an extracellular domain comprising an anti-CD22 target binding moiety,a hinge domain and a transmembrane domain derived from CD28,a cytoplasmic domain comprising a CD28 costimulatory domain and a CD3ζ activation domain; and(ii) the second polynucleotide encodes an anti-cluster of differentiation 19 (CD 19) CAR comprising:an extracellular domain comprising an anti-CD19 target binding moiety,a hinge domain and a transmembrane domain derived from CD28 or CD8a,a cytoplasmic domain comprising a 4-1BB costimulatory domain and a CD3(^ activation domain.

[0076] In above-described embodiments of the polynucleotide or the pair of polynucleotides, each target binding moiety is independently an antibody or antigen-binding fragment thereof, or a peptide.

[0077] In above-described embodiments of the polynucleotide or the pair of polynucleotides, the antibody or antigen-binding fragment thereof is selected from a single chain variable fragment (scFv), Fab, Fab’, F(ab’)2, Fv fragment, disulfide-linked Fv (dsFv), diabody, VHH, VNAR, singledomain antibody (sdAb), nanobody, dAb fragment, Fd' fragment, and Fd fragment.

[0078] In above-described embodiments of the polynucleotide or the pair of polynucleotides, the anti-CD 19 target binding moiety is an anti-CD19 antibody or antigen-binding fragment thereof.

[0079] In above-described embodiments of the polynucleotide or the pair of polynucleotides, the anti-CD 19 antibody or antigen-binding fragment thereof is derived from FMC63, HD37, CAT, budoprutug, coltuximab, denintuzumab, duvortuxizumab, emfizatamab, englumafusp alfa, inebilizumab, loncastuximab, obexelimab, tafasitamab, taplitumomab, or zeripatamig.

[0080] In above-described embodiments of the polynucleotide or the pair of polynucleotides, the anti-CD 19 target binding moiety is an anti-CD 19 scFv.

[0081] In above-described embodiments of the polynucleotide or the pair of polynucleotides, the anti-CD 19 scFv is derived from FMC63.Attorney Docket No: 243734.000231

[0082] In above-described embodiments of the polynucleotide or the pair of polynucleotides, the anti-CD19 scFv comprises an HCDR1, an HCDR2, and an HCDR3 contained within a VH comprising the amino acid sequence EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYY NSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVT VSS (SEQ ID NO: 20), or an amino acid sequence having at least 80% identity thereto; and / or an LCDR1, an LCDR2, and an LCDR3 contained within a VL comprising the amino acid sequence DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPS RFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT (SEQ ID NO: 18), or an amino acid sequence having at least 80% identity thereto.

[0083] In above-described embodiments of the polynucleotide or the pair of polynucleotides, the anti-CD19 scFv comprises an HCDR1 comprising the amino acid sequence DYGVS (SEQ ID NO: 127), an HCDR2 comprising the amino acid sequence VIWGSETTYYNSALKS (SEQ ID NO: 128), and an HCDR3 comprising the amino acid sequence HYYYGGSYAMDY (SEQ ID NO: 129); and / or an LCDR1 comprising the amino acid sequence RASQDISKYLN (SEQ ID NO: 130), an LCDR2 comprising the amino acid sequence HTSRLHS (SEQ ID NO: 131), and an LCDR3 comprising the amino acid sequence QQGNTLPYT (SEQ ID NO: 132).

[0084] In above-described embodiments of the polynucleotide or the pair of polynucleotides, the anti-CD19 scFv comprises a VH comprising the amino acid sequence EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYY NSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVT VSS (SEQ ID NO: 20), or an amino acid sequence having at least 80% identity thereto; and / or a VL comprising the amino acid sequence DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPS RFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT (SEQ ID NO: 18), or an amino acid sequence having at least 80% identity thereto.

[0085] In above-described embodiments of the polynucleotide or the pair of polynucleotides, the nucleotide sequence encoding the VH of the anti-CD19 scFv comprises the sequence of SEQ ID NO: 4, or a nucleotide sequence having at least 80% identity thereto; and / or the nucleotide sequence encoding the VL of the anti-CD19 scFv comprises the sequence of SEQ ID NO: 2, or a nucleotide sequence having at least 80% identity thereto.Attorney Docket No: 243734.000231

[0086] In above-described embodiments of the polynucleotide or the pair of polynucleotides, the anti-CD22 target binding moiety is an anti-CD22 antibody or antigen-binding fragment thereof.

[0087] In above-described embodiments of the polynucleotide or the pair of polynucleotides, the anti-CD22 antibody or antigen-binding fragment thereof is derived from m971, LT22, or 16P.

[0088] In above-described embodiments of the polynucleotide or the pair of polynucleotides, the anti-CD22 target binding moiety is an anti-CD22 scFv.

[0089] In above-described embodiments of the polynucleotide or the pair of polynucleotides, the anti-CD22 scFv is derived from m971.

[0090] In above-described embodiments of the polynucleotide or the pair of polynucleotides, the anti-CD22 scFv comprises an HCDR1, an HCDR2, and an HCDR3 contained within a VH comprising the amino acid sequence QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSK WYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIWGQG TMVTVSS (SEQ ID NO: 26), or an amino acid sequence having at least 80% identity thereto; and / or an LCDR1, an LCDR2, and an LCDR3 contained within a VL comprising the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIKR (SEQ ID NO: 27), or an amino acid sequence having at least 80% identity thereto.

[0091] In above-described embodiments of the polynucleotide or the pair of polynucleotides, the anti-CD22 scFv comprises an HCDR1 comprising the amino acid sequence GDSVSSNSAA (SEQ ID NO: 117), an HCDR2 comprising the amino acid sequence TYYRSKWYN (SEQ ID NO: 118), and an HCDR3 comprising the amino acid sequence AREVTGDLEDAFDI (SEQ ID NO: 119); and / or an LCDR1 comprising the amino acid sequence QTIWS (SEQ ID NO: 120), an LCDR2 comprising the amino acid sequence AAS, and an LCDR3 comprising the amino acid sequence QQSYSIPQT (SEQ ID NO: 122).

[0092] In above-described embodiments of the polynucleotide or the pair of polynucleotides, the anti-CD22 scFv comprises a VH comprising the amino acid sequence QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSK WYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIWGQGAttorney Docket No: 243734.000231TMVTVSS (SEQ ID NO: 26), or an amino acid sequence having at least 80% identity thereto; and / or a VL comprising the amino acid sequence DIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIKR (SEQ ID NO: 27), or an amino acid sequence having at least 80% identity thereto.

[0093] In above-described embodiments of the polynucleotide or the pair of polynucleotides, the anti-CD22 scFv comprises a nucleotide sequence encoding the VH of the anti-CD22 scFv comprises the sequence of SEQ ID NO: 10, or SEQ ID NO: 35, or a nucleotide sequence having at least 80% identity thereto; wherein the nucleotide sequence encoding the VL of the anti-CD22 scFv comprises the sequence of SEQ ID NO: 12, or SEQ ID NO: 37, or a nucleotide sequence having at least 80% identity thereto.

[0094] In above-described embodiments of the polynucleotide or the pair of polynucleotides, the scFv further comprises a linker between the VH and VL.

[0095] In above-described embodiments of the polynucleotide or the pair of polynucleotides, the linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 19), GGGGS (SEQ ID NO: 91), GGGGSGGGGS (SEQ ID NO: 92), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 93), KESGSVSSEQLAQFRSLD (SEQ ID NO: 94), EGKSSGSGSESKST (SEQ ID NO: 95), EGKSSGSGSESKSTQ (SEQ ID NO: 96), GSTSGSGKSSEGKG (SEQ ID NO: 97), SSADDAKKDDAKKDDAKKDDAKKDG (SEQ ID NO: 98), EGKSSGSGSESKVD (SEQ ID NO: 99), ESGSVSSEELAFRSLD (SEQ ID NO: 100), or an amino acid sequence having at least 80% sequence identity thereto.

[0096] In above-described embodiments of the polynucleotide or the pair of polynucleotides, the linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 19), or an amino acid sequence having at least 80% sequence identity thereto.

[0097] In above-described embodiments of the polynucleotide or the pair of polynucleotides, the nucleotide sequence encoding the linker comprises the nucleotide sequence GGCGGCGGAGGAAGCGGGGGCGGAGGATCTGGTGGTGGTGGATCT (SEQ ID NO: 3), GGTGGCGGAGGTAGTGGTGGTGGCGGAAGTGGCGGAGGTGGAAGC (SEQ ID NO: 11), GGCGGCGGAGGAAGCGGAGGCGGAGGATCTGGTGGTGGTGGATCT (SEQ ID NO: 32), or GGAGGCGGAGGTTCTGGTGGCGGAGGAAGTGGTGGCGGCGGATCT (SEQ ID NO: 36), or a nucleotide sequence having at least 80% sequence identity thereto.Attorney Docket No: 243734.000231

[0098] In above-described embodiments of the polynucleotide or the pair of polynucleotides, the anti-CD19 scFv comprises the amino acid sequence DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSG VPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGGSGGGGSGGGGGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIW GSETTYYNSALKSRLTIIKDNSKSOVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYW GQGTSVTVSS (SEQ ID NO: 61) or EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSAL KSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQK PDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYT FGGGTKLEIT (SEQ ID NO: 133), or an amino acid sequence having at least 80% sequence identity thereto.

[0099] In above-described embodiments of the polynucleotide or the pair of polynucleotides, the nucleotide sequence encoding the anti-CD19 scFv comprises the sequence SEQ ID NO: 62, SEQ ID NO: 65, SEQ ID NO: 134, or SEQ ID NO: 135, or a nucleotide sequence having at least 80% identity thereto.

[0100] In above-described embodiments of the polynucleotide or the pair of polynucleotides, the anti-CD22 scFv comprises the amino acid sequence QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDIWGQGTMVTVSSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNW YQQRPGKAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSY SIPQTFGQGTKLEIKR (SEQ ID NO: 63) or DIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSG VPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIKRGGGGSGGGGSGGGGSGGGGSQVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDA FDIWGQGTMVTVSS (SEQ ID NO: 136), or an amino acid sequence having at least 80% sequence identity thereto.Attorney Docket No: 243734.000231

[0101] In above-described embodiments of the polynucleotide or the pair of polynucleotides, the nucleotide sequence encoding the anti-CD22 scFv comprises the sequence SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 137, SEQ ID NO: 138, or a nucleotide sequence having at least 80% identity thereto.

[0102] In above-described embodiments of the polynucleotide or the pair of polynucleotides, the CD28 costimulatory domain comprises the amino acid sequence RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 30), or an amino acid sequence having at least 80% sequence identity thereto.

[0103] In above-described embodiments of the polynucleotide or the pair of polynucleotides, the nucleotide sequence encoding the CD28 costimulatory domain comprises the sequence CGAAGCAAGCGGAGCCGGCTGCTGCACTCCGACTACATGAACATGACCCCTAGACG GCCTGGACCTACCAGAAAGCACTACCAGCCTTACGCTCCTCCTAGAGACTTCGCCGC CTACCGGTCC (SEQ ID NO: 15), or CGAAGCAAGCGGAGCCGGCTGCTGCACAGCGACTACATGAACATGACCCCTAGACG GCCCGGACCAACCAGAAAGCACTACCAGCCTTACGCTCCTCCTAGAGATTTCGCCGC CTACCGGTCC (SEQ ID NO: 40), or a nucleotide sequence having at least 80% sequence identity thereto.

[0104] In above-described embodiments of the polynucleotide or the pair of polynucleotides, the 4-1BB costimulatory domain comprises the amino acid sequence KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 23), or an amino acid sequence having at least 80% sequence identity thereto.

[0105] In above-described embodiments of the polynucleotide or the pair of polynucleotides, the nucleotide sequence encoding the 4-1BB costimulatory domain comprises the sequence AAGCGGGGCAGAAAGAAACTGCTCTACATCTTCAAGCAGCCCTTCATGCGGCCCGT GCAGACCACACAAGAGGAAGATGGCTGCTCCTGCAGATTCCCCGAGGAAGAAGAA GGCGGCTGCGAGCTG (SEQ ID NO: 7), or a nucleotide sequence having at least 80% sequence identity thereto.

[0106] In above-described embodiments of the polynucleotide or the pair of polynucleotides, the CD3ζ activation domain comprises the amino acid sequence RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGAttorney Docket No: 243734.000231LYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO: 24), or an amino acid sequence having at least 80% sequence identity thereto.

[0107] In above-described embodiments of the polynucleotide or the pair of polynucleotides, the nucleotide encoding the CD3ζ activation domain comprises the sequence AGAGTGAAGTTCAGCAGATCCGCCGACGCTCCTGCCTATCAGCAGGGCCAAAACCA GCTGTACAACGAGCTGAACCTGGGGAGAAGAGAAGAGTACGACGTGCTGGACAAG CGGAGAGGCAGAGATCCTGAAATGGGCGGCAAGCCCAGACGGAAGAATCCTCAAG AGGGCCTGTATAATGAGCTGCAGAAAGACAAGATGGCCGAGGCCTACAGCGAGATC GGAATGAAGGGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTGTACCAGGGCC TGAGCACCGCCACCAAGGATACCTATGATGCCCTGCACATGCAGGCCCTGCCTCCAA GA (SEQ ID NO: 8), AGAGTGAAATTCTCCAGAAGCGCTGACGCCCCAGCTTACCAGCAGGGACAGAATCA GCTCTATAACGAACTGAATCTCGGCAGGCGCGAGGAATATGATGTGCTGGATAAGA GGCGCGGCAGGGACCCAGAGATGGGAGGAAAGCCTCGGCGGAAGAACCCACAAGA AGGACTTTACAACGAACTGCAAAAGGATAAGATGGCAGAAGCTTACTCCGAGATTG GCATGAAGGGCGAACGTCGGAGAGGAAAAGGCCACGACGGACTCTATCAGGGACT GTCCACAGCCACAAAGGACACATACGACGCACTCCATATGCAGGCTCTCCCACCTA GA (SEQ ID NO: 16), or AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAACC AGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGACAAG AGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACCCTCAGG AAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACAGTGAGATT GGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTTTACCAGGGTCT CAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAGGCCCTGCCCCCTCGC (SEQ ID NO: 41), or a nucleotide sequence having at least 80% sequence identity thereto.

[0108] In above-described embodiments of the polynucleotide or the pair of polynucleotides, the CD8a transmembrane domain comprises the amino acid sequence IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 22), IYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLH (SEQ ID NO: 46), or IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 124), or an amino acid sequence having at least 80% sequence identity thereto.Attorney Docket No: 243734.000231

[0109] In above-described embodiments of the polynucleotide or the pair of polynucleotides, the nucleotide sequence encoding the CD8a transmembrane domain comprises the sequence ATCTACATCTGGGCCCCTCTGGCTGGAACATGTGGCGTGCTGCTGCTGAGCCTGGTC ATCACCCTGTATTGC (SEQ ID NO: 6), ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTA TCACCCTTTACTGC (SEQ ID NO: 39), ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTA TCACCCTTTACTGCCGAAGCAAGCGGAGCCGGCTGCTGCACTAA (SEQ ID NO: 45), ATCTACATCTGGGCCCCTCTGGCTGGAACATGTGGCGTGCTGCTGCTGAGCCTGGTC ATCACC (SEQ ID NO: 123), or ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGGTTA TCACC (SEQ ID NO: 126), or a nucleotide sequence having at least 80% sequence identity thereto.

[0110] In above-described embodiments of the polynucleotide or the pair of polynucleotides, the CD28 transmembrane domain comprises the amino acid sequence FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 29) or FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLH (SEQ ID NO: 48), or an amino acid sequence having at least 80% sequence identity thereto.

[0111] In above-described embodiments of the polynucleotide or the pair of polynucleotides, the nucleotide encoding the CD28 transmembrane domain comprises the sequence TTCTGGGTGCTCGTTGTTGTTGGCGGCGTGCTGGCCTGTTATAGCCTGCTGGTTACCG TGGCCTTCATCATCTTTTGGGTC (SEQ ID NO: 14), TTTTGGGTCTTGGTGGTTGTGGGCGGCGTACTGGCCTGCTACTCCCTGCTAGTCACCG TCGCGTTCATCATCTTCTGGGTC (SEQ ID NO: 34), or TTCTGGGTGCTCGTTGTTGTTGGCGGCGTGCTGGCCTGTTACAGCCTGCTGGTTACCG TGGCCTTCATCATCTTTTGGGTC (SEQ ID NO: 43), or TTCTGGGTGCTCGTTGTTGTTGGCGGCGTGCTGGCCTGTTACAGCCTGCTGGTTACCG TGGCCTTCATCATCTTTTGGGTCCGAAGCAAGCGGAGCCGGCTGCTGCAC (SEQ ID NO: 47), or a nucleotide sequence having at least 80% sequence identity thereto.

[0112] In above-described embodiments of the polynucleotide or the pair of polynucleotides, the CD8a stalk hinge domain comprises the amino acid sequenceAttorney Docket No: 243734.000231TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 21), or an amino acid sequence having at least 80% sequence identity thereto.

[0113] In above-described embodiments of the polynucleotide or the pair of polynucleotides, the nucleotide sequence encoding the CD8a stalk hinge domain comprises the sequence ACAACAACCCCTGCTCCTCGGCCTCCTACACCAGCTCCTACAATTGCCAGCCAGCCA CTGTCTCTGAGGCCCGAAGCTTGTAGACCTGCTGCTGGCGGAGCCGTGCATACAAGA GGACTGGATTTCGCCTGCGAC (SEQ ID NO: 5), or ACCACGACGCCAGCGCCGCGACCACCAACGCCGGCGCCCACCATCGCGTCGCAGCC CCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCACACGA GGGGGCTGGACTTCGCCTGTGAT (SEQ ID NO: 38), or a nucleotide sequence having at least 80% sequence identity thereto.

[0114] In above-described embodiments of the polynucleotide or the pair of polynucleotides, the CD28 hinge domain comprises the amino acid sequence IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 28), or an amino acid sequence having at least 80% sequence identity thereto.

[0115] In above-described embodiments of the polynucleotide or the pair of polynucleotides, the nucleotide sequence encoding the CD28 hinge domain comprises the sequence ATCGAAGTGATGTACCCTCCTCCTTACCTGGACAACGAGAAGTCCAACGGCACCATC ATCCACGTGAAGGGCAAGCACCTGTGTCCTTCTCCACTGTTCCCCGGACCAAGCAAG CCT (SEQ ID NO: 13), ATCGAGGTGATGTACCCACCGCCTTACCTGGACAACGAGAAAAGCAACGGCACCAT CATTCACGTGAAGGGCAAACACCTGTGCCCATCTCCTCTGTTCCCTGGACCATCCAA GCCC (SEQ ID NO: 33), or ATCGAAGTGATGTACCCGCCTCCTTACCTGGACAACGAGAAGTCCAACGGCACCAT CATCCACGTGAAGGGAAAGCACCTGTGTCCTTCTCCACTGTTCCCCGGACCTAGCAA GCCT (SEQ ID NO: 42), or a nucleotide sequence having at least 80% sequence identity thereto.

[0116] In some embodiments of the polynucleotide or the pair of polynucleotides, the anti-CD22 CAR comprises the amino acid sequence of any of SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, or SEQ ID NO: 83, or an amino acid sequence having at least 80% sequence identity thereto.Attorney Docket No: 243734.000231

[0117] In some embodiments of the polynucleotide or the pair of polynucleotides, the anti-CD22 CAR comprises the amino acid sequence SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, or SEQ ID NO: 83.

[0118] In some embodiments of the polynucleotide or the pair of polynucleotides, the anti-CD22 CAR comprises the amino acid sequence SEQ ID NO: 79.

[0119] In some embodiments of the polynucleotide or the pair of polynucleotides, the nucleotide sequence encoding the anti-CD22 CAR comprises the sequence of any of SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, or SEQ ID NO: 82, or a nucleotide sequence having at least 80% sequence identity thereto.

[0120] In some embodiments of the polynucleotide or the pair of polynucleotides, the nucleotide sequence encoding the anti-CD22 CAR comprises the sequence of any one of SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, or SEQ ID NO: 82.

[0121] In some embodiments of the polynucleotide or the pair of polynucleotides, the nucleotide sequence encoding the anti-CD22 CAR comprises SEQ ID NO: 78.

[0122] In some embodiments of the polynucleotide or the pair of polynucleotides, the anti-CD19 CAR comprises the amino acid sequence of SEQ ID NO: 88 or SEQ ID NO: 90, or an amino acid sequence having at least 80% sequence identity thereto.

[0123] In some embodiments of the polynucleotide or the pair of polynucleotides, the anti-CD19 CAR comprises the amino acid sequence of SEQ ID NO: 88 or SEQ ID NO: 90.

[0124] In some embodiments of the polynucleotide or the pair of polynucleotides, the antiCD 19 CAR comprises the amino acid sequence SEQ ID NO: 90.

[0125] In some embodiments of the polynucleotide or the pair of polynucleotides, the nucleotide sequence encoding the anti-CD19 CAR comprises the sequence of SEQ ID NO: 87 or SEQ ID NO: 89, or a nucleotide sequence having at least 80% sequence identity thereto.

[0126] In some embodiments of the polynucleotide or the pair of polynucleotides, the nucleotide sequence encoding the CD19 CAR comprises the sequence of SEQ ID NO: 89.

[0127] In some embodiments of the polynucleotide or the pair of polynucleotides, the anti-CD19 CAR comprises the amino acid sequence SEQ ID NO: 85, or an amino acid sequence having at least 80% sequence identity thereto.

[0128] In some embodiments of the polynucleotide or the pair of polynucleotides, the anti-CD19 CAR comprises the amino acid sequence SEQ ID NO: 85.Attorney Docket No: 243734.000231

[0129] In some embodiments of the polynucleotide or the pair of polynucleotides, the nucleotide sequence encoding the CD 19 CAR comprises the sequence of SEQ ID NO: 84 or SEQ ID NO: 86, or a nucleotide sequence having at least 80% sequence identity thereto.

[0130] In some embodiments of the polynucleotide or the pair of polynucleotides, the nucleotide sequence encoding the CD 19 CAR comprises the sequence of SEQ ID NO: 84 or SEQ ID NO: 86.

[0131] In some embodiments of the polynucleotide or the pair of polynucleotides, each extracellular domain further comprises a leader sequence.

[0132] In some embodiments of the polynucleotide or the pair of polynucleotides, each leader sequence is independently derived from CD8a or a human immunoglobulin heavy chain variable region.

[0133] In some embodiments of the polynucleotide or the pair of polynucleotides, the CD8a leader sequence comprises the amino acid sequence MALPVTALLLPLALLLHAARP (SEQ ID NO: 17), or an amino acid sequence having at least 80% sequence identity thereto.

[0134] In some embodiments of the polynucleotide or the pair of polynucleotides, the nucleotide sequence encoding the CD8a leader sequence comprises the sequence ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTTCTGCATGCCGCCA GACCT (SEQ ID NO: 1), ATGGCACTGCCAGTTACTGCCCTGCTGCTCCCACTTGCACTGCTGCTTCATGCTGCTA GACCT (SEQ ID NO: 31), or a nucleotide sequence having at least 80% sequence identity thereto.

[0135] In some embodiments of the polynucleotide or the pair of polynucleotides, the human immunoglobulin heavy chain variable region leader sequence comprises the amino acid sequence MDWIWRILFLVGAATGAHS (SEQ ID NO: 57), or an amino acid sequence having at least 80% sequence identity thereto.

[0136] In some embodiments of the polynucleotide or the pair of polynucleotides, the nucleotide sequence encoding the human immunoglobulin heavy chain variable region leader sequence comprises the sequence ATGGATTGGATCTGGCGGATCCTGTTCCTTGTGGGAGCTGCTACAGGCGCCCATTCT(SEQ ID NO: 51), or a nucleotide sequence having at least 80% sequence identity thereto.

[0137] In some embodiments of the polynucleotide or the pair of polynucleotides, the polynucleotide further encodes CD20.Attorney Docket No: 243734.000231

[0138] In some embodiments of the polynucleotide or the pair of polynucleotides, the nucleotide sequence encoding CD20 is present adjacent to the nucleotide sequence encoding the CAR comprising a signaling domain.

[0139] In some embodiments of the polynucleotide or the pair of polynucleotides, the nucleotide sequence encoding CD20 is present 5’ to the nucleotide sequence encoding the anti-CD22 CAR.

[0140] In some embodiments of the polynucleotide or the pair of polynucleotides, the CD20 comprises the amino acid sequence MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRESKTLGAVQIMN GLFHIALGGLLMIPAGIYAPICVTVWYPLWGGIMYIISGSLLAATEKNSRKCLVKGKMIM NSLSLFAAISGMILSIMDILNIKISHFLKMESLNFIRAHTPYINIYNCEPANPSEKNSPSTQY CYSIQSLFLGILSVMLIFAFFQELVIAGIVENEWKRTCSRPKSNIVLLSAEEKKEQTIEIKEE VVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSSPGSG (SEQ ID NO: 55), or an amino acid sequence having at least 80% sequence identity thereto.

[0141] In some embodiments of the polynucleotide or the pair of polynucleotides, the nucleotide sequence encoding the CD20 comprises the sequence SEQ ID NO: 49, or a nucleotide sequence having at least 80% sequence identity thereto.

[0142] In some embodiments of the polynucleotide or the pair of polynucleotides, the polynucleotide further comprises a separation sequence between the two CARs.

[0143] In some embodiments of the polynucleotide or the pair of polynucleotides, the polynucleotide further comprises a separation sequence between CD20 and its adjacent CAR.

[0144] In some embodiments of the polynucleotide or the pair of polynucleotides, the separation sequence is selected from GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 25), EGRGSLLTCGDVEENPGP (SEQ ID NO: 56), AEGRGSLLTCGDVEENPGP (SEQ ID NO: 44), GSGSRVTELLYRMKRAETYCPRPLLAIHPTEARHKQKIVAPVKQLLNFDLLKLAGDVES NPGP (SEQ ID NO: 52), LLCFLLLLLSGDVELNPGP (SEQ ID NO: 53), HHFMFLLLLLAGDIELNPGP (SEQ ID NO: 58), WFLVLLSFILSGDIEVNPGP (SEQ ID NO: 59), KNCAMYMLLLSGDVETNPGP (SEQ ID NO: 67), MVISQLMLKLAGDVEENPGP (SEQ ID NO: 69), GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 71), GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 73), or D-X-E-X-NPGP (SEQ ID NO: 75),Attorney Docket No: 243734.000231in which X is any amino acid residue, or an amino acid sequence having at least 80% sequence identity thereto.

[0145] In some embodiments of the polynucleotide or the pair of polynucleotides, the separation sequence the amino acid sequence GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 25), or EGRGSLLTCGDVEENPGP (SEQ ID NO: 56), or an amino acid sequence having at least 80% sequence identity thereto.

[0146] In some embodiments of the polynucleotide or the pair of polynucleotides, the nucleotide encoding the separation sequence comprises the sequence GGTTCTGGCGAAGGCAGAGGCTCTCTGCTGACATGCGGAGATGTGGAAGAGAACCC CGGACCT (SEQ ID NO: 9), GAAGGCAGAGGCTCTCTGCTGACATGCGGAGATGTGGAAGAGAACCCCGGACCT(SEQ ID NO: 125), or GAAGGCAGAGGTTCTCTGCTGACATGCGGCGACGTGGAAGAGAACCCTGGACCT(SEQ ID NO: 50), or a nucleotide sequence having at least 80% sequence identity thereto.

[0147] In some embodiments of the polynucleotide or the pair of polynucleotides, the polynucleotide comprises or consisting of the nucleotide sequence of any one of SEQ ID NO: 72 or SEQ ID NO: 74, or a nucleotide sequence having at least 80% sequence identity thereto.

[0148] In some embodiments of the polynucleotide or the pair of polynucleotides, the polynucleotide comprises or consisting of the nucleotide sequence of any one of SEQ ID NO: 72 or SEQ ID NO: 74.

[0149] In some embodiments of the polynucleotide or the pair of polynucleotides, the polynucleotide comprises or consisting of the nucleotide sequence of SEQ ID NO: 74.

[0150] In some embodiments of the polynucleotide or the pair of polynucleotides, the polynucleotide is a DNA molecule.

[0151] In some embodiments of the polynucleotide or the pair of polynucleotides, the polynucleotide is an RNA molecule.

[0152] In one aspect, provided herein is a recombinant vector comprising the polynucleotide of the present disclosure or the pair of polynucleotides of the present disclosure.

[0153] In one aspect, provided herein is a pair of recombinant vectors comprising the pair of polynucleotides of the present disclosure.

[0154] In some embodiments, the vector is a viral vector.Attorney Docket No: 243734.000231

[0155] In some embodiments, the viral vector is a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated virus vector, an alphaviral vector, a herpes virus vector, a baculoviral vector, or a vaccinia virus vector.

[0156] In some embodiments, the viral vector is a lentiviral vector.

[0157] In some embodiments, the vector is a non-viral vector.

[0158] In some embodiments, the non-viral vector is a minicircle plasmid, a Sleeping Beauty transposon, a PiggyBac transposon, or a single or double stranded DNA molecule that is used as a template for homology directed repair (HDR) based gene editing.

[0159] In some embodiments, the vector is a bicistronic vector.

[0160] In one aspect, provided herein is a dual-targeting chimeric antigen receptor (CAR) system which is encoded by the polynucleotide of the present disclosure or the pair of polynucleotides of the present disclosure.

[0161] In one aspect, provided herein is a chimeric antigen receptor (CAR) which is encoded by the polynucleotide of the present disclosure or the pair of polynucleotides of the present disclosure.

[0162] In one aspect, provided herein is an isolated host cell comprising the polynucleotide of the present disclosure, the pair of polynucleotides of any one of the present disclosure, the recombinant vector of the present disclosure, the pair of recombinant vectors of the present disclosure, the dual-targeting chimeric antigen receptor (CAR) system of the present disclosure, or the CAR the present disclosure.

[0163] In some embodiments, the host cell is an immune cell.

[0164] In some embodiments, the host cell is a T cell, a nature killer (NK) cell, or a macrophage.

[0165] In some embodiments, the host cell is a T cell.

[0166] In some embodiments, the host cell is a CD8+T-cell, a CD4+T-cell, a cytotoxic T-cell, an αβ T-cell receptor (TCR) T-cell, an invariant natural killer T (iNKT) cell, a γδ T-cell, a memory T-cell, a memory stem T-cell (TSCM), a naive T-cell, an effector T-cell, a T-helper cell, or a regulatory T-cell (Treg).

[0167] In some embodiments, the host cell is a natural killer (NK) cell.

[0168] In some embodiments, the host cell has been activated and / or expanded ex vivo.

[0169] In some embodiments, the host cell is an allogeneic cell.Attorney Docket No: 243734.000231

[0170] In some embodiments, the host cell is an autologous cell.

[0171] In some embodiments, the host cell is isolated from a subject having a tumor, one or more cells of the tumor express CD 19 and / or CD22.

[0172] In some embodiments, the tumor is a hematological malignancy.

[0173] In some embodiments, the hematological malignancy is acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), B-cell lymphoma, B-cell acute lymphoblastic leukemia (BALL), chronic lymphocytic leukemia (CLL), acute myelogenous leukemia (AML), mantle cell lymphoma (MCL), multiple myeloma (MM), small lymphocytic lymphoma (SLL), and / or hairy cell leukemia.

[0174] In some embodiments, the host cell is derived from a blood sample, a marrow sample, a tissue sample, or a tumor sample.

[0175] In one aspect, provided herein is a pharmaceutical composition comprising the isolated host cell of the present disclosure and a pharmaceutically acceptable carrier and / or excipient.

[0176] In one aspect, provided herein is a method of generating the isolated host cell of the present disclosure, said method comprising genetically modifying the host cell with the polynucleotide of the present disclosure, the pair of polynucleotides of the present disclosure, the recombinant vector of the present disclosure or the pair of recombinant vectors of the present disclosure.

[0177] In some embodiments of the method, the vector is a viral vector, and the genetic modification is conducted by a transduction using said vector.

[0178] In some embodiments of the method, the genetic modification is conducted ex vivo.

[0179] In one aspect, provided herein is a method for killing a tumor cell expressing CD 19 and / or CD22, said method comprising contacting said cell with the host cell(s) of the present disclosure or the pharmaceutical composition of the present disclosure.

[0180] In one aspect, provided herein is a method for treating a tumor in a subject in need thereof, wherein one or more cells of the tumor express CD 19 and / or CD22, said method comprising administering to the subject a therapeutically effective amount of the host cell(s) of the present disclosure or the pharmaceutical composition of the present disclosure.

[0181] In some embodiments, the tumor is a hematological malignancy.

[0182] In some embodiments, the hematological malignancy is acute lymphoblastic leukemia (ALL), B-cell lymphoma, B-cell acute lymphoblastic leukemia (BALL), chronic lymphocyticAttorney Docket No: 243734.000231leukemia (CLL), acute myelogenous leukemia (AML), mantle cell lymphoma (MCL), multiple myeloma (MM), small lymphocytic lymphoma (SLL), and / or hairy cell leukemia.

[0183] In some embodiments, the method comprises:a) isolating T cells, NK cells, iNKT cells, or macrophages from the subject or generating T-cells, NK cells, iNKT cells, or macrophages from stem cells;b) genetically modifying said T cells, NK cells, iNKT cells, macrophages, or stem cells ex vivo with the polynucleotide of any one of the present disclosure, the pair of polynucleotides of the present disclosure, the vector of the present disclosure, or the pair of recombinant vectors of the present disclosure;c) optionally, expanding and / or activating said T cells, NK cells, iNKT cells, or macrophages before, after, or during step (b); andd) introducing the genetically modified T cells, NK cells, iNKT cells, or macrophages into the subject.

[0184] In some embodiments, the stem cells comprise induced pluripotent stem cells (iPSCs).

[0185] In some embodiments, the subject is human.

[0186] In one aspect, provided herein is a polynucleotide encoding a dual-targeting chimeric antigen receptor (CAR) system comprising two CARs, wherein each CAR comprises an extracellular domain comprising a means for binding to a target, wherein optionally, one of the CARs lacks a means for activating effector function of an immune cell; and

[0187] wherein one extracellular domain comprises a means for binding to cluster of differentiation 19 (CD 19) and the other extracellular domain comprises a means for binding to cluster of differentiation 22 (CD22).

[0188] In one aspect, provided herein is a pair of polynucleotides encoding a dual-targeting chimeric antigen receptor (CAR) system comprising two CARs, wherein the first polynucleotide encodes a first CAR of the dual-targeting CAR system, and the second polynucleotide encodes a second CAR of the dual-targeting CAR system, wherein each CAR comprises an extracellular domain comprising a means for binding to a target; wherein optionally, one of the CARs lacks a means for activating effector function of an immune cell; and wherein one extracellular domain comprises a means for binding to cluster of differentiation 19 (CD 19) and the other extracellular domain comprises a means for binding to cluster of differentiation 22 (CD22).Attorney Docket No: 243734.000231

[0189] In some embodiments, one of the CARs lacks a means for activating effector function of an immune cell.

[0190] In some embodiments, the CAR lacking the means for activating effector function of an immune cell further lacks a means for boosting effector function of the immune cell.

[0191] In one aspect, provided herein is a polynucleotide encoding a dual-targeting chimeric antigen receptor (CAR) system comprising two CARs, wherein the two CARs comprise:(i) an anti-cluster of differentiation 22 (CD22) CAR comprising:an extracellular domain comprising a means for binding to CD22,a hinge domain and a transmembrane domain derived from CD28,a cytoplasmic domain comprising a CD28 costimulatory domain and a CD3ζ activation domain; and(ii) an anti-cluster of differentiation 19 (CD 19) CAR comprisingan extracellular domain comprising a means for binding to CD 19,a hinge domain and a transmembrane domain derived from CD28 or CD8a,a cytoplasmic domain comprising a 4- IBB costimulatory domain and lacking a means for activating effector function of an immune cell.

[0192] In one aspect, provided herein is a pair of polynucleotides encoding a du al -targeting chimeric antigen receptor (CAR) system comprising two CARs, wherein(i) the first polynucleotide encodes an anti-cluster of differentiation 22 (CD22) CAR comprising:an extracellular domain comprising a means for binding to CD22,a hinge domain and a transmembrane domain derived from CD28,a cytoplasmic domain comprising a CD28 costimulatory domain and a CD3ζ activation domain; and(ii) the second polynucleotide encodes an anti-cluster of differentiation 19 (CD 19) CAR comprisingan extracellular domain comprising a means for binding to CD 19,a hinge domain and a transmembrane domain derived from CD28 or CD8a,a cytoplasmic domain comprising a 4-1BB costimulatory domain and lacking a means for activating effector function of an immune cell.Attorney Docket No: 243734.000231

[0193] In one aspect, provided herein is a polynucleotide encoding a dual -targeting chimeric antigen receptor (CAR) system comprising two CARs, wherein one CAR binds to cluster of differentiation 19 (CD 19) and the other CAR binds to cluster of differentiation 22 (CD22), the improvement comprising one of the CARs lacking an activation domain.

[0194] In one aspect, provided herein is a pair of polynucleotides encoding a dual-targeting chimeric antigen receptor (CAR) system comprising two CARs, wherein the first polynucleotide encodes a first CAR of the dual-targeting CAR system, and the second polynucleotide encodes a second CAR of the dual-targeting CAR system, wherein the first CAR binds to cluster of differentiation 19 (CD 19) and the second CAR binds to cluster of differentiation 22 (CD22), the improvement comprising one of the CARs lacking an activation domain.BRIEF DESCRIPTION OF THE DRAWINGS

[0195] Figure 1 depicts a scheme of lentiviral vector LV-MND-CD22co-CD19d-CD28H. The lentiviral vector is a 3rdgeneration self-inactivating lentiviral vector encoding an expression cassette containing the CD22-CAR, a 2 A peptide, and the CD19-CAR. LTR: long terminal repeat; ALTR (delta LTR): inactive LTR; \p: packing signal; cPPT: central polypurine tract; RRE: Rev response element; MND: myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer-binding site substituted (MND) promoter.

[0196] Figures 2A-2B depict a scheme of evaluated CAR constructs and lentiviral vectors.Figure 2A depicts CAR constructs. Figure 2B depicts lentiviral vectors. FL: full length; TM: transmembrane domain.

[0197] Figures 3A-3B depict the generation of CD19-CD22-CAR T cells. CAR T cells were generated from peripheral blood CD4 / CD8-selected T cells by standard lentiviral transduction. CAR expression was determined by flow cytometric analysis. Figure 3A depicts representative flow cytometric plots. Figure 3B depicts summary data represented as the percentage (%) of CAR positive cells for each of the analyzed CAR constructs. (n=3 independent donors; mean ± SD, Two-way analysis of variance (ANOVA), ****p<0.0001.

[0198] Figures 4A-4B depict the functional characterization of CD19-CD22-CAR T cells.Figure 4A depicts the results of coculture assay of CAR and nontransduced (NT) T-cell populations with wild-type (WT) BV173 cells. Interferon gamma (IFNy) production was measured by enzyme-linked immunosorbent assay (ELISA), n=9 (3 donors in technical triplicates), meanAttorney Docket No: 243734.000231±SD, two-way ANOVA, ****p<0.0001. Figure 4B depicts the results of a luciferase-based cytotoxicity assay performed at an effectontarget (E: T) ratio of 2:1 and 0.5:1 with the indicated targets (WT, CD19 knockout (KO), CD22KO BV173 cells and Kgla cells Neg (Ctrl), n=9 (3 donors in technical triplicates), mean ±SEM, Two-way ANOVA, ****p<0.0001 vs NT for antigenpositive targets (WT, CD19KO, CD22KO) except for 22-19-8TM T cells (0.0043).

[0199] Figures 5A-5D depict that CD19-CD22-CAR T cells have antitumor activity in vivo.Figure 5A depicts a scheme of animal experiment. NGS mice received an admixture of CD19KO and CD22KO BV173 cells and on day 7 a single dose of IxlO6CAR or NT T cells intravenously (i.v.) Figure 5B depicts the quantitative bioluminescence data (n=3 donors, 5 mice per group per donor for NT, 19-22-FL, 22-19-18TM; n=2 donors, 5 mice per group per donor for 22-19-FL, 22-19-8TM). Figure 5C depicts statistical analysis (Two-way ANOVA). The quantitative bioluminescence data until day 14 (Week 1-2), day 15 to 28 (Week 3-4), and day 29 to 42 (Week 5-6) were combined. Figure 5D depicts results of the statistical analysis for week 5-6, NS = no statistically significant difference.

