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  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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  • C12N2510/00—Genetically modified cells

Definitions

• the present invention relates to the field of biotechnology, namely, to an isolated alternative intracellular signaling domain of a chimeric antigen receptor (CAR) and a chimeric antigen receptor (CAR) comprising this signaling domain.
• the invention also relates to a nucleic acid encoding an alternative intracellular signal domain of a chimeric antigen receptor and a nucleic acid encoding a chimeric antigen receptor with the above signaling domain, an expression vector, a delivery vector, as well as a genetically modified cell that includes the above chimeric antigen receptor, and the method of obtaining this cell.
• Chimeric antigen receptors are recombinant protein molecules that target cellular 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 / SO 952791509000211).
• CAR generally consists of several domains that perform specific functions.
• the extracellular domain of CAR is most often represented by a single-chain variable fragment of an antibody specific for a tumor antigen (single-chain variable fragment, scFv) and provides recognition of a target independent of the molecules of the major histocompatibility complex.
• Intracellular domains are responsible for signaling when binding to a target antigen and for efficient activation of CAR expressing immune cells. “The intracellular part of the receptor consists of the C03 (-chain (POP-zeta chain) and additional co-stimulatory domains, most often derived from natural proteins CD28 and 4-1BB.
• CD28 - CD3 ((POP-zeta chain)) CAR receptor can provide an earlier than in the case of using 4-1BB - CD3z (POP-zeta chain) CAR, activation of CAR-T lymphocytes due to the release of cytokines 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 from: http: // www.nature.com/doifinder/10.1038/nrd4597), which in turn may provide increased therapeutic efficacy.
• a moderate decrease in the level of activation of CAR-T cells and the release of cytokines, while maintaining the rate of transmission of the activation signal through the co-stimulatory domain based on CD28 without loss of cytotoxic activity, will significantly improve the therapeutic properties of adoptive immunotherapy of oncological diseases based on expressing CD28 - CD3 (POP-zeta chains ) CAR receptor of immune cells.
• TCR T-cell receptors
• MHC major histocompatibility complex
• Zap-70 kinase interacts with ITAMs phosphorylated at two sites in CD3z (CBS- zeta chain) using Src-homology 2 (SH2) domains Binding of Zap-70 to CDZz (POS-zeta chain) leads to a change in the kinase conformation and its autophosphorylation at amino acid residues Y315 and Y319. Lck-mediated phosphorylation and trans-autophosphorylation leads to activation of Zap70.After activation, Zap70 stops interacting with CDZz (POP-zeta chain) and associates with
• SUBSTITUTE SHEET (RULE 26) plasma membrane, where, in turn, 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.
• Zap-70 is the most important participant in the CAR signaling pathway; therefore, the regulation of the activity of this kinase may represent one of the ways of regulating the level of CAR-T activation, which directly affects their viability, persistence, and functional activity.
• Numerous studies have revealed the main structural and functional elements of the system of interaction between ZAP70 and ITAM motives [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.
• integrin beta 3 fragments of the amino acid sequence of integrin beta 3 (ITGB3) (cytoplasmic tail 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 US A. 1996 Oct 15; 93 (21): 11819-24; Durrant TN, van den Bosch MT, Hers I, Integrin II6b3 outside-in signaling, Blood. 2017 Oct 5; 130 (14): 1607-1619].
• the inventors have surprisingly found that the hybrid signaling domains of the cytoplasmic part of the chimeric antigen receptor (CAR), including instead of one or two immunoreceptor tyrosine-basic activation motifs (ITAM) selected from ITAM1, ITAM2 and ITAM3, in the intracellular signaling domain of the chimeric antigen receptor (CAR) (eg, CD3 / (POP-zeta)) peptide fragments of the amino acid sequence of integrin beta 3 (ITGB3) stimulate CAR-dependent activation of genetically modified immune cells expressing the corresponding genetic construct.
• ITAM immunoreceptor tyrosine-basic activation motifs
• the present invention relates to an isolated alternative intracellular signaling domain of a chimeric antigen receptor (CAR), wherein one or two immunoreceptor tyrosine-basic activation motifs (ITAMs) selected from ITAM1, ITAM2 and ITAM3 are replaced with a fragment of the amino acid sequence of integrin beta 3 (ITGB3).
• CAR chimeric antigen receptor
• ITAMs immunoreceptor tyrosine-basic activation motifs
• the alternative intracellular signaling domain of the chimeric antigen receptor is characterized in that one immunoreceptor tyrosine-basic activation motif (TSAM) selected from ITAMl, ITAM2, and ITAM3 is replaced with a fragment of the beta 3 integrin amino acid sequence (ITGB3).
• TSAM immunoreceptor tyrosine-basic activation motif
• the alternate intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized in that ITAM1 is replaced by ITGB3.
• the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized in that ITAM2 is replaced by ITGB3.
• the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized in that ITAM3 is replaced by ITGB3.
• the alternative intracellular signaling domain of the chimeric antigen receptor is characterized in that two immunoreceptor tyrosine-basic activation motifs (ITAM) selected from ITAMl, ITAM2, and ITAM3 are replaced with a fragment of the amino acid sequence of integrin beta 3 (ITGB3).
• ITAM immunoreceptor tyrosine-basic activation motifs
• the alternate intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized in that ITAM2 and ITAM3 are replaced by two ITGB3.
• the alternate intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized in that ITAMl and ITAM3 are replaced by two ITGB3.
• the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized in that ITAMl and ITAM2 are replaced by two ITGB3.
• the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized in that ITAMl has the amino acid sequence APAYKQGQNQLYNELNLGRREEYDVLDKR (SEQ ID NO: 1).
• the alternative intracellular signaling domain of the chimeric antigen receptor is characterized in that ITAM2 has the amino acid sequence PRRKNPQEGLYNELQKDKMAEAYSEIGM (SEQ ID NO: 2).
• the alternative intracellular signaling domain of the chimeric antigen receptor is characterized in that ITAM3 has the amino acid sequence ERRRGKGHDGLYQGLSTATKDTYDALHMQ (SEQ ID NO: 3).
• 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 1 or 2 conservative substitutions.
• the alternative intracellular signaling domain of the chimeric antigen receptor is characterized in that ITGB3 has the amino acid sequence DTANNPLYKEATSTFTNITYRGT (SEQ ID NO: 4) with any 1 conservative substitution.
• the alternative intracellular signaling domain of the chimeric antigen receptor is characterized in that ITGB3 has the amino acid sequence DTANNPLYKEATSTFTNITYRGT (SEQ ID NO: 4) with any 2 conservative substitutions.
• an alternative intracellular chimeric antigen receptor (CAR) signaling domain is characterized in that ITGB3 has the amino acid sequence DTANNPLYKEATSTFTNITYRGT (SEQ ID NO: 4).
• the chimeric antigen receptor (CAR) alternate intracellular signaling domain has the amino acid sequence
• the chimeric antigen receptor (CAR) alternate intracellular signaling domain has the amino acid sequence
• RVKFSRSADDTANNPLYKEATSTFTNITYRGTRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEI GMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 6 or has an amino acid sequence of at least 6 amino acids with ID NO: 6) or has an amino acid sequence of at least 6 amino acids with ID NO: 6 amino acid residues at least 95% identical to ID NOs in comparison
• the chimeric antigen receptor (CAR) alternate intracellular signaling domain has the amino acid sequence
• the chimeric antigen receptor (CAR) alternate intracellular signaling domain has the amino acid sequence
• RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKDTANNPLYKEATSTFTNITY RGTKGDTANNPLYKEATSTFTNITYRGTALPPR (SEQ ID NO: 8) or has an amino acid sequence that is at least 95% identical to the amino acid sequence ID NO: 8 and SEQ amino acid residues at least 95% identical to the ID NO:
• the chimeric antigen receptor (CAR) alternate intracellular signaling domain has the amino acid sequence
• RVKFSRSADDTANNPLYKEATSTFTNITYRGTRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEI GMKGDTANNPLYKEATSTFTNITYRGTALPPR (SEQ ID NO: 9) or has an amino acid sequence that is at least 95% identical to the amino acid sequence with SEQ ID NO: 9 amino acid residues in comparison with SEQ ID NO: 9.
• the chimeric antigen receptor (CAR) alternate intracellular signaling domain has the amino acid sequence
• SUBSTITUTE SHEET (RULE 26) is identical to the amino acid sequence of SEQ ID NO: 10 and differs from SEQ ID NO: 10 only in non-conservative amino acid residues.
• the present invention relates to an isolated alternative intracellular signaling domain of a chimeric antigen receptor (CAR) that includes one or two immunoreceptor tyrosine-basic activation motifs (ITAM) selected from ITAMl, ITAM2 and ITAM3, and one or two amino acid sequence fragments integrin beta 3 (ITGB3), with the total number of ITAM and ITGB3 being three.
• CAR chimeric antigen receptor
• ITAM immunoreceptor tyrosine-basic activation motifs
• ITGB3 amino acid sequence fragments integrin beta 3
• the alternative intracellular signaling domain of the chimeric antigen receptor is characterized in that it includes two immunoreceptor tyrosine-basic activation motifs (ITAM) selected from ITAMl, ITAM2 and ITAM3, and one fragment of the amino acid sequence of integrin beta 3 (ITGB3).
• ITAM immunoreceptor tyrosine-basic activation motifs
• the alternative intracellular signaling domain of the chimeric antigen receptor is characterized by comprising ITAM2, IT M3, and ITGB3.
• an alternative intracellular signaling that includes ITAMl, ITAM3, and ITGB3.
• the alternative intracellular signaling domain of the chimeric antigen receptor is characterized by comprising ITAMl, ITAM2, and ITGB3.
• the alternative intracellular signaling domain of the chimeric antigen receptor is characterized by comprising one immunoreceptor tyrosine-basic activation motif (ITAM) selected from ITAMl, ITAM2, and ITAM3, and two fragments of the amino acid sequence of integrin beta 3 (ITGB3).
• ITAM immunoreceptor tyrosine-basic activation motif
• the alternative intracellular signaling domain of the chimeric antigen receptor is characterized by comprising ITAMl and two fragments of the beta 3 integrin amino acid sequence (ITGB3).
• the alternative intracellular signaling domain of the chimeric antigen receptor is characterized by comprising ITAM2 and two fragments of the beta 3 integrin amino acid sequence (ITGB3).
