CN112442508B - Chimeric antigen receptor targeting CD22 and CD19 and application thereof - Google Patents

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

Info

Publication number
CN112442508B
CN112442508B CN201910823106.4A CN201910823106A CN112442508B CN 112442508 B CN112442508 B CN 112442508B CN 201910823106 A CN201910823106 A CN 201910823106A CN 112442508 B CN112442508 B CN 112442508B
Authority
CN
China
Prior art keywords
ser
gly
thr
ala
leu
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201910823106.4A
Other languages
Chinese (zh)
Other versions
CN112442508A (en
Inventor
王建祥
王敏
张瑜
徐颖茜
饶青
廖晓龙
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Heyuan Biotechnology Tianjin Co ltd
Original Assignee
Heyuan Biotechnology Tianjin Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Heyuan Biotechnology Tianjin Co ltd filed Critical Heyuan Biotechnology Tianjin Co ltd
Priority to CN201910823106.4A priority Critical patent/CN112442508B/en
Publication of CN112442508A publication Critical patent/CN112442508A/en
Application granted granted Critical
Publication of CN112442508B publication Critical patent/CN112442508B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment

Abstract

The present invention discloses nucleic acid molecules encoding chimeric antigen receptors targeting CD22 and CD19, said chimeric antigen receptors comprising an extracellular region, a transmembrane region and an intracellular signaling region, wherein said extracellular region encoded thereby comprises CD22 and CD19 binding domains, said CD22 and CD19 binding domains consisting of an antibody single chain variable region fragment of CD22 and an antibody single chain variable region 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 the amino acid sequence shown in SEQ ID No.9, the amino acid sequence shown in SEQ ID No.10, the amino acid sequence shown in SEQ ID No.11 or the amino acid sequence shown in SEQ ID No. 12. The chimeric antigen receptor of the invention can be used for CD19 + And CD22 + Treatment of B-cell hematological tumors, in combination 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 medicine, in particular to a chimeric antigen receptor targeting CD22 and CD19 and application thereof.
Background
Chimeric antigen receptor (chimeric antigen receptor, CAR) modified T cells are widely appreciated and used in tumor therapy as an immunotherapeutic strategy. The structure of a CAR generally consists of four parts, an extracellular targeted junction region (often a single chain antibody with antigen recognition function), a hinge region, a transmembrane region and an intracellular signal transduction region. CARs are currently classified into one generation (without costimulatory molecules), two generation (with one costimulatory molecule), and three generation (with two costimulatory molecules) based on the number of costimulatory molecules added to the intracellular signaling region. Currently the most widely used is the second generation CAR.
B-cell malignancies such as B-cell lymphocytic leukemia (B-ALL) and lymphoma are malignant diseases caused by abnormal clonal proliferation of B-lymphocytes, and although the current chemotherapy effect is remarkable, 15% of pediatric B-ALL and 60% of adult B-ALL patients have poor prognosis due to drug resistance to chemotherapy drugs; lymphoma patients still have 15% relapse after treatment with the first-line regimen, and these patients still have 50% final relapse after hematopoietic stem cell transplantation. Therefore, finding effective cellular immunotherapies to treat B-cell malignancies has been a hotspot in research in the hematology community.
CD19 and CD22 are antigen molecules specific to the surface of B lymphocytes. CD19 is 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 after differentiation of B cells into plasma cells CD22 is no longer expressed; 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 positive for CD22; almost ALL B precursor cell acute lymphoblastic leukemia (B-ALL) expresses CD22; chronic lymphocytic leukemia, hairy Cell Leukemia (HCL) also have high levels of CD22 expression. Similarly to the CD19 antigen, CD22 is also B cell restricted expressed, not expressed in other parenchymal cells, nor expressed in hematopoietic stem cells, and thus has been a major therapeutic target in B cell malignancies because of its high specificity as a B cell tumor antigen.
