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

Abstract

The invention discloses a preparation method of a natural ligand-mediated multi-target recognition-regulatable gene-engineered immune cell. In order to solve the problem of efficacy and safety of CAR-T therapy, the inventors constructed the target recognition part of growth factors and CAR-immune cells and activation domains separately. In tumor-targeted therapy, tag molecules are used to modify growth factors As a regulatory protein, by constructing a tag-specific CAR on immune cells, a new type of multi-target recognition and regulated genetically engineered immune cells can be prepared, so as to achieve stable recognition of tumor-related antigen multi-target points and improve its safety and controllability. , in order to overcome the difficulty that tandem antibody CAR-T cells are dangerous in the treatment process.

Description

Natural ligand-mediated multi-target recognition modulates the mechanism of genetically engineered immune cells backup method

technical field

The invention relates to the use of natural ligands to mediate genetically engineered immune cells for improving the multi-target recognition activity and safety controllability of anti-tumor immunotherapy.

Background technique

In recent years, cancer immunotherapy has attracted much attention and is the focus of the field of cancer treatment. In December 2011, Nature and the Journal of Clinical Oncology, the top journal of clinical oncology, respectively published a review article with the same title "Cancerimmunotherapy comes of age", evaluating that immunotherapy is the only method in existing technology that can completely remove cancer cells. It makes up for the shortcomings of traditional therapy, and is considered to be the most active and promising treatment method in the comprehensive treatment model of tumors in the 21st century.

Chimeric Antigen Receptor-T (Chimeric Antigen Receptor-T, CAR-T) cell technology is to genetically modify T cells to specifically recognize tumor-specific or related antigens and exert a toxic effect on tumor cells, thereby exerting an anti-tumor effect. Immunotherapy techniques. The concept of CAR-T immunotherapy was first proposed by Dotti (1) and others. It has been nearly 30 years since the breakthrough progress in the treatment of leukemia in clinical trials. After continuous scientific research and clinical transformation, CAR-T technology has experienced 4 years. Generation Innovation (2). However, most of the current clinical applications are still second-generation CAR molecular structures. Commonly used co-stimulatory molecules include CD28, CD137, OX40, etc. Compared with the classic T cell receptor (TCR)-human leukocyte antigen (HLA) complex recognition mode, the recognition of tumor cells by CAR molecules depends on the specificity of the antibody, so it is simpler and more efficient. It is clear and avoids the problem of tumor immune escape caused by the down-regulation of HLA molecule expression.

CAR is divided into three parts: the intracellular signal peptide region, the transmembrane region and the extracellular antigen binding region: the extracellular binding antigen region binds the antigen to exert specificity. The transmembrane region of CAR is composed of signal molecules, such as CD3, CD3ζ, CD4, CD28, etc. The intracellular signal peptide region includes T cell receptor CD3ζ chain or tyrosine on FcεRIγ, etc. CAR-modified T cells are T cells isolated from peripheral blood cells of patients, and then genetically engineered to transfect CAR into T cells to form chimeric antigen receptor T cells (3).

T cells are modified by lentivirus transfection of CAR gene, and the modified T cells carry antigens that can specifically recognize tumor cells, and have the ability to self-replicate and kill tumor cells. Therefore, CAR-T specifically recognizes tumor cells, targets and kills tumor cells, and reduces the influence of the tumor microenvironment. CAR-T is not only not affected by the restriction of MHC, but also recognizes multiple antigens, and there is no risk of TCR mismatch. CAR-T can also directly inhibit the growth of tumor cells. CAR-T secretes cellular factors such as interferon gamma (IFN-γ), thereby inducing macrophages to secrete interleukin 6 (IL-6) and interleukin 10 (IL-10). , Interleukin 12 (IL-12) inhibits the growth of tumor cells (4). In addition to its own unique killing effect, CAR-T cells can also activate the body's immune system and play an anti-tumor effect.

Compared with the rapid progress of CAR-T cell therapy in hematological tumors, the clinical effect of CAR-T cells in the treatment of solid tumors is unsatisfactory. Compared with hematological tumors, solid tumors have complex tissue components and lack ideal specific targets. In order to achieve the desired therapeutic effect, as many as 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 them, targets such as epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2) and mesothelin (MSLN) are more developed. .

