CN113735978B - Chimeric antigen receptor targeting CD19, preparation method and application thereof - Google Patents

Abstract

The invention discloses a chimeric antigen receptor targeting CD19, a preparation method and application thereof. The chimeric antigen receptor of the invention contains a fully human single chain antibody against CD 19. Compared with the CAR-T cells constructed by using the murine single-chain antibody aiming at CD19 which is clinically used at present, the CAR-T cells can obviously reduce immunogenicity and can be used for treating recurrent CD19+ leukemia.

Description

Chimeric antigen receptor targeting CD19, preparation method and application thereof

Technical Field

The invention relates to the field of biotechnology, in particular to a chimeric antigen receptor targeting CD19, a preparation method and application thereof.

Background

The principle of immunotherapy represented by CAR-T (Chimeric Antigen Receptor T-Cell, chimeric antigen receptor-modified T cells) is that the T cells extracted from a patient are modified by genetic engineering means to form CAR-T cells, and the T cells can specifically recognize tumor surface-related antigens (tumor Cell markers) to target and kill tumors. Compared with the conventional immune cells, the targeting, killing activity and persistence of the CAR-T cells are higher, and the tumor local immunosuppression microenvironment can be overcome and the host immune tolerance state can be broken. The modified immune cell therapy represented by CAR-T cells has remarkable curative effects on the treatment of acute leukemia and non-Hodgkin's lymphoma, and is considered as one of the most promising tumor treatment modes.

CAR-T immunotherapy is one of the most subverted technologies at present, and all countries in Europe and America and Asia are actively developing clinical trials of the therapy, wherein the maximum of 204 clinical trial projects are developed in China, and 50% of the clinical trials of the global CAR-T therapy are occupied. The university of pennsylvania hospital, philadelphia childhood hospital, the american cancer institute (NCI), fred Hutchinson cancer research center, the commemorative stonelen cancer center, the seattle childhood hospital, and the like are the earliest research institutions for CART cell therapy. Emerging international biopharmaceutical companies such as Juno, kite, celgene, cellectis, bluebird, traditional international pharmaceutical megaly such as Nohua, bai-Shi-Guibao, condunia, qiangsheng, ji-De and the like have invested a great deal of capital and manpower into the development of CAR-T therapies, wherein the novel targeted CD19CART drug products KTE-C19 and CTL-019 developed by KITE and Nohua are approved by FDA in sequence because of outstanding curative effects.

However, to date, the prevalence of fever or neurotoxic side effects caused by cytokine release syndrome in CD 19-targeted CAR-T products presents a major challenge for the broad clinical use and clinical treatment of the product. The rise and application of the synthetic biology technology provide more safe and efficient CAR-T development choices for CAR-T immunotherapy of tumors, and provide a hint for developing safer CAR-T new therapies and medicines.

Disclosure of Invention

The present invention provides a CD 19-targeting chimeric antigen receptor comprising, in order: an extracellular antigen binding domain, a spacer domain, a transmembrane domain, a costimulatory signaling domain, and a primary signaling domain.

Preferably, the extracellular antigen-binding domain is an scFv that specifically binds CD 19; the amino acid sequence of the scFv is shown as SEQ ID NO. 1.

Preferably, the spacer domain is an IgG4hinge region; preferably, the amino acid sequence of the IgG4hinge region is shown in SEQ ID NO. 2.

Preferably, the transmembrane domain is the transmembrane region of CD 28; preferably, the amino acid sequence of the transmembrane region of CD28 is shown in SEQ ID NO. 3.

Preferably, the costimulatory signaling domain is the intracellular signaling domain of 4-1 BB; preferably, the amino acid sequence of the intracellular signaling domain of 4-1BB is shown in SEQ ID NO. 4.

Preferably, the primary signaling domain is the intracellular signaling domain of cd3ζ; preferably, the amino acid sequence of the intracellular signaling domain of cd3ζ is shown as sequence of SEQ ID No. 5.

Preferably, the chimeric antigen receptor further comprises a signal peptide at its N-terminus; preferably, the signal peptide is a granulocyte-macrophage colony stimulating factor receptor 2 signal peptide.

Preferably, the chimeric antigen receptor further comprises a self-cleaving peptide at its C-terminus; preferably, the self-cleaving peptide is T2A. The self-cleaving peptide is linked to the C-terminus of the primary signaling domain.

The amino acid sequence of T2A is shown in SEQ ID NO. 7.

Preferably, the chimeric antigen receptor further comprises a cell surface marker at its C-terminus.

Cell surface markers are used to determine whether a chimeric antigen receptor is expressed, in particular, the cell surface markers are used to determine transduction or engineering of the cell for expression of the chimeric antigen receptor, e.g., a truncated form of a cell surface receptor, e.g., truncated EGFR (tgfr). In some aspects, the marker comprises all or part (e.g., truncated form) of CD34, NGFR, or an epidermal growth factor receptor (e.g., tgfr) or a functional variant thereof.

Preferably, the cell surface marker is a fragment of EGFR (tgfr) comprising extracellular domain III and extracellular domain IV, preferably the amino acid sequence of tgfr is shown in SEQ ID No. 8.

Preferably, the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO. 6.

Chimeric antigen receptors of embodiments of the invention may comprise synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexylcarboxylic acid, norleucine, α -aminon-decanoic acid, homoserine, S-acetamidomethyl-cysteine, trans-3-hydroxyproline and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β -phenylserine, β -hydroxyphenylalanine, phenylglycine, α -naphthylalanine, cyclohexylalanine, cyclohexylglycine, indoline-2-carboxylic acid, 1,2,3, 4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic acid monoamide, N ' -benzyl-N ' -methyl-lysine, N ' -dibenzyl-lysine, 6-hydroxylysine, ornithine, α -aminocyclopentane carboxylic acid, α -aminocyclohexylcarboxylic acid, α -aminocycloheptane carboxylic acid, α - (2-amino-2-norcamphene) -carboxylic acid, α, γ -diaminobutyric acid, α, β -diaminopropionic acid, homophenylalanine, and α -tert-butylglycine.

The chimeric antigen receptor of embodiments of the invention may be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized via, for example, a disulfide bridge, or converted to an acid addition salt and/or optionally dimerized or multimerized.

Chimeric antigen receptors of embodiments of the invention may be obtained by methods known in the art. Chimeric antigen receptors can be made by any suitable method for making a polypeptide or protein. Suitable methods for de novo synthesis of polypeptides and proteins are known in the art. In addition, chimeric antigen receptors can be produced recombinantly using nucleic acids described herein using standard recombinant methods such as those described in Green and Sambrook, molecular Cloning: A Laboratory Manual (4 th edition), cold Spring Harbor Laboratory Press (2012). Alternatively, the chimeric antigen receptor described herein can be synthesized commercially by a company. In this regard, the chimeric antigen receptor of the invention may be synthetic and/or recombinant.

The present invention provides a nucleic acid encoding the chimeric antigen receptor described above.

Further, the nucleic acid further comprises a promoter, and/or a cleavage site located after the promoter, and/or a kozak sequence located after the cleavage site.

Still further, a promoter is located at the N-terminus of the nucleic acid, the promoter being a promoter that elongates factor 1 alpha; preferably, the promoter sequence is shown in SEQ ID NO. 9.

Still further, the cleavage site is a recognition site for Pac I; preferably, the sequence of the cleavage site is shown in positions 1-8 of SEQ ID NO. 10.

Further, the kozak sequence is shown in positions 9-14 of SEQ ID NO. 10.

Preferably, the coding sequence of the signal peptide in the nucleic acid is shown in positions 15-78 of SEQ ID NO. 10.

Preferably, the coding sequence of the scFv in the nucleic acid is shown in 79-822 of SEQ ID NO. 10.

Preferably, the coding sequence of the spacer domain in the nucleic acid is shown in positions 823-846 of SEQ ID NO. 10.