• the alternative intracellular signaling domain of the chimeric antigen receptor is characterized by comprising ITAM3 and two fragments of the beta 3 integrin amino acid sequence (ITGB3).
• the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized in that ITAMl has the amino acid sequence APAYKQGQNQLYNELNLGRREEYDVLDKR (SEQ ID NO: 1).
• an alternative intracellular signaling domain of a chimeric antigen receptor is characterized in that ITAM2 has the amino acid sequence PRRKNPQEGLYNELQKDKMAEAYSEIGM (SEQ ID NO: 2).
• the alternative intracellular signaling domain of the chimeric antigen receptor is characterized in that ITAM3 has the amino acid sequence ERRRGKGHDGLYQGLSTATKDTYDALHMQ (SEQ ID NO: 3).
• an alternative intracellular signaling domain of a chimeric antigen receptor is characterized in that ITGB3 has the amino acid sequence DTANNPLYKEATSTFTNITYRGT
• SUBSTITUTE SHEET (RULE 26) (SEQ ID NO: 4) or amino acid sequence
• DTANNPLYKEATSTFTNITYRGT (SEQ ID NO: 4) with any 1 or 2 conservative substitutions.
• the alternative intracellular signaling domain of the chimeric antigen receptor is characterized in that ITGB3 has the amino acid sequence DTANNPLYKEATSTFTNITYRGT (SEQ ID NO: 4) with any 1 conservative substitution.
• an alternative intracellular chimeric antigen receptor (CAR) signaling domain is characterized in that ITGB3 has the amino acid sequence DTANNPLYKEATSTFTNITYRGT (SEQ ID NO: 4) with any 2 conservative substitutions.
• the alternative intracellular signaling domain of the chimeric antigen receptor is characterized in that ITGB3 has the amino acid sequence DTANNPLYKEATSTFTNITYRGT (SEQ ID NO: 4).
• the alternate intracellular signaling domain of the chimeric antigen receptor (CAR) has the amino acid sequence
• the chimeric antigen receptor (CAR) alternate intracellular signaling domain has the amino acid sequence
• RVKFSRSADDTANNPLYKEATSTFTNITYRGTRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEI GMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 6 or has an amino acid sequence that is at least 95% amino acid in amino acid sequence identical with ID NO 6 in comparison with ID NO: 6) or has an amino acid sequence that is at least 95% identical in amino acid sequence to ID NO 6 and has no amino acid sequence that is at least 95% identical in amino acid sequence to ID SEQ.
• the chimeric antigen receptor (CAR) alternate intracellular signaling domain has the amino acid sequence
• the chimeric antigen receptor (CAR) alternate intracellular signaling domain has the amino acid sequence
• RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKDTANNPLYKEATSTF TNITYRGTKGDTANNPLYKEATSTFTNITYRGTALPPR (SEQ ID NO: 8) or has an amino acid sequence that is at least 95% identical to the amino acid sequence ID NO: 8 in comparison with SEQ and SEQ amino acid residues at least 95% identical to ID NO:
• the chimeric antigen receptor (CAR) alternate intracellular signaling domain has the amino acid sequence
• the chimeric antigen receptor (CAR) alternate intracellular signaling domain has the amino acid sequence
• RVKFSRSADDTANNPLYKEATSTFTNITYRGTRGRDPEMGGKDTANNPLYKEATSTFTNITYRGTKGE RRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 10) or has an amino acid sequence that is at least 95% identical in amino acid sequence to NO amino acid sequence ID: 10 amino acid residues in SEQ
• the present invention relates to an isolated nucleic acid that encodes the aforementioned alternative intracellular signaling domain.
• the present invention relates to an isolated chimeric antigen receptor (CAR) comprising the aforementioned alternative intracellular signaling domain.
• CAR chimeric antigen receptor
• an isolated chimeric antigen receptor in some embodiments, is provided.
• an isolated chimeric antigen receptor in some embodiments, is provided.
• CAR is characterized in that the antigen binding domain that is specific for the tumor antigen is Fab or scFv.
• an isolated chimeric antigen receptor in some embodiments, is provided.
• CAR is characterized in that the antigen-binding domain that is specific for the tumor antigen is scFv.
• an isolated chimeric antigen receptor in some embodiments, is provided.
• CAR is characterized in that the antigen-binding domain is specific for a tumor antigen, which is selected from the group: CD19, CD20, CD123 or BCMA.
• an isolated chimeric antigen receptor is characterized in that the antigen binding domain is specific for the tumor antigen CD19.
• an isolated chimeric antigen receptor is characterized in that the antigen binding domain that is specific for the tumor antigen CD19 has the amino acid sequence
• an isolated chimeric antigen receptor is characterized in that the transmembrane domain is a transmembrane domain of a protein selected from the group consisting of an alpha, beta, or zeta chain of a T cell receptor, CD28, POP epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.
• an isolated chimeric antigen receptor is characterized in that wherein the transmembrane domain is CD8.
• an isolated chimeric antigen receptor is characterized in that the CD8 transmembrane domain has the amino acid sequence shown
• an isolated chimeric antigen receptor is characterized in that the co-stimulatory domain is a functional signaling domain of a protein selected from the group consisting of 0X40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CDlla / CD18), ICOS (CD278) and 4-1BB (CD137).
• an isolated chimeric antigen receptor is characterized in that the co-stimulatory domain is CD28 or 4-1BB.
• an isolated chimeric antigen receptor is characterized in that the co-stimulatory domain is CD28.
• an isolated chimeric antigen receptor is characterized in that the CD28 co-stimulatory domain has the amino acid sequence
• an isolated chimeric antigen receptor in some embodiments, is provided.
• CAR is characterized in that the co-stimulatory domain is 4-1BB.
• an isolated chimeric antigen receptor in some embodiments, is provided.
• an isolated chimeric antigen receptor in some embodiments, is provided.
• CAR is characterized in that it additionally contains a leader sequence.
• an isolated chimeric antigen receptor in some embodiments, is provided.
• CAR has an amino acid sequence represented by an amino acid sequence that is selected from the group: SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26, or has an amino acid sequence that is at least 95% identical to the amino acid sequence selected from the group: SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26, and is different from the amino acid sequence , which is selected from the group: SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26, only in non-conservative amino acid residues.
• Chimeric antigen receptor 19CAR-T-CD28 - a1bcc13dzeta (ITGB3-ITAM2-ITAM3) has the amino acid sequence
• Chimeric antigen receptor 19CAR-T-CD28-a11 cc13dzeta (ITAM1-ITGB3-ITAM3) has the amino acid sequence
• Chimeric antigen receptor 19CAR-T-CD28 - AI cc13dzeta (ITGB3-ITGB3-ITAM3) has the amino acid sequence
• ITAM2- ITGB3 has the amino acid sequence
• Chimeric antigenic receptor 19CAR-T- 41bb - altcd3fl3eTa (ITGB3- ITAM2- ITAM3) has the amino acid sequence
• Chimeric antigen receptor 19CAR-T- 41bb - altcd3fl3eTa (ITGB3- ITGB3- ITAM3) has the amino acid sequence
• ITAM2- ITGB3 has the amino acid sequence
• the present invention relates to an isolated nucleic acid that encodes the above isolated chimeric antigen receptor (CAR).
• CAR chimeric antigen receptor
• the present invention relates to an expression vector comprising the aforementioned nucleic acid.
• the present invention relates to a delivery vector comprising the above nucleic acid.
• the vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adeno-associated viral vector, adenoviral vector, or retroviral vector.
• the vector further comprises a promoter.
• the vector is an in vitro transcribed vector.
• the nucleic acid sequence further comprises a poly (A) sequence.
• the nucleic acid sequence further comprises a 3'UTR.
• the present invention relates to a method for producing a genetically modified cell that contains the above chimeric antigen receptor (CAR), comprising transforming the cell with the above vector.
• CAR chimeric antigen receptor
• the present invention relates to a genetically modified cell that contains the above chimeric antigen receptor (CAR), which is obtained by the above method.
• CAR chimeric antigen receptor
• the cell is a T lymphocyte.
• the cell is a CD8 + T cell.
• the cell is an NK cell.
• the cell is used as a drug.
• the cell is used as a drug for tumor diseases.
• the cell is used as a drug for diseases where the tumor is cancer.
• FIG. 1 Map of the pCDH-19CAR-T-ITGB3-ITGB3-eGFP vector.
• FIG. 2 Schematic representation of the 19CAR-T-ITGB3-ITGB3-eGFP expression cassette.
• FIG. 3 Vector map pCDH-19CAR-T-ITGB3-ITGB3-ITGB3-eGFP.
• FIG. 4 Schematic representation of the 19CAR-T-ITGB3-ITGB3-ITGB3-eGFP expression cassette.
• FIG. 5 Vector map pCDH-19CAR-T-ITAMl-ITGB3-ITGB3-eGFP.
• FIG. b Schematic representation of the 19CAR-T-ITAMl-ITGB3-ITGB3-eGFP expression cassette.
• FIG. 7 Vector map pCDH-19CAR-T-ITAMl-ITAM2-ITGB3-eGFP.
• FIG. 8 Schematic representation of the 19CAR-T-ITAM1-ITAM2-ITGB3-eGFP expression cassette.
• FIG. 9 Map of the pCDH-CAR-T-GFP vector.
• FIG. 10 Schematic representation of the pCDH-CAR-T-GFP expression cassette.
• FIG. 11 Vector map pCDH-19CAR-T-ITAMl-ITGB3-ITAM2-eGFP.
• FIG. 12 Schematic representation of the expression cassette_rSEN-19CAR-T-ITAMl-ITGB3-ITAM2-eGFP.
• FIG. 13 Vector map pCDH-19CAR-T-ITGB3-ITAM2-ITGB3-eGFP.
• FIG. 14 Schematic representation of the pCDH-19CAR-T-ITGB3-ITAM2-ITGB3-eGFP expression cassette.
• FIG. 15 Vector map pCDH-19CAR-T_CD28-ITGB3-ITGB3-eGFP.
• FIG. 16 Schematic representation of the pCDH-19CAR-T_CD28-ITGB3-ITGB3-eGFP expression cassette.
• FIG. 17 Vector map pCDH-19CAR-T-CD28_ITGB3-ITGB3-ITGB3-eGFP.
• FIG. 18 Schematic representation of the pCDH-19CAR-T-CD28_ITGB3-ITGB3-ITGB3-eGFP expression cassette.