The studies that have been reported so far usually use a single target, but the traditional single target CAR-T has many disadvantages, such as: prone to immune evasion. The difficulty faced by CAR-T cells in the treatment of tumors is the problem of therapeutic targets. Selecting an appropriate target for complex solid tumors is more difficult than for hematological tumors, for two main reasons. First, in hematological tumors, such as ALL, almost all tumor cells express CD19 or CD20, so CAR-T cell therapy targeting these two antigens is expected to eliminate all tumor cells, thereby bringing about the cure of hematological tumors. In contrast, the composition of cells in solid tumor tissues is complex, and different tumor cells often have different protein expression profiles, and it is difficult for a single target to cover all tumor cells. Both theoretical and practical experience show that even if tumor cells expressing a certain antigen can be effectively eliminated, there will still be a large number of antigen-negative tumor cells remaining, and this incomplete phenotype coverage cannot meet the requirements of tumor cure. Secondly, it is difficult to find a tumor-specific antigen target in solid tumors. Most of the existing CAR-T cell therapy experiments on solid tumors select antigens that are specifically highly expressed in tumors, and these antigens are often still in some normal tissues. There will be low levels of expression, such as HER2, EGFR, etc. This greatly increases the possibility of CAR-T cells attacking normal tissues in the treatment of solid tumors, which is called targeted toxicity. With the enhancement of the anti-tumor function of CAR-T cells, the harm of off-target side effects will also increase. In response to these problems, the current effective solution is to adopt a multi-target strategy, that is, the combination of multi-target CAR-T cells. The combination of multi-target CAR-T cells can seek coverage of a higher proportion of tumor cells. The coverage of multiple targets can increase the proportion of CAR-T cells killing tumor cells, and the elimination of a higher proportion of tumor cells is expected to improve the survival of patients. beneficial. This multi-target (dual-target, even multi-target) CAR-T cell therapy strategy is also used in the treatment of B-cell leukemia. For example, CAR-T cell therapy targeting both CD19 and CD20 is considered to be effective in preventing CD19 negative. Relapse of Leukemia (5)(6). Multiple target coverage can be achieved by infusion of mixed CAR-T cells. In addition, it can also be achieved by integrating two (or more) single chain antibody (single chain antibody, scFv) segments targeting different antigens on the CAR molecule (7), that is, the method of tandem antibodies, while the dual target In the preparation process of CAR-immune cells, the target recognition function of CAR-immune cells is usually reduced due to the mismatch or steric hindrance of the tandemly expressed antibodies on the cell surface, and even serious off-target toxicity is caused. The use of natural ligands of cell growth factors to recognize multiple tumor-associated antigens has been reported. However, the use of growth factors as the direct recognition site of CAR-immune cells usually causes serious complications due to the inability to distinguish tumor tissues from normal tissues. Off-target toxicity. Therefore, it is an important idea of the method to select suitable natural ligands of growth factors recognized by tumor-associated antigens, and on this basis, to construct a regulated CAR-T therapy strategy that separates tumor-targeted recognition and CAR-T cell downstream signal transduction. .

B-cell activating factor (BLyS), a member of the tumor necrosis factor (TNF) family that regulates B cell maturation, survival and function, is overexpressed in a variety of autoimmune diseases. BLyS is a typical type II transmembrane protein with the N-terminus in the envelope, lacking the signal peptide sequence, and the C-terminus outside the cell. BLyS is a monoglycoprotein with a molecular weight of 31.3kD consisting of 285 amino acids, in which the 1-46 amino acids at the N-terminal are the intracellular domain, the 47-73 amino acids are the hydrophobic transmembrane domain, and the 74-285 amino acids are the cellular domain. In the outer region, amino acids 133-285 are the main functional regions. BLyS may have a glycosylation site (N242), and there is no disulfide bond. The homology between human and mouse is 93%. BLyS has a strong tropism for B cells. As a costimulatory factor of lymphocytes, BLyS has a strong stimulating effect on activated B cells to promote proliferation and differentiation. Increased expression of BLyS and its receptors have been identified in a number of B-cell malignancies.

The biological effects of BLyS are mediated by three cell surface receptors called B-cell activating factor receptors (BAFF-R), transmembrane activators and calcium regulators that interact with cyclophilin ligands Transmembrane activator and CAML interactor (TACI) and B cell maturation antigen (BCMA), both of which are mainly expressed on the surface of B cells, are type III membrane proteins. The extracellular region of the receptor has cysteine-rich regions (CRDs) that can form receptor-ligand structural motifs, of which BAFF-R may be a specific receptor that exists only on B cells, while TACI and BCMA are also Receptor for APRIL, another member of the TNF superfamily (8).

Traditional CAR-T cells usually target a single tumor-associated antigen, so in the process of tumor treatment, they usually face the problem of tumor antigen escape. The biology of BLyS used in this study is mediated by three cell surface receptors, which can target and recognize multiple tumor-associated antigen receptor molecules. It is multispecific and can reduce tumor antigen escape and mismatch to a certain extent. and the possibility of off-target. In addition, compared with the existing technology, the target recognition molecule BLyS is a naturally occurring ligand molecule in the human body and has no immunogenicity, which increases the safety of CAR-T cells during the treatment process. There are no reports on chimeric antigen receptor (CAR) targeting BLyS.

A proliferation-inducing ligand (APRIL), also known as TNFSF13, is one of the important members of the tumor necrosis factor family. APRIL is lowly expressed in most normal tissues and is mainly expressed on a variety of immune cells, such as Dendritic cells, macrophages, monocytes, T lymphocytes, etc. APRIL mainly promotes and participates in the proliferation, differentiation and survival of lymphocytes by binding to receptors, as well as inducing the production of immunoglobulins and class switching. In recent years, more and more studies have shown that APRIL is involved in the pathogenesis of various autoimmune diseases (9).

APRIL contains 250 amino acids and is a type II transmembrane protein. It is divided into an intracellular domain (N-terminal, no signal peptide sequence), a transmembrane domain (amino acids 29-49) and an extracellular domain (amino acids 50-250). There are two forms of membrane-bound with a relative molecular mass of about 27 000 and an extracellular soluble form with a relative molecular mass of about 17 000. APRIL can combine with itself to form a homotrimer, or it can form a heterotrimer with TNFSF13B, a member of the same family, namely B-cell activation factor (BAFF) (one BAFF binds two APRILs or two BAFF binds 1 APRIL). In addition, APRIL can also combine with the protein TWEAK of the same family and its upstream gene is located to form a type II transmembrane protein.

As a cytokine with multiple functions, APRIL must bind to receptors on its target cells to exert its biological effects. There are currently two clear receptors for APRIL, TACI and BCMA. TACI is mainly expressed on the surface of some B cells and activated T cells, while BCMA is expressed on the surface of mature B cells. Both are shared receptors of APRIL and BAFF, and both belong to the tumor necrosis factor receptor family, which can be related to tumor necrosis factor. Protein (TRAF) binding to promote activation of anti-apoptotic and proliferative signaling pathways. APRIL can promote and participate in various functions such as lymphocyte proliferation, differentiation, survival, and induction of immunoglobulin production and class switching by binding to receptors. Although it has been reported to use APRIL as the recognition domain of CAR for the preparation of traditional hematological tumor treatment CAR-T cells (10), APRIL-based CAR may cause serious target toxicity, such as B cell exhaustion or neural Therefore, optimizing the targeting strategy of APRIL and constructing regulated CAR-T cells can solve this potential problem.