Preferably, the coding sequence of the transmembrane domain in the nucleic acid is shown in SEQ ID NO.10 at positions 847-939.

Preferably, the coding sequence of the costimulatory signaling domain in the nucleic acid is as shown in SEQ ID NO.10 at positions 940-1068.

Preferably, the coding sequence of the primary signalling domain in said nucleic acid is as shown in SEQ ID NO.10 at positions 1069-1404.

Preferably, the nucleotide sequence of T2A is shown at positions 1405-1476 of SEQ ID NO. 10.

Preferably, the nucleotide sequence of tEGFR is shown at positions 1477-2550 in SEQ ID NO. 10.

Preferably, the coding sequence of the chimeric antigen receptor in the nucleic acid is shown in positions 15-2585 of SEQ ID NO. 10.

Preferably, the nucleic acid comprises the nucleotide sequence shown as SEQ ID NO. 10.

The present invention provides a vector comprising the nucleic acid as described above.

In embodiments, the vector of the invention may be any suitable vector and may be used to transform or transfect any suitable host cell. Suitable vectors include those designed for proliferation and amplification or for expression or both, such as plasmids and viruses. The carrier may be selected from: pUC series (Fermentas Life Sciences, glen Burnie, MD), pBluescript series (Stratagene, laJolla, calif.), pET series (Novagen, madison, wis.), pGEX series (Pharmacia Biotech, uppsala, sweden) and pEX series (Clontech, palo Alto, calif.). Phage vectors such as λGT10, λGT11, λ ZapII (Stratagene), λEMBL4, and λNM1149 can also be used. Examples of vectors include pEUK-Cl, pMAM and pMAMneo (Clontech). Viruses that may be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. In general, suitable vectors include an origin of replication, a promoter sequence, a convenient restriction enzyme site, and one or more selectable markers that function in at least one organism (e.g., WO01/96584; WO01/29058; and U.S. Pat. No.6,326,193).

The present invention provides a recombinant microorganism comprising a nucleic acid as described above or a vector as described above; preferably, the recombinant microorganism is a recombinant virus.

The present invention provides an engineered immune cell that expresses the chimeric antigen receptor described above; preferably, the immune cell contains a nucleic acid as described above or a vector as described above.

The present invention provides an immune cell population comprising the engineered immune cells described previously; preferably, the population of immune cells further comprises unmodified or incompletely engineered immune cells.

The present invention provides a method of preparing an engineered immune cell as described above, comprising introducing a nucleic acid as described above or a vector as described above into an immune cell.

Several suitable methods are known in the art for introducing nucleic acids and corresponding vectors into host cells. Methods include, but are not limited to, calcium phosphate transfection, electroporation, lipofection, the use of transfection reagents such as cationic lipids, biolistically mediated or multimeric mediated transfer.

The immune cells of the invention may be Peripheral Blood Lymphocytes (PBLs) or Peripheral Blood Mononuclear Cells (PBMCs). The immune cells of the invention may be B cells, natural Killer (NK) cells or T cells.

The T cell of the invention may be any T cell, such as a cultured T cell, e.g. a primary T cell; or T cells from a cultured T cell line, such as Jurkat, supT1, etc.; or T cells obtained from a mammal. If obtained from a mammal, the T cells may be obtained from a variety of sources including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissues or fluids. T cells may also be enriched or purified. The T cells may be human T cells. The T cells may be T cells isolated from a human. T cells may be any type of T cell and may be at any stage of development, including but not limited to cd4+/cd8+ double positive T cells, cd4+ helper T cells (e.g., th1 and Th2 cells), cd8+ T cells (e.g., cytotoxic T cells), tumor infiltrating cells, memory T cells, naive T cells, and the like. The T cells may be cd8+ T cells or cd4+ T cells.

The present invention provides a composition comprising the chimeric antigen receptor described above, the nucleic acid described above, the vector described above, the recombinant microorganism described above, the engineered immune cell described above, the population of immune cells described above.

Preferably, the composition comprises a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers are carriers that have no deleterious side effects or toxicity under the conditions of use. Methods of preparing the administrable drug are known or obvious to those skilled in the art and are described in more detail, for example, in Remington, the Science and Practice ofPharmacy, 22 nd edition, pharmaceutical Press (2012).

The choice of vector will be determined in part by the particular nucleic acid, vector, immune cell of the invention and the particular method used to administer the nucleic acid, vector, immune cell of the invention. Thus, there are a variety of suitable formulations of the pharmaceutical compositions of the present invention. Suitable formulations may include any of those for parenteral, subcutaneous, intravenous, intramuscular, intraarterial, intrathecal, intratumoral or intraperitoneal administration. More than one route may be used to administer the nucleic acids, vectors, immune cells of the invention, and in some cases, a particular route may provide a more direct and more effective response than another route.

The pharmaceutical compositions of the invention may also include other pharmaceutically active agents or drugs such as chemotherapeutic agents, e.g., asparaginase, busulfan, carboplatin, cisplatin, daunomycin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate, taxol, rituximab, vinca alkaloids, vincristine, and the like, administered in combination with the nucleic acids, vectors, immune cells of the invention.

When the nucleic acids, vectors, immune cells of the invention are administered with one or more additional therapeutic agents, the one or more additional therapeutic agents may be co-administered to the mammal. By "co-administration" is meant administration of one or more additional therapeutic agents and a nucleic acid, vector, immune cell of the invention in sufficiently close temporal proximity that the nucleic acid, vector, immune cell of the invention can enhance the effect of the one or more additional therapeutic agents, and vice versa. In this regard, the nucleic acid, vector, immune cell of the invention may be administered first, followed by administration of one or more additional therapeutic agents, and vice versa. Alternatively, the nucleic acid, vector, immune cell and one or more additional therapeutic agents of the invention may be administered simultaneously.

The invention provides a kit comprising the chimeric antigen receptor described above, the nucleic acid described above, the vector described above, the recombinant microorganism described above, the engineered immune cell described above, the population of immune cells described above, the composition described above.

The present invention provides an application comprising an application as set forth in any one of the following:

1) Use of the chimeric antigen receptor described above, the nucleic acid described above, the vector described above, the recombinant microorganism described above, the immune cell described above, the population of immune cells described above, the composition described above, for the preparation of a medicament for the treatment of a disease or disorder associated with CD19 expression;

2) Use of the chimeric antigen receptor described above for the preparation of the nucleic acid described above, the vector described above, the recombinant microorganism described above, the immune cell described above, the population of immune cells described above, the composition described above, the kit described above;

3) The use of the nucleic acid as described above for the preparation of the vector as described above, the recombinant microorganism as described above, the immune cell as described above, the population of immune cells as described above, the composition as described above, the kit as described above;

4) The use of the aforementioned vector for the preparation of the aforementioned recombinant microorganism, the aforementioned immune cell population, the aforementioned composition, the aforementioned kit;

5) The use of the recombinant microorganism described above for the preparation of the immune cell described above, the population of immune cells described above, the composition described above, the kit described above;

6) The use of the immune cells described above for the preparation of the immune cell population described above, the composition described above, the kit described above;

7) The use of the population of immune cells described above for the preparation of the composition described above, the kit described above;

8) Use of a composition as described above for the preparation of a kit as described above.

The present invention provides a method of treating a disease or disorder associated with CD19 expression comprising administering to a subject in need thereof a nucleic acid as described above, a vector as described above, an immune cell as described above, a population of immune cells as described above, a composition as described above.

Diseases or disorders associated with CD19 expression of the present invention include neoplasms, autoimmune and inflammatory diseases, infectious diseases.

Further, the tumors include B-cell malignancies, such as B-cell leukemias and lymphomas, including B-cell Chronic Lymphocytic Leukemia (CLL), acute Lymphocytic Leukemia (ALL), prolymphocytic leukemia, hairy cell leukemia, common acute lymphoblastic leukemia, non-hodgkin lymphoma, diffuse large B-cell lymphoma (DLBCL), multiple myeloma, follicular lymphoma, spleen and marginal zone lymphoma, mantle cell lymphoma, indolent B-cell lymphoma, hodgkin lymphoma.