[0200] Figures 6A-6D depict that 19-22-FL and 22-19-28 TM T cells have antitumor activity in a rechallenge experiment. Figure 6A depicts a scheme of the animal experiment. On day 11 post initial CAR T-cell injection, mice received a 2nddose of an admixture of CD19KO and CD22KO BV173-ffluc cells. Figure 6B depicts the quantitative bioluminescence data. Figure 6C depicts statistical analysis (Two-way ANOVA). The quantitative bioluminescence data until day 14 (Week 1-2), day 15 to 28 (Week 3-4), and day 29 to 42 (Week 5-7) were combined. Figure 6D depicts results of the statistical analysis for weeks 2-4 and 5-7.

[0201] Figures 7A-7D depict that CD19-CD22-CAR T cells have antitumor activity in an acute lymphoblastic leukemia (ALL) patient-derived xenograft (PDX) model. Figure 7A depicts a scheme of the animal experiment. NGS mice received PDX-B-ALL-ffluc cells and on day 7 a single dose of IxlO6CAR or NT T cells i.v. Figure 7B depicts quantitative bioluminescence data (n=2 donors, 5 mice per group). Figure 7C depicts statistical analysis (Two-way ANOVA). The quantitative bioluminescence data until day 14 (Week 1-2), day 15 to 28 (Week 3-4), and day 29 to 42 (Week 5-6) were combined. Figure 7D depicts results of the statistical analysis for weeks 3-4 and 5-6, NS = no statistically significant difference.

[0202] Figures 8A-8D depict that CD19-CD22-CAR T cells have antitumor activity in the CD22KO BV173 model. Figure 8A depicts a schematic of animal experiment. NGS mice receivedAttorney Docket No: 243734.000231CD22KO BV173-ffluc cells and on day 7 a single dose of 1x106CAR or NT T cells i.v. Figure 8B depicts the quantitative bioluminescence data (n=2 donors, 5 mice per group). Figure 8C depicts the statistical analysis (Two-way ANOVA). The quantitative bioluminescence data until day 14 (Week 1-2), day 15 to 28 (Week 3-4), and day 29 to 42 (Week 5-6) were combined. Figure 8D depicts results of the statistical analysis for weeks 3-4 and 5-6, NS = not significant.

[0203] Figures 9A-9F display a panel of successfully generated CD22-CAR lentiviral constructs. Figure 9A depicts a schematic representation of CD22-CAR lentiviral constructs, with varying hinge and transmembrane domains. Figure 9B depicts the flow cytometry-based detection of CAR T cell transduction. Figure 9C depicts the expansion after CAR T cell transduction, represented as fold change from baseline cell count. Figure 9D depicts the production of IFN-y and Figure 9E depicts the production of IL-2 measured by ELISA after 24 hours co-culture of CD22-CAR T cells and either antigen-negative (KglA) or antigen-positive (BV173) tumor cells at 2:1 effectortarget (E: T) ratio (n=3 donors). Figure 9F depicts a luciferase-based cytotoxicity assay of CD22-CAR T cells after 24 hours co-culture at 0.5:1 effectortarget (E: T) ratio (n=2 donors), ns = no statistically significant difference (p > 0.05), ** p < 0.01, **** p < 0.0001 according to 2-way ANOVA with Tukey’s multiple comparison test. Figures 9D-9F display results correspond to CAR constructs CD8, CD28, M.8, and M.28 in order of appearance.

[0204] Figures 10A-10G depict the characterization and in vitro activity of the optimized CD22-CAR T cells. Figure 10A depicts a schematic representation of optimized CD22-CAR T cells containing either CD8 or CD28 hinge / transmembrane domain and corresponding negative control ACAR constructs. Figure 10B depicts the flow cytometry-based transduction efficiency and vector copy number of optimized CD22-CAR T cells. Figure 10C depicts the expansion of CD22-CAR T cells after transduction, relative to baseline cell count. Figure 10D depicts the immunophenotype of nontransduced (NT) and CD22-CAR T cells, including CD4 / CD8 ratio and relative proportions of CCR7+ / CC45RO+central memory, CCR77CD45RO+effector memory, CCR77CD45RO" TEMRA (CD45RO-negative terminally differentiated effector memory T cells re-expressing CD45RA), and CCR7+ / CD45ROnaive T cell populations (n=5 donors, error bars indicate mean with standard deviation). Figures 10E and 10F depict cytokine production measured by IFN-y (Figure 10E) or IL-2 (Figure 10F) ELISA after 24 hours co-culture at 2:1 effectortarget ratio in the presence of CD22-negative (KglA, BV173 CD22 knockout [KO]) or CD22-positive (BV173 wild type [WT]) targets (n=3 donors). Figures 10E and 10F display resultsAttorney Docket No: 243734.000231correspond to CAR constructs ACD8, ACD28, CD8, and CD28 in order of appearance. Figure 10G depicts a luciferase-based 24 hours cytotoxicity assay at varying effectortarget (E: T) ratios in the presence of KglA or BV173 tumor cells, ns = no statistically significant difference (p > 0.05), **** p < 0.0001 according to 2-way ANOVA with Tukey’s multiple comparison test.

[0205] Figures 11A-11E show in vivo anti -tumor activity of CD22-CAR T cells. Figure 11A depicts a schematic of experimental design. NSG mice were injected intravenously (i.v.) with 3xl06luciferase-labeled BV173 tumor cells (Tumor.ffLuc). After 7 days, 3xl06T cells were injected i.v. in the following groups: nontransduced, control AC AR, CD22-CAR with CD8 hinge / transmembrane (CD22-CAR-CD8) and CD22-CAR with CD28 hinge / transmembrane (CD22-CAR-CD28) (n=10 per group). Tumors were monitored via bioluminescence imaging.Figure 11B depicts a Kaplan-Meier survival curve, displayed by assigned treatment group. Figure 11C depicts bioluminescence data quantifying tumor burden, lines represent individual animals within each treatment group. Figure 11D depicts the design of tumor rechallenge experiment. On Day 102 after initial tumor injection, surviving mice (CD22-CAR-CD8 n=4, CD22 CAR-CD28 n=7) were rechallenged with a second injection of tumor cells, and compared to a new group of untreated mice (n=5). Figure HE depicts tumor burden as assessed by bioluminescence imaging after rechallenging with no additional T cell infusions. Survival curve comparisons used log-rank test, ns = no statistically significant difference (p > 0.05), **** p < 0.0001.DETAILED DESCRIPTION

[0206] The present disclosure provides, among other aspects, chimeric antigen receptors (CARs) that target cluster of differentiation 19 (CD 19) and / or cluster of differentiation 22 (CD22). The present disclosure further provides T-cells or other immune cells expressing the anti-CD19 and / or CD22 CARs disclosed herein.

[0207] Currently, it is unclear how to best target CD 19- and / or CD22-positive malignancies. CAR-expressing cells targeting CD19 and / or CD22 of the present disclosure could potentially target a broad range of CD 19- and / or CD22-positive malignancies and non-malignant diseases that are caused by CD 19- and / or CD22-positive cells of the B-cell lineage.

[0208] CARs are primarily comprised of 1) a target binding moiety such as, but not limited to, a single-chain variable fragment (scFv) derived from a target specific monoclonal antibody, and 2) an activation domain (e.g., a lymphocyte activation domain) such as, but not limited to, the -Attorney Docket No: 243734.000231chain (zeta- chain) from the T-cell receptor CD3. These two regions are fused together via a transmembrane domain. A hinge domain is usually required to provide more flexibility and accessibility between the target binding moiety and the transmembrane domain. Upon transduction, the cell, e.g., lymphocyte, expresses the CAR on its surface, and upon contact and ligation with the target antigen, it signals through the activation domain (e.g., CD3^ chain) inducing cytotoxicity and cellular activation.

[0209] CAR constructs may also include costimulatory polypeptides to boost the CAR-induced immune response. The most commonly used costimulating molecules include CD28 and 4- IBB, which promote both T-cell proliferation and cell survival. Another example of a costimulatory domain is a MyD88 / CD40 molecule that can be used with or without the use of a separate dimerization agent. Additional CAR constructs may also include no activation domains (e.g., CD3(^, CD28, and 4-1BB), which further used to develop dual-targeting chimeric antigen receptor (CAR) systems and bicistronic vectors comprising the CAR systems.

[0210] In one aspect, the disclosure as described herein provides a bicistronic lentiviral vector that encodes a CD19- and CD22- CAR separated by a separation sequence (e.g., 2A sequence) to generate CD19-CD22-specific CAR T cells for the adoptive immunotherapy of malignant and non-malignant diseases. The design of a bicistronic viral vector as illustrated herein may comprise one of the CARs lacking an activation domain (e.g., a lymphocyte activation domain such as a CD3(^ activation domain).Definitions

[0211] The term “chimeric antigen receptor” or “CAR” as used herein is defined as an artificial cell-surface receptor that is capable of binding to an antigen expressed by undesired cells, for example, cluster of differentiation 19 (CD 19) and / or cluster of differentiation 22 (CD22). An undesired cell may include, but not limited to, a malignant cell or a cell that produces antibodies that cause autoimmune diseases. A CAR described herein may comprise an extracellular targetbinding domain, a transmembrane domain, and optionally, a cytoplasmic domain comprising an activation domain (e.g., a lymphocyte activation domain) and / or at least one costimulatory signaling domain, all in a combination that is not naturally found together on a single protein. A “dual-targeting chimeric antigen receptor (CAR) system” described herein may comprise two CARs which can bind to two antigens, for example, CD 19 and CD22. The CARs of the present disclosure can be used with lymphocytes such as T-cells and natural killer (NK) cells.Attorney Docket No: 243734.000231

[0212] The terms “T cell”, “T-cell”, and “T lymphocyte” are interchangeable and used synonymously herein. As used herein, T-cell includes thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. A T-cell can be a T helper (Th) cell, for example a T helper 1 (Thl) or a T helper 2 (Th2) cell. The T-cell can be a helper T-cell (HTL; CD4+T-cell) CD4+T-cell, a cytotoxic T-cell (CTL; CD8+T-cell), a tumor infiltrating cytotoxic T-cell (TIL; CD8+T-cell), CD4+CD8+T-cell, or any other subset of T-cells. Other illustrative populations of T-cells suitable for use in particular embodiments include naive T-cells and memory T-cells. Also included are “NKT cells,” which refer to a specialized population of T-cells that express a semi-invariant a0 T-cell receptor, but also express a variety of molecular markers that are typically associated with NK cells, such as NK1.1. NKT cells include NK 1.1+and NK1. T, as well as CD4+, CD4, CD8+, and CD8 cells. The TCR on NKT cells is unique in that it recognizes glycolipid antigens presented by the MHC I-like molecule CD Id. NKT cells can have either protective or deleterious effects due to their abilities to produce cytokines that promote either inflammation or immune tolerance. Also included are “gamma-delta T-cells (y8 T-cells),” which refer to a specialized population that to a small subset of T-cells possessing a distinct TCR on their surface, and unlike the majority of T-cells in which the TCR is composed of two glycoprotein chains designated a- and 0-TCR chains, the TCR in y8 T-cells is made up of a y-chain and a 8-chain. y8 T-cells can play a role in immunosurveillance and immunoregulation, and were found to be an important source of IL- 17 and to induce robust CD8+cytotoxic T-cell response. Also included are “regulatory T-cells” or “Tregs” which refer to T-cells that suppress an abnormal or excessive immune response and play a role in immune tolerance. Tregs cells are typically transcription factor Foxp3 -positive CD4+T-cells and can also include transcription factor Foxp3 -negative regulatory T-cells that are IL- 10-producing CD4+T-cells.

[0213] The terms “natural killer cell” and “NK cell” are interchangeable and used synonymously herein. As used herein, NK cell refers to a differentiated lymphocyte with a CD 16+, CD56+, and / or CD57+TCR- phenotype. NK cells are characterized by their ability to bind to and kill cells that fail to express “self’ MHC / HLA antigens by the activation of specific cytolytic enzymes, the ability to kill tumor cells or other diseased cells that express a ligand for NK activating receptors, and the ability to release protein molecules called cytokines that stimulate or inhibit the immune response.Attorney Docket No: 243734.000231

[0214] As used herein, the term “antigen” refers to any agent (e.g., protein, peptide, polysaccharide, glycoprotein, glycolipid, nucleic acid, portions thereof, or combinations thereof) capable of being bound by a T-cell receptor. An antigen is also able to provoke an immune response. An example of an immune response may involve, without limitation, antibody production, or the activation of specific immunologically competent cells, or both. A skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be synthesized or can be derived from a biological sample, or can be a macromolecule besides a polypeptide. Such a biological sample can include, but is not limited to, a tissue sample, a tumor sample, a cell, or a fluid with other biological components, organisms, subunits of proteins / antigens, or killed or inactivated whole cells or lysates. In some embodiments, an antigen described herein is cluster of differentiation 19 (CD 19) and / or cluster of differentiation 22 (CD22).

[0215] The term “target binding moiety” refers to a target-specific binding element that may be any ligand that binds to an antigen of interest (e.g., CD 19 and / or CD22), or a polypeptide or fragment thereof, wherein the ligand is either naturally-derived or synthetic. Examples of target binding moieties include, but are not limited to, antibodies; polypeptides derived from antibodies, such as, for example, single chain variable fragments (scFv), Fab, Fab’, F(ab’)2, and Fv fragments; polypeptides derived from T-cell receptors (TCRs), such as, for example, TCR variable domains; secreted factors (e.g., cytokines, growth factors) that can be artificially fused to signaling domains (e g., “zytokines”); and any ligand or receptor fragment (e.g., CD27, NKG2D) that binds to the antigen of interest. Combinatorial libraries can also be used to identify peptides binding with high affinity to a therapeutic target.

[0216] The terms “antibody” and “antibodies” refer to monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, single-chain Fvs (scFv), single chain antibodies, Fab fragments, F(ab’) fragments, disulfide-linked Fvs (sdFv), intrabodies, minibodies, diabodies and anti-idiotypic (anti -Id) antibodies (including, e.g., anti-Id antibodies to antigen-specific TCR), and epitope-binding fragments of any of the above. The terms “antibody” and “antibodies” also refer to covalent diabodies such as those disclosed in U. S. Pat. Appl. Pub. No. 2007 / 0004909 and Ig-DARTS such as those disclosed in U. S. Pat. Appl. Pub. No.2009 / 0060910, each of which is incorporated herein by reference in its entirety. Antibodies useful as a TCR-binding molecule include immunoglobulin molecules and immunologically activeAttorney Docket No: 243734.000231fragments of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgMl, IgM2, IgAl, and IgA2) or subclass.

[0217] The term “host cell” means any cell that contains a heterologous nucleic acid. The heterologous nucleic acid can be a vector (e.g., an expression vector). For example, a host cell can be a cell from any organism that is selected, modified, transformed, grown, used, or manipulated in any way, for the production of a substance by the cell, for example the expression by the cell of a gene, a DNA or RNA sequence, a protein, or an enzyme. The host cell may be selected based on the vector backbone and the desired result. By way of example, a plasmid or cosmid can be introduced into a prokaryote host cell for replication of several types of vectors. Bacterial cells such as, but not limited to, DH5a, JM109, KCB, SURE® Competent Cells, and SOLOP ACK Gold Cells, can be used as host cells for vector replication and / or expression. Additionally, bacterial cells such as E. coli LE392 can be used as host cells for phage viruses. Eukaryotic cells that can be used as host cells include, but are not limited to, yeast (e.g., YPH499, YPH500, and YPH501), insects and mammals. Examples of mammalian eukaryotic host cells include, but are not limited to, HeLa, NIH3T3, Jurkat, 293, COS, CHO, Saos, and PC 12 cells. In certain embodiments, the host cell is autologous. In certain embodiments, the host cell is allogenic.

[0218] Host cells of the present disclosure include T-cells and NK cells that contain DNA or RNA sequences encoding a CAR or CAR system and expressing the CAR or CAR system on the cell surface. Such host cells may be used for enhancing T-cell or NK cell activity, and treatment of tumors.

[0219] The terms “activation” or “stimulation” mean to induce a change in the biologic state by which the cells (e.g., T-cells and NK cells) express activation markers, produce cytokines, proliferate, and / or become cytotoxic to target cells. All of these changes can be produced by primary stimulatory signals. Costimulatory signals can amplify the magnitude of the primary signals and suppress cell death following initial stimulation, thereby resulting in a more durable activation state and thus a higher cytotoxic capacity. A “costimulatory signal” refers to a signal, which, in combination with a primary signal such as TCR / CD3 ligation, leads to T-cell and / or NK cell proliferation and / or upregulation or downregulation of key molecules.

[0220] The term “proliferation” refers to an increase in cell division, either symmetric or asymmetric division of cells. The term “expansion” refers to the outcome of cell division and cellAttorney Docket No: 243734.000231death.

[0221] The term “differentiation” refers to a method of decreasing the potency or proliferation of a cell or moving the cell to a more developmentally restricted state.

[0222] The terms “express” and “expression” mean allowing or causing the information in a gene or DNA sequence to become produced, for example producing a protein by activating the cellular functions involved in transcription and translation of a corresponding gene or DNA sequence. A DNA sequence is expressed in or by a cell to form an “expression product” such as a protein. The expression product itself, e.g., the resulting protein, may also be said to be “expressed” by the cell. An expression product can be characterized as intracellular, extracellular, or transmembrane.

[0223] The term “transfection” means the introduction of a “foreign” (i.e., extrinsic, or extracellular) nucleic acid into a cell using recombinant DNA technology. The term “genetic modification” means the introduction of a “foreign” (i.e., extrinsic, or extracellular) gene, DNA or RNA sequence to a host cell, so that the host cell will express the introduced gene or sequence to produce a desired substance, typically a protein or enzyme coded by the introduced gene or sequence. The introduced gene or sequence may also be called a “cloned” or “foreign” gene or sequence, and may include regulatory or control sequences operably linked to a polynucleotide encoding an expression product of interest (e.g., a CAR), such as start, stop, promoter, signal, secretion, or other sequences which can be used by a cell’s genetic machinery. The gene or sequence may include nonfunctional sequences or sequences with no known function. A host cell that receives and expresses introduced DNA or RNA has been “genetically engineered.” The DNA or RNA introduced to a host cell can come from any source, including cells of the same genus or species as the host cell, or from a different genus or species.

[0224] The term “transduction” means the introduction of a foreign nucleic acid into a cell using a viral vector.

[0225] The term “genetically modified” or “genetically engineered” refers to the addition of extra genetic material in the form of DNA or RNA into a cell.

[0226] As used herein, the term “derivative” or “variant” in the context of proteins or polypeptides (e.g., CAR constructs or domains thereof) refers to: (a) a polypeptide that has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to the polypeptide it is a derivative or variant of; (b) a polypeptide encoded by a nucleotideAttorney Docket No: 243734.000231sequence that has at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%>, 90%, 95%, 98%, or 99% sequence identity to a nucleotide sequence encoding the polypeptide it is a derivative or variant of; (c) a polypeptide that contains 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acid mutations (i.e., additions, deletions, and / or substitutions) relative to the polypeptide it is a derivative or variant of; (d) a polypeptide encoded by nucleic acids that can hybridize under high, moderate, or typical stringency hybridization conditions to nucleic acids encoding the polypeptide it is a derivative or variant of; (e) a polypeptide encoded by a nucleotide sequence that can hybridize under high, moderate, or typical stringency hybridization conditions to a nucleotide sequence encoding a fragment of the polypeptide, it is a derivative or variant of, of at least 20 contiguous amino acids, at least 30 contiguous amino acids, at least 40 contiguous amino acids, at least 50 contiguous amino acids, at least 75 contiguous amino acids, at least 100 contiguous amino acids, at least 125 contiguous amino acids, or at least 150 contiguous amino acids; or (f) a fragment of the polypeptide it is a derivative or variant of.

[0227] Percent sequence identity can be determined using any method known to one of skill in the art. In a specific embodiment, the percent identity is determined using the “Best Fit” or “Gap” program of the Sequence Analysis Software Package (Version 10; Genetics Computer Group, Inc., University of Wisconsin Biotechnology Center, Madison, Wisconsin). Information regarding hybridization conditions (e.g., high, moderate, and typical stringency conditions) have been described, see, e.g., U. S. Pat. Appl. Pub. No. US 2005 / 0048549 (e.g., paragraphs 72-73), the content of which is incorporated herein by reference in its entirety.

[0228] The terms “vector,” “cloning vector”, and “expression vector” mean the vehicle by which a DNA or RNA sequence (e.g., a foreign gene) can be introduced into a host cell, so as to genetically modify the host cell and promote expression (e.g., transcription and translation) of the introduced sequence. Vectors include plasmids, synthesized RNA and DNA molecules, phages, viruses, etc. In certain embodiments, the vector is a viral vector such as, but not limited to, a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated virus vector, an alphaviral vector, a herpes virus vector, a baculoviral vector, and a vaccinia virus vector. In some embodiments, the viral vector is a lentiviral vector.

[0229] The term “regulatory element” refers to any cis-acting genetic element that controls some aspect of the expression of nucleic acid sequences. In some embodiments, the term “promoter” comprises essentially the minimal sequences required to initiate transcription. In someAttorney Docket No: 243734.000231embodiments, the term “promoter” includes the sequences to start transcription, and in addition, also include sequences that can upregulate or downregulate transcription, commonly termed “enhancer elements” and “repressor elements,” respectively.

[0230] As used herein, the terms “operatively linked” or “operably linked”, or similar phrases, when used in reference to nucleic acids or amino acids, refer to the operational linkage of nucleic acid sequences or amino acid sequence, respectively, placed in functional relationships with each other. For example, an operatively linked promoter, enhancer element(s), open reading frame, 5’ and 3’ UTR, and terminator sequences can result in the accurate production of a nucleic acid molecule (e.g., RNA). In some embodiments, operatively linked nucleic acid elements result in the transcription of an open reading frame and, ultimately, the production of a polypeptide (i.e., expression of the open reading frame). As another example, an operatively linked peptide is one in which the functional domains are placed at appropriate distances from each other to impart the intended function of each domain.

[0231] The term “enhance” or “promote” or “increase” or “expand” or “improve” refers generally to the ability of a composition contemplated herein to produce, elicit, or cause a greater physiological response (i.e., downstream effects) compared to the response caused by either vehicle or a control molecule / composition. A measurable physiological response may include an increase in T-cell expansion, activation, effector function, persistence, and / or tumor cell killing ability, among others apparent from the understanding in the art and the description herein. In certain embodiments, an “increased” or “enhanced” amount can be a “statistically significant” amount, and may include an increase that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the response produced by vehicle or a control composition.

[0232] By “decrease” or “lower” or “lessen” or “reduce” or “abate” is generally meant the ability of a composition contemplated herein to produce, elicit, or cause a lesser physiological response (i.e., downstream effects) compared to the response caused by either vehicle or a control molecule / composition. In certain embodiments, a “decrease” or “reduced” amount can be a “statistically significant” amount, and may include a decrease that is 1.1, 1.2, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30 or more times (e.g., 500, 1000 times) (including all integers and decimal points in between and above 1, e.g., 1.5, 1.6, 1.7. 1.8, etc.) the response (reference response) produced by vehicle, a control composition, or the response in a particular cell lineage.Attorney Docket No: 243734.000231

[0233] The term “treat” or “treatment” of a state, disorder, or condition include: (1 ) preventing, delaying, or reducing the incidence and / or likelihood of the appearance of at least one clinical or sub-clinical symptom of the state, disorder, or condition developing in a subject that may be afflicted with or predisposed to the state, disorder, or condition, but does not yet experience or display clinical or subclinical symptoms of the state, disorder, or condition; or (2) inhibiting the state, disorder, or condition, i.e., arresting, reducing, or delaying the development of the disease or a relapse thereof or at least one clinical or sub-clinical symptom thereof; or (3) relieving the disease, i.e., causing regression of the state, disorder or condition or at least one of its clinical or sub-clinical symptoms. The benefit to a subject to be treated is either statistically significant or at least perceptible to the patient or to the physician.

[0234] The term “effective” applied to dose or amount refers to that quantity of a compound or pharmaceutical composition that is sufficient to result in a desired activity upon administration to a subject in need thereof. When a combination of active ingredients is administered, the effective amount of the combination may or may not include amounts of each ingredient that would have been effective if administered individually. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular drug or drugs employed, the mode or route of administration, and the like.

[0235] The phrase “pharmaceutically acceptable,” as used in connection with compositions described herein, refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to a mammal (e.g., a human). Preferably, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U. S. Pharmacopeia or other generally recognized pharmacopeia for use in mammals, and more particularly in humans.

[0236] The term “protein” as used herein encompasses all kinds of naturally occurring and synthetic proteins, including protein fragments of all lengths, fusion proteins and modified proteins including, without limitation, glycoproteins, as well as all other types of modified proteins (e.g., proteins resulting from phosphorylation, acetylation, myristoylation, palmitoylation, glycosylation, oxidation, formylation, amidation, polyglutamylation, ADP-ribosylation, pegylation, biotinylation, etc.).

[0237] The terms “nucleic acid,” “nucleotide,” and “polynucleotide” encompass both DNAAttorney Docket No: 243734.000231and RNA unless specified otherwise. By a “nucleic acid sequence” or “nucleotide sequence” is meant the nucleic acid sequence encoding an amino acid, the term may also refer to the nucleic acid sequence including the portion coding for any amino acids added as an artifact of cloning, including any amino acids coded for by linkers.

[0238] The terms “patient,” “individual,” “subject,” and “animal” are used interchangeably herein and refer to mammals including, without limitation, human and veterinary animals (e.g., cats, dogs, cows, horses, sheep, pigs, etc.) and experimental animal models. In a preferred embodiment, the subject is a human.

[0239] The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which a compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions. Alternatively, the carrier can be a solid dosage form carrier including, but not limited to, one or more of a binder (for compressed pills), a glidant, an encapsulating agent, a flavorant, and a colorant. Suitable pharmaceutical carriers are described in “Remington’s Pharmaceutical Sciences” by E. W. Martin.

[0240] Singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, a reference to “a method” includes one or more methods, and / or steps of the type described herein and / or which will become apparent to those persons skilled in the art upon reading this disclosure.

[0241] The term “about” or “approximately” includes being within a statistically meaningful range of a value. Such a range can be within an order of magnitude, preferably within 50%, more preferably within 20%, still more preferably within 10%, and even more preferably within 5% of a given value or range. The allowable variation encompassed by the term “about” or “approximately” depends on the particular system under study, and can be readily appreciated by one of ordinary skill in the art.

[0242] If aspects of the disclosure are described as “comprising” a feature, or versions there of (e.g., comprise), embodiments also are contemplated “consisting of’ or “consisting essentially of’ the feature.

[0243] The practice of the present disclosure employs, unless otherwise indicated,Attorney Docket No: 243734.000231conventional techniques of statistical analysis, molecular biology (including recombinant techniques), microbiology, cell biology, and biochemistry, which are within the skill of the art. Such tools and techniques are described in detail in e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual. 3rded. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York; Ausubel et al. eds. (2005) Current Protocols in Molecular Biology. John Wiley and Sons, Inc.: Hoboken, NJ; Bonifacino et al. eds. (2005) Current Protocols in Cell Biology. John Wiley and Sons, Inc.: Hoboken, NJ; Coligan et al. eds. (2005) Current Protocols in Immunology, John Wiley and Sons, Inc.: Hoboken, NJ; Coico et al. eds. (2005) Current Protocols in Microbiology, John Wiley and Sons, Inc.: Hoboken, NJ; Coligan et al. eds. (2005) Current Protocols in Protein Science, John Wiley and Sons, Inc.: Hoboken, NJ; and Enna et al. eds. (2005) Current Protocols in Pharmacology, John Wiley and Sons, Inc.: Hoboken, NJ. Additional techniques are explained, e g., in U. S. Patent No. 7,912,698 and U. S. Patent Appl. Pub. Nos.2011 / 0202322 and 2011 / 0307437.

[0244] The technology illustratively described herein suitably may be practiced in the absence of any element(s) not specifically disclosed herein.

[0245] The terms and expressions which have been employed are used as terms of description and not of limitation, and use of such terms and expressions do not exclude any equivalents of the features shown and described or portions thereof, and various modifications are possible within the scope of the technology claimed.Chimeric Antigen Receptors

[0246] In certain aspects, the present disclosure provides chimeric antigen receptors (CARs) that target cluster of differentiation 19 (CD19) and / or cluster of differentiation 22 (CD22).

[0247] CD 19 is a member of the immunoglobulin superfamily, primarily expressed on B lineage cells and follicular dendritic cells. CD 19 serves two primary functions in human B cells: firstly, it acts as an adaptor protein, recruiting cytoplasmic signaling proteins to the membrane; secondly, it operates within the CD19 / CD21 complex to lower the threshold for B cell receptor signaling pathways.

[0248] CD22 is a member of the sialic acid-binding immunoglobulin-like lectin (Siglec) family of proteins. This molecule is expressed at a high level on the surface of mature B cells compared to immature B cells. As an inhibitory receptor for B cell receptor (BCR) signaling, it plays a regulatory role in preventing over-activation of the immune system.Attorney Docket No: 243734.000231

[0249] Both CD19 and CD22 have been established as promising targets for the treatment of various diseases, including hematological cancers such as leukemia. However, the effectiveness of a treatment targeting only CD 19 or only CD22 may be compromised due to, for example, tumor antigen escape, leading to reduced treatment efficacy.

[0250] In certain aspects, the present invention provides a polynucleotide encoding a dualtargeting chimeric antigen receptor (CAR) system comprising two CARs, wherein each CAR comprises an extracellular domain comprising a target binding moiety; wherein optionally one of the CARs lacks an activation domain (e.g., a lymphocyte activation domain); and wherein one target binding moiety binds CD 19 and the other target binding moiety binds to CD22.

[0251] In certain aspects, the present invention provides a pair of polynucleotides encoding a dual -targeting chimeric antigen receptor (CAR) system comprising two CARs, wherein the first polynucleotide encodes a first CAR of the dual-targeting CAR system, and the second polynucleotide encodes a second CAR of the dual-targeting CAR system, and wherein each CAR comprises an extracellular domain comprising a target binding moiety; wherein optionally one of the CARs lacks an activation domain (e.g., a lymphocyte activation domain); and wherein one target binding moiety binds to CD 19 and the other target binding moiety binds to CD22.

[0252] In certain aspects, the dual-targeting CAR system comprises an anti-CD22 CAR comprising an activation domain (e.g., a lymphocyte activation domain) and an anti-CD19 CAR lacking an activation domain (e.g., a lymphocyte activation domain).

[0253] In certain aspects, the dual-targeting CAR system comprises an anti-CD19 CAR comprising an activation domain (e.g., a lymphocyte activation domain) and an anti-CD22 CAR lacking an activation domain (e.g., a lymphocyte activation domain).

[0254] In certain aspects, the dual-targeting CAR system comprises an anti-CD22 CAR comprising an activation domain (e.g., a lymphocyte activation domain) and an anti-CD19 CAR lacking an activation domain (e.g., a lymphocyte activation domain) and a costimulatory domain.

[0255] In certain aspects, the dual -targeting CAR system comprises an anti-CD19 CAR comprising an activation domain (e.g., a lymphocyte activation domain) and an anti-CD22 CAR lacking an activation domain (e.g., a lymphocyte activation domain) and a costimulatory domain.

[0256] In certain aspects, the present disclosure provides a polynucleotide encoding a CAR comprising: a) an extracellular target-binding domain comprising a target binding moiety which binds to CD22; b) a hinge domain; c) a transmembrane domain; d) a costimulatory domain; and e)Attorney Docket No: 243734.000231optionally an activation domain (e.g., a lymphocyte activation domain).

[0257] In certain aspects, the present disclosure provides a polynucleotide encoding a CAR comprising: a) an extracellular target-binding domain comprising a target binding moiety which binds to CD22; b) a hinge domain; c) a transmembrane domain; d) a costimulatory domain; and e) an activation domain (e.g., a lymphocyte activation domain).

[0258] In certain aspects, the present disclosure provides a polynucleotide encoding a CAR comprising: a) an extracellular target-binding domain comprising a target binding moiety which binds to CD22; b) a hinge domain; c) a transmembrane domain; and d) a costimulatory domain.

[0259] In certain aspects, the present disclosure provides a polynucleotide encoding a CAR comprising: a) an extracellular target-binding domain comprising a target binding moiety which binds to CD 19; b) a hinge domain; c) a transmembrane domain; d) a costimulatory domain; and e) optionally an activation domain (e.g., a lymphocyte activation domain).

[0260] In certain aspects, the present disclosure provides a polynucleotide encoding a CAR comprising: a) an extracellular target-binding domain comprising a target binding moiety which binds to CD 19; b) a hinge domain; c) a transmembrane domain; and d) a costimulatory domain.

[0261] In certain aspects, the present disclosure provides a polynucleotide encoding a CAR comprising: a) an extracellular target-binding domain comprising a target binding moiety which binds to CD 19; b) a hinge domain; c) a transmembrane domain; d) a costimulatory domain; and e) an activation domain (e.g., a lymphocyte activation domain).

[0262] In certain aspects, the present disclosure provides a polynucleotide encoding a CAR comprising: a) an extracellular target-binding domain comprising a target binding moiety which binds to CD22; b) a hinge domain; c) a transmembrane domain; d) optionally a costimulatory domain; and e) optionally an activation domain (e.g., a lymphocyte activation domain).

[0263] In certain aspects, the present disclosure provides a polynucleotide encoding a CAR comprising: a) an extracellular target-binding domain comprising a target binding moiety which binds to CD22; b) a hinge domain; and c) a transmembrane domain.

[0264] In certain aspects, the present disclosure provides a polynucleotide encoding a CAR comprising: a) an extracellular target-binding domain comprising a target binding moiety which binds to CD 19; b) a hinge domain; c) a transmembrane domain; d) optionally a costimulatory domain; and e) optionally an activation domain (e.g., a lymphocyte activation domain).

[0265] In certain aspects, the present disclosure provides a polynucleotide encoding a CARAttorney Docket No: 243734.000231comprising: a) an extracellular target-binding domain comprising a target binding moiety which binds to CD 19; b) a hinge domain; and c) a transmembrane domain.

[0266] In certain aspects, the present disclosure provides a polynucleotide encoding a chimeric antigen receptor (CAR) comprising an extracellular domain comprising a target binding moiety, wherein the CAR lacks an activation domain (e.g., a lymphocyte activation domain); and wherein the target binding moiety binds to CD22.

[0267] In certain aspects, the present disclosure provides a polynucleotide encoding a chimeric antigen receptor (CAR) comprising an extracellular domain comprising a target binding moiety, wherein the CAR lacks an activation domain (e.g., a lymphocyte activation domain); and wherein the target binding moiety binds to CD 19.

[0268] In various embodiments, the polynucleotide encoding a CAR or a pair of polynucleotides encoding a dual-targeting CAR system comprises a DNA molecule. In various embodiments, the polynucleotide encoding a CAR or a pair of polynucleotides encoding a dual-targeting CAR system comprises an RNA molecule.

[0269] In one aspect, the present disclosure provides CAR polypeptides encoded by a polynucleotide or a pair of polynucleotides described above.Extracellular Target-Binding Domain

[0270] In certain aspects, CARs of the present disclosure comprise an extracellular targetbinding domain, wherein the extracellular target-binding domain comprises a target binding moiety.

[0271] The choice of target binding moiety depends upon the type and number of antigens that define the surface of a target cell. For example, the target binding moiety may be chosen to recognize an antigen that acts as a cell surface marker on target cells associated with a particular disease state. In certain embodiments, the CARs of the present disclosure can be genetically modified to target a tumor antigen of interest by way of engineering a desired target binding moiety that specifically binds to an antigen (e.g., on a tumor cell).

[0272] In certain embodiments, the antigen that is targeted by the extracellular target-binding domain is cluster of differentiation 19 (CD 19) and / or cluster of differentiation 22 (CD22).

[0273] In certain embodiments, the target binding moiety can be monomeric or multimeric (e.g., homodimeric or heterodimeric), or associated with multiple proteins in a non-covalent complex. In some embodiments, the target binding moiety comprises an target binding peptide,Attorney Docket No: 243734.000231polypeptide, or functional variant thereof, that binds to an antigen. In some embodiments, the target binding polypeptide is an antibody or an antibody fragment that binds to an antigen. Target binding moieties may comprise antibodies and / or antibody fragments such as monoclonal antibodies, multispecific antibodies, chimeric antibodies, single-chain Fvs (scFvs), single chain antibodies, Fab fragments, F(ab’) fragments, disulfide-linked Fvs (dsFvs), intrabodies, minibodies, single domain antibody variable domains, nanobodies (VHHs), diabodies, and anti -idiotypic (anti-id) antibodies (including, e.g., anti-Id antibodies to target specific TCR), and epitope-binding fragments of any of the above. Antibodies and / or antibody fragments may be derived from murine antibodies, rabbit antibodies, human antibodies, fully humanized antibodies, camelid antibody variable domains and humanized versions, shark antibody variable domains and humanized versions, and camelized antibody variable domains.

[0274] In some embodiments, the target binding moiety is a single-chain Fv (scFv). In some embodiments, an scFv described herein may comprise from N-terminus to C-terminus: VH - linker - VL. In some embodiments, an scFv described herein may comprise from N-terminus to C-terminus: VL - linker - VH.

[0275] In some embodiments, the scFv comprises a linker between the VH and VL. Nonlimiting examples of the linker sequences that may be used in the scFvs described herein include GGGGSGGGGSGGGGS ((G4S)3; SEQ ID NO: 19), GGGGS (SEQ ID NO: 91), (G4S)2 (SEQ ID NO: 92), (G4S)4 (SEQ ID NO: 93), KESGSVSSEQLAQFRSLD (SEQ ID NO: 94), EGKSSGSGSESKST (SEQ ID NO: 95), EGKSSGSGSESKSTQ (SEQ ID NO: 96), GSTSGSGKSSEGKG (SEQ ID NO: 97), SSADDAKKDDAKKDDAKKDDAKKDG (SEQ ID NO: 98), EGKSSGSGSESKVD (SEQ ID NO: 99), and ESGSVSSEELAFRSLD (SEQ ID NO: 100), or functional variants thereof. Additional linkers include those described in, e.g., Whitlow and Filpula, Methods, Volume 2, Issue 2, April 1991, Pages 97-105, the content of which is incorporated herein by reference in its entirety.

[0276] In some embodiments, the linker sequence comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 19), or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 19. In certain embodiments, the nucleotide sequence that encodes the linker sequence comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 19, or aAttorney Docket No: 243734.000231variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 19. In certain embodiments, the nucleotide sequence that encodes the linker sequence comprises the nucleotide sequence set forth in SEQ ID NO: 3, 11, 32, or 36, or a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 3, 11, 32, or 36. In certain embodiments, the linker sequence comprises the amino acid sequence set forth in SEQ ID NO: 19. In certain embodiments, the nucleotide sequence that encodes the linker sequence comprises the nucleotide sequence set forth in SEQ ID NO: 3, 11, 32, or 36.

[0277] In certain embodiments, the target binding moiety comprises a polypeptide or functional variant thereof that binds to CD 19 and / or CD22. In certain embodiments, the target binding moiety is an antibody or an antibody fragment that binds to CD 19 and / or CD22. In certain embodiments, the target binding moiety is a single chain variable fragment (scFv) that binds to CD19 and / or CD22 (anti-CD19 and / or CD22 scFv). In some embodiments, the anti-CD19 and / or CD22 scFv is derived from an mAb specific for the cluster of differentiation 19 (CD19) and / or cluster of differentiation 22 (CD22).

[0278] In some embodiments, the anti-CD19 scFv is derived from a CD 19 specific mAb FMC63, HD37, CAT19, budoprutug, coltuximab, denintuzumab, duvortuxizumab, emfizatamab, englumafusp alfa, inebilizumab, loncastuximab, obexelimab, tafasitamab, taplitumomab, or zeripatamig, or an antibody that competes for binding human CD 19 and / or mouse CD 19 with such an antibody; or a functional variant thereof. In some embodiments, the anti-CD19 scFv is derived from a CD 19 specific mAb FMC63.

[0279] In some embodiments, the anti-CD19 scFv comprises within the heavy chain variable region (VH) the following complementarity determining regions (CDRs): a heavy chain CDR1 (HCDR1) comprising the amino acid sequence DYGVS set forth in SEQ ID NO: 127; a heavy chain CDR2 (HCDR2) comprising the amino acid sequence VIWGSETTYYNSALKS set forth in SEQ ID NO: 128; and a heavy chain CDR3 (HCDR3) comprising the amino acid sequence HYYYGGSYAMDY set forth in SEQ ID NO: 129.

