CN109265550B - BCMA antibodies, chimeric antigen receptors and drugs - Google Patents

Abstract

The invention discloses a BCMA antibody, a chimeric antigen receptor and a medicament, and relates to the technical field of cellular immunotherapy. The BCMA antibody provided by the invention has heavy chain CDR regions shown in SEQ ID NO.6-8 and light chain CDR regions shown in SEQ ID NO.14-16, or has heavy chain CDR regions shown in SEQ ID NO.38-40 and light chain CDR regions shown in SEQ ID NO. 46-48; the BCMA antibody has the capacity of specifically binding BCMA protein, and the chimeric antigen receptor T cells prepared by the BCMA antibody have specific killing effect on target cells positive to the BCMA protein, and can be used for preparing medicaments for treating or preventing tumors.

Description

BCMA antibodies, chimeric antigen receptors and drugs

Technical Field

The invention relates to the technical field of cellular immunotherapy, and particularly relates to a BCMA antibody, a chimeric antigen receptor and a medicament.

Background

Multiple Myeloma (MM) is a malignant tumor characterized by massive proliferation of clonal plasma cells. MM treatment can induce remission at present, but almost all patients eventually relapse and die. Although some monoclonal antibodies have shown promise in pre-clinical studies and early clinical trials to treat MM, no consensus has been achieved. Clearly, new immunotherapies for MM are clearly highly desirable, and the development of effective antigen-specific adoptive T cell therapies for this disease would be a major advance. T cells can be genetically modified to express a Chimeric Antigen Receptor (CAR) that includes a fusion protein of an antigen recognition moiety and a T cell activation domain. For B-line malignancies, the most commonly used is an adoptive T cell approach against CD19 CAR. anti-CD 19-CAR transduced T cells cured leukemia and lymphoma in mice, some patients also had remission in early clinical trials of adoptively infused anti-CD 19-CAR transduced T cells, but at the same time, T cells transduced with anti-CD 19 CARs also cleared normal B cells and unfortunately, CD19 is rarely expressed in malignant plasma cells of MM, so treatment of MM with CAR-expressing T cells would require the search for other better targets.

One candidate Antigen for immunotherapy of MM is the B Cell Maturation Antigen (B Cell Maturation Antigen, BCMA, CD 269). BCMA RNA is detected ubiquitously in MM cells, and BCMA protein is detected on the plasma cell surface of multiple myeloma patients. BCMA is a member of the tumor necrosis factor receptor (TNF) superfamily that binds B cell activating factor (BAFF) and proliferation-inducing ligand (APRIL). It has been reported that BCMA is expressed mainly by plasma cells and a part of mature B cells in normal cells, but not on most B cells and other organs. BCMA deficient mice appear all normal, seem healthy, and have normal B cell numbers, but plasma cells do not survive long term. Thus, BCMA would be a suitable target antigen for the treatment of MM with CAR expressing T cells.

However, there are currently fewer classes of antibodies directed against BCMA.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a BCMA antibody, which can be specifically combined with BCMA protein and can be used for preparing chimeric antigen receptor T cells targeting the BCMA protein.

Another object of the present invention is to provide a chimeric antigen receptor targeting BCMA protein, which can target BCMA protein, and T cells expressing the chimeric antigen receptor can specifically kill target cells positive to BCMA.

Another object of the present invention is to provide a chimeric antigen receptor T cell targeting BCMA protein, which can specifically kill BCMA positive target cells, and can be used for treating BCMA positive tumors.

Another object of the present invention is to provide a nucleic acid molecule.

It is another object of the present invention to provide a vector.

Another object of the present invention is to provide a recombinant cell.

The invention also aims to provide a medicament for treating tumors, which can be used for treating or preventing tumors with positive BCMA.

The invention is realized by the following steps:

the invention takes human BCMA (hBCMA) protein as antigen, combines phage library display technology, and elutriates to obtain 4 scFV antibodies which are not reported in the prior art. Experiments prove that the 4 scFV antibodies have the capacity of combining BCMA protein, and the chimeric antigen receptor T cells prepared by the scFV antibodies have specific killing effect on target cells positive to the BCMA protein. Therefore, the 4 scFV antibodies can be used for preparing chimeric antigen receptor T cells targeting BCMA protein and preparing tumors positive for BCMA.

Based on this, in one aspect, the present invention provides a BCMA antibody, wherein the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region are shown as SEQ ID No.6, SEQ ID No.7 and SEQ ID No.8, respectively, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region are shown as SEQ ID No.14, SEQ ID No.15 and SEQ ID No.16, respectively;

or the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region of the BCMA antibody are respectively shown as SEQ ID NO.38, SEQ ID NO.39 and SEQ ID NO.40, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region of the BCMA antibody are respectively shown as SEQ ID NO.46, SEQ ID NO.47 and SEQ ID NO. 48.

Further, in some embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the BCMA antibody is shown as SEQ ID No.5, and the amino acid sequence of the light chain variable region of the BCMA antibody is shown as SEQ ID No. 13;

or the amino acid sequence of the heavy chain variable region of the BCMA antibody is shown in SEQ ID NO.37, and the amino acid sequence of the light chain variable region of the BCMA antibody is shown in SEQ ID NO. 45.

Antibodies having the above-described CDR sequences can specifically bind to BCMA protein, which is used to prepare chimeric antigen receptor T cells that target BCMA protein.

In addition, the BCMA antibody provided by the invention is used for preparing a detection reagent for detecting BCMA protein, or a label is added on the antibody provided by the invention for detecting BCMA protein, and the invention belongs to the protection scope of the invention.

Further, in some embodiments of the invention, the BCMA antibody is one of a full length antibody, F (ab ') 2, Fab', Fab, Fv, and scFv.

On the basis of the heavy chain variable region and the light chain variable region of the BCMA antibody provided by the invention, the antibody type of any one of full-length antibody, F (ab ') 2, Fab', Fab, Fv and scFv which can be combined with BCMA protein can be easily constructed by a person skilled in the art; any type of antibody, so long as it contains the heavy chain CDR sequences and/or light chain CDR sequences, is within the scope of the present invention.

In another aspect, the present invention provides a chimeric antigen receptor targeting BCMA protein comprising the heavy chain variable region and the light chain variable region of said BCMA antibody targeting BCMA protein as described above.

Further, in some embodiments of the invention, the chimeric antigen receptor further has one or a combination of several of the following elements:

a signal peptide, a linker, a hinge region, a CD8 a transmembrane domain, a 4-1BB costimulatory signaling region, and a CD3 zeta signaling domain.

Further, in some embodiments of the present invention, the amino acid sequence of the signal peptide is shown in SEQ ID NO. 73.

Further, in some embodiments of the present invention, the amino acid sequence of the above linker is shown in SEQ ID NO. 74.

Further, in some embodiments of the present invention, the amino acid sequence of the hinge region (hinge) is represented by SEQ ID NO. 75.

Further, in some embodiments of the invention, the amino acid sequence of the transmembrane domain of CD8 a is shown as seq id No. 76.

Further, in some embodiments of the present invention, the amino acid sequence of the aforementioned 4-1BB co-stimulatory signaling region is represented by SEQ ID NO. 77.

Further, in some embodiments of the invention, the amino acid sequence of the CD3 zeta signaling domain is set forth in SEQ ID No. 78.

It is noted that in other embodiments, the combined class and sequence of the signal peptide, linker, hinge region, CD8 a transmembrane domain, 4-1BB costimulatory signaling region, and CD3 zeta signaling domain may be altered by one skilled in the art according to the actual circumstances or needs, and any type of alteration may be used, so long as the chimeric antigen receptor has the CDR sequence or light chain variable region sequence of the light chain variable region of the above-described antibody of the present invention and/or the CDR sequence or heavy chain variable region sequence of the heavy chain variable region of the above-described antibody, which fall within the scope of the present invention.

Further, in some embodiments of the invention, a signal peptide, the light chain variable region of the above antibody, a linker, the heavy chain variable region of the above antibody, a hinge region, a CD8 a transmembrane domain, a 4-1BB costimulatory signaling region, and a CD3 zeta signaling domain are in serial order to comprise the chimeric antigen receptor.

The chimeric antigen receptor can target BCMA protein, and T cells expressing the chimeric antigen receptor can specifically kill target cells with positive BCMA.

In another aspect, the invention provides an isolated nucleic acid molecule encoding an antibody as described above, or encoding a chimeric antigen receptor as described above.

In another aspect, the present invention provides a vector comprising the nucleic acid molecule described above.

In another aspect, the invention provides a recombinant cell comprising a nucleic acid molecule encoding the chimeric antigen receptor described above, or the vector described above.

In another aspect, the present invention provides a chimeric antigen receptor T cell targeting BCMA protein, which expresses the chimeric antigen receptor described above.

The T cell can specifically kill target cells with positive BCMA, and can be used for treating tumors with positive BCMA.

In another aspect, the present invention provides a medicament for treating tumors, which comprises the chimeric antigen receptor T cell described above.

Further, in some embodiments of the invention, the tumor is a BCMA positive tumor.

Further, in some embodiments of the invention, BCMA-positive tumor cells include: MM1S, H929, etc.

