CN112552414A - LILRB4 and B7-H3 double-targeting chimeric antigen receptor and application thereof - Google Patents

LILRB4 and B7-H3 double-targeting chimeric antigen receptor and application thereof Download PDF

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CN112552414A
CN112552414A CN202011596313.XA CN202011596313A CN112552414A CN 112552414 A CN112552414 A CN 112552414A CN 202011596313 A CN202011596313 A CN 202011596313A CN 112552414 A CN112552414 A CN 112552414A
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朱建高
杨文君
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Zhejiang Compvss Biotechnology Co ltd
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Abstract

The invention relates to the field of chimeric antigen receptors, and discloses a chimeric antigen receptor. The chimeric antigen receptor comprises an anti-LILRB 4 single-chain antibody, an anti-B7-H3 single-chain antibody, a human CD8 hinge transmembrane region, a human 4-1BB intracellular region, a human CD3 zeta intracellular region, a human P2A peptide and a human IFN full length which are connected in sequence. Cytological experiment results show that the T cells expressing the chimeric antigen receptor have killing effect on tumor cells better than that of single-target same type CAR-T. Animal experiment results show that the T cell expressing the chimeric antigen receptor can obviously prolong the survival rate of animals, and the effect is better than that of single-target similar CAR-T. The invention also discloses application of the LILRB4 and B7-H3 double-targeting chimeric antigen receptor in preparation of a medicament for treating acute myeloid leukemia.

Description

LILRB4 and B7-H3 double-targeting chimeric antigen receptor and application thereof
Technical Field
The invention relates to the technical field of chimeric antigen receptors, in particular to a LILRB4 and B7-H3 double-targeting chimeric antigen receptor and application thereof.
Background
Chimeric antigen receptor T (CAR-T) cell therapy is becoming a promising immunotherapeutic strategy due to its significant efficacy in hematological tumors. CAR-T cell refers to a T cell that has been artificially genetically engineered to express a Chimeric Antigen Receptor (CAR) on the cell surface. CARs are a core component of CAR-T, conferring on T cells the ability to recognize tumor surface Tumor Associated Antigens (TAAs) in an histocompatibility antigen (HLA) -independent manner. Thus, CAR-T cells are able to specifically target tumor cells expressing TAA surface markers in vivo and kill tumor cells by activating a T cell immune response. In 2010, the first B-cell lymphoma patient received CAR-T cell therapy targeting CD19 and achieved exciting positive results. Since then, an increasing number of research groups have demonstrated the safety and efficacy of CAR-T technology in the treatment of B-cell hematological tumors through clinical trials.
Acute Myeloid Leukemia (AML) is the most common form of Leukemia in adults with the highest mortality rate and remains a therapeutic challenge. Currently, chemotherapy is still the main treatment for acute myeloid leukemia, and allogeneic hematopoietic stem cell transplantation (allo-HSCT) is bridged after patients reach complete remission. The method can completely relieve partial AML patients, and the Disease-free survival (DFS) of 5 years of AML patients receiving allo-HSCT can reach 40-50%. However, approximately 43% of patients relapse after receiving primary chemotherapy, while only half of relapsing patients achieve complete remission with less than 15% survival in the case of relapses. Furthermore, complete remission is not achieved in 18% of patients after multiple chemotherapy-induced events.
There are currently over 1000 CAR-T related clinical trials documented worldwide, and three CAR-T products approved by the FDA for marketing for the treatment of acute B-cell leukemia (B-ALL), diffuse large B-cell lymphoma (DLBCL), and Mantle Cell Lymphoma (MCL), respectively. However, due to the heterogeneity of myeloid hematological tumors, current CAR-T therapy is not ideal for the treatment of AML. CAR-T therapies that have been developed to target targets such as CD33, CD123, etc., have been prevalent in clinical applications with varying degrees of hematopoietic damage. The reason for this is that, primarily, the targets are expressed to varying degrees in normal hematopoietic progenitor cells, so that the persistence of CAR-T cells in vivo may cause irreversible off-target toxicity to the hematopoietic system.
CAR-T cell therapy in clinical practice, another important problem to be solved is relapse. Of the acute B-lymphoblastic leukemia patients who received CD19 CAR-T therapy and achieved complete remission, approximately 30-50% eventually relapsed, with most patients relapsing within 1 year after the first treatment. Recurrence was divided into two cases, CD 19-positive tumor recurrence and CD 19-negative tumor recurrence. In the cases receiving CD19 CAR-T treatment, more than 60% of relapses were CD19 negative, i.e. caused by loss of CD19 surface antigen by tumor cells.
Therefore, there is a need in the art to develop a CAR-T product that can both chase after acute myeloid leukemia tumor cells that develop antigen escape, and that does not cause irreversible off-target toxicity to the hematopoietic system.
Disclosure of Invention
One of the purposes of the invention is to provide a chimeric antigen receptor double-targeted by LILRB4 and B7-H3.
In order to achieve the purpose, the invention adopts the technical scheme that: a LILRB4 and B7-H3 dual-targeted chimeric antigen receptor, the amino acid sequence of which comprises:
sequentially connected anti-LILRB 4 single-chain antibody, anti-B7-H3 single-chain antibody, human CD8 hinge transmembrane region, human 4-1BB intracellular region, human CD3 zeta intracellular region, human P2A peptide and human IFN protein peptide; or
The chimeric antigen receptor has the amino acid sequence with one or more amino acid substitutions, deletions or additions and has similar biological activity.
Preferably, the chimeric antigen receptor comprises a first functional protein or a second functional protein; or
A fusion protein obtained by connecting a label to the N end or/and the C end of the first functional protein or the second functional protein;
the amino acid sequence of the first functional protein is shown as SEQ ID No.4 or SEQ ID No.5 or SEQ ID No. 6;
the amino acid sequence of the second functional protein is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence of the first functional protein, and the second functional protein has the same biological activity as the first functional protein.
Specifically, the amino acid sequence of the anti-LILRB 4 single-chain antibody is the amino acid sequence from 22 th to 280 th positions in SEQ ID NO.4 or an amino acid sequence with similar biological activity; and/or
The amino acid sequence of the anti-B7-H3 single-chain antibody is the amino acid sequence at position 296-525 in SEQ ID NO.4 or an amino acid sequence with similar biological activity; and/or
The amino acid sequence of the human CD8 hinge transmembrane region is the amino acid sequence at position 526-594 of SEQ ID NO.4 or an amino acid sequence with similar biological activity; and/or
The amino acid sequence of the human 4-1BB intracellular domain is the amino acid sequence at position 595-641 of SEQ ID NO.4 or an amino acid sequence with similar biological activity; and/or
The amino acid sequence of the intracellular region of human CD3 zeta is the amino acid sequence at position 642-760 in SEQ ID NO.4 or has similar biological activity with the amino acid sequence; and/or
The amino acid sequence of the human P2A peptide is shown as 761-779 in SEQ ID NO.4 or an amino acid sequence with similar biological activity;
the human IFN is human IFN alpha 2a or human IFN alpha 2b or human IFN beta;
the amino acid sequence of the human IFN alpha 2b is shown as amino acid 780-967 of SEQ ID NO. 4; or
(ii) an amino acid sequence having similar biological activity as the human IFN α 2 b;
the amino acid sequence of the human IFN alpha 2a is shown as amino acid 780-967 of SEQ ID NO. 5; or
(ii) an amino acid sequence having similar biological activity as the human IFN α 2 a;
the amino acid sequence of the human IFN beta is shown as the amino acid 780-966 of SEQ ID NO 6; or
An amino acid sequence having similar biological activity as the human IFN β;
amino acids with similar biological activity refer to families of amino acid residues with similar side chains, including amino acids with basic side chains, amino acids with acidic side chains, amino acids with uncharged polar side chains, amino acids with nonpolar side chains, amino acids with β -branched side chains, and amino acids with aromatic side chains.
Preferably, the amino acid sequence of the signal peptide is the amino acid sequence from position 1 to 21 of SEQ ID NO.4 or an amino acid sequence having similar biological activity thereto.
The second objective of the invention is to provide a polynucleotide sequence.
In order to achieve the purpose, the invention adopts the technical scheme that: a polynucleotide sequence encoding the chimeric antigen receptor described above.
Preferably, a first gene sequence or a second gene sequence is included; or
A third gene sequence obtained by hybridizing the first gene sequence or the second gene sequence with a nucleotide sequence;
the first gene sequence is shown as SEQ ID No.1 or SEQ ID No.2 or SEQ ID No. 3;
the second gene sequence is a nucleotide sequence with identity of more than 75% with the first gene sequence.
Specifically, the coding sequence of the anti-LILRB 4 single-chain antibody is shown as the polynucleotide sequence at position 64-840 of SEQ ID NO. 1; and/or
The coding sequence of the anti-B7-H3 single-chain antibody is shown as the sequence of the polynucleotide at the 886-1575 position of SEQ ID NO. 1; and/or
The coding sequence of the human CD8 hinge transmembrane region is shown as the polynucleotide sequence at the 1576-1782 site in SEQ ID NO. 1; and/or
The coding sequence of the human 4-1BB intracellular region is shown as the polynucleotide sequence at 1783-1923 in SEQ ID NO. 1; and/or
The coding sequence of the intracellular region of human CD3 zeta is shown as the polynucleotide sequence at the 1924-2280 th site in SEQ ID NO. 1; and/or
The coding sequence of the human P2A peptide is shown as the polynucleotide sequence at 2281-2337 th site in SEQ ID NO: 1; and/or
The human IFN full-length sequence is the full-length sequence of any one gene of human IFN alpha 2a, human IFN alpha 2b and human IFN beta, or oIFN alpha 2a, oIFN alpha 2b and oIFN beta obtained by gene optimization of human IFN alpha 2a, human IFN alpha 2b or human IFN beta respectively.
