CN109265550B - BCMA Antibodies, Chimeric Antigen Receptors and Drugs - Google Patents

Abstract

The invention discloses a BCMA antibody, a chimeric antigen receptor and a medicine, and relates to the technical field of cellular immunotherapy. The BCMA antibody provided by the present invention has the heavy chain CDR region shown in SEQ ID NO.6-8 and the light chain CDR region shown in SEQ ID NO.14-16, or, has SEQ ID NO.38- The heavy chain CDR region shown in 40 and the light chain CDR region shown in SEQ ID NO.46-48; the ability of the described BCMA antibody to specifically bind to the BCMA protein, the chimera prepared using the described BCMA antibody The antigen receptor T cells have specific killing effect on BCMA protein-positive target cells, and can be used to prepare medicines 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, in particular to a BCMA antibody, a chimeric antigen receptor and a drug.

Background technique

Multiple myeloma (MM) is a malignant tumor characterized by massive proliferation of clonal plasma cells. Current MM therapy induces remission, but almost all patients still eventually relapse and die. Although some monoclonal antibodies have shown promise in the treatment of MM in preclinical studies and early clinical trials, they have not been consistent. Clearly, new immunotherapies for MM are clearly much needed, and the development of effective antigen-specific adoptive T-cell therapy for the disease would be a major advance. T cells can be genetically modified to express a chimeric antigen receptor (CAR), which includes a fusion protein of an antigen recognition moiety and a T cell activation domain. For B-lineage malignancies, the adoptive T-cell approach with anti-CD19 CAR is most commonly used. Anti-CD19-CAR-transduced T cells cured leukemia and lymphoma in mice, and some patients also achieved remission in early clinical trials of adoptively infused anti-CD19-CAR-transduced T cells, but at the same time, anti-CD19-CAR-transduced T cells CD19CAR-transduced T cells also deplete normal B cells, and unfortunately CD19 is rarely expressed in malignant plasma cells in MM, so treating MM with CAR-expressing T cells will require finding other better targets.

One candidate antigen for immunotherapy of MM is B Cell Maturation Antigen (BCMA, CD269). BCMA RNA is ubiquitously detected in MM cells, and BCMA protein can be detected on the surface of plasma cells from patients with multiple myeloma. 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 in normal cells, BCMA is mainly expressed by plasma cells and a subset of mature B cells, but not expressed on most B cells and other organs. The BCMA-deficient mice looked normal, seemed healthy, and had normal numbers of B cells, but the plasma cells did not survive long-term. Therefore, 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 against BCMA.

In view of this, the present invention is proposed.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a BCMA antibody, which can specifically bind to BCMA protein and can be used to prepare chimeric antigen receptor T cells targeting 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 BCMA-positive target cells.

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 to treat BCMA-positive tumors.

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

Another object of the present invention is to provide a carrier.

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

Another object of the present invention is to provide a medicament for treating tumors, which can be used for treating or preventing BCMA-positive tumors.

The present invention is realized in this way:

In the present invention, human BCMA (hBCMA) protein is used as an antigen, combined with phage library display technology, and four scFV antibodies that have not been reported in the prior art are obtained by panning. Experiments have verified that the four scFV antibodies have the ability to bind BCMA protein, and the chimeric antigen receptor T cells prepared by using the scFV antibodies have specific killing effect on target cells that are positive for BCMA protein. Therefore, the four scFV antibodies can be used to generate chimeric antigen receptor T cells targeting BCMA protein and to generate BCMA-positive tumors.

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

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

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

Alternatively, 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.

The antibody with the above-mentioned CDR sequence can specifically bind to BCMA protein, which is used to prepare chimeric antigen receptor T cells targeting BCMA protein.

In addition, using the BCMA antibody provided by the present invention to prepare a detection reagent for detecting BCMA protein, or adding a label to the antibody provided by the present invention for detecting BCMA protein, belong to the protection scope of the present invention.

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

On the basis of the variable region of the heavy chain and the variable region of the light chain of the BCMA antibody provided by the present invention, it is easy for those skilled in the art to construct a full-length antibody, F(ab')2, Fab that can bind to the BCMA protein. ', Fab, Fv and scFv of any type of antibody; no matter what type of antibody, as long as it contains the above-mentioned heavy chain CDR sequence and/or light chain CDR sequence, it belongs to the protection scope of the present invention.

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

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

Signal peptide, linker, hinge region, CD8α transmembrane domain, 4-1BB costimulatory signaling region and CD3ζ signaling domain.

