CN114805611A - Chimeric antigen receptor targeting terminal mannose and application thereof - Google Patents

Chimeric antigen receptor targeting terminal mannose and application thereof Download PDF

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CN114805611A
CN114805611A CN202210741551.8A CN202210741551A CN114805611A CN 114805611 A CN114805611 A CN 114805611A CN 202210741551 A CN202210741551 A CN 202210741551A CN 114805611 A CN114805611 A CN 114805611A
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杨旸
刘学欢
朱圣淋
杨宇
周伸奥
张全
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Shanghai Hanyuan Biotechnology Co.,Ltd.
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Shanghai Yinghui Medical Instrument Co ltd
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Abstract

The invention provides a chimeric antigen receptor targeting terminal mannose and application thereof. In particular to a chimeric antigen receptor, which comprises a mannose binding molecule, a hinge region, a transmembrane region, an intracellular costimulatory region and an optional signal transduction region, and optionally further comprises a signal peptide sequence. The immune cells expressing the chimeric antigen can effectively inhibit tumor growth and recurrence.

Description

Chimeric antigen receptor targeting terminal mannose and application thereof
Technical Field
The invention relates to the field of cell therapy, in particular to a mannose-targeting chimeric antigen receptor and application thereof.
Background
With the development of biotechnology, cancer diseases have come up with new approaches to cellular immunotherapy. Cell therapy products currently on the market or under investigation are mainly focused on the area of hematological tumors, however the number of patients afflicted with solid tumors is significantly higher than that of hematological malignancies. Although some cellular immunotherapy aiming at the field of solid tumors is in the preclinical/clinical stage at present, the research and development process in the field of solid tumors is hindered due to insufficient target specificity, large target difference among organs, limited development difficulty of new targets, poor malignant tumor treatment effect, easy occurrence of serious toxic and side effects in the treatment process and the like, and no cell therapy product for the solid tumors is approved up to now.
Disclosure of Invention
The chimeric antigen receptor targeting terminal mannose provided by the invention specifically recognizes an N-sugar structure containing terminal mannose on an antigen/ligand at a tumor cell or a lesion site.
In one aspect, provided herein is a chimeric antigen receptor comprising a mannose binding molecule, a hinge region, a transmembrane region, and an intracellular region, optionally further comprising a signal peptide sequence.
In one or more embodiments, the mannose binding molecule comprises one or more of: a mannose-binding protein or a mannose-binding domain thereof, an antibody or antigen-binding fragment thereof that specifically binds mannose, an aptamer that specifically binds mannose, or a derivative thereof.
In one or more embodiments, the mannose binding protein is a mannose lectin.
In one or more embodiments, the mannose binding protein has:
(1) the sequence shown as SEQ ID NO. 2, or
(2) A sequence having at least 60% (e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%) sequence identity to SEQ ID NO. 2 and retaining its mannose binding activity.
In one or more embodiments, the sequence of item (2) has the mannose binding motifs WGG, RL 2 Q, EGP, wherein L is 2 1-3 arbitrary amino acids. L is 2 Preferably 2 arbitrary amino acids.
In one or more embodiments, the sequence of item (2) has 1-5 mannose binding motifs WGGL 1 RL 2 QL 3 EGP, wherein L 1 Is 12 to 18 arbitrary amino acids, L 2 1 to 3 arbitrary amino acids, L 3 Is 20-25 arbitrary amino acids.
In one or more embodiments, the sequence of item (2) has 1-2 of the mannose binding motifs.
In one or more embodiments, L 1 Is 15 arbitrary amino acids, L 2 Is 2 arbitrary amino acids, L 3 Is 24 arbitrary amino acids.
In one or more embodiments, (2) is a sequence having at least 60% (e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%) sequence identity to SEQ ID No. 2 and having the same amino acids as SEQ ID No. 2 at least 12 or at least 13 or at least 16 or all of its positions corresponding to SEQ ID No. 2 selected from the group consisting of: 10, 11, 12, 28, 31, 56, 57, 58, 77, 78, 79, 95, 98, 123, 124, 125 th bit.
In one or more embodiments, (2) has the sequence set forth in any one of SEQ ID NOs 17-27.
In one or more embodiments, the mannose binding domain of a mannose binding protein comprises the mannose binding motifs WGG, RL 2 Q, EGP, wherein L is 2 1-3 arbitrary amino acids. L is 2 Preferably 2 arbitrary amino acids.
In one or more of the entitiesIn embodiments, the mannose binding domain of a mannose binding protein comprises a mannose binding motif, WGGL 1 RL 2 QL 3 EGP, wherein L 1 Is 12 to 18 arbitrary amino acids, L 2 1 to 3 arbitrary amino acids, L 3 Is 20-25 arbitrary amino acids. Preferably, L 1 Is 15 arbitrary amino acids, L 2 Is 2 arbitrary amino acids, L 3 Is 24 arbitrary amino acids.
In one or more embodiments, the mannose binding domain of a mannose binding protein is a fragment of SEQ ID No. 2 comprising at least 12 or at least 13 or at least 16 or all of the sites selected from: 10, 11, 12, 28, 31, 56, 57, 58, 77, 78, 79, 95, 98, 123, 124, 125 th bit.
In one or more embodiments, the chimeric antigen receptor comprises, in order from the N-terminus to the C-terminus, a mannose binding molecule, a hinge region, a transmembrane region, and an intracellular region.
In one or more embodiments, the hinge region comprises a hinge region or extracellular region derived sequence selected from CTLA4, CD28, CD7, IgA1, IgA2, IgG1, IgG4, IgD, CD8 α, PD-1. The hinge region is preferably a CD8 a hinge region.
In one or more embodiments, the amino acid sequence of the hinge region is set forth in SEQ ID NO 4.
In one or more embodiments, the transmembrane region comprises a transmembrane domain selected from the group consisting of TCR α, TCR β, TCR γ, TCR δ, CD4, CD8 α, CD28, CD3 ζ, CD3 ε, CD3 γ, CD3 δ, CD45, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, and CD 154. The transmembrane region is preferably the CD28 transmembrane domain.
In one or more embodiments, the amino acid sequence of the transmembrane region is set forth in SEQ ID NO 5.
In one or more embodiments, the intracellular region comprises an intracellular costimulatory region and/or a signaling region.
In one or more embodiments, the intracellular co-stimulatory region comprises a co-stimulatory region from a protein selected from the group consisting of: MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, signaling lymphocyte activating molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CDS, ICAM-1, LFA-1(CD11 30/CD 30), 4-1BB (CD137), B30-H30, CDS, ICAM-1, ICOS (CD278), GITR, BAFFR, LIGHT, HVEM (LIGHT TR), KIRDS 30, SLAMF 30, NKp30 (KLRF 30), NKp30, CD30 alpha, CD30 beta, IL2 30 gamma, ITIL 7 alpha, VLA 30, VLITGA 30, GAITGA 30, GAITGB, 30, GAITGB, 30, CD30, CD30, GAITGB, 30, CD30, GAITGB, 30, CD30, 6855, CD30, CD30, CD30, GAITGB, CD30, CD 6855, CD30, CD 6855, CD30, CD 6855, 30, 6855, CD30, 6855, GAITGB, GAITGA, 30, CD 6855, CD 68511, 6855, 68511, 6855, CD 6855, 30, CD30, GAITGB, 30, GAITGB, CD30, CD30, GAITGA, CD30, CD 6855, 30, CD30, 2B4) CD84, CD96 (tactle), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55), PSGL1, CD100(SEMA4D), CD69, SLAMF6(NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG/Cbp, CD19a, and ligands that bind specifically to CD 83. The intracellular co-stimulatory region is preferably a CD28 co-stimulatory domain.
In one or more embodiments, the amino acid sequence of the intracellular co-stimulatory region is shown as SEQ ID NO 6.
In one or more embodiments, the signaling region comprises a signaling domain selected from BCR, fceri, fcyri, fcyriia, fcyriiia, CD3 ζ. The signaling region is preferably a CD3 zeta signaling domain.
In one or more embodiments, the amino acid sequence of the signaling region is set forth in SEQ ID NO 7.
The invention also provides a nucleic acid molecule comprising a sequence selected from:
(1) a coding sequence for a chimeric antigen receptor according to any embodiment herein;
(2) the complement of (1);
(3) a fragment of 5 to 50bp of any one of (1) or (2).
In one or more embodiments, the fragment is a primer.
In one or more embodiments, the coding sequence is DNA or RNA.
The invention also provides a nucleic acid construct comprising a nucleic acid molecule according to any of the embodiments of the invention.
In one or more embodiments, the nucleic acid construct is a vector, such as a cloning vector, an expression vector, or an integration vector.
In another aspect of the invention, there is provided a host cell which:
(1) comprising and/or expressing a chimeric antigen receptor as described in any embodiment herein, and/or
(2) Comprising a nucleic acid molecule and/or a nucleic acid construct as described herein.
In one or more embodiments, the cell is an immune cell.
In another aspect, the present invention provides a pharmaceutical composition comprising pharmaceutically acceptable excipients and:
(1) the chimeric antigen receptor and/or coding sequence thereof, as described in any embodiment herein, or
(2) A nucleic acid molecule, nucleic acid construct or cell as described in any of the embodiments herein.
In one or more embodiments, the pharmaceutical composition is for treating a tumor.
In one or more embodiments, the tumor is selected from one or more of the following: gastric cancer, thyroid tumor, gallbladder cancer, cholangiocarcinoma, lung cancer, melanoma, head and neck cancer, breast cancer, ovarian cancer, cervical cancer, liver cancer, colorectal cancer, brain glioma, pancreatic cancer, bladder cancer, prostate cancer, renal cancer, osteosarcoma.
In another aspect, the invention provides the use of an agent for the preparation of an activated immune cell, said agent comprising:
(1) the chimeric antigen receptor and/or coding sequence thereof, as described in any embodiment herein, or
(2) A nucleic acid molecule, nucleic acid construct or cell according to any of the embodiments herein,
in another aspect, the invention provides the use of an agent for the manufacture of a medicament for the treatment of a tumour, said agent comprising: (1) a chimeric antigen receptor and/or coding sequence thereof as described in any embodiment herein, or (2) a nucleic acid molecule, nucleic acid construct or cell as described in any embodiment herein.
The present invention also provides a method of treating or preventing a tumor, the method comprising administering to a patient in need thereof a therapeutically effective amount of a cell or a pharmaceutical composition according to any embodiment of the present invention.
In one or more embodiments, the cells or pharmaceutical composition are administered by intravenous injection.
The invention has the advantages that: different from other chimeric antigen receptor related products, the antigen or ligand recognized by the extracellular antigen/ligand binding domain of the chimeric antigen receptor of the invention is not a protein, but an N-sugar structure containing terminal mannose, and the special binding target has high recognition strength and low variability, and the degree of target interference generated by factors such as gene mutation is far lower than that of the traditional protein target.
Drawings
FIG. 1, schematic plasmid and protein structure of CEGA 23-CAR. A, a pSLenti-CEGA23-CAR-P2A-EGFP recombinant plasmid structure schematic diagram. B, structural schematic diagram of CEGA23-CAR protein expressed by the plasmid in cells.
FIG. 2, fluorescent micrograph of lentivirus infected HEK293T cells.
Figure 3, positive signal for CD 8T lymphocytes.
FIG. 4 EGFP-positive signal of CAR-T cells.
