Alternative intracellular signaling domains of chimeric antigen receptors

Abstract

The present invention relates to the field of biotechnology, specifically to isolated alternative intracellular signaling domains of chimeric antigen receptors (CARs) and chimeric antigen receptors (CARs) comprising said signaling domains. The present invention also relates to nucleic acids encoding alternative intracellular signaling domains of chimeric antigen receptors, and nucleic acids encoding chimeric antigen receptors having the above-mentioned signaling domains, expression vectors, delivery vectors, and also to genetically engineered cells comprising the above-mentioned chimeric antigen receptors, as well as methods for producing said cells.

Description

[Technical Field]

[0001] The present invention relates to the field of biotechnology, specifically to isolated alternative intracellular signaling domains of chimeric antigen receptors (CARs) and chimeric antigen receptors (CARs) comprising said signaling domains. The present invention also relates to nucleic acids encoding the alternative intracellular signaling domains of chimeric antigen receptors, and nucleic acids encoding chimeric antigen receptors having the above-mentioned signaling domains, expression vectors, delivery vectors, and genetically engineered cells comprising the above-mentioned chimeric antigen receptors, and methods for producing said cells. [Background technology]

[0002] Chimeric antigen receptors (CARs) are recombinant protein molecules that target cell surface antigens (Sadelain M, Brentjens R, Riviere I. The promise and potential pitfalls of chimeric antigen receptors. Curr Opin Immunol [Internet]. 2009;21:215–23. Available from: http: / / linkinghub.elsevier.com / retrieve / pii / S0952791509000211).

[0003] CARs generally consist of several domains that perform specific functions. The extracellular domain of CARs is usually represented by a single-chain variable fragment (single-chain variable fragment, scFv) of an antibody specific for a tumor antigen, providing target recognition independent of major histocompatibility complex molecules. The intracellular domain is responsible for signal transduction and effective activation of CAR-expressing immune cells upon binding to the target antigen. The intracellular part of the receptor consists of the CD3ζ chain (CD3-zeta chain) and additional costimulatory domains, usually derived from the natural proteins CD28 and 4-1BB.The presence of a costimulatory domain in the CAR receptor has been found to significantly enhance the activity of CAR-expressing immune cells compared to the activity of CARs whose intracellular portion consists only of the CD3ζ chain (CD3 zeta chain) (Maher J, Brentjens RJ, Gunset G, Riviere I, Sadelain M. Human T-lymphocyte cytotoxicity and proliferation directed by a single chimeric TCRζ / CD28 receptor. Nat Biotechnol [Internet]. 2002;20:70-5. Available at: http: / / www.nature.com / articles / nbt0102-70; Imai C, Mihara K, Andreansky M, Nicholson IC, Pui CH, Geiger TL et al. Chimeric receptors with 4-1BB signaling capacity provoke potent cytotoxicity against acute lymphoblastic leukemia. Leukemia [Internet].2004;18:676~84.: Available from http: / / www.nature.com / articles / 2403302; Kowolik CM, Topp MS, Gonzalez S, Pfeiffer T, Olivares S, Gonzalez N, et al. CD28 costimulation provided through a CD19-specific chimeric antigen receptor enhances in vivo persistence and antitumor efficacy of adoptively transferred T cells.Cancer Res.2006;66:10995~1004).

[0004] Both variations in CAR receptor design have shown remarkable antitumor activity and high potency in preclinical and clinical trials (Sadelain M, Brentjens R, Riviere I. The Basic Principles of Chimeric Antigen Receptor Design. Cancer Discovery [Internet]. 2013;3:388-98. Available at: http: / / cancerdiscovery.aacrjournals.org / lookup / doi / 10.1158 / 2159-8290.CD-12-0548). Notable differences have been shown between CAR receptors containing the CD28 costimulatory domain and those containing the 4-1BB costimulatory domain.

[0005] Results from clinical trials using CD19-specific CAR-T therapy to treat lymphoproliferative disorders have shown that the 4-1BB-CD3ζ (CD3-zeta chain) CAR results in longer persistence of CAR-T lymphocytes in vivo (more than 6 months for most patients) and its use allows for a less pronounced cytokine release syndrome compared to the use of CD28-CD3ζ CAR (CD3-zeta chain) (Davila ML, Riviere I, Wang X, Bartido S, Park J, Curran K et al. Efficacy and Toxicity Management of 19-28z CAR T Cell Therapy in B Cell Acute Lymphoblastic Leukemia. Sci Transl Med [Internet]. 2014;6:224ra25-224ra25. Available from: http: / / stm.sciencemag.org / cgi / doi / 10.1126 / scitranslmed.3008226; Maude SL. Chimeric Antigen Receptor T Cells for Sustained Remissions in Leukemia.N Engl J Med[Internet].2014;371:1507~17.: http: / / www.nejm.org / doi / abs / 10.1056 / NEJMoa1407222%5Cn Available from http: / / www.ncbi.nlm.nih.gov / pubmed / 25317870; Zhang T, Cao L, Xie J, Shi N, Zhang Z, Luo Z, et al. Efficiency of CD19 chimeric antigen receptor-modified T cells for treatment of B cell malignancies in phase I clinical trials: a meta-analysis.Oncotarget[Internet].2015;6:33961~71.: Available from http: / / www.oncotarget.com / fulltext / 5582).

[0006] Accumulating data indicate that the CD28-CD3ζ (CD3-zeta chain) CAR receptor can result in faster activation of CAR-T lymphocytes compared to the use of the 4-1BB-CD3ζ (CD3-zeta chain) CAR due to cytokine release and a pronounced cytotoxic effect (van der Stegen SJC, Hamieh M, Sadelain M. The pharmacology of second-generation chimeric antigen receptors. Nat Rev Drug Discov [Internet]. Nature Publishing Group; 2015; 14:499-509.: available at http: / / www.nature.com / doifinder / 10.1038 / nrd4597), which in turn may result in improved therapeutic efficacy.

[0007] A moderate reduction in CAR-T cell activation levels and cytokine release, while maintaining the transduction rate of activation signals through the CD28-based costimulatory domain without losing cytotoxic activity, allows for a significant improvement in the therapeutic properties for adoptive immunotherapy of tumor diseases based on CD28-CD3ζ (CD3-zeta chain) CAR receptor-expressing immune cells.

[0008] Interaction of the natural T cell receptor (TCR) with components of the major histocompatibility complex (MHC) further leads to binding of the CD8 coreceptor and activation of the Lck tyrosine kinase. The latter phosphorylates a peptide sequence within CD3ζ (CD3-zeta chain), i.e., an immunoreceptor tyrosine-based activation motif (ITAM). Zap-70 kinase interacts with the doubly phosphorylated ITAM within CD3ζ (CD3-zeta chain) via its Src-homology 2 (SH2) domain. Binding of Zap-70 to CD3ζ (CD3-zeta chain) leads to a conformational change in the kinase and its autophosphorylation at amino acid residues Y315 and Y319. Lck-mediated phosphorylation and trans-autophosphorylation induce Zap70 activation. Following activation, Zap70 ceases its interaction with CD3ζ (CD3-zeta chain) and associates with the plasma membrane, where it then phosphorylates other substrate proteins, including LAT20 [Yamasaki S, Takamatsu M, Iwashima M, The kinase, SH3, and SH2 domains of Lck play critical roles in T-cell activation after ZAP-70 membrane localization, Mol Cell Biol. 1996 Dec;16(12):7151-60; Visco C, Magistrelli G, Bosotti R, Perego R, Rusconi L, Toma S, Zamai M, Acuto O, Isacchi A, Activation of Zap-70 tyrosine kinase due to a structural rearrangement induced by tyrosine phosphorylation and / or ITAM binding, Biochemistry. 2000 Mar 14;39(10):2784-91].

[0009] Thus, Zap-70 is a crucial member of the CAR signaling pathway; therefore, modulation of the activity of this kinase could be an approach to regulate the level of CAR-T activation, which directly impacts CAR-T survival, persistence, and functional activity. Numerous studies have elucidated the key structural and functional elements of the ZAP70-ITAM interaction system [Katz ZB, Novotna L, Blount A, Lillemeier BF, A cycle of Zap70 kinase activation and release from the TCR amplifies and disperses antigenic stimuli, Nat Immunol. 2017 Jan;18(1):86-95; Deindl S, Kadlecek TA, Cao X, Kuriyan J, Weiss A, Stability of an autoinhibitory interface in the structure of the tyrosine kinase ZAP-70 impacts T cell receptor response, Proc Natl Acad Sci USA. 2009 Dec 8;106(49):20699-704; Szabo M, Czompoly T, Kvell K, Talaber G, Bartis D, Nemeth P, Berki T, Boldizsar F, Fine-tuning of proximal TCR signaling by ZAP-70 tyrosine residues in Jurkat cells, Int Immunol. 2012 February;24(2):79~87.].

[0010] Modulation of CAR activity may be possible through the creation of hybrid peptide substrates of Zap-70 that differ from the native ITAM in the CD3ζ (CD3-zeta chain)-based CAR activation domain. One candidate is a fragment of the amino acid sequence of integrin beta 3 (ITGB3) [Liu XY, Timmons S, Lin YZ, Hawiger J., Identification of a functionally important sequence in the cytoplasmic tail of integrin beta 3 by using cell-permeable peptide analogs, Proc Natl Acad Sci USA. 1996 Oct 15;93(21):11819-24; Durrant TN, van den Bosch MT, Hers I. Integrin αIIbβ3 outside-in signaling, Blood. 2017 Oct 5;130(14):1607-1619].

[0011] The authors of the present invention have unexpectedly discovered that a hybrid signaling domain of the cytoplasmic portion of a chimeric antigen receptor (CAR), which comprises a peptide fragment of the amino acid sequence of integrin beta 3 (ITGB3) in place of one or two immunoreceptor tyrosine-based activation motifs (ITAMs) selected from ITAM1, ITAM2, and ITAM3 in the intracellular signaling domain (e.g., CD3ζ (CD3-zeta)) of the chimeric antigen receptor (CAR), stimulates CAR-dependent activation of genetically modified immune cells expressing the corresponding gene construct. Summary of the Invention [Means for solving the problem]

[0012] In one aspect, the present invention relates to an isolated alternative intracellular signaling domain of a chimeric antigen receptor (CAR), in which one or two immunoreceptor tyrosine-based activation motifs (ITAMs) selected from ITAM1, ITAM2, and ITAM3 are replaced with a fragment of the amino acid sequence of integrin beta 3 (ITGB3).

[0013] In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized in that one immunoreceptor tyrosine-based activation motif (ITAM) selected from ITAM1, ITAM2, and ITAM3 is replaced with a fragment of the amino acid sequence of integrin beta 3 (ITGB3).

[0014] In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized by a replacement of ITAM1 with ITGB3. In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized by a replacement of ITAM2 with ITGB3.

[0015] In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized by a replacement of ITAM3 with ITGB3. In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized in that two immunoreceptor tyrosine-based activation motifs (ITAMs) selected from ITAM1, ITAM2, and ITAM3 are replaced with a fragment of the amino acid sequence of integrin beta 3 (ITGB3).

[0016] In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized by a replacement of ITAM2 and ITAM3 with two ITGB3s. In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized by a replacement of ITAM1 and ITAM3 with two ITGB3s.

[0017] In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized by a replacement of ITAM1 and ITAM2 with two ITGB3s. In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized in that ITAM1 has the amino acid sequence APAYKQGQNQLYNELNLGRREEYDVLDKR (SEQ ID NO: 1).

[0018] In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized in that ITAM2 has the amino acid sequence PRRKNPQEGLYNELQKDKMAEAYSEIGM (SEQ ID NO: 2).

[0019] In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized in that the ITAM3 has the amino acid sequence ERRRGKGHDGLYQGLSTATKDTYDALHMQ (SEQ ID NO: 3).

[0020] In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized in that ITGB3 has the amino acid sequence DTANNPLYKEATSTFTNITYRGT (SEQ ID NO: 4) or the amino acid sequence DTANNPLYKEATSTFTNITYRGT (SEQ ID NO: 4) with any one or two conservative substitutions.

[0021] In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized in that ITGB3 has the amino acid sequence DTANNPLYKEATSTFTNITYRGT (SEQ ID NO: 4) with any one conservative substitution.

[0022] In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized in that ITGB3 has the amino acid sequence DTANNPLYKEATSTFTNITYRGT (SEQ ID NO: 4) with any two conservative substitutions.

[0023] In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized in that ITGB3 has the amino acid sequence DTANNPLYKEATSTFTNITYRGT (SEQ ID NO: 4).

[0024] In some aspects, the alternative intracellular signaling domain of a chimeric antigen receptor (CAR) has the amino acid sequence RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGDTANNPLYKEATSTFTNITYRGTALPPR (SEQ ID NO:5), or has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:5 and differs from SEQ ID NO:5 only in non-conserved amino acid residues.

[0025] In some aspects, the alternative intracellular signaling domain of a chimeric antigen receptor (CAR) has the amino acid sequence RVKFSRSADDTANNPLYKEATSTFTNITYRGTRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:6), or has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:6 and differs from SEQ ID NO:6 only in non-conserved amino acid residues.

[0026] In some aspects, the alternative intracellular signaling domain of a chimeric antigen receptor (CAR) has the amino acid sequence RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKDTANNPLYKEATSTFTNITYRGTKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:7), or has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:7 and differs from SEQ ID NO:7 only in non-conserved amino acid residues.

[0027] In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) has the amino acid sequence RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKDTANNPLYKEATSTFTNITYRGTKGDTANNPLYKEATSTFTNITYRGTALPPR (SEQ ID NO:8), or has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:8 and differs from SEQ ID NO:8 only in non-conserved amino acid residues.

[0028] In some aspects, the alternative intracellular signaling domain of a chimeric antigen receptor (CAR) has the amino acid sequence RVKFSRSADDTANNPLYKEATSTFTNITYRGTRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGDTANNPLYKEATSTFTNITYRGTALPPR (SEQ ID NO:9), or has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:9 and differs from SEQ ID NO:9 only in non-conserved amino acid residues.

[0029] In some aspects, the alternative intracellular signaling domain of a chimeric antigen receptor (CAR) has the amino acid sequence RVKFSRSADDTANNPLYKEATSTFTNITYRGTRGRDPEMGGKDTANNPLYKEATSTFTNITYRGTKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 10), or has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 10 and differs from SEQ ID NO: 10 only in non-conserved amino acid residues.

[0030] In one aspect, the present invention relates to an isolated alternative intracellular signaling domain of a chimeric antigen receptor (CAR), comprising one or two immunoreceptor tyrosine-based activation motifs (ITAMs) selected from ITAM1, ITAM2, and ITAM3, and one or two fragments of the amino acid sequence of integrin beta 3 (ITGB3), wherein the total number of ITAMs and ITGB3s is three.

[0031] In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized in that it comprises two immunoreceptor tyrosine-based activation motifs (ITAMs) selected from ITAM1, ITAM2, and ITAM3, and a fragment of the amino acid sequence of integrin beta 3 (ITGB3).

[0032] In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized by comprising ITAM2, ITAM3, and ITGB3. In some embodiments, the alternative intracellular signaling domains include ITAM1, ITAM3, and ITGB3.

[0033] In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized by comprising ITAM1, ITAM2, and ITGB3. In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized in that it comprises one immunoreceptor tyrosine-based activation motif (ITAM) selected from ITAM1, ITAM2, and ITAM3, and two fragments of the amino acid sequence of integrin beta 3 (ITGB3).

[0034] In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized in that it comprises two fragments of the amino acid sequences of ITAM1 and integrin beta 3 (ITGB3).

