CN111454909A

CN111454909A - Preparation method of natural ligand-mediated multi-target recognition controllable genetically engineered immune cells

Info

Publication number: CN111454909A
Application number: CN202010073487.1A
Authority: CN
Inventors: 曹宇; 李书宏; 赵丽君; 郭建; 王莹莹; 王志东
Original assignee: Peking University Shenzhen Graduate School
Current assignee: Peking University Shenzhen Graduate School
Priority date: 2019-01-22
Filing date: 2020-01-22
Publication date: 2020-07-28

Abstract

The invention discloses a preparation method of natural ligand-mediated multi-target recognition controllable genetically engineered immune cells. In order to solve the problems of effectiveness and safety of CAR-T treatment, the inventor separately constructs a recognition part of a growth factor on a target spot and CAR-immune cells and an activation structure domain, in the tumor targeted treatment, a tag molecule is adopted to modify the growth factor as a regulatory protein, and a tag specificity CAR is constructed on the immune cells to prepare novel multi-target recognition controllable genetic engineering immune cells, so that the safety controllability of the multi-target recognition controllable genetic engineering immune cells is improved while the multi-target stable recognition of tumor-related antigens is realized, and the difficulty that the CAR-T cells of a tandem antibody have high risk in the treatment process is overcome.

Description

Preparation method of natural ligand-mediated multi-target recognition controllable genetically engineered immune cells

Technical Field

The invention relates to a method for improving multi-target recognition activity and safety controllability of anti-tumor immunotherapy by adopting natural ligand mediated genetic engineering immunocytes.

Background

In recent years, cancer immunotherapy has attracted much attention and is a focus in the field of cancer therapy. In 12 months of 2011, the Journal of national and Clinical tumor of top grade respectively publishes the review article of cancer comates of the same topic, and the evaluation of immunotherapy is the only method which has the possibility of completely eliminating cancer cells in the existing technology, and the method makes up the defects of the traditional therapy and is considered as a most active and most promising treatment means in the comprehensive tumor treatment mode in twenty-first century.

The CAR-T immunotherapy concept is firstly proposed by Dotti (1) and the like, has been developed for nearly 30 years by achieving breakthrough progress in treating leukemia through current clinical trials, and through continuous scientific research and clinical transformation, the CAR-T technology has undergone 4 generations of innovation (2), but the majority of clinical applications at present are still the second generation of CAR molecular structures, and the same stimulatory molecules commonly used are CD28, CD137, OX40 and the like.

CAR is divided into intracellular signal peptide, transmembrane and extracellular antigen binding region 3 parts: the extracellular antigen-binding region exerts specificity for binding to an antigen. The transmembrane region of the CAR consists of a signal molecule, such as CD3, CD3 ζ, CD4, CD28, and the like. Intracellular signaling peptide regions include tyrosine, etc., on the zeta chain of the T cell receptor CD3 or FcRI gamma. CAR-modified T cells are T cells separated from peripheral blood cells of a patient and then transfected into T cells using genetic engineering techniques to form chimeric antigen receptor T cells (3).

CAR-T not only is not influenced by MHC restriction, but also recognizes antigen diversification, and does not have TCR mismatch risk, CAR-T can also directly inhibit the growth of tumor cells, and factors of CAR-T secretion cells such as gamma interferon (IFN-gamma) induce macrophages to secrete interleukin 6(I L-6), interleukin 10(I L-10) and interleukin 12(I L-12) to inhibit the growth of tumor cells (4).

In order to achieve the desired therapeutic effect, up to 22 different targets (the number of which even exceeds the number of hematological tumors) are currently being tried for CAR-T cell therapy of solid tumors, among which a comparatively large number of targets such as Epidermal Growth Factor Receptor (EGFR), human epidermal growth factor receptor 2 (HER 2), and mesothelin (MS L N) are being developed.

The present invention relates to a method for treating a tumor, and more particularly to a method for treating a tumor, wherein the method comprises the steps of constructing a tumor-targeting antigen.

B lymphocyte stimulator (B-cell activating factor, B L yS) is a member of the Tumor Necrosis Factor (TNF) family that regulates B cell maturation, survival and function, and is overexpressed in various autoimmune diseases B L yS is a typical type II transmembrane protein, with the N-terminus enclosed, lacking a signal peptide sequence, and the C-terminus extracellular B L yS is a monomeric glycoprotein consisting of 285 amino acids and having a molecular weight of 31.3kD, wherein the amino acids 1 to 46 at the N-terminus are the intracellular domain, the amino acids 47 to 73 are the hydrophobic transmembrane domain, the amino acids 74 to 285 are the extracellular domain, the amino acids 133 to 285 are the main domain in which it functions, B L yS may have a glycosylation site (N242), no disulfide bond exists, human and mouse homology is 93%, B L yS has a strong B cell tropism, as a co-stimulatory factor for lymphocytes, B L yS has a strong effect on the proliferation of activated B cells and on the stimulation of B cell differentiation, and its malignant tumor receptor L has been identified.

The biological effects of B L yS are mediated by three cell surface receptors, known as the B-cell activator receptor (BAFF-R), the transmembrane activator and calcium modulator and cyclophilin ligand interaction (TACI) and the B-cell maturation antibody (BCMA), all of which are predominantly expressed on the surface of B-cells, all type III membrane proteins, the extracellular region of the receptor has cysteine-rich regions (CRDs) that form the receptor-ligand motif, where BAFF-R may be a specific receptor present only on B-cells, while TACI and BCMA are receptors for APRI L, another member of the TNF superfamily (8).

In addition, compared with the prior art, the targeted recognition molecule B L yS is a ligand molecule naturally existing in a human body, has no immunogenicity, and increases the safety of the CAR-T cell in the treatment process.

A proliferation-inducing ligand (APRI L), also known as TNFSF13, is one of the important members of the tumor necrosis factor family, APRI L is low expressed in most normal tissues and mainly expressed on various immune cells, such as dendritic cells, macrophages, monocytes, T lymphocytes and the like, APRI L mainly promotes and participates in the proliferation, differentiation and survival of lymphocytes and induces the generation and class switching of immunoglobulin and various functions through the binding with receptors, and more researches in recent years show that APRI L participates in the onset of various autoimmune diseases (9).

APRI L contains 250 amino acids and is a type II transmembrane protein, which is divided into an intracellular region (N-terminal, without signal peptide sequence), a transmembrane region (amino acids 29-49) and an extracellular region (amino acids 50-250), and has two forms of a membrane-bound form with a relative molecular mass of about 27000 and an extracellular-soluble form with a relative molecular mass of about 17000. APRI L can be bound by itself to form a homotrimer, or can be bound by the same family member TNFSF 3B, namely B-cell activating factor (B-cell activating factor, BAFF), to form a heterotrimer (1 BAFF binds to 2 APRI L or 2 BAFF binds to 1 APRI L). furthermore, APRI L can also be bound by the same family protein TWEAK located upstream of the same family member to form a type II transmembrane protein.

There are two receptors currently identified as APRI L, TACI and BCMA. TACI is expressed primarily on the surface of a portion of B cells and activated T cells, while BCMA is expressed on the surface of mature B cells, both of which are common receptors to APRI L and BAFF, and both belong to the tumor necrosis factor receptor family, and both of which bind to tumor necrosis factor-related protein (TRAF), promote activated anti-apoptosis, and promote proliferative signaling pathways, APRI L can promote and participate in various functions such as lymphocyte proliferation, differentiation, survival, and induction of immunoglobulin production and class switching by binding to the receptor, although there are reports of using APRI L as the recognition domain of a CAR for the preparation of conventional hematologic tumor therapy CAR-T cells (10), APRI L-based CARs may cause serious targeted toxicity, such as B cell failure or neurotoxicity, and so optimizing APRI L to construct a targeted CAR-T cell-regulatory potential problem.

