CN109293773A

CN109293773A - Target antibody, Chimeric antigen receptor and the drug of CD38 albumen

Info

Publication number: CN109293773A
Application number: CN201811121365.4A
Authority: CN
Inventors: 晁瑞华; 刘明耀; 杜冰; 席在喜
Original assignee: Shanghai Bioray Biotechnology Co Ltd
Current assignee: Shanghai Bioray Biotechnology Co Ltd
Priority date: 2018-09-25
Filing date: 2018-09-25
Publication date: 2019-02-01
Anticipated expiration: 2038-09-25
Also published as: CN109293773B

Abstract

The invention discloses a kind of antibody, Chimeric antigen receptor and drugs for targeting CD38 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.6-8 and SEQ ID NO.14-16, either, have light chain CDR region shown in heavy chain CDR region shown in SEQ ID NO.22-24 and SEQ ID NO.30-32；The antibody has the ability of specific binding CD38 albumen, has the fragmentation effect of specificity to the target cell that CD38 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 CD38 albumen

Technical field

The present invention relates to cellular immunotherapy technical fields, in particular to a kind of antibody for targeting 38 albumen, are fitted into 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, is malignant plasma cell One of the most common type type in disease, is apt to occur in person in middle and old age, and patient is often accompanied by ostalgia, hypercalcinemia, anaemia, kidney damage.Although Using classic chemotherapy and hematopoietic stem cell transplantation and targeted drug treatment myeloma, so that MM treatment can induce and alleviate, but Nearly all patient finally can still recur and death, and one of major reason is exactly with progression of disease, normal immunological ball egg White generation is suppressed, therefore is easy to appear various bacterial infections.Although some monoclonal antibodies in preclinical study and The hope for the treatment of MM is shown in early studies in man, but does not obtain consistency approval.The T of technique for gene engineering processing is thin Born of the same parents combine tumour antibody specific recognition and costimulatory signal, specific for tumour antigen identification, increasing with non-MHC dependence Grow and kill ability.In recent years, T cell (the chimeric antigen of the Chimeric antigen receptor modification of MM antigentic specificity Receptor, CAR-T cell) it studies and also carries out and obtain first-stage success successively, CAR-T cell therapy becomes effectively treatment MM's New departure.T cell can be expressed Chimeric antigen receptor (CAR) by genetic modification, and this receptor includes antigen recognition portion and T thin The fusion protein of born of the same parents' activation domain.For B system malignant tumour, the most commonly used is the adoptive T cell methods of anti-CD19CAR. The T cell of anti-CD19-CAR transduction has cured leukaemia and lymthoma in mouse, and some patients are also in the anti-of infusion of adopting Alleviated in the early studies in man of the T cell of CD19-CAR transduction, and most of B cell mark egg on MM tumour cell White expression quantity is atomic, or even does not express, therefore needs to find novel targets for the CAR-T of MM treatment.

In addition to B cell maturation antigen (B Cell Maturation Antigen, BCMA, CD269) this candidate antigens Outside, multiple myeloma cells wide expression CD38 albumen.CD38 is also referred to as cricoid ADP- ribose hydrolase, is a kind of sugar Albumen.CD38 molecule wide expression also has expression in hemocytoblast, in NK cell, T cell, B cell etc..It has been reported that Compared with normal plasma cells, the expression of myeloma cell CD38 is obviously increased, so that CD38, which becomes, treats the important of this cancer Target spot.However, less for the antibody isotype of CD38 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 CD38 albumen, which can specifically bind CD38 albumen, use In the Chimeric antigen receptor T cell of preparation targeting CD38 albumen.

Another object of the present invention is to provide a kind of Chimeric antigen receptors for targeting CD38 albumen, can target CD38 egg White, the T cell for expressing the Chimeric antigen receptor can the target cell be positive of specific killing CD38.

Another object of the present invention is to provide a kind of Chimeric antigen receptor T cells for targeting CD38 albumen, which can The target cell for specifically killing the CD38 positive, can be used for treating the tumour of the CD38 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 CD38 The tumour being positive.

The present invention is implemented as follows:

The present invention is using people CD38 (hCD38) 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 CD38 albumen, adopt There is the killing of specificity to imitate the target cell that CD38 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 CD38 albumen and is used to prepare The tumour that CD38 is positive.

Based on this, on the one hand, the present invention provides it is a kind of target CD38 albumen antibody, the CDR1 of heavy chain variable region, For the amino acid sequence of CDR2 and CDR3 respectively as shown in SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, light chain can Become the amino acid sequence of CDR1, CDR2 and the CDR3 in area respectively such as SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16 It is shown；

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.22, SEQ ID NO.23, shown in SEQ ID NO.24, the amino acid sequence of CDR1, CDR2 and CDR3 of light chain variable region Respectively as shown in SEQ ID NO.30, SEQ ID NO.31, SEQ ID NO.32.

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.5, the amino acid sequence of the light chain variable region of the antibody is as shown in SEQ ID NO.13；

Alternatively, the amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO.21, the antibody it is light The amino acid sequence of chain variable region is as shown in SEQ ID NO.29.

Antibody with above-mentioned CDR sequence can specifically bind CD38 albumen, be used to prepare the embedding of targeting CD38 albumen Close antigen receptor T cell.

In addition, antibody provided by the invention to be used to prepare to the detection reagent of detection CD38 albumen, or mentioned in the present invention Addition label is belonged to the scope of protection of the present invention for detecting CD38 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 CD38 albumen On the basis of variable region and light chain variable region, easy building obtain in combination with the full length antibody of CD38 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 CD38 albumen, contain above-mentioned targeting The heavy chain variable region and light chain variable region of the antibody of CD38 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 CD38 albumen, and the T cell for expressing the Chimeric antigen receptor can specific killing The target cell that CD38 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 CD38 albumen, expression has above-mentioned Chimeric antigen receptor.