Preferably, the B-cell malignancy is relapsed cd19+ leukemia.

Exemplary autoimmune and inflammatory diseases include multiple sclerosis, rheumatoid arthritis, and Systemic Lupus Erythematosus (SLE).

The invention has the advantages and beneficial effects that:

the invention constructs the CAR-T by utilizing the fully human scFv targeting CD19, and compared with the murine scFv, the CAR-T has lower immunogenicity, and is particularly suitable for recurrent patients of CD19+ leukemia tolerant to the murine CAR-T.

Drawings

FIG. 1 shows a specific structural schematic diagram of a humanized H19-CAR gene;

FIG. 2 shows a schematic diagram of a specific structure of a murine M19-CAR gene;

FIG. 3 shows a graph of CAR-T cell expansion;

FIG. 4 shows a statistical plot of CAR transfection efficiency;

FIG. 5 shows a graph of the results of the killing capacity of CAR-T cells on Nalm-6 cells;

FIG. 6 shows a statistical plot of the results of CAR-T cell cytokine secretion;

FIG. 7 shows an in vivo animal imaging;

FIG. 8 shows a statistical plot of fluorescence intensity.

Detailed Description

The invention will be described in detail below with reference to the accompanying drawings in combination with examples. It should be noted that, those skilled in the art should understand that the drawings and the embodiments of the present invention are only for illustrative purposes, and should not limit the present invention in any way. The embodiments and features of the embodiments in this application may be combined with each other without contradiction.

EXAMPLE 1 CAR-T construction

1. Construction of lentiviral vectors

The scfv fragment of fully human or murine CD19 was cloned into a second generation CAR structural backbone with 4-1BB and CD3 zeta, and the extracellular tEGFR domain was linked by T2A to yield the designation H19-CAR (fully human) or M19-CAR (murine). The H19-CAR gene has a specific structure of anti-HCD 19 scFv-IgG4 range-CD 28TM-4-1BB-CD3ζ -T2A-tEGFR, and the M19-CAR gene has a specific structure of anti-MCD 19 scFv-IgG4 range-CD 28TM-4-1BB-CD3ζ -T2A-tEGFR (see FIGS. 1 and 2). Wherein the anti HCD19 scFv is a coding sequence for an anti-fully human CD19 single chain variable fragment (scFv), the anti mcd19 scFv is a coding sequence for an anti-murine CD19 single chain variable fragment (scFv), the IgG4 range is the hinge region, the CD28TM is the transmembrane region, the 4-1BB is the costimulatory domain, and the CD3 ζ is the intracellular signaling domain, the tgfr extracellular region is expressed by a T2A peptide linkage so that expression of the CAR is detected after viral transduction.

The coding sequence of the H19-CAR gene coding chain is 15-2585 of SEQ ID NO.10, 1-8 of SEQ ID NO.10 is the recognition site of Pac I, 15-78 of SEQ ID NO.10 is the coding sequence of signal peptide, 79-822 of full human anti-CD 19 single chain variable fragment (scFv), 823-846 of hinge sequence, 847-939 of CD28TM gene sequence, 940-1068 of 4-1BB gene sequence, 1069-1404 of CD3 zeta gene sequence, 1405-1476 of T2A gene sequence, 1477-2550 of tEGFR gene sequence. The amino acid sequence of the protein H19-CAR is shown in SEQ ID NO.6 in the sequence table.

The H19-CAR gene expression vector (lentiviral expression vector) is synthesized by a complete sequence, and is named as Senl-H19, and the sequence is shown as SEQ ID NO. 13. Senl-H19 contains the coding region of the H19-CAR gene shown as 79-2550 in SEQ ID NO.10, and can express a protein H19-CAR with the amino acid sequence shown as SEQ ID NO.6 in a sequence table in cells.

The constructed lentiviral expression vector is used for lentiviral packaging after being identified by Pac I and SpeI enzyme digestion and the sequence determination are correct.

The scfv of the fully human CD19 in the Senl_H2 19 is replaced by the scfv of the murine CD19 (the amino acid sequence is shown as SEQ ID NO.12, the nucleotide sequence is shown as SEQ ID NO. 11), and the Senl_M19 expression vector is prepared.

The constructed lentiviral expression vector is used for lentiviral packaging after being identified by Pac I and SpeI enzyme digestion and the sequence determination are correct.

2. Preparation of lentiviruses

The 293FT cell culture flask (T175) which has grown to 80% -90% is prepared from 37 ℃ and 5% CO ₂ Is removed from the cell culture chamber and 2mL of TrypLE is added ^TM After digestion of EXPRESS, washed cells were collected and added to a 9X 10 cell culture dish every 145mm ⁶ Adding 20mL of DMEM medium (Thermo Fisher) into each cell, shaking gently, adding 37 ℃ and 5% CO ₂ Culturing in an incubator.

After 24H, the above solutions were mixed uniformly in an amount of 375. Mu.l serum-free DMEM medium, 15. Mu.g lentiviral expression vector Senl_H2 19 or Senl_M19, 7.5. Mu.g packaging plasmid encoding Rev protein psPAX2, 3.75. Mu.g envelope plasmid encoding VSV-G protein pMD2.G per 145mm dish, to form solution A, 315. Mu.l serum-free DMEM medium was added with 60. Mu.l PEI transfection reagent, mixed uniformly to form solution B, and then solution B was added to solution A to mix uniformly, and left at room temperature for 20min. 293FT cells used to package the virus were purified from 5% CO at 37 ℃ ₂ Is taken out of the cell culture box, the mixed solution is added into each plate on average, is gently shaken evenly and is put into 5 percent CO at 37 DEG C ₂ In an incubator. After 4h-6h, the old medium was discarded, cells were washed with 5mL of warmed PBS, and 20mL of fresh warmed DMEM medium containing 10% fetal bovine serum was added, and the mixture was placed in 5% CO at 37 ℃C ₂ Culturing in an incubator. Culturing for 48-72 hr, collecting culture supernatant as virus stock solution, collecting stock solution into 50ml centrifuge tube, centrifuging at 2000r/min for 5min to remove cell sedimentThe virus stock was then filtered through a 0.45 μm filter using a 50ml syringe into a new 50ml centrifuge tube and centrifuged at 4℃and 18300g for 2h. This is the virus concentrate. The virus concentrate was dispensed at 100. Mu.l/tube, and 10. Mu.l was left for virus titer measurement. And (5) placing the packaged concentrated solution at the temperature of-80 ℃ for preservation.

3. Preparation of Peripheral Blood Mononuclear Cells (PBMC)

The peripheral blood may be a healthy donor or the peripheral blood of the patient itself.

Peripheral blood was mixed with sterile physiological saline 1:1, mixing and diluting, slowly adding into mononuclear cell separating liquid (Oriental Huahui) (diluted blood: separating liquid=2:1), centrifuging for 20min, increasing 7 and decreasing 4, and performing density gradient centrifugation. After centrifugation, the white membrane layer is gently sucked out and transferred into a new 50mL centrifuge tube, 800g of sterile physiological saline is added for resuspension, centrifugation is carried out at 4 ℃ for 5min, supernatant is discarded after centrifugation, a vortex oscillator is used for shaking off the cell precipitation layer, sterile physiological saline is added for fixing the volume to 50mL, the centrifuge tube is turned upside down, the mixture is transferred into the centrifuge tube after uniform mixing, and centrifugation is carried out at 800g and 4 ℃ for 5min, so that the obtained precipitate is mononuclear cells.