• FIG. 19 Vector map pCDH-19CAR-T-CD28_ITAMl-ITGB3-ITGB3-eGFP.
• FIG. 20 Schematic representation of the pCDH-19CAR-T-CD28_ITAMl-ITGB3-ITGB3-eGFP expression cassette.
• FIG. 21 Vector map pCDH-19CAR-T-CD28_ITAMl-ITAM2-ITGB3-eGFP.
• FIG. 22 Schematic representation of the pCDH-19CAR-T-CD28_ITAMl-ITAM2-ITGB3-eGFP expression cassette.
• FIG. 23 Map of the vector pCDH-19CAR-T-CD28_ITAMl-ITGB3-ITAM2-eGFP.
• FIG. 24 Schematic representation of the pCDH-19CAR-T-CD28_ITAMl-ITGB3-ITAM2-eGFP expression cassette.
• FIG. 25 Vector map pCDH-19CAR-T-CD28_ ITGB3-ITAM2-ITGB3-eGFP.
• FIG. 26 Schematic representation of the pCDH-19CAR-T-CD28 ITGB3-ITAM2-ITGB3-eGFP expression cassette.
• FIG. 27 The level of GFP expression in viable T-lymphocytes after transduction with lentiviral drugs, 72 hours,%.
• FIG. 28 Evaluation of the cytotoxic activity of CAR-T-lymphocytes expressing control CAR-ITAMx3 and experimental CAR-ITAMx2-ITGB3xl when co-cultured with target cells.
• FIG. 29 Comparison of the functional activity of the genetic constructs of the control CAR-ITAMx3 and experimental CAR-ITAMx2-ITGB3xl for the ability to activate the NFAT signaling cascade in the Jurkat ATCRab NFAT-GFP reporter cell line, 72 hours.
• mammal any animal classified as a mammal, including primates, humans, rodents, canines, felines, cattle, small ruminants, horses, pigs, etc.
• isolated means modified or removed from the natural state.
• a nucleic acid or peptide naturally present in an animal is not “isolated”, but the same nucleic acid or peptide, partially or completely separated from materials accompanying them in their natural state, is “isolated”.
• the isolated nucleic acid or protein may exist in substantially purified form or may exist in a non-natural environment such as, for example, a genetically modified cell.
• identity or “homology” should be interpreted as meaning the percentage of amino acid residues in a candidate sequence that are identical to the residues of the corresponding sequence to which it is compared, after sequence comparison and introduction of "gaps" if necessary to achieve
• SUBSTITUTE SHEET (RULE 26) maximum percent identity for the entire sequence and excluding any conservative substitutions as part of sequence identity. Neither the N- or C-terminal extension nor the insertional segments should be construed as diminishing identity or homology. Comparison methods and computer programs are well known. Sequence identity can be determined using sequence analysis software (eg, Sequence Analysis Software Package, Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Ave., Madison, WI 53705). This software is suitable for such sequences by determining the degree of homology for a variety of substitutions, deletions (elimination) and other modifications.
• sequence analysis software eg, Sequence Analysis Software Package, Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Ave., Madison, WI 53705
• the term "antigen” or “Ad” is defined as a molecule that provokes an immune response. This immune response may involve the production of antibodies or the activation of specific immunologically competent T cells, or both. It will be understood by those skilled in the art that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. In addition, antigens can be obtained from recombinant or genomic DNA. Those of skill in the art will understand that any DNA containing nucleotide sequences or an incomplete nucleotide sequence encoding a protein that elicits an immune response thus encodes an “antigen” as the term is used herein.
• an antigen does not necessarily encode exclusively the full-length nucleotide sequence of a gene. It is quite obvious that the present invention, as non-limiting examples, relates to the use of partial nucleotide sequences of several genes, and that these nucleotide sequences are arranged in various combinations to stimulate the desired immune response. In addition, those skilled in the art will understand that an antigen does not necessarily encode a “gene”. It is quite obvious that the antigen can be synthesized or obtained from a biological sample. Such a biological sample can include, but are not limited to, a tissue sample, a tumor sample, a cell, or a biological fluid.
• Disease is a state of animal health where the animal cannot maintain homeostasis and where, if the disease is not alleviated, the animal's health continues to deteriorate.
• an "impairment" in an animal is a health condition in which the animal is able to maintain homeostasis, but the animal's health condition is less favorable than it would be in the absence of impairment. If left untreated, the disorder does not necessarily impair the animal's health.
• malignant neoplasm is defined as a disease characterized by the rapid and uncontrolled growth of abnormal cells. Malignant cells can spread locally or through the bloodstream and lymphatic system to other parts of the body. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer, pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancers, lymphoma, leukemia , lung cancer, myeloma, multiple myeloma, and the like.
• SUBSTITUTE SHEET (RULE 26) Malignant neoplasms that can be treated include tumors that are not vascularized or substantially unvascularized as well as vascularized tumors.
• Malignant neoplasms may include non-solid tumors (such as hematologic tumors such as leukemias and lymphomas) or may include solid tumors.
• Types of malignancies to be treated with CARs of the invention include, but are not limited to, carcinoma, blastoma, and sarcoma, and specific leukemias or lymphoid malignancies, benign and malignant tumors, and malignant neoplasms such as sarcomas, carcinomas, and melanomas. Also included are adult tumors / malignancies and pediatric tumors / malignant neoplasms.
• Hematologic malignancies are malignant neoplasms of the blood or bone marrow.
• hematologic (or hematogenous) malignant neoplasms include leukemias, including acute leukemias (such as acute lymphocytic leukemia, acute myelocytic leukemia, acute myelogenous leukemia, and myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukelocytic leukemias) (such as granular leukemia, chronic myelogenous leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin's lymphoma (slow-growing and high-grade forms), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, myelodyspeloid leukemia syndrome, hairy cells and lymph nodes.
• Solid tumors are abnormal tissue tumors, usually without cysts or areas of fluid. Solid tumors can be benign or malignant. The different types of solid tumors are named after the type of cells that form them (for example, sarcomas, carcinomas and lymphomas). Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovium, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, subclinic cancer, lymph gland cancer lungs, ovarian cancer, prostate cancer, hepatic cell carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid cancer, papillary thyroid cancer, pheochromocytoma, sebaceous gland carcinoma, papillary
• endogenous refers to any material from an organism, cell, tissue, or system, or material produced in an organism, cell, tissue, or system.
• SUBSTITUTE SHEET (RULE 26)
• exogenous refers to any material that is introduced into an organism, cell, tissue or system, or produced outside an organism, cell, tissue, or system.
• expression is defined as the transcription and / or translation of a particular nucleotide sequence triggered by its promoter.
• tumor antigen or overexpression of a tumor antigen is intended to mean an abnormal level of expression of a tumor antigen in a cell from a disease focus, such as a solid tumor, in a particular tissue or organ of a patient, relative to the level of expression in a normal cell from that tissue or organ.
• a disease focus such as a solid tumor
• Patients with solid tumors or hematologic malignancies characterized by overexpression of the tumor antigen can be identified by standard assays known in the art.
• patient refers to any animal or its cells, in vitro or in situ, amenable to the methods described herein.
• the patient or individual is a human.
• polynucleotide is defined as a chain of nucleotides.
• nucleic acids are polymers of nucleotides.
• nucleic acids and polynucleotides are used interchangeably.
• nucleic acids are polynucleotides that can be hydrolyzed to monomeric "nucleotides”.
• Monomeric nucleotides can be hydrolyzed to nucleosides.
• polynucleotides include, but are not limited to, all nucleic acid sequences obtainable by any means available in the art, including, but not limited to, recombinant methods, i. E. cloning nucleic acid sequences from a recombinant library or the genome of a cell; using conventional cloning and PCR technology, and the like, and synthetic methods.
• peptide As used herein, the terms “peptide”, “polypeptide” and “protein” are used interchangeably and refer to a compound composed of amino acid residues covalently linked by peptide bonds.
• the protein or peptide must contain at least two amino acids, and there is no limitation on the maximum number of amino acids that the protein or peptide sequence can contain.
• Polypeptides include any peptide or protein containing two or more amino acids linked to each other by peptide bonds.
• the term also refers to short chains, also commonly referred to in the art, such as peptides, oligopeptides, and oligomers, and longer chains, generally referred to in the art as proteins, of which many types exist.
• Polypeptides include, but are not limited to, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variant polypeptides, modified polypeptides, derivatives, analogs, fusion proteins. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.
• CAR chimeric antigen receptor
• the intracellular signaling domain of CAR is responsible for the activation of at least one normal effector function of the immune system cell into which the CAR is placed.
• effector function refers to the specialized function of a cell.
• the effector function of a T cell for example, can be cytolytic activity or helper activity, including cytokine secretion.
• intracellular signaling domain refers to the portion of a protein that signals an effector function and causes the cell to perform a specialized function.
• intracellular signaling domain is intended to include any truncated portion of the intracellular signaling domain sufficient to signal effector function.
• intracellular signaling domains for use in CARs of the invention include cytoplasmic sequences of a T cell receptor (TCR) and co-receptors that act together to initiate signal transduction after antigen receptor binding, as well as any derivative or variant of these sequences, and any synthetic sequence, having the same functional ability.
• TCR T cell receptor
• T cell activation is mediated by two different classes of cytoplasmic sequences mediating signal transduction: initiating antigen-dependent primary activation via TCRs (primary cytoplasmic sequences mediating signal transduction) and acting in an antigen-independent manner to provide a secondary or costimulatory signal ( secondary cytoplasmic sequences mediating signal transmission).
• Primary cytoplasmic signaling sequences regulate the primary activation of the TCR complex in a stimulatory or inhibitory manner.
• Primary cytoplasmic sequences that mediate signal transduction, acting in a stimulatory manner may contain signaling motifs known as immunoreceptor tyrosine activation motifs (immunoreceptor tyrosine-basic activation motif) or ITAM.
• ITAMs including primary cytoplasmic signaling sequences include those derived from TCR-zeta, FcR-gamma, FcR-beta, CP3-gamma, CP3-delta, POP-epsilon, CD8, CD22, CD79a, CD79b, and CD66d. It is particularly preferred if the cytoplasmic signaling mediating molecule in the CAR contains a signaling mediating cytoplasmic sequence derived from POP-zeta.
• the CAR cytoplasmic domain can be engineered to contain the POP-zeta signaling domain alone or in combination with any other desired cytoplasmic domains useful in CAR conditions.