Further, in some embodiments of the present invention, the amino acid sequence of the above 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 above hinge region is shown in SEQ ID NO.75.

Further, in some embodiments of the present invention, the amino acid sequence of the CD8α transmembrane domain is shown in SEQ ID NO.76.

Further, in some embodiments of the present invention, the amino acid sequence of the above-mentioned 4-1BB costimulatory signaling region is shown in SEQ ID NO.77.

Further, in some embodiments of the present invention, the amino acid sequence of the above-mentioned CD3ζ signaling domain is shown in SEQ ID NO.78.

It should be noted that, in other embodiments, those skilled in the art can change the signal peptide, linker, hinge region, CD8α transmembrane domain, 4-1BB costimulatory signaling region and CD3ζ signaling structure according to actual conditions or needs The combination type and sequence of the domains, regardless of the form of change, as 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-mentioned antibody of the present invention, and/or the above-mentioned antibody. The CDR sequences of the heavy chain variable region or the heavy chain variable region sequences belong to the protection scope of the present invention.

Further, in some embodiments of the present invention, the signal peptide, the light chain variable region of the above-mentioned antibody, the linker, the heavy chain variable region of the above-mentioned antibody, the hinge region, the CD8α transmembrane domain, the 4-1BB costimulatory signaling The chimeric antigen receptor is composed of the chimeric antigen receptor in sequential tandem with the CD3ζ signaling domain.

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

In another aspect, the present invention provides an isolated nucleic acid molecule encoding the above-mentioned antibody, or which encodes the above-mentioned chimeric antigen receptor.

In another aspect, the present invention provides a vector containing the above-mentioned nucleic acid molecule.

In another aspect, the present invention provides a recombinant cell containing the nucleic acid molecule encoding the above-mentioned chimeric antigen receptor, or the above-mentioned vector.

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

The T cells can specifically kill BCMA-positive target cells and can be used to treat BCMA-positive tumors.

In another aspect, the present invention provides a medicament for treating tumors, which contains the above-mentioned chimeric antigen receptor T cells.

Further, in some embodiments of the present invention, the above-mentioned tumor is a BCMA-positive tumor.

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

This drug can be used to treat or prevent BCMA-positive tumors.

Further, in some embodiments of the present invention, the above-mentioned drugs also contain pharmaceutically acceptable excipients.

Description of drawings

In order to illustrate the technical solutions of the embodiments of the present invention more clearly, the following briefly introduces the accompanying drawings used in the embodiments. It should be understood that the following drawings only show some embodiments of the present invention, and therefore do not It should be regarded as a limitation of the scope, and for those of ordinary skill in the art, other related drawings can also be obtained according to these drawings without any creative effort.

Figure 1 shows the detection results of the binding force of BCMA antibody 2077/2082/2073/2079 to human BCMA protein.

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

Figure 3 shows the positive rate of CAR-BCMA after 72Hrs infection of CD4+CD8+ T cells by flow cytometry.

Figure 4A shows the change of target cell ratio with time when T cells expressing CAR-BCMA (CAR-BCMA-2073-T, CAR-BCMA-2079-T) were co-cultured with target cells H929 when the effector-target ratio was 5/1. Flow chart.

Figure 4B shows the change of the ratio of target cells with time when T cells expressing CAR-BCMA (CAR-BCMA-2077-T, CAR-BCMA-2082-T) were co-cultured with target cells H929 when the effector-target ratio was 5/1. Flow chart.

Figure 5 shows T cells expressing CAR-BCMA (CAR-BCMA-2073-T, CAR-BCMA-2077-T, CAR-BCMA-2079-The CAR-BCMA-2082-T) when the effector-target ratio is 5/1. ) co-cultured with target cells H929, the statistical results of the proportion of target cells over time.

Figure 6A shows that T cells expressing CAR-BCMA (CAR-BCMA-2073-T, CAR-BCMA-2079-T) were co-cultured with target cells K562-BCMA cells when the effector-target ratio was 7/1. Flow chart of time change.

Figure 6B shows the co-culture of CAR-BCMA-expressing T cells (CAR-BCMA-2077-T, CAR-BCMA-2082-T) and target cells K562-BCMA cells when the effector-target ratio is 7/1. Flow chart of time change.

Figure 7. Statistical results of the ratio of target cells over time when T cells expressing CAR-BCMA were co-cultured with target cells K562-BCMA cells when the effector-target ratio was 7/1.

Figure 8 shows the detection of cytokine release of T cells expressing targeting BCMA chimeric antigen receptors after being stimulated and activated by BCMA-positive cells.

