CN112442508A - Chimeric antigen receptor targeting CD22 and CD19 and application thereof - Google Patents

Chimeric antigen receptor targeting CD22 and CD19 and application thereof Download PDF

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CN112442508A
CN112442508A CN201910823106.4A CN201910823106A CN112442508A CN 112442508 A CN112442508 A CN 112442508A CN 201910823106 A CN201910823106 A CN 201910823106A CN 112442508 A CN112442508 A CN 112442508A
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王建祥
王敏
张瑜
徐颖茜
饶青
廖晓龙
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Heyuan Biotechnology Tianjin Co ltd
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Institute of Hematology and Blood Diseases Hospital of CAMS and PUMC
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Abstract

The present invention discloses nucleic acid molecules encoding a chimeric antigen receptor targeting CD22 and CD19, said chimeric antigen receptor comprising an extracellular region, a transmembrane region and an intracellular signaling region, encoding said extracellular region comprising a CD22 and CD19 binding domain, said CD22 and CD19 binding domain consisting of an antibody single chain variable fragment of CD22 and an antibody single chain variable fragment of CD 19; the antibody single-chain variable region fragment of CD22 and the antibody single-chain variable region fragment of CD19 have the amino acid sequences shown in SEQ ID No.9, SEQ ID No.10, SEQ ID No.11 or SEQ ID No.12The sequence of the amino acid sequences shown. The chimeric antigen receptor of the present invention can be used for CD19+And CD22+Treatment of B cell hematologic tumors, and combination therapy with CD19CAR-T cells or CD22CAR-T cells.

Description

Chimeric antigen receptor targeting CD22 and CD19 and application thereof
Technical Field
The invention relates to the technical field of biological medicines, in particular to a chimeric antigen receptor targeting CD22 and CD19 and application thereof.
Background
Chimeric Antigen Receptor (CAR) modified T cells are widely regarded and applied in tumor therapy as an immunotherapy strategy. The structure of a CAR generally consists of an extracellular targeting junction region (often a single chain antibody with antigen recognition), a hinge region, a transmembrane region, and an intracellular signaling region. CARs are currently classified into primary (no co-stimulatory molecule), secondary (containing one co-stimulatory molecule), and tertiary (containing two co-stimulatory molecules) depending on the number of co-stimulatory molecules added to the intracellular signaling region. Currently the most widely used are the second generation CARs.
B cell malignancies such as B cell lymphocytic leukemia (B-ALL) and lymphomas are malignant diseases caused by abnormal clonal proliferation of B lymphocytes, and despite the significant effect of current chemotherapy, 15% of children B-ALL and 60% of adult B-ALL patients have poor prognosis due to resistance to chemotherapeutic drugs; lymphoma patients still had 15% relapse after treatment with the first-line regimen, and these patients had 50% final relapse after hematopoietic stem cell transplantation. Therefore, the search for effective cellular immunotherapies to treat B-cell malignancies has been a focus of research in the hematology community.
CD19 and CD22 are antigen molecules specific to the surface of B lymphocytes. CD19 was expressed on the surface of almost ALL B-ALL and lymphoma cells, on the surface of B lineage normal hematopoietic cells, and not in normal non-hematopoietic tissues. CD22 is a B-lineage differentiation antigen expressed at various stages of B cell development, and B cells do not express CD22 after differentiating into plasma cells; 60% -80% of B cell malignant tumors express CD22, and more than 90% of Diffuse Large B Cell Lymphomas (DLBCLs) and Follicular Lymphomas (FLs) are CD22 positive; nearly ALL B precursor cell acute lymphoblastic leukemia (B-ALL) expresses CD 22; chronic lymphocytic leukemia, Hairy Cell Leukemia (HCL) also has high levels of CD22 expression. Similar to the CD19 antigen, CD22 is also B cell restricted, not expressed in other parenchymal cells, nor in hematopoietic stem cells, and therefore has high specificity as a B cell tumor antigen and has become a major therapeutic target in B cell malignancies.
At present, although CD19-CAR-T has better curative effect in treatment of refractory and relapsed B-ALL, the patients still have abnormity such as CD19 antigen mutation or loss in treatment, so that CD19-CAR-T cells cannot recognize and kill B-ALL cells, and the disease relapse is caused. CD22 and CD19 have extensive co-expression on the surface of tumor cells, and CD22 remains after the loss of CD19 antigen. Therefore, the CAR-T cells aiming at double targets of CD19 and CD22 can effectively avoid antigen variation, reduce relapse and improve the curative effect of resisting B-series malignant tumor.
However, it was found that if antigen-binding moieties targeting CD19 and CD22 are inserted into the vector by simple ligation, their killing effect after expression is poor, and therefore a specific ligation of CD19 and CD22 antigen-binding moieties is needed to improve their tumor cell killing effect.
Disclosure of Invention
In one aspect of the invention, aiming at relapse or ineffectiveness caused by escape mechanisms of B-ALL and the like during treatment of CD19CAR-T in the prior art, the invention provides a dual-target chimeric antigen receptor targeting CD22 and CD19 simultaneously and application thereof.
The technical scheme provided by the invention is as follows:
a nucleic acid molecule encoding a chimeric antigen receptor targeting CD22 and CD19, said chimeric antigen receptor comprising an extracellular region, a transmembrane region, and an intracellular signaling region, encoding said extracellular region comprising a CD22 and CD19 binding domain, said CD22 and CD19 binding domain consisting of an antibody single chain variable fragment of CD22 and an antibody single chain variable fragment of CD 19;
the antibody single-chain variable region fragment of CD22 and the antibody single-chain variable region fragment of CD19 are arranged according to the sequence of an amino acid sequence shown in SEQ ID No.9, an amino acid sequence shown in SEQ ID No.10, an amino acid sequence shown in SEQ ID No.11 or an amino acid sequence shown in SEQ ID No. 12.
In the present invention, CD22+Tumor cells and CD19+After the tumor cells are combined with the CAR of 19-22 shown in SEQ ID No.1, the CAR of 22-19 shown in SEQ ID No.2, the CAR of 19 gamma 22 shown in SEQ ID No.3 or the CAR of 22 gamma 19 shown in SEQ ID No.4, the corresponding CAR-T cells can be activated to generate a cytotoxic effect; whereas cells not expressing either CD22 or CD19 were unable to activate CAR-T cells to generate a response. Therefore, the antibody is expressed by CD19scFv-CD22scFv (shown in SEQ ID No. 1), CD22scFv-CD19scFv (shown in SEQ ID No. 2) and CD19VL-CD22VL-CD22VH-CD19VH(shown in SEQ ID No. 3) and CD22VL-CD19VL-CD19VH-CD22VH(shown in SEQ ID No. 4) is a CAR-T cell prepared by an antigen recognition region and capable of recognizing and killing CD22+Tumor cells and CD19+At the same time, the tumor cells do not produce off-target effect on cells which do not express CD19 and CD 22.
In the present invention, the inventors also found that the connection and arrangement order of the CD22scFv and the CD19scFv greatly influence the tumor cell killing effect of the combination. For example, in one embodiment of the present invention, the inventors have demonstrated that the killing effect of the 22-19CAR-T linkage is more pronounced.
The sequences of the CD22scFv and CD19scFv of the present invention can be prepared by a conventional method based on the description of SEQ ID Nos. 1 to 4. For example, the methods described in patent No. ZL201510233748.0 and patent application No. 201810549259. X.
In the present invention, the CD19scFv-CD22scFv, CD22scFv-CD19scFv, CD19V may be usedL-CD22VL-CD22VH-CD19VHAnd CD22VL-CD19VL-CD19VH-CD22VHThe amino acid sequence of (a) is subjected to random or engineered point mutation in a suitable manner, for example, for the purpose of obtaining better affinity and/or dissociation properties, and such mutated amino acid sequences are included in the scope of the present invention.
In the present invention, the light and heavy chains of the same or different antibodies of the CD22scFv and the CD19scFv are connected by a linker peptide, and the linker peptide or the combination of the linker peptides with any suitable length or suitable property can achieve the object of the present invention.
In the present invention, the nucleic acid molecule may encode a signal peptide. The signal peptide can guide the antigen recognition region and the hinge region to transfer to the outside of the cell. Any suitable signal peptide or combination of signal peptides can accomplish the objectives of the present invention.
Preferably, in one embodiment of the invention, the extracellular region encoded by the nucleic acid molecule of the invention further comprises a signal peptide constructed at the amino terminus of the chimeric antigen receptor or an amino acid sequence having 90-99% identity to the signal peptide, the signal peptide being the signal peptide sequence in CD8 α or GM-CSF.
More preferably, the signal peptide is the signal peptide shown as SEQ ID NO. 13.
In one embodiment of the invention, said CD22 and CD19 binding domains encoded by the nucleic acid molecules of the invention are linked to said transmembrane region encoded thereby by a hinge region. Any suitable hinge region sequence may accomplish the objects of the present invention. Preferably, in one embodiment of the present invention, the hinge region is CD8 α.
In the present invention, the nucleic acid molecule also encodes a transmembrane domain. Any suitable transmembrane domain can accomplish the objects of the present invention. Preferably, in one embodiment of the invention, the transmembrane region is a transmembrane domain of a protein selected from the group consisting of: an α, β, or zeta chain of a T cell receptor, CD3 ∈ CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, or CD 154.
In the present invention, the intracellular signaling region encoded by the nucleic acid molecule further comprises a co-stimulatory factor.
Preferably, the co-stimulatory factor is one or several of the functional signal domains obtained by the amino acid sequence selected from the following proteins or from 90-99% identity to said proteins: MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, lymphocyte activation signaling molecules, activated NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CDS, ICAM-1, LFA-1, 4-1BB, B27-H27, CD278, GITR, BAFFR, LIGHT, HVEM, KIRDS 27, SLAMF 27, NKp 27, CD27 alpha, CD27 beta, IL2 27 gamma, IL7 27 alpha, ITGA 27, VLA 27, CD27 alpha, CD27, CD 3649, CD27, ITGA 27, VLA 27, CD27, GAITGB, GAITCD 27, GAITGL 27, GAITGB, CD27, GAITGL 27, CD27, GAITGB 11, CD27, GAITGB 27, GAITGL 27, CD27, GAITGB 11, CD27, GAITGL 27, CD27, GAITGB 11, CD27, GAITGL 27, GAITGB 11, CD27, GAITGL 27, CD27, GAITGB 11, CD 27.
More preferably, in one embodiment of the invention, the co-stimulatory factor is CD28 or 4-1BB or an amino acid sequence with 90-99% identity thereto.
Also, the nucleic acid molecules of the invention encode any suitable intracellular signaling domain. Can be an amino acid sequence with which the intracellular signaling structure of CD3 ζ has 90-99% identity.
Preferably, the chimeric antigen receptor encoded by the nucleic acid molecule of the invention is CD19scFv-CD22scFv, CD22scFv-CD19scFv, CD19VL-CD22VL-CD22VH-CD19VHAnd CD22VL-CD19VL-CD19VH-CD22VHThe structure formed by serially connecting the antigen recognition region, the CD8 alpha hinge region, the transmembrane region, and the 4-1BB and CD3 zeta intracellular signal domains is a signal transduction domain, and the sequence of the signal transduction domain is shown as SEQ ID NO. 5-8.
In addition, any peptide chain, which may be an oligopeptide or a polypeptide, may be inserted as a spacer at a suitable position between the above antigen recognition region, hinge region, transmembrane region and intracellular signal region.