4. Isolation of T cells

After centrifugation, a cell sorting solution (DPBS+0.5% HSA) was added to the mononuclear cell pellet (1X 10) ⁷ Mu.l of cell sorting solution was added to each cell and magnetic beads (MASC) (1X 10) ⁷ Adding 20 μl of sorting magnetic beads into individual cells), adding sorting magnetic beads (CD 3 sorting magnetic beads are used as peripheral blood sorting magnetic beads of healthy donors, and peripheral blood of patients can be CD3 sorting magnetic beads, CD4 and CD8 sorting magnetic beads and the like according to flow detection results), blowing, sucking, mixing uniformly, placing into a refrigerator at 4deg.C, incubating for 15min in the absence of light, and incubating every 1×10 after incubation is completed ⁷ Adding 1-2 mL of cell sorting solution into the PBMC, uniformly mixing, and centrifuging at 4 ℃ for 5min. According to 1X 10 ⁸ PBMC were added with 500. Mu.L buffer and the cell pellet was blow-mixed. The cell suspension was then added to the prepared magnetic column and allowed to drop freely, and after the liquid was dropped, 3mL of cell sorting liquid was added to wash the magnetic column and repeated 2 times. The cells left in the magnetic column are positive cells. Remove the column from the rack and add 5mL of cellsAnd (3) the sorting liquid forcefully presses buffer liquid in the magnetic column by using a piston in the magnetic column, and the positive cells in the magnetic column are washed into a centrifuge tube, so that the sorting positive cells are obtained.

5. Activation culture of T cells

Mixing the centrifugal tubes with the sorted positive cells uniformly, and centrifuging for 5min at 4 ℃ under 800 g. After centrifugation, the supernatant was discarded, resuspended in TexMACS GMP serum-free Medium (MACS) containing 200UI/mL IL-2, and activated beads were added at a ratio of T cells to activated beads CD3/CD28 (Thermo Fisher) 1:1, placed at 37℃and 5% CO ₂ The cell culture chamber is used for culturing.

Unless otherwise indicated, the following cell cultures were TexMACS GMP serum-free medium containing 200UI/mL IL-2.

5. T cell preparation of CD 19-targeting CAR

T cells were prepared according to the above-described method for T cell isolation and activation. Activating selected T cells with Dynabeads CD3/CD28 activating magnetic beads, and placing into 37 deg.C and 5% CO ₂ Culturing in a cell incubator (Day 0), adding the above prepared virus concentrate into T cells after culturing for 48 hr, centrifuging for 2 hr (2000 rpm,35 deg.C, increasing speed 4, decreasing speed 4) for slow virus transfection, and placing into 37 deg.C, 5% CO after transfection ₂ Cell incubators were incubated, day5 sampled, and car+ ratio was detected by flow cytometry. Day12 to Day14 cells were harvested, medium was removed by washing, and cells were resuspended using 0.9% saline containing 1% hsa to obtain T cells expressing CD 19-targeted CARs, the CAR-T cells expressing fully human scFv were designated senl_h19 cells, and the CAR-T cells expressing murine scFv were designated senl_m19 cells.

6. Results

The amplification curve of the CAR-T cells is shown in FIG. 3 and the CAR transfection efficiency is shown in FIG. 4. There was no significant difference in fold expansion between Senl_H2 cells and Senl_M19 cells compared to control Senl_M19 cells, and fold expansion was > 100 fold when cultured for 14 days. When cultured for 14 days, the CAR positive rate of the Senl_H2 cells is 52% + -0.14%, and has no significant difference from the CAR positive rate of the Senl_M19 of 53.77% + -12.2%. The results indicate that scFv of human origin had no significant effect on cell growth and CAR transfection compared to scFv of murine origin.

Example 2 in vitro functional study of CAR-T cells

1. Step (a)

Cells were harvested on day14 of culture as effector cells and Nalm-6 cells (purchased from the pool of derivative cells) as target cells.

Target cells were first collected, centrifuged at 2000rpm for 5min, and DPBS resuspended at 2X 10 ⁶ The 2. Mu.M CFSE working solution was added in an amount of 100. Mu.l per cell, and the mixture was gently mixed. Incubate at 37℃for 20min in the absence of light. After the incubation was completed, staining was terminated by adding 5 volumes of complete medium (1640 containing 10% FBS), centrifuging at 2000rpm for 5min, resuspension with complete medium, incubation at 37℃in the absence of light for 10min, centrifuging at 2000rpm for 5min after the incubation was completed, resuspension of cells with complete medium, and adjusting the target cell density to 1X 10 ⁶ Mu.l of each well was added to a 96-well plate at a well density of 1X 10 ⁵ And/or holes. Then adding a proper amount of effector cells into target cells according to different effect target ratios (E: T=0.3:1, 1:1, 3:1, 10:1), incubating for 4 hours after mixing, and detecting the cell killing ratio by flow cytometry. Cells were stained with 2 μl 7AAD and anexin V and incubated for 15min in the dark. The killing rate (%) of CFSE-labeled target cells was the sum of the ratio of Annexin V+/7AAD-, annexin V+/7AAD+ and Annexin V-/7 AAD+.

In addition, target cells were collected and the cell density was adjusted to 1X 10 ⁶ Mu.l of each well was added to a 96-well plate at a well density of 1X 10 ⁵ And (3) adding an appropriate amount of effector cells to each target cell of each well according to the ratio of effector cells E to T=3:1, uniformly mixing and incubating for 18 hours, and taking a supernatant to detect cytokine secretion (LEGENDplex HU Th1/Th2 Panel (8-plex) (Biolegend).

2. Results

The results show that, as shown in FIGS. 5 and 6, both the Senl_H19 cells and the Senl_M19 cells have strong killing power to Nalm-6 cells, and the effective target ratio is 0.3: 1. 1: 1. 3: 1. 10:1, the killing mean of Senl_H2 cells on Nalm-6 cells was 17.8%, 43.6%, 76.5% and 91.1%, respectively; the average killing of Nalm-6 cells by Senl_M19 cells was 16.9%, 39.8%, 60.1% and 89.0%, respectively. IFN-gamma concentrations in supernatant of senl_H2 and senl_M19 cells were 3550.28pg/ml and 2205.11pg/ml, respectively; TNF- α was 470.86pg/ml and 505.48pg/ml, respectively; IL-5 concentrations were 1182.50pg/ml and 1483.29pg/ml, respectively; IL-13 concentrations were 3112.10pg/ml and 3253.56pg/ml, respectively; IL-2 concentrations were 1328.96pg/ml and 1286.70pg/ml, respectively; IL6 concentrations were 9.89 and 13.45pg/ml, respectively; IL-10 concentrations were 31.34pg/ml and 45.04pg/ml, respectively; the IL-4 concentration was 48.73pg/ml and 30.26pg/ml, respectively.

Example 3 in vivo functional study of CAR-T cells

1. Step (a)

A mouse tumor model was constructed with human B-lymphocytic leukemia cells Raji-luc. Will be 5X 10 ⁵ The Raji-luc is injected into tail vein of NCG mice (Jiangsu Ji Yikang Biotechnology Co., ltd.) with age of 6-8 weeks and weight of 20-24g, and after tumor formation is detected by living body imaging, cells are returned for treatment.

Mice were randomly divided into 2 groups: a Senl_H2 cell group and a Senl_M19 cell group, 6 cells each. Senl_H2 cells and Senl_M19 cells were prepared by the CAR-T cell culture method described above, and cells harvested on day14 of culture were used as the Senl_H2 cells, senl_M19 cells and T cell controls used in this example. Each mouse was injected 1×10 by tail vein ⁷ Individual car+ cells/cell. Mice survival was monitored daily and tumor growth characteristics were observed with a small animal in vivo imager (IVIS luminea, series III, PE) and luminescence signals were collected every 7 days.

2. Results

Fig. 7 is a photograph of a living animal by an imager, and fig. 8 is a fluorescence intensity chart. Mice survived all within 28 days after dosing. The clinical symptoms of the mice in the Senl_H2 cell group and the Senl_M19 cell group are obviously improved, and adverse reactions related or unrelated to medicines do not occur during the test, which indicates that the Senl_H2 cell and the Senl_M19 cell have the function of resisting lymphoblastic leukemia in the mice.