The invention also aims to provide a chimeric antigen receptor T cell double-targeted by LILRB4 and B7-H3. The LILRB4 and B7-H3 double-targeted chimeric antigen receptor T cell is prepared by introducing the gene sequence coding the chimeric antigen receptor into the T cell.
In order to achieve the purpose, the invention adopts the technical scheme that: a chimeric antigen receptor T cell double-targeted by LILRB4 and B7-H3, wherein the chimeric antigen receptor of any one of claims 1-3 is stably expressed in the T cell.
The LILRB4-B7H3-CAR-IFN polynucleotide sequence is obtained by adding a gene-optimized full-length coding sequence of human IFN to the C-terminal end of the LILRB4-B7H3-CAR sequence. Wherein the IFN gene sequence can be the full-length sequence of any one gene among human IFN alpha 2a, human IFN alpha 2b and human IFN beta. The human IFN alpha 2a and human IFN alpha 2b amino acid sequence is highly similar, only in the 23 rd amino acid difference (human IFN alpha 2a 23 rd amino acid is K, human IFN alpha 2b 23 rd amino acid is R, belonging to conservative substitution). Human IFN beta also belongs to type I interferon, has similar biological functions compared with the two types I interferon, and has the functions of inducing tumor cell apoptosis and regulating immune cell activity. Often in clinical research IFN alpha 2 and IFN beta mutual replacement use. Animal experiments show that after a full-length fragment of human IFN alpha 2B gene is added at the C terminal of the B7H3-CAR, the CAR-T cell expressing the B7H3-CAR-IFN alpha 2B has stronger tumor killing capacity in animals compared with the B7H3-CAR sequence. The applicants therefore believe that any of the three genes described above can act synergistically on CAR-T cells.
The human CD8 hinge transmembrane region suitable for use in the present invention can be the various human CD8 hinge transmembrane region sequences commonly used in the art for CARs. As an illustrative example, the amino acid sequence of the human CD8 alpha hinge transmembrane region of the present invention is shown as amino acids 526 and 594 of SEQ ID NO. 4.
The human 4-1BB intracellular domain suitable for use in the present invention may be any of the various human 4-1BB intracellular domains known in the art for CAR. As an illustrative example, the amino acid sequence of the intracellular domain of human 4-1BB for use in the present invention is shown in SEQ ID NO 4 at position 595-641.
The intracellular domain of human CD3 ζ suitable for use in the present invention may be various intracellular domains of human CD3 ζ conventionally used in CARs in the art. As an illustrative example, the amino acid sequence of the intracellular domain of human CD3 ζ is shown as amino acids 642-760 of SEQ ID NO. 4.
P2A peptides suitable for use in the invention can be various self-cleaving sequences conventionally used in the art for CARs. As an illustrative example, the amino acid sequence of the P2A peptide is shown as amino acids 761-779 of SEQ ID NO. 4. The invention also includes mutants of the CAR shown in SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO. 6. 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 (e.g., activating T cells) of the CAR. Sequence identity between two aligned sequences can be calculated using, for example, BLASTp from NCBI.
Mutants also include: an amino acid sequence having one or several mutations (insertions, deletions or substitutions) in the amino acid sequence shown in SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, 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 invention adopts the gene sequences of an anti-LILRB 4 single-chain antibody (specifically scFv derived from clone No. 293623) and an anti-B7-H3 single-chain antibody (specifically scFv derived from clone No. C11D5.3), or the scFv sequences constructed by the antibody obtained by screening through a phage display technology. The NCBI GenBank database was searched for information on the human CD8 hinge transmembrane region, the human 4-1BB intracellular region, the human CD3 zeta intracellular region, the P2A peptide and the full-length cDNA sequence of the human IFN gene (including the full-length cDNA sequences of the human IFN alpha 2a, human IFN alpha 2b and human IFN beta genes). The cDNA full-length sequences of the human IFN genes are subjected to gene optimization respectively to obtain IFN full-length sequences (oIFN, including oIFN alpha 2a, oIFN alpha 2b and oIFN beta) with highest expression efficiency in human T cells.
The invention synthesizes the gene segment of the chimeric antigen receptor anti-LILRB 4 scFv-B7-H3scFv-CD8TM-4-1BB-CD3 zeta-oIFN through the whole gene and inserts the gene segment into a retrovirus vector. The recombinant plasmid packages the virus in ECO cells, infects T cells, and causes the T cells to express the chimeric antigen receptor. The transduction method of the present invention for modifying T lymphocytes with the chimeric antigen receptor gene is based on a retrovirus transduction method. The method has the advantages of high transduction efficiency, stable expression of exogenous genes, high batch stability, shortened time for in vitro culture of T lymphocytes to reach clinical level, and the like. The transduced nucleic acid is expressed on the surface of the CAR-T cell by transcription and translation. The proportion of retrovirus-infected T lymphocytes and the expression of cell surface CAR can be calculated by flow cytometry by measuring the amount of protein L bound to the kappa chain of the anti-B7-H3 single chain antibody. The invention transduces T lymphocytes through retrovirus, and the proportion of the obtained CAR positive T lymphocytes is up to 80%. In vitro enzyme-linked immunosorbent assay (ELISA) detection shows that CAR-T cells can secrete a large amount of IFN to the culture supernatant, which indicates that retrovirus successfully transduces T cells and expresses secretory IFN. The killing function of CAR-T cells on specific tumor cells can be detected by CFSE labeling experiments. The CAR-T cells prepared by the invention have strong killing function on LILRB4 or B7-H3 positive tumor cells, and the killing efficiency is over 80 percent under the condition that the effective target ratio is 1 to 1. At an effective target ratio of 1 to 27, the killing efficiency still exceeds 30%. In animal experiments, the LILRB4-B7H3-CAR-IFN T cells were able to kill U266 tumor cells (LILRB4+ B7H3+) transplanted into animals more effectively and durably than CAR-T cells targeting LILRB4 alone.
The invention also aims to provide application of the LILRB4 and B7-H3 double-targeted chimeric antigen receptor T cell, which is characterized in that the LILRB4 and B7-H3 double-targeted chimeric antigen receptor T cell is used for preparing a medicament for treating acute myelocytic leukemia.
The invention constructs a CAR expressing LILRB4 and B7-H3 double-target scFv at the same time for the first time and is used for preparing CAR-T cells. Meanwhile, a human IFN full-length gene is added at the C-terminal of the CAR, and the CAR-T cell which simultaneously expresses the CAR and releases secretory human IFN protein is obtained. Compared with the prior art, the invention has the beneficial effects that:
1) both LILRB4 and B7-H3 demonstrated high expression in tumor tissues of AML patients, and the expression level was positively correlated with prognosis, suggesting that it has important functions in tumor development, and CAR-T cells targeting these two TAAs can effectively inhibit tumor growth;
2) LILRB4 and B7-H3 were slightly different in differently typed AML, LILRB4 was mainly expressed in monocytic AML (M4/M5), while B7-H3 was highly expressed in M3 and M5-typed AML, and NMP 1-mutated AML, the complementary expression profiles of the two TAAs were more favorable to prevent antigen escape;
3) LILRB4 and B7-H3 both have the property of immune checkpoints, while targeting two antigens may have additional immune activation functions;
4) the CAR-T cells of LILRB4 and B7-H3 are doubly targeted, and the synergistic effect of IFN is matched, so that microenvironment immune cells can be activated to the maximum extent, and the antigen immune escape effect is prevented. Among them, IFN-potentiated fourth generation CARs are also the original design of this team. Cytokines can modulate the immune microenvironment around the tumor tissue while acting as a third signal, further increasing the level of CAR-T cell response. Cytokines include interleukins, interferons, tumor necrosis factor superfamily, colony stimulating factors, chemokines, growth factors, etc., and are many hundreds in variety. The selection of type I interferon as the third signal is based on the results of the previous studies and a great deal of previous work by the applicant. Firstly, the I-type interferon is the earliest interferon type researched at present, and the physiological function and potential side effect of the I-type interferon are deeply and comprehensively known; secondly, the I-type interferon has multiple regulation effects, on one hand, the I-type interferon can directly induce tumor cell apoptosis, and on the other hand, the I-type interferon can also regulate the activity of T cells; finally, artificially prepared type I interferon recombinant proteins, such as INF α 2a, IFN α 2b, IFN β, and the like, have been clinically applied to various tumor treatments, but since directly injected interferon recombinant proteins have short half-lives in vivo and do not easily reach focal sites, the combined use of type I interferons with CAR-T cell therapy is advantageous to maximize the biological functions of IFN α 2b at the right time and place. The human IFN gene sequence can be the full-length sequence of any one gene among human IFN alpha 2a, human IFN alpha 2b and human IFN beta. The design is ingenious in that the CAR gene and the IFN gene are separated by P2A peptide, so that CAR and secretory IFN protein can be expressed simultaneously. At the same time when the CAR-T cell reaches the tumor focus and activates the CAR gene, the P2A peptide is hydrolyzed under the action of intracellular protease to release free IFN which is secreted to the outside of the cell to play an immune activation function. The expression of IFN is regulated by CAR gene, so that the IFN activity can be released at the focus position, and the effect of precise synergy can be achieved.