[0035] In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized in that it comprises two fragments of the amino acid sequences of ITAM2 and integrin beta 3 (ITGB3).

[0036] In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized in that it comprises two fragments of the amino acid sequences of ITAM3 and integrin beta 3 (ITGB3).

[0037] In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized in that ITAM1 has the amino acid sequence APAYKQGQNQLYNELNLGRREEYDVLDKR (SEQ ID NO: 1).

[0038] In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized in that ITAM2 has the amino acid sequence PRRKNPQEGLYNELQKDKMAEAYSEIGM (SEQ ID NO: 2).

[0039] In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized in that the ITAM3 has the amino acid sequence ERRRGKGHDGLYQGLSTATKDTYDALHMQ (SEQ ID NO: 3).

[0040] In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized in that ITGB3 has the amino acid sequence DTANNPLYKEATSTFTNITYRGT (SEQ ID NO: 4) or the amino acid sequence DTANNPLYKEATSTFTNITYRGT (SEQ ID NO: 4) with any one or two conservative substitutions.

[0041] In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized in that ITGB3 has the amino acid sequence DTANNPLYKEATSTFTNITYRGT (SEQ ID NO: 4) with any one conservative substitution.

[0042] In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized in that ITGB3 has the amino acid sequence DTANNPLYKEATSTFTNITYRGT (SEQ ID NO: 4) with any two conservative substitutions.

[0043] In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized in that ITGB3 has the amino acid sequence DTANNPLYKEATSTFTNITYRGT (SEQ ID NO: 4).

[0044] In some aspects, the alternative intracellular signaling domain of a chimeric antigen receptor (CAR) has the amino acid sequence RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGDTANNPLYKEATSTFTNITYRGTALPPR (SEQ ID NO:5), or has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:5 and differs from SEQ ID NO:5 only in non-conserved amino acid residues.

[0045] In some aspects, the alternative intracellular signaling domain of a chimeric antigen receptor (CAR) has the amino acid sequence RVKFSRSADDTANNPLYKEATSTFTNITYRGTRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:6), or has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:6 and differs from SEQ ID NO:6 only in non-conserved amino acid residues.

[0046] In some aspects, the alternative intracellular signaling domain of a chimeric antigen receptor (CAR) has the amino acid sequence RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKDTANNPLYKEATSTFTNITYRGTKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:7), or has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:7 and differs from SEQ ID NO:7 only in non-conserved amino acid residues.

[0047] In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) has the amino acid sequence RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKDTANNPLYKEATSTFTNITYRGTKGDTANNPLYKEATSTFTNITYRGTALPPR (SEQ ID NO:8), or has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:8 and differs from SEQ ID NO:8 only in non-conserved amino acid residues.

[0048] In some aspects, the alternative intracellular signaling domain of a chimeric antigen receptor (CAR) has the amino acid sequence RVKFSRSADDTANNPLYKEATSTFTNITYRGTRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGDTANNPLYKEATSTFTNITYRGTALPPR (SEQ ID NO:9), or has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:9 and differs from SEQ ID NO:9 only in non-conserved amino acid residues.

[0049] In some aspects, the alternative intracellular signaling domain of a chimeric antigen receptor (CAR) has the amino acid sequence RVKFSRSADDTANNPLYKEATSTFTNITYRGTRGRDPEMGGKDTANNPLYKEATSTFTNITYRGTKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 10), or has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 10 and differs from SEQ ID NO: 10 only in non-conserved amino acid residues.

[0050] In one aspect, the invention relates to an isolated nucleic acid encoding the alternative intracellular signaling domain described above. In one aspect, the present invention relates to an isolated chimeric antigen receptor (CAR) comprising an alternative intracellular signaling domain as described above.

[0051] In some embodiments, the isolated chimeric antigen receptor (CAR) comprises: a) an antigen-binding domain specific for a tumor antigen; b) transmembrane domain; c) costimulatory domain; d) Alternative intracellular signaling domains The present invention is characterized by comprising:

[0052] In some aspects, the isolated chimeric antigen receptor (CAR) is characterized in that the antigen-binding domain specific for the tumor antigen is a Fab or scFv. In some aspects, the isolated chimeric antigen receptor (CAR) is characterized in that the antigen-binding domain that is specific for a tumor antigen is an scFv.

[0053] In some aspects, the isolated chimeric antigen receptor (CAR) is characterized in that the antigen binding domain is specific for a tumor antigen selected from the group of CD19, CD20, CD123, or BCMA.

[0054] In some aspects, the isolated chimeric antigen receptor (CAR) is characterized in that the antigen binding domain is specific for the CD19 tumor antigen. In some aspects, the isolated chimeric antigen receptor (CAR) is characterized in that the antigen binding domain specific for a CD19 tumor antigen has the amino acid sequence DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS (SEQ ID NO: 11).

[0055] In some aspects, the isolated chimeric antigen receptor (CAR) is characterized in that the transmembrane domain is a transmembrane domain of a protein selected from the group consisting of the T-cell receptor-alpha, -beta, or -zeta chains, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.

[0056] In some aspects, the isolated chimeric antigen receptor (CAR) is characterized in that the transmembrane domain is CD8. In some aspects, the isolated chimeric antigen receptor (CAR) is characterized in that the transmembrane domain of CD8 has an amino acid sequence represented by IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 12).

[0057] In some aspects, the isolated chimeric antigen receptor (CAR) is characterized in that the costimulatory domain is a functional signaling domain of a protein selected from the group consisting of OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137).

[0058] In some aspects, the isolated chimeric antigen receptor (CAR) is characterized in that the costimulatory domain is CD28 or 4-1BB. In some aspects, the isolated chimeric antigen receptor (CAR) is characterized in that the costimulatory domain is CD28.

[0059] In some aspects, the isolated chimeric antigen receptor (CAR) is characterized in that the CD28 costimulatory domain has the amino acid sequence AAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 13).

[0060] In some aspects, the isolated chimeric antigen receptor (CAR) is characterized in that the costimulatory domain is 4-1BB. In some aspects, the isolated chimeric antigen receptor (CAR) is characterized in that the 4-1BB costimulatory domain has an amino acid sequence represented by KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 14).

[0061] In some aspects, the isolated chimeric antigen receptor (CAR) is characterized in that it further comprises a leader sequence. In some aspects, the isolated chimeric antigen receptor (CAR) comprises: having an amino acid sequence represented by an amino acid sequence selected from the group of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26; or It has an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26, and differs from the amino acid sequence selected from the group of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 only by non-conserved amino acid residues.

[0062] The chimeric antigen receptor 19 CAR-T-CD28-altcd3zeta (ITGB3-ITAM2-ITAM3) has the amino acid sequence

[0063] [ka]

[0064] It has. The chimeric antigen receptor 19 CAR-T-CD28-altcd3 zeta (ITAM1-ITGB3-ITAM3) has the amino acid sequence

[0065] [ka]

[0066] It has. The chimeric antigen receptor 19 CAR-T-CD28-altcd3zeta (ITAM1-ITAM2-ITGB3) has the amino acid sequence

[0067] [ka]

[0068] It has. The chimeric antigen receptor 19 CAR-T-CD28-altcd3zeta (ITGB3-ITGB3-ITAM3) has the amino acid sequence

[0069] [ka]

[0070] It has. The chimeric antigen receptor 19 CAR-T-CD28-altcd3 zeta (ITAM1-ITGB3-ITGB3) has the amino acid sequence

[0071] [ka]

[0072] It has. The chimeric antigen receptor 19 CAR-T-CD28-altcd3zeta (ITGB3-ITAM2-ITGB3) has the amino acid sequence

[0073] [ka]

[0074] It has. The chimeric antigen receptor 19CAR-T-41bb-altcd3zeta (ITGB3-ITAM2-ITAM3) has the amino acid sequence

[0075] [ka]

[0076] It has. The chimeric antigen receptor 19CAR-T-41bb-altcd3zeta (ITAM1-ITGB3-ITAM3) has the amino acid sequence

[0077] [ka]

[0078] It has. The chimeric antigen receptor 19CAR-T-41bb-altcd3zeta (ITAM1-ITAM2-ITGB3) has the amino acid sequence

[0079] [ka]

[0080] It has. The chimeric antigen receptor 19CAR-T-41bb-altcd3zeta (ITGB3-ITGB3-ITAM3) has the amino acid sequence

[0081] [ka]

[0082] It has. The chimeric antigen receptor 19CAR-T-41bb-altcd3zeta (ITAM1-ITGB3-ITGB3) has the amino acid sequence

[0083] [ka]

[0084] It has. The chimeric antigen receptor 19CAR-T-41bb-altcd3zeta (ITGB3-ITAM2-ITGB3) has the amino acid sequence

[0085] [ka]

[0086] It has. In one aspect, the present invention relates to an isolated nucleic acid encoding the above-described isolated chimeric antigen receptor (CAR).

[0087] In one aspect, the invention relates to an expression vector comprising the above nucleic acid. In one aspect, the invention relates to a delivery vector comprising the above nucleic acid. In some aspects, the vector is selected from the group consisting of DNA, RNA, a plasmid, a lentiviral vector, an adeno-associated viral vector, an adenoviral vector, or a retroviral vector.

[0088] In some aspects, the vector further comprises a promoter. In some embodiments, the vector is an in vitro transcription vector. In some embodiments, the nucleic acid sequence further comprises a poly(A) sequence.

[0089] In some embodiments, the nucleic acid sequence further comprises a 3' UTR. In one aspect, the present invention relates to a method for producing a genetically modified cell comprising the above chimeric antigen receptor (CAR), the method comprising the step of transforming a cell according to the above method.

[0090] In one aspect, the present invention relates to a genetically modified cell comprising the above chimeric antigen receptor (CAR), wherein the genetically modified cell is produced by the above method. In some embodiments, the cell is a T lymphocyte.

[0091] In some embodiments, the cells are CD8+ T cells. In some embodiments, the cells are NK cells. In some aspects, the cells are used as a medicament.

[0092] In some aspects, the cells are used as a medicament for a tumor disease. In some aspects, the cells are used as a medicament for a disease in which the tumor is cancer. [Brief explanation of the drawings]

[0093] [Figure 1] FIG. 1 is a diagram of the map of the vector pCDH-19CAR-T-ITGB3-ITGB3-eGFP. [Figure 2] Schematic representation of the expression cassette 19CAR-T-ITGB3-ITGB3-eGFP. [Figure 3] FIG. 1 is a diagram of the map of the vector pCDH-19CAR-T-ITGB3-ITGB3-ITGB3-eGFP. [Figure 4] Schematic representation of the expression cassette 19CAR-T-ITGB3-ITGB3-ITGB3-eGFP. [Figure 5] FIG. 1 is a diagram of the map of the vector pCDH-19CAR-T-ITAM1-ITGB3-ITGB3-eGFP. [Figure 6] Schematic representation of the expression cassette 19CAR-T-ITAM1-ITGB3-ITGB3-eGFP. [Figure 7]FIG. 1 is a diagram of the map of the vector pCDH-19CAR-T-ITAM1-ITAM2-ITGB3-eGFP. [Figure 8] Schematic representation of the expression cassette 19CAR-T-ITAM1-ITAM2-ITGB3-eGFP. [Figure 9] FIG. 1 is a diagram of the map of the vector pCDH-CAR-T-GFP. [Figure 10] Schematic representation of the expression cassette pCDH-CAR-T-GFP. [Figure 11] FIG. 1 is a diagram of the map of the vector pCDH-19CAR-T-ITAM1-ITGB3-ITAM2-eGFP. [Figure 12] Schematic representation of the expression cassette pCDH-19CAR-T-ITAM1-ITGB3-ITAM2-eGFP. [Figure 13] FIG. 1 is a diagram of the map of the vector pCDH-19CAR-T-ITGB3-ITAM2-ITGB3-eGFP. [Figure 14] Schematic representation of the expression cassette pCDH-19CAR-T-ITGB3-ITAM2-ITGB3-eGFP. [Figure 15] FIG. 1 is a diagram of the map of the vector pCDH-19CAR-T_CD28-ITGB3-ITGB3-eGFP. [Figure 16] Schematic representation of the expression cassette pCDH-19CAR-T_CD28-ITGB3-ITGB3-eGFP. [Figure 17] FIG. 1 is a diagram of the map of the vector pCDH-19CAR-T-CD28_ITGB3-ITGB3-ITGB3-eGFP. [Figure 18] Schematic representation of the expression cassette pCDH-19CAR-T-CD28_ITGB3-ITGB3-ITGB3-eGFP. [Figure 19] FIG. 1 is a diagram of the map of the vector pCDH-19CAR-T-CD28_ITAM1-ITGB3-ITGB3-eGFP. [Figure 20] Schematic representation of the expression cassette pCDH-19CAR-T-CD28_ITAM1-ITGB3-ITGB3-eGFP. [Figure 21] FIG. 1 is a diagram of the map of the vector pCDH-19CAR-T-CD28_ITAM1-ITAM2-ITGB3-eGFP. [Figure 22] Schematic representation of the expression cassette pCDH-19CAR-T-CD28_ITAM1-ITAM2-ITGB3-eGFP. [Figure 23] FIG. 1 is a diagram of the map of the vector pCDH-19CAR-T-CD28_ITAM1-ITGB3-ITAM2-eGFP. [Figure 24] Schematic representation of the expression cassette pCDH-19CAR-T-CD28_ITAM1-ITGB3-ITAM2-eGFP. [Figure 25] FIG. 1 is a diagram of the map of the vector pCDH-19CAR-T-CD28_ITGB3-ITAM2-ITGB3-eGFP. [Figure 26] Schematic representation of the expression cassette pCDH-19CAR-T-CD28_ITGB3-ITAM2-ITGB3-eGFP.

[0094] [Table 1] [Figure 27] GFP expression levels in viable T lymphocytes after lentiviral drug transduction, 72 h, %,. [Figure 28] Figure 1 shows the measurement of cytotoxic activity of CAR-T lymphocytes expressing control CAR-ITAM x3 and experimental CAR-ITAM x2-ITGB3 x1 when co-cultured with target cells. A shows the trend of the percentage of T lymphocytes / CAR-T lymphocytes and Raji cells when co-cultured for 0 and 72 hours; B shows the concentration of IFN-γ in the culture medium when T lymphocytes / CAR-T lymphocytes and target cells are co-cultured for 72 hours; C shows the concentration of IL2 in the culture medium when T lymphocytes / CAR-T lymphocytes and target cells are co-cultured for 72 hours. [Figure 29]FIG. 10 shows a comparison of the functional activity of control CAR-ITAMx3 and experimental CAR-ITAMx2-ITGB3x1 gene constructs in terms of their ability to induce the NFAT signaling cascade in the JurkatΔTCRabNFAT-GFP reporter cell line at 72 hours. DETAILED DESCRIPTION OF THE INVENTION

[0095] Definitions and General Methods Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0096] Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, the classifications and methods of the present invention relating to cell culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, synthetic organic chemistry, medicinal and pharmaceutical chemistry, and protein and nucleic acid hybridization and chemistry described herein are well known to those of skill in the art and are widely used in the art. Enzymatic reactions and purification methods are performed according to manufacturer's instructions as common in the art or as described herein.

[0097] "Mammal" refers to any animal classified as a mammal, including primates, humans, rodents, dogs, cats, cows, miniature cows, horses, pigs, etc. "Isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide naturally occurring in an animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein may exist in a substantially purified form or may exist in a non-native environment, such as, for example, a genetically modified cell.