At present, although the CD19-CAR-T has better curative effect in the treatment of refractory and recurrent B-ALL, the abnormality such as CD19 antigen mutation or loss and the like still occurs in the treatment of patients, so that the CD19-CAR-T cells can not recognize and kill the B-ALL cells, and the disease is recurrent. CD22 and CD19 have extensive co-expression on the surface of tumor cells, and CD22 remains after loss of CD19 antigen. Therefore, the CAR-T cells aiming at the 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 has been found that if the antigen binding portions targeting CD19 and CD22 are inserted into the vector by simple ligation, their killing effect after expression is poor, and thus it is necessary to find a specific ligation of the antigen binding portions of CD19 and CD22 to enhance their killing effect on tumor cells.
Disclosure of Invention
In one aspect, the invention provides a dual-target chimeric antigen receptor for simultaneously targeting CD22 and CD19 and application thereof, aiming at relapse or inefficiency caused by escape mechanism of B-ALL and the like during CD19CAR-T treatment in the prior art.
The technical scheme provided by the invention is as follows:
a nucleic acid molecule encoding a chimeric antigen receptor that targets CD22 and CD19, said chimeric antigen receptor comprising an extracellular region, a transmembrane region, and an intracellular signaling region, wherein said extracellular region encoded thereby comprises CD22 and CD19 binding domains, said CD22 and CD19 binding domains consisting of an antibody single chain variable region fragment of CD22 and an antibody single chain variable region 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 the amino acid sequence shown in SEQ ID No.9, the amino acid sequence shown in SEQ ID No.10, the amino acid sequence shown in SEQ ID No.11 or the amino acid sequence shown in SEQ ID No. 12.
In the present invention, CD22 + Tumor cells and CD19 + Tumor cells and 19-22CAR shown in SEQ ID No.1, 22-19CAR shown in SEQ ID No.2, SEQThe CAR of 19 gamma 22 shown in ID No.3 or the CAR of 22 gamma 19 shown in SEQ ID No.4 can activate corresponding CAR-T cells after being combined to generate cytotoxic effect; whereas cells that do not express neither CD22 nor CD19 are unable to activate CAR-T cells to respond. Thus, the CD19scFv-CD22scFv (shown in SEQ ID No. 1), CD22scFv-CD19scFv (shown in SEQ ID No. 2), CD19V L -CD22V L -CD22V H -CD19V H (shown in SEQ ID No. 3) and CD22V L -CD19V L -CD19V H -CD22V H (shown in SEQ ID No. 4) CAR-T cells prepared for the antigen recognition region recognize and kill CD22 + Tumor cells and CD19 + While tumor cells, no off-target effect is produced on cells that do not express CD19 and CD22.
In the present invention, the inventors have also found that the order of attachment of CD22scFv and CD19scFv has a great influence on the effect of killing tumor cells produced by the combination thereof. For example, in one embodiment of the present invention, the inventors have demonstrated that the killing effect of this 22-19CAR-T connection is more pronounced.
The sequences of the CD22scFv and the CD19scFv can be prepared according to the conventional method according to the description of SEQ ID No. 1-4. For example, the method described in patent No. ZL201510233748.0 and patent No. 201810549259. X.
In the present invention, the CD19scFv-CD22scFv, CD22scFv-CD19scFv, CD19V may be L -CD22V L -CD22V H -CD19V H And CD22V L -CD19V L -CD19V H -CD22V H Random or engineered point mutations of the amino acid sequence in a suitable manner may be aimed, for example, at achieving better affinity and/or dissociation properties, and these mutated amino acid sequences are included within 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 connecting peptide, and any connecting peptide or combination of connecting peptides with a suitable length or a 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 may direct the transfer of the antigen recognition and hinge regions to the outside of the cell. Any suitable signal peptide or combination of signal peptides may be used to achieve the objects of the 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, which is the signal peptide sequence in CD 8. Alpha. Or GM-CSF.
More preferably, the signal peptide is as shown in SEQ ID NO. 13.
In one embodiment of the invention, the CD22 and CD19 binding domains encoded by the nucleic acid molecules of the invention are linked to the transmembrane region encoded thereby by a hinge region. Any suitable hinge region sequence may achieve the objects of the invention. Preferably, in one embodiment of the present invention, the hinge region is CD8 alpha.
In the present invention, the nucleic acid molecule also encodes a transmembrane domain. Any suitable transmembrane domain can achieve 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: the α, β or ζ chain, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137 or CD154 of the T cell receptor.