Drawings
FIG. 1 is a schematic representation of the sequence of CAR; ScFv: a single chain antibody variable region; hinge: a CD8 hinge region; TM: the CD8 transmembrane domain. lilrb4-CAR T; b. b7h3-CAR T; lilrb4-CAR-IFN T; lilrb4-B7H3-CAR-IFN T;
FIG. 2A, flow cytometry analysis shows the positive rate of CAR-T cell surface Protein L, i.e. the expression efficiency of CAR, for the CD4+ and CD8+ subpopulations 3 days after retroviral infection of T cells; b, detecting the content of IFN alpha 2 in the supernatant of the CAR-T cell culture medium after retroviral infection by enzyme-linked immunosorbent assay (ELISA);
FIG. 3 shows the detection of target cell lysis rate by CFSE labeling after co-culture of CAR-T cells and target cells at different effective target ratios;
FIG. 4 is a graph of D-luciferin sodium salt imaging after tail vein injection of CAR-T cells in U266 tumor transplantation model to observe tumor cell residues in mice; a, main experimental process; b, pictures show sodium salt imaging results of mice of each group. And C, counting survival curves of the mice of each group at different time points.
Detailed Description
The present invention will be further described with reference to the following 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.
Example 1:
1) determination of LILRB4-B7H3-CAR-IFN alpha 2B T gene sequence and construction of retroviral vector:
the scFv sequence of LILRB4 was derived from clone # 293623; the scFv sequence of B7H3 was derived from clone number C11D5.3. From NCBI website database search for human CD8 hinge transmembrane region, human 4-1BB intracellular region, human CD3 ζ intracellular region and human IFN alpha 2b full-length cDNA sequence information. The full-length cDNA sequence of the wild-type human IFN alpha 2b gene is called nIFN alpha 2 b. Codon optimization is carried out on the nIFN alpha 2b sequence on website http:// sg.idtdna.com/site to obtain oIFN alpha 2b, and the better suitability for human cell expression under the condition of unchanged coding amino acid sequence is ensured.
The full-length polynucleotide sequence of LILRB4-B7H3-CAR-IFN alpha 2B is obtained according to the sequence of LILRB4 scFv, linker, B7-H3scFv, human CD8 hinge transmembrane region, human 4-1BB intracellular region, human CD3 zeta intracellular region, P2A peptide and oIFN alpha 2B. Meanwhile, LILRB4-CAR comprising only LILRB4 scFv, human CD8 hinge transmembrane region, human 4-1BB intracellular region, human CD3 zeta intracellular region, LILRB4-CAR comprising LILRB4 scFv, human CD8 hinge transmembrane region, human 4-1BB intracellular region, human CD3 zeta intracellular region, P2A peptide, human IFN full-length LILRB4-CAR-IFN, and three full-length polynucleotide sequences comprising B7-H3scFv, human CD8 hinge transmembrane region, human 4-1BB intracellular region, human CD3 intracellular region, P2 zeta 2A peptide, human IFN full-length B7H3-CAR-IFN were constructed as controls. A CAR targeting CD19 (CTR-CAR) was also constructed as a negative control. The full-length polynucleotide sequence and the amino acid sequence information of the LILRB4-B7H3-CAR-IFN alpha 2B are shown in a nucleotide sequence table (SEQ ID NO.1, SEQ ID NO. 4). The LILRB4-CAR full-length polynucleotide sequence is shown in SEQ ID NO. 8. The LILRB4-CAR-IFN full-length polynucleotide sequence is shown in SEQ ID NO. 9. The full-length polynucleotide sequence of the B7H3-CAR-IFN is shown as SEQ ID NO. 10. The sequence of the full-length polynucleotide of the CTR-CAR is shown as SEQ ID NO. 7. All the above polynucleotides were synthesized by Scutellaria Biotech, Inc., cloned in pUC57 vector, and sequenced again.
The nucleotide sequences of the above various CARs were double-digested with NotI (NEB) and EcoRI (NEB), ligated by T4 ligase (NEB), inserted into the NotI-EcoRI site of retrovirus (MP71), and transformed into competent E.coli (DH 5. alpha.).
The plasmids were extracted and purified using a plasmid purification kit from Qiagen, and the various CAR plasmids obtained above were subjected to retroviral packaging experiments. Finally 5 retroviruses expressing CARs of different sequences were obtained.
The plasmid map constructed in this step is shown in FIG. 1.
2) Establishment of retroviral packaging and toxigenic strains:
using the retroviral vectors expressing various CARs prepared in step 1), 5 retroviruses were packaged separately according to the following method:
1. day 1: phoenix Ecotropic (ECO) cells should be less than 20 passages, but not overgrown. At 0.6X 106Laying the cells in a density plate of per ml, adding 10ml of DMEM medium into a 10cm dish, fully and uniformly mixing the cells, and culturing the cells at 37 ℃ overnight;
2. day 2: the ECO cell fusion degree reaches about 90 percent for transformationDyeing (generally, paving for 14-18h or so); preparation of plasmid MP 71-12.5. mu.g of target Gene, 1.25M CaCl2 250μl，H2O1 ml, the total volume is 1.25 ml; in another tube, an equal volume of 2 × HBS to the plasmid complex was added, and the plasmid complex was vortexed for 20 s. The mixture was gently added to the ECO dish edge to edge, incubated at 37 ℃ for 4h, medium removed, washed once with PBS, and re-added with pre-warmed fresh medium.
3. Day 4: after transfection for 48h, the supernatant was collected and filtered through a 0.45um filter to obtain a retrovirus solution, which was stored at-80 ℃.
4. Establishing an toxigenic strain: the obtained retrovirus infects HY268 cells, and after two days of infection, flow cell sorting is carried out, and the cell strain with the highest secretory retrovirus titer and derived from single cells is screened and stored for a long time. The cell strain can be used for preparing retrovirus supernatant in a large scale for preparing CAR-T cells by gene transduction.
3) Retrovirus infects human T cells:
1. the frozen healthy human peripheral blood PBMC were thawed and cell density was adjusted to 1-2X 106/ml using 10% FBS in RPMI-1640 complete medium.
PBMC is collected from Ficoll separating liquid (tertiary Tianjin), and is separated by a magnetic bead method to obtain purer CD3+ T cells, and clinical-grade Dynabeads Human T Expander CD3/CD28 magnetic beads (Invitrogen) are added according to the ratio of the magnetic beads to the CD3+ cells of 3:1 to activate the T cells.
The day after T cell activation, the non-tissue treated plates were coated with Retronectin (Takara) diluted with PBS to a final concentration of 15. mu.g/ml, 1.2ml per well of 6-well plates. Protected from light and kept at 4 ℃ overnight for use.
And 4, after the T cells are activated and cultured for two days, taking out the coated 6-hole plate, sucking away the coating solution, and adding PBS to wash the plate once.
5. Adding the retrovirus liquid prepared in the step 2) into the holes, adding 5-6ml of the retrovirus liquid into each hole, centrifuging at 32 ℃ and 2000 Xg for 2 h. 3ml of fresh complete medium containing hIL-2(500U/ml) was added to each well and incubation was continued for 1 day.
6. After the cells are infected, the density of the cells is observed every day, and T cell culture solution containing IL-2100U/ml is supplemented timely to maintain the density of the T cells at about 5 multiplied by 105/ml, so that the cells can be conveniently expanded.
7. Thus, CAR-T cells (LILRB4-B7H3-CAR-IFN T, LILRB4-CAR-IFN T, LILRB4-CAR T, B7H3-CAR-IFN T and CTR-CAR T) infected with the five retroviruses prepared in step 2), respectively, were obtained.
4) The enzyme-linked immunosorbent assay detects the proportion of T lymphocytes after infection and the expression of surface CAR protein and IFN alpha 2b protein:
since the light chain of the anti-B7-H3 single chain antibody is kappa chain capable of binding Protein L, we used FACS methods to elucidate the proportion of CAR-positive T lymphocytes and expression of CAR Protein by detecting biotin-labeled Protein L bound to CAR-T cells.
Centrifuging respectively to collect two CAR-T cells (LILRB4-B7H3-CAR-IFN T, LILRB4-CAR T, experimental group) and Control T cell (CTR-CAR T, Control group) prepared in step 3) 72 hours after infection, washing 1 time with 1% BSA-PBS, discarding supernatant, adding biotin (biotin) -labeled protein L antibody, washing 3 times with 1% BSA-PBS after keeping out of the sun for 30min, and resuspending; adding PE-labeled avidin (Streptavidin), washing with 1% BSA-PBS after 10min in dark, and resuspending; and finally, detecting the fluorescence intensity of the PE by a flow cytometer.
Fig. 2a shows that the positive rate of Protein L (CAR) in both CD4+ T cells and CD8+ T cells reached 80% 3 days after T cells were infected with the retrovirus prepared in step 3).
IFN alpha 2b and IFN alpha 2a belong to IFN alpha 2 subfamily, have high sequence homology, through ELISA detection supernatant IFN alpha 2 content, can verify IFN alpha 2b expression level. Taking the five CAR-T cells obtained in the step 3) as test cells, and operating according to the following steps: test cells were collected 72 hours after infection by centrifugation, and the cultured supernatant was collected and assayed for IFN α 2 content by ELISA (Biolegend).
In FIG. 2, B shows ELISA results, the content of IFN alpha 2B in the supernatant of LILRB4-B7H3-CAR-IFN T cells is significantly higher than that in the supernatant of CTR-CAR T and LILRB4-CAR T cells. The results confirmed that LILRB4-B7H3-CAR-IFN T cells can express secreted IFN alpha 2B.