The method for producing the nucleic acid molecule can be produced by a known technique such as chemical synthesis or PCR amplification based on the nucleotide sequence of the domains such as the antigen recognition region, the hinge region, the transmembrane region, and the intracellular signal region. In general, the codons encoding the amino acids of the domains described above can be optimized to optimize their expression in a host cell. The information on the above-mentioned nucleotide sequence can be obtained by searching a database such as a known literature or NCBI (https:// www.ncbi.nlm.nih.gov /).
In one embodiment of the present invention, the mouse anti-human CD22 monoclonal antibody hybridoma cell line (HIB22) and mouse anti-human CD19 monoclonal antibody hybridoma cell line (HIB19) used by the inventors were developed by the applicant, the hospital for hematological diseases (institute of hematology, chinese medical academy of sciences), and the monoclonal antibodies produced by the hybridomas are commercially available and can be used for the detection and diagnosis of CD 22-positive and CD 19-positive acute lymphoblastic leukemia and lymphoma, respectively.
In another aspect of the invention, there is provided a chimeric antigen receptor encoded by the nucleic acid molecule described above.
The extracellular region of the above chimeric antigen receptor comprises CD22 and CD19 binding domains, the CD22 and CD19 binding domains consist of an antibody single chain variable fragment of CD22 and an antibody single chain variable fragment of CD 19;
the antibody single-chain variable region fragment of CD22 and the antibody single-chain variable region fragment of CD19 are arranged according to the sequence of an amino acid sequence shown in SEQ ID No.9, an amino acid sequence shown in SEQ ID No.10, an amino acid sequence shown in SEQ ID No.11 or an amino acid sequence shown in SEQ ID No. 12.
Preferably, the chimeric antigen receptor of the present invention is CD19scFv-CD22scFv, CD22scFv-CD19scFv, or CD19VL-CD22VL-CD22VH-CD19VHOr CD22VL-CD19VL-CD19VH-CD22VHThe structure formed by serially connecting the antigen recognition region, the CD8 alpha hinge region, the transmembrane region, and the 4-1BB and CD3 zeta intracellular signal domains is a signal conduction domain, and the amino acid sequence is shown as SEQ ID NO.1-4 of the sequence table.
In another aspect of the invention, there is provided a vector comprising the nucleic acid molecule described above.
In the present invention, the vector may be a linear vector or a cyclic vector. The vector may be a non-viral vector such as a plasmid, a viral vector, or a vector using a transposon. The vector can contain regulatory sequences such as a promoter, a terminator and the like, and marker sequences such as a drug resistance gene, a reporter gene and the like. In addition, the vector may also comprise a sequence encoding a suicide gene, and the number of CAR-T cells in vivo can be controlled by administering a substance that activates the suicide gene according to the course of treatment.
Examples of the viral vector include a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector. In one embodiment of the invention, a lentiviral expression vector is used.
In another aspect of the invention, a cell is provided, said cell comprising a nucleic acid molecule as described above, a chimeric antigen receptor as described above or a vector as described above.
In one embodiment of the present invention, the cell is a human T cell. The T cell can be obtained from body fluid such as blood, bone marrow, etc., tissue such as spleen, thymus, lymph, etc., or cancer tissue such as primary tumor, metastatic tumor, cancerous ascites, etc., by separation and purification. Also, the T cell may be CD4+T cell, CD8+T cells, α β T cells, or γ δ T cells. The T cells may be replaced by NK cells in a suitable manner, which is also considered to be comprised within the scope of the present invention.
In another aspect of the invention, the invention provides an application of the nucleic acid molecule in preparing a B cell blood tumor resisting medicine.
In another aspect of the invention, the invention provides an application of the chimeric antigen receptor in preparing a B cell blood tumor resisting medicine.
In another aspect of the invention, the invention provides an application of the carrier in preparing a B cell blood tumor resisting medicine.
In another aspect of the invention, the invention provides an application of the cell in preparing a B cell blood tumor resisting medicine.
Preferably, the application is the application in preparing anti-B cell blood tumor medicines with wide co-expression of CD19 and CD22 or relapse or ineffectiveness due to antigen variation after treatment of CD19 CAR-T.
The above-mentioned ineffectiveness means that after the patient is treated by the CD19CAR-T, the patient has no reactivity to the CD19CAR-T due to the loss, mutation and the like of CD19 antigen, and the treatment is ineffective.
Such B cell hematologic tumors include, but are not limited to, B cell lymphoma, Acute Lymphocytic Leukemia (ALL), etc., as long as they express CD19 and CD22 during the pathological process. Preferably, the B cell hematological tumor is a B cell lymphoma or acute B lymphocyte leukemia.
In another aspect of the present invention, a pharmaceutical composition is provided, which comprises the above nucleic acid molecule, the above chimeric antigen receptor, the above vector or the above cell, and a pharmaceutically acceptable carrier.
The pharmaceutical composition of the present invention may contain, in addition to the above-mentioned components, any pharmaceutically acceptable additives, for example, physiological saline, cell culture medium, glucose, water for injection, glycerol, ethanol, and a combination thereof, a stabilizer, a surfactant, a preservative, an isotonic agent, and the like.
Likewise, the pharmaceutical compositions of the present invention may also be used in combination with other suitable anti-cancer agents. For example, vincristine, daunorubicin, asparaginase, cyclophosphamide, prednisone, etc.
Preferably, the pharmaceutical composition of the invention further comprises a nucleic acid molecule encoding a chimeric antigen receptor of CD19 and CD22, a chimeric antigen receptor of CD19 and CD22, a vector comprising a nucleic acid molecule encoding a chimeric antigen receptor of CD19 and CD22 or a cell comprising a chimeric antigen receptor of CD19 and CD 22.
The nucleic acid molecule encoding the chimeric antigen receptor of CD19 and CD22, the chimeric antigen receptor of CD19 and CD22, the vector comprising the nucleic acid molecule encoding the chimeric antigen receptor of CD19 and CD22, or the cell comprising the chimeric antigen receptor of CD19 and CD22 described above may be any suitable nucleic acid molecule encoding the chimeric antigen receptor of CD19 and CD22, the chimeric antigen receptor of CD19 and CD22, the vector comprising the nucleic acid molecule encoding the chimeric antigen receptor of CD19 and CD22, or the cell comprising the chimeric antigen receptor of CD19 and CD 22.
In another aspect of the invention, there is provided a use of the above-mentioned nucleic acid molecule in the treatment of a B cell hematological tumor.
In another aspect of the invention, there is provided a use of the chimeric antigen receptor described above for the treatment of a B cell hematological tumor.
In another aspect of the invention, there is provided a use of the above vector for treating a B cell hematological tumor.
In another aspect of the invention, there is provided a use of the above cell in the treatment of a B cell hematological tumor.
In another aspect of the invention, the invention provides a use of the above pharmaceutical composition in the treatment of B cell hematological tumors.
The invention has the beneficial effects that:
the invention synthesizes and rearranges the light and heavy chains of mouse anti-human CD22scFv and mouse anti-human CD19scFv by nucleic acid molecule chemical synthesis or PCR technology to obtain CD19scFv-CD22scFv, CD22scFv-CD19scFv and CD19VL-CD22VL-CD22VH-CD19VHAnd CD22VL-CD19VL-CD19VH-CD22VHAnd cloning into slow virus expression vector containing signal peptide and CD8 alpha-4-1 BB-CD3 zeta, and packing into slow virus expression vector carrying CD19scFv-CD22scFv-CD8 alpha-4-1 BB-CD3 zeta, CD22scFv-CD19scFv-CD8 alpha-4-1 BB-CD3 zeta, and CD19V zetaL-CD22VL-CD22VH-CD19VH-CD8 alpha-4-1 BB-CD3 zeta or CD22VL-CD19VL-CD19VH-CD22VH-a lentiviral vector encoding a gene encoding CD8 α -4-1BB-CD3 ζ. T cells are infected with lentivirus, and the T cells express the chimeric antigen receptor. The chimeric antigen receptor modified T cell pair CD19 is proved by detecting cytokines secreted by the T cell through flow cytometry, degranulation analysis experiment and ELISA+、CD22+And CD19+/CD22+The B cell lymphoma cell and B cell lymphocytic leukemia cell have strong killing effect, almost have no killing effect on cells which do not express CD19 and CD22, and effectively preventOff-target effects are observed. The chimeric antigen receptor of the present invention can be used for CD19+And CD22+Treatment of B cell hematologic tumors, and combination therapy with CD19CAR-T cells or CD22CAR-T cells.