[0098] The term "identity" or "homology" is understood to mean the percentage of amino acid residues in a candidate sequence that are identical with those of the corresponding sequence to which it is compared, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent identity for the entire sequence, and not counting any conservative substitutions as part of the sequence identity. Neither N- nor C-terminal extensions nor insertions shall be construed as reducing identity or homology. Alignment methods and computer programs are well known in the art. Sequence identity can be measured using sequence analysis software (e.g., Sequence Analysis Software Package, Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Ave., Madison, WI 53705). This software matches similar sequences by assigning degrees of homology to various substitutions, deletions (eliminations), and other modifications.

[0099] As used herein, the term "antigen" or "Ag" is defined as a molecule that elicits an immune response. This immune response may include either or both antibody production and activation of specific immunocompetent T cells. Those skilled in the art will understand that virtually any macromolecule, including any protein or peptide, can function as an antigen. Furthermore, antigens may be derived from recombinant or genomic DNA. Those skilled in the art will understand that any DNA containing a nucleotide sequence or partial nucleotide sequence encoding a protein that elicits an immune response therefore encodes an "antigen" as that term is used herein. Furthermore, those skilled in the art will understand that an antigen need not be encoded solely by the full-length nucleotide sequence of a gene. It is readily apparent that the present invention relates to the use of partial nucleotide sequences of two or more genes, by way of non-limiting example, and that these nucleotide sequences can be arranged in various combinations to elicit a desired immune response. Furthermore, those skilled in the art will understand that an antigen need not be encoded by a "gene" at all. It is readily apparent that an antigen may be synthetic or derived from a biological sample. Such biological samples may include, by way of non-limiting example, tissue samples, tumor samples, cells, or biological fluids.

[0100] A "disease" is a condition in an animal's health in which the animal is unable to maintain homeostasis and the animal's health will continue to deteriorate unless the disease is ameliorated. In contrast, a "disorder" in an animal is one in which the animal is able to maintain homeostasis, but the animal's health state is less favorable than it would be without the disorder. If left untreated, the disorder does not necessarily cause a further deterioration in the animal's health state.

[0101] As used herein, the term "malignant neoplasm" is defined as a disease characterized by the rapid and uncontrollable growth of abnormal cells. Malignant cells may spread locally or to other parts of the organism via the bloodstream and lymphatic system. Examples of various malignant neoplasms include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, renal cancer, liver cancer, brain malignancies, lymphoma, leukemia, lung cancer, myeloma, multiple myeloma, etc.

[0102] Malignant neoplasms that can be treated include tumors that are not or have not yet been substantially vascularized, as well as vascularized tumors. Malignant neoplasms can include non-solid tumors (such as hematological tumors, for example, leukemia and lymphoma) or solid tumors. Types of malignant neoplasms treated with the CAR of the present invention include, but are not limited to, carcinomas, blastomas, and sarcomas, as well as certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignant tumors, such as sarcomas, carcinomas, and melanomas. Adult tumors / cancers and pediatric tumors / malignant neoplasms are also included.

[0103] Hematological malignancies are malignant neoplasms of the blood or bone marrow. Examples of hematological (or hematopoietic) malignancies include acute leukemia (e.g., acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia, myeloblastic, promyelocytic, myelomonocytic, monocytic leukemia, and erythroleukemia), chronic leukemia (e.g., chronic myelocytic (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), including leukemia, polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (indolent and high-grade), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.

[0104] A solid tumor is an abnormal mass of tissue that usually does not contain cysts or fluid areas. Solid tumors can be benign or malignant. Various types of solid tumors are named for the type of cells that form them (e.g., sarcoma, carcinoma, and lymphoma). Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid malignancies, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, cholangiocarcinoma, choriocarcinoma, and Wilms' tumor. , cervical cancer, testicular cancer, seminoma, bladder cancer, melanoma, and CNS tumors (e.g., gliomas (such as brain stem gliomas and mixed gliomas)), glioblastoma (also known as glioblastoma multiforme), astrocytoma, CNS lymphoma, germinoma, medulloblastoma, schwannoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, neuroblastoma, retinoblastoma, and brain metastases.

[0105] As used herein, the term "endogenous" refers to any material that is derived from or naturally produced within an organism, cell, tissue, or system.

[0106] As used herein, the term "exogenous" refers to any material that is introduced into an organism, cell, tissue or system, or that is produced outside of an organism, cell, tissue or system. As used herein, the term "expression" is defined as the transcription and / or translation of a particular nucleotide sequence driven by its promoter.

[0107] The term "overexpressed" tumor antigen or "overexpression" of a tumor antigen is intended to refer to an abnormal level of expression of a tumor antigen in cells from a lesion, such as a solid tumor, within a particular tissue or organ of a patient, compared to the level of expression in normal cells derived from that tissue or organ. Patients with solid tumors or hematologic malignancies characterized by tumor antigen overexpression can be determined by standard assays known in the art.

[0108] The terms "patient," "individual," and the like are used interchangeably herein and refer to any animal or cells thereof amenable to the methods described herein, whether in vitro or in situ. In certain non-limiting embodiments, the patient or individual is a human.

[0109] As used herein, the term "polynucleotide" is defined as a chain of nucleotides. Furthermore, a nucleic acid is a polymer of nucleotides. Therefore, as used herein, the terms nucleic acid and polynucleotide are interchangeable. Those skilled in the art have the general knowledge that a nucleic acid is a polynucleotide that can be hydrolyzed into monomeric "nucleotides." Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotides include, but are not limited to, all nucleic acid sequences obtained by any means available in the art, including recombinant means, i.e., cloning nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning techniques and PCR, etc., and synthetic means.

[0110] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide necessarily contains at least two amino acids, with no limit on the maximum number of amino acids that can comprise a protein's or peptide's sequence. A polypeptide includes any peptide or protein containing two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both shorter chains, also commonly referred to in the art as peptides, oligopeptides, and oligomers, and longer chains, commonly referred to in the art as proteins, many of which exist. "Polypeptide" includes, inter alia, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, and fusion proteins. A polypeptide includes natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0111] Detailed Description of the Invention Isolated alternative intracellular signaling domains of chimeric antigen receptors (CARs) The intracellular signaling domain of the CAR, or in other words, the cytoplasmic domain of the present invention, is responsible for activating at least one of the normal effector functions of the immune cell in which the CAR is placed. The term "effector function" refers to the specialized function of a cell. The effector function of a T cell can be, for example, cytolytic activity or helper activity, including cytokine secretion. Thus, the term "intracellular signaling domain" refers to the portion of a protein that transmits an effector function signal and causes the cell to perform a specialized function. Usually, the entire intracellular signaling domain can be used, but in many cases, the entire chain is not necessarily used. When a truncated portion of the intracellular signaling domain is used, such a truncated portion can be used instead of the intact chain, as long as it transmits the effector function signal. Thus, the term "intracellular signaling domain" is meant to include any truncated portion of the intracellular signaling domain that is sufficient to transmit the effector function signal.

[0112] Preferred examples of intracellular signaling domains for use in CARs according to the present invention include the cytoplasmic sequences of the T cell receptor (TCR) and co-receptors that act in concert to initiate signal transduction following antigen receptor binding, as well as any derivatives or variants of these sequences and any synthetic sequences that have the same functional capacity.

[0113] It is known that signals generated through the TCR alone are insufficient for full activation of T cells, and that secondary or costimulatory signals are also required. Thus, T cell activation can be said to be mediated by two classes of cytoplasmic signaling sequences: those that initiate antigen-dependent primary activation through the TCR (primary cytoplasmic signaling sequences) and those that act in an antigen-independent manner to provide secondary or costimulatory signals (secondary cytoplasmic signaling sequences).

[0114] Primary cytoplasmic signaling sequences regulate the primary activation of the TCR complex in either a stimulatory or inhibitory manner. Primary cytoplasmic signaling sequences that act in a stimulatory manner can contain signaling motifs known as immunoreceptor tyrosine-based activation motifs, or ITAMs.

[0115] Examples of ITAMs containing primary cytoplasmic signaling sequences include those derived from TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD8, CD22, CD79a, CD79b, and CD66d. It is particularly preferred that the cytoplasmic signaling molecule in the CAR contains a cytoplasmic signaling sequence derived from CD3 zeta.

[0116] The cytoplasmic domain of a CAR can be designed to include the CD3-zeta signaling domain by itself or in combination with any other desired cytoplasmic domain useful in the context of a CAR.

[0117] The CD3-zeta signaling domain has the amino acid sequence RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0118] The upper signaling domain contains three ITAMs (immunoreceptor tyrosine-based activation motifs), specifically ITAM1, ITAM2 and ITAM3. ITAM1 has the amino acid sequence represented by APAYKQGQNQLYNELNLGRREEYDVLDKR (SEQ ID NO: 1).

[0119] ITAM2 has the amino acid sequence represented by PRRKNPQEGLYNELQKDKMAEAYSEIGM (SEQ ID NO: 2). ITAM3 has the amino acid sequence represented by ERRRGKGHDGLYQGLSTATKDTYDALHMQ (SEQ ID NO: 3).

[0120] In one aspect, the present invention relates to an isolated alternative intracellular signaling domain of a chimeric antigen receptor (CAR), in which one or two immunoreceptor tyrosine-based activation motifs (ITAMs) selected from ITAM1, ITAM2, and ITAM3 are replaced with a fragment of the amino acid sequence of integrin beta 3 (ITGB3).

[0121] In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized in that one immunoreceptor tyrosine-based activation motif (ITAM) selected from ITAM1, ITAM2, and ITAM3 is replaced with a fragment of the amino acid sequence of integrin beta 3 (ITGB3).

[0122] In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized by a replacement of ITAM1 with ITGB3. In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized by a replacement of ITAM2 with ITGB3.

[0123] In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized by a replacement of ITAM3 with ITGB3. In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized in that two immunoreceptor tyrosine-based activation motifs (ITAMs) selected from ITAM1, ITAM2, and ITAM3 are replaced with a fragment of the amino acid sequence of integrin beta 3 (ITGB3).

[0124] In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized by a replacement of ITAM2 and ITAM3 with two ITGB3s. In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized by a replacement of ITAM1 and ITAM3 with two ITGB3s.

[0125] In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized by a replacement of ITAM1 and ITAM2 with two ITGB3s. In one aspect, the present invention relates to an isolated alternative intracellular signaling domain of a chimeric antigen receptor (CAR), comprising one or two immunoreceptor tyrosine-based activation motifs (ITAMs) selected from ITAM1, ITAM2, and ITAM3, and one or two fragments of the amino acid sequence of integrin beta 3 (ITGB3), wherein the total number of ITAMs and ITGB3s is three.

[0126] In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized in that it comprises two immunoreceptor tyrosine-based activation motifs (ITAMs) selected from ITAM1, ITAM2, and ITAM3, and a fragment of the amino acid sequence of integrin beta 3 (ITGB3).

[0127] In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized by comprising ITAM2, ITAM3, and ITGB3. In some embodiments, the alternative intracellular signaling domains include ITAM1, ITAM3, and ITGB3.

[0128] In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized by comprising ITAM1, ITAM2, and ITGB3. In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized in that it comprises one immunoreceptor tyrosine-based activation motif (ITAM) selected from ITAM1, ITAM2, and ITAM3, and two fragments of the amino acid sequence of integrin beta 3 (ITGB3).

[0129] In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized in that it comprises two fragments of the amino acid sequences of ITAM1 and integrin beta 3 (ITGB3).

[0130] In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized in that it comprises two fragments of the amino acid sequences of ITAM2 and integrin beta 3 (ITGB3).

[0131] In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized in that it comprises two fragments of the amino acid sequences of ITAM3 and integrin beta 3 (ITGB3).

[0132] In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized in that ITGB3 has an amino acid sequence represented by DTANNPLYKEATSTFTNITYRGT (SEQ ID NO: 4) or an amino acid sequence represented by DTANNPLYKEATSTFTNITYRGT (SEQ ID NO: 4) with any one or two conservative substitutions.

[0133] In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized in that ITGB3 has an amino acid sequence represented by DTANNPLYKEATSTFTNITYRGT (SEQ ID NO: 4) with any one conservative substitution.

[0134] In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized in that ITGB3 has an amino acid sequence represented by DTANNPLYKEATSTFTNITYRGT (SEQ ID NO: 4) with any two conservative substitutions.

[0135] In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized in that ITGB3 has an amino acid sequence represented by DTANNPLYKEATSTFTNITYRGT (SEQ ID NO: 4).

[0136] Conservative amino acid substitutions refer to the replacement of one amino acid with another amino acid of similar structure, and such substitutions do not result in changes to the properties of ITGB3. Conservative substitutions are shown in Table A under "preferred substitutions." If such substitutions result in altered biological activity, then further significant modifications may be introduced, which are referred to in Table A as "exemplary substitutions."

[0137] [Table 2]

[0138] In some aspects, the alternative intracellular signaling domain of a chimeric antigen receptor (CAR) has the amino acid sequence RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGDTANNPLYKEATSTFTNITYRGTALPPR (SEQ ID NO:5), or has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:5 and differs from SEQ ID NO:5 only in non-conserved amino acid residues.

[0139] In some aspects, the alternative intracellular signaling domain of a chimeric antigen receptor (CAR) has the amino acid sequence RVKFSRSADDTANNPLYKEATSTFTNITYRGTRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:6), or has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:6 and differs from SEQ ID NO:6 only in non-conserved amino acid residues.

[0140] In some aspects, the alternative intracellular signaling domain of a chimeric antigen receptor (CAR) has the amino acid sequence RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKDTANNPLYKEATSTFTNITYRGTKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO:7), or has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:7 and differs from SEQ ID NO:7 only in non-conserved amino acid residues.

[0141] In some aspects, the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) has the amino acid sequence RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKDTANNPLYKEATSTFTNITYRGTKGDTANNPLYKEATSTFTNITYRGTALPPR (SEQ ID NO:8), or has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:8 and differs from SEQ ID NO:8 only in non-conserved amino acid residues.

[0142] In some aspects, the alternative intracellular signaling domain of a chimeric antigen receptor (CAR) has the amino acid sequence RVKFSRSADDTANNPLYKEATSTFTNITYRGTRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGDTANNPLYKEATSTFTNITYRGTALPPR (SEQ ID NO:9), or has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:9 and differs from SEQ ID NO:9 only in non-conserved amino acid residues.

[0143] In some aspects, the alternative intracellular signaling domain of a chimeric antigen receptor (CAR) has the amino acid sequence RVKFSRSADDTANNPLYKEATSTFTNITYRGTRGRDPEMGGKDTANNPLYKEATSTFTNITYRGTKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 10), or has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 10 and differs from SEQ ID NO: 10 only in non-conserved amino acid residues.

[0144] Chimeric antigen receptor (CAR) In one aspect, the present invention relates to an isolated chimeric antigen receptor (CAR) comprising an alternative intracellular signaling domain as described above.

[0145] In some embodiments, the isolated chimeric antigen receptor (CAR) comprises: a) an antigen-binding domain specific for a tumor antigen; b) transmembrane domain; c) costimulatory domain; d) Alternative intracellular signaling domains The present invention is characterized by comprising:

[0146] The present invention relates to a chimeric antigen receptor (CAR) comprising an extracellular domain and an intracellular domain. The extracellular domain contains a target-specific binding element, also referred to as an antigen-binding domain. The intracellular or cytoplasmic domain contains a costimulatory signaling region and a zeta chain portion. The costimulatory signaling region refers to the portion of the CAR that contains the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule, other than an antigen receptor or its ligand, that is required for an efficient lymphocyte response to an antigen.

[0147] A spacer domain can be incorporated between the extracellular domain and the transmembrane domain of a CAR, or between the cytoplasmic domain and the transmembrane domain of a CAR. As used herein, the term "spacer domain" typically refers to any oligopeptide or polypeptide that functions to link the transmembrane domain to either the extracellular domain or the cytoplasmic domain in a polypeptide chain. A spacer domain can contain up to 300 amino acids, preferably 10-100 amino acids, and most preferably 25-50 amino acids.

