CN109293773B - Antibodies, chimeric antigen receptors and drugs targeting CD38 protein - Google Patents

Abstract

The invention discloses an antibody targeting CD38 protein, a chimeric antigen receptor and a medicament, and relates to the technical field of cellular immunotherapy. The antibody provided by the invention has heavy chain CDR regions shown in SEQ ID NO.6-8 and light chain CDR regions shown in SEQ ID NO.14-16, or has heavy chain CDR regions shown in SEQ ID NO.22-24 and light chain CDR regions shown in SEQ ID NO. 30-32; the antibody has the capacity of specifically binding CD38 protein, and the chimeric antigen receptor T cell prepared by the antibody has specific killing effect on the target cell positive to the CD38 protein, and can be used for preparing medicaments for treating or preventing tumors.

Description

Antibodies, chimeric antigen receptors and drugs targeting CD38 protein

Technical Field

The invention relates to the technical field of cellular immunotherapy, and particularly relates to a 38 protein-targeted antibody, a chimeric antigen receptor and a medicament.

Background

Multiple Myeloma (MM) is a malignant tumor characterized by massive proliferation of clonal plasma cells, is the most common type of malignant plasma cell disease, and is better developed in middle-aged and old people, and patients are often accompanied by bone pain, hypercalcemia, anemia and kidney damage. Despite the use of traditional chemotherapy and hematopoietic stem cell transplantation and targeted drug therapy for myeloma, which results in induced remission, almost all patients eventually relapse and die, one of the important reasons being that as the disease progresses, the production of normal immunoglobulins is suppressed and thus various bacterial infections are easily seen. Although some monoclonal antibodies have shown promise in preclinical studies and early clinical trials for treatment of MM, there is no consistent acceptance. The T cells treated by the genetic engineering technology combine tumor antibody specific recognition and co-stimulation signals, and have the capabilities of MHC-independent tumor antigen specific recognition, proliferation and killing. In recent years, the study of chimeric antigen receptor-modified T cells (CAR-T cells) specific to MM antigens has also been carried out in succession and with preliminary success, CAR-T cell therapy has become a new approach for the effective treatment of MM. T cells can be genetically modified to express a Chimeric Antigen Receptor (CAR) that includes a fusion protein of an antigen recognition moiety and a T cell activation domain. For B-line malignancies, the most commonly used is an adoptive T cell approach against CD19 CAR. anti-CD 19-CAR transduced T cells cured leukemia and lymphoma in mice, some patients also achieved remission in early clinical trials of adoptively infused anti-CD 19-CAR transduced T cells, while most B cell marker proteins were expressed in minimal or no amounts on MM tumor cells, thus CAR-T therapy for MM needed to find new targets.

In addition to the candidate Antigen, the B Cell Maturation Antigen (BCMA, CD269), multiple myeloma cells widely express CD38 protein. CD38 is also known as a cyclic ADP-ribose hydrolase and is a glycoprotein. The CD38 molecule is widely expressed in hematopoietic progenitor cells, and is also expressed in NK cells, T cells, B cells, and the like. The literature reports that the expression of CD38 in myeloma cells is significantly increased compared to normal plasma cells, making CD38 an important target for the treatment of this cancer. However, there are currently fewer classes of antibodies directed to CD 38.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide an antibody targeting CD38 protein, which can be specifically combined with CD38 protein and is used for preparing chimeric antigen receptor T cells targeting CD38 protein.

Another objective of the present invention is to provide a chimeric antigen receptor targeting CD38 protein, which can target CD38 protein, and T cells expressing the chimeric antigen receptor can specifically kill target cells positive for CD 38.

Another objective of the present invention is to provide a chimeric antigen receptor T cell targeting CD38 protein, which can specifically kill CD38 positive target cells, and can be used for treating CD38 positive tumors.

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

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

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

The invention also aims to provide a medicament for treating tumors, which can be used for treating or preventing tumors positive to CD 38.

The invention is realized by the following steps:

the invention takes human CD38(hCD38) protein as antigen, combines phage library display technology, and elutes to obtain 4 scFV antibodies which are not reported in the prior art. Experiments prove that the 4 scFV antibodies have the capacity of combining CD38 protein, and the chimeric antigen receptor T cells prepared by the scFV antibodies have specific killing effect on target cells with positive CD38 protein. Therefore, the 4 scFV antibodies can be used for preparing chimeric antigen receptor T cells targeting CD38 protein and preparing tumors positive for CD 38.