FIG. 5, killing effect of CAR-T cells on HCG-27 target cells.
FIG. 6, killing effect of CAR-T cells on AGS target cells.
FIG. 7, killing effect of CAR-T cells on PC-9 target cells.
FIG. 8, killing effect of CAR-T cells on A549 target cells.
FIG. 9, killing effect of CAR-T cells on RKO target cells.
Figure 10, killing effect of CAR-T cells on SW948 target cells.
FIG. 11, killing effect of CAR-T cells on HOS target cells.
FIG. 12, killing effect of CAR-T cells on U-2OS target cells.
FIG. 13 IL-2 secretion levels after incubation of CAR-T cells with tumor target cells.
FIG. 14 IFN-gamma secretion levels after incubation of CAR-T cells with tumor target cells.
FIG. 15 TNF-alpha secretion levels after incubation of CAR-T cells with tumor target cells.
FIG. 16, in vivo killing of CAR-T cells HCG-27 assay.
Figure 17, in vivo killing of CAR-T cells a549 assay.
Figure 18, in vivo killing of CAR-T cells SW948 assay.
FIG. 19, alignment of CEGA23 with other native sequences, shows a conserved mannose binding motif.
FIG. 20, an example of the structure of an N-sugar containing a terminal mannose.
Detailed Description
The inventors found that the mannose binding molecule as an extracellular ligand binding domain of a Chimeric Antigen Receptor (CAR), and the preparation of immune cells expressing the CAR, can effectively inhibit tumor growth and recurrence. Accordingly, provided herein are Chimeric Antigen Receptors (CARs) that target mannose. The CAR contains an optional signal peptide sequence, an antigen recognition region, i.e., a mannose binding molecule as described herein, an optional linker, a hinge region, a transmembrane region, and an intracellular region. Wherein the intracellular region comprises one or more intracellular co-stimulatory domains and/or one or more intracellular signaling domains. The "hinge region", "transmembrane region" and "intracellular region" herein may each be selected from the sequences of hinge, transmembrane and intracellular regions known in the CAR-T art.
Herein, mannose mainly refers to terminal mannose of a sugar chain, particularly terminal mannose in an N-sugar structure. N-sugar structures containing terminal mannose include, but are not limited to: linear mannose, branched trio-mannose, branched tetraoligo-mannose, branched penta-oligo-mannose, branched hexa-oligo-mannose, branched hepta-mannose, high mannose (-Asn), one-touch complex carbohydrate, two-touch complex carbohydrate, three-touch complex carbohydrate, four-touch complex carbohydrate, multiple-touch complex carbohydrate, four-touch hybrid carbohydrate, multiple-touch hybrid carbohydrate. Fig. 20 shows an example of an N-sugar structure containing a terminal mannose.
The Chimeric Antigen Receptor (CAR) of the present invention comprises a mannose binding molecule, a hinge region, a transmembrane region, an intracellular costimulatory region.
Herein, a "binding molecule" for mannose is a protein or nucleic acid that specifically binds mannose, including, but not limited to, mannose binding proteins, antibodies, antigen binding fragments of antibodies, heavy chain antibodies, nanobodies, minibodies, affibodies, target binding regions of receptors, ligands, cytokines, and chemokines. Herein, the term "antibody" includes monoclonal antibodies (including full length antibodies having an immunoglobulin Fc region), antibody compositions having polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies), diabodies and single chain molecules, and antibody fragments, particularly antigen-binding fragments, e.g., Fab, F (ab') 2, and Fv.
Molecules that can serve as mannose binding molecules include, but are not limited to, i) a natural or artificially engineered mannose binding protein (mannose lectin), ii) a mannose binding domain of a natural or artificially engineered mannose binding protein (mannose lectin), iii) an antibody or antibody fragment that specifically binds mannose, iv) an aptamer or aptamer derivative that specifically binds mannose.
Any mannose-binding molecule capable of recognizing a terminal mannose of a sugar chain shown in FIG. 19 can be used as the mannose-binding molecule herein.
In some embodiments, the mannose-binding molecule may be a mannose-binding protein or a mannose-binding domain thereof. In some embodiments, the mannose-binding molecule is CEGA23 shown in SEQ ID No. 2, or another native sequence having a similar mannose-binding domain as CEGA 23.
The inventor researches and discovers that the mannose binding domain of CEGA23 comprises mannose binding motifs WGG, RL 2 Q and EGP, wherein L 2 1-3 arbitrary amino acids. Further, mannose of CEGA23The binding domain comprises a conserved mannose binding motif WGGL 1 RL 2 QL 3 EGP. WGG, RL in this motif 2 Q, EGP these 3 sites form a specific spatial conformation in the protein, which together constitute the active binding site. In embodiments of the invention, the mannose binding molecule may have one or more (e.g. 1, 2, 3, 4, 5, 6) of said mannose binding motifs, wherein L is 1 Each independently is 12-18 arbitrary amino acids, L 2 Each independently is 1-3 arbitrary amino acids, L 3 Each independently is 20-25 arbitrary amino acids. Preferably, L 1 Is 15 arbitrary amino acids, L 2 Is 2 arbitrary amino acids, L 3 Is 24 arbitrary amino acids. FIG. 19 shows an alignment of SEQ ID NO 2 to SEQ ID NO 17 with the boxes identifying the motif amino acids.
In a further embodiment, the mannose binding domain of CEGA23 is a fragment comprising at least 12 or at least 13 or at least 16 or all of the positions selected from SEQ ID No. 2: 10, 11, 12, 28, 31, 56, 57, 58, 77, 78, 79, 95, 98, 123, 124, 125; preferably comprises amino acids 10, 11, 12, 28, 31, 56, 57, 58, 77, 78, 79, 95, 98, 123, 124 and 125, or amino acids 10, 11, 12, 28, 31, 77, 78, 79, 95, 98, 123, 124 and 125, or amino acids 56, 57, 58, 77, 78, 79, 95, 98, 123, 124 and 125 of SEQ ID NO 2. Exemplary other native sequences include:
lectin OAA as shown in SEQ ID NO 17 is derived fromPseudomonas sp.(BG 5) having amino acids at positions 10, 11, 12, 28, 31, 56, 57, 58, 77, 78, 79, 95, 98, 123, 124, 125 of SEQ ID NO: 2;
the Lectin ESA-2 shown in SEQ ID NO 18 is derived fromNostocales cyanobacterium(HT-58-2) having amino acids at positions 10, 11, 12, 28, 31, 56, 57, 58, 77, 78, 79, 95, 98, 123, 124, 125 of SEQ ID NO: 2;
lectin ESA-2 as shown in SEQ ID NO 19 was derived fromAquimarina sp. (TRL 1) having the sequence 10, 11, 12, 28, 31, 56, 57, 58, 77, 78, 79, 95, 98, 123, 124, SEQ ID NO:2,An amino acid at position 125;
the protein shown in SEQ ID NO. 20 is derived fromHerpetosiphon aurantiacus(ATCC 23779/DSM 785/114-95) having the amino acids at positions 10, 11, 12, 28, 31, 56, 57, 58, 77, 78, 79, 95, 98, 123, 124, 125 of SEQ ID NO: 2;
the Lectin OAA shown in SEQ ID NO:21 is derived from:Chloroflexia bacterium (SDU 3-3) having amino acids at positions 10, 11, 12, 28, 31, 56, 57, 58, 77, 78, 79, 95, 98, 123, 124, 125 of SEQ ID NO: 2;
the myxohemagglutinin (Myxo hemagglutinin) shown in SEQ ID NO. 22 is derived from:Nostoc sp. (PCC 7107) having amino acids at positions 10, 11, 12, 28, 31, 56, 57, 58, 77, 78, 79, 95, 98, 123, 124, 125 of SEQ ID NO: 2;
lectin SfL-2 shown in SEQ ID NO. 23 was derived fromSolieria filiformis(Red alga) (Euhymenia filiformis) having amino acids at positions 10, 11, 12, 28, 31, 56, 57, 58, 77, 78, 79, 95, 98, 123, 124, 125 of SEQ ID NO: 2;
the protein shown in SEQ ID NO. 24 is derived fromLyngbya sp. (strain PCC 8106) having amino acids at positions 10, 11, 12, 28, 31, 56, 57, 58, 77, 78, 79, 95, 98, 123, 124, 125 of SEQ ID NO: 2;
the Myxobacterial hemagglutinin (Myxobacterial hemagglutinin) shown in SEQ ID NO. 25 is derived fromStigmatella aurantiaca (strain DW4/3-1) having amino acids at positions 10, 11, 12, 28, 31, 77, 78, 79, 95, 98, 123, 124, 125 of SEQ ID NO: 2;
the Ricin B-type lectin domain protein (Ricin B-type lectin domain-binding protein) shown in SEQ ID NO:26 is derived fromHyalangium minutumAmino acids 56, 57, 58, 77, 78, 79, 95, 98, 123, 124, 125 of SEQ ID NO 2;
lectin ESA-2 as shown in SEQ ID NO 27 was derived fromJanthinobacterium agaricidamnosum(NBRC 102515 = DSM 9628) having amino acids at positions 10, 11, 12, 28, 31, 77, 78, 79, 95, 98, 123, 124, 125 of SEQ ID NO: 2.
One skilled in the art can alter one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids of a mannose-binding molecule of the invention to obtain a variant of the mannose-binding molecule without substantially affecting the activity of the mannose-binding molecule (e.g., mannose-binding activity). These variants include (but are not limited to): deletion, insertion and/or substitution of one or more (usually 1 to 50, preferably 1 to 30, more preferably 1 to 20, most preferably 1 to 10) amino acids, and addition of one or several (usually up to 20, preferably up to 10, more preferably up to 5) amino acids at the C-terminus and/or N-terminus. Conservative substitutions with amino acids of similar or similar properties are not known in the art to alter the function of the protein. Amino acid residues that can be conservatively substituted are well known in the art. Such substituted amino acid residues may or may not be encoded by the genetic code. Also, for example, the addition of one or several amino acids at the C-terminus and/or N-terminus does not generally alter the function of the protein. All of which are considered to be included within the scope of the present invention.
The mannose binding molecules described herein include variants of CEGA23 that have at least 60% (e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%) sequence identity to CEGA23 as set forth in SEQ ID No. 2 and retain its mannose binding activity. Such variants typically comprise the mannose binding domain or mannose binding motif of the CEGA23 variant. According to the inventors' discovery, the mannose binding motif comprises your WGGL 1 RL 2 QL 3 EGP, wherein L 1 Is 12 to 18 arbitrary amino acids, L 2 1 to 3 arbitrary amino acids, L 3 Is 20-25 arbitrary amino acids. The CEGA23 variant may have 1-5 (e.g., 1-2) of the mannose binding motifs.
In particular, such CEGA23 variants have at least 60% (e.g., at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, at least 99%) sequence identity to SEQ ID No. 2 and have a sequence of identical amino acids to SEQ ID No. 2 at least 12 or at least 13 or at least 16 or all of its positions corresponding to SEQ ID No. 2 selected from the group consisting of: 10, 11, 12, 28, 31, 56, 57, 58, 77, 78, 79, 95, 98, 123, 124, 125 th bit. Preferably, the positions are positions corresponding to positions 10, 11, 12, 28, 31, 56, 57, 58, 77, 78, 79, 95, 98, 123, 124 and 125 of SEQ ID NO. 2, or positions 10, 11, 12, 28, 31, 77, 78, 79, 95, 98, 123, 124 and 125, or positions 56, 57, 58, 77, 78, 79, 95, 98, 123, 124 and 125.