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FIG. 1 shows the lentiviral expression vectors of examples of the present invention, CD19scFv-CD22scFv-CD8 α -4-1BB-CD3 ζ, CD22scFv-CD19scFv-CD8 α -4-1BB-CD3 ζ, CD19VL-CD22VL-CD22VH-CD19VH-CD8 alpha-4-1 BB-CD3 zeta and CD22VL-CD19VL-CD19VH-CD22VHThe electrophoretic identification picture of the restriction endonuclease cut fragment of CD8 alpha-4-1 BB-CD3 zeta in the nucleic acid molecular weight marker lane of 15kb in FIG. A; 2 is a DNA fragment (2314bp) and a vector fragment (7228bp) encoding CD19scFv-CD22scFv-CD8 α -4-1BB-CD3 ζ obtained by double digestion of a lentivirus expression plasmid CD19scFv-CD22scFv-CD8 α -4-1BB-CD3 ζ with endonucleases Nhe I and Not I, 3 is a DNA fragment (2314bp) and a vector fragment (7228bp) encoding CD22scFv-CD19scFv-CD8 α -4-1BB-CD3 ζ obtained by double digestion of a lentivirus expression plasmid CD22scFv-CD19scFv-CD8 α -4-1BB-CD3 ζ with endonucleases Nhe I and Not I, and in FIG. B, 4 is a 15kb nucleic acid molecular weight marker lane; 5 is the double digestion of the lentivirus expression plasmid CD19V with endonucleases Nhe I and Not IL-CD22VL-CD22VH-CD19VHCD19V encoded by-CD 8 alpha-4-1 BB-CD3 zetaL-CD22VL-CD22VH-CD19VHDNA fragment (2233bp) and vector fragment (7234bp) of-CD 8 alpha-4-1 BB-CD3 zeta, 6 is lentiviral expression plasmid CD22V double-digested with endonucleases NheI and Not IL-CD19VL-CD19VH-CD22VHCD22V encoded by-CD 8 alpha-4-1 BB-CD3 zetaL-CD19VL-CD19VH-CD22VH-DNA fragment (2233bp) and vector fragment (7234bp) of CD8 α -4-1BB-CD3 ζ;
FIG. 2 is a schematic diagram of an example of a CD22scFv-CD19scFv-CD8 α -4-1BB-CD3 ζ lentivirus expression vector in which the counterclockwise sequence is a forward gene segment and the clockwise sequence is a reverse gene segment;
FIG. 3 shows an embodiment of the present invention using flow cytometryThe double target point CD19scFv-CD22scFv-CD8 alpha-4-1 BB-CD3 zeta, CD22scFv-CD19scFv-CD8 alpha-4-1 BB-CD3 zeta, CD19V zetaL-CD22VL-CD22VH-CD19VH-CD8 alpha-4-1 BB-CD3 zeta and CD22VL-CD19VL-CD19VH-CD22VHResults of expression of CAR molecules in CD8 a-4-1 BB-CD3 ζ -modified T cells (denoted 19-22CAR-T, 22-19CAR-T, 19 gamma 22CAR-T, and 22 gamma 19CAR-T, respectively), while showing expression results of CAR molecules in single-target CD19scFv-CD8 a-4-1 BB-CD3 ζ and CD22scFv-CD8 a-4-1 BB-CD3 ζ -modified T cells (denoted 19CAR-T and 22CAR-T, respectively), to compare the functions of single-target and dual-target CAR-T, GFP as expression of a marker protein carried by the vector, F (ab')2The expression of the rabbit anti-mouse IgG marker CD22scFv and CD19scFv on the surface of the T cell;
FIG. 4 shows the detection of target cells used in the examples of the present invention by flow cytometry: the expression result graphs of CD19 and CD22 target antigen molecules in a Burkitt lymphoma cell line Namalwa, a chronic myelocytic leukemia cell line MV4-11-CD19 infected with a CD19 antigen molecule, a chronic myelocytic leukemia cell line MV4-11-CD22 infected with a CD22 antigen molecule and a wild type MV4-11 cell, wherein A is the positive expression rate of the CD19 target antigen molecule; b is the positive rate of CD22 target antigen molecule expression;
FIG. 5 is a graph showing the results of flow cytometry for detecting the survival rate of tumor cells remaining after co-culturing T cells and target cells according to the present invention, wherein vector-T is a control group of T cells transfected with empty vector; 19CAR-T for CD19scFv-CD8 alpha-4-1 BB-CD3 zeta modified T cells, 22CAR-T for CD22scFv-CD8 alpha-4-1 BB-CD3 zeta modified T cells, 19-22CAR-T for CD19scFv-CD22scFv-CD8 alpha-4-1 BB-CD3 zeta modified T cells, 22CAR-T for CD22scFv-CD19scFv-CD8 alpha-4-1 BB-CD3 zeta modified T cells, 19 gamma 22CAR-T for CD19VL-CD22VL-CD22VH-CD19VHExperimental group of-CD 8 alpha-4-1 BB-CD3 zeta-modified T cells, 22 gamma 19CAR-T being CD22VL-CD19VL-CD19VH-CD22VH-experimental panel of CD8 α -4-1BB-CD3 ζ modified T cells; a and B are vector-T, 19CAR-T, 22CAR-T, 19-22 respectively when the target cell is a Namalwa cell lineGraphs of the survival of tumor cells remaining after 24 hours of co-culture of CAR-T and 22-19CAR-T with target cells at effective-to-target ratios of 1:8, 1:4, 1:2, and 1:1, respectively, and a flow chart of the survival of tumor cells remaining after 48 hours of co-culture at effective-to-target ratios of 1: 1; c and D are MV4-11-CD19 cell line respectively, the residual tumor cell survival rate after co-culturing vector-T, 19CAR-T, 22CAR-T, 19-22CAR-T and 22-19CAR-T with target cells at effective target ratio of 1:8, 1:4, 1:2 and 1:1 respectively for 24 hours, and the residual tumor cell survival rate after co-culturing at effective target ratio of 1:1 for 48 hours are shown in a flow chart; e and F are graphs of the survival of tumor cells remaining after co-culturing vector-T, 19CAR-T, 22CAR-T, 19-22CAR-T, and 22-19CAR-T with target cells at effective target ratios of 1:8, 1:4, 1:2, and 1:1, respectively, for 24 hours when the target cells are MV4-11-CD22 cell lines, and flow charts of the survival of tumor cells remaining after co-culturing at effective target ratios of 1:1 for 48 hours, respectively; g and H are graphs of the survival rate of the residual tumor cells after 24 hours of co-culture of Vec-T, 19-22CAR-T and 22-19CAR-T with the target cells at effective target ratios of 1:8, 1:4, 1:2 and 1:1, respectively, when the target cells are wild-type MV4-11 cell lines, and a flow chart of the survival rate of the residual tumor cells after 48 hours of co-culture at an effective target ratio of 1:1, respectively; i is a graph of the survival rate of tumor cells remaining after co-culturing vector-T, 19 gamma 22CAR-T and 22 gamma 19CAR-T with target cells at effective target ratios of 1:2, 1:1 and 2:1, respectively, for 24 hours when the target cells are MV4-11-CD19 and MV4-11-CD22 mixed at a ratio of 1: 1; j is a graph of the survival rate of tumor cells remaining after 24 hours of co-culture of vecor-T, 19 gamma 22CAR-T and 22 gamma 19CAR-T with target cells at effective target ratios of 1:2, 1:1 and 2:1, respectively, when the target cells were MV 4-11;
FIG. 6 is a graph of the results of degranulation measurements of killing by vecor-T and CAR-T on Namalwa, MV4-11-CD19, MV4-11-CD22 and MV4-11 (effective target ratio 1:1) in examples of the present invention, wherein CAR-T in A is 19CAR-T, 22CAR-T, 19-22CAR-T and 22-19CAR-T, and CAR-T in B is 19 gamma 22CAR-T and 22 gamma 19CAR-T, respectively;
FIG. 7 is a graph of the results of the levels of cytokines TNF- α (A) IL-2(B) and IFN- γ (C) released by T cells 48 hours after vecor-T, 19CAR-T, 22CAR-T, 19-22CAR-T and 22-19CAR-T were co-cultured with four target cells Namalwa, MV4-11-CD19, MV4-11-CD22 and MV4-11, respectively, at an effective target ratio of 1:1 in accordance with an example of the present invention;
FIG. 8 is a graph showing the results of detecting the expression of CD22 and CD19 target antigen molecules in Bone Marrow Mononuclear Cells (BMMCs) of B-ALL patients by flow cytometry, wherein P1 to P4 represent patient numbers;
FIG. 9 is a graph showing the results of flow assays of residual tumor cell viability after culturing BMMCs in patients with 22-19CAR-T and 19-22CAR-T and B-ALL at an effective target ratio of 1:4 for 48 hours according to example of the present invention;
FIG. 10 is a graph of the results of a degranulation assay of the killing effect of 22-19CAR-T and 19-22CAR-T on BMMCs of ALL patients in an example of the invention;
FIG. 11 is a graph showing the results of evaluation of the effects of 22-19CAR-T and 19-22CAR-T in mice in examples of the present invention, wherein 6-8 week NOD/SCID female mice were randomly divided into two groups and injected via tail vein on day 0 to 5X 106The Namalwa cells were injected into the tail vein at 1X 10 times on days 5, 9 and 12, respectively7vec-T cells or CAR-T cells (as shown in a), mice were monitored weekly for weight change after T cell injection (as shown in B), tumor burden plot is shown in panel C, mouse survival curve is shown in D, and survival was calculated using SPSS software.
DESCRIPTION OF THE SEQUENCES
SEQ ID NO.1 is the amino acid sequence of CD19scFv-CD22scFv-CD8 alpha-4-1 BB-CD3 zeta of the present invention;
SEQ ID NO.2 is the amino acid sequence of CD22scFv-CD19scFv-CD8 alpha-4-1 BB-CD3 zeta of the present invention;
SEQ ID NO.3 is CD19V of the present inventionL-CD22VL-CD22VH-CD19VH-the amino acid sequence CD8 α -4-1BB-CD3 ζ;
SEQ ID NO.4 is CD22V of the present inventionL-CD19VL-CD19VH-CD22VH-the amino acid sequence CD8 α -4-1BB-CD3 ζ;
SEQ ID NO.5 is a nucleic acid sequence of the CD19scFv-CD22scFv-CD8 α -4-1BB-CD3 ζ of the present invention;
SEQ ID NO.6 is a nucleic acid sequence of the CD22scFv-CD19scFv-CD8 α -4-1BB-CD3 ζ of the present invention;
SEQ ID NO.7 is CD19V of the present inventionL-CD22VL-CD22VH-CD19VH-the nucleic acid sequence CD8 α -4-1BB-CD3 ζ;
SEQ ID NO.8 is CD22V of the present inventionL-CD19VL-CD19VH-CD22VH-the nucleic acid sequence CD8 α -4-1BB-CD3 ζ;
SEQ ID NO.9 is the amino acid sequence of the antigen recognition region of CD19scFv-CD22scFv-CD8 alpha-4-1 BB-CD3 zeta of the present invention;
SEQ ID NO.10 is the amino acid sequence of the antigen recognition region of CD22scFv-CD19scFv-CD8 alpha-4-1 BB-CD3 zeta of the present invention;
SEQ ID NO.11 is CD19V of the present inventionL-CD22VL-CD22VH-CD19VH-the amino acid sequence of the antigen recognition region CD8 α -4-1BB-CD3 ζ;
SEQ ID NO.12 is CD22V of the present inventionL-CD19VL-CD19VH-CD22VH-the amino acid sequence of the antigen recognition region CD8 α -4-1BB-CD3 ζ;
SEQ ID No.13 is the amino acid sequence of the signal peptide in the chimeric antigen receptor targeting CD19 and CD22 according to the present invention;
SEQ ID No.14 is the amino acid sequence of CD8 alpha-4-1 BB-CD3 zeta in the chimeric antigen receptor targeting CD19 and CD22 according to the present invention.
Detailed Description
The invention discloses chimeric antigen receptors targeting CD22 and CD19 and application thereof, and can be realized by appropriately modifying process parameters by referring to the content in the text. It is expressly intended that all such alterations and modifications which are obvious to those skilled in the art are deemed to be incorporated herein by reference, and that the techniques of the invention may be practiced and applied by those skilled in the art without departing from the spirit, scope and range of equivalents of the invention.
In the present invention, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art.
In order to make those skilled in the art better understand the technical solution of the present invention, the following detailed description of the present invention is provided with reference to specific embodiments.
Example 1: construction of a Dual-target chimeric antigen receptor vector
1. The plasmid containing the CD8 alpha-4-1 BB-CD3 zeta fragment constructed by the inventor in the previous period is cut by BamH I and EcoR I endonucleases to obtain the CD8 alpha-4-1 BB-CD3 zeta fragment, and the amino acid sequence of the fragment is shown in SEQ ID NO. 5. The plasmid containing the CD8 alpha-4-1 BB-CD3 zeta fragment can be prepared by any suitable method in the prior art, for example, the patent with the patent number ZL 201510233748.0.
2. The synthesized CD22scFv-CD19scFv fragment, CD19scFv-CD22scFv, CD19VL-CD22VL-CD22VH-CD19VHAnd CD22VL-CD19VL-CD19VH-CD22VHThe fragments are respectively connected with a target vector, and the constructed CD22scFv-CD19scFv-CD8 alpha-4-1 BB-CD3 zeta CAR (22-19CAR), CD19scFv-CD22scFv-CD8 alpha-4-1 BB-CD3 zeta CAR (19-22CAR), CD19VL-CD22VL-CD22VH-CD19VH-CD8 alpha-4-1 BB-CD3 zeta (19 gamma 22CAR-T) and CD22VL-CD19VL-CD19VH-CD22VHThe target vector of-CD 8 alpha-4-1 BB-CD3 zeta (22 gamma 19CAR-T) was identified by digestion with endonucleases Nhe I and Not I. The result is shown in FIG. 1, and the enzyme digestion result shows that the positive clone contains the target band and the sequencing identification is correct. For example, the CD22scFv-CD19scFv-CD8 α -4-1BB-CD3 ζ CAR vector schematic is shown in FIG. 2.