[0280] In some embodiments, the anti-CD19 scFv comprises within the light chain variable region (VL) the following complementarity determining regions (CDRs): a light chain CDR1Attorney Docket No: 243734.000231(LCDR1) comprising the amino acid sequence RASQDISKYLN set forth in SEQ ID NO: 130; a light chain CDR2 (LCDR2) comprising the amino acid sequence HTSRLHS set forth in SEQ ID NO: 131; and a light chain CDR3 (LCDR3) comprising the amino acid sequence QQGNTLPYT set forth in SEQ ID NO: 132.

[0281] In some embodiments, the anti-CD19 scFv comprises within the heavy chain variable region (VH) the following complementarity determining regions (CDRs): a heavy chain CDR1 (HCDR1) comprising the amino acid sequence DYGVS set forth in SEQ ID NO: 127; a heavy chain CDR2 (HCDR2) comprising the amino acid sequence VIWGSETTYYNSALKS set forth in SEQ ID NO: 128; and a heavy chain CDR3 (HCDR3) comprising the amino acid sequence HYYYGGSYAMDY set forth in SEQ ID NO: 129; and comprises within the light chain variable region (VL) the following complementarity determining regions (CDRs): a light chain CDR1 (LCDR1) comprising the amino acid sequence RASQDISKYLN set forth in SEQ ID NO: 130; a light chain CDR2 (LCDR2) comprising the amino acid sequence HTSRLHS set forth in SEQ ID NO: 131; and a light chain CDR3 (LCDR3) comprising the amino acid sequence QQGNTLPYT set forth in in SEQ ID NO: 132.

[0282] In some embodiments, the anti-CD19 scFv comprises a heavy chain variable domain (VH) comprising the amino acid sequence set forth in SEQ ID NO: 20, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 20. In certain embodiments, the nucleotide sequence that encodes the VH of the anti-CD19 scFv comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 20, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 20. In certain embodiments, the nucleotide sequence that encodes the VH of the anti-CD19 scFv comprises the nucleotide sequence set forth in SEQ ID NO: 4, or a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 4. In certain embodiments, the VH of the anti-CD19 scFv comprises the amino acid sequence set forth in SEQ ID NO: 20. In certain embodiments, the nucleotideAttorney Docket No: 243734.000231sequence that encodes the VH of the anti-CD19 scFv comprises the nucleotide sequence set forth in SEQ ID NO: 4.

[0283] In some embodiments, the anti-CD19 scFv comprises a light chain variable domain (VL) comprising the amino acid sequence set forth in SEQ ID NO: 18, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 18. In certain embodiments, the nucleotide sequence that encodes the VL of the anti-CD19 scFv comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 18, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 18. In certain embodiments, the nucleotide sequence that encodes the VL of the anti-CD19 scFv comprises the nucleotide sequence set forth in SEQ ID NO: 2, or a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 2. In certain embodiments, the VL of the anti-CD19 scFv comprises the amino acid sequence set forth in SEQ ID NO: 18. In certain embodiments, the nucleotide sequence that encodes the VL of the anti-CD19 scFv comprises the nucleotide sequence set forth in SEQ ID NO: 2.

[0284] In some embodiments, the anti-CD19 scFv comprises the amino acid sequence set forth in SEQ ID NO: 61, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 61. In certain embodiments, the nucleotide sequence that encodes the anti-CD19 scFv comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 61, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 61. In certain embodiments, the nucleotide sequence that encodes the anti-CD19 scFv comprises the nucleotide sequence set forth in SEQ ID NO: 62 or 65, or a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at leastAttorney Docket No: 243734.00023198%, or at least 99% sequence identity with SEQ ID NO: 62 or 65. In certain embodiments, the anti-CD19 scFv comprises the amino acid sequence set forth in SEQ ID NO: 61. In certain embodiments, the nucleotide sequence that encodes the anti-CD19 scFv comprises the nucleotide sequence set forth in SEQ ID NO: 62 or 65.

[0285] In some embodiments, the anti-CD19 scFv comprises the amino acid sequence set forth in SEQ ID NO: 133, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 133. In certain embodiments, the nucleotide sequence that encodes the anti-CD19 scFv comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 133, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 133. In certain embodiments, the nucleotide sequence that encodes the anti-CD19 scFv comprises the nucleotide sequence set forth in SEQ ID NO: 134 or 135, or a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 134 or 135. In certain embodiments, the anti-CD19 scFv comprises the amino acid sequence set forth in SEQ ID NO: 133. In certain embodiments, the nucleotide sequence that encodes the anti-CD19 scFv comprises the nucleotide sequence set forth in SEQ ID NO: 134 or 135.

[0286] In some embodiments, the anti-CD22 scFv is derived from a CD22 specific mAb m971, m972, LT22, 16P, or an antibody that competes for binding human CD22 and / or mouse CD22 with such an antibody; or a functional variant thereof. In some embodiments, the anti-CD22 scFv is derived from a CD22 specific mAb m971. Other non-limiting examples of anti-CD22 moi eties such as anti-CD22 antibodies are described in U. S. Pat. Nos US 8,591,889 and US 10,543,263, which are incorporated herein by reference in their entirety.

[0287] In some embodiments, anti-CD22 scFv comprises within the heavy chain variable region (VH) the following complementarity determining regions (CDRs): a heavy chain CDR1 (HCDR1) comprising the amino acid sequence GDSVSSNSAA set forth in SEQ ID NO: 117, a heavy chain CDR2 (HCDR2) comprising the amino acid sequence TYYRSKWYN set forth inAttorney Docket No: 243734.000231SEQ ID NO: 118, and a heavy chain CDR3 (HCDR3) comprising the amino acid sequence AREVTGDLEDAFDI set forth in SEQ ID NO: 119.

[0288] In some embodiments, anti-CD22 scFv comprises within the light chain variable region (VL) the following complementarity determining regions (CDRs): a light chain CDR1 (LCDR1) comprising the amino acid sequence QTIWS set forth in SEQ ID NO: 120; a light chain CDR2 (LCDR2) comprising the amino acid sequence AAS; and a light chain CDR3 (LCDR3) comprising the amino acid sequence QQSYSIPQT set forth in SEQ ID NO: 122.

[0289] In some embodiments, anti-CD22 scFv comprises within the heavy chain variable region (VH) the following complementarity determining regions (CDRs): a heavy chain CDR1 (HCDR1) comprising the amino acid sequence GDSVSSNSAA set forth in SEQ ID NO: 117, a heavy chain CDR2 (HCDR2) comprising the amino acid sequence TYYRSKWYN set forth in SEQ ID NO: 118, and a heavy chain CDR3 (HCDR3) comprising the amino acid sequence AREVTGDLEDAFDI set forth in SEQ ID NO: 119; and comprises within the light chain variable region (VL) the following complementarity determining regions (CDRs): a light chain CDR1 (LCDR1) comprising the amino acid sequence QTIWS set forth in SEQ ID NO: 120; a light chain CDR2 (LCDR2) comprising the amino acid sequence AAS; and a light chain CDR3 (LCDR3) comprising the amino acid sequence QQSYSIPQT set forth in SEQ ID NO: 122.

[0290] In some embodiments, anti-CD22 scFv comprises a heavy chain variable domain (VH) comprising the amino acid sequence set forth in SEQ ID NO: 26, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 26. In certain embodiments, the nucleotide sequence that encodes the VH of anti-CD22 scFv comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 26, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 26. In certain embodiments, the nucleotide sequence that encodes the VH of the anti-CD22 scFv comprises the nucleotide sequence set forth in SEQ ID NO: 10 or 35, or a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 10 or 35. In certain embodiments, the VH of anti-CD22 scFv comprises theAttorney Docket No: 243734.000231amino acid sequence set forth in SEQ ID NO: 26. In certain embodiments, the nucleotide sequence that encodes the VH of the anti-CD19 scFv comprises the nucleotide sequence set forth in SEQ ID NO: 10 or 35.

[0291] In some embodiments, anti-CD22 scFv comprises a light chain variable domain (VL) comprising the amino acid sequence set forth in SEQ ID NO: 27, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 27. In certain embodiments, the nucleotide sequence that encodes the VL of anti-CD22 scFv comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 27, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 27. In certain embodiments, the nucleotide sequence that encodes the VL of the anti-CD22 scFv comprises the nucleotide sequence set forth in SEQ ID NO: 12 or 37, or a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 12 or 37. In certain embodiments, the VL of anti-CD22 scFv comprises the amino acid sequence set forth in SEQ ID NO: 27. In certain embodiments, the nucleotide sequence that encodes the VL of the anti-CD22 scFv comprises the nucleotide sequence set forth in SEQ ID NO: 12 or 37.

[0292] In some embodiments, the anti-CD22 scFv comprises the amino acid sequence set forth in SEQ ID NO: 63, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 63. In certain embodiments, the nucleotide sequence that encodes the anti-CD22 scFv comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 63, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 63. In certain embodiments, the nucleotide sequence that encodes the anti-CD22 scFv comprises the nucleotide sequence set forth in SEQ ID NO: 64 or 66, or a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, atAttorney Docket No: 243734.000231least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 64 or 66. In certain embodiments, the anti-CD22 scFv comprises the amino acid sequence set forth in SEQ ID NO: 63. In certain embodiments, the nucleotide sequence that encodes the anti-CD22scFv comprises the nucleotide sequence set forth in SEQ ID NO: 64 or 66.

[0293] In some embodiments, the anti-CD22 scFv comprises the amino acid sequence set forth in SEQ ID NO: 136, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 136. In certain embodiments, the nucleotide sequence that encodes the anti-CD22 scFv comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 136, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 136. In certain embodiments, the nucleotide sequence that encodes the anti-CD22 scFv comprises the nucleotide sequence set forth in SEQ ID NO: 137 or 138, or a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 137 or 138. In certain embodiments, the anti-CD22 scFv comprises the amino acid sequence set forth in SEQ ID NO: 136. In certain embodiments, the nucleotide sequence that encodes the anti-CD22 scFv comprises the nucleotide sequence set forth in SEQ ID NO: 137 or 138.Leader Sequence

[0294] In certain aspects, the CAR of the present disclosure comprises a leader sequence. The leader sequence may be positioned amino-terminal to the extracellular target-binding domain. The leader sequence may be optionally cleaved from the target binding moiety during cellular processing and localization of the CAR to the cellular membrane.

[0295] In some embodiments, the leader sequence may be derived from human CD8a. In some embodiments, the leader sequence comprises the amino acid sequence set forth in SEQ ID NO: 17 or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 17. In certain embodiments, theAttorney Docket No: 243734.000231nucleotide sequence encoding the leader sequence comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 17, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 17. In certain embodiments, the nucleotide sequence encoding the leader sequence comprises the sequence set forth in SEQ ID NO: 1 or 31, or a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity with SEQ ID NO: 1 or 31. In certain embodiments, the leader sequence comprises the amino acid sequence of SEQ ID NO: 17. In certain embodiments, the nucleotide sequence encoding the leader sequence comprises the nucleotide sequence set forth in SEQ ID NO: 1 or 31.

[0296] In some embodiments, the leader sequence may be derived from human immunoglobulin heavy chain variable region. In some embodiments, the leader sequence comprises the amino acid sequence set forth in SEQ ID NO: 57 or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 57. In certain embodiments, the nucleotide sequence encoding the leader sequence comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 57, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 57. In certain embodiments, the nucleotide sequence encoding the leader sequence comprises the sequence set forth in SEQ ID NO: 51, or a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 51. In certain embodiments, the leader sequence comprises the amino acid sequence of SEQ ID NO: 57. In certain embodiments, the nucleotide sequence encoding the leader sequence comprises the nucleotide sequence set forth in SEQ ID NO: 51.Hinge DomainAttorney Docket No: 243734.000231

[0297] In certain embodiments, CARs described herein further comprise a hinge domain between the extracellular target binding domain and the transmembrane domain, wherein the target binding moiety, hinge domain, and the transmembrane domain are in frame with each other.

[0298] A hinge domain can comprise any oligo- or polypeptide that functions to link the target binding moiety to the transmembrane domain. A hinge domain can be used to provide more flexibility and accessibility for the target binding moiety. A hinge domain may comprise up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids. A hinge domain may be derived from all or part of naturally occurring molecules, such as from all or part of the extracellular region of CD8a stalk, CD4, or CD28, or from all or part of an antibody constant region. Alternatively, the hinge domain may be a synthetic sequence that corresponds to a naturally occurring linker region sequence, or may be an entirely synthetic linker region sequence. Nonlimiting examples of hinge domains which may be used in accordance with the disclosure include a part of human CD8a chain, partial extracellular domain of CD28, FcyRIIIa receptor, an Ig hinge such as IgG, IgM, IgA, IgD, and IgE,, or functional fragment thereof. In some embodiments, additional linking amino acids are added to the hinge region to ensure that the target binding moiety is an optimal distance from the transmembrane domain. In some embodiments, when the hinge domain is derived from an Ig, the hinge may be mutated to prevent Fc receptor binding.

[0299] In some embodiments, the hinge domain may be derived from CD8a, CD28, or an immunoglobulin (IgG). For example, the IgG hinge may be from IgGl, IgG2, IgG3, IgG4, IgMl, IgM2, IgAl, IgA2, IgD, IgE, or a chimera thereof. In some embodiments, the hinge domain may be derived from CD8a stalk, CD28, or IgG4.

[0300] In some embodiments, each CAR of the dual-targeting CAR system of the present disclosure comprises a hinge domain independently derived from CD8a stalk, CD28, IgGl, IgG2, IgG3, or IgG4. In some embodiments, the hinge domains of the dual-targeting CAR system of the present disclosure are the same or different.

[0301] In some embodiments, a CAR of the dual-targeting CAR system of the present disclosure comprises a hinge domain derived from CD8a stalk or CD28. In some embodiments, a CAR of the dual -targeting CAR system of the present disclosure comprises a hinge domain derived from CD8a stalk. In some embodiments, a CAR of the dual-targeting CAR system of the present disclosure comprises a hinge domain derived from CD28. In some embodiments, both CARs of the dual-targeting CAR system of the present disclosure comprise a hinge domain derived fromAttorney Docket No: 243734.000231CD8a stalk. In some embodiments, both CARs of the dual -targeting CAR system of the present disclosure comprise a hinge domain derived from CD28.

[0302] In certain embodiments, the hinge domain comprises an immunoglobulin IgG hinge or functional fragment thereof. In certain embodiments, the IgG hinge is from IgGl, IgG2, IgG3, IgG4, IgMl, IgM2, IgAl, IgA2, IgD, IgE, or a chimera thereof. In certain embodiments, the hinge domain comprises the CHI, CH2, CH3, and / or hinge region of the immunoglobulin. In certain embodiments, the hinge domain comprises the core hinge region of the immunoglobulin. The term “core hinge” can be used interchangeably with the term “short hinge” (a.k.a. “SH”). Non-limiting examples of suitable hinge domains are the core immunoglobulin hinge regions listed in Table 1 (see also Wypych et al., JBC 2008 283(23): 16194-16205, which is incorporated herein by reference in its entirety for all purposes). In certain embodiments, the hinge domain is a fragment of the immunoglobulin hinge.Table 1. Amino Acid Sequence of Short Hinge Regions of IgG ImmunoglobulinsIgG Subtype Short Hinge Sequence SEQ ID NO IgGl EPKSCDKTHTCPPCP SEQ ID NO 101 IgGl DLEPKSCDKTHTCPPCPDPK SEQ ID NO 102 IgG2 ERKCCVECPPCP SEQ ID NO 103 IgG3 ELKTPLGDTTHTCPRCP(EPKSCDTPPPCPRCP)3SEQ ID NO 104IgG4 ESKYGPPCPSCP SEQ ID NO 105

[0303] In certain embodiments, the hinge domain comprises an IgG4 hinge, or a variant thereof. In certain embodiments, the hinge domain comprises the short hinge structure of IgGl, IgG2, IgG3, or IgG4 or a variant thereof.

[0304] In certain embodiments, hinge domain comprises a short hinge region and comprises the amino acid sequence set forth in SEQ ID NO: 101, 102, 103, 104, or 105, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%>, at least 85%, at least 90%>, at least 95%, at least 96%>, at least 97%, at least 98%o, or at least 99%o sequence identity with SEQ ID NO: 101, 102, 103, 104, or 105. In certain embodiments, the nucleotide sequence encoding the hinge comprising the short hinge region comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 101, 102, 103, 104, or 105, or a variant thereof having at least 50%>, at least 55%>, at least 60%>, at least 65%>, at least 70%, at least 75%, at least 80%>, at least 85%, at least 90%>, at least 95%, at least 96%>, at least 97%, at least 98%o, or at least 99% sequence identity with SEQ ID NO: 101, 102, 103, 104, or 105. InAttorney Docket No: 243734.000231certain embodiments, hinge domain comprises a short hinge region and comprises the amino acid sequence set forth in SEQ ID NO: 101, 102, 103, 104, or 105.

[0305] In some embodiments, the hinge domain is derived from CD8a stalk or complete or partial sequences of the CD8a stalk, which are also called CD8a hinge. In some embodiments, the hinge domain derived from CD8a stalk comprises the amino acid sequence set forth in SEQ ID NO: 21, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 21. In certain embodiments, the nucleotide sequence that encodes the CD8a stalk hinge domain comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 21, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 21. In certain embodiments, the nucleotide sequence that encodes the CD8a stalk hinge domain comprises the nucleotide sequence set forth in SEQ ID NO: 5 or 38, or a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 5 or 38. In certain embodiments, the CD8a stalk hinge domain comprises the amino acid sequence set forth in SEQ ID NO: 21. In certain embodiments, the nucleotide sequence that encodes the CD8a stalk hinge domain comprises the nucleotide sequence set forth in SEQ ID NO: 5 or 38.

[0306] In some embodiments, the hinge domain is derived from CD28. In some embodiments, the hinge domain derived from CD28 hinge domain comprises the amino acid sequence set forth in SEQ ID NO: 28, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%. or at least 99% sequence identity with SEQ ID NO: 28. In certain embodiments, the nucleotide sequence that encodes the CD28 hinge domain comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 28, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 28. In certain embodiments, the nucleotide sequence that encodes the CD28 hinge domain comprises the nucleotide sequence set forth in SEQ ID NO: 13,Attorney Docket No: 243734.00023133, or 42, or a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 13, 33, or 42. In certain embodiments, the CD28 hinge domain comprises the amino acid sequence set forth in SEQ ID NO: 28. In certain embodiments, the nucleotide sequence that encodes the CD28 hinge domain comprises the nucleotide sequence set forth in SEQ ID NO: 13, 33, or 42.

[0307] In some embodiments, the hinge domain is derived from IgG4. In some embodiments, the hinge domain derived from IgG4 hinge domain comprises the amino acid sequence set forth in SEQ ID NO: 60, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 60. In certain embodiments, the nucleotide sequence that encodes the IgG4 hinge domain comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 60, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 60. In certain embodiments, the nucleotide sequence that encodes the CD28 hinge domain comprises the nucleotide sequence set forth in SEQ ID NO: 54, or a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 54. In certain embodiments, the CD28 hinge domain comprises the amino acid sequence set forth in SEQ ID NO: 60. In certain embodiments, the nucleotide sequence that encodes the CD28 hinge domain comprises the nucleotide sequence set forth in SEQ ID NO: 54.

[0308] In some embodiments, in addition to the sequences described above, the hinge domain can comprise additional linker amino acids to allow for extra flexibility and / or accessibility. Transmembrane Domain

[0309] In certain aspects, the CARs of the present disclosure comprise a transmembrane domain, fused in frame between the extracellular target-binding domain and the cytoplasmic domain.

[0310] The transmembrane domain may be derived from the protein contributing to the extracellular target-binding domain, the protein contributing the signaling or co-signaling domain,Attorney Docket No: 243734.000231or by a totally different protein. In some instances, the transmembrane domain can be selected or modified by amino acid substitution, deletions, or insertions to minimize interactions with other members of the CAR complex. In some instances, the transmembrane domain can be selected or modified by amino acid substitution, deletions, or insertions to avoid-binding of proteins naturally associated with the transmembrane domain. In certain embodiments, the transmembrane domain includes additional amino acids to allow for flexibility and / or optimal distance between the domains connected to the transmembrane domain.

[0311] The transmembrane domain may be derived either from a natural or from a synthetic source. Where the source is natural, the domain may be derived from any membrane-bound or transmembrane protein. Non-limiting examples of transmembrane domains of particular use in this disclosure may be derived from (i.e., comprise at least the transmembrane region(s) of the alpha (α), beta (β), or zeta (ζ) chain of the T-cell receptor, CD28, CD3 epsilon (CD3s), CD45, CD4, CD5, CD8, CD8a, CD9, CD16, CD22, CD33, CD37, CD40, CD64, CD80, CD86, CD134 (0X40), CD 137, or CD 154. Alternatively, the transmembrane domain may be synthetic, in which case it will comprise predominantly hydrophobic residues such as leucine and valine. For example, a triplet of phenylalanine, tryptophan, and / or valine can be found at each end of a synthetic transmembrane domain.

[0312] In certain embodiments, it will be desirable to utilize a transmembrane domain which contains a cysteine residue capable of forming disulfide bonds, so that the resulting chimeric antigen receptor will be able to form disulfide-linked dimers with a neighboring chimeric antigen receptor.

[0313] In certain embodiments, a CAR of the dual -targeting CAR system of the present disclosure comprises a transmembrane domain derived from CD8a or CD28 which contains a cysteine residue capable of forming disulfide bonds. This enables the resulting chimeric protein to form disulfide-linked heterodimers with a neighboring CAR of the dual-targeting CAR system target a different antigen, or disulfide-linked homodimers with a neighboring CAR of the dualtargeting CAR system target the same antigen.

[0314] In certain embodiments, it will be desirable to utilize the transmembrane domain of the eta (η), or FcεRIγ chains which contain a cysteine residue capable of disulfide bonding, so that the resulting chimeric protein will be able to form disulfide linked dimers with itself, or with unmodified versions of theζ, η, or FcεRIγ chains or related proteins. In some instances, theAttorney Docket No: 243734.000231transmembrane domain will be selected or modified by amino acid substitution to avoid-binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex. In other cases, it will be desirable to employ the transmembrane domain of ζ, η, or FcεRIγ and -β, MB1 (Igα.), B29, or CD3- γ, ζ, or η, in order to retain physical association with other members of the receptor complex.

[0315] In some embodiments, each CAR of the dual-targeting CAR system of the present disclosure comprises a transmembrane domain independently derived from α, β or ζ chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD8a, CD9, CD16, CD22, CD33, CD37, CD40, CD64, CD80, CD86, CD134 (0X40), CD137, or CD154.

[0316] In some embodiments, the transmembrane domains of the dual-targeting CAR system of the present disclosure are the same or different.

[0317] In some embodiments, a CAR of the dual-targeting CAR system of the present disclosure comprises a transmembrane domain derived from CD8a or CD28. In some embodiments, a CAR of the dual-targeting CAR system of the present disclosure comprises a transmembrane domain derived from CD8a. In some embodiments, a CAR of the dual-targeting CAR system of the present disclosure comprises a transmembrane domain derived from CD28. In some embodiments, both CARs of the dual-targeting CAR system of the present disclosure comprise a transmembrane domain derived from CD8a. In some embodiments, both CARs of the dual-targeting CAR system of the present disclosure comprise a transmembrane domain derived from CD28.

[0318] In some embodiments, a CAR of the dual-targeting CAR system of the present disclosure comprises a transmembrane domain derived from CD8a or CD28 that facilitates heterodimerization through disulfide formation with the transmembrane domain of the other CAR of the dual-targeting CAR system.

[0319] In certain embodiments, the transmembrane domain in the CAR of the disclosure is derived from the CD28 transmembrane domain. In certain embodiments, the CD28 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 19, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 19. In certain embodiments, the nucleotide sequence that encodes the CD28 transmembrane domain comprises the nucleotide sequence that encodes theAttorney Docket No: 243734.000231amino acid sequence of SEQ ID NO: 29, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 29. In certain embodiments, the nucleotide sequence that encodes the CD28 transmembrane domain comprises the nucleotide sequence set forth in SEQ ID NO: 14, 34, or 43, or a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 14, 34, or 43. In certain embodiments, the CD28 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 29. In certain embodiments, the nucleotide sequence that encodes the CD28 transmembrane domain comprises the nucleotide sequence set forth in SEQ ID NO: 14, 34, or 43.

[0320] In certain embodiments, the CD28 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 48, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 48. In certain embodiments, the nucleotide sequence that encodes the CD28 transmembrane domain comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 48, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 48. In certain embodiments, the nucleotide sequence that encodes the CD28 transmembrane domain comprises the nucleotide sequence set forth in SEQ ID NO: 47, or a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 47. In certain embodiments, the CD28 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 48. In certain embodiments, the nucleotide sequence that encodes the CD28 transmembrane domain comprises the nucleotide sequence set forth in SEQ ID NO: 47.

[0321] In certain embodiments, the transmembrane domain in the CAR of the disclosure is derived from the CD8a transmembrane domain. In certain embodiments, the CD8a transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 22, or aAttorney Docket No: 243734.000231variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 22. In certain embodiments, the nucleotide sequence that encodes the CD8a transmembrane domain comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 22, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 22. In certain embodiments, the nucleotide sequence that encodes the CD8a transmembrane domain comprises the nucleotide sequence set forth in SEQ ID NO: 6 or 39, or a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 6 or 39. In certain embodiments, the CD8a transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 22. In certain embodiments, the nucleotide sequence that encodes the CD8a transmembrane domain comprises the nucleotide sequence set forth in SEQ ID NO: 6 or 39.

[0322] In certain embodiments, the CD8α transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 46, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 46. In certain embodiments, the nucleotide sequence that encodes the CD8a transmembrane domain comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 46, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 46. In certain embodiments, the nucleotide sequence that encodes the CD8a transmembrane domain comprises the nucleotide sequence set forth in SEQ ID NO: 45, or a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 45. In certain embodiments, the CD8a transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 46. In certain embodiments, the nucleotide sequence thatAttorney Docket No: 243734.000231encodes the CD8a transmembrane domain comprises the nucleotide sequence set forth in SEQ ID NO: 45.

[0323] In certain embodiments, the transmembrane domain in the CAR of the disclosure is derived from the CD8a transmembrane domain. In certain embodiments, the CD8a transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 124, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 124. In certain embodiments, the nucleotide sequence that encodes the CD8a transmembrane domain comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 124, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 124. In certain embodiments, the nucleotide sequence that encodes the CD8a transmembrane domain comprises the nucleotide sequence set forth in SEQ ID NO: 123 or 126, or a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 123 or 126. In certain embodiments, the CD8a transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 124. In certain embodiments, the nucleotide sequence that encodes the CD8α transmembrane domain comprises the nucleotide sequence set forth in SEQ ID NO: 123 or 126.

[0324] In certain aspects, the CARs of the present disclosure comprise a hinge domain and a transmembrane domain. In some embodiments, CARs of the present disclosure comprise a hinge domain derived from CD8a stalk, CD4, CD28, IgGl, IgG2, IgG3, IgG4 IgMl, IgM2, IgAl, IgA2, IgD, and / or IgE and the transmembrane domain derived from α, β or ζ chain of the T-cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD8a, CD9, CD 16, CD22, CD33, CD37, CD40, CD64, CD80, CD86, CD134 (0X40), CD137, and / or CD154. In some embodiments, CARs of the present disclosure comprise a hinge domain derived from CD8a stalk, CD28, or IgGl and the transmembrane domain derived from CD8a, CD28, CD8, CD4, CD3( / , CD40, CD 134 (0X40), or CD7. In certain embodiments, the CARs of the present disclosure comprise a hinge domain derived from CD8α stalk and a transmembrane domain derived from CD8α. In certain embodiments, the CD8a hinge domain and the CD8a transmembrane domain comprises the amino acid sequence setAttorney Docket No: 243734.000231forth in SEQ ID NO: 107, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 107. In certain embodiments, the nucleotide sequence that encodes the CD8a hinge domain and the CD8a transmembrane domain comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 107, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, sequence identity with SEQ ID NO: 107. In certain embodiments, the nucleotide sequence that encodes the CD8a hinge domain and the CD8a transmembrane domain comprises the nucleotide sequence set forth in SEQ ID NO: 106 or 110, or a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 106 or 110. In certain embodiments, the CD8a hinge domain and the CD8a transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 107. In certain embodiments, the nucleotide sequence that encodes the CD8a hinge domain and the CD8a transmembrane domain comprises the nucleotide sequence set forth in SEQ ID NO: 106 or 110.

[0325] In certain embodiments, the CARs of the present disclosure comprise a hinge domain derived from CD28 and a transmembrane domain derived from CD28. In certain embodiments, the CD28 hinge domain and the CD28 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 109, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 109. In certain embodiments, the nucleotide sequence that encodes the CD28 hinge domain and the CD28 transmembrane domain comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 109, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 109. In certain embodiments, the nucleotide sequence that encodes the CD28 hinge domain and the CD28 transmembrane domain comprises the nucleotide sequence set forth in SEQ ID NO: 108, 111, or 116, or a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at leastAttorney Docket No: 243734.00023170%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 108, 111, or 116. In certain embodiments, the CD28 hinge domain and the CD28 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 109. In certain embodiments, the nucleotide sequence that encodes the CD28 hinge domain and the CD28 transmembrane domain comprises the nucleotide sequence set forth in SEQ ID NO: 108, 111, or 116.

[0326] In certain embodiments, the CARs of the present disclosure comprise a hinge domain derived from m!gG4 and a transmembrane domain derived from CD28. In certain embodiments, the mIgG4 hinge domain and the CD28 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 113 or 115, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 113 or 115. In certain embodiments, the nucleotide sequence that encodes the mIgG4 hinge domain and the CD28 transmembrane domain comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 113 or 115, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 113 or 115. In certain embodiments, the nucleotide sequence that encodes the mIgG4 hinge domain and the CD28 transmembrane domain comprises the nucleotide sequence set forth in SEQ ID NO: 112 or 114, or a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 112 or 114. In certain embodiments, the mIgG4 hinge domain and the CD28 transmembrane domain comprises the amino acid sequence set forth in SEQ ID NO: 113 or 115. In certain embodiments, the nucleotide sequence that encodes the mIgG4 hinge domain and the CD28 transmembrane domain comprises the nucleotide sequence set forth in SEQ ID NO: 112 or 114.Cytoplasmic Domain

[0327] In certain aspects, CARs of the present disclosure comprise a cytoplasmic domain, which comprises one or more costimulatory domains and / or one or more activation domains. The cytoplasmic domain, which comprises one or more costimulatory domains and / or one or more activation domains, is responsible for activation of at least one of the normal effector functions ofAttorney Docket No: 243734.000231the lymphocyte in which the CAR has been placed in. The term “effector function” refers to a specialized function of a cell. Effector function of a T-cell, for example, may be cytolytic activity or helper activity including the secretion of cytokines. Thus, the term “activation domain” refers to the portion of a protein which transduces the effector function signal and directs the cell to perform a specialized function. While usually the entire activation domain is present, in many cases it is not necessary to use the entire chain. To the extent that a truncated portion of the intracellular activation domain is used, such truncated portion may be used in place of the intact chain as long as it transduces the effector function signal. The term intracellular activation domain is thus meant to include any truncated portion of the activation domain sufficient to transduce the effector function signal.

[0328] In some embodiments, the activation domain described herein is a lymphocyte activation domain.

[0329] Non-limiting examples of activation domains which can be used in the CARs of the present disclosure include, e.g., activation domains derived from CD3ζ, CD3δ, CD3ε, CD3γ, ZAP70, DAP10, DAP12, Fc epsilon receptor I γ chain (FCER1G), CD226, NKG2C, NKG2D, NKG2E, CD79A, FcR β, CD5, CD22, CD66d, or CD79B. In some embodiments, the CAR of the present disclosure comprises an activation domain derived from CD3ζ.

[0330] In certain embodiments, the activation domain in the CAR of the disclosure is designed to comprise an activation domain derived from CD3(^. In certain embodiments, the CD3ζ activation domain comprises the amino acid sequence set forth in SEQ ID NO: 24, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 24. In certain embodiments, the nucleotide sequence that encodes the CD3(^ activation domain comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 24, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 24. In certain embodiments, the nucleotide sequence that encodes the CD3ζ activation domain comprises the nucleotide sequence set forth in SEQ ID NO: 8, 16, or 41, or a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at leastAttorney Docket No: 243734.00023199% sequence identity with SEQ ID NO: 8, 16, or 41. In certain embodiments, the CD3ζ activation domain comprises the amino acid sequence set forth in SEQ ID NO: 24. In certain embodiments, the nucleotide sequence that encodes the CD3(^ activation domain comprises the nucleotide sequence set forth in SEQ ID NO: 8, 16, or 41.

[0331] Non-limiting examples of costimulatory domains which can be used in the CARs of the present disclosure include, those derived from CD28, 4-lBB(CD137), CD27, CD40, CD134 (0X40), CD226, CD79A, ICOS, MyD88, BTLA, CD27, CD30, GITR, HVEM, IL-2Rβ, or the STAT3-binding YXXQ wherein X is any amino acid, or any combination thereof. In some embodiments, the CAR of the present disclosure comprises one costimulatory domain. In some embodiments, the CAR of the present disclosure comprises a costimulatory domain derived from CD28. In some embodiments, the CAR of the present disclosure comprises a costimulatory domain derived from 4- IBB.

[0332] In some embodiments, each CAR of the dual-targeting CAR system of the present disclosure comprises a costimulatory domain independently derived from CD28, 4-lBB(CD137), CD27, CD40, CD134 (0X40), CD226, CD79A, ICOS, MyD88, BTLA, CD27, CD30, GITR, HVEM, IL-2Rβ, or the STAT3-binding YXXQ wherein X is any amino acid, or any combination thereof. In some embodiments, the costimulatory domains of the dual-targeting CAR system of the present disclosure are the same or different.

[0333] In some embodiments, a CAR of the dual-targeting CAR system of the present disclosure comprises a costimulatory domain derived from CD28 or 4-1BB. In some embodiments, one CAR of the dual-targeting CAR system of the present disclosure comprises a costimulatory domain derived from CD28 and the other CAR of the dual-targeting CAR system of the present disclosure comprises a costimulatory domain derived from 4-1BB.

[0334] In some embodiments, a CAR of the present disclosure comprises two or more costimulatory domains. In certain embodiments, a CAR of the present disclosure comprises two, three, four, five, six or more costimulatory domains. In some embodiments, the costimulatory domain present in one CAR is different from the costimulatory domain present in the other CAR. For example, the anti-CD19 CAR of the present disclosure may comprise a costimulatory domain derived from 4-1BB and the anti-CD22 CAR of the present disclosure may comprise a costimulatory domain derived from CD28.Attorney Docket No: 243734.000231

[0335] In certain embodiments, the CARs of the present disclosure comprise a cytoplasmic domain, which comprises an activation domain, a MyD88 polypeptide or functional fragment thereof, and a CD40 cytoplasmic polypeptide region or a functional fragment thereof. In certain embodiments, the CAR lacks the CD40 transmembrane and / or CD40 extracellular domains. In certain embodiments, the CAR includes the CD40 transmembrane domain. In certain embodiments, the CAR includes the CD40 transmembrane domain and a portion of the CD40 extracellular domain, wherein the CD40 extracellular domain does not interact with natural or synthetic ligands of CD40.

[0336] In certain embodiments, the activation domain is separated from the MyD88 polypeptide or functional fragment thereof and / or the CD40 cytoplasmic polypeptide region or a functional fragment thereof. In certain embodiments, the activation domain is separated from the MyD88 polypeptide or functional fragment thereof and / or the CD40 cytoplasmic polypeptide region or a functional fragment thereof by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids.

[0337] In some embodiments, the activation domain(s) and costimulatory domain(s) can be in any order. In some embodiments, the activation domain is upstream of the costimulatory domains. In some embodiments, the activation domain is downstream from the costimulatory domains. In the cases where two or more costimulatory domains are included, the order of the costimulatory domains could be switched.

[0338] In some embodiments, the costimulatory domain derived from CD28 comprises the amino acid sequence set forth in SEQ ID NO: 30, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 30. In certain embodiments, the nucleotide sequence that encodes the CD28 costimulatory domain comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 30, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 30. In certain embodiments, the nucleotide sequence that encodes the CD28 costimulatory domain comprises the nucleotide sequence set forth in SEQ ID NO: 15 or 40, or a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%,Attorney Docket No: 243734.000231at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 15 or 40. In certain embodiments, the CD28 costimulatory domain comprises the amino acid sequence set forth in SEQ ID NO: 30. In certain embodiments, the nucleotide sequence that encodes the CD28 costimulatory domain comprises the nucleotide sequence set forth in SEQ ID NO: 15 or 40.

[0339] In some embodiments, the costimulatory domain derived from 4- IBB comprises the amino acid sequence set forth in SEQ ID NO: 23, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 23. In certain embodiments, the nucleotide sequence that encodes the 4-1BB costimulatory domain comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 23, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 23. In certain embodiments, the nucleotide sequence that encodes the 4-1BB costimulatory domain comprises the nucleotide sequence set forth in SEQ ID NO: 7, or a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 7. In certain embodiments, the 4-1BB costimulatory domain comprises the amino acid sequence set forth in SEQ ID NO: 23. In certain embodiments, the nucleotide sequence that encodes the 4- IBB costimulatory domain comprises the nucleotide sequence set forth in SEQ ID NO: 7.Additional Genes

[0340] In addition to the CAR construct, the polynucleotide may further comprise at least one additional gene that encodes an additional peptide. Examples of additional genes can include a transduced host cell selection marker, an in vivo tracking marker, a cytokine, a suicide gene, or some other functional gene. In certain embodiments, the functional additional gene can induce the expression of another molecule. In certain embodiments, the functional additional gene can increase the safety of the CAR(s). For example, the CAR construct may comprise an additional gene which is CD20. As another example, the CAR construct may comprise an additional geneAttorney Docket No: 243734.000231which is truncated CD 19 (tCD19). The tCD19 can be used as a tag. Expression of tCD19 may also help determine transduction efficiency.

[0341] Other examples of additional genes include genes that encode polypeptides with a biological function; examples include, but are not limited to, cytokines, chimeric cytokine receptors, dominant negative receptors, safety switches (such as, without limitation, CD20, truncated EGFR or HER2, inducible caspase 9 molecules). As another example, the CAR construct may comprise an additional gene which is a synNotch receptor. Once activated, the synNotch receptor can induce the expression of a target gene (e.g., a second CAR and / or bispecific molecule).

[0342] In certain embodiments, the CAR construct described herein comprises at least one additional gene (i.e., a second gene). In certain embodiments, the CAR construct described herein comprises one second gene. In other embodiments, the CAR construct described herein comprises two additional genes (i.e., a third gene). In yet another embodiment, the CAR construct described herein comprises three additional genes (i.e., a fourth gene). In certain embodiments, the additional genes are separated from each other and the CAR construct. For example, they may be separated by 2A sequences and / or an internal ribosomal entry sites (IRES). In certain examples, the CAR can be at any position of the polynucleotide chain (for example construct A: CAR, second gene, third gene, fourth gene; construct B: second gene, CAR, third gene, fourth gene; etc.)

[0343] Non-limiting examples of classes of additional genes that can be used to increase the effector function of CAR containing host cells, include (a) secretable cytokines (e.g., but not limited to, IL-7, IL-12, IL-15, and / or IL-18), (b) membrane bound cytokines (e.g., but not limited to, IL- 15), (c) chimeric cytokine receptors (e.g., but not limited to, IL-2 / IL-7, or IL-4 / IL-7), (d) constitutive active cytokine receptors (e.g., but not limited to, C7R), (e) dominant negative receptors (DNR; e.g., but not limited to TGFRII DNR), (f) ligands of costimulatory molecules (e.g., but not limited to, CD80, and / or 4-1BBL), (g) nuclear factor of activated T-cells (NFATs) (e.g., but not limited to, NFATcl, NFATc2, NFATc3, NFATc4, and / or NFAT5), (h) antibodies, including fragments thereof and bispecific antibodies (e.g., but not limited to, bispecific T-cell engagers (BiTEs)), or (i) a second CAR.

[0344] In certain embodiments, the additional gene may be regulated by an NFAT dependent promoter. Activation of the T-cell or other lymphocyte leads to activation of the transcription factor NF AT resulting in the induction of the expression of the protein encoded by the gene linkedAttorney Docket No: 243734.000231with the NFAT dependent promoter. One or more members of the NFAT family (i.e., NFATcl, NFATc2, NFATc3, NFATc4, and NFAT5) is expressed in most cells of the immune system. NFAT-dependent promoters and enhancers tend to have three to five NFAT binding sites.