• the signaling domain of POP-zeta has the amino acid sequence
• the above signaling domain contains 3 ITAMs (immunoreceptor tyrosine activation motifs), namely ITAM1, ITAM2 and ITAM3.
• ITAM1 has the amino acid sequence represented by APAYKQGQNQLYNELNLGRREEYDVLDKR (SEQ ID NO: 1).
• 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).
• the present invention relates to an isolated alternative intracellular signaling domain of a chimeric antigen receptor (CAR), wherein one or two immunoreceptor tyrosine-basic activation motifs (ITAMs) selected from ITAM1, ITAM2 and ITAM3 are replaced with a fragment of the amino acid sequence of integrin beta 3 (ITGB3).
• CAR chimeric antigen receptor
• ITAMs immunoreceptor tyrosine-basic activation motifs
• the alternative intracellular signaling domain of the chimeric antigen receptor is characterized in that one immunoreceptor tyrosine-basic activation motif (ITAM) selected from ITAM1, ITAM2, and ITAM3 is replaced with a fragment of the beta 3 integrin amino acid sequence (ITGB3).
• ITAM immunoreceptor tyrosine-basic activation motif
• the alternate intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized in that ITAM1 is replaced by ITGB3.
• the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized in that ITAM2 is replaced by ITGB3.
• the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized in that ITAM3 is replaced by ITGB3.
• an alternative intracellular signaling domain of a chimeric antigen receptor is characterized in that two immunoreceptor tyrosine-basic activation motifs (ITAM), selected from ITAM1, ITAM2, and ITAM3, are replaced with a fragment of the beta 3 integrin amino acid sequence (ITGB3).
• ITAM immunoreceptor tyrosine-basic activation motifs
• the alternate intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized in that ITAM2 and ITAM3 are replaced by two ITGB3.
• the alternate intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized in that ITAM1 and ITAM3 are replaced by two ITGB3.
• the alternate intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized in that ITAM1 and ITAM2 are replaced by two ITGB3.
• the present invention relates to an isolated alternative intracellular signaling domain of a chimeric antigen receptor (CAR) that includes one or two immunoreceptor tyrosine-base activation motifs (ITAM) selected from ITAMl, ITAM2 and ITAM3, and one or two fragments
• CAR chimeric antigen receptor
• ITAM immunoreceptor tyrosine-base activation motifs
• SUBSTITUTE SHEET (RULE 26) the amino acid sequence of integrin beta 3 (ITGB3), with the total number of ITAM and ITGB3 being three.
• the alternative intracellular signaling domain of the chimeric antigen receptor is characterized in that it includes two immunoreceptor tyrosine-basic activation motifs (ITAM) selected from ITAM1, ITAM2, and ITAM3, and one fragment of the amino acid sequence of integrin beta 3 (ITGB3).
• ITAM immunoreceptor tyrosine-basic activation motifs
• the chimeric antigen receptor (CAR) alternate intracellular signaling domain is characterized by comprising ITAM2, ITAM3, and ITGB3.
• an alternative intracellular signaling that includes ITAM1, ITAM3, and ITGB3.
• the alternative intracellular signaling domain of the chimeric antigen receptor is characterized by comprising ITAMl, ITAM2, and ITGB3.
• the alternative intracellular signaling domain of the chimeric antigen receptor is characterized by comprising one immunoreceptor tyrosine-basic activation motif (ITAM) selected from ITAMl, ITAM2, and ITAM3, and two fragments of the amino acid sequence of integrin beta 3 (ITGB3).
• ITAM immunoreceptor tyrosine-basic activation motif
• the alternative intracellular signaling domain of the chimeric antigen receptor is characterized in that it includes ITAMl and and two fragments of the amino acid sequence of integrin beta 3 (ITGB3).
• the alternative intracellular signaling domain of the chimeric antigen receptor is characterized by comprising ITAM2 and two fragments of the amino acid sequence of integrin beta 3 (ITGB3).
• the alternate intracellular signaling domain of the chimeric antigen receptor is characterized by comprising ITAM3 and two fragments of the amino acid sequence of integrin beta 3 (ITGB3).
• the alternative intracellular signaling domain of the chimeric antigen receptor (CAR) is characterized in that ITGB3 has the amino acid sequence represented by DT NNPLYKEATSTFTNITYRGT SEQ ID NO: 4) or the amino acid sequence represented by DTANNPLYKEATSTFTNITYRGT SEQ ID NO: 4) with any 1 or 2 conservative substitutions.
• the alternative intracellular signaling domain of the chimeric antigen receptor is characterized in that ITGB3 has the amino acid sequence represented by DTANNPLYKEATSTFTNITYRGT (SEQ ID NO: 4) with any 1 conservative substitution.
• the alternative intracellular signaling domain of the chimeric antigen receptor is characterized in that ITGB3 has the amino acid sequence represented by DTANNPLYKEATSTFTNITYRGT (SEQ ID NO: 4) with any 2 conservative substitutions.
• the alternative intracellular signaling domain of the chimeric antigen receptor is characterized in that ITGB3 has the amino acid sequence represented by DTANNPLYKEATSTFTNITYRGT (SEQ ID NO: 4).
• Amino acid conservative substitutions are understood to mean the replacement of one amino acid by another with similar structure. This replacement does not change the properties of ITGB3.
• the chimeric antigen receptor (CAR) alternate intracellular signaling domain has the amino acid sequence
• the chimeric antigen receptor (CAR) alternate intracellular signaling domain has the amino acid sequence
• RVKFSRSADDTANNPLYKEATSTFTNITYRGTRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEI GMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR SEQ ID NO: 6 or has an amino acid sequence of at least 6 amino acids with ID NO: 6) or has an amino acid sequence of at least 6 amino acids with ID NO: 6 amino acid residues at least 95% identical to ID NOs in comparison
• the alternate intracellular signaling domain of the chimeric antigen receptor (CAR) has the amino acid sequence
• the alternate intracellular signaling domain of the chimeric antigen receptor (CAR) has the amino acid sequence
• RVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKDTANNPLYKEATSTFTNITY RGTKGDTANNPLYKEATSTFTNITYRGTALPPR (SEQ ID NO: 8) or has an amino acid sequence that is at least 95% identical to the amino acid sequence ID NO: 8 and SEQ amino acid residues at least 95% identical to the ID NO:
• the chimeric antigen receptor (CAR) alternate intracellular signaling domain has the amino acid sequence
• RVKFSRSADDTANNPLYKEATSTFTNITYRGTRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEI GMKGDTANNPLYKEATSTFTNITYRGTALPPR (SEQ ID NO: 9) or has an amino acid sequence that is at least 95% identical to the amino acid sequence with SEQ ID NO: 9 amino acid residues in comparison with SEQ ID NO: 9.
• the chimeric antigen receptor (CAR) alternate intracellular signaling domain has the amino acid sequence
• RVKFSRSADDTANNPLYKEATSTFTNITYRGTRGRDPEMGGKDTANNPLYKEATSTFTNITYRGTKGE RRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 10) or has an amino acid sequence that is at least 95% identical in amino acid sequence NO: 10 amino acid residues to SEQ ID NO: 10.
• the present invention relates to an isolated chimeric antigen receptor (CAR) comprising the aforementioned alternative intracellular signaling domain.
• CAR chimeric antigen receptor
• an isolated chimeric antigen receptor is characterized by comprising:
• the present invention relates to a chimeric antigen receptor
• CAR cytoplasmic domain
• the extracellular domain contains a target-specific binding member, otherwise referred to as an antigen binding domain.
• the intracellular domain or, otherwise, the cytoplasmic domain contains the co-stimulatory signaling region and part of the zeta chain.
• the co-stimulatory signaling region refers to the part of the CAR containing the intracellular domain
• Co-stimulatory molecules are cell surface molecules, other than antigen receptors or their ligands, required for an effective lymphocyte response to an antigen.
• a spacer domain can be inserted between the extracellular domain and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR.
• the term "spacer domain” generally means any oligo or polypeptide that functions by linking a transmembrane domain to an extracellular domain or cytoplasmic domain in a polypeptide chain.
• the spacer domain may contain up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids.
• a CAR of the invention comprises an extracellular domain comprising an antigen binding domain; transmembrane domain and cytoplasmic domain.
• a transmembrane domain is used that is naturally associated with one of the domains in the CAR.
• the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different proteins on the membrane surface to minimize interactions with other components of the receptor complex.
• antibody binding domain of an antibody or
• an “antigen-binding portion” of an antibody or an “antigen-binding fragment” refers to one or more antibody fragments that retain the ability to specifically bind an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of linking fragments included in the term
• An "antigen binding portion" of an antibody includes:
• Fab fragment a monovalent fragment consisting of the VL, VH, CL and CH1 domains
• CDR complementarity determining region
• the two regions of the Fv fragment, VL and VH are encoded by different genes, they can be combined using recombinant methods using a synthetic linker, which makes it possible to obtain them in the form of a single protein chain in which the VL and VH regions are paired to form monovalent molecules (known as single chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879 - 5883). It is assumed that such single-chain molecules are also included in the term "antigennegative part" of the antibody. Such antibody fragments are obtained using conventional methods known
• variable refers to the fact that certain segments of the variable domains differ widely in sequence among antibodies.
• the V domain mediates antigen binding and determines the specificity of a particular antibody for its particular antigen.
• variability is unevenly distributed over the 110 amino acid variable domains.
• V regions are composed of invariant fragments called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme variability called “hypervariable regions” or CDRs.
• FRs framework regions
• hypervariable regions in each chain are held together in close proximity by FRs and with the hypervariable regions of the other chain contribute to the formation of the antigen-binding site of antibodies.
• hypervariable region refers to the amino acid residues of an antibody that are responsible for antigen binding.
• the hypervariable region contains amino acid residues from the "complementarity determining region" or "CDR" and / or such residues from the "hypervariable loop".
• affinity maturation it may also be preferable to alter one or more amino acid residues of the CDR regions in order to increase the binding affinity for the target epitope.
• affinity maturation is known as "affinity maturation” and in some cases can be performed in connection with humanization, for example, in situations where humanization of an antibody leads to a decrease in the specificity or affinity of binding, and it is not possible to sufficiently improve the specificity or affinity of binding by using only reverse mutations.
• Various methods of affinity maturation are known in the art, for example, 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 proposed by Wu et al. Proc Natl Acad Sci USA 95: 6037 6042 (1998).