Example 2: preparation of double-target chimeric antigen receptor lentivirus modified T cell
1. 22-19CAR, 19-22CAR, 19 gamma 22CAR and 22 gamma 19CAR expression plasmids and packaging plasmids PRSV-Rev, pMDlg-PRRE and pMD.2G are respectively extracted by using an EndoFree Plasmid Maxi Plasmid extraction kit (QIAGEN). Each CAR plasmid and packaging plasmid (four plasmids) was replaced with 12.2: 4.11: 8.75: 3.5 ratios were transfected with PEI transfection reagent (polyscience Inc.) (see PEI transfection reagent for details). The fresh culture solution is replaced 12 hours after transfection, then virus supernatants are respectively collected 24 hours and 48 hours, centrifuged for 15 minutes at 4 ℃, 3000rpm, filtered by a 0.45 mu m filter, ultracentrifuged for 10 times at 50000g and 4 ℃, 1.5 hours, and then stored at-80 ℃.
Preparation of T cells: 10ml of fresh healthy human peripheral blood was taken, and T cells were extracted using RosetteSep T cell enrichment Cocktail (Stemcell Co.) and Ficoll-Paque PLUS (GE Healthcare Co.) (the specific procedure was according to RosetteSep T cell enrichment Cocktail instructions). According to cell: magnetic beads are 1: anti-CD 3/CD28 magnetic beads (Gibco) were added at a ratio of 1, and the T cells were cultured for 24 hours before transfection.
3. Lentivirus infected T cells and culture of infected T cells: taking out virus supernatant at-80 deg.C, thawing at room temperature, and adding 1 × 10 of the virus supernatant6T cells were added to 100. mu.l of virus supernatant and Polybrene was added to a final concentration of 8. mu.g/ml. Centrifuge at 32 ℃ and 1800rpm for 1.5 hours, transfer to 5% CO2And culturing at 37 ℃ in an incubator.
4. Flow cytometry detects the positive rate of CAR-modified T cells: cells were collected and labeled rabbit anti-mouse IgG F (ab')2Antibody, flow cytometry analysis of T cells F (ab')2And expression of GFP. As shown in FIG. 3, the positive rate of CAR-T was 70% or more.
Experimental example 1: killing effect of chimeric antigen receptors 22-19CAR, 19-22-CAR, 19 gamma 22CAR and 22 gamma 19CAR lentivirus-modified T cells on leukemia cells
1. Expression level of CD22 in hematological tumor cell lines:
namalwa, MV4-11 cell lines were purchased from ATCC, USA, MV4-11-CD19 and MV4-11-CD22 were monoclonal cell lines selected after MV4-11 cell lines were infected with CD19 and CD22, respectively. After the respective cultivation, 5X 10 of each sample was aspirated5After washing the cell suspension with PBS for 2 times, the cell suspension was labeled with PE anti-human CD19 monoclonal antibody and APC anti-human CD22 monoclonal antibody (Biolegend), respectively, and the labeled PE-isotype and APC-isotype were used as control groups, and incubated on ice for 30 minutes. The expression levels of CD19 and CD22 were measured using flow cytometry and the results are shown in figure 4. Wherein A is the expression positive rate of the CD19 target antigen molecule; b is the positive rate of CD22 target antigen molecule expression; the CD19 positive rate of Namalwa and MV4-11-CD19 is more than 95%, the CD22 positive rate of Namalwa and MV4-11-CD22 is more than 95%, and the wild type MV4-11 hardly expresses CD19 and CD 22.
Flow assay of residual tumor cells after coculture of CAR-modified T cells with Namalwa, MV4-11-CD19, MV4-11-CD22 and wild-type MV4-11 cell line:
the cells were aligned at 2X 105Cell/well inoculation 24-well culture plates, 2.5X 10 added separately4(E:T＝1:8)、5×104(E:T＝1:4)、1×105(E:T＝1:2)、2×105(E:T＝1:1)、4×105(E: T ═ 2:1), concentration of CAR-T cells, and transfection of empty vector T cells without CAR (vector-T) were repeated as a control group, and co-cultured in an incubator. The co-cultured cells were labeled with Namalwa, MV4-11-CD19, MV4-11-CD22 and a wild-type MV4-11 cell line with PE anti-human CD19 monoclonal antibody and APC anti-human CD22 monoclonal antibody (Biolegend), and T cells were labeled with PE-Cy7 anti-human CD3 monoclonal antibody (Biolegend), and the residual cells were detected by flow cytometry. The results are shown in FIG. 5, 1) compared to vecor-T, 19CAR-T, 22CAR-T, 19-22CAR-T and 22-19CAR-T all were able to significantly kill CD19+CD22+Target cell Namalwa (A, B of fig. 5); 2) compared with vecor-T and 22CAR-T, 19-22CAR-T and 22-19CAR-T all significantly killed MV4-11-CD19 target cells (C, D of FIG. 5); 3) compared with vecor-T and 19CAR-T, 22CAR-T, 19-22CAR-T and 22-19CAR-T all significantly killed MV4-11-CD22 target cells (E, F of FIG. 5); 4) similar to vecor-T, 19-22CAR-T and 22-19CAR-T did not kill CD19/CD22 double negative target cell MV4-11 (FIG. 5, G, H); 5) both the 19 gamma 22CAR-T and 22 gamma 19CAR-T significantly killed MV4-11-CD19 and MV4-11-CD22 target cells (I of FIG. 5) as compared to vecor-T; 6) similar to vecor-T, 19 gamma 22CAR-T and 22 gamma 19CAR-T did not kill CD19/CD22 double negative target cells MV4-11 (J of FIG. 5). The results demonstrate that 19-22CAR-T, 22-19CAR-T, 19 gamma 22CAR-T and 22 gamma 19CAR-T have significant dual-target advantages over CD19+、CD22+And CD19+/CD22+The target cells have high-efficiency specific killing effect. In the above experiment, the killing effect of 22-19CAR-T is more remarkable.
3. Degranulation assay analysis of activation of CAR-modified T cells:
CAR-T and vecor-T cells were contacted with Namalwa, MV4-11-CD19, MV4-11-CD22 and wild type, respectivelyMV4-11 cell line is co-cultured according to an effective target ratio of 1:1, and anti-CD107a antibody and monensin are added into a co-culture system; detection of GFP 4h later using flow cytometer+Expression level of cell surface CD107 a. The results are shown in FIG. 6 at A, B, 1) and Namalwa (CD 19) as the target cell+CD22+) After co-culture, the degranulation levels of the 19CAR-T, 22CAR-T, 19-22CAR-T, 22-19CAR-T, 19 gamma 22CAR-T and 22 gamma 19CAR-T groups were above 10% on average, and the degranulation level of vecor-T was below 2%; 2) after coculture with target cells MV4-11-CD19, the degranulation levels of the 19CAR-T, 19-22CAR-T, 22-19CAR-T, 19 gamma 22CAR-T and 22 gamma 19CAR-T groups were above 25% on average, while the degranulation levels of vecor-T and 22CAR-T were below 2%; 3) the degranulation levels of the 22CAR-T, 19-22CAR-T, 22-19CAR-T, 19 gamma 22CAR-T and 22 gamma 19CAR-T groups were above 15% and the degranulation levels of vecor-T and 19CAR-T were below 2% after coculture with target cell MV4-11-CD 22; 4) after coculture with wild type MV4-11, vecor-T, 19CAR-T, 22CAR-T, 19-22CAR-T, 19 gamma 22CAR-T and 22 gamma 19CAR-T had a degranulation mean below 2%, demonstrating that 19-22CAR-T, 22-19CAR-T, 19 gamma 22CAR-T and 22 gamma 19CAR-T can significantly and specifically activate CD19+Or CD22+The target cell of (1).
ELISA for the detection of the levels of the cytokines IFN-. gamma., TNF-. alpha.and IL-2 in the supernatant of the co-culture of lymphoma cell lines with CAR-T cells:
namalwa, MV4-11-CD19, MV4-11-CD22 and wild type MV4-11 cell lines were separately cultured in a 2X 10 manner5Cells/well were seeded in 24-well plates. At 2X 10 per hole5The cells were added with CAR-T, vecor-T cells, and the culture medium was supplemented to 1ml for co-culture in an incubator for 24 hours. ELISA detection kit (R) adopting human IFN-gamma, TNF-alpha and IL-2&Company D), and detecting the co-culture supernatant (see the description of ELISA detection kit for specific steps). The results are shown in FIG. 7, 1) and CD19+CD22+After the target cells are cultured together with Namalwa, the release levels of three factors of 19CAR-T, 19-22CAR-T and 22-19CAR-T groups are obviously increased compared with vecor-T; the secretion levels of TNF-alpha were significantly higher in the 19-22CAR-T and 22-19CAR-T groups than in the 19CAR-T group, and IL-2 was significantly higher in the 19-22CAR-T and 22-19CAR-T groups than in the 22CAR-T group. 2) Co-culture with MV4-11-CD19After feeding, the release levels of the three factors of the 19CAR-T, 19-22CAR-T and 22-19CAR-T groups were significantly higher than those of vecor-T and 22 CAR-T. 3) After co-culture with MV4-11-CD22, the levels of TNF- α and IFN- γ release were significantly higher in the 22CAR-T, 19-22CAR-T and 22-19CAR-T groups than in the vecor-T and 19 CAR-T; while the secretion level of IL-2 showed that the 19-22CAR-T and 22-19CAR-T groups were superior to the 22CAR-T group. 4) The release levels of the three factors vecor-T, 19CAR-T, 22CAR-T, 19-22CAR-T were lower after co-culture with wild type MV 4-11. Demonstrating that 19-22CAR-T and 22-19CAR-T can specifically activate CD19+、CD22+And CD19+CD22+And release cytokines at levels superior to 19CAR-T or 22 CAR-T.
Expression levels of Bone Marrow Mononuclear Cells (BMMCs) CD19 and CD22 in ALL patients:
the patient specimens were obtained from the hematological hospital of the academy of medical sciences of China and informed consent of the patients was obtained. After separating BMMCs by Ficoll gradient centrifugation, each sample was aspirated at 5X 105After washing the cell suspension with PBS 2 times, the cell suspension was labeled with PE anti-human CD19 monoclonal antibody, APC anti-human CD22 monoclonal antibody (Biolegend), PE-isotype, and APC-isotype (control), and incubated on ice for 30 minutes. The ratio and intensity of BMMCs expressing CD19 and CD22 of each patient were examined by flow cytometry, as shown in FIG. 8, in which P1-P4 represent patients No. 1-4.
Flow assay of residual tumor cells after coculture of CAR-modified T cells with BMMCs from ALL patients:
pressing the cells at 4X 105Cell/well inoculation 24-well culture plates, 1X 10 added separately5CAR-modified T cells of (E: T ═ 1:4) were transfected with empty vector T cells (vec-T) containing no CAR as a control group and co-cultured in an incubator for 48 h. The co-cultured cells were labeled with leukemia cells of ALL patients with PE anti-human CD19 mab and APC anti-human CD22 mab (Biolegend), and T cells with APC-Cy7 anti-human CD3 mab (Biolegend), and residual cells were detected by flow cytometry. The results are shown in FIG. 9 and show that CAR-T and CD19+/CD22+Patient 1-4 BMMCs co-cultured for 48 hours, 19-22CAR-T group retained CD19, respectively+/CD22+The cells retained CD19 for 3.03%, 0.24%, 1.34%, 2.87%, 22-19CAR-T groups respectively+/CD22+The cells were 4.10%, 0.45%, 1.68%, 3.89%, and the control retention was 46.58%, 63.95%, 23.76%, 44.88%. As can be seen from the above results, CAR-T vs. CD19+/CD22+The leukemia primary BMMCs have killing effect.