Vascular Endothelial Growth Factor (VEGF), a specific heparin-binding growth factor for vascular endothelial cells, induces angiogenesis in vivo. It is secreted by some tumor cells, and can promote endothelial cell proliferation by means of binding with correspondent receptor on the vascular endothelium, at the same time can raise vascular permeability to make endothelial cell migrate, induce tumor angiogenesis and maintain tumor growth, so that it is the most strong angiogenesis factor discovered at present, and is related to several physiological and pathological processes (11). There are currently 7 VEGF families, VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and placental growth factor (PIGF). The human VEGF-A gene is located on chromosome 6p21.3, is a single gene, has a full length of 14kb, and consists of 8 exons and 7 introns. The coded product is 34-45 kD homodimer glycoprotein. VEGF is cut at the transcription level to generate 5 isomers which are named as VEGF-AA145, VEGF-A165, VEGF-A121, VEGF-A189 and VEGF-A206 according to the length of amino acid. Wherein VEGF-A121 is a weakly acidic polypeptide which does not bind to heparin, VEGF-A165 is a basic protein which has low affinity to heparin, and both are secreted in a soluble, freely diffusing form and readily reach target cells; VEGF-A145, VEGF-A165 and VEGF-A206 have high affinity with heparin, and are secreted to bind to the cell surface or cell matrix, and belong to cell-related isoforms. VEGF-A has been shown to be an endothelial cell selective mitogen, which can increase the cytoplasmic Ca2+ concentration of endothelial cells and increase the permeability of capillaries (mainly veins and venules behind capillaries) to macromolecular substances, can make the endothelial cells in a slender shape and stimulate the replication thereof from various ways, stimulate the glucose transport into the endothelial cells, promote the displacement of endothelial cells, murine monocytes and fetal bovine osteoblasts, change the gene activation pattern of the endothelial cells, up-regulate the expression of plasminogen activators (including urokinase type and tissue type) and PAI-I inhibitors thereof, and induce the expression of other endothelial cell interstitial proteases, collagenase and tissue factor, in addition to the increase of the cytoplasmic Ca2+ concentration of endothelial cells and the increase of the permeability of capillaries to macromolecular substances. VEGF-A mediates many physiological and pathological angiogenesis, and its expression is also enhanced during tissue angiogenesis. Cells in which the embryogenic tissue is in a differentiated state express more than adult and fully differentiated cells. Under physiological conditions, VEGF-A may be expressed at high levels in the placenta, in many embryonic tissues and in some normal tissues of adults with physiological vascular proliferation (e.g., proliferating endometrium). In addition, low levels of expression are also found in normal glomerular cells, cardiomyocytes, prostate epithelium, seminal fluid and certain epithelial cells of the adrenal cortex and lungs in animals and adults. In pathological conditions, there is an overexpression of VEGF-A in the synovium cells of healing skin wounds, psoriasis, delayed hypersensitivity reactions, rheumatoid arthritis.

The receptors of the VEGF family discovered so far are classified into tyrosine kinase receptors VEGFR1(Flt1), VEGFR2(KDR/Klk1), VEGFR3(Flt4) and non-tyrosine kinase receptors neurofibrillary proteins (NRP1 and NRP2), Flt1, KDR, Flt4 are Receptor tyrosine protein kinases (RTK), both of which are mainly expressed on vascular endothelial cells, the latter being mainly present in lymphatic endothelial cells, NP1 and NP2 are non-tyrosine protein kinase transmembrane receptors, containing long extracellular segments and short intracellular segments, not only expressed in endothelial cells, but also expressed in some tumor cells the main biological functions of VEGF are achieved by VEGFR2, VEGFR2 and after VEGF binding dimerized and the tyrosine residues within themselves phosphorylated, VEGFR2 have been self-phosphorylated at sites confirmed, Tyr1054, Tyr951, Tyr996, Tyr1059, Tyr1175 has been shown to be widely used as a clinical targeting Receptor for vascular endothelial cell signaling, VEGF Receptor targeting VEGF-kinase, such as a Receptor targeting Receptor tyrosine kinase, VEGF-binding protein targeting Receptor targeting gene targeting genes, VEGF-targeting various tumor cells, VEGF-targeting genes, VEGF-targeting VEGF-targeting genes, VEGF-targeting VEGF-targeting VEGF-targeting genes, VEGF-targeting genes, VEGF-targeting, VEGF-targeting, VEGF-targeting.

Reference to the literature

1.Dotti G,Savoldo B,&Brenner M(2009)Fifteen years of gene therapybased on chimeric antigen receptors:"are we nearly there yet？".Hum Gene Ther20(11):1229-1239.

2.Chmielewski M,Hombach AA,&Abken H(2014)Of CARs and TRUCKs:chimericantigen receptor(CAR)T cells engineered with an inducible cytokine tomodulate the tumor stroma. Immunol Rev 257(1):83-90.

3.Chen F,et al.(2015)Construction of Anti-CD20 Single-Chain Antibody-CD28-CD137-TCRzeta Recombinant Genetic Modified T Cells and its TreatmentEffect on B Cell Lymphoma.Med Sci Monit 21:2110-2115.

4.Spear P,Barber A,Rynda-Apple A,&Sentman CL(2012)Chimeric antigenreceptor T cells shape myeloid cell function within the tumormicroenvironment through IFN-gamma and GM-CSF.J Immunol 188(12):6389-6398.

5.Zah E,Lin MY,Silva-Benedict A,Jensen MC,&Chen YY(2016)T CellsExpressing CD19/CD20 Bispecific Chimeric Antigen Receptors Prevent AntigenEscape by Malignant B Cells.Cancer Immunol Res 4(6):498-508.

6.Schneider D,et al.(2017)A tandem CD19/CD20 CAR lentiviral vectordrives on-target and off-target antigen modulation in leukemia cell lines.JImmunother Cancer 5:42.

7.Feng KC,et al.(2017)Cocktail treatment with EGFR-specific andCD133-specific chimeric antigen receptor-modified T cells in a patient withadvanced cholangiocarcinoma.J Hematol Oncol 10(1):4.

8.Bossen C&Schneider P(2006)BAFF,APRIL and their receptors:structure,function and signaling.Semin Immunol 18(5):263-275.

9.Liu XG&Hou M(2013)Immune thrombocytopenia and B-cell-activatingfactor/a proliferation-inducing ligand.Semin Hematol 50Suppl 1:S89-99.

10.Lee L,et al.(2018)An APRIL-based chimeric antigen receptor fordual targeting of BCMA and TACI in multiple myeloma.Blood 131(7):746-758.

11.Lange C,Storkebaum E,de Almodovar CR,Dewerchin M,&Carmeliet P(2016)Vascular endothelial growth factor:a neurovascular target inneurological diseases.Nat Rev Neurol 12(8):439-454.

12.Niederman TM,et al.(2002)Antitumor activity of cytotoxic Tlymphocytes engineered to target vascular endothelial growth factorreceptors.Proc Natl Acad Sci USA 99(10):7009-7014.

Disclosure of Invention

In order to solve the problems of effectiveness and safety of CAR-T treatment, the inventor of the invention separately constructs a recognition part of a natural ligand (B L yS, APRI L and VEGF) for tumor targeting and an activation structure domain of a CAR-immune cell, adopts a tag molecule to modify a growth factor natural ligand as a regulatory protein in the tumor targeting treatment, and prepares a novel multi-target recognition controllable genetically engineered immune cell by constructing a tag specific CAR on the immune cell, thereby realizing the stable recognition of multiple targets of tumor-related antigens and improving the safety controllability of the cells while the multi-target recognition of the tumor-related antigens is realized, so as to overcome the problem that the traditional tandem CAR structure combines the tumor recognition domain and the activation domain of the immune cell together, thereby causing the risks of off-target toxicity and the like easily generated in the treatment process of the genetically engineered immune cell.

The technical scheme adopted by the invention is as follows:

an anti-tumor CAR-immune cell composition comprising CAR-immune cells and a regulatory protein constructed based on a natural ligand,

the natural ligand is a naturally-occurring ligand molecule which can be specifically combined with a tumor or a receptor related to the tissue surface of the tumor;

the CAR domain of the CAR-immune cell is linked to a recognition molecule for specific recognition of a tag molecule;

the regulatory protein has a natural ligand molecule that recognizes a plurality of tumor or tissue-associated antigens thereof and a tag molecule that specifically binds to the recognition molecule of the CAR extracellular recognition domain of the CAR-immune cell.

In some examples, the regulatory protein has a native ligand molecule that recognizes at least 2, 3, or 4 tumor or tissue-associated antigens thereof. The number of natural ligands on the regulatory protein is increased, so that the natural ligands can be more tightly combined with tumor cells, the specificity of the natural ligands on the tumor cells can be improved, and the risk problems of off-target toxicity and the like can be reduced or avoided in the using process.

In some examples, the natural ligand in the regulatory protein that recognizes the tumor or tissue-associated antigen thereof is at least one of B L yS, APRI L, and VEGF.

In some examples, the natural ligand in the regulatory protein that recognizes a tumor antigen is B L yS, targeting the extracellular binding domains of BAFF-R, BCMA and TACI.

In some examples, the natural ligand in the regulatory protein that recognizes a tumor antigen is APRI L, targeting the extracellular binding domains of BCMA and TACI.

In some examples, the natural ligand in the regulatory protein that recognizes a tumor antigen is VEGF, targeting the extracellular binding domains of VEGFR1, VEGFR2, and VEGFR 3.

In some examples, the natural ligand molecule and the tag molecule of the regulatory protein are coupled via a hydrophilic bifunctional linker.

In some examples, the linker is a connecting peptide.