Based on the above, in one aspect, the invention provides an antibody targeting a CD38 protein, wherein the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region are respectively shown in SEQ ID No.6, SEQ ID No.7 and SEQ ID No.8, and the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region are respectively shown in SEQ ID No.14, SEQ ID No.15 and SEQ ID No. 16;

or the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region of the antibody are respectively shown in SEQ ID NO.22, SEQ ID NO.23 and SEQ ID NO.24, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region of the antibody are respectively shown in SEQ ID NO.30, SEQ ID NO.31 and 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 represented by SEQ ID No.5, and the amino acid sequence of the light chain variable region of the antibody is represented by SEQ ID No. 13;

or the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO.21, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 29.

Antibodies having the CDR sequences described above can specifically bind to CD38 protein, which is used to prepare chimeric antigen receptor T cells that target CD38 protein.

In addition, the antibody provided by the invention is used for preparing a detection reagent for detecting CD38 protein, or a label is added on the antibody provided by the invention for detecting CD38 protein, and the invention belongs to the protection scope of the invention.

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

For those skilled in the art, on the basis of the heavy chain variable region and the light chain variable region of the antibody provided by the invention, which can specifically bind to the CD38 protein, any one of full-length antibody, F (ab ') 2, Fab', Fab, Fv and scFv, which can bind to the CD38 protein, can be easily constructed; any type of antibody, so long as it contains the heavy chain CDR sequences and/or light chain CDR sequences, is within the scope of the present invention.

In another aspect, the present invention provides a chimeric antigen receptor targeting CD38 protein, comprising the heavy chain variable region and the light chain variable region of the antibody targeting CD38 protein as described above.

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

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

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

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

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

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

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

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

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

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

The chimeric antigen receptor can target CD38 protein, and T cells expressing the chimeric antigen receptor can kill CD38 positive target cells specifically.

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

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

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

In another aspect, the present invention provides a chimeric antigen receptor T cell targeting CD38 protein, which expresses the chimeric antigen receptor described above.

The T cell can kill the target cell with positive CD38 specifically, and can be used for treating tumor with positive CD 38.

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

Further, in some embodiments of the invention, the tumor is a tumor positive for CD 38.

Further, in some embodiments of the invention, tumor cells positive for CD38 include: 1S, U266, RPMI8226 and the like.

The medicine can be used for treating or preventing tumor with positive CD 38.

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

Drawings

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

FIG. 1 shows the results of the phase ELISA.

FIG. 2 is a schematic diagram of CAR-CD38 structure.

FIG. 3 is a flow cytometer showing CAR-CD38 positivity after infection of 72Hrs with CD4+ CD8+ T cells.

FIG. 4A is a flow chart of the change in target cell ratio over time of CAR-CD38 expressing T cells (CAR-CD38-2427-T, CAR-CD38-2430-T) co-cultured with the target cell MM.1S at an effective target ratio of 3/1.

FIG. 4B is a flow chart of the change in target cell ratio over time in the co-culture of CAR-CD38 expressing T cells (CAR-CD38-2446-T, CAR-CD38-2453-T) with the target cell MM.1S at an effective target ratio of 3/1.

FIG. 5 is a graph of the change in target cell ratio with time in co-culture of CAR-CD38 expressing T cells (CAR-CD38-2427-T, CAR-CD38-2430-T, CAR-CD38-2446-T and CAR-CD38-2453-T) with the target cell MM.1S at an effective target ratio of 3/1.

FIG. 6 shows the results of cytokine (INF-. gamma.and TNF-. alpha.) release assays from T cells expressing chimeric antigen receptor targeting CD38 after activation by stimulation with CD38 positive cells.

Detailed Description

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

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

Example 1

1 phage library panning

The biotinylated hCD38 protein was used as the panning antigen for the fully human antibody library, phage antibodies were first blocked with blocking solution (PBST/5% skim milk powder) at room temperature for 2h, and the amount of phage added was 2 × 10¹²phage, then 10. mu.g antigen, incubated for 1h at room temperature, after which 50. mu.l pre-blocked

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

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

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

2 screening of Single chain antibody Positive clones against CD38 Using phase Elisa

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

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

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

(4) The Costar-9018 enzyme-labeled plate was coated with 0.5. mu.g/ml of hCD38 antigen, blocked overnight at 4 ℃ in 3% BSA, the collected phage supernatant was added, and incubated at 4 ℃ for 2 h.

