CN109293772A - Target antibody, Chimeric antigen receptor and the drug of BCMA albumen - Google Patents

Target antibody, Chimeric antigen receptor and the drug of BCMA albumen Download PDF

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CN109293772A
CN109293772A CN201811117340.7A CN201811117340A CN109293772A CN 109293772 A CN109293772 A CN 109293772A CN 201811117340 A CN201811117340 A CN 201811117340A CN 109293772 A CN109293772 A CN 109293772A
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CN109293772B (en
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晁瑞华
刘明耀
杜冰
席在喜
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Shanghai Bioray Biotechnology Co Ltd
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Shanghai Bioray Biotechnology Co Ltd
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Abstract

The invention discloses a kind of antibody, Chimeric antigen receptor and drugs for targeting BCMA albumen, are related to cellular immunotherapy technical field.Antibody provided by the invention has light chain CDR region shown in heavy chain CDR region shown in SEQ ID NO.22-24 and SEQ ID NO.30-32, either, have light chain CDR region shown in heavy chain CDR region shown in SEQ ID NO.54-56 and SEQ ID NO.62-64；The antibody has the ability of specific binding BCMA albumen, has the fragmentation effect of specificity to the target cell that BCMA albumen is positive using the Chimeric antigen receptor T cell of the Antibody preparation, can be used for preparing the drug for treating or preventing tumour.

Description

Target antibody, Chimeric antigen receptor and the drug of BCMA albumen
Technical field
The present invention relates to cellular immunotherapy technical field, in particular to a kind of antibody for targeting BCMA albumen, embedding Close antigen receptor and drug.
Background technique
Huppert's disease (MM) is the malignant tumour characterized by a large amount of hyperplasia of Clonal thick liquid cell.MM treatment at present can Inducer remission, but nearly all patient finally can still recur and death.Although some monoclonal antibodies are in preclinical study Hope with treatment MM is shown in early studies in man, but does not obtain consistency and still may be used.Obviously, the new of MM is immunized Treatment is clearly in demand, and it will be great for developing the effective adoptive T cell therapy of antigentic specificity for the disease Progress.T cell can be expressed Chimeric antigen receptor (CAR) by genetic modification, and this receptor includes antigen recognition portion and T cell The fusion protein of activation domain.For B system malignant tumour, the most commonly used is the adoptive T cell methods of anti-CD19 CAR.It is anti- The T cell of CD19-CAR transduction has cured leukaemia and lymthoma in mouse, and some patients are also in the anti-CD19- for infusion of adopting Alleviated in the early studies in man of the T cell of CAR transduction, but simultaneously, can also be removed with the T cell that anti-CD19 CAR transduces Fall normal B cell, and unfortunately, CD19 is seldom expressed in the malignant plasma cell of MM, therefore thin with the T that CAR is expressed Born of the same parents, which treat MM, will need to find other better targets.
A kind of candidate antigens of the immunization therapy of MM be B cell maturation antigen (B Cell Maturation Antigen, BCMA, CD269).BCMA RNA generally detects that the thick liquid cell surface of multiple myeloma patients can be detected in MM cell BCMA albumen.BCMA is the member of Tumor Necrosis Factor Receptors (TNF) superfamily, in combination with B cell activity factor (BAFF) and Proliferation-inducing ligand (APRIL).It is reported that BCMA is mainly by thick liquid cell and a part of mature B cell table in normal cell It reaches, and is not expressed in most of B cell and other organs.The mouse that BCMA lacks, which seems, to be all gone well, it appears that very Health, and B cell quantity is normal, but thick liquid cell is unable to long-term surviving.Therefore, BCMA, which will be for treatment, has CAR table Up to the suitable target antigen of the MM of T cell.
However, less for the antibody isotype of BCMA at present.
In consideration of it, the present invention is specifically proposed.
Summary of the invention
The purpose of the present invention is to provide the antibody of targeting BCMA albumen, which can specifically bind BCMA albumen, use In the Chimeric antigen receptor T cell of preparation targeting BCMA albumen.
Another object of the present invention is to provide a kind of Chimeric antigen receptors for targeting BCMA albumen, can target BCMA egg White, the T cell for expressing the Chimeric antigen receptor can the target cell be positive of specific killing BCMA.
Another object of the present invention is to provide a kind of Chimeric antigen receptor T cells for targeting BCMA albumen, which can The target cell for specifically killing the BCMA positive, can be used for treating the tumour of the BCMA positive.
Another object of the present invention is to provide a kind of nucleic acid molecules.
Another object of the present invention is to provide a kind of carriers.
Another object of the present invention is to provide a kind of recombinant cells.
Another object of the present invention is to provide a kind of drug for treating tumour, the medicament can be used to treat or prevention BCMA The tumour being positive.
The present invention is implemented as follows:
The present invention is using people BCMA (hBCMA) albumen as antigen, and in conjunction with phage library display technique, elutriation obtains 4 now The scFV antibody for thering is technology not report.And experiments verify that 4 scFV antibody all have the ability in conjunction with BCMA albumen, adopt There is the killing of specificity to imitate the target cell that BCMA albumen is positive with the Chimeric antigen receptor T cell of the scFV Antibody preparation Fruit.Therefore, which can be used for preparing the Chimeric antigen receptor T cell of targeting BCMA albumen and is used to prepare The tumour that BCMA is positive.