The optional signal peptide on the CAR can be selected as desired. In general, a signal peptide is a peptide sequence that targets a polypeptide to a desired site in a cell. The signal peptide targets the polypeptide to the secretory pathway of the cell and will allow the polypeptide to integrate and anchor to the lipid bilayer; the signal peptide may also be a membrane-localized signal peptide. Exemplary signal peptides such as the CD8 signal peptide, CD28 signal peptide, CD4 signal peptide, or light chain signal peptide, the sequences of which are within the knowledge of one skilled in the art. CD28 signal peptides suitable for use in the present invention can be the various CD28 signal peptide sequences commonly used in the art for CARs. In certain embodiments, the amino acid sequence of the CD28 signal peptide comprises the sequence set forth in SEQ ID NO. 1.
The hinge region of the chimeric antigen receptor is located between the extracellular antigen-binding region and the transmembrane region, is a segment of amino acids that is typically found between two domains of a protein, and can allow flexibility of the protein and movement of the two domains relative to each other. The hinge region may be a hinge region of a naturally occurring protein or a portion thereof. The hinge region of an antibody (such as an IgG, IgA, IgM, IgE, or IgD antibody) may also be used for the chimeric antigen receptors described herein. Non-naturally occurring peptides may also be used as the hinge region of the chimeric antigen receptor described herein. Illustratively, the hinge region of the CAR is selected from a CD8 a hinge region, an IgD hinge region, an IgG1 Fc CH2CH3 hinge region, or an IgG4 Fc CH2CH3 hinge region, the sequences of which are within the knowledge of one skilled in the art. Suitable CD8 a hinge regions for use in the invention can be various CD8 a hinge region sequences commonly used in the art for CARs. In certain embodiments, the CD8 a hinge region comprises the sequence set forth in SEQ ID NO. 4.
The transmembrane region of the chimeric antibody receptor may form an alpha helix, a complex of more than one alpha helix, a beta barrel, or any other stable structure capable of spanning the domain of the cellular phospholipid bilayer. The transmembrane region may be of natural or synthetic origin. The transmembrane region may be selected from the transmembrane regions of the following proteins: CD3 epsilon, CD4, CD5, CD8 alpha, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, alpha, beta or zeta chain of T cell receptor. The human CD28 transmembrane region suitable for use in the present invention can be the various CD28 transmembrane region sequences commonly used in the art for CARs. In certain embodiments, the amino acid sequence of the CD28 transmembrane region comprises the sequence set forth in SEQ ID NO 5.
The intracellular signaling domain is responsible for the activation of at least one normal effector function of immune effector cells expressing the chimeric antigen receptor. For example, the effector function of a T cell may be cytolytic activity or helper activity, including secretion of cytokines. While the entire intracellular signaling domain can generally be utilized, in many cases, the use of the entire chain is not necessary. For use of a truncated portion of an intracellular signaling domain, such a truncated portion can be used in place of the entire chain, so long as it transduces effector function signals. Thus, an intracellular signaling region includes any truncated form of an intracellular signaling domain sufficient to transduce an effector function signal. The intracellular signaling domain of the CAR can be selected as desired, including but not limited to intracellular signaling domains derived from at least one of CD3 ζ, FcR γ (FCER1G), FcR β (fcepsilon Rib), CD3 γ, CD3 δ, CD3 epsilon, CD5, CD22, CD79a, CD79b, and CD66 d. Preferably, the intracellular signaling region is derived from the CD3 ζ intracellular signaling region. Further, the intracellular signaling region of CD3 ζ has the amino acid sequence shown in SEQ ID NO. 7.
In addition to stimulation of antigen-specific signals, many immune effector cells also require co-stimulation to promote cell proliferation, differentiation and survival, as well as to activate effector functions of the cells. The "co-stimulatory domain" may be the cytoplasmic portion of the co-stimulatory molecule. The term "co-stimulatory molecule" refers to an associated binding partner on an immune cell (such as a T cell) that specifically binds to a co-stimulatory ligand, thereby mediating a co-stimulatory response by the immune cell, such as, but not limited to, proliferation and survival. Suitable intracellular co-stimulatory domains may be selected as desired, including intracellular domains with co-stimulatory signaling molecules, such as at least one of those derived from 4-1BB, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54, CD83, OX40, CD137, CD134, CD150, CD152, CD223, CD270, PD-L2, PD-L1, CD278, DAP10, LAT, NKD2C, SLP76, TRIM, fcepsilon RI γ, MyD88, and 41 BBL. In certain embodiments, the amino acid sequence of the CD28 co-stimulatory domain comprises the sequence set forth in SEQ ID NO 6.
The above-mentioned portions forming the chimeric antigen receptor of the present invention, such as the CD8 signal peptide, the anti-MSLN nanobody, the CD8 hinge region, the CD28 transmembrane region, the CD28 costimulatory domain, the CD3 ζ intracellular signal domain, etc., may be directly linked to each other or may be linked through a linker sequence. The linker sequence may be one known in the art to be suitable for use with antibodies, for example, a G and S containing linker sequence. Typically, the linker contains one or more motifs which repeat back and forth. For example, the motif may be GGGS, GGGGS, SSSSG, GSGSA and GGSGG. Preferably, the motifs are adjacent in the linker sequence with no intervening amino acid residues between the repeats. The linker sequence may comprise 1, 2, 3, 4 or 5 repeat motifs. The linker may be 3 to 25 amino acid residues in length, for example 3 to 15, 5 to 15, 10 to 20 amino acid residues. In certain embodiments, the linker sequence is a polyglycine linker sequence. The number of glycines in the linker sequence is not particularly limited, and is usually 2 to 20, such as 2 to 15, 2 to 10, 2 to 8. In addition to glycine and serine, other known amino acid residues may be contained in the linker, such as alanine (a), leucine (L), threonine (T), glutamic acid (E), phenylalanine (F), arginine (R), glutamine (Q), and the like. In certain embodiments, the linker sequence is a (GGGGS) n linker, where n is an integer from 1 to 5. In certain embodiments, the linker comprises the sequence set forth in SEQ ID NO 3.
The invention also includes mutants of the CAR of any embodiment. These mutants include: an amino acid sequence that has at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97% sequence identity to the CAR and retains the biological activity of the CAR (e.g., recognizes terminal mannose and activates cells). Sequence identity between two aligned sequences can be calculated using, for example, BLASTp from NCBI.
In an exemplary embodiment, the CAR comprises, in order from N-terminus to C-terminus, a CD28 signal peptide, a CEGA23 protein, a CD8 a hinge region, a CD28 transmembrane domain, a CD28 costimulatory domain, and a CD3 zeta signaling domain. In specific embodiments, an exemplary CAR having the structure described above is shown in SEQ ID NO 15.
It will be appreciated that in gene cloning procedures it is often necessary to design appropriate cleavage sites which will introduce one or more irrelevant residues at the end of the expressed amino acid sequence without affecting the activity of the sequence of interest. In order to construct a fusion protein, facilitate expression of a recombinant protein, obtain a recombinant protein that is automatically secreted outside of a host cell, or facilitate purification of a recombinant protein, it is often necessary to add some amino acids to the N-terminus, C-terminus, or other suitable regions within the recombinant protein, for example, including, but not limited to, suitable linker peptides, signal peptides, leader peptides, terminal extensions, and the like. Thus, the amino-terminus or the carboxy-terminus of a CAR of the invention may also contain one or more polypeptide fragments as protein tags. Any suitable label may be used herein. For example, the tag may be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ε, B, gE, and Ty 1. These tags can be used to purify proteins.
The antigen recognition region in the CAR of the invention may also be a variant of the aforementioned mannose binding molecule. Furthermore, other portions of the CAR may also be subject to sequence changes, resulting in a mutant that has at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97% sequence identity to the CAR and retains the biological activity of the CAR (e.g., activated T cells). Sequence identity between two aligned sequences can be calculated using, for example, BLASTp by NCBI.
Mutants also include: an amino acid sequence having one or several mutations (insertions, deletions or substitutions) in the amino acid sequence of the CAR according to any of the embodiments, while still retaining the biological activity of the CAR. The number of mutations usually means within 1-10, such as 1-8, 1-5 or 1-3. The substitution is preferably a conservative substitution. For example, conservative substitutions with amino acids of similar or similar properties are not typically used in the art to alter the function of a protein or polypeptide. "amino acids with similar or analogous properties" include, for example, families of amino acid residues with analogous side chains, including amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), 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). Thus, substitution of one or more sites with another amino acid residue from the same side chain species in the polypeptide of the invention will not substantially affect its activity.
The present invention includes polynucleotides encoding the fusion proteins of the present invention. The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand. The invention also includes degenerate variants of the polynucleotide sequences encoding the fusion proteins, i.e., nucleotide sequences which encode the same amino acid sequence but differ in nucleotide sequence.
Thus, the present invention also relates to polynucleotides which hybridize to the above-described polynucleotide sequences and which have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the polynucleotides of the present invention. In the present invention, "stringent conditions" mean: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 XSSC, 0.1% SDS,60 ℃; or (2) denaturant is added during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42 ℃ and the like; or (3) hybridization occurs only when the identity between two sequences is at least 90% or more, preferably 95% or more. Also, the polynucleotides that hybridize to the mature polypeptide encode polypeptides having the same biological functions and activities as the mature polypeptide.
The full-length nucleotide sequence or a fragment thereof of the binding molecule of the present invention can be obtained by PCR amplification, recombinant methods, or synthetic methods. One possibility is to use synthetic methods to synthesize the sequence of interest, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them. Alternatively, the coding sequence for the heavy chain and an expression tag (e.g., 6His) can be fused together to form a fusion protein. The sequence of the CAR can also be obtained as above. Alternatively, the full length of the CAR can be obtained by obtaining the sequences of the various portions of the CAR (signal peptide, antigen recognition region, hinge region, transmembrane region, or intracellular region) as described above and then ligating them together.
Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. The biomolecules (nucleic acids, proteins, etc.) to which the present invention relates include biomolecules in an isolated form. At present, DNA sequences encoding the proteins of the present invention (or fragments or derivatives thereof) have been obtained completely by chemical synthesis. The DNA sequence may then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. Furthermore, mutations can also be introduced into the protein sequences of the invention by chemical synthesis. The parts of the CAR can be cloned sequentially into the vector or can be integrated into a full-length CAR and then cloned.
The invention also relates to nucleic acid constructs comprising the polynucleotide sequences described herein, and one or more control sequences operably linked to these sequences. The polynucleotide sequences of the invention may be manipulated in a variety of ways to ensure expression of the antibody or CAR. The nucleic acid construct may be manipulated prior to insertion into the vector depending on the expression vector or requirements. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.
In certain embodiments, the nucleic acid construct is a vector, such as a cloning vector, an expression vector, and an integration vector. Expression of a polynucleotide sequence of the invention is typically achieved by operably linking the polynucleotide sequence of the invention to an expression vector. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters that may be used to regulate the expression of the desired nucleic acid sequence. The integration vector contains the components for integrating the target sequence into the genome of the cell. These vectors may be used to transform an appropriate host cell so that it can express the protein. Vectors typically contain sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences. The sequences (collectively referred to as "flanking sequences" in certain embodiments) typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence containing donor and acceptor splice sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for insertion of a nucleic acid encoding an antibody to be expressed, and a selectable marker element.
