CN117247459A - Engineered immune cells targeting CD69 and uses thereof - Google Patents

Engineered immune cells targeting CD69 and uses thereof Download PDF

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CN117247459A
CN117247459A CN202210655239.7A CN202210655239A CN117247459A CN 117247459 A CN117247459 A CN 117247459A CN 202210655239 A CN202210655239 A CN 202210655239A CN 117247459 A CN117247459 A CN 117247459A
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李国坤
张静
孙慧芳
任江涛
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Nanjing Bioheng Biotech Co Ltd
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Nanjing Bioheng Biotech Co Ltd
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Abstract

The invention provides engineered immune cells targeting CD69 and uses thereof. Specifically, the invention provides a chimeric antigen receptor targeting CD69 and an engineering immune cell comprising the chimeric antigen receptor. The invention also provides the use of the engineered immune cells targeting CD69 in the preparation of a medicament for the treatment/prevention/diagnosis of cancer, infection or autoimmune disease.

Description

Engineered immune cells targeting CD69 and uses thereof
Technical Field
The present invention belongs to the field of immunotherapy. More specifically, the invention relates to an engineered immune cell targeting CD69 and uses thereof.
Background
Currently, CAR T therapy has achieved tremendous success in the field of hematological tumors, and as understanding of its function, mechanism, etc. continues to deepen, CAR T is also gradually breaking through in the field of solid tumors. Universal CAR T products continue to be of interest to researchers due to their potential low cost, off-the-shelf advantages, but universal CAR T always faces some challenges compared to autologous CAR T.
Among the challenges faced by the generic CAR T, poor in vivo persistence is one of the most important issues. There are generally two current solutions, one is to knock out genes in CAR-T cells that cause immune rejection in the host, such as TCR, HLA-class I genes, etc., which would improve the persistence of CAR-T cells to some extent; another approach is to purposefully eliminate or inhibit NK cells and activated T cells in a host, thereby reducing the rejection and killing of exogenous CAR-T cells by these immune cells and improving the persistence of CAR-T cells.
CD69 is a member of the C-type lectin receptor family, which is not normally expressed in resting T cells. When T cells receive a stimulation signal via a TCR, the cell surface synthesizes and expresses a number of novel glycoproteins, such as CD25, CD69, CD71, etc., with CD69 being the earliest expressed. Thus, CD69 is often a marker for early expression of activated T cells. Furthermore, as with T cells, CD69 is not expressed in NK cells in a quiescent state, but rather is expressed rapidly after NK cells are stimulated. Thus, CD69 is also a marker of NK cell activation.
Therefore, the invention provides an engineered immune cell expressing a chimeric antigen receptor targeting CD69, which has the capability of eliminating tumor cells, patient self NK cells and activated T cells, and can remarkably improve the killing function on target cells. In addition, in autoimmune diseases and Graft Versus Host Disease (GVHD) and Host Versus Graft Disease (HVGD) accompanied by organ transplantation, there are also phenomena such as T cell overactivation and abnormal activation, and the like, and CD 69-targeted engineered immune cells can be used for the treatment and control of these conditions as well.
Disclosure of Invention
The invention aims to provide an engineered immune cell targeting CD69, which can be used for killing NK cells and activated T cells of a patient in cell immunotherapy, so that the immune rejection of the patient is reduced, and the effect of cell therapy is enhanced. At the same time, it is also useful for the treatment and control of autoimmune diseases and GVHD and HVGD associated with organ transplantation.
Thus, in a first aspect, the invention provides a chimeric antigen receptor comprising an antibody that targets CD69, a transmembrane domain, and an intracellular signaling domain.
In one embodiment, the antibody of the invention is an intact antibody, fab ', F (ab') 2, fv fragment, scFv antibody fragment, linear antibody, sdAb, or nanobody.
In one embodiment, the CD 69-targeting antibody comprises CDR-H1 as shown in SEQ ID NO. 1, CDR-H2 as shown in SEQ ID NO. 2, CDR-H3 as shown in SEQ ID NO. 3, CDR-L1 as shown in SEQ ID NO. 4, CDR-L2 as shown in SEQ ID NO. 5, CDR-L3 as shown in SEQ ID NO. 6. Preferably, the CD 69-targeting antibody comprises a heavy chain variable region sequence having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID No. 7 and a light chain variable region sequence having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID No. 8. More preferably, the CD 69-targeting antibody comprises a sequence having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID No. 9, or the nucleotide sequence shown in SEQ ID No. 10.
In one embodiment, the chimeric antigen receptor further comprises an antibody that targets a second target. In a specific embodiment, the second target is selected from the group consisting of: TSHR, CD7, CD19, CD123, CD22, BAFF-R, CD, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, GPRC5D, tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B H3, KIT, IL-13Ra2, mesothelin, IL-lRa, PSCA, PRSS21, VEGFR2, lewis Y, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha ERBB2 (Her 2/neu), MUC1, EGFR, NCAM, claudin18.2, prostase, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gproo, bcr-abl, tyrosinase, ephA2, fucosyl GMl, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD 2, folate receptor beta, TEM1/CD248, TEM7R, CLDN6, GPRC5D, CXORF, CD97, CD179a, ALK, polysialic acid, PLAC1, globoH, NY-BR-1 UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, legumain, HPV E6, E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, tie 2, MAD-CT-1, MAD-CT-2, fos associated antigen 1, p53 mutant, prostate specific protein, survivin and telomerase, PCTA-l/Galectin 8, melanA/MARTL Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS 2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin Bl, MYCN, rhoC, TRP-2, CYP1B 1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79B, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, PD1, PDL2, tgfβ, APRIL, NKG2D, or any combination thereof; preferably, the second target is selected from: CD7, CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD138, CD171, MUC1, AFP, folate receptor α, CEA, PSCA, PSMA, her, EGFR, IL13Ra2, GD2, NKG2D, EGFRvIII, CS1, BCMA, mesothelin, cluadin18.2, ROR1, NY-ESO-1, MAGE-A4, or any combination thereof.
In one embodiment, the second target is CD19, which comprises CDR-H1 as shown in SEQ ID NO. 11, CDR-H2 as shown in SEQ ID NO. 12, CDR-H3 as shown in SEQ ID NO. 13, CDR-L1 as shown in SEQ ID NO. 14, CDR-L2 as shown in SEQ ID NO. 15, CDR-L3 as shown in SEQ ID NO. 16. Preferably, the CD 19-targeting antibody comprises a sequence identical to SEQ ID NO: 17. 21, 23 and a heavy chain variable region sequence having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to any one of SEQ ID NOs: 18. 22, 24, and a light chain variable region sequence having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid sequence set forth in any one of figures. More preferably, the CD19 targeting antibody comprises a sequence having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOS.19, 25-28, or the nucleotide sequence set forth in any one of SEQ ID NOS.20, 29-32.
In one embodiment, the antibody targeting CD69 and the antibody targeting the second target in the chimeric antigen receptor are in tandem or parallel.
In one embodiment, the chimeric antigen receptor comprises: (1) An antigen binding region comprising an antibody that targets CD69 and an antibody that targets a second target; (2) a transmembrane domain; and (3) an intracellular signaling domain. In another embodiment, the chimeric antigen receptor of the invention comprises: (1) A first unit structure targeting CD69 comprising an antibody that targets CD69, a transmembrane domain, and an intracellular signaling domain; and (2) a second unit structure that targets a second target, comprising an antibody that targets the second target, a transmembrane domain, and an intracellular signaling domain.
In one embodiment, the intracellular signaling domain comprises a costimulatory domain and/or a primary signaling domain.
In one embodiment, the costimulatory domain is selected from the costimulatory signaling domains of the following proteins: LTB, CD94, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18, CD27, CD28, CD30, CD40, CD54, CD83, CD134 (OX 40), CD137 (4-1 BB), CD270 (HVEM), CD272 (BTLA), CD276 (B7-H3), CD278 (ICOS), CD357 (GITR), DAP10, DAP12, LAT, NKG2C, SLP, PD-1, LIGHT, TRIM, ZAP, or any combination thereof. Preferably, the co-stimulatory domain is selected from 4-1BB, CD28, CD27, OX40, CD278 or any combination thereof.
In one embodiment, the primary signaling domain is selected from the signaling domains of the following proteins: fcrγ, fcrβ, cd3γ, cd3δ, cd3ε, cd3ζ, CD22, CD79a, CD79b, CD66d, or any combination thereof. Preferably, the primary signaling domain comprises a signaling domain of cd3ζ.
In one embodiment, the transmembrane domain is selected from the transmembrane domains of the following proteins: TCR alpha chain, TCR beta chain, TCR gamma chain, TCR delta chain, cd3ζ subunit, cd3ε subunit, cd3γ subunit, cd3δ subunit, CD45, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or any combination thereof. Preferably, the transmembrane domain is selected from the group consisting of the transmembrane domain of CD8 a, CD4, CD28 or CD 278.
In a second aspect, the invention provides nucleic acid molecules encoding the chimeric antigen receptors described above, as well as vectors comprising the nucleic acid molecules.
In a third aspect, the invention provides an engineered immune cell comprising the above nucleic acid molecule or vector, or expressing the above chimeric antigen receptor.