Vascular endothelial growth factor (VEGF), a heparin-binding growth factor specific for vascular endothelial cells, can induce angiogenesis in vivo. It is secreted by some tumor cells, and promotes endothelial cell proliferation by binding to the corresponding receptors on the vascular endothelium. At the same time, it can increase vascular permeability to allow endothelial cells to migrate, induce tumor angiogenesis, and maintain tumor growth. The most potent angiogenic factor involved in many physiological and pathological processes (11). There are currently seven VEGF families, namely VEGF-A, VEGF-B, VEGF-C, VEGF-D, VEGF-E, VEGF-F, and placenta growth factor (PIGF). The human VEGF-A gene is located on chromosome 6p21.3. It is a single gene with a total length of 14kb and consists of 8 exons and 7 introns. The encoded product is a homodimeric glycoprotein of 34-45kD. VEGF can be cleaved at the transcriptional level to produce five isomers, which are named VEGF-AA145, VEGF-A165, VEGF-A121, VEGF-A189 and VEGF-A206 according to the amino acid length. Among them, VEGF-A121 is a weakly acidic polypeptide that does not bind to heparin, and VEGF-A165 is a basic protein with low affinity to heparin. Both are secreted in a soluble and freely diffusible form and are easy to reach target cells; VEGF-A145, VEGF-A165 and VEGF-A206 have high affinity with heparin, and bind to cell surface or cell matrix after secretion, and belong to cell-associated isoforms. At present, it has been confirmed that VEGF-A is a selective mitogen of endothelial cells. In addition to increasing the concentration of Ca2+ in the cytoplasm of endothelial cells and increasing the permeability of microvessels (mainly post-capillary veins and venules) to macromolecular substances, It can still make endothelial cells elongated in various ways and stimulate their replication, stimulate the transport of glucose into endothelial cells, promote the translocation of endothelial cells, mouse monocytes and fetal bovine osteoblasts, and can change the activation of endothelial cells. pattern, up-regulates the expression of plasminogen activator (including urokinase-type and tissue-type) and its inhibitor PAI-I, and induces the expression of other endothelial cell proteases, interstitial collagenase and tissue factor. VEGF-A mediates many physiological and pathological angiogenesis, and its expression is also enhanced when tissue blood vessels proliferate. Cells in a differentiated state of embryonic developing tissues have higher expression than adult and fully differentiated cells. Under physiological conditions, VEGF-A can be expressed at high levels in the placenta, many embryonic tissues and some adult normal tissues with physiological vascular proliferation (such as proliferative endometrium). In addition, it is also expressed at low levels in normal glomerular cells, cardiomyocytes, prostate epithelium, semen, and certain epithelial cells of adrenal cortex and lung in animals and adults. Pathologically, VEGF-A is overexpressed in the synovial layer cells of healing skin wounds, psoriasis, delayed allergic reactions, and rheumatoid arthritis.

The currently discovered receptors of the VEGF family are divided into tyrosine kinase receptors VEGFR1 (Flt1), VEGFR2 (KDR/Klk1), VEGFR3 (Flt4) and non-tyrosine kinase receptors: neuropilin (NRP1 and NRP2) . Flt1, KDR, and Flt4 are receptor tyrosine kinases (RTKs). The former two are mainly expressed on vascular endothelial cells, while the latter are mainly found in lymphatic endothelial cells. NP1 and NP2 are non-tyrosine protein kinase transmembrane receptors, which contain long extracellular segments and short intracellular segments, and are expressed not only in endothelial cells, but also in some tumor cells. The main biological function of VEGF is achieved through VEGFR2. VEGFR2 and VEGF bind to dimerization and the intracellular tyrosine residues themselves are phosphorylated. VEGFR2 has 6 self-phosphorylated sites confirmed, Tyr1054, Tyr951, Tyr996, Tyr1059, Tyr1175, Tyr1214. The functions of most of these sites are unknown, but it has been confirmed that Tyr951 is involved in the binding of src homology 2, SH2, and Tyr1175 is the binding site of phosphatase C-y (PLC-y). Numerous clinical studies have shown that various diseases in which the VEGF/VEGFR signaling pathway plays a central role in angiogenesis, such as cancer, various VEGF signaling inhibitors, including anti-VEGF neutralizing antibodies and VEGFR kinase/multikinase Inhibitors have been successfully developed and are now widely used in clinical practice. Although it has been reported that VEGF-A165 is used as the recognition domain of CAR for the preparation of CAR-T cells that traditionally target the blood vessels of solid tumors (12), the traditional CAR based on VEGF-A may cause severe on-target toxicity , such as cardiovascular failure and other complications, in view of this, we decided to optimize the regulated CAR-T cell strategy of targeting VEGF.

references

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2. Chmielewski M, Hombach AA, & Abken H (2014) Of CARs and TRUCKs: chimericantigen receptor (CAR) T cells engineered with an inducible cytokine to modulate 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 Treatment Effect 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 and CD133-specific chimeric antigen receptor-modified T cells in a patient with advanced 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-activating factor/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 factor receptors. Proc Natl Acad Sci USA 99(10):7009-7014.