• An antigen binding domain of an antibody of the invention that “binds” a target antigen is an antigen binding domain of an antibody that binds an antigen with sufficient affinity such that the antigen binding domain of an antibody can be used as a diagnostic and / or therapeutic agent when targeting a protein or cell, or tissue that expresses the antigen and cross-reacts slightly with other proteins.
• analytical methods sorting of fluorescently activated cells (FACS), radioimmunoprecipitation (R1A) or ELISA (ELISA), the degree of binding of the antigen-binding domain of an antibody to a protein that is not a “target” (with a “non-target protein”) is less than 10% of binding the antigen-binding domain of the antibody to a specific target protein.
• the term “specific binding” or the expressions “specifically binds to” or “specific to” a particular polypeptide or epitope on a particular target polypeptide means binding that is markedly (measurable) different from a non-specific interaction.
• a CAR of the invention comprises a target specific binding member, otherwise referred to as an antigen binding domain.
• the choice of domain depends on the type and number of ligands that determine the surface of the target glucose.
• an antigen binding domain can be selected to recognize a ligand that acts as a cell surface marker on target cells associated with a particular disease state.
• examples of cell surface markers that can act as ligands for the antigen-binding domain in the CAR of the invention include markers associated with cancer cells.
• a CAR of the invention can be engineered to target a tumor antigen of interest by constructing a desired antigen binding domain that specifically binds to the antigen on a tumor cell.
• tumor antigen or “hyperproliferative disorder antigen” or “hyperproliferative disorder associated antigen” refers to antigens typical of particular hyperproliferative disorders, such as cancer.
• the antigens described herein are included by way of example only. The list is not intended to be limiting, and additional examples will be apparent to those skilled in the art.
• Tumor antigens are proteins produced by tumor cells that elicit an immune response, specifically T-cell mediated immune responses.
• the choice of antigen binding domain according to the invention will depend on the particular type of cancer to be treated.
• Tumor antigens are well known in the art and include, for example, glioma-associated antigen, carcinoembryonic antigen (CEA), human p-chorionic gonadotropin, alpha-fetolrotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RUI, RU2 (AS), intestinal carboxylesterase, mut hsp70-2, M-CSF, prostasis, prostate specific antigen (PSA), PAP, NY-ESO-1, LAGE-la, p53, protein , PSMA, Her2 / neu, survivin and telomerase, prostate carcinoma antigen-1 (PCTA-1), MAGE, ELF
• the tumor antigen comprises one or more epitopes of tumor antigens associated with a cancer.
• Malignant tumors express a number of proteins that can serve as target antigens for the immune response. These molecules include, but are not limited to, tissue-specific antigens such as MART-1, tyrosinase and GP 100 in melanoma and prostatic acid phosphatase (PAP) and prostate specific antigen (PSA) in prostate cancer.
• Other target molecules belong to a group of molecules involved in transformation, such as the HER-2 / Neu / ErbB-2 oncogene.
• Another group of target antigens are oncofetal antigens such as carcinoembryonic antigen (CEA).
• B-cell lymphoma an immunoglobulin of a tumor-specific idiotype forms, in essence, a tumor-specific immunoglobulin antigen that is unique to an individual tumor.
• B-cell differentiation antigens such as CD19, CD20, and CD37 are other candidates for target antigens in B-cell lymphoma. Some of these antigens (CEA, HER-2, CD19,
• SUBSTITUTE SHEET (RULE 26) CD20, idiotype) have been used with limited success as targets for passive immunotherapy using monoclonal antibodies.
• the type of tumor antigen described in the invention can also be a tumor specific antigen (TSA) or a tumor associated antigen (TAA).
• TSA is unique to tumor cells and is not present on other cells in the body.
• the TAA antigen is not unique to tumor cells and is also expressed on normal cells under conditions that prevent the induction of a state of immunological tolerance to the antigen. Expression of antigen in a tumor can occur under conditions that allow the immune system to respond to the antigen.
• TAA may be antigens expressed on normal cells during fetal development, when the immune system is immature and unable to respond, or they may be antigens normally present at extremely low levels on normal cells but expressed at much higher levels on tumor cells ...
• TSA or TAA antigens include the following: differentiation antigens such as MART-1 / MelanA (MART-1), gplOO (Pmel 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multilinear antigens such as MAGE -1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens such as CEA; overexpressed oncogenes and mutant tumor suppressor genes such as p53, Ras, HER-2 / neu; unique tumor antigens resulting from chromosomal translocations, such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens such as the Epstein-Barr virus EBVA antigens and the Eb and E7 antigens of the human papillomavirus (HPV).
• differentiation antigens such as MART-1 / MelanA (MART-1), gp
• an isolated chimeric antigen receptor in some embodiments, is provided.
• CAR is characterized in that the antigen-binding domain is specific for a tumor antigen, which is selected from the group: CD19, CD20, CD123 or BCMA.
• an isolated chimeric antigen receptor in some embodiments, is provided.
• CAR is characterized in that the antigen-binding domain is specific for the tumor antigen CD19.
• an isolated chimeric antigen receptor in some embodiments, is provided.
• CAR is characterized in that the antigen binding domain that is specific for the tumor antigen is Fab or scFv.
• an isolated chimeric antigen receptor in some embodiments, is provided.
• CAR is characterized in that the antigen-binding domain that is specific for the tumor antigen is scFv.
• an isolated chimeric antigen receptor in some embodiments, is provided.
• CAR is characterized in that the antigen-binding domain, which is specific for the tumor antigen CD19, has the amino acid sequence shown
• SUBSTITUTE SHEET (RULE 26) LKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSS (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 in non-conserved amino acid residues.
• An antigen binding domain with the amino acid sequence of SEQ ID NO: 11 consists of:
• variable domain of the light chain with the amino acid sequence
• variable domain of the heavy chain with the amino acid sequence
• a co-stimulatory domain or co-stimulatory signaling region refers to the portion of a CAR containing the intracellular domain of a co-stimulatory molecule.
• a co-stimulatory molecule is a cell surface molecule other than an antigen receptor or its ligands required for an effective lymphocyte response to an antigen. Examples of such molecules include CD27, CD28, 4-1BB (CD137), 0X40, CD30, ICAM-1, CDS, CD40, PD-1, ICOS (CD278), functionally associated lymphocyte antigen-1 (LFA-1, CDlla / CDl8), CD2, CD7, LIGHT, NKG2C, B7-H3 and a ligand that specifically binds to CD83, and the like.
• an isolated chimeric antigen receptor is characterized in that the co-stimulatory domain is a functional signaling domain of a protein selected from the group consisting of 0X40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CDlla / CD18), ICOS (CD278) and 4-1BB (CD137).
• the signaling cytoplasmic sequences in the cytoplasmic signaling portion of the CAR can be linked to each other in a random or ad hoc order.
• the compound can form a short oligo or polypeptide linker, preferably 2 to 10 amino acids in length.
• the glycine-serine doublet is a suitable linker.
• the CAR cytoplasmic domain is engineered to contain the aforementioned alternative intracellular signaling domain and the CD28 co-stimulatory signaling domain.
• the CAR cytoplasmic domain is constructed containing the aforementioned alternative intracellular signaling domain and the 4-1BB co-stimulatory signaling domain.
• the cytoplasmic domain is constructed comprising the aforementioned alternative intracellular signaling domain and the CD28 and 4-1BB co-stimulatory domain.
• an isolated chimeric antigen receptor is characterized in that the CD28 co-stimulatory domain has the amino acid sequence shown
• an isolated chimeric antigen receptor is characterized in that the 4-1BB co-stimulatory domain has the amino acid sequence represented by KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 14).
• a CAR can be constructed to contain a transmembrane domain fused to the extracellular domain of the CAR.
• a transmembrane domain is used that is naturally associated with one of the domains in the CAR.
• the transmembrane domain can be selected or modified by amino acid substitution to avoid binding of such domains to the transmembrane domains of the same or different proteins on the membrane surface to minimize interactions with other components of the receptor complex.
• the transmembrane domain can be obtained from a natural or synthetic source. If the source is natural, the domain can be obtained from any membrane-bound or transmembrane protein. Transmembrane regions for specific use in this invention can be obtained from (i.e., obtained containing at least transmembrane regions) alpha, beta or zeta chains of the T cell receptor, CD28, CD3 epsilon, CD45, CD4, CD5 , CD8, CD9, CD1 b, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154. Alternatively, the transmembrane domain may be synthetic, in which case it will contain predominantly hydrophobic residues such as leucine and valine. Preferably, a triplet of phenylalanine, tryptophan and valine will be found at each end of the synthetic transmembrane domain.
• an isolated chimeric antigen receptor is characterized in that the transmembrane domain is a transmembrane domain of a protein selected from the group consisting of an alpha, beta, or zeta chain of a T cell receptor, CD28, POP epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 and CD154.
• an isolated chimeric antigen receptor is characterized in that wherein the transmembrane domain is CD8.
• an isolated chimeric antigen receptor is characterized in that the CD8 transmembrane domain has the amino acid sequence IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 12).
• a short oligo or polypeptide linker preferably 2 to 10 amino acids in length, can form a link between the transmembrane domain and the CAR cytoplasmic signaling domain.
• the glycine-serine doublet is a suitable linker.
• the CAR transmembrane domain of the invention comprises a CD8 hinge domain.
• the CD8 hinge domain has the amino acid sequence TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 35).
• an isolated chimeric antigen receptor is characterized by further comprising a leader sequence.
• the isolated chimeric antigen receptor has an amino acid sequence represented by an amino acid sequence selected from the group: SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26, or has an amino acid sequence that is at least 95% identical to an amino acid sequence selected from the group: SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26, and has differences in comparison with the amino acid sequence, which is selected from the group: SEQ ID NO: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26, only in non-conserved amino acid residues.