It should be noted that the above-mentioned embodiments are merely preferred embodiments of the present invention, and are not intended to limit the present invention, but various modifications and variations of the present invention will be apparent to those skilled in the art. It will be understood by those skilled in the art that any modifications, equivalent substitutions, improvements, etc. that fall within the spirit and principles of the present invention are intended to be included within the scope of the present invention.

Sequence listing

<110> Hebei Senson Biotech Co., ltd

<120> CD 19-targeted chimeric antigen receptor, preparation method and application thereof

<141> 2021-04-22

<160> 13

<170> SIPOSequenceListing 1.0

<210> 1

<211> 248

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 1

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Val Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Ser Ser Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Gly Thr Tyr Tyr Tyr Gly Thr Arg Val Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro

130 135 140

Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg

145 150 155 160

Ser Ser Lys Ser Leu Gln Asn Val Asn Gly Asn Thr Tyr Leu Tyr Trp

165 170 175

Phe Gln Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Arg Met

180 185 190

Ser Asn Leu Asn Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser

195 200 205

Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe

210 215 220

Ala Val Tyr Tyr Cys Met Gln His Leu Glu Tyr Pro Ile Thr Phe Gly

225 230 235 240

Ala Gly Thr Lys Leu Glu Ile Lys

245

<210> 2

<211> 13

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 2

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Met

1 5 10

<210> 3

<211> 27

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 3

Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu

1 5 10 15

Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val

20 25

<210> 4

<211> 42

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 4

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 5

<211> 112

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 5

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 6

<211> 823

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 6

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Val Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Ile Asn Pro Tyr Asn Asp Gly Thr Lys Tyr Asn Glu Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Ser Ser Asp Lys Ser Ile Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Gly Thr Tyr Tyr Tyr Gly Thr Arg Val Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly

115 120 125

Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ser Pro

130 135 140

Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg

145 150 155 160

Ser Ser Lys Ser Leu Gln Asn Val Asn Gly Asn Thr Tyr Leu Tyr Trp

165 170 175

Phe Gln Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Arg Met

180 185 190

Ser Asn Leu Asn Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser

195 200 205

Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe

210 215 220

Ala Val Tyr Tyr Cys Met Gln His Leu Glu Tyr Pro Ile Thr Phe Gly

225 230 235 240

Ala Gly Thr Lys Leu Glu Ile Lys Glu Ser Lys Tyr Gly Pro Pro Cys

245 250 255

Pro Pro Cys Pro Met Phe Trp Val Leu Val Val Val Gly Gly Val Leu

260 265 270

Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val

275 280 285

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

290 295 300

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

305 310 315 320

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg

325 330 335

Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn

340 345 350

Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg

355 360 365

Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro

370 375 380

Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala

385 390 395 400

Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His

405 410 415

Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp

420 425 430

Ala Leu His Met Gln Ala Leu Pro Pro Arg Leu Glu Gly Gly Gly Glu

435 440 445

Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly

450 455 460

Pro Arg Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro

465 470 475 480

His Pro Ala Phe Leu Leu Ile Pro Arg Lys Val Cys Asn Gly Ile Gly

485 490 495

Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys

500 505 510

His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro

515 520 525

Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro

530 535 540

Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu

545 550 555 560

Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu

565 570 575

Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser

580 585 590

Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu

595 600 605

Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu

610 615 620

Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly

625 630 635 640

Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala

645 650 655

Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly

660 665 670

Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg

675 680 685

Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe

690 695 700

Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln

705 710 715 720

Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln

725 730 735

Cys Ala His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala

740 745 750

Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala

755 760 765

Gly His Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr

770 775 780

Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser

785 790 795 800

Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu Leu Leu Val Val Ala

805 810 815

Leu Gly Ile Gly Leu Phe Met

820

<210> 7

<211> 24

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 7

Leu Glu Gly Gly Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp

1 5 10 15

Val Glu Glu Asn Pro Gly Pro Arg

20

<210> 8

<211> 357

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 8

Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro

1 5 10 15

Ala Phe Leu Leu Ile Pro Arg Lys Val Cys Asn Gly Ile Gly Ile Gly

20 25 30

Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile Lys His Phe

35 40 45

Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu Pro Val Ala

50 55 60

Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp Pro Gln Glu

65 70 75 80

Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe Leu Leu Ile

85 90 95

Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe Glu Asn Leu

100 105 110

Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe Ser Leu Ala

115 120 125

Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser Leu Lys Glu

130 135 140

Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn Leu Cys Tyr

145 150 155 160

Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser Gly Gln Lys

165 170 175

Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys Ala Thr Gly

180 185 190

Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp Gly Pro Glu

195 200 205

Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly Arg Glu Cys

210 215 220

Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu Phe Val Glu

225 230 235 240

Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro Gln Ala Met

245 250 255

Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile Gln Cys Ala

260 265 270

His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro Ala Gly Val

275 280 285

Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp Ala Gly His

290 295 300

Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys Thr Gly Pro

305 310 315 320

Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro Ser Ile Ala

325 330 335

Thr Gly Met Val Gly Ala Leu Leu Leu Leu Leu Val Val Ala Leu Gly

340 345 350

Ile Gly Leu Phe Met

355

<210> 9

<211> 1180

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg 60

ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt 120

gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca 180

gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc 240

gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt 300

acttccacct ggctgcagta cgtgattctt gatcccgagc ttcggggttg gaagtgggtg 360

ggagagttcg aggccttgcg cttaaggagc cccttcgcct cgtgcttgag ttgaggcctg 420

gcctgggcgc tggggccgcc gcgtgcgaat ctggtggcac cttcgcgcct gtctcgctgc 480

tttcgataag tctctagcca tttaaaattt ttgatgacct gctgcgacgc tttttttctg 540

gcaagatagt cttgtaaatg cgggccaaga tctgcacact ggtatttcgg tttttggggc 600

cgcgggcggc gacggggccc gtgcgtccca gcgcacatgt tcggcgaggc ggggcctgcg 660

agcgcggcca ccgagaatcg gacgggggta gtctcaagct ggccggcctg ctctggtgcc 720

tggcctcgcg ccgccgtgta tcgccccgcc ctgggcggca aggctggccc ggtcggcacc 780

agtagcgtga gcggaaagat ggccgcttcc cggccctgct gcagggagct caaaatggag 840

gacgcggcgc tcgggagagc gggcgggtga gtcacccaca caaaggaaaa gggcctttcc 900

gtcctcagcc gtcgcttcat gtgactccac ggagtaccgg gcgccgtcca ggcacctcga 960

ttagttctcg agcttttgga gtacgtcgtc tttaggttgg ggggaggggt tttatgcgat 1020

ggagtttccc cacactgagt gggtggagac tgaagttagg ccagcttggc acttgatgta 1080

attctccttg gaatttgccc tttttgagtt tggatcttgg ttcattctca agcctcagac 1140

agtggttcaa agtttttttc ttccatttca ggtgtcgtga 1180

<210> 10

<211> 2550

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

ttaattaagc caccatgctg ctgctggtga ccagcctgct gctgtgcgag ctgccccacc 60

ccgcctttct gctgatccga ggtgcagctg gtggagagcg gcggcggcct ggtgaagccc 120

ggcggcagcc tgaagctgag ctgcgccgcc agcggctaca ccttcaccag ctacgtgatg 180

cactgggtga gacaggcccc cggcaagggc ctggagtgga tcggctacat caacccctac 240

aacgacggca ccaagtacaa cgagaagttc cagggcagag tgaccatcag cagcgacaag 300

agcatcagca ccgcctacat ggagctgagc agcctgagaa gcgaggacac cgccatgtac 360

tactgcgcca gaggcaccta ctactacggc accagagtgt tcgactactg gggccagggc 420

accctggtga ccgtgagcag cggtggtggt ggttctggcg gcggcggctc cggtggtggt 480

ggttctgaca tcgtgatgac ccagagcccc gccaccctga gcctgagccc cggcgagaga 540

gccaccctga gctgcagaag cagcaagagc ctgcagaacg tgaacggcaa cacctacctg 600

tactggttcc agcagaagcc cggccagagc ccccagctgc tgatctacag aatgagcaac 660

ctgaacagcg gcgtgcccga cagattcagc ggcagcggca gcggcaccga gttcaccctg 720

accatcagca gcctggagcc cgaggacttc gccgtgtact actgcatgca gcacctggag 780

taccccatca ccttcggcgc cggcaccaag ctggagatca aggaatctaa gtacggaccg 840

ccctgccccc cttgccctat gttctgggtg ctggtggtgg tcggaggcgt gctggcctgc 900

tacagcctgc tggtcaccgt ggccttcatc atcttttggg tgaaacgggg cagaaagaaa 960

ctcctgtata tattcaaaca accatttatg agaccagtac aaactactca agaggaagat 1020

ggctgtagct gccgatttcc agaagaagaa gaaggaggat gtgaactgcg ggtgaagttc 1080

agcagaagcg ccgacgcccc tgcctaccag cagggccaga atcagctgta caacgagctg 1140

aacctgggca gaagggaaga gtacgacgtc ctggataagc ggagaggccg ggaccctgag 1200

atgggcggca agcctcggcg gaagaacccc caggaaggcc tgtataacga actgcagaaa 1260

gacaagatgg ccgaggccta cagcgagatc ggcatgaagg gcgagcggag gcggggcaag 1320

ggccacgacg gcctgtatca gggcctgtcc accgccacca aggataccta cgacgccctg 1380

cacatgcagg ccctgccccc aaggctcgag ggcggcggag agggcagagg aagtcttcta 1440

acatgcggtg acgtggagga gaatcccggc cctaggatgc ttctcctggt gacaagcctt 1500

ctgctctgtg agttaccaca cccagcattc ctcctgatcc cacgcaaagt gtgtaacgga 1560

ataggtattg gtgaatttaa agactcactc tccataaatg ctacgaatat taaacacttc 1620

aaaaactgca cctccatcag tggcgatctc cacatcctgc cggtggcatt taggggtgac 1680

tccttcacac atactcctcc tctggatcca caggaactgg atattctgaa aaccgtaaag 1740

gaaatcacag ggtttttgct gattcaggct tggcctgaaa acaggacgga cctccatgcc 1800

tttgagaacc tagaaatcat acgcggcagg accaagcaac atggtcagtt ttctcttgca 1860

gtcgtcagcc tgaacataac atccttggga ttacgctccc tcaaggagat aagtgatgga 1920

gatgtgataa tttcaggaaa caaaaatttg tgctatgcaa atacaataaa ctggaaaaaa 1980

ctgtttggga cctccggtca gaaaaccaaa attataagca acagaggtga aaacagctgc 2040

aaggccacag gccaggtctg ccatgccttg tgctcccccg agggctgctg gggcccggag 2100

cccagggact gcgtctcttg ccggaatgtc agccgaggca gggaatgcgt ggacaagtgc 2160

aaccttctgg agggtgagcc aagggagttt gtggagaact ctgagtgcat acagtgccac 2220

ccagagtgcc tgcctcaggc catgaacatc acctgcacag gacggggacc agacaactgt 2280

atccagtgtg cccactacat tgacggcccc cactgcgtca agacctgccc ggcaggagtc 2340

atgggagaaa acaacaccct ggtctggaag tacgcagacg ccggccatgt gtgccacctg 2400

tgccatccaa actgcaccta cggatgcact gggccaggtc ttgaaggctg tccaacgaat 2460

gggcctaaga tcccgtccat cgccactggg atggtggggg ccctcctctt gctgctggtg 2520

gtggccctgg ggatcggcct cttcatgtga 2550

<210> 11

<211> 735

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

gacatccaga tgacccagac cacctccagc ctgagcgcca gcctgggcga ccgggtgacc 60

atcagctgcc gggccagcca ggacatcagc aagtacctga actggtatca gcagaagccc 120

gacggcaccg tcaagctgct gatctaccac accagccggc tgcacagcgg cgtgcccagc 180

cggtttagcg gcagcggctc cggcaccgac tacagcctga ccatctccaa cctggaacag 240

gaagatatcg ccacctactt ttgccagcag ggcaacacac tgccctacac ctttggcggc 300

ggaacaaagc tggaaatcac cggcagcacc tccggcagcg gcaagcctgg cagcggcgag 360

ggcagcacca agggcgaggt gaagctgcag gaaagcggcc ctggcctggt ggcccccagc 420

cagagcctga gcgtgacctg caccgtgagc ggcgtgagcc tgcccgacta cggcgtgagc 480

tggatccggc agccccccag gaagggcctg gaatggctgg gcgtgatctg gggcagcgag 540

accacctact acaacagcgc cctgaagagc cggctgacca tcatcaagga caacagcaag 600

agccaggtgt tcctgaagat gaacagcctg cagaccgacg acaccgccat ctactactgc 660

gccaagcact actactacgg cggcagctac gccatggact actggggcca gggcaccagc 720

gtgaccgtga gcagc 735

<210> 12

<211> 245

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 12

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Ser Thr Ser Gly

100 105 110

Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys Gly Glu Val Lys

115 120 125

Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser

130 135 140

Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser

145 150 155 160

Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile

165 170 175

Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu

180 185 190

Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn

195 200 205

Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr

210 215 220

Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser

225 230 235 240

Val Thr Val Ser Ser

245

<210> 13

<211> 9919

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

actagtgtcg acaatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt 60

aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct 120

attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt 180

tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac 240

gcaaccccca ctggttgggg cattgccacc aactgtcagc tcctttccgg gactttcgct 300

ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca 360

ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaagct gacgtccttt 420

ccatggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc 480

ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct 540

cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg 600

cctggaattc gagctcggta cctttaagac caatgactta caaggcagct gtagatctta 660

gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa cgaagacaag 720

atctgctttt tgcttgtact gggtctctct ggttagacca gatctgagcc tgggagctct 780

ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag 840

tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt 900

cagtgtggaa aatctctagc agtagtagtt catgtcatct tattattcag tatttataac 960

ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt tattgcagct tataatggtt 1020

acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta 1080

gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggctctag ctatcccgcc 1140

cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg 1200

ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca 1260

gaagtagtga ggaggctttt ttggaggcct agggacgtac ccaattcgcc ctatagtgag 1320

tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc 1380

gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa 1440

gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atgggacgcg 1500

ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca 1560

cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc 1620

gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct 1680

ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg 1740

ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc 1800

ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg 1860

attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg 1920

aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg 1980

gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat 2040

aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc 2100

gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa 2160

cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac 2220

tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga 2280

tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag 2340

agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca 2400

cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca 2460

tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa 2520

ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc 2580

tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa 2640

cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag 2700

actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct 2760

ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac 2820

tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa 2880

ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt 2940

aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat 3000

ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg 3060

agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc 3120

ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg 3180

tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag 3240

cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact 3300

ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg 3360

gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc 3420

ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg 3480

aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg 3540

cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag 3600

ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc 3660

gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct 3720

ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc 3780

ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc 3840

gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac 3900

cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact 3960

ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc 4020

aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat 4080

ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac cctcactaaa 4140

gggaacaaaa gctggagctg caagcttaat gtagtcttat gcaatactct tgtagtcttg 4200

caacatggta acgatgagtt agcaacatgc cttacaagga gagaaaaagc accgtgcatg 4260

ccgattggtg gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac agacgggtct 4320