In one embodiment, the immune cells are T cells, B cells, macrophages, dendritic cells, monocytes, NK cells, NKT cells, and/or the like. Preferably, the immune cells are T cells, NK cells or NKT cells. More preferably, the T cell is CD4 + CD8 + T cells, CD4 + T cells, CD8 + T cells, CD4 - CD8 - T cells, tumor infiltrating cells, memory T cells, naive T cells, γδ -T cells and/or αβ -T cells.
In one embodiment, the expression of at least one MHC-related gene is inhibited or silenced in the engineered immune cell. The MHC related gene is selected from HLA-A, HLA-B, HLA-C, B2M, HLA-DPA, HLA-DQ, HLA-DRA, TAP1, TAP2, LMP7, RFX5, RFXAP, RFXANK, CIITA or any combination thereof, preferably HLA-A, HLA-B, HLA-C, B2M, RFX, RFXAP, RFXANK, CIITA or any combination thereof.
In one embodiment, the expression of at least one TCR/CD3 associated gene is inhibited or silenced in the engineered immune cell. The TCR/CD3 gene is selected from TRAC, TRBC, CD3 gamma, cd3 delta, cd3 epsilon, cd3ζ, or any combination thereof.
In a preferred embodiment, the expression of at least one TCR/CD3 gene and at least one MHC-related gene of the engineered immune cell is inhibited or silenced, wherein the at least one TCR/CD3 gene is selected from TRAC, TRBC or any combination thereof, and the at least one MHC-related gene is B2M, RFX, RFXAP, RFXANK, CIITA or any combination thereof. In one embodiment, the expression of TRAC or TRBC, and B2M of the engineered immune cell is inhibited or silenced. In one embodiment, the expression of TRAC or TRBC, and CIITA, of the engineered immune cell is inhibited or silenced. In a preferred embodiment, the expression of TRAC or TRBC, B2M and CIITA of the engineered immune cells is inhibited or silenced. In a preferred embodiment, the expression of TRAC or TRBC, B2M and RFX5 of the engineered immune cells is inhibited or silenced.
In one embodiment, expression of one or more of the following genes is also inhibited or silenced in the engineered immune cell: CD52, GR, dCK and immune checkpoint genes such as PD1, LAG3, TIM3, CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, TNFRSF10B, TNFRSF10A, CASP, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL ST, EIF2AK4, CSK, PAG1, SIT, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2 and GUCY1B3. Preferably, the engineered immune cell is inhibited or silenced with CD52, dCK, PD1, LAG3, TIM3, CTLA4, TIGIT, or any combination thereof.
In one embodiment, the engineered immune cell also expresses a cell suicide element. Preferably, the cell suicide element is located at the N-terminus or C-terminus of the chimeric antigen receptor, and the self-cleaving element comprises a 2A peptide or an IRES peptide, preferably P2A and T2A. More preferably, the cell suicide element is selected from the group consisting of: HSV-TK, iCasp9, ΔCD20, mTMPK, ΔCD19, RQR8, EGFRt, or any combination thereof.
In a fourth aspect, the invention provides a pharmaceutical composition comprising an engineered immune cell of the invention as described above and one or more pharmaceutically acceptable excipients.
In a fifth aspect, the present invention provides the use of the chimeric antigen receptor or the engineered immune cell or the pharmaceutical composition described above in the manufacture of a medicament for the treatment/prevention/diagnosis of cancer, infection or autoimmune disease, in the manufacture of a medicament for the treatment/alleviation/elimination of GVHD or HVGD.
In one embodiment, the cancer is selected from: brain glioma, blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and CNS cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colon and rectal cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer, glioblastoma (GBM), liver cancer, hepatoma, intraepithelial tumors, renal cancer, laryngeal cancer, liver tumors, lung cancer, lymphoma, melanoma, myeloma, neuroblastoma, oral cancer, ovarian cancer, pancreatic cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, rectal cancer, cancer of the respiratory system, salivary gland cancer, skin cancer, squamous cell cancer, gastric cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, malignant tumors of the urinary system vulvar cancer and other carcinomas and sarcomas, and B-cell lymphomas, mantle cell lymphomas, AIDS-related lymphomas, and Waldenstrom's macroglobulinemia, chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), B-cell prolymphocytic leukemia, blast plasmacytoid dendritic cell tumor, burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, chronic Myelogenous Leukemia (CML), malignant lymphoproliferative diseases, MALT lymphomas, hairy cell leukemia, marginal zone lymphomas, multiple myeloma, myelodysplasia, plasmablastoid lymphomas, pre-leukemia, plasmacytoid dendritic cell tumors, post-transplant lymphoproliferative disorder (PTLD).
Detailed Description
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
Chimeric antigen receptor
As used herein, the term "chimeric antigen receptor" refers to an artificially constructed hybrid polypeptide whose basic structure comprises an antigen binding domain (e.g., an antigen binding portion of an antibody), a transmembrane domain, and an intracellular signaling domain. Chimeric antigen receptors are capable of redirecting the specificity and reactivity of T cells and other immune cells to a selected target in a non-MHC-restricted manner using the antigen binding properties of the antigen binding domain. non-MHC-restricted antigen recognition gives T cells expressing chimeric antigen receptors the ability to recognize antigen independent of antigen processing, thus bypassing the primary mechanism of tumor escape.
The invention provides a chimeric antigen receptor comprising an antibody that targets CD69, a transmembrane domain, and an intracellular signaling domain. In one embodiment, the antibody described in the present invention is an intact antibody, fab ', F (ab') 2, fv fragment, scFv antibody fragment, linear antibody, sdAb, or nanobody.
In one embodiment, the CD 69-targeting antibody comprises CDR-H1 as shown in SEQ ID NO. 1, CDR-H2 as shown in SEQ ID NO. 2, CDR-H3 as shown in SEQ ID NO. 3, CDR-L1 as shown in SEQ ID NO. 4, CDR-L2 as shown in SEQ ID NO. 5, CDR-L3 as shown in SEQ ID NO. 6. Preferably, the CD 69-targeting antibody comprises a heavy chain variable region sequence having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID No. 7 and a light chain variable region sequence having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID No. 8. More preferably, the CD 69-targeting antibody comprises a sequence having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID No. 9, or the nucleotide sequence shown in SEQ ID No. 10.
As used herein, the term "antibody" has the broadest sense as understood by those skilled in the art and includes monoclonal antibodies (including intact antibodies), polyclonal antibodies, multivalent antibodies, and antibody fragments or synthetic polypeptides carrying one or more CDR sequences that are capable of exhibiting the desired biological activity. The antibodies of the invention may be of any class (e.g., igG, igE, igM, igD, igA, etc.) or subclass (e.g., igG1, igG2a, igG3, igG4, igA1, igA2, etc.). The antibodies of the invention also include recombinant antibodies, human antibodies, humanized antibodies, camelid antibodies, murine antibodies, chimeric antibodies, and antigen binding portions thereof.
The term "functional variant" or "functional fragment" refers to a variant that substantially comprises the amino acid sequence of a parent but that contains at least one amino acid modification (i.e., substitution, deletion, or insertion) as compared to the parent amino acid sequence, provided that the variant retains the biological activity of the parent amino acid sequence. In one embodiment, the amino acid modification is preferably a conservative modification.
As used herein, the term "conservative modification" refers to an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody or antibody fragment containing the amino acid sequence. These conservative modifications include amino acid substitutions, additions and deletions. Modifications may be introduced into the chimeric antigen receptor or Fc fusion polypeptide of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Conservative modifications may be chosen, for example, based on polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the similarity of the amphipathic nature of the residues involved.
Thus, a "functional variant" or "functional fragment" has at least 75%, preferably at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a parent amino acid sequence and retains biological activity, e.g., binding activity, of the parent amino acid.
As used herein, the term "sequence identity" refers to the degree to which two (nucleotide or amino acid) sequences have identical residues at identical positions in an alignment, and is typically expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Thus, two copies with identical sequences have 100% identity.
In another embodiment, the chimeric antigen receptor further comprises an antibody that targets a second target. In one embodiment, the antibody that targets CD69 and the antibody that targets the second target are in tandem or parallel. In this context, "tandem" refers to the attachment of an antibody targeting CD69 and an antibody targeting a second target through a linker into the antigen binding region of a chimeric antigen receptor and sharing the same transmembrane domain and intracellular signaling domain. In other words, in the case of "tandem", the chimeric antigen receptor of the present invention comprises: (1) An antigen binding region comprising an antibody that targets CD69 and an antibody that targets a second target; (2) a transmembrane domain; and (3) an intracellular signaling domain. Those skilled in the art will appreciate that in tandem format, the antibody targeting CD69 and the antibody targeting the second target may be linked in any order by a linker. Also, when both antibodies are scFv, they may also be linked (from N-terminus to C-terminus) in the following order, for example: VL1-VL 2-linker-VH 2-VH1, VL2-VL 1-linker-VH 1-VH2, VH1-VH 2-linker-VL 2-VL1, VH12-VH 1-linker-VL 1-VL2. In this context, "parallel" means that the CD 69-targeting antibody and the second target-targeting antibody are located in two different unit structures, each linked to a transmembrane domain and an intracellular signaling domain, respectively, which may be located in the same vector (e.g., by linking the two unit structures via a 2A peptide so that they are expressed separately), or in different vectors (e.g., each vector comprises one CD 69-or second target-targeting unit structure, and then the two vectors are introduced together into an immune cell). In other words, in the case of "parallel", the chimeric antigen receptor of the present invention comprises: (1) A first unit structure targeting CD69 comprising an antibody that targets CD69, a transmembrane domain, and an intracellular signaling domain; and (2) a second unit structure that targets a second target, comprising an antibody that targets the second target, a transmembrane domain, and an intracellular signaling domain. Optionally, the first unit structure and the second unit structure are located on the same carrier or on different carriers.