Figure 9 shows the treatment of BCMA-positive tumors by T cells expressing targeting BCMA chimeric antigen receptor in a mouse myeloma transplantation model.

Detailed ways

In order to make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not indicated in the examples, it is carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used without the manufacturer's indication are conventional products that can be purchased from the market.

The features and performances of the present invention will be further described in detail below in conjunction with the embodiments.

Example 1

1 Phage library panning

M-280Streptavidin磁珠，室温孵育30min。Biotinylated hBCMA protein was used as panning antigen for a fully human antibody library. First, the phage antibody was blocked with blocking solution (PBST/5% nonfat dry milk) at room temperature for 2 hours. The input amount of phage was 2×10 ¹² phage, then 10 μg of antigen was added, and incubated at room temperature for 1 hour. After incubation, 50 μl of pre-blocked

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

The unbound phages were first washed with PBST, then the phages bound to the magnetic beads were eluted with 0.1M HCl-Glycine, and then the eluate was neutralized with Tris-HCl, and some phages were taken to infect the large intestine in the logarithmic growth phase. Bacillus TG1, the collected phages were used for the next round of panning.

The screening intensity of each round was gradually increased, and the panning was terminated when the enrichment reached more than 100 times.

2 Use phage Elisa to screen positive clones of anti-BCMA single chain antibody

(1) The phage-infected TG1 monoclones after four rounds of panning were selected and inoculated in a 96-well plate, and the medium was 2YT (containing 2% glucose, 100 μg/ml Ampicilline).

(2) After overnight culture at 37°C and 250 rpm, the cells were transferred to a new medium, cultured to the logarithmic growth phase, and then M13K07 helper phage was added, and the cells were infected at 37°C for 1 h.

(3) Centrifuge at 4000 rpm for 15 min, use 2YT (containing 100 μg/ml Ampicilline, 70 μg/ml Kanamycin) medium to culture overnight at 30°C, and centrifuge to collect the phage supernatant for ELISA to identify clones.

(4) Costar-9018 ELISA plate was coated with 0.5 μg/ml hBCMA antigen, blocked with 3% BSA at 4°C overnight, added to the collected phage supernatant, and incubated at 4°C for 2 h.

(5) After washing off unbound phage, add Ml3 Bacteriophage antibody (HRP), and incubate at 4°C for 1 h. After washing, TMB chromogenic solution was added to develop color, and the reaction was terminated with 2M HCl.

(6) Use a microplate reader to read at 450 nm, select clones with an OD ₄₅₀ >1.5 for sequencing, and perform Germline analysis and PTMs site analysis on the sequences. After excluding molecules with potential development risks, a total of 4 hBCMA-binding proteins were obtained. scFv single chain antibody (hereinafter may also be referred to as BCMA antibody).

The four scFv single chain antibodies are:

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

Its heavy chain variable region amino acid sequence is: SEQ ID NO.5, and 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: SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, and 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: SEQ ID NO.13, and the corresponding nucleotide coding sequence is: SEQ ID NO.9;

The VL-CDR1, VL-CDR2 and VL-CDR3 amino acid sequences of the light chain variable region are respectively: SEQ ID NO.14, SEQID NO.15, SEQ ID NO.16, and 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 as antibody 2077)

Its heavy chain variable region amino acid sequence is: SEQ ID NO.21, and the corresponding nucleotide coding sequence is: SEQ ID NO.17;

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

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

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

Anti-BCMA single chain antibody 3 (designated as antibody 2079)

The amino acid sequences of the heavy chain variable region are respectively: SEQ ID NO.37, and the corresponding nucleotide coding sequence is: SEQID NO.33;

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

The amino acid sequence of the light chain variable region is: SEQ ID NO.45, and the corresponding nucleotide coding sequence is: SEQ ID NO.41;

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

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

Its heavy chain variable region amino acid sequence is: SEQ ID NO.53, and its corresponding nucleotide sequence is: SEQ ID NO.49;

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

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

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

Example 2

Detect the binding ability of the four BCMA antibodies obtained in Example 1, namely 2073, 2077, 2079 or 2082, to hBCMA protein

Detection method:

(1) hBCMA protein coating, diluted from 1μg/ml, 3-fold gradient dilution, a total of 8 gradients, the concentrations are 1μg/ml, 333ng/ml, 111ng/ml, 37ng/ml, 12.3ng/ml, 4.1 ng/ml, 1.37ng/ml and 0.45ng/ml, respectively, were coated with 100 μl hBCMA protein dilution solution on Costar-9018 microtiter plate, overnight at 4°C.