In the present invention, the intracellular signal transduction 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 an amino acid sequence selected from the group consisting of the following proteins or having 90-99% identity to said proteins: MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, lymphocyte activation signal molecules, activated NK cell receptors, BTLA, toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDs, ICAM-1, LFA-1, 4-1BB, B7-H3, CD278, GITR, BAFFR, LIGHT, HVEM, KIRDS2, SLAMF7, NKp80, NKp44, NKp30, NKp46, CD19, CD4, CD8 α, CD8 β, IL2rβ, IL2rγ, IL7rα, ITGA4, VLA1, CD49 α, IA4, CD49D, ITGA6, VLA6, CD49f, ITGAD, CD11d, ITGAE, CD, ITGAL, CD11 α, ITGAM, CD11B, ITGAX, CD c, CD29, ITGB1, ITGB2, CD18, ITGB7, g2D, NKG2C, TNFR, CD226, CD96, CD69, CD6, SLP 162, CD 36, CD 35, CD 36, or CD 28.
More preferably, in one embodiment of the invention, the co-stimulatory factor is CD28 or 4-1BB or an amino acid sequence having 90-99% identity thereto.
At the same time, the nucleic acid molecules of the invention also encode any suitable intracellular signaling domain. May be an amino acid sequence to which the intracellular signal structure of CD3 zeta has 90-99% identity.
Preferably, the chimeric antigen receptor encoded by the nucleic acid molecule of the invention is a polypeptide of the invention in the form of CD19scFv-CD22scFv, CD22scFv-CD19scFv, CD19V L -CD22V L -CD22V H -CD19V H And CD22V L -CD19V L -CD19V H -CD22V H The antigen recognition region, the CD8 alpha hinge region and the transmembrane region, and the 4-1BB and CD3 zeta intracellular signal domain are connected in series to form a structure which is a signal transduction domain, and the sequence of the structure 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 antigen recognition region, the hinge region, the transmembrane region, and the intracellular signal region.
The method for producing a nucleic acid molecule can be produced by known techniques such as chemical synthesis or PCR amplification based on the base sequences of the domains such as the antigen recognition region, hinge region, transmembrane region and intracellular signal region. In general, codons encoding amino acids of the above domains can be optimized to optimize their expression in a host cell. The information on the above base sequence can be obtained by searching a database such as 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 (HIB 22) and the mouse anti-human CD19 monoclonal antibody hybridoma cell line (HIB 19) used by the inventors were developed by the applicant, the national academy of medical science blood disease hospital (national academy of medical science blood institute), and monoclonal antibodies produced by using the hybridomas were commercialized 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 chimeric antigen receptor comprises a CD22 and a CD19 binding domain, wherein the CD22 and the CD19 binding domain consist of an antibody single-chain variable region fragment of CD22 and an antibody single-chain variable region 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 the amino acid sequence shown in SEQ ID No.9, the amino acid sequence shown in SEQ ID No.10, the amino acid sequence shown in SEQ ID No.11 or the amino acid sequence shown in SEQ ID No. 12.
Preferably, the chimeric antigen receptor of the invention is a CD19scFv-CD22scFv, CD22scFv-CD19scFv, CD19V L -CD22V L -CD22V H -CD19V H Or CD22V L -CD19V L -CD19V H -CD22V H The antigen recognition region, the CD8 alpha hinge region and the transmembrane region, and the 4-1BB and CD3 zeta intracellular signal domain are connected in series to form a structure which is a signal transduction domain, and the amino acid sequence is shown as a sequence table SEQ ID NO. 1-4.
In another aspect of the invention, there is provided a vector comprising the nucleic acid molecule described above.
In the present invention, the carrier may be a linear carrier or a cyclic carrier. 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 promoters, terminators and the like, and marker sequences such as drug resistance genes, reporter genes and the like. In addition, the vectors described above may also contain sequences encoding suicide genes, and the number of CAR-T cells in vivo may be controlled by administering a substance that activates the suicide gene, depending on the course of treatment.
The viral vectors may be retrovirus vectors, lentivirus vectors, adenovirus vectors, adeno-associated virus vectors, and the like. In one embodiment of the invention, lentiviral expression vectors are used.