In some examples, the linker peptide is selected from a linear polypeptide, an β -fold peptide, or a coiled-coil peptide.

In some examples, the hydrophilic bifunctional linker is selected from a molecule that can be attached by a click reaction.

In some examples, the tag molecule is selected from one of FITC, biotin, His tag, Flag tag, Myc tag, GCN4 tag, HA tag, V5 tag, Chitosan Binding Protein (CBP) tag, Maltose Binding Protein (MBP), glutathione-S-transferase (GST), and DNA Aptamer tag. The tag molecules can be selected according to specific conditions, and when the regulatory protein is a fusion protein, the polypeptide tag molecules are preferably selected to facilitate fusion expression. Particularly, the regulatory protein is expressed by means of fusion protein, and the used tag molecule is a tag molecule for separating and purifying protein and is beneficial to purifying the fusion protein.

In some examples, the tag-specific recognition molecule in the CAR-immune cell is selected from a single chain antibody, a protein ligand, a short peptide molecule, or a nucleic acid molecule to specifically recognize and capture the tag molecule on the regulatory protein. In the presence of regulatory protein, the CAR-immune cell can mediate the specific binding with the tumor cell through the tag molecule on the regulatory protein, so as to achieve the purpose of specifically killing the tumor cell.

In some examples, the CAR-immune cell is constructed using a method selected from lentivirus, adenovirus, adeno-associated virus, transposon technology, mRNA transfection, nanogenes, Zinc Finger nucleic, TA L EN, and CRISPR/case 9.

In some examples, the regulatory protein is expressed by fusion by means of gene recombination, or by means of random chemical coupling and site-specific chemical bonding, or other methods.

In some examples, the immune cell is selected from the group consisting of a T cell, a natural killer cell, a macrophage, a tumor immune cell, and a stem cell-induced immune cell.

In some examples, the T cell type is selected from CD4, CD8, Treg, Th17, Th9, Tfr, Tfh, Tfc, Tph, Trm.

In some examples, the hinge region in the CAR structure is selected from at least one of the immunoglobulin family or the tumor necrosis factor superfamily.

In some examples, the CAR-immune cell further comprises at least one of co-stimulatory factors CD28, 4-1BB, OX-40, CD27, ICOS in the CAR structure.

In some examples, the regulatory protein is selected from the group consisting of APRI L-Myc protein, Myc-APRI L protein, VEGF121-Myc protein, Myc-VEGF121 protein, VEGF165-Myc protein, Myc-VEGF165 protein, B L0 yS-F L1 AG protein, F L2 AG-B L3 yS protein, APRI L-F L AG protein, F L AG-APRI L protein, VEGF121-F L AG protein, F L AG-VEGF121 protein, VEGF165-F L AG protein, F L AG-VEGF165 protein.

The invention has the beneficial effects that:

according to the genetically engineered immune cell, the tag molecule is connected with the CAR structure domain, and the regulatory protein connected with the tag is targeted by the tag, preferably the regulatory protein has two or more specific recognition receptors, for example, the receptors of B L yS comprise three types, namely BCMA, BAFF-R and TACI, and through the mediation of the regulatory protein, a plurality of related antigen receptor molecules on a tumor cell can be targeted and recognized, so that the genetically engineered immune cell has multiple specificities, the purpose of recognizing a plurality of targets by one protein function domain is achieved, the possibility of escape, mismatching and off-target of the tumor antigen can be reduced to a certain extent, and the safety and the effectiveness of CAR-immune cell (such as CAR-T cell) treatment are improved to a certain extent.

The genetically engineered immune cells of the invention produce regulatable CAR-immune cells, such as CAR-T, by constructing regulatory molecules based on short peptides and/or chemical tag molecules, and combining CARs that target the short peptides or chemical tag molecules. By injecting the fusion protein connected with the tag in vitro, the CAR-T treatment effect can be controlled by adjusting the injection time and amount, and the safety is improved.

Drawings

FIG. 1 is a schematic representation of a regulatory protein molecule designed and constructed from natural ligands in combination with CAR-T cells and tumor surface receptors;

FIG. 2 is a schematic structural diagram of a tumor recognition regulatory molecule;

FIG. 3 is a schematic diagram of amidation chemical coupling for preparing tumor recognition regulatory molecules;

FIG. 4 is a schematic diagram of the preparation of tumor recognition regulatory molecules by click chemistry coupling;

FIG. 5 shows the results of enzyme-cutting electrophoresis and immunoblotting of different regulatory molecules constructed by B L yS with thrombin;

FIG. 6 is a schematic structural diagram of a short peptide epitope PNE-targeted CAR;

FIG. 7 shows the expression of cell surface receptors of different hematologic tumors;

FIG. 8 shows the results of flow cytometry analysis of the binding of different regulatory molecules constructed from B L yS to different hematological tumor cells;

FIG. 9 is the result of the killing of different hematologic tumor cells by CAR-T cells mediated by different regulatory molecules constructed from B L yS;

figure 10B L yS constructed different regulatory molecules mediated cytokine release analysis of CAR-T cells on tumor killing;

FIG. 11 shows that B L yS constructed regulatory molecules mediate the analysis of the inhibitory activity of CAR-T cells on tumor Nalm6 in mice;

FIG. 12B L yS constructed regulatory molecules mediated the analysis of the inhibitory activity of CAR-T cells on tumor RPMI8226 in mice.

Detailed Description

The following further illustrates the technical solution of the present invention in connection with an exemplary experiment.

The invention takes natural ligands B L yS, APRI L and VEGF as recognition molecules of tumor cells and microenvironment to construct regulatory molecules capable of recognizing antigen proteins on the surfaces of blood tumors and solid tumors, further introduces tag molecules and tag-targeted CAR-immune cells, and prepares a multi-target recognition natural ligand-mediated regulatory CAR-immune cell strategy (figure 1).

The technical scheme of the invention is further explained by combining experiments.

Construction of regulatory protein (switch molecule)

1. Construction of fusion protein expression vectors

The method comprises the steps of preparing regulatory protein by adopting a biological fusion method, wherein the structure of an expression vector is shown in figure 1, taking B L yS as an example, introducing a His label and a thrombin cutting site at the N end of a Myc-BlyS or B L yS-Myc target gene by a PCR method, carrying out double enzyme digestion by adopting restriction enzyme, cloning the Myc-BlyS or B L yS-Myc target gene on a pET32a (+) vector by adopting T4 DNA ligase, carrying out conversion on a DH5 α competent cell, coating a single clone, carrying out sequencing identification, carrying out secretory expression in a mammalian cell, carrying out PCR on the target gene of the regulatory protein by adopting the restriction enzyme double enzyme digestion, cloning the T4 DNA ligase on a pFase vector, carrying out conversion on the DH5 α competent cell, coating the plate, and carrying out sequencing identification by selecting the single clone.

Wherein, the B L yS-Myc protein sequence is shown in SEQ ID NO. 1, and the Myc-B L yS protein sequence is shown in SEQ ID NO. 2.

Besides, the regulatory protein can be prepared by a chemical coupling method, also taking B L yS as an example, a His tag and a thrombin cutting site are introduced into a target gene of B L yS at the N end by a PCR method, after double cutting by MscI and XhoI, T4 DNA ligase is adopted to clone into a pET32a (+) vector, the vector is plated after being transformed into DH5 α competent cells, and a single clone is picked for sequencing identification, wherein the structure of the expression vector is shown in FIG. 2.

Similarly, other regulatory protein sequences were constructed as follows:

APRI L-Myc protein sequences such asSEQ ID No.: 3 is shown in the specification;

the Myc-APRI L protein sequence is shown in SEQ ID NO. 4;

VEGF121-Myc protein sequence such asSEQ ID No.: 5 is shown in the specification;

the Myc-VEGF121 protein sequence is as set forth in SEQ ID No.: 6 is shown in the specification;

VEGF165-Myc protein sequences such asSEQ ID No.: 7 is shown in the specification;

the Myc-VEGF165 protein sequence is as set forth in SEQ ID No.: 8 is shown in the specification;

b L yS-F L AG protein sequence asSEQ ID No.: 9 is shown in the figure;

the protein sequence of F L AG-B L yS is shown as SEQ ID NO. 10;

APRI L-F L AG protein sequence asSEQ ID No.: 11 is shown in the figure;

f L AG-APRI L protein sequence asSEQ ID No.: 12 is shown in the specification;

VEGF121-F L AG protein sequence such asSEQ ID No.: 13 is shown in the figure;

f L AG-VEGF121 protein sequence such asSEQ ID No.: 14 is shown in the figure;

VEGF165-F L AG protein sequence such asSEQ ID No.: 15 is shown in the figure;

f L AG-VEGF165 protein sequence such asSEQ ID No.: shown at 16.