(2) After blocking with 3% BSA at room temperature for 2 hours, add BCMA antibody, ie, the phage supernatant (10^10 pfu) corresponding to 2073, 2077, 2079 or 2082, and incubate at room temperature for 2 hours.

(3) After washing off unbound phage, add Ml3 Bacteriophage antibody (HRP), and incubate at 4°C for 1 h. After washing, TMB chromogenic solution was added to develop color, and the reaction was terminated with 2M HCl.

(4) Read at 450 nm with a microplate reader, and the results are shown in Figure 1.

It can be seen from the results in Figure 1 that the phage expressing BCMA antibody 2073, 2077, 2079 or 2082 has good binding ability to hBCMA protein, showing an S curve, showing a dose-effect relationship, indicating that the four BCMAs obtained in Example 1 The antibodies, ie, 2073, 2077, 2079 and 2082, all have the ability to bind to hBCMA protein.

Example 3

Construction of Chimeric Antigen Receptor Expression Vector

Build method:

(1) Whole gene synthesis: signal peptide (nucleotide 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 (nucleic acid sequence SEQ ID NO. 74) ID NO.68, amino acid sequence SEQ ID NO.75), CD8α 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.76) sequence SEQ ID NO. 70, amino acid sequence SEQ ID NO. 77) and CD3ζ signaling domain (nucleic acid sequence SEQ ID NO. 71, amino acid sequence SEQ ID NO. 78). The above sequences are connected in sequence. 4 kinds of chimeric antigen receptor expression cassettes were obtained respectively, named respectively: 2073 chimeric antigen receptor expression cassette (full-length nucleotide sequence as shown in SEQ ID NO.65, amino acid sequence as shown in SEQ ID NO.:72) shown), 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 nuclear The nucleotide sequence is shown in SEQ ID NO.80, the amino acid sequence is shown in SEQ ID NO.83) and the 2082 chimeric antigen receptor expression cassette (the full-length nucleotide sequence is shown in SEQ ID NO.81, the amino acid sequence is shown in SEQ ID NO.81) as shown in SEQ ID NO. 84).

The Kozac sequence was introduced at the front end of each expression cassette, and the structure of the expression cassette was shown in Figure 2.

(2) After the sequence of the chimeric antigen receptor expression cassette was synthesized by the whole gene, it was connected to the empty vector pCDH-EF1-MSC-T2A-copGFP through the XbaI/EcoRI restriction site to obtain the chimeric antigen receptor expression vector; The 4 chimeric antigen receptor expression vectors were named after being sequenced and verified to be correct:

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

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

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

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

Example 4

Preparation of strains containing chimeric antigen receptor expression vector

method:

(1) Take out the DH5α competent cells in a -80°C refrigerator and thaw on ice.

(2) Add 5ng of plasmid to the competent cells, mix gently, and place on ice for 5 minutes.

The plasmids are: 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.

(3) Heat shock at 42°C for 90 seconds and on ice for 30 minutes.

(4) Add 0.5 ml of non-resistant LB and incubate for 30 minutes at 37° C. and 180 rpm.

(5) Spread onto ampicillin-resistant plates.

(6) Invert overnight culture at 37°C.

(7) Pick a single clone and culture it in ampicillin-resistant LB at 37° C. and 200 rpm for 9-12 hours.

(8) Add glycerol to the bacterial solution, the final concentration of glycerol is 10%, and the bacteria are stored in a refrigerator at -80°C for later use, which can be used for subsequent large-scale extraction of plasmids.

(9) After culturing the above strains in LB in large quantities, use a plasmid extraction kit (Beijing Tiangen Biochemical Technology Co., Ltd. endotoxin-free plasmid extraction kit) to extract plasmids for use in infection. The plasmid extraction method can be carried out according to the instructions.

Example 5

virus packaging

Cells were transfected by PEI method. 24 hours before transfection, 293T cells were trypsinized, 4 × 10 ⁶ 293T cells were plated in a 10cm cell culture dish, and the cells were cultured in DMEM medium containing 10% FBS in a 37°C 5% CO2 incubator without Over 24 hours, cells can be transfected when they reach 60-80% density.