The T cell can specifically kill the target cell that CD38 is positive, and can be used for treating the tumour of the CD38 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 CD38 is positive.

Further, in some embodiments of the present invention, the tumour cell that CD38 is positive includes: MM.1S, The cells such as U266, RPMI8226.

The medicament can be used to treat or the tumour that is positive of prevention CD38.

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 be phage ELISA qualification result.

Fig. 2 is CAR-CD38 structural schematic diagram.

Fig. 3 is CAR-CD38 positive rate after FCM results show CD4+CD8+T cell infection 72Hrs.

Fig. 4 A is effect target ratio when being 3/1, expresses T cell (CAR-CD38-2427-T, CAR-CD38- of CAR-CD38 It 2430-T) is co-cultured with target cell MM.1S, the streaming figure that target ration changes over time.

Fig. 4 B is effect target ratio when being 3/1, expresses T cell (CAR-CD38-2446-T, CAR-CD38- of CAR-CD38 It 2453-T) is co-cultured with target cell MM.1S, the streaming figure that target ration changes over time.

Fig. 5 is effect target ratio when being 3/1, expresses T cell (CAR-CD38-2427-T, CAR-CD38-2430- of CAR-CD38 T, CAR-CD38-2446-T and CAR-CD38-2453-T) it is co-cultured with target cell MM.1S, what target ration changed over time Schematic diagram.

Fig. 6 is the T cell of expression targeting CD38 Chimeric antigen receptor in the cell after the stimulation activation of CD38 positive cell The factor (INF- γ and TNF-α) discharges testing result.

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 hCD38 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 AntiCD3 McAb 8 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 hCD38 antigen coat Costar-9018 ELISA Plate of 0.5 μ g/ml, 3%BSA4 DEG C is closed overnight, is added The bacteriophage supernatant of collection, 4 DEG C of incubation 2h.

(5) Ml3Bacteriophage antibody (HRP) is added after washing away unbonded bacteriophage, 4 DEG C of incubation 1h.After washing The colour developing of TMB developing solution is added, is terminated and is reacted with 2M HCl.

(6) it is read with microplate reader in 450nm, selects the clone of OD450 > 1.5 to be sequenced (see Fig. 1), sequence is carried out Germline analysis and PTMs Locus Analysis in Shoots have been obtained 4 in conjunction with hCD38 after excluding the molecule for having potential development risk The scFv single-chain antibody (being alternatively referred to as CD38 antibody hereinafter) of protein characteristic.

It is respectively:

8 single-chain antibody 1 (being named as antibody 2427) of AntiCD3 McAb:

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；

8 single-chain antibody 2 (being named as antibody 2430) of AntiCD3 McAb

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。

8 single-chain antibody 3 (being named as antibody 2446) of AntiCD3 McAb

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；

8 single-chain antibody 4 (being named as antibody 2453) of AntiCD3 McAb

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

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), CD38 antibody's light chain variable region (2427,2430,2446 or 2453 light chain variable regions), linker (nucleic acid sequence SEQ ID NO.67, amino acid sequence SEQ ID NO.74), (2427,2430,2446 or 2453 heavy chain can for CD38 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).

Said elements sequence is sequentially connected, 4 kinds of Chimeric antigen receptor expression cassettes is respectively obtained, it is embedding to be respectively designated as 2427 Closing antigen receptor expression cassette, (full length nucleotide sequence is as shown in SEQ ID NO.65, amino acid sequence such as SEQ ID NO.72 institute Show), (full length nucleotide sequence is as shown in SEQ ID NO.79, amino acid sequence such as SEQ for 2430 Chimeric antigen receptor expression cassettes Shown in ID NO.82), (full length nucleotide sequence is as shown in SEQ ID NO.80, amino acid for 2446 Chimeric antigen receptor expression cassettes Sequence is as shown in SEQ ID NO.83) and 2453 Chimeric antigen receptor expression cassettes (full length nucleotide sequence such as SEQ ID NO.81 Shown, 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-CD38-2427-copGFP, light chain variable region and heavy chain variable region containing antibody 2427；

PCDH-EF1-CAR-CD38-2430-copGFP, light chain variable region and heavy chain variable region containing antibody 2430；

PCDH-EF1-CAR-CD38-2446-copGFP, light chain variable region and heavy chain variable region containing antibody 2446；

PCDH-EF1-CAR-CD38-2453-copGFP, light chain variable region and heavy chain variable region containing antibody 2453.

Embodiment 3

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 is: pCDH-EF1-CAR-CD38-2427-copGFP, pCDH-EF1-CAR-CD38-2430-copGFP, PCDH-EF1-CAR-CD38-2446-copGFP or be pCDH-EF1-CAR-CD3-2453-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 4

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-CD38-2427-copGFP, pCDH-EF1-CAR-CD38-2430- are taken CopGFP, pCDH-EF1-CAR-CD38-2446-copGFP or pCDH-EF1-CAR-CD38-2453-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) obtain, is mixed, 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:

2427 virus liquids (containing 2427 Chimeric antigen receptor expression cassettes), 2430 virus liquids (contain 2430 Chimeric antigen receptor tables Up to box), 2446 virus liquids (contain 2446 Chimeric antigen receptor expression cassettes), 2453 virus liquids (express containing 2453 Chimeric antigen receptors Box), empty carrier virus liquid.