[0148] In one embodiment, a CAR of the present invention comprises an extracellular domain comprising an antigen-binding domain, a transmembrane domain, and a cytoplasmic domain. In one embodiment, a transmembrane domain that is naturally associated with one of the domains in the CAR is used.

[0149] In another aspect, the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domain to transmembrane domains of surface proteins of the same or different membranes, thereby minimizing interactions with other members of the receptor complex.

[0150] antigen-binding domain The terms "antigen-binding domain" of an antibody or "antigen-binding portion" or "antigen-binding fragment" of an antibody (or simply "antibody portion" or "antibody fragment"), as used herein, refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-size antibody. Examples of binding fragments encompassed by the term "antigen-binding portion" of an antibody include the following: (i) a monovalent fragment, a Fab fragment, consisting of the VL, VH, CL and CH1 domains; (ii) a bivalent fragment, the F(ab')2 fragment, containing two Fab fragments linked by a disulfide bridge at the hinge region; (iii) an Fd fragment consisting of a VH domain and a CH1 domain; (iv) an Fv fragment consisting of the VL and VH domains in a single arm of an antibody; (v) dAb fragments composed of VH / VHH domains (Ward et al., (1989) Nature 341:544-546); and (vi) an isolated complementarity determining region (CDR).

[0151] In addition, the two regions of an Fv fragment, VL and VH, can be encoded by separate genes and linked using recombinant methods using a synthetic linker that allows them to be combined into a single protein chain, in which the VL and VH regions pair to form a monovalent molecule (known as a single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain molecules are also contemplated as encompassed within the term "antigen-binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened in the same manner as intact antibodies.

[0152] The term "variable" refers to the fact that certain portions of variable domains vary significantly in sequence among antibodies. V domains mediate antigen binding and determine the specificity of a particular antibody for its specific antigen. However, variability is not evenly distributed across the 110-amino acid span of variable domains. Instead, V regions are composed of invariant segments of 15-30 amino acids called framework regions (FRs), separated by shorter regions of hypervariability called "hypervariable regions" or CDRs. Naturally occurring heavy and light chain variable domains each contain four FR regions, primarily in a beta-sheet configuration, connected by three hypervariable regions, which form connecting loops and, in some cases, form part of the beta-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, together with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies.

[0153] As used herein, the term "hypervariable region" refers to the amino acid residues of an antibody that are responsible for antigen binding. Hypervariable regions generally comprise amino acid residues from the "complementarity determining regions" or "CDRs" and / or from the "hypervariable loops".

[0154] In certain cases, it may be desirable to modify one or more CDR amino acid residues to improve binding affinity for the target epitope. This is known as "affinity maturation" and may be performed as needed in conjunction with humanization, for example, in situations where antibody humanization results in a decrease in binding specificity or affinity and back mutation alone is not sufficient to improve binding specificity or affinity. Various affinity maturation methods are known in the art, such as the in vitro scanning saturation mutagenesis method described by Burks et al., Proc Natl Acad Sci USA, 94:412-417 (1997), and the stepwise in vitro affinity maturation method described by Wu et al., Proc Natl Acad Sci USA, 95:6037-6042 (1998).

[0155] An antibody antigen-binding domain according to the present invention that "binds to" a target antigen refers to an antibody antigen-binding domain that binds to the antigen with sufficient affinity and that cross-reacts to a small extent with other proteins such that it can be used as a diagnostic and / or therapeutic agent in targeting proteins, cells, or tissues that express the antigen. Analytical methods: The extent of binding of the antibody antigen-binding domain to "non-target" proteins is less than about 10% of the binding of the antibody antigen-binding domain to a specific target protein, as determined by fluorescence-activated cell sorting (FACS), radioimmunoprecipitation assay (RIA), or ELISA. With respect to the binding of an antibody antigen-binding domain to a target molecule, the term "specific binding," or the phrase "specifically binds to" or "specific for" a particular polypeptide or epitope on a particular polypeptide target, refers to binding that is clearly (measurably) distinct from non-specific interactions.

[0156] In one embodiment, the CAR of the present invention comprises a target-specific binding element, alternatively referred to as an antigen-binding domain. The choice of domain depends on the type and number of ligands that define the surface of the target cell. For example, the antigen-binding domain can be selected to recognize a ligand that functions as a cell surface marker on the target cell associated with a particular disease state. Thus, examples of cell surface markers that can function as ligands for the domain of the antigen-binding domain in the CAR of the present invention include those associated with malignant cells.

[0157] In one embodiment, the CAR of the present invention can be engineered to target a tumor antigen of interest by engineering a desired antigen-binding domain that specifically binds to the antigen on tumor cells. In the context of the present invention, "tumor antigen" or "hyperproliferative disorder antigen" or "antigen associated with hyperproliferative disorder" refers to an antigen that is common to a specific hyperproliferative disorder, such as a malignant neoplasm. The antigens discussed herein are included merely as examples. This list is not intended to be exhaustive, and further examples will be readily apparent to those skilled in the art.

[0158] Tumor antigens are proteins produced by tumor cells that elicit an immune response, particularly a T cell-mediated immune response. The choice of antigen-binding domain of the present invention depends on the specific type of malignant neoplasm to be treated. Tumor antigens are well known in the art and include, for example, glioma-associated antigens, carcinoembryonic antigen (CEA), β-human chorionic gonadotropin, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostase, prostate-specific antigen (PSA), PAP, NY-ESO-1, LAGE-1a, p53, prostein, PSMA, Her2 / neu, survivin and telomerase, prostate cancer tumor antigen-1 (PCTA-1), MAGE, ELF2M, neutrophil elastase, ephrin B2, CD22, insulin growth factor (IGF)-I, IGF-II, IGF receptor, and mesothelin.

[0159] In one embodiment, the tumor antigen comprises one or more antigenic tumor epitopes associated with malignant tumors. Malignant tumors express several proteins that can serve as target antigens for immune responses. These molecules include, but are not limited to, tissue-specific antigens such as MART-1, tyrosinase, and GP100 in melanoma, and prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA) in prostate cancer. Other target molecules belong to the group of transformation-related molecules, such as the oncogene HER-2 / Neu / ErbB-2. Yet another group of target antigens is oncofetal antigens, such as carcinoembryonic antigen (CEA). In B-cell lymphomas, tumor-specific idiotypic immunoglobulins constitute truly tumor-specific immunoglobulin antigens that are unique to individual tumors. B-cell differentiation antigens, such as CD19, CD20, and CD37, are other candidates for target antigens in B-cell lymphomas. Some of these antigens (CEA, HER-2, CD19, CD20, idiotype) have been used with some success as targets for passive immunotherapy using monoclonal antibodies.

[0160] The type of tumor antigen described in the present invention can also be a tumor-specific antigen (TSA) or a tumor-associated antigen (TAA). TSAs are unique to tumor cells and do not occur on other cells in the organism. TAA antigens are not unique to tumor cells and are also expressed on normal cells under conditions that prevent the induction of a state of immune tolerance to the antigen. Antigen expression in tumors can occur under conditions that allow the immune system to respond to the antigen. TAA can be an antigen expressed on normal cells during fetal development, when the immune system is immature and unable to respond, or TAA can be an antigen that is normally present at very low levels on normal cells but is expressed at much higher levels on tumor cells.

[0161] Non-limiting examples of TSA or TAA antigens include differentiation antigens such as MART-1 / MelanA (MART-1), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, and p15; overexpressed fetal antigens such as CEA; overexpressed oncogenes and mutated tumor suppressor genes such as p53, Ras, and HER-2 / neu; unique tumor antigens resulting from chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, and MYL-RAR; and viral antigens such as Epstein-Barr virus antigen EBVA and human papillomavirus (HPV) antigens E6 and E7. Other large protein-based antigens include TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3 / CA 27.29 / BCAA, CA 195, CA 242, CA-50, CAM43, CD68 / P1, CO-029, FGF-5, G250, Ga733 / EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB / 70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 / Mac-2 binding protein / cyclophilin C-related protein, TAAL6, TAG72, TLP, and TPS.

[0162] In some aspects, the isolated chimeric antigen receptor (CAR) is characterized in that the antigen binding domain is specific for a tumor antigen selected from the group of CD19, CD20, CD123, or BCMA.

[0163] In some aspects, the isolated chimeric antigen receptor (CAR) is characterized in that the antigen binding domain is specific for the CD19 tumor antigen. In some aspects, the isolated chimeric antigen receptor (CAR) is characterized in that the antigen-binding domain specific for the tumor antigen is a Fab or scFv.

[0164] In some aspects, the isolated chimeric antigen receptor (CAR) is characterized in that the antigen-binding domain that is specific for a tumor antigen is an scFv. In some aspects, the isolated chimeric antigen receptor (CAR) is characterized in that an antigen binding domain that is specific for a CD19 tumor antigen has an amino acid sequence represented by DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSGKPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS (SEQ ID NO: 11), or has an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 11 and differs from SEQ ID NO: 11 only at non-conserved amino acid residues.

[0165] The antigen-binding domain having the amino acid sequence of SEQ ID NO: 11 is: a) a light chain variable domain having the amino acid sequence DIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEIT; b) a linker with the amino acid sequence GSTSGSGKPGSGEGSTKG; c) a heavy chain variable domain having the amino acid sequence EVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETTYYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS; It consists of:

[0166] Costimulatory domain A costimulatory domain or costimulatory signaling region refers to a portion of a CAR that contains the intracellular domain of a costimulatory molecule. A costimulatory molecule is a cell surface molecule other than an antigen receptor or its ligand that is required for an efficient lymphocyte response to an antigen. Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, CD30, ICAM-1, CDS, CD40, PD-1, ICOS (CD278), lymphocyte function-associated antigen 1 (LFA-1, CD11a / CD18), CD2, CD7, LIGHT, NKG2C, B7-H3, and ligands that specifically bind to CD83.

[0167] In some aspects, the isolated chimeric antigen receptor (CAR) is characterized in that the costimulatory domain is a functional signaling domain of a protein selected from the group consisting of OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137).

[0168] The cytoplasmic signaling sequences within the cytoplasmic signaling portion of the CAR can be linked to each other in random or specified order. If necessary, a short oligopeptide or polypeptide linker, preferably 2-10 amino acids in length, can form the linkage. Specifically, a glycine-serine doublet is a suitable linker.

[0169] In one embodiment, the cytoplasmic domain of the CAR is engineered to include an alternative intracellular signaling domain as described above and the costimulatory signaling domain of CD28. In another embodiment, the cytoplasmic domain of the CAR is engineered to contain an alternative intracellular signaling domain as described above and the costimulatory signaling domain of 4-1BB.

[0170] In yet another embodiment, the cytoplasmic domain is engineered to include the alternative intracellular signaling domains described above and the costimulatory domains of CD28 and 4-1BB. In some aspects, the isolated chimeric antigen receptor (CAR) is characterized in that the costimulatory domain of CD28 has the amino acid sequence represented by AAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 13).

[0171] In some aspects, the isolated chimeric antigen receptor (CAR) is characterized in that the costimulatory domain of 4-1BB has an amino acid sequence represented by KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 14).

[0172] Transmembrane domain Regarding the transmembrane domain, CAR can be engineered to include a transmembrane domain fused to the extracellular domain of CAR.In one embodiment, the transmembrane domain that naturally associates with one of the domains in CAR is used.In some cases, the transmembrane domain can be selected or modified by amino acid substitution to prevent the domain from binding to the transmembrane domain of the same or different membrane surface protein, thereby minimizing the interaction with other members of the receptor complex.

[0173] Transmembrane domains can be derived from either natural or synthetic sources. If the source is natural, the domain can be derived from any membrane-bound or transmembrane protein. Transmembrane regions for particular use in the present invention can be derived from (i.e., can be engineered to include at least the transmembrane regions of) the alpha, beta, or zeta chains of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD154. Alternatively, the transmembrane domain can be synthetic, in which case it will contain predominantly hydrophobic residues such as leucine and valine. Preferably, triplets of phenylalanine, tryptophan, and valine will be found at both ends of a synthetic transmembrane domain.

[0174] In some aspects, the isolated chimeric antigen receptor (CAR) is characterized in that the transmembrane domain is a transmembrane domain of a protein selected from the group consisting of the T-cell receptor-alpha, -beta, or -zeta chains, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.

[0175] In some aspects, the isolated chimeric antigen receptor (CAR) is characterized in that the transmembrane domain is CD8. In some aspects, the isolated chimeric antigen receptor (CAR) is characterized in that the transmembrane domain of CD8 has the amino acid sequence IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 12).

[0176] Optionally, a short oligopeptide or polypeptide linker, preferably between 2 and 10 amino acids in length, can form the link between the transmembrane and cytoplasmic signaling domains of the CAR. Specifically, a glycine-serine doublet provides a suitable linker.

[0177] In some cases, the transmembrane domain of a CAR according to the present invention comprises the hinge domain of CD8. In one embodiment, the hinge domain of CD8 has the amino acid sequence TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 35).

[0178] In some aspects, the isolated chimeric antigen receptor (CAR) is characterized in that it further comprises a leader sequence. In some aspects, the isolated chimeric antigen receptor (CAR) comprises: having an amino acid sequence represented by an amino acid sequence selected from the group of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26; or It has an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26, and differs from the amino acid sequence selected from the group of SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 only by non-conserved amino acid residues.

[0179] The chimeric antigen receptor 19 CAR-T-CD28-altcd3zeta (ITGB3-ITAM2-ITAM3) has the amino acid sequence

[0180] [ka]

[0181] It has. The chimeric antigen receptor 19 CAR-T-CD28-altcd3 zeta (ITAM1-ITGB3-ITAM3) has the amino acid sequence

[0182] [ka]

[0183] It has. The chimeric antigen receptor 19 CAR-T-CD28-altcd3zeta (ITAM1-ITAM2-ITGB3) has the amino acid sequence

[0184] [ka]

[0185] It has. The chimeric antigen receptor 19 CAR-T-CD28-altcd3 zeta (ITGB3-ITGB3-ITAM3) has the amino acid sequence

[0186] [ka]

[0187] It has. The chimeric antigen receptor 19 CAR-T-CD28-altcd3 zeta (ITAM1-ITGB3-ITGB3) has the amino acid sequence

[0188] [ka]

[0189] It has. The chimeric antigen receptor 19 CAR-T-CD28-altcd3zeta (ITGB3-ITAM2-ITGB3) has the amino acid sequence

[0190] [ka]

[0191] It has. The chimeric antigen receptor 19CAR-T-41bb-altcd3zeta (ITGB3-ITAM2-ITAM3) has the amino acid sequence

[0192] [ka]

[0193] It has. The chimeric antigen receptor 19CAR-T-41bb-altcd3zeta (ITAM1-ITGB3-ITAM3) has the amino acid sequence

[0194] [ka]

[0195] It has. The chimeric antigen receptor 19CAR-T-41bb-altcd3zeta (ITAM1-ITAM2-ITGB3) has the amino acid sequence

[0196] [ka]

[0197] It has. The chimeric antigen receptor 19CAR-T-41bb-altcd3zeta (ITGB3-ITGB3-ITAM3) has the amino acid sequence

[0198] [ka]

[0199] It has. The chimeric antigen receptor 19CAR-T-41bb-altcd3zeta (ITAM1-ITGB3-ITGB3) has the amino acid sequence

[0200] [ka]

[0201] It has. The chimeric antigen receptor 19CAR-T-41bb-altcd3zeta (ITGB3-ITAM2-ITGB3) has the amino acid sequence

[0202] [ka]

[0203] It has. nucleic acid molecule The terms "nucleic acid," "nucleic sequence," "nucleic acid sequence," "polynucleotide," "oligonucleotide," "polynucleotide sequence," and "nucleotide sequence," used interchangeably herein, refer to an ordered sequence of nucleotides, modified or unmodified, defining a fragment or region of a nucleic acid, containing or not containing non-naturally occurring nucleotides, and being either double-stranded DNA or RNA, single-stranded DNA or RNA, or a transcription product of said DNA.