In one embodiment, the second target of the invention is selected from the group consisting of: CD2, CD3, CD4, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD30, CD33, CD37, CD38, CD40L, CD44, CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD126, CD138, CD171, CD 179a, DR4, DR5, TAC, TEM1/CD248, VEGF, GUCY2C, EGP40, EGP-2, EGP-4, CD133, IFNAR1, DLL3, kappa light chain, TIM3, TSHR, CD19, BAFF-R, CLL-1, EGFRvIII, tEGFR, GD2, GD3, BCMA, tn antigen, PSMA, ROR1, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B H3, KIT, IL-13Ra2, IL-llRa, IL-22Ra, IL-2, mesothelin, PSCA, PRSS21, VEGFR2, lewis Y, PDGFR-beta, SSEA-4, AFP, folate receptor alpha, erbB2 (Her 2/neu), erbB3, erbB4, MUC1, MUC16, EGFR, CS1, NCAM, claudin18.2, c-Met, prostase, PAP, ELF M, ephrin B2, IGF-I receptor, CAIX, LMP2, gpl00, bcr-abl, tyrosinase, ephA2, fucosyl GMl, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD 2, folate receptor beta, TEM7R, CLDN6, GPRC5D, CXORF61, ALK, polysialic acid, PLAC1, globoH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, MAGE-A3, MAGE-A6, legumain, HPV E6, E7, ETV6-AML, sperm protein 17, XAGE1, tie 2, MAD-CT-1, MAD-CT-2, fos associated antigen 1, p53 mutant, PSA, survivin and telomerase, PCTA-l/Galectin 8, melanA/MARTl, ras mutant, hTERT, sarcoma breakpoint, ML-IAP, TMPRSS2 ETS fusion gene, NA17, PAX3, androgen receptor, progesterone receptor, cyclin Bl, MYCN, rhoC, TRP-2, CYP1B 1, BORIS, SART3, PAX5, OY-TES 1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79B, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST, EMR2, LY75, GPC3, FCRL5, IGLL1, PD1, PDL2, TGF beta, APRIL, NKG2D, NKG D ligand, and/or pathogen-specific antigen, biotinylated molecule, molecules expressed by HIV, HCV, HBV and/or other pathogens; and/or neoepitopes or neoantigens. Preferably, the second target is selected from the group consisting of CD7, CD19, CD20, CD22, CD30, CD33, CD38, CD123, CD138, CD171, MUC1, AFP, folate receptor α, CEA, PSCA, PSMA, her2, EGFR, IL13Ra2, GD2, NKG2D, claudin18.2, ROR1, egfrvlll, CS1, BCMA, GPRC5D and mesothelin, more preferably from the group consisting of CD19, claudin18.2 and BCMA.
In one embodiment, the second target is CD19. Preferably, the CD 19-targeting antibody comprises a CDR-H1 as shown in SEQ ID NO. 11, a CDR-H2 as shown in SEQ ID NO. 12, a CDR-H3 as shown in SEQ ID NO. 13, a CDR-L1 as shown in SEQ ID NO. 14, a CDR-L2 as shown in SEQ ID NO. 15, a CDR-L3 as shown in SEQ ID NO. 16. Preferably, the CD 19-targeting antibody comprises a sequence identical to SEQ ID NO: 17. 21, 23 and a heavy chain variable region sequence having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to any one of SEQ ID NOs: 18. 22, 24, and a light chain variable region sequence having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid sequence set forth in any one of figures. More preferably, the CD19 targeting antibody comprises a sequence having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid sequence set forth in any one of SEQ ID NOS.19, 25-28, or the nucleotide sequence set forth in any one of SEQ ID NOS.20, 29-32.
As used herein, the term "transmembrane domain" refers to a polypeptide structure that enables the expression of a chimeric antigen receptor on the surface of an immune cell (e.g., a lymphocyte, NK cell, or NKT cell) and directs the cellular response of the immune cell against a target cell. The transmembrane domain may be natural or synthetic, and may be derived from any membrane-bound or transmembrane protein. The transmembrane domain is capable of signaling when the chimeric receptor polypeptide binds to a target antigen. Particularly suitable transmembrane domains for use in the present invention may be derived from, for example, a TCR alpha chain, a TCR beta chain, a TCR gamma chain, a TCR delta chain, a cd3ζ subunit, a cd3ε subunit, a cd3γ subunit, a cd3δ subunit, CD45, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or any combination thereof. Preferably, the transmembrane domain is selected from the group consisting of the transmembrane domain of CD8 a, CD4, CD28 or CD 278. More preferably, the transmembrane domain is a CD 8. Alpha. Transmembrane domain having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO. 39, or to SEQ ID NO. 40 or 41.
In one embodiment, the chimeric antigen receptor of the invention can further comprise a hinge region between the antibody and the transmembrane domain. As used herein, the term "hinge region" generally refers to any oligopeptide or polypeptide that functions to connect a transmembrane domain to an antigen binding region. In particular, the hinge region serves to provide greater flexibility and accessibility to the antibody. The hinge region may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. The hinge region may be derived from all or part of a natural molecule, such as from all or part of the extracellular region of CD8, CD4 or CD28, or from all or part of the antibody constant region. Alternatively, the hinge region may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or may be a fully synthetic hinge sequence. Preferably, the hinge region comprises a portion of the hinge region of a CD8 alpha chain having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO. 45, or to SEQ ID NO. 46 or 47.
As used herein, the term "intracellular signaling domain" refers to a portion of a protein that transduces an effector function signal and directs a cell to perform a specified function, comprising a co-stimulatory domain and/or a primary signaling domain. Intracellular signaling domains are responsible for intracellular signaling after antigen binding by the antigen binding region, leading to activation of immune cells and immune responses. In other words, the intracellular signaling domain is responsible for activating at least one of the normal effector functions of immune cells in which the chimeric antigen receptor is expressed. For example, the effector function of a T cell may be cytolytic activity or helper activity, including secretion of cytokines.
In one embodiment, the primary signaling domain comprised by the chimeric antigen receptor of the invention may be cytoplasmic sequences of T cell receptor and co-receptor that function together to elicit signaling upon antigen receptor binding, as well as any derivatives or variants of these sequences and any synthetic sequences with the same or similar functions. The primary signaling domain comprises two different types of cytoplasmic signal sequences: those that elicit antigen-dependent primary activation, and those that act in an antigen-independent manner to provide a secondary or co-stimulatory signal. The primary cytoplasmic signal sequence can contain a number of immune receptor tyrosine activation motifs (ITAMs). The primary signaling domains of the invention include, but are not limited to, those derived from fcrγ, fcrβ, cd3γ, cd3δ, cd3ε, cd3ζ, CD22, CD79a, CD79b, CD66d, or any combination thereof. Preferably, the primary signaling domain comprises a signaling domain of cd3ζ. More preferably, the primary signalling domain has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid shown in SEQ ID NO. 36, or to the sequence of SEQ ID NO. 37 or 38.
In one embodiment, the chimeric antigen receptor of the invention further comprises one or more co-stimulatory domains. The co-stimulatory domain may be an intracellular functional signaling domain from a co-stimulatory molecule, which may comprise the whole intracellular portion of the co-stimulatory molecule, or a functional fragment thereof. "costimulatory molecule" refers to a cognate binding partner that specifically binds to a costimulatory ligand on a T cell, thereby mediating a costimulatory response (e.g., proliferation) of the T cell. Costimulatory molecules include, but are not limited to, MHC class 1 molecules, BTLA and Toll ligand receptors. Non-limiting examples of costimulatory domains of the present invention include, but are not limited to, costimulatory signaling domains derived from the following proteins: TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18, CD27, CD28, CD30, CD40, CD54, CD83, CD134 (OX 40), CD137 (4-1 BB), CD270 (HVEM), CD272 (BTLA), CD276 (B7-H3), CD278 (ICOS), CD357 (GITR), DAP10, DAP12, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM, ZAP, or any combination thereof. Preferably, the co-stimulatory domain is selected from 4-1BB, CD28, CD27, OX40, CD278 or any combination thereof. More preferably, the costimulatory domain is 4-1BB, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO. 33, or to the amino acid sequence shown in SEQ ID NO. 34 or 35.
In some embodiments, the CAR of the invention further comprises a signal peptide selected from the group consisting of signal peptides of: CD8 alpha, igG1, GM-CSFR alpha, igG4, or any combination thereof. Preferably, the signal peptide comprises a signal peptide of CD8 a. More preferably, the signal peptide has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID NO. 42, or to the nucleotide sequence shown in SEQ ID NO. 43 or 44.
In some embodiments, the CARs of the invention further comprise a linker for spacing any domain/region described herein. For example, the linker can be located between the signal peptide and the antibody, between the VH and VL of the antibody, between the antibody and the hinge region, between the hinge region and the transmembrane domain, flanking or on the N-or C-region of the costimulatory domain, and/or between the transmembrane domain and the primary signaling domain. The linker may be a peptide of about 6 to about 40 amino acids in length, or about 6 to about 25 amino acids in length. Linker sequences commonly used in the art include, for example, SEQ ID NO. 48 or SEQ ID NO. 49.