5) Detecting the specific killing effect of the CAR-T cells on the tumor cells by a CFSE labeling method:
CFSE (CFDA-SE) is a cell staining reagent that can fluorescently label living cells, can easily penetrate cell membranes, covalently bind to intracellular proteins in living cells, and release green fluorescence after hydrolysis. The tumor cells can be labeled and quantified by using the CFSE (fluorescent quantitative electron microscope) living cell labeling principle, so that the killing efficiency of the CAR-T cells on tumor target cells can be detected. The specific method comprises the following steps: the target cells were divided equally into two groups and adjusted to the same cell density. Staining with low and high concentrations of CFSE, respectively, wherein the high concentration stained target cells are co-cultured with non-stained immune cells in a certain ratio. After a period of incubation, the high concentration stained target cell tube (along with immune cells) is mixed with the low concentration stained target cell tube in equal amounts. Finally, the killing rate of CAR T cells against target cells was calculated by comparing the percentage of target cells in the CFSE low concentration-labeled group and the CFSE high concentration-labeled group. The method comprises the following specific steps:
1. the logarithmic phase of THP-1 cells was centrifuged at 300-500g for 1-5min and the supernatant was removed. Resuspending the cells in PBS and adjusting the cell density to (1-2). times.107One per ml.
2. The cell density was (1-2). times.107The HepG2 cell suspension per ml was divided equally into two portions, one designated CFSE high-labeled cells and the other designated CFSE low-labeled cells. CFSE low labeled cells were stained with low concentration CFSE (0.5. mu.M) and CFSE high labeled cells were labeled with high concentration CFSE (5. mu.M). The dyeing method specifically comprises the following steps: CFSE dye (Invitrogen) was added to the tube at the indicated concentration and incubated for 10min at 37 ℃ in the absence of light.
3. Complete medium stop marker was added at least 2 volumes cold and centrifuged at 300-500g for 5 min.
4. The supernatant was removed, the cell pellet was collected and washed 2 times with complete medium.
5. Stained THP-1 cells were seeded into 96-well plates, CFSE high-labeled group (CFSE high-labeled cells + T cells): each well was inoculated with THP-1 cells (5X 10)4One by 100 mul), adding different amounts of various CAR-T cells respectively to ensure that the number ratio of the CAR-T cells to the THP-1 cells is 1:1, 1:3, 1:9 and 1:27 respectively; CFSE Low Mark group (only CFSE Low Mark)Note cell): each well was inoculated with THP-1 cells (5X 10)4Pieces/100 μ l) were cultured separately and made up to the same volume with complete medium. CFSE high-labeled cell wells that were not co-cultured with T cells were also set as a control group.
6. After incubation for 6 hours at 37 ℃, all cells in the CFSE high-labeled group and CFSE low-labeled group were mixed at a ratio of 1:1, and the mixed cells were designated as the experimental group mixed cells. All cells in the control group (only CFSE high-labeled cells) and the CFSE low-labeled cells were collected at the same time, mixed at a ratio of 1:1, and the mixed cells were marked as control mixed cells.
7. And (4) detecting the fluorescence value of each group of FITC single channels by using a flow-type computer.
8. Analysis of target cell lysis rate: after the machine is operated by a flow type, two FITC positive peaks, namely CFSE high-labeled cell peaks and CFSE low-labeled cell peaks, are detected, and the target cell proportion of a CFSE high-labeled group and a CFSE low-labeled group is measured. The killing rate (%) of T cells against target cells was then calculated according to the following formula:
the killing rate (%) of T cells against target cells was 100% - (% CFSE high labeled cells in the mixed cells of the experimental group/% CFSE low labeled cells in the mixed cells of the experimental group)/(% CFSE high labeled cells in the mixed cells of the control group/% CFSE low labeled cells in the mixed cells of the control group) × 100%.
The results of the experiment are shown in fig. 3 and table 1. The results show that: after the LILRB4-B7H3-CAR-IFN T cells and the target cells THP-1 are co-cultured according to different effective target ratios, the cell lysis rate reaches over 90 percent when the effective target ratio is 1: 1; when the effective target ratio is 1:27, the cell lysis rate is still over 30 percent. The killing effect is slightly better than that of LILRB4-CAR-IFN T, and far better than that of B7H3-CAR-IFN T and CTR CAR T.
Table 1 lysis (%) of CAR T cell killing tumor cells.
Figure BDA0002869046270000161
6) Tumor transplantation model detection of tumor killing effect of CAR-T cells in animals:
the tail vein of B-NDG Severe combined immunodeficiency mice (Baiosaccae panel) was inoculated with fluorescein-labeled human myeloma cells U266-luc (Shanghai Jing anti). The inoculation amount is 1 multiplied by 1070.3 ml. The groups were randomly divided into 3 groups, namely CTR-CAR T, LILRB4-CAR T cell control group, and LILRB4-B7H3-CAR-IFN T cell group, each of which was 6 mice. (A in FIG. 4)
2. After 15 days of tumor cell inoculation, different types of CAR-T cells were injected into tail vein of mice respectively, and the amount of injected CAR-T cells was 5X 106CAR+T/0.3ml。
3. Sodium salt imaging was performed by intraperitoneal injection of 3mg of D-luciferin into mice 7, 14 and 21 days after CAR-T cell injection, respectively. The number of residual tumor cells in the mice was observed, and the fluorescein intensity (photon density) was counted.
In FIG. 4B shows that there was a significant reduction in human myeloma cell residues in mice injected with LILRB4-B7H3-CAR-IFN T compared to the CTR-CAR T control group and LILRB4-CAR T. The better effect of the LILRB4-B7H3-CAR-IFN T cells on killing the tumor is shown. C in FIG. 4 shows that around 40 days after CAR-T injection, mice injected with LILRB4-B7H3-CAR T cells had much lower mortality than mice injected with LILRB4-CAR T cells and CTR-CAR T cells.
The raw materials and equipment used in the invention are common raw materials and equipment in the field if not specified; the methods used in the present invention are conventional in the art unless otherwise specified.
While one embodiment of the present invention has been described in detail, the description is only a preferred embodiment of the present invention and should not be taken as limiting the scope of the invention. All equivalent changes and modifications made within the scope of the present invention shall fall within the scope of the present invention.
Sequence listing
<110> Zhejiang Kangbaiyu Biotechnology Ltd
<120> LILRB4 and B7-H3 double-targeted chimeric antigen receptor and application thereof
<141> 2020-12-29
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agcaccgtgt acctgcagat gaacaatctg agagccgagg acaccgggat ctactactgt 420
accggcacaa gatacggaag cagcctggac tactggggcc aggggacaag cgtgacagtg 480
agctccggcg gcgggggttc tgacattgtg atgagccaga gcccctcctc cctggcagtg 540
agcgtgggag aaaaagtgac catgagctgc aagagcagcc agaacctgtt ttacagcacc 600
aaccagaaaa actacctggc ctggtaccag cagaagcccg gccagtctcc caagctgctg 660
atttattggg ccagcacaag agagagcggc gtgcccgaca gattcaccgg aagcggcagc 720
ggaacagcct tcaccctgac tatcagcagc gtgaaagctg aggacctggc cgtgtactac 780
tgtcagcagt actacaacta cccactgacc ttcggcgcag gcaccaagct ggagctgaag 840
ggcggcgggg gttctggtgg cggcggcagc ggcggtggag gatcagacat tgtgatgacc 900
cagagccaca aatttatgag caccagcatt ggagcccgcg tgagcattac ctgcaaggcc 960
agccaggacg tgagaaccgc cgtggcctgg taccagcaga aacccggcca gagccccaaa 1020
ctgctgatct acagcgccag ctacagatac accggcgtgc ccgaccgctt caccggaagc 1080
ggaagcggaa ccgacttcac cttcaccatc agcagcgtgc aggctgaaga cctggccgtg 1140
tactactgcc agcagcacta cggaaccccc ccctggacct tcggaggagg caccaaactg 1200
gaaatcaaag gtggcggcgg cagcgaggtg cagctggtgg aaagcggggg aggactggtg 1260
aagcccggag gaagcctgaa gctgagctgc gaggcaagca gatttacatt cagcagctac 1320
gccatgagct gggtgagaca gacacccgag aagagactgg agtgggtggc agccatcagc 1380