The medicine can be used for treating or preventing tumor with positive BCMA.

Further, in some embodiments of the present invention, the above-mentioned medicament further comprises a pharmaceutically acceptable excipient.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

Fig. 1 shows the results of the binding assay between BCMA antibody 2077/2082/2073/2079 and human BCMA protein.

FIG. 2 is a schematic diagram of the CAR-BCMA structure.

FIG. 3 shows the CAR-BCMA positivity after infection of 72Hrs with CD4+ CD8+ T cells by flow cytometry.

FIG. 4A is a flow chart of the change in target cell ratio over time in coculture of CAR-BCMA expressing T cells (CAR-BCMA-2073-T, CAR-BCMA-2079-T) with target cell H929 at an effective target ratio of 5/1.

FIG. 4B is a flow chart of the change in target cell ratio over time in coculture of CAR-BCMA expressing T cells (CAR-BCMA-2077-T, CAR-BCMA-2082-T) with target cell H929 at an effective target ratio of 5/1.

FIG. 5 is a graph of statistics of The change in target cell ratio over time in co-culture of CAR-BCMA expressing T cells (CAR-BCMA-2073-T, CAR-BCMA-2077-T, CAR-BCMA-2079-The CAR-BCMA-2082-T) with target cell H929 at an effective target ratio of 5/1.

FIG. 6A is a flow chart of the change in target cell ratio over time in coculture of CAR-BCMA expressing T cells (CAR-BCMA-2073-T, CAR-BCMA-2079-T) with target cell K562-BCMA cells at an effective target ratio of 7/1.

FIG. 6B is a flow chart of the change in target cell ratio over time in co-culture of CAR-BCMA expressing T cells (CAR-BCMA-2077-T, CAR-BCMA-2082-T) with target cell K562-BCMA cells at an effective target ratio of 7/1.

FIG. 7 is a graph of statistics of the change in target cell ratio over time for CAR-BCMA expressing T cells co-cultured with target cell K562-BCMA cells at an effective target ratio of 7/1.

Figure 8 is a cytokine release assay of T cells expressing a targeting BCMA chimeric antigen receptor after activation by BCMA positive cell stimulation.

Fig. 9 is a graph of BCMA positive tumor treatment with T cells expressing a targeting BCMA chimeric antigen receptor in a mouse myeloma transplantation model.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

1 phage library panning

First, blocking phage antibody for 2h with blocking solution (PBST/5% skim milk powder) at room temperature, the input amount of phage is 2 × 10¹²phage, then 10. mu.g antigen, incubated for 1h at room temperature, after which 50. mu.l pre-blocked

M-280Streptavidin magnetic beads, incubated at room temperature for 30 min.

Washing off the unbound phage by PBST, eluting the phage bound to the magnetic beads by 0.1M HCl-Glycine, neutralizing the eluate with Tris-HCl, collecting part of phage to infect Escherichia coli TG1 in logarithmic growth phase, and collecting phage for next round of panning.

Gradually increasing the screening intensity of each round, and stopping the elutriation when the enrichment degree reaches more than 100 times.

2 screening of anti-BCMA Single chain antibody Positive clones Using phase Elisa

(1) A single clone of TGl infected with phage after four rounds of panning was selected and inoculated into a 96-well plate in 2YT medium (containing 2% glucose, 100. mu.g/ml Ampicilline).

(2) After being cultured overnight at 37 ℃ and 250rpm, the culture medium is transferred to a new culture medium, after the culture is carried out until logarithmic growth phase, M13K07 helper phage is added, and the culture medium is statically infected for 1h at 37 ℃.

(3) The cells were centrifuged at 4000rpm for 15min, cultured overnight at 30 ℃ using 2YT (containing 100. mu.g/ml Ampicilline and 70. mu.g/ml Kanamycin), and the phage supernatant was centrifuged to identify clones by ELISA.

(4) The Costar-9018 enzyme-labeled plate is coated with 0.5 mu g/ml of hBCMA antigen, 3% BSA is sealed overnight at 4 ℃, the collected phage supernatant is added, and incubation is carried out for 2h at 4 ℃.

(5) Unbound phage were washed away and Ml3 Bacteriophage antibody (HRP) was added and incubated for 1h at 4 ℃. After washing, TMB developing solution is added for developing color, and the reaction is stopped by 2M HCl.

(6) Reading at 450nm with a microplate reader, and selecting OD₄₅₀>1.5 clones were sequenced, and Germline analysis and PTMs site analysis were performed on the sequences, and 4 scFv single-chain antibodies (hereinafter also referred to as BCMA antibodies) having the property of binding to hBCMA protein were obtained in total after eliminating molecules at potential development risk.

The four scFv single chain antibodies were:

anti-BCMA single chain antibody 1 (designated antibody 2073):

the heavy chain variable region amino acid sequence is as follows: SEQ ID NO.5, the corresponding nucleotide coding sequence is: SEQ ID No. 1;

the amino acid sequences of VH-CDR1, VH-CDR2 and VH-CDR3 of the heavy chain variable region are respectively as follows: SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, the corresponding nucleotide coding sequence is: SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO. 4;

the amino acid sequence of the light chain variable region is as follows: SEQ ID NO.13, the corresponding nucleotide coding sequence is: SEQ ID No. 9;

the light chain variable region has the amino acid sequences of VL-CDR1, VL-CDR2 and VL-CDR 3: SEQ ID No.14, SEQ ID No.15, SEQ ID No.16, the corresponding nucleotide coding sequence is: SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO. 12;

anti-BCMA Single-chain antibody 2 (designated antibody 2077)

The heavy chain variable region amino acid sequence is as follows: SEQ ID NO.21, the corresponding nucleotide coding sequence is: SEQ ID No. 17;

the amino acid sequences of the VH-CDR1, VH-CDR2 and VH-CDR3 of the heavy chain variable region are respectively as follows: SEQ ID NO.22, SEQ ID NO.23 and SEQ ID NO.24, wherein the corresponding nucleotide coding sequences are as follows: SEQ ID No.18, SEQ ID No.19, SEQ ID No. 20;

the amino acid sequence of the light chain variable region is as follows: SEQ ID NO.29, corresponding nucleotide coding sequence: SEQ ID No. 25;

the amino acid sequences of the VL-CDR1, VL-CDR2 and VL-CDR3 in the light chain variable region are respectively as follows: SEQ ID NO.30, SEQ ID NO.31, SEQ ID NO.32, the corresponding nucleotide coding sequence being: SEQ ID NO.26, SEQ ID NO.27, SEQ ID NO. 28.

anti-BCMA Single-chain antibody 3 (designated antibody 2079)

The amino acid sequences of the heavy chain variable regions are respectively as follows: SEQ ID NO.37, corresponding to the nucleotide coding sequence: SEQ ID No. 33;

the amino acid sequences of the VH-CDR1, VH-CDR2 and VH-CDR3 of the heavy chain variable region are respectively as follows: SEQ ID NO.38, SEQ ID NO.39, SEQ ID NO.40, the corresponding nucleotide coding sequences are: SEQ ID No.34, SEQ ID No.35, SEQ ID No. 36;

the amino acid sequence of the light chain variable region is as follows: SEQ ID No.45, corresponding nucleotide coding sequence: SEQ ID No. 41;

the amino acid sequences of the VL-CDR1, VL-CDR2 and VL-CDR3 in the light chain variable region are respectively as follows: SEQ ID NO.46, SEQ ID NO.47, SEQ ID NO.48, the corresponding nucleotide coding sequences are: SEQ ID NO.42, SEQ ID NO.43, SEQ ID NO. 44;

anti-BCMA single-chain antibody 4 (designated as antibody 2082)

The heavy chain variable region amino acid sequence is as follows: SEQ ID No.53, which corresponds to the nucleotide sequence: SEQ ID No. 49;

the amino acid sequences of the VH-CDR1, VH-CDR2 and VH-CDR3 of the heavy chain variable region are respectively as follows: SEQ ID NO.54, SEQ ID NO.55, SEQ ID NO.56, the corresponding nucleotide coding sequences are: SEQ ID No.50, SEQ ID No.51, SEQ ID No. 52;

the amino acid sequence of the light chain variable region is as follows: SEQ ID NO.61, the corresponding nucleotide coding sequence is: SEQ ID No. 57;

the amino acid sequences of the VL-CDR1, VL-CDR2 and VL-CDR3 in the light chain variable region are respectively as follows: SEQ ID NO.62, SEQ ID NO.63, SEQ ID NO.64, the corresponding nucleotide coding sequences are: SEQ ID NO.58, SEQ ID NO.59, SEQ ID NO. 60.

Example 2

Detecting the binding force of 4 BCMA antibodies (2073, 2077, 2079 or 2082) obtained in example 1 and hBCMA protein

The detection method comprises the following steps:

(1) hBCMA protein was coated, diluted from 1. mu.g/ml, 3-fold gradient dilutions, 8 gradients at 1. mu.g/ml, 333ng/ml, 111ng/ml, 37ng/ml, 12.3ng/ml, 4.1ng/ml, 1.37ng/ml, 0.45ng/ml, Costar-9018 plates were coated with 100. mu.l hBCMA protein dilutions, overnight at 4 ℃.