Based on this, on the one hand, the present invention provides it is a kind of target BCMA albumen antibody, the CDR1 of heavy chain variable region, The amino acid sequence of CDR2 and CDR3 is respectively as shown in SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24, light chain The amino acid sequence of CDR1, CDR2 and CDR3 of variable region are respectively such as SEQ ID NO.30, SEQ ID NO.31, SEQ ID Shown in NO.32；
Alternatively, the amino acid sequence of CDR1, CDR2 and CDR3 of the heavy chain variable region of the antibody are respectively such as SEQ ID NO.54, SEQ ID NO.55, shown in SEQ ID NO.56, the amino acid sequence of CDR1, CDR2 and CDR3 of light chain variable region Respectively as shown in SEQ ID NO.62, SEQ ID NO.63, SEQ ID NO.64.
Further, in some embodiments of the present invention, the amino acid sequence of the heavy chain variable region of the antibody is such as Shown in SEQ ID NO.21, the amino acid sequence of the light chain variable region of the antibody is as shown in SEQ ID NO.29；
Alternatively, the amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO.53, the antibody it is light The amino acid sequence of chain variable region is as shown in SEQ ID NO.61.
Antibody with above-mentioned CDR sequence can specifically bind BCMA albumen, be used to prepare the embedding of targeting BCMA albumen Close antigen receptor T cell.
In addition, antibody provided by the invention to be used to prepare to the detection reagent of detection BCMA albumen, or mentioned in the present invention Addition label is belonged to the scope of protection of the present invention for detecting BCMA albumen on the antibody of confession.
Further, in some embodiments of the present invention, the antibody be full length antibody, F (ab ') 2, Fab ', One of Fab, Fv and scFv.
To those skilled in the art, the present invention provides the heavy chains for the antibody that can specifically bind BCMA albumen On the basis of variable region and light chain variable region, easy building obtain in combination with the full length antibody of BCMA albumen, F (ab ') 2, Fab ', Any Antibody types in Fab, Fv and scFv；No matter what type of antibody, as long as contain above-mentioned heavy CDR sequences And/or CDR sequence, it all belongs to the scope of protection of the present invention.
On the other hand, the present invention provides a kind of Chimeric antigen receptor for targeting BCMA albumen, contain above-mentioned targeting The heavy chain variable region and light chain variable region of the antibody of BCMA albumen.
Further, in some embodiments of the present invention, which also has one of following element Or several combination:
Signal peptide, linker, hinge area, CD8 α transmembrane domain, 4-1BB costimulatory signal conducting region and CD3 ζ signal pass Transduction domain.
Further, in some embodiments of the present invention, the amino acid sequence of above-mentioned signal peptide such as SEQ ID NO.73 It is shown.
Further, in some embodiments of the present invention, the amino acid sequence of above-mentioned linker such as SEQ ID NO.74 It is shown.
Further, in some embodiments of the present invention, the amino acid sequence such as SEQ ID of above-mentioned hinge area (hinge) Shown in NO.75.
Further, in some embodiments of the present invention, the amino acid sequence such as SEQ of above-mentioned CD8 α transmembrane domain Shown in ID NO.76.
Further, in some embodiments of the present invention, the amino acid sequence of above-mentioned 4-1BB costimulatory signal conducting region As shown in SEQ ID NO.77.
Further, in some embodiments of the present invention, the amino acid sequence of above-mentioned CD3 ζ signal transduction structural domain is such as Shown in SEQ ID NO.78.
It should be noted that in other examples, those skilled in the art can according to the actual situation or needs change Varying signal peptide, linker, hinge area, CD8 α transmembrane domain, 4-1BB costimulatory signal conducting region and CD3 ζ signal transduction structure The combination sort and sequence in domain, no matter the change based on which kind of form, as long as the Chimeric antigen receptor has the present invention above-mentioned anti- The CDR sequence of the heavy chain variable region of the CDR sequence or light-chain variable sequence and/or above-mentioned antibody of the light chain variable region of body or Weight chain variabl area sequence all belongs to the scope of protection of the present invention.
Further, in some embodiments of the present invention, signal peptide, the light chain variable region of above-mentioned antibody, linker, on State heavy chain variable region, hinge area, CD8 α transmembrane domain, 4-1BB costimulatory signal conducting region and the CD3 ζ signal transduction of antibody Structural domain is constituted the Chimeric antigen receptor in a manner of being sequentially connected in series.
The Chimeric antigen receptor can target BCMA albumen, and the T cell for expressing the Chimeric antigen receptor can specific killing The target cell that BCMA is positive.
On the other hand, the present invention provides a kind of isolated nucleic acid molecules, encode on above-mentioned antibody or its coding The Chimeric antigen receptor stated.
On the other hand, the present invention provides a kind of carriers, contain above-mentioned nucleic acid molecules.
On the other hand, the present invention provides a kind of recombinant cell, contain the nucleic acid point for encoding above-mentioned Chimeric antigen receptor Son or above-mentioned carrier.
On the other hand, the present invention provides a kind of Chimeric antigen receptor T cell for targeting BCMA albumen, expression has above-mentioned Chimeric antigen receptor.
The T cell can specifically kill the target cell that BCMA is positive, and can be used for treating the tumour of the BCMA positive.
On the other hand, the present invention provides a kind of drugs for treating tumour, thin containing above-mentioned Chimeric antigen receptor T Born of the same parents.
Further, in some embodiments of the present invention, above-mentioned tumour is the tumour that BCMA is positive.
Further, in some embodiments of the present invention, the tumour cell that BCMA is positive includes: MM1S and NCI- H929。
The medicament can be used to treat or the tumour that is positive of prevention BCMA.