gacatggatt ggacgaacca ctgaattgcc gcattgcaga gatattgtat ttaagtgcct 4380

agctcgatac ataaacgggt ctctctggtt agaccagatc tgagcctggg agctctctgg 4440

ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt 4500

gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt 4560

gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg gaaaccagag 4620

gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg cgaggggcgg 4680

cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag agatgggtgc 4740

gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat tcggttaagg 4800

ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag ggagctagaa 4860

cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca aatactggga 4920

cagctacaac catcccttca gacaggatca gaagaactta gatcattata taatacagta 4980

gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga agctttagac 5040

aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc cgctgatctt 5100

cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata aatataaagt 5160

agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag tggtgcagag 5220

agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag cagcaggaag 5280

cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat tgtctggtat 5340

agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc tgttgcaact 5400

cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa gatacctaaa 5460

ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca ccactgctgt 5520

gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc acacgacctg 5580

gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct taattgaaga 5640

atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata aatgggcaag 5700

tttgtggaat tggtttaaca taacaaattg gctgtggtat ataaaattat tcataatgat 5760

agtaggaggc ttggtaggtt taagaatagt ttttgctgta ctttctatag tgaatagagt 5820

taggcaggga tattcaccat tatcgtttca gacccacctc ccaaccccga ggggacccga 5880

caggcccgaa ggaatagaag aagaaggtgg agagagagac agagacagat ccattcgatt 5940

agtgaacgga tctcgacggt atcggttaac ttttaaaaga aaagggggga ttggggggta 6000

cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca 6060

aaaacaaatt acaaaaattc aaaattttat cgatgtcgac gataagcttt gcaaagatgg 6120

ataaagtttt aaacagagag gaatctttgc agctaatgga ccttctaggt cttgaaagga 6180

gtgggaattg gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag 6240

aagttggggg gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac 6300

tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat 6360

ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag 6420

gtaagtgccg tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg 6480

ccttgaatta cttccacctg gctgcagtac gtgattcttg atcccgagct tcggggttgg 6540

aagtgggtgg gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt 6600

tgaggcctgg cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg 6660

tctcgctgct ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct 6720

ttttttctgg caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt 6780

ttttggggcc gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg 6840

gggcctgcga gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc 6900

tctggtgcct ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg 6960

gtcggcacca gtagcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc 7020

aaaatggagg acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag 7080

ggcctttccg tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag 7140

gcacctcgat tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt 7200

ttatgcgatg gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca 7260

cttgatgtaa ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa 7320

gcctcagaca gtggttcaaa gtttttttct tccatttcag gtgtcgtgat taattaagcc 7380

accatgctgc tgctggtgac cagcctgctg ctgtgcgagc tgccccaccc cgcctttctg 7440

ctgatccgag gtgcagctgg tggagagcgg cggcggcctg gtgaagcccg gcggcagcct 7500

gaagctgagc tgcgccgcca gcggctacac cttcaccagc tacgtgatgc actgggtgag 7560

acaggccccc ggcaagggcc tggagtggat cggctacatc aacccctaca acgacggcac 7620

caagtacaac gagaagttcc agggcagagt gaccatcagc agcgacaaga gcatcagcac 7680

cgcctacatg gagctgagca gcctgagaag cgaggacacc gccatgtact actgcgccag 7740

aggcacctac tactacggca ccagagtgtt cgactactgg ggccagggca ccctggtgac 7800

cgtgagcagc ggtggtggtg gttctggcgg cggcggctcc ggtggtggtg gttctgacat 7860

cgtgatgacc cagagccccg ccaccctgag cctgagcccc ggcgagagag ccaccctgag 7920

ctgcagaagc agcaagagcc tgcagaacgt gaacggcaac acctacctgt actggttcca 7980

gcagaagccc ggccagagcc cccagctgct gatctacaga atgagcaacc tgaacagcgg 8040

cgtgcccgac agattcagcg gcagcggcag cggcaccgag ttcaccctga ccatcagcag 8100

cctggagccc gaggacttcg ccgtgtacta ctgcatgcag cacctggagt accccatcac 8160

cttcggcgcc ggcaccaagc tggagatcaa ggaatctaag tacggaccgc cctgcccccc 8220

ttgccctatg ttctgggtgc tggtggtggt cggaggcgtg ctggcctgct acagcctgct 8280

ggtcaccgtg gccttcatca tcttttgggt gaaacggggc agaaagaaac tcctgtatat 8340

attcaaacaa ccatttatga gaccagtaca aactactcaa gaggaagatg gctgtagctg 8400

ccgatttcca gaagaagaag aaggaggatg tgaactgcgg gtgaagttca gcagaagcgc 8460

cgacgcccct gcctaccagc agggccagaa tcagctgtac aacgagctga acctgggcag 8520

aagggaagag tacgacgtcc tggataagcg gagaggccgg gaccctgaga tgggcggcaa 8580

gcctcggcgg aagaaccccc aggaaggcct gtataacgaa ctgcagaaag acaagatggc 8640

cgaggcctac agcgagatcg gcatgaaggg cgagcggagg cggggcaagg gccacgacgg 8700

cctgtatcag ggcctgtcca ccgccaccaa ggatacctac gacgccctgc acatgcaggc 8760

cctgccccca aggctcgagg gcggcggaga gggcagagga agtcttctaa catgcggtga 8820

cgtggaggag aatcccggcc ctaggatgct tctcctggtg acaagccttc tgctctgtga 8880

gttaccacac ccagcattcc tcctgatccc acgcaaagtg tgtaacggaa taggtattgg 8940

tgaatttaaa gactcactct ccataaatgc tacgaatatt aaacacttca aaaactgcac 9000

ctccatcagt ggcgatctcc acatcctgcc ggtggcattt aggggtgact ccttcacaca 9060

tactcctcct ctggatccac aggaactgga tattctgaaa accgtaaagg aaatcacagg 9120

gtttttgctg attcaggctt ggcctgaaaa caggacggac ctccatgcct ttgagaacct 9180

agaaatcata cgcggcagga ccaagcaaca tggtcagttt tctcttgcag tcgtcagcct 9240

gaacataaca tccttgggat tacgctccct caaggagata agtgatggag atgtgataat 9300

ttcaggaaac aaaaatttgt gctatgcaaa tacaataaac tggaaaaaac tgtttgggac 9360

ctccggtcag aaaaccaaaa ttataagcaa cagaggtgaa aacagctgca aggccacagg 9420

ccaggtctgc catgccttgt gctcccccga gggctgctgg ggcccggagc ccagggactg 9480

cgtctcttgc cggaatgtca gccgaggcag ggaatgcgtg gacaagtgca accttctgga 9540

gggtgagcca agggagtttg tggagaactc tgagtgcata cagtgccacc cagagtgcct 9600

gcctcaggcc atgaacatca cctgcacagg acggggacca gacaactgta tccagtgtgc 9660

ccactacatt gacggccccc actgcgtcaa gacctgcccg gcaggagtca tgggagaaaa 9720

caacaccctg gtctggaagt acgcagacgc cggccatgtg tgccacctgt gccatccaaa 9780

ctgcacctac ggatgcactg ggccaggtct tgaaggctgt ccaacgaatg ggcctaagat 9840

cccgtccatc gccactggga tggtgggggc cctcctcttg ctgctggtgg tggccctggg 9900

gatcggcctc ttcatgtga 9919

Claims (40)

1. A CD 19-targeting chimeric antigen receptor comprising, in order: an extracellular antigen binding domain, a spacer domain, a transmembrane domain, a costimulatory signaling domain, and a primary signaling domain; the extracellular antigen-binding domain is an scFv that specifically binds CD 19; the amino acid sequence of the scFv is shown as SEQ ID NO.1 or SEQ ID NO. 12;

the spacer domain is an IgG4hinge region; the amino acid sequence of the IgG4hinge region is shown in SEQ ID NO. 2;

the transmembrane domain is the transmembrane region of CD 28; the amino acid sequence of the transmembrane region of the CD28 is shown in SEQ ID NO. 3;

the costimulatory signaling domain is the intracellular signaling domain of 4-1 BB; the amino acid sequence of the intracellular signal transduction domain of the 4-1BB is shown as SEQ ID NO. 4;

the primary signaling domain is the intracellular signaling domain of cd3ζ; the amino acid sequence of the intracellular signaling domain of CD3 zeta is shown as the sequence of SEQ ID NO. 5.