Specific steps are as follows:

(1) Plasmid, PEI, DMEM medium were placed at room temperature for 5min;

(2) Take 450 μl of DMEM into a 1.5 ml EP tube, add 50 μl of PEI (1 μg/μl) and mix, and let stand for 5 minutes at room temperature;

(3) Take 10 μg of 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), 10μg psPAX2, 5μg pMD2.G, add DMEM to 500μl, mix well, let stand at room temperature for 5min;

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

(5) Slowly drop 1 ml of DNA/PEI mixture into a 293T petri dish, mix gently, and incubate at 37°C for 6-8 hours;

(6) Discard the original medium, replace with fresh medium, and put it into a 37°C incubator to continue incubation;

(7) After 48 hours of replacing the medium, collect the medium, then add 10ml of fresh medium to each dish to continue culturing, collect the supernatant again after 24 hours, and mix with the supernatant collected in 48 hours;

(8) Centrifuge at 4000g for 10min at 4°C to remove cell debris;

(9) the supernatant obtained by filtration with a 0.45 μm filter;

(10) the filtered supernatant is subjected to tangential flow filtration;

(11) Transfer the virus supernatant after tangential flow filtration into an ultracentrifuge tube, centrifuge at 25,000 rpm for 2 hours, resuspend the virus precipitate obtained after ultracentrifugation with serum-free medium, and gently pipette until it is completely dissolved to obtain the virus The virus liquids obtained by using different vectors are named respectively:

2073 virus fluid (including 2073 chimeric antigen receptor expression cassette), 2077 virus fluid (including 2077 chimeric antigen receptor expression cassette), 2079 virus fluid (including 2079 chimeric antigen receptor expression cassette), 2082 virus fluid (including 2079 chimeric antigen receptor expression cassette) 2080 chimeric antigen receptor expression cassette), empty vector virus liquid;

(12) Distribute each virus solution, store it in a -80°C refrigerator, and reserve 5-10 μl of virus concentrate for titer determination.

Example 6

Virus titer determination

method:

293T cells were digested and counted, and a cell suspension was prepared in DMEM medium containing 10% FBS. The cell density was adjusted to 4×10 ⁵ /ml, and 0.5 ml of the cell suspension was added to each well of a 24-well culture plate. After 8 hours of cell adherent culture, 1μl, 10μl, 20μl, 30μl, 50μl of virus solution diluted 100 times was infected, and the medium was changed after 24 hours, and the positive rate of 293T cells was detected by flow cytometry after 48 hours.

Centrifuge, resuspend and adjust the cell density to 1×10 ⁶ /ml, add biotin-BCMA antigen to 50 μl, the final concentration is 1 μg/ml, after incubation for 30 min, wash once with DPBS, resuspend, and stain with secondary antibody APC-Streptavidin (purchased). 30min from BD), washed once, resuspended in DPBS, and detected by flow cytometry.

Example 7

Preparation of chimeric antigen receptor T cells targeting human BCMA antigen

1 Isolation of PBMC from human peripheral blood mononuclear cells

Use an anticoagulant tube (purchased from BD) to collect about 25 ml of peripheral blood, add it to the lymphocyte separation solution according to the volume ratio of 1:1, and centrifuge the gradient for 25 minutes. Peripheral blood mononuclear cells PBMC.

2CD4+CD8+T cell enrichment and activation

Resuspend PBMC, adjust the density to 1×10 ⁵ /μl, add 10 μl of CD4/CD8 magnetic beads to 50 μl of cell suspension, and obtain CD4+CD8+ T cells by magnetic pole separation.

The obtained 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 Miltenyi, 10 μl/ml). The concentration of IL-2 was 200IU/ml. After 24 hours of activation, the medium was changed, and the culture was continued with a complete medium containing 200 IU/ml of IL-2.

3 Lentiviral infection

Adjust the cell density of T cells to 1×10 ⁶ /ml, according to MOI=10, with the virus liquid (2073 virus liquid, 2077 virus liquid, 2079 virus liquid or 2082 virus liquid) obtained in Example 6 after infection and activation for 48 hours. For T cells, the medium was changed after 24 hours, and IL-2 200IU/ml was added continuously. Different virus solutions were used to obtain different T cells that could express chimeric antigen receptors targeting human BCMA antigen; they were named CAR-BCMA respectively. -2073-T, CAR-BCMA-2077-T, CAR-BCMA-2079-T, CAR-BCMA-2082-T.