ggcggaggaa gatataccta ctaccccgac agcatgaagg ggagattcac aatcagcaga 1440
gacaacgcta agaacttcct gtacctgcag atgagcagcc tgagaagcga ggacacagca 1500
atgtactact gcgccaggca ctatgacggc tacctggact actggggcca gggcaccacc 1560
ctgaccgtgt catccactac aactccagca cccagacccc ctacacctgc tccaactatc 1620
gcaagtcagc ccctgtcact gcgccctgaa gcctgtcgcc ctgctgccgg gggagctgtg 1680
catactcggg gactggactt tgcctgtgat atctacatct gggcgccctt ggccgggact 1740
tgtggggtcc ttctcctgtc actggttatc accctttact gcaggttcag tgtcgtgaag 1800
agaggccgga agaagctgct gtacatcttc aagcagcctt tcatgaggcc cgtgcagact 1860
acccaggagg aagatggatg cagctgtaga ttccctgaag aggaggaagg aggctgtgag 1920
ctgagagtga agttctcccg aagcgcagat gccccagcct atcagcaggg acagaatcag 1980
ctgtacaacg agctgaacct gggaagacgg gaggaatacg atgtgctgga caaaaggcgg 2040
ggcagagatc ctgagatggg cggcaaacca agacggaaga acccccagga aggtctgtat 2100
aatgagctgc agaaagacaa gatggctgag gcctactcag aaatcgggat gaagggcgaa 2160
agaaggagag gaaaaggcca cgacggactg taccaggggc tgagtacagc aacaaaagac 2220
acctatgacg ctctgcacat gcaggctctg ccaccaagac gagctaaacg aggctcaggc 2280
gcgacgaact ttagtttgct gaagcaagct ggggatgtag aggaaaatcc gggtcccatg 2340
gccctgacct tcgccctgct ggtggccctg ctggtcctga gctgcaagag ctcctgcagc 2400
gtggggtgcg acctgcccca gacccacagc ctgggctcca gaagaaccct gatgctgctg 2460
gcccagatga gaaaaatcag tctgttcagc tgcctgaaag acagacacga ctttggcttc 2520
cctcaggagg aatttggaaa ccagttccag aaggccgaaa ccatccccgt gctgcacgag 2580
atgatccagc agatcttcaa cctgttctcc accaaagata gcagcgcagc ctgggacgaa 2640
accctgctgg acaagttcta caccgagctg taccagcagc tgaacgacct ggaggcctgc 2700
gtgatccagg gcgtgggagt gaccgagaca ccactgatga aagaggatag cattctggcc 2760
gtgaggaaat acttccagag aatcaccctg tacctgaaag agaaaaagta cagtccctgc 2820
gcctgggagg tggtgagagc cgagatcatg agaagcttca gcctgagcac caatctgcag 2880
gaaagcctga gaagcaagga gtga 2904
<210> 3
<211> 2901
<212> DNA
<213> Artificial Synthesis
<400> 3
atggctctgc ctgtgaccgc cctgctgctg cctctggctc tgctgctgca cgccgctcgg 60
cctatggctc tgcctgtgac cgccctgctg ctgcctctgg ctctgctgct gcacgccgct 120
cggcctgagg tgaacctgga ggagagcggg ggggggctgg tgcagcctgg aggaagtatg 180
aagctgagct gtattgccag cggattcaca tttagcaact attggatgaa ctgggtgagg 240
cagagtcccg agaagggact ggagtgggtg gcagaaatta gactgaagta caacaactac 300
gccacacact acgcagaaag cgtgaagggg agattcacca tcagcagaga cgatagcaag 360
agcaccgtgt acctgcagat gaacaatctg agagccgagg acaccgggat ctactactgt 420
accggcacaa gatacggaag cagcctggac tactggggcc aggggacaag cgtgacagtg 480
agctccggcg gcgggggttc tgacattgtg atgagccaga gcccctcctc cctggcagtg 540
agcgtgggag aaaaagtgac catgagctgc aagagcagcc agaacctgtt ttacagcacc 600
aaccagaaaa actacctggc ctggtaccag cagaagcccg gccagtctcc caagctgctg 660
atttattggg ccagcacaag agagagcggc gtgcccgaca gattcaccgg aagcggcagc 720
ggaacagcct tcaccctgac tatcagcagc gtgaaagctg aggacctggc cgtgtactac 780
tgtcagcagt actacaacta cccactgacc ttcggcgcag gcaccaagct ggagctgaag 840
ggcggcgggg gttctggtgg cggcggcagc ggcggtggag gatcagacat tgtgatgacc 900
cagagccaca aatttatgag caccagcatt ggagcccgcg tgagcattac ctgcaaggcc 960
agccaggacg tgagaaccgc cgtggcctgg taccagcaga aacccggcca gagccccaaa 1020
ctgctgatct acagcgccag ctacagatac accggcgtgc ccgaccgctt caccggaagc 1080
ggaagcggaa ccgacttcac cttcaccatc agcagcgtgc aggctgaaga cctggccgtg 1140
tactactgcc agcagcacta cggaaccccc ccctggacct tcggaggagg caccaaactg 1200
gaaatcaaag gtggcggcgg cagcgaggtg cagctggtgg aaagcggggg aggactggtg 1260
aagcccggag gaagcctgaa gctgagctgc gaggcaagca gatttacatt cagcagctac 1320
gccatgagct gggtgagaca gacacccgag aagagactgg agtgggtggc agccatcagc 1380
ggcggaggaa gatataccta ctaccccgac agcatgaagg ggagattcac aatcagcaga 1440
gacaacgcta agaacttcct gtacctgcag atgagcagcc tgagaagcga ggacacagca 1500
atgtactact gcgccaggca ctatgacggc tacctggact actggggcca gggcaccacc 1560
ctgaccgtgt catccactac aactccagca cccagacccc ctacacctgc tccaactatc 1620
gcaagtcagc ccctgtcact gcgccctgaa gcctgtcgcc ctgctgccgg gggagctgtg 1680
catactcggg gactggactt tgcctgtgat atctacatct gggcgccctt ggccgggact 1740
tgtggggtcc ttctcctgtc actggttatc accctttact gcaggttcag tgtcgtgaag 1800
agaggccgga agaagctgct gtacatcttc aagcagcctt tcatgaggcc cgtgcagact 1860
acccaggagg aagatggatg cagctgtaga ttccctgaag aggaggaagg aggctgtgag 1920
ctgagagtga agttctcccg aagcgcagat gccccagcct atcagcaggg acagaatcag 1980
ctgtacaacg agctgaacct gggaagacgg gaggaatacg atgtgctgga caaaaggcgg 2040
ggcagagatc ctgagatggg cggcaaacca agacggaaga acccccagga aggtctgtat 2100
aatgagctgc agaaagacaa gatggctgag gcctactcag aaatcgggat gaagggcgaa 2160
agaaggagag gaaaaggcca cgacggactg taccaggggc tgagtacagc aacaaaagac 2220
acctatgacg ctctgcacat gcaggctctg ccaccaagac gagctaaacg aggctcaggc 2280
gcgacgaact ttagtttgct gaagcaagct ggggatgtag aggaaaatcc gggtcccatg 2340
actaataaat gcctgcttca gatcgccttg ctgctttgtt tcagcacaac tgcactgtca 2400
atgtcttata acctgctcgg gtttctccag agaagctcca attttcagtg tcagaaactg 2460
ctttggcagc tgaacggccg cttggaatac tgcctgaaag acagaatgaa cttcgatatc 2520
ccggaagaga taaaacagct gcagcaattt cagaaggagg atgcggcctt gaccatttac 2580
gagatgcttc aaaacatatt tgcaatcttc cggcaggact cttcctcaac cgggtggaat 2640
gaaaccatcg tggaaaatct cctcgcgaat gtctaccacc agatcaacca tcttaagacc 2700
gttttggagg agaagcttga gaaggaggac ttcacccgcg ggaaacttat gtcttcactg 2760
cacttgaagc gctactacgg tcggattctc cattacctga aagccaagga gtactcccac 2820
tgcgcctgga caatcgtccg ggtggagatc ctgaggaact tctacttcat taatcgcctg 2880
actgggtatc tgaggaactg a 2901
<210> 4
<211> 967
<212> PRT
<213> Artificial Synthesis
<400> 4
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 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro
20 25 30
Leu Ala Leu Leu Leu His Ala Ala Arg Pro Glu Val Asn Leu Glu Glu
35 40 45
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Met Lys Leu Ser Cys
50 55 60
Ile Ala Ser Gly Phe Thr Phe Ser Asn Tyr Trp Met Asn Trp Val Arg
65 70 75 80
Gln Ser Pro Glu Lys Gly Leu Glu Trp Val Ala Glu Ile Arg Leu Lys
85 90 95
Tyr Asn Asn Tyr Ala Thr His Tyr Ala Glu Ser Val Lys Gly Arg Phe
100 105 110
Thr Ile Ser Arg Asp Asp Ser Lys Ser Thr Val Tyr Leu Gln Met Asn
115 120 125
Asn Leu Arg Ala Glu Asp Thr Gly Ile Tyr Tyr Cys Thr Gly Thr Arg
130 135 140
Tyr Gly Ser Ser Leu Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val
145 150 155 160
Ser Ser Gly Gly Gly Gly Ser Asp Ile Val Met Ser Gln Ser Pro Ser
165 170 175
Ser Leu Ala Val Ser Val Gly Glu Lys Val Thr Met Ser Cys Lys Ser
180 185 190
Ser Gln Asn Leu Phe Tyr Ser Thr Asn Gln Lys Asn Tyr Leu Ala Trp
195 200 205
Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala
210 215 220
Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser
225 230 235 240
Gly Thr Ala Phe Thr Leu Thr Ile Ser Ser Val Lys Ala Glu Asp Leu
245 250 255
Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Asn Tyr Pro Leu Thr Phe Gly
260 265 270
Ala Gly Thr Lys Leu Glu Leu Lys Gly Gly Gly Gly Ser Gly Gly Gly
275 280 285
Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser His Lys
290 295 300
Phe Met Ser Thr Ser Ile Gly Ala Arg Val Ser Ile Thr Cys Lys Ala
305 310 315 320
Ser Gln Asp Val Arg Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly
325 330 335
Gln Ser Pro Lys Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly
340 345 350
Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe
355 360 365
Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln
370 375 380
Gln His Tyr Gly Thr Pro Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu
385 390 395 400
Glu Ile Lys Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly
405 410 415
Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Lys Leu Ser Cys Glu Ala
420 425 430
Ser Arg Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Thr
435 440 445
Pro Glu Lys Arg Leu Glu Trp Val Ala Ala Ile Ser Gly Gly Gly Arg
450 455 460
Tyr Thr Tyr Tyr Pro Asp Ser Met Lys Gly Arg Phe Thr Ile Ser Arg
465 470 475 480
Asp Asn Ala Lys Asn Phe Leu Tyr Leu Gln Met Ser Ser Leu Arg Ser
485 490 495
Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg His Tyr Asp Gly Tyr Leu
500 505 510
Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Thr Thr Thr
515 520 525
Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro
530 535 540
Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val
545 550 555 560
His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro
565 570 575
Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu
580 585 590