[0204] It is also necessary to include herein that the present invention does not relate to nucleotide sequences in their natural chromosomal environment, i.e., in their natural state. The sequences of the present invention are isolated and / or purified, i.e., sampled directly or indirectly, for example, by copying, and their environment is at least partially modified. Therefore, it is also necessary to refer herein to isolated nucleic acids obtained by recombinant genetics, for example, by host cells, or obtained by chemical synthesis.

[0205] An "isolated" nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one nucleic acid molecule-contaminant to which it is normally associated in the natural source of the protein's (polypeptide's) nucleic acid. An isolated nucleic acid molecule is different from the form or set in which it is found under natural conditions. Thus, an isolated nucleic acid molecule is different from the nucleic acid molecule present in a cell under natural conditions. However, an isolated nucleic acid molecule includes a nucleic acid molecule located in a cell in which the protein (polypeptide) is normally expressed, for example, if the nucleic acid molecule has a chromosomal location that differs from its location in the cell under natural conditions.

[0206] Unless otherwise indicated, the term nucleotide sequence includes its complement. Thus, a nucleic acid having a particular sequence should be understood to encompass its complementary strand, with its complementary sequence.

[0207] In the context of the present invention, the following abbreviations for commonly occurring nucleic acid bases are used: "A" for adenine, "C" for cytosine, "G" for guanine, "T" for thymine (5-methyluracil), which is absent in RNA but occurs in DNA in place of uracil, and "U" for uracil, which occurs in RNA in place of thymine.

[0208] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or RNA can also include introns, in that a nucleotide sequence that encodes a protein may in some versions contain introns.

[0209] "Encoding" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, either a predetermined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a predetermined sequence of amino acids, and the biological properties resulting therefrom. Thus, a gene encodes a protein when transcription and translation of its corresponding mRNA results in the production of that protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand used as a template for transcription of a gene or cDNA can be referred to as encoding the protein or other product of that gene or cDNA.

[0210] For example, nucleic acid sequences encoding desired molecules can be obtained using recombinant methods known in the art, such as by screening libraries from cells which express the gene, by deriving the gene from a vector known to contain it, or by isolating it directly from cells and tissues containing it using standard techniques. Alternatively, the gene of interest can be made synthetically, more usually rather than cloned.

[0211] In one aspect, the invention relates to an isolated nucleic acid encoding the alternative intracellular signaling domain described above. In one aspect, the present invention relates to an isolated nucleic acid encoding the above-mentioned isolated chimeric antigen receptor (CAR) comprising the above-mentioned alternative intracellular signaling domain.

[0212] vector As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. In some embodiments, a vector is a plasmid, i.e., a circular double-stranded molecule of DNA into which additional DNA segments can be incorporated by an enzymatic ligation reaction. In some embodiments, a vector is a viral vector, in which case additional DNA segments can be incorporated into the viral genome by an enzymatic ligation reaction. In some embodiments, vectors are capable of autonomous replication in genetically modified cells into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors having a bacterial origin of replication site). In further embodiments, vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, thereby replicating along with the host genes. Furthermore, certain vectors are capable of inducing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors").

[0213] As used herein, the term "expression control sequence" refers to polynucleotide sequences necessary for the expression and processing of coding sequences into which they are incorporated by enzymatic ligation. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and, if desired, sequences that enhance protein secretion. The nature of such control sequences varies depending on the host organism. Eukaryotic cells include promoters, polyadenylation signals, and enhancers. The term "control sequence" is intended to include at least all components whose presence is essential for expression and processing, and may also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences.

[0214] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader sequence is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; and a ribosome binding site is operably linked to a coding sequence if it is configured so as to promote translation. Typically, "operably linked" means that the linked DNA sequences are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers need not be contiguous.

[0215] By "delivery vector" or "delivery vectors" is intended any delivery vector that can be used in the present invention to contact a cell (i.e., "contact") or deliver (i.e., "introduce") the required agents / chemicals and molecules (proteins or nucleic acids) into a cell or intracellular compartment. Delivery vectors include, but are not limited to, liposomal delivery vectors, viral delivery vectors, drug delivery vectors, chemical carriers, polymeric carriers, lipoplexes, polyplexes, dendrimers, microbubbles (ultrasound contrast agents), nanoparticles, emulsions, or other suitable introduction vectors. These delivery vectors allow for the delivery of molecules, chemicals, macromolecules (genes, proteins), or other vectors such as plasmids, peptides. In these cases, the delivery vector is a molecular carrier. By "delivery vector" or "delivery vectors" is also intended delivery methods for performing transfection.

[0216] "Expression vector" refers to a vector containing a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. Expression vectors contain the appropriate cis-acting elements for expression; other elements for expression may be supplied by elements of a genetically engineered cell or in an in vitro expression system. Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate a recombinant polynucleotide.

[0217] In short, the expression of natural or synthetic nucleic acid encoding CAR is usually carried out by operably linking the nucleic acid encoding CAR polypeptide or a part thereof to a promoter, and incorporating the construct into an expression vector.The vector can be suitable for replication and integration into eukaryotic cells.Usually, cloning vectors contain transcription and translation terminators, initiation sequences, and promoters that are useful for regulating the expression of desired nucleic acid sequences.

[0218] As used herein, the term "promoter" is defined as a DNA sequence recognized by the synthetic machinery of a cell or introduced synthetic machinery necessary to initiate the specific transcription of a polynucleotide sequence.

[0219] As used herein, the term "promoter / regulatory sequence" refers to a nucleic acid sequence that is necessary for the expression of a gene product operably linked to the promoter / regulatory sequence. In some instances, this sequence may be a core promoter sequence, and in other instances, this sequence may also include an enhancer sequence and other regulatory elements that are necessary for the expression of the gene product. The promoter / regulatory sequence may, for example, express the gene product in a tissue-specific manner.

[0220] A "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell under most or all physiological conditions of the cell.

[0221] An "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or specifying a gene product, causes the gene product to be produced in a cell substantially only when an inducer corresponding to the promoter is present in the cell.

[0222] A "tissue-specific" promoter is a nucleotide sequence that, when operably linked to a polynucleotide that encodes or specifies a gene product, causes the gene product to be produced in a cell substantially only if the cell is a cell of the tissue type corresponding to the promoter.

[0223] One example of a suitable promoter is the immediate-early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is the elongation factor 1 alpha (EF-1a) promoter. However, other constitutive promoter sequences can also be used, including, but not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus immediate-early promoter, and Rous sarcoma virus promoter, as well as human gene promoters, such as the actin gene promoter, myosin gene promoter, hemoglobin gene promoter, and creatine kinase gene promoter. Furthermore, the present invention need not be limited to the use of constitutive promoters. Inducible promoters are also contemplated as part of the present invention. The use of an inducible promoter provides a molecular switch that can turn on expression of a polynucleotide sequence to which it is operably linked when such expression is desired, or turn off expression when expression is not desired. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid gene promoters, progesterone gene promoters, and tetracycline gene promoters.

[0224] Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. Typically, these are located in the region 30–110 bp upstream of the start site, although some promoters have recently been shown to contain functional elements downstream of the start site. The spacing between promoter elements is often flexible, so that if elements are inverted or moved relative to one another, the promoter is prevented from functioning. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased to 50 bp before activity begins to decline. Depending on the promoter, individual elements appear to function either cooperatively or independently to activate transcription.

[0225] The present invention relates to a DNA construct comprising a CAR sequence, wherein the CAR sequence comprises a nucleic acid sequence of an antigen-binding domain operably linked to a nucleic acid sequence of an intracellular domain. Examples of intracellular domains that can be used in CARs according to the present invention include, but are not limited to, the alternative intracellular signaling domains described above, the costimulatory domains of CD28 and / or 4-1BB, etc.; in some cases, the CAR can comprise any combination of the alternative intracellular signaling domains described above, the costimulatory domain of CD28, the costimulatory domain of 4-1BB, etc.

[0226] In one embodiment, a CAR according to the invention comprises an anti-CD19 scFv, the hinge and transmembrane domains of human CD8, and the costimulatory domain of 4-1BB, as well as an alternative intracellular signaling domain as described above.

[0227] In one embodiment, a CAR according to the invention comprises an anti-CD19 scFv, the hinge and transmembrane domains of human CD8, and the costimulatory domain of CD28, as well as an alternative intracellular signaling domain as described above.

[0228] The expression constructs of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery techniques. Methods for gene delivery are known in the art. For example, see U.S. Patent Nos. 5,399,346, 5,580,859, and 5,589,466, which are incorporated herein by reference in their entirety. In another aspect, the present invention relates to a gene therapy vector.

[0229] Nucleic acid can be cloned into several types of vectors.For example, nucleic acid can be cloned into vectors, including but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids.Particularly interesting vectors include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0230] Furthermore, expression vectors can be delivered to cells in the form of viral vectors.Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals.Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses.Generally, suitable vectors contain a replication origin that is functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (for example, WO01 / 96584; WO01 / 29058; and U.S. Patent No. 6,326,193).

[0231] Several virus-based systems have been developed for gene transfer into mammalian cells. For example, retroviruses provide a suitable platform for gene delivery systems. A selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to target cells either in vivo or ex vivo. Several retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Several adenoviral vectors are known in the art. In one embodiment, lentiviral vectors are used.

[0232] The present invention also provides a vector into which the DNA of the present invention is inserted.Vector derived from retroviruses such as lentiviruses is suitable for achieving long-term gene transfer, since it allows long-term stable integration of transgenes and their proliferation in daughter cells.Lentivirus vectors have an additional advantage over vectors derived from oncoretroviruses such as murine leukemia viruses in that they can transduce non-proliferating cells such as hepatocytes.Lentivirus vectors also have the additional advantage of low immunogenicity.

[0233] " Lentivirus " as used herein refers to a genus of the Retroviridae family.Lentivirus is unique among retroviruses in that it can infect non-dividing cells; it can deliver a significant amount of genetic information as DNA into host cells, and as a result, it is one of the most efficient methods for implementing gene delivery vectors.HIV, SIV, and FIV are examples of lentivirus.Vector derived from lentivirus provides a means to achieve significant levels of gene transfer in vivo.

[0234] To evaluate the expression of a CAR polypeptide or a portion thereof, the expression vector introduced into the cell can also contain either a selection marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a population of cells transfected or infected with a viral vector. In another aspect, the selection marker can carry a separate fragment of DNA and can be used in co-transfection techniques. Both the selection marker and the reporter gene can be flanked by appropriate regulatory sequences to enable expression in the host cell. Useful selection markers include, for example, antibiotic resistance genes, such as neo.

[0235] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. Generally, reporter genes are genes not present in or expressed by an organism or tissue and encode a polypeptide whose expression is manifested by some easily detectable property, such as enzymatic activity. Expression of the reporter gene is assayed at an appropriate time after DNA is introduced into recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or the green fluorescent protein gene (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well known and may be prepared using known techniques or commercially available. Generally, a construct with the minimal 5' flanking region that exhibits the highest expression level of the reporter gene is identified as the promoter. Such promoter regions can be linked to a reporter gene and used to evaluate agents for their ability to modulate promoter-driven transcription.

[0236] The present invention relates to a vector comprising a nucleic acid molecule encoding the above-mentioned chimeric antigen receptor (CAR) according to the present invention. In one aspect, the present invention relates to an expression vector comprising the above-mentioned nucleic acid encoding the above-mentioned chimeric antigen receptor (CAR) according to the present invention.

[0237] In one aspect, the present invention relates to a delivery vector comprising the above-mentioned nucleic acid encoding the above-mentioned chimeric antigen receptor (CAR) according to the present invention. In some aspects, the vector is selected from the group consisting of DNA, RNA, a plasmid, a lentiviral vector, an adeno-associated viral vector, an adenoviral vector, or a retroviral vector.

[0238] In some aspects, the vector further comprises a promoter. In some embodiments, the vector is an in vitro transcription vector. In some embodiments, the nucleic acid sequence further comprises a poly(A) sequence.

[0239] In some embodiments, the nucleic acid sequence further comprises a 3' UTR. Host cells and methods for producing same As used herein, the term "genetically modified cells" (or "recombinant host cells") refers to cells into which a recombinant expression vector has been introduced. The present invention relates to "genetically modified cells," which can, for example, contain the above vectors according to the present invention. It should be understood that "genetically modified cells" and "recombinant host cells" refer not only to the particular claimed cells, but also to the progeny of such cells. Such progeny may not actually be identical to the parent cell, as modifications may occur in subsequent generations due to either mutations or environmental influences; however, such cells are still included within the scope of the term "genetically modified cells" as used herein.

[0240] In one aspect, the present invention relates to a method for producing a "genetically modified cell" comprising the above chimeric antigen receptor (CAR), comprising the step of transforming a cell by the method described above.

[0241] Methods for incorporating and expressing genes in cells are known in the art.In the context of expression vectors, the vector can be easily inserted into host cells, such as mammalian, bacterial, yeast or insect cells, by any method known in the art.For example, the expression vector can be transfected into host cells by physical, chemical or biological methods.

[0242] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc. Methods for generating cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method for introducing polynucleotides into host cells is transfection using calcium phosphate.

[0243] Biological methods for introducing a target polynucleotide into a host cell include the use of DNA vectors and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex viruses, adenoviruses, and adeno-associated viruses, etc. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.

[0244] Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (e.g., an artificial membrane vesicle).

[0245] When a non-viral delivery system is utilized, an exemplary delivery vehicle is a liposome. The use of lipid formulations for the introduction of nucleic acids into host cells (in vitro, ex vivo, or in vivo) is contemplated. In another aspect, the nucleic acid can be linked to a lipid. The lipid-linked nucleic acid can be encapsulated in the aqueous interior of the liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linker molecule connected to both the liposome and the oligonucleotide, enclosed in the liposome, complexed with the liposome, dispersed in a solution containing lipids, mixed with lipids, or combined with lipids; the lipid-linked nucleic acid can be contained as a suspension in the lipid, contained with or complexed to micelles, or otherwise linked to lipids. The lipid, lipid / DNA, or lipid / expression vector-associated compositions are not limited to any particular structure in solution. For example, they can exist in a bilayer structure, as micelles, or in a "collapsed" structure. They may also be simply dispersed in solution, and sometimes form aggregates that are not uniform in size or shape.Lipid is a fatty substance, and can be naturally occurring lipid or synthetic lipid.For example, lipid includes the lipid droplets that naturally occur in cytoplasm, and the class of compounds that contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0246] Suitable lipids for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine ("DMPC") can be purchased from Sigma, St. Louis, MO; dicetyl phosphate ("DCP") can be purchased from K&K Laboratories (Plainview, NY); cholesterol ("Choi") can be purchased from Calbiochem-Behring; dimyristyl phosphatidylglycerol ("DMPG") and other lipids can be purchased from Avanti Polar Lipids, Inc. (Birmingham, AL). Lipid stock solutions in chloroform or chloroform / methanol can be stored at approximately -20°C. Chloroform evaporates more readily than methanol, so it is used as the sole solvent. "Liposome" is a general term that encompasses a variety of unilamellar and multilamellar lipid vesicles formed by the formation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. Liposomes form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before forming a closed structure, encapsulating water and dissolved solutes between the lipid bilayers (Ghosh et al., 191 Glycobiology 5;505-10). However, compositions with structures in solution that differ from the normal vesicular structure are also included. For example, lipids can assume a micellar structure or simply exist as heterogeneous aggregates of lipid molecules. Lipofectamine-nucleic acid complexes are also included.