Further, in some embodiments of the present invention, said medicine also contains pharmaceutically acceptable auxiliary material.
Detailed description of the invention
In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached Figure is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not construed as pair The restriction of range for those of ordinary skill in the art without creative efforts, can also be according to this A little attached drawings obtain other relevant attached drawings.
Fig. 1 is the binding force testing result of BCMA antibody 2077/2082/2073/2079 and the BCMA albumen of people.
Fig. 2 is CAR-BCMA structural schematic diagram.
Fig. 3 is CAR-BCMA positive rate after FCM results show CD4+CD8+T cell infection 72Hrs.
Fig. 4 A is effect target ratio when being 5/1, expresses T cell (CAR-BCMA-2073-T, the CAR-BCMA- of CAR-BCMA It 2079-T) is co-cultured with target cell H929, the streaming figure that target ration changes over time.
Fig. 4 B is effect target ratio when being 5/1, expresses T cell (CAR-BCMA-2077-T, the CAR-BCMA- of CAR-BCMA It 2082-T) is co-cultured with target cell H929, the streaming figure that target ration changes over time.
Fig. 5 is effect target ratio when being 5/1, expresses T cell (CAR-BCMA-2073-T, the CAR-BCMA-2077- of CAR-BCMA T, CAR-BCMA-2079-The CAR-BCMA-2082-T) it is co-cultured with target cell H929, what target ration changed over time Statistical results chart.
Fig. 6 A is effect target ratio when being 7/1, expresses T cell (CAR-BCMA-2073-T, the CAR-BCMA- of CAR-BCMA It 2079-T) is co-cultured with target cell K562-BCMA cell, the streaming figure that target ration changes over time.
Fig. 6 B is effect target ratio when being 7/1, expresses T cell (CAR-BCMA-2077-T, the CAR-BCMA- of CAR-BCMA It 2082-T) is co-cultured with target cell K562-BCMA cell, the streaming figure that target ration changes over time.
When Fig. 7 effect target ratio is 7/1, the T cell and target cell K562-BCMA cell for expressing CAR-BCMA are co-cultured, and target is thin The statistical results chart that born of the same parents' ratio changes over time.
Fig. 8 is the T cell of expression targeting BCMA Chimeric antigen receptor in the cell after the stimulation activation of BCMA positive cell Factor release detection.
Fig. 9 is that the T cell of expression targeting BCMA Chimeric antigen receptor is positive to BCMA in mouse myeloma transplantation model The treatment condition of tumor.
Specific embodiment
It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer builds The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase Product.
Feature and performance of the invention are described in further detail with reference to embodiments.
Embodiment 1
1 phage library elutriation
Use biotinylated hBCMA albumen as the elutriation antigen in human antibody library.Confining liquid (PBST/ is used first 5% skimmed milk power) room temperature closing phage antibody 2h, the input amount of bacteriophage is 2 × 1012Then it is anti-that 10 μ g are added in phage Original is incubated at room temperature 1h, it is pre- closed that 50 μ l is added after incubationM-280 Streptavidin magnetic bead, room temperature are incubated Educate 30min.
Unbonded bacteriophage, then HCl-Glycine elution of bound the biting on magnetic bead with 0.1M are first washed away with PBST Thallus takes the e. coli tg1 of part Phage Infection logarithmic growth phase later in Tris-HCl and eluent, collection Bacteriophage is used for next round elutriation.
The proof strength of every wheel is gradually increased, when degree of enrichment reaches 100 times or more, terminates elutriation.
2 screen the single-chain antibody positive colony of anti-BCMA using phage Elisa
(1) the TGl monoclonal of the Phage Infection after selecting four-wheel elutriation, is inoculated in 96 orifice plates, culture medium 2YT (containing 2%glucose, 100 μ g/ml Ampicilline).
(2) 37 DEG C, 250rpm be forwarded to after being incubated overnight in new culture medium, culture is to being added after logarithmic growth phase M13K07 helper phage, 37 DEG C static to infect 1h.
(3) 4000rpm is centrifuged 15min, uses 2YT (containing 100 μ g/ml Ampicilline, 70 μ g/ml Kanamycin) 30 DEG C of culture medium are incubated overnight, centrifuging and taking bacteriophage supernatant, carry out ELISA identification clone.
(4) with the hBCMA antigen coat Costar-9018 ELISA Plate of 0.5 μ g/ml, 4 DEG C of 3%BSA are closed overnight, are added The bacteriophage supernatant of collection, 4 DEG C of incubation 2h.
(5) Ml3 Bacteriophage antibody (HRP) is added after washing away unbonded bacteriophage, 4 DEG C of incubation 1h.Washing The colour developing of TMB developing solution is added afterwards, is terminated and is reacted with 2M HCl.
(6) it is read with microplate reader in 450nm, selects OD450> 1.5 clones are sequenced, and carry out Germline points to sequence Analysis and PTMs Locus Analysis in Shoots have been obtained 4 in conjunction with hBCMA protein characteristic after excluding the molecule for having potential development risk ScFv single-chain antibody (is alternatively referred to as BCMA antibody) hereinafter.