(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 5

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-CD38 antigen in/ml, 50 μ l, it is final concentration of 1ug/ml, after being incubated for 30min, DPBS cleaning is primary, is resuspended, dye secondary antibody APC-Streptavidin (being purchased from BD) 30min, cleaning DPBS is resuspended after primary, flow cytometer detection.

Embodiment 6

The T cell of the Chimeric antigen receptor of preparation targeting people CD38 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.

2CD4+CD8+T cell enrichment 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 CD38 antigen The T cell of original receptor；It is respectively designated as CAR-CD38-2427-T, CAR-CD38-2430-T, CAR-CD38-2446-T or CAR- CD38-2453-T。

Chimeric antigen receptor (CAR-CD38) expression of 4 detection targeting people CD38 antigens

During the cultivation process, 72 hours after virus infection T cell (CAR-CD38-2427-T, CAR-CD38-2430- are taken T, CAR-CD38-2446-T or CAR-CD38-2453-T), being centrifuged, be resuspended and adjusting cell density is 1 × 10⁶In/ml, 50 μ l Add biotin-CD38 antigen, final concentration of 0.2ug/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-CD38.

As a result see Fig. 3, as the result is shown CAR-CD38-2427-T, CAR-CD38-2430-T, CAR-CD38-2446-T or CAR-CD38-2453-T has the Chimeric antigen receptor of expression targeting CD38, and the positive rate of CAR-CD38-2427-T is The positive rate that the positive rate of 94.5%, CAR-CD38-2430-T are 92.2%, CAR-CD38-2446-T is 79.2%, CAR- The positive rate of CD38-2453-T is 37.6%.

Embodiment 7

Co culture system in vitro detects the tumor-killing effect of CAR-CD38T, target cell MM.1S.

Take infection after 72 hours Chimeric antigen receptor T cell (CAR-CD38-2427-T, CAR-CD38-2430-T, CAR-CD38-2446-T or CAR-CD38-2453-T) and CD38+ tumour cell MM.1S, count, adjust cell density be 1 × 10⁶/ ml is co-cultured, it may be assumed that T cell 1 × 10 according to effect target ratio 3:1⁶, MM.1S 3.3 × 10⁵, control cell is to feel without virus The CD4+CD8+T cell for contaminating processing, is denoted as Ctrl-T cell.MM.1S cell is detected total thin in 0H, 18H, 42H, 72H respectively Ratio in born of the same parents marks target cell, streaming using antibody A PC-conjugated Human CD38/TNFRSF17 Antibody Detection, is as a result shown in Fig. 4 A and Fig. 4 B；Cell killing efficacy is counted simultaneously, as a result sees Fig. 5.

The results show that in CAR-CD38-2427-T, CAR-CD38-2430-T, CAR-CD38-2446-T or CAR-CD38- In the presence of 2453-T, as time increases, the ratio of CD38+ target cell MM.1S is significantly reduced, 0-72H when Between in section, CAR-CD38-2427-T group: target cell is reduced to 15.2% by 29.1%；CAR-CD38-2430-T group: target cell 30.7% is reduced to by 35.1%；CAR-CD38-2446-T group: target cell is reduced to 5.25% by 31.3%；CAR-CD38- 2453-T group: target cell is reduced to 4.76% by 29.1%.Thus illustrate, 4 kinds of Chimeric antigen receptor T cell CAR-CD38- 2427-T, CAR-CD38-2430-T, CAR-CD38-2446-T and CAR-CD38-2453-T can target CD38+ tumour cell MM.1S causes to kill to tumour cell.

Embodiment 8

ELISA method detection Cytokine Expression Level

Method:

Preparation of samples: 72 hours after infection T cell (CAR-CD38-2427-T, CAR-CD38-2430-T, CAR- are taken CD38-2446-T or CAR-CD38-2453-T) and CD38+ tumour cell RAJI, it counts, adjusting cell density is 1 × 10⁶/ Ml is co-cultured, i.e. T cell 1 × 10 according to effect target ratio (E:T) 1:1⁶, RAJI 1 × 10⁶, control cell be without virus infection at The CD4+CD8+T cell of reason, 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. 6.

From fig. 6 it can be seen that Chimeric antigen receptor T cell CAR-CD38-2427-T, CAR- of 4 kinds of targeting CD38 CD38-2430-T, CAR-CD38-2446-T and CAR-CD38-2453-T can generate the INF- γ and TNF-α of high concentration, high In control group (Ctrl-T).

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 CD38 albumen are targeted

<160> 84

<170> PatentIn version 3.5

<210> 1

<211> 351

<212> DNA

<213>artificial sequence

<400> 1

caggtgcagc tgcaggagtc gggcgcagga ctgttgaagc cttcggagac cctgtccctc 60

acctgcgctg tctatggtgg gtcctccagt ggtgactact ggagctggat ccgccagccc 120

ccagggaagg ggctggagtg gattggggaa atcaatcata gtggaatcac caactacaac 180

ccgtccctca agagtcgagt caccatatca ctagacacgt ccaagaatca gttctccctg 240

aagttgaagt ctgtgaccgc cgcggacacg gctgtctatt actgtgcgag agaacggggt 300

aacaatggta tggacgtctg gggccaaggc accctggtca ccgtctcgag t 351

<210> 2

<211> 15

<212> DNA

<213>artificial sequence

<400> 2

ggtgactact ggagc 15

<210> 3

<211> 48

<212> DNA

<213>artificial sequence

<400> 3

gaaatcaatc atagtggaat caccaactac aacccgtccc tcaagagt 48

<210> 4

<211> 27

<212> DNA

<213>artificial sequence

<400> 4

gaacggggta acaatggtat ggacgtc 27

<210> 5

<211> 117

<212> PRT

<213>artificial sequence

<400> 5

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

1 5 10 15

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

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

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

65 70 75 80

Lys Leu Lys Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Glu Arg Gly Asn Asn Gly Met Asp Val Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 6