[0345] In certain embodiments, the functional additional gene can be a suicide gene. A suicide gene is a recombinant gene that will cause the host cell in which the gene is expressed to undergo programmed cell death or antibody-mediated clearance at a desired time. Suicide genes can function to increase the safety of the CAR. In another embodiment, the additional gene is an inducible suicide gene. Non-limiting examples of suicide genes include i) molecules that are expressed on the cell surface and can be targeted with a clinical grade monoclonal antibody including CD20, EGFR or a fragment thereof, HER2 or a fragment thereof, and ii) inducible suicide genes (e.g., but not limited to inducible caspase 9 (see Straathof et al. (2005) Blood.105(11): 4247-4254; U. S. Pat. Appl. Pub. No. 2011 / 0286980, each of which is incorporated herein by reference in its entirety for all purposes)).

[0346] In certain aspects, CARs of the present disclosure may be regulated by a safety switch. As used herein, the term “safety switch” refers to any mechanism that is capable of removing or inhibiting the effect of a CAR from a system (e g., a culture or a subject). Safety switches can function to increase the safety of the CAR.

[0347] The function of the safety switch may be inducible. Non-limiting examples of safety switches include (a) molecules that are expressed on the cell surface and can be targeted with a clinical grade monoclonal antibody including CD20, EGFR or a fragment thereof, HER2 or a fragment thereof, and (b) inducible suicide genes (e g., but not limited to, herpes simplex virus thymidine kinase (HSV-TK) and inducible caspase 9 (see Straathof et al. (2005) Blood. 105(11): 4247-4254; U. S. Pat. Appl. Pub. No. 2011 / 0286980, each of which is incorporated herein by reference in its entirety for all purposes).

[0348] In some embodiments, the safety switch is a CD20 polypeptide. Expression of human CD20 on the cell surface presents an attractive strategy for a safety switch. Cells that express CD20 can be rapidly eliminated with the FDA approved monoclonal antibody rituximab through complement-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity (see e.g., Griffioen, M., et al. Haematologica 94, 1316-1320 (2009), which is incorporated herein by reference in its entirety for all purposes). Rituximab is an anti-CD20 monoclonal antibody that has been FDA approved for Chronic Lymphocytic Leukemia (CLL) and Non-Hodgkin’s LymphomaAttorney Docket No: 243734.000231(NHL), among others (Storz, U. MAbs 6, 820-837 (2014), which is incorporated herein by reference in its entirety for all purposes). The CD20 safety switch is non-immunogenic and can function as a reporter / selection marker in addition to a safety switch (Bonifant, C. L., et al. Mol Ther 24, 1615-1626 (2016); van Loenen, M. M., et al. Gene Ther 20, 861-867 (2013); each of which is incorporated herein by reference in its entirety for all purposes).

[0349] In some embodiments, the additional gene sequence may be derived from CD20. In some embodiments, the CD20 sequence comprises the amino acid sequence set forth in SEQ ID NO: 55 or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 55. In certain embodiments, the nucleotide sequence encoding the CD20 sequence comprises the nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:55, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 55. In certain embodiments, the nucleotide sequence encoding the CD20 sequence comprises the sequence set forth in SEQ ID NO: 49, or a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 49. In certain embodiments, the CD20 sequence comprises the amino acid sequence of SEQ ID NO: 55. In certain embodiments, the nucleotide sequence encoding the CD20 sequence comprises the nucleotide sequence set forth in SEQ ID NO: 49.

[0350] In some embodiments, the sequence encoding an additional gene is operably linked to the sequence encoding CAR via a separation sequence encoding a self-cleaving peptide and / or an Internal Ribosome Entry Site (IRES) as disclosed herein. In some embodiments, the sequence encoding an anti-CD19 CAR is operably linked to the sequence encoding an anti-CD22 CAR via a sequence encoding a self-cleaving peptide and / or an Internal Ribosome Entry Site (IRES) as disclosed herein. The self-cleaving peptide can link two sequences encoding two CAR polypeptides in one bicistronic vector and allow the translation of both polypeptides in cells.

[0351] Non-limiting examples of self-cleaving peptide sequences include Thoseaasigna virus 2A (T2A; GSGEGRGSLLTCGDVEENPGP, SEQ ID NO: 25; AEGRGSLLTCGDVEENPGP, SEQ ID NO: 44; or EGRGSLLTCGDVEENPGP, SEQ ID NO: 56); the foot and mouth diseaseAttorney Docket No: 243734.000231virus (FMDV) 2A sequence (F2A; GSGSRVTELLYRMKRAETYCPRPLLAIHPTEARHKQKIVAPVKQLLNFDLLKLAGDVES NPGP, SEQ ID NO: 52); Sponge (Amphimedon queenslandica) 2A sequence (LLCFLLLLLSGDVELNPGP, SEQ ID NO: 53; or HHFMFLLLLLAGDIELNPGP, SEQ ID NO: 58); acorn worm 2A sequence (Saccoglossus kowalevskii) (WFLVLLSFILSGDIEVNPGP, SEQ ID NO: 59); amphioxus (Branchiostoma floridae) 2A sequence (KNCAMYMLLLSGDVETNPGP, SEQ ID NO: 67; or MVISQLMLKLAGDVEENPGP, SEQ ID NO: 69); porcine teschovirus- 1 2A sequence (P2A; GSGATNFSLLKQAGDVEENPGP, SEQ ID NO: 71); and equine rhinitis A virus 2A sequence (E2A; GSGQCTNYALLKLAGDVESNPGP, SEQ ID NO: 73). In some embodiments, the separation sequence is a naturally occurring or synthetic sequence. In certain embodiments, the separation sequence includes the 2A consensus sequence D-X-E-X-NPGP (SEQ ID NO: 75), in which X is any amino acid residue. In certain embodiments, the separation sequence includes GSG at N-terminus of the separation sequence to improve cleavage efficiency 13. Further non-limiting examples of separation sequences are described in Wang, Y. et al. 2A self-cleaving peptide-based multi-gene expression system in the silkworm Bombyx mori. Sci. Rep. 5, 16273; doi: 10.1038 / srep 16273 (2015) which is incorporated herein by reference in its entirety.

[0352] Alternatively, an Internal Ribosome Entry Site (IRES) may be used to link the CARs and / or the additional gene. IRES is an RNA element that allows for translation initiation in a capindependent manner. IRES can link two coding sequences in one bicistronic vector and allow the translation of both proteins in cells.

[0353] In some embodiments, the self-cleaving 2A peptide is a T2A peptide and comprises the amino acid sequence set forth in SEQ ID NO: 25 or 56. In some embodiments, the sequence encoding the T2A peptide comprises the nucleotide sequence SEQ ID NO: 9, 125, or 50.

[0354] In certain embodiments, the host cells can be genetically modified to express not only CARs as disclosed herein but to also express fusion protein with signaling activity (e.g., costimulation, T-cell activation). These fusion proteins can improve host cell activation and / or responsiveness. In certain embodiments, the fusion protein can enhance the host cell’s response to the target antigen. In certain embodiments, the fusion protein can impart resistance to suppression signals.Attorney Docket No: 243734.000231

[0355] In certain embodiments, fusion proteins can comprise portions of CD4, CD8a, CD28, portions of a T-cell receptor, or a target binding moiety (e.g., scFv) linked to a MyD88, CD40, and / or other signaling molecules.

[0356] In certain embodiments, the fusion protein comprises an extracellular target-binding domain (as disclosed above), a transmembrane domain (as described above), and a cytoplasmic domain, wherein the cytoplasmic domain comprises at least one costimulatory protein (as described above). In certain embodiments, the costimulatory fusion protein does not comprise a lymphocyte activation domain (e.g., CD3ζ). In certain embodiments, the at least one costimulatory protein can be a MyD88 polypeptide or functional fragment thereof, and / or a CD40 cytoplasmic polypeptide region or a functional fragment thereof.

[0357] In certain embodiments, the fusion protein comprises an extracellular domain (such as, but not limited to CD 19, CD34), a transmembrane domain (as described above), and a cytoplasmic domain, wherein the cytoplasmic domain comprises at least one co-stimulatory protein (as described above). In certain embodiments, the fusion protein does not comprise a lymphocyte activation domain (e.g., CD3ζ). In certain embodiments, the at least one portion of the fusion protein can be a MyD88 polypeptide or functional fragment thereof, and / or a CD40 cytoplasmic polypeptide region or a functional fragment thereof.

[0358] Non-limiting examples of fusion proteins include the constructs described in the publication of Int. Pat. Appl. Pub. Nos WO2019222579 and WO2016073875, which are incorporated herein by reference in its entirety for all purposes.

[0359] In certain embodiments, the fusion proteins are introduced into the host cell on a separate vector from the CAR. In certain embodiments, the fusion proteins are introduced into the host cell on the same vector as the CAR. In certain embodiments, the fusion proteins are introduced into the host cell on the same vector as the CAR but separated by a separation sequence such as 2A.Non-Limiting Examples of CARs

[0360] In certain aspects, the present invention provides a polynucleotide encoding a dualtargeting chimeric antigen receptor (CAR) system comprising two CARs, wherein the two CARs comprise:(i) an anti-cluster of differentiation 22 (CD22) CAR comprising:an extracellular domain comprising an anti-CD22 target binding moiety,Attorney Docket No: 243734.000231a hinge domain and a transmembrane domain derived from CD28, a cytoplasmic domain comprising a CD28 costimulatory domain and a CD3ζ activation domain; and(ii) an anti-cluster of differentiation 19 (CD 19) CAR comprising:an extracellular domain comprising an anti-CD19 target binding moiety, a hinge domain and a transmembrane domain derived from CD28 or CD8u, a cytoplasmic domain comprising a 4- IBB costimulatory domain and lacking an activation domain.

[0361] In certain aspects, the present invention provides a polynucleotide encoding a dualtargeting chimeric antigen receptor (CAR) system comprising two CARs, wherein the two CARs comprise:(i) an anti-cluster of differentiation 22 (CD22) CAR comprising:an extracellular domain comprising an anti-CD22 target binding moiety, a hinge domain and a transmembrane domain derived from CD28, a cytoplasmic domain comprising a CD28 costimulatory domain and a CD3ζ activation domain; and(ii) an anti-cluster of differentiation 19 (CD 19) CAR comprising:an extracellular domain comprising an anti-CD19 target binding moiety, a hinge domain and a transmembrane domain derived from CD28, a cytoplasmic domain comprising a 4-1BB costimulatory domain and lacking an activation domain.

[0362] In certain aspects, the present invention provides a polynucleotide encoding a dualtargeting chimeric antigen receptor (CAR) system comprising two CARs, wherein the two CARs comprise:(i) an anti-cluster of differentiation 22 (CD22) CAR comprising:an extracellular domain comprising an anti-CD22 target binding moiety, a hinge domain and a transmembrane domain derived from CD28, a cytoplasmic domain comprising a CD28 costimulatory domain and a CD3ζ activation domain; and(ii) an anti-cluster of differentiation 19 (CD 19) CAR comprising:an extracellular domain comprising an anti-CD19 target binding moiety,Attorney Docket No: 243734.000231a hinge domain and a transmembrane domain derived from CD8a, a cytoplasmic domain comprising a 4- IBB costimulatory domain and lacking an activation domain.

[0363] In certain aspects, the present invention provides a pair of polynucleotides encoding a dual -targeting chimeric antigen receptor (CAR) system comprising two CARs, wherein:(i) the first polynucleotide encodes an anti-cluster of differentiation 22 (CD22) CAR comprising:an extracellular domain comprising an anti-CD22 target binding moiety, a hinge domain and a transmembrane domain derived from CD28, a cytoplasmic domain comprising a CD28 costimulatory domain and a CD3ζ activation domain; and(ii) the second polynucleotide encodes an anti-cluster of differentiation 19 (CD 19) CAR comprising:an extracellular domain comprising an anti-CD19 target binding moiety, a hinge domain and a transmembrane domain derived from CD28 or CD8a, a cytoplasmic domain comprising a 4- IBB costimulatory domain and lacking an activation domain.

[0364] In certain aspects, the present invention provides a pair of polynucleotides encoding a dual-targeting chimeric antigen receptor (CAR) system comprising two CARs, wherein:(i) the first polynucleotide encodes an anti-cluster of differentiation 22 (CD22) CAR comprising:an extracellular domain comprising an anti-CD22 target binding moiety, a hinge domain and a transmembrane domain derived from CD28, a cytoplasmic domain comprising a CD28 costimulatory domain and a CD3ζ activation domain; and(ii) the second polynucleotide encodes an anti-cluster of differentiation 19 (CD 19) CAR comprising:an extracellular domain comprising an anti-CD19 target binding moiety, a hinge domain and a transmembrane domain derived from CD28, a cytoplasmic domain comprising a 4- IBB costimulatory domain and lacking an activation domain.Attorney Docket No: 243734.000231

[0365] In certain aspects, the present invention provides a pair of polynucleotides encoding a dual-targeting chimeric antigen receptor (CAR) system comprising two CARs, wherein:(i) the first polynucleotide encodes an anti-cluster of differentiation 22 (CD22) CAR comprising:an extracellular domain comprising an anti-CD22 target binding moiety, a hinge domain and a transmembrane domain derived from CD28, a cytoplasmic domain comprising a CD28 costimulatory domain and a CD3ζ activation domain; and(ii) the second polynucleotide encodes an anti-cluster of differentiation 19 (CD 19) CAR comprising:an extracellular domain comprising an anti-CD19 target binding moiety, a hinge domain and a transmembrane domain derived from CD8a, a cytoplasmic domain comprising a 4- IBB costimulatory domain and lacking an activation domain.

[0366] In certain aspects, the present invention provides a polynucleotide encoding a dualtargeting chimeric antigen receptor (CAR) system comprising two CARs, wherein the two CARs comprise:(i) an anti-cluster of differentiation- 19 (CD19) CAR comprising:an extracellular domain comprising an anti-CD19 target binding moiety, a hinge domain and a transmembrane domain derived from CD28, a cytoplasmic domain comprising a CD28 costimulatory domain and a CD3ζ activation domain; and(ii) an anti-cluster of differentiation 22 (CD22) CAR comprising:an extracellular domain comprising an anti-CD22 target binding moiety, a hinge domain and a transmembrane domain derived from CD28 or CD8a, a cytoplasmic domain comprising a 4- IBB costimulatory domain and lacking an activation domain.

[0367] In certain aspects, the present invention provides a polynucleotide encoding a dualtargeting chimeric antigen receptor (CAR) system comprising two CARs, wherein the two CARs comprise:(i) an anti-cluster of differentiation- 19 (CD 19) CAR comprising:Attorney Docket No: 243734.000231an extracellular domain comprising an anti-CD19 target binding moiety, a hinge domain and a transmembrane domain derived from CD28, a cytoplasmic domain comprising a CD28 costimulatory domain and a CD3ζ activation domain; and(ii) an anti-cluster of differentiation 22 (CD22) CAR comprising:an extracellular domain comprising an anti-CD22 target binding moiety, a hinge domain and a transmembrane domain derived from CD28, a cytoplasmic domain comprising a 4-1BB costimulatory domain and lacking an activation domain.

[0368] In certain aspects, the present invention provides a polynucleotide encoding a dualtargeting chimeric antigen receptor (CAR) system comprising two CARs, wherein the two CARs comprise:(i) an anti-cluster of differentiation- 19 (CD19) CAR comprising:an extracellular domain comprising an anti-CD19 target binding moiety, a hinge domain and a transmembrane domain derived from CD28, a cytoplasmic domain comprising a CD28 costimulatory domain and a CD3ζ activation domain; and(ii) an anti-cluster of differentiation 22 (CD22) CAR comprising:an extracellular domain comprising an anti-CD22 target binding moiety, a hinge domain and a transmembrane domain derived from CD8a, a cytoplasmic domain comprising a 4- IBB costimulatory domain and lacking an activation domain.

[0369] In certain aspects, the present invention provides a pair of polynucleotides encoding a dual-targeting chimeric antigen receptor (CAR) system comprising two CARs, wherein:(i) the first polynucleotide encodes an anti-cluster of differentiation- 19 (CD 19) CAR comprising:an extracellular domain comprising an anti-CD19 target binding moiety, a hinge domain and a transmembrane domain derived from CD28, a cytoplasmic domain comprising a CD28 costimulatory domain and a CD3ζ activation domain; andAttorney Docket No: 243734.000231(ii) the second polynucleotide encodes an anti-cluster of differentiation 22 (CD22) CAR comprising:an extracellular domain comprising an anti-CD22 target binding moiety, a hinge domain and a transmembrane domain derived from CD28 or CD8a, a cytoplasmic domain comprising a 4-1BB costimulatory domain and lacking an activation domain.

[0370] In certain aspects, the present invention provides a pair of polynucleotides encoding a dual-targeting chimeric antigen receptor (CAR) system comprising two CARs, wherein:(i) the first polynucleotide encodes an anti-cluster of differentiation- 19 (CD 19) CAR comprising:an extracellular domain comprising an anti-CD19 target binding moiety, a hinge domain and a transmembrane domain derived from CD28, a cytoplasmic domain comprising a CD28 costimulatory domain and a CD3ζ activation domain; and(ii) the second polynucleotide encodes an anti-cluster of differentiation 22 (CD22) CAR comprising:an extracellular domain comprising an anti-CD22 target binding moiety, a hinge domain and a transmembrane domain derived from CD28, a cytoplasmic domain comprising a 4-1BB costimulatory domain and lacking an activation domain.

[0371] In certain aspects, the present invention provides a pair of polynucleotides encoding a dual -targeting chimeric antigen receptor (CAR) system comprising two CARs, wherein:(i) the first polynucleotide encodes an anti-cluster of differentiation-19 (CD 19) CAR comprising:an extracellular domain comprising an anti-CD19 target binding moiety, a hinge domain and a transmembrane domain derived from CD28, a cytoplasmic domain comprising a CD28 costimulatory domain and a CD3ζ activation domain; and(ii) the second polynucleotide encodes an anti-cluster of differentiation 22 (CD22) CAR comprising:an extracellular domain comprising an anti-CD22 target binding moiety,Attorney Docket No: 243734.000231a hinge domain and a transmembrane domain derived from CD8a, a cytoplasmic domain comprising a 4-1BB costimulatory domain and lacking an activation domain.

[0372] In certain aspects, the present invention provides a polynucleotide encoding a dualtargeting chimeric antigen receptor (CAR) system comprising two CARs, wherein the two CARs comprise:(i) an anti-cluster of differentiation 22 (CD22) CAR comprising:an extracellular domain comprising an anti-CD22 target binding moiety, a hinge domain and a transmembrane domain derived from CD28, a cytoplasmic domain comprising a CD28 costimulatory domain and a CD3ζ activation domain; and(ii) an anti-cluster of differentiation 19 (CD 19) CAR comprising:an extracellular domain comprising an anti-CD19 target binding moiety, a hinge domain and a transmembrane domain derived from CD28 or CD8a, a cytoplasmic domain comprising a 4-1BB costimulatory domain and a CD3(^ activation domain.

[0373] In certain aspects, the present invention provides a pair of polynucleotides encoding a dual -targeting chimeric antigen receptor (CAR) system comprising two CARs, wherein:(i) an anti-cluster of differentiation 22 (CD22) CAR comprising:an extracellular domain comprising an anti-CD22 target binding moiety, a hinge domain and a transmembrane domain derived from CD28, a cytoplasmic domain comprising a CD28 costimulatory domain and a CD3ζ activation domain; and(ii) an anti-cluster of differentiation 19 (CD 19) CAR comprising:an extracellular domain comprising an anti-CD19 target binding moiety, a hinge domain and a transmembrane domain derived from CD28 or CD8a, a cytoplasmic domain comprising a 4-1BB costimulatory domain and a CD3^ activation domain.

[0374] In certain aspects, the present invention provides a polynucleotide encoding a dualtargeting chimeric antigen receptor (CAR) system comprising two CARs, wherein the two CARs comprise:Attorney Docket No: 243734.000231(i) an anti-cluster of differentiation 22 (CD22) CAR comprising:an extracellular domain comprising a means for binding to CD22,a hinge domain and a transmembrane domain derived from CD28,a cytoplasmic domain comprising a CD28 costimulatory domain and a CD3ζ activation domain; and(ii) an anti-cluster of differentiation 19 (CD 19) CAR comprisingan extracellular domain comprising a means for binding to CD 19,a hinge domain and a transmembrane domain derived from CD28 or CD8a,a cytoplasmic domain comprising a 4- IBB costimulatory domain and lacking a means for activating effector function of an immune cell.

[0375] In certain aspects, the present invention provides a pair of polynucleotides encoding a dual-targeting chimeric antigen receptor (CAR) system comprising two CARs, wherein:(i) the first polynucleotide encodes an anti-cluster of differentiation 22 (CD22) CAR comprising:an extracellular domain comprising a means for binding to CD22,a hinge domain and a transmembrane domain derived from CD28,a cytoplasmic domain comprising a CD28 costimulatory domain and a CD3ζ activation domain; and(ii) the second polynucleotide encodes an anti-cluster of differentiation 19 (CD 19) CAR comprisingan extracellular domain comprising a means for binding to CD 19,a hinge domain and a transmembrane domain derived from CD28 or CD8a,a cytoplasmic domain comprising a 4- IBB costimulatory domain and lacking a means for activating effector function of an immune cell.

[0376] In certain aspects, the present invention provides a polynucleotide encoding a dualtargeting chimeric antigen receptor (CAR) system comprising two CARs, wherein one CAR binds to cluster of differentiation 19 (CD 19) and the other CAR binds to cluster of differentiation 22 (CD22), the improvement comprising one of the CARs lacking an activation domain.

[0377] In certain aspects, the present invention provides a pair of polynucleotides encoding a dual -targeting chimeric antigen receptor (CAR) system comprising two CARs, wherein the first polynucleotide encodes a first CAR of the dual-targeting CAR system, and the secondAttorney Docket No: 243734.000231polynucleotide encodes a second CAR of the dual -targeting CAR system, wherein the first CAR binds to cluster of differentiation 19 (CD 19) and the second CAR binds to cluster of differentiation 22 (CD22), the improvement comprising one of the CARs lacking an activation domain.

[0378] In certain embodiments, a polynucleotide encoding a dual-targeting chimeric antigen receptor (CAR) system comprising two CARs, wherein the polynucleotide comprises from 5’ to 3’: anti-CD19 CAR - separation sequence - anti-CD22 CAR.

[0379] In certain embodiments, a polynucleotide encoding a dual-targeting chimeric antigen receptor (CAR) system comprising two CARs, wherein the polynucleotide comprises from 5’ to 3 ’: anti-CD22 CAR - separation sequence - anti-CD 19 CAR. The anti-CD 19 CAR and ani-CD22 CAR may include any of the CAR constructs as illustrated herein.

[0380] In certain embodiments, an anti-CD 19 CAR of the disclosure comprises an extracellular binding domain comprising the amino acid sequence of SEQ ID NO: 61, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 61. In certain embodiments, the extracellular binding domain of an anti-CD19 CAR of the disclosure is encoded by a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 61, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 61. In certain embodiments, the nucleotide sequence that encodes the extracellular binding domain of an anti-CD 19 CAR of the disclosure comprises the nucleotide sequence set forth in SEQ ID NO: 62 or 65, or a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 62 or 65. In certain embodiments, an anti-CD19 CAR of the disclosure comprises an extracellular binding domain comprising the amino acid sequence set forth in SEQ ID NO: 61. In certain embodiments, the nucleotide sequence that encodes the extracellular binding domain of an anti-CD19 CAR of the disclosure comprises the nucleotide sequence set forth in SEQ ID NO: 62 or 65.

[0381] In certain embodiments, an anti-CD19 CAR of the disclosure comprises an extracellular binding domain comprising the amino acid sequence of SEQ ID NO: 133, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, atAttorney Docket No: 243734.000231least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 133. In certain embodiments, the extracellular binding domain of an anti-CD19 CAR of the disclosure is encoded by a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 133, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 133. In certain embodiments, the nucleotide sequence that encodes the extracellular binding domain of an anti-CD19 CAR of the disclosure comprises the nucleotide sequence set forth in SEQ ID NO: 134 or 135, or a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 134 or 135. In certain embodiments, an anti-CD19 CAR of the disclosure comprises an extracellular binding domain comprising the amino acid sequence set forth in SEQ ID NO: 133. In certain embodiments, the nucleotide sequence that encodes the extracellular binding domain of an anti-CD19 CAR of the disclosure comprises the nucleotide sequence set forth in SEQ ID NO: 134 or 135.

[0382] In certain embodiments, an anti-CD22 CAR of the disclosure comprises an extracellular binding domain comprising the amino acid sequence of SEQ ID NO: 63, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 63. In certain embodiments, the extracellular binding domain of an anti-CD22 CAR of the disclosure is encoded by a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 63, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 63. In certain embodiments, the nucleotide sequence that encodes the extracellular binding domain of an anti-CD22 CAR of the disclosure comprises the nucleotide sequence set forth in SEQ ID NO: 64 or 66, or a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 64 or 66. In certain embodiments, an anti-CD22 CAR of the disclosure comprises an extracellularAttorney Docket No: 243734.000231binding domain comprising the amino acid sequence set forth in SEQ ID NO: 63. In certain embodiments, the nucleotide sequence that encodes the extracellular binding domain of an anti-CD22 CAR of the disclosure comprises the nucleotide sequence set forth in SEQ ID NO: 64 or 66.

[0383] In certain embodiments, an anti-CD22 CAR of the disclosure comprises an extracellular binding domain comprising the amino acid sequence of SEQ ID NO: 136, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 136. In certain embodiments, the extracellular binding domain of an anti-CD22 CAR of the disclosure is encoded by a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 136, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 136. In certain embodiments, the nucleotide sequence that encodes the extracellular binding domain of an anti-CD22 CAR of the disclosure comprises the nucleotide sequence set forth in SEQ ID NO: 137 or 138, or a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 137 or 138. In certain embodiments, an anti-CD22 CAR of the disclosure comprises an extracellular binding domain comprising the amino acid sequence set forth in SEQ ID NO: 136. In certain embodiments, the nucleotide sequence that encodes the extracellular binding domain of an anti-CD22 CAR of the disclosure comprises the nucleotide sequence set forth in SEQ ID NO: 137 or 138.

[0384] In certain embodiments, an anti-CD19 CAR of the disclosure comprises the amino acid sequence of SEQ ID NO: 85, 88, or 90, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 85, 88, or 90. In certain embodiments, an anti-CD19 CAR of the disclosure is encoded by a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 85, 88, or 90, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 85, 88, or 90. In certain embodiments, theAttorney Docket No: 243734.000231nucleotide sequence that encodes an anti-CD19 CAR of the disclosure comprises the nucleotide sequence set forth in SEQ ID NO: 84, 86, 87, or 89, or a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 84, 86, 87, or 89. In certain embodiments, an anti-CD19 CAR of the disclosure comprises an amino acid sequence set forth in SEQ ID NO: 85, 88, or 90. In certain embodiments, the nucleotide sequence that encodes an anti-CD19 CAR of the disclosure comprises the nucleotide sequence set forth in SEQ ID NO: 84, 86, 87, or 89.

[0385] In certain embodiments, an anti-CD22 CAR of the disclosure comprises the amino acid sequence of SEQ ID NO: 77, 79, 81, or 83, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 77, 79, 81, or 83. In certain embodiments, an anti-CD22 CAR of the disclosure is encoded by a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 77, 79, 81, or 83, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 77, 79, 81, or 83. In certain embodiments, the nucleotide sequence that encodes an anti-CD22 CAR of the disclosure comprises the nucleotide sequence set forth in SEQ ID NO: 76, 78, 80, or 82, or a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 76, 78, 80, or 82. In certain embodiments, an anti-CD22 CAR of the disclosure comprises an amino acid sequence set forth in SEQ ID NO: 77, 79, 81, or 83. In certain embodiments, the nucleotide sequence that encodes an anti-CD22 CAR of the disclosure comprises the nucleotide sequence set forth in SEQ ID NO: 76, 78, 80, or 82.

[0386] In certain embodiments, the nucleotide sequence that encodes a dual -targeting antiCD 19 and CD22 CAR of the disclosure comprises the nucleotide sequence set forth in SEQ ID NO: 68, or a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 68. In certain embodiments,Attorney Docket No: 243734.000231the nucleotide sequence that encodes a dual -targeting anti-CD19 and CD22 CAR of the disclosure comprises the nucleotide sequence set forth in SEQ ID NO: 68.

[0387] In certain embodiments, the nucleotide sequence that encodes a dual-targeting antiCD 19 and CD22 CAR of the disclosure comprises the nucleotide sequence set forth in SEQ ID NO: 70, or a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 70. In certain embodiments, the nucleotide sequence that encodes a dual -targeting an anti-CD19 and CD22 CAR of the disclosure comprises the nucleotide sequence set forth in SEQ ID NO: 70.

[0388] In certain embodiments, the nucleotide sequence that encodes a dual-targeting antiCD 19 and CD22 CAR of the disclosure comprises the nucleotide sequence set forth in SEQ ID NO: 72, or a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 72. In certain embodiments, the nucleotide sequence that encodes a dual -targeting anti-CD19 and CD22 CAR of the disclosure comprises the nucleotide sequence set forth in SEQ ID NO: 72.

[0389] In certain embodiments, the nucleotide sequence that encodes a dual-targeting antiCD 19 and CD22 CAR of the disclosure comprises the nucleotide sequence set forth in SEQ ID NO: 74, or a nucleotide sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 74. In certain embodiments, the nucleotide sequence that encodes a dual -targeting anti-CD19 and CD22 CAR of the disclosure comprises the nucleotide sequence set forth in SEQ ID NO: 74.Vectors

[0390] The present disclosure provides recombinant vectors comprising a polynucleotide encoding a CAR comprising polynucleotides encoding the proteins disclosed above. In certain embodiments, recombinant vectors comprise a polynucleotide or a pair of polynucleotides encoding a dual-targeting CAR system comprising polynucleotides encoding the proteins disclosed above. In certain embodiments, the present disclosure provides a pair of recombinant vectors comprising a pair of polynucleotides encoding a dual-targeting CAR system describedAttorney Docket No: 243734.000231herein. In certain embodiments, a polynucleotide described herein is operatively linked to at least one regulatory element for expression of the CAR.

[0391] In certain embodiments, recombinant vectors of the disclosure comprise the nucleotide sequence of SEQ ID NO: 68, 70, 72, or 74, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 68, 70, 72, or 74. In certain embodiments, recombinant vectors of the disclosure comprise the nucleotide sequence of SEQ ID NO: 68, 70, 72, or 74.

[0392] In certain embodiments, recombinant vectors of the disclosure comprise the nucleotide sequence of SEQ ID NO: 72 or 74, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 72 or 74.

[0393] In certain embodiments, recombinant vectors of the disclosure comprise the nucleotide sequence of SEQ ID NO: 74, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 74.

[0394] In certain embodiments, recombinant vectors of the disclosure comprise the nucleotide sequence of SEQ ID NO: 84, 86, 87, or 89, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 84, 86, 87, or 89. In certain embodiments, recombinant vectors of the disclosure comprise the nucleotide sequence of SEQ ID NO: 84, 86, 87, or 89.

[0395] In certain embodiments, recombinant vectors of the disclosure comprise the nucleotide sequence of SEQ ID NO: 76, 78, 80, or 82, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 76, 78, 80, or 82. In certain embodiments, recombinant vectors of the disclosure comprise the nucleotide sequence of SEQ ID NO: 76, 78, 80, or 82.

[0396] In certain embodiments, a pair of recombinant vectors of the disclosure comprises a recombinant vector comprising the nucleotide sequence of SEQ ID NO: 84, 86, 87, or 89, or aAttorney Docket No: 243734.000231variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 84, 86, 87, or 89 and a recombinant vector comprising the nucleotide sequence of SEQ ID NO: 76, 78, 80, or 82, or a variant thereof having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with SEQ ID NO: 76, 78, 80, or 82.

[0397] In certain embodiments, the recombinant vector comprises a polynucleotide encoding a CAR, wherein the polynucleotide is operatively linked to at least one additional gene. In some embodiments, the additional gene is a CD20.

[0398] In certain embodiments, the vector is a viral vector. In certain embodiments, the viral vector can be, but is not limited to, a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated virus vector, an alphaviral vector, a herpes virus vector, a baculoviral vector, and a vaccinia virus vector. In some embodiments, the viral vector is a lentiviral vector.

[0399] In some embodiments, the vector is a non-viral vector. The viral vector may be a plasmid or a transposon (such as a PiggyBac or a Sleeping Beauty transposon). In some embodiments, the non-viral vector can be, but is not limited to, a minicircle plasmid, a Sleeping Beauty transposon, a PiggyBac transposon, or a single or double stranded DNA molecule that is used as a template for homology directed repair (HDR) based gene editing.

[0400] In certain embodiments, the polynucleotide encoding the CAR is operably linked to at least a regulatory element. The regulatory element can be capable of mediating expression of the CAR in the host cell. Regulatory elements include, but are not limited to, promoters, enhancers, initiation sites, polyadenylation (poly A) tails, IRES elements, response elements, and termination signals. In certain embodiments, the regulatory element regulates CAR expression. In certain embodiments, the regulatory element increased the expression of the CAR. In certain embodiments, the regulatory element increased the expression of the CAR once the host cell is activated. In certain embodiments, the regulatory element decreases expression of the CAR. In certain embodiments, the regulatory element decreases expression of the CAR once the host cell is activated.CAR-Modified Host CellsAttorney Docket No: 243734.000231

[0401] In one aspect, the present disclosure provides an isolated host cell comprising a polynucleotide, a pair of polynucleotides, a recombinant vector, or a pair of recombinant vectors described herein. In one aspect, the present disclosure provides an isolated host cell comprising a CAR described herein. In one aspect, the present disclosure provides an isolated host cell comprising a dual-targeting CAR system described herein. In some embodiments, the CAR targets cluster of differentiation 19 (CD 19) and / or cluster of differentiation 22 (CD22).

[0402] In various embodiments, the host cell is an immune cell. The immune cell may be a T-cell, a natural killer (NK) cell, or a macrophage.

[0403] In various embodiments, the host cell is a T-cell. T-cells may include, but are not limited to, thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. A T-cell can be a T helper (Th) cell, for example a T helper 1 (Thl) or a T helper 2 (Th2) cell. The T-cell can be a helper T-cell (HTL; CD4+T-cell) CD4+T-cell, a cytotoxic T-cell (CTL; CD8+T-cell), a tumor infiltrating cytotoxic T-cell (TIL; CD8+T-cell), CD4+CD8+T-cell, or any other subset of T-cells. Other illustrative populations of T-cells suitable for use in particular embodiments include naive T-cells memory T-cells, andNKT cells.

[0404] In some embodiments, the T-cell is selected from a CD8+T-cell, a CD4+T-cell, a cytotoxic T-cell, an αβ T-cell receptor (TCR) T-cell, an invariant natural killer T (iNKT) cell, a natural killer T (NKT) cell, a y8 T-cell, a memory T-cell, a memory T-cell, a memory stem T-cell (TSCM), a naive T-cell, an effector T-cell, a T-helper cell, and a regulatory T-cell (Treg).

[0405] In various embodiments, the host cell is an NK cell. NK cell refers to a differentiated lymphocyte with a CD3-CD16+, CD3-CD56+, CD16+CD56+and / or CD57+TCR- phenotype.

[0406] In various embodiments, the host cell has been activated and / or expanded ex vivo.

[0407] In various embodiments, the host cell is an allogeneic cell. In various embodiments, the host cell is an autologous cell.

[0408] In some embodiments, the host cell is isolated from a subject having a tumor, wherein the one or more cells of the tumor express CD 19 and / or CD22. In some embodiments, the host cell is isolated from a subject having a tumor, wherein the one or more cells of the tumor express CD19 and CD22. In some embodiments, the host cell has dual-antigen costimulation and specificity.

[0409] In certain embodiments, the host cell is isolated from a subject having a cancer, wherein one or more cells of the cancer cells express CD19 and / or CD22. In certain embodiments, the hostAttorney Docket No: 243734.000231cell is isolated from a subject having a cancer, wherein one or more cells of the cancer cells express CD 19 and CD22. In some embodiments, the cancer is a hematological cancer. In some embodiments, the cancer is a cancer of B cell origin.

[0410] In some embodiments, the tumor is a malignant tumor.

[0411] In some embodiments, the tumor is a hematological malignancy. Non-limiting examples of the hematological malignancy may include leukemia and lymphoma. In some embodiments, the hematological malignancy is acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), B-cell lymphoma, B-cell acute lymphoblastic leukemia (BALL), chronic lymphocytic leukemia (CLL), diffuse large B cell lymphoma (DLBCL), mantle cell lymphoma (MCL), multiple myeloma (MM), small lymphocytic lymphoma (SLL), T-cell acute Lymphoid Leukemia (TALL), and / or hairy cell leukemia.

[0412] Additional non-limiting examples of leukemia or lymphoma associated with CD 19 and / or CD22 expression include acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), ALK+ large B-cell lymphoma, B-cell lymphoma, B-cell acute lymphoblastic leukemia (BALL), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt’s lymphoma, chronic myeloid leukemia, chronic lymphocytic leukemia (CLL), diffuse large B cell lymphoma (DLBCL) such as, but not limited to, primary DLCBL of the CNS; primary cutaneous DLBCL leg type; T-cell / histiocyte rich large B-cell lymphoma; or EBV+DLBCL of the elderly, DLBCL associated with chronic inflammation, extraosseous plasmocytoma, follicular lymphoma, Hodgkin lymphoma, hairy cell leukemia, intravascular large B-cell lymphoma, lymphoplasmacytic lymphoma, large B-cell lymphoma arising in HHV8-associated multicenric Castleman disease, lymphomatoid granulomatosis, mantle cell lymphoma (MCL), multiple myeloma (MM), malignant lymphoproliferative conditions, MALT lymphoma (extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue), Marginal zone lymphoma, pediatric follicular lymphoma, myelodysplasia and myelodysplastic syndrome, nodal marginal zone lymphoma, non-Hodgkin lymphoma, plasmablastic lymphoma, plasma cell myeloma, pediatric nodal marginal zone lymphoma, primary effusion lymphoma, primary cutaneous follicle center lymphoma, plasmacytoid dendritic cell neoplasm, small lymphocytic lymphoma (SLL), solitary plasmocytoma of bone, small cell- or a large cell- follicular lymphoma, splenic marginal zone lymphoma, splenic lymphoma / leukemia (e.g., unclassifiable), splenic diffuse red pulp small B-cell lymphoma, primary mediastinal (theymic) large B-cell lymphoma, WaldenstromAttorney Docket No: 243734.000231macroglobulinemia, T-cell acute Lymphoid Leukemia (TALL), unclassifiable (e.g., with features intermediate between DLBCL and Burkitt lymphoma or intermediate between DLBCL and classical Hodgkin lymphoma), and “preleukemia” which includes a diverse set of hematological conditions united by ineffective production (or dysplasia) of myeloid blood cells, and to disease associated with B-cell antigen- (e.g., CD19 and / or CD22) expression include, but not limited to atypical or non-classical cancers, malignancies, precancerous conditions, or proliferative diseases expressing B-cell antigen (e.g., CD19 and / or CD22); and any combination thereof.

[0413] In some examples, the hematological malignancy is a B-cell malignancy. Examples include, but are not limited to, non-Hodgkin lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia (HCL), primary central nervous system (CNS) lymphoma, and primary intraocular lymphoma.

[0414] In some examples, the hematological malignancy is a T-cell malignancy. Examples include, but are not limited to, T-lymphoblastic lymphoma / leukemia, peripheral T-cell lymphoma (e g., cutaneous T-cell lymphoma, adult T-cell leukemia, angioimmunoblastic T-cell lymphoma, extranodal natural killer / T-cell lymphoma, enteropathy-associated intestinal T-cell lymphoma (EATL), anaplastic large cell lymphoma (ALCL), or peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS)).

[0415] In some embodiments, the host cell is derived from a blood sample, a marrow sample, a tissue sample, or a tumor sample.