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

During the culture, the T cells 72 hours after virus infection were taken, CAR-BCMA-2073-T, CAR-BCMA-2077-T, CAR-BCMA-2079-T, CAR-BCMA-2082-T), centrifuged, resuspended Suspend and adjust the cell density to 1×10 ⁶ /ml, add biotin-BCMA antigen to 50 μl, the final concentration is 0.2 μg/ml, after incubation for 30 min, wash once with DPBS, resuspend, stain with secondary antibody APC-Streptavidin (purchased from BD) ) 30min, washed once and resuspended in DPBS, and the positive rate of CAR-BCMA was detected by flow cytometry.

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

Example 8

The tumor-killing effect of T cells targeting the chimeric antigen receptor of human BCMA antigen was detected by co-culture in vitro, and the target cell was NCI-H929.

Take 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 at 72 hours after infection, and count them , adjust the cell density to 1*10 ⁶ /ml, and co-culture according to the effect-target ratio of 5:1, namely: T cells 1*10 ⁶ , NCI-H929 2×10 ⁵ , control cells are CD4+ without virus infection CD8+T cells, denoted as Ctrl-T cells. The proportion of NCI-H929 cells in total cells was detected at 0H, 24H, and 48H respectively, and the target cells were labeled with the antibody APC-conjugated Human BCMA/TNFRSF17 Antibody, and the results were shown in Figure 4A and Figure 4B; The results are shown in Figure 5.

The results showed that in the presence of CAR-BCMA-2073-T, CAR-BCMA-2077-T, CAR-BCMA-2079-T or CAR-BCMA-2082-T, the target cells NCI- The proportion of H929 was significantly reduced; CAR-BCMA-2073-T: decreased from 15.4% to 0.435%; CAR-BCMA-2079-T: decreased from 11.3% to 6.63%; CAR-BCMA-2077-T: decreased from 19.6% to 0.628%; CAR-BCMA-2082-T: from 18.5% to 0.185%. This shows that the four chimeric antigen receptor T cells CAR-BCMA-2073-T, CAR-BCMA-2077-T, CAR-BCMA-2079-T and CAR-BCMA-2082-T can kill well Target cell NCI-H929.

Example 9

The tumor-killing effect of CAR-BCMAT was detected by co-culture in vitro, and the target cell was K562-BCMA.

T cells (CAR-BCMA-2073-T, CAR-BCMA-2077-T, CAR-BCMA-2079-T or CAR-BCMA-2082-T) and BCMA-overexpressing cells K562-BCMA were taken 72 hours after infection , counted, adjusted the cell density to 1×10 ⁶ /ml, and co-cultured according to the effect-target ratio of 7:1, namely: T cells 1×10 ⁶ , K562-BCMA 1.5×10 ⁵ , control cells were not treated with virus liquid CD4+CD8+T cells, denoted as Ctrl-T cells. The proportion of K562-BCMA cells in total cells was detected at 0H, 24H, and 48H, respectively, and the target cells were labeled with the antibody APC-conjugated Human BCMA/TNFRSF17 Antibody. The results are shown in Figure 6A and 6B, and the cell killing effect was calculated. The results are shown in Figure 6 7.

The results showed that in the presence of CAR-BCMA-2073-T, CAR-BCMA-2077-T, CAR-BCMA-2079-T or CAR-BCMA-2082-T, the target cells K562- The proportion of BCMA was significantly reduced. In the time period of 0-48h, the proportion of target cells K562-BCMA decreased as follows: CAR-BCMA-2073-T: from 8.69% to 6.1%; CAR-BCMA-2079-T : from 9.28% to 8.69%; CAR-BCMA-2077-T: from 8.95% to 1.03%; CAR-BCMA-2082-T: from 11.2% to 0.676%; thus, the 4 chimeric antigens Recipient T cells CAR-BCMA-2073-T, CAR-BCMA-2077-T, CAR-BCMA-2079-T and CAR-BCMA-2082-T can kill BCMAT-positive target cells K562-BCMA well .

Example 10

Detection of Cytokine Expression Levels by ELISA

method:

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, counted, adjusted the cell density to 1×10 ⁶ /ml, and co-cultured according to the effect-target ratio (E:T) of 1:1, that is, T cells 1×10 ⁶ , NCI-H929 1×10 ⁶ , control cells were untreated CD4+CD8+T cells treated with virus infection were denoted as Ctrl-T cells. The supernatant was collected at 24H and stored at -80°C for later use.

Detection: Cytokines INF-γ and TNF-α were detected by CBA method, according to the instructions. The results are shown in Figure 8.

As can be seen from the figure, the INF- The contents of γ and TNF-α were higher than the control (Ctrl-T).