Engineered immune cells and methods of making same
The present invention provides an engineered immune cell comprising the chimeric antigen receptor described above or a nucleic acid molecule encoding the same.
The present invention also provides a composition comprising: (1) A first population of engineered immune cells expressing a first structural unit comprising an antibody targeting CD69, a transmembrane domain, and an intracellular signaling domain; and (2) a second population of engineered immune cells expressing a second unit structure comprising an antibody that targets a second target, a transmembrane domain, and an intracellular signaling domain. In this embodiment, the first engineered immune cell population and the second engineered immune cell population may be administered to the subject simultaneously or sequentially.
As used herein, the term "immune cell" refers to any cell of the immune system that has one or more effector functions (e.g., cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and/or CDC). For example, the immune cells may be T cells, macrophages, dendritic cells, monocytes, NK cells and/or NKT cells, or immune cells derived from stem cells, such as adult stem cells, embryonic stem cells, cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells, hematopoietic stem cells, or the like. Preferably, the immune cells are T cells. The T cell may be any T cell, such as an in vitro cultured T cell, e.g. a primary T cell, or a T cell from an in vitro cultured T cell line, e.g. Jurkat, supT1, etc., or a T cell obtained from a subject. Examples of subjects include humans, dogs, cats, mice, rats and transgenic species thereof. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, and from an infected site Tissue in place, ascites, pleural effusion, spleen tissue and tumors. T cells may also be concentrated or purified. The T cells may be any type of T cell and may be at any stage of development, including but not limited to CD4 + CD8 + T cells, CD4 + T cells (e.g., th1 and Th2 cells), cd8+ T cells (e.g., cytotoxic T cells), CD4 - CD8 - T cells, tumor infiltrating cells, memory T cells, naive T cells, γδ -T cells, αβ -T cells, and the like. In a preferred embodiment, the immune cell is a human T cell. T cells can be obtained from the blood of a subject using a variety of techniques known to those skilled in the art, such as Ficoll isolation. In the present invention, immune cells are engineered to express chimeric antigen receptors and Fc fusion polypeptides.
The nucleic acid molecule sequence encoding the chimeric antigen receptor of the invention can be introduced into immune cells using conventional methods known in the art (e.g., by transduction, transfection, transformation, etc.) to express the chimeric antigen receptor.
In one embodiment, the first unit structure targeting CD69 is located on the same vector as the second unit structure targeting the second target, e.g., by 2A peptide ligation, such that the two unit structures can be expressed independently without affecting each other. As used herein, the term "2A peptide" is a cis-hydrolase acting element (chisels), originally found in Foot and Mouth Disease Virus (FMDV). The average length of the 2A peptide is 18-22 amino acids. At the time of protein translation, the 2A peptide can be cleaved from its last 2 amino acid C-terminal end by ribosome jump. Specifically, the peptide chain association between glycine and proline is impaired at the 2A site, and can initiate ribosome jump to begin translation from codon 2, thereby allowing independent expression of 2 proteins in 1 transcriptional unit. This 2A peptide-mediated cleavage is widely present in eukaryotic animal cells. The higher cleavage efficiency of the 2A peptide and the capability of promoting the balanced expression of upstream and downstream genes can be utilized to improve the expression efficiency of the heteromultimeric protein (such as cell surface receptor, cytokine, immunoglobulin and the like). Common 2A peptides include, but are not limited to, P2A, T2A, E2A, F a, and the like.
As used herein, the term "vector" is a nucleic acid molecule that serves as a mediator for the transfer of (foreign) genetic material into an immune cell, where it may for example be replicated and/or expressed.
In one embodiment, vectors of the invention include, but are not limited to, linear nucleic acid molecules (e.g., DNA or RNA), plasmids, viruses (e.g., retroviruses, lentiviruses, adenoviruses, vaccinia viruses, rous sarcoma viruses (RSV, polyomaviruses, adeno-associated viruses (AAV), etc.), phages, phagemids, cosmids, and artificial chromosomes (including BACs and YACs). The vectors themselves are typically nucleotide sequences, typically DNA sequences comprising an insert (transgene) and larger sequences that serve as the "backbone" of the vector.
In one embodiment, the engineered immune cells of the invention further comprise inhibition or silencing of expression of at least one MHC-associated gene, e.g., inhibition or silencing of expression of at least one MHC gene, or inhibition or silencing of expression of a gene that interacts with or modulates expression of at least one MHC gene.
In one embodiment, inhibition or silencing of MHC-related gene expression refers to inhibiting or silencing expression of one or more genes selected from the group consisting of: HLA-A, HLA-B, HLA-C, B2M, HLA-DPA, HLA-DQ, HLA-DRA, TAP1, TAP2, LMP7, RFX5, RFXAP, RFXANK, CIITA or any combination thereof, preferably selected from HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, CIITA or any combination thereof.
In one embodiment, the engineered immune cells of the invention further comprise inhibition or silencing of expression of at least one TCR/CD3 gene.
In one embodiment, inhibiting or silencing expression of at least one TCR/CD3 gene refers to inhibiting or silencing expression of one or more genes selected from the group consisting of: TRAC, TRBC, CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta.
In one embodiment, in addition to the MHC-related gene and optionally the TCR/CD3 gene, the engineered immune cells of the invention may further comprise inhibited or silenced expression of at least one gene selected from the group consisting of: CD52, GR, dCK and immune checkpoint genes such as PD1, LAG3, TIM3, CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, TNFRSF10B, TNFRSF10A, CASP, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL ST, EIF2AK4, CSK, PAG1, SIT, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2 and GUCY1B3.
In one embodiment, the engineered immune cell also expresses a cell suicide element. Preferably, the cell suicide element is located at the N-terminus or C-terminus of the CAR, and the self-cleaving element comprises a 2A peptide or an IRES peptide, preferably P2A and T2A. More preferably, the cell suicide element is selected from the group consisting of: HSV-TK, iCasp9, ΔCD20, mTMPK, ΔCD19, RQR8, EGFRt, or any combination thereof.
Methods of inhibiting gene expression or silencing genes are well known to those skilled in the art and include, but are not limited to, inactivation of genes, for example, by meganucleases, zinc finger nucleases, TALE nucleases or Cas enzymes in CRISPR systems, mediated DNA breaks, or by antisense oligonucleotides, RNAi, shRNA, etc.
Pharmaceutical compositions and kits
The invention also provides a pharmaceutical composition comprising the engineered immune cell of the invention as an active agent, and one or more pharmaceutically acceptable excipients. Thus, the invention also covers the use of said engineered immune cells in the preparation of a pharmaceutical composition or medicament.
As used herein, the term "pharmaceutically acceptable excipient" refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and active ingredient (i.e., capable of eliciting a desired therapeutic effect without causing any undesired local or systemic effects). Examples of pharmaceutically acceptable excipients include, but are not limited to, fillers, binders, disintegrants, coatings, adsorbents, anti-adherent agents, glidants, antioxidants, flavoring agents, colorants, sweeteners, solvents, co-solvents, buffers, chelating agents, surfactants, diluents, wetting agents, preservatives, emulsifiers, coating agents, isotonicity agents, absorption delaying agents, stabilizers, and tonicity adjusting agents. The selection of suitable excipients is known to those skilled in the art to prepare the desired pharmaceutical compositions of the present invention. Exemplary excipients for use in the pharmaceutical compositions of the present invention include saline, buffered saline, dextrose, and water. In general, the choice of suitable excipients depends inter alia on the active agent used, the disease to be treated and the desired dosage form of the pharmaceutical composition.
The pharmaceutical composition according to the present invention may be suitable for administration by a variety of routes. Typically, administration is accomplished parenterally. Parenteral delivery methods include topical, intra-arterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual, or intranasal administration.
The pharmaceutical compositions according to the invention can also be prepared in various forms, such as solid, liquid, gaseous or lyophilized forms, in particular in the form of ointments, creams, transdermal patches, gels, powders, tablets, solutions, aerosols, granules, pills, suspensions, emulsions, capsules, syrups, elixirs, extracts, tinctures or fluid extract, or in a form which is particularly suitable for the desired method of administration. Processes known in the present invention for producing a medicament may include, for example, conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical compositions comprising immune cells such as described herein are typically provided in solution, and preferably comprise a pharmaceutically acceptable buffer.
The pharmaceutical composition according to the invention may also be administered in combination with one or more other agents suitable for the treatment and/or prevention of the disease to be treated. Preferred examples of agents suitable for use in combination include known anticancer drugs such as cisplatin, maytansine derivatives, rapamycin (rachelmycin), calicheamicin (calicheamicin), docetaxel, etoposide, gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium porphyrin II (sorfimer sodiumphotofrin II), temozolomide, topotecan, glucuronic acid Qu Meisha t (trimetreate glucuronate), orlistat E (auristatin E), vincristine and doxorubicin; peptide cytotoxins, such as ricin, diphtheria toxin, pseudomonas bacterial exotoxin A, DNA enzyme and rnase; radionuclides such as iodine 131, rhenium 186, indium 111, iridium 90, bismuth 210 and 213, actinium 225, and astatine 213; prodrugs, such as antibody directed enzyme prodrugs; immunostimulants such as IL-2, chemokines such as IL-8, platelet factor 4, melanoma growth stimulatory proteins, and the like; antibodies or fragments thereof, such as anti-CD 3 antibodies or fragments thereof, complement activators, heterologous protein domains, homoprotein domains, viral/bacterial protein domains and viral/bacterial peptides.