• the chimeric antigen receptor 19CAR-T- CD28 - a1bsbZdzeta (ITGB3- ITAM2- ITAM3) has an amino acid sequence LLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKL LIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSG KPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETT YYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPA PRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCAAAI EVMYPPPYLDNEKSNGTI
• the chimeric antigen receptor 19CAR-T-CD28 - a1bcc! 3dzeta (ITGB3 - ITGB3 - ITAM3) has the amino acid sequence
• ITAM2- ITGB3 has an amino acid sequence MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKL LIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSG KPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETT YYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPA PRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCAAAI EVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSK RSRLL
• Chimeric antigen receptor 19CAR-T- 41bb - altcd3 fl3 eTa (ITGB3- ITAM2- ITAM3) has the amino acid sequence
• the chimeric antigen receptor 19CAR-T- 41bb - a1bsDZdzeta (ITGB3- ITGB3- ITAM3) has the amino acid sequence
• ITAM2- ITGB3 has an amino acid sequence MLLLVTSLLLCELPHPAFLLIPDIQMTQTTSSLSASLGDRVTISCRASQDISKYLNWYQQKPDGTVKL LIYHTSRLHSGVPSRFSGSGSGTDYSLTISNLEQEDIATYFCQQGNTLPYTFGGGTKLEITGSTSGSG KPGSGEGSTKGEVKLQESGPGLVAPSQSLSVTCTVSGVSLPDYGVSWIRQPPRKGLEWLGVIWGSETT YYNSALKSRLTIIKDNSKSQVFLKMNSLQTDDTAIYYCAKHYYYGGSYAMDYWGQGTSVTVSSTTTPA PRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGR KKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEEEGGCELRVKFSRSADDTANNPLYKEATSTFTNITYR GTRGRDPEMG
• nucleic acid denotes a clear sequence of nucleotides, modified or not modified defining a fragment or region of nucleic acid, whether or not containing unnatural nucleotides and is either double-stranded DNA or RNA, or single-stranded DNA or RNA, or transcription products of said DNA.
• this invention does not relate to nucleotide sequences in their natural chromosomal environment, i.e. in a natural state.
• the sequences of the present invention have been isolated and / or purified, i. E. were taken straight
• SUBSTITUTE SHEET (RULE 26) or indirectly, such as by copying, where their environment has been at least partially modified.
• isolated nucleic acids obtained by genetic recombination, for example, using host cells (host cells), or obtained by chemical synthesis.
• nucleic acid molecule is a nucleic acid molecule that has been identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of a protein (polypeptide) nucleic acid.
• An isolated nucleic acid molecule differs from the form or set in which it naturally occurs. Thus, the isolated nucleic acid molecule differs from the nucleic acid molecule that exists naturally in cells.
• an isolated nucleic acid molecule includes a nucleic acid molecule found in cells in which a protein (polypeptide) is normally expressed, for example, if the nucleic acid molecule has a different localization in the chromosome from its localization in cells in natural conditions.
• nucleotide sequence encompasses its complement, unless otherwise indicated.
• a nucleic acid having a specific sequence is to be understood as encompassing its complementary strand with its complementary sequence.
• A refers to adenine
• C refers to cytosine
• G refers to guanine
• T refers to thymine (5-methyluracil)
• RNA takes the place of uracil in DNA
• and refers to uracil (takes the place of thymine in RNA).
• nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and encoding the same amino acid sequence.
• the phrase “a nucleotide sequence encoding a protein or RNA may also include introns in case the nucleotide sequence encoding a protein may in some versions contain introns.
• Coding refers to the inherent property of specific nucleotide sequences in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as templates for the synthesis of other polymers and macromolecules in biological processes that have a specific sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a specific amino acid sequence and, as a result, biological properties.
• a gene encodes a protein if, as a result of transcription and translation of mRNA corresponding to this gene, a protein is produced in a cell or other biological system.
• a coding strand the nucleotide sequence of which is identical to the sequence of mRNA and which is usually presented in sequence lists, and a non-coding strand used as a template for transcription of a gene or cDNA, can be referred to as coding for a protein or other product of this gene or cDNA.
• Nucleic acid sequences encoding the desired molecules can be obtained using recombinant techniques known in the art, such as, for example, screening libraries from cells expressing the gene, isolating the gene from a vector known to include it, or isolating it directly from
• SUBSTITUTE SHEET (RULE 26) containing it cells and tissues using standard methods.
• the gene of interest can often be obtained synthetically rather than cloned.
• the present invention relates to an isolated nucleic acid that encodes the aforementioned alternative intracellular signaling domain.
• the present invention provides an isolated nucleic acid that encodes the aforementioned isolated chimeric antigen receptor (CAR), which comprises the aforementioned alternative intracellular signaling domain.
• CAR isolated chimeric antigen receptor
• vector means a nucleic acid molecule capable of transporting another nucleic acid to which it is linked.
• the vector is a plasmid, i. E. circular double-stranded DNA molecule, into which additional DNA segments can be inserted using the enzymatic ligation reaction.
• the vector is a viral vector in which additional DNA segments can be inserted by enzymatic ligation into the viral genome.
• vectors are capable of autonomous replication in a genetically modified cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
• vectors eg, non-episomal mammalian vectors
• vectors can be integrated into the genome of a host cell upon introduction into a host cell, and thus replicate with the host genome.
• some vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply “expression vectors”).
• control sequence means polynucleotide sequences that are required to influence the expression and processing of the coding sequences to which they are inserted by an enzymatic ligation reaction.
• Expression control sequences include the corresponding transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and full denylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that increase protein stability; and, if desired, sequences that enhance protein secretion.
• the nature of such control sequences differs depending on the host organism. Eukaryotic cells contain promoters, polyadenylation signals, and enhancers.
• control sequences includes at least all components that are essential for expression and processing, and may also include additional components whose presence is useful, for example, leader sequences and fusion cell sequences.
• a nucleic acid is “operably linked” if it is in a functional relationship with another nucleotide sequence.
• DNA of a presequence or secretory leader is operably linked to DNA of 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;
• the ribosome binding site is operably linked to a coding sequence if located so that it can facilitate translation.
• "operably linked” means that the linked DNA sequences are contiguous, and in the case of a secretory leader sequence, are contiguous and in frame. However, enhancers do not have to be contiguous.
• delivery vector or “delivery vectors” is meant any delivery vector that can be used in the present invention to bring into contact with a cell (i.e., “contacting”) or deliver into cells or intracellular compartments (i.e. . "Introduction") agents / chemicals and molecules (proteins or nucleic acids) required in the present invention. It includes, but is 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 appropriate transfer vectors.
• delivery vectors enable the delivery of molecules, chemicals, macromolecules (genes, proteins) or other vectors such as plasmids, peptides. In these cases, delivery vectors are molecular carriers.
• delivery vector or “delivery vectors” are also meant delivery methods for performing transfection.
• “Expression vector” refers to a vector containing a recombinant polynucleotide containing expression control sequences operably linked to a nucleotide sequence to be expressed.
• the expression vector contains the appropriate cis-active elements for expression; other elements for expression can be supplemented with elements of a genetically modified cell or in an in vitro expression system.
• Expression vectors include all vectors known in the art, such as cosmids, plasmids (eg, non-enveloped or contained in liposomes), and viruses (eg, lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) comprising a recombinant polynucleotide.
• expression of natural or synthetic nucleic acids encoding CARs is typically achieved by functional coupling of a nucleic acid encoding a CAR polypeptide or portion thereof with a promoter and inserting the construct into an expression vector.
• the vectors can be suitable for replication and integration into eukaryotic cells.
• Typical cloning vectors contain transcriptional and translation terminators, initiation sequences, and promoters useful to regulate the expression of the desired nucleic acid sequence.
• promoter is defined as a DNA sequence recognized by a synthetic apparatus of a cell or an embedded synthetic apparatus necessary for
• SUBSTITUTE SHEET (RULE 26) initiating a specific transcription of the polynucleotide sequence.
• promoter / regulatory sequence means a nucleic acid sequence necessary for the expression of a gene product operably linked to a promoter / regulatory sequence. In some cases, this sequence may be the core sequence of the promoter, and in other cases, this sequence may also include an enhancer sequence and other regulatory elements necessary for expression of the gene product.
• the promoter / regulatory sequence for example, can be a sequence expressing a gene product in a tissue-specific manner.
• a "constitutive" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, causes the production of the gene product in the cell under most or all of the physiological conditions in the cell.
• an "inducible" promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, causes the production of a gene product in a cell, essentially only when an inducing factor corresponding to the promoter is present in the cell.
• tissue-specific promoter is a nucleotide sequence that, when operably linked to a polynucleotide encoding or defining a gene product, causes the production of a gene product in a cell, essentially only when the cell is a tissue cell of the type corresponding to the promoter.
• a suitable promoter is the cytomegalovirus (CMV) immediate early promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of driving high levels of expression of any polynucleotide sequence operably linked thereto.
• CMV cytomegalovirus
• EF-la elongation factor-1a
• constitutive promoter sequences can also be used, including, but not limited to, monkey virus 40 early promoter (SV40), mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat promoter [LTR], the MoMuLV promoter, the avian leukemia virus promoter, the Epstein-Barr virus immediate early promoter, the Rous sarcoma virus promoter, as well as human gene promoters, by way of non-limiting examples, such as the actin gene promoter, myosin gene promoter, hemoglobin gene promoter and creatine kinase gene promoter.
• the invention should not be limited to the use of constitutive promoters. Inducible promoters are also included as part of the invention.
• an inducible promoter provides a molecular "switch" capable of triggering expression of the polynucleotide sequence to which it is operably linked, if such expression is desired, or terminating expression if expression is not desired.
• inducible promoters include, but are not limited to, a metallothionein promoter, glucocorticoid gene promoters, a progesterone gene promoter, and a tetracycline gene promoter.
• SUBSTITUTE SHEET (RULE 26) Additional promoter elements, such as enhancers, regulate the frequency of transcription initiation. As a rule, they are localized in the area of 30-110 bp. above the start site, although a number of promoters have recently been shown to contain functional elements below the start site. Often the spacing between promoter elements is flexible in such a way as to prevent the function of the promoter when the elements are inverted or displaced relative to each other. In the thymidine kinase (tk) promoter, the spacing between promoter elements can be increased up to 50 bp. before the start of the decrease in activity. It is believed that, depending on the promoter, individual elements can function together or independently to activate transcription.
• tk thymidine kinase
• the present invention relates to a DNA construct comprising CAR sequences, wherein the sequence comprises an antigen-binding domain nucleic acid sequence operably linked to an intracellular domain nucleic acid sequence.
• An example of an intracellular domain that can be used in a CAR of the invention includes, but is not limited to, the aforementioned alternative intracellular signaling domain, a CD28 and / or 4-1BB co-stimulatory domain, and the like, in some cases, the CAR may contain any a combination of the above alternative intracellular signaling domain, CD28 co-stimulatory domain, 4-1BB co-stimulatory domain, and the like.