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

(6) A total of 4 scFv single-chain antibodies (hereinafter also referred to as CD38 antibodies) with the property of binding to hCD38 protein were obtained by using an enzyme reader to read at 450nm, selecting clones with OD450>1.5 (see FIG. 1) for sequencing, and performing Germline analysis and PTMs site analysis on the sequences, excluding molecules with potential development risk.

The method comprises the following steps:

anti-CD 38 single chain antibody 1 (designated antibody 2427):

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

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

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

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

anti-CD 38 Single chain antibody 2 (named antibody 2430)

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

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

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

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

anti-CD 38 Single chain antibody 3 (named antibody 2446)

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

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

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

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

anti-CD 38 single chain antibody 4 (named antibody 2453)

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

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

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

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

Example 2

Construction of chimeric antigen receptor expression vectors

The construction method comprises the following steps:

(1) and (3) total gene synthesis: signal peptide (SEQ ID No.66, SEQ ID No.73), light chain variable region of CD38 antibody (light chain variable region of 2427, 2430, 2446 or 2453), linker (SEQ ID No.67, SEQ ID No.74), heavy chain variable region of CD38 antibody (heavy chain variable region of 2427, 2430, 2446 or 2453), hinge region (hinge) (SEQ ID No.68, SEQ ID No.75), CD8 a transmembrane domain (TM) (SEQ ID No.69, SEQ ID No.76), 4-1BB costimulatory signaling region (SEQ ID No.70, SEQ ID No.77) and CD3 ζ transmembrane signaling domain (SEQ ID No.71, SEQ ID No. 78).

The above element sequences are connected in sequence to respectively obtain 4 chimeric antigen receptor expression cassettes which are respectively named 2427 chimeric antigen receptor expression cassettes (the full-length nucleotide sequence is shown as SEQ ID No.65, and the amino acid sequence is shown as SEQ ID No. 72), 2430 chimeric antigen receptor expression cassettes (the full-length nucleotide sequence is shown as SEQ ID No.79, and the amino acid sequence is shown as SEQ ID No. 82), 2446 chimeric antigen receptor expression cassettes (the full-length nucleotide sequence is shown as SEQ ID No.80, and the amino acid sequence is shown as SEQ ID No. 83), and 2453 chimeric antigen receptor expression cassettes (the full-length nucleotide sequence is shown as SEQ ID No.81, and the amino acid sequence is shown as SEQ ID No. 84).

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

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

pCDH-EF1-CAR-CD38-2427-copGFP, light and heavy chain variable regions comprising antibody 2427;

pCDH-EF1-CAR-CD38-2430-copGFP containing the light and heavy chain variable regions of antibody 2430;

pCDH-EF1-CAR-CD38-2446-copGFP comprising the light chain variable region and the heavy chain variable region of antibody 2446;

pCDH-EF1-CAR-CD38-2453-copGFP, light chain variable region and heavy chain variable region comprising antibody 2453.

Example 3

Preparation of strains containing chimeric antigen receptor expression vectors

The method comprises the following steps:

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

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

The plasmids were: pCDH-EF1-CAR-CD38-2427-copGFP, pCDH-EF1-CAR-CD38-2430-copGFP, pCDH-EF1-CAR-CD38-2446-copGFP or pCDH-EF1-CAR-CD 3-2453-copGFP.

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

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

(5) Spread onto ampicillin resistant plates.

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

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

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

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

Example 4

Virus package

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

The method comprises the following specific steps:

(1) plasmid, PEI, DMEM medium were left at room temperature for 5 min.

(2) Mu.l of DMEM was put in a 1.5ml EP tube, 50. mu.l of PEI (1. mu.g/. mu.l) was added thereto and mixed well, followed by standing at room temperature for 5 min.