The type of vector is not limited, and for example, plasmids, phagemids, phage derivatives, animal viruses and cosmids may be changed depending on the host cell to be introduced. Viral vector technology is well known in the art and is described, for example, in Sambrook et al (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and Molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.
To assess the expression of the CAR polypeptide or portion thereof, the vector introduced into the cells can also comprise either or both of a selectable marker gene or a reporter gene to facilitate identification and selection of expressing cells from a population of cells sought to be transfected or infected by the viral vector.
Host cells suitable for introduction of the nucleic acid constructs described herein can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells, in particular immune cells, preferably immune effector cells. Representative examples are: escherichia coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf 9; CHO, COS7, 293 cells, etc.
An "immune effector cell" is an immune cell that can perform an immune effector function. In some embodiments, the immune effector cell performs ADCC effector function. Examples of immune effector cells that mediate ADCC include Peripheral Blood Mononuclear Cells (PBMCs), Natural Killer (NK) cells, monocytes, cytotoxic T cells, neutrophils, and eosinophils. Preferably, the immune effector cell is selected from: culturing at least one of differentiated immune cells, T lymphocytes, NK cells, Peripheral Blood Mononuclear Cells (PBMCs) and hematopoietic stem cells from pluripotent stem cells or embryonic stem cells. More preferably, the immune effector cell is a T lymphocyte (homo T cell). T cells suitable for use in the present invention may be of various types from various sources. In some embodiments, the T cell may be a CD8+ T cell. In some embodiments, the T cells produce IL-2, IFN, and/or TNF when expressing the chimeric antigen receptor and binding to the target cell. In some embodiments, CD8+ T cells lyse target cells when expressing chimeric antigen receptors and binding to the target cells.
T cells may be derived from PBMCs of B cell malignancies patients. In certain embodiments, after T cells are obtained, activation may be stimulated with an appropriate amount (e.g., 30-800 ng/ml) of CD3 and CD28 antibodies, and then incubated in a medium containing an appropriate amount (e.g., 30-800 IU/ml) of IL2 for use.
Methods for introducing nucleic acids or vectors into mammalian cells are known in the art, and the vectors can be transferred into the cells by physical, chemical, or biological methods. When the host is prokaryotic, such as E.coli, competent cells capable of DNA uptake can be harvested after the exponential growth phase and treated by the CaCl2 method using procedures well known in the art. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, and the like. In some embodiments, the transduced or transfected immune effector cells are propagated ex vivo following introduction of the nucleic acid or vector.
The obtained transformant can be cultured by a conventional method to express the antibody or CAR encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used. The culturing is performed under conditions suitable for growth of the host cell. In embodiments where an inducible promoter is used to express the CAR, after the host cells are grown to an appropriate cell density, the selected promoter is induced by a suitable method (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.
The polypeptide in the above method may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods using its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.
All aspects of the CARs, coding sequences, nucleic acid constructs, and cells described herein are useful in the preparation of medicaments for the prevention or treatment of various conditions and diseases described herein, which are diseases or conditions associated with terminal mannose on the surface of a cell, referring to diseases, such as tumors, caused directly or indirectly by aberrant expression of terminal mannose on the surface of a cell.
The invention also includes a class of cell therapies comprising genetically modifying immune cells to express a CAR described herein, and injecting the cells into a recipient in need thereof. The injected cells are capable of killing tumor cells of the recipient.
The CAR-modified cells of the invention can be administered alone or as a pharmaceutical composition in combination with a diluent and/or with other components such as relevant cytokines or cell populations. Accordingly, the invention also provides pharmaceutical compositions comprising CAR-modified cells made by the methods described herein and a pharmaceutically acceptable excipient.
In the present invention, a "pharmaceutically acceptable adjuvant" is a pharmaceutically or comestibly acceptable carrier, solvent, suspension or excipient for delivering the CAR-modified immune cells of the invention to an animal or human. Herein, pharmaceutically acceptable excipients are non-toxic to the recipient of the composition at the dosages and concentrations employed. Various types of carriers or excipients commonly used in the delivery of immune cells in therapy known in the art may be included. Exemplary excipients may be liquids or solids, including but not limited to: pH adjusters, surfactants, carbohydrates, adjuvants, antioxidants, chelating agents, ionic strength enhancers, preservatives, carriers, glidants, sweeteners, dyes/colorants, flavoring agents, wetting agents, dispersants, suspending agents, stabilizers, isotonic agents, solvents or emulsifiers. In some embodiments, the pharmaceutically acceptable excipients may include one or more inactive ingredients, including but not limited to: stabilizers, preservatives, additives, adjuvants, sprays, compressed air or other suitable gases, or other suitable inactive ingredients in combination with the pharmaceutically effective compound. See, e.g., REMINGTON' S PHARMACEUTICAL SCIENCES, 18 th edition, eds. A.R. Genrmo, 1990, Mack Publishing Company. The optimal pharmaceutical composition can be determined depending on the intended route of administration, mode of delivery and the desired dosage.
The pharmaceutical compositions of the present invention may be selected for parenteral delivery, for inhalation or for delivery through the alimentary canal (such as orally), for example for intravenous infusion delivery. The preparation of such compositions is within the skill of the art. Other pharmaceutical compositions will be apparent to those skilled in the art, including formulations comprising immune cells, particularly immune cells (e.g., T cells), in sustained or controlled release delivery formulations. The pharmaceutical compositions of the present invention may also be administered in a manner suitable for the disease to be treated (or prevented).
Pharmaceutical compositions for in vivo administration are generally provided in the form of sterile preparations. Sterilization is achieved by filtration through sterile filtration membranes. Compositions for parenteral administration may be stored in lyophilized form or in solution (e.g., a cryopreserved formulation). Parenteral compositions are typically placed in a container having a sterile access port, such as an intravenous solution strip or vial having a stopper pierceable by a hypodermic injection needle.
Once formulated, the pharmaceutical compositions are stored in sterile vials as solutions, suspensions, gels, emulsions, solids, crystals, frozen stock, or as dehydrated or lyophilized powders. The pharmaceutical formulations (e.g., frozen formulations) can be stored in a ready-to-use form or in a form for further formulation prior to administration. For example, pharmaceutical compositions suitable for delivery as described herein can be cryopreserved formulations that can withstand long-distance transport without damaging the cells. In addition to the cells themselves, cryopreservation preparations typically include components such as cell cryopreservation solution, Human Serum Albumin (HSA), and the like. Prior to administration (e.g., intravenous infusion), the cryopreserved pharmaceutical composition is cryopreserved (e.g., in liquid nitrogen). The cryopreserved formulation can be infused into a patient directly after thawing or formulated as an infusion composition. The composition and concentration of conventional cryopreservation fluids are known to those skilled in the art. For example, the cryopreservation solution or infusion composition may further comprise dimethyl sulfoxide, sodium chloride, glucose, sodium acetate, potassium chloride or magnesium chloride, etc., the concentration of which can be determined by one skilled in the art (e.g., an experienced physician) according to the condition of the cell, disease, patient, etc.
In some embodiments of the invention, the CAR-containing cells of the invention or compositions thereof can be combined with other therapies known in the art.
"patient," "subject," "individual," and the like are used interchangeably herein and refer to a living organism, such as a mammal, that can elicit an immune response. Examples include, but are not limited to, humans, dogs, cats, mice, rats, and transgenic species thereof.
In particular embodiments, the invention accomplishes CAR molecule preparation by PCR or homologous recombination by linking the nucleotide sequence of the extracellular antigen/ligand binding domain (e.g., CEGA 23) to other sequences, ultimately forming a gene molecule encoding a CAR, and inserting the gene molecule encoding the CAR into a retroviral vector. CAR-T cells were prepared using a retroviral system to transduce CARs into CD3 and CD28 antibody-activated T cells. CAR expression and cell proliferation capacity in CAR-T cells was verified by FACS. And tested the efficiency of killing CAR-T cells in vitro against target cells expressing terminal mannose-containing N-carbohydrate targets and cytokine expression under stimulation by target cells expressing terminal mannose-containing N-carbohydrate targets. The detection of the anti-tumor capacity and the tumor recurrence inhibiting capacity of the CAR-T cells in tumor-bearing mice is proved.
The present invention is described in further detail by referring to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Accordingly, the present invention should in no way be construed as limited to the following examples, but rather should be construed to include any and all variations which become apparent in light of the teachings provided herein. The methods and reagents used in the examples are, unless otherwise indicated, conventional in the art.
Examples
Assay device and reagent
BD flow cytometer Aria III; cytiva surface plasma resonance physical and chemical analyzer Biacore 4000; a ThermoFisher electroporator Neon; sartorius bioreactor Biostat B; leica fluorescent display DMi 8; cytiva high content Analyzer IN Cell Analyzer 2500 HS; ThermoFisher automated fluorescence cytometer (Countess II FL); a ThermoFisher dynamic endotoxin detector Multiskan ET; ThermoFisher multifunctional microplate reader Varioskan LUX; ThermoFisher carbon dioxide incubator thermo 1501; gibco Mass selection magnetic frame CTS-DynaMag-Magnet; a Beckman Coulter standard centrifuge Avanti j-e; a ThermoFisher desktop refrigerated centrifuge FRESCO 17; eppendorf cryocentrifuge 5415D; agilent capillary electrophoresis apparatus ZAG M5320 AA; genscript fast moisture transfer instrument eBlot 1. unit; bruker multimode small animal In-Vivo imager In-Vivo Xtreme; biozone small SPF laboratory animal IVC system samrt rack; ZHEJIANG FUXIA secondary biosafety cabinet BSC-1000 IIA 2.
Gibco Dynabeads ™ Mouse T-Activator CD3/CD28 for T Cell Expansion and Activation; gibco Dynabeads "capture Human T-Activator CD3/CD28 for T Cell Expansion and Activation; invitrogen Dynabeads chamber FlowComp chamber human CD4 kit; invitrogen Dynabeads fragment FlowComp fragment human CD8 kit; invitrogen Dynabeads "FlowComp" mouse CD4 kit; invitrogen Dynabeads "FlowComp" mouse CD8 kit; a ThermoFisher MagniSort ™ human NK cell enrichment kit; invitrogen Neon ™ transfection System 100. mu.L kit; invitrogen Lipofectamine ™ 2000/3000 Transfection Reagent; performing special effects on fetal calf serum by the Procell; the Procell RPMI-1640/RPMI-1640 has no phenol red; a Procell DPBS; a Procell L-glutamine solution; stemcell mTeSR Plus; the Promega Killing assay kit; preprotech recombinant human interleukin 2; biolegend CD19/CD45/CD3/CD4/CD8/CD69 antibodies; BD Matrixgel/Matrixgel high concentration; a four-upright cypress human mononuclear cell separation solution; the NEST cell culture factory; BD EDTA anticoagulation tube; ThermoFisher Spinner Double Side arms flashes; nunc porous cell culture plates; greiner U-bottom 96 well plates.
Example 1 preparation of Gene sequences of the chimeric antigen receptor CEGA23-CAR targeting terminal mannose
Genes encoding the CD28 signal peptide, the CEGA23 protein targeting the terminal mannose, the linker sequence, the CD8 a hinge region, the CD28 transmembrane domain, the CD28 costimulatory domain, and the CD3 zeta signaling domain were prepared separately. The amino acid sequences of the sequences are respectively shown as SEQ ID NO 1-7; the coding gene sequences are respectively shown in SEQ ID NO 8-14.