2. The chimeric antigen receptor according to claim 1, further comprising the following components: self-cleaving peptides, cell surface markers.

3. The chimeric antigen receptor according to claim 2, wherein the self-cleaving peptide is T2A; the amino acid sequence of the T2A is shown in SEQ ID NO. 7.

4. The chimeric antigen receptor according to claim 2, wherein the cell surface marker is tgfr; the amino acid sequence of the tEGFR is shown as SEQ ID NO. 8.

5. The chimeric antigen receptor according to claim 1, wherein the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID No. 6.

6. A nucleic acid encoding the chimeric antigen receptor of claim 1.

7. The nucleic acid of claim 6, further comprising a promoter, and/or a cleavage site located after the promoter, and/or a kozak sequence located after the cleavage site.

8. The nucleic acid of claim 7, wherein the promoter is located at the N-terminus of the nucleic acid, the promoter being a promoter that extends factor 1 a; the sequence of the promoter is shown as SEQ ID NO. 9.

9. The nucleic acid of claim 7, wherein the cleavage site is a recognition site for Pac I; the sequence of the enzyme cutting site is shown in the 1 st-8 th positions of SEQ ID No. 10.

10. The nucleic acid of claim 7, wherein the kozak sequence is set forth in positions 9-14 of SEQ ID No. 10.

11. The nucleic acid of claim 7, wherein the coding sequence of the signal peptide in said nucleic acid is set forth in positions 15-78 of SEQ ID No. 10.

12. The nucleic acid of claim 7, wherein the scFv in the nucleic acid has a coding sequence set forth in positions 79-822 of SEQ ID No. 10.

13. The nucleic acid of claim 7, wherein the coding sequence of the spacer domain in said nucleic acid is set forth in positions 823-846 of SEQ ID No. 10.

14. The nucleic acid of claim 7, wherein the coding sequence of the transmembrane domain in said nucleic acid is set forth in SEQ ID No.10 at positions 847-939.

15. The nucleic acid of claim 7, wherein the coding sequence of the costimulatory signaling domain in the nucleic acid is as set forth in SEQ ID No.10 at positions 940-1068.

16. The nucleic acid of claim 7, wherein the coding sequence of the primary signaling domain in said nucleic acid is set forth in SEQ ID No.10 at positions 1069-1404.

17. The nucleic acid of claim 7, wherein the coding sequence for the chimeric antigen receptor in said nucleic acid is set forth in positions 15-2585 of SEQ ID No. 10.

18. The nucleic acid of claim 7, wherein the coding sequence of T2A in said nucleic acid is as set forth in SEQ ID No.10 at positions 1405-1476.

19. The nucleic acid of claim 7, wherein the coding sequence for tgfr in said nucleic acid is set forth in SEQ ID No.10 at positions 1477-2550.

20. The nucleic acid of claim 7, wherein the nucleic acid comprises the nucleotide sequence set forth in SEQ ID No. 10.

21. A vector comprising the nucleic acid of any one of claims 6-20.

22. The vector of claim 21, wherein the vector sequence is set forth in SEQ ID No. 13.

23. A recombinant microorganism comprising the nucleic acid of any one of claims 6-20 or the vector of claim 21 or 22.

24. The recombinant microorganism according to claim 23, wherein the recombinant microorganism is a recombinant virus.

25. An engineered immune cell expressing the chimeric antigen receptor of any one of claims 1-5.

26. The engineered immune cell of claim 25, wherein the immune cell contains a nucleic acid as described in any one of claims 6-20 or a vector as described in claim 21 or 22.

27. A population of immune cells comprising the engineered immune cell of claim 25 or 26.

28. The population of immune cells of claim 27, wherein the population of immune cells further comprises unmodified or incompletely engineered immune cells.

29. A method of preparing an engineered immune cell of claim 25 or 26, comprising introducing the nucleic acid of any one of claims 6-20 or the vector of claim 21 or 22 into an immune cell.

30. A composition comprising the chimeric antigen receptor of any one of claims 1-5, the nucleic acid of any one of claims 6-20, the vector of claim 21 or 22, the recombinant microorganism of claim 23 or 24, the engineered immune cell of claim 25 or 26, the population of immune cells of claim 27 or 28.

31. The composition of claim 30, wherein the composition further comprises a pharmaceutically acceptable carrier.

32. A kit comprising the chimeric antigen receptor of any one of claims 1-5, the nucleic acid of any one of claims 6-20, the vector of claim 21 or 22, the recombinant microorganism of claim 23 or 24, the engineered immune cell of claim 25 or 26, the population of immune cells of claim 27 or 28, the composition of claim 30.

33. Use of the chimeric antigen receptor of any one of claims 1-5, the nucleic acid of any one of claims 6-20, the vector of claim 21 or 22, the recombinant microorganism of claim 23 or 24, the engineered immune cell of claim 25 or 26, the population of immune cells of claim 27 or 28, the composition of claim 30 in the manufacture of a medicament for treating a disease or disorder associated with CD19 expression.

34. The use according to claim 33, wherein the diseases or disorders associated with CD19 expression include neoplasms, autoimmune and inflammatory diseases, infectious diseases.

35. The use of claim 34, wherein the tumor comprises a B cell malignancy.

36. The use of claim 35, wherein the B-cell malignancy comprises B-cell leukemia and lymphoma.

37. The use according to claim 36, wherein the B-cell leukemia comprises B-cell chronic lymphocytic leukemia, acute lymphocytic leukemia, prolymphocytic leukemia, hairy cell leukemia, common acute lymphoblastic leukemia, non-acute lymphoblastic leukemia.

38. The use according to claim 36, wherein said lymphomas comprise non-hodgkin's lymphomas, diffuse large B-cell lymphomas, multiple myelomas, follicular lymphomas, splenic and marginal band lymphomas, mantle cell lymphomas, indolent B-cell lymphomas, hodgkin's lymphomas.

39. The use of claim 37, wherein the B-cell malignancy is relapsed cd19+ leukemia.

40. An application comprising any one of:

1) Use of the chimeric antigen receptor of any one of claims 1-5 in the preparation of the nucleic acid of any one of claims 6-20, the vector of claim 21 or 22, the recombinant microorganism of claim 23 or 24, the engineered immune cell of claim 25 or 26, the population of immune cells of claim 27 or 28, the composition of claim 30, the kit of claim 32;

2) Use of the nucleic acid of any one of claims 6-20 in the preparation of the vector of claim 21 or 22, the recombinant microorganism of claim 23 or 24, the engineered immune cell of claim 25 or 26, the population of immune cells of claim 27 or 28, the composition of claim 30, the kit of claim 32;

3) Use of the vector of claim 21 or 22 in the preparation of the recombinant microorganism of claim 23 or 24, the engineered immune cell of claim 25 or 26, the population of immune cells of claim 27 or 28, the composition of claim 30, the kit of claim 32;

4) Use of a recombinant microorganism according to claim 23 or 24 for the preparation of an engineered immune cell according to claim 25 or 26, an immune cell population according to claim 27 or 28, a composition according to claim 30, a kit according to claim 32;

5) Use of an immune cell of claim 25 or 26 in the preparation of an immune cell population of claim 27 or 28, a composition of claim 30, a kit of claim 32;

6) Use of the population of immune cells of claim 27 or 28 in the preparation of the composition of claim 30, the kit of claim 32;

7) Use of the composition of claim 30 for the preparation of the kit of claim 32.

Images