SUMMARY OF THE INVENTION

In order to solve the problem of efficacy and safety of CAR-T therapy, the inventors of the present invention separately constructed the tumor-targeted recognition part of natural ligands (BLyS, APRIL and VEGF) and the activation domain of CAR-immune cells. In tumor targeted therapy, tag molecules are used to modify the natural ligands of growth factors as regulatory proteins. By constructing tag-specific CARs on immune cells, a new type of multi-target recognition and regulatable gene-engineered immune cells are prepared, so as to achieve tumor-related While stably identifying multiple antigen targets, it can improve its safety and controllability to overcome the traditional tandem CAR structure that combines the tumor recognition domain with the immune cell activation domain, resulting in off-target toxicity that genetically engineered immune cells are prone to produce during treatment. and other risk issues.

The technical scheme adopted by the present invention is:

A composition of anti-tumor CAR-immune cells, including CAR-immune cells and regulatory proteins constructed based on natural ligands,

The natural ligand is a naturally occurring ligand molecule that can specifically bind to a tumor or its tissue surface-related receptor;

The CAR domain of the CAR-immune cell is connected with a recognition molecule, and the recognition molecule is used to specifically recognize the tag molecule;

The regulatory protein has natural ligand molecules and tag molecules that can recognize a variety of tumors or their tissue-associated antigens, and the tag molecules can be specific to the recognition molecules of the CAR extracellular recognition domain of the CAR-immune cells combine.

In some examples, the regulatory protein has a natural ligand molecule that recognizes at least 2, 3, or 4 tumor or tissue-associated antigens thereof. The increase in the number of natural ligands on the regulatory protein makes it more closely combined with tumor cells, which is conducive to increasing its specificity to tumors, and is conducive to reducing or avoiding dangerous problems such as off-target toxicity during use.

In some examples, the natural ligand in the regulatory protein that recognizes a tumor or its tissue-associated antigen is at least one of BLyS, APRIL, and VEGF.

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

In some examples, the natural ligand in the regulatory protein that recognizes the tumor antigen is APRIL, which targets the extracellular binding domains of BCMA and TACI.

In some examples, the natural ligand in the regulatory protein that recognizes the tumor antigen is VEGF, targeting the extracellular binding domains of VEGFRl, VEGFR2, and VEGFR3.

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

In some examples, the linker is a linker peptide.

In some examples, the linker peptide is selected from a linear polypeptide, a beta-sheet peptide, or a coiled-coil peptide.

In some examples, the hydrophilic bifunctional linker is selected from molecules that can be linked via a click reaction.

In some examples, the tag molecule is selected from the group consisting of FITC, biotin, His tag, Flag tag, Myc tag, GCN4 tag, HA tag, V5 tag, chitosan binding protein (CBP) tag, maltose binding protein (MBP), gluten One of the tathione-S-transferase (GST) and DNA Aptamer tags. The tag molecule can be selected according to the specific situation. When the regulatory protein is a fusion protein, a polypeptide tag molecule is preferred, which is convenient for fusion expression. In particular, the regulatory protein is expressed in the form of a fusion protein, and the tag molecule used is the tag molecule used for protein separation and purification, which is beneficial to the purification of the fusion protein.

In some examples, the tag-specific recognition molecules in the CAR-immune cells are selected from single-chain antibodies, protein ligands, short peptide molecules or nucleic acid molecules to specifically recognize and capture the tag molecules on regulatory proteins. In the presence of regulatory proteins, CAR-immune cells can mediate their specific binding to tumor cells through the tag molecules on the regulatory proteins, so as to specifically kill tumor cells.

In some instances, the CAR-immune cells are constructed using a method selected from lentivirus, adenovirus, adeno-associated virus, transposon technology, mRNA transfection, nanogene, Zinc Finger Nuclease, TALEN, and CRISPR/Cas9. It can also be constructed using the general method.

In some instances, the regulatory proteins are fused and expressed by gene recombination, or prepared by random chemical coupling and site-specific chemical bonding, or other methods.

In some examples, the immune cells are selected from T cells, natural killer cells, macrophages, tumor immune cells, and stem cell-induced immune cells.

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 structure of the CAR-immune cells further includes at least one of costimulatory factors CD28, 4-1BB, OX-40, CD27, and ICOS.

In some examples, the regulatory protein is selected from the group consisting of: APRIL-Myc protein, Myc-APRIL protein, VEGF121-Myc protein, Myc-VEGF121 protein, VEGF165-Myc protein, Myc-VEGF165 protein, BLyS-FLAG protein, FLAG-BLyS protein, APRIL-FLAG protein, FLAG-APRIL protein, VEGF121-FLAG protein, FLAG-VEGF121 protein, VEGF165-FLAG protein, FLAG-VEGF165 protein.

The beneficial effects of the present invention are:

The genetically engineered immune cells of the present invention connect the tag molecule to the CAR domain, and then use the tag to target the regulatory protein to which the tag is attached. Preferably, these regulatory proteins have two or more specific recognition receptors, such as The receptors of BLyS include BCMA, BAFF-R, and TACI. Through the mediation of regulatory proteins, it can target and recognize multiple related antigen receptor molecules on tumor cells, with multi-specificity to achieve a protein functional domain recognition. The purpose of multiple targets can reduce the possibility of tumor antigen escape, mismatch and off-target to a certain extent, and improve the safety and efficacy of CAR-immune cell (such as CAR-T cell) therapy to a certain extent.

The genetically engineered immune cells of the present invention can prepare regulated CAR-immune cells by constructing regulatory molecules based on short peptides and/or chemically tagged molecules, and combining with CARs targeting short peptides or chemically tagged molecules, such as CAR-T. By injecting the fusion protein with this tag in vitro, the effect of CAR-T treatment can be controlled by adjusting the injection time and amount, which increases safety.