<211> 5

<212> PRT

<213>artificial sequence

<400> 6

Gly Asp Tyr Trp Ser

1 5

<210> 7

<211> 16

<212> PRT

<213>artificial sequence

<400> 7

Glu Ile Asn His Ser Gly Ile Thr Asn Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 8

<211> 9

<212> PRT

<213>artificial sequence

<400> 8

Glu Arg Gly Asn Asn Gly Met Asp Val

1 5

<210> 9

<211> 330

<212> DNA

<213>artificial sequence

<400> 9

caggctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc 60

tcttgttctg gaagcagctc caacatcgga agtaattatg tatactggta ccagcagctc 120

ccaggaacgg cccccaaact cctcatctat aggaataatc agcggcccgc aggggtccct 180

gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg 240

tccgaggatg aggctgatta ctactgtgca gcatgggatg acagtgtgag tggttgggtg 300

ttcggcggag gcacccagct gaccgtcctc 330

<210> 10

<211> 39

<212> DNA

<213>artificial sequence

<400> 10

tctggaagca gctccaacat cggaagtaat tatgtatac 39

<210> 11

<211> 21

<212> DNA

<213>artificial sequence

<400> 11

aggaataatc agcggcccgc a 21

<210> 12

<211> 33

<212> DNA

<213>artificial sequence

<400> 12

gcagcatggg atgacagtgt gagtggttgg gtg 33

<210> 13

<211> 110

<212> PRT

<213>artificial sequence

<400> 13

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

1 5 10 15

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

20 25 30

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

35 40 45

Ile Tyr Arg Asn Asn Gln Arg Pro Ala Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Val

85 90 95

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

100 105 110

<210> 14

<211> 13

<212> PRT

<213>artificial sequence

<400> 14

Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Tyr Val Tyr

1 5 10

<210> 15

<211> 7

<212> PRT

<213>artificial sequence

<400> 15

Arg Asn Asn Gln Arg Pro Ala

1 5

<210> 16

<211> 11

<212> PRT

<213>artificial sequence

<400> 16

Ala Ala Trp Asp Asp Ser Val Ser Gly Trp Val

1 5 10

<210> 17

<211> 351

<212> DNA

<213>artificial sequence

<400> 17

caggtgcagc tgcaggagtg gggcgcagga ctgttgaagc cttcggagac cctgtccctc 60

acctgcgctg tctatggtgg gtcctccagt ggtgactact ggagctggat ccgccagccc 120

ccagggaagg ggctggagtg gattggggaa atcaatcata gtggaagcac caactacaac 180

ccgtccctca agagtcgagt caccatatca ctagacacgt ccaagaatca gttctccctg 240

aagttgaggt ctgtgaccgc cgcggacacg gctgtgtatt actgtgcgag agaacggggt 300

aacaatggta tggacgtctg gggccaaggc accctggtca ctgtctcgag t 351

<210> 18

<211> 15

<212> DNA

<213>artificial sequence

<400> 18

ggtgactact ggagc 15

<210> 19

<211> 48

<212> DNA

<213>artificial sequence

<400> 19

gaaatcaatc atagtggaag caccaactac aacccgtccc tcaagagt 48

<210> 20

<211> 27

<212> DNA

<213>artificial sequence

<400> 20

gaacggggta acaatggtat ggacgtc 27

<210> 21

<211> 117

<212> PRT

<213>artificial sequence

<400> 21

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

1 5 10 15

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

20 25 30

Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile

35 40 45

Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

50 55 60

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

65 70 75 80

Lys Leu Arg Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

85 90 95

Arg Glu Arg Gly Asn Asn Gly Met Asp Val Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 22