In another aspect of the invention, there is provided a cell comprising the nucleic acid molecule described above, the chimeric antigen receptor described above or the vector described above.
In one embodiment of the invention, the cells are human T cells. The T cells can be derived from body fluid such as blood and bone marrow, or from tissue such as spleen, thymus, lymph, or cancer tissue such as primary tumor, metastatic tumor, cancerous ascites, etc., and can be obtained by separation and purification. Meanwhile, the T cells may be CD4 + T cells, CD8 + T cells, αβ T cells, or γδ T cells. The T cells may be replaced in a suitable manner with NK cells, which are also considered to be included within the scope of the present invention.
In another aspect, the invention provides an application of the nucleic acid molecule in preparing anti-B cell blood tumor drugs.
In another aspect, the invention provides an application of the chimeric antigen receptor in preparing anti-B cell blood tumor drugs.
In another aspect, the invention provides an application of the vector in preparation of anti-B cell blood tumor drugs.
In another aspect, the invention provides an application of the cell in preparing an anti-B cell blood tumor drug.
Preferably, the above-mentioned application is the application in preparing anti-B cell hematological tumor drugs that are widely co-expressed with CD19 and CD22 in patients or that relapse or fail due to antigenic variation after treatment with CD19 CAR-T.
The above-mentioned invalidation means that the patient is not responsive to CD19CAR-T due to the occurrence of loss, mutation, etc. of CD19 antigen after the patient is treated with CD19CAR-T, and thus the treatment invalidation occurs.
The above-mentioned B-cell hematological tumors include, but are not limited to, B-cell lymphomas, acute Lymphoblastic Leukemia (ALL), etc., as long as they express CD19 and CD22 during pathological processes. Preferably, the B-cell hematological tumor is a B-cell lymphoma or acute B-cell leukemia.
In another aspect of the invention, a pharmaceutical composition is provided comprising the nucleic acid molecule described above, the chimeric antigen receptor described above, the vector described above or the cell described above, and a pharmaceutically acceptable carrier.
The pharmaceutical composition of the present invention may contain any pharmaceutically acceptable additive such as physiological saline, cell culture medium, glucose, water for injection, glycerin, ethanol, and combinations thereof, stabilizers, surfactants, preservatives, isotonic agents, and the like, in addition to the above-mentioned components.
Likewise, the pharmaceutical compositions of the present invention may be used in combination with other suitable anticancer agents. For example vincristine, daunorubicin, asparaginase, cyclophosphamide, prednisone, etc.
Preferably, the pharmaceutical composition of the invention further comprises a nucleic acid molecule encoding a CD19 and CD22 chimeric antigen receptor, a vector comprising a nucleic acid molecule encoding a CD19 and CD22 chimeric antigen receptor or a cell comprising a CD19 and CD22 chimeric antigen receptor.
The above-described nucleic acid molecule encoding a CD19 and CD22 chimeric antigen receptor, a vector comprising a nucleic acid molecule encoding a CD19 and CD22 chimeric antigen receptor, or a cell comprising a CD19 and CD22 chimeric antigen receptor may be any suitable nucleic acid molecule encoding a CD19 and CD22 chimeric antigen receptor, a vector comprising a nucleic acid molecule encoding a CD19 and CD22 chimeric antigen receptor, or a cell comprising a CD19 and CD22 chimeric antigen receptor.
In another aspect, the invention provides the use of the nucleic acid molecule in the treatment of B cell hematological tumors.
In another aspect, the invention provides the use of the chimeric antigen receptor in the treatment of B-cell hematological tumors.
In another aspect, the invention provides the use of the vector in the treatment of B cell hematological tumors.
In another aspect, the invention provides the use of the above cell in the treatment of B cell hematological neoplasms.
In another aspect, the invention provides the application of the pharmaceutical composition in treating B cell hematological tumors.