Example 11

NSG mice were used, and the tumor cells used were the BCMA-positive cell line MM.1S (denoted as NCI-MM.1S-LUC) stably expressing firefly luciferase. The therapeutically injected effector cells contained CAR-BCMA T cells (CAR-BCMA-2077-T or CAR-BCMA-2082-T), and the control group was the saline group and the virus-uninfected CD4+CD8+ T cell group. 3 days after the successful modeling, effector cells were injected into the tail vein of 5×10 ⁶ / mouse, and images were taken by the IVS in vivo imaging system every 7 days after the injection to display the tumor growth, weigh the weight of the mice, and observe the survival of the mice. situation and record. The results are shown in Figure 9.

It can be seen from Figure 9 that the fluorescence area of mice injected with CAR-BCMA-2077-T or CAR-BCMA-2082-T was significantly reduced on days 3 and 7, indicating that tumor cells were significantly reduced and the surface , CAR-BCMA-2077-T or CAR-BCMA-2082-T expressing chimeric antigen receptor T cells targeting BCMA can target BCMA, effectively kill BCMA-expressing tumor cells, and achieve the purpose of treating tumors.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

SEQUENCE LISTING

<110> East China Normal University, Shanghai Bangyao 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 sequences

<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 sequences

<400> 2

agcaacagtg ctgcttggaa c 21

<210> 3

<211> 54

<212> DNA

<213> Artificial sequences

<400> 3

aggacatact acaggtccaa gtggtataat gattatgcat tatctgtgaa aagt 54

<210> 4

<211> 21

<212> DNA

<213> Artificial sequences

<400> 4

ggcgccagct cgtttgacta c 21

<210> 5

<211> 119

<212> PRT

<213> Artificial sequences

<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 sequences

<400> 6

Ser Asn Ser Ala Ala Trp Asn

1 5

<210> 7

<211> 18

<212> PRT

<213> Artificial sequences

<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 sequences

<400> 8

Gly Ala Ser Ser Phe Asp Tyr

1 5

<210> 9

<211> 339

<212> DNA

<213> Artificial sequences

<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 sequences

<400> 10

aagtccagcc agaatgtttt atacagctcc aacaatagga actacttagc t 51

<210> 11

<211> 21

<212> DNA

<213> Artificial sequences

<400> 11

tgggcatcta cccgggagtc c 21

<210> 12

<211> 27

<212> DNA

<213> Artificial sequences

<400> 12

cagcaatatt atggttctgt tgtcact 27

<210> 13

<211> 113

<212> PRT

<213> Artificial sequences

<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 sequences

<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 sequences

<400> 15

Trp Ala Ser Thr Arg Glu Ser

1 5

<210> 16

<211> 9

<212> PRT

<213> Artificial sequences

<400> 16

Gln Gln Tyr Tyr Gly Ser Val Val Thr

1 5

<210> 17

<211> 378

<212> DNA

<213> Artificial sequences

<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 sequences

<400> 18

agcaacagtg ctgcttggaa c 21

<210> 19

<211> 54

<212> DNA

<213> Artificial sequences

<400> 19

aggacatact acaggtccaa gtggtataat gattatgcag tatctgtgaa aagt 54

<210> 20

<211> 42

<212> DNA

<213> Artificial sequences

<400> 20

ttagcctacg aagtacgctc cacagactgg tacttcgatc tc 42

<210> 21

<211> 126

<212> PRT

<213> Artificial sequences

<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 sequences

<400> 22

Ser Asn Ser Ala Ala Trp Asn

1 5

<210> 23

<211> 18

<212> PRT

<213> Artificial sequences

<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 sequences

<400> 24

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

1 5 10

<210> 25

<211> 330

<212> DNA

<213> Artificial sequences

<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 sequences

<400> 26

tctggaagca gctccaacat cggaaataat gctgtaaac 39

<210> 27

<211> 21

<212> DNA

<213> Artificial sequences

<400> 27

tatgatgatc tgctgccctc a 21

<210> 28

<211> 33

<212> DNA

<213> Artificial sequences

<400> 28

gcagcatggg atgacagcct gaatggttgg gtg 33

<210> 29

<211> 110

<212> PRT

<213> Artificial sequences

<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 sequences

<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 sequences

<400> 31

Tyr Asp Asp Leu Leu Pro Ser

1 5

<210> 32

<211> 21

<212> PRT

<213> Artificial sequences

<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 sequences

<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 sequences

<400> 34

agcaacagtg ctgcttggaa c 21

<210> 35

<211> 54

<212> DNA

<213> Artificial sequences

<400> 35

aggacatact acaggtccac gtggtataat gattatgcag tatctgtgaa aagt 54

<210> 36

<211> 24

<212> DNA

<213> Artificial sequences

<400> 36

aggggaactg gaacccttga ctac 24

<210> 37

<211> 119

<212> PRT

<213> Artificial sequences

<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 Pro Ser 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 sequences