[0416] In one aspect, the present disclosure provides a method of generating an isolated host cell described herein. The method includes genetically modifying the host cell with polynucleotide(s) encoding a CAR or CAR system and optionally an additional gene (e.g., CD20). In some embodiments, the host cell is genetically modified with a recombinant vector described herein. In specific embodiments, the vector is a viral vector, and the genetic modification is conducted by a transduction using the viral vector. The genetically modifying step may be conducted in vivo or ex vivo. In some embodiments, the genetically modifying step is conducted ex vivo. The method may further include activation and / or expansion of the host cell ex vivo before, after, and / or during the genetic modification.Isolation / EnrichmentAttorney Docket No: 243734.000231

[0417] The host cells may be autologous / autogeneic (“self’) or non-autologous (“non-self,” e.g., allogeneic, syngeneic, or xenogeneic). In certain embodiments, the host cells are obtained from a mammalian subject. In other embodiments, the host cells are obtained from a primate subject. In certain embodiments, the host cells are obtained from a human subject.

[0418] Lymphocytes can be obtained from sources such as, but not limited to, peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus issue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. Lymphocytes may also be generated by differentiation of stem cells. In certain embodiments, lymphocytes can be obtained from blood collected from a subject using techniques generally known to the skilled person, such as sedimentation, e.g., FICOLL™ separation.

[0419] In certain embodiments, cells from the circulating blood of a subject are obtained by apheresis. An apheresis device typically contains lymphocytes, including T-cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In certain embodiments, the cells collected by apheresis may be washed to remove the plasma fraction and to place the cells in an appropriate buffer or media for subsequent processing. The cells can be washed with PBS or with another suitable solution that lacks calcium, magnesium, and most, if not all other, divalent cations. A washing step may be accomplished by methods known to those in the art, such as, but not limited to, using a semiautomated flowthrough centrifuge (e.g., Cobe 2991 cell processor, or the Baxter CytoMate). After washing, the cells may be resuspended in a variety of biocompatible buffers, cell culture medias, or other saline solution with or without buffer.

[0420] In certain embodiments, host cells can be isolated from peripheral blood mononuclear cells (PBMCs) by lysing the red blood cells and depleting the monocytes. As a non-limiting example, the cells can be sorted by centrifugation through a PERCOLL™ gradient. In certain embodiments, after isolation of PBMC, both cytotoxic and helper T lymphocytes can be sorted into naive, memory, and effector T-cell subpopulations either before or after activation, expansion, and / or genetic modification.

[0421] In certain embodiments, T lymphocytes can be enriched. For example, a specific subpopulation of T lymphocytes, expressing one or more markers such as, but not limited to, CD3, CD4, CD8, CD14, CD15, CD16, CD19, CD27, CD28, CD34, CD36, CD45RA, CD45RO, CD56, CD62, CD62L, CD122, CD123, CD127, CD235a, CCR7, HLA-DR or a combination thereof usingAttorney Docket No: 243734.000231either positive or negative selection techniques. In certain embodiments, the T lymphocytes for use in the compositions of the disclosure do not express or do not substantially express one or more of the following markers: CD57, CD244, CD160, PD-1, CTLA4, TIM3, and LAG3.

[0422] In certain embodiments, NK cells can be enriched. For example, a specific subpopulation of T lymphocytes, expressing one or more markers such as, but not limited to, CD2, CD16, CD56, CD57, CD94, CD122, or a combination thereof using either positive or negative selection techniques.Stimulation / Activation

[0423] In order to reach sufficient therapeutic doses of host cell compositions, host cells are often subjected to one or more rounds of stimulation / activation. In certain embodiments, a method of producing host cells for administration to a subject comprises stimulating the host cells to become activated in the presence of one or more stimulatory signals or agents (e.g., compound, small molecule, e.g., small organic molecule, nucleic acid, polypeptide, or a fragment, isoform, variant, analog, or derivative thereof). In certain embodiments, a method of producing host cells for administration to a subject comprises stimulating the host cells to become activated and to proliferate in the presence of one or more stimulatory signals or agents.

[0424] Host cells (e.g., T lymphocytes, NK cells, and macrophages) can be activated by inducing a change in their biologic state by which the cells express activation markers, produce cytokines, proliferate, and / or become cytotoxic to target cells. All these changes can be produced by primary stimulatory signals. Costimulatory signals amplify the magnitude of the primary signals and suppress cell death following initial stimulation resulting in a more durable activation state and thus a higher cytotoxic capacity.

[0425] T cells can be activated generally using methods as described, for example, in U. S. Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; and 6,867,041, each of which is incorporated herein by reference in its entirety.

[0426] In certain embodiments, the T-cell based host cells can be activated by binding to an agent that activates CD3q.

[0427] In other embodiments, a CD2-binding agent may be used to provide a primary stimulation signal to the T-cells. For example, and not by limitation, CD2 agents include CD2 ligands and anti-CD2 antibodies, e.g., the T1 1.3 antibody in combination with the T1 1.1 or T1 1.2Attorney Docket No: 243734.000231antibody (Meuer, S. C. et al. (1984) Cell 36:897-906) and the 9.6 antibody (which recognizes the same epitope as TI 1.1) in combination with the 9-1 antibody (Yang, S. Y. et al. (1986) J. Immunol.137: 1097-1100). Other antibodies which bind to the same epitopes as any of the above-described antibodies can also be used.

[0428] In certain embodiments, the host cells are activated by administering phorbol myristate acetate (PMA) and ionomycine. In certain embodiments, the host cells are activated by administering an appropriate antigen that induces activation and then expansion. In certain embodiments, PMA, ionomycin, and / or appropriate antigen are administered with CD3 induce activation and / or expansion.

[0429] In general, the activating agents used in the present disclosure includes, but is not limited to, an antibody, a fragment thereof and a proteinaceous binding molecule with antibodylike functions. Examples of (recombinant) antibody fragments are Fab fragments, Fv fragments, single-chain Fv fragments (scFvs), a divalent antibody fragment such as an (Fab)2’ -fragment, diabodies, triabodies (Iliades, P., et al., FEBS Lett (1997) 409, 437-441), decabodies (Stone, E., et al., Journal of Immunological Methods (2007) 318, 88-94) and other domain antibodies (Holt, L. J., et al., Trends Biotechnol. (2003), 21, 11, 484-490). The divalent antibody fragment may be an (Fab)2’ -fragment, or a divalent single-chain Fv fragment while the monovalent antibody fragment may be selected from the group consisting of a Fab fragment, an Fv fragment, and a single-chain Fv fragment (scFv).

[0430] In certain embodiments, one or more binding sites of the CD3(^ agents may be a bivalent proteinaceous artificial binding molecule such as a dimeric lipocalin mutein (i.e., duocalin). In certain embodiments the receptor binding reagent may have a single second binding site, (i.e., monovalent). Examples of monovalent agents include, but are not limited to, a monovalent antibody fragment, a proteinaceous binding molecule with antibody-like binding properties or an MHC molecule. Examples of monovalent antibody fragments include, but are not limited to a Fab fragment, an Fv fragment, and a single-chain Fv fragment (scFv), including a divalent single-chain Fv fragment.

[0431] The agent that specifically binds CD3 includes, but is not limited to, an anti-CD3-antibody, a divalent antibody fragment of an anti-CD3 antibody, a monovalent antibody fragment of an anti-CD3-antibody, and a proteinaceous CD3-binding molecule with antibody-like binding properties. A proteinaceous CD3-binding molecule with antibody-like binding properties can beAttorney Docket No: 243734.000231an aptamer, a mutein based on a polypeptide of the lipocalin family, a glubody, a protein based on the ankyrin scaffold, a protein based on the crystalline scaffold, an adnectin, and an avimer. It also can be coupled to a bead.

[0432] In certain embodiments, the activating agent (e.g., CD3-binding agents) can be present in a concentration of about 0.1 pg / ml to about 10 pg / ml. In certain embodiments, the activating agent (e.g., CD3-binding agents) can be present in a concentration of about 0.2 pg / ml to about 9 pg / ml, about 0.3 pg / ml to about 8 pg / ml, about 0.4 pg / ml to about 7 pg / ml, about 0.5 pg / ml to about 6 pg / ml, about 0.6 pg / ml to about 5 pg / ml, about 0.7 pg / ml to about 4 pg / ml, about 0.8 pg / ml to about 3 pg / ml, or about 0.9 pg / ml to about 2 pg / ml. In certain embodiments, the activating agent (e.g., CD3-binding agents) is administered at a concentration of about 0.1 pg / ml, about 0.2 pg / ml, about 0.3 pg / ml, about 0.4 pg / ml, about 0.5 pg / ml, about 0.6 pg / ml, about 0.7 pg / ml, about 0.8 pM, about 0.9 pg / ml, about 1 pg / ml, about 2 pg / ml, about 3 pg / ml, about 4 pM, about 5 pg / ml, about 6 pg / ml, about 7 pg / ml, about 8 pg / ml, about 9 pg / ml, or about 10 pg / ml. In certain embodiments, the CD3-binding agents can be present in a concentration of 1 pg / ml.

[0433] NK cells can be activated generally using methods as described, for example, in U. S. Patents 7,803,376, 6,949,520, 6,693,086, 8,834,900, 9,404,083, 9,464,274, 7,435,596, 8,026,097, 8,877,182; U. S. Patent Applications US2004 / 0058445, US2007 / 0160578, US2013 / 0011376, US2015 / 0118207, US2015 / 0037887; and Int. Pat. Appl. Pub. No WO2016 / 122147, each of which is incorporated herein by reference in its entirety.

[0434] In certain embodiments, the NK based host cells can be activated by, for example and not limitation, inhibition of inhibitory receptors on NK cells (e.g., KIR2DL1, KIR2DL2 / 3, KIR2DL4, KIR2DL5A, KIR2DL5B, KIR3DL1, KIR3DL2, KIR3DL3, LILRB1, NKG2A, NKG2C, NKG2E orLILRB5 receptor).

[0435] In certain embodiments, the NK based host cells can be activated by, for example and not limitation, feeder cells (e.g., native K562 cells or K562 cells that are genetically modified to express 4-1 BBL and cytokines such as IL 15 or IL21).

[0436] In other embodiments, interferons or macrophage-derived cytokines can be used to activate NK cells. For example and not limitation, such interferons include but are not limited to interferon alpha and interferon gamma, and such cytokines include but are not limited to IL- 15, IL-2, IL-21.Attorney Docket No: 243734.000231

[0437] In certain embodiments, the NK activating agent can be present in a concentration of about 0.1 pg / ml to about 10 pg / ml. In certain embodiments, the NK activating agent can be present in a concentration of about 0.2 pg / ml to about 9 pg / ml, about 0.3 pg / ml to about 8 pg / ml, about 0.4 pg / ml to about 7 pg / ml, about 0.5 pg / ml to about 6 pg / ml, about 0.6 pg / ml to about 5 pg / ml, about 0.7 pg / ml to about 4 pg / ml, about 0.8 pg / ml to about 3 pg / ml, or about 0.9 pg / ml to about 2 pg / ml. In certain embodiments, the NK activating agent is administered at a concentration of about 0.1 pg / ml, about 0.2 pg / ml, about 0.3 pg / ml, about 0.4 pg / ml, about 0.5 pg / ml, about 0.6 pg / ml, about 0.7 pg / ml, about 0.8 pM, about 0.9 pg / ml, about 1 pg / ml, about 2 pg / ml, about 3 pg / ml, about 4 pM, about 5 pg / ml, about 6 pg / ml, about 7 pg / ml, about 8 pg / ml, about 9 pg / ml, or about 10 pg / ml. In certain embodiments, the NK activating agent can be present in a concentration of 1 pg / ml.

[0438] In certain embodiments, the activating agent is attached to a solid support such as, but not limited to, a bead, an absorbent polymer present in culture plate or well or other matrices such as, but not limited to, Sepharose or glass; may be expressed (such as in native or recombinant forms) on cell surface of natural or recombinant cell line by means known to those skilled in the art.

[0439] In certain embodiments, the host cells expressing a dual-targeting CAR system of the present disclosure produce cytokines such as but not limited to IL-2 and IFNy. In certain embodiments, the host cells expressing a dual-targeting CAR system of the present disclosure produce elevated levels of IL-2, as compared to control or non-transduced cells. In certain embodiments, the host cells expressing a dual-targeting CAR system of the present disclosure produce elevated levels of IFNy, as compared to control or non-transduced cells.Polynucleotide Transfer

[0440] In certain embodiments, the host cells are genetically modified to express a CAR or CAR system described above. The host cells can be genetically modified after stimulation / activation. In certain embodiments, the host cells are modified within 12 hours, 16 hours, 24 hours, 36 hours, or 48 hours of stimulation / activation. In certain embodiments, the cells are modified within 16 to 24 hours after stimulation / activation. In certain embodiments, the host cells are modified within 24 hours.

[0441] In order to genetically modify the host cell to express the CAR or CAR system, the CAR polynucleotide construct must be transferred into the host cell. Polynucleotide transfer mayAttorney Docket No: 243734.000231be via viral or non-viral gene methods. Suitable methods for polynucleotide delivery for use with the current methods include any method known by those of skill in the art, by which a polynucleotide can be introduced into an organelle, cell, tissue, or organism.

[0442] In some embodiments, polynucleotides are transferred to the cell in a non-viral vector. In some embodiments, the non-viral vector is a transposon. Exemplary transposons that can be used in the present disclosure include, but are not limited to, a sleeping beauty transposon and a PiggyBac transposon. In some embodiments, the non-viral vector is a minicircle plasmid or a single or double stranded DNA molecule that is used as a template for homology directed repair (HDR) based gene editing.

[0443] Nucleic acid vaccines can be used to transfer CAR polynucleotides into the host cells. Such vaccines include, but are not limited to non-viral polynucleotide vectors, “naked” DNA and RNA, and viral vectors. Methods of genetically modifying cells with these vaccines, and for optimizing the expression of genes included in these vaccines are known to those of skill in the art.

[0444] In certain embodiments, the host cells can be genetically modified by methods ordinarily used by one of skill in the art. In certain embodiments, the host cells can be transduced via retroviral transduction. References describing retroviral transduction of genes are Anderson et al., U. S. Pat. No. 5,399,346; Mann et al., Cell 33: 153 (1983); Temin et al., U. S. Pat. No. 4,650,764; Temin et al., U. S. Pat. No. 4,980,289; Markowitz et al., J. Virol. 62:1120 (1988); Temin et al., U. S. Pat. No. 5,124,263; Int. Pat. Appl. Pub. No. WO 95 / 07358, published Mar. 16, 1995, by Dougherty et al.; and Kuo et al., Blood 82:845 (1993), each of which is incorporated herein by reference in its entirety.

[0445] One method of genetic modification includes ex vivo modification. Various methods are available for transfecting cells and tissues removed from a subject via ex vivo modification. For example, retroviral gene transfer in vitro can be used to genetically modified cells removed from the subject and the cell transferred back into the subject. See e.g., Wilson et al., Science, 244:1344-1346, 1989 and Nabel et al., Science, 244(4910): 1342-1344, 1989, both of which are incorporated herein by reference in their entity. In certain embodiments, the host cells may be removed from the subject and transfected ex vivo using the polynucleotides (e.g., expression vectors) of the disclosure. In certain embodiments, the host cells obtained from the subject can beAttorney Docket No: 243734.000231transfected or transduced with the polynucleotides (eg., expression vectors) of the disclosure and then administered back to the subject.

[0446] Another method of gene transfer includes injection. In certain embodiments, a cell or a polynucleotide or a viral vector may be delivered to a cell, tissue, or organism via one or more injections (e.g., a needle injection). Non-limiting methods of injection include injection of a composition (e.g., a saline based composition). Polynucleotides can also be introduced by direct microinjection. Non-limiting sites of injection include, subcutaneous, intradermal, intramuscular, intranodal (allows for direct delivery of antigen to lymphoid tissues), intravenous, intraprostatic, intratumor, intralymphatic (allows direct administration of DCs), and intraperitoneal. It is understood that proper site of injection preparation is necessary (e.g., shaving of the site of injection to observe proper needle placement).

[0447] Electroporation is another method of polynucleotide delivery. See e.g., Potter et al., (1984) Proc. Nat’l Acad. Sci. USA, 81, 7161-7165 and Tur-Kaspa et al., (1986) Mol. Cell Biol., 6, 716-718, both of which are incorporated herein in their entirety for all purposes. Electroporation involves the exposure of a suspension of cells and DNA to a high-voltage electric discharge. In certain embodiments, cell wall-degrading enzymes, such as pectin-degrading enzymes, can be employed to render the host cells more susceptible to genetic modification by electroporation than untreated cells. See e.g., U. S. Pat. No. 5,384,253, incorporated herein by reference in its entirety for all purposes.

[0448] In vivo electroporation involves a basic injection technique in which a vector is injected intradermally in a subject. Electrodes then apply electrical pulses to the intradermal site causing the cells localized there (e.g., resident dermal dendritic cells), to take up the vector. These tumor antigen-expressing dendritic cells activated by local inflammation can then migrate to lymphnodes.

[0449] Methods of electroporation for use with this disclosure include, for example, Sardesai, N. Y., and Weiner, D. B., Current Opinion in Immunotherapy 23:421-9 (2011) and Ferraro, B. et al., Human Vaccines 7: 120-127 (2011), both of which are hereby incorporated by reference herein in their entirety for all purposes.

[0450] Additional methods of polynucleotide transfer include liposome-mediated transfection (e.g., polynucleotide entrapped in a lipid complex suspended in an excess of aqueous solution. See e.g., Ghosh and Bachhawat, (1991) In: Liver Diseases, Targeted Diagnosis and Therapy UsingAttorney Docket No: 243734.000231Specific Receptors and Ligands, pp. 87-104). Also contemplated is a polynucleotide complexed with Lipofectamine, or Superfect); DEAE-dextran (e.g., a polynucleotide is delivered into a cell using DEAE-dextran followed by polyethylene glycol. See e.g., Gopal, T. V., Mol Cell Biol. 1985 May; 5(5): 1188-90); calcium phosphate (e.g., polynucleotide is introduced to the cells using calcium phosphate precipitation. See e g., Graham and van der Eb, (1973) Virology, 52, 456-467; Chen and Okayama, Mol. Cell Biol., 7(8):2745-2752, 1987), and Rippe et al., Mol. Cell Biol., 10:689-695, 1990); sonication loading (introduction of a polynucleotide by direct sonic loading. See e.g., Fechheimer et al., (1987) Proc. Nat’l Acad. Sci. USA, 84, 8463-8467); microprojectile bombardment (e.g., one or more particles may be coated with at least one polynucleotide and delivered into cells by a propelling force. See e.g., U. S. Pat. No. 5,550,318; U. S. Pat. No.5,538,880; U. S. Pat. No. 5,610,042; and Int. Pat. Appl. Pub. No. WO 94 / 09699; Klein et al., (1987) Nature, 327, 70-73, Yang et al., (1990) Proc. Nat’l Acad. Sci. USA, 87, 9568-9572); and receptor-mediated transfection (e.g., selective uptake of macromolecules by receptor-mediated endocytosis that will be occurring in a target cell using cell type-specific distribution of various receptors. See e.g., Wu and Wu, (1987) J. Biol. Chem., 262, 4429-4432; Wagner et al., Proc. Natl. Acad. Sci. USA, 87(9):3410-3414, 1990; Perales et al., Proc. Natl. Acad. Sci. USA, 91:4086-4090, 1994; Myers, EPO 0273085; Wu and Wu, Adv. Drug Delivery Rev., 12:159-167, 1993; Nicolau et al., (1987) Methods Enzymol., 149, 157-176), each reference cited here is incorporated by reference in their entirety for all purposes.

[0451] In further embodiments, host cells are genetically modified using gene editing with homology-directed repair (HDR). Homology-directed repair (HDR) is a mechanism used by cells to repair double strand DNA breaks. In HDR, a donor polynucleotide with homology to the site of the double strand DNA break is used as a template to repair the cleaved DNA sequence, resulting in the transfer of genetic information from the donor polynucleotide to the DNA. As such, new nucleic acid material may be inserted or copied into a target DNA cleavage site. Double strand DNA breaks in host cells may be induced by a site-specific nuclease. The term “site-specific nuclease” as used herein refers to a nuclease capable of specifically recognizing and cleaving a nucleic acid (DNA or RNA) sequence. Suitable site-specific nucleases for use in the present disclosure include, but are not limited to, RNA-guided endonuclease (e.g., CRISPR-associated (Cas) proteins), zinc finger nuclease, a TALEN nuclease, or mega-TALEN nuclease. For example, a site-specific nuclease (e.g., a Cas9 + guide RNA) capable of inducing a double strand break in aAttorney Docket No: 243734.000231target DNA sequence is introduced to a host cell, along with a donor polynucleotide encoding a CAR of the present disclosure and optionally an additional protein (e.g., CD20).Expansion / Proliferation

[0452] After the host cells are activated and transduced, the cells are cultured to proliferate. T-cells may be cultured for at least 1, 2, 3, 4, 5, 6, or 7 days, at least 2 weeks, at least 1, 2, 3, 4, 5, or 6 months or more with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more rounds of expansion.[00453J Agents that can be used for the expansion of T-cells can include interleukins, such as IL-2, IL-7, IL-15, or IL-21 (see for example Cornish et al. 2006, Blood. 108(2):600-8, Bazdar and Sieg, 2007, Journal of Virology, 2007, 81(22): 12670-12674, Battalia et al, 2013, Immunology, 139(1): 109-120). Other illustrative examples for agents that may be used for the expansion of T-cells are agents that bind to CD8, CD45 or CD90, such as aCD8, aCD45 or aCD90 antibodies. Illustrative examples of T-cell population include antigen-specific T-cells, T helper cells, cytotoxic T-cells, memory T-cell (an illustrative example of memory T-cells are CD62L+CD8+specific central memory T-cells), or regulatory T-cells (an illustrative example of Treg are CD4+CD25+CD45RA+Treg cells).

[0454] Additional agents that can be used to expand T lymphocytes include methods as described, for example, in U. S. Patents 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; and 6,867,041, each of which is incorporated herein by reference in its entirety.

[0455] In certain embodiments, the agent(s) used for expansion (e.g., IL-2) are administered at about 20 units / ml to about 200 units / ml. In certain embodiments, the agent(s) used for expansion (e.g., IL-2) are administered at about 25 units / ml to about 190 units / ml, about 30 units / ml to about 180 units / ml, about 35 units / ml to about 170 units / ml, about 40 units / ml to about 160 units / ml, about 45 units / ml to about 150 units / ml, about 50 units / ml to about 140 units / ml, about 55 units / ml to about 130 units / ml, about 60 units / ml to about 120 units / ml, about 65 units / ml to about 110 units / ml, about 70 units / ml to about 100 units / ml, about 75 units / ml to about 95 units / ml, or about 80 units / ml to about 90 units / ml. In certain embodiments, the agent(s) used for expansion (e.g., IL-2) are administered at about 20 units / ml, about 25 units / ml, about 30 units / ml, 35 units / ml, 40 units / ml, 45 units / ml, about 50 units / ml, about 55 units / ml, about 60 units / ml, about 65 units / ml, about 70 units / ml, about 75 units / ml, about 80 units / ml, about 85 units / ml, about 90 units / ml, aboutAttorney Docket No: 243734.00023195 units / ml, about 100 units / ml, about 105 units / ml, about 110 units / ml, about 115 units / ml, about 120 units / ml, about 125 units / ml, about 130 units / ml, about 135 units / ml, about 140 units / ml, about 145 units / ml, about 150 units / ml, about 155 units / ml, about 160 units / ml, about 165 units / ml, about 170 units / ml, about 175 units / ml, about 180 units / ml, about 185 units / ml, about 190 units / ml, about 195 units / ml, or about 200 units / ml. In certain embodiments, the agent(s) used for expansion (e.g., IL-2) are administered at about 5 mg / ml to about 10 ng / ml. In certain embodiments, the agent(s) used for expansion (e.g., IL-2) are administered at about 5.5 ng / ml to about 9.5 ng / ml, about 6 ng / ml to about 9 ng / ml, about 6.5 ng / ml to about 8.5 ng / ml, or about 7 ng / ml to about 8 ng / ml. In certain embodiments, the agent(s) used for expansion (e.g., IL-2) are administered at about 5 ng / ml, 6 ng / ml, 7 ng / ml, 8 ng / ml, 9, ng / ml, or 10 ng / ml.

[0456] After the host cells are activated and transduced, the cells are cultured to proliferate. NK cells may be cultured for at least 1, 2, 3, 4, 5, 6, or 7 days, at least 2 weeks, at least 1, 2, 3, 4, 5, or 6 months or more with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more rounds of expansion.

[0457] Agents that can be used for the expansion of natural killer cells can include agents that bind to CD 16 or CD56, such as for example aCD16 or aCD56 antibodies. In certain embodiments, the binding agent includes antibodies (see for example Hoshino et al, Blood. 1991 Dec. 15; 78(12):3232-40.). Other agents that may be used for expansion of NK cells may be IL-15 (see for example Vitale et al. 2002. The Anatomical Record. 266:87-92, which is hereby incorporated by reference in its entirety for all purposes).

[0458] Conditions appropriate for T-cell culture include an appropriate media (e g., Minimal Essential Media (MEM), RPMI Media 1640, Lonza RPMI 1640, Advanced RPMI, Clicks, AIM-V, DMEM, a-MEM, F-12, TexMACS, X-Vivo 15, and X-Vivo 20, Optimizer, with added amino acids, sodium pyruvate, and vitamins, either serum-free or supplemented with an appropriate amount of serum (or plasma) or a defined set of hormones, and / or an amount of cytokine(s) sufficient for the growth and expansion).

[0459] Examples of other additives for host cell expansion include, but are not limited to, surfactant, piasmanate, pH buffers such as HEPES, and reducing agents such as N-acetyl-cysteine and 2-mercaptoethanol, antibiotics (e.g., penicillin and streptomycin), are included only in experimental cultures, not in cultures of cells that are to be infused into a subject. The target cells are maintained under conditions necessary to support growth, for example, an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air plus 5% CO2).Attorney Docket No: 243734.000231

[0460] In certain embodiments, host cells of the present disclosure may be modified such that the expression of an endogenous TCR, MHC molecule, or other immunogenic molecule is decreased or eliminated. When allogeneic cells are used, rejection of the therapeutic cells may be a concern as it may cause serious complications such as the graft-versus-host disease (GvHD). Although not wishing to be bound by theory, immunogenic molecules (e.g., endogenous TCRs and / or MHC molecules) are typically expressed on the cell surface and are involved in self vs nonself discrimination. Decreasing or eliminating the expression of such molecules may reduce or eliminate the ability of the therapeutic cells to cause GvHD.

[0461] In certain embodiments, expression of an endogenous TCR in the host cells is decreased or eliminated. In a particular embodiment, expression of an endogenous TCR (e.g., αβ TCR) in the host cells is decreased or eliminated. Expression of the endogenous TCR may be decreased or eliminated by disrupting the TRAC locus, TCR beta constant locus, and / or CD3 locus. In certain embodiments, expression of an endogenous TCR may be decreased or eliminated by disrupting one or more of the TRAC, TRBC1, TRBC2, CD3E, CD3G, and / or CD3D locus.

[0462] In certain embodiments, expression of one or more endogenous MHC molecules in the host cells is decreased or eliminated. Modified MHC molecule may be an MHC class I or class II molecule. In certain embodiments, expression of an endogenous MHC molecule may be decreased or eliminated by disrupting one or more of the MHC, β2M, TAP1, TAP2, CIITA, RFX5, RFXAP, and / or RFXANK locus.

[0463] Expression of the endogenous TCR, an MHC molecule, and / or any other immunogenic molecule in the host cell can be disrupted using genome editing techniques such as Clustered regularly interspaced short palindromic repeats (CRISPR) / Cas, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and Meganucleases. These genome editing methods may disrupt a target gene by entirely knocking out all of its output or partially knocking down its expression. In a particular embodiment, expression of the endogenous TCR, an MHC molecule and / or any other immunogenic molecule in the host cell is disrupted using the CRISPR / Cas technique.Pharmaceutical Compositions

[0464] In some embodiments, the compositions comprise one or more polypeptides of the CARs and other related molecules (e.g., second CAR or bispecific molecule), polynucleotides,Attorney Docket No: 243734.000231vectors comprising same, and cell compositions, as disclosed herein. Compositions of the present disclosure include, but are not limited to pharmaceutical compositions.

[0465] In one aspect, the present disclosure provides a pharmaceutical composition comprising a polynucleotide or a recombinant vector described herein, and a pharmaceutically accepted carrier and / or excipient.

[0466] In another aspect, the present disclosure provides pharmaceutical composition comprising the CAR-modified host cells described herein and a pharmaceutically acceptable carrier and / or excipient. In some embodiments, the host cells are modified with a CD 19 and / or CD22-binding CAR or CAR system.

[0467] In another aspect, the present disclosure provides pharmaceutical composition comprising host cells modified with a CD 19 and / or CD22-binding CAR or CAR system and a pharmaceutically acceptable carrier and / or excipient.

[0468] Examples of pharmaceutical carriers include but are not limited to sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions.

[0469] Compositions comprising CAR-modified host cells disclosed herein may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose or dextrans, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0470] Compositions comprising CAR-modified host cells disclosed herein may comprise one or more of the following: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer’s solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.Attorney Docket No: 243734.000231

[0471] In some embodiments, the compositions are formulated for parenteral administration, e.g., intravascular (intravenous or intraarterial), intraperitoneal, intratumoral, intraventricular, intrapleural, or intramuscular administration. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. An injectable pharmaceutical composition is preferably sterile. In some embodiments, the composition is reconstituted from a lyophilized preparation prior to administration.

[0472] In some embodiments, the CAR-modified host cells may be mixed with substances that adhere or penetrate then prior to their administration, e.g., but not limited to, nanoparticles.Therapeutic Methods

[0473] In one aspect, the present disclosure provides a method for treating a tumor in a subject in need thereof, wherein one or more cells of the tumor express CD 19 and / or CD22. A therapeutically effective amount of the CAR-modified host cells described herein or the pharmaceutical composition comprising the host cells is administered to the subject.

[0474] The term “tumor” refers to a benign or malignant abnormal growth of tissue. The term “tumor” includes cancer. Examples of tumors are, but not limited to, the soft tissue tumors (e.g., lymphomas), and tumors of the blood and blood-forming organs (e.g., leukemias), and solid tumors, which is one that grows in an anatomical site outside the bloodstream (e.g., carcinomas). Examples of cancer include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma (e.g., osteosarcoma or rhabdomyosarcoma), and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer (e.g., epithelial squamous cell cancer), adenosquamous cell carcinoma, lung cancer (e.g., including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung), cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer (e.g., including gastrointestinal cancer, pancreatic cancer), cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, primary or metastatic melanoma, multiple myeloma and B-cell lymphoma, non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, brain (e.g., high grade glioma, diffuse pontine glioma, ependymoma, neuroblastoma, or glioblastoma), as well as head and neck cancer, and associated metastases. Additional examples of tumors can be found in The Merck Manual of Diagnosis andAttorney Docket No: 243734.000231Therapy, 19th Edition, § on Hematology and Oncology, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3); The Merck Manual of Diagnosis and Therapy, 20th Edition, § on Hematology and Oncology, published by Merck Sharp & Dohme Corp., 2018 (ISBN 978-0-911-91042-1) (2018 digital online edition at internet website of Merck Manuals); and SEER Program Coding and Staging Manual 2016, each of which is incorporated by reference in its entirety for all purposes.

[0475] In some embodiments, host cells modified with a CD 19 and / or CD22-binding CAR or CAR system, or pharmaceutical compositions thereof can be used in a method for killing a tumor cell expressing CD 19 and / or CD22. In some embodiments, the tumor cell is in a subject in need thereof.

[0476] In some embodiments, host cells modified with a CD19 and / or CD22-binding CAR, or pharmaceutical compositions thereof, are administered to a subject to treat a tumor expressing CD 19 and / or CD22. In some embodiments, host cells modified with a CD 19 and / or CD22-binding CAR, or pharmaceutical compositions thereof, are administered to a subject to treat a tumor expressing CD 19 and CD22. In some embodiments, the tumor is a hematological cancer. In some embodiments, the hematological cancer is a cancer of B cell origin.

[0477] Without wishing to bound by theory, a CAR of the dual-targeting CAR system of the present disclosure may form a heterodimer with the other CAR of the dual-targeting CAR system enabling the dual-targeting CAR system to recognize one or more cells of the tumor expressing CD 19 and / or CD22 and kill those target cells.

[0478] In some embodiments, the tumor is a malignant tumor.

[0479] In some embodiments, the tumor is a hematological malignancy. Non-limiting examples of the hematological malignancy may include leukemia and lymphoma. In some embodiments, the hematological malignancy is acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), B-cell lymphoma, B-cell acute lymphoblastic leukemia (BALL), chronic lymphocytic leukemia (CLL), diffuse large B cell lymphoma (DLBCL), mantle cell lymphoma (MCL), multiple myeloma (MM), small lymphocytic lymphoma (SLL), T-cell acute Lymphoid Leukemia (TALL), and / or hairy cell leukemia.

[0480] Additional non-limiting examples of leukemia or lymphoma associated with CD19 and / or CD22 expression include acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), ALK+ large B-cell lymphoma, B-cell lymphoma, B-cell acute lymphoblasticAttorney Docket No: 243734.000231leukemia (BALL), B cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm, Burkitt’s lymphoma, chronic myeloid leukemia, chronic lymphocytic leukemia (CLL), diffuse large B cell lymphoma (DLBCL) such as, but not limited to, primary DLCBL of the CNS; primary cutaneous DLBCL leg type; T-cell / histiocyte rich large B-cell lymphoma; or EBV+DLBCL of the elderly, DLBCL associated with chronic inflammation, extraosseous plasmocytoma, follicular lymphoma, Hodgkin lymphoma, hairy cell leukemia, intravascular large B-cell lymphoma, lymphoplasmacytic lymphoma, large B-cell lymphoma arising in HHV8-associated multicenric Castleman disease, lymphomatoid granulomatosis, mantle cell lymphoma (MCL), multiple myeloma (MM), malignant lymphoproliferative conditions, MALT lymphoma (extranodal marginal zone lymphoma of mucosa-associated lymphoid tissue), Marginal zone lymphoma, pediatric follicular lymphoma, myelodysplasia and myelodysplastic syndrome, nodal marginal zone lymphoma, non-Hodgkin lymphoma, plasmablastic lymphoma, plasma cell myeloma, pediatric nodal marginal zone lymphoma, primary effusion lymphoma, primary cutaneous follicle center lymphoma, plasmacytoid dendritic cell neoplasm, small lymphocytic lymphoma (SLL), solitary plasmocytoma of bone, small cell- or a large cell-follicular lymphoma, splenic marginal zone lymphoma, splenic lymphoma / leukemia (e.g., unclassifiable), splenic diffuse red pulp small B-cell lymphoma, primary mediastinal (theymic) large B-cell lymphoma, Waldenstrom macroglobulinemia, T-cell acute Lymphoid Leukemia (TALL), unclassifiable (e.g., with features intermediate between DLBCL and Burkitt lymphoma or intermediate between DLBCL and classical Hodgkin lymphoma), and “preleukemia” which includes a diverse set of hematological conditions united by ineffective production (or dysplasia) of myeloid blood cells, and to disease associated with B-cell antigen- (e.g., CD19 and / or CD22) expression include, but not limited to atypical or non-classical cancers, malignancies, precancerous conditions, or proliferative diseases expressing B-cell antigen (e.g., CD19 and / or CD22); and any combination thereof.

[0481] In some examples, the hematological malignancy is a B-cell malignancy. Examples include, but are not limited to, non-Hodgkin lymphoma, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma, Burkitt lymphoma, lymphoplasmacytic lymphoma, hairy cell leukemia (HCL), primary central nervous system (CNS) lymphoma, and primary intraocular lymphoma.Attorney Docket No: 243734.000231

[0482] In some examples, the hematological malignancy is a T-cell malignancy. Examples include, but are not limited to, T-lymphoblastic lymphoma / leukemia, peripheral T-cell lymphoma (e.g., cutaneous T-cell lymphoma, adult T-cell leukemia, angioimmunoblastic T-cell lymphoma, extranodal natural killer / T-cell lymphoma, enteropathy-associated intestinal T-cell lymphoma (EATL), anaplastic large cell lymphoma (ALCL), or peripheral T-cell lymphoma, not otherwise specified (PTCL, NOS)).

[0483] In cases where the CAR-modified host cells also express a CD20 polypeptide, the method may further include administering an anti-CD20 antibody to the subject for removal of the isolated host cells. The anti-CD20 antibody is administered in an amount effective for sufficient removal of the isolated host cells from the subject. In some embodiments, the anti-CD20 antibody is administered in an amount effective for removal of more than 50% of the isolated host cells from the subject. For example, the anti-CD20 antibody may be administered in an amount effective for removal of more than 55%, more than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85%, more than 90%, more than 95%, more than 98%, more than 99%, or about 100% of the isolated host cells from the subject. The anti-CD20 antibody may be administered in an amount effective for removal of about 50% to about 70%, about 60% to about 80%, about 70% to about 90%, or about 80% to about 100% of the isolated host cells from the subject.

[0484] Non-limiting examples of anti-CD20 antibodies that can be used for removal the isolated host cells include rituximab, ibritumomab tiuxetan, Tositumomab, ofatumumab, ocrelizumab, TRU-015, veltuzumab, AME-133v, PRO131921, and obinutuzumab. In some embodiments, the anti-CD20 antibody is rituximab.

[0485] In some embodiments, the therapeutic method of the present disclosure includes one or more of the following steps: (a) isolating immune cells from the subject or donor; (b) modifying the immune cells ex vivo with a polynucleotide encoding a CAR and optionally an additional protein, a second CAR and / or a bispecific molecule, or a recombinant vector comprising the same; (c) optionally, expanding and / or activating the modified immune cells before, after, and / or during step (b); (d) introducing a therapeutically effective amount of the modified immune cells into the subject, and (e) in cases when the modified immune cells comprise the CD20 suicide switch, optionally, administering an anti-CD20 antibody to the subject, wherein the anti-CD20 antibodyAttorney Docket No: 243734.000231is administered in an amount effective for removal of the modified immune cells from the subject. The immune cells may be T-cells and / or NK cells.

[0486] In some of the embodiments, the method of treating a tumor in a subject in need thereof, wherein one or more cells of the tumor express CD19 and / or CD22, includes one or more of the following steps: (a) isolating T cells, NK cells, iNKT cells, or macrophages from the subject or generating T-cells, NK cells, iNKT cells, or macrophages from stem cells; (b) genetically modifying the T cells, NK cells, iNKT cells, macrophages ex vivo with the polynucleotide of any one of those described above or the vector of any one of those described above; (c) optionally, expanding and / or activating the T cells, NK cells, iNKT cells, or macrophages before, after, or during step (b); and (d) introducing the genetically modified T cells, NK cells, iNKT cells, macrophages into the subject.

[0487] In some embodiments, the stem cells comprise induced pluripotent stem cells (iPSCs).

[0488] In some embodiments, the modified host cell is an autologous cell. In some embodiments, the modified host cell is an allogeneic cell. In cases where the host cell is isolated from a donor, the method may further include a method to prevent graft-versus-host disease (GvHD) and the host cell rejection.

[0489] In some embodiments of any of the therapeutic methods described above, the composition is administered in a therapeutically effective amount. The dosages of the composition administered in the methods of the disclosure will vary widely, depending upon the subject’s physical parameters, the frequency of administration, the manner of administration, the clearance rate, and the like. The initial dose may be larger, and might be followed by smaller maintenance doses. The dose may be administered as infrequently as weekly or biweekly, or fractionated into smaller doses and administered daily, semi-weekly, etc., to maintain an effective dosage level. It is contemplated that a variety of doses will be effective to achieve in vivo persistence of modified host cells. It is also contemplated that a variety of doses will be effective to improve in vivo effector function of modified host cells.

[0490] In some embodiments, composition comprising the modified host cells manufactured by the methods described herein may be administered at a dosage of 102 to 1010 cells / kg body weight, 105 to 109 cells / kg body weight, 105 to 108 cells / kg body weight, 105 to 107 cells / kg body weight, 107 to 109 cells / kg body weight, or 107 to 108 cells / kg body weight, including allAttorney Docket No: 243734.000231integer values within those ranges. The number of modified host cells will depend on the therapeutic use for which the composition is intended for.

[0491] Modified host cells may be administered multiple times at dosages listed above. The modified host cells may be allogeneic, syngeneic, xenogeneic, or autologous to the patient undergoing therapy.

[0492] The compositions and methods described in the present disclosure may be utilized in conjunction with other types of therapy for tumors, such as chemotherapy, surgery, radiation, gene therapy, and so forth.