The beneficial effects of the invention are as follows:
the invention synthesizes and rearranges the light and heavy chains of the mouse anti-human CD22scFv and the mouse anti-human CD19scFv by the nucleic acid molecule chemical synthesis or PCR technology to obtain CD19scFv-CD22scFv, CD22scFv-CD19scFv and CD19V L -CD22V L -CD22V H -CD19V H And CD22V L -CD19V L -CD19V H -CD22V H And cloned into a lentiviral expression vector containing a signal peptide and CD8 alpha-4-1 BB-CD3 zeta, packaged to carry CD19scFv-CD22scFv-CD8 alpha-4-1 BB-CD3 zeta, CD22scFv-CD19scFv-CD8 alpha-4-1 BB-CD3 zeta, CD19V L -CD22V L -CD22V H -CD19V H -CD8 alpha-4-1 BB-CD3 zeta or CD22V L -CD19V L -CD19V H -CD22V H -lentiviral vector of the gene encoding CD8 alpha-4-1 BB-CD3 zeta. T cells are infected with lentiviruses to express the chimeric antigen receptor. The chimeric antigen receptor modified T cell pair CD19 is proved by flow cytometry, degranulation analysis experiments and ELISA detection of cytokines secreted by the T cells + 、CD22 + CD19 + /CD22 + The B cell lymphoma cells and the B cell lymphoblastic leukemia cells have strong killing effect, almost have no killing effect on cells which do not express CD19 and CD22, and effectively prevent off-target effect. The chimeric antigen receptor of the invention can be used for CD19 + And CD22 + Treatment of B-cell hematological tumors, in combination with CD19CAR-T cells or CD22CAR-T cells.
Drawings
FIG. 1 shows the lentiviral expression vector CD19scFv-CD22scFv-CD 8. Alpha. -4-1B in the examples of the present inventionB-CD3ζ、CD22scFv-CD19scFv-CD8α-4-1BB-CD3ζ、CD19V L -CD22V L -CD22V H -CD19V H -CD8 alpha-4-1 BB-CD3 zeta and CD22V L -CD19V L -CD19V H -CD22V H -CD8 a-4-1 BB-cd3ζ restriction enzyme fragment electrophoresis identification map, panel a, 1 being a 15kb nucleic acid molecular weight marker lane; 2 is a DNA fragment (2314 bp) and a vector fragment (7228 bp) encoding CD19scFv-CD22scFv-CD8 alpha-4-1 BB-CD3 zeta obtained by double digestion of lentiviral expression plasmid CD19scFv-CD22scFv-CD8 alpha-4-1 BB-CD3 zeta with endonucleases NheI and Not I, 3 is a DNA fragment (2314 bp) and a vector fragment (7228 bp) encoding CD22scFv-CD19scFv-CD8 alpha-4-1 BB-CD3 zeta obtained by double digestion of lentiviral expression plasmid CD22scFv-CD19scFv-CD8 alpha-1 BB-CD3 zeta with endonucleases NheI and Not I, 4 is a 15kb nucleic acid molecular weight marker lane in FIG. B; 5 is a lentivirus expression plasmid CD19V digested with the endonucleases NheI and NotI L -CD22V L -CD22V H -CD19V H -CD8 alpha-4-1 BB-CD3 zeta resulting in encoded CD19V L -CD22V L -CD22V H -CD19V H DNA fragment (2233 bp) and vector fragment (7234 bp) of-CD 8 alpha-4-1 BB-CD3 zeta, 6 is a lentivirus expression plasmid CD22V digested with the endonucleases NheI and Not I L -CD19V L -CD19V H -CD22V H -CD8 alpha-4-1 BB-CD3 zeta resulting in encoded CD22V L -CD19V L -CD19V H -CD22V H -a DNA fragment (2233 bp) and a vector fragment (7234 bp) of CD8 a-4-1 BB-CD3 ζ;
FIG. 2 is a schematic diagram of an example of a CD22scFv-CD19scFv-CD8 alpha-4-1 BB-CD3 zeta lentiviral expression vector, wherein the anticlockwise sequence is a forward gene fragment and the clockwise sequence is a reverse gene fragment;
FIG. 3 shows the detection of double target CD19scFv-CD22scFv-CD8 alpha-4-1 BB-CD3 zeta, CD22scFv-CD19scFv-CD8 alpha-4-1 BB-CD3 zeta, CD19V constructed in the examples of the present invention by flow cytometry L -CD22V L -CD22V H -CD19V H -CD8 alpha-4-1 BB-CD3 zeta and CD22V L -CD19V L -CD19V H -CD22V H Results of expression of CAR molecules in CD8 a-4-1 BB-CD3 zeta modified T cells (19-22 CAR-T, 22-19CAR-T, 19 gamma 22CAR-T and 22 gamma 19CAR-T, simultaneously displaying the expression results of CAR molecules in single-target CD19scFv-CD8 alpha-4-1 BB-CD3 zeta and CD22scFv-CD8 alpha-4-1 BB-CD3 zeta modified T cells (respectively represented by 19CAR-T and 22 CAR-T) to compare the functions of single-target and double-target CAR-T, GFP is the expression of a marker protein carried by a vector, and F (ab') 2 Labeling the expression of CD22scFv and CD19scFv on T cell surfaces for rabbit anti-mouse IgG;