• a CAR of the invention comprises an anti-CD19 scFv, a human CD8 hinge and transmembrane domain, and a 4-1BB co-stimulatory domain and the above-mentioned alternative intracellular signaling domain.
• a CAR of the invention comprises an anti-CD19 scFv, a human CD8 hinge and transmembrane domain, and a CD28 co-stimulatory domain and the aforementioned alternative intracellular signaling domain.
• the expression constructs of the present invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery methods. Methods for delivering genes are known in the art. See, for example, US Patent Nos. 5,399,346, 5,580,859, 5,589,466, incorporated herein by reference in their entirety.
• the invention relates to a gene therapy vector.
• Nucleic acid can be cloned into some types of vectors.
• a nucleic acid can be cloned into a vector, including, but not limited to, a plasmid, phagemid, phage derivative, animal virus, and cosmid.
• Specific vectors of interest include expression vectors, replication vectors, vectors for generating probes, and vectors for sequencing.
• the expression vector can be delivered to the cell in the form of a viral vector.
• Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other manuals on virology and molecular biology.
• Viruses useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes virus, and lentiviruses.
• a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, suitable restriction endonuclease sites and one or more
• SUBSTITUTE SHEET (RULE 26) selective markers (for example, WO 01/96584; 0 01/29058 and US patent N * 6326193).
• retroviruses provide a suitable platform for gene delivery systems.
• the selected gene can be inserted into a vector and packaged into retroviral particles using methods known in the art.
• the recombinant virus can then be isolated and delivered to the individual's cells in vivo or ex vivo.
• retroviral systems are known in the art.
• adenoviral vectors are used.
• a number of adenoviral vectors are known in the art.
• lentiviral vectors are used.
• the present invention also relates to vectors into which the DNA of the present invention is inserted.
• Retrovirus-based vectors such as lentivirus are suitable tools for achieving long-term gene transfer because they enable long-term stable integration of the transgene and its multiplication in daughter cells.
• Lentiviral 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. They also have the added advantage of low immunogenicity.
• lentivirus refers to the genus Retroviridae. Lentiviruses are unique among retroviruses because they able to infect non-dividing cells; they can deliver a significant amount of genetic information in the form of DNA to a host cell, thus they represent one of the most efficient ways to implement a gene delivery vector. HIV, SIV and FIV are examples of lentiviruses. Vectors derived from lentiviruses represent a means of achieving significant levels of gene transfer in vivo.
• an expression vector for insertion into a cell may also contain a selectable marker gene, or a reporter gene, or both to facilitate the identification and selection of expressing cells from a population of cells to be transfected or infected with viral vectors.
• the selectable marker can carry a separate DNA fragment and can be used in a cotransfection method. Both selectable markers and reporter genes can be flanked by appropriate regulatory sequences, allowing expression in host cells. Useful selectable markers include, for example, antibiotic resistance genes such as peo, etc. P .
• Reporter genes are used to identify potentially transfected cells and to assess the functionality of regulatory sequences.
• a reporter gene is a gene that is not present or expressed in an organism or tissue and encodes a polypeptide, the expression of which is manifested in some easily identifiable property, for example, enzymatic activity. The expression of the reporter gene is analyzed at an appropriate time point after DNA insertion into recipient cells.
• Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or the green fluorescent protein (GFP) gene (e.g., Ui-Tei et al.,
• SUBSTITUTE SHEET (RULE 26) 2000 FEBS Letters 479: 79-82).
• Suitable expression systems are well known and can be obtained using known methods or from a commercial source.
• the construct with the minimum 5'-flanking region showing the highest level of reporter gene expression is defined as a promoter.
• Such promoter regions can be linked to a reporter gene and used to assess the ability to modulate promoter-driven transcription.
• the present invention relates to vectors containing nucleic acid molecules that encode the above chimeric antigen receptor (CAR) of the invention.
• the present invention relates to an expression vector comprising the aforementioned nucleic acid that encodes the aforementioned chimeric antigen receptor (CAR) of the invention.
• CAR chimeric antigen receptor
• the present invention relates to a delivery vector comprising the aforementioned nucleic acid that encodes the aforementioned chimeric antigen receptor (CAR) of the invention.
• CAR chimeric antigen receptor
• the vector is selected from the group consisting of DNA, RNA, plasmid, lentiviral vector, adeno-associated viral vector, adenoviral vector, or retroviral vector.
• the vector further comprises a promoter.
• the vector is an in vitro transcribed vector.
• the nucleic acid sequence further comprises a poly (A) sequence.
• the nucleic acid sequence further comprises a 3'UTR.
• genetically modified cell means a cell into which a recombinant expression vector has been introduced.
• the present invention relates to "genetically modified cells", which may include, for example, a vector in accordance with the present invention described above. It should be understood that “genetically modified cell” and “recombinant host cell” mean not only the specific claimed cell, but also the progeny of such a cell. Since modifications may occur in subsequent generations due to mutation or environmental influences, such offspring may not actually be identical to the parent cell, but such cells are still included within the scope of the term “genetically modified cell” as used herein.
• the present invention relates to a method for producing a "genetically modified cell” that contains the above chimeric antigen receptor (CAR), comprising transforming the cell with the above vector.
• CAR chimeric antigen receptor
• an expression vector the vector can be easily inserted into a host cell, for example, a mammalian, bacterial, yeast or insect cell, by any method known in the art.
• a host cell for example, a mammalian, bacterial, yeast or insect cell
• an expression vector can be transfected into a host cell by physical, chemical, or biological means.
• SUBSTITUTE SHEET (RULE 26) Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing 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). The preferred method of introducing a polynucleotide into a host cell is by calcium phosphate transfection.
• Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors.
• Viral vectors, and especially retroviral vectors have become the most widely used method for inserting genes into mammalian cells, for example, human cells.
• Other viral vectors can be derived from lentivirus, poxviruses, herpes simplex virus I, adenoviruses and adeno-associated viruses, and the like. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.
• Chemical methods for introducing a polynucleotide into a host cell include colloidal dispersion systems such as complexes of macromolecules, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
• colloidal dispersion systems such as complexes of macromolecules, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
• An example of a colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome (eg, an artificial membrane agent).
• lipid formulations is intended to introduce nucleic acids into a host cell (in vitro, ex vivo, or in vivo).
• nucleic acid can be combined with a lipid.
• Lipid-bound nucleic acid can be encapsulated in the aqueous interior of the liposome, can be distributed within the lipid bilayer of the liposome, attached to the liposome by a linker molecule coupled to both the liposome and the oligonucleotide, embedded in the liposome, complexed with the liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, it may be contained in a suspension in a lipid, contained or complexed with a micelle, or it may otherwise be combined with a lipid.
• Compositions associated with lipids / lipids / DNA or lipids / expression vector are not limited to any particular structure in solution.
• Lipids are fatty substances that can be natural or synthetic lipids.
• lipids include fat droplets naturally occurring in the cytoplasm, as well as a class of compounds containing long chain aliphatic hydrocarbons and derivatives thereof such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.
• Suitable lipids can be obtained from commercial sources.
• DMPC dimyristyl phosphatidylcholine
• DCP dicetyl phosphate
• Choi cholesterol
• DMPG dimyristyl phosphatidylglycerol
• Stock solutions of lipids in chloroform or chloroform / methanol can be stored at approximately -20 ° C.
• Liposome is a general term that includes a variety of unilamellar and multilayer lipid agents produced by the formation of closed lipid bilayers or aggregates. Liposomes can be characterized as having a vesicular structure with a phospholipid bilayer membrane and an internal aqueous medium. Multilayer liposomes have multiple lipid layers separated by an aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution.
• Lipid components undergo self-rearrangement to form closed structures and retain water and solutes between lipid bilayers (Ghosh et al., 191 Glycobiology 5; 505-10).
• compositions having structures other than the normal vesicular structure in solution are also included.
• lipids can have a micellar structure or simply exist as heterogeneous aggregates of lipid molecules. Also includes lipofectamine-nucleic acid complexes.
• assays can be performed to confirm the presence of a recombinant DNA sequence in a host cell.
• assays include, for example, "molecular biology” assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR, and PCR; "Biochemical” assays, such as determining the presence or absence of a particular peptide, for example, by immunological methods (ELISA and Western blotting), or through the assays described herein, to determine the means included in the scope of the invention.
• the present invention relates to a genetically modified cell that contains the above chimeric antigen receptor (CAR), which is obtained by the above method.
• CAR chimeric antigen receptor
• the embodiment according to the invention also provides a genetically modified cell containing any of the recombinant expression vectors described herein.
• the term "genetically modified cell” refers to any type of cell that may contain a recombinant expression vector according to the invention.
• the genetically modified cell can be eukaryotic, for example, a cell of a plant, animal, fungus or algae, or it can be a prokaryotic cell, for example, a bacterial or protozoan cell.
• the genetically modified cell can be a cultured cell or a primary cell, i. E. isolated directly from the body, for example, humans.
• the genetically modified cell can be an adherent cell or a suspension cell, i. E. a cell that grows in suspension.
• Suitable host cells are known in the art and include, for example, E. coli DH5a cells, Chinese hamster ovary cells, VERO monkey cells, COS cells, HEK293 cells, and the like.
• the host cell can be a prokaryotic cell, for example an OH5or cell.
• the genetically modified cell can be a mammalian cell.
• the genetically modified cell can be a human cell. While a genetically modified cell can be any type of cell, can come from any type of tissue, and can be at any stage of development, a genetically modified cell can
• SUBSTITUTE SHEET (RULE 26) be a peripheral blood lymphocyte (PBL) or peripheral blood mononuclear cell (PBMC).
• the genetically modified cell can be a T cell.
• the genetically modified cell can be an NK cell.
• a T cell can be any T cell, such as a cultured T cell, for example, a primary T cell, or a T cell from a cultured T cell line, for example, Jurkat, SupTl, etc., or T cell derived from a mammal. If the T cell is obtained from a mammal, then it can be obtained from a variety of sources, including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. T cells can also be enriched or purified.
• the T cell can be a human T cell.
• the T cell can be a T cell isolated from the human body.
• the T cell can be any type of T cell and can be at any stage of development, including, but not limited to, CD4 + / CD8 + double positive T cells, CD4 + T helper cells, for example, Thi and Th2 cells, CD8 + T cells (eg, cytotoxic T cells), tumor infiltrating cells, memory T cells, naive T cells, and the like.