<211> 5

<212> PRT

<213>artificial sequence

<400> 22

Gly Asp Tyr Trp Ser

1 5

<210> 23

<211> 16

<212> PRT

<213>artificial sequence

<400> 23

Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys Ser

1 5 10 15

<210> 24

<211> 9

<212> PRT

<213>artificial sequence

<400> 24

Glu Arg Gly Asn Asn Gly Met Asp Val

1 5

<210> 25

<211> 330

<212> DNA

<213>artificial sequence

<400> 25

caggcagggc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc 60

tcttgttctg gaagcagctc caacatcgga agtaattatg tatactggta ccagcagctc 120

ccaggaacgg cccccaaact cctcatctat aggaataatc agcggccctc aggggtccct 180

gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg 240

tccgaggatg aggctgatta ttactgtgca gcatgggatg acagcctgag tggttgggtg 300

ttcggcggag gcacccagct gaccgccctc 330

<210> 26

<211> 39

<212> DNA

<213>artificial sequence

<400> 26

tctggaagca gctccaacat cggaagtaat tatgtatac 39

<210> 27

<211> 21

<212> DNA

<213>artificial sequence

<400> 27

aggaataatc agcggccctc a 21

<210> 28

<211> 33

<212> DNA

<213>artificial sequence

<400> 28

gcagcatggg atgacagcct gagtggttgg gtg 33

<210> 29

<211> 110

<212> PRT

<213>artificial sequence

<400> 29

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

1 5 10 15

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

20 25 30

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

35 40 45

Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser

50 55 60

Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg

65 70 75 80

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

85 90 95

Ser Gly Trp Val Phe Gly Gly Gly Thr Gln Leu Thr Ala Leu

100 105 110

<210> 30

<211> 13

<212> PRT

<213>artificial sequence

<400> 30

Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn Tyr Val Tyr

1 5 10

<210> 31

<211> 7

<212> PRT

<213>artificial sequence

<400> 31

Arg Asn Asn Gln Arg Pro Ser

1 5

<210> 32

<211> 11

<212> PRT

<213>artificial sequence

<400> 32

Ala Ala Trp Asp Asp Ser Leu Ser Gly Trp Val

1 5 10

<210> 33

<211> 360

<212> DNA

<213>artificial sequence

<400> 33

gaggtgcagc tggtgcagtc tggagcagag gtgaaaaagc ccggggagtc tctgaagatc 60

tcctgtaagg gttctggata cagctttacc agctactgga tcggctgggt gcgccagatg 120

cccgggaaag gcctggagtg gatggggatc atctatcctg gtgactctga taccagatac 180

agcccgtcct tccaaggcca ggtcaccatc tcagccgaca agtccatcag caccgcctac 240

ctgcagtgga gcagcctgaa ggcctcggac accgccatgt attactgtgc gagagttagc 300

agtggctggc cctactacat ggacgtctgg ggcaaaggga ccacggtcac cgtctcgagt 360

<210> 34

<211> 15

<212> DNA

<213>artificial sequence

<400> 34

agctactgga tcggc 15

<210> 35

<211> 51

<212> DNA

<213>artificial sequence

<400> 35

atcatctatc ctggtgactc tgataccaga tacagcccgt ccttccaagg c 51

<210> 36

<211> 33

<212> DNA

<213>artificial sequence

<400> 36

gttagcagtg gctggcccta ctacatggac gtc 33

<210> 37

<211> 120

<212> PRT

<213>artificial sequence

<400> 37

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

1 5 10 15

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

20 25 30

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

35 40 45

Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe

50 55 60

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

65 70 75 80

Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys

85 90 95

Ala Arg Val Ser Ser Gly Trp Pro Tyr Tyr Met Asp Val Trp Gly Lys

100 105 110

Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 38

<211> 5

<212> PRT

<213>artificial sequence

<400> 38

Ser Tyr Trp Ile Gly

1 5

<210> 39

<211> 17

<212> PRT

<213>artificial sequence

<400> 39

Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe Gln

1 5 10 15

Gly

<210> 40

<211> 11

<212> PRT

<213>artificial sequence

<400> 40

Val Ser Ser Gly Trp Pro Tyr Tyr Met Asp Val

1 5 10

<210> 41

<211> 321

<212> DNA

<213>artificial sequence

<400> 41

gccatccgga tgacccagtc tccatcttcc gtgtctgcat ctgtgggaga cagagtcacc 60

atcacttgtc gggcgagtca gggtattagc aactggttag cctggtatca gcagaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180

aggttcagcg gcagtggatc tgggacagat ttcactctca ccgtcagcag cctgcagcct 240

gaagattttg caacttacta ttgtcaacag gctaccagtt tccccctaac tttcggcgga 300

gggaccaagg tggaaatcaa a 321

<210> 42

<211> 33

<212> DNA

<213>artificial sequence

<400> 42

cgggcgagtc agggtattag caactggtta gcc 33

<210> 43

<211> 21

<212> DNA

<213>artificial sequence

<400> 43

gctgcatcca gtttgcaaag t 21

<210> 44

<211> 27

<212> DNA

<213>artificial sequence

<400> 44

caacaggcta ccagtttccc cctaact 27

<210> 45

<211> 107

<212> PRT

<213>artificial sequence

<400> 45

Ala Ile Arg Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Asn Trp

20 25 30

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

35 40 45

Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Val Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Thr Ser Phe Pro Leu