Use of the same
The invention also provides application of the chimeric antigen receptor or the engineered immune cell or the pharmaceutical composition in preparing medicines for treating/preventing/diagnosing cancers, infections or autoimmune diseases. The invention also provides the use of the chimeric antigen receptor or the engineered immune cell or the pharmaceutical composition in treating/alleviating GVHD or HVGD.
In one embodiment, the disease is a cancer associated with CD69 or second target expression. For example, the cancers include, but are not limited to: brain glioma, blastoma, sarcoma, leukemia, basal cell carcinoma, biliary tract cancer, bladder cancer, bone cancer, brain and CNS cancer, breast cancer, peritoneal cancer, cervical cancer, choriocarcinoma, colon and rectal cancer, connective tissue cancer, cancer of the digestive system, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer (including gastrointestinal cancer), glioblastoma (GBM), liver cancer, hepatoma, intraepithelial tumors, renal cancer, laryngeal cancer, liver tumors, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, adenoid lung cancer and squamous lung cancer), lymphomas (including Hodgkin's lymphoma and non-Hodgkin's lymphoma), melanomas, myelomas, neuroblastomas, oral cancer (e.g., lip, tongue, mouth and throat), ovarian cancer, pancreatic cancer, prostate cancer retinoblastoma, rhabdomyosarcoma, rectal cancer, cancers of the respiratory system, salivary gland carcinoma, skin cancer, squamous cell carcinoma, gastric cancer, testicular cancer, thyroid cancer, uterine or endometrial cancer, malignant tumors of the urinary system, vulvar cancer, and other carcinomas and sarcomas, as well as B-cell lymphomas (including low-grade/follicular non-hodgkin lymphoma (NHL), small Lymphocytic (SL) NHL, intermediate-grade/follicular NHL, intermediate-grade diffuse NHL, higher immunocytogenic NHL, higher lymphoblastic NHL, higher small-grade non-cracked cellular NHL, large-tumor mass NHL), mantle cell lymphoma, AIDS-related lymphoma, and Waldenstrom's macroglobulinemia, chronic Lymphocytic Leukemia (CLL), acute Lymphoblastic Leukemia (ALL), B-cell acute lymphoblastic leukemia (B-ALL), T-cell acute lymphoblastic leukemia (T-ALL), B-cell prolymphocytic leukemia, blast plasmacytoid dendritic cell tumor, burkitt's lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, chronic Myelogenous Leukemia (CML), malignant lymphoproliferative disease, MALT lymphoma, hairy cell leukemia, marginal zone lymphoma, multiple myeloma, myelodysplasia, plasmablasts, pre-leukemia, plasmacytoid dendritic cell tumor, and post-transplant lymphoproliferative disorder (PTLD); and other diseases associated with target expression. Preferably, the disease that can be treated with the engineered immune cells or pharmaceutical compositions of the invention is selected from: leukemia, lymphoma, multiple myeloma, brain glioma, pancreatic cancer, gastric cancer, etc.
The invention will be described in detail below with reference to the drawings in connection with embodiments. It should be noted that, those skilled in the art should understand that the drawings and the embodiments of the present invention are only for illustration, and should not limit the present invention in any way. The embodiments and features of the embodiments in this application may be combined with each other without contradiction.
Drawings
FIG. 1 shows the expression of CD69 and CD19 in Jurkat, nalm6, K562 tumor cell lines, with unbraain as a blank control and isotype as a isotype control.
Figure 2 shows the overall expression levels of single chain antibodies in different CAR T cells.
Figure 3 shows the in vitro killing of Jurkat (a), nalm6 (B), K562 (C) tumor cells by different CAR T cells.
Fig. 4 shows the release levels of cytokines IL2 (a) and ifnγ (B) after co-culture of different CAR T cells with target cells.
Detailed Description
Example 1 tumor cell line validation
CD69 expression in Jurkat, nalm6, K562 cell lines was detected by flow cytometry using the antibody PE anti-human CD69 anti-body (bioleged, cat. 310906), respectively. CD19 expression in Jurkat, nalm6, K562 cell lines was detected by flow cytometry using the antibody APC anti-human CD19 anti-body (bioleged, cat. 302212), respectively. The results are shown in FIG. 1. It can be seen that Jurkat expresses CD69 but does not express CD19 and thus serves as a CD69 positive target cell; nalm6 does not express CD69 but expresses CD19 and thus serves as a CD19 positive target cell; k562 expresses little CD69 and no CD19 and thus serves as a non-target cell.
Example 2 preparation of CD 69-targeting CAR T cells and verification of their function
2.1 preparation of CAR T cells
Genes encoding the following proteins were synthesized separately: CD 8. Alpha. Signal peptide (SEQ ID NO: 42), CD69 scFv (SEQ ID NO: 9), CD19 scFv (SEQ ID NO: 19), CD 8. Alpha. Hinge (SEQ ID NO: 45), CD 8. Alpha. Transmembrane region (SEQ ID NO: 42), 4-1BB co-stimulatory domain (SEQ ID NO: 33), CD3 zeta signaling domain (SEQ ID NO: 36), linker (SEQ ID NO: 49), T2A (SEQ ID NO: 50) were synthesized according to the CAR structure given in Table 1.
This was cloned into pLVX vector (Public Protein/Plasmid Library (PPL), cat# PPL00157-4 a) and CD69 CAR, dual CAR, bi CAR and CD19 CAR plasmids were constructed, respectively. The correct insertion of the target sequence was confirmed by sequencing.
TABLE 1 Structure in different CAR plasmids
The plasmids are packed into lentiviruses, and activated T cells are further transfected, so that corresponding CAR T cells are obtained.
At 37℃and 5% CO 2 After 10 days of incubation, the expression level of single-chain antibodies in CAR T cells was detected by flow cytometry using FITC-Rabbit anti-mouse IgG, F (ab') specific (jackson immunoresearch, cat. No. 315-095-006), and the results are shown in FIG. 2. It can be seen that the single-chain antibody in the CAR T cells prepared according to the present invention can be expressed normally.
2.2 detection of the killing effect of CAR T cells on target cells
At 1X 10 4 Concentration of individual cells/well target cells Jurkat, nalm6 or non-target cells K562 were plated in 96 well plates, then NT cells and each CAR T cell were plated in 96 well plates at 16:1, 8:1, 4:1, 2:1 effective target ratios for co-cultivation, and fluorescence values were determined using an enzyme-labeled instrument after 8 hours. According to the calculation formula: (mean fluorescence of target cells-mean fluorescence of sample)/mean fluorescence of target cells×100%, and the killing efficiency was calculated, and the result is shown in fig. 3.
It can be seen that at various effective target ratios, the CD69 CAR T, dual CAR T, bi CAR T cells of the invention had a strong killing effect on Jurkat cells, and their effect was significantly stronger than CD19 CAR T and NT. Notably, when the effective target ratio is 2:1, the killing effect of Dual CAR T cells and Bi CAR T cells on Jurkat was significantly higher than CD69 CAR-T cells. Likewise, dual CAR T, bi CAR T, CD19 CAR T cells also had strong killing effects on Nalm6 cells, and their effects were significantly stronger than CD69 CAR T and NT. In addition, each CAR T cell had very weak killing of non-target cell K562 cells, indicating that each CAR T cell exhibited specific killing only to cells expressing CD69 or CD 19.
2.3 detection of cytokine Release levels of CAR T cells
At 1X 10 5 Concentration of individual cells/well target cells Jurkat, nalm6 and non-target cells K562 cells were plated in 96 well plates and Dual CAR T, bi CAR T cells, CD1 were added at a ratio of 1:1, respectively9 CAR T cells, CD69 CAR T cells, and NT cells (negative control), cell co-culture supernatant was collected after 18-24 hours of co-culture.
The content of IL2 and IFNγ in the co-culture supernatant was measured using the Human IL-2DuoSet ELISA Kit (R & D systems, cat. DY 202) and Human IFN-gamma DuoSet ELISA Kit (R & D systems, cat. DY 285), respectively, and the results are shown in FIG. 4.
It can be seen that the release levels of both cytokines IL2 and ifnγ are significantly increased after co-culture of CAR T cells of the invention with target cells.
The above results indicate that CD 69-targeted CAR T cells of the invention are capable of effectively producing specific killing of target cells in vitro. In addition, dual CAR T cells and Bi CAR T cells targeting CD19 and CD69 simultaneously had significantly higher killing activity against Jurkat than CD69 CAR T cells, indicating that this Dual target design was able to increase killing of target cells relative to single targets.
It should be noted that the above-mentioned embodiments are merely preferred embodiments of the present invention, and are not intended to limit the present invention, but various modifications and variations of the present invention will be apparent to those skilled in the art. It will be understood by those skilled in the art that any modifications, equivalent substitutions, improvements, etc. that fall within the spirit and principles of the present invention are intended to be included within the scope of the present invention.