(2) Blocking with 3% BSA at room temperature for 2h, adding BCMA antibody, i.e., 2073, 2077, 2079 or 2082 corresponding phage supernatant (10^10pfu), and incubating at room temperature for 2 h.

(3) Unbound phage were washed away and Ml3 Bacteriophage antibody (HRP) was added and incubated for 1h at 4 ℃. After washing, TMB developing solution is added for developing color, and the reaction is stopped by 2M HCl.

(4) The reading was done at 450nm using a microplate reader, and the results are shown in FIG. 1.

As can be seen from the results in fig. 1, the phage expressing BCMA antibodies 2073, 2077, 2079 or 2082 has good binding ability to hBCMA protein, shows S curve, and shows dose-response relationship, indicating that 4 BCMA antibodies obtained in example 1, i.e., 2073, 2077, 2079 and 2082, all have binding ability to hBCMA protein.

Example 3

Construction of chimeric antigen receptor expression vectors

The construction method comprises the following steps:

(1) and (3) total gene synthesis: signal peptide (nucleic acid sequence SEQ ID No.66, amino acid sequence SEQ ID No.73), BCMA antibody light chain variable region (light chain variable region of 2073, 2077, 2079 or 2082), linker (nucleic acid sequence SEQ ID No.67, amino acid sequence SEQ ID No.74), BCMA antibody heavy chain variable region (heavy chain variable region of 2073, 2077, 2079 or 2082), hinge region (hinge) (nucleic acid sequence SEQ ID No.68, amino acid sequence SEQ ID No.75), CD8 a transmembrane domain (TM) (nucleic acid sequence SEQ ID No.69, amino acid sequence SEQ ID No.76), 4-1BB costimulatory signaling region (nucleic acid sequence SEQ ID No.70, amino acid sequence SEQ ID No.77), and CD3 ζ signaling domain (nucleic acid sequence SEQ ID No.71, amino acid sequence SEQ ID No. 78). The above sequences are connected in sequence. 4 chimeric antigen receptor expression cassettes were obtained, respectively, and named: 2073 chimeric antigen receptor expression cassette (full length nucleotide sequence shown in SEQ ID NO.65, amino acid sequence shown in SEQ ID NO. 72), 2077 chimeric antigen receptor expression cassette (full length nucleotide sequence shown in SEQ ID NO.79, amino acid sequence shown in SEQ ID NO. 82), 2079 chimeric antigen receptor expression cassette (full length nucleotide sequence shown in SEQ ID NO.80, amino acid sequence shown in SEQ ID NO. 83) and 2082 chimeric antigen receptor expression cassette (full length nucleotide sequence shown in SEQ ID NO.81, amino acid sequence shown in SEQ ID NO. 84).

And Kozac sequence was introduced into the head of each cassette, the cassette structure is shown in FIG. 2.

(2) After synthesizing the sequence of the chimeric antigen receptor expression cassette by the whole gene, connecting the sequence to an empty vector pCDH-EF1-MSC-T2A-copGFP through an XbaI/EcoRI enzyme cutting site to obtain a chimeric antigen receptor expression vector; obtaining 4 chimeric antigen receptor expression vectors, and after the chimeric antigen receptor expression vectors are verified to be correct by sequencing, respectively naming the chimeric antigen receptor expression vectors as:

pCDH-EF1-CAR-BCMA-2073-copGFP containing the light chain variable region and the heavy chain variable region of antibody 2073;

pCDH-EF1-CAR-BCMA-2077-copGFP containing the light chain variable region and the heavy chain variable region of antibody 2077;

pCDH-EF1-CAR-BCMA-2079-copGFP containing the light chain variable region and the heavy chain variable region of antibody 2079;

pCDH-EF1-CAR-BCMA-2082-copGFP, light and heavy chain variable regions comprising antibody 2082.

Example 4

Preparation of strains containing chimeric antigen receptor expression vectors

The method comprises the following steps:

(1) DH 5. alpha. was made competent in the-80 ℃ freezer and thawed on ice.

(2) Add 5ng plasmid to the competence, mix gently, ice for 5 minutes.

The plasmids were: pCDH-EF1-CAR-BCMA-2073-copGFP, pCDH-EF1-CAR-BCMA-2077-copGFP, pCDH-EF1-CAR-BCMA-2079-copGFP or pCDH-EF 1-CAR-BCMA-2082-copGFP.

(3) Heat shock at 42 ℃ for 90 seconds and ice for 30 minutes.

(4) 0.5ml of non-resistant LB was added and incubated at 37 ℃ and 180rpm for 30 minutes.

(5) Spread onto ampicillin resistant plates.

(6) The cells were cultured overnight at 37 ℃ by inversion.

(7) The single colonies were picked and cultured in ampicillin-resistant LB at 37 ℃ and 200rpm for 9 to 12 hours.

(8) Adding glycerol into the bacterial liquid, wherein the final concentration of the glycerol is 10%, and storing the strain in a refrigerator at-80 ℃ for later use, and the method can be used for subsequent mass extraction of plasmids.

(9) After the strains are cultured in LB in large quantity, a plasmid extraction kit (endotoxin-free plasmid extraction kit of Beijing Tiangen Biochemical technology Co., Ltd.) is used for extracting plasmids for infection. The plasmid extraction method is carried out according to the instruction.

Example 5

Virus package

24 hours before transfection, 293T cells were trypsinized, 4 × 10⁶The 293T cells of (1) were plated in a 10cm cell culture dish, and the cells were cultured in DMEM medium containing 10% FBS at 37 ℃ in a 5% CO2 incubator for not more than 24 hours, and were transfected when the cells reached a density of 60-80%.

The method comprises the following specific steps:

(1) placing plasmid, PEI and DMEM medium at room temperature for 5 min;

(2) taking 450 μ l of DMEM in a 1.5ml EP tube, adding 50 μ l of PEI (1 μ g/μ l) and mixing uniformly, and standing for 5min at room temperature;

(3) taking 10 μ g plasmid (pCDH-EF1-CAR-BCMA-2073-copGFP, pCDH-EF1-CAR-BCMA-2077-copGFP, pCDH-EF1-CAR-BCMA-2079-copGFP or pCDH-EF1-CAR-BCMA-2082-copGFP), 10 μ g psPAX2, 5 μ g pMD2.G, adding DMEM to 500 μ l, mixing, standing at room temperature for 5 min;

(4) adding the prepared PEI-DMEM solution in the step (2) into the DMEM containing the plasmids obtained in the step (3), uniformly mixing, and standing at room temperature for 20 min; obtaining a DNA/PEI mixture;

(5) slowly dropping 1ml of DNA/PEI mixture into a 293T culture dish, gently mixing uniformly, and incubating for 6-8h at 37 ℃ in an incubator;

(6) discarding the original culture medium, replacing the fresh culture medium, and putting the culture medium into a 37 ℃ incubator for continuous incubation;

(7) after the culture medium is replaced for 48 hours, collecting the culture medium, adding 10ml of fresh culture medium into each dish to continue culturing, and collecting the supernatant again after 24 hours and mixing the supernatant with the supernatant collected for 48 hours;

(8) centrifuging at 4 deg.C and 4000g for 10min to remove cell debris;

(9) the resulting supernatant was filtered through a 0.45 μm filter;

(10) subjecting the filtered supernatant to tangential flow filtration;

(11) transferring the virus supernatant subjected to tangential flow filtration into an ultracentrifuge tube, centrifuging at 25000rpm for 2h, carrying out heavy suspension on a virus precipitate obtained after ultracentrifuge by using a serum-free culture medium, lightly blowing and beating until the virus precipitate is completely dissolved to obtain virus liquid, wherein the virus liquid obtained by adopting different vectors is respectively named as:

2073 virus fluid (containing 2073 chimeric antigen receptor expression cassette), 2077 virus fluid (containing 2077 chimeric antigen receptor expression cassette), 2079 virus fluid (containing 2079 chimeric antigen receptor expression cassette), 2082 virus fluid (containing 2080 chimeric antigen receptor expression cassette), and empty carrier virus fluid;

(12) subpackaging each virus solution, storing in a refrigerator at-80 deg.C, and reserving 5-10 μ l virus concentrate for titer determination.

Example 6

Viral titer determination

The method comprises the following steps:

293T cells were digested and counted, and prepared into cell suspension using DMEM medium containing 10% FBS, and cell density was adjusted to 4 × 10⁵Perml, 0.5ml of cell suspension was added to each well of a 24-well plate. After 8 hours of cell adherent culture, 1 mul, 10 mul, 20 mul, 30 mul and 50 mul of virus solution diluted by 100 times are infected, the solution is changed after 24 hours, and the positive rate of 293T cells is detected by flow type after 48 hours.

Centrifuging, resuspending and adjusting the cell density to 1 × 10⁶Adding biotin-BCMA antigen into 50 μ l/ml, incubating for 30min, washing DPBS once, resuspending, staining secondary antibody APC-Streptavidin (purchased from BD) for 30min, resuspending DPBS after washing once, and detecting by flow.