The signal peptide, the CEGA23 protein targeting the terminal mannose, a linker sequence, a CD8 alpha hinge region, a CD28 transmembrane domain, a CD28 costimulatory domain and a CD3 zeta signal transduction domain are sequentially connected together from the 5 'end to the 3' end by a PCR method to obtain the coding gene of the chimeric antigen receptor CEGA23-CAR targeting the terminal mannose. The amino acid sequence of the CEGA23-CAR is shown as SEQ ID NO. 15, and the coding sequence is shown as SEQ ID NO. 16.
Example 2 construction of the pSLenti-CEGA23-CAR-P2A-EGFP recombinant plasmid
The gene encoding CEGA23-CAR was inserted between the BamHI and EcoRI cleavage sites of the pSLenti-P2A-EGFP vector using T4 ligase, and after the pSLenti-P2A-EGFP vector EF1 α, using EF1 α as a promoter. When the coding gene of the CEGA23-CAR is inserted into a pSLenti-P2A-EGFP vector, the 5 'end of the coding gene of the CEGA23-CAR is added with an initiation codon ATG which is connected with a BamH I enzyme cutting site in the pSLenti-P2A-EGFP vector, and the 3' end of the coding gene of the CEGA23-CAR is connected with an EcoR I enzyme cutting site in the pSLenti-P2A-EGFP vector. Transferring the ligation product into an escherichia coli competent cell DH5 alpha, coating the transformed bacterial liquid in LB solid flat half containing ampicillin for overnight culture at 37 ℃, and carrying out positive clone PCR screening, plasmid extraction and plasmid sequencing identification on the monoclonal colony in the flat plate the next day. And (3) performing gel electrophoresis detection on the PCR product and performing sequencing and identification on the plasmid to obtain the correct pSLenti-CEGA23-CAR-P2A-EGFP recombinant plasmid, wherein the size and the gene sequence of the target fragment are all met. FIG. 1, A shows the pSLenti-CEGA23-CAR-P2A-EGFP recombinant plasmid structure diagram. FIG. 1, B shows a schematic structural diagram of CEGA23-CAR protein expressed by a plasmid in cells.
Example 3 recombinant lentivirus construction
The pSLenti-CEGA23-CAR-P2A-EGFP recombinant plasmid, the packaging plasmid psPAX2 and the envelope plasmid pMD2G are co-transfected into cultured HEK293T cells, the supernatant containing viruses is harvested at 72h, the supernatant is filtered by a 0.45 mu m filter membrane, 1/10 volume of Takara lentivirus concentrated solution Lenti-X Concentrator is added into the filtered virus supernatant, then the mixed solution is placed on ice for incubation for 12-24h, and after the incubation is finished, a ThermoFis desktop centrifuge is used for centrifugation, wherein the centrifugation parameters are 1500g, 45min and 4 ℃. After the centrifugation is finished, discarding the supernatant, removing the liquid remained on the tube wall as much as possible, adding 1/10 volumes of virus preservation solution, and lightly blowing to resuspend the precipitate; then, part of the virus was taken for gradient dilution, HEK293T cells were infected, and virus titer was determined to be 6.15X10 by FACS fluorescence detection 8 TU/mL (BD Asia III as an instrument used in FACS, for details, see instructions for instruments), fluorescent display of lentivirus-infected HEK293T cellsThe micrographs are shown in FIG. 2. The obtained recombinant lentivirus was aliquoted at 100. mu.L/cell and stored in an ultra-low temperature freezer at-80 ℃.
Example 4 preparation of chimeric antigen receptor targeting terminal mannose (CEGA 23-CAR) T cells
Peripheral Blood Mononuclear Cell (PBMC) isolation: peripheral blood mononuclear cells were obtained using the STEMCELL Technologies brand easy Sep chamber Direct Human PBMC Isolation Kit. The specific operation method refers to the kit instruction.
Isolation of antigen-specific T lymphocytes by immunomagnetic bead: CD8 positive T lymphocytes were obtained using the STEMCELL Technologies brand easy Sep chamber Human Na meive CD8+ T Cell Isolation Kit II. The specific operation method refers to the kit instruction. A portion of the cells were FACS tested for CD8 positive signals, as shown in FIG. 3, with a CD8 positive rate of greater than 95%.
Activation and proliferation of CD8 positive T lymphocytes: diluting the CD3 and CD28 antibodies to 1 mug/mL and 2 mug/mL by PBS, adding the diluted antibodies into a six-hole plate for hole plate coating, adding 1mL of diluent into each hole, and placing the six-hole plate in a refrigerator at 4 ℃ for coating overnight; the following day, the isolated CD8 positive T cells were diluted to 2X10 in RPMI-1640 medium with 10% FBS and 1% double antibody 6 Per mL, the coating solution in the six-well plate was removed, 2mL of diluted T cells were added to each well, and the T cells were cultured at 37 ℃ in a 5% CO2 incubator for 48 hours.
Lentivirus transfection method for preparing T lymphocytes targeting terminal mannose: collecting the successfully activated and proliferated T cells, and centrifuging, wherein the centrifugation parameters are set to 300g, 5min and RT; centrifuging, removing supernatant, carrying out heavy suspension by using fresh RPMI-1640 medium containing 8. mu.g/mL Polybrene, and adding the heavy suspension into each well of a six-well plate; adding CEGA23-CAR expressing lentivirus to each well of a six-well plate (MOI values set to 20 according to lentivirus titer); culturing virus-infected T cells in a 5% CO2 culture box at 37 deg.C for 8-12h, replacing the culture medium with fresh RPMI-1640 medium containing 300IU/mL IL-2, and culturing in a 5% CO2 culture box at 37 deg.C; after 2 days of culture, a part of cells were taken and FACS-detected for EGFP positive signals, and the results are shown in FIG. 4, in which the cell positive rate was 90% or more. The remaining cells were continued in expansion culture to obtain sufficient CEGA23-CAR expressing T cells for subsequent efficacy validation experiments.
Example 5 in vitro tumor cell killing assay of chimeric antigen receptor T cells targeting terminal mannose
The in vitro tumor killing effects of the prepared terminal mannose-targeted chimeric antigen receptor T cells (CEGA 23-CAR T), T cells not transduced by lentivirus (CTL T) were compared: t cells and tumor target cells were treated according to Effector: different ratios of Target cells (efficiency: Target cell number ratio of 1:1, 1:2, 1:5 and 1: 10) were co-cultured in vitro in 96-well plates, 150. mu.L of medium containing 7X10 was added to each well 4 Individual target cells (HCG-27, AGS, PC-9, A549, RKO, SW948, HOS, U-2 OS); putting a 96-well plate in a 37 ℃ and 5% CO2 incubator for culture, after 4-8 h, using Promega brand CytoTox 96 Non-Radioactive Cytoxicity Assay kit, and calculating the Cytotoxicity percentage by measuring the content of LDH in the supernatant of the culture medium (tomor lysine% = LDH experimental release/LDH maximum release x100%, the specific operation method of the Cytoxicity Assay please refer to the kit specification); the results are shown in fig. 5-12, which indicate that the tumor killing effect of the chimeric antigen receptor T cell targeting terminal mannose is obviously higher than that of the negative control group.
Treating the tumor cells with Endoglycosidase H (Endo H, specific cleavage high mannose structure) in advance, and then co-culturing the tumor cells with different T lymphocytes to detect the in-vitro tumor killing effect of the T cells: culturing tumor cells in a six-hole plate, adding Roche brand endoglycosidase H into a culture medium, culturing the six-hole plate in a 37 ℃ and 5% CO2 incubator for 24 hours, and then performing an in-vitro tumor killing experiment on the T lymphocytes according to the method; the results are shown in FIGS. 5-12, which indicate that the chimeric antigen receptor T cells targeting terminal mannose lose the killing effect on Endo H-treated tumor cells.
Example 6 detection of secretion levels of chimeric antigen receptor T cell cytokines targeting terminal mannose
The prepared terminal mannose-targeted chimeric antigen receptor T cells (CEGA 23-CAR T) and non-lentivirus-transduced T cells (CTL T) were mixed with tumor cells HCG-27, AGS, PC-9, A549, RKO, SW948, HOS, U-2OS according to the following Effector: the cells were CO-cultured at 37 ℃ and 5% CO2 for 24h under the condition that the Target cell ratio was 2:1, and then the secretion effects of cytokines IL-2, IFN-gamma and TNF-alpha were detected by using an ELISA detection kit of human IL-2, IFN-gamma and TNF-alpha brand. For the specific operation of ELISA, refer to the kit instructions. The results are shown in FIGS. 13-15, which indicate that the cytokine secretion level of the chimeric antigen receptor T cell targeting terminal mannose after incubation with the tumor target cell is significantly higher than that of the negative control group.
Tumor cells are treated by Endoglycosidase H (Endo H, specific cleavage high mannose structure) in advance, and then are co-cultured with different T lymphocytes to detect IL-2, IFN-gamma and TNF-alpha secretion effects of cytokines, and the results are shown in figures 13-15, which shows that the cytokine secretion level and a negative control group have no obvious difference after the chimeric antigen receptor T cells targeting terminal mannose and the tumor target cells treated by the Endo H are incubated.
Example 7 detection of the anti-tumor Capacity of chimeric antigen receptor T cells targeting terminal mannose in tumor-bearing mice
Tumor target cell HCG-27 (5 x 10) 6 ）、A549（1x10 7 ）、SW948（5x10 6 ) Immunization of immunodeficient NSG mice (NOD-SCID IL2 rg-/-) was performed subcutaneously and the subcutaneous tumor volume of the mice was measured weekly by injection of 1X 10 at day 16 into the tail vein of the mice 6 Non-lentivirus-transduced T cells (Non-transduced T) or 1X 10 6 Injecting PBS (MOCK) into a blank control group of terminal mannose-targeted chimeric antigen receptor T cells (CEGA 23-CAR T), and then continuously measuring the subcutaneous tumor volume of the mice every week to determine the inhibition effect of the terminal mannose-targeted chimeric antigen receptor T cells on the tumor growth; the results are shown in fig. 16-18, which indicate that the growth inhibition effect of the prepared chimeric antigen receptor T cells targeting terminal mannose on tumor cells is obviously better than that of the negative control group.