Sequence listing
<110> Nanjing North Hengheng Biotechnology Co., ltd
<120> CD 69-targeting engineered immune cells and uses thereof
<130> BHCN55
<160> 51
<170> SIPOSequenceListing 1.0
<210> 1
<211> 5
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 1
Ser Tyr Asp Met His
1 5
<210> 2
<211> 17
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 2
Trp Ile Asn Pro Tyr Ser Gly Asn Thr Asn Tyr Ala Gln Lys Phe Gln
1 5 10 15
Gly
<210> 3
<211> 14
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 3
Met Tyr Tyr Asp Lys Asp Tyr Leu Ser Trp Gly Thr Asp Ser
1 5 10
<210> 4
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 4
Ser Gly Asp Ser Leu Gly Ser Lys Tyr Val Tyr
1 5 10
<210> 5
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 5
Gly Asp Ser Lys Arg Pro Ser
1 5
<210> 6
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 6
Gly Ser Tyr Thr Thr Gly Ala Lys Ser His Val
1 5 10
<210> 7
<211> 123
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 7
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Asp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asn Pro Tyr Ser Gly Asn Thr Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Met Tyr Tyr Asp Lys Asp Tyr Leu Ser Trp Gly Thr Asp Ser
100 105 110
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 8
<211> 109
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 8
Asp Ile Glu Leu Thr Gln Pro Pro Ser Val Ser Val Ala Pro Gly Gln
1 5 10 15
Thr Ala Arg Ile Ser Cys Ser Gly Asp Ser Leu Gly Ser Lys Tyr Val
20 25 30
Tyr Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Val Val Ile Tyr
35 40 45
Gly Asp Ser Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser
50 55 60
Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Glu
65 70 75 80
Asp Glu Ala Asp Tyr Tyr Cys Gly Ser Tyr Thr Thr Gly Ala Lys Ser
85 90 95
His Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly
100 105
<210> 9
<211> 247
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 9
Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Asp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Ile Asn Pro Tyr Ser Gly Asn Thr Asn Tyr Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Met Tyr Tyr Asp Lys Asp Tyr Leu Ser Trp Gly Thr Asp Ser
100 105 110
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser
115 120 125
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln
130 135 140
Pro Pro Ser Val Ser Val Ala Pro Gly Gln Thr Ala Arg Ile Ser Cys
145 150 155 160
Ser Gly Asp Ser Leu Gly Ser Lys Tyr Val Tyr Trp Tyr Gln Gln Lys
165 170 175
Pro Gly Gln Ala Pro Val Val Val Ile Tyr Gly Asp Ser Lys Arg Pro
180 185 190
Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser Asn Ser Gly Asn Thr Ala
195 200 205
Thr Leu Thr Ile Ser Gly Thr Gln Ala Glu Asp Glu Ala Asp Tyr Tyr
210 215 220
Cys Gly Ser Tyr Thr Thr Gly Ala Lys Ser His Val Phe Gly Gly Gly
225 230 235 240
Thr Lys Leu Thr Val Leu Gly
245
<210> 10
<211> 741
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
caggtgcagc tggtgcagag cggcgccgag gtgaagaagc ctggcgccag cgtgaaggtg 60
agctgcaagg cctccggcta cacctttacc tcctacgaca tgcactgggt gaggcaggcc 120
cctggccagg gactggagtg gatgggctgg atcaacccct actccggcaa cacaaattac 180
gcccagaagt ttcagggcag ggtgacaatg accagagaca caagcatctc cacagcctac 240
atggagctga gcagcctgag gagcgaggac acagccgtgt actactgcgc cagaatgtac 300
tacgacaagg attacctgag ctggggcacc gacagctggg gccagggaac cctggtgaca 360
gtgagttccg gcggcggcgg aagcggagga ggaggatctg gcggcggcgg tagcgacatc 420
gagctgaccc agccccctag cgtgtccgtg gccccaggac agacagccag gatcagctgt 480
tccggcgaca gcctgggcag caagtacgtg tactggtacc agcagaagcc tggccaggcc 540
cctgtggtgg tcatctatgg cgatagcaag aggcccagcg gcatccctga gagattcagc 600
ggctccaata gcggcaacac cgccacactg accatctccg gcacacaggc cgaggatgag 660
gccgactact actgcggcag ctacacaacc ggcgccaaga gccacgtgtt tggcggcggc 720
acaaagctga cagtgctggg c 741
<210> 11
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 11
Gly Val Ser Leu Pro Asp Tyr
1 5
<210> 12
<211> 5
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 12
Trp Gly Ser Glu Thr
1 5
<210> 13
<211> 12
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 13
His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr
1 5 10
<210> 14
<211> 11
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 14
Arg Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn
1 5 10
<210> 15
<211> 7
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 15
His Thr Ser Arg Leu His Ser
1 5
<210> 16
<211> 9
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 16
Gln Gln Gly Asn Thr Leu Pro Tyr Thr
1 5
<210> 17
<211> 120
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 17
Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln
1 5 10 15
Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr
20 25 30
Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu
35 40 45
Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys
50 55 60
Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu
65 70 75 80
Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala
85 90 95
Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Ser Val Thr Val Ser Ser
115 120
<210> 18
<211> 107
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 18
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
35 40 45
Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr
100 105
<210> 19
<211> 242
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 19
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
35 40 45
Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu
115 120 125
Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys
130 135 140
Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg
145 150 155 160
Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser
165 170 175
Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile
180 185 190
Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln
195 200 205
Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly
210 215 220
Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val
225 230 235 240
Ser Ser
<210> 20
<211> 725
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 20
gacatccaga tgacacagac caccagcagc ctgtccgcca gcctgggcga cagggtgacc 60
atctcctgta gagccagcca ggacatcagc aagtacctga actggtacca gcaaaagccc 120
gatggcaccg tgaagctgct gatctaccac acatccaggc tgcactccgg cgtgcccagc 180
aggttctccg gcagcggaag cggcaccgat tactccctga ccatcagcaa tctggagcag 240
gaggacatcg ccacatactt ctgccagcag ggcaatacac tgccctacac atttggcggc 300
ggaacaaagc tggagatcac cggcggcggc ggcagcggag gaggcggaag cggcggagga 360
ggaagcgagg tgaagctgca ggagtccggc cctggcctgg tggctccttc ccagagcctg 420
tccgtgacat gcacagtgtc cggcgtgagc ctgcccgact acggcgtgag ctggatcaga 480
cagcctccta gaaagggcct ggagtggctg ggcgtgatct ggggctccga gaccacctac 540
tacaacagcg ccctgaagag caggctgaca atcatcaagg ataactccaa gagccaggtg 600
tttctgaaga tgaacagcct gcagaccgat gatacagcca tctactactg tgccaagcac 660
tactactacg gcggctccta cgccatggac tactggggcc agggcacctc cgtgaccgtg 720
tccag 725
<210> 21
<211> 120
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 21
Gln Val Thr Leu Lys Glu Ser Gly Pro Val Leu Val Lys Pro Thr Glu
1 5 10 15
Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr
20 25 30
Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu
35 40 45
Ala Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys
50 55 60
Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Ser Gln Val Val Leu
65 70 75 80
Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala
85 90 95
Arg His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Met Val Thr Val Ser Ser
115 120
<210> 22
<211> 106
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 22
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Leu
35 40 45
Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Gln
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
100 105
<210> 23
<211> 120
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 23
Gln Val Thr Leu Lys Glu Ser Gly Pro Val Leu Val Lys Pro Thr Glu
1 5 10 15
Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr
20 25 30
Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu
35 40 45
Ala Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys
50 55 60
Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Ser Gln Val Val Leu
65 70 75 80
Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala
85 90 95
Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Met Val Thr Val Ser Ser
115 120
<210> 24
<211> 106
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 24
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile
100 105
<210> 25
<211> 241
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 25
Gln Val Thr Leu Lys Glu Ser Gly Pro Val Leu Val Lys Pro Thr Glu
1 5 10 15
Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr
20 25 30
Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu
35 40 45
Ala Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys
50 55 60
Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Ser Gln Val Val Leu
65 70 75 80
Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala
85 90 95
Arg His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser
130 135 140
Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala
145 150 155 160
Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly
165 170 175
Lys Ala Pro Lys Leu Leu Leu Tyr His Thr Ser Arg Leu His Ser Gly
180 185 190
Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr Leu
195 200 205
Thr Ile Ser Ser Leu Gln Gln Glu Asp Phe Ala Thr Tyr Tyr Cys Gln
210 215 220
Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu
225 230 235 240
Ile
<210> 26
<211> 240
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 26
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Leu
35 40 45
Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Arg Phe Ser Gly Ser
50 55 60
Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Gln Glu
65 70 75 80
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr Thr
85 90 95
Phe Gly Gln Gly Thr Lys Val Glu Ile Gly Gly Gly Gly Ser Gly Gly
100 105 110
Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Thr Leu Lys Glu Ser Gly
115 120 125
Pro Val Leu Val Lys Pro Thr Glu Thr Leu Thr Leu Thr Cys Thr Val
130 135 140
Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro
145 150 155 160
Pro Gly Lys Ala Leu Glu Trp Leu Ala Val Ile Trp Gly Ser Glu Thr
165 170 175
Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp
180 185 190
Thr Ser Lys Ser Gln Val Val Leu Thr Met Thr Asn Met Asp Pro Val
195 200 205