Description of drawings

Figure 1 is a schematic diagram of the combination of regulatory protein molecules designed and constructed by natural ligands with CAR-T cells and tumor surface receptors;

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

Figure 3 is a schematic diagram of the preparation of tumor recognition regulatory molecules by amidation chemical coupling;

Figure 4 is a schematic diagram of the preparation of tumor recognition regulatory molecules by click chemical coupling;

Fig. 5 is the result of enzyme digestion electrophoresis and immunoblotting verification of different regulatory molecules constructed by BLyS using thrombin;

Figure 6 is a schematic structural diagram of a short peptide epitope PNE targeting CAR;

Figure 7 is the cell surface receptor expression of different blood tumors;

Figure 8 is a flow cytometric analysis result of the binding of different regulatory molecules constructed by BLyS to different hematological tumor cells;

Figure 9 shows the results of killing different blood tumor cells mediated by different regulatory molecules constructed by BLyS mediated by CAR-T cells;

Figure 10 Analysis of cytokine release by different regulatory molecules constructed by BLyS to mediate CAR-T cell killing of tumors;

Figure 11: Analysis of the inhibitory activity of CAR-T cells mediated by regulatory molecules constructed by BLyS on tumor Nalm6 in mice;

Figure 12: Analysis of the inhibitory activity of CAR-T cells mediated by regulatory molecules constructed by BLyS on tumor RPMI8226 in mice.

Detailed ways

The technical solutions of the present invention are further described below in conjunction with exemplary experiments.

The present invention uses natural ligands BLyS, APRIL and VEGF as the recognition molecules of tumor cells and microenvironment, constructs regulatory molecules that can recognize the surface antigen proteins of hematological tumors and solid tumors, and further introduces tag molecules and tag-targeted CAR-immune cells, A natural ligand-mediated regulatable CAR-immune cell strategy for multi-target recognition was prepared (Figure 1).

The technical scheme of the present invention is further described below in conjunction with experiments.

1. Construction of regulatory proteins (switch molecules)

1. Construction of fusion protein expression vector

The regulatory protein is prepared by the method of biological fusion, and the structure of the expression vector is shown in Figure 1. Taking BLyS as an example, the Myc-BlyS or BLyS-Myc target gene was introduced into the N-terminal His tag and a thrombin cleavage site by PCR. After double restriction endonuclease digestion, T4 DNA ligase was used to clone into pET32a(+) vector, transformed into DH5α competent cells, plated, and single clones were picked for sequencing and identification. In addition, in order to achieve secretory expression in mammalian cells, the target gene of the regulatory protein can also be cloned into the pFuse vector with T4 DNA ligase after double restriction endonuclease digestion by PCR method, and transformed into DH5α Competent cells were plated and single clones were picked for sequencing and identification.

Wherein, the BLyS-Myc protein sequence is shown in SEQ ID NO.: 1; the Myc-BLyS protein sequence is shown in SEQ ID NO.: 2.

In addition, chemical coupling methods can be used to prepare regulatory proteins. Also taking BLyS as an example, the target gene of BLyS is introduced into the N-terminal His tag and thrombin cleavage site by PCR, and MscI and XhoI are used for double digestion. Then, it was cloned into pET32a(+) vector using T4 DNA ligase, transformed into DH5α competent cells and plated, and single clones were picked for sequencing and identification. The structure of the expression vector is shown in Figure 2.

Similarly, other regulatory protein sequences were constructed, as follows:

The APRIL-Myc protein sequence is shown in SEQ ID NO.: 3;

Myc-APRIL protein sequence is shown in SEQ ID NO.: 4;

VEGF121-Myc protein sequence is shown in SEQ ID NO.: 5;

The Myc-VEGF121 protein sequence is shown in SEQ ID NO.: 6;

VEGF165-Myc protein sequence is shown in SEQ ID NO.: 7;

The Myc-VEGF165 protein sequence is shown in SEQ ID NO.: 8;

The BLyS-FLAG protein sequence is shown in SEQ ID NO.: 9;

The FLAG-BLyS protein sequence is shown in SEQ ID NO.: 10;

The APRIL-FLAG protein sequence is shown in SEQ ID NO.: 11;

The FLAG-APRIL protein sequence is shown in SEQ ID NO.: 12;

The VEGF121-FLAG protein sequence is shown in SEQ ID NO.: 13;

The FLAG-VEGF121 protein sequence is shown in SEQ ID NO.: 14;

The VEGF165-FLAG protein sequence is shown in SEQ ID NO.: 15;

The FLAG-VEGF165 protein sequence is shown in SEQ ID NO.:16.

2. Expression and purification of fusion protein

The constructed plasmids were transfected into BL21, Rosetta gami(DE3)pLysS or Tuner(DE3)pLysS. After plating, single clones were picked and cultured overnight, and inoculated into 500mL fresh TB medium at 1:100 until the OD ₆₀₀ value reached 2.0 At ~2.4, IPTG was induced for 20 hours. The cells were collected by centrifugation at 4°C, weighed, and stored frozen at -80°C. Add Lysis Buffer 10 times the volume of the bacterial cell weight, and then ultrasonically disrupt the cells until the bacterial solution is no longer viscous. After centrifuging the cell lysate at 4°C for 1 hour, the supernatant was filtered through a 0.45 μm filter. The supernatant of bacterial cell lysis was purified by nickel column, and Wash Buffer was selected to wash the impurity protein on the nickel column. Then use Elution Buffer to elute the target protein. The eluted target protein was dialyzed with Tris-HCl/NaCl buffer, and then digested with thrombin at room temperature for 16 hours. Finally, Endotoxin Removal Agarose Resin resin filler was used to remove endotoxin, and then Endotoxin detection system (GenScript, L00350) was used to detect endotoxin content to ensure that endotoxin content was below 5EU/kg.