Degranulation assay after coculture of CAR-modified T cells with BMMCs of ALL patients activation of CAR-modified T cells was analyzed:
respectively carrying out co-culture on the CAR-T and vecor-T cells and BMMCs of patients 1-4 according to an effective target ratio of 1:1, and adding an anti-CD107a antibody and monensin into a co-culture system; 4h later, CD3 detection by flow cytometry+Expression level of cell surface CD107 a. The results are shown in FIG. 10, and show that the activation percentages of 19-22CAR-T cells in the CAR-T and No.1-4 patient coculture system are 35.53%, 40.37%, 31.37% and 30.20%, respectively; the percent 22-19CAR-T cell activation was 51.21%, 33.04%, 18.01%, 50.23%, respectively. In the co-culture system of vec-T and patients No.1-4, the activation percentages of T cells are 3.07%, 1.73%, 3.43% and 2.40%, respectively. From the above results, it can be shown that CAR-T and vecor-T have significant differences in activation.
CAR-modified T cells in CD19+/CD22+Role in lymphoma mouse model:
selecting 6-8 weeks NOD/SCID female mice, randomly dividing into two groups, and injecting Namalwa cells 5 × 10 via tail vein6A/only; on days 5, 9 and 12 after transplantation, 1X 10 was injected via tail vein7The vecor-T cells or CAR-T cells (see FIG. 11A), compared to the control group, did not significantly lose body weight upon injection of CAR-T, suggesting that CAR-T treatment had no significant toxic side effects on the mice (see FIG. 11B). Median survival was 25 days, 36 days and 37 days in vecor-T, 19-22CAR-T and 22-19CAR-T groups, respectively, tumor burden was plotted as C in FIG. 11, tumor burden was significantly lower in CAR-T group than in vec-T group, survival curves were plotted as D in FIG. 11, and differences in survival were calculated for the two groups: both the 19-22CAR-T group and the 22-19CAR-T group can significantly prolong the survival of miceThere was a significant statistical difference (p ═ 0.0011) compared to the control group. The median survival and tumor burden results were combined to show that the therapeutic effect of 22-19CAR-T was slightly better than that of 19-22 CAR-T.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> hematological disease Hospital of Chinese medical science (institute of hematology of Chinese medical science)
<120> chimeric antigen receptor targeting CD22 and CD19 and application thereof
<130> 2019
<160> 14
<170> SIPOSequenceListing 1.0
<210> 1
<211> 744
<212> PRT
<213> Artificial
<400> 1
Asp Ile Val Leu Thr Gln Ser Pro Lys Phe Met Ser Thr Ser Val Gly
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Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Pro Leu Ile
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Tyr Ser Ala Thr Tyr Arg Asn Ser Gly Val Pro Asp Arg Phe Thr Gly
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Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Asn Val Gln Ser
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Lys Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Arg Tyr Pro Tyr
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Thr Ser Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly
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Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln
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Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser Ser Val Lys Ile Ser
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Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr Trp Met Asn Trp Val
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Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Gln Ile Tyr Pro
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Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys Gly Gln Ala Thr
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Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Gly
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Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg Lys Thr Ile
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Ser Ser Val Val Asp Phe Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr
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Leu Thr Val Ser Ser Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu
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Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Asp Ile
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Glu Leu Thr Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly Glu Lys
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Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser Ser Asn
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Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro
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Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp
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Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Val Ser
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Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln Ser Tyr
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Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg
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Val Lys Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser
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Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Asp Phe Ser Ile Ser Trp
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Met Asn Trp Val Arg Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly
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Arg Ile Tyr Pro Gly Asp Gly Asp Ser Asn Tyr Asn Gly Lys Phe Glu
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Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met
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Gln Leu Ser Gly Leu Thr Ser Val Asp Ser Ala Val Tyr Phe Cys Ala
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Arg Thr Thr Thr Met Ile Ala Leu Tyr Ala Met Asp Tyr Trp Gly Gln
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Gly Thr Thr Val Thr Val Ser Ser Glu Phe Thr Thr Thr Pro Ala Pro
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Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu
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Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg
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Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly
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Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu
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Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu
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Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly
705 710 715 720
Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu
725 730 735
His Met Gln Ala Leu Pro Pro Arg
740
<210> 2
<211> 744
<212> PRT
<213> Artificial
<400> 2
Asp Ile Glu Leu Thr Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly
1 5 10 15
Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser
20 25 30
Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Val Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln
85 90 95
Ser Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile
100 105 110
Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
115 120 125
Ser Gln Val Lys Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly
130 135 140
Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Asp Phe Ser Ile
145 150 155 160
Ser Trp Met Asn Trp Val Arg Gln Arg Pro Gly Gln Gly Leu Glu Trp
165 170 175
Ile Gly Arg Ile Tyr Pro Gly Asp Gly Asp Ser Asn Tyr Asn Gly Lys
180 185 190
Phe Glu Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala
195 200 205
Tyr Met Gln Leu Ser Gly Leu Thr Ser Val Asp Ser Ala Val Tyr Phe
210 215 220
Cys Ala Arg Thr Thr Thr Met Ile Ala Leu Tyr Ala Met Asp Tyr Trp
225 230 235 240
Gly Gln Gly Thr Thr Val Thr Val Ser Ser Glu Ala Ala Ala Lys Glu
245 250 255
Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala
260 265 270
Ala Ala Lys Asp Ile Val Leu Thr Gln Ser Pro Lys Phe Met Ser Thr
275 280 285
Ser Val Gly Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val
290 295 300
Gly Thr Asn Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys
305 310 315 320
Pro Leu Ile Tyr Ser Ala Thr Tyr Arg Asn Ser Gly Val Pro Asp Arg
325 330 335
Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Asn
340 345 350
Val Gln Ser Lys Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Arg
355 360 365
Tyr Pro Tyr Thr Ser Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Gly
370 375 380
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val
385 390 395 400
Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser Ser Val
405 410 415
Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr Trp Met
420 425 430
Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Gln
435 440 445
Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys Gly
450 455 460
Gln Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln
465 470 475 480
Leu Ser Gly Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg
485 490 495
Lys Thr Ile Ser Ser Val Val Asp Phe Tyr Phe Asp Tyr Trp Gly Gln
500 505 510
Gly Thr Thr Leu Thr Val Ser Ser Glu Phe Thr Thr Thr Pro Ala Pro
515 520 525
Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu
530 535 540
Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg
545 550 555 560
Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly
565 570 575
Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Lys Arg
580 585 590
Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro
595 600 605
Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu
610 615 620
Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala
625 630 635 640
Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu
645 650 655
Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly
660 665 670
Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu
675 680 685
Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser
690 695 700
Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly
705 710 715 720
Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu
725 730 735
His Met Gln Ala Leu Pro Pro Arg
740
<210> 3
<211> 717
<212> PRT
<213> Artificial
<400> 3
Asp Ile Val Leu Thr Gln Ser Pro Lys Phe Met Ser Thr Ser Val Gly
1 5 10 15
Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Pro Leu Ile
35 40 45
Tyr Ser Ala Thr Tyr Arg Asn Ser Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Asn Val Gln Ser
65 70 75 80
Lys Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Arg Tyr Pro Tyr
85 90 95
Thr Ser Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly
100 105 110
Ser Asp Ile Glu Leu Thr Gln Ser Pro Ser Ser Leu Ala Val Ser Val
115 120 125
Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr
130 135 140
Ser Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly
145 150 155 160
Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly
165 170 175
Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
180 185 190
Thr Val Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln
195 200 205
Gln Ser Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu
210 215 220
Ile Lys Arg Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu
225 230 235 240
Gly Ser Thr Lys Gly Gln Val Lys Leu Gln Gln Ser Gly Pro Glu Leu
245 250 255
Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr
260 265 270
Asp Phe Ser Ile Ser Trp Met Asn Trp Val Arg Gln Arg Pro Gly Gln