2. Expression and purification of fusion proteins

The constructed plasmids were transfected into B L21, Rosetta gami (DE3) p L ysS or Tuner (DE3) p L ysS, plated, picked out monoclonals for overnight culture, and inoculated into 500m L fresh TB medium at a ratio of 1: 100 until OD reached OD 3₆₀₀When the value reaches 2.0-2.4, inducing with IPTG for 20 hours, centrifugally collecting thalli at 4 ℃, freezing and storing at-80 ℃ after weighing, adding L sys Buffer with the volume 10 times of the weight of the thalli, then breaking cells by ultrasound until the bacterial liquid is not sticky, centrifugally separating the cell lysate for 1 hour at 4 ℃, filtering the supernatant by adopting a 0.45 mu m filter membrane, purifying the supernatant after cell lysate by adopting a nickel column,the eluted target protein is dialyzed by Tris-HCl/NaCl Buffer solution, thrombin is added for digestion at room temperature for 16 hours, after the completion of the digestion is verified by SDS-PAGE electrophoresis, the digested label is removed by a nickel column, finally, Endotoxin is removed by using an Endotoxin Removal Agarose Resin filler, and then Endotoxin content is detected by using an Endotoxin detection system (GenScript, &. lTtT translation = L "&. lTtT/T &. gTt00350) to ensure that the Endotoxin content is below 5/kg.

The fusion protein can also be secreted and expressed in mammalian cells, and 293F suspension cells are inoculated into cell culture shake flasks and incubated for 24h at 37 ℃, 150rpm and 5% carbon dioxide concentration in a shaking incubator until the cell density reaches 2 × 10⁶And m L, incubating a mixture of a fusion protein expression vector constructed by the transfection pFase vector and PEI MAX for 72 hours in a shaking incubator with 37 ℃, 150rpm and 5% carbon dioxide concentration, centrifuging, collecting supernatant, separating and purifying a nickel column, performing enzyme digestion for 16 hours at room temperature by adopting thrombin, and verifying the enzyme digestion effect by SDS-PAGE electrophoresis.

3. Preparation of chemically coupled proteins

The natural purified B L yS protein can be subjected to random amidation coupling by adopting small molecules which modify Myc tags and introduce succinyl groups, so as to construct randomly coupled regulatory protein (shown in figure 4), and other chemical coupling methods can also be adopted to construct regulatory protein, such as a mixed anhydride method, a diazotization method, a carbonyl diimidazole method and other chemical coupling methods.

For mutant B L yS protein with introduced unnatural amino acids, Myc short peptide tags with cycloalkynyl groups can be synthesized simultaneously, and site-specific modified regulatory protein (as shown in FIG. 5) can be prepared by copper-free click chemistry.

SDS-PAGE and Western Blot to verify fusion proteins

Adding a proper amount of purified protein molecules into a reduction sample loading buffer solution with the volume being one third of that of a protein sample, uniformly mixing the protein molecules by vortex, centrifuging the protein sample after boiling the sample for 10 minutes in a metal bath, adopting 12% separation gel and 5% concentrated gel, fixing a gel plate in a mold, pouring an electrophoresis buffer solution into the gel plate to observe whether the solution leaks or not, pulling out a comb, sequentially loading the sample, supplementing the sample loading buffer solution to a scale mark, regulating the voltage to 80V to start electrophoresis, regulating the voltage to 120V after the sample enters the separation gel to observe the electrophoresis condition, soaking a membrane, a filter paper and a sponge in an electrotransfer buffer solution before use, sequentially placing one piece of gel in the sequence of a white plate-sponge-filter paper-membrane-gel-filter paper-sponge-blackboard (ensuring no bubbles), placing the gel on ice, connecting a power supply 220mA, 2h, dyeing the other piece of gel by Coomassie brilliant blue, decoloring by clear water to observe the protein electrophoresis condition, taking out the membrane after the membrane is finished, cutting the membrane into a proper size, labeling protein molecular weight standard, adding a degreasing solution (5% milk powder + 0.12% tpen), rinsing, observing the protein electrophoresis condition, incubating the protein electrophoresis condition for 10min, incubating, and rinsing the gel for 10 times of a proper times of a MyST 3 times, and shaking for a proper time, and displaying the result of a proper amount of a MyST 3 times of a MyST 3, and a MyST 3 times of a MyS/2 times of a MyS, and a normal temperature, and a MyST 3 times of a normal temperature, and a normal temperature.

Production of CAR-T cells

1. Vector construction

Taking lentivirus vector pE L PS as an example for constructing the CAR targeting Myc and F L AG, taking Myc targeting antibody 9E10 and F L AG targeting antibody FBT as an example, the variable region of the antibody is introduced into the structure of the second-generation CAR by adopting a mode of V L-connecting peptide-VH and VH-connecting peptide-V L, the conventional second-generation CAR structure comprises an EF1 α promoter, a signal peptide molecule, a single-chain antibody, a hinge region, a transmembrane region, a costimulatory molecule and a CD3zeta structure region, the constructed CAR is cloned to pE L PS vector by using T4 DNA ligase after double enzyme digestion by adopting BamHI and SalI, or the vector is constructed by adopting a homologous recombination kit, and the structure of the lentivirus expression vector is shown in figure 6.

Representative CAR structures are as follows:

pE L PS-CAR-anti-Myc9E10(V L-VH) -BBZ protein sequence asSEQ ID No.: 17 is shown;

pE L PS-CAR-anti-Myc9E10 (VH-V L) -BBZ protein sequence asSEQ ID No.: 18 is shown in the figure;

pE L PS-CAR-anti-F L AG FBT (V L-VH) -BBZ protein sequence asSEQ ID No.: 19 is shown in the figure;

pE L PS-CAR-anti-F L AG FBT (VH-V L) -BBZ protein sequence asSEQ ID No.: 20 is shown in the figure;

pE L PS-CAR-anti-F L AG M2(V L-VH) -BBZ protein sequence asSEQ ID No.: 21 is shown in the figure;

pE L PS-CAR-anti-F L AG M2(VH-V L) -BBZ protein sequence asSEQ ID No.: 22, respectively.

2. Preparation of lentiviruses

293FT cells were cultured in DMEM medium (10% FBS, 1% penicillin streptomycin diabody, 1% nonessential amino acids, 1% sodium pyruvate, 1% L-glutamine) and after the cells were attached, the cells were changed to antibiotic-free DMEM medium when the density reached 80% 90%, plasmids were mixed in 0.3m L OPTI-MEM according to the ratio of pE L PS-CAR: pMD L g pRRE: pRSV-Rev: pMD2.G ═ 2.25:1.8:1.8:0.75(μ g), 20 μ L lipofectamine 2000 was added to 0.3m L OPTI-MEM, the two were mixed after standing for 5min and vortexed to mix them uniformly, and after standing for 15min at room temperature, the cells were dropped into 293FT cells, after culturing for 6 hours in a constant temperature incubator, new antibiotic-free DMEM medium was replaced at 37 ℃ with 5% CO₂The culture was further incubated for 48 hours. Collecting all supernatant, centrifuging at 4 deg.C and 1500rpm for 10min, discarding cell precipitate to obtain packaged virus, and storing at-80 deg.C.

3. Extraction of human PBMC

Taking a proper amount of Ficoll-Poque in a centrifuge tube (the ratio of blood to Ficoll-Poque is 2:1 is appropriate), slowly and obliquely adding fresh blood taken from healthy volunteers into the centrifuge tube filled with Ficoll-Poque to enable cell suspension to be suspended on layering liquid, centrifuging for 30min at a horizontal centrifuge of 27 ℃ and 2000rpm (the rising speed is 9 and the falling speed is 0), taking out the centrifuge tube after centrifugation is finished, observing and dividing the centrifuge tube into 4 layers, wherein the uppermost layer is plasma containing partial platelets, the second layer is a thin white film layer which is mainly mononuclear cells and also contains a small amount of platelets, the third layer is liquid layer separation, the fourth layer is granulocytes and erythrocytes, which are settled at the bottom of the tube, the granulocytes are tightly attached to the erythrocytes to form a thin white film, namely PBMC is sucked by a pipette, the upper layer is sucked by a pipette (can be centrifuged and stored after centrifugation), carefully sucking the white film in the tube to stratify, adding the white film into two centrifuge tubes (the same volume as possible), adding RPMI-24 min, adding the RPMI to a constant temperature culture medium, adding the RPMI-24 min, adding the supernatant into a constant temperature culture medium after centrifugation is 27-24 min, adding the supernatant after centrifugation, centrifuging for centrifugation, adding the supernatant for centrifugation at a low speed is 2-10 ℃ for centrifugation, adding the centrifugation for 30min, and the centrifugation, adding the centrifugation, the centrifugation for 10 ℃ of 27-10, adding the centrifugation of 27 min, adding the low speed is 2000 ℃ for centrifugation, and.