This four scFv single-chain antibodies are respectively:
Anti- BCMA single-chain antibody 1 (being named as antibody 2073):
Its heavy chain variable amino acid sequence are as follows: SEQ ID NO.5, corresponding nucleotide coding sequence are as follows: SEQ ID NO.1；
The amino acid sequence of VH-CDR1, VH-CDR2 and VH-CDR3 of its heavy chain variable region be respectively as follows: SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, corresponding nucleotide coding sequence are as follows: SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4；
Its chain variable region amino acid sequence are as follows: SEQ ID NO.13, corresponding nucleotide coding sequence are as follows: SEQ ID NO.9；
VL-CDR1, VL-CDR2 and VL-CDR3 amino acid sequence of light chain variable region are respectively as follows: SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, corresponding nucleotide coding sequence are as follows: SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12；
Anti- BCMA single-chain antibody 2 (being named as antibody 2077)
Its heavy chain variable amino acid sequence are as follows: SEQ ID NO.21, corresponding nucleotide coding sequence are as follows: SEQ ID NO.17；
VH-CDR1, VH-CDR2 and VH-CDR3 amino acid sequence of its heavy chain variable region be respectively as follows: SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24, corresponding nucleotide coding sequence are as follows: SEQ ID NO.18, SEQ ID NO.19,SEQ ID NO.20；
Its chain variable region amino acid sequence are as follows: SEQ ID NO.29, corresponding nucleotide coding sequence are as follows: SEQ ID NO.25；
VL-CDR1, VL-CDR2 and VL-CDR3 amino acid sequence of its light chain variable region be respectively as follows: SEQ ID NO.30, SEQ ID NO.31, SEQ ID NO.32, corresponding nucleotide coding sequence are as follows: SEQ ID NO.26, SEQ ID NO.27, SEQ ID NO.28。
Anti- BCMA single-chain antibody 3 (being named as antibody 2079)
Its heavy chain variable amino acid sequence is respectively as follows: SEQ ID NO.37, corresponding nucleotide coding sequence are as follows: SEQ ID NO.33；
VH-CDR1, VH-CDR2 and VH-CDR3 amino acid sequence of its heavy chain variable region be respectively as follows: SEQ ID NO.38, SEQ ID NO.39, SEQ ID NO.40, corresponding nucleotide coding sequence are respectively as follows: SEQ ID NO.34, SEQ ID NO.35,SEQ ID NO.36；
Its chain variable region amino acid sequence are as follows: SEQ ID NO.45, corresponding nucleotide coding sequence are as follows: SEQ ID NO.41；
VL-CDR1, VL-CDR2 and VL-CDR3 amino acid sequence of its light chain variable region be respectively as follows: SEQ ID NO.46, SEQ ID NO.47, SEQ ID NO.48, corresponding nucleotide coding sequence are respectively as follows: SEQ ID NO.42, SEQ ID NO.43,SEQ ID NO.44；
Anti- BCMA single-chain antibody 4 (being named as antibody 2082)
Its heavy chain variable amino acid sequence are as follows: SEQ ID NO.53, corresponding nucleotide sequence are as follows: SEQ ID NO.49；
VH-CDR1, VH-CDR2 and VH-CDR3 amino acid sequence of its heavy chain variable region be respectively as follows: SEQ ID NO.54, SEQ ID NO.55, SEQ ID NO.56, corresponding nucleotide coding sequence are respectively as follows: SEQ ID NO.50, SEQ ID NO.51,SEQ ID NO.52；
Its chain variable region amino acid sequence are as follows: SEQ ID NO.61, corresponding nucleotide coding sequence are as follows: SEQ ID NO.57；
VL-CDR1, VL-CDR2 and VL-CDR3 amino acid sequence of its light chain variable region be respectively as follows: SEQ ID NO.62, SEQ ID NO.63, SEQ ID NO.64, corresponding nucleotide coding sequence are respectively as follows: SEQ ID NO.58, SEQ ID NO.59、SEQ ID NO.60。
Embodiment 2
The binding force of 4 BCMA antibody i.e. 2073,2077,2079 or 2082 and hBCMA albumen that detection embodiment 1 obtains
Detection method:
(1) hBCMA albumen is coated with, and is diluted since the 1 μ g/ml, 3 times of gradient dilutions, totally 8 gradients, concentration are respectively 1 μ G/ml, 333ng/ml, 111ng/ml, 37ng/ml, 12.3ng/ml, 4.1ng/ml, 1.37ng/ml, 0.45ng/ml are used respectively 100 μ l hBCMA diluted protein solutions are coated with Costar-9018 ELISA Plate, and 4 DEG C overnight.
(2) it is corresponded on bacteriophage with addition BCMA antibody i.e. 2073,2077,2079 or 2082 after 3%BSA room temperature closing 2h (10^10pfu) clearly is incubated at room temperature 2h.
(3) Ml3 Bacteriophage antibody (HRP) is added after washing away unbonded bacteriophage, 4 DEG C of incubation 1h.Washing The colour developing of TMB developing solution is added afterwards, is terminated and is reacted with 2M HCl.
(4) it is read with microplate reader in 450nm, the result is shown in Figure 1.
Result can be seen that bacteriophage and the hBCMA that expression has BCMA antibody 2073,2077,2079 or 2082 from Fig. 1 The binding ability of albumen is good, shows S curve, shows dose-effect relationship, illustrates that 4 BCMA that embodiment 1 obtains are anti- Body i.e. 2073,2077,2079 and 2082 all have the binding ability with hBCMA albumen.