• the T cell can be a CD8 + T cell or a CD4 + T cell.
• T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from the site of infection, ascitic fluid, pleural effusion, spleen tissue, and tumors. In certain embodiments of the present invention, any number of 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 an individual using any number of methods known to those skilled in the art, such as ficoll separation. In one preferred embodiment, cells from the circulating blood of an individual are obtained by apheresis.
• the apheresis product contains lymphocytes, including T cells, monocytes, granulocytes, B cells, other nuclear leukocytes, erythrocytes, and platelets.
• the cells harvested by apheresis can be washed to remove a plasma fraction and to place the cells in an appropriate buffer or medium for subsequent processing steps.
• the cells are washed with phosphate buffered saline (PBS).
• the wash solution is free of calcium and may be free of magnesium, or may be free of many, if not all, divalent cations.
• the initial activation steps in the absence of calcium lead to enhanced activation.
• the washing step can be performed by methods known in the art, such as using a semi-automatic "flow-through” centrifuge (eg, Cobe 2991 cell processor, Baxter CytoMate, or Haemonetics Cell Saver 5) according to the manufacturer's instructions.
• a semi-automatic "flow-through” centrifuge eg, Cobe 2991 cell processor, Baxter CytoMate, or Haemonetics Cell Saver 5
• the cells can be resuspended in a variety of biocompatible buffers, such as, for example, Ca2 + free, Mg2 + free PBS, Plasmalyte A, or other saline with or without buffer.
• biocompatible buffers such as, for example, Ca2 + free, Mg2 + free PBS, Plasmalyte A, or other saline with or without buffer.
• the genetically modified cell is a T lymphocyte.
• the genetically modified cell is a CD8 + T cell.
• the genetically modified cell is a G cell.
• the genetically modified cell is used as a medicine.
• the genetically modified cell is used as a drug for neoplastic diseases.
• the genetically modified cell is used as a medicine for diseases where the tumor is cancer.
• the required gene segments were obtained from oligonucleotides created by chemical synthesis. Gene segments from 300 to 4000 kb in length, which are flanked by unique restriction sites, were collected by annealing and ligation of oligonucleotides, including PCR amplification and subsequent cloning through the indicated restriction sites. The DNA sequences of the subcloned gene fragments were confirmed by DNA sequencing.
• the fusion of genes containing the described antibody chains, as described below, was carried out by PCR and / or synthesis and assembly of genes using known recombination methods and procedures by linking the appropriate segments of nucleic acids, for example, using unique restriction sites in the corresponding vectors.
• the subcloned nucleotide sequences were confirmed by DNA sequencing.
• large numbers of plasmids were generated by obtaining plasmids from transformed E. coli cultures.
• DNA sequences were determined by Sanger sequencing.
• Infomax's Vector NTI Advance suite, version 8.0, and SnapGene Viewer were used to create, map, analyze, annotate and illustrate sequences.
• a fragment of three repeats of ITGB3 was collected from synthetic oligonucleotides (10 pieces, 59 bp each), as a result of two rounds of amplification, an amplicon of 308 bp was obtained.
• the unchanged scFv part (Leader-SVL-GS-linker-SVH-4-lBB CD3) was amplified, to which the specified number of ITGB3 fragments (1, 2, or 3, depending on the number of ITGB3 fragments) was sutured by splicing-PCR (SOE) on the design features of a particular vector).
• SOE splicing-PCR
• a fragment of three repeats of ITGB3 was collected from synthetic oligonucleotides (10 pieces, 59 bp each), as a result of two rounds of amplification, an amplicon of 308 bp was obtained.
• the unchanged scFv part (Leader-SVL-GS-linker-SVH-CD28-CD3) was amplified, to which the specified number of ITGB3 fragments (1, 2, or 3, depending on the number of ITGB3 fragments) was sutured by the splicing-PCR method (SOE). design features of a particular vector).
• SOE splicing-PCR method
• Synthetic 60 bp oligonucleotides were used to obtain a block of three repeating fragments of ITGB3. each, forming a completely overlapping sequence of a gene region that is part of the chimeric structure CD8 4-1BB altCD3 zeta or CD8 CD28 altCD3 zeta. The assembly of the gene was performed by a two-round PCR method.
• SUBSTITUTE SHEET (RULE 26) as a template vector pCDH-CAR-T-EGFP, with the participation of specific oligonucleotide primers, limiting the unchanged part of 19CAR-T-ITAM1.
• the splicing PCR method was used to stitch the gene regions with the ITGB3 and ITAM2 blocks.
• cassettes with the CD28 co-stimulatory domain were obtained, namely:
• the resulting cassettes were integrated into the pCDH-eGFP plasmid vector at specific restriction sites, where T2A elements and an open reading frame of green fluorescent protein (eGFP) were placed in the same reading frame as an expression marker.
• eGFP green fluorescent protein
• CAR includes the co-stimulatory domain 4-1BB and an alternative intracellular signaling domain of the chimeric antigen receptor with a different set of ITGB3 and ITAM, namely:
• CAR includes the co-stimulatory domain of CD28 and an alternative intracellular signaling domain of the chimeric antigen receptor with a different set of ITGB3 and ITAM, namely: vector pCDH-19CAR-T_CD28-ITGB3-ITGB3-eGFP (Fig. 15);
• T-lymphocytes were concentrated in 1 ml of culture medium (RPMI-1640 with L-glutamine (Biolot, 1.3.4.1) + 10% fetal calf blood serum (Gibco, 26140079) + 100U IL2
• T lymphocytes were transferred to a T75 suspension cell culture flask (Eppendorf, 0030711025) with 15 ml of culture medium.
• To activate T-lymphocytes 300 ⁇ l of Dynabeads Human T-Activator CD3 / CD28 for T Cell Expansion and Activation (Thermofisher, 11132D) were added. The culture bottle was placed for 72 hours in a C0 2 incubator (37 ° C, 5% C0 2).
• lentiviral preparations For transduction of human T-lymphocytes, two lentiviral preparations were used: the first containing genes anti-CD19-CAR-ITAMx3 and GFP (control) and the second preparation (experimental), which is selected from the group of vectors obtained in example 3 (Fig. 1, Fig. Fig. 3, Fig. 5, Fig. 7, Fig. 11, Fig. 13, Fig. 15, Fig. 17, Fig. 19, Fig. 21, Fig. 23, Fig. 25). In both preparations, the concentration of lentiviral particles (LVP / ml) was determined by ELISA. The transduction was delivered in a 15 ml bioreactor format (TPP, 87017).
• TPP 15 ml bioreactor format
• Protamine sulfate (OOO Ellara) at a concentration of 50 ⁇ g / ml was used as a transduction activator.
• 5x10 5 activated T-lymphocytes, a lentiviral preparation (in the amount of 2x10 5 LVP / cell), protamine sulfate and 10 ml of culture medium were introduced into the bioreactor, the bioreactor was left for 5 hours in a Multitron shaker (110 RPM; 37 ° C; 5% CO2) ... After 5 hours, the bioreactor was removed and centrifuged for 5 minutes at 1500 rpm, 24 ° C.
• the supernatant was removed, the pellet was resuspended in 2 ml of culture medium, transferred to a 6-well suspension culture plate (Eppendorf, 0030720016) and placed in a COr incubator (37 ° C, 5% COg) Transduction analysis was performed after 72 hours.
• the efficiency of T-lymphocyte transduction was assessed by the signal level of the reporter protein GFP; the efficiency was assessed by flow cytometry.
• the dye propidium iodide was used for the analysis of cell viability.
• the cytotoxic activity of the obtained CAR-T lymphocytes was assessed in the presence of target cells expressing the CD19 receptor by direct co-cultivation.
• a well of a 48-well plate for suspension cultures Eppendorf, 0030723015
• 1 ml of culture medium RPMI-1640 with L-glutamine (Biolot, 1.3.4.1) + 10% fetal calf blood serum (Gibco, 26140079)
• 10 000 CAR-T lymphocytes and 5,000 target cells Raji.
• the incubation time was 72 hours a C0 2 incubator (37 ° C, 5% C0 2).
• the analysis was performed on a flow cytometer, and the ratio of CD3 positive and CD3 negative cells was estimated.
• BD Cytometric Bead Array was used.
• the presence of CARs on the cell surface upon interaction with the antigen activates the NFAT signaling cascade.
• the use of the Jurkat ATCRab NFAT-GFP reporter cell line made it possible to screen the anti-CD19-CAR-ITAMx3 genetic constructs (control) and a preparation of the experimental genetic construct, which is selected from the group of vectors in FIG. 1, Fig. 3, FIG. 5, Fig. 7, Fig. 11, Fig. 13, Fig. 15, Fig. 17, Fig. 19, Fig. 21, Fig. 23, Fig. 25). Transfection of the reporter cell line was performed by electroporation (electroporator: Neon, Thermo Scientific, USA).
• Raji cells were used as target cells expressing the CD19 receptor, incubation was 6 hours in a CO2 incubator (37 ° C, 5% CO2). The analysis was performed on a flow cytometer, the intensity of the luminescence of the GFP reporter protein was estimated (Fig. 27).
• CAR-T were obtained expressing the control CAR-ITAMx3, and experimental CAR-T, which expressed one of the CAR variants (in accordance with a selected vector from the group of vectors indicated in Fig. 1, Fig. 3, Fig. 5, Fig. 7, Fig. 11, Fig. 13, Fig. 15, Fig. 17, Fig. 19, Fig. 21, Fig. 23, Fig. 25), namely:
• CAR-T In direct co-cultivation of experimental CAR-T, where CAR includes a signaling domain with only ITGB3x2 or only ITGB3x3 (“CAR- (co-stimulatory domain 4-1BB) - (signaling domain with ITGB3x2) >>;
• CAR- (co-stimulatory domain 4-1BB) - (signaling domain with ITGB3x3) CD19 + target cells of the Raji line (2: 1) showed low cytotoxic activity compared to control CAR-ITAMx3.
• CAR-ITAMx2-ITGB3xl co-stimulatory domain 4-1BB
• CAR-ITAMxl-ITGB3x2 (“CAR- (co-stimulatory domain 4-1BB) - (signaling domain with ITAMxl-ITGB3x2)”; “CAR- (co-stimulatory domain CD28) - (signaling domain with ITAMxl-

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