[0247] Regardless of the method used to introduce exogenous nucleic acid into host cells or otherwise expose the cells to the inhibitors of the present invention, various assays can be performed to confirm the presence of the recombinant DNA sequence in the host cells. Such assays include "molecular biological" assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR and PCR; and "biochemical" assays, such as detecting the presence or absence of specific peptides by immunological means (ELISA and Western blotting) or by the assays described herein to identify agents within the scope of the present invention.

[0248] In one aspect, the present invention relates to a genetically modified cell comprising the above chimeric antigen receptor (CAR) produced by the above method. Aspects of the present invention further provide genetically modified cells containing any of the recombinant expression vectors described herein. As used herein, the term "genetically modified cells" refers to any type of cell capable of containing a recombinant expression vector according to the present invention. Genetically modified cells can be eukaryotic cells, such as plant, animal, fungal, or algal cells, or prokaryotic cells, such as bacterial or protozoan cells. Genetically modified cells can be cultured cells or primary cells, i.e., cells isolated directly from an organism, such as a human. Genetically modified cells can be adherent cells or suspension cells, i.e., cells that grow in suspension. Suitable host cells are known in the art and include, for example, DH5α E. coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, and the like. When amplifying or replicating a recombinant expression vector, the host cell is preferably a prokaryotic cell, e.g., DH5α cells. When producing a recombinant CAR, the genetically modified cell can be a mammalian cell. The genetically modified cell can also be a human cell. The genetically modified cells can be of any cell type, can be derived from any type of tissue, and can be at any stage of development, while the genetically modified cells can be peripheral blood lymphocytes (PBLs) or peripheral blood mononuclear cells (PBMCs). The genetically modified cells can be T cells. The genetically modified cells can be natural killer (NK) cells.

[0249] For purposes of the present invention, T cells can be any T cell, such as a cultured T cell, e.g., a primary T cell, or a T cell from a cultured T cell line, e.g., Jurkat, SupT1, etc., or a T cell obtained from a mammal. If obtained from a mammal, the T cells can be obtained from a number of sources, including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. The T cells can also be enriched or purified. The T cells can be human T cells. The T cells can be T cells isolated from a human organism. The T cells can be any type of T cell and at any stage of development, including CD4 T cells, ...+ / CD8 + Double positive T cells, CD4 + Helper T cells, e.g., Th1 and Th2 cells, CD8 + These include, but are not limited to, T cells (e.g., cytotoxic T cells), tumor-infiltrating lymphocytes, memory T cells, naive T cells, etc. T cells include CD8 + T cells may also contain CD4 + It can also be T cells.

[0250] T cells can be obtained from several sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from an infection site, ascites, pleural effusion, spleen tissue, and tumors. In certain embodiments of the present invention, several T cell lines available in the art can be used. In certain embodiments of the present invention, T cells can be obtained from a unit of blood collected from a subject using several techniques known to those skilled in the art, such as Ficoll separation. In a preferred embodiment, cells from an individual's circulating blood are obtained by apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated leukocytes, red blood cells, and platelets. In one embodiment, cells collected by apheresis can be washed to remove the plasma fraction and place the cells in an appropriate buffer or medium for subsequent processing steps. In one embodiment of the present invention, the cells are washed with phosphate-buffered saline (PBS). In alternative embodiments, the wash solution lacks calcium and may lack magnesium, or may lack many, if not all, divalent cations. Furthermore, surprisingly, an initial activation step in the absence of calcium leads to enhanced activation. As those skilled in the art will appreciate, the wash step can be performed by methods known in the art, such as by using a semi-automated "flow-through" centrifuge (e.g., a Cell Processor Cobe 2991, Baxter CytoMate, or Haemonetics Cell Saver 5) according to the manufacturer's instructions. After washing, the cells can be resuspended in various biocompatible buffers, such as Ca2+-free, Mg2+-free PBS, Plasmalyte A, or other saline solutions with or without buffering agents. Alternatively, undesirable components of the apheresis sample can be removed, and the cells can be resuspended directly in culture medium.

[0251] In some embodiments, the genetically modified cells are T lymphocytes. In some embodiments, the genetically modified cells are CD8+ T cells. In some embodiments, the genetically modified cells are NK cells.

[0252] In some aspects, the genetically modified cells are used as a medicament. In some aspects, the genetically modified cells are used as a medicament for a tumor disease. In some aspects, the genetically modified cells are used as a medicine for diseases in which the tumor is cancer. Without being limited thereto, the present specification includes the following aspects. [Aspect 1] An alternative intracellular signaling domain of a chimeric antigen receptor (CAR) in which one or two immunoreceptor tyrosine-based activation motifs (ITAMs) selected from ITAM1, ITAM2, and ITAM3 are replaced with a fragment of the amino acid sequence of integrin beta 3 (ITGB3). [Aspect 2] 2. The alternative intracellular signaling domain of a chimeric antigen receptor (CAR) according to embodiment 1, wherein one immunoreceptor tyrosine-based activation motif (ITAM) selected from ITAM1, ITAM2 and ITAM3 is replaced with a fragment of the amino acid sequence of integrin beta 3 (ITGB3). [Aspect 3] 3. The alternative intracellular signaling domain of the chimeric antigen receptor (CAR) of embodiment 2, wherein ITAM1 is replaced with ITGB3. [Aspect 4] 3. The alternative intracellular signaling domain of the chimeric antigen receptor (CAR) of embodiment 2, wherein ITAM2 is replaced with ITGB3. [Aspect 5] 3. The alternative intracellular signaling domain of the chimeric antigen receptor (CAR) of embodiment 2, wherein ITAM3 is replaced with ITGB3. [Aspect 6] 2. The alternative intracellular signaling domain of a chimeric antigen receptor (CAR) according to embodiment 1, wherein two immunoreceptor tyrosine-based activation motifs (ITAMs) selected from ITAM1, ITAM2 and ITAM3 are replaced with a fragment of the amino acid sequence of integrin beta 3 (ITGB3). [Aspect 7] 7. The alternative intracellular signaling domain of the chimeric antigen receptor (CAR) of embodiment 6, wherein ITAM2 and ITAM3 are replaced with two ITGB3s. [Aspect 8] 7. The alternative intracellular signaling domain of the chimeric antigen receptor (CAR) according to embodiment 6, wherein ITAM1 and ITAM3 are replaced with two ITGB3s. [Aspect 9] 7. The alternative intracellular signaling domain of the chimeric antigen receptor (CAR) according to embodiment 6, wherein ITAM1 and ITAM2 are replaced with two ITGB3s. [Aspect 10] 2. The alternative intracellular signaling domain of the chimeric antigen receptor (CAR) of embodiment 1, wherein ITAM1 has the amino acid sequence represented by SEQ ID NO:1. [Aspect 11] 2. The alternative intracellular signaling domain of the chimeric antigen receptor (CAR) of embodiment 1, wherein ITAM2 has the amino acid sequence represented by SEQ ID NO:2. [Aspect 12] 2. The alternative intracellular signaling domain of the chimeric antigen receptor (CAR) of embodiment 1, wherein ITAM3 has the amino acid sequence represented by SEQ ID NO:3. [Aspect 13] 2. The alternative intracellular signaling domain of a chimeric antigen receptor (CAR) of embodiment 1, wherein ITGB3 has an amino acid sequence represented by SEQ ID NO:4, or an amino acid sequence represented by SEQ ID NO:4 with any one or two conservative substitutions. [Aspect 14] 14. The alternative intracellular signaling domain of a chimeric antigen receptor (CAR) according to embodiment 13, wherein ITGB3 has an amino acid sequence represented by SEQ ID NO: 4 with any one conservative substitution. [Aspect 15] 14. The alternative intracellular signaling domain of a chimeric antigen receptor (CAR) according to embodiment 13, wherein ITGB3 has an amino acid sequence represented by SEQ ID NO: 4 with any two conservative substitutions. [Aspect 16] 14. The alternative intracellular signaling domain of the chimeric antigen receptor (CAR) of embodiment 13, wherein ITGB3 has the amino acid sequence represented by SEQ ID NO:4. [Aspect 17] 3. The alternative intracellular signaling domain of a chimeric antigen receptor (CAR) of embodiment 2, having an amino acid sequence represented by SEQ ID NO:5, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:5 and differs from SEQ ID NO:5 only in non-conserved amino acid residues. [Aspect 18] 3. The alternative intracellular signaling domain of a chimeric antigen receptor (CAR) of embodiment 2, having an amino acid sequence represented by SEQ ID NO:6, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:6 and differs from SEQ ID NO:6 only in non-conserved amino acid residues. [Aspect 19] 3. The alternative intracellular signaling domain of a chimeric antigen receptor (CAR) of embodiment 2, having an amino acid sequence represented by SEQ ID NO:7, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:7 and differs from SEQ ID NO:7 only in non-conserved amino acid residues. [Aspect 20] 7. The alternative intracellular signaling domain of a chimeric antigen receptor (CAR) according to embodiment 6, having an amino acid sequence represented by SEQ ID NO: 8, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 8 and differs from SEQ ID NO: 8 only in non-conserved amino acid residues. [Aspect 21] 7. The alternative intracellular signaling domain of a chimeric antigen receptor (CAR) according to embodiment 6, having an amino acid sequence represented by SEQ ID NO:9, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:9 and differs from SEQ ID NO:9 only in non-conserved amino acid residues. [Aspect 22] 7. The alternative intracellular signaling domain of a chimeric antigen receptor (CAR) according to embodiment 6, having an amino acid sequence represented by SEQ ID NO: 10, or having an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 10 and differs from SEQ ID NO: 10 only in non-conserved amino acid residues. [Aspect 23] 1. An isolated alternative intracellular signaling domain of a chimeric antigen receptor (CAR), comprising: comprising one or two immunoreceptor tyrosine-based activation motifs (ITAMs) selected from ITAM1, ITAM2, and ITAM3, and one or two fragments of the amino acid sequence of integrin beta 3 (ITGB3); The total number of ITAM and ITGB3 is 3. Alternative intracellular signaling domains of chimeric antigen receptors (CARs). [Aspect 24] 24. An alternative intracellular signaling domain of a chimeric antigen receptor (CAR) according to embodiment 23, comprising two immunoreceptor tyrosine-based activation motifs (ITAMs) selected from ITAM1, ITAM2 and ITAM3, and a fragment of the amino acid sequence of integrin beta 3 (ITGB3). [Aspect 25] 25. An alternative intracellular signaling domain of the chimeric antigen receptor (CAR) of embodiment 24, comprising ITAM2, ITAM3 and ITGB3. [Aspect 26] 25. An alternative intracellular signaling domain of the chimeric antigen receptor (CAR) of embodiment 24, comprising ITAM1, ITAM3 and ITGB3. [Aspect 27] 25. An alternative intracellular signaling domain of the chimeric antigen receptor (CAR) of embodiment 24, comprising ITAM1, ITAM2 and ITGB3. [Aspect 28] 24. An alternative intracellular signaling domain of a chimeric antigen receptor (CAR) according to embodiment 23, comprising one immunoreceptor tyrosine-based activation motif (ITAM) selected from ITAM1, ITAM2 and ITAM3, and two fragments of the amino acid sequence of integrin beta 3 (ITGB3). [Aspect 29] 29. The alternative intracellular signaling domain of the chimeric antigen receptor (CAR) according to embodiment 28, comprising two fragments of the amino acid sequences of ITAM1 and integrin beta 3 (ITGB3). [Aspect 30] 29. The alternative intracellular signaling domain of the chimeric antigen receptor (CAR) of embodiment 28, comprising two fragments of the amino acid sequences of ITAM2 and integrin beta 3 (ITGB3). [Aspect 31] 29. The alternative intracellular signaling domain of the chimeric antigen receptor (CAR) of embodiment 28, comprising two fragments of the amino acid sequences of ITAM3 and integrin beta 3 (ITGB3). [Aspect 32] 24. The alternative intracellular signaling domain of the chimeric antigen receptor (CAR) of embodiment 23, wherein ITAM1 has the amino acid sequence represented by SEQ ID NO:1. [Aspect 33] 24. The alternative intracellular signaling domain of the chimeric antigen receptor (CAR) of embodiment 23, wherein ITAM2 has the amino acid sequence represented by SEQ ID NO:2. [Aspect 34] 24. The alternative intracellular signaling domain of the chimeric antigen receptor (CAR) of embodiment 23, wherein ITAM3 has the amino acid sequence represented by SEQ ID NO:3. [Aspect 35] 24. The alternative intracellular signaling domain of a chimeric antigen receptor (CAR) according to embodiment 23, wherein ITGB3 has an amino acid sequence represented by SEQ ID NO: 4, or an amino acid sequence represented by SEQ ID NO: 4 with any one or two conservative substitutions. [Aspect 36] 24. The alternative intracellular signaling domain of a chimeric antigen receptor (CAR) according to embodiment 23, wherein ITGB3 has an amino acid sequence represented by SEQ ID NO: 4 with any one conservative substitution. [Aspect 37] 24. The alternative intracellular signaling domain of a chimeric antigen receptor (CAR) according to embodiment 23, wherein ITGB3 has an amino acid sequence represented by SEQ ID NO: 4 with any two conservative substitutions. [Aspect 38] 24. The alternative intracellular signaling domain of the chimeric antigen receptor (CAR) of embodiment 23, wherein ITGB3 has the amino acid sequence represented by SEQ ID NO:4. [Aspect 39] 25. The alternative intracellular signaling domain of a chimeric antigen receptor (CAR) according to embodiment 24, having an amino acid sequence represented by SEQ ID NO:5, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:5 and differs from SEQ ID NO:5 only in non-conserved amino acid residues. [Aspect 40] 25. The alternative intracellular signaling domain of a chimeric antigen receptor (CAR) according to embodiment 24, having an amino acid sequence represented by SEQ ID NO:6, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:6 and differs from SEQ ID NO:6 only in non-conserved amino acid residues. [Aspect 41] 25. The alternative intracellular signaling domain of a chimeric antigen receptor (CAR) according to embodiment 24, having an amino acid sequence represented by SEQ ID NO:7, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:7 and differs from SEQ ID NO:7 only in non-conserved amino acid residues. [Aspect 42] 29. The alternative intracellular signaling domain of a chimeric antigen receptor (CAR) according to embodiment 28, having an amino acid sequence represented by SEQ ID NO: 8, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 8 and differs from SEQ ID NO: 8 only in non-conserved amino acid residues. [Aspect 43] 29. The alternative intracellular signaling domain of a chimeric antigen receptor (CAR) according to embodiment 28, having an amino acid sequence represented by SEQ ID NO:9, or an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:9 and differs from SEQ ID NO:9 only in non-conserved amino acid residues. [Aspect 44] 29. The alternative intracellular signaling domain of a chimeric antigen receptor (CAR) according to embodiment 28, having an amino acid sequence represented by SEQ ID NO: 10, or having an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 10 and differs from SEQ ID NO: 10 only in non-conserved amino acid residues. [Aspect 45] 45. An isolated nucleic acid encoding an alternative intracellular signalling domain according to embodiments 1 to 44. [Aspect 46] 45. An isolated chimeric antigen receptor (CAR) comprising an alternative intracellular signaling domain according to embodiments 1 to 44. [Aspect 47] a) an antigen-binding domain specific for a tumor antigen; b) transmembrane domain; c) costimulatory domain; d) an alternative intracellular signaling domain according to any one of embodiments 1 to 44. 47. The isolated chimeric antigen receptor (CAR) of embodiment 46, comprising: [Aspect 48] 48. The isolated chimeric antigen receptor (CAR) of embodiment 47, wherein the antigen-binding domain specific for the tumor antigen is a Fab or scFv. [Aspect 49] 49. The isolated chimeric antigen receptor (CAR) of embodiment 48, wherein the antigen-binding domain specific for the tumor antigen is an scFv. [Aspect 50] 48. The isolated chimeric antigen receptor (CAR) of embodiment 47, wherein the antigen-binding domain is specific for a tumor antigen selected from the group of CD19, CD20, CD123 or BCMA. [Aspect 51] 51. The isolated chimeric antigen receptor (CAR) of embodiment 50, wherein the antigen-binding domain is specific for the CD19 tumor antigen. [Aspect 52] 52. The isolated chimeric antigen receptor (CAR) of embodiment 51, wherein the antigen-binding domain specific for the CD19 tumor antigen has the amino acid sequence represented by SEQ ID NO: 11. [Aspect 53] 49. The isolated chimeric antigen receptor (CAR) of embodiment 48, wherein the transmembrane domain is a transmembrane domain of a protein selected from the group consisting of the T-cell receptor-alpha, -beta, or -zeta chain, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. [Aspect 54] 54. The isolated chimeric antigen receptor (CAR) of embodiment 53, wherein the transmembrane domain is CD8. [Aspect 55] 55. The isolated chimeric antigen receptor (CAR) of embodiment 54, wherein the CD8 transmembrane domain has the amino acid sequence represented by SEQ ID NO: 12. [Aspect 56] 48. The isolated chimeric antigen receptor (CAR) of embodiment 47, wherein the costimulatory domain is a functional signaling domain of a protein selected from the group consisting of OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137). [Aspect 57] 57. The isolated chimeric antigen receptor (CAR) of embodiment 56, wherein the costimulatory domain is CD28 or 4-1BB. [Aspect 58] 58. The isolated chimeric antigen receptor (CAR) of embodiment 57, wherein the costimulatory domain is CD28. [Aspect 59] 59. The isolated chimeric antigen receptor (CAR) of embodiment 58, wherein the CD28 costimulatory domain has the amino acid sequence represented by SEQ ID NO: 13. [Aspect 60] 57. The isolated chimeric antigen receptor (CAR) of embodiment 56, wherein the costimulatory domain is 4-1BB. [Aspect 61] 61. The isolated chimeric antigen receptor (CAR) of embodiment 60, wherein the 4-1BB costimulatory domain has the amino acid sequence represented by SEQ ID NO: 14. [Aspect 62] 47. The isolated chimeric antigen receptor (CAR) of embodiment 46, further comprising a leader sequence. [Aspect 63] having an amino acid sequence represented by an amino acid sequence selected from the group of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26; or having an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26, and that differs from the amino acid sequence selected from the group of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 only in non-conserved amino acid residues; 47. The isolated chimeric antigen receptor (CAR) of embodiment 46. [Aspect 64] 64. An isolated nucleic acid encoding the isolated chimeric antigen receptor (CAR) of any one of embodiments 46 to 63. [Aspect 65] 65. An expression vector comprising the nucleic acid of embodiment 64. [Aspect 66] A delivery vector comprising the nucleic acid of embodiment 64. [Aspect 67] 67. The vector of embodiment 65 or 66, wherein the vector is selected from the group consisting of a DNA, an RNA, a plasmid, a lentiviral vector, an adeno-associated viral vector, an adenoviral vector, or a retroviral vector. [Aspect 68] 67. The vector of embodiment 65 or 66, further comprising a promoter. [Aspect 69] 67. The vector of embodiment 65 or 66, which is an in vitro transcription vector. [Aspect 70] 67. The vector of embodiment 65 or 66, wherein the nucleic acid sequence further comprises a poly(A) sequence. [Aspect 71] 67. The vector of embodiment 65 or 66, wherein the nucleic acid sequence further comprises a 3' UTR. [Aspect 72] A method for producing a genetically modified cell comprising a chimeric antigen receptor (CAR) according to any one of aspects 46 to 63, the method comprising the step of transforming a cell with a vector according to any one of aspects 65 to 71. [Aspect 73] A genetically modified cell comprising the chimeric antigen receptor (CAR) of any one of embodiments 46 to 63, wherein the genetically modified cell is produced using the method of embodiment 72. [Aspect 74] 74. The cell of embodiment 73, which is a T lymphocyte. [Aspect 75] 74. The cell of embodiment 73, which is a CD8+ T cell. [Aspect 76] 74. The cell of embodiment 73, which is a NK cell. [Aspect 77] 74. The cell of embodiment 73, for use as a medicament. [Aspect 78] 78. The cell according to embodiment 77, for use as a medicament for a tumor disease. [Aspect 79] 79. The cell according to embodiment 78, for use as a medicament for a disease in which the tumor is cancer. [Example]