Embodiment 3
Construct Chimeric antigen receptor expression vector
Construction method:
(1) full genome synthesizes: signal peptide (nucleic acid sequence SEQ ID NO.66, amino acid sequence SEQ ID NO.73), BCMA antibody's light chain variable region (2073,2077,2079 or 2082 light chain variable regions), linker (nucleic acid sequence SEQ ID NO.67, amino acid sequence SEQ ID NO.74), (2073,2077,2079 or 2082 heavy chain can for BCMA antibody heavy chain variable region Become area), hinge area (hinge) (nucleic acid sequence SEQ ID NO.68, amino acid sequence SEQ ID NO.75), the transmembrane structure CD8 α Domain (TM) (nucleic acid sequence SEQ ID NO.69, amino acid sequence SEQ ID NO.76), 4-1BB costimulatory signal conducting region (core Acid sequence SEQ ID NO.70, amino acid sequence SEQ ID NO.77) and CD3 ζ signal transduction structural domain (nucleic acid sequence SEQ ID NO.71, amino acid sequence SEQ ID NO.78).Above-mentioned sequence is sequentially connected.Respectively obtain 4 kinds of Chimeric antigen receptor tables Up to box, be respectively designated as: (full length nucleotide sequence is as shown in SEQ ID NO.65, amino for 2073 Chimeric antigen receptor expression cassettes Acid sequence is as shown in SEQ ID NO.72), 2077 Chimeric antigen receptor expression cassettes (full length nucleotide sequence such as SEQ ID NO.79 Shown, amino acid sequence is as shown in SEQ ID NO.82), 2079 Chimeric antigen receptor expression cassettes (full length nucleotide sequence such as SEQ Shown in ID NO.80, amino acid sequence is as shown in SEQ ID NO.83) and 2082 Chimeric antigen receptor expression cassette (full length nucleotides Sequence as shown in SEQ ID NO.81, amino acid sequence is as shown in SEQ ID NO.84).
And Kozac sequence is introduced in each expression cassette front end, expression box structure is as shown in Figure 2.
(2) after the sequence of full genome synthesis Chimeric antigen receptor expression cassette, sky is connected by XbaI/EcoRI restriction enzyme site On carrier pCDH-EF1-MSC-T2A-copGFP, Chimeric antigen receptor expression vector is obtained；Obtain 4 Chimeric antigen receptor tables Up to carrier, after sequence verification is correct, it is respectively designated as:
PCDH-EF1-CAR-BCMA-2073-copGFP, light chain variable region and heavy chain variable region containing antibody 2073；
PCDH-EF1-CAR-BCMA-2077-copGFP, light chain variable region and heavy chain variable region containing antibody 2077；
PCDH-EF1-CAR-BCMA-2079-copGFP, light chain variable region and heavy chain variable region containing antibody 2079；
PCDH-EF1-CAR-BCMA-2082-copGFP, light chain variable region and heavy chain variable region containing antibody 2082.
Embodiment 4
Prepare the strain of the expression vector containing Chimeric antigen receptor
Method:
(1) DH5 α competence is taken out in -80 DEG C of refrigerators, thawed on ice.
(2) add 5ng plasmid in competence, mix gently, 5 minutes on ice.
Plasmid: pCDH-EF1-CAR-BCMA-2073-copGFP, pCDH-EF1-CAR-BCMA-2077-copGFP, PCDH-EF1-CAR-BCMA-2079-copGFP or be pCDH-EF1-CAR-BCMA-2082-copGFP.
(3) 42 DEG C thermal shock 90 seconds, on ice 30 minutes.
(4) add the LB of 0.5ml non-resistant, 37 DEG C, 180rpm is cultivated 30 minutes.
(5) it is coated on the plate of ammonia benzyl resistance.
(6) 37 DEG C of inversions are incubated overnight.
(7) monoclonal is chosen, 37 DEG C in the LB of ammonia benzyl resistance, 200rpm is cultivated 9-12 hours.
(8) glycerol adding in bacterium solution, final glycerol concentration 10%, -80 DEG C of refrigerators save strain, spare, can be used for subsequent big Amount extracts plasmid.
(9) by above-mentioned strain in LB after mass propgation, using plasmid extraction kit, (Beijing Tiangeng biochemical technology is limited Company's endotoxin-free plasmid extraction agent box) extracting plasmid, in case infection uses.Plasmid extraction method by specification is It can.
Embodiment 5
Virus packaging
PEI method transfects cell.The preceding 24 hours pancreatin digestion 293T cell of transfection, 4 × 106293T cell be layered on one In 10cm Tissue Culture Dish, cell is cultivated in 37 DEG C of 5%CO2 incubators, is not surpassed in the DMEM culture medium containing 10%FBS 24 hours are spent, can be transfected when cell reaches 60-80% density.