SEQUENCE LISTING
<110> Shanghai fluorescence medical instruments Ltd
<120> terminal mannose-targeted chimeric antigen receptor and application thereof
<130> 218756 1CNCN
<160> 27
<170> PatentIn version 3.5
<210> 1
<211> 21
<212> PRT
<213> Artificial Sequence
<220>
<223> CD28 Signal peptide
<400> 1
Met Leu Arg Leu Leu Leu Ala Leu Asn Leu Phe Pro Ser Ile Gln Val
1 5 10 15
Thr Gly Gly Ser Ser
20
<210> 2
<211> 133
<212> PRT
<213> Artificial Sequence
<220>
<223> CEGA23
<400> 2
Met Ser Lys Tyr Ala Val Ala Asn Gln Trp Gly Gly Ser Ser Ala Pro
1 5 10 15
Trp His Pro Gly Gly Thr Trp Val Leu Gly Ala Arg Asp Asn Gln Asn
20 25 30
Val Val Ala Ile Glu Ile Lys Ser Gly Asp Gly Gly Lys Ser Phe Thr
35 40 45
Gly Thr Met Thr Tyr Ala Gly Glu Gly Pro Ile Gly Phe Lys Ala Gln
50 55 60
Arg Thr Gly Gln Asn Gln Tyr Asn Val Glu Asn Gln Trp Gly Gly Asn
65 70 75 80
Asp Ala Pro Trp His Pro Gly Gly Lys Trp Val Ile Gly Gly Arg Asp
85 90 95
Asn Gln Asn Val Ile Ala Leu Ser Val Thr Ser Ser Asp Gly Gly Lys
100 105 110
Asn Leu Ser Gly Thr Asn Thr Tyr Ala Asn Glu Gly Pro Ile Gly Phe
115 120 125
Arg Gly Gln Ile Glu
130
<210> 3
<211> 18
<212> PRT
<213> Artificial Sequence
<220>
<223> linker
<400> 3
Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr
1 5 10 15
Lys Gly
<210> 4
<211> 24
<212> PRT
<213> Artificial Sequence
<220>
<223> CD8 alpha hinge region
<400> 4
Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr
1 5 10 15
Arg Gly Leu Asp Phe Ala Cys Asp
20
<210> 5
<211> 31
<212> PRT
<213> Artificial Sequence
<220>
<223> CD28 transmembrane domain
<400> 5
Ser Lys Pro Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys
1 5 10 15
Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg
20 25 30
<210> 6
<211> 40
<212> PRT
<213> Artificial Sequence
<220>
<223> CD28 costimulatory domain
<400> 6
Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro
1 5 10 15
Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro
20 25 30
Arg Asp Phe Ala Ala Tyr Arg Ser
35 40
<210> 7
<211> 118
<212> PRT
<213> Artificial Sequence
<220>
<223> Signal transduction Domain
<400> 7
Ala Ser Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr
1 5 10 15
Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg
20 25 30
Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met
35 40 45
Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn
50 55 60
Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met
65 70 75 80
Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly
85 90 95
Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala
100 105 110
Leu Pro Pro Arg Ala Ser
115
<210> 8
<211> 63
<212> DNA
<213> Artificial Sequence
<220>
<223> CD28 Signal peptide
<400> 8
atgctcaggc tgctcttggc tctcaactta ttcccttcaa ttcaagtaac aggagggtct 60
tcg 63
<210> 9
<211> 399
<212> DNA
<213> Artificial Sequence
<220>
<223> CEGA23
<400> 9
atgtcaaaat atgcagtagc taatcaatgg ggcggcagca gcgcaccgtg gcacccgggt 60
ggcacttggg tgctgggtgc gcgtgacaat cagaacgttg ttgcgattga gatcaaatcc 120
ggtgatggtg gcaagtcgtt caccggtacg atgacctacg cgggtgaggg tccgattggc 180
ttcaaagcgc agcgtaccgg tcaaaaccag tataacgtgg aaaatcaatg gggtggaaac 240
gacgctccgt ggcatccagg tggcaagtgg gtcatcggcg gtcgcgacaa ccaaaacgtg 300
atcgccctga gcgttaccag ctctgatggc ggcaagaact tgtccggcac caatacgtac 360
gctaatgaag gtccgatcgg ctttcgtggt cagattgag 399
<210> 10
<211> 54
<212> DNA
<213> Artificial Sequence
<220>
<223> linker
<400> 10
ggctccacct ctggatccgg caagcccgga tctggcgagg gatccaccaa gggc 54
<210> 11
<211> 72
<212> DNA
<213> Artificial Sequence
<220>
<223> hinge region
<400> 11
ctgcgacctg aggcttgtcg accagcagcc ggaggcgcag tgcacacgag ggggctggac 60
ttcgcctgtg at 72
<210> 12
<211> 93
<212> DNA
<213> Artificial Sequence
<220>
<223> CD28 transmembrane domain
<400> 12
tctaagccct tttgggtgct ggtggtggtt ggtggagtcc tggcttgcta tagcttgcta 60
gtaacagtgg cctttattat tttctgggtg agg 93
<210> 13
<211> 120
<212> DNA
<213> Artificial Sequence
<220>
<223> CD28 costimulatory domain
<400> 13
agtaagagga gcaggctcct gcacagtgac tacatgaaca tgactcccag gcggcccgga 60
cccacccgca agcattacca gccctatgcc ccaccacgcg acttcgcagc ctatcgctcc 120
<210> 14
<211> 354
<212> DNA
<213> Artificial Sequence
<220>
<223> Signal transduction Domain
<400> 14
gctagcctga gagtgaagtt cagcaggagc gcagacgccc ccgcgtacca gcagggccag 60
aaccagctct ataacgagct caatctagga cgaagagagg agtacgatgt tttggacaag 120
agacgtggcc gggaccctga gatgggggga aagccgcaga gaaggaagaa ccctcaggaa 180
ggcctgtaca atgaactgca gaaagataag atggcggagg cctacagtga gattgggatg 240
aaaggcgagc gccggagggg caaggggcac gatggccttt accagggtct cagtacagcc 300
accaaggaca cctacgacgc ccttcacatg caggccctgc cccctcgcgc tagc 354
<210> 15
<211> 389
<212> PRT
<213> Artificial Sequence
<220>
<223> CAR
<400> 15
Met Leu Arg Leu Leu Leu Ala Leu Asn Leu Phe Pro Ser Ile Gln Val
1 5 10 15
Thr Gly Gly Ser Ser Met Ser Lys Tyr Ala Val Ala Asn Gln Trp Gly
20 25 30
Gly Ser Ser Ala Pro Trp His Pro Gly Gly Thr Trp Val Leu Gly Ala
35 40 45
Arg Asp Asn Gln Asn Val Val Ala Ile Glu Ile Lys Ser Gly Asp Gly
50 55 60
Gly Lys Ser Phe Thr Gly Thr Met Thr Tyr Ala Gly Glu Gly Pro Ile
65 70 75 80
Gly Phe Lys Ala Gln Arg Thr Gly Gln Asn Gln Tyr Asn Val Glu Asn
85 90 95
Gln Trp Gly Gly Asn Asp Ala Pro Trp His Pro Gly Gly Lys Trp Val
100 105 110
Ile Gly Gly Arg Asp Asn Gln Asn Val Ile Ala Leu Ser Val Thr Ser
115 120 125
Ser Asp Gly Gly Lys Asn Leu Ser Gly Thr Asn Thr Tyr Ala Asn Glu
130 135 140
Gly Pro Ile Gly Phe Arg Gly Gln Ile Glu Gly Ser Thr Ser Gly Ser
145 150 155 160
Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Leu Arg Pro Glu
165 170 175
Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp
180 185 190
Phe Ala Cys Asp Arg Arg Pro Pro Ser Lys Pro Phe Trp Val Leu Val
195 200 205
Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala
210 215 220
Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser Arg Leu Leu His Ser
225 230 235 240
Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly Pro Thr Arg Lys His
245 250 255
Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala Ala Tyr Arg Ser Ala
260 265 270
Ser Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln
275 280 285
Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu
290 295 300
Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly
305 310 315 320
Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu
325 330 335
Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys
340 345 350
Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu
355 360 365
Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu
370 375 380
Pro Pro Arg Ala Ser
385
<210> 16
<211> 1167
<212> DNA
<213> Artificial Sequence
<220>
<223> CAR
<400> 16
atgctcaggc tgctcttggc tctcaactta ttcccttcaa ttcaagtaac aggagggtct 60
tcgatgtcaa aatatgcagt agctaatcaa tggggcggca gcagcgcacc gtggcacccg 120
ggtggcactt gggtgctggg tgcgcgtgac aatcagaacg ttgttgcgat tgagatcaaa 180
tccggtgatg gtggcaagtc gttcaccggt acgatgacct acgcgggtga gggtccgatt 240
ggcttcaaag cgcagcgtac cggtcaaaac cagtataacg tggaaaatca atggggtgga 300
aacgacgctc cgtggcatcc aggtggcaag tgggtcatcg gcggtcgcga caaccaaaac 360
gtgatcgccc tgagcgttac cagctctgat ggcggcaaga acttgtccgg caccaatacg 420
tacgctaatg aaggtccgat cggctttcgt ggtcagattg agggctccac ctctggatcc 480
ggcaagcccg gatctggcga gggatccacc aagggcctgc gacctgaggc ttgtcgacca 540
gcagccggag gcgcagtgca cacgaggggg ctggacttcg cctgtgatag aagacctcct 600
tctaagccct tttgggtgct ggtggtggtt ggtggagtcc tggcttgcta tagcttgcta 660
gtaacagtgg cctttattat tttctgggtg aggagtaaga ggagcaggct cctgcacagt 720
gactacatga acatgactcc caggcggccc ggacccaccc gcaagcatta ccagccctat 780
gccccaccac gcgacttcgc agcctatcgc tccgctagcc tgagagtgaa gttcagcagg 840
agcgcagacg cccccgcgta ccagcagggc cagaaccagc tctataacga gctcaatcta 900
ggacgaagag aggagtacga tgttttggac aagagacgtg gccgggaccc tgagatgggg 960
ggaaagccgc agagaaggaa gaaccctcag gaaggcctgt acaatgaact gcagaaagat 1020
aagatggcgg aggcctacag tgagattggg atgaaaggcg agcgccggag gggcaagggg 1080
cacgatggcc tttaccaggg tctcagtaca gccaccaagg acacctacga cgcccttcac 1140
atgcaggccc tgccccctcg cgctagc 1167
<210> 17
<211> 133
<212> PRT
<213> Pseudomonas sp. 6G5
<400> 17
Met Ser Lys Tyr Ala Val Ala Asn Gln Trp Gly Gly Ser Ser Ala Pro
1 5 10 15
Trp His Pro Gly Gly Thr Trp Val Leu Gly Ala Arg Asp Asn Gln Asn
20 25 30
Val Val Ala Ile Asp Ile Lys Ser Gly Asp Gly Gly Lys Thr Phe Thr
35 40 45
Gly Thr Met Thr Tyr Ala Gly Glu Gly Pro Ile Gly Phe Lys Ala Gln
50 55 60
Arg Thr Gly Gln Asn Gln Tyr Asn Val Glu Asn Gln Trp Gly Gly Asn
65 70 75 80
Asp Ala Pro Trp His Pro Gly Gly Lys Trp Val Ile Gly Gly Arg Asp
85 90 95
Asn Gln Asn Val Ile Ala Leu Ser Val Thr Ser Asn Asp Gly Gly Lys
100 105 110
Asn Leu Ser Gly Thr Asn Thr Tyr Ala Asn Glu Gly Pro Ile Gly Phe
115 120 125
Arg Gly Gln Ile Glu
130
<210> 18
<211> 275
<212> PRT
<213> Unknown
<220>
<223> Nostocales cyanobacterium
<400> 18
Met Thr Thr Thr Lys Thr Ala Asn Asn Leu Tyr Asn Val Glu Asn Gln
1 5 10 15
Trp Gly Gly Thr Ser Ala Pro Trp Asn Pro Gly Gly Ala Trp Val Ile
20 25 30
Gly Ala Arg Ala Asn Gln Arg Val Val Ala Leu Lys Val Thr Ser Ser
35 40 45
Asp Asn Gly Lys Thr Leu Thr Gly Thr Thr Thr Tyr Ala Gly Glu Gly
50 55 60
Pro Ile Gly Phe Arg Ala Thr Leu Thr Asp Ser Ser Asp Thr Tyr Thr
65 70 75 80
Val Glu Asn Gln Trp Gly Gly Ser Ser Ala Pro Trp Asn Pro Gly Gly
85 90 95
Thr Trp Val Leu Gly Ser Arg Gly Asn Gln Asn Val Val Ala Ile Asp
100 105 110
Ile Thr Ser Ser Asp Asp Gly Asn Thr Leu Thr Gly Thr Ile Thr Tyr
115 120 125
Ala Gly Glu Gly Pro Ile Gly Phe Lys Ser Ala Val Val Asp Gly Gly
130 135 140
Val Tyr Thr Val Glu Asn Gln Trp Gly Gly Ser Ser Ala Pro Trp Asn
145 150 155 160
Pro Gly Gly Ile Trp Val Leu Gly Ser Arg Gly Asn Gln Asn Val Val
165 170 175
Ala Ile Asp Ile Thr Ser Ser Asp Asp Gly Asn Thr Leu Thr Gly Thr
180 185 190
Ile Thr Tyr Ala Gly Glu Gly Pro Ile Gly Phe Lys Gly Lys Ile Phe
195 200 205
Gly Ser Asn Asn Tyr Thr Val Asp Asn Gln Trp Gly Gly Asn Thr Ala
210 215 220
Pro Trp Asn Pro Gly Gly Ile Trp Leu Ile Gly Gly Arg Val Gly Gln
225 230 235 240
Asn Val Val Ala Leu Asn Val Thr Ser Ser Asp Gly Gly Lys Thr Leu
245 250 255
Thr Gly Thr Thr Thr Tyr Lys Gly Glu Gly Pro Ile Gly Phe Arg Ala
260 265 270
Thr Gln Ile
275
<210> 19
<211> 276
<212> PRT
<213> Unknown
<220>
<223> Aquimarina sp.