85 90 95

Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105

<210> 46

<211> 11

<212> PRT

<213>artificial sequence

<400> 46

Arg Ala Ser Gln Gly Ile Ser Asn Trp Leu Ala

1 5 10

<210> 47

<211> 7

<212> PRT

<213>artificial sequence

<400> 47

Ala Ala Ser Ser Leu Gln Ser

1 5

<210> 48

<211> 9

<212> PRT

<213>artificial sequence

<400> 48

Gln Gln Ala Thr Ser Phe Pro Leu Thr

1 5

<210> 49

<211> 366

<212> DNA

<213>artificial sequence

<400> 49

gaggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtt 60

tcctgcaagg catctggata caccttcacc agctactata tgcactgggt gcgacaggcc 120

cctggacaag ggcttgagtg gatgggaata atcaacccta gtggtggtag cacaagctac 180

gcacagaagt tccagggcag agtcaccatg accagggaca cgtccacgag cacagtctac 240

atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gagagactac 300

ggtgactacg ggaggtacta cggtatggac gtctggggcc aagggaccac ggtcaccgtc 360

tcgagt 366

<210> 50

<211> 15

<212> DNA

<213>artificial sequence

<400> 50

agctactata tgcac 15

<210> 51

<211> 51

<212> DNA

<213>artificial sequence

<400> 51

ataatcaacc ctagtggtgg tagcacaagc tacgcacaga agttccaggg c 51

<210> 52

<211> 39

<212> DNA

<213>artificial sequence

<400> 52

gactacggtg actacgggag gtactacggt atggacgtc 39

<210> 53

<211> 122

<212> PRT

<213>artificial sequence

<400> 53

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

1 5 10 15

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

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

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

85 90 95

Ala Arg Asp Tyr Gly Asp Tyr Gly Arg Tyr Tyr Gly Met Asp Val Trp

100 105 110

Gly Gln Gly Thr Thr Val Thr Val Ser Ser

115 120

<210> 54

<211> 5

<212> PRT

<213>artificial sequence

<400> 54

Ser Tyr Tyr Met His

1 5

<210> 55

<211> 17

<212> PRT

<213>artificial sequence

<400> 55

Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe Gln

1 5 10 15

Gly

<210> 56

<211> 13

<212> PRT

<213>artificial sequence

<400> 56

Asp Tyr Gly Asp Tyr Gly Arg Tyr Tyr Gly Met Asp Val

1 5 10

<210> 57

<211> 330

<212> DNA

<213>artificial sequence

<400> 57

cagtctgccc tgactcagcc tgcctccgtg tctgggtctc ctggacagtc gatcaccatc 60

tcctgcactg gaaccaacag tgacgttggt gtttataact atgtctcctg gtaccaacag 120

tatccaggca aagcccccaa actcatgatt tatgatgtca gtgagcggcc ctcaggggtt 180

tctaatcgct tctctggctc caagtctgtc aacacggcct ccctgaccat ctctgggctc 240

caggctgagg acgaggctga ttattactgc agctcatata caagcagcaa cactgatgtc 300

ttcggaactg gcaccaaagt gaccgtcctc 330

<210> 58

<211> 42

<212> DNA

<213>artificial sequence

<400> 58

actggaacca acagtgacgt tggtgtttat aactatgtct cc 42

<210> 59

<211> 21

<212> DNA

<213>artificial sequence

<400> 59

gatgtcagtg agcggccctc a 21

<210> 60

<211> 30

<212> DNA

<213>artificial sequence

<400> 60

agctcatata caagcagcaa cactgatgtc 30

<210> 61

<211> 110

<212> PRT

<213>artificial sequence

<400> 61

Gln Ser Ala 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 Asn Ser Asp Val Gly Val Tyr

20 25 30

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

35 40 45

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

50 55 60

Ser Gly Ser Lys Ser Val 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 Tyr Thr Ser Ser

85 90 95

Asn Thr Asp Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu

100 105 110

<210> 62

<211> 14

<212> PRT

<213>artificial sequence

<400> 62

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

1 5 10

<210> 63

<211> 7

<212> PRT

<213>artificial sequence

<400> 63

Asp Val Ser Glu Arg Pro Ser

1 5

<210> 64

<211> 10

<212> PRT

<213>artificial sequence

<400> 64

Ser Ser Tyr Thr Ser Ser Asn Thr Asp Val

1 5 10

<210> 65

<211> 1470

<212> DNA

<213>artificial sequence

<400> 65

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60

atcccacagg ctgtgctgac tcagccaccc tcagcgtctg ggacccccgg gcagagggtc 120

accatctctt gttctggaag cagctccaac atcggaagta attatgtata ctggtaccag 180

cagctcccag gaacggcccc caaactcctc atctatagga ataatcagcg gcccgcaggg 240

gtccctgacc gattctctgg ctccaagtct ggcacctcag cctccctggc catcagtggg 300

ctccggtccg aggatgaggc tgattactac tgtgcagcat gggatgacag tgtgagtggt 360

tgggtgttcg gcggaggcac ccagctgacc gtcctcggct ccacctctgg atccggcaag 420

cccggatctg gcgagggatc caccaagggc caggtgcagc tgcaggagtc gggcgcagga 480

ctgttgaagc cttcggagac cctgtccctc acctgcgctg tctatggtgg gtcctccagt 540

ggtgactact ggagctggat ccgccagccc ccagggaagg ggctggagtg gattggggaa 600

atcaatcata gtggaatcac caactacaac ccgtccctca agagtcgagt caccatatca 660

ctagacacgt ccaagaatca gttctccctg aagttgaagt ctgtgaccgc cgcggacacg 720

gctgtctatt actgtgcgag agaacggggt aacaatggta tggacgtctg gggccaaggc 780

accctggtca ccgtctcgag taccacgacg ccagcgccgc gaccaccaac accggcgccc 840

accatcgcgt cacagcccct gtccctgcgc ccagaggcgt gccggccagc ggcggggggc 900

gcagtgcaca cgagggggct ggacttcgcc tgtgatatct acatctgggc gcccttggcc 960

gggacttgtg gggtccttct cctgtcactg gttatcaccc tttactgcaa acggggcaga 1020

aagaaactcc tgtatatatt caaacaacca tttatgagac cagtacaaac tactcaagag 1080

gaagatggct gtagctgccg atttccagaa gaagaagaag gaggatgtga actgagagtg 1140

aagttcagca ggagcgcaga cgcccccgcg tacaagcagg gccagaacca gctctataac 1200

gagctcaatc taggacgaag agaggagtac gatgttttgg acaagagacg tggccgggac 1260

cctgagatgg ggggaaagcc gagaaggaag aaccctcagg aaggcctgta caatgaactg 1320

cagaaagata agatggcgga ggcctacagt gagattggga tgaaaggcga gcgccggagg 1380

ggcaaggggc acgatggcct ttaccagggt ctcagtacag ccaccaagga cacctacgac 1440

gcccttcaca tgcaggccct gccccctcgc 1470

<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> 490

<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 Gln Ala Val Leu Thr Gln Pro Pro Ser Ala