Example 7

T cells for making chimeric antigen receptors targeting human BCMA antigens

1 isolation of PBMC from human peripheral blood mononuclear cells

Approximately 25ml of peripheral blood was collected using an anticoagulation tube (purchased from BD) according to 1: adding the mixture into lymphocyte separating medium according to the volume ratio of 1, carrying out gradient centrifugation for 25min, taking leucoderma cells after centrifugation, and washing twice by using DPBS to obtain the PBMC of the human peripheral blood mononuclear cells.

2CD4+ CD8+ T cell enrichment and activation

Resuspending PBMC at density 1 × 10⁵Mu.l of each CD4/CD8 magnetic bead was added to 50. mu.l of the cell suspension, and CD4+ CD8+ T cells were obtained by magnetic separation.

The resulting CD4+ CD8+ T cells were cultured in AIM-V complete medium containing 10% FBS, and the T cells were activated with anti-human CD3/CD28 antibody (purchased from Gentianchang, 10. mu.l/ml) at an IL-2 concentration of 200 IU/ml. After 24 hours of activation, the medium was changed and the culture was continued using complete medium containing IL-2200 IU/ml.

3 Lentiviral infection

Regulating T cell density to 1 × 10⁶(ii)/ml, wherein the virus solution obtained in example 6 (2073 virus solution, 2077 virus solution, 2079 virus solution or 2082 virus solution) is used for infecting T cells after 48 hours of activation according to MOI of 10, the solution is changed after 24 hours, IL-2200 IU/ml is continuously added, and different virus solutions are adopted to obtain different T cells capable of expressing chimeric antigen receptors targeting human BCMA antigen; named CAR-BCMA-2073-T, CAR-BCMA-2077-T, CAR-BCMA-2079-T, CAR-BCMA-2082-T, respectively.

4 detection of chimeric antigen receptor targeting human BCMA antigen (CAR-BCMA) expression

During the culture process, T cells CAR-BCMA-2073-T, CAR-BCMA-2077-T, CAR-BCMA-2079-T, CAR-BCMA-2082-T72 hours after virus infection are taken, centrifuged, resuspended and adjusted to a cell density of 1 × 10⁶Adding biotin-BCMA antigen into 50 mu l of the solution per ml, incubating for 30min, washing DPBS once, resuspending, staining secondary antibody APC-Streptavidin (purchased from BD) for 30min, resuspending DPBS after washing once, and detecting the positive rate of CAR-BCMA by flow.

The results are shown in figure 3, and show that the CAR-BCMA-2073-T, CAR-BCMA-2077-T, CAR-BCMA-2079-T and CAR-BCMA-2082-T all express chimeric antigen receptors targeting BCMA, and the positive rates of the chimeric antigen receptors reach 45.1%, 61.5%, 22.3% and 78.5% respectively.

Example 8

In vitro co-culture was performed to detect the tumor killing effect of T cells targeting the chimeric antigen receptor of human BCMA antigen, the target cell was NCI-H929.

Collecting T cells (CAR-BCMA-2073-T, CAR-BCMA-2077-T, CAR-BCMA-2079-T or CAR-BCMA-2082-T) and BCMA + tumor cell NCI-H929 72hr after infection, counting, and regulating cell density to 1 × 10⁶Per ml, co-cultured according to a target-effective ratio of 5:1, namely: t cells 1 x 10⁶，NCI-H929 2×10⁵Control cells were CD4+ CD8+ T cells untreated with viral infection and scored as Ctrl-T cells. The proportion of NCI-H929 cells in total cells was measured at 0H, 24H, 48H, respectively, and the target cells were labeled with the Antibody APC-conjugated Human BCMA/TNFRSF17Antibody, and flow-assayed, and the results are shown in FIGS. 4A and 4B; meanwhile, the cell killing effect is counted, and the result is shown in figure 5.

The results show that in the presence of CAR-BCMA-2073-T, CAR-BCMA-2077-T, CAR-BCMA-2079-T or CAR-BCMA-2082-T, the proportion of target cells NCI-H929 decreases significantly with increasing time; CAR-BCMA-2073-T: reduced from 15.4% to 0.435%; CAR-BCMA-2079-T: reduced from 11.3% to 6.63%; CAR-BCMA-2077-T: decrease from 19.6% to 0.628%; CAR-BCMA-2082-T: from 18.5% to 0.185%. Thus, 4 chimeric antigen receptor T cells CAR-BCMA-2073-T, CAR-BCMA-2077-T, CAR-BCMA-2079-T and CAR-BCMA-2082-T all can kill target cell NCI-H929 well.

Example 9

And (3) detecting the tumor killing effect of the CAR-BCMAT by in-vitro co-culture, wherein the target cell is K562-BCMA.

Collecting T cells (CAR-BCMA-2073-T, CAR-BCMA-2077-T, CAR-BCMA-2079-T or CAR-BCMA-2082-T) 72hr after infection and BCMA-over-expressing cell K562-BCMA, counting, and adjusting cell density to 1 × 10⁶/ml, co-cultured according to an effective target ratio of 7:1, i.e. T cells 1 × 10⁶，K562-BCMA 1.5×10⁵Control cells were CD4+ CD8+ T cells untreated with virus fluid and scored as Ctrl-T cells. The ratio of K562-BCMA cells in total cells was determined at 0H, 24H, 48H, respectively, and the target cells were labeled with the Antibody APC-conjugated Human BCMA/TNFRSF17AntibodyThe results are shown in fig. 6A and 6B, while the cell killing effect is counted, and the results are shown in fig. 7.

The results show that in the presence of CAR-BCMA-2073-T, CAR-BCMA-2077-T, CAR-BCMA-2079-T or CAR-BCMA-2082-T, the proportion of target cell K562-BCMA decreases significantly with time, and over a period of 0-48h the proportion of target cell K562-BCMA decreases by an amount of respectively: CAR-BCMA-2073-T: reduced from 8.69% to 6.1%; CAR-BCMA-2079-T: reduced from 9.28% to 8.69%; CAR-BCMA-2077-T: reduced from 8.95% to 1.03%; CAR-BCMA-2082-T: reduced from 11.2% to 0.676%; therefore, 4 chimeric antigen receptor T cells CAR-BCMA-2073-T, CAR-BCMA-2077-T, CAR-BCMA-2079-T and CAR-BCMA-2082-T can well kill target cells K562-BCMA which are positive to BCMAT.

Example 10

ELISA method for detecting cytokine expression level

The method comprises the following steps:

sample preparation, T cells (CAR-BCMA-2073-T, CAR-BCMA-2077-T, CAR-BCMA-2079-T or CAR-BCMA-2082-T) and BCMA + tumor cells NCI-H929 were taken 72 hours after infection, counted, and cell density was adjusted to 1 × 10⁶/ml, co-cultured according to an effective target ratio (E: T)1:1, i.e. T cells 1 × 10⁶，NCI-H929 1×10⁶Control cells were CD4+ CD8+ T cells untreated with viral infection and scored as Ctrl-T cells. The supernatant was collected at 24H and stored at-80 ℃ until use.

And (3) detection: cytokines INF-gamma, TNF-alpha were detected using the CBA method, as described in the instructions. The results are shown in FIG. 8.

As can be seen from the figure, the INF-gamma and TNF-alpha contents of 4 chimeric antigen receptor T cells CAR-BCMA-2073-T, CAR-BCMA-2077-T, CAR-BCMA-2079-T and CAR-BCMA-2082-T were all higher than the control (Ctrl-T).

Example 11

Using NSG mice, the tumor cells used were MM.1S (designated NCI-MM.1S-LUC), a BCMA positive cell line stably expressing firefly luciferase. The treatment-injected effector cells contain CAR-BCMA T cells (CAR-BCMA-2077-T or CAR-BCMA-2082-T), and the control group is physiological saline group, CD4+ CD8+ T cells without virus infection3 days after successful modeling, tail vein injection of effector cells 5 × 10⁶Mice were imaged by IVS in vivo imaging system every 7 days after injection to show tumor growth, and mice were weighed and observed for survival and recorded. The results are shown in FIG. 9.