The fusion protein can also be secreted and expressed in mammalian cells. Inoculate 293F suspension cells into cell culture shake flasks and incubate at 37°C, 150rpm, and 5% carbon dioxide in a shaker incubator for 24h until the cell density reaches 2×10 ⁶ /mL, the mixture of the fusion protein expression vector constructed by transfecting the pFuse vector and PEI MAX was incubated at 37°C, 150rpm, and 5% carbon dioxide concentration in a shaking incubator for 72h, and the supernatant was collected by centrifugation. Thrombin was used for digestion at room temperature for 16 hours, and the digestion effect was verified by SDS-PAGE electrophoresis.

3. Preparation of Chemically Conjugated Proteins

The above-mentioned protein expression and purification system is used to separate and purify the natural BLyS protein, or construct a mutant BLyS protein, that is, select a non-functional site on the BLyS protein, and specifically introduce a non-natural amino acid, such as phenylalanine with an azide group. , after further purification of the passed protein, enzymatic digestion, removal of His tag and treatment of endotoxin. For the naturally purified BLyS protein, a small molecule modified with the Myc tag and the introduction of a succinyl group can be used for random amidation coupling to construct a randomly coupled regulatory protein (as shown in Figure 4), or other chemical couples can be used. Conjugation methods, such as mixed acid anhydride method, diazotization method, carbonyl diimidazole method and other chemical coupling methods to construct regulatory proteins.

For mutant BLyS proteins introduced with unnatural amino acids, Myc short peptide tags with cycloalkynyl groups can be synthesized simultaneously, and site-specifically modified regulatory proteins can be prepared by copper-free click chemistry (as shown in Figure 5). Other site-specific modification chemical conjugation methods can also be used, such as engineered cysteine, enzyme-assisted linkage, carbohydrate recombination and carbohydrate conjugation, amino-terminal engineered serine, linkage to Fab nucleotide binding sites, native cysteine Acid-rebridging allosite-specific conjugation method to construct regulatory proteins.

4. SDS-PAGE and Western Blot to verify the fusion protein

Take an appropriate amount of purified protein molecules, add one-third of the protein sample volume of reducing loading buffer, vortex to mix well, and then centrifuge. The samples were boiled in a metal bath for 10 minutes and centrifuged. Use 12% separating gel and 5% stacking gel to fix the gel plate in the mold, pour the electrophoresis buffer into the electrophoresis buffer to observe whether there is leakage, remove the comb, and load the samples in turn; add the sample buffer to the scale line, adjust the voltage Start electrophoresis at 80V, adjust the voltage to 120V after the sample enters the separation gel, and observe the electrophoresis situation; soak the membrane, filter paper, and sponge in the electrotransfer buffer before use, and a piece of glue follows the whiteboard-sponge-filter paper-membrane-glue- Put the filter paper-sponge-blackboard in sequence (make sure there are no air bubbles), put it on ice, and connect the power supply to 220mA for 2h. Another piece of gel was stained with Coomassie brilliant blue and destained with water to observe the protein electrophoresis. After transfer, the membrane was taken out, cut to an appropriate size, and labeled with protein molecular weight standards. A blocking solution (5% nonfat dry milk + 0.12% tween20 TBST) was added, and the cells were blocked at room temperature for 1 h. Wash with TBST shaking for 3 minutes. The primary antibody was incubated with shaking at room temperature for 2 h. Rinse the NC membrane with an appropriate amount of TBST for 10 min/time, 3 times in total. Incubate the secondary antibody with shaking at room temperature for 1 h, and rinse with an appropriate amount of TBST for 10 minutes/time, a total of 3 times. Gel imager development. The results are shown in Figure 3. BLyS/Myc and Myc/BLyS showed the correct molecular weight and good purity before and after thrombin digestion. The tagged BLyS protein was further verified by immunoblotting.

2. Preparation of CAR-T cells

1. Vector construction

Taking the lentiviral vector pELPS to construct a CAR targeting Myc and FLAG as an example, taking the Myc targeting antibody 9E10 and FLAG targeting antibody FBT as an example, the variable region of the antibody adopts VL-linking peptide-VH and VH-linking peptide- The VL method is introduced into the structure of the second-generation CAR. The conventional second-generation CAR structure includes EF1α promoter, signal peptide molecule, single-chain antibody, hinge region, transmembrane region, costimulatory molecule and CD3zeta domain. The constructed CAR was double digested with BamHI and SalI and then cloned into the pELPS vector using T4 DNA ligase, or the vector was constructed using a homologous recombination kit. The structure of the lentiviral expression vector is shown in Figure 6.

A representative CAR structure is as follows:

The protein sequence of pELPS-CAR-anti-Myc9E10(VL-VH)-BBZ is shown in SEQ ID NO.: 17;

The protein sequence of pELPS-CAR-anti-Myc 9E10(VH-VL)-BBZ is shown in SEQ ID NO.: 18;

The protein sequence of pELPS-CAR-anti-FLAG FBT(VL-VH)-BBZ is shown in SEQ ID NO.: 19;

The protein sequence of pELPS-CAR-anti-FLAG FBT(VH-VL)-BBZ is shown in SEQ ID NO.: 20;

The protein sequence of pELPS-CAR-anti-FLAG M2(VL-VH)-BBZ is shown in SEQ ID NO.: 21;

The protein sequence of pELPS-CAR-anti-FLAG M2(VH-VL)-BBZ is shown in SEQ ID NO.:22.