275 280 285
Gly Leu Glu Trp Ile Gly Arg Ile Tyr Pro Gly Asp Gly Asp Ser Asn
290 295 300
Tyr Asn Gly Lys Phe Glu Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser
305 310 315 320
Ser Ser Thr Ala Tyr Met Gln Leu Ser Gly Leu Thr Ser Val Asp Ser
325 330 335
Ala Val Tyr Phe Cys Ala Arg Thr Thr Thr Met Ile Ala Leu Tyr Ala
340 345 350
Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly
355 360 365
Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg
370 375 380
Pro Gly Ser Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe
385 390 395 400
Ser Ser Tyr Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu
405 410 415
Glu Trp Ile Gly Gln Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn
420 425 430
Gly Lys Phe Lys Gly Gln Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser
435 440 445
Thr Ala Tyr Met Gln Leu Ser Gly Leu Thr Ser Glu Asp Ser Ala Val
450 455 460
Tyr Phe Cys Ala Arg Lys Thr Ile Ser Ser Val Val Asp Phe Tyr Phe
465 470 475 480
Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Glu Phe Thr
485 490 495
Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser
500 505 510
Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly
515 520 525
Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp
530 535 540
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
545 550 555 560
Thr Leu Tyr Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
565 570 575
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
580 585 590
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
595 600 605
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
610 615 620
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
625 630 635 640
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
645 650 655
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
660 665 670
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
675 680 685
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
690 695 700
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
705 710 715
<210> 4
<211> 717
<212> PRT
<213> Artificial
<400> 4
Asp Ile Glu Leu Thr Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly
1 5 10 15
Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser
20 25 30
Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Val Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln
85 90 95
Ser Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile
100 105 110
Lys Arg Gly Gly Gly Gly Ser Asp Ile Val Leu Thr Gln Ser Pro Lys
115 120 125
Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser Val Thr Cys Lys Ala
130 135 140
Ser Gln Asn Val Gly Thr Asn Val Ala Trp Tyr Gln Gln Lys Pro Gly
145 150 155 160
Gln Ser Pro Lys Pro Leu Ile Tyr Ser Ala Thr Tyr Arg Asn Ser Gly
165 170 175
Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
180 185 190
Thr Ile Thr Asn Val Gln Ser Lys Asp Leu Ala Asp Tyr Phe Cys Gln
195 200 205
Gln Tyr Asn Arg Tyr Pro Tyr Thr Ser Gly Gly Gly Thr Lys Leu Glu
210 215 220
Ile Lys Arg Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu
225 230 235 240
Gly Ser Thr Lys Gly Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu
245 250 255
Val Arg Pro Gly Ser Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr
260 265 270
Ala Phe Ser Ser Tyr Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln
275 280 285
Gly Leu Glu Trp Ile Gly Gln Ile Tyr Pro Gly Asp Gly Asp Thr Asn
290 295 300
Tyr Asn Gly Lys Phe Lys Gly Gln Ala Thr Leu Thr Ala Asp Lys Ser
305 310 315 320
Ser Ser Thr Ala Tyr Met Gln Leu Ser Gly Leu Thr Ser Glu Asp Ser
325 330 335
Ala Val Tyr Phe Cys Ala Arg Lys Thr Ile Ser Ser Val Val Asp Phe
340 345 350
Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly
355 360 365
Gly Gly Gly Ser Gln Val Lys Leu Gln Gln Ser Gly Pro Glu Leu Val
370 375 380
Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Asp
385 390 395 400
Phe Ser Ile Ser Trp Met Asn Trp Val Arg Gln Arg Pro Gly Gln Gly
405 410 415
Leu Glu Trp Ile Gly Arg Ile Tyr Pro Gly Asp Gly Asp Ser Asn Tyr
420 425 430
Asn Gly Lys Phe Glu Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser
435 440 445
Ser Thr Ala Tyr Met Gln Leu Ser Gly Leu Thr Ser Val Asp Ser Ala
450 455 460
Val Tyr Phe Cys Ala Arg Thr Thr Thr Met Ile Ala Leu Tyr Ala Met
465 470 475 480
Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Glu Phe Thr
485 490 495
Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser
500 505 510
Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly
515 520 525
Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp
530 535 540
Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile
545 550 555 560
Thr Leu Tyr Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln
565 570 575
Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser
580 585 590
Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
595 600 605
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln
610 615 620
Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu
625 630 635 640
Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg
645 650 655
Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met
660 665 670
Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
675 680 685
Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp
690 695 700
Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
705 710 715
<210> 5
<211> 2232
<212> DNA
<213> Artificial
<400> 5
gatattgtgc tgacccagag ccccaagttc atgagcacca gcgtgggcga tagagtgagc 60
gtgacctgca aggcaagcca gaacgtggga acaaacgtgg cctggtacca acagaaaccc 120
ggccaaagcc ctaagcccct gatttacagc gccacctaca gaaatagcgg cgtgcccgac 180
agatttacag gaagcggcag cggaaccgat ttcacactga ccatcaccaa cgtgcagagc 240
aaagacctgg ccgactactt ctgccagcag tacaacagat acccctacac cagcggagga 300
ggaacaaagc tggagatcaa gagaggtggt ggtggttctg gcggcggcgg ctccggtggt 360
ggtggttctc aagtgcaact gcaacagagc ggagccgaac tggtgagacc cggaagcagc 420
gtgaagatca gctgcaaggc ttccggctac gcctttagca gctactggat gaactgggtg 480
aagcagagac ctggacaggg actggaatgg atcggccaga tttaccctgg agacggcgac 540
acaaactaca acggcaagtt caagggccaa gctacactga ccgccgacaa aagcagcagc 600
accgcctata tgcagctgag cggactgacc agcgaagata gcgctgtgta cttctgcgcc 660
agaaagacca tcagcagcgt ggtggacttc tacttcgact actggggaca aggcaccacc 720
ctgacagtga gcagcgaagc cgctgctaag gaagccgctg ctaaggaagc cgctgctaag 780
gaagccgctg ctaaggaagc cgctgctaag gacattgagc tcacccagtc tccatcctcc 840
ctagctgtgt cagttggaga gaaggttact atgagctgca agtccagtca gagcctttta 900
tatagtagca atcaaaagaa ctatttggcc tggtaccagc agaaaccagg gcagtctcct 960
aaactgctga tttactgggc atccactagg gaatctgggg tccctgatcg cttcacaggc 1020
agtggatctg ggacagattt cactctcacc gtcagcagtg tgaaggctga agacctggca 1080
gtttattact gtcagcaatc ttatagttat ccattcacat tcggctcggg caccaagctg 1140
gaaatcaaac ggggtggtgg tggttctggc ggcggcggct ccggtggtgg tggttctcag 1200
gtcaaactgc agcagtcagg acctgaactg gtgaagcctg gggcctcagt gaagatttcc 1260
tgcaaagctt ctggctacga tttcagtatt tcttggatga actgggtgag gcagaggcct 1320
ggacagggtc ttgagtggat tggacggatt tatcctggag atggagatag taactacaat 1380
gggaagttcg agggcaaggc cacactgact gcagacaaat cctccagcac agcctacatg 1440
cagctcagcg gcctgacctc tgtggactct gcggtctatt tttgtgcaag aaccaccact 1500
atgattgccc tctatgctat ggactactgg ggccaaggga ccacggtcac cgtctcctca 1560
gaattcacca cgacgccagc gccgcgacca ccaacaccgg cgcccaccat cgcgtcgcag 1620
cccctgtccc tgcgcccaga ggcgtgccgg ccagcggcgg ggggcgcagt gcacacgagg 1680
gggctggact tcgcctgtga tatctacatc tgggcgccct tggccgggac ttgtggggtc 1740
cttctcctgt cactggttat caccctttac aaacggggca gaaagaaact cctgtatata 1800
ttcaaacaac catttatgag accagtacaa actactcaag aggaagatgg ctgtagctgc 1860
cgatttccag aagaagaaga aggaggatgt gaactgagag tgaagttcag caggagcgca 1920
gacgcccccg cgtaccagca gggccagaac cagctctata acgagctcaa tctaggacga 1980
agagaggagt acgatgtttt ggacaagaga cgtggccggg accctgagat ggggggaaag 2040
ccgagaagga agaaccctca ggaaggcctg tacaatgaac tgcagaaaga taagatggcg 2100
gaggcctaca gtgagattgg gatgaaaggc gagcgccgga ggggcaaggg gcacgatggc 2160
ctttaccagg gtctcagtac agccaccaag gacacctacg acgcccttca catgcaggcc 2220
ctgccccctc gc 2232
<210> 6
<211> 2232
<212> DNA
<213> Artificial
<400> 6
gacattgagc tcacccagtc tccatcctcc ctagctgtgt cagttggaga gaaggttact 60
atgagctgca agtccagtca gagcctttta tatagtagca atcaaaagaa ctatttggcc 120
tggtaccagc agaaaccagg gcagtctcct aaactgctga tttactgggc atccactagg 180
gaatctgggg tccctgatcg cttcacaggc agtggatctg ggacagattt cactctcacc 240
gtcagcagtg tgaaggctga agacctggca gtttattact gtcagcaatc ttatagttat 300
ccattcacat tcggctcggg caccaagctg gaaatcaaac ggggtggtgg tggttctggc 360
ggcggcggct ccggtggtgg tggttctcag gtcaaactgc agcagtcagg acctgaactg 420
gtgaagcctg gggcctcagt gaagatttcc tgcaaagctt ctggctacga tttcagtatt 480
tcttggatga actgggtgag gcagaggcct ggacagggtc ttgagtggat tggacggatt 540
tatcctggag atggagatag taactacaat gggaagttcg agggcaaggc cacactgact 600
gcagacaaat cctccagcac agcctacatg cagctcagcg gcctgacctc tgtggactct 660
gcggtctatt tttgtgcaag aaccaccact atgattgccc tctatgctat ggactactgg 720
ggccaaggga ccacggtcac cgtctcctca gaagccgctg ctaaggaagc cgctgctaag 780
gaagccgctg ctaaggaagc cgctgctaag gaagccgctg ctaaggatat tgtgctgacc 840
cagagcccca agttcatgag caccagcgtg ggcgatagag tgagcgtgac ctgcaaggca 900
agccagaacg tgggaacaaa cgtggcctgg taccaacaga aacccggcca aagccctaag 960
cccctgattt acagcgccac ctacagaaat agcggcgtgc ccgacagatt tacaggaagc 1020
ggcagcggaa ccgatttcac actgaccatc accaacgtgc agagcaaaga cctggccgac 1080
tacttctgcc agcagtacaa cagatacccc tacaccagcg gaggaggaac aaagctggag 1140
atcaagagag gtggtggtgg ttctggcggc ggcggctccg gtggtggtgg ttctcaagtg 1200
caactgcaac agagcggagc cgaactggtg agacccggaa gcagcgtgaa gatcagctgc 1260
aaggcttccg gctacgcctt tagcagctac tggatgaact gggtgaagca gagacctgga 1320
cagggactgg aatggatcgg ccagatttac cctggagacg gcgacacaaa ctacaacggc 1380
aagttcaagg gccaagctac actgaccgcc gacaaaagca gcagcaccgc ctatatgcag 1440
ctgagcggac tgaccagcga agatagcgct gtgtacttct gcgccagaaa gaccatcagc 1500
agcgtggtgg acttctactt cgactactgg ggacaaggca ccaccctgac agtgagcagc 1560
gaattcacca cgacgccagc gccgcgacca ccaacaccgg cgcccaccat cgcgtcgcag 1620
cccctgtccc tgcgcccaga ggcgtgccgg ccagcggcgg ggggcgcagt gcacacgagg 1680
gggctggact tcgcctgtga tatctacatc tgggcgccct tggccgggac ttgtggggtc 1740
cttctcctgt cactggttat caccctttac aaacggggca gaaagaaact cctgtatata 1800
ttcaaacaac catttatgag accagtacaa actactcaag aggaagatgg ctgtagctgc 1860
cgatttccag aagaagaaga aggaggatgt gaactgagag tgaagttcag caggagcgca 1920
gacgcccccg cgtaccagca gggccagaac cagctctata acgagctcaa tctaggacga 1980
agagaggagt acgatgtttt ggacaagaga cgtggccggg accctgagat ggggggaaag 2040
ccgagaagga agaaccctca ggaaggcctg tacaatgaac tgcagaaaga taagatggcg 2100
gaggcctaca gtgagattgg gatgaaaggc gagcgccgga ggggcaaggg gcacgatggc 2160
ctttaccagg gtctcagtac agccaccaag gacacctacg acgcccttca catgcaggcc 2220
ctgccccctc gc 2232
<210> 7
<211> 2151
<212> DNA
<213> Artificial
<400> 7
gatattgtgc tgacccagag ccccaagttc atgagcacca gcgtgggcga tagagtgagc 60
gtgacctgca aggcaagcca gaacgtggga acaaacgtgg cctggtacca acagaaaccc 120
ggccaaagcc ctaagcccct gatttacagc gccacctaca gaaatagcgg cgtgcccgac 180
agatttacag gaagcggcag cggaaccgat ttcacactga ccatcaccaa cgtgcagagc 240
aaagacctgg ccgactactt ctgccagcag tacaacagat acccctacac cagcggagga 300
ggaacaaagc tggagatcaa gagaggcggc ggaggttctg acattgagct cacccagtct 360
ccatcctccc tagctgtgtc agttggagag aaggttacta tgagctgcaa gtccagtcag 420
agccttttat atagtagcaa tcaaaagaac tatttggcct ggtaccagca gaaaccaggg 480
cagtctccta aactgctgat ttactgggca tccactaggg aatctggggt ccctgatcgc 540
ttcacaggca gtggatctgg gacagatttc actctcaccg tcagcagtgt gaaggctgaa 600
gacctggcag tttattactg tcagcaatct tatagttatc cattcacatt cggctcgggc 660
accaagctgg aaatcaaacg gggcagcaca agcggctctg gcaagcctgg atctggcgag 720