<400> 19
Met Ala Ile Tyr Gln Val Gln Asn Gln Trp Gly Gly Asn Ser Ala Pro
1 5 10 15
Trp His Ala Gly Gly Thr Trp Val Leu Gly Gly Arg Asp Asn Gln Asn
20 25 30
Val Val Ala Ile Asp Ile Lys Ser Gly Asp Gly Gly Arg Thr Phe Ser
35 40 45
Gly Thr Met Thr Tyr Glu Gly Glu Gly Pro Ile Gly Phe Lys Ala Ile
50 55 60
Gln Ile Ala Gly Asn Asn Tyr Ser Val Glu Asn Gln Trp Gly Gly Ala
65 70 75 80
Ser Ala Pro Trp His Pro Gly Gly Asn Trp Ile Ile Gly Gly Arg Asn
85 90 95
Gly Gln Asn Val Ile Glu Leu Asn Val Thr Ala Glu Ser Gly Ser Ala
100 105 110
Asn Leu Glu Gly Thr Met Lys Tyr Ala Gly Glu Gly Pro Ile Gly Phe
115 120 125
Lys Gly Gln Glu Thr Val Gly Ser Ser Tyr Ser Ile Glu Asn Gln Trp
130 135 140
Gly Gly Ala Ser Ala Pro Trp His Pro Gly Gly Thr Phe Val Leu Gly
145 150 155 160
Ala Arg Glu Asn Gln Asn Pro Val Ala Tyr Asp Ile Gln Ser Thr Asp
165 170 175
Gly Gly Lys Thr Phe Thr Gly Thr Met Thr Tyr Ala Gly Glu Gly Pro
180 185 190
Ile Gly Phe Arg Ala Ile Gln Thr Ala Gly Asn Asn Tyr Ala Ala Glu
195 200 205
Asn Gln Trp Gly Gly Ala Ser Ala Pro Trp His Pro Gly Gly Asn Leu
210 215 220
Val Ile Gly Ala Arg Val Asn Gln Asn Val Val Gln Leu Lys Ile Asn
225 230 235 240
Ser Asn Asp Asn Gly Glu Thr Phe Ser Gly Glu Met Thr Tyr Leu Gly
245 250 255
Glu Gly Pro Ile Gly Val Lys Ala Val Leu Ser Ser Arg Val Leu Ser
260 265 270
Gly Ala Thr Ser
275
<210> 20
<211> 133
<212> PRT
<213> Herpetosiphon aurantiacus
<400> 20
Met Ser Val Tyr Trp Val Glu Asn Gln Trp Gly Gly Asp Ser Ala Pro
1 5 10 15
Trp His Pro Gly Gly Thr Trp Val Leu Gly Ala Arg Asp Asn Gln Asn
20 25 30
Val Val Ala Ile Asn Ile Ser Ser Ala Asp Asn Gly Gln Thr Phe Thr
35 40 45
Gly Thr Met Thr Tyr Ile Asn Glu Gly Pro Ile Gly Phe Arg Ala Thr
50 55 60
Arg Thr Ser Ala Asn Asn Tyr Ala Val Glu Asn Gln Trp Gly Gly Asp
65 70 75 80
Ser Ala Pro Trp His Pro Gly Gly His Trp Ile Ile Gly Thr Arg Asp
85 90 95
Asn Gln Asn Pro Val Asn Leu Asp Val Asp Ser Arg Asp Gly Gly Gln
100 105 110
Thr Leu Asn Gly Thr Met Val Tyr Ala Gly Glu Gly Pro Ile Gly Phe
115 120 125
Arg Gly Lys Leu Gln
130
<210> 21
<211> 133
<212> PRT
<213> Unknown
<220>
<223> Chloroflexia bacterium
<400> 21
Met Ala Thr Tyr Ala Val Glu Asn Gln Trp Gly Gly Pro Asp Ala Pro
1 5 10 15
Trp His Ala Gly Gly Thr Trp Val Leu Gly Ala Arg Ser Glu Gln Ser
20 25 30
Val Val Ala Ile Asp Ile Ser Ser Ser Asp Gly Gly Asp Ser Phe Phe
35 40 45
Gly Thr Met Thr Tyr Ala Asn Glu Gly Pro Ile Gly Leu Arg Ala Thr
50 55 60
Leu Leu Asn Gly Asn Ser Tyr Asn Val Glu Asn Gln Trp Gly Gly Ser
65 70 75 80
Asn Ala Pro Trp His Pro Gly Gly Thr Trp Val Ile Gly Gly Arg Asp
85 90 95
Asn Gln His Val Ile Glu Leu His Val Ser Gly Asp Gly Asp Val Leu
100 105 110
Asp Gly Thr Asn Thr Tyr Val Gly Glu Gly Pro Ile Gly Phe His Gly
115 120 125
Val Leu Glu Ser Ala
130
<210> 22
<211> 274
<212> PRT
<213> Unknown
<220>
<223> Nostoc sp
<400> 22
Met Thr Ala Thr Ala Thr Ile Ser Asn Leu Tyr Ile Ala Gln Asn Gln
1 5 10 15
Trp Gly Gly Ser Ser Ala Pro Trp Asn Pro Gly Gly Ala Trp Val Ile
20 25 30
Gly Ala Arg Ser Asn Gln Arg Val Val Ala Leu Lys Val Thr Ser Ser
35 40 45
Asp Asn Gly Lys Thr Leu Asn Gly Thr Met Thr Tyr Ala Gly Glu Gly
50 55 60
Pro Ile Gly Phe Arg Gly Thr Leu Thr Thr Ser Asp Thr Tyr Lys Val
65 70 75 80
Glu Asn Gln Trp Gly Gly Ser Ser Ala Pro Trp Asn Pro Gly Gly Asn
85 90 95
Trp Ile Leu Gly Cys Arg Gly Asn Gln Asn Val Val Ala Ile Asp Ile
100 105 110
Thr Ser Asn Asp Gly Gly Asn Thr Leu Asn Gly Thr Ile Thr Tyr Ala
115 120 125
Gly Glu Gly Pro Ile Gly Phe Lys Ser Ala Ala Ala Asn Gly Ser Val
130 135 140
Tyr Thr Val Glu Asn Gln Trp Gly Gly Ala Ser Ala Pro Trp Asn Pro
145 150 155 160
Gly Gly Thr Trp Ala Leu Gly Cys Arg Asp Asn Gln Asn Val Val Ala
165 170 175
Ile Asn Val Thr Ser Asn Asp Gly Gly Lys Thr Leu Thr Gly Thr Asn
180 185 190
Thr Tyr Ala Gly Glu Gly Pro Ile Gly Phe Arg Gly Asn Leu Leu Gly
195 200 205
Ser Asn Asn Tyr Thr Val Glu Asn Gln Trp Gly Gly Ala Ser Ala Pro
210 215 220
Trp Asn Ala Gly Gly Thr Trp Val Ile Gly Cys Arg Ala Gly Gln Asn
225 230 235 240
Ala Val Ala Ile Asn Val Thr Ser Asn Asp Gly Gly Lys Thr Phe Thr
245 250 255
Gly Thr Met Thr Tyr Ala Gly Glu Gly Pro Ile Gly Phe Arg Ala Thr
260 265 270
Lys Ile
<210> 23
<211> 267
<212> PRT
<213> Solieria filliformis
<400> 23
Gly Arg Tyr Thr Val Gln Asn Gln Trp Gly Gly Ser Ser Ala Pro Trp
1 5 10 15
Asn Asp Ala Gly Val Phe Val Leu Gly Gly Arg Ala Asn Gln Asn Val
20 25 30
Met Ala Ile Asp Val Ser Ser Ser Asp Gly Gly Lys Thr Leu Thr Gly
35 40 45
Thr Met Thr Tyr Ser Gly Glu Gly Pro Ile Gly Phe Lys Gly Thr Arg
50 55 60
Arg Gly Glu Ser Asn Asn Tyr Glu Val Glu Asn Gln Trp Gly Gly Ser
65 70 75 80
Ser Ala Pro Trp His Pro Ala Gly Thr Phe Val Ile Gly Ser Arg Ser
85 90 95
Gly Gln Ala Val Val Ala Met Asn Val Thr Ser His Asp Gly Gly Lys
100 105 110
Thr Leu Ser Gly His Met Thr Tyr Glu Asn Glu Gly Pro Ile Gly Phe
115 120 125
Lys Gly Thr Gln Ala Glu Gly Asp Thr Tyr Asn Val Glu Asn Gln Trp
130 135 140
Gly Gly Ser Ser Ala Pro Trp Asn Lys Ala Gly Val Trp Ala Leu Gly
145 150 155 160
Ser Arg Ala Ser Gln Gly Val Val Lys Leu Asp Val Ser Ser Ser Asp
165 170 175
Gly Gly Lys Thr Leu Thr Gly Thr Met Gln Tyr Gln Asn Glu Gly Pro
180 185 190
Ile Gly Phe Arg Gly Thr Leu Thr Gly Ala Asn Asn Tyr Lys Ala Glu
195 200 205
Asn Gln Trp Gly Gly Ser Ser Gly Ala Trp Asn Pro Ala Gly Leu Trp
210 215 220
Leu Ile Gly Asp Arg His Asn Gln Asn Ile Ile Gly Val Lys Val Thr
225 230 235 240
Ser Asp Asp Asn Gly Lys Thr Leu Glu Gly Thr Cys Thr Tyr Tyr Arg
245 250 255
Glu Gly Pro Ile Gly Phe Lys Gly Val Ala Asn
260 265
<210> 24
<211> 256
<212> PRT
<213> Unknown
<220>
<223> Lyngbya sp.