2. Preparation of Lentivirus

293FT cells were cultured in DMEM medium (10% FBS, 1% penicillin-streptomycin double antibody, 1% non-essential amino acids, 1% sodium pyruvate, 1% L-glutamine), and the cells adhered to a density of 80 At 90%, cells were changed to antibiotic-free DMEM medium. Plasmids were mixed in 0.3mL OPTI-MEM according to the ratio of pELPS-CAR:pMDLgpRRE:pRSV-Rev:pMD2.G=2.25:1.8:1.8:0.75(μg), 20μL lipofectamine 2000 was added to 0.3mL OPTI-MEM, and let stand After 5 min, the two were mixed and vortexed to make the mixture evenly mixed. After standing at room temperature for 15 min, the mixture was added dropwise to the 293FT cells. After the cells were cultured in a constant temperature incubator for 6 hours, they were replaced with new antibiotic-free DMEM medium and cultured for another 48 hours at 37 °C, 5% CO ₂ . All supernatants were collected, centrifuged at 1500 rpm for 10 min at 4°C, and the cell pellet was discarded. The supernatant was the packaged virus, which was stored at -80°C for later use.

3. Extraction of Human PBMCs

Take an appropriate amount of Ficoll-Poque into a centrifuge tube (the ratio of blood to Ficoll-Poque is preferably 2:1). Fresh blood from healthy volunteers was slowly slanted into a centrifuge tube containing Ficoll-Poque to resuspend the cell suspension on the layered fluid. Centrifuge with a horizontal centrifuge at 27° C. and 2000 rpm for 30 min (the increase speed is 9, and the speed decrease is 0). After centrifugation, take out the centrifuge tube and observe that it is divided into 4 layers: the uppermost layer is plasma, containing some platelets; the second layer is a thin buffy coat, mainly mononuclear cells, and a small amount of platelets are mixed; the third layer is The liquid layer is separated; the fourth layer is granulocytes and erythrocytes. The erythrocytes settle at the bottom of the tube, while the granulocytes are closely attached to the erythrocytes to form a thin tunica albuginea, namely PBMC. Aspirate the upper serum with a Pasteur pipette (it can be stored in separate packs after centrifugation), and then carefully aspirate the buffy coat layer and add it to two centrifuge tubes (with the same volume as possible). Add RPMI-1640 medium to 50mL, and mix by inversion. Centrifuge at 27°C and 2000rpm in a horizontal centrifuge for 10min (the rising speed is 9 and the falling speed is 9). The low-speed centrifugation is beneficial to remove the remaining platelets in the cell suspension, and the supernatant is removed after centrifugation. Add AIM-V medium at 27°C, centrifuge again at 2000rpm for 10min, and remove the supernatant after centrifugation. Resuspend with 15mL AIM-V medium and add to T75 culture flask. Incubate at 37°C for more than 1 h, the purpose of this step is to remove adherent cells. After that, the supernatant was sucked into a centrifuge tube, and fresh RPMI-1640 medium was added to culture at a constant temperature of 37°C until use.

4. PBMC cryopreservation

1×10 ⁸ PBMC cells were resuspended in 700 μL of cryopreservation solution, and the resuspended solution was transferred to a cryopreservation tube, and the cryopreservation tube was placed in a programmed cooling box. Long-term storage in liquid nitrogen.

5. Activation of T cells

The cryopreserved PBMCs taken out from liquid nitrogen were quickly thawed in a 37°C water bath, 10 mL of AIM-V medium was added at 24°C, and centrifuged at 1500 rpm for 5 minutes. After centrifugation, the supernatant was aspirated, and then resuspended in an appropriate amount of AIM-V medium (if there are particles, the supernatant can be transferred). Take an appropriate amount of magnetic beads according to the ratio of 30 μL magnetic beads per 10 ⁶ cells (vortex before use), and wash the magnetic beads with 2-3 mL of PBS containing 5% FBS (the magnetic beads will be adsorbed on the sidewall). After removing the supernatant from the magnetic beads, wash again with AIM-V medium. After T cells were counted, they were diluted with culture medium to 1×10 ⁶ /mL, and then magnetic bead suspension was added. IL-2 was added, the initial concentration of IL-2 was 2.6×10 ⁵ IU/mL, and the final concentration was adjusted to 300 IU/mL.

6. T Cell Transduction

The T cells activated for 24h were centrifuged at 1500rpm for 5 minutes, further resuspended in RPMI-1640 medium, counted using a hemocytometer and trypan blue, and an appropriate amount of cell suspension was mixed with the virus solution and added. 10 mg/mL of polybrene to a final concentration of 8 μg/mL and 300 IU/mL of IL-2. Centrifuge at 1000g for 90 minutes at 33°C and incubate overnight in a constant temperature incubator at 37°C. The next day, the cells were centrifuged and supplemented with fresh culture medium in a 37°C incubator to continue culturing, and the anti-tumor activity was detected after the expression was stable.

3. Functional verification

1. Regulation of protein binding activity

Blood tumor cells from different sources were collected and verified for the expression of BCMA, BAFFR, TACI and BLyS respectively (Figure 7), thereby confirming that Nalm6, RPMI8226 and IM-9 were positive cells, and Jurkat was negative cells. Tumor cells were further collected, incubated at 4°C for 2 hours with 200nM regulatory protein, washed 3 times with flow cytometry washing solution, and incubated with APC-anti-Myc-labeled secondary antibody at 4°C for 1 hour, and then analyzed by flow cytometry. analyze. The results are shown in Figure 8. The results show that, on the one hand, BLyS has a broad-spectrum tumor targeting recognition ability, and has a good specific recognition activity for leukemia, lymphoma and myeloma, etc. The recognition ability mainly depends on the BLyS protein. The multi-target binding function of BAFF-R, TACI and BCMA; on the other hand, the experimental results can clearly show that the modification of the tag molecule on the C-terminus of BLyS will cause a certain degree of non-specific binding activity of negative cells, while BLyS The N-terminal modified fusion protein showed good binding activity with unexpected effects.