As can be seen from fig. 9, the fluorescence area of mice receiving CAR-BCMA-2077-T or CAR-BCMA-2082-T injection was significantly reduced on day 3 and day 7, indicating that tumor cells were significantly reduced, and the chimeric antigen receptor T cells CAR-BCMA-2077-T or CAR-BCMA-2082-T expressing targeting BCMA could target BCMA, effectively killing BCMA-expressing tumor cells, and achieving the goal of tumor treatment.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> university of east China, Shanghai Yao Biotechnology Co., Ltd

<120> BCMA antibodies, chimeric antigen receptors and drugs

<160>84

<170>PatentIn version 3.5

<210>1

<211>357

<212>DNA

<213> Artificial sequence

<400>1

caggtacagc tgcagcagtc aggtccagga ctggtgaagc cctcgcagac cctctcactc 60

acctgtgcca tctccgggga cagtgtctct agcaacagtg ctgcttggaa ctggatcagg 120

cagtccccat cgagaggcct tgagtggctg ggaaggacat actacaggtc caagtggtat 180

aatgattatg cattatctgt gaaaagtcga ataaccatca acccagacac atccaagaac 240

cagttctccc tgcagctgaa ctctgtgact cccgaggaca cggctgtgta ttactgtgca 300

agaggcgcca gctcgtttga ctactggggc cagggaaccc tggtcaccgt ctcgagt 357

<210>2

<211>21

<212>DNA

<213> Artificial sequence

<400>2

agcaacagtg ctgcttggaa c 21

<210>3

<211>54

<212>DNA

<213> Artificial sequence

<400>3

aggacatact acaggtccaa gtggtataat gattatgcat tatctgtgaa aagt 54

<210>4

<211>21

<212>DNA

<213> Artificial sequence

<400>4

ggcgccagct cgtttgacta c 21

<210>5

<211>119

<212>PRT

<213> Artificial sequence

<400>5

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn

20 25 30

Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu

35 40 45

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

50 55 60

Leu Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn

65 70 75 80

Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val

85 90 95

Tyr Tyr Cys Ala Arg Gly Ala Ser Ser Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210>6

<211>7

<212>PRT

<213> Artificial sequence

<400>6

Ser Asn Ser Ala Ala Trp Asn

1 5

<210>7

<211>18

<212>PRT

<213> Artificial sequence

<400>7

Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Leu Ser Val

1 5 10 15

Lys Ser

<210>8

<211>7

<212>PRT

<213> Artificial sequence

<400>8

Gly Ala Ser Ser Phe Asp Tyr

1 5

<210>9

<211>339

<212>DNA

<213> Artificial sequence

<400>9

gacatcgtga tgacccagtc tccagactcc ctggctgtgt ctctgggcga gagggccacc 60

atcaattgca agtccagcca gaatgtttta tacagctcca acaataggaa ctacttagct 120

tggtaccaac agaaacctgg acagcctcct aagctgctca tttactgggc atctacccgg 180

gagtccgggg tccctgaccg attcagtggc agcgggtctg ggacagattt cactctcacc 240

atcagcagcc tgcaggctga agatgtggca gtttattact gtcagcaata ttatggttct 300

gttgtcactt tcggcggagg gaccaaggtg gaaatcaaa 339

<210>10

<211>51

<212>DNA

<213> Artificial sequence

<400>10

aagtccagcc agaatgtttt atacagctcc aacaatagga actacttagc t 51

<210>11

<211>21

<212>DNA

<213> Artificial sequence

<400>11

tgggcatcta cccgggagtc c 21

<210>12

<211>27

<212>DNA

<213> Artificial sequence

<400>12

cagcaatatt atggttctgt tgtcact 27

<210>13

<211>113

<212>PRT

<213> Artificial sequence

<400>13

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

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Asn Val Leu Tyr Ser

20 25 30

Ser Asn Asn Arg Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln

85 90 95

Tyr Tyr Gly Ser Val Val Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

100 105 110

Lys

<210>14

<211>17

<212>PRT

<213> Artificial sequence

<400>14

Lys Ser Ser Gln Asn Val Leu Tyr Ser Ser Asn Asn Arg Asn Tyr Leu

1 5 10 15

Ala

<210>15

<211>7

<212>PRT

<213> Artificial sequence

<400>15

Trp Ala Ser Thr Arg Glu Ser

1 5

<210>16

<211>9

<212>PRT

<213> Artificial sequence

<400>16

Gln Gln Tyr Tyr Gly Ser Val Val Thr

1 5

<210>17

<211>378

<212>DNA

<213> Artificial sequence

<400>17

caggtacagc tgcagcagtc aggtccagga ctggtgaagc cctcgcagac cctctcactc 60

acctgtgcca tctccgggga cagtgtctct agcaacagtg ctgcttggaa ctggatcagg 120

cagtccccat cgagaggcct tgagtggctg ggaaggacat actacaggtc caagtggtat 180

aatgattatg cagtatctgt gaaaagtcga ataaccatca acccagacac atccaagaac 240

cagttctccc tgcagctgaa ctctgtgact cccgaggaca cggctgtgta ttactgtgca 300

aggttagcct acgaagtacg ctccacagac tggtacttcg atctctgggg ccgtggcacc 360

ctggtcaccg tctcgagt 378

<210>18

<211>21

<212>DNA

<213> Artificial sequence

<400>18

agcaacagtg ctgcttggaa c 21

<210>19

<211>54

<212>DNA

<213> Artificial sequence

<400>19

aggacatact acaggtccaa gtggtataat gattatgcag tatctgtgaa aagt 54

<210>20

<211>42

<212>DNA

<213> Artificial sequence

<400>20

ttagcctacg aagtacgctc cacagactgg tacttcgatc tc 42

<210>21

<211>126

<212>PRT

<213> Artificial sequence

<400>21

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn

20 25 30

Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu

35 40 45

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

50 55 60

Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn

65 70 75 80

Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val

85 90 95

Tyr Tyr Cys Ala Arg Leu Ala Tyr Glu Val Arg Ser Thr Asp Trp Tyr

100 105 110

Phe Asp Leu Trp Gly Arg Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210>22

<211>7

<212>PRT

<213> Artificial sequence

<400>22

Ser Asn Ser Ala Ala Trp Asn

1 5

<210>23

<211>18

<212>PRT

<213> Artificial sequence

<400>23

Arg Thr Tyr Tyr Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Val Ser Val

1 5 10 15

Lys Ser

<210>24

<211>14

<212>PRT

<213> Artificial sequence

<400>24

Leu Ala Tyr Glu Val Arg Ser Thr Asp Trp Tyr Phe Asp Leu

1 510

<210>25

<211>330

<212>DNA

<213> Artificial sequence

<400>25

cagcctgtgc tgactcagcc accctcggtg tctgaagccc ccaggcagag ggtcaccatc 60

tcctgttctg gaagcagctc caacatcgga aataatgctg taaactggta ccagcagctc 120

ccaggaaagg ctcccaaact cctcatctat tatgatgatc tgctgccctc aggggtctct 180

gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccag 240

tctgaggatg aggctgatta ttactgtgca gcatgggatg acagcctgaa tggttgggtg 300

ttcggcggag ggaccaagct gaccgtccta 330

<210>26

<211>39

<212>DNA

<213> Artificial sequence

<400>26

tctggaagca gctccaacat cggaaataat gctgtaaac 39

<210>27

<211>21

<212>DNA

<213> Artificial sequence

<400>27

tatgatgatc tgctgccctc a 21

<210>28

<211>33

<212>DNA

<213> Artificial sequence

<400>28

gcagcatggg atgacagcctgaatggttgg gtg 33

<210>29

<211>110

<212>PRT

<213> Artificial sequence

<400>29

Gln Pro Val Leu Thr Gln Pro Pro Ser Val Ser Glu Ala Pro Arg Gln

1 5 10 15

Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn

20 25 30

Ala Val Asn Trp Tyr Gln Gln Leu Pro Gly Lys Ala Pro Lys Leu Leu

35 40 45

Ile Tyr Tyr Asp Asp Leu Leu Pro Ser Gly Val Ser Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu

85 90 95

Asn Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu

100 105 110

<210>30

<211>13

<212>PRT

<213> Artificial sequence

<400>30

Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn Ala Val Asn

1 5 10

<210>31

<211>7

<212>PRT

<213> Artificial sequence

<400>31

Tyr Asp Asp Leu Leu Pro Ser

1 5

<210>32

<211>21

<212>PRT

<213> Artificial sequence

<400>32

Ala Ala Trp Asp Asp Ser Leu Asn Gly Trp Val Phe Gly Gly Gly Thr

1 5 10 15

Lys Leu Thr Val Leu

20

<210>33

<211>357

<212>DNA

<213> Artificial sequence

<400>33

caggtacagc tgcagcagtc aggtccagga ctggtaaagc cctcgcagac cctctcactc 60

acctgtgcca tctccgggga cagtgtctct agcaacagtg ctgcttggaa ctggatcagg 120

cagtccccat cgagaggcct tgagtggctg ggaaggacat actacaggtc cacgtggtat 180

aatgattatg cagtatctgt gaaaagtcga ataaccatta acccagacac atccaagaac 240

cagttctccc tgcagctgaa ctctgtgact cccgaggaca cggctgtgta ttactgtgca 300

aggggaactg gaacccttga ctactggggc cagggaaccc tggtcaccgt ctcgagt 357

<210>34

<211>21

<212>DNA

<213> Artificial sequence

<400>34

agcaacagtg ctgcttggaa c 21

<210>35

<211>54

<212>DNA

<213> Artificial sequence

<400>35

aggacatact acaggtccac gtggtataat gattatgcag tatctgtgaa aagt 54

<210>36

<211>24

<212>DNA

<213> Artificial sequence

<400>36

aggggaactg gaacccttga ctac 24

<210>37

<211>119

<212>PRT

<213> Artificial sequence

<400>37

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

1 5 10 15

Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly Asp Ser Val Ser Ser Asn

20 25 30

Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser ProSer Arg Gly Leu Glu

35 40 45

Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Thr Trp Tyr Asn Asp Tyr Ala

50 55 60

Val Ser Val Lys Ser Arg Ile Thr Ile Asn Pro Asp Thr Ser Lys Asn

65 70 75 80

Gln Phe Ser Leu Gln Leu Asn Ser Val Thr Pro Glu Asp Thr Ala Val

85 90 95

Tyr Tyr Cys Ala Arg Gly Thr Gly Thr Leu Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser

115

<210>38

<211>7

<212>PRT

<213> Artificial sequence

<400>38

Ser Asn Ser Ala Ala Trp Asn

1 5

<210>39

<211>18

<212>PRT

<213> Artificial sequence

<400>39

Arg Thr Tyr Tyr Arg Ser Thr Trp Tyr Asn Asp Tyr Ala Val Ser Val

1 5 10 15

Lys Ser

<210>40

<211>8

<212>PRT

<213> Artificial sequence

<400>40

Arg Gly Thr Gly Thr Leu Asp Tyr

1 5

<210>41

<211>330

<212>DNA

<213> Artificial sequence

<400>41

cagtctgccc tgactcagcc tgcctccgtg tctgggtctc ctggacagtc gatcaccatg 60

tcctgcactg gaaccagcag tgacgttggt ggttataagt atgtctcctg gtaccaacaa 120

cacccaggca aagcccccca actcatgatt catgatgtcc gtgagcggcc ctcaggggtt 180

tctaatcgct tctctggctc caagtctggc aacacggcct ccctgaccat ctctgggctc 240

caggctgagg acgaggctga ttattactgc agctcattta caaacaacag cacttttgtc 300

ttcggaactg ggaccaaagt caccgtccta 330

<210>42

<211>42

<212>DNA

<213> Artificial sequence

<400>42

actggaacca gcagtgacgt tggtggttat aagtatgtct cc 42

<210>43

<211>21

<212>DNA

<213> Artificial sequence

<400>43

gatgtccgtg agcggccctc a 21

<210>44

<211>30

<212>DNA

<213> Artificial sequence

<400>44

agctcattta caaacaacag cacttttgtc 30

<210>45

<211>110

<212>PRT

<213> Artificial sequence

<400>45

Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Met Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr

20 25 30

Lys Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Gln Leu

35 40 45

Met Ile His Asp Val Arg Glu Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

65 70 75 80

Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Phe Thr Asn Asn

85 90 95

Ser Thr Phe Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu

100 105 110

<210>46

<211>14

<212>PRT

<213> Artificial sequence

<400>46

Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Lys Tyr Val Ser

1 5 10

<210>47

<211>7

<212>PRT

<213> Artificial sequence

<400>47

Asp Val Arg Glu Arg Pro Ser

1 5

<210>48

<211>10

<212>PRT

<213> Artificial sequence

<400>48

Ser Ser Phe Thr Asn Asn Ser Thr Phe Val

1 5 10

<210>49

<211>366

<212>DNA

<213> Artificial sequence

<400>49

caggtacagc tgcagcagtc aggtccagca ttggtgaagc cctcgcagac cctctcactc 60

acctgtgtca tctccgggga ctctgtctct agcaacagtg cttcttggac ctggatcagg 120

cagtcccctt cgagaggcct tgagtggctgggaaggacgt accgtcgggc cgacaggtgg 180

tattatgatt atgcactctc gctgaatagt cgactaacca tcaatccaga cacatccaaa 240

aaccatttcg ccctgcacct gacctctgtg actcccgagg acacggctgt ttattactgt 300

tcaagagaat attggggagg ttcttttgat gtctggggcc aagggaccac ggtcaccgtc 360

tcgagt 366

<210>50

<211>21

<212>DNA

<213> Artificial sequence

<400>50

agcaacagtg cttcttggac c 21

<210>51

<211>57

<212>DNA

<213> Artificial sequence

<400>51

aggacgtacc gtcgggccga caggtggtat tatgattatg cactctcgct gaatagt 57

<210>52

<211>27

<212>DNA

<213> Artificial sequence

<400>52

gaatattggg gaggttcttt tgatgtc 27

<210>53

<211>122

<212>PRT

<213> Artificial sequence

<400>53

Gln Val Gln Leu Gln Gln Ser Gly Pro Ala Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Val Ile Ser Gly Asp Ser Val Ser Ser Asn

20 25 30

Ser Ala Ser Trp Thr Trp Ile Arg Gln Ser Pro Ser Arg Gly Leu Glu

35 40 45

Trp Leu Gly Arg Thr Tyr Arg Arg Ala Asp Arg Trp Tyr Tyr Asp Tyr

50 55 60

Ala Leu Ser Leu Asn Ser Arg Leu Thr Ile Asn Pro Asp Thr Ser Lys

65 70 75 80

Asn His Phe Ala Leu His Leu Thr Ser Val Thr Pro Glu Asp Thr Ala

85 90 95

Val Tyr Tyr Cys Ser Arg Glu Tyr Trp Gly Gly Ser Phe Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210>54

<211>7

<212>PRT

<213> Artificial sequence

<400>54

Ser Asn Ser Ala Ser Trp Thr

1 5

<210>55

<211>19

<212>PRT

<213> Artificial sequence

<400>55

Arg Thr Tyr Arg Arg Ala Asp Arg Trp Tyr Tyr Asp Tyr Ala Leu Ser

1 5 10 15

Leu Asn Ser

<210>56

<211>9

<212>PRT

<213> Artificial sequence

<400>56

Glu Tyr Trp Gly Gly Ser Phe Asp Val

1 5

<210>57

<211>333

<212>DNA

<213> Artificial sequence

<400>57

cagcctgtgc tgactcagcc tgcctccgtg tctgggtcgc ctggacagtc gatcaccatc 60

tcctgcactg gaaccagcag tgacgttggt ggttatgact atgtctcctg gtaccaacaa 120

cacccaggca aagcccccaa actcatgctt tatgaggtca ataatcggcc ctcaggggtt 180

tctaatcgct tctctggctc caagtctggc aacacggcct ccctgaccat ctctgggctc 240

caggctgagg acgagggtgc ttattactgc agctcatata caagcagcaa cactcatgtg 300

gtattcggcg gaggcaccca gctgaccgtc ctc 333

<210>58

<211>42

<212>DNA

<213> Artificial sequence

<400>58

actggaacca gcagtgacgt tggtggttat gactatgtct cc 42

<210>59

<211>21

<212>DNA

<213> Artificial sequence

<400>59

gaggtcaata atcggccctc a 21

<210>60

<211>33

<212>DNA

<213> Artificial sequence

<400>60

agctcatata caagcagcaa cactcatgtg gta 33

<210>61

<211>111

<212>PRT

<213> Artificial sequence

<400>61

Gln Pro Val Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln

1 5 10 15

Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr

20 25 30

Asp Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu

35 40 45

Met Leu Tyr Glu Val Asn Asn Arg Pro Ser Gly Val Ser Asn Arg Phe

50 55 60

Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu

6570 75 80

Gln Ala Glu Asp Glu Gly Ala Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser

85 90 95

Asn Thr His Val Val Phe Gly Gly Gly Thr Gln Leu Thr Val Leu

100 105 110

<210>62

<211>14

<212>PRT

<213> Artificial sequence

<400>62

Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr Asp Tyr Val Ser

1 5 10

<210>63

<211>7

<212>PRT

<213> Artificial sequence

<400>63

Glu Val Asn Asn Arg Pro Ser

1 5

<210>64

<211>11

<212>PRT

<213> Artificial sequence

<400>64

Ser Ser Tyr Thr Ser Ser Asn Thr His Val Val

1 5 10

<210>65

<211>1485

<212>DNA

<213> Artificial sequence

<400>65

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60

atcccagaca tcgtgatgac ccagtctcca gactccctgg ctgtgtctct gggcgagagg 120

gccaccatca attgcaagtc cagccagaat gttttataca gctccaacaa taggaactac 180

ttagcttggt accaacagaa acctggacag cctcctaagc tgctcattta ctgggcatct 240

acccgggagt ccggggtccc tgaccgattc agtggcagcg ggtctgggac agatttcact 300

ctcaccatca gcagcctgca ggctgaagat gtggcagttt attactgtca gcaatattat 360

ggttctgttg tcactttcgg cggagggacc aaggtggaaa tcaaaggctc cacctctgga 420

tccggcaagc ccggatctgg cgagggatcc accaagggcc aggtacagct gcagcagtca 480

ggtccaggac tggtgaagcc ctcgcagacc ctctcactca cctgtgccat ctccggggac 540

agtgtctcta gcaacagtgc tgcttggaac tggatcaggc agtccccatc gagaggcctt 600

gagtggctgg gaaggacata ctacaggtcc aagtggtata atgattatgc attatctgtg 660

aaaagtcgaa taaccatcaa cccagacaca tccaagaacc agttctccct gcagctgaac 720

tctgtgactc ccgaggacac ggctgtgtat tactgtgcaa gaggcgccag ctcgtttgac 780

tactggggcc agggaaccct ggtcaccgtc tcgagtacca cgacgccagc gccgcgacca 840

ccaacaccgg cgcccaccat cgcgtcacag cccctgtccc tgcgcccaga ggcgtgccgg 900

ccagcggcgg ggggcgcagt gcacacgagg gggctggact tcgcctgtga tatctacatc 960

tgggcgccct tggccgggac ttgtggggtc cttctcctgt cactggttat caccctttac 1020

tgcaaacggg gcagaaagaa actcctgtat atattcaaac aaccatttat gagaccagta 1080

caaactactc aagaggaaga tggctgtagc tgccgatttc cagaagaaga agaaggagga 1140

tgtgaactga gagtgaagtt cagcaggagc gcagacgccc ccgcgtacaa gcagggccag 1200

aaccagctct ataacgagct caatctagga cgaagagagg agtacgatgt tttggacaag 1260

agacgtggcc gggaccctga gatgggggga aagccgagaa ggaagaaccc tcaggaaggc 1320

ctgtacaatg aactgcagaa agataagatg gcggaggcct acagtgagat tgggatgaaa 1380

ggcgagcgcc ggaggggcaa ggggcacgat ggcctttacc agggtctcag tacagccacc 1440

aaggacacct acgacgccct tcacatgcag gccctgcccc ctcgc 1485

<210>66

<211>66

<212>DNA

<213> Artificial sequence

<400>66

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60

atccca 66

<210>67

<211>54

<212>DNA

<213> Artificial sequence

<400>67

ggctccacct ctggatccgg caagcccgga tctggcgagg gatccaccaa gggc 54

<210>68

<211>135

<212>DNA

<213> Artificial sequence

<400>68

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc acagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgat 135