[0253] The following examples are provided for a better understanding of the present invention. These examples are for illustrative purposes only and should not be construed as limiting the scope of the invention in any way.

[0254] All publications, patents, and patent applications cited herein are incorporated herein by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those skilled in the art in light of the teachings of the present invention that certain changes and modifications can be made to the invention without departing from the spirit or scope of the accompanying embodiments.

[0255] Materials and General Methods Recombinant DNA Techniques DNA manipulations were performed according to standard techniques as described by Sambrook J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biological reagents were used according to the manufacturer's instructions.

[0256] Gene synthesis Desired gene segments were prepared from chemically synthesized oligonucleotides. Gene segments of 300–4000 kb in length, flanked by unique restriction sites, were assembled by annealing and ligation of oligonucleotides, including PCR amplification, and then cloned via the indicated restriction sites. The DNA sequences of the subcloned gene fragments were confirmed by DNA sequencing.

[0257] Fusion genes comprising the antibody chains described below were generated by PCR and / or gene synthesis and assembled using known recombinant methods and techniques by joining the appropriate nucleic acid segments, for example, using unique restriction sites in the corresponding vectors. The subcloned nucleic acid sequences were verified by DNA sequencing. For transient transfection, larger quantities of plasmids were prepared by plasmid preparation from transformed E. coli cultures.

[0258] DNA sequence determination The DNA sequence was determined by Sanger sequencing. DNA and protein sequence analysis and sequence data management Infomax's Vector NTI Advance suite version 8.0 and SnapGene Viewer were used for sequence generation, mapping, analysis, annotation and illustration.

[0259] Cloning protocol for preparing vectors carrying the costimulatory domain of 4-1BB The three ITGB3 repeat fragments were assembled from synthetic oligonucleotides (10 of 59 bp each); after two rounds of amplification, a 308-bp amplicon was prepared. The constant portion of the scFv (leader-SVL-GS-linker-SVH-4-1BB CD3) was then amplified, and a predetermined number of ITGB3 fragments (1, 2, or 3, depending on the structural characteristics of the individual vector) were ligated to it by splicing PCR (SOE, splicing by overlap extension). The resulting product was cloned into the previously linearized vector pCDH-eGFP using the XbaI / NotI sites. The resulting construct was verified by sequencing the entire cassette.

[0260] Cloning protocol for preparing vectors containing the costimulatory domain of CD28 The three ITGB3 repeat fragments were assembled from synthetic oligonucleotides (10 of 59 bp each); after two rounds of amplification, a 308-bp amplicon was prepared. The constant portion of the scFv (leader-SVL-GS-linker-SVH-CD28-CD3) was then amplified, and a predetermined number of ITGB3 fragments (1, 2, or 3, depending on the structural characteristics of the individual vector) were ligated to it by splicing PCR (SOE, splicing by overlap extension). The resulting product was cloned into the previously linearized vector pCDH-eGFP using the XbaI / NotI sites. The resulting construct was verified by sequencing the entire cassette.

[0261] Example 1 Preparation of synthetic ITGB3 gene sequence. To prepare blocks of three repeat fragments of ITGB3, synthetic oligonucleotides of 60 bp each were used, which form perfectly overlapping sequences of gene regions within the chimeric constructs CD8 4-1BB altCD3 zeta or CD8 CD28 altCD3 zeta. The genes were assembled by two-round PCR.

[0262] Example 2 Sequence of the codon-optimized ITGB3 fragment. Three different variations of the nucleotide sequence encoding ITGB3 were used to position the ITGB3 fragment in the 19CAR-T cassette to prevent possible intragenic recombination. These sequence variations are given below.

[0263] Variation 1 gatactgccaacaacccactttacaaagaagctacatccaccttcaccaatataacctacagaggtact DTANNPLYKEATSTFTNITYR GT Variation 2 gacaccgctaacaatcctctgtataaagaggcaaccagcacttttacaaacattacttataggggaacc DTANNPLYKEATSTFTNITYR GT Variation 3 gatacagcaaataaccccttgtacaaggaagccacttctacattcactaatatcacctatcgcggcaca DTANNPLYKEATSTFTNITYR GT Example 3 Cloning of different sequence variations of cassettes 19CAR-T-ITGB3 and 19CAR-T-CD28-ITGB3.

[0264] To clone the cassette 19CAR-T-ITGB3-ITGB3-ITGB3 (Figure 4), the constant region of the gene was generated by PCR amplification using the vector pCDH-CAR-T-EGFP as a template, where specific oligonucleotide primers restrict the constant portion of the cassette 19CAR-T. The region of the 19CAR-T gene and the block of three ITGB3 fragments were fused by splicing PCR (SOE, splicing by overlap extension).

[0265] To clone the cassette 19CAR-T-ITGB3-ITGB3 (Figure 2), a region of the gene was similarly prepared by PCR amplification using the validated pCDH-19CAR-T-ITGB3-ITGB3-ITGB3 as a template, which was restricted by specific primers and included the ITGB3-ITGB3 repeat.

[0266] To clone the cassette 19CAR-T-ITAM1-ITGB3-ITGB3 (Figure 6), the vector pCDH-CAR-T-EGFP was used as a template to generate the constant region of the gene by PCR amplification, where specific oligonucleotide primers were used to limit the constant portion of the 19CAR-T-ITAM1 gene region, which was then fused with the ITGB3-ITGB3 block by splicing PCR.

[0267] To clone the cassette 19CAR-T-ITAM1-ITAM2-ITGB3 (Figure 8), the vector pCDH-CAR-T-EGFP was used as a template to generate the constant region of the gene by PCR amplification, where specific oligonucleotide primers were used to limit the constant portion of the 19CAR-T-ITAM1-ITAM2 gene region, which was then fused with the ITGB3 block by splicing PCR.

[0268] To clone the cassette 19CAR-T-ITGB3-ITAM2-ITGB3 (Figure 14), the vector pCDH-CAR-T-EGFP was used as a template to generate the constant region of the gene by PCR amplification, where specific oligonucleotide primers were used to confine the constant portion with 19CAR-T. The gene region was fused with two ITGB3 and ITAM2 blocks by splicing PCR.

[0269] To clone the cassette 19CAR-T-ITAM1-ITGB3-ITAM2 (Figure 12), the vector pCDH-CAR-T-EGFP was used as a template to generate the constant region of the gene by PCR amplification, where specific oligonucleotide primers were used to limit the constant portion of 19CAR-T-ITAM1. The gene region was fused with the ITGB3 and ITAM2 blocks by splicing PCR.

[0270] Similarly, a cassette comprising the costimulatory domain of CD28, in particular: expression cassette pCDH-19CAR-T_CD28-ITGB3-ITGB3-eGFP (Figure 16); expression cassette pCDH-19CAR-T-CD28_ITGB3-ITGB3-ITGB3-eGFP (Figure 18); expression cassette pCDH-19CAR-T-CD28_ITAM1-ITGB3-ITGB3-eGFP (Figure 20); expression cassette pCDH-19CAR-T-CD28_ITAM1-ITAM2-ITGB3-eGFP (Figure 22); expression cassette pCDH-19CAR-T-CD28_ITAM1-ITGB3-ITAM2-eGFP (Figure 24); Expression cassette pCDH-19CAR-T-CD28_ ITGB3-ITAM2-ITGB3-eGFP (Figure 26) was prepared.

[0271] The resulting cassette was integrated into the plasmid vector pCDH-eGFP via specific restriction sites; the open reading frames of the T2A element and green fluorescent protein (eGFP) as an expression marker were placed in the same reading frame.

[0272] As a result, vectors in which the CAR comprises the costimulatory domain of 4-1BB and alternative intracellular signaling domains of chimeric antigen receptors with different combinations of ITGB3 and ITAM are specifically vector pCDH-19CAR-T-ITGB3-ITGB3-eGFP ( Figure 1 ); vector pCDH-19CAR-T-ITGB3-ITGB3-ITGB3-eGFP ( Figure 3 ); vector pCDH-19CAR-T-ITAM1-ITGB3-ITGB3-eGFP ( Figure 5 ); vector pCDH-19CAR-T-ITAM1-ITAM2-ITGB3-eGFP ( Figure 7 ); vector pCDH-19CAR-T-ITAM1-ITGB3-ITAM2-eGFP ( Figure 11 ); vector pCDH-19CAR-T-ITGB3-ITAM2-ITGB3-eGFP (Figure 13), was produced.

[0273] Furthermore, vectors in which the CAR comprises the costimulatory domain of CD28 and alternative intracellular signaling domains of chimeric antigen receptors with different combinations of ITGB3 and ITAM are particularly vector pCDH-19CAR-T_CD28-ITGB3-ITGB3-eGFP ( Figure 15 ); vector pCDH-19CAR-T-CD28_ITGB3-ITGB3-ITGB3-eGFP (Figure 17); vector pCDH-19CAR-T-CD28_ITAM1-ITGB3-ITGB3-eGFP (Figure 19); vector pCDH-19CAR-T-CD28_ITAM1-ITAM2-ITGB3-eGFP (Figure 21); vector pCDH-19CAR-T-CD28_ITAM1-ITGB3-ITAM2-eGFP (Figure 23); vector pCDH-19CAR-T-CD28_ITGB3-ITAM2-ITGB3-eGFP (Figure 25), was produced.

[0274] Example 4 Isolation and activation of T cells. Twenty-seven milliliters of venous peripheral blood was collected from a conditionally healthy volunteer donor into a standard Vacutainer tube containing a sterile solution of heparin sodium. To prepare the mononuclear cell fraction, 3 ml of Ficoll solution (1.077 g / L density gradient) (PanEco, P052n) layered with the venous blood was transferred to a sterile 15 ml centrifuge tube (Corning, 430791). The tube was capped and centrifuged at 1500 rpm for 18 minutes at 24°C. The mononuclear cell layer was collected from the phase interface. To obtain a population of T cells (CD3+), negative selection immunomagnetic separation was performed using the EasySep Human T Cell Enrichment Kit (Stemcell, 19051) according to the manufacturer's instructions. One round of immunomagnetic separation was performed.

[0275] The resulting T lymphocytes were concentrated in 1 ml of culture medium (RPMI-1640 supplemented with L-glutamine (Biolot, 1.3.4.1) + 10% fetal bovine serum (Gibco, 26140079) + 100 U IL2 (Ronkoleikin, LLC "NPK BIOTECH"). T lymphocyte concentrations were determined using a Countess® II FL cell counter and viability analyzer (Thermofisher, AMQAF1000). 12 × 10 6 T lymphocytes were transferred to a T75 culture flask (Eppendorf, 0030711025) for suspension cell culture supplemented with 15 ml of culture medium. 300 ml of Dynabeads Human T-Activator CD3 / CD28 for T Cell Expansion and Activation (Thermofisher, 11132D) was added to activate the T lymphocytes. The culture vial was placed in a CO2 incubator (37°C, 5% CO2) for 72 hours.