Tyr Cys Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr
595 600 605
Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu
610 615 620
Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu
625 630 635 640
Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln
645 650 655
Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu
660 665 670
Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly
675 680 685
Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln
690 695 700
Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu
705 710 715 720
Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr
725 730 735
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro
740 745 750
Arg Arg Ala Lys Arg Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys
755 760 765
Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Ala Leu Thr Phe
770 775 780
Ala Leu Leu Val Ala Leu Leu Val Leu Ser Cys Lys Ser Ser Cys Ser
785 790 795 800
Val Gly Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr
805 810 815
Leu Met Leu Leu Ala Gln Met Arg Arg Ile Ser Leu Phe Ser Cys Leu
820 825 830
Lys Asp Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln
835 840 845
Phe Gln Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln
850 855 860
Ile Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu
865 870 875 880
Thr Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp
885 890 895
Leu Glu Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu
900 905 910
Met Lys Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile
915 920 925
Thr Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val
930 935 940
Val Arg Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln
945 950 955 960
Glu Ser Leu Arg Ser Lys Glu
965
<210> 5
<211> 967
<212> PRT
<213> Artificial Synthesis
<400> 5
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 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro
20 25 30
Leu Ala Leu Leu Leu His Ala Ala Arg Pro Glu Val Asn Leu Glu Glu
35 40 45
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Met Lys Leu Ser Cys
50 55 60
Ile Ala Ser Gly Phe Thr Phe Ser Asn Tyr Trp Met Asn Trp Val Arg
65 70 75 80
Gln Ser Pro Glu Lys Gly Leu Glu Trp Val Ala Glu Ile Arg Leu Lys
85 90 95
Tyr Asn Asn Tyr Ala Thr His Tyr Ala Glu Ser Val Lys Gly Arg Phe
100 105 110
Thr Ile Ser Arg Asp Asp Ser Lys Ser Thr Val Tyr Leu Gln Met Asn
115 120 125
Asn Leu Arg Ala Glu Asp Thr Gly Ile Tyr Tyr Cys Thr Gly Thr Arg
130 135 140
Tyr Gly Ser Ser Leu Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val
145 150 155 160
Ser Ser Gly Gly Gly Gly Ser Asp Ile Val Met Ser Gln Ser Pro Ser
165 170 175
Ser Leu Ala Val Ser Val Gly Glu Lys Val Thr Met Ser Cys Lys Ser
180 185 190
Ser Gln Asn Leu Phe Tyr Ser Thr Asn Gln Lys Asn Tyr Leu Ala Trp
195 200 205
Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala
210 215 220
Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser
225 230 235 240
Gly Thr Ala Phe Thr Leu Thr Ile Ser Ser Val Lys Ala Glu Asp Leu
245 250 255
Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Asn Tyr Pro Leu Thr Phe Gly
260 265 270
Ala Gly Thr Lys Leu Glu Leu Lys Gly Gly Gly Gly Ser Gly Gly Gly
275 280 285
Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser His Lys
290 295 300
Phe Met Ser Thr Ser Ile Gly Ala Arg Val Ser Ile Thr Cys Lys Ala
305 310 315 320
Ser Gln Asp Val Arg Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly
325 330 335
Gln Ser Pro Lys Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly
340 345 350
Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe
355 360 365
Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln
370 375 380
Gln His Tyr Gly Thr Pro Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu
385 390 395 400
Glu Ile Lys Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly
405 410 415
Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Lys Leu Ser Cys Glu Ala
420 425 430
Ser Arg Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Thr
435 440 445
Pro Glu Lys Arg Leu Glu Trp Val Ala Ala Ile Ser Gly Gly Gly Arg
450 455 460
Tyr Thr Tyr Tyr Pro Asp Ser Met Lys Gly Arg Phe Thr Ile Ser Arg
465 470 475 480
Asp Asn Ala Lys Asn Phe Leu Tyr Leu Gln Met Ser Ser Leu Arg Ser
485 490 495
Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg His Tyr Asp Gly Tyr Leu
500 505 510
Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Thr Thr Thr
515 520 525
Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro
530 535 540
Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val
545 550 555 560
His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro
565 570 575
Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu
580 585 590
Tyr Cys Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr
595 600 605
Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu
610 615 620
Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu
625 630 635 640
Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln
645 650 655
Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu
660 665 670
Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly
675 680 685
Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln
690 695 700
Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu
705 710 715 720
Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr
725 730 735
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro
740 745 750
Arg Arg Ala Lys Arg Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys
755 760 765
Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Ala Leu Thr Phe
770 775 780
Ala Leu Leu Val Ala Leu Leu Val Leu Ser Cys Lys Ser Ser Cys Ser
785 790 795 800
Val Gly Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr
805 810 815
Leu Met Leu Leu Ala Gln Met Arg Lys Ile Ser Leu Phe Ser Cys Leu
820 825 830
Lys Asp Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln
835 840 845
Phe Gln Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln
850 855 860
Ile Phe Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu
865 870 875 880
Thr Leu Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp
885 890 895
Leu Glu Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu
900 905 910
Met Lys Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile
915 920 925
Thr Leu Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val
930 935 940
Val Arg Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln
945 950 955 960
Glu Ser Leu Arg Ser Lys Glu
965
<210> 6
<211> 966
<212> PRT
<213> Artificial Synthesis
<400> 6
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 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro
20 25 30
Leu Ala Leu Leu Leu His Ala Ala Arg Pro Glu Val Asn Leu Glu Glu
35 40 45
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Met Lys Leu Ser Cys
50 55 60
Ile Ala Ser Gly Phe Thr Phe Ser Asn Tyr Trp Met Asn Trp Val Arg
65 70 75 80
Gln Ser Pro Glu Lys Gly Leu Glu Trp Val Ala Glu Ile Arg Leu Lys
85 90 95
Tyr Asn Asn Tyr Ala Thr His Tyr Ala Glu Ser Val Lys Gly Arg Phe
100 105 110
Thr Ile Ser Arg Asp Asp Ser Lys Ser Thr Val Tyr Leu Gln Met Asn
115 120 125
Asn Leu Arg Ala Glu Asp Thr Gly Ile Tyr Tyr Cys Thr Gly Thr Arg
130 135 140
Tyr Gly Ser Ser Leu Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val
145 150 155 160
Ser Ser Gly Gly Gly Gly Ser Asp Ile Val Met Ser Gln Ser Pro Ser
165 170 175
Ser Leu Ala Val Ser Val Gly Glu Lys Val Thr Met Ser Cys Lys Ser
180 185 190
Ser Gln Asn Leu Phe Tyr Ser Thr Asn Gln Lys Asn Tyr Leu Ala Trp
195 200 205
Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala
210 215 220
Ser Thr Arg Glu Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser
225 230 235 240
Gly Thr Ala Phe Thr Leu Thr Ile Ser Ser Val Lys Ala Glu Asp Leu
245 250 255
Ala Val Tyr Tyr Cys Gln Gln Tyr Tyr Asn Tyr Pro Leu Thr Phe Gly
260 265 270
Ala Gly Thr Lys Leu Glu Leu Lys Gly Gly Gly Gly Ser Gly Gly Gly
275 280 285
Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser His Lys
290 295 300
Phe Met Ser Thr Ser Ile Gly Ala Arg Val Ser Ile Thr Cys Lys Ala
305 310 315 320
Ser Gln Asp Val Arg Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly
325 330 335
Gln Ser Pro Lys Leu Leu Ile Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly
340 345 350
Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Phe
355 360 365
Thr Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln
370 375 380