Freezing and storing PBMC (peripheral blood mononuclear cell)

1×10⁸And (3) resuspending the PBMC cells into 700 mu L of freezing medium, transferring the resuspension solution into a freezing tube, putting the freezing tube into a programmed cooling box, transferring the programmed cooling box into an ultra-low temperature refrigerator at minus 80 ℃, and transferring the box into liquid nitrogen for long-term storage after 24 hours.

Activation of T cells

Rapidly thawing frozen PBMC taken out of liquid nitrogen in 37 deg.C water bath, adding 10m L AIM-V culture medium, centrifuging at 24 deg.C and 1500rpm for 5min, sucking supernatant after centrifugation, and re-suspending with appropriate amount of AIM-V culture medium (if there are particles, supernatant can be transferred)⁶ Cell size 30 μ L beads (using front vortex) an appropriate amount of beads was washed with 2-3 m L PBS containing 5% FBS (beads were attached to the side wall), after the supernatant was aspirated, the cells were washed with AIM-V medium, and the cells were diluted with medium after T cell countingTo 1 × 10⁶Adding a magnetic bead suspension after the concentration of the mixture is/m L, adding I L-2, wherein the initial concentration of I L-2 is 2.6 × 10⁵IU/m L, adjusted to a final concentration of 300IU/m L.

T cell transduction

The activated 24h T cells are centrifuged for 5 minutes at 1500rpm, further resuspended in RPMI-1640 medium, counted using a hemocytometer and trypan blue, the appropriate amount of cell suspension is mixed with virus solution, 10mg/m L polybrene (polybrene) is added to the mixture to obtain a final concentration of 8 mug/m L and 300IU/m L of I L-2, centrifuged for 90 minutes at 33 ℃ and 1000g, and cultured overnight in a 37 ℃ incubator, the cells are centrifuged the next day, then supplemented with fresh culture solution, the incubator is kept at 37 ℃ for further culture, and the cells are used for detecting the anti-tumor activity after the cells are stably expressed.

Third, function verification

1. Modulating protein binding activity

The results are shown in FIG. 8, the results show that B L yS has broad-spectrum tumor target recognition capability and good specific recognition activity for leukemia, lymphoma, myeloma and the like, the recognition capability mainly depends on the multi-target binding function of B L yS protein on BAFF-R, TACI and BCMA, and the experimental results can clearly show that the modification of the tag molecule on the C end of B L yS can cause certain degree of negative cell binding activity, and the binding effect of B L is good, so that the binding effect of B L is unexpected, and the binding effect of B L yS is good.

2. Cytotoxicity test:

rough cell counting, determining approximate cell volume (mainly to determine the minimum volume, should take as many cells as possible to avoid loss of cell volume due to experimental manipulationSufficient) tumor cells were labeled with fluorescein CFSE, resuspended for accurate cell count and diluted to a defined cell density to a final concentration of 0.2 × 10⁶At a concentration of L m, and weighing sufficient CAR-T cells to obtain a final concentration of 2 × 10⁶M L tumor cells were mixed with CAR-T cells at a ratio of 1:10 (in this case at a concentration of 0.1 × 10)⁶Tumor cells/m L, 1 × 10⁶The method comprises the steps of preparing gradient regulatory protein solutions with the highest concentration of 100nM, 10-fold dilution and the lowest concentration of 1 pM., adding protein solutions with different concentrations into each well, 10 mu L per well, setting a negative control and a positive control at the same time, wherein the negative control condition is that no fusion protein solution is added, the positive control condition is that no T cell and fusion protein solution are added, placing a 96-well plate after the completion of the sample addition in a 37 ℃ constant temperature incubator for incubation for more than 24h, half an hour before the completion of the culture, adding lysis solution 10 mu L to the positive control group in advance, after the completion of the incubation, centrifuging the 96-well plate with a horizontal centrifuge of 2000rpm for 5min, correspondingly transferring the solution in the 96-well plate after the centrifugation to another 96-well U-plate (55 mu L), centrifuging the horizontal centrifuge of 2000rpm for 5min, taking 2U-well U-well plates, correspondingly transferring 30 mu L, adding a test buffer into a substrate MIX, uniformly mixing, adding 30 mu L per well into each well, adding the above mentioned mixture after 20-30 rpm, centrifuging for 5min, taking 2U-well, using a MyyBCC/mC, and using a MyC-mC analyzer with a high-absorbance and a MyC-pH meter to obtain a high-specific killing effect on a high-killing effect on a tumor cell killing effect of a tumor cell, wherein the high-killing effect of a MyC killing cell killing ligand is shown by adopting a high-killing ligand of a MyC receptor, and a MyC killing ligand of a MyC-killing ligand of a MyC cell killing ligand of a.

3. Cytokine release assay

1 × 10⁵Tumor cells were seeded in 24-well plates, with two duplicate wells per target cell after CAR-T cells were washed with PBS, according to 1 × 10⁶Cell density of/m L diluted in T cell expansion Medium without I L-2 at 1 × 10⁶The T cells were seeded in culture wells containing target cells 10nM of B L yS/Myc and Myc/B L yS were added to the corresponding wells, respectively, at 37 deg.C, 5% CO₂After 24h of culture in an incubator, collecting supernatant, and detecting the content of IFN-gamma and TNF- α in different samples by adopting IFN-gamma and TNF- α E L ISA kits, wherein the result is shown in figure 10. the cell factor secretion is a mark of the effect of CAR-T cells, after analyzing the cell factor release measurement result, B L yS/Myc and Myc/B L yS are found to mediate CAR-T cells to kill tumors, show remarkably enhanced cell factor secretion, and have good CAR-T cell mediated activation effect on tumor cells expressing BCMA, BAFFR and TACI.

4. Animal experiments (determination of the in vivo tumor killing ability of T cells;

to establish a tumor model, mice were injected caudal vein with Nalm6 and RPMI8226 tumor cells bearing luciferase, and after tumors grew and dispersed well in NSG mice, the mice were divided into three groups and injected intravenously (i.v) with the corresponding CAR-T cells (30 × 10)⁶CAR-T cells/mice for tumor models), and using 0.5mg/kg/dose of B L yS/Myc and Myc/B L yS for daily injection into mice, and using IVIS fluorescence imaging system to regularly observe the distribution of tumor cells in NSG mice, the results are shown in FIG. 11, B L yS/Myc-mediated CAR-T cells can effectively inhibit proliferation and amplification of Nalm6 leukemia tumor cells, furthermore, Myc/B L yS-mediated CAR-T cells can also effectively inhibit proliferation and amplification of RPMI8226 myeloma cells (FIG. 12), thereby further proving that the natural ligand-constructed regulatable and controllable-T cells can be used for constructing CAR cells for targeting different receptor expressions to realize effective inhibition activities on different tumors.

In conclusion, the functional verification results show that the regulatory switch molecules constructed by using B L yS as the targeting molecules can mediate CAR-T cells, so that the effective killing control effect on blood tumors expressing different tumor-associated antigens is realized, and the safety and effectiveness of the regulatory switch molecules are greatly improved compared with the traditional CAR-T therapy from different points of view.

<110> Shenzhen institute of university of Beijing

<120> preparation method of natural ligand mediated multi-target point recognition controllable gene engineering immune cell

<160>22

<210>1<211>180<212> PRT <213> Artificial sequence <400>1

Ala Val Gln Gly Pro Glu Glu Thr Val Thr Gln Asp Cys Leu Gln Leu Ile AlaAsp Ser Glu Thr Pro Thr Ile Gln Lys Gly Ser Tyr Thr Phe Val Pro Trp Leu LeuSer Phe Lys Arg Gly Ser Ala Leu Glu Glu Lys Glu Asn Lys Ile Leu Val Lys GluThr Gly Tyr Phe Phe Ile Tyr Gly Gln Val Leu Tyr Thr Asp Lys Thr Tyr Ala MetGly His Leu Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu Leu Ser Leu ValThr Leu Phe Arg Cys Ile Gln Asn Met Pro Glu Thr Leu Pro Asn Asn Ser Cys TyrSer Ala Gly Ile Ala Lys Leu Glu Glu Gly Asp Glu Leu Gln Leu Ala Ile Pro ArgGlu Asn Ala Gln Ile Ser Leu Asp Gly Asp Val Thr Phe Phe Gly Ala Leu Lys LeuLeu Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys GlyGlu Gln Lys Leu Ile Ser Glu Glu Asp Leu