Asp Thr Ala Thr Tyr Tyr Cys Ala Arg His Tyr Tyr Tyr Gly Gly Ser
210 215 220
Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser
225 230 235 240
<210> 27
<211> 241
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 27
Gln Val Thr Leu Lys Glu Ser Gly Pro Val Leu Val Lys Pro Thr Glu
1 5 10 15
Thr Leu Thr Leu Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr
20 25 30
Gly Val Ser Trp Ile Arg Gln Pro Pro Gly Lys Ala Leu Glu Trp Leu
35 40 45
Ala Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys
50 55 60
Ser Arg Leu Thr Ile Ser Lys Asp Thr Ser Lys Ser Gln Val Val Leu
65 70 75 80
Thr Met Thr Asn Met Asp Pro Val Asp Thr Ala Thr Tyr Tyr Cys Ala
85 90 95
Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Met Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser
130 135 140
Ser Val Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala
145 150 155 160
Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly
165 170 175
Lys Ala Pro Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly
180 185 190
Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
195 200 205
Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln
210 215 220
Gln Gly Asn Thr Leu Pro Tyr Thr Phe Gly Gln Gly Thr Arg Leu Glu
225 230 235 240
Ile
<210> 28
<211> 241
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 28
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Gly Asn Thr Leu Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Gly Gly Gly Gly Ser Gly
100 105 110
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Thr Leu Lys Glu Ser
115 120 125
Gly Pro Val Leu Val Lys Pro Thr Glu Thr Leu Thr Leu Thr Cys Thr
130 135 140
Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln
145 150 155 160
Pro Pro Gly Lys Ala Leu Glu Trp Leu Ala Val Ile Trp Gly Ser Glu
165 170 175
Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ser Lys
180 185 190
Asp Thr Ser Lys Ser Gln Val Val Leu Thr Met Thr Asn Met Asp Pro
195 200 205
Val Asp Thr Ala Thr Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly
210 215 220
Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Met Val Thr Val Ser
225 230 235 240
Ser
<210> 29
<211> 723
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 29
caggtaacct tgaaagaatc aggcccagtc ctggtaaagc cgacggagac attgacgctg 60
acctgcacag tcagtggagt gtcattgccc gattatggtg tctcttggat tcgccagcct 120
cctggcaagg ctcttgaatg gttggctgtc atctggggca gtgagactac ttattataac 180
agtgcgttga aatcccggct gactattagt aaagatactt ccaaaagcca agtagtcctc 240
actatgacaa acatggatcc tgtagacacg gctacttatt attgtgcacg gcattactac 300
tacggcggct cttatgctat ggattactgg ggtcaaggaa caatggttac tgtttcatct 360
ggcggcgggg ggtcaggtgg gggtggttcc gggggtggtg gtagtgacat ccaaatgaca 420
caatctccga gtagtctctc cgcaagtgtg ggcgatcgag ttacaattac ttgtcgagcc 480
tcacaggaca tatctaagta cttgaattgg taccaacaga agccggggaa ggcaccaaag 540
ttgctgctgt atcacactag tagattgcat agtggggtgc cctcaagatt cagtggaagt 600
ggatcaggca ctgattacac tttgactatt agtagtctgc aacaggaaga cttcgcaacg 660
tactattgtc agcaaggtaa caccttgccg tacacattcg gtcaaggtac caaagtggaa 720
att 723
<210> 30
<211> 723
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 30
gatattcaga tgacccaaag tccgtctagc ttgtcagcct ccgtagggga cagagttacg 60
ataacttgta gagcttcaca ggatatatca aagtacctta attggtatca acaaaagcct 120
ggtaaagccc cgaagcttct cctttaccat acgtctcgac tccactccgg tgtcccttct 180
cgcttcagtg gttctgggag tgggaccgac tatacattga ccatctcttc tttgcaacaa 240
gaggacttcg ctacgtatta ctgtcagcaa ggtaatactc ttccgtatac tttcggacaa 300
ggcacgaaag tagaaattgg tggtggcggt tcaggcgggg gtggaagcgg ggggggtggg 360
tctcaggtca ccttgaagga atctggacct gttctggtga agcctacaga aactttgaca 420
ctgacatgca ccgtctctgg tgtatcattg ccggactacg gtgtgtcatg gattaggcaa 480
ccgcctggaa aagccttgga atggctcgcc gtcatctggg ggtctgagac gacctattac 540
aactcagcgt tgaagtcacg cctcacgatt tcaaaggaca cgtctaaatc acaagtggtt 600
ttgacaatga ctaacatgga cccggtcgat acggctactt actactgcgc tcgccactat 660
tattatgggg ggtcctatgc gatggactat tggggacagg gcacaatggt gacagtctca 720
agc 723
<210> 31
<211> 723
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 31
caggtgacat tgaaagagtc cggacctgtt ctcgtcaaac cgacggagac gctcaccctt 60
acatgcacgg tcagtggcgt ctcactgcca gattatgggg tttcttggat aagacagccc 120
cctggcaagg cccttgaatg gcttgcggtc atctggggtt ctgaaacaac atattataac 180
agtgcactca aatcacgcct tacgatttca aaggatacaa gtaaatctca agttgttctg 240
accatgacga atatggatcc agtggacacc gcgacgtact attgcgcgaa acattactac 300
tacggcggaa gctacgctat ggactattgg ggacaaggaa cgatggtcac tgtctctagc 360
ggcgggggtg gaagtggagg agggggatca gggggaggag gaagtgacat tcaaatgacg 420
cagtctccta gttcagtctc tgcatctgtg ggcgacagag tcacgattac ctgtagggca 480
agccaggata tctctaagta ccttaattgg tatcaacaaa aaccagggaa ggccccgaag 540
cttcttatct accatacaag ccgactccat agtggggttc cctcacgatt tagcgggtct 600
ggctctggta cagacttcac tctcactatt agtagcctgc aaccagagga cttcgcaacc 660
tactactgcc agcagggaaa cactcttcca tatacattcg gacagggaac ccggctcgag 720
att 723
<210> 32
<211> 723
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 32
gatatacaaa tgacgcagtc tccatcatcc gtatccgcca gcgtaggaga tagagtcacc 60
atcacatgtc gcgcgagtca ggacatcagc aaatacctca attggtacca gcaaaagccc 120
ggcaaggctc caaaattgct gatataccat acgagtaggt tgcacagtgg agtacccagt 180
agattttctg ggtcagggag tggcacagat ttcacgctga caataagtag tcttcaacct 240
gaagatttcg caacctatta ctgtcaacaa ggtaatacgc tgccgtacac gtttggtcag 300
ggtacgaggc tcgaaatcgg tggcggggga agtggaggtg gtggatctgg gggtgggggc 360
tctcaagtta ctttgaaaga gagtggacca gtgctcgtaa agcctacgga gacgcttacc 420
cttacgtgca cagtatctgg ggtgagtctc ccagattacg gtgtaagttg gatacgccag 480
ccgccgggga aagccttgga atggctcgca gtgatttggg gatccgaaac tacctattac 540
aatagcgccc ttaaatccag gctgactatt tccaaagaca cgagcaaatc ccaggtcgtc 600
cttaccatga caaacatgga cccagttgac accgctacct actactgtgc gaaacattat 660
tactacggtg ggagctatgc tatggactac tggggccaag gcacaatggt cactgttagt 720
agc 723
<210> 33
<211> 40
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 33
Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg
1 5 10 15
Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro
20 25 30
Glu Glu Glu Glu Gly Gly Cys Glu
35 40
<210> 34
<211> 126
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 34
aagagaggca ggaagaagct gctgtacatc ttcaagcagc ctttcatgag acctgtgcag 60
accacccagg aggaggacgg ctgtagctgc agattccccg aggaggagga gggcggctgt 120
gagctc 126
<210> 35
<211> 126
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 35
aaaaggggca gaaagaagct gctctacatc ttcaaacagc cttttatgag gcccgtgcag 60
accacacagg aggaggatgg ctgctcctgt agattccccg aagaggagga gggaggctgt 120
gagctg 126
<210> 36
<211> 113
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 36
Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln
1 5 10 15
Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu
20 25 30
Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly
35 40 45
Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln
50 55 60
Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu
65 70 75 80
Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr
85 90 95
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro
100 105 110
Arg
<210> 37
<211> 336
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 37
agagtgaagt tctccagaag cgccgatgcc cctgcctaca agcagggcca gaatcagctg 60
tacaatgagc tgaatctggg caggagagag gagtacgatg tgctggataa gaggagaggc 120
agggaccctg agatgggcgg caagcccagg agaaagaacc ctcaggaggg cctgtacaat 180
gaactgcaga aggacaagat ggccgaggcc tactccgaga tcggcatgaa gggcgagagg 240
aggaggggca agggccacga tggcctgtac cagggcctgt ccacagccac caaggacacc 300
tacgatgccc tgcacatgca ggccctgcct cccagg 336
<210> 38
<211> 336
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 38
agagtgaagt ttagcaggtc cgccgacgcc cctgcctata agcagggcca aaatcagctg 60
tataatgagc tgaacctggg caggcgggag gagtacgacg tgctggataa aaggagaggc 120
cgggaccctg agatgggagg caagcctaga agaaagaatc cccaggaggg cctctacaat 180
gaactccaga aggacaaaat ggccgaggct tacagcgaga tcggcatgaa aggcgagaga 240
aggaggggca aaggccacga tggactgtac cagggactgt ccacagctac aaaggatacc 300
tacgacgccc tgcacatgca agccctgccc cccaga 336
<210> 39
<211> 25
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 39
Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu
1 5 10 15
Ser Leu Val Ile Thr Leu Tyr Cys Lys
20 25
<210> 40
<211> 72
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 40
atctacatct gggcccctct ggccggcaca tgtggcgtgc tgctgctgag cctggtcatt 60
acactgtact gt 72
<210> 41
<211> 72
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 41
atctacattt gggcccccct ggccggcacc tgcggagtgc tgctgctctc cctggtcatt 60
accctgtact gt 72
<210> 42
<211> 21
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 42
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro
20
<210> 43
<211> 63
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 43
atggccctgc ctgtgaccgc cctgctgctg cctctggccc tgctgctcca cgccgctagg 60
cct 63
<210> 44
<211> 63
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 44
atggccctgc ccgtgacagc cctgctgctg ccactggccc tgctcctgca cgccgctaga 60
ccc 63
<210> 45
<211> 45
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 45
Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
1 5 10 15
Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly
20 25 30
Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
35 40 45
<210> 46
<211> 135
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 46
accacaacac ctgcccccag gccccctaca cctgccccta caatcgcctc ccagcctctg 60
tccctgagac ccgaggcctg cagacctgcc gccggaggag ctgtgcacac aagaggcctg 120
gatttcgcct gcgat 135
<210> 47
<211> 135
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 47
accaccacac ctgccccaag acctcctacc cctgccccca ccatcgcctc ccaacctctg 60
tccctcaggc ctgaggcctg taggcccgcc gctggaggag ctgtccacac cagaggcctg 120
gactttgcct gtgac 135
<210> 48
<211> 18
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 48
Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr
1 5 10 15
Lys Gly
<210> 49
<211> 15
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 49
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 50
<211> 18
<212> PRT
<213> Artificial sequence (Artificial Sequence)
<400> 50
Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro
1 5 10 15
Gly Pro
<210> 51
<211> 54
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 51
gagggcagag gaagtcttct aacatgcggt gacgtggagg agaatcccgg ccct 54

Claims (22)

1. A chimeric antigen receptor comprising an antibody that targets CD69, a transmembrane domain, and an intracellular signaling domain.