<210>69

<211>72

<212>DNA

<213> Artificial sequence

<400>69

atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60

accctttact gc 72

<210>70

<211>126

<212>DNA

<213> Artificial sequence

<400>70

aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagtacaa 60

actactcaag aggaagatgg ctgtagctgc cgatttccag aagaagaaga aggaggatgt 120

gaactg 126

<210>71

<211>336

<212>DNA

<213> Artificial sequence

<400>71

agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 60

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 180

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 240

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 300

tacgacgccc ttcacatgca ggccctgccc cctcgc 336

<210>72

<211>495

<212>PRT

<213> Artificial sequence

<400>72

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 Asp Ile Val Met Thr Gln Ser Pro Asp Ser

20 25 30

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

35 40 45

Gln Asn Val Leu Tyr Ser Ser Asn Asn Arg Asn Tyr Leu Ala Trp Tyr

50 55 60

Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser

65 70 75 80

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

85 90 95

Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala

100 105 110

Val Tyr Tyr Cys Gln Gln Tyr Tyr Gly Ser Val Val Thr Phe Gly Gly

115 120125

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

130 135 140

Gly Ser Gly Glu Gly Ser Thr Lys Gly Gln Val Gln Leu Gln Gln Ser

145 150 155 160

Gly Pro Gly Leu Val Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys Ala

165 170 175

Ile Ser Gly Asp Ser Val Ser Ser Asn Ser Ala Ala Trp Asn Trp Ile

180 185 190

Arg Gln Ser Pro Ser Arg Gly Leu Glu Trp Leu Gly Arg Thr Tyr Tyr

195 200 205

Arg Ser Lys Trp Tyr Asn Asp Tyr Ala Leu Ser Val Lys Ser Arg Ile

210 215 220

Thr Ile Asn Pro Asp Thr Ser Lys Asn Gln Phe Ser Leu Gln Leu Asn

225 230 235 240

Ser Val Thr Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Ala

245 250 255

Ser Ser Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

260 265 270

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

275 280285

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

290 295 300

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile

305 310 315 320

Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val

325 330 335

Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe

340 345 350

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490 495

<210>73

<211>22

<212>PRT

<213> Artificial sequence

<400>73

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

20

<210>74

<211>18

<212>PRT

<213> Artificial sequence

<400>74

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

1 5 10 15

Lys Gly

<210>75

<211>45

<212>PRT

<213> Artificial sequence

<400>75

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

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

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp

35 40 45

<210>76

<211>24

<212>PRT

<213> Artificial sequence

<400>76

Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu

1 5 10 15

Ser Leu Val Ile Thr Leu Tyr Cys

20

<210>77

<211>42

<212>PRT

<213> Artificial sequence

<400>77

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 GlyCys Glu Leu

35 40

<210>78

<211>112

<212>PRT

<213> Artificial sequence

<400>78

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys 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>79