<210>2<211>180<212> PRT <213> Artificial sequence <400>2

Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Gly Ser Thr Ser Gly Ser Gly LysPro Gly Ser Gly Glu Gly Ser Thr Lys Gly Ala Val Gln Gly Pro Glu Glu Thr ValThr Gln Asp Cys Leu Gln Leu Ile Ala Asp Ser Glu Thr Pro Thr Ile Gln Lys GlySer Tyr Thr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser Ala Leu Glu GluLys Glu Asn Lys Ile Leu Val Lys Glu Thr Gly Tyr Phe Phe Ile Tyr Gly Gln ValLeu Tyr Thr Asp Lys Thr Tyr Ala Met Gly His Leu Ile Gln Arg Lys Lys Val HisVal Phe Gly Asp Glu Leu Ser Leu Val Thr Leu Phe Arg Cys Ile Gln Asn Met ProGlu Thr Leu Pro Asn Asn Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu GlyAsp Glu Leu Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Leu Asp Gly AspVal Thr Phe Phe Gly Ala Leu Lys Leu Leu

<210>3<211>174<212> PRT <213> Artificial sequence <400>3

Ala Val Leu Thr Gln Lys Gln Lys Lys Gln His Ser Val Leu His Leu Val ProIle Asn Ala Thr Ser Lys Asp Asp Ser Asp Val Thr Glu Val Met Trp Gln Pro AlaLeu Arg Arg Gly Arg Gly Leu Gln Ala Gln Gly Tyr Gly Val Arg Ile Gln Asp AlaGly Val Tyr Leu Leu Tyr Ser Gln Val Leu Phe Gln Asp Val Thr Phe Thr Met GlyGln Val Val Ser Arg Glu Gly Gln Gly Arg Gln Glu Thr Leu Phe Arg Cys Ile ArgSer Met Pro Ser His Pro Asp Arg Ala Tyr Asn Ser Cys Tyr Ser Ala Gly Val PheHis Leu His Gln Gly Asp Ile Leu Ser Val Ile Ile Pro Arg Ala Arg Ala Lys LeuAsn Leu Ser Pro His Gly Thr Phe Leu Gly Phe Val Lys Leu Gly Ser Thr Ser GlySer Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Gln Lys Leu Ile SerGlu Glu Asp Leu

<210>4<211>174<212> PRT <213> Artificial sequence <400>4

Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Gly Ser Thr Ser Gly Ser Gly LysPro Gly Ser Gly Glu Gly Ser Thr Lys Gly Ala Val Leu Thr Gln Lys Gln Lys LysGln His Ser Val Leu His Leu Val Pro Ile Asn Ala Thr Ser Lys Asp Asp Ser AspVal Thr Glu Val Met Trp Gln Pro Ala Leu Arg Arg Gly Arg Gly Leu Gln Ala GlnGly Tyr Gly Val Arg Ile Gln Asp Ala Gly Val Tyr Leu Leu Tyr Ser Gln Val LeuPhe Gln Asp Val Thr Phe Thr Met Gly Gln Val Val Ser Arg Glu Gly Gln Gly ArgGln Glu ThrLeu Phe Arg Cys Ile Arg Ser Met Pro Ser His Pro Asp Arg Ala TyrAsn Ser Cys Tyr Ser Ala Gly Val Phe His Leu His Gln Gly Asp Ile Leu Ser ValIle Ile Pro Arg Ala Arg Ala Lys Leu Asn Leu Ser Pro His Gly Thr Phe Leu GlyPhe Val Lys Leu

<210>5<211>149<212> PRT <213> Artificial sequence <400>5

Ala Pro Met Ala Glu Gly Gly Gly Gln Asn His His Glu Val Val Lys Phe MetAsp Val Tyr Gln Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val Asp Ile Phe GlnGlu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser Cys Val Pro Leu Met ArgCys Gly Gly Cys Cys Asn Asp Glu Gly Leu Glu Cys Val Pro Thr Glu Glu Ser AsnIle Thr Met Gln Ile Met Arg Ile Lys Pro His Gln Gly Gln His Ile Gly Glu MetSer Phe Leu Gln His Asn Lys Cys Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg GlnGlu Lys Cys Asp Lys Pro Arg Arg Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly SerGly Glu Gly Ser Thr Lys Gly Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu

<210>6<211>149<212> PRT <213> Artificial sequence <400>6

Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Gly Ser Thr Ser Gly Ser Gly LysPro Gly Ser Gly Glu Gly Ser Thr Lys Gly Ala Pro Met Ala Glu Gly Gly Gly GlnAsn His His Glu Val Val Lys Phe Met Asp Val Tyr Gln Arg Ser Tyr Cys His ProIle Glu Thr Leu Val Asp Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr Ile PheLys Pro Ser Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly LeuGlu Cys Val Pro Thr Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg Ile Lys ProHis Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys Glu CysArg Pro Lys Lys Asp Arg Ala Arg Gln Glu Lys Cys Asp Lys Pro Arg Arg

<210>7<211>193<212> PRT <213> Artificial sequence <400>7

Ala Pro Met Ala Glu Gly Gly Gly Gln Asn His His Glu Val Val Lys Phe MetAsp Val Tyr Gln Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val Asp Ile Phe GlnGlu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser Cys Val Pro Leu Met ArgCys Gly Gly Cys Cys Asn Asp Glu Gly Leu Glu Cys Val Pro Thr Glu Glu Ser AsnIle Thr Met Gln Ile Met Arg Ile Lys Pro His Gln Gly Gln His Ile Gly Glu MetSer Phe Leu Gln His Asn Lys Cys Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg GlnGlu Asn Pro Cys Gly Pro Cys Ser Glu Arg Arg Lys His Leu Phe Val Gln Asp ProGln Thr Cys Lys Cys Ser Cys Lys Asn Thr Asp Ser Arg Cys Lys Ala Arg Gln LeuGlu Leu Asn Glu Arg Thr Cys Arg Cys Asp Lys Pro Arg Arg Gly Ser Thr Ser GlySer Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Gln Lys Leu Ile SerGlu Glu Asp Leu

<210>8<211>193<212> PRT <213> Artificial sequence <400>8

Glu Gln Lys Leu Ile Ser Glu Glu Asp Leu Gly Ser Thr Ser Gly Ser Gly LysPro Gly Ser Gly Glu Gly Ser Thr Lys Gly Ala Pro Met Ala Glu Gly Gly Gly GlnAsn His His Glu Val Val Lys Phe Met Asp Val Tyr Gln Arg Ser Tyr Cys His ProIle Glu Thr Leu Val Asp Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr Ile PheLys Pro Ser Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly LeuGlu Cys Val Pro Thr Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg Ile Lys ProHis Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys Glu CysArg Pro Lys Lys Asp Arg Ala Arg Gln Glu Asn Pro Cys Gly Pro Cys Ser Glu ArgArg Lys His Leu Phe Val Gln Asp Pro Gln Thr Cys Lys Cys Ser Cys Lys Asn ThrAsp Ser Arg Cys Lys Ala Arg Gln Leu Glu Leu Asn Glu Arg Thr Cys Arg Cys AspLys Pro Arg Arg

<210>9<211>178<212> PRT <213> Artificial sequence <400>9

AlaVal Gln Gly Pro Glu Glu Thr Val Thr Gln Asp Cys Leu Gln Leu Ile AlaAsp Ser Glu Thr Pro Thr Ile Gln Lys Gly Ser Tyr Thr Phe Val Pro Trp Leu LeuSer Phe Lys Arg Gly Ser Ala Leu Glu Glu Lys Glu Asn Lys Ile Leu Val Lys GluThr Gly Tyr Phe Phe Ile Tyr Gly Gln Val Leu Tyr Thr Asp Lys Thr Tyr Ala MetGly His Leu Ile Gln Arg Lys Lys Val His Val Phe Gly Asp Glu Leu Ser Leu ValThr Leu Phe Arg Cys Ile Gln Asn Met Pro Glu Thr Leu Pro Asn Asn Ser Cys TyrSer Ala Gly Ile Ala Lys Leu Glu Glu Gly Asp Glu Leu Gln Leu Ala Ile Pro ArgGlu Asn Ala Gln Ile Ser Leu Asp Gly Asp Val Thr Phe Phe Gly Ala Leu Lys LeuLeu Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys GlyAsp Tyr Lys Asp Asp Asp Asp Lys

<210>10<211>178<212> PRT <213> Artificial sequence <400>10

Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ser Thr Ser Gly Ser Gly Lys Pro GlySer Gly Glu Gly Ser Thr Lys Gly Ala Val Gln Gly Pro Glu Glu Thr Val Thr GlnAsp Cys Leu Gln Leu Ile Ala Asp Ser Glu Thr Pro Thr Ile Gln Lys Gly Ser TyrThr Phe Val Pro Trp Leu Leu Ser Phe Lys Arg Gly Ser Ala Leu Glu Glu Lys GluAsn Lys Ile Leu Val Lys Glu Thr Gly Tyr Phe Phe Ile Tyr Gly Gln Val Leu TyrThr Asp Lys Thr Tyr Ala Met Gly His Leu Ile Gln Arg Lys Lys Val His Val PheGly Asp Glu Leu Ser Leu Val Thr Leu Phe Arg Cys Ile Gln Asn Met Pro Glu ThrLeu Pro Asn Asn Ser Cys Tyr Ser Ala Gly Ile Ala Lys Leu Glu Glu Gly Asp GluLeu Gln Leu Ala Ile Pro Arg Glu Asn Ala Gln Ile Ser Leu Asp Gly Asp Val ThrPhe Phe Gly Ala Leu Lys Leu Leu