[0493] It is also contemplated that when used to treat various diseases / disorders, the compositions and methods of the present disclosure can be utilized with other therapeutic methods / agents suitable for the same or similar diseases / disorders. Such other therapeutic methods / agents can be co-administered (simultaneously or sequentially) to generate additive or synergistic effects. Suitable therapeutically effective dosages for each agent may be lowered due to the additive action or synergy.

[0494] In some embodiments of any of the above therapeutic methods, the method further comprises administering to the subject one or more additional compounds selected from the group consisting of immuno-suppressives, biologicals, probiotics, prebiotics, and cytokines (e.g., IFN or IL-2).

[0495] As a non-limiting example, the disclosure can be combined with other therapies that block inflammation (e.g., via blockage of IL1, INFa / p, IL6, TNF, IL23, etc.).

[0496] The methods and compositions of the disclosure can be combined with other immunomodulatory treatments such as, e.g., therapeutic vaccines (including but not limited to GV AX, DC-based vaccines, etc.), checkpoint inhibitors (including but not limited to agents that block CTLA4, PD1, LAG3, TIM3, etc.) or activators (including but not limited to agents that enhance 4- IBB, 0X40, etc.). The methods of the disclosure can be also combined with other treatments that possess the ability to modulate NKT function or stability, including but not limited to CD Id, CD Id-fusion proteins, CD Id dimers or larger polymers of CD Id either unloaded or loaded with antigens, CD 1 d-chimeric antigen receptors (CDld-CAR), or any other of the five known CD1 isomers existing in humans (CDla, CDlb, CDlc, CDle). The methods of the disclosure can also be combined with other treatments such as midostaurin, enasidenib, or a combination thereof.Attorney Docket No: 243734.000231

[0497] Therapeutic methods of the disclosure can be combined with additional immunotherapies and therapies. For example, when used for treating tumors, the compositions of the disclosure can be used in combination with conventional therapies, such as, e.g., surgery, radiotherapy, chemotherapy, or combinations thereof, depending on type of the tumor, patient condition, other health issues, and a variety of factors. In certain aspects, other therapeutic agents useful for combination tumor therapy with the inhibitors of the disclosure include anti-angiogenic agents. Many anti-angiogenic agents have been identified and are known in the art, including, e.g., TNP-470, platelet factor 4, thrombospondin- 1, tissue inhibitors of metalloproteases (TIMP1 and TIMP2), prolactin (16-Kd fragment), angiostatin (38-Kd fragment of plasminogen), endostatin, bFGF soluble receptor, transforming growth factor beta, interferon alpha, soluble KDR and FLT-1 receptors, placental proliferin-related protein, as well as those listed by Carmeliet and Jain (2000). In one embodiment, the modified host cells of the disclosure can be used in combination with a VEGF antagonist or a VEGF receptor antagonist such as anti-VEGF antibodies, VEGF variants, soluble VEGF receptor fragments, aptamers capable of blocking VEGF or VEGFR, neutralizing anti-VEGFR antibodies, inhibitors of VEGFR tyrosine kinases and any combinations thereof (e.g., anti-hVEGF antibody A4.6.1, bevacizumab or ranibizumab).

[0498] Non-limiting examples of chemotherapeutic compounds which can be used in combination treatments of the present disclosure include, for example, aminoglutethimide, amsacrine, anastrozole, asparaginase, azacitidine, beg, bicalutamide, bleomycin, buserelin, busulfan, campothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, clodronate, colchicine, cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin, daunorubicin, decitabine, dienestrol, diethylstilbestrol, docetaxel, doxorubicin, epirubicin, estradiol, estramnustine, etoposide, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide, gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide, imatinib, interferon, irinotecan, ironotecan, letrozole, leucovorin, leuprolide, levamisole, lomustine, mechlorethamine, medroxyprogesterone, megestrol, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin, paclitaxel, pamidronate, pentostatin, plicamycin, porfimer, procarbazine, raltitrexed, rituximab, streptozocin, suramin, tamoxifen, temozolomide, teniposide, testosterone, thioguanine, thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine, vincristine, vindesine, and vinorelbine.Attorney Docket No: 243734.000231

[0499] These chemotherapeutic compounds may be categorized by their mechanism of action into, for example, following groups: anti-metabolites / anti-tumor agents, such as pyrimidine analogs (5-fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine) and purine analogs, folate antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin and 2-chlorodeoxyadenosine (cladribine)); antiproliferative / antimitotic agents including natural products such as vinca alkaloids (vinblastine, vincristine, and vinorelbine); microtubule disruptors such as taxane (paclitaxel, docetaxel), vincristin, vinblastin, nocodazole, epothilones and navelbine, epidipodophyllotoxins (etoposide, teniposide); DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, cytoxan, dactinomycin, daunorubicin, doxorubicin, epirubicin, hexamethyhnelamineoxaliplatin, iphosphamide, melphalan, merchlorehtamine, mitomycin, mitoxantrone, nitrosourea, plicamycin, procarbazine, taxol, taxotere, teniposide, triethylenethiophosphoramide and etoposide (VP16)); antibiotics such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycins, plicamycin (mithramycin) and mitomycin; enzymes (L-asparaginase which systemically metabolizes L-asparagine and deprives cells which do not have the capacity to synthesize their own asparagine); antiplatelet agents; antiproliferative / antimitotic alkylating agents such as nitrogen mustards (mechlorethamine, cyclophosphamide and analogs, melphalan, chlorambucil), ethylenimines and methylmelamines (hexamethylmelamine and thiotepa), alkyl sulfonates-busulfan, nitrosoureas (carmustine (BCNU) and analogs, streptozocin), trazenes-dacarbazinine (DTIC); antiproliferative / antimitotic antimetabolites such as folic acid analogs (methotrexate); platinum coordination complexes (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogs (estrogen, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts and other inhibitors of thrombin); fibrinolytic agents (such as tissue plasminogen activator, streptokinase and urokinase), aspirin, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agents; antisecretory agents (breveldin); immunosuppressives (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); anti -angiogenic compounds (e.g., TNP-470, genistein, bevacizumab) and growth factor inhibitors (e.g., fibroblast growth factor (FGF) inhibitors); angiotensin receptor blocker; nitric oxide donors; anti-sense oligonucleotides; antibodies (trastuzumab); cell cycleAttorney Docket No: 243734.000231inhibitors and differentiation inducers (tretinoin); mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin and mitoxantrone, topotecan, irinotecan); corticosteroids (cortisone, dexamethasone, hydrocortisone, methylpednisolone, prednisone, and prenisolone); growth factor signal transduction kinase inhibitors; mitochondrial dysfunction inducers; caspase activators; and chromatin disruptors.

[0500] In various embodiments of the methods described herein, the subject is a human. The subject may be a juvenile or an adult, of any age or sex.

[0501] In accordance with the present disclosure there may be numerous tools and techniques within the skill of the art, such as those commonly used in molecular biology, pharmacology, and microbiology. Such tools and techniques are described in detail in e.g., Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press: Cold Spring Harbor, New York; Ausubel et al. eds. (2005) Current Protocols in Molecular Biology. John Wiley and Sons, Inc.: Hoboken, NJ; Bonifacino et al. eds. (2005) Current Protocols in Cell Biology. John Wiley and Sons, Inc.: Hoboken, NJ; Coligan et al. eds. (2005) Current Protocols in Immunology, John Wiley and Sons, Inc.: Hoboken, NJ; Coico et al. eds. (2005) Current Protocols in Microbiology, John Wiley and Sons, Inc.: Hoboken, NJ; Coligan et al. eds. (2005) Current Protocols in Protein Science, John Wiley and Sons, Inc.: Hoboken, NJ; and Enna et al. eds. (2005) Current Protocols in Pharmacology, John Wiley and Sons, Inc.: Hoboken, NJ.EXAMPLES

[0502] The following examples are provided to further describe some of the embodiments disclosed herein. The examples are intended to illustrate, not to limit, the disclosed embodiments.Example 1. Description of CD22-specific and CD19-specific chimeric antigen receptors.

[0503] CD22-specific chimeric antigen receptor (CD22-CAR) described herein consists of the anti-CD22 single chain antibody fragment (scFv) m971, the partial ectodomain of CD28, which functions as a hinge, the transmembrane (TM) domain of CD28, and the costimulatory / signaling domains of CD28 and CD3(^ (Q. The CD19-CAR consists of the anti-CD19 scFv FMC63, the partial ectodomain of CD28, which functions as a hinge, the TM domain of CD28, and the costimulatory domain of 4 IBB. The CARs are encoded by a recombinant lentiviral vector (LV-MND-CD22co-CD19d-CD28H) that is composed of self-inactivating (SIN) 3’ partially deletedAttorney Docket No: 243734.000231viral LTRs. The expression cassette of the LV is under the control of the MND promoter (myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primerbinding site substituted) and consists of the CD22-CAR, a 2A peptide, and the CD19-CAR (Figure 1)Single chain variable fragments used as CAR antigen binding domains

[0504] CD19-CAR: The CD19-CAR antigen binding domain consists of the anti-CD19 scFv FMC63t2-4

[0505] CD22-CAR: The CD22-CAR antigen binding domain consists of the anti-CD22 scFv m9715’7.Example 2. Introduction to CAR and lentiviral selection for the generation of CD19-CD22-CAR T cells.

[0506] The aim of this phase I clinical study was to address the emergence of antigen-loss variants through a dual -targeting approach, evaluating the safety and efficacy of a CD19-CD22-CAR T cell product described herein. A bicistronic lentiviral vector which can produce a uniform T cell product in which all transduced T cells express both CD 19- and CD22-CARs was developed. This strategy was selected to promote effective antigen recognition by both CARs.

[0507] A CD22-CAR with a CD28 hinge / TM CD28(^ signaling domain and a CD19-CAR with a CD8a hinge / TM and a 41BB(^ signaling domain (Figure 2A) were generated. In addition, two CD19-CARs were generated that lacked the signaling domain and had a hinge / TM that either matched (28TM) or did not match (8TM) the CD22-CAR (Figure 2A). To evaluate the activity of these CARs, four bicistronic vectors were generated (Figure 2B).Example 3. Generation and characterization of CD19-CD22-CAR T cells in vitro.

[0508] A lentivirus-transduction protocol was used to generate CD19-CD22-CAR T cells. Briefly, CD4- / CD8-positive T cells were selected from an apheresis product, activated, transduced with LVs, and expanded in cytokines (IL7 / IL15) for 7 to 10 days. Using this protocol, four CD19-CD22-CAR T-cell populations (19-22-FL, 22-19-FL, 22-19-8TM, 22-19-28TM) and nontransduced (NT) T cells were generated. CAR expression varied between 26.5% - 57.5% (Figures 3 A and 3B).Attorney Docket No: 243734.000231

[0509] To evaluate cytokine secretion, T cells were incubated with tumor cells for 18-24 hours. All CD19-CD22-CAR T-cell populations recognized CD19+ / CD22+targets as judged by significant IFNy production (Figure 4A). To determine their ability to target CD19- and / or CD22-positive target cells, cytotoxicity assays with CD19+ / CD22+(wild type [WT]), CD19 / CD22' (CD22 knockout [KO]), and CD197CD22+(CD19KO) BV173 cells were conducted. CD19’ / CD22' Kgla cells served as a negative control (Neg Ctrl). All bispecific CD19-CD22-CAR T-cell populations eliminated CD19+and / or CD22+target cells at an effector to target (E: T) ratio of 2: 1 (Figure 4B). At an E: T ratio of 0:5, 22-19-8TM T cells showed decreased killing of CD19+(CD22 KO) target cells. However, this only reached statistical significance for 19-22-FL vs 22-19-8TM T cells (p=0.46; Figure 4B).

[0510] In summary, the in vitro studies demonstrated that CD19-CD22-CAR T cells were successfully generated. They also highlight that only one of the CARs has to contain a signaling domain to achieve target antigen recognition through both CARs.Example 4. CD19-CD22-CAR T cells have antitumor activity in vivo,

[0511] To evaluate the antitumor activity of the generated CD19-CD22-CART cells, aBV173 ALL xenograft mouse model was used in which immunodeficient NSG mice (NOD scid gamma mice) were injected with firefly luciferase-expressing BV173 (BV173-ffluc) cells to allow for noninvasive bioluminescence imaging. NSG mice were injected with an admixture of CD19KO and CD22KO BV173-ffluc cells and on day 7 received an intravenous (i.v.) injection of a subtherapeutic CAR T-cell dose (106CAR+T cells) (Figure 5A); mice injected with NT T cells served as controls. All CD19-CD22-CAR T-cell populations had significant antitumor activity compared to NT cells (Figures 5B-5D). The 22-19-28TM T cell population achieved improved tumor control in comparison to the other evaluated CD19-CD22-CAR T-cell populations which reached significance for 19-22-FL and 22-19-8TM (Figures 5B-5D).

[0512] Only mice treated with CD19-CD22-CAR T cells that were generated with 19-22-FL or 22-19-28TM LVs resulted in long term survivors that were tumor free. These mice were rechallenged with a 2nddose of an admixture of CD19KO and CD22KO BV173-ffluc cells on day 111 post initial CAR T-cell injection (Figure 6A). Naive, untreated mice served as controls. While both CD19-CD22-CAR T-cell populations delayed tumor growth in comparison to naive mice, 22-19-28TM T cells had superior activity in comparison to 19-22-FL T cells (Figures 6A-6D).Attorney Docket No: 243734.000231

[0513] Based on the results of the in vivo experiments, CD19-CD22-CAR T cells that were generated with 19-22-FL or 22-19-28TM LVs were selected for evaluation in an ALL patient derived xenograft (PDX) model.Example 5. CD19-CD22-CAR T cells have antitumor activity in an ALL PDX model.

[0514] For this study, a patient derived xenograft (PDX) model was used that originated from a near haploid B-ALL with IGH-CRLF2 fusion that expressed both CD 19 and CD22 antigens and was genetically modified to express YFP-ffluc (PDX-B-ALL-ffluc). NSG mice were injected with 1x106PDX B-ALL-ffluc cells on day 0 and on day 7 received an i.v. injection of a subtherapeutic CAR T-cell dose (106CAR+T cells) (Figure 7A); mice injected with NT T cells served as controls. Both CD19-CD22-CAR T cell populations had significant antitumor activity compared to NT cells and there was no significant difference between them (Figures 7B-7D).

[0515] In summary, the data demonstrates that CD19-CD22-CAR T cells generated with a LV in which only one of CARs has a C, signaling domain (22-19-28TM) have at least comparable antitumor activity than CD19-CD22-CAR T cells that are generated with LVs that encode two CARs that each contain signaling domains. Since increased, signaling is associated with decreased CAR T-cell functionality12, CD19-CD22-CAR T cells transduced with the 22-19-28TM LV were selected for further clinical study.Example 6. Comparison of CD22-19-CAR and CD19-CAR T cells in vivo.

[0516] One concern of expressing a CD22-CAR in CD19-CAR T cells is that it diminishes the antitumor activity of CD19-CAR T cells. To evaluate this experimentally, the antitumor activity of the standard CD19-CAR with a 41BB.(^ signaling domain (19-FL) versus the selected CD19-CD22-CAR T-cell product was compared in the CD22KO BV173 cell model. This CD19-CAR T-cell product was selected since it has shown potent antitumor activity in early phase testing in pediatric patients with CD19-positive ALL.1NSG mice were injected with CD22KO BV173-ffluc cells and on day 7 received an i.v. injection of a subtherapeutic CAR T-cell dose (106CAR+ T cells) (Figure 8A); mice injected with NT T cells served as controls. All CD19-CD22-CAR and CD19-CAR T-cell populations had significant antitumor activity compared to NT cells (Figures 8B-8D). In addition, the antitumor activity of CD19-CD22-CAR T cells was significantly prolonged when compared to that of CD19-CAR T cells (Figures 8B-8D). Thus, CD22-CARAttorney Docket No: 243734.000231expression in the developed CD19-CD22-CAR T-cell product did not diminish the antitumor activity of the expressed CD19-CAR.Conclusion

[0517] In summary, the comprehensive preclinical studies described herein demonstrated that the selected CD19-CD22-CAR T cell product (22-19-28TM) has potent antitumor activity in relevant preclinical models. Thus, early phase clinical testing is warranted in pediatric patients with recurrent / refractory CD19+and / or CD22+ALL.Example 7. CD22-CARs with varying hinge and transmembrane domains are expressed using lentiviral vectors and recognize CD22+tumor cells.

[0518] A variety of approaches have been attempted to target CD22 with CAR T cells for patients with B cell lineage hematologic malignancies. Because CD22 is a target with relatively low antigen density, this approach incorporates CD28 costimulation to enhance initial antitumor activity. A panel of lentiviral constructs were generated to evaluate various hinge and transmembrane configurations, each containing a CD20 detection sequence, m971 -based CD22 single chain variable fragment, CD28 costimulatory domain, and CD3^ activation domain, with varying hinge and transmembrane configurations (Figure 9A). These CD22-CAR constructs successfully expressed in donor human T cells (Figure 9B) and expanded in culture after transduction (Figure 9C). When co-cultured with CD22-positive tumor cells (BV173), CD22-CAR T cells produced IFNy (Figure 9D) and IL-2 (Figure 9E), with no significant fFNy or IL-2 production in the presence of CD22-negative tumor cells (Kgl A) or media control. CD22-CAR T cells demonstrated specific antitumor activity in a luciferase-based cytotoxicity assay (Figure 9F).The constructs containing CD8a (CD22-CAR-CD8) and CD28 (CD22CAR-CD28) hinge and transmembrane components were selected for further development.Example 8. Optimized CD22-CAR T cell constructs have robust antigen-specific antitumor activity in vitro.

[0519] The CD22-CAR constructs containing either CD8 or CD28 hinge / transmembrane domains were optimized, including removal of the CD20 tag (Figure 10A). Corresponding ACAR (delta CAR) constructs lacking costimulatory and activation domains were designed to serve as negative controls (Figure 10A). These optimized CD22-C AR vectors efficiently transduced donorAttorney Docket No: 243734.000231human T cells (Figure 10B) and expanded well in culture (Figure IOC). The phenotype of transduced CD22-CAR T cells was similar to that of nontransduced T cells and ACAR controls (Figure 10D). When cultured with CD22-positive tumor cells, CD22-CAR T cells containing either a CD8 or CD28 hinge / transmembrane domain displayed significant IFNy (Figure 10E) and IL-2 (Figure 10F) production, which was not seen in the presence of CD22-negative tumor cells or with ACAR constructs. Optimized CD22-CAR constructs produced specific antitumor activity at a range of effector to target ratios against CD22+positive tumor cells (Figure 10G).Example 9. CD22-CAR T cells have robust and durable in vivo antitumor activity.

[0520] Having established in vitro activity of CD22-CAR T cells, the leading candidates (CD22-CAR-CD8 and CD22-CAR-CD28) were evaluated in vivo. In this model, NSG mice were injected with luciferase-labeled BV173 tumor cells (Tumor.ffluc), followed by infusion of CD22-CAR or control T cells (Figure HA). Both CD22-CAR treatment groups provided survival advantage in comparison to controls (Figure 11B), with CD22-CD8 and CD22-CD28- CAR T cells demonstrating robust antitumor activity and sustained disease control (Figure 11C). Having established in vivo activity, surviving animals in the CD22-CAR-CD8 and CD22-CAR-CD28 treatment groups were then challenged with another exposure to Tumor.ffluc cells to assess for the presence of persisting functional CAR T cells (Figure 11D). The CD22-CAR-CD8 group had slower tumor growth than untreated animals, but all eventually had tumor progression (Figure HE). In contrast, animals in the group previously treated with CD22-CAR-CD28 T cells all experienced either transient or sustained disease control. Based on robust initial antitumor activity and evidence of superior functional persistence, the CD22-CAR-CD28 construct was selected for incorporation into the CD19-CD22 dual targeting approach.References1. Talleur AC, Qudeimat A, Metais JY, Langfitt D, Mamcarz E, Crawford JC, Huang S, Cheng C, Hurley C, Madden R, Sharma A, Suliman A, Srinivasan A, Velasquez MP, Obeng EA, Willis C, Akel S, Karol SE, Inaba H, Bragg A, Zheng W, Z...

Claims

1. Attorney Docket No: 243734.000231Claims1. A polynucleotide encoding a dual-targeting chimeric antigen receptor (CAR) system comprising two CARs, wherein each CAR comprises an extracellular domain comprising a target binding moiety;wherein optionally, one of the CARs lacks an activation domain; andwherein one target binding moiety binds to cluster of differentiation 19 (CD 19) and the other target binding moiety binds to cluster of differentiation 22 (CD22).

2. A pair of polynucleotides encoding a dual-targeting chimeric antigen receptor (CAR) system comprising two CARs, wherein the first polynucleotide encodes a first CAR of the dualtargeting CAR system, and the second polynucleotide encodes a second CAR of the dualtargeting CAR system,wherein each CAR comprises an extracellular domain comprising a target binding moiety; wherein optionally, one of the CARs lacks an activation domain; andwherein one target binding moiety binds to cluster of differentiation 19 (CD 19) and the other target binding moiety binds to cluster of differentiation 22 (CD22).

3. A polynucleotide encoding a chimeric antigen receptor (CAR) comprising an extracellular domain comprising a target binding moiety, wherein the CAR lacks an activation domain; and wherein the target binding moiety binds to cluster of differentiation 19 (CD 19) or cluster of differentiation 22 (CD22).

4. The polynucleotide of claim 1 or the pair of polynucleotides of claim 2, wherein one of the CARs lacks an activation domain.

5. The polynucleotide or the pair of polynucleotides of claim 4, wherein the CAR lacking the activation domain further lacks a costimulatory domain.

6. The polynucleotide of any one of claims 1 and 3-5, or the pair of polynucleotides of any one of claims 2, 4, and 5, wherein each target binding moiety is independently an antibody or antigen-binding fragment thereof, or a peptide.Attorney Docket No: 243734.0002317. The polynucleotide or the pair of polynucleotides of claim 6, wherein the antibody or antigenbinding fragment thereof is selected from a single chain variable fragment (scFv), Fab, Fab’, F(ab’)2, Fv fragment, disulfide-linked Fv (dsFv), diabody, VHH, VNAR, single-domain antibody (sdAb), nanobody, dAb fragment, Fd’ fragment, and Fd fragment.

8. The polynucleotide of any one of claims 1 and 3-7 or the pair of polynucleotides of any one of claims 2 and 4-7, wherein the anti-CD19 target binding moiety is an anti-CD19 antibody or antigen-binding fragment thereof.

9. The polynucleotide or the pair of polynucleotides of claim 8, wherein the anti-CD19 antibody or antigen-binding fragment thereof is derived from FMC63, HD37, CAT19, budoprutug, coltuximab, denintuzumab, duvortuxizumab, emfizatamab, englumafusp alfa, inebilizumab, loncastuximab, obexelimab, tafasitamab, taplitumomab, or zeripatamig.

10. The polynucleotide of any one of claims 1 and 3-9 or the pair of polynucleotides of any one of claims 2 and 4-9, wherein the anti-CD19 target binding moiety is an anti-CD19 scFv.

11. The polynucleotide or the pair of polynucleotides of claim 10, wherein the anti-CD19 scFv is derived from FMC63.

12. The polynucleotide or the pair of polynucleotides of claim 11, wherein the anti-CD19 scFv comprises a heavy chain complementarity determining region 1 (HCDR1), an HCDR2, and an HCDR3 contained within a heavy chain variable domain (VH) comprising the amino acid sequence EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETT YYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQ GTSVTVSS (SEQ ID NO: 20), or an amino acid sequence having at least 80% identity thereto; and / or a light chain complementarity determining region 1 (LCDR1), an LCDR2, and an LCDR3 contained within a light chain variable domain (VL) comprising the amino acid sequenceAttorney Docket No: 243734.000231DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSG VPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT (SEQ ID NO: 18), or an amino acid sequence having at least 80% identity thereto.

13. The polynucleotide or the pair of polynucleotides of claim 12, wherein the anti-CD19 scFv comprises an HCDR1 comprising the amino acid sequence DYGVS (SEQ ID NO: 127), an HCDR2 comprising the amino acid sequence VIWGSETTYYNSALKS (SEQ ID NO: 128), and an HCDR3 comprising the amino acid sequence HYYYGGSYAMDY (SEQ ID NO: 129); and / or an LCDR1 comprising the amino acid sequence RASQDISKYLN (SEQ ID NO: 130), an LCDR2 comprising the amino acid sequence HTSRLHS (SEQ ID NO: 131), and an LCDR3 comprising the amino acid sequence QQGNTLPYT (SEQ ID NO: 132).

14. The polynucleotide or the pair of polynucleotides of any one of claims 11-13, wherein the antiCD 19 scFv comprises a VH comprising the amino acid sequence EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETT YYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQ GTSVTVSS (SEQ ID NO: 20), or an amino acid sequence having at least 80% identity thereto; and / or a VL comprising the amino acid sequence DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSG VPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT (SEQ ID NO: 18), or an amino acid sequence having at least 80% identity thereto.

15. The polynucleotide or the pair of polynucleotides of any one of claims 11-14, wherein the nucleotide sequence encoding the VH of the anti-CD19 scFv comprises the sequence of SEQ ID NO: 4, or a nucleotide sequence having at least 80% identity thereto; and / or the nucleotide sequence encoding the VL of the anti-CD19 scFv comprises the sequence of SEQ ID NO: 2, or a nucleotide sequence having at least 80% identity thereto.

16. The polynucleotide of any one of claims 1 and 3-15 or the pair of polynucleotides of any one of claims 2 and 4-15, wherein the anti-CD22 target binding moiety is an anti-CD22 antibody or antigen-binding fragment thereof.Attorney Docket No: 243734.00023117. The polynucleotide or the pair of polynucleotides of claim 16, wherein the anti-CD22 antibody or antigen-binding fragment thereof is derived from m971, LT22, or 16P.

18. The polynucleotide of any one of claims 1 and 3-17 or the pair of polynucleotides of any one of claims 2 and 4-17, wherein the anti-CD22 target binding moiety is an anti-CD22 scFv.

19. The polynucleotide or the pair of polynucleotides of claim 18, wherein the anti-CD22 scFv is derived from m971.

20. The polynucleotide or the pair of polynucleotides of claim 19, wherein the anti-CD22 scFv comprises an HCDR1, an HCDR2, and an HCDR3 contained within a VH comprising the amino acid sequence QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRS KWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDI WGQGTMVTVSS (SEQ ID NO: 26), or an amino acid sequence having at least 80% identity thereto; and / or an LCDR1, an LCDR2, and an LCDR3 contained within a VL comprising the amino acid sequence DIQMTQ SP S SL S AS VGDRVTITCRASQTIWS YLNWYQQRPGKAPNLLIYAAS SLQ SG VPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIKR (SEQ ID NO: 27), or an amino acid sequence having at least 80% identity thereto.

21. The polynucleotide or the pair of polynucleotides of claim 20, wherein the anti-CD22 scFv comprises an HCDR1 comprising the amino acid sequence GDSVSSNSAA (SEQ ID NO: 117), an HCDR2 comprising the amino acid sequence TYYRSKWYN (SEQ ID NO: 118), and an HCDR3 comprising the amino acid sequence AREVTGDLEDAFDI (SEQ ID NO: 119); and / or an LCDR1 comprising the amino acid sequence QTIWS (SEQ ID NO: 120), an LCDR2 comprising the amino acid sequence AAS, and an LCDR3 comprising the amino acid sequence QQSYSIPQT (SEQ ID NO: 122).Attorney Docket No: 243734.00023122. The polynucleotide or the pair of polynucleotides of any one of claims 19-21, wherein the anti- CD22 scFv comprises a VH comprising the amino acid sequence QVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRS KWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDI WGQGTMVTVSS (SEQ ID NO: 26), or an amino acid sequence having at least 80% identity thereto; and / or a VL comprising the amino acid sequenceDIQMTQ SP S SL S AS VGDRVTITCRASQTIWS YLNWYQQRPGKAPNLLIYAAS SLQ SG VPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIKR (SEQ ID NO: 27), or an amino acid sequence having at least 80% identity thereto.

23. The polynucleotide or the pair of polynucleotides of any one of claims 19-22, wherein the anti- CD22 scFv comprises a nucleotide sequence encoding the VH of the anti-CD22 scFv comprises the sequence of SEQ ID NO: 10, or SEQ ID NO: 35, or a nucleotide sequence having at least 80% identity thereto; wherein the nucleotide sequence encoding the VL of the anti- CD22 scFv comprises the sequence of SEQ ID NO: 12, or SEQ ID NO: 37, or a nucleotide sequence having at least 80% identity thereto.

24. The polynucleotide or the pair of polynucleotides of any one of claims 10-23, wherein the scFv further comprises a linker between the VH and VL.

25. The polynucleotide or the pair of polynucleotides of claim 24, wherein the linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 19), GGGGS (SEQ ID NO: 91), GGGGSGGGGS (SEQ ID NO: 92), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 93), KESGSVSSEQLAQFRSLD (SEQ ID NO: 94), EGKSSGSGSESKST (SEQ ID NO: 95), EGKSSGSGSESKSTQ (SEQ ID NO: 96), GSTSGSGKSSEGKG (SEQ ID NO: 97), SSADDAKKDDAKKDDAKKDDAKKDG (SEQ ID NO: 98), EGKSSGSGSESKVD (SEQ ID NO: 99), ESGSVSSEELAFRSLD (SEQ ID NO: 100), or an amino acid sequence having at least 80% sequence identity thereto.Attorney Docket No: 243734.00023126. The polynucleotide or the pair of polynucleotides of claim 24 or claim 25, wherein the linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 19), or an amino acid sequence having at least 80% sequence identity thereto.

27. The polynucleotide or the pair of polynucleotides of claim 26, wherein the nucleotide sequence encoding the linker comprises the nucleotide sequence GGCGGCGGAGGAAGCGGGGGCGGAGGATCTGGTGGTGGTGGATCT (SEQ ID NO: 3), GGTGGCGGAGGTAGTGGTGGTGGCGGAAGTGGCGGAGGTGGAAGC (SEQ ID NO: 11), GGCGGCGGAGGAAGCGGAGGCGGAGGATCTGGTGGTGGTGGATCT (SEQ ID NO: 32), or GGAGGCGGAGGTTCTGGTGGCGGAGGAAGTGGTGGCGGCGGATCT (SEQ ID NO: 36), or a nucleotide sequence having at least 80% sequence identity thereto.

28. The polynucleotide or the pair of polynucleotides of any one of claims 11-15 and 24-27, wherein the anti-CD19 scFv comprises the amino acid sequence DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLH SGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGGG5GGGG5GGGG5EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGL EWLGVIWGSETTYYNSALKSRLTIIKDNSKSOVFLKMNSLQTDDTAIYYCAKHYYY GGSYAMDYWGQGTSVTVSS (SEQ ID NO: 61) or EVKLOESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETT YYNSALKSRLTUKDNSKSOVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGO GTSVTVSSGGGG GGGG5GGGG5DIQMTQTTSSLSASLGDRVTISCRASQDISKYL NWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFC QQGNTLPYTFGGGTKLEIT (SEQ ID NO: 133), or an amino acid sequence having at least 80% sequence identity thereto.

29. The polynucleotide or the pair of polynucleotides of claim 28, wherein the nucleotide sequence encoding the anti-CD19 scFv comprises the sequence GACATCCAGATGACCCAGACAACCAGCAGCCTGTCTGCCAGCCTGGGCGAT AGAGTGACCATCAGCTGTAGAGCCAGCCAGGACATCAGCAAGTACCTGAAC TGGTATCAGCAGAAACCCGACGGCACCGTGAAGCTGCTGATCTACCACACCAttorney Docket No: 243734.000231AGCAGACTGCACAGCGGCGTGCCAAGCAGATTTTCTGGCAGCGGCTCTGGC ACCGACTACAGCCTGACAATCAGCAACCTGGAACAAGAGGATATCGCTACC TACTTCTGCCAGCAAGGCAACACCCTGCCTTACACCTTTGGCGGAGGCACC AAGCTGGAAATCACAGGCGGCGGAGGAAGCGGGGGCGGAGGATCTGGTGGTGGT GGA TCTGAAGTGAAACTGCAAGAGTCTGGCCCTGGCCTGGTGGCCCCATCTCAAT CTCTGAGCGTGACCTGTACCGTCAGCGGAGTGTCCCTGCCTGATTATGGCGTGTC CTGGATCCGGCAGCCTCCTAGAAAAGGCCTGGAATGGCTGGGCGTGATCTGGGG CAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTGACCATCATCAA GGACAACTCCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGA CACCGCCATCTACTATTGCGCCAAGCACTACTACTACGGCGGCAGCTACGCCATG GATTATTGGGGCCAGGGCACCAGCGTGACCGTGTCTAGT (SEQ ID NO: 62), GACATCCAGATGACCCAGACAACCAGCAGCCTGTCTGCCAGCCTGGGCGAT AGAGTGACCATCAGCTGTAGAGCCAGCCAGGACATCAGCAAGTACCTGAAC TGGTATCAGCAGAAACCCGACGGCACCGTGAAGCTGCTGATCTACCACACC AGCAGACTGCACAGCGGCGTGCCAAGCAGATTTTCTGGCAGCGGCTCTGGC ACCGACTACAGCCTGACAATCAGCAACCTGGAACAAGAGGATATCGCTACC TACTTCTGCCAGCAAGGCAACACCCTGCCTTACACCTTTGGCGGAGGCACC AAGCTGGAAATCACAGGCGGCGGAGGAAGCGGAGGCGGAGGATCTGGTGGTGGT GGATCTGAAGTGAAACTGCAAGAGTCTGGCCCTGGCCTGGTGGCCCCATCTCAAT CTCTGAGCGTGACCTGTACCGTCAGCGGAGTGTCCCTGCCTGATTATGGCGTGTC CTGGATCCGGCAGCCTCCTAGAAAAGGCCTGGAATGGCTGGGCGTGATCTGGGG CAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTGACCATCATCAA GGACAACTCCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGA CACCGCCATCTACTATTGCGCCAAGCACTACTACTACGGCGGCAGCTACGCCATG GATTATTGGGGCCAGGGCACCAGCGTGACCGTGTCTAGT (SEQ ID NO: 65), GAAGTGAAACTGCAAGAGTCTGGCCCTGGCCTGGTGGCCCCATCTCAATCTCTG AGCGTGACCTGTACCGTCAGCGGAGTGTCCCTGCCTGATTATGGCGTGTCCTGGA TCCGGCAGCCTCCTAGAAAAGGCCTGGAATGGCTGGGCGTGATCTGGGGCAGCG AGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTGACCATCATCAAGGACA ACTCCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCG CCATCTACTATTGCGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGATTAAttorney Docket No: 243734.000231TTGGGGCCAGGGCACCAGCGTGACCGTGTCTAGTGGCGGCGGAGGAAGCGGGGGCGGAGGATCTGGTGGTGGTGGATCTGACATCCAGATGACCCAGACAACCAGCA GCCTGTCTGCCAGCCTGGGCGATAGAGTGACCATCAGCTGTAGAGCCAGCC AGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAACCCGACGGCACCG TGAAGCTGCTGATCTACCACACCAGCAGACTGCACAGCGGCGTGCCAAGCA GATTTTCTGGCAGCGGCTCTGGCACCGACTACAGCCTGACAATCAGCAACC TGGAACAAGAGGATATCGCTACCTACTTCTGCCAGCAAGGCAACACCCTGC CTTACACCTTTGGCGGAGGCACCAAGCTGGAAATCACA (SEQ ID NO: 134), or GAAGTGAAACTGCAAGAGTCTGGCCCTGGCCTGGTGGCCCCATCTCAATCTCTG AGCGTGACCTGTACCGTCAGCGGAGTGTCCCTGCCTGATTATGGCGTGTCCTGGA TCCGGCAGCCTCCTAGAAAAGGCCTGGAATGGCTGGGCGTGATCTGGGGCAGCG AGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTGACCATCATCAAGGACA ACTCCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCG CCATCTACTATTGCGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGATTA TTGGGGCCAGGGCACCAGCGTGACCGTGTCTAGTGGCGGCGGAGGAAGCGGAGGCGGAGGATCTGGTGGTGGTGGATCTGACATCCAGATGACCCAGACAACCAGCA GCCTGTCTGCCAGCCTGGGCGATAGAGTGACCATCAGCTGTAGAGCCAGCC AGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAACCCGACGGCACCG TGAAGCTGCTGATCTACCACACCAGCAGACTGCACAGCGGCGTGCCAAGCA GATTTTCTGGCAGCGGCTCTGGCACCGACTACAGCCTGACAATCAGCAACC TGGAACAAGAGGATATCGCTACCTACTTCTGCCAGCAAGGCAACACCCTGC CTTACACCTTTGGCGGAGGCACCAAGCTGGAAATCACA (SEQ ID NO 135), or or a nucleotide sequence having at least 80% identity thereto.

30. The polynucleotide or the pair of polynucleotides of any one of claims 19-27, wherein the anti- CD22 scFv comprises the amino acid sequence OVOLQOSGPGLVKPSOTLSLTCAISGDSVSSNSAAWNWIROSPSRGLEWLGRTYYRS KWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDI WGQGTMVTVSSGGGG5GGGG5GGGGADIOMTOSPSSLSASVGDRVTITCRASOTI WSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAEDFA TYYCQQSYSIPQTFGQGTKLEIKR (SEQ ID NO 63) orAttorney Docket No: 243734.000231DIQMTQSPSSLSASVGDRVTTTCRASQTTWSYLNWYQQRPGKAPNLLIYAASSLQ SGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIKRGG GG5GGGG5GGGG5QV0LQ0SGPGLVKPS0TLSLTCAISGDSVSSNSAAWNWIR0SPS RGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNOFSLQLNSVTPEDTAVYYCA REVTGDLEDAFDIWGQGTMVTVSS (SEQ ID NO: 136), or an amino acid sequence having at least 80% sequence identity thereto.