20 25 30

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

35 40 45

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

50 55 60

Thr Ala Pro Lys Leu Leu Ile Tyr Arg Asn Asn Gln Arg Pro Ala Gly

65 70 75 80

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

85 90 95

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

100 105 110

Ala Trp Asp Asp Ser Val Ser Gly Trp Val Phe Gly Gly Gly Thr Gln

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 Glu Ser Gly Ala Gly

145 150 155 160

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

165 170 175

Gly Ser Ser Ser Gly Asp Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly

180 185 190

Lys Gly Leu Glu Trp Ile Gly Glu Ile Asn His Ser Gly Ile Thr Asn

195 200 205

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

210 215 220

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

225 230 235 240

Ala Val Tyr Tyr Cys Ala Arg Glu Arg Gly Asn Asn Gly Met Asp Val

245 250 255

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

260 265 270

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

275 280 285

Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<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> 1470

<212> DNA

<213>artificial sequence

<400> 79

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60

atcccacagg cagggctgac tcagccaccc tcagcgtctg ggacccccgg gcagagggtc 120

accatctctt gttctggaag cagctccaac atcggaagta attatgtata ctggtaccag 180

cagctcccag gaacggcccc caaactcctc atctatagga ataatcagcg gccctcaggg 240

gtccctgacc gattctctgg ctccaagtct ggcacctcag cctccctggc catcagtggg 300

ctccggtccg aggatgaggc tgattattac tgtgcagcat gggatgacag cctgagtggt 360

tgggtgttcg gcggaggcac ccagctgacc gccctcggct ccacctctgg atccggcaag 420

cccggatctg gcgagggatc caccaagggc caggtgcagc tgcaggagtg gggcgcagga 480

ctgttgaagc cttcggagac cctgtccctc acctgcgctg tctatggtgg gtcctccagt 540

ggtgactact ggagctggat ccgccagccc ccagggaagg ggctggagtg gattggggaa 600

atcaatcata gtggaagcac caactacaac ccgtccctca agagtcgagt caccatatca 660

ctagacacgt ccaagaatca gttctccctg aagttgaggt ctgtgaccgc cgcggacacg 720

gctgtgtatt actgtgcgag agaacggggt aacaatggta tggacgtctg gggccaaggc 780

accctggtca ctgtctcgag taccacgacg ccagcgccgc gaccaccaac accggcgccc 840

accatcgcgt cacagcccct gtccctgcgc ccagaggcgt gccggccagc ggcggggggc 900

gcagtgcaca cgagggggct ggacttcgcc tgtgatatct acatctgggc gcccttggcc 960

gggacttgtg gggtccttct cctgtcactg gttatcaccc tttactgcaa acggggcaga 1020

aagaaactcc tgtatatatt caaacaacca tttatgagac cagtacaaac tactcaagag 1080

gaagatggct gtagctgccg atttccagaa gaagaagaag gaggatgtga actgagagtg 1140

aagttcagca ggagcgcaga cgcccccgcg tacaagcagg gccagaacca gctctataac 1200

gagctcaatc taggacgaag agaggagtac gatgttttgg acaagagacg tggccgggac 1260

cctgagatgg ggggaaagcc gagaaggaag aaccctcagg aaggcctgta caatgaactg 1320

cagaaagata agatggcgga ggcctacagt gagattggga tgaaaggcga gcgccggagg 1380

ggcaaggggc acgatggcct ttaccagggt ctcagtacag ccaccaagga cacctacgac 1440

gcccttcaca tgcaggccct gccccctcgc 1470

<210> 80

<211> 1470

<212> DNA

<213>artificial sequence

<400> 80

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60

atcccagcca tccggatgac ccagtctcca tcttccgtgt ctgcatctgt gggagacaga 120

gtcaccatca cttgtcgggc gagtcagggt attagcaact ggttagcctg gtatcagcag 180

aaaccaggga aagcccctaa gctcctgatc tatgctgcat ccagtttgca aagtggggtc 240

ccatcaaggt tcagcggcag tggatctggg acagatttca ctctcaccgt cagcagcctg 300

cagcctgaag attttgcaac ttactattgt caacaggcta ccagtttccc cctaactttc 360

ggcggaggga ccaaggtgga aatcaaaggc tccacctctg gatccggcaa gcccggatct 420

ggcgagggat ccaccaaggg cgaggtgcag ctggtgcagt ctggagcaga ggtgaaaaag 480

cccggggagt ctctgaagat ctcctgtaag ggttctggat acagctttac cagctactgg 540

atcggctggg tgcgccagat gcccgggaaa ggcctggagt ggatggggat catctatcct 600

ggtgactctg ataccagata cagcccgtcc ttccaaggcc aggtcaccat ctcagccgac 660

aagtccatca gcaccgccta cctgcagtgg agcagcctga aggcctcgga caccgccatg 720

tattactgtg cgagagttag cagtggctgg ccctactaca tggacgtctg gggcaaaggg 780

accacggtca ccgtctcgag taccacgacg ccagcgccgc gaccaccaac accggcgccc 840

accatcgcgt cacagcccct gtccctgcgc ccagaggcgt gccggccagc ggcggggggc 900

gcagtgcaca cgagggggct ggacttcgcc tgtgatatct acatctgggc gcccttggcc 960

gggacttgtg gggtccttct cctgtcactg gttatcaccc tttactgcaa acggggcaga 1020

aagaaactcc tgtatatatt caaacaacca tttatgagac cagtacaaac tactcaagag 1080

gaagatggct gtagctgccg atttccagaa gaagaagaag gaggatgtga actgagagtg 1140

aagttcagca ggagcgcaga cgcccccgcg tacaagcagg gccagaacca gctctataac 1200

gagctcaatc taggacgaag agaggagtac gatgttttgg acaagagacg tggccgggac 1260

cctgagatgg ggggaaagcc gagaaggaag aaccctcagg aaggcctgta caatgaactg 1320

cagaaagata agatggcgga ggcctacagt gagattggga tgaaaggcga gcgccggagg 1380

ggcaaggggc acgatggcct ttaccagggt ctcagtacag ccaccaagga cacctacgac 1440

gcccttcaca tgcaggccct gccccctcgc 1470

<210> 81

<211> 1485

<212> DNA

<213>artificial sequence

<400> 81

atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 60

atcccacagt ctgccctgac tcagcctgcc tccgtgtctg ggtctcctgg acagtcgatc 120

accatctcct gcactggaac caacagtgac gttggtgttt ataactatgt ctcctggtac 180

caacagtatc caggcaaagc ccccaaactc atgatttatg atgtcagtga gcggccctca 240

ggggtttcta atcgcttctc tggctccaag tctgtcaaca cggcctccct gaccatctct 300

gggctccagg ctgaggacga ggctgattat tactgcagct catatacaag cagcaacact 360

gatgtcttcg gaactggcac caaagtgacc gtcctcggct ccacctctgg atccggcaag 420