(3) Mu.g of plasmid (pCDH-EF1-CAR-CD38-2427-copGFP, pCDH-EF1-CAR-CD38-2430-copGFP, pCDH-EF1-CAR-CD38-2446-copGFP or pCDH-EF1-CAR-CD38-2453-copGFP) was taken, 10. mu.g of psPAX2, 5. mu.g of pMD2.G, DMEM was added to 500. mu.l, mixed well, and left to stand at room temperature for 5 min.

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

(5) 1ml of the DNA/PEI mixture was slowly dropped into 293T petri dishes, gently mixed, and incubated at 37 ℃ for 6-8h in an incubator.

(6) Discarding the original culture medium, replacing the fresh culture medium, and placing the culture medium into a 37 ℃ incubator for continuous incubation.

(7) After 48 hours of medium exchange, the medium was collected, then 10ml of fresh medium was added to each dish to continue the culture, and after 24 hours, the supernatant was collected again and mixed with the supernatant collected for 48 hours.

(8) The cells were centrifuged at 4000g for 10min at 4 ℃ to remove cell debris.

(9) The resulting supernatant was filtered through a 0.45 μm filter.

(10) The filtered supernatant was subjected to tangential flow filtration.

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

2427 viral fluid (containing 2427 chimeric antigen receptor expression cassette), 2430 viral fluid (containing 2430 chimeric antigen receptor expression cassette), 2446 viral fluid (containing 2446 chimeric antigen receptor expression cassette), 2453 viral fluid (containing 2453 chimeric antigen receptor expression cassette), and empty carrier viral fluid.

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

Example 5

Viral titer determination

The method comprises the following steps:

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

Centrifuging, resuspending and adjusting the cell density to 1 × 10⁶Adding biotin-CD38 antigen into 50 μ l per ml, incubating for 30min, washing DPBS once, resuspending, staining secondary antibody APC-Streptavidin (purchased from BD) for 30min, resuspending DPBS after washing once, and detecting by flow.

Example 6

T cell for preparing chimeric antigen receptor targeting human CD38 antigen

1 isolation of PBMC from human peripheral blood mononuclear cells

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

2CD4+ CD8+ T cell enrichment and activation

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

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

3 Lentiviral infection

Regulating T cell density to 1 × 10⁶The virus solution obtained in example 4 (2427 virus solution, 2430 virus solution, 2446 virus solution or 2453 virus solution) is used for infecting T cells after 48 hours of activation according to MOI of 10, the solution is changed after 24 hours, IL-2200 IU/ml is continuously added, and different virus solutions are adopted to obtain different T cells capable of expressing chimeric antigen receptors targeting human CD38 antigen; are named CAR-CD38-2427-T, CAR-CD38-2430-T, CAR-CD38-2446-T or CAR-CD38-2453-T, respectively.

4 detection of expression of chimeric antigen receptor targeting human CD38 antigen (CAR-CD38)

During the culture process, T cells (CAR-CD38-2427-T, CAR-CD38-2430-T, CAR-CD38-2446-T or CAR-CD38-2453-T) 72 hours after virus infection are taken, centrifuged, resuspended and the cell density is adjusted to 1 × 10⁶Adding biotin-CD38 antigen into 50 μ l per ml to a final concentration of 0.2ug/ml, incubating for 30min, washing DPBS once, re-suspending, staining secondary antibody APC-Streptavidin (purchased from BD) for 30min, re-suspending DPBS after washing once, and detecting the positive rate of CAR-CD38 by flow-type assay.

The results are shown in FIG. 3, and show that the CAR-CD38-2427-T, CAR-CD38-2430-T, CAR-CD38-2446-T or CAR-CD38-2453-T both express chimeric antigen receptor targeting CD38, the positive rate of CAR-CD38-2427-T is 94.5%, the positive rate of CAR-CD38-2430-T is 92.2%, the positive rate of CAR-CD38-2446-T is 79.2%, and the positive rate of CAR-CD38-2453-T is 37.6%.

Example 7

In vitro co-culture tested the tumor killing effect of CAR-CD38T, with mm.1s as the target cell.