<210>11<211>172<212> PRT <213> Artificial sequence <400>11

Ala Val Leu Thr Gln Lys Gln Lys Lys Gln His Ser Val Leu His Leu Val ProIle Asn Ala Thr Ser Lys Asp Asp Ser Asp Val Thr Glu Val Met Trp Gln Pro AlaLeu Arg Arg Gly Arg Gly Leu Gln Ala Gln Gly Tyr Gly Val Arg Ile Gln Asp AlaGly Val Tyr Leu Leu Tyr Ser Gln Val Leu Phe Gln Asp Val Thr Phe Thr Met GlyGln Val Val Ser Arg Glu Gly Gln Gly Arg Gln Glu Thr Leu Phe Arg Cys Ile ArgSer Met Pro Ser His Pro Asp Arg Ala Tyr Asn Ser Cys Tyr Ser Ala Gly Val PheHis Leu His Gln Gly Asp Ile Leu Ser Val Ile Ile Pro Arg Ala Arg Ala Lys LeuAsn Leu Ser Pro His Gly Thr Phe Leu Gly Phe Val Lys Leu Gly Ser Thr Ser GlySer Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Asp Tyr Lys Asp Asp AspAsp Lys

<210>12<211>172<212> PRT <213> Artificial sequence <400>12

Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ser Thr Ser Gly Ser Gly Lys Pro GlySer Gly Glu Gly Ser Thr Lys Gly Ala Val Leu Thr Gln Lys Gln Lys Lys Gln HisSer Val Leu His Leu Val Pro Ile Asn Ala Thr Ser Lys Asp Asp Ser Asp Val ThrGlu Val Met Trp Gln Pro Ala Leu Arg Arg Gly Arg Gly Leu Gln Ala Gln Gly TyrGly Val Arg Ile Gln Asp Ala Gly Val Tyr Leu Leu Tyr Ser Gln Val Leu Phe GlnAsp Val Thr Phe Thr Met Gly Gln Val Val Ser Arg Glu Gly Gln Gly Arg Gln GluThr Leu Phe Arg Cys Ile Arg Ser Met Pro Ser His Pro Asp Arg Ala Tyr Asn SerCys Tyr Ser Ala Gly Val Phe His Leu His Gln Gly Asp Ile Leu Ser Val Ile IlePro Arg Ala Arg Ala Lys Leu Asn Leu Ser Pro His Gly Thr Phe Leu Gly Phe ValLys Leu

<210>13<211>147<212> PRT <213> Artificial sequence <400>13

Ala Pro Met Ala Glu Gly Gly Gly Gln Asn His His Glu Val Val Lys Phe MetAsp Val Tyr Gln Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val Asp Ile Phe GlnGlu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser Cys Val Pro Leu Met ArgCys Gly Gly Cys Cys Asn Asp Glu Gly Leu Glu Cys Val Pro Thr Glu Glu Ser AsnIle Thr MetGln Ile Met Arg Ile Lys Pro His Gln Gly Gln His Ile Gly Glu MetSer Phe Leu Gln His Asn Lys Cys Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg GlnGlu Lys Cys Asp Lys Pro Arg Arg Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly SerGly Glu Gly Ser Thr Lys Gly Asp Tyr Lys Asp Asp Asp Asp Lys

<210>14<211>147<212> PRT <213> Artificial sequence <400>14

Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ser Thr Ser Gly Ser Gly Lys Pro GlySer Gly Glu Gly Ser Thr Lys Gly Ala Pro Met Ala Glu Gly Gly Gly Gln Asn HisHis Glu Val Val Lys Phe Met Asp Val Tyr Gln Arg Ser Tyr Cys His Pro Ile GluThr Leu Val Asp Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys ProSer Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu Glu CysVal Pro Thr Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg Ile Lys Pro His GlnGly Gln His Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys Glu Cys Arg ProLys Lys Asp Arg Ala Arg Gln Glu Lys Cys Asp Lys Pro Arg Arg

<210>15<211>191<212> PRT <213> Artificial sequence <400>15

Ala Pro Met Ala Glu Gly Gly Gly Gln Asn His His Glu Val Val Lys Phe MetAsp Val Tyr Gln Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val Asp Ile Phe GlnGlu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser Cys Val Pro Leu Met ArgCys Gly Gly Cys Cys Asn Asp Glu Gly Leu Glu Cys Val Pro Thr Glu Glu Ser AsnIle Thr Met Gln Ile Met Arg Ile Lys Pro His Gln Gly Gln His Ile Gly Glu MetSer Phe Leu Gln His Asn Lys Cys Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg GlnGlu Asn Pro Cys Gly Pro Cys Ser Glu Arg Arg Lys His Leu Phe Val Gln Asp ProGln Thr Cys Lys Cys Ser Cys Lys Asn Thr Asp Ser Arg Cys Lys Ala Arg Gln LeuGlu Leu Asn Glu Arg Thr Cys Arg Cys Asp Lys Pro Arg Arg Gly Ser Thr Ser GlySer Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Asp Tyr Lys Asp Asp AspAsp Lys

<210>16<211>191<212> PRT <213> Artificial sequence <400>16

Asp Tyr Lys Asp Asp Asp Asp Lys Gly Ser Thr Ser Gly Ser Gly Lys Pro GlySer Gly Glu Gly Ser Thr Lys Gly Ala Pro Met Ala Glu Gly Gly Gly Gln Asn HisHis Glu Val Val Lys Phe Met Asp Val Tyr Gln Arg Ser Tyr Cys His Pro Ile GluThr Leu Val Asp Ile Phe Gln Glu Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys ProSer Cys Val Pro Leu Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu Glu CysVal Pro Thr Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg Ile Lys Pro His GlnGly Gln His Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys Glu Cys Arg ProLys Lys Asp Arg Ala Arg Gln Glu Asn Pro Cys Gly Pro Cys Ser Glu Arg Arg LysHis Leu Phe Val Gln Asp Pro Gln Thr Cys Lys Cys Ser Cys Lys Asn Thr Asp SerArg Cys Lys Ala Arg Gln Leu Glu Leu Asn Glu Arg Thr Cys Arg Cys Asp Lys ProArg Arg

<210>17<211>497<212> PRT <213> Artificial sequence <400>17

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His AlaAla Arg Pro Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu GlyGln Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Asp Asn Tyr Gly Phe SerPhe Met Asn Trp Phe Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr AlaIle Ser Asn Arg Gly Ser Gly Val Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly ThrAsp Phe Ser Leu Asn Ile His Pro Val Glu Glu Asp Asp Pro Ala Met Tyr Phe CysGln Gln Thr Lys Glu Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile LysGly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val His LeuVal Glu Ser Gly Gly Asp Leu Val Lys Pro Gly Gly Ser Leu Lys Leu Ser Cys AlaAla Ser Gly Phe Thr Phe Ser His Tyr Gly MetSer Trp Val Arg Gln Thr Pro AspLys Arg Leu Glu Trp Val Ala Thr Ile Gly Ser Arg Gly Thr Tyr Thr His Tyr ProAsp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Asp Lys Asn Ala Leu TyrLeu Gln Met Asn Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala Arg ArgSer Glu Phe Tyr Tyr Tyr Gly Asn Thr Tyr Tyr Tyr Ser Ala Met Asp Tyr Trp GlyGln Gly Ala Ser Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro ThrPro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg ProAla Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr IleTrp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr LeuTyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met ArgPro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu GluGlu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala TyrLys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu TyrAsp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg ArgLys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu AlaTyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly LeuTyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln AlaLeu Pro Pro Arg