2. The chimeric antigen receptor according to claim 1, wherein the antibody targeting CD69 comprises CDR-H1 as shown in SEQ ID No. 1, CDR-H2 as shown in SEQ ID No. 2, CDR-H3 as shown in SEQ ID No. 3, CDR-L1 as shown in SEQ ID No. 4, CDR-L2 as shown in SEQ ID No. 5, CDR-L3 as shown in SEQ ID No. 6.
3. The chimeric antigen receptor according to claim 2, wherein the CD 69-targeting antibody comprises a heavy chain variable region sequence having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID No. 7 and a light chain variable region sequence having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid sequence shown in SEQ ID No. 8.
4. The chimeric antigen receptor according to claim 1, wherein the chimeric antigen receptor further comprises an antibody that targets a second target; wherein the second target is selected from the group consisting of: TSHR, CD7, CD19, CD123, CD22, BAFF-R, CD, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, GPRC5D, tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B H3, KIT, IL-13Ra2, mesothelin, IL-lRa, PSCA, PRSS21, VEGFR2, lewis Y, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor alpha, ERBB2, MUC1, EGFR, NCAM, claudin18.2, prostase, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gproo, bcr-abl, tyrosinase, ephA2, fucosyl GMl, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD 2, folate receptor beta, TEM1/CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD179a, ALK, polysialic acid, PLAC1, globoH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, legumain, HPV E6, E7, MAGE Al, ETV6-AML, sperm protein 17, XAGE1, tie 2, MAD-CT-1, MAD-CT-2, fos associated antigen 1, p53 mutant, prostate specific protein, survivin and telomerase, PCTA-l/Galectin 8, melanA/MARTl, ras mutant, hTERT, sarcoma breakpoint, fos associated antigen 1, p53 mutant, prostate specific protein, survivin and telomerase ML-IAP, ERG, NA17, PAX3, androgen receptor, cyclin Bl, MYCN, rhoC, TRP-2, CYP1B 1, BORIS, SART3, PAX5, OY-TES 1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79B, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGFL 1, PD1, CD79a, PDL1, PDL2, tgfβ, APRIL, NKG2D, or any combination thereof.
5. The chimeric antigen receptor according to claim 4, wherein the chimeric antigen receptor further comprises an antibody targeting CD19, which comprises CDR-H1 as shown in SEQ ID NO. 11, CDR-H2 as shown in SEQ ID NO. 12, CDR-H3 as shown in SEQ ID NO. 13, CDR-L1 as shown in SEQ ID NO. 14, CDR-L2 as shown in SEQ ID NO. 15, CDR-L3 as shown in SEQ ID NO. 16.
6. The chimeric antigen receptor of claim 5, wherein the CD 19-targeting antibody comprises a sequence identical to SEQ ID NO: 17. 21, 23 and a heavy chain variable region sequence having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to any one of SEQ ID NOs: 18. 22, 24, and a light chain variable region sequence having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity to the amino acid sequence set forth in any one of figures.
7. The chimeric antigen receptor according to claim 4, wherein the antibody that targets CD69 and the antibody that targets the second target in the chimeric antigen receptor are in tandem, the chimeric antigen receptor comprising: (1) An antigen binding region comprising an antibody that targets CD69 and an antibody that targets a second target; (2) a transmembrane domain; and (3) an intracellular signaling domain.
8. The chimeric antigen receptor according to claim 4, wherein the antibody targeting CD69 and the antibody targeting the second target in the chimeric antigen receptor are in parallel, the chimeric antigen receptor comprising: (1) A first unit structure targeting CD69 comprising an antibody that targets CD69, a transmembrane domain, and an intracellular signaling domain; and (2) a second unit structure that targets a second target, comprising an antibody that targets the second target, a transmembrane domain, and an intracellular signaling domain.
9. The chimeric antigen receptor of any one of claims 1-8, wherein the antibody is an intact antibody, fab ', F (ab') 2, fv fragment, scFv antibody fragment, linear antibody, sdAb, or nanobody.
10. The chimeric antigen receptor of claim 1 or 4, the intracellular signaling domain comprising a costimulatory domain and/or a primary signaling domain.
11. The chimeric antigen receptor according to claim 10, wherein the costimulatory domain is selected from the group consisting of costimulatory signaling domains of: LTB, CD94, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18, CD27, CD28, CD30, CD40, CD54, CD83, CD134, CD137, CD270, CD272, CD276, CD278, CD357, DAP10, DAP12, LAT, NKG2C, SLP, PD-1, LIGHT, TRIM, ZAP70, or any combination thereof.
12. The chimeric antigen receptor according to claim 10, wherein the primary signaling domain is selected from the signaling domains of: fcrγ, fcrβ, cd3γ, cd3δ, cd3ε, cd3ζ, CD22, CD79a, CD79b, CD66d, or any combination thereof.
13. The chimeric antigen receptor according to claim 1 or 4, wherein the transmembrane domain is selected from the group consisting of the transmembrane domains of: TCR alpha chain, TCR beta chain, TCR gamma chain, TCR delta chain, cd3ζ subunit, cd3ε subunit, cd3γ subunit, cd3δ subunit, CD45, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or any combination thereof.
14. A nucleic acid molecule encoding the chimeric antigen receptor of any one of claims 1-13.
15. A vector comprising the nucleic acid molecule of claim 14.
16. An engineered immune cell comprising the nucleic acid molecule of claim 14 or the vector of claim 15, or expressing the chimeric antigen receptor of any one of claims 1-13.
17. The engineered immune cell of claim 16, wherein the immune cell is a T cell, B cell, macrophage, dendritic cell, monocyte, NK cell, and/or NKT cell.
18. The engineered immune cell of claim 17, wherein the T cell is CD4 + CD8 + T cells, CD4 + T cells, CD8 + T cells, memory T cells, CD4 - CD8 - T cells, tumor infiltrating cells, naive T cells, γδ -T cells and/or αβ -T cells.
19. The engineered immune cell of claim 16, wherein expression of at least one MHC-related gene and/or at least one TCR/CD 3-related gene is inhibited or silenced.
20. The engineered immune cell of claim 19, the MHC-related gene is selected from HLA-A, HLa-B, HLA-C, B2M, HLA-DPA, HLa-DQ, HLa-DRA, TAP1, TAP2, LMP7, RFX5, RFXAP, RFXANK, CIITA, or any combination thereof, preferably HLA-A, HLa-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, CIITA, or any combination thereof; the TCR/CD3 gene is selected from TRAC, TRBC, CD3 gamma, cd3 delta, cd3 epsilon, cd3ζ, or any combination thereof.
21. A pharmaceutical composition comprising the engineered immune cell of any one of claims 15-20 and one or more pharmaceutically acceptable carriers and/or excipients.
22. Use of the chimeric antigen receptor of any one of claims 1-13 or the engineered immune cell of any one of claims 15-20 or the pharmaceutical composition of claim 21 in the manufacture of a medicament for the treatment/prevention/diagnosis of cancer, infection or autoimmune disease, or for the treatment/alleviation/elimination of GVHD or HVGD.
CN202210655239.7A 2022-06-10 2022-06-10 Engineered immune cells targeting CD69 and uses thereof Pending CN117247459A (en)

Priority Applications (1)

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CN202210655239.7A CN117247459A (en) 2022-06-10 2022-06-10 Engineered immune cells targeting CD69 and uses thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210655239.7A CN117247459A (en) 2022-06-10 2022-06-10 Engineered immune cells targeting CD69 and uses thereof

Publications (1)

Publication Number Publication Date
CN117247459A true CN117247459A (en) 2023-12-19

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Country Status (1)

Country Link
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