ggctctacca agggccaggt caaactgcag cagtcaggac ctgaactggt gaagcctggg 780
gcctcagtga agatttcctg caaagcttct ggctacgatt tcagtatttc ttggatgaac 840
tgggtgaggc agaggcctgg acagggtctt gagtggattg gacggattta tcctggagat 900
ggagatagta actacaatgg gaagttcgag ggcaaggcca cactgactgc agacaaatcc 960
tccagcacag cctacatgca gctcagcggc ctgacctctg tggactctgc ggtctatttt 1020
tgtgcaagaa ccaccactat gattgccctc tatgctatgg actactgggg ccaagggacc 1080
acggtcaccg tctcctcagg cggcggaggt tctcaagtgc aactgcaaca gagcggagcc 1140
gaactggtga gacccggaag cagcgtgaag atcagctgca aggcttccgg ctacgccttt 1200
agcagctact ggatgaactg ggtgaagcag agacctggac agggactgga atggatcggc 1260
cagatttacc ctggagacgg cgacacaaac tacaacggca agttcaaggg ccaagctaca 1320
ctgaccgccg acaaaagcag cagcaccgcc tatatgcagc tgagcggact gaccagcgaa 1380
gatagcgctg tgtacttctg cgccagaaag accatcagca gcgtggtgga cttctacttc 1440
gactactggg gacaaggcac caccctgaca gtgagcagcg aattcaccac gacgccagcg 1500
ccgcgaccac caacaccggc gcccaccatc gcgtcgcagc ccctgtccct gcgcccagag 1560
gcgtgccggc cagcggcggg gggcgcagtg cacacgaggg ggctggactt cgcctgtgat 1620
atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 1680
accctttaca aacggggcag aaagaaactc ctgtatatat tcaaacaacc atttatgaga 1740
ccagtacaaa ctactcaaga ggaagatggc tgtagctgcc gatttccaga agaagaagaa 1800
ggaggatgtg aactgagagt gaagttcagc aggagcgcag acgcccccgc gtaccagcag 1860
ggccagaacc agctctataa cgagctcaat ctaggacgaa gagaggagta cgatgttttg 1920
gacaagagac gtggccggga ccctgagatg gggggaaagc cgagaaggaa gaaccctcag 1980
gaaggcctgt acaatgaact gcagaaagat aagatggcgg aggcctacag tgagattggg 2040
atgaaaggcg agcgccggag gggcaagggg cacgatggcc tttaccaggg tctcagtaca 2100
gccaccaagg acacctacga cgcccttcac atgcaggccc tgccccctcg c 2151
<210> 8
<211> 2151
<212> DNA
<213> Artificial
<400> 8
gacattgagc tcacccagtc tccatcctcc ctagctgtgt cagttggaga gaaggttact 60
atgagctgca agtccagtca gagcctttta tatagtagca atcaaaagaa ctatttggcc 120
tggtaccagc agaaaccagg gcagtctcct aaactgctga tttactgggc atccactagg 180
gaatctgggg tccctgatcg cttcacaggc agtggatctg ggacagattt cactctcacc 240
gtcagcagtg tgaaggctga agacctggca gtttattact gtcagcaatc ttatagttat 300
ccattcacat tcggctcggg caccaagctg gaaatcaaac ggggcggcgg aggttctgat 360
attgtgctga cccagagccc caagttcatg agcaccagcg tgggcgatag agtgagcgtg 420
acctgcaagg caagccagaa cgtgggaaca aacgtggcct ggtaccaaca gaaacccggc 480
caaagcccta agcccctgat ttacagcgcc acctacagaa atagcggcgt gcccgacaga 540
tttacaggaa gcggcagcgg aaccgatttc acactgacca tcaccaacgt gcagagcaaa 600
gacctggccg actacttctg ccagcagtac aacagatacc cctacaccag cggaggagga 660
acaaagctgg agatcaagag aggcagcaca agcggctctg gcaagcctgg atctggcgag 720
ggctctacca agggccaagt gcaactgcaa cagagcggag ccgaactggt gagacccgga 780
agcagcgtga agatcagctg caaggcttcc ggctacgcct ttagcagcta ctggatgaac 840
tgggtgaagc agagacctgg acagggactg gaatggatcg gccagattta ccctggagac 900
ggcgacacaa actacaacgg caagttcaag ggccaagcta cactgaccgc cgacaaaagc 960
agcagcaccg cctatatgca gctgagcgga ctgaccagcg aagatagcgc tgtgtacttc 1020
tgcgccagaa agaccatcag cagcgtggtg gacttctact tcgactactg gggacaaggc 1080
accaccctga cagtgagcag cggcggcgga ggttctcagg tcaaactgca gcagtcagga 1140
cctgaactgg tgaagcctgg ggcctcagtg aagatttcct gcaaagcttc tggctacgat 1200
ttcagtattt cttggatgaa ctgggtgagg cagaggcctg gacagggtct tgagtggatt 1260
ggacggattt atcctggaga tggagatagt aactacaatg ggaagttcga gggcaaggcc 1320
acactgactg cagacaaatc ctccagcaca gcctacatgc agctcagcgg cctgacctct 1380
gtggactctg cggtctattt ttgtgcaaga accaccacta tgattgccct ctatgctatg 1440
gactactggg gccaagggac cacggtcacc gtctcctcag aattcaccac gacgccagcg 1500
ccgcgaccac caacaccggc gcccaccatc gcgtcgcagc ccctgtccct gcgcccagag 1560
gcgtgccggc cagcggcggg gggcgcagtg cacacgaggg ggctggactt cgcctgtgat 1620
atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 1680
accctttaca aacggggcag aaagaaactc ctgtatatat tcaaacaacc atttatgaga 1740
ccagtacaaa ctactcaaga ggaagatggc tgtagctgcc gatttccaga agaagaagaa 1800
ggaggatgtg aactgagagt gaagttcagc aggagcgcag acgcccccgc gtaccagcag 1860
ggccagaacc agctctataa cgagctcaat ctaggacgaa gagaggagta cgatgttttg 1920
gacaagagac gtggccggga ccctgagatg gggggaaagc cgagaaggaa gaaccctcag 1980
gaaggcctgt acaatgaact gcagaaagat aagatggcgg aggcctacag tgagattggg 2040
atgaaaggcg agcgccggag gggcaagggg cacgatggcc tttaccaggg tctcagtaca 2100
gccaccaagg acacctacga cgcccttcac atgcaggccc tgccccctcg c 2151
<210> 9
<211> 520
<212> PRT
<213> Artificial
<400> 9
Asp Ile Val Leu Thr Gln Ser Pro Lys Phe Met Ser Thr Ser Val Gly
1 5 10 15
Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Pro Leu Ile
35 40 45
Tyr Ser Ala Thr Tyr Arg Asn Ser Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Asn Val Gln Ser
65 70 75 80
Lys Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Arg Tyr Pro Tyr
85 90 95
Thr Ser Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly
100 105 110
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln
115 120 125
Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser Ser Val Lys Ile Ser
130 135 140
Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr Trp Met Asn Trp Val
145 150 155 160
Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Gln Ile Tyr Pro
165 170 175
Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys Gly Gln Ala Thr
180 185 190
Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln Leu Ser Gly
195 200 205
Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg Lys Thr Ile
210 215 220
Ser Ser Val Val Asp Phe Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr
225 230 235 240
Leu Thr Val Ser Ser Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu
245 250 255
Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Asp Ile
260 265 270
Glu Leu Thr Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly Glu Lys
275 280 285
Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser Ser Asn
290 295 300
Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro
305 310 315 320
Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val Pro Asp
325 330 335
Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Val Ser
340 345 350
Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln Ser Tyr
355 360 365
Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys Arg
370 375 380
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln
385 390 395 400
Val Lys Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala Ser
405 410 415
Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Asp Phe Ser Ile Ser Trp
420 425 430
Met Asn Trp Val Arg Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly
435 440 445
Arg Ile Tyr Pro Gly Asp Gly Asp Ser Asn Tyr Asn Gly Lys Phe Glu
450 455 460
Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met
465 470 475 480
Gln Leu Ser Gly Leu Thr Ser Val Asp Ser Ala Val Tyr Phe Cys Ala
485 490 495
Arg Thr Thr Thr Met Ile Ala Leu Tyr Ala Met Asp Tyr Trp Gly Gln
500 505 510
Gly Thr Thr Val Thr Val Ser Ser
515 520
<210> 10
<211> 520
<212> PRT
<213> Artificial
<400> 10
Asp Ile Glu Leu Thr Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly
1 5 10 15
Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser
20 25 30
Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Val Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln
85 90 95
Ser Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile
100 105 110
Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
115 120 125
Ser Gln Val Lys Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly
130 135 140
Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Asp Phe Ser Ile
145 150 155 160
Ser Trp Met Asn Trp Val Arg Gln Arg Pro Gly Gln Gly Leu Glu Trp
165 170 175
Ile Gly Arg Ile Tyr Pro Gly Asp Gly Asp Ser Asn Tyr Asn Gly Lys
180 185 190
Phe Glu Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala
195 200 205
Tyr Met Gln Leu Ser Gly Leu Thr Ser Val Asp Ser Ala Val Tyr Phe
210 215 220
Cys Ala Arg Thr Thr Thr Met Ile Ala Leu Tyr Ala Met Asp Tyr Trp
225 230 235 240
Gly Gln Gly Thr Thr Val Thr Val Ser Ser Glu Ala Ala Ala Lys Glu
245 250 255
Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala
260 265 270
Ala Ala Lys Asp Ile Val Leu Thr Gln Ser Pro Lys Phe Met Ser Thr
275 280 285
Ser Val Gly Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val
290 295 300
Gly Thr Asn Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys
305 310 315 320
Pro Leu Ile Tyr Ser Ala Thr Tyr Arg Asn Ser Gly Val Pro Asp Arg
325 330 335
Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Asn
340 345 350
Val Gln Ser Lys Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Arg
355 360 365
Tyr Pro Tyr Thr Ser Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Gly
370 375 380
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val
385 390 395 400
Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ser Ser Val
405 410 415
Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Tyr Trp Met
420 425 430
Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile Gly Gln
435 440 445
Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe Lys Gly
450 455 460
Gln Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr Met Gln
465 470 475 480
Leu Ser Gly Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys Ala Arg
485 490 495
Lys Thr Ile Ser Ser Val Val Asp Phe Tyr Phe Asp Tyr Trp Gly Gln
500 505 510
Gly Thr Thr Leu Thr Val Ser Ser
515 520
<210> 11
<211> 493
<212> PRT
<213> Artificial
<400> 11
Asp Ile Val Leu Thr Gln Ser Pro Lys Phe Met Ser Thr Ser Val Gly
1 5 10 15
Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn Val Gly Thr Asn
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Pro Leu Ile
35 40 45
Tyr Ser Ala Thr Tyr Arg Asn Ser Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Asn Val Gln Ser
65 70 75 80
Lys Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Asn Arg Tyr Pro Tyr
85 90 95
Thr Ser Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly
100 105 110
Ser Asp Ile Glu Leu Thr Gln Ser Pro Ser Ser Leu Ala Val Ser Val
115 120 125
Gly Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr
130 135 140
Ser Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly
145 150 155 160
Gln Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly
165 170 175
Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
180 185 190
Thr Val Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln
195 200 205
Gln Ser Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu
210 215 220
Ile Lys Arg Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu
225 230 235 240
Gly Ser Thr Lys Gly Gln Val Lys Leu Gln Gln Ser Gly Pro Glu Leu
245 250 255
Val Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr
260 265 270
Asp Phe Ser Ile Ser Trp Met Asn Trp Val Arg Gln Arg Pro Gly Gln
275 280 285
Gly Leu Glu Trp Ile Gly Arg Ile Tyr Pro Gly Asp Gly Asp Ser Asn
290 295 300
Tyr Asn Gly Lys Phe Glu Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser
305 310 315 320
Ser Ser Thr Ala Tyr Met Gln Leu Ser Gly Leu Thr Ser Val Asp Ser
325 330 335
Ala Val Tyr Phe Cys Ala Arg Thr Thr Thr Met Ile Ala Leu Tyr Ala
340 345 350
Met Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly
355 360 365
Gly Gly Ser Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Val Arg
370 375 380
Pro Gly Ser Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe
385 390 395 400
Ser Ser Tyr Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu
405 410 415
Glu Trp Ile Gly Gln Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn
420 425 430
Gly Lys Phe Lys Gly Gln Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser
435 440 445
Thr Ala Tyr Met Gln Leu Ser Gly Leu Thr Ser Glu Asp Ser Ala Val
450 455 460
Tyr Phe Cys Ala Arg Lys Thr Ile Ser Ser Val Val Asp Phe Tyr Phe