Gln His Tyr Gly Thr Pro Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu
385 390 395 400
Glu Ile Lys Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly
405 410 415
Gly Gly Leu Val Lys Pro Gly Gly Ser Leu Lys Leu Ser Cys Glu Ala
420 425 430
Ser Arg Phe Thr Phe Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Thr
435 440 445
Pro Glu Lys Arg Leu Glu Trp Val Ala Ala Ile Ser Gly Gly Gly Arg
450 455 460
Tyr Thr Tyr Tyr Pro Asp Ser Met Lys Gly Arg Phe Thr Ile Ser Arg
465 470 475 480
Asp Asn Ala Lys Asn Phe Leu Tyr Leu Gln Met Ser Ser Leu Arg Ser
485 490 495
Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg His Tyr Asp Gly Tyr Leu
500 505 510
Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Thr Thr Thr
515 520 525
Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro
530 535 540
Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val
545 550 555 560
His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro
565 570 575
Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu
580 585 590
Tyr Cys Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr
595 600 605
Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu
610 615 620
Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu
625 630 635 640
Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln
645 650 655
Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu
660 665 670
Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly
675 680 685
Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln
690 695 700
Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu
705 710 715 720
Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr
725 730 735
Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro
740 745 750
Arg Arg Ala Lys Arg Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys
755 760 765
Gln Ala Gly Asp Val Glu Glu Asn Pro Gly Pro Met Thr Asn Lys Cys
770 775 780
Leu Leu Gln Ile Ala Leu Leu Leu Cys Phe Ser Thr Thr Ala Leu Ser
785 790 795 800
Met Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Ser Asn Phe Gln
805 810 815
Cys Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys Leu
820 825 830
Lys Asp Arg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu Gln
835 840 845
Gln Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu Gln
850 855 860
Asn Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp Asn
865 870 875 880
Glu Thr Ile Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln Ile Asn
885 890 895
His Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe Thr
900 905 910
Arg Gly Lys Leu Met Ser Ser Leu His Leu Lys Arg Tyr Tyr Gly Arg
915 920 925
Ile Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp Thr
930 935 940
Ile Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn Arg Leu
945 950 955 960
Thr Gly Tyr Leu Arg Asn
965
<210> 7
<211> 1506
<212> DNA
<213> Artificial Synthesis
<400> 7
atggctctgc ctgtgaccgc cctgctgctg cctctggctc tgctgctgca cgccgctcgg 60
cctagctacg tgctgaccca gcccccctcc gtgagcgtgg cacctggaaa aacagccaga 120
atctcctgcg gaggaaacaa catcggaacc aagaacgtgc actggtacca gcagaaaccc 180
ggacaggccc ccgtgctggt ggtgtacgcc gacagcgacc gccccagcgg aatcccagag 240
agattcagcg gcagcaacag cggaaacacc gccaccctga ccatcagcag agtggaagtg 300
ggagacgaag ccgactatta ttgccaggtg tgggactccg tgagctatca cgtggtgttc 360
ggcggaggaa caacactgac agtgctgggg ggcggcgggg gttctggtgg cggcggcagc 420
ggcggtggag gatcacaggt gcagctggtg gaaagtggcg gcggcgtggt gcagcccgga 480
ggaagcctga gactgagctg cgcccccagc ggcttcgtgt tcagatccta tggcatgcac 540
tgggtgagac agacacctgg caaagggctg gagtgggtga gtctgatttg gcacgacggc 600
agcaaccggt tctacgccga cagcgtgaag ggcagattca ccattagcag agacaacagc 660
aaaaacacac tgtatctgca gatgaacagc ctgagagccg aagacaccgc catgtatttc 720
tgcgctaggg agagactgat cgccgcccct gccgccttcg acctgtgggg acagggcacc 780
ctggtgaccg tgtccagcac tacaactcca gcacccagac cccctacacc tgctccaact 840
atcgcaagtc agcccctgtc actgcgccct gaagcctgtc gccctgctgc cgggggagct 900
gtgcatactc ggggactgga ctttgcctgt gatatctaca tctgggcgcc cttggccggg 960
acttgtgggg tccttctcct gtcactggtt atcacccttt actgcaggtt cagtgtcgtg 1020
aagagaggcc ggaagaagct gctgtacatc ttcaagcagc ctttcatgag gcccgtgcag 1080
actacccagg aggaagatgg atgcagctgt agattccctg aagaggagga aggaggctgt 1140
gagctgagag tgaagttctc ccgaagcgca gatgccccag cctatcagca gggacagaat 1200
cagctgtaca acgagctgaa cctgggaaga cgggaggaat acgatgtgct ggacaaaagg 1260
cggggcagag atcctgagat gggcggcaaa ccaagacgga agaaccccca ggaaggtctg 1320
tataatgagc tgcagaaaga caagatggct gaggcctact cagaaatcgg gatgaagggc 1380
gaaagaagga gaggaaaagg ccacgacgga ctgtaccagg ggctgagtac agcaacaaaa 1440
gacacctatg acgctctgca catgcaggct ctgccaccaa gacgagctaa acgaggctca 1500
ggctga 1506
<210> 8
<211> 2115
<212> DNA
<213> Artificial Synthesis
<400> 8
atggctctgc ctgtgaccgc cctgctgctg cctctggctc tgctgctgca cgccgctcgg 60
cctatgatcc ccaccttcac cgccctgctg tgcctgggcc tgagcctggg acctagaacc 120
cacatgcagg ccggccccct gcccaagcct accctgtggg ctgagcccgg cagcgtgatc 180
agctggggca actccgtgac catttggtgc cagggaaccc tggaggctag agagtacaga 240
ctggacaagg aggagagccc cgccccctgg gatagacaga accccctgga gcccaagaac 300
aaggctagat tcagcatccc cagcatgacc gaagactacg ccggaagata taggtgctac 360
tatagaagcc ccgtgggctg gagccagccc agcgatccac tggaactggt gatgacagga 420
gcctacagca aacccaccct gagcgccctg cccagccctc tggtgaccag cggaaagagc 480
gtgaccctgc tgtgtcagag cagaagcccc atggacacct ttctgctgat caaagagaga 540
gccgcccacc ccctgctgca cctgagaagc gaacacggag cccagcagca tcaggccgag 600
ttccccatga gcccagtgac aagcgtgcac ggcggcacct acagatgctt cagcagccac 660
ggcttctccc actacctgct gagccacccc agcgaccccc tggaactgat cgtgagcggc 720
agcctggaag gccctagacc cagtcccacc agaagcgtga gcaccgccgc cggacccgag 780
gatcagccac tgatgcccac cggatccgtg ccccatagcg gcctgaggag acactgggaa 840
gtgctgatcg gcgtgctggt ggtgagcatc ctgctgctga gcctgctgct gttcctgctg 900
ctgcagcact ggagacaggg gaagcataga accctggctc agagacaggc cgattttcag 960
agaccccctg gcgccgctga gcccgaacct aaagacgggg gcctgcagag aagaagcagc 1020
cccgccgccg acgtgcaggg agaaaacttc tgcgccgccg tgaagaacac ccagcccgaa 1080
gacggcgtgg aaatggacac cagacagagc ccacatgacg aagaccccca ggccgtgacc 1140
tacgccaagg tgaagcacag cagacccaga agagagatgg ccagcccccc cagcccactg 1200
tctggcgaat ttctggacac caaggacaga caggctgaag aagacagaca gatggacacc 1260
gaagccgccg cctccgaggc ccctcaggat gtgacctacg ctagactgca ctccttcacc 1320
ctgaggcaga aggccacaga acccccaccc tcccaggaag gcgccagccc tgctgaaccc 1380
tccgtgtacg ccaccctggc catccacact acaactccag cacccagacc ccctacacct 1440
gctccaacta tcgcaagtca gcccctgtca ctgcgccctg aagcctgtcg ccctgctgcc 1500
gggggagctg tgcatactcg gggactggac tttgcctgtg atatctacat ctgggcgccc 1560
ttggccggga cttgtggggt ccttctcctg tcactggtta tcacccttta ctgcaggttc 1620
agtgtcgtga agagaggccg gaagaagctg ctgtacatct tcaagcagcc tttcatgagg 1680
cccgtgcaga ctacccagga ggaagatgga tgcagctgta gattccctga agaggaggaa 1740
ggaggctgtg agctgagagt gaagttctcc cgaagcgcag atgccccagc ctatcagcag 1800
ggacagaatc agctgtacaa cgagctgaac ctgggaagac gggaggaata cgatgtgctg 1860
gacaaaaggc ggggcagaga tcctgagatg ggcggcaaac caagacggaa gaacccccag 1920
gaaggtctgt ataatgagct gcagaaagac aagatggctg aggcctactc agaaatcggg 1980
atgaagggcg aaagaaggag aggaaaaggc cacgacggac tgtaccaggg gctgagtaca 2040
gcaacaaaag acacctatga cgctctgcac atgcaggctc tgccaccaag acgagctaaa 2100
cgaggctcag gctga 2115
<210> 9
<211> 2736
<212> DNA
<213> Artificial Synthesis
<400> 9
atggctctgc ctgtgaccgc cctgctgctg cctctggctc tgctgctgca cgccgctcgg 60
cctatgatcc ccaccttcac cgccctgctg tgcctgggcc tgagcctggg acctagaacc 120
cacatgcagg ccggccccct gcccaagcct accctgtggg ctgagcccgg cagcgtgatc 180
agctggggca actccgtgac catttggtgc cagggaaccc tggaggctag agagtacaga 240
ctggacaagg aggagagccc cgccccctgg gatagacaga accccctgga gcccaagaac 300
aaggctagat tcagcatccc cagcatgacc gaagactacg ccggaagata taggtgctac 360
tatagaagcc ccgtgggctg gagccagccc agcgatccac tggaactggt gatgacagga 420
gcctacagca aacccaccct gagcgccctg cccagccctc tggtgaccag cggaaagagc 480
gtgaccctgc tgtgtcagag cagaagcccc atggacacct ttctgctgat caaagagaga 540
gccgcccacc ccctgctgca cctgagaagc gaacacggag cccagcagca tcaggccgag 600
ttccccatga gcccagtgac aagcgtgcac ggcggcacct acagatgctt cagcagccac 660
ggcttctccc actacctgct gagccacccc agcgaccccc tggaactgat cgtgagcggc 720
agcctggaag gccctagacc cagtcccacc agaagcgtga gcaccgccgc cggacccgag 780
gatcagccac tgatgcccac cggatccgtg ccccatagcg gcctgaggag acactgggaa 840
gtgctgatcg gcgtgctggt ggtgagcatc ctgctgctga gcctgctgct gttcctgctg 900