<210>18<211>497<212> PRT <213> Artificial sequence <400>18

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His AlaAla Arg Pro Glu Val His Leu Val Glu Ser Gly Gly Asp Leu Val Lys Pro Gly GlySer Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser His Tyr Gly Met SerTrp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val Ala Thr Ile Gly Ser ArgGly Thr Tyr Thr His Tyr Pro Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg AspAsn Asp Lys Asn Ala Leu Tyr Leu Gln Met Asn Ser Leu Lys Ser GluAsp Thr AlaMet Tyr Tyr Cys Ala Arg Arg Ser Glu Phe Tyr Tyr Tyr Gly Asn Thr Tyr Tyr TyrSer Ala Met Asp Tyr Trp Gly Gln Gly Ala Ser Val Thr Val Ser Ser Gly Gly GlyGly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Leu Thr Gln SerPro Ala Ser Leu Ala Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Arg Ala SerGlu Ser Val Asp Asn Tyr Gly Phe Ser Phe Met Asn Trp Phe Gln Gln Lys Pro GlyGln Pro Pro Lys Leu Leu Ile Tyr Ala Ile Ser Asn Arg Gly Ser Gly Val Pro AlaArg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser Leu Asn Ile His Pro Val GluGlu Asp Asp Pro Ala Met Tyr Phe Cys Gln Gln Thr Lys Glu Val Pro Trp Thr PheGly Gly Gly Thr Lys Leu Glu Ile Lys Thr Thr Thr Pro Ala Pro Arg Pro Pro ThrPro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg ProAla Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr IleTrp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr LeuTyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met ArgPro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu GluGlu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala TyrLys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu TyrAsp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg ArgLys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu AlaTyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly LeuTyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln AlaLeu Pro Pro Arg

<210>19<211>485<212> PRT <213> Artificial sequence <400>19

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His AlaAla Arg Pro Asp Ile Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Phe GlyGln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser Asp Gly GlnThr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser Pro Lys Arg Leu Ile TyrLeu Val Ser Lys Leu Asp Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser GlyThr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr TyrCys Trp Gln Gly Thr His Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu IleLys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val GlnLeu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly Ala Ser Val Lys Leu Ser CysThr Ala Ser Gly Phe Asn Ile Lys Asp Tyr Tyr Met His Trp Val Lys Gln Arg ThrGlu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro Glu Asp Gly Glu Thr Lys TyrAla Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr Ala Asp Thr Ser Ser Lys Thr AlaTyr Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys Ala ArgLeu Lys Gly Gly Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Thr Thr ThrPro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser LeuArg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu AspPhe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu LeuLeu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr IlePhe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys SerCys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser ArgSer Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu AsnLeu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro GluMet Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu GlnLys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg ArgGly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr TyrAsp Ala Leu His Met Gln Ala Leu Pro Pro Arg

<210>20<211>485<212> PRT <213> Artificial sequence <400>20

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His AlaAla Arg Pro Glu Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Lys Pro Gly AlaSer Val Lys Leu Ser Cys Thr Ala Ser Gly Phe Asn Ile Lys Asp Tyr Tyr Met HisTrp Val Lys Gln Arg Thr Glu Gln Gly Leu Glu Trp Ile Gly Arg Ile Asp Pro GluAsp Gly Glu Thr Lys Tyr Ala Pro Lys Phe Gln Gly Lys Ala Thr Ile Thr Ala AspThr Ser Ser Lys Thr Ala Tyr Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr AlaVal Tyr Tyr Cys Ala Arg Leu Lys Gly Gly Tyr Trp Gly Gln Gly Thr Thr Leu ThrVal Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser AspIle Val Met Thr Gln Thr Pro Leu Thr Leu Ser Val Thr Phe Gly Gln Pro Ala SerIle Ser Cys Lys Ser Ser Gln Ser Leu Leu Asp Ser Asp Gly Gln Thr Tyr Leu AsnTrp Leu Leu Gln Arg Pro Gly Gln Ser Pro Lys Arg Leu Ile Tyr Leu Val Ser LysLeu Asp Ser Gly Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe ThrLeu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Trp Gln GlyThr His Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Thr Thr ThrPro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser LeuArg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu AspPhe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu LeuLeu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr IlePhe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys SerCys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser ArgSer Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu AsnLeu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro GluMet Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu GlnLys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg ArgGly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr TyrAsp Ala Leu His Met Gln Ala Leu Pro Pro Arg

<210>21<211>488<212> PRT <213> Artificial sequence <400>21

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His AlaAla Arg Pro Asp Val Leu Met Thr Gln Ala Pro Leu Thr Leu Pro Val Ser Leu GlyAsp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ala Ile Val His Ala Asn Gly AsnThr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Ala Leu Leu Ile TyrLys Val Ala Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser GlyThr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr TyrCys Phe Gln Gly Ala His Ala Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu IleLys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val GlnLeu Gln Gln Ser Gly Gly Glu Leu Ala Lys Pro Gly Ala Ser Val Lys Met Ser CysLys Ser Ser Gly Tyr Thr Phe Thr Ala Tyr Ala Ile His Trp Ala Lys Gln Ala AlaGly Ala Gly Leu Glu Trp Ile Gly Tyr Ile Ala Pro Ala Ala Gly Ala Ala Ala TyrAsn Ala Ala Phe Lys Gly Lys Ala Thr Leu Ala Ala Asp Lys Ser Ser Ser Thr AlaTyr Met Ala Ala Ala Ala Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys Ala ArgAla Ala Ala Ala Gly Ala Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser SerThr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln ProLeu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr ArgGly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys GlyVal Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys LeuLeu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu AspGly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val LysPhe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr AsnGlu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly ArgAsp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr AsnGlu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly GluArg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr LysAsp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

<210>22<211>488<212> PRT <213> Artificial sequence <400>22

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His AlaAla Arg Pro Glu Val Gln Leu Gln Gln Ser Gly Gly Glu Leu Ala Lys Pro Gly AlaSer Val Lys Met Ser Cys Lys Ser Ser Gly Tyr Thr Phe Thr Ala Tyr Ala Ile HisTrp Ala Lys Gln Ala Ala Gly Ala Gly Leu Glu Trp Ile Gly Tyr Ile Ala Pro AlaAla Gly Ala Ala Ala Tyr Asn Ala Ala Phe Lys Gly Lys Ala Thr Leu Ala Ala AspLys Ser Ser Ser Thr Ala Tyr Met Ala Ala Ala Ala Leu Thr Ser Glu Asp Ser AlaVal Tyr Tyr Cys Ala Arg Ala Ala Ala Ala Gly Ala Asp Tyr Trp Gly Gln Gly ThrThr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly GlyGly Ser Asp Val Leu Met Thr Gln Ala Pro Leu Thr Leu Pro Val Ser Leu Gly AspGln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ala Ile Val His Ala Asn Gly Asn ThrTyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser Pro Ala Leu Leu Ile Tyr LysVal Ala Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly ThrAsp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr CysPhe Gln Gly Ala His Ala Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile LysThr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln ProLeu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr ArgGly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys GlyVal Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys LeuLeu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu AspGly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val LysPhe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr AsnGlu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly ArgAsp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr AsnGlu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly GluArg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr LysAsp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

Claims

1. An anti-tumor CAR-immune cell composition comprising a CAR-immune cell and a natural ligand-based regulatory protein, wherein:

2. The composition of claim 1, wherein the natural ligand of the regulatory protein that recognizes the tumor or tissue-associated antigen thereof is at least one of B L yS, APRI L, and VEGF.

3. The composition according to claim 1 or 2, characterized in that: the label molecule is selected from one of FITC, biotin, His label, Flag label, Myc label, GCN4 label, HA label, V5 label, Chitosan Binding Protein (CBP) label, Maltose Binding Protein (MBP), glutathione-S-transferase (GST) and DNA Aptamer label.

4. The composition according to any one of claims 1 to 3, characterized in that:

the natural ligand molecule and the label molecule of the regulatory protein are coupled through a hydrophilic bifunctional linker;

preferably, the hydrophilic bifunctional linker is a connecting peptide;

preferably, the linker peptide is selected from the group consisting of a linear polypeptide, an β -fold peptide, or a coiled-coil peptide.

5. The composition according to any one of claims 1 to 4, characterized in that: the tag-specific recognition molecule in the CAR-immune cell is selected from a single chain antibody, a protein ligand, a short peptide molecule, or a nucleic acid molecule.

6. The composition according to any one of claims 1 to 5, characterized in that: the CAR structure also comprises co-stimulatory factors CD28, 4-1BB, OX-40, CD27, ICOS and at least one of immunoglobulin family or tumor necrosis factor superfamily.

7. The composition according to any one of claims 1 to 6, characterized in that: the immune cell is selected from the group consisting of a T cell, a natural killer cell, a macrophage, a tumor immune cell, and a stem cell-induced immune cell.

8. The composition according to any one of claims 1 to 7, characterized in that: the regulatory protein has at least 2 natural ligand molecules that recognize a variety of tumor or tissue-associated antigens thereof.

9. The composition according to any one of claims 1 to 4, characterized in that: the regulatory protein is expressed in a fusion way in a gene recombination way; or by means of random chemical coupling and site-specific chemical bonding.

10. Use of a multi-target recognition regulatable genetically engineered immune cell composition as defined in any one of claims 1 to 9 for the preparation of an anti-tumor medicament.