<400> 38

Ser Asn Ser Ala Ala Trp Asn

1 5

<210> 39

<211> 18

<212> PRT

<213> Artificial sequences

<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 sequences

<400> 40

Arg Gly Thr Gly Thr Leu Asp Tyr

1 5

<210> 41

<211> 330

<212> DNA

<213> Artificial sequences

<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 sequences

<400> 42

actggaacca gcagtgacgt tggtggttat aagtatgtct cc 42

<210> 43

<211> 21

<212> DNA

<213> Artificial sequences

<400> 43

gatgtccgtg agcggccctc a 21

<210> 44

<211> 30

<212> DNA

<213> Artificial sequences

<400> 44

agctcattta caaacaacag cacttttgtc 30

<210> 45

<211> 110

<212> PRT

<213> Artificial sequences

<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 sequences

<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 sequences

<400> 47

Asp Val Arg Glu Arg Pro Ser

1 5

<210> 48

<211> 10

<212> PRT

<213> Artificial sequences

<400> 48

Ser Ser Phe Thr Asn Asn Ser Thr Phe Val

1 5 10

<210> 49

<211> 366

<212> DNA

<213> Artificial sequences

<400> 49

caggtacagc tgcagcagtc aggtccagca ttggtgaagc cctcgcagac cctctcactc 60

acctgtgtca tctccgggga ctctgtctct agcaacagtg cttcttggac ctggatcagg 120

cagtcccctt cgagaggcct tgagtggctg ggaaggacgt 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 sequences

<400> 50

agcaacagtg cttcttggac c 21

<210> 51

<211> 57

<212> DNA

<213> Artificial sequences

<400> 51

aggacgtacc gtcgggccga caggtggtat tatgattatg cactctcgct gaatagt 57

<210> 52

<211> 27

<212> DNA

<213> Artificial sequences

<400> 52

gaatattggg gaggttcttt tgatgtc 27

<210> 53

<211> 122

<212> PRT

<213> Artificial sequences

<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 sequences

<400> 54

Ser Asn Ser Ala Ser Trp Thr

1 5

<210> 55

<211> 19

<212> PRT

<213> Artificial sequences

<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 sequences

<400> 56

Glu Tyr Trp Gly Gly Ser Phe Asp Val

1 5

<210> 57

<211> 333

<212> DNA

<213> Artificial sequences

<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 sequences

<400> 58

actggaacca gcagtgacgt tggtggttat gactatgtct cc 42

<210> 59

<211> 21

<212> DNA

<213> Artificial sequences

<400> 59

gaggtcaata atcggccctc a 21

<210> 60

<211> 33

<212> DNA

<213> Artificial sequences

<400> 60

agctcatata caagcagcaa cactcatgtg gta 33

<210> 61

<211> 111

<212> PRT

<213> Artificial sequences

<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

65 70 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 sequences

<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 sequences

<400> 63

Glu Val Asn Asn Arg Pro Ser

1 5

<210> 64

<211> 11

<212> PRT

<213> Artificial sequences

<400> 64

Ser Ser Tyr Thr Ser Ser Asn Thr His Val Val

1 5 10

<210> 65

<211> 1485

<212> DNA

<213> Artificial sequences

<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 sequences

<400> 66

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60

atccca 66

<210> 67

<211> 54

<212> DNA

<213> Artificial sequences

<400> 67

ggctccacct ctggatccgg caagcccgga tctggcgagg gatccaccaa gggc 54

<210> 68

<211> 135

<212> DNA

<213> Artificial sequences

<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 sequences

<400> 69

atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60

accctttact gc 72

<210> 70

<211> 126

<212> DNA

<213> Artificial sequences

<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 sequences

<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 sequences

<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

115 120 125

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

275 280 285

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 sequences

<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 sequences

<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 sequences

<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 sequences

<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 sequences

<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 Gly Cys Glu Leu

35 40

<210> 78

<211> 112

<212> PRT

<213> Artificial sequences

<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 sequences

<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 aaactcctgt atatattcaa 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 gagatgggggg 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 sequences

<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 sequences

<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 sequences

<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 sequences

<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 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

145 150 155 160

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 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 sequences

<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

145 150 155 160

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 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

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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