ctgcagcact ggagacaggg gaagcataga accctggctc agagacaggc cgattttcag 960
agaccccctg gcgccgctga gcccgaacct aaagacgggg gcctgcagag aagaagcagc 1020
cccgccgccg acgtgcaggg agaaaacttc tgcgccgccg tgaagaacac ccagcccgaa 1080
gacggcgtgg aaatggacac cagacagagc ccacatgacg aagaccccca ggccgtgacc 1140
tacgccaagg tgaagcacag cagacccaga agagagatgg ccagcccccc cagcccactg 1200
tctggcgaat ttctggacac caaggacaga caggctgaag aagacagaca gatggacacc 1260
gaagccgccg cctccgaggc ccctcaggat gtgacctacg ctagactgca ctccttcacc 1320
ctgaggcaga aggccacaga acccccaccc tcccaggaag gcgccagccc tgctgaaccc 1380
tccgtgtacg ccaccctggc catccacact acaactccag cacccagacc ccctacacct 1440
gctccaacta tcgcaagtca gcccctgtca ctgcgccctg aagcctgtcg ccctgctgcc 1500
gggggagctg tgcatactcg gggactggac tttgcctgtg atatctacat ctgggcgccc 1560
ttggccggga cttgtggggt ccttctcctg tcactggtta tcacccttta ctgcaggttc 1620
agtgtcgtga agagaggccg gaagaagctg ctgtacatct tcaagcagcc tttcatgagg 1680
cccgtgcaga ctacccagga ggaagatgga tgcagctgta gattccctga agaggaggaa 1740
ggaggctgtg agctgagagt gaagttctcc cgaagcgcag atgccccagc ctatcagcag 1800
ggacagaatc agctgtacaa cgagctgaac ctgggaagac gggaggaata cgatgtgctg 1860
gacaaaaggc ggggcagaga tcctgagatg ggcggcaaac caagacggaa gaacccccag 1920
gaaggtctgt ataatgagct gcagaaagac aagatggctg aggcctactc agaaatcggg 1980
atgaagggcg aaagaaggag aggaaaaggc cacgacggac tgtaccaggg gctgagtaca 2040
gcaacaaaag acacctatga cgctctgcac atgcaggctc tgccaccaag acgagctaaa 2100
cgaggctcag gcgcgacgaa ctttagtttg ctgaagcaag ctggggatgt agaggaaaat 2160
ccgggtccca tggccctgac cttcgccctg ctggtggccc tgctggtcct gagctgcaag 2220
agctcctgca gcgtggggtg cgacctgccc cagacccaca gcctgggctc cagaagaacc 2280
ctgatgctgc tggcccagat gagaagaatc agtctgttca gctgcctgaa agacagacac 2340
gactttggct tccctcagga ggaatttgga aaccagttcc agaaggccga aaccatcccc 2400
gtgctgcacg agatgatcca gcagatcttc aacctgttct ccaccaaaga tagcagcgca 2460
gcctgggacg aaaccctgct ggacaagttc tacaccgagc tgtaccagca gctgaacgac 2520
ctggaggcct gcgtgatcca gggcgtggga gtgaccgaga caccactgat gaaagaggat 2580
agcattctgg ccgtgaggaa atacttccag agaatcaccc tgtacctgaa agagaaaaag 2640
tacagtccct gcgcctggga ggtggtgaga gccgagatca tgagaagctt cagcctgagc 2700
accaatctgc aggaaagcct gagaagcaag gagtga 2736
<210> 10
<211> 2994
<212> DNA
<213> Artificial Synthesis
<400> 10
atggctctgc ctgtgaccgc cctgctgctg cctctggctc tgctgctgca cgccgctcgg 60
cctatgctga gacggagggg gagccccggg atgggagtgc atgtgggagc cgccctgggg 120
gctctgtggt tctgcctgac cggggccctg gaggtgcagg tgcctgagga ccccgtggtg 180
gccctggtgg gaactgatgc caccctgtgc tgttcttttt ctcccgagcc tgggttttct 240
ctggctcagc tgaacctgat ttggcagctg accgacacca agcagctggt gcactccttt 300
gccgaggggc aggaccaggg ctccgcctat gccaacagga ccgccctgtt ccccgacctg 360
ctggcccagg gaaacgcctc cctgcgcctg cagagagtga gagtggctga tgagggcagc 420
tttacctgct ttgtgtccat cagagacttc ggcagcgccg ccgtgtccct gcaggtggct 480
gctccttact ccaagcccag catgaccctg gagcccaaca aggatctgag acccggagac 540
accgtgacca ttacctgcag cagctaccag gggtatcctg aagccgaagt gttttggcag 600
gacggccagg gagtgcccct gacaggcaac gtgaccacca gccagatggc caatgagcag 660
ggactgttcg acgtgcacag catcctgaga gtggtgctgg gagccaatgg cacctacagc 720
tgcctggtga gaaaccccgt gctgcagcag gacgcccaca gcagcgtgac catcacaccc 780
cagaggagcc ccaccggcgc cgtggaggtg caggtgcccg aagaccccgt ggtggctctg 840
gtgggaacag acgccaccct gagatgcagc ttcagcccag agcctggctt cagcctggcc 900
cagctgaacc tgatctggca gctgacagac accaaacagc tggtgcatag cttcaccgag 960
ggcagagacc agggcagcgc ctacgccaac agaaccgccc tgtttcccga cctgctggct 1020
cagggcaacg cctctctgag actgcagaga gtgagggtgg ctgacgaagg cagcttcaca 1080
tgctttgtgt ctatcagaga ctttggcagc gccgctgtga gcctgcaggt ggccgctcct 1140
tacagcaagc cctccatgac cctggaaccc aacaaggacc tgaggcccgg cgacaccgtg 1200
actattacct gcagtagcta cagaggatat ccagaggccg aagtgttctg gcaggacggg 1260
cagggagtgc ctctgacagg caatgtgacc acctcccaga tggccaacga gcagggactg 1320
tttgacgtgc actccgtgct gagggtggtg ctgggcgcca acggcactta ctcctgtctg 1380
gtgcggaatc ctgtgctgca gcaggatgcc cacggcagcg tgaccattac agggcagccc 1440
atgaccttcc cccccgaagc cctgtgggtg actgtgggac tgagcgtgtg tctgatcgcc 1500
ctgctggtgg ccctggcctt tgtgtgttgg agaaagatta agcagtcatg cgaggaggag 1560
aacgccggcg ccgaggatca ggacggcgaa ggagagggca gcaagaccgc cctgcagccc 1620
ctgaagcact ccgactctaa ggaggatgac ggacaggaga ttgccactac aactccagca 1680
cccagacccc ctacacctgc tccaactatc gcaagtcagc ccctgtcact gcgccctgaa 1740
gcctgtcgcc ctgctgccgg gggagctgtg catactcggg gactggactt tgcctgtgat 1800
atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 1860
accctttact gcaggttcag tgtcgtgaag agaggccgga agaagctgct gtacatcttc 1920
aagcagcctt tcatgaggcc cgtgcagact acccaggagg aagatggatg cagctgtaga 1980
ttccctgaag aggaggaagg aggctgtgag ctgagagtga agttctcccg aagcgcagat 2040
gccccagcct atcagcaggg acagaatcag ctgtacaacg agctgaacct gggaagacgg 2100
gaggaatacg atgtgctgga caaaaggcgg ggcagagatc ctgagatggg cggcaaacca 2160
agacggaaga acccccagga aggtctgtat aatgagctgc agaaagacaa gatggctgag 2220
gcctactcag aaatcgggat gaagggcgaa agaaggagag gaaaaggcca cgacggactg 2280
taccaggggc tgagtacagc aacaaaagac acctatgacg ctctgcacat gcaggctctg 2340
ccaccaagac gagctaaacg aggctcaggc gcgacgaact ttagtttgct gaagcaagct 2400
ggggatgtag aggaaaatcc gggtcccatg gccctgacct tcgccctgct ggtggccctg 2460
ctggtcctga gctgcaagag ctcctgcagc gtggggtgcg acctgcccca gacccacagc 2520
ctgggctcca gaagaaccct gatgctgctg gcccagatga gaagaatcag tctgttcagc 2580
tgcctgaaag acagacacga ctttggcttc cctcaggagg aatttggaaa ccagttccag 2640
aaggccgaaa ccatccccgt gctgcacgag atgatccagc agatcttcaa cctgttctcc 2700
accaaagata gcagcgcagc ctgggacgaa accctgctgg acaagttcta caccgagctg 2760
taccagcagc tgaacgacct ggaggcctgc gtgatccagg gcgtgggagt gaccgagaca 2820
ccactgatga aagaggatag cattctggcc gtgaggaaat acttccagag aatcaccctg 2880
tacctgaaag agaaaaagta cagtccctgc gcctgggagg tggtgagagc cgagatcatg 2940
agaagcttca gcctgagcac caatctgcag gaaagcctga gaagcaagga gtga 2994

Claims (7)

1. A LILRB4 and B7-H3 double-targeted chimeric antigen receptor, wherein the amino acid sequence of the chimeric antigen receptor comprises:
sequentially connected anti-LILRB 4 single-chain antibody, anti-B7-H3 single-chain antibody, human CD8 hinge transmembrane region, human 4-1BB intracellular region, human CD3 zeta intracellular region, human P2A peptide and human IFN protein peptide; or
The chimeric antigen receptor has the amino acid sequence with one or more amino acid substitutions, deletions or additions and has similar biological activity.
2. The chimeric antigen receptor according to claim 1, wherein the N-terminus of the coding sequence of the anti-LILRB 4 single-chain antibody further comprises a signal peptide.
3. The chimeric antigen receptor according to claim 1, wherein said chimeric antigen receptor comprises a first functional protein or a second functional protein; or
A fusion protein obtained by connecting a label to the N end or/and the C end of the first functional protein or the second functional protein;
the amino acid sequence of the first functional protein is shown as SEQ ID No.4 or SEQ ID No.5 or SEQ ID No. 6;
the amino acid sequence of the second functional protein is obtained by substituting and/or deleting and/or adding one or more amino acid residues of the amino acid sequence of the first functional protein, and the second functional protein has the same biological activity as the first functional protein.
4. A polynucleotide sequence, characterised in that the polynucleotide sequence encodes a chimeric antigen receptor according to any one of claims 1 to 3.
5. A polynucleotide sequence according to claim 4 comprising a first gene sequence or a second gene sequence; or
A third gene sequence obtained by hybridizing the first gene sequence or the second gene sequence with a nucleotide sequence;
the first gene sequence is shown as SEQ ID No.1 or SEQ ID No.2 or SEQ ID No. 3;
the second gene sequence is a nucleotide sequence with identity of more than 75% with the first gene sequence.
6. A chimeric antigen receptor T cell double-targeted by LILRB4 and B7-H3, wherein the chimeric antigen receptor of any one of claims 1-3 is stably expressed in the T cell.
7. Use of a chimeric antigen receptor T cell double-targeted by LILRB4 and B7-H3 for the preparation of a medicament for the treatment of acute myeloid leukemia according to claim 6.
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