<400> 24
Met Ser Thr Ala Pro Trp His Glu Gly Gly Lys Trp Val Ile Gly Gly
1 5 10 15
Arg Ser Asn Gln Asn Val Val Ala Ile Asn Val Lys Ser Gly Asp Asn
20 25 30
Gly Lys Thr Leu Asn Gly Thr Met Thr Tyr Ala Gly Glu Gly Pro Ile
35 40 45
Gly Phe Arg Ala Thr Leu Ser Gly Ser Asn Asn Tyr Met Val Glu Asn
50 55 60
Gln Trp Gly Gly Ser Ser Ala Pro Trp His Pro Gly Gly Gln Trp Val
65 70 75 80
Leu Gly Tyr Arg Thr Asp Gln Asn Val Val Glu Leu Asp Leu Lys Ser
85 90 95
Glu Asp Gly Gly Gln Thr Leu Asn Gly Thr Met Thr Tyr Gln Gly Glu
100 105 110
Gly Pro Ile Gly Phe Lys Ala Ala Met Ala Glu Gly Tyr Ala Tyr Thr
115 120 125
Val Glu Asn Gln Trp Gly Gly Ser Ser Ala Pro Trp Asn Glu Gly Gly
130 135 140
Thr Leu Val Leu Gly Ser Arg Asn Asn Gln Lys Val Val Ala Ile Asp
145 150 155 160
Ile Gln Ser Gly Asp Asn Gly Lys Thr Leu Asn Gly Thr Met Thr Tyr
165 170 175
His Gly Glu Gly Pro Ile Gly Phe Arg Ala Thr Leu Ser Gly Ser Asn
180 185 190
Asn Tyr Met Val Glu Asn Gln Trp Gly Gly Ser Ser Ala Pro Trp His
195 200 205
Pro Gly Gly Gln Trp Ile Ile Gly Tyr Arg Glu Asn Gln Asn Val Val
210 215 220
Ala Leu Asn Ile Asn Ser Asn Asp Glu Gly Thr Thr Leu Asn Gly Thr
225 230 235 240
Met Thr Tyr Gln Gly Glu Gly Pro Ile Gly Phe Lys Gly Ser Leu Met
245 250 255
<210> 25
<211> 267
<212> PRT
<213> Stigmatella aurantiaca
<400> 25
Met Ser Leu Tyr Gln Val Gln Asn Gln Trp Gly Gly Gln Ser Ala Ala
1 5 10 15
Trp Asn Pro Gly Gly Met Trp Ala Ile Gly Asn Arg Pro Asn Gln Asn
20 25 30
Val Ile Ala Leu Asn Leu Lys Ser Thr Asp Gly Gly Lys Thr Leu Thr
35 40 45
Gly Thr Met Thr Tyr Ala Gly Glu Gln Ala Ile Gly Val Gln Ala Ala
50 55 60
Gln Ala Gly Thr Asn Ser Tyr Thr Val Gln Asn Gln Trp Gly Gly Ser
65 70 75 80
Ser Ala Pro Trp Gln Pro Gly Gly Ser Trp Ile Leu Gly Asp Arg Pro
85 90 95
Asn Gln Ser Val Val Ala Ile Asp Ile Thr Ser Thr Asp Gly Gly Arg
100 105 110
Thr Leu Thr Gly Thr Ile Thr Tyr Ala Gly Glu Asn Pro Ile Gly Phe
115 120 125
Lys Ala Glu Gln Ser Ala Gly Gly Met Tyr Ser Val Gln Asn Gln Trp
130 135 140
Gly Gly Ser Ser Ala Ala Trp Gln Gln Gly Gly Ala Trp Val Val Gly
145 150 155 160
Ala Arg Gln Asn Gln Ser Val Val Ala Ile Lys Ala Thr Ser Thr Asp
165 170 175
Gly Gly Lys Thr Leu Thr Gly Thr Met Thr Tyr Ser Gly Glu Gly Ala
180 185 190
Ile Gly Phe Lys Ala Thr Leu Ser Gly Asp Asn Thr Tyr Thr Val Gln
195 200 205
Asn Gln Trp Gly Gly Ala Ser Ala Pro Trp Gln Pro Gly Gly Gln Trp
210 215 220
Ile Leu Gly Ala Arg Lys Gly Gln Gly Val Ile Ala Ile Asp Val Thr
225 230 235 240
Ser Asn Asp Gly Gly Lys Thr Leu Ala Gly Thr Met Thr Tyr Ala Gly
245 250 255
Glu Gly Pro Ile Gly Phe Arg Gly Thr Leu Asn
260 265
<210> 26
<211> 273
<212> PRT
<213> Unknown
<220>
<223> Hyalangium minutum
<400> 26
Met Leu Gly Gly Arg Pro Asn Gln Ser Val Val Ala Ile Gln Val Lys
1 5 10 15
Ser Gln Asp Asp Gly Gln Thr Leu Thr Gly Thr Met Thr Tyr Asn Gly
20 25 30
Glu Gly Pro Ile Gly Phe Arg Ala Lys Ala Thr Gly Asn Asn Gln Tyr
35 40 45
Ala Val Glu Asn Gln Trp Gly Gly Ala Ser Ala Pro Trp Gln Pro Gly
50 55 60
Gly Thr Trp Val Ile Gly Gly Arg Ser Gly Gln Ala Val Val Ala Leu
65 70 75 80
Asp Val Lys Ser Ala Asp Gln Gly Lys Ser Leu Ser Gly Thr Val Thr
85 90 95
Tyr Lys Gly Glu Gly Pro Ile Ser Phe Lys Gly Met Leu Gly Ser Gly
100 105 110
Ala Pro Ala Ser Ala Ser Ala Pro Ala Pro Ala Pro Ala Pro Ala Pro
115 120 125
Ala Pro Ala Ser Tyr Val Val Leu Ile Ala Lys His Ser Gly Lys Val
130 135 140
Val Gly Val Ala Gly Gln Ser Lys Asp Asn Gly Ala Pro Val Ile Gln
145 150 155 160
Trp Ala Pro Ser Lys Thr Asp Asn Glu Lys Trp Ile Val Glu Pro Ala
165 170 175
Ala Asp Gly Tyr Val Ile Leu Lys Ala Met His Ser Gly Lys Val Leu
180 185 190
Asn Val Ser Gly Asn Ser Lys Thr Pro Gly Ala Lys Val Val Gln Trp
195 200 205
Pro Gln Ser Gly Thr Asp Asn Glu Lys Trp Lys Ile Asp Ser Thr Pro
210 215 220
Asp Gly Phe Val Thr Leu Thr Ala Lys His Ser Gly Gln Val Leu Asn
225 230 235 240
Val Ser Gly Asn Ser Lys Ala Asp Gly Gly Glu Leu Val Gln Trp Pro
245 250 255
Lys Ser Gly Thr Asp Asn Glu Lys Phe Lys Leu Val Lys Met Pro Leu
260 265 270
Asn
<210> 27
<211> 143
<212> PRT
<213> Janthinobacterium agaricidamnosum
<220>
<221> misc_feature
<222> (137)..(138)
<223> Xaa can be any naturally occurring amino acid
<400> 27
Met Ser Lys Thr Ser Lys Ser Ala Asn Asn Leu His His Val Lys Asn
1 5 10 15
Gln Trp Gly Gly Pro Ser Ala Pro Trp Asn Glu Gly Gly Val Trp Val
20 25 30
Leu Gly Gly Arg Ser Gly Gln Asn Val Ala Ala Leu Asn Ile Asn Ser
35 40 45
Ala Asp Gly Gly Asn Thr Phe Thr Gly Ala Met Lys Tyr Val Gly Glu
50 55 60
Gly Gln Ile Gly Phe Arg Ala Thr Leu Thr Gln Ser Asn Thr Tyr Leu
65 70 75 80
Val Glu Asn Gln Trp Gly Gly Asp Ser Ala Pro Trp Asn Pro Gly Gly
85 90 95
Thr Trp Val Ile Gly Gly Arg Ser Asn Gln Asn Val Val Ala Leu Asn
100 105 110
Val Glu Ser Ser Asp Gly Gly Asn Thr Leu Ala Gly Ser Met Ser Tyr
115 120 125
Asn Gly Glu Gly Pro Ile Gly Phe Xaa Xaa Thr His Leu Ile Asn
130 135 140

Claims (13)

1. A chimeric antigen receptor comprising, in order, a mannose binding molecule, a hinge region, a transmembrane region, and an intracellular region, wherein the intracellular region comprises an intracellular costimulatory region and a signaling region,
the mannose binding protein is shown as SEQ ID NO. 2,
the amino acid sequence of the hinge region is shown as SEQ ID NO. 4,
the amino acid sequence of the transmembrane region is shown as SEQ ID NO. 5,
the amino acid sequence of the intracellular costimulatory region is shown as SEQ ID NO 6, an
The amino acid sequence of the signal conduction region is shown as SEQ ID NO. 7.
2. The chimeric antigen receptor according to claim 1, further comprising a signal peptide at the N-terminus as shown in SEQ ID No. 1 and a linker between the mannose binding protein and the hinge region as shown in SEQ ID No. 3.
3. A nucleic acid molecule comprising a sequence selected from:
(1) a coding sequence for the chimeric antigen receptor of claim 1 or 2;
(2) the complementary sequence of (1).
4. A nucleic acid construct comprising the nucleic acid molecule of claim 3.
5. The nucleic acid construct of claim 4, wherein said nucleic acid construct is a vector.
6. A host cell, said host cell:
(1) expressing the chimeric antigen receptor of claim 1 or 2, and/or
(2) Comprising the nucleic acid molecule of claim 3 and/or the nucleic acid construct of claim 4 or 5.
7. The host cell of claim 6, wherein the host cell is an immune cell.
8. The host cell of claim 6, wherein the host cell is a T cell.
9. A pharmaceutical composition comprising pharmaceutically acceptable excipients and:
(1) the chimeric antigen receptor of claim 1 or 2, or
(2) The nucleic acid molecule of claim 3, the nucleic acid construct of claim 4 or 5, or the host cell of claim 6 or 7 or 8.
10. The pharmaceutical composition of claim 9, wherein the pharmaceutical composition is for use in the treatment of a tumor selected from one or more of the following: gastric cancer, thyroid tumor, gallbladder cancer, cholangiocarcinoma, lung cancer, melanoma, head and neck cancer, breast cancer, ovarian cancer, cervical cancer, liver cancer, colorectal cancer, brain glioma, pancreatic cancer, bladder cancer, prostate cancer, renal cancer, osteosarcoma.
11. Use of an agent for the preparation of an activated immune cell, said agent comprising:
(1) the chimeric antigen receptor of claim 1 or 2, or
(2) The nucleic acid molecule of claim 3, the nucleic acid construct of claim 4 or 5, or the cell of claim 6 or 7 or 8.
12. The use of claim 11, wherein the immune cell is a T cell.
13. Use of an agent for the manufacture of a medicament for the treatment of a tumour, said agent comprising:
(1) the chimeric antigen receptor of claim 1 or 2, or
(2) The nucleic acid molecule of claim 3, the nucleic acid construct of claim 4 or 5, or the cell of claim 6 or 7 or 8.
CN202210741551.8A 2022-06-28 2022-06-28 Chimeric antigen receptor targeting terminal mannose and application thereof Pending CN114805611A (en)

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