31. The polynucleotide or the pair of polynucleotides of claim 30, wherein the nucleotide sequence encoding the anti-CD22 scFv comprises the sequence CAGGTCCAGCTGCAGCAATCTGGACCAGGACTGGTCAAGCCCTCTCAGACCCTGAGCCTGACATGTGCCATCTCCGGCGATAGCGTGTCCAGCAATTCTGCCGCCTGGAACTGGATCAGACAGTCCCCTAGCAGAGGCCTCGAGTGGCTCGGCAGAACCTACTATCGGAGCAAGTGGTACAACGACTACGCCGTGTCCGTGAAGTCCAGGATCACCATCAATCCCGACACCAGCAAGAACCAGTTCTCCCTGCAGCTGAACAGCGTGACACCTGAGGATACCGCCGTGTACTACTGTGCCAGAGAAGTGACCGGCGACCTGGAAGATGCCTTCGATATCTGGGGACAGGGAACCATGGTCACAGTGTCATCTGGTGGCGGAGGTAGTGGTGGCGGAAGTGGCGGAGGTGGAAGCGACATTCAGATGACTC AGTCCCCAAGCAGCCTGAGCGCCTCCGTGGGAGACAGAGTGACAATTACAT GCCGGGCCAGCCAGACCATCTGGTCCTATCTCAATTGGTATCAACAGAGGC CCGGCAAGGCCCCTAACCTGCTTATCTATGCCGCCTCTAGCCTGCAGTCTG GCGTGCCATCTAGATTCAGCGGAAGAGGCTCCGGCACAGATTTCACCCTGA CCATTAGCTCACTGCAGGCCGAAGATTTCGCCACCTATTACTGTCAGCAGTC CTACAGCATCCCTCAGACCTTCGGCCAGGGAACAAAGCTCGAGATCAAGCGG (SEQ ID NO: 64), CAGGTTCAGCTGCAGCAGTCTGGCCCTGGCCTGGTTAAGCCTAGCCAGACACTG AGCCTGACCTGTGCCATCTCTGGCGATAGCGTGTCCAGCAATAGCGCCGCCTGG AACTGGATCAGACAGAGCCCTTCTAGAGGCCTGGAATGGCTGGGCAGAACCTAC TACCGGTCCAAGTGGTACAACGACTACGCCGTGTCCGTGAAGTCCCGGATCACC ATCAATCCCGACACCAGCAAGAACCAGTTCAGCCTCCAGCTGAACAGCGTGACC CCTGAGGATACCGCCGTGTACTACTGCGCCAGAGAAGTGACAGGCGATCTGGAA GATGCCTTCGACATCTGGGGCCAGGGCACAATGGTCACAGTTTCTAGCGGAGGCAttorney Docket No: 243734.000231GGAGGTTCTGGTGGCGGAGGAAGTGGTGGCGGCGGATCTGATATCCAGATGACA CAGAGCCCCTCCAGCCTGTCTGCCAGCGTGGGAGATAGAGTGACCATCACC TGTAGAGCCAGCCAGACCATCTGGTCCTACCTGAATTGGTATCAGCAGAGG CCCGGCAAGGCCCCTAACCTGCTGATCTATGCTGCCAGCTCTCTGCAGTCC GGCGTGCCAAGCAGATTTTCCGGAAGAGGCAGCGGCACCGACTTCACCCTG ACAATTTCTAGCCTGCAAGCCGAGGACTTCGCCACCTACTACTGCCAGCAG TCCTACAGCATCCCTCAGACCTTTGGCCAGGGGACCAAGCTGGAAATCAAG AGA (SEQ ID NO: 66), GACATTCAGATGACTCAGTCCCCAAGCAGCCTGAGCGCCTCCGTGGGAGAC AGAGTGACAATTACATGCCGGGCCAGCCAGACCATCTGGTCCTATCTCAAT TGGTATCAACAGAGGCCCGGCAAGGCCCCTAACCTGCTTATCTATGCCGCC TCTAGCCTGCAGTCTGGCGTGCCATCTAGATTCAGCGGAAGAGGCTCCGGC ACAGATTTCACCCTGACCATTAGCTCACTGCAGGCCGAAGATTTCGCCACCT ATTACTGTCAGCAGTCCTACAGCATCCCTCAGACCTTCGGCCAGGGAACAA AGCTCGAGATCAAGCGGGGTGGCGGAGGTAGTGGTGGTGGCGGAAGTGGCGGA GGTGGAAGCCAGGTCCAGCTGCAGCAATCTGGACCAGGACTGGTCAAGCCCTCT CAGACCCTGAGCCTGACATGTGCCATCTCCGGCGATAGCGTGTCCAGCAATTCTG CCGCCTGGAACTGGATCAGACAGTCCCCTAGCAGAGGCCTCGAGTGGCTCGGCA GAACCTACTATCGGAGCAAGTGGTACAACGACTACGCCGTGTCCGTGAAGTCCA GGATCACCATCAATCCCGACACCAGCAAGAACCAGTTCTCCCTGCAGCTGAACA GCGTGACACCTGAGGATACCGCCGTGTACTACTGTGCCAGAGAAGTGACCGGCG ACCTGGAAGATGCCTTCGATATCTGGGGACAGGGAACCATGGTCACAGTGTCAT CT (SEQ ID NO: 137), or GATATCCAGATGACACAGAGCCCCTCCAGCCTGTCTGCCAGCGTGGGAGAT AGAGTGACCATCACCTGTAGAGCCAGCCAGACCATCTGGTCCTACCTGAAT TGGTATCAGCAGAGGCCCGGCAAGGCCCCTAACCTGCTGATCTATGCTGCC AGCTCTCTGCAGTCCGGCGTGCCAAGCAGATTTTCCGGAAGAGGCAGCGGC ACCGACTTCACCCTGACAATTTCTAGCCTGCAAGCCGAGGACTTCGCCACCT ACTACTGCCAGCAGTCCTACAGCATCCCTCAGACCTTTGGCCAGGGGACCA AGCTGGAAATCAAGAGAGGAGGCGGAGGTTCTGGTGGCGGAGGAAGTGGTGGCG GCGGATCTCAGGTTCAGCTGCAGCAGTCTGGCCCTGGCCTGGTTAAGCCTAGCCAAttorney Docket No: 243734.000231GACACTGAGCCTGACCTGTGCCATCTCTGGCGATAGCGTGTCCAGCAATAGCGC CGCCTGGAACTGGATCAGACAGAGCCCTTCTAGAGGCCTGGAATGGCTGGGCAG AACCTACTACCGGTCCAAGTGGTACAACGACTACGCCGTGTCCGTGAAGTCCCG GATCACCATCAATCCCGACACCAGCAAGAACCAGTTCAGCCTCCAGCTGAACAG CGTGACCCCTGAGGATACCGCCGTGTACTACTGCGCCAGAGAAGTGACAGGCGA TCTGGAAGATGCCTTCGACATCTGGGGCCAGGGCACAATGGTCACAGTTTCTAGC(SEQ ID NO: 138), or a nucleotide sequence having at least 80% identity thereto.

32. The polynucleotide of any one of claims 1, 3, 4, and 6-31 or the pair of polynucleotides of any one of claims 2, 4, and 6-31, wherein each CAR comprises a cytoplasmic domain comprising a costimulatory domain.

33. The polynucleotide or the pair of polynucleotides of claim 32, wherein the costimulatory domain present in one CAR is different from the costimulatory domain present in the other CAR.

34. The polynucleotide or the pair of polynucleotides of claim 32 or claim 33, wherein each costimulatory domain is independently derived from CD28, 4-1BB (CD137), CD27, CD40, CD134 (0X40), CD226, CD79A, ICOS, BTLA, CD30, GITR, HVEM, IL-2Rβ, a STAT3- binding YXXQ in which X is any amino acid, or MyD88, or any combination thereof.

35. The polynucleotide of or the pair of polynucleotides of claim 32 or claim 33, wherein each costimulatory domain is independently derived from CD28 or 4-1BB.

36. The polynucleotide or the pair of polynucleotides of any one of claims 32-35, wherein the costimulatory domain of the anti-CD22 CAR is derived from CD28; and / or the costimulatory domain of the anti-CD19 CAR is derived from 4- IBB.

37. The polynucleotide or the pair of polynucleotides of any one of claims 32-35, wherein the costimulatory domain of the anti-CD19 CAR is derived from CD28; and / or the costimulatory domain of the anti-CD22 CAR is derived from 4- IBB.Attorney Docket No: 243734.00023138. The polynucleotide or the pair of polynucleotides of any one of claims 34-37, wherein the CD28 costimulatory domain comprises the amino acid sequence RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 30), or an amino acid sequence having at least 80% sequence identity thereto.

39. The polynucleotide or the pair of polynucleotides of claim 38, wherein the nucleotide sequence encoding the CD28 costimulatory domain comprises the sequence CGAAGCAAGCGGAGCCGGCTGCTGCACTCCGACTACATGAACATGACCCCTAGA CGGCCTGGACCTACCAGAAAGCACTACCAGCCTTACGCTCCTCCTAGAGACTTCG CCGCCTACCGGTCC (SEQ ID NO: 15), or CGAAGCAAGCGGAGCCGGCTGCTGCACAGCGACTACATGAACATGACCCCTAGA CGGCCCGGACCAACCAGAAAGCACTACCAGCCTTACGCTCCTCCTAGAGATTTC GCCGCCTACCGGTCC (SEQ ID NO: 40), or a nucleotide sequence having at least 80% sequence identity thereto.

40. The polynucleotide or the pair of polynucleotides of any one of claims 34-37, wherein the 4- 1BB costimulatory domain comprises the amino acid sequence KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 23), or an amino acid sequence having at least 80% sequence identity thereto.

41. The polynucleotide or the pair of polynucleotides of claim 40, wherein the nucleotide sequence encoding the 4- IBB costimulatory domain comprises the sequence AAGCGGGGCAGAAAGAAACTGCTCTACATCTTCAAGCAGCCCTTCATGCGGCCC GTGCAGACCACACAAGAGGAAGATGGCTGCTCCTGCAGATTCCCCGAGGAAGAA GAAGGCGGCTGCGAGCTG (SEQ ID NO: 7), or a nucleotide sequence having at least 80% sequence identity thereto.

42. The polynucleotide of any one of claims 1 and 4-41 or the pair of polynucleotides of any one of claims 2 and 4-41, wherein the anti-CD22 CAR comprises an activation domain.Attorney Docket No: 243734.00023143. The polynucleotide of any one of claims 1 and 4-42 or the pair of polynucleotides of any one of claims 2 and 4-42, wherein the activation domain is derived from CD3(^, CD38, CD3s, CD3y, zeta-chain-associated protein kinase (ZAP) 70 (ZAP70), DNAX-activating protein (DAP) 10 (DAP10), DAP12, Fc epsilon receptor I y chain (FCER1G), CD226, NKG2C, NKG2D, NKG2E, FcR, CD5, CD22, CD66d, CD79B, or CD79A44. The polynucleotide of any one of claims 1 and 4-43 or the pair of polynucleotides of any one of claims 2 and 4-43, wherein the activation domain is derived from CD3ζ.

45. The polynucleotide or the pair of polynucleotides of claim 43 or claim 44, wherein the CD3^ activation domain comprises the amino acid sequence RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR (SEQ ID NO: 24), or an amino acid sequence having at least 80% sequence identity thereto.

46. The polynucleotide or the pair of polynucleotides of claim 45, wherein the nucleotide encoding the CD3ζ activation domain comprises the sequence AGAGTGAAGTTCAGCAGATCCGCCGACGCTCCTGCCTATCAGCAGGGCCAAAAC CAGCTGTACAACGAGCTGAACCTGGGGAGAAGAGAAGAGTACGACGTGCTGGA CAAGCGGAGAGGCAGAGATCCTGAAATGGGCGGCAAGCCCAGACGGAAGAATC CTCAAGAGGGCCTGTATAATGAGCTGCAGAAAGACAAGATGGCCGAGGCCTACA GCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTG TACCAGGGCCTGAGCACCGCCACCAAGGATACCTATGATGCCCTGCACATGCAG GCCCTGCCTCCAAGA (SEQ ID NO: 8), AGAGTGAAATTCTCCAGAAGCGCTGACGCCCCAGCTTACCAGCAGGGACAGAAT CAGCTCTATAACGAACTGAATCTCGGCAGGCGCGAGGAATATGATGTGCTGGAT AAGAGGCGCGGCAGGGACCCAGAGATGGGAGGAAAGCCTCGGCGGAAGAACCC ACAAGAAGGACTTTACAACGAACTGCAAAAGGATAAGATGGCAGAAGCTTACTC CGAGATTGGCATGAAGGGCGAACGTCGGAGAGGAAAAGGCCACGACGGACTCT ATCAGGGACTGTCCACAGCCACAAAGGACACATACGACGCACTCCATATGCAGGAttorney Docket No: 243734.000231CTCTCCCACCTAGA (SEQ ID NO: 16), or AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAA CCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGA CAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACC CTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACA GTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTT TACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAG GCCCTGCCCCCTCGC (SEQ ID NO: 41), or a nucleotide sequence having at least 80% sequence identity thereto.

47. The polynucleotide of any one of claims 1 and 3-46 or the pair of polynucleotides of any one of claims 2 and 4-46, wherein each CAR further comprises a transmembrane domain.

48. The polynucleotide of any one of claims 1 and 3-47 or the pair of polynucleotides of any one of claims 2 and 4-47, wherein each transmembrane domain is independently derived from a, or chain of the T-cell receptor, CD8a, CD28, CD8, CD3 epsilon, CD45, CD4, CD3(^, CD5, CD9, CD16, CD22, CD33, CD37, CD40, CD64, CD80, CD86, CD134 (0X40), CD137, or CD 154, or CD7, or a combination thereof.

49. The polynucleotide of any one of claims 1 and 3-48 or the pair of polynucleotides of any one of claims 2 and 4-48, wherein each transmembrane domain is independently derived from CD8ot or CD28.

50. The polynucleotide of any one of claims 1 and 3-49 or the pair of polynucleotides of any one of claims 2 and 4-49, wherein the transmembrane domain of the anti-CD22 CAR is derived from CD28; and / or the transmembrane domain of the anti-CD19 CAR is derived from CD28.

51. The polynucleotide of any one of claims 1 and 3-49 or the pair of polynucleotides of any one of claims 2 and 4-49, wherein the transmembrane domain of the anti-CD22 CAR is derived from CD28; and / or the transmembrane domain of the anti-CD19 CAR is derived from CD8a.Attorney Docket No: 243734.00023152. The polynucleotide or the pair of polynucleotides of any one of claims 48, 49, and 51, wherein the CD8a transmembrane domain comprises the amino acid sequence IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 22), IYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLH (SEQ ID NO: 46), or IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 124), or an amino acid sequence having at least 80% sequence identity thereto.

53. The polynucleotide or the pair of polynucleotides of claim 52, wherein the nucleotide sequence encoding the CD8a transmembrane domain comprises the sequence ATCTACATCTGGGCCCCTCTGGCTGGAACATGTGGCGTGCTGCTGCTGAGCCTGG TCATCACCCTGTATTGC (SEQ ID NO: 6), ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGG TTATCACCCTTTACTGC (SEQ ID NO: 39), ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGG TTATCACCCTTTACTGCCGAAGCAAGCGGAGCCGGCTGCTGCACTAA (SEQ ID NO: 45), ATCTACATCTGGGCCCCTCTGGCTGGAACATGTGGCGTGCTGCTGCTGAGCCTGG TCATCACC (SEQ ID NO: 123), or ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGG TTATCACC (SEQ ID NO: 126), or a nucleotide sequence having at least 80% sequence identity thereto.

54. The polynucleotide or the pair of polynucleotides of any one of claims 48-51, wherein the CD28 transmembrane domain comprises the amino acid sequence FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 29), or FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLH (SEQ ID NO: 48), or an amino acid sequence having at least 80% sequence identity thereto.

55. The polynucleotide or the pair of polynucleotides of claim 54, wherein the nucleotide encoding the CD28 transmembrane domain comprises the sequence TTCTGGGTGCTCGTTGTTGTTGGCGGCGTGCTGGCCTGTTATAGCCTGCTGGTTACAttorney Docket No: 243734.000231CGTGGCCTTCATCATCTTTTGGGTC (SEQ ID NO: 14), TTTTGGGTCTTGGTGGTTGTGGGCGGCGTACTGGCCTGCTACTCCCTGCTAGTCA CCGTCGCGTTCATCATCTTCTGGGTC (SEQ ID NO: 34), TTCTGGGTGCTCGTTGTTGTTGGCGGCGTGCTGGCCTGTTACAGCCTGCTGGTTAC CGTGGCCTTCATCATCTTTTGGGTC (SEQ ID NO: 43), or TTCTGGGTGCTCGTTGTTGTTGGCGGCGTGCTGGCCTGTTACAGCCTGCTGGTTAC CGTGGCCTTCATCATCTTTTGGGTCCGAAGCAAGCGGAGCCGGCTGCTGCAC(SEQ ID NO: 47), or a nucleotide sequence having at least 80% sequence identity thereto.

56. The polynucleotide or the pair of polynucleotides of any one of claims 47-55, wherein each extracellular domain further comprises a hinge domain between the target-binding moiety and the transmembrane domain.

57. The polynucleotide or the pair of polynucleotides of claim 56, wherein each hinge domain is independently derived from CD8a stalk; CD28; CD4; or IgGl, IgG2, IgG3, IgG4, IgMl, IgM2, IgAl, IgA2, IgD, or IgE, or a chimera thereof; or any combination thereof.

58. The polynucleotide or the pair of polynucleotides of claim 56 or claim 57, wherein each hinge domain is independently derived from CD8a stalk or CD28.

59. The polynucleotide or the pair of polynucleotides of any one of claims 56-58, wherein the hinge domain of the anti-CD22 CAR is derived from CD28, and / or the hinge domain of the anti-CD19 CAR is derived from CD28.

60. The polynucleotide or the pair of polynucleotides of any one of claims 56-58, wherein the hinge domain of the anti-CD22 CAR is derived from CD28, and / or the hinge domain of the anti-CD19 CAR is derived from CD8a stalk.

61. The polynucleotide or the pair of polynucleotides of any one of claims 57, 58, and 60, wherein the CD8a stalk hinge domain comprises the amino acid sequenceAttorney Docket No: 243734.000231TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 21), or an amino acid sequence having at least 80% sequence identity thereto.

62. The polynucleotide or the pair of polynucleotides of claim 61, wherein the nucleotide sequence encoding the CD8a stalk hinge domain comprises the sequence ACAACAACCCCTGCTCCTCGGCCTCCTACACCAGCTCCTACAATTGCCAGCCAGC CACTGTCTCTGAGGCCCGAAGCTTGTAGACCTGCTGCTGGCGGAGCCGTGCATAC AAGAGGACTGGATTTCGCCTGCGAC (SEQ ID NO: 5), or ACCACGACGCCAGCGCCGCGACCACCAACGCCGGCGCCCACCATCGCGTCGCAG CCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCAC ACGAGGGGGCTGGACTTCGCCTGTGAT (SEQ ID NO: 38), or a nucleotide sequence having at least 80% sequence identity thereto.

63. The polynucleotide or the pair of polynucleotides of any one of claims 57-60, wherein the CD28 hinge domain comprises the amino acid sequence IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 28), or an amino acid sequence having at least 80% sequence identity thereto.

64. The polynucleotide or the pair of polynucleotides of claim 63, wherein the nucleotide sequence encoding the CD28 hinge domain comprises the sequence ATCGAAGTGATGTACCCTCCTCCTTACCTGGACAACGAGAAGTCCAACGGCACC ATCATCCACGTGAAGGGCAAGCACCTGTGTCCTTCTCCACTGTTCCCCGGACCAA GCAAGCCT (SEQ ID NO: 13), ATCGAGGTGATGTACCCACCGCCTTACCTGGACAACGAGAAAAGCAACGGCACC ATCATTCACGTGAAGGGCAAACACCTGTGCCCATCTCCTCTGTTCCCTGGACCAT CCAAGCCC (SEQ ID NO: 33), or ATCGAAGTGATGTACCCGCCTCCTTACCTGGACAACGAGAAGTCCAACGGCACC ATCATCCACGTGAAGGGAAAGCACCTGTGTCCTTCTCCACTGTTCCCCGGACCTA GCAAGCCT (SEQ ID NO: 42), or a nucleotide sequence having at least 80% sequence identity thereto.Attorney Docket No: 243734.00023165. The polynucleotide or the pair of polynucleotides of claim 57, wherein the IgG4 hinge domain comprises the amino acid sequence ESKYGPPCPSCPAPEFEGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFN WYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSS IEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSL GK (SEQ ID NO: 60), or an amino acid sequence having at least 80% sequence identity thereto.

66. The polynucleotide or the pair of polynucleotides of claim 65, wherein the nucleotide sequence encoding the IgG4 hinge domain comprises the sequence GAGTCTAAGTACGGCCCTCCTTGTCCTAGCTGCCCCGCTCCTGAATTTGAAGGCG GCCCTTCCGTGTTCCTGTTTCCTCCAAAGCCTAAGGACACCCTGATGATCAGCAG AACCCCTGAAGTGACCTGCGTGGTGGTGGACGTGTCCCAAGAGGATCCTGAGGT GCAGTTCAATTGGTACGTGGACGGCGTGGAAGTGCACAACGCCAAGACCAAGCC TAGAGAGGAACAGTTCCAGAGCACCTACAGAGTGGTGTCCGTGCTGACAGTGCT GCACCAGGATTGGCTGAACGGCAAAGAGTACAAGTGCAAGGTGTCCAACAAGG GCCTGCCTAGCAGCATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCAAGA GAACCCCAGGTGTACACACTGCCTCCAAGCCAAGAGGAAATGACCAAGAACCA GGTGTCCCTGACCTGCCTGGTCAAGGGCTTCTACCCTTCCGATATCGCCGTGGAA TGGGAGAGCAATGGCCAGCCTGAGAACAACTACAAGACCACACCTCCTGTGCTG GACAGCGACGGCTCATTCTTCCTGTACAGCAGACTGACCGTGGACAAGAGCAGA TGGCAAGAGGGCAACGTGTTCAGCTGCAGCGTGATGCACGAGGCCCTGCACAAC CACTACACCCAGAAGTCTCTGAGCCTGAGCCTCGGCAAG (SEQ ID NO: 54), or a nucleotide sequence having at least 80% sequence identity thereto.

67. A polynucleotide encoding a dual-targeting chimeric antigen receptor (CAR) system comprising two CARs, wherein the two CARs comprise:(i) an anti-cluster of differentiation 22 (CD22) CAR comprising:an extracellular domain comprising an anti-CD22 target binding moiety,a hinge domain and a transmembrane domain derived from CD28,Attorney Docket No: 243734.000231a cytoplasmic domain comprising a CD28 costimulatory domain and a CD3ζ activation domain; and(ii) an anti-cluster of differentiation 19 (CD 19) CAR comprisingan extracellular domain comprising an anti-CD19 target binding moiety,a hinge domain and a transmembrane domain derived from CD28 or CD8a,a cytoplasmic domain comprising a 4- IBB costimulatory domain and lacking an activation domain.

68. A pair of polynucleotides encoding a dual -targeting chimeric antigen receptor (CAR) system comprising two CARs, wherein(i) the first polynucleotide encodes an anti-cluster of differentiation 22 (CD22) CAR comprising:an extracellular domain comprising an anti-CD22 target binding moiety,a hinge domain and a transmembrane domain derived from CD28,a cytoplasmic domain comprising a CD28 costimulatory domain and a CD3ζ activation domain; and(ii) the second polynucleotide encodes an anti-cluster of differentiation 19 (CD 19) CAR comprisingan extracellular domain comprising an anti-CD19 target binding moiety,a hinge domain and a transmembrane domain derived from CD28 or CD8a,a cytoplasmic domain comprising a 4- IBB costimulatory domain and lacking an activation domain.

69. A polynucleotide encoding a dual-targeting chimeric antigen receptor (CAR) system comprising two CARs, wherein the two CARs comprise:(i) an anti-cluster of differentiation 22 (CD22) CAR comprising:an extracellular domain comprising an anti-CD22 target binding moiety,a hinge domain and a transmembrane domain derived from CD28,a cytoplasmic domain comprising a CD28 costimulatory domain and a CD3ζ activation domain; and(ii) an anti-cluster of differentiation 19 (CD 19) CAR comprisingAttorney Docket No: 243734.000231an extracellular domain comprising an anti-CD19 target binding moiety,a hinge domain and a transmembrane domain derived from CD28 or CD8a,a cytoplasmic domain comprising a 4-1BB costimulatory domain and a CD3(^ activation domain.

70. A pair of polynucleotides encoding a dual-targeting chimeric antigen receptor (CAR) system comprising two CARs, wherein(i) the first polynucleotide encodes an anti-cluster of differentiation 22 (CD22) CAR comprising:an extracellular domain comprising an anti-CD22 target binding moiety,a hinge domain and a transmembrane domain derived from CD28,a cytoplasmic domain comprising a CD28 costimulatory domain and a CD3ζ activation domain; and(ii) the second polynucleotide encodes an anti-cluster of differentiation 19 (CD 19) CAR comprisingan extracellular domain comprising an anti-CD19 target binding moiety,a hinge domain and a transmembrane domain derived from CD28 or CD8a,a cytoplasmic domain comprising a 4-1BB costimulatory domain and a CD3(^ activation domain.

71. The polynucleotide of claim 67 or claim 69 or the pair of polynucleotides of claim 68 or claim 70, wherein each target binding moiety is independently an antibody or antigen-binding fragment thereof, or a peptide.

72. The polynucleotide or the pair of polynucleotides of claim 71, wherein the antibody or antigenbinding fragment thereof is selected from a single chain variable fragment (scFv), Fab, Fab', F(ab')2, Fv fragment, disulfide-linked Fv (dsFv), diabody, VHH, VNAR, single-domain antibody (sdAb), nanobody, dAb fragment, Fd' fragment, and Fd fragment.Attorney Docket No: 243734.00023173. The polynucleotide of any one of claims 67, 69, 71, and 72 or the pair of polynucleotides of any one of claims 68 and 70-72, wherein the anti-CD19 target binding moiety is an anti-CD19 antibody or antigen-binding fragment thereof.

74. The polynucleotide or the pair of polynucleotides of claim 73, wherein the anti-CD19 antibody or antigen-binding fragment thereof is derived from FMC63, HD37, CAT, budoprutug, coltuximab, denintuzumab, duvortuxizumab, emfizatamab, englumafusp alfa, inebilizumab, loncastuximab, obexelimab, tafasitamab, taplitumomab, or zeripatamig.

75. The polynucleotide of any one of claims 67, 69, and 71-74 or the pair of polynucleotides of any one of claims 68 and 70-74, wherein the anti-CD19 target binding moiety is an anti-CD19 scFv.

76. The polynucleotide or the pair of polynucleotides of claim 75, wherein the anti-CD19 scFv is derived from FMC63.

77. The polynucleotide or the pair of polynucleotides of claim 76, wherein the anti-CD19 scFv comprises an HCDR1, an HCDR2, and an HCDR3 contained within a VH comprising the amino acid sequence EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETT YYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQ GTSVTVSS (SEQ ID NO: 20), or an amino acid sequence having at least 80% identity thereto; and / or an LCDR1, an LCDR2, and an LCDR3 contained within a VL comprising the amino acid sequence DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSG VPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT (SEQ ID NO: 18), or an amino acid sequence having at least 80% identity thereto.

78. The polynucleotide or the pair of polynucleotides of claim 77, wherein the anti-CD19 scFv comprises an HCDR1 comprising the amino acid sequence DYGVS (SEQ ID NO: 127), an HCDR2 comprising the amino acid sequence VIWGSETTYYNSALKS (SEQ ID NO: 128),Attorney Docket No: 243734.000231and anHCDR3 comprising the amino acid sequence HYYYGGSYAMDY (SEQ ID NO: 129); and / or an LCDR1 comprising the amino acid sequence RASQDISKYLN (SEQ ID NO: 130), an LCDR2 comprising the amino acid sequence HTSRLHS (SEQ ID NO: 131), and an LCDR3 comprising the amino acid sequence QQGNTLPYT (SEQ ID NO: 132).

79. The polynucleotide or the pair of polynucleotides of any one of claims 76-78, wherein the antiCD 19 scFv comprises a VH comprising the amino acid sequence EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETT YYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQ GTSVTVSS (SEQ ID NO: 20), or an amino acid sequence having at least 80% identity thereto; and / or a VL comprising the amino acid sequence DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSG VPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT (SEQ ID NO: 18), or an amino acid sequence having at least 80% identity thereto.

80. The polynucleotide or the pair of polynucleotides of any one of claims 76-79, wherein the nucleotide sequence encoding the VH of the anti-CD19 scFv comprises the sequence of SEQ ID NO: 4, or a nucleotide sequence having at least 80% identity thereto; and / or the nucleotide sequence encoding the VL of the anti-CD19 scFv comprises the sequence of SEQ ID NO: 2, or a nucleotide sequence having at least 80% identity thereto.

81. The polynucleotide of any one of claims 67, 69, and 73-80 or the pair of polynucleotides of any one of claims 68 and 70-78, wherein the anti-CD22 target binding moiety is an anti-CD22 antibody or antigen-binding fragment thereof.

82. The polynucleotide or the pair of polynucleotides of claim 81, wherein the anti-CD22 antibody or antigen-binding fragment thereof is derived from m971, LT22, or 16P.

83. The polynucleotide of any one of claims 67, 69, and 71-82 or the pair of polynucleotides of any one of claims 68 and 70-80, wherein the anti-CD22 target binding moiety is an anti-CD22 scFv.Attorney Docket No: 243734.00023184. The polynucleotide or the pair of polynucleotides of claim 83, wherein the anti-CD22 scFv is derived from m971.

85. The polynucleotide or the pair of polynucleotides of claim 84, wherein the anti-CD22 scFv comprises an HCDR1, an HCDR2, and an HCDR3 contained within a VH comprising the amino acid sequence Q VQLQQ SGPGLVKP SQTLSLTC AISGD S VS SNS AAWNWIRQ SP SRGLEWLGRTYYRS KWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDI WGQGTMVTVSS (SEQ ID NO: 26), or an amino acid sequence having at least 80% identity thereto; and / or an LCDR1, an LCDR2, and an LCDR3 contained within a VL comprising the amino acid sequence DIQMTQ SP S SL S AS VGDRVTITCRASQTIWS YLNWYQQRPGKAPNLLIYAAS SLQ SG VPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIKR (SEQ ID NO: 27), or an amino acid sequence having at least 80% identity thereto.

86. The polynucleotide or the pair of polynucleotides of claim 85, wherein the anti-CD22 scFv comprises an HCDR1 comprising the amino acid sequence GDSVSSNSAA (SEQ ID NO: 117), an HCDR2 comprising the amino acid sequence TYYRSKWYN (SEQ ID NO: 118), and an HCDR3 comprising the amino acid sequence AREVTGDLEDAFDI (SEQ ID NO: 119); and / or an LCDR1 comprising the amino acid sequence QTIWS (SEQ ID NO: 120), an LCDR2 comprising the amino acid sequence AAS, and an LCDR3 comprising the amino acid sequence QQSYSIPQT (SEQ ID NO: 122).

87. The polynucleotide or the pair of polynucleotides of any one of claims 83-86, wherein the anti- CD22 scFv comprises a VH comprising the amino acid sequenceQ VQLQQ SGPGLVKP SQTLSLTC AISGD S VS SNS AAWNWIRQ SP SRGLEWLGRTYYRS KWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDI WGQGTMVTVSS (SEQ ID NO: 26), or an amino acid sequence having at least 80% identity thereto; and / or a VL comprising the amino acid sequenceAttorney Docket No: 243734.000231DIQMTQ SP S SL S A S VGDRVTITCR A SQTIW S YLNW YQQRPGK APNLLI Y A A S SLQ SG VPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIKR (SEQ ID NO: 27), or an amino acid sequence having at least 80% identity thereto.

88. The polynucleotide or the pair of polynucleotides of any one of claims 83-87, wherein the anti- CD22 scFv comprises a nucleotide sequence encoding the VH of the anti-CD22 scFv comprises the sequence of SEQ ID NO: 10, or SEQ ID NO: 35, or a nucleotide sequence having at least 80% identity thereto; wherein the nucleotide sequence encoding the VL of the anti- CD22 scFv comprises the sequence of SEQ ID NO: 12, or SEQ ID NO: 37, or a nucleotide sequence having at least 80% identity thereto.

89. The polynucleotide or the pair of polynucleotides of any one of claims 76-88, wherein the scFv further comprises a linker between the VH and VL.

90. The polynucleotide or the pair of polynucleotides of claim 89, wherein the linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 19), GGGGS (SEQ ID NO: 91), GGGGSGGGGS (SEQ ID NO: 92), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 93), KESGSVSSEQLAQFRSLD (SEQ ID NO: 94), EGKSSGSGSESKST (SEQ ID NO: 95), EGKSSGSGSESKSTQ (SEQ ID NO: 96), GSTSGSGKSSEGKG (SEQ ID NO: 97), SSADDAKKDDAKKDDAKKDDAKKDG (SEQ ID NO: 98), EGKSSGSGSESKVD (SEQ ID NO: 99), ESGSVSSEELAFRSLD (SEQ ID NO: 100), or an amino acid sequence having at least 80% sequence identity thereto.

91. The polynucleotide or the pair of polynucleotides of claim 89 or claim 90, wherein the linker comprises the amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 19), or an amino acid sequence having at least 80% sequence identity thereto.

92. The polynucleotide or the pair of polynucleotides of claim 91, wherein the nucleotide sequence encoding the linker comprises the nucleotide sequence GGCGGCGGAGGAAGCGGGGGCGGAGGATCTGGTGGTGGTGGATCT (SEQ ID NO: 3), GGTGGCGGAGGTAGTGGTGGTGGCGGAAGTGGCGGAGGTGGAAGC (SEQ IDAttorney Docket No: 243734.000231NO: 11), GGCGGCGGAGGAAGCGGAGGCGGAGGATCTGGTGGTGGTGGATCT (SEQ ID NO: 32), or GGAGGCGGAGGTTCTGGTGGCGGAGGAAGTGGTGGCGGCGGATCT (SEQ ID NO: 36), or a nucleotide sequence having at least 80% sequence identity thereto.

93. The polynucleotide or the pair of polynucleotides of any one of claims 76-80 and 89-90, wherein the anti-CD19 scFv comprises the amino acid sequence DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLH SGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGGG GAGGGY S'GGGYESEVKLOESGPGLVAPSOSLSVTCTVSGVSLPDYGVSWIROPPRKGL EWLGVIWGSETTYYNSALKSRLTIIKDNSKSOVFLKMNSLQTDDTAIYYCAKHYYY GGSYAMDYWGQGTSVTVSS (SEQ ID NO: 61) or EVKLQESGPGLVAPSOSLSVTCTVSGVSLPDYGVSWIROPPRKGLEWLGVIWGSETT YYNSALKSRLTUKDNSKSOVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGO GISyiySSGGGG GGGG5GGGG5DIQMTQTTSSLSASLGDRVTISCRASQDISKYL NWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFC QQGNTLPYTFGGGTKLEIT (SEQ ID NO: 133), or an amino acid sequence having at least 80% sequence identity thereto.

94. The polynucleotide or the pair of polynucleotides of claim 93, wherein the nucleotide sequence encoding the anti-CD19 scFv comprises the sequence GACATCCAGATGACCCAGACAACCAGCAGCCTGTCTGCCAGCCTGGGCGAT AGAGTGACCATCAGCTGTAGAGCCAGCCAGGACATCAGCAAGTACCTGAAC TGGTATCAGCAGAAACCCGACGGCACCGTGAAGCTGCTGATCTACCACACC AGCAGACTGCACAGCGGCGTGCCAAGCAGATTTTCTGGCAGCGGCTCTGGC ACCGACTACAGCCTGACAATCAGCAACCTGGAACAAGAGGATATCGCTACC TACTTCTGCCAGCAAGGCAACACCCTGCCTTACACCTTTGGCGGAGGCACC AAGCTGGAAATCACAGGCGGCGGAGGAAGCGGGGGCGGAGGATCTGGTGGTGGT GG47C7GAAGTGAAACTGCAAGAGTCTGGCCCTGGCCTGGTGGCCCCATCTCAAT CTCTGAGCGTGACCTGTACCGTCAGCGGAGTGTCCCTGCCTGATTATGGCGTGTC CTGGATCCGGCAGCCTCCTAGAAAAGGCCTGGAATGGCTGGGCGTGATCTGGGG CAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTGACCATCATCAAAttorney Docket No: 243734.000231GGACAACTCCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGA CACCGCCATCTACTATTGCGCCAAGCACTACTACTACGGCGGCAGCTACGCCATG GATTATTGGGGCCAGGGCACCAGCGTGACCGTGTCTAGT (SEQ ID NO: 62), GACATCCAGATGACCCAGACAACCAGCAGCCTGTCTGCCAGCCTGGGCGAT AGAGTGACCATCAGCTGTAGAGCCAGCCAGGACATCAGCAAGTACCTGAAC TGGTATCAGCAGAAACCCGACGGCACCGTGAAGCTGCTGATCTACCACACC AGCAGACTGCACAGCGGCGTGCCAAGCAGATTTTCTGGCAGCGGCTCTGGC ACCGACTACAGCCTGACAATCAGCAACCTGGAACAAGAGGATATCGCTACC TACTTCTGCCAGCAAGGCAACACCCTGCCTTACACCTTTGGCGGAGGCACC AAGCTGGAAATCACAGGCGGCGGAGGAAGCGGAGGCGGAGGATCTGGTGGTGGT GG4TCTGAAGTGAAACTGCAAGAGTCTGGCCCTGGCCTGGTGGCCCCATCTCAAT CTCTGAGCGTGACCTGTACCGTCAGCGGAGTGTCCCTGCCTGATTATGGCGTGTC CTGGATCCGGCAGCCTCCTAGAAAAGGCCTGGAATGGCTGGGCGTGATCTGGGG CAGCGAGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTGACCATCATCAA GGACAACTCCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGA CACCGCCATCTACTATTGCGCCAAGCACTACTACTACGGCGGCAGCTACGCCATG GATTATTGGGGCCAGGGCACCAGCGTGACCGTGTCTAGT (SEQ ID NO: 65), GAAGTGAAACTGCAAGAGTCTGGCCCTGGCCTGGTGGCCCCATCTCAATCTCTG AGCGTGACCTGTACCGTCAGCGGAGTGTCCCTGCCTGATTATGGCGTGTCCTGGA TCCGGCAGCCTCCTAGAAAAGGCCTGGAATGGCTGGGCGTGATCTGGGGCAGCG AGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTGACCATCATCAAGGACA ACTCCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCG CCATCTACTATTGCGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGATTA TTGGGGCCAGGGCACCAGCGTGACCGTGTCTAGTGGCGGCGG^GGA4GCGGGGG CGGAGGATCTGGTGGTGGTGGATCTGACATCCAGATGACCCAGACAACCAGCA GCCTGTCTGCCAGCCTGGGCGATAGAGTGACCATCAGCTGTAGAGCCAGCC AGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAACCCGACGGCACCG TGAAGCTGCTGATCTACCACACCAGCAGACTGCACAGCGGCGTGCCAAGCA GATTTTCTGGCAGCGGCTCTGGCACCGACTACAGCCTGACAATCAGCAACC TGGAACAAGAGGATATCGCTACCTACTTCTGCCAGCAAGGCAACACCCTGC CTTACACCTTTGGCGGAGGCACCAAGCTGGAAATCACA (SEQ ID NO 134), orAttorney Docket No: 243734.000231GAAGTGAAACTGCAAGAGTCTGGCCCTGGCCTGGTGGCCCCATCTCAATCTCTG AGCGTGACCTGTACCGTCAGCGGAGTGTCCCTGCCTGATTATGGCGTGTCCTGGA TCCGGCAGCCTCCTAGAAAAGGCCTGGAATGGCTGGGCGTGATCTGGGGCAGCG AGACAACCTACTACAACAGCGCCCTGAAGTCCCGGCTGACCATCATCAAGGACA ACTCCAAGAGCCAGGTGTTCCTGAAGATGAACAGCCTGCAGACCGACGACACCG CCATCTACTATTGCGCCAAGCACTACTACTACGGCGGCAGCTACGCCATGGATTA TTGGGGCCAGGGCACCAGCGTGACCGTGTCTAGTGGCGGCGG4GGA4GCGG^GG CGG^GG^ZCZGGZGGZGGZGG^ZCZGACATCCAGATGACCCAGACAACCAGCA GCCTGTCTGCCAGCCTGGGCGATAGAGTGACCATCAGCTGTAGAGCCAGCC AGGACATCAGCAAGTACCTGAACTGGTATCAGCAGAAACCCGACGGCACCG TGAAGCTGCTGATCTACCACACCAGCAGACTGCACAGCGGCGTGCCAAGCA GATTTTCTGGCAGCGGCTCTGGCACCGACTACAGCCTGACAATCAGCAACC TGGAACAAGAGGATATCGCTACCTACTTCTGCCAGCAAGGCAACACCCTGC CTTACACCTTTGGCGGAGGCACCAAGCTGGAAATCACA (SEQ ID NO: 135), or a nucleotide sequence having at least 80% identity thereto.