[0276] All work was carried out under sterile conditions in a biosafety class 2 airflow cabinet. Example 5 Transduction of activated T lymphocytes with lentiviral drugs.

[0277] Two lentiviral drugs were used to transduce human T lymphocytes: the first drug (control) containing the genes for anti-CD19-CAR-ITAMx3 and GFP, and the second drug (experimental drug) selected from the group of vectors produced in Example 3 (Figures 1, 3, 5, 7, 11, 13, 15, 19, 21, 23, and 25). The concentration of lentiviral particles (LVP / ml) in both drugs was determined by ELISA. Transduction was performed in a 15 ml bioreactor (TPP, 87017) format. Protamine sulfate (LLC Ellara) at a concentration of 50 μg / ml was used as a transduction activator. 5×10 5 Activated T lymphocytes, lentiviral drug (2 × 10 5LVP / cell), protamine sulfate, and 10 ml of culture medium were added to the bioreactor, which was then placed on a Multitron shaker (110 rpm; 37°C; 5% CO2) for 5 hours. After 5 hours, the bioreactor was removed and centrifuged at 1500 rpm and 24°C for 5 minutes. The supernatant was removed, and the pellet was resuspended in 2 ml of culture medium. The pellet was transferred to a 6-well suspension culture plate (Eppendorf, 0030720016) and placed in a CO2 incubator (37°C, 5% CO2). Transduction assays were performed after 72 hours. The efficiency of T lymphocyte transduction was determined based on the signal level of the GFP reporter protein, and the efficiency was assessed by flow cytometry. Cell viability was analyzed using propidium iodide dye.

[0278] The cytotoxic activity of the generated CART lymphocytes was determined in direct coculture with target cells expressing the CD19 receptor. 10,000 CART lymphocytes and 5,000 target cells (Raji) were transferred to each well of a 48-well suspension culture plate (Eppendorf, 0030723015) in 1 ml of culture medium (RPMI-1640 supplemented with L-glutamine (Biolot, 1.3.4.1) and 10% fetal bovine serum (Gibco, 26140079)). The incubation period was 72 hours in a CO2 incubator (37°C, 5% CO2). Analysis was performed using a flow cytometer to determine the ratio of CD3-positive to CD3-negative cells.

[0279] Cytokine levels in the culture medium were analyzed using the BD Cytometric Bead Array (CBA) Human Th1 / Th2 Cytokine Kit II (BD, 551809) according to the manufacturer's instructions.

[0280] The presence of CAR on the cell surface during the interaction process with antigen triggers the activation of the NFAT signaling cascade. ΔTCRabThe use of the NFAT-GFP reporter cell line allowed us to screen the anti-CD19-CAR-ITAMx3 (control) gene construct and experimental gene constructs selected from the group of vectors shown in Figures 1, 3, 5, 7, 11, 13, 15, 17, 19, 21, 23, and 25. The reporter cell line was transfected by electroporation (electroporation device: Neon, Thermo Scientific, USA). Raji cells were used as target cells expressing the CD19 receptor, and the incubation time was 6 hours in a CO2 incubator (37°C, 5% CO2). Analysis was performed using a flow cytometer to measure the glow intensity of the GFP reporter protein (Figure 27).

[0281] Example 6 result. CAR T expressing control CAR-ITAMx3 and experimental CAR T expressing one of the CAR variants (by a selected vector from the group of vectors shown in Figure 1, Figure 3, Figure 5, Figure 7, Figure 11, Figure 13, Figure 15, Figure 17, Figure 19, Figure 21, Figure 23, Figure 25) by transduction with recombinant lentiviral vectors, in particular: CAR-(costimulatory domain of 4-1BB)-(signaling domain with ITGB3×2); CAR-(costimulatory domain of 4-1BB)-(signaling domain with ITGB3×3); CAR-(costimulatory domain of 4-1BB)-(signaling domain with ITAM × 2-ITGB3 × 1); CAR-(costimulatory domain of 4-1BB)-(signaling domain with ITAM × 1-ITGB3 × 2); CAR-(costimulatory domain of CD28)-(signaling domain with ITGB3×2); CAR-(costimulatory domain of CD28)-(signaling domain with ITGB3×3); CAR-(costimulatory domain of CD28)-(signaling domain with ITAM × 2-ITGB3 × 1); CAR-(CD28 costimulatory domain)-(signaling domain with ITAMx1-ITGB3x2) And, we created.

[0282] The level of GFP expression in experimental cells (17.6%) was comparable to that of control GFP (15.63%). In direct co-culture of experimental CAR-Ts containing only ITGB3x2 or only ITGB3x3 (CAR-(costimulatory domain of 4-1BB)-(signaling domain with ITGB3x2); CAR-(costimulatory domain of 4-1BB)-(signaling domain with ITGB3x3); CAR-(costimulatory domain of CD28)-(signaling domain with ITGB3x2); CAR-(costimulatory domain of CD28)-(signaling domain with ITGB3x3)) with Raji CD19+ target cells (2:1), reduced cytotoxic activity was observed compared to that of the control CAR-ITAMx3.

[0283] Therefore, the above CAR-Ts in which the CAR contains a signaling domain with only ITGB3x2 or only ITGB3x3 were rejected, but these are not the subject of the present invention.

[0284] In direct co-culture of the various experimental CAR-ITAMx2-ITGB3x1 (CAR-(costimulatory domain 4-1BB)-(signaling domain with ITAMx1-ITGB3x2)); CAR-(costimulatory domain CD28)-(signaling domain with ITAMx2-ITGB3x1)) with target Raji CD19+ cells (2:1), comparable cytotoxic activity was observed compared with the control CAR-ITAMx3. Culture of both CAR cell variations was accompanied by the secretion of the inflammatory cytokines IL-2 and IFN-γ, characteristic of CAR-T lymphocytes (Figure 28(A, B, C)).

[0285] Similarly, in direct co-culture of the various experimental CAR-ITAMx1-ITGB3x2 constructs (CAR-(costimulatory domain 4-1BB)-(signaling domain with ITAMx1-ITGB3x2); CAR-(costimulatory domain CD28)-(signaling domain with ITAMx1-ITGB3x2)) with target RajiCD19+ cells (2:1), comparable cytotoxic activity was observed compared to the control CAR-ITAMx3 construct.

[0286] Jurkat expressing control CAR-ITAM × 3 ΔTCRab Addition of target (Raji CD19+) cells to the NFAT-GFP reporter cell line and various experimental constructs, CAR-ITAMx1-ITGB3x2 (CAR-(costimulatory domain 4-1BB)-(signaling domain with ITAMx1-ITGB3x2)); CAR-(costimulatory domain CD28)-(signaling domain with ITAMx1-ITGB3x2)), induces CAR-mediated activation of the NFAT signaling cascade. Thus, the presence of a synthetic activation domain within the CAR receptor does not significantly reduce CAR-dependent activation of cells expressing the corresponding gene construct (Figure 29).

[0287] As above, Jurkat cells expressing control CAR-ITAMx3 ΔTCRab Addition of target (Raji CD19+) cells to the NFAT-GFP reporter cell line and various experimental CAR-ITAMx1-ITGB3x2 ("CAR-(costimulatory domain 4-1BB)-(signaling domain with ITAMx1-ITGB3x2)"; "CAR-(costimulatory domain CD28)-(signaling domain with ITAMx1-ITGB3x2)") induces CAR-mediated activation of the NFAT signaling cascade. Thus, the presence of a synthetic activation domain within the CAR receptor does not significantly reduce CAR-dependent activation of cells expressing the corresponding gene construct.

Claims

1. 1. An isolated chimeric antigen receptor (CAR) comprising an alternative intracellular signaling domain of the chimeric antigen receptor (CAR), In the alternative intracellular signaling domain of the chimeric antigen receptor (CAR), one or two immunoreceptor tyrosine-based activation motifs (ITAMs) selected from ITAM1, ITAM2, and ITAM3 are replaced with the amino acid sequence of integrin beta 3 (ITGB3), wherein ITGB3 has the amino acid sequence represented by SEQ ID NO:

4. The isolated chimeric antigen receptor (CAR).

2. 2. The isolated chimeric antigen receptor (CAR) of claim 1, wherein in the alternative intracellular signaling domain of the chimeric antigen receptor (CAR), one immunoreceptor tyrosine-based activation motif (ITAM) selected from ITAM1, ITAM2, and ITAM3 is substituted with the amino acid sequence of integrin beta 3 (ITGB3).

3. 2. The isolated chimeric antigen receptor (CAR) of claim 1, wherein in the alternative intracellular signaling domain of the chimeric antigen receptor (CAR), two immunoreceptor tyrosine-based activation motifs (ITAMs) selected from ITAM1, ITAM2, and ITAM3 are replaced with the amino acid sequence of integrin beta 3 (ITGB3).

4. ITAM2 and ITAM3 are replaced with two ITGB3s; or ITAM1 and ITAM3 are replaced with two ITGB3s; or ITAM1 and ITAM2 are replaced by two ITGB3s, The isolated chimeric antigen receptor (CAR) of claim 1.

5. the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) The amino acid sequence represented by SEQ ID NO: 5; or The amino acid sequence represented by SEQ ID NO: 6; or The amino acid sequence represented by SEQ ID NO: 7; 3. The isolated chimeric antigen receptor (CAR) of claim 2, comprising:

6. the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) The amino acid sequence represented by SEQ ID NO: 8; or The amino acid sequence represented by SEQ ID NO: 9; or The amino acid sequence represented by SEQ ID NO: 10 4. The isolated chimeric antigen receptor (CAR) of claim 3, comprising:

7. the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) comprises one or two immunoreceptor tyrosine-based activation motifs (ITAMs) selected from ITAM1, ITAM2, and ITAM3, and one or two amino acid sequences of integrin beta 3 (ITGB3), and the total number of ITAMs and ITGB3s is 3; The isolated chimeric antigen receptor (CAR) of claim 1.

8. the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) comprises two immunoreceptor tyrosine-based activation motifs (ITAMs) selected from ITAM1, ITAM2, and ITAM3, and one of the amino acid sequences of integrin beta 3 (ITGB3); The isolated chimeric antigen receptor (CAR) of claim 7.

9. the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) comprising ITAM2, ITAM3 and ITGB3; or comprising ITAM1, ITAM3 and ITGB3; or including ITAM1, ITAM2 and ITGB3, The isolated chimeric antigen receptor (CAR) of claim 8.

10. the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) comprises one immunoreceptor tyrosine-based activation motif (ITAM) selected from ITAM1, ITAM2, and ITAM3, and two amino acid sequences of integrin beta 3 (ITGB3); The isolated chimeric antigen receptor (CAR) of claim 7.

11. the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) or comprising two amino acid sequences of ITAM1 and integrin beta 3 (ITGB3); or or comprising two amino acid sequences of ITAM2 and integrin beta 3 (ITGB3); or It contains two amino acid sequences: ITAM3 and integrin beta 3 (ITGB3), The isolated chimeric antigen receptor (CAR) of claim 10.

12. 2. The isolated chimeric antigen receptor (CAR) of claim 1, wherein ITAM1 has the amino acid sequence represented by SEQ ID NO:

1.

13. 2. The isolated chimeric antigen receptor (CAR) of claim 1, wherein ITAM2 has the amino acid sequence represented by SEQ ID NO:

2.

14. 2. The isolated chimeric antigen receptor (CAR) of claim 1, wherein ITAM3 has the amino acid sequence represented by SEQ ID NO:

3.

15. The alternative intracellular signaling domain of the chimeric antigen receptor (CAR) has the amino acid sequence represented by SEQ ID NO: 5; or the amino acid sequence represented by SEQ ID NO: 6; or the amino acid sequence represented by SEQ ID NO:

7. The isolated chimeric antigen receptor (CAR) of claim 8.

16. The alternative intracellular signaling domain of the chimeric antigen receptor (CAR) has the amino acid sequence represented by SEQ ID NO: 8; or the amino acid sequence represented by SEQ ID NO: 9; or the amino acid sequence represented by SEQ ID NO:

10. The isolated chimeric antigen receptor (CAR) of claim 10.

17. a) an antigen-binding domain specific for a tumor antigen; b) a transmembrane domain; c) a costimulatory domain; d) an alternative intracellular signaling domain of a chimeric antigen receptor (CAR), in which one or two immunoreceptor tyrosine-based activation motifs (ITAMs) selected from ITAM1, ITAM2, and ITAM3 are replaced with the amino acid sequence of integrin beta 3 (ITGB3), wherein ITGB3 has the amino acid sequence represented by SEQ ID NO: 4; The isolated chimeric antigen receptor (CAR) of any one of claims 1 to 16, comprising:

18. 18. The isolated chimeric antigen receptor (CAR) of claim 17, wherein the antigen-binding domain specific for the tumor antigen is a Fab or scFv.

19. 18. The isolated chimeric antigen receptor (CAR) of claim 17, wherein the antigen binding domain is specific for a tumor antigen selected from the group of CD19, CD20, CD123, or BCMA.

20. 20. The isolated chimeric antigen receptor (CAR) of claim 19, wherein the antigen-binding domain specific for the CD19 tumor antigen has the amino acid sequence represented by SEQ ID NO:

11.

21. 19. The isolated chimeric antigen receptor (CAR) of claim 18, wherein the transmembrane domain is a transmembrane domain of a protein selected from the group consisting of T-cell receptor-alpha, -beta, or -zeta chains, CD28, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.

22. 22. The isolated chimeric antigen receptor (CAR) of claim 21, wherein the CD8 transmembrane domain has the amino acid sequence represented by SEQ ID NO:

12.

23. The isolated chimeric antigen receptor (CAR) of claim 17, wherein the costimulatory domain is a functional signaling domain of a protein selected from the group consisting of OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), and 4-1BB (CD137).

24. 24. The isolated chimeric antigen receptor (CAR) of claim 23, wherein the CD28 costimulatory domain has the amino acid sequence represented by SEQ ID NO:

13.

25. 24. The isolated chimeric antigen receptor (CAR) of claim 23, wherein the 4-1BB costimulatory domain has the amino acid sequence represented by SEQ ID NO:

14.

26. The isolated chimeric antigen receptor (CAR) of any one of claims 1 to 16, further comprising a leader sequence.

27. The isolated chimeric antigen receptor (CAR) of any one of claims 1 to 16, having an amino acid sequence represented by an amino acid sequence selected from the group of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26.

28. An isolated nucleic acid encoding the isolated chimeric antigen receptor (CAR) of any one of claims 1 to 27.

29. 29. An expression vector comprising the nucleic acid of claim 28.

30. 30. The expression vector of claim 29, selected from the group consisting of DNA, RNA, a plasmid, a lentiviral vector, an adeno-associated viral vector, an adenoviral vector, or a retroviral vector.

31. further comprising a promoter, or an in vitro transcription vector, or the nucleic acid sequence further comprises a poly(A) sequence, or the nucleic acid sequence further comprises a 3'UTR; 30. The expression vector of claim 29.

32. 30. An in vitro method for producing a genetically modified cell comprising the chimeric antigen receptor (CAR) of any one of claims 1 to 27, the method comprising the step of transforming a cell with the expression vector of claim 29.

33. A genetically modified cell comprising the chimeric antigen receptor (CAR) of any one of claims 1 to 27, wherein the genetically modified cell is produced using the method of claim 32.

34. are T lymphocytes; or are CD8+ T cells; or NK cells, The cell of claim 33.

35. The cell of claim 33 for use as a medicament.

36. The cell of claim 35 for use as a medicament for a tumor disease.

37. The cell of claim 36, which is used as a drug for a disease in which the tumor is cancer.

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