Specific step is as follows:
(1) by plasmid, PEI, DMEM culture medium is placed in room temperature 5min；
(2) it takes 450 μ l of DMEM in 1.5mlEP pipe, adds 50 μ l PEI (1 μ g/ μ l) mixing, be stored at room temperature 5min；
(3) 10 μ g plasmid (pCDH-EF1-CAR-BCMA-2073copGFP, pCDH-EF1-CAR-BCMA-2077- are taken CopGFP, pCDH-EF1-CAR-BCMA-2079-copGFP or pCDH-EF1-CAR-BCMA-2082-copGFP), 10 μ g DMEM to 500 μ l is added in psPAX2,5 μ g pMD2.G, mixes, is stored at room temperature 5min；
(4) the PEI-DMEM solution of (2) the step of preparing is added in the DMEM containing plasmid that step (3) obtains, is mixed It is even, it is stored at room temperature 20min；Obtain DNA/PEI mixture；
(5) 1ml DNA/PEI mixture is slowly instilled in 293T culture dish, is mixed gently, 37 DEG C of incubators are incubated for 6- 8h hours；
(6) original culture medium is discarded, fresh culture is replaced, is put into 37 DEG C of incubators and continues to be incubated for；
(7) after replacing culture medium 48 hours, culture medium is collected, every ware adds 10ml fresh culture and continues to train later Support, for 24 hours after collect supernatant again, with 48 hours collect supernatant mix；
(8) 4 DEG C, 4000g is centrifuged 10min, removes cell fragment；
(9) supernatant being obtained by filtration with 0.45 μm of filter；
(10) filtered supernatant is subjected to tangential flow filtration；
(11) viral supernatants after tangential flow filtration are transferred in ultracentrifugation pipe, 25000rpm is centrifuged 2h, uses serum-free Culture medium is resuspended to the viral pellet obtained from after is surpassed, and gently piping and druming obtains virus liquid, using difference up to being completely dissolved The virus liquid that carrier obtains is respectively designated as:
2073 virus liquids (containing 2073 Chimeric antigen receptor expression cassettes), 2077 virus liquids (contain 2077 Chimeric antigen receptor tables Up to box), 2079 virus liquids (contain 2079 Chimeric antigen receptor expression cassettes), 2082 virus liquids (express containing 2080 Chimeric antigen receptors Box)；
(12) each virus liquid is dispensed, is placed in -80 DEG C of refrigerators and saves, and reserved 5-10 μ l viral concentration liquid carries out titre survey It is fixed.
Embodiment 6
Virus titer measurement
Method:
293T cell is digested and counted, cell suspension is made with the DMEM culture medium containing 10%FBS, adjustment cell density is 4×1050.5ml cell suspension is added into every hole of 24 well culture plates by/ml.After cell adhere-wall culture 8 hours, infection dilution 100 times of 1 μ l of virus liquid, 10 μ l, 20 μ l, 30 μ l, 50 μ l, change liquid after 24 hours, flow cytometer detection 293T cell sun after 48 hours Property rate.
Being centrifuged, be resuspended and adjusting cell density is 1 × 106Add biotin-BCMA antigen, final concentration of 1 μ in/ml, 50 μ l G/ml, after being incubated for 30min, DPBS cleaning is primary, is resuspended, dye secondary antibody APC-Streptavidin (being purchased from BD) 30min, cleaning one DPBS is resuspended after secondary, flow cytometer detection.
Embodiment 7
The T cell of the Chimeric antigen receptor of preparation targeting people BCMA antigen
The separation of 1 human peripheral blood mononuclear cell PBMC
Using anticoagulant tube (being purchased from BD) acquisition peripheral blood about 25ml, separation of lymphocytes is added to according to the volume ratio of 1:1 In liquid, gradient centrifugation 25min takes tunica albuginea confluent monolayer cells after centrifugation, washed twice with DPBS, obtains human peripheral blood mononuclear cell PBMC.
2 CD4+CD8+T cell enrichments and activation
PBMC is resuspended, adjustment density is 1 × 105/ μ l, according in 50 μ l cell suspensions be added each 10 μ l of CD4/CD8 magnetic bead, Pass through the isolated CD4+CD8+T cell of magnetic pole.
The AIM-V complete medium culture containing 10%FBS is added, with anti-human CD3/ in obtained CD4+CD8+T cell CD28 antibody (being purchased from U.S. day Ni, 10 μ l/ml) activating T cell, IL-2 concentration is 200IU/ml.After activation 24 hours, liquid is changed, is made Continue to cultivate with the complete medium containing IL-2 200IU/ml.
3 slow-virus infections
Regulatory T-cell cell density is 1 × 106/ ml, by MOI=10, (2073 diseases of the virus liquid obtained in embodiment 6 Venom, 2077 virus liquids, 2079 virus liquids or 2082 virus liquids) T cell after activation 48 hours is infected, liquid is changed after 24 hours, IL-2 200IU/ml is persistently added, using different virus liquids, obtains the different inosculating antibodies for expressing targeting people BCMA antigen The T cell of original receptor；It is respectively designated as CAR-BCMA-2073-T, CAR-BCMA-2077-T, CAR-BCMA-2079-T, CAR- BCMA-2082-T。
Chimeric antigen receptor (CAR-BCMA) expression of 4 detection targeting people BCMA antigens
During the cultivation process, 72 hours after virus infection T cells CAR-BCMA-2073-T, CAR-BCMA-2077- are taken T, CAR-BCMA-2079-T, CAR-BCMA-2082-T), being centrifuged, be resuspended and adjusting cell density is 1 × 106In/ml, 50 μ l Add biotin-BCMA antigen, final concentration of 0.2 μ g/ml, after being incubated for 30min, DPBS cleaning is primary, is resuspended, and contaminates secondary antibody APC- Streptavidin (being purchased from BD) 30min cleans primary rear DPBS and is resuspended, the positive rate of flow cytometer detection CAR-BCMA.
As a result see Fig. 3, as the result is shown CAR-BCMA-2073-T, CAR-BCMA-2077-T, CAR-BCMA-2079-T, CAR-BCMA-2082-T has a Chimeric antigen receptor of expression targeting BCMA, positive rate respectively reached 45.1%, 61.5%, 22.3%, 78.5%.
Embodiment 8
The tumor-killing effect of the T cell of the Chimeric antigen receptor of co culture system in vitro detection targeting people BCMA antigen, target are thin Born of the same parents are NCI-H929.