2. Cytotoxicity test:

Rough cell count to determine the approximate amount of cells (mainly to determine the minimum amount, as many cells should be taken as possible to avoid insufficient cell amount due to losses caused by experimental operations). After the tumor cells were labeled with fluorescein CFSE, they were resuspended for accurate cell counting, and then diluted to a certain cell density, so that the final concentration was 0.2 × 10 ⁶ cells/mL; after a sufficient amount of CAR-T cells were resuspended and quantified, Its final concentration was 2×10 ⁶ cells/mL. Tumor cells and CAR-T cells were mixed at a ratio of 1:10 (at this time, the concentration was 0.1×10 ⁶ tumor cells/mL, 1×10 ⁶ T cells/mL, and the effector-target ratio was 10:1). Prepare a gradient-regulated protein solution with a maximum concentration of 100 nM, 10-fold dilution, and a minimum concentration of 1 pM. Protein solutions of different concentrations were added to each well, 10 μL per well. At the same time, a negative control and a positive control were set. The negative control condition was without the addition of fusion protein solution, and the positive control condition was without the addition of T cells and fusion protein solution. The 96-well plate after the addition of samples was placed in a constant temperature incubator at 37°C and incubated for more than 24h. Half an hour before the end of the culture, 10 μL of lysate was added to the positive control group in advance. After the incubation, the 96-well plate was centrifuged at 2000 rpm in a horizontal centrifuge for 5 min. The centrifuged solution in the 96-well plate was transferred to another 96-well U-shaped plate (55 μL), and centrifuged at 2000 rpm in a horizontal centrifuge for 5 min. Take two 96-well U-shaped plates and transfer 30 μL correspondingly. The assay buffer was added to the substrate MIX and mixed. Add 30 μL of the above mixture to each well. After incubation at room temperature for 20-30 min, stop buffer was added, and then the absorbance value was detected at 490 nm with a microplate reader; for suspended tumor cells, after labeling with CFSE, the same operation was performed with CAR-T cells and regulatory proteins. After incubating in a 37°C constant temperature incubator for 24 hours, after staining with 7-AAD, the number of target cells was analyzed by a high-throughput flow cytometer. Graphpad Prism was used to analyze the experimental results as shown in Figure 9. Although both BLyS/Myc and Myc/BLyS can effectively kill different blood tumor cells, BLyS/Myc showed non-specific killing activity against negative cells, while Myc/BLyS The killing activity is more specific. In addition, BLyS/Myc and Myc/BLyS have good killing activities against tumor cells expressing BCMA, BAFFR and TACI, which benefit from the high affinity and specificity of natural ligands for different receptors. bring efficiency.

3. Cytokine Release Assay

1×10 ⁵ tumor cells were seeded into a 24-well plate, and two duplicate wells were set for each target cell. CAR-T cells were washed with PBS, diluted into T cell expansion medium without IL-2 at a cell density of 1×10 ⁶ /mL, and seeded at 1×10 ⁶ T cells in a culture containing target cells in the hole. 10nM BLyS/Myc and Myc/BLyS were added to the corresponding wells respectively, and after culturing for 24 h at 37°C in a 5% CO ₂ incubator, the supernatant was collected, and IFN-γ and TNF-α ELISA kits were used to detect IFN-γ in different samples. The content of γ and TNF-α, the results are shown in FIG. 10 . As mentioned above, cytokine secretion is a hallmark of CAR-T cells functioning. After analyzing the results of cytokine release assays, we found that BLyS/Myc and Myc/BLyS showed significantly enhanced cytokine secretion while mediating CAR-T cell killing of tumors, and showed significant enhancement of cytokine secretion in tumors expressing BCMA, BAFFR and TACI. All cells have good CAR-T cell-mediated activation effect.

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

NSG mice (6-8 weeks old) were housed and treated under specific pathogen-free conditions. In order to establish a tumor model, mice were injected with Nalm6 and RPMI8226 tumor cells with luciferase through tail vein. After the tumors grew and dispersed well in NSG mice, the mice were divided into three groups and injected intravenously (iv) corresponding to of CAR-T cells (30×10 ⁶ CAR-T cells/mouse for tumor model), and daily injections of BLyS/Myc and Myc/BLyS at 0.5mg/kg/dose into mice, and IVIS fluorescence The imaging system regularly observes the distribution of tumor cells in NSG mice. The results are shown in Figure 11, BLyS/Myc-mediated CAR-T cells can effectively inhibit the proliferation and expansion of Nalm6 leukemia tumor cells. In addition, Myc/BLyS-mediated CAR-T cells can also effectively inhibit the proliferation and expansion of RPMI8226 myeloma cells (Figure 12), which further proves that regulatable CAR-T cells constructed with natural ligands can be used to construct It is used to target tumor cells expressing different receptors to achieve effective inhibitory activity against different tumors.

In conclusion, the functional verification results show that the regulatory switch molecule constructed by using BLyS as a targeting molecule can mediate CAR-T cells to achieve effective killing and control effects on hematological tumors expressing different tumor-associated antigens. Compared with traditional CAR-T therapy, the safety and efficacy have been greatly improved. The combination of regulatable molecules and CAR-T cells has largely solved the uncontrollability of traditional CAR-T cells in patients. The safety of T therapy during use. At the same time, the multi-target recognition strategy using natural ligand molecules effectively avoids the problem of immune escape in single-target recognition, and increases the effective killing of tumor cells by CAR-T cells.

<110> Peking University Shenzhen Graduate School

<120> Preparation method of natural ligand-mediated multi-target recognition and regulated genetically engineered immune cells

<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 Thr Leu 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

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 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 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 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 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 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 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 Met Ser 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 A sn 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 Glu Asp 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 P he 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 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 G ln 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 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 G ly 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 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 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 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 T yr 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 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 GlyGlyGly 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 T yr 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 (10)

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

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.

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.

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