cccggatctg gcgagggatc caccaagggc gaggtgcagc tggtgcagtc tggggctgag 480

gtgaagaagc ctggggcctc agtgaaggtt tcctgcaagg catctggata caccttcacc 540

agctactata tgcactgggt gcgacaggcc cctggacaag ggcttgagtg gatgggaata 600

atcaacccta gtggtggtag cacaagctac gcacagaagt tccagggcag agtcaccatg 660

accagggaca cgtccacgag cacagtctac atggagctga gcagcctgag atctgaggac 720

acggccgtgt attactgtgc gagagactac ggtgactacg ggaggtacta cggtatggac 780

gtctggggcc aagggaccac 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> 82

<211> 490

<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 Ala Gly Leu Thr Gln Pro Pro Ser Ala

20 25 30

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

35 40 45

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

50 55 60

Thr Ala Pro Lys Leu Leu Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

Leu Thr Ala 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 Glu Trp Gly Ala Gly

145 150 155 160

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

165 170 175

Gly Ser Ser Ser Gly Asp Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly

180 185 190

Lys Gly Leu Glu Trp Ile Gly Glu Ile Asn His Ser Gly Ser Thr Asn

195 200 205

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

210 215 220

Lys Asn Gln Phe Ser Leu Lys Leu Arg Ser Val Thr Ala Ala Asp Thr

225 230 235 240

Ala Val Tyr Tyr Cys Ala Arg Glu Arg Gly Asn Asn Gly Met Asp Val

245 250 255

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

260 265 270

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

275 280 285

Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 83

<211> 490

<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 Ala Ile Arg Met Thr Gln Ser Pro Ser Ser

20 25 30

Val Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Val Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln

100 105 110

Ala Thr Ser Phe Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile

115 120 125

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

130 135 140

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

145 150 155 160

Pro Gly Glu Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe

165 170 175

Thr Ser Tyr Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu

180 185 190

Glu Trp Met Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser

195 200 205

Pro Ser Phe Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser

210 215 220

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

225 230 235 240

Tyr Tyr Cys Ala Arg Val Ser Ser Gly Trp Pro Tyr Tyr Met Asp Val

245 250 255

Trp Gly Lys Gly Thr Thr Val Thr Val Ser Ser Thr Thr Thr Pro Ala

260 265 270

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

275 280 285

Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

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

465 470 475 480

Ala Leu His Met Gln Ala Leu Pro Pro Arg

485 490

<210> 84

<211> 495

<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 Ile Ser Cys Thr Gly Thr Asn

35 40 45

Ser Asp Val Gly Val Tyr Asn Tyr Val Ser Trp Tyr Gln Gln Tyr Pro

50 55 60

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

65 70 75 80

Gly Val Ser Asn Arg Phe Ser Gly Ser Lys Ser Val 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 Tyr Thr Ser Ser Asn Thr Asp 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 Glu Val Gln Leu Val Gln Ser Gly Ala Glu

145 150 155 160

Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly

165 170 175

Tyr Thr Phe Thr Ser Tyr Tyr Met His Trp Val Arg Gln Ala Pro Gly

180 185 190

Gln Gly Leu Glu Trp Met Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr

195 200 205

Ser Tyr Ala Gln Lys Phe Gln Gly Arg Val Thr Met Thr Arg Asp Thr

210 215 220

Ser Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp

225 230 235 240

Thr Ala Val Tyr Tyr Cys Ala Arg Asp Tyr Gly Asp Tyr Gly Arg Tyr

245 250 255

Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr 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

Claims

1. a kind of antibody for targeting CD38 albumen, which is characterized in that the amino acid of CDR1, CDR2 and CDR3 of its heavy chain variable region Sequence respectively as shown in SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, CDR1, CDR2 of light chain variable region and The amino acid sequence of CDR3 is respectively as shown in SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16；

Alternatively, the amino acid sequence of CDR1, CDR2 and CDR3 of the heavy chain variable region of the antibody respectively as SEQ ID NO.22, Shown in SEQ ID NO.23, SEQ ID NO.24, the amino acid sequence of CDR1, CDR2 and CDR3 of light chain variable region are respectively such as SEQ ID NO.30, SEQ ID NO.31, shown in SEQ ID NO.32.

2. the antibody of targeting CD38 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.5, the amino acid sequence of the light chain variable region of the antibody such as SEQ ID NO.13 institute Show；

Alternatively, the amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO.21, the light chain of the antibody can Become the amino acid sequence in area as shown in SEQ ID NO.29.

3. it is according to claim 1 or 2 targeting CD38 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 CD38 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 CD38 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 CD38 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.