Take 72 hours after infecting T cells (CAR-BCMA-2073-T, CAR-BCMA-2077-T, CAR-BCMA-2079-T Or CAR-BCMA-2082-T) and BCMA+ tumour cell NCI-H929, it counts, adjusting cell density is 1 × 106/ ml, according to effect Target ratio 5:1 is co-cultured, it may be assumed that T cell 1 × 106, NCI-H929 2 × 105, control cell is the CD4+ handled without virus infection CD8+T cell is denoted as Ctrl-T cell.Ratio of the NCI-H929 cell in total cell is detected in 0H, 24H, 48H respectively, is made Target cell is marked with antibody A PC-conjugated Human BCMA/TNFRSF17 Antibody, as a result flow cytometer detection is shown in figure 4A and Fig. 4 B；Cell killing efficacy is counted simultaneously, as a result sees Fig. 5.
The results show that in CAR-BCMA-2073-T, CAR-BCMA-2077-T, CAR-BCMA-2079-T or CAR-BCMA- In the presence of 2082-T, as time increases, the ratio of target cell NCI-H929 is significantly reduced；CAR-BCMA-2073- T: it reduces from 15.4% to 0.435%；CAR-BCMA-2079-T: it reduces from 11.3% to 6.63%；CAR-BCMA-2077-T: It reduces from 19.6% to 0.628%；CAR-BCMA-2082-T: it reduces from 18.5% to 0.185%.Thus illustrate, 4 kinds chimeric Antigen receptor T cell CAR-BCMA-2073-T, CAR-BCMA-2077-T, CAR-BCMA-2079-T and CAR-BCMA-2082-T Target cell NCI-H929 can be killed well.
Embodiment 10
Co culture system in vitro detects the tumor-killing effect of CAR-BCMAT, target cell K562-BCMA.
Take 72 hours after infecting T cells (CAR-BCMA-2073-T, CAR-BCMA-2077-T, CAR-BCMA-2079-T Or CAR-BCMA-2082-T) and be overexpressed BCMA cell K562-BCMA, count, adjust cell density be 1 × 106/ ml, is pressed It is co-cultured according to effect target ratio 7:1, it may be assumed that T cell 1 × 106, K562-BCMA 1.5 × 105, control cell is to handle without virus liquid CD4+CD8+T cell is denoted as Ctrl-T cell.Ratio of the K562-BCMA cell in total cell is detected in 0H, 24H, 48H respectively Example, using antibody A PC-conjugated Human BCMA/TNFRSF17 Antibody mark target cell, as a result see Fig. 6 A and Fig. 6 B, while Cell killing efficacy is counted, as a result see Fig. 7.
The results show that in CAR-BCMA-2073-T, CAR-BCMA-2077-T, CAR-BCMA-2079-T or CAR-BCMA- In the presence of 2082-T, as time increases, the ratio of target cell K562-BCMA is significantly reduced, in the time of 0-48h In section, the ratio of target cell K562-BCMA, which reduces amplitude, is respectively: CAR-BCMA-2073-T: reducing from 8.69% to 6.1%； CAR-BCMA-2079-T: it reduces from 9.28% to 8.69%；CAR-BCMA-2077-T: it reduces from 8.95% to 1.03%； CAR-BCMA-2082-T: it reduces from 11.2% to 0.676%；Thus illustrate, 4 kinds of Chimeric antigen receptor T cell CAR-BCMA- 2073-T, CAR-BCMA-2077-T, CAR-BCMA-2079-T and CAR-BCMA-2082-T can kill well BCMAT Positive target cell K562-BCMA.
Embodiment 11
ELISA method detection Cytokine Expression Level
Method:
Preparation of samples: 72 hours after infection T cells (CAR-BCMA-2073-T, CAR-BCMA-2077-T, CAR- are taken BCMA-2079-T or CAR-BCMA-2082-T) and BCMA+ tumour cell NCI-H929, count, adjust cell density be 1 × 106/ ml is co-cultured, i.e. T cell 1 × 10 according to effect target ratio (E:T) 1:16, NCI-H929 1 × 106, control cell is without disease The CD4+CD8+T cell of venom processing, is denoted as Ctrl-T cell.Supernatant is collected in 24H, -80 DEG C save backup.
Detection: cell factor INF- γ, TNF-α are detected using CBA method, are carried out to specifications.As a result see Fig. 8.
It can be seen from the figure that 4 kinds of Chimeric antigen receptors T cell CAR-BCMA-2073-T, CAR-BCMA-2077-T, The INF- γ and TNF-α content of CAR-BCMA-2079-T and CAR-BCMA-2082-T is above control (Ctrl-T).
Embodiment 12
Using NSG mouse, the tumour cell used is the BCMA positive cell line of expressing luciferase stably MM.1S (is denoted as NCI-MM.1S-LUC).Treat the T cell (CAR-BCMA-2077- that the effector cell of injection contains CAR-BCMA T or CAR-BCMA-2082-T), control group is the CD4+CD8+T groups of cells of uninfecting virus.3 days tail vein notes after modeling successfully Penetrate effector cell 5 × 106/ mouse is taken pictures imaging by IVS living imaging system, shows tumour growth feelings for every 7 days after injection Condition, weighs the weight of mouse, while observing the survival condition and record of mouse.As a result see Fig. 9.