95. The polynucleotide or the pair of polynucleotides of any one of claims 84-92, wherein the anti- CD22 scFv comprises the amino acid sequence QVOLQOSGPGLVKPSOTLSLTCAISGDSVSSNSAAWNWIROSPSRGLEWLGRTYYRS KWYNDYAVSVKSRITINPDTSKNQFSLQLNSVTPEDTAVYYCAREVTGDLEDAFDI WGQGTMVTVSSGGGG5GGGG5GGGGADIOMTOSPSSLSASVGDRVTITCRASOTI WSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAEDFA TYYCQQSYSIPQTFGQGTKLEIKR (SEQ ID NO 63) or DIQMTQSPSSLSASVGDRVTITCRASQTIWSYLNWYQQRPGKAPNLLIYAASSLQSGVPSRFSGRGSGTDFTLTISSLQAEDFATYYCQQSYSIPQTFGQGTKLEIKRGG GG5GGGG5GGGG5OVOLQOSGPGLVKPSOTLSLTCAISGDSVSSNSAAWNWIROSPS RGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNOFSLQLNSVTPEDTAVYYCA REVTGDLEDAFDIWGOGTMVTVSS (SEQ ID NO: 136), or an amino acid sequence having at least 80% sequence identity thereto.Attorney Docket No: 243734.00023196. The polynucleotide or the pair of polynucleotides of claim 95, wherein the nucleotide sequence encoding the anti-CD22 scFv comprises the sequence CAGGTCCAGCTGCAGCAATCTGGACCAGGACTGGTCAAGCCCTCTCAGACCCTG AGCCTGACATGTGCCATCTCCGGCGATAGCGTGTCCAGCAATTCTGCCGCCTGGA ACTGGATCAGACAGTCCCCTAGCAGAGGCCTCGAGTGGCTCGGCAGAACCTACT ATCGGAGCAAGTGGTACAACGACTACGCCGTGTCCGTGAAGTCCAGGATCACCA TCAATCCCGACACCAGCAAGAACCAGTTCTCCCTGCAGCTGAACAGCGTGACAC CTGAGGATACCGCCGTGTACTACTGTGCCAGAGAAGTGACCGGCGACCTGGAAG ATGCCTTCGATATCTGGGGACAGGGAACCATGGTCACAGTGTCATCTGG7GGCG GAGGTAGTGGTGGTGGCGGAAGTGGCGGAGGTGGAAGCGACATTCAGATGACTC AGTCCCCAAGCAGCCTGAGCGCCTCCGTGGGAGACAGAGTGACAATTACAT GCCGGGCCAGCCAGACCATCTGGTCCTATCTCAATTGGTATCAACAGAGGC CCGGCAAGGCCCCTAACCTGCTTATCTATGCCGCCTCTAGCCTGCAGTCTG GCGTGCCATCTAGATTCAGCGGAAGAGGCTCCGGCACAGATTTCACCCTGA CCATTAGCTCACTGCAGGCCGAAGATTTCGCCACCTATTACTGTCAGCAGTC CTACAGCATCCCTCAGACCTTCGGCCAGGGAACAAAGCTCGAGATCAAGCGG (SEQ ID NO: 64), CAGGTTCAGCTGCAGCAGTCTGGCCCTGGCCTGGTTAAGCCTAGCCAGACACTG AGCCTGACCTGTGCCATCTCTGGCGATAGCGTGTCCAGCAATAGCGCCGCCTGG AACTGGATCAGACAGAGCCCTTCTAGAGGCCTGGAATGGCTGGGCAGAACCTAC TACCGGTCCAAGTGGTACAACGACTACGCCGTGTCCGTGAAGTCCCGGATCACC ATCAATCCCGACACCAGCAAGAACCAGTTCAGCCTCCAGCTGAACAGCGTGACC CCTGAGGATACCGCCGTGTACTACTGCGCCAGAGAAGTGACAGGCGATCTGGAA GATGCCTTCGACATCTGGGGCCAGGGCACAATGGTCACAGTTTCTAGCGGAGGC GGAGGTTCTGGTGGCGGAGGAAGTGGTGGCGGCGGATCTGATATCCAGATGACA CAGAGCCCCTCCAGCCTGTCTGCCAGCGTGGGAGATAGAGTGACCATCACC TGTAGAGCCAGCCAGACCATCTGGTCCTACCTGAATTGGTATCAGCAGAGG CCCGGCAAGGCCCCTAACCTGCTGATCTATGCTGCCAGCTCTCTGCAGTCC GGCGTGCCAAGCAGATTTTCCGGAAGAGGCAGCGGCACCGACTTCACCCTG ACAATTTCTAGCCTGCAAGCCGAGGACTTCGCCACCTACTACTGCCAGCAG TCCTACAGCATCCCTCAGACCTTTGGCCAGGGGACCAAGCTGGAAATCAAGAttorney Docket No: 243734.000231AGA (SEQ ID NO: 66), GACATTCAGATGACTCAGTCCCCAAGCAGCCTGAGCGCCTCCGTGGGAGAC AGAGTGACAATTACATGCCGGGCCAGCCAGACCATCTGGTCCTATCTCAAT TGGTATCAACAGAGGCCCGGCAAGGCCCCTAACCTGCTTATCTATGCCGCC TCTAGCCTGCAGTCTGGCGTGCCATCTAGATTCAGCGGAAGAGGCTCCGGC ACAGATTTCACCCTGACCATTAGCTCACTGCAGGCCGAAGATTTCGCCACCT ATTACTGTCAGCAGTCCTACAGCATCCCTCAGACCTTCGGCCAGGGAACAA AGCTCGAGATCAAGCGGGGTGGCGGAGGTAGTGGTGGTGGCGGAAGTGGCGGA GG7GG44GGCAGGTCCAGCTGCAGCAATCTGGACCAGGACTGGTCAAGCCCTCT CAGACCCTGAGCCTGACATGTGCCATCTCCGGCGATAGCGTGTCCAGCAATTCTG CCGCCTGGAACTGGATCAGACAGTCCCCTAGCAGAGGCCTCGAGTGGCTCGGCA GAACCTACTATCGGAGCAAGTGGTACAACGACTACGCCGTGTCCGTGAAGTCCA GGATCACCATCAATCCCGACACCAGCAAGAACCAGTTCTCCCTGCAGCTGAACA GCGTGACACCTGAGGATACCGCCGTGTACTACTGTGCCAGAGAAGTGACCGGCG ACCTGGAAGATGCCTTCGATATCTGGGGACAGGGAACCATGGTCACAGTGTCAT CT (SEQ ID NO: 137), GATATCCAGATGACACAGAGCCCCTCCAGCCTGTCTGCCAGCGTGGGAGAT AGAGTGACCATCACCTGTAGAGCCAGCCAGACCATCTGGTCCTACCTGAAT TGGTATCAGCAGAGGCCCGGCAAGGCCCCTAACCTGCTGATCTATGCTGCC AGCTCTCTGCAGTCCGGCGTGCCAAGCAGATTTTCCGGAAGAGGCAGCGGC ACCGACTTCACCCTGACAATTTCTAGCCTGCAAGCCGAGGACTTCGCCACCT ACTACTGCCAGCAGTCCTACAGCATCCCTCAGACCTTTGGCCAGGGGACCA AGCTGGAAATCAAGAGAGGAGGCGGAGGTTCTGGTGGCGGAGGAAGTGGTGGCG GCGGATCZCAGGTTCAGCTGCAGCAGTCTGGCCCTGGCCTGGTTAAGCCTAGCCA GACACTGAGCCTGACCTGTGCCATCTCTGGCGATAGCGTGTCCAGCAATAGCGC CGCCTGGAACTGGATCAGACAGAGCCCTTCTAGAGGCCTGGAATGGCTGGGCAG AACCTACTACCGGTCCAAGTGGTACAACGACTACGCCGTGTCCGTGAAGTCCCG GATCACCATCAATCCCGACACCAGCAAGAACCAGTTCAGCCTCCAGCTGAACAG CGTGACCCCTGAGGATACCGCCGTGTACTACTGCGCCAGAGAAGTGACAGGCGA TCTGGAAGATGCCTTCGACATCTGGGGCCAGGGCACAATGGTCACAGTTTCTAGC(SEQ ID NO: 138), or a nucleotide sequence having at least 80% identity thereto.Attorney Docket No: 243734.00023197. The polynucleotide of any one of claims 67, 69, and 71-96 or the pair of polynucleotides of any one of claims 68 and 70-96, wherein the CD28 costimulatory domain comprises the amino acid sequence RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS (SEQ ID NO: 30), or an amino acid sequence having at least 80% sequence identity thereto.

98. The polynucleotide or the pair of polynucleotides of claim 97, wherein the nucleotide sequence encoding the CD28 costimulatory domain comprises the sequence CGAAGCAAGCGGAGCCGGCTGCTGCACTCCGACTACATGAACATGACCCCTAGA CGGCCTGGACCTACCAGAAAGCACTACCAGCCTTACGCTCCTCCTAGAGACTTCG CCGCCTACCGGTCC (SEQ ID NO: 15), or CGAAGCAAGCGGAGCCGGCTGCTGCACAGCGACTACATGAACATGACCCCTAGA CGGCCCGGACCAACCAGAAAGCACTACCAGCCTTACGCTCCTCCTAGAGATTTC GCCGCCTACCGGTCC (SEQ ID NO: 40), or a nucleotide sequence having at least 80% sequence identity thereto.

99. The polynucleotide of any one of claims 67, 69, and 71-98 or the pair of polynucleotides of any one of claims 68 and 70-98, wherein the 4- IBB costimulatory domain comprises the amino acid sequence KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 23), or an amino acid sequence having at least 80% sequence identity thereto.

100. The polynucleotide or the pair of polynucleotides of claim 99, wherein the nucleotide sequence encoding the 4- IBB costimulatory domain comprises the sequence AAGCGGGGCAGAAAGAAACTGCTCTACATCTTCAAGCAGCCCTTCATGCGGCCC GTGCAGACCACACAAGAGGAAGATGGCTGCTCCTGCAGATTCCCCGAGGAAGAA GAAGGCGGCTGCGAGCTG (SEQ ID NO: 7), or a nucleotide sequence having at least 80% sequence identity thereto.

101. The polynucleotide of any one of claims 67, 69, and 71-100 or the pair of polynucleotides of any one of claims 68 and 70-100, wherein the CD3ζ activation domain comprises the amino acid sequenceAttorney Docket No: 243734.000231RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQ EGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQAL PPR (SEQ ID NO: 24), or an amino acid sequence having at least 80% sequence identity thereto.

102. The polynucleotide or the pair of polynucleotides of claim 101, wherein the nucleotide encoding the CD3ζ activation domain comprises the sequence AGAGTGAAGTTCAGCAGATCCGCCGACGCTCCTGCCTATCAGCAGGGCCAAAAC CAGCTGTACAACGAGCTGAACCTGGGGAGAAGAGAAGAGTACGACGTGCTGGA CAAGCGGAGAGGCAGAGATCCTGAAATGGGCGGCAAGCCCAGACGGAAGAATC CTCAAGAGGGCCTGTATAATGAGCTGCAGAAAGACAAGATGGCCGAGGCCTACA GCGAGATCGGAATGAAGGGCGAGCGCAGAAGAGGCAAGGGACACGATGGACTG TACCAGGGCCTGAGCACCGCCACCAAGGATACCTATGATGCCCTGCACATGCAG GCCCTGCCTCCAAGA (SEQ ID NO: 8), AGAGTGAAATTCTCCAGAAGCGCTGACGCCCCAGCTTACCAGCAGGGACAGAAT CAGCTCTATAACGAACTGAATCTCGGCAGGCGCGAGGAATATGATGTGCTGGAT AAGAGGCGCGGCAGGGACCCAGAGATGGGAGGAAAGCCTCGGCGGAAGAACCC ACAAGAAGGACTTTACAACGAACTGCAAAAGGATAAGATGGCAGAAGCTTACTC CGAGATTGGCATGAAGGGCGAACGTCGGAGAGGAAAAGGCCACGACGGACTCT ATCAGGGACTGTCCACAGCCACAAAGGACACATACGACGCACTCCATATGCAGG CTCTCCCACCTAGA (SEQ ID NO: 16), or AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCCGCGTACCAGCAGGGCCAGAA CCAGCTCTATAACGAGCTCAATCTAGGACGAAGAGAGGAGTACGATGTTTTGGA CAAGAGACGTGGCCGGGACCCTGAGATGGGGGGAAAGCCGAGAAGGAAGAACC CTCAGGAAGGCCTGTACAATGAACTGCAGAAAGATAAGATGGCGGAGGCCTACA GTGAGATTGGGATGAAAGGCGAGCGCCGGAGGGGCAAGGGGCACGATGGCCTT TACCAGGGTCTCAGTACAGCCACCAAGGACACCTACGACGCCCTTCACATGCAG GCCCTGCCCCCTCGC (SEQ ID NO: 41), or a nucleotide sequence having at least 80% sequence identity thereto.Attorney Docket No: 243734.000231103. The polynucleotide of any one of claims 67, 69, and 71-102 or the pair of polynucleotides of any one of claims 68 and 70-102, wherein the CD8a transmembrane domain comprises the amino acid sequence IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 22), IYIWAPLAGTCGVLLLSLVITLYCRSKRSRLLH (SEQ ID NO: 46), or IYIWAPLAGTCGVLLLSLVIT (SEQ ID NO: 124), or an amino acid sequence having at least 80% sequence identity thereto.

104. The polynucleotide or the pair of polynucleotides of claim 103, wherein the nucleotide sequence encoding the CD8a transmembrane domain comprises the sequence ATCTACATCTGGGCCCCTCTGGCTGGAACATGTGGCGTGCTGCTGCTGAGCCTGG TCATCACCCTGTATTGC (SEQ ID NO: 6), ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGG TTATCACCCTTTACTGC (SEQ ID NO: 39), ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGG TTATCACCCTTTACTGCCGAAGCAAGCGGAGCCGGCTGCTGCACTAA (SEQ ID NO: 45), ATCTACATCTGGGCCCCTCTGGCTGGAACATGTGGCGTGCTGCTGCTGAGCCTGG TCATCACC (SEQ ID NO: 123), or ATCTACATCTGGGCGCCCTTGGCCGGGACTTGTGGGGTCCTTCTCCTGTCACTGG TTATCACC (SEQ ID NO: 126), or a nucleotide sequence having at least 80% sequence identity thereto.

105. The polynucleotide of any one of claims 67, 69, and 71-104 or the pair of polynucleotides of any one of claims 68 and 70-104, wherein the CD28 transmembrane domain comprises the amino acid sequence FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 29) or FWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLH (SEQ ID NO: 48), or an amino acid sequence having at least 80% sequence identity thereto.

106. The polynucleotide or the pair of polynucleotides of claim 105, wherein the nucleotide encoding the CD28 transmembrane domain comprises the sequence TTCTGGGTGCTCGTTGTTGTTGGCGGCGTGCTGGCCTGTTATAGCCTGCTGGTTACAttorney Docket No: 243734.000231CGTGGCCTTCATCATCTTTTGGGTC (SEQ ID NO: 14), TTTTGGGTCTTGGTGGTTGTGGGCGGCGTACTGGCCTGCTACTCCCTGCTAGTCA CCGTCGCGTTCATCATCTTCTGGGTC (SEQ ID NO: 34), or TTCTGGGTGCTCGTTGTTGTTGGCGGCGTGCTGGCCTGTTACAGCCTGCTGGTTAC CGTGGCCTTCATCATCTTTTGGGTC (SEQ ID NO: 43), or TTCTGGGTGCTCGTTGTTGTTGGCGGCGTGCTGGCCTGTTACAGCCTGCTGGTTAC CGTGGCCTTCATCATCTTTTGGGTCCGAAGCAAGCGGAGCCGGCTGCTGCAC(SEQ ID NO: 47), or a nucleotide sequence having at least 80% sequence identity thereto.

107. The polynucleotide of any one of claims 67, 69, and 71-106 or the pair of polynucleotides of any one of claims 68 and 70-106, wherein the CD8u stalk hinge domain comprises the amino acid sequence TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 21), or an amino acid sequence having at least 80% sequence identity thereto.

108. The polynucleotide or the pair of polynucleotides of claim 107, wherein the nucleotide sequence encoding the CD8a stalk hinge domain comprises the sequence ACAACAACCCCTGCTCCTCGGCCTCCTACACCAGCTCCTACAATTGCCAGCCAGC CACTGTCTCTGAGGCCCGAAGCTTGTAGACCTGCTGCTGGCGGAGCCGTGCATAC AAGAGGACTGGATTTCGCCTGCGAC (SEQ ID NO: 5), or ACCACGACGCCAGCGCCGCGACCACCAACGCCGGCGCCCACCATCGCGTCGCAG CCCCTGTCCCTGCGCCCAGAGGCGTGCCGGCCAGCGGCGGGGGGCGCAGTGCAC ACGAGGGGGCTGGACTTCGCCTGTGAT (SEQ ID NO: 38), or a nucleotide sequence having at least 80% sequence identity thereto.

109. The polynucleotide of any one of claims 67, 69, and 71-108 or the pair of polynucleotides of any one of claims 68 and 70-108, wherein the CD28 hinge domain comprises the amino acid sequence IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 28), or an amino acid sequence having at least 80% sequence identity thereto.

110. The polynucleotide or the pair of polynucleotides of claim 109, wherein the nucleotide sequence encoding the CD28 hinge domain comprises the sequenceAttorney Docket No: 243734.000231ATCGAAGTGATGTACCCTCCTCCTTACCTGGACAACGAGAAGTCCAACGGCACC ATCATCCACGTGAAGGGCAAGCACCTGTGTCCTTCTCCACTGTTCCCCGGACCAA GCAAGCCT (SEQ ID NO: 13), ATCGAGGTGATGTACCCACCGCCTTACCTGGACAACGAGAAAAGCAACGGCACC ATCATTCACGTGAAGGGCAAACACCTGTGCCCATCTCCTCTGTTCCCTGGACCAT CCAAGCCC (SEQ ID NO: 33), or ATCGAAGTGATGTACCCGCCTCCTTACCTGGACAACGAGAAGTCCAACGGCACC ATCATCCACGTGAAGGGAAAGCACCTGTGTCCTTCTCCACTGTTCCCCGGACCTA GCAAGCCT (SEQ ID NO: 42), or a nucleotide sequence having at least 80% sequence identity thereto.

111. The polynucleotide of any one of claims 1, 67, and 69 or the pair of polynucleotides of any one of claims 2, 68, and 70, wherein the anti-CD22 CAR comprises the amino acid sequence of any of SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, or SEQ ID NO: 83, or an amino acid sequence having at least 80% sequence identity thereto.

112. The polynucleotide of any one of claims 1, 67, 69, and 111 or the pair of polynucleotides of any one of claims 2, 68, 70, and 111, wherein the anti-CD22 CAR comprises the amino acid sequence SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, or SEQ ID NO: 83.

113. The polynucleotide of any one of claims 1, 67, 69, and 111-112 or the pair of polynucleotides of any one of claims 2, 68, 70, and 111-112, wherein the anti-CD22 CAR comprises the amino acid sequence SEQ ID NO: 79.

114. The polynucleotide of any one of claims 1, 67, 69, and 111-113 or the pair of polynucleotides of any one of claims 2, 68, 70, and 111-113, wherein the nucleotide sequence encoding the anti-CD22 CAR comprises the sequence of any of SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, or SEQ ID NO: 82, or a nucleotide sequence having at least 80% sequence identity thereto.Attorney Docket No: 243734.000231115. The polynucleotide of any one of claims 1, 67, 69, and 111-114 or the pair of polynucleotides of any one of claims 2, 68, 70, and 111-114, wherein the nucleotide sequence encoding the anti-CD22 CAR comprises the sequence of any one of SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, or SEQ ID NO: 82.

116. The polynucleotide of any one of claims 1, 67, 69, and 111-115 or the pair of polynucleotides of any one of claims 2, 68, 70, and 111-115, wherein the nucleotide sequence encoding the anti-CD22 CAR comprises SEQ ID NO: 78.

117. The polynucleotide of any one of claims 1, 3, 67, and 111-116 or the pair of polynucleotides of any one of claims 2, 68, and 111-116, wherein the anti-CD19 CAR comprises the amino acid sequence of SEQ ID NO: 88 or SEQ ID NO: 90, or an amino acid sequence having at least 80% sequence identity thereto.

118. The polynucleotide of any one of claims 1, 3, 67, and 111-117 or the pair of polynucleotides of any one of claims 2, 68, and 111-117, wherein the anti-CD19 CAR comprises the amino acid sequence of SEQ ID NO: 88 or SEQ ID NO: 90.

119. The polynucleotide of any one of claims 1, 3, 67, and 111-118 or the pair of polynucleotides of any one of claims 2, 68, and 111-118, wherein the anti-CD19 CAR comprises the amino acid sequence SEQ ID NO: 90.

120. The polynucleotide of any one of claims 1, 3, 67, and 111-119, or the pair of polynucleotides of any one of claims 2, 68, and 111-120, wherein the nucleotide sequence encoding the anti-CD19 CAR comprises the sequence of SEQ ID NO: 87 or SEQ ID NO: 89, or a nucleotide sequence having at least 80% sequence identity thereto.

121. The polynucleotide of any one of claims 1, 3, 67, and 111-120 or the pair of polynucleotides of any one of claims 2, 68, and 111-120, wherein the nucleotide sequence encoding the CD 19 CAR comprises the sequence of SEQ ID NO: 89.Attorney Docket No: 243734.000231122. The polynucleotide of any one of claims 1, 69, and 111-116 or the pair of polynucleotides of any one of claims 2, 70, and 111-116, wherein the anti-CD19 CAR comprises the amino acid sequence SEQ ID NO: 85, or an amino acid sequence having at least 80% sequence identity thereto.

123. The polynucleotide of any one of claims 1, 69, 111-116, and 120 or the pair of polynucleotides of any one of claims 2, 70, 111-116, and 120, wherein the anti-CD19 CAR comprises the amino acid sequence SEQ ID NO: 85.

124. The polynucleotide of any one of claims 1, 69, 111-116, and 122-123 or the pair of polynucleotides of any one of claims 2, 70, 111-116, and 122-123, wherein the nucleotide sequence encoding the CD19 CAR comprises the sequence of SEQ ID NO: 84 or SEQ ID NO: 86, or a nucleotide sequence having at least 80% sequence identity thereto.

125. The polynucleotide of any one of claims 1, 69, 111-116, and 122-124 or the pair of polynucleotides of any one of claims 2, 70, 111-116, and 122-124, wherein the nucleotide sequence encoding the CD19 CAR comprises the sequence of SEQ ID NO: 84 or SEQ ID NO: 86.

126. The polynucleotide of any one of claims 1, 3-67, 69, and 111-125 or the pair of polynucleotides of any one of claims 2, 4-66, 68, 70, and 111-125, wherein each extracellular domain further comprises a leader sequence.

127. The polynucleotide or the pair of polynucleotides of claim 126, wherein each leader sequence is independently derived from CD8a or a human immunoglobulin heavy chain variable region.

128. The polynucleotide or the pair of polynucleotides of claim 127, wherein the CD8a leader sequence comprises the amino acid sequence MALPVTALLLPLALLLHAARP (SEQ ID NO: 17), or an amino acid sequence having at least 80% sequence identity thereto.Attorney Docket No: 243734.000231129. The polynucleotide or the pair of polynucleotides of claim 128, wherein the nucleotide sequence encoding the CD8a leader sequence comprises the sequence ATGGCTCTGCCTGTGACAGCTCTGCTGCTGCCTCTGGCTCTGCTTCTGCATGCCGC CAGACCT (SEQ ID NO: 1), ATGGCACTGCCAGTTACTGCCCTGCTGCTCCCACTTGCACTGCTGCTTCATGCTG CTAGACCT (SEQ ID NO: 31), or a nucleotide sequence having at least 80% sequence identity thereto.

130. The polynucleotide or the pair of polynucleotides of claim 127, wherein the human immunoglobulin heavy chain variable region leader sequence comprises the amino acid sequence MDWIWRILFLVGAATGAHS (SEQ ID NO: 57), or an amino acid sequence having at least 80% sequence identity thereto.

131. The polynucleotide or the pair of polynucleotides of claim 130, wherein the nucleotide sequence encoding the human immunoglobulin heavy chain variable region leader sequence comprises the sequence ATGGATTGGATCTGGCGGATCCTGTTCCTTGTGGGAGCTGCTACAGGCGCCCATT CT (SEQ ID NO: 51), or a nucleotide sequence having at least 80% sequence identity thereto.

132. The polynucleotide of any one of claims 1, 3-67, 69, and 111-131 or the pair of polynucleotides of any one of claims 2, 4-66, 68, 70, and 111-131, wherein the polynucleotide further encodes CD20.

133. The polynucleotide or the pair of polynucleotides of claim 132, wherein the nucleotide sequence encoding CD20 is present adjacent to the nucleotide sequence encoding the CAR comprising a signaling domain.

134. The polynucleotide or the pair of polynucleotides of claim 133, wherein the nucleotide sequence encoding CD20 is present 5’ to the nucleotide sequence encoding the anti-CD22 CAR.Attorney Docket No: 243734.000231135. The polynucleotide or the pair of polynucleotides of any one of claims 132-134, wherein the CD20 comprises the amino acid sequence MTTPRNSVNGTFPAEPMKGPIAMQSGPKPLFRRMSSLVGPTQSFFMRESKTLGAVQI MNGLFHIALGGLLMIPAGIYAPICVTVWYPLWGGIMYIISGSLLAATEKNSRKCLVK GKMIMNSLSLFAAISGMILSIMDILNIKISHFLKMESLNFIRAHTPYINIYNCEPANPSE KNSPSTQYCYSIQSLFLGILSVMLIFAFFQELVIAGIVENEWKRTCSRPKSNIVLLSAEE KKEQTIEIKEEVVGLTETSSQPKNEEDIEIIPIQEEEEEETETNFPEPPQDQESSPIENDSS PGSG (SEQ ID NO: 55), or an amino acid sequence having at least 80% sequence identity thereto.

136. The polynucleotide or the pair of polynucleotides of claim 135, wherein the nucleotide sequence encoding the CD20 comprises the sequence ATGACCACACCTCGGAATAGCGTGAACGGCACATTCCCCGCCGAGCCTATGAAG GGACCTATCGCCATGCAGAGCGGCCCCAAGCCTCTGTTTAGACGGATGTCTAGC CTCGTGGGCCCCACACAGAGCTTTTTCATGAGAGAGAGCAAGACCCTGGGCGCC GTGCAGATCATGAACGGCCTGTTTCACATTGCCCTCGGCGGCCTGCTGATGATCC CTGCCGGAATCTATGCCCCTATCTGCGTGACCGTGTGGTATCCTCTGTGGGGCGG CATCATGTACATCATCTCTGGATCTCTGCTGGCCGCCACCGAGAAGAACAGCAG AAAGTGTCTGGTCAAGGGCAAGATGATCATGAATAGCCTGAGCCTGTTCGCCGC CATCAGCGGCATGATCCTGAGCATCATGGATATCCTGAATATCAAGATCAGCCA CTTCCTGAAGATGGAAAGCCTGAACTTCATCAGGGCCCACACACCTTACATCAA CATCTACAACTGCGAGCCCGCCAATCCTAGCGAGAAGAATAGCCCCAGCACACA GTACTGCTACTCTATCCAGAGCCTGTTTCTGGGCATCCTGAGCGTGATGCTGATC TTCGCATTCTTCCAAGAGCTGGTTATCGCCGGCATCGTGGAAAACGAGTGGAAG CGGACCTGCAGCAGACCCAAGAGCAACATCGTGCTGCTGAGCGCCGAGGAAAA GAAAGAGCAGACCATCGAGATCAAAGAGGAAGTCGTCGGCCTGACCGAGACAA GCAGCCAGCCTAAGAACGAAGAGGACATTGAGATCATCCCCATCCAAGAAGAG GAAGAAGAAGAGACTGAGACAAACTTCCCCGAGCCTCCTCAGGACCAAGAGAG CAGCCCCATTGAGAACGATAGCAGCCCTGGCTCTGGC (SEQ ID NO: 49), or a nucleotide sequence having at least 80% sequence identity thereto.Attorney Docket No: 243734.000231137. The polynucleotide or the pair of polynucleotides of any one of claims 1, 3-67, 69, and 111-136 or the pair of polynucleotides of any one of claims 2, 4-66, 68, 70, and 111-136, wherein the polynucleotide further comprises a separation sequence between the two CARs.

138. The polynucleotide or the pair of polynucleotides of any one of claims 1, 3-67, 69, and 111-137 or the pair of polynucleotides of any one of claims 2, 4-66, 68, 70, and 111-137, wherein the polynucleotide further comprises a separation sequence between CD20 and its adjacent CAR.

139. The polynucleotide or the pair of polynucleotides of claim 137 or claim 138, wherein the separation sequence is selected from GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 25), EGRGSLLTCGDVEENPGP (SEQ ID NO: 56), AEGRGSLLTCGDVEENPGP (SEQ ID NO: 44), GSGSRVTELLYRMKRAETYCPRPLLAIHPTEARHKQKIVAPVKQLLNFDLLKLAGDV ESNPGP (SEQ ID NO: 52), LLCFLLLLLSGDVELNPGP (SEQ ID NO: 53), HHFMFLLLLLAGDIELNPGP (SEQ ID NO: 58), WFLVLLSFILSGDIEVNPGP (SEQ ID NO: 59), KNCAMYMLLLSGDVETNPGP (SEQ ID NO: 67), MVISQLMLKLAGDVEENPGP (SEQ ID NO: 69), GSGATNFSLLKQAGDVEENPGP (SEQ ID NO: 71), GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 73), or D-X-E-X- NPGP (SEQ ID NO: 75), in which X is any amino acid residue, or an amino acid sequence having at least 80% sequence identity thereto.

140. The polynucleotide or the pair of polynucleotides of any one of claims 137-139, wherein the separation sequence the amino acid sequence GSGEGRGSLLTCGDVEENPGP (SEQ ID NO: 25), or EGRGSLLTCGDVEENPGP (SEQ ID NO: 56), or an amino acid sequence having at least 80% sequence identity thereto.

141. The polynucleotide or the pair of polynucleotides of claim 140, wherein the nucleotide encoding the separation sequence comprises the sequence GGTTCTGGCGAAGGCAGAGGCTCTCTGCTGACATGCGGAGATGTGGAAGAGAAC CCCGGACCT (SEQ ID NO: 9),Attorney Docket No: 243734.000231GAAGGCAGAGGCTCTCTGCTGACATGCGGAGATGTGGAAGAGAACCCCGGACCT(SEQ ID NO: 125), or GAAGGCAGAGGTTCTCTGCTGACATGCGGCGACGTGGAAGAGAACCCTGGACCT(SEQ ID NO: 50), or a nucleotide sequence having at least 80% sequence identity thereto.

142. The polynucleotide of any one of claims 1, 3-67, 69, and 111-141 or the pair of polynucleotides of any one of claims 2, 4-66, 68, 70, and 111-141, wherein the polynucleotide comprises or consisting of the nucleotide sequence of any one of SEQ ID NO: 72 or SEQ ID NO: 74, or a nucleotide sequence having at least 80% sequence identity thereto.

143. The polynucleotide of any one of claims 1, 3-67, 69, and 111-142 or the pair of polynucleotides of any one of claims 2, 4-66, 68, 70, and 111-142, wherein the polynucleotide comprises or consisting of the nucleotide sequence of any one of SEQ ID NO: 72 or SEQ ID NO: 74.

144. The polynucleotide of any one of claims 1, 3-67, 69, and 111-143 or the pair of polynucleotides of any one of claims 2, 4-66, 68, 70, and 111-143, wherein the polynucleotide comprises or consisting of the nucleotide sequence of SEQ ID NO: 74.

145. The polynucleotide of any one of claims 1, 3-67, 69, and 111-144 or the pair of polynucleotides of any one of claims 2, 4-66, 68, 70, and 111-144, which is a DNA molecule.

146. The polynucleotide of any one of claims 1, 3-67, 69, and 111-144 or the pair of polynucleotides of any one of claims 2, 4-66, 68, 70, and 111-144, which is an RNA molecule.

147. A recombinant vector comprising the polynucleotide of any one of claims 1, 3-67, 69, and 111-146 or the pair of polynucleotides of any one of claims 2, 4-66, 68, 70, and 111-146.

148. A pair of recombinant vectors comprising the pair of polynucleotides of any one of claims 2, 4-66, 68, 70, and 111-146.Attorney Docket No: 243734.000231149. The recombinant vector of claim 147 or the pair of recombinant vectors of claim 148, wherein the vector is a viral vector.

150. The recombinant vector or pair of recombinant vectors of claim 149, wherein the viral vector is a retroviral vector, a lentiviral vector, an adenoviral vector, an adeno-associated virus vector, an alphaviral vector, a herpes virus vector, a baculoviral vector, or a vaccinia virus vector.

151. The recombinant vector or pair of recombinant vectors of claim 150, wherein the viral vector is a lentiviral vector.

152. The recombinant vector of claim 147 or the pair of recombinant vectors of claim 148, wherein the vector is a non-viral vector.

153. The recombinant vector or pair of recombinant vectors of claim 152, wherein the non-viral vector is a minicircle plasmid, a Sleeping Beauty transposon, a PiggyBac transposon, or a single or double stranded DNA molecule that is used as a template for homology directed repair (HDR) based gene editing.

154. The recombinant vector of any one of claims 147-153 or the pair of recombinant vectors of any one of claims 148-153 comprises a bicistronic vector.

155. A dual-targeting chimeric antigen receptor (CAR) system which is encoded by the polynucleotide of any one of claims 1, 4-67, 69, and 111-146 or the pair of polynucleotides of any one of claims 2, 4-66, 68, 70, and 111-146.

156. A chimeric antigen receptor (CAR) which is encoded by the polynucleotide of any one of claims 1, 3-67, 69, and 111-146 or the pair of polynucleotides of any one of claims 2, 4-66, 68, 70, and 111-146.Attorney Docket No: 243734.000231157. An isolated host cell comprising the polynucleotide of any one of claims 1, 3-67, 69, and 111-146, the pair of polynucleotides of any one of claims 2, 4-66, 68, 70, and 111-146, the recombinant vector of any one of claims 147-154, the pair of recombinant vectors of any one of claims 147-154, the dual-targeting chimeric antigen receptor (CAR) system of claim 155, or the CAR of claim 156.

158. The isolated host cell of claim 157, wherein the host cell is an immune cell.

159. The isolated host cell of claim 157 or claim 158, wherein the host cell is a T cell, a nature killer (NK) cell, or a macrophage.

160. The isolated host cell of claim 157-159, wherein the host cell is a T cell.

161. The isolated host cell of claim 160, wherein the host cell is a CD81T-cell, a CD4 T-cell, a cytotoxic T-cell, an aP T-cell receptor (TCR) T-cell, an invariant natural killer T (iNKT) cell, a y8 T-cell, a memory T-cell, a memory stem T-cell (TSCM), a naive T-cell, an effector T-cell, a T-helper cell, or a regulatory T-cell (Treg).

162. The isolated host cell of any one of claims 157-161, wherein the host cell is a natural killer (NK) cell.

163. The isolated host cell of any one of claims 157-162, wherein the host cell has been activated and / or expanded ex vivo.

164. The isolated host cell of any one of claims 157-163, wherein the host cell is an allogeneic cell.

165. The isolated host cell of any one of claims 157-163, wherein the host cell is an autologous cell.Attorney Docket No: 243734.000231166. The isolated host cell of claim 164 or claim 165, wherein the host cell is isolated from a subject having a tumor, wherein one or more cells of the tumor express CD19 and / or CD22.

167. The isolated host cell of claim 166, wherein the tumor is a hematological malignancy.

168. The isolated host cell of claim 167, wherein the hematological malignancy is acute lymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), B-cell lymphoma, B- cell acute lymphoblastic leukemia (BALL), chronic lymphocytic leukemia (CLL), acute myelogenous leukemia (AML), mantle cell lymphoma (MCL), multiple myeloma (MM), small lymphocytic lymphoma (SLL), and / or hairy cell leukemia.

169. The isolated host cell of any one of claims 157-168, wherein the host cell is derived from a blood sample, a marrow sample, a tissue sample, or a tumor sample.

170. A pharmaceutical composition comprising the isolated host cell of any one of claims 157- 169 and a pharmaceutically acceptable carrier and / or excipient.

171. A method of generating the isolated host cell of any one of claims 157-169, said method comprising genetically modifying the host cell with the polynucleotide of any one of claims 1, 3-67, 69, and 111-146, the pair of polynucleotides of any one of claims 2, 4-66, 68, 70, and 111-146, the recombinant vector of any one of claims 147-154 or the pair of recombinant vectors of any one of claims 148-154.

172. The method of claim 171, wherein the vector is a viral vector, and the genetic modification is conducted by a transduction using said vector.

173. The method of any claim 171 or claim 172, wherein the genetic modification is conducted ex vivo.Attorney Docket No: 243734.000231174. A method for killing a tumor cell expressing CD19 and / or CD22, said method comprising contacting said cell with the host cell(s) of any one of claims 157-169 or the pharmaceutical composition of claim 170.

175. A method for treating a tumor in a subject in need thereof, wherein one or more cells of the tumor express CD19 and / or CD22, said method comprising administering to the subject a therapeutically effective amount of the host cell(s) of any one of claims 157-169 or the pharmaceutical composition of claim 170.

176. The method of claim 175, wherein the tumor is a hematological malignancy.

177. The method of claim 176, wherein the hematological malignancy is acute lymphoblastic leukemia (ALL), B-cell lymphoma, B-cell acute lymphoblastic leukemia (BALL), chronic lymphocytic leukemia (CLL), acute myelogenous leukemia (AML), mantle cell lymphoma (MCL), multiple myeloma (MM), small lymphocytic lymphoma (SLL), and / or hairy cell leukemia.

178. The method of any one of claims 175-177, the method comprising:a) isolating T cells, NK cells, iNKT cells, or macrophages from the subject or generating T- cells, NK cells, iNKT cells, or macrophages from stem cells;b) genetically modifying said T cells, NK cells, iNKT cells, macrophages, or stem cells ex vivo with the polynucleotide of any one of claims 1, 3-67, 69, and 111-146, the pair of polynucleotides of any one of claims 2, 4-66, 68, 70, and 111-146, the vector of any one of claims 147 and 149-154, or the pair of recombinant vectors of any one of claims 148-154; c) optionally, expanding and / or activating said T cells, NK cells, iNKT cells, or macrophages before, after, or during step (b); andd) introducing the genetically modified T cells, NK cells, iNKT cells, or macrophages into the subject.

179. The method of claim 178, wherein the stem cells comprise induced pluripotent stem cells (iPSCs).Attorney Docket No: 243734.000231180. The method of any one of claims 175-179, wherein the subject is human.

181. A polynucleotide encoding a dual-targeting chimeric antigen receptor (CAR) system comprising two CARs, wherein each CAR comprises an extracellular domain comprising a means for binding to a target,wherein optionally, one of the CARs lacks a means for activating effector function of an immune cell; andwherein one extracellular domain comprises a means for binding to cluster of differentiation 19 (CD19) and the other extracellular domain comprises a means for binding to cluster of differentiation 22 (CD22).

182. A pair of polynucleotides encoding a dual-targeting chimeric antigen receptor (CAR) system comprising two CARs, wherein the first polynucleotide encodes a first CAR of the dual-targeting CAR system, and the second polynucleotide encodes a second CAR of the dualtargeting CAR system,wherein each CAR comprises an extracellular domain comprising a means for binding to a target;wherein optionally, one of the CARs lacks a means for activating effector function of an immune cell; andwherein one extracellular domain comprises a means for binding to cluster of differentiation 19 (CD 19) and the other extracellular domain comprises a means for binding to cluster of differentiation 22 (CD22).

183. The polynucleotide of claim 181 or the pair of polynucleotides of claim 182, wherein one of the CARs lacks a means for activating effector function of an immune cell.

184. The polynucleotide or the pair of polynucleotides of claim 183, wherein the CAR lacking the means for activating effector function of an immune cell further lacks a means for boosting effector function of the immune cell.Attorney Docket No: 243734.000231185. A polynucleotide encoding a dual -targeting chimeric antigen receptor (CAR) system comprising two CARs, wherein the two CARs comprise:(i) an anti-cluster of differentiation 22 (CD22) CAR comprising:an extracellular domain comprising a means for binding to CD22,a hinge domain and a transmembrane domain derived from CD28,a cytoplasmic domain comprising a CD28 costimulatory domain and a CD3ζ activation domain; and(ii) an anti-cluster of differentiation 19 (CD 19) CAR comprisingan extracellular domain comprising a means for binding to CD 19,a hinge domain and a transmembrane domain derived from CD28 or CD8a,a cytoplasmic domain comprising a 4- IBB costimulatory domain and lacking a means for activating effector function of an immune cell.

186. A pair of polynucleotides encoding a dual -targeting chimeric antigen receptor (CAR) system comprising two CARs, wherein(i) the first polynucleotide encodes an anti-cluster of differentiation 22 (CD22) CAR comprising:an extracellular domain comprising a means for binding to CD22,a hinge domain and a transmembrane domain derived from CD28,a cytoplasmic domain comprising a CD28 costimulatory domain and a CD3ζ activation domain; and(ii) the second polynucleotide encodes an anti-cluster of differentiation 19 (CD 19) CAR comprisingan extracellular domain comprising a means for binding to CD 19,a hinge domain and a transmembrane domain derived from CD28 or CD8a,a cytoplasmic domain comprising a 4-1BB costimulatory domain and lacking a means for activating effector function of an immune cell.

187. A polynucleotide encoding a dual-targeting chimeric antigen receptor (CAR) system comprising two CARs, wherein one CAR binds to cluster of differentiation 19 (CD 19) and theAttorney Docket No: 243734.000231other CAR binds to cluster of differentiation 22 (CD22), the improvement comprising one of the CARs lacking an activation domain.

188. A pair of polynucleotides encoding a dual -targeting chimeric antigen receptor (CAR) system comprising two CARs, wherein the first polynucleotide encodes a first CAR of the dual-targeting CAR system, and the second polynucleotide encodes a second CAR of the dualtargeting CAR system, wherein the first CAR binds to cluster of differentiation 19 (CD 19) and the second CAR binds to cluster of differentiation 22 (CD22), the improvement comprising one of the CARs lacking an activation domain.

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