465 470 475 480
Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser
485 490
<210> 12
<211> 493
<212> PRT
<213> Artificial
<400> 12
Asp Ile Glu Leu Thr Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly
1 5 10 15
Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser
20 25 30
Ser Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln
35 40 45
Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val
50 55 60
Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr
65 70 75 80
Val Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln
85 90 95
Ser Tyr Ser Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile
100 105 110
Lys Arg Gly Gly Gly Gly Ser Asp Ile Val Leu Thr Gln Ser Pro Lys
115 120 125
Phe Met Ser Thr Ser Val Gly Asp Arg Val Ser Val Thr Cys Lys Ala
130 135 140
Ser Gln Asn Val Gly Thr Asn Val Ala Trp Tyr Gln Gln Lys Pro Gly
145 150 155 160
Gln Ser Pro Lys Pro Leu Ile Tyr Ser Ala Thr Tyr Arg Asn Ser Gly
165 170 175
Val Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
180 185 190
Thr Ile Thr Asn Val Gln Ser Lys Asp Leu Ala Asp Tyr Phe Cys Gln
195 200 205
Gln Tyr Asn Arg Tyr Pro Tyr Thr Ser Gly Gly Gly Thr Lys Leu Glu
210 215 220
Ile Lys Arg Gly Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu
225 230 235 240
Gly Ser Thr Lys Gly Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu
245 250 255
Val Arg Pro Gly Ser Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr
260 265 270
Ala Phe Ser Ser Tyr Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln
275 280 285
Gly Leu Glu Trp Ile Gly Gln Ile Tyr Pro Gly Asp Gly Asp Thr Asn
290 295 300
Tyr Asn Gly Lys Phe Lys Gly Gln Ala Thr Leu Thr Ala Asp Lys Ser
305 310 315 320
Ser Ser Thr Ala Tyr Met Gln Leu Ser Gly Leu Thr Ser Glu Asp Ser
325 330 335
Ala Val Tyr Phe Cys Ala Arg Lys Thr Ile Ser Ser Val Val Asp Phe
340 345 350
Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly
355 360 365
Gly Gly Gly Ser Gln Val Lys Leu Gln Gln Ser Gly Pro Glu Leu Val
370 375 380
Lys Pro Gly Ala Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Asp
385 390 395 400
Phe Ser Ile Ser Trp Met Asn Trp Val Arg Gln Arg Pro Gly Gln Gly
405 410 415
Leu Glu Trp Ile Gly Arg Ile Tyr Pro Gly Asp Gly Asp Ser Asn Tyr
420 425 430
Asn Gly Lys Phe Glu Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser
435 440 445
Ser Thr Ala Tyr Met Gln Leu Ser Gly Leu Thr Ser Val Asp Ser Ala
450 455 460
Val Tyr Phe Cys Ala Arg Thr Thr Thr Met Ile Ala Leu Tyr Ala Met
465 470 475 480
Asp Tyr Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
485 490
<210> 13
<211> 21
<212> PRT
<213> Homo sapiens
<400> 13
Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu
1 5 10 15
His Ala Ala Arg Pro
20
<210> 14
<211> 222
<212> PRT
<213> Artificial
<400> 14
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 Ile Tyr Ile
35 40 45
Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val
50 55 60
Ile Thr Leu Tyr Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys
65 70 75 80
Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys
85 90 95
Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val
100 105 110
Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn
115 120 125
Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val
130 135 140
Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg
145 150 155 160
Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys
165 170 175
Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg
180 185 190
Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys
195 200 205
Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
210 215 220

Claims (17)

1. A nucleic acid molecule encoding a chimeric antigen receptor targeting CD22 and CD19, said chimeric antigen receptor comprising an extracellular region, a transmembrane region and an intracellular signaling region, characterized in that it encodes said extracellular region comprising a CD22 and CD19 binding domain, said CD22 and CD19 binding domain consisting of an antibody single chain variable fragment of CD22 and an antibody single chain variable fragment of CD 19;
the antibody single-chain variable region fragment of CD22 and the antibody single-chain variable region fragment of CD19 are arranged according to the sequence of an amino acid sequence shown in SEQ ID No.9, an amino acid sequence shown in SEQ ID No.10, an amino acid sequence shown in SEQ ID No.11 or an amino acid sequence shown in SEQ ID No. 12.
2. The nucleic acid molecule according to claim 1, wherein said extracellular domain encoded thereby further comprises a signal peptide, or an amino acid sequence with 90-99% identity thereto, constructed at the amino terminus of said chimeric antigen receptor, said signal peptide being the signal peptide sequence in CD8 a or GM-CSF, preferably the signal peptide as shown in SEQ ID No. 13.
3. The nucleic acid molecule according to claim 1, characterized in that the CD22 and CD19 binding domains it encodes are linked to the transmembrane region it encodes by a hinge region, preferably a hinge region sequence in CD8 a; the transmembrane region is the transmembrane domain of a protein selected from the group consisting of: an alpha, beta, or zeta chain of a T cell receptor, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, 4-1BB, or CD 154.
4. The nucleic acid molecule of claim 1, wherein said intracellular signaling region encoded thereby further comprises a costimulatory factor.
5. The nucleic acid molecule according to claim 4, wherein said co-stimulatory factor is one or several of the functional signal domains obtained by the amino acid sequence selected from the following proteins or from 90-99% identity to said proteins: MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, lymphocyte activation signaling molecules, activated NK cell receptors, BTLA, Toll ligand receptors, OX40, CD2, CD7, CD27, CDS, ICAM-1, LFA-1, 4-1BB, B27-H27, CD278, GITR, BAFFR, LIGHT, HVEM, KIRDS 27, SLAMF 27, NKp 27, CD27 alpha, CD27 beta, IL2 27 gamma, IL7 27 alpha, ITGA 27, VLA 27, CD27 alpha, CD27, CD 3649, CD27, ITGA 27, VLA 27, CD27, GAITGB, GAITCD 27, GAITGL 27, GAITGB, CD27, GAITGL 27, CD27, GAITGB 11, CD27, GAITGB 27, GAITGL 27, CD27, GAITGB 11, CD27, GAITGL 27, CD27, GAITGB 11, CD27, GAITGL 27, GAITGB 11, CD27, GAITGL 27, CD27, GAITGB 11, CD 27.
6. The nucleic acid molecule of claim 5, wherein the co-stimulatory factor is CD28 or 4-1BB or an amino acid sequence with 90-99% identity thereto.
7. The nucleic acid molecule of claim 1, wherein the sequence of said nucleic acid molecule is as set forth in SEQ ID No.5, SEQ ID No.6, SEQ ID No.7 or SEQ ID No. 8.
8. A chimeric antigen receptor targeting CD22 and CD19, wherein said chimeric antigen receptor is encoded by the nucleic acid molecule of any one of claims 1 to 7.
9. The chimeric antigen receptor according to claim 8, wherein the amino acid sequence of the chimeric antigen receptor is shown as SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 or SEQ ID No. 4.
10. A vector comprising the nucleic acid molecule of any one of claims 1 to 7.
11. A cell comprising a nucleic acid molecule according to any one of claims 1 to 7, a chimeric antigen receptor according to claim 8 or 9 or a vector according to claim 10.
12. Use of a nucleic acid molecule according to any one of claims 1 to 7, a chimeric antigen receptor according to claim 8 or 9, a vector according to claim 10 or a cell according to claim 11 for the preparation of a medicament against a B-cell hematological tumor, preferably a B-cell lymphoma or an acute B-lymphocytic leukemia.
13. The use of claim 12, wherein the use is in the preparation of a medicament for treating a B cell hematological tumor in which the patient expresses both CD19 and CD22 or relapses or is ineffective due to antigenic variation following CD19CAR-T treatment.
14. A pharmaceutical composition comprising a nucleic acid molecule according to any one of claims 1 to 7, a chimeric antigen receptor according to claim 8 or 9, a vector according to claim 10 or a cell according to claim 11, and a pharmaceutically acceptable carrier.
15. The pharmaceutical composition of claim 14, further comprising a nucleic acid molecule encoding a chimeric antigen receptor of CD19 and CD22, a chimeric antigen receptor of CD19 and CD22, a vector comprising a nucleic acid molecule encoding a chimeric antigen receptor of CD19, CD22, or a cell comprising a chimeric antigen receptor of CD19, CD 22.
16. Use of a nucleic acid molecule according to any one of claims 1 to 7, a chimeric antigen receptor according to claim 8 or 9, a vector according to claim 10 or a cell according to claim 11 or a pharmaceutical composition according to claim 14 or 15 for the treatment of a B-cell hematological tumor.
17. A method of treating a B-cell hematological tumor, wherein a nucleic acid molecule according to any one of claims 1 to 7, a chimeric antigen receptor according to claim 8 or 9, a vector according to claim 10, a cell according to claim 11 or a pharmaceutical composition according to claim 14 or 15 is used in said method.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113527518A (en) * 2021-07-19 2021-10-22 广州百暨基因科技有限公司 Bispecific chimeric antigen receptor targeting CD22 and CD19 and application thereof
CN114149506A (en) * 2021-12-16 2022-03-08 华道(上海)生物医药有限公司 anti-CD 22 antibody and application thereof

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120121614A1 (en) * 2009-03-16 2012-05-17 Jeff Lion Methods and compositions for bi-specific targeting of cd19/cd22
CN104788573A (en) * 2015-05-08 2015-07-22 中国医学科学院血液病医院（血液学研究所） Chimeric antigen receptor hCD19scFv-CD8a-CD-28-CD3zata and application thereof
CN108504668A (en) * 2018-05-23 2018-09-07 上海恒润达生生物科技有限公司 Target CD19 and CD22 Chimeric antigen receptors and application thereof
CN108715859A (en) * 2018-05-31 2018-10-30 中国医学科学院血液病医院（血液学研究所） Target Chimeric antigen receptor and its application of CD22
CN110157738A (en) * 2018-02-13 2019-08-23 亘喜生物科技（上海）有限公司 Target engineering immunocyte and its application of CD19 and CD22

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20120121614A1 (en) * 2009-03-16 2012-05-17 Jeff Lion Methods and compositions for bi-specific targeting of cd19/cd22
CN104788573A (en) * 2015-05-08 2015-07-22 中国医学科学院血液病医院（血液学研究所） Chimeric antigen receptor hCD19scFv-CD8a-CD-28-CD3zata and application thereof
CN110157738A (en) * 2018-02-13 2019-08-23 亘喜生物科技（上海）有限公司 Target engineering immunocyte and its application of CD19 and CD22
CN108504668A (en) * 2018-05-23 2018-09-07 上海恒润达生生物科技有限公司 Target CD19 and CD22 Chimeric antigen receptors and application thereof
CN108715859A (en) * 2018-05-31 2018-10-30 中国医学科学院血液病医院（血液学研究所） Target Chimeric antigen receptor and its application of CD22

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HAIYING QIN等: "Preclinical Development of Bivalent Chimeric Antigen Receptors Targeting Both CD19 and CD22", 《MOLECULAR THERAPY: ONCOLYTICS》 *
SONIA GUEDAN等: "Engineering and Design of Chimeric Antigen Receptors", 《MOLECULAR THERAPY: METHODS & CLINICAL DEVELOPMENT》 *
TERRY J. FRY等: "CD22-CAR T Cells Induce Remissions in CD19-CAR Naïve and Resistant B-ALL", 《NATURE MEDICINE》 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113527518A (en) * 2021-07-19 2021-10-22 广州百暨基因科技有限公司 Bispecific chimeric antigen receptor targeting CD22 and CD19 and application thereof
CN114149506A (en) * 2021-12-16 2022-03-08 华道(上海)生物医药有限公司 anti-CD 22 antibody and application thereof

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