<211>1497

<212>DNA

<213> Artificial sequence

<400>79

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60

atcccacagc ctgtgctgac tcagccaccc tcggtgtctg aagcccccag gcagagggtc 120

accatctcct gttctggaag cagctccaac atcggaaata atgctgtaaa ctggtaccag 180

cagctcccag gaaaggctcc caaactcctc atctattatg atgatctgct gccctcaggg 240

gtctctgacc gattctctgg ctccaagtct ggcacctcag cctccctggc catcagtggg 300

ctccagtctg aggatgaggc tgattattac tgtgcagcat gggatgacag cctgaatggt 360

tgggtgttcg gcggagggac caagctgacc gtcctaggct ccacctctgg atccggcaag 420

cccggatctg gcgagggatc caccaagggc caggtacagc tgcagcagtc aggtccagga 480

ctggtgaagc cctcgcagac cctctcactc acctgtgcca tctccgggga cagtgtctct 540

agcaacagtg ctgcttggaa ctggatcagg cagtccccat cgagaggcct tgagtggctg 600

ggaaggacat actacaggtc caagtggtat aatgattatg cagtatctgt gaaaagtcga 660

ataaccatca acccagacac atccaagaac cagttctccc tgcagctgaa ctctgtgact 720

cccgaggaca cggctgtgta ttactgtgca aggttagcct acgaagtacg ctccacagac 780

tggtacttcg atctctgggg ccgtggcacc ctggtcaccg tctcgagtac cacgacgcca 840

gcgccgcgac caccaacacc ggcgcccacc atcgcgtcac agcccctgtc cctgcgccca 900

gaggcgtgcc ggccagcggc ggggggcgca gtgcacacga gggggctgga cttcgcctgt 960

gatatctaca tctgggcgcc cttggccggg acttgtgggg tccttctcct gtcactggtt 1020

atcacccttt actgcaaacg gggcagaaag aaactcctgtatatattcaa acaaccattt 1080

atgagaccag tacaaactac tcaagaggaa gatggctgta gctgccgatt tccagaagaa 1140

gaagaaggag gatgtgaact gagagtgaag ttcagcagga gcgcagacgc ccccgcgtac 1200

aagcagggcc agaaccagct ctataacgag ctcaatctag gacgaagaga ggagtacgat 1260

gttttggaca agagacgtgg ccgggaccct gagatggggg gaaagccgag aaggaagaac 1320

cctcaggaag gcctgtacaa tgaactgcag aaagataaga tggcggaggc ctacagtgag 1380

attgggatga aaggcgagcg ccggaggggc aaggggcacg atggccttta ccagggtctc 1440

agtacagcca ccaaggacac ctacgacgcc cttcacatgc aggccctgcc ccctcgc 1497

<210>80

<211>1476

<212>DNA

<213> Artificial sequence

<400>80

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60

atcccacagt ctgccctgac tcagcctgcc tccgtgtctg ggtctcctgg acagtcgatc 120

accatgtcct gcactggaac cagcagtgac gttggtggtt ataagtatgt ctcctggtac 180

caacaacacc caggcaaagc cccccaactc atgattcatg atgtccgtga gcggccctca 240

ggggtttcta atcgcttctc tggctccaag tctggcaaca cggcctccct gaccatctct 300

gggctccagg ctgaggacga ggctgattat tactgcagct catttacaaa caacagcact 360

tttgtcttcg gaactgggac caaagtcacc gtcctaggct ccacctctgg atccggcaag 420

cccggatctg gcgagggatc caccaagggc caggtacagc tgcagcagtc aggtccagga 480

ctggtaaagc cctcgcagac cctctcactc acctgtgcca tctccgggga cagtgtctct 540

agcaacagtg ctgcttggaa ctggatcagg cagtccccat cgagaggcct tgagtggctg 600

ggaaggacat actacaggtc cacgtggtat aatgattatg cagtatctgt gaaaagtcga 660

ataaccatta acccagacac atccaagaac cagttctccc tgcagctgaa ctctgtgact 720

cccgaggaca cggctgtgta ttactgtgca aggggaactg gaacccttga ctactggggc 780

cagggaaccc tggtcaccgt ctcgagtacc acgacgccag cgccgcgacc accaacaccg 840

gcgcccacca tcgcgtcaca gcccctgtcc ctgcgcccag aggcgtgccg gccagcggcg 900

gggggcgcag tgcacacgag ggggctggac ttcgcctgtg atatctacat ctgggcgccc 960

ttggccggga cttgtggggt ccttctcctg tcactggtta tcacccttta ctgcaaacgg 1020

ggcagaaaga aactcctgta tatattcaaa caaccattta tgagaccagt acaaactact 1080

caagaggaag atggctgtag ctgccgattt ccagaagaag aagaaggagg atgtgaactg 1140

agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 1200

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 1260

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 1320

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 1380

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 1440

tacgacgccc ttcacatgca ggccctgccc cctcgc 1476

<210>81

<211>1476

<212>DNA

<213> Artificial sequence

<400>81

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60

atcccacagt ctgccctgac tcagcctgcc tccgtgtctg ggtctcctgg acagtcgatc 120

accatgtcct gcactggaac cagcagtgac gttggtggtt ataagtatgt ctcctggtac 180

caacaacacc caggcaaagc cccccaactc atgattcatg atgtccgtga gcggccctca 240

ggggtttcta atcgcttctc tggctccaag tctggcaaca cggcctccct gaccatctct 300

gggctccagg ctgaggacga ggctgattat tactgcagct catttacaaa caacagcact 360

tttgtcttcg gaactgggac caaagtcacc gtcctaggct ccacctctgg atccggcaag 420

cccggatctg gcgagggatc caccaagggc caggtacagc tgcagcagtc aggtccagga 480

ctggtaaagc cctcgcagac cctctcactc acctgtgcca tctccgggga cagtgtctct 540

agcaacagtg ctgcttggaa ctggatcagg cagtccccat cgagaggcct tgagtggctg 600

ggaaggacat actacaggtc cacgtggtat aatgattatg cagtatctgt gaaaagtcga 660

ataaccatta acccagacac atccaagaac cagttctccc tgcagctgaa ctctgtgact 720

cccgaggaca cggctgtgta ttactgtgca aggggaactg gaacccttga ctactggggc 780

cagggaaccc tggtcaccgt ctcgagtacc acgacgccag cgccgcgacc accaacaccg 840

gcgcccacca tcgcgtcaca gcccctgtcc ctgcgcccag aggcgtgccg gccagcggcg 900

gggggcgcag tgcacacgag ggggctggac ttcgcctgtg atatctacat ctgggcgccc 960

ttggccggga cttgtggggt ccttctcctg tcactggtta tcacccttta ctgcaaacgg 1020

ggcagaaaga aactcctgta tatattcaaa caaccattta tgagaccagt acaaactact 1080

caagaggaag atggctgtag ctgccgattt ccagaagaag aagaaggagg atgtgaactg 1140

agagtgaagt tcagcaggag cgcagacgcc cccgcgtaca agcagggcca gaaccagctc 1200

tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 1260

cgggaccctg agatgggggg aaagccgaga aggaagaacc ctcaggaagg cctgtacaat 1320

gaactgcaga aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc 1380

cggaggggca aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc 1440

tacgacgccc ttcacatgca ggccctgccc cctcgc 1476

<210>82

<211>499

<212>PRT

<213> Artificial sequence

<400>82

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 Gln Pro Val Leu Thr Gln Pro Pro Ser Val

20 25 30

Ser Glu Ala Pro Arg Gln Arg Val Thr Ile Ser Cys Ser Gly Ser Ser

35 40 45

Ser Asn Ile Gly Asn Asn Ala Val Asn Trp Tyr Gln Gln Leu Pro Gly

50 55 60

Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Asp Asp Leu Leu Pro Ser Gly

65 70 75 80

Val Ser Asp Arg Phe Ser Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu

85 90 95

Ala Ile Ser Gly Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala

100 105 110

Ala Trp Asp Asp Ser Leu Asn Gly Trp Val Phe Gly Gly Gly Thr Lys

115 120 125

Leu Thr Val Leu Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly

130 135 140

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

145 150 155 160

Leu Val Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly

165 170 175

Asp Ser Val Ser Ser Asn Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser

180 185 190

Pro Ser Arg Gly Leu Glu Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Lys

195 200 205

Trp Tyr Asn Asp Tyr Ala Val Ser Val Lys Ser Arg Ile Thr Ile Asn

210 215 220

Pro Asp Thr Ser Lys Asn Gln Phe Ser Leu Gln Leu Asn Ser Val Thr

225 230 235 240

Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Leu Ala Tyr Glu Val

245 250 255

Arg Ser Thr Asp Trp Tyr Phe Asp Leu Trp Gly Arg Gly Thr Leu Val

260 265 270

Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala

275 280 285

Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg

290 295 300

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

305 310 315 320

Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu

325 330 335

Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu

340 345 350

Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln

355 360 365

Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly

370 375 380

Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys

450 455 460

Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu

465 470 475 480

Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu

485 490 495

Pro Pro Arg

<210>83

<211>492

<212>PRT

<213> Artificial sequence

<400>83

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 Gln Ser Ala Leu Thr Gln Pro Ala Ser Val

20 2530

Ser Gly Ser Pro Gly Gln Ser Ile Thr Met Ser Cys Thr Gly Thr Ser

35 40 45

Ser Asp Val Gly Gly Tyr Lys Tyr Val Ser Trp Tyr Gln Gln His Pro

50 55 60

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

65 70 75 80

Gly Val Ser Asn Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser

85 90 95

Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys

100 105 110

Ser Ser Phe Thr Asn Asn Ser Thr Phe Val Phe Gly Thr Gly Thr Lys

115 120 125

Val Thr Val Leu Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly

130 135 140

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

145 150 155 160

Leu Val Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly

165 170 175

Asp Ser Val Ser Ser Asn Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser

180 185190

Pro Ser Arg Gly Leu Glu Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Thr

195 200 205

Trp Tyr Asn Asp Tyr Ala Val Ser Val Lys Ser Arg Ile Thr Ile Asn

210 215 220

Pro Asp Thr Ser Lys Asn Gln Phe Ser Leu Gln Leu Asn Ser Val Thr

225 230 235 240

Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Thr Gly Thr Leu

245 250 255

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr

260 265 270

Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro

275 280 285

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

290 295 300

His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro

305 310 315 320

Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

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

485 490

<210>84

<211>492

<212>PRT

<213> Artificial sequence

<400>84

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 Gln Ser Ala Leu Thr Gln Pro Ala Ser Val

20 25 30

Ser Gly Ser Pro Gly Gln Ser Ile Thr Met Ser Cys Thr Gly Thr Ser

35 40 45

Ser Asp Val Gly Gly Tyr Lys Tyr Val Ser Trp Tyr Gln Gln His Pro

50 55 60

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

65 70 75 80

Gly Val Ser Asn Arg Phe Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser

85 90 95

Leu Thr Ile Ser Gly Leu Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys

100 105 110

Ser Ser Phe Thr Asn Asn Ser Thr Phe Val Phe Gly Thr Gly Thr Lys

115 120 125

Val Thr Val Leu Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly

130 135 140

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

145 150155 160

Leu Val Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys Ala Ile Ser Gly

165 170 175

Asp Ser Val Ser Ser Asn Ser Ala Ala Trp Asn Trp Ile Arg Gln Ser

180 185 190

Pro Ser Arg Gly Leu Glu Trp Leu Gly Arg Thr Tyr Tyr Arg Ser Thr

195 200 205

Trp Tyr Asn Asp Tyr Ala Val Ser Val Lys Ser Arg Ile Thr Ile Asn

210 215 220

Pro Asp Thr Ser Lys Asn Gln Phe Ser Leu Gln Leu Asn Ser Val Thr

225 230 235 240

Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Thr Gly Thr Leu

245 250 255

Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr

260 265 270

Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro

275 280 285

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

290 295 300

His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro

305 310315 320

Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475480

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

485 490

Claims (10)

1. A BCMA antibody is characterized in that the amino acid sequences of CDR1, CDR2 and CDR3 in a heavy chain variable region are respectively shown as SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, and the amino acid sequences of CDR1, CDR2 and CDR3 in a light chain variable region are respectively shown as SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO. 16;

or the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region of the BCMA antibody are respectively shown in SEQ ID NO.38, SEQ ID NO.39 and SEQ ID NO.40, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region of the BCMA antibody are respectively shown in SEQ ID NO.46, SEQ ID NO.47 and SEQ ID NO. 48.

2. The BCMA antibody according to claim 1, wherein the amino acid sequence of the heavy chain variable region of the BCMA antibody is shown as SEQ ID No.5, and the amino acid sequence of the light chain variable region of the BCMA antibody is shown as SEQ ID No. 13;

or the amino acid sequence of the heavy chain variable region of the BCMA antibody is shown in SEQ ID NO.37, and the amino acid sequence of the light chain variable region of the BCMA antibody is shown in SEQ ID NO. 45.

3. The BCMA antibody according to claim 1 or 2, characterized in that said BCMA antibody is one of a full length antibody, F (ab ') 2, Fab', Fab, Fv and scFv.

4. A chimeric antigen receptor targeting BCMA protein comprising the heavy chain variable region and the light chain variable region of the BCMA antibody according to any one of claims 1 to 3.

5. The chimeric antigen receptor according to claim 4, further comprising one or a combination of the following elements:

a signal peptide, a linker, a hinge region, a CD8 a transmembrane domain, a 4-1BB costimulatory signaling region, and a CD3 zeta signaling domain.

6. An isolated nucleic acid molecule encoding the BCMA antibody according to any one of claims 1 to 3, or encoding the chimeric antigen receptor according to claim 4 or 5.

7. A vector comprising the nucleic acid molecule of claim 6.

8. A recombinant cell comprising a nucleic acid molecule encoding the chimeric antigen receptor of claim 4 or 5, or the vector of claim 7.

9. A chimeric antigen receptor T cell targeting BCMA protein, which expresses the chimeric antigen receptor of claim 4 or 5.

10. A medicament for treating tumors, which comprises the chimeric antigen receptor T cell according to claim 9.

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