Collecting chimeric antigen receptor T cells (CAR-CD38-2427-T, CAR-CD38-2430-T, CAR-CD38-2446-T or CAR-CD38-2453-T) and CD38+ tumor cells MM.1S 72h after infection, counting, and regulating cell density to 1 × 10⁶/ml, co-cultured according to an effective target ratio of 3:1, i.e., T cells 1 × 10⁶，MM.1S 3.3×10⁵Control cells were CD4+ CD8+ T cells untreated with viral infection and scored as Ctrl-T cells. At 0H, 18H, 42H respectively72H assay of the proportion of MM.1S cells in total cells, labeling of target cells with the Antibody APC-conjugated Human CD38/TNFRSF17 Antibody, flow assay, results are shown in FIG. 4A and FIG. 4B; meanwhile, the cell killing effect is counted, and the result is shown in figure 5.

The results show that in the presence of CAR-CD38-2427-T, CAR-CD38-2430-T, CAR-CD38-2446-T or CAR-CD38-2453-T, the proportion of CD38+ target cell mm.1s decreases significantly with increasing time, and over the period of 0-72H, the CAR-CD38-2427-T group: the target cells are reduced from 29.1% to 15.2%; CAR-CD38-2430-T group: the target cells are reduced from 35.1% to 30.7%; CAR-CD38-2446-T group: the target cells are reduced from 31.3% to 5.25%; CAR-CD38-2453-T group: the target cells are reduced from 29.1% to 4.76%. Thus, 4 chimeric antigen receptor T cells CAR-CD38-2427-T, CAR-CD38-2430-T, CAR-CD38-2446-T and CAR-CD38-2453-T can target CD38+ tumor cell MM.1S and kill the tumor cell.

Example 8

ELISA method for detecting cytokine expression level

The method comprises the following steps:

sample preparation by taking T cells (CAR-CD38-2427-T, CAR-CD38-2430-T, CAR-CD38-2446-T or CAR-CD38-2453-T) and CD38+ tumor cells RAJI 72 hours after infection, counting, and adjusting cell density to 1 × 10⁶/ml, co-cultured according to an effective target ratio (E: T)1:1, i.e. T cells 1 × 10⁶，RAJI 1×10⁶Control cells were CD4+ CD8+ T cells untreated with viral infection and scored as Ctrl-T cells. The supernatant was collected at 24H and stored at-80 ℃ until use.

And (3) detection: cytokines INF-gamma, TNF-alpha were detected using the CBA method, as described in the instructions. The results are shown in FIG. 6.

As can be seen in FIG. 6, 4 chimeric antigen receptor T-cells CAR-CD38-2427-T, CAR-CD38-2430-T, CAR-CD38-2446-T targeting CD38 and CAR-CD38-2453-T all produced high concentrations of INF- γ and TNF- α, higher than the control (Ctrl-T).

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

SEQUENCE LISTING

<110> Shanghai Yao Biotechnology Ltd

<120> antibodies, chimeric antigen receptors and drugs targeting CD38 protein

<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

1 510 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 1015

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 7580

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 ValSer 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

15 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 GluGlu 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

370375 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 (10)

1. An antibody targeting CD38 protein is characterized in that the amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region are respectively shown as SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, and the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region are respectively shown as SEQ ID NO.14, SEQ ID NO.15 and SEQ ID NO. 16;

or the amino acid sequences of CDR1, CDR2 and CDR3 of the heavy chain variable region of the antibody are respectively shown in SEQ ID No.22, SEQ ID No.23 and SEQ ID No.24, and the amino acid sequences of CDR1, CDR2 and CDR3 of the light chain variable region of the antibody are respectively shown in SEQ ID No.30, SEQ ID No.31 and SEQ ID No. 32.

2. The antibody targeting the CD38 protein according to claim 1, wherein the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID No.5, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID No. 13;

or the amino acid sequence of the heavy chain variable region of the antibody is shown as SEQ ID NO.21, and the amino acid sequence of the light chain variable region of the antibody is shown as SEQ ID NO. 29.

3. The antibody targeting the CD38 protein according to claim 1 or 2, wherein the antibody is one of a full length antibody, F (ab ') 2, Fab', Fab, Fv and scFv.

4. A chimeric antigen receptor targeting CD38 protein, comprising the heavy chain variable region and the light chain variable region of an antibody targeting CD38 protein according to any one of claims 1 to 3.

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

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

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

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

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

9. A chimeric antigen receptor T cell targeting CD38 protein, wherein said T cell expresses the chimeric antigen receptor of claim 4 or 5.

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

Images