From fig. 9, it can be seen that receiving the mouse after CAR-BCMA-2077-T or CAR-BCMA-2082-T injection, the 3rd It and the 7th day its fluorescence area substantially reduce, and illustrate that tumour cell significantly reduces, surface, the chimeric antigen of expression targeting BCMA Recipient T cells CAR-BCMA-2077-T or CAR-BCMA-2082-T can target BCMA, effectively the tumour of killing expression BCMA Cell is, it can be achieved that treatment tumour purpose.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
SEQUENCE LISTING
<110>Shanghai Bang Yao Biotechnology Co., Ltd
<120>antibody, Chimeric antigen receptor and the drug of BCMA albumen are targeted
<160> 84
<170> PatentIn version 3.5
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cagttctccc tgcagctgaa ctctgtgact cccgaggaca cggctgtgta ttactgtgca 300
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<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 5 10
<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 atgacagcct gaatggttgg 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 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 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 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 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
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 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
115 120 125
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 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 Gly Cys 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 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 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 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 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
145 150 155 160
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 kind of antibody for targeting BCMA albumen, which is characterized in that the amino acid of CDR1, CDR2 and CDR3 of its heavy chain variable region Sequence is respectively as shown in SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24, CDR1, CDR2 of light chain variable region With the amino acid sequence of CDR3 respectively as shown in SEQ ID NO.30, SEQ ID NO.31, SEQ ID NO.32；
Alternatively, the amino acid sequence of CDR1, CDR2 and CDR3 of the heavy chain variable region of the antibody respectively as SEQ ID NO.54, Shown in SEQ ID NO.55, SEQ ID NO.56, the amino acid sequence of CDR1, CDR2 and CDR3 of light chain variable region are respectively such as SEQ ID NO.62, SEQ ID NO.63, shown in SEQ ID NO.64.
2. the antibody of targeting BCMA albumen according to claim 1, which is characterized in that the heavy chain variable region of the antibody Amino acid sequence is as shown in SEQ ID NO.21, the amino acid sequence of the light chain variable region of the antibody such as SEQ ID NO.29 institute Show；
Alternatively, the amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO.53, the light chain of the antibody can Become the amino acid sequence in area as shown in SEQ ID NO.61.
3. it is according to claim 1 or 2 targeting BCMA albumen antibody, which is characterized in that the antibody be full length antibody, One of F (ab ') 2, Fab ', Fab, Fv and scFv.
4. a kind of Chimeric antigen receptor for targeting BCMA albumen, which is characterized in that it is described in any item that it contains claim 1-3 Target the heavy chain variable region and light chain variable region of the antibody of BCMA albumen.
5. Chimeric antigen receptor according to claim 4, which is characterized in that the Chimeric antigen receptor also has following element One or more of combination:
Signal peptide, linker, hinge area, CD8 α transmembrane domain, 4-1BB costimulatory signal conducting region and CD3 ζ signal transduction knot Structure domain.
6. a kind of isolated nucleic acid molecules, which is characterized in that it encodes the described in any item antibody of claim 1-3, Huo Zheqi Encode Chimeric antigen receptor described in claim 4 or 5.
7. a kind of carrier, which is characterized in that it contains nucleic acid molecules as claimed in claim 6.
8. a kind of recombinant cell, which is characterized in that it contains the nucleic acid for encoding Chimeric antigen receptor described in claim 4 or 5 Molecule or carrier as claimed in claim 7.
9. a kind of Chimeric antigen receptor T cell for targeting BCMA albumen, which is characterized in that its expression has the right to require described in 4 or 5 Chimeric antigen receptor.
10. a kind of drug for treating tumour, which is characterized in that it contains Chimeric antigen receptor T cell as claimed in claim 9.
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CN112979807B (en) * 2019-12-17 2022-07-26 深圳市菲鹏生物治疗股份有限公司 BCMA binding antibodies and uses thereof
CN112979807A (en) * 2019-12-17 2021-06-18 深圳市菲鹏生物治疗股份有限公司 BCMA binding antibodies and uses thereof
CN113061185A (en) * 2020-01-02 2021-07-02 益科思特(北京)医药科技发展有限公司 Preparation method and application of BCMA antibody
CN113061185B (en) * 2020-01-02 2023-08-08 益科思特(北京)医药科技发展有限公司 Preparation method and application of BCMA antibody
CN115279792A (en) * 2020-03-13 2022-11-01 酷罗赛尔公司 Antibodies and chimeric antigen receptors specific for BCMA
CN114539403B (en) * 2020-11-27 2023-07-14 缔码生物科技(武汉)有限公司 Rabbit recombinant monoclonal antibody targeting human BCMA protein and application thereof
CN114539403A (en) * 2020-11-27 2022-05-27 缔码生物科技(武汉)有限公司 Rabbit recombinant monoclonal antibody of targeted human BCMA protein and application
CN113173991A (en) * 2020-12-31 2021-07-27 北京艺妙神州医药科技有限公司 anti-BCMA antibody and application thereof
CN114763383A (en) * 2021-01-13 2022-07-19 博生吉医药科技(苏州)有限公司 Monoclonal antibody targeting human BCMA and application thereof
CN114790462B (en) * 2021-01-25 2023-10-31 中国医学科学院血液病医院(中国医学科学院血液学研究所) Chimeric antigen receptor targeting BCMA and application thereof
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CN114181311B (en) * 2021-12-20 2023-06-20 华东师范大学 Fully human anti-DLL 3 scFv and application thereof in CART cell therapy
CN114181311A (en) * 2021-12-20 2022-03-15 华东师范大学 Fully human anti-DLL 3scFv and application thereof in CART cell treatment
CN116063521A (en) * 2022-10-31 2023-05-05 北京奇迈永华生物科技有限公司 Antibodies targeting BCMA and chimeric antigen receptor composed thereof
CN116063521B (en) * 2022-10-31 2024-05-14 北京奇迈永华生物科技有限公司 Antibodies targeting BCMA and chimeric antigen receptor composed thereof

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