FIG. 4 shows the detection of target cells used in the examples of the present invention by flow cytometry: the Burkitt lymphoma cell line Namalwa, the chronic granulocytic leukemia cell line MV4-11-CD19 infected with CD19 antigen molecule, the chronic granulocytic leukemia cell line MV4-11-CD22 infected with CD22 antigen molecule, and the expression results of CD19 and CD22 target antigen molecules in wild type MV4-11 cells, wherein A is the positive rate of expression of CD19 target antigen molecule; b is the positive rate of expression of the CD22 target antigen molecule;
FIG. 5 is a graph showing the result of detecting the survival rate of tumor cells remained after co-culturing T cells and target cells in the example of the present invention by flow cytometry, wherein vector-T is a control group of T cells transfected with empty vector; 19CAR-T is CD19scFv-CD8 alpha-4-1 BB-CD3 zeta modified T cell experimental group, 22CAR-T is CD22scFv-CD8 alpha-4-1 BB-CD3 zeta modified T cell experimental group, 19-22CAR-T is CD19scFv-CD22scFv-CD8 alpha-4-1 BB-CD3 zeta modified T cell experimental group, 22-19CAR-T is CD22scFv-CD19scFv-CD8 alpha-4-1 BB-CD3 zeta modified T cell experimental group, 19CAR-T is CD19V experimental group L -CD22V L -CD22V H -CD19V H Experimental group of-CD 8 alpha-4-1 BB-CD3 zeta modified T cells, 22 gamma 19CAR-T is CD22V L -CD19V L -CD19V H -CD22V H -an experimental set of cd8α -4-1BB-cd3ζ modified T cells; a and B are graphs showing the survival rate of tumor cells remaining after co-culturing vector-T, 19CAR-T,22CAR-T,19-22CAR-T and 22-19CAR-T with target cells for 24 hours at effective target ratios of 1:8, 1:4, 1:2 and 1:1, respectively, and a flow chart showing the survival rate of tumor cells remaining after co-culturing for 48 hours at effective target ratios of 1:1, respectively, when the target cells are Namalwa cell lines; where C and D are MV4-11-CD19 cell lines, respectively, vector-T, 19CAR-T,22CAR-T,19-22CAR-T and 22-19CAR-T are each effectiveTarget ratios 1:8, 1:4, 1:2 and 1:1 were co-cultured with target cells for 24 hours, and residual tumor cell viability flow charts after 48 hours of co-culture at an effective target ratio of 1:1; e and F are graphs showing the results of tumor cell viability 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 a flow chart showing the tumor cell viability remaining after co-culturing for 48 hours at effective target ratios of 1:1, respectively; g and H are graphs showing the results of tumor cell viability remaining after co-culturing Vec-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, and residual tumor cell viability after co-culturing at effective target ratios of 1:1 for 48 hours, respectively, when the target cells are wild-type MV4-11 cell lines; i is a graph showing the survival rate of tumor cells remained after co-culturing vector-T, 19 gamma 22CAR-T and 22 gamma 19CAR-T with target cells for 24 hours at effective target ratios of 1:2, 1:1 and 2:1 respectively when the target cells are MV4-11-CD19 and MV4-11-CD22 mixed according to the ratio of 1:1; j is a graph showing the survival rate of tumor cells remained after the vecor-T, the 19 gamma 22CAR-T and the 22 gamma 19CAR-T are co-cultured with target cells for 24 hours according to the effective target ratios of 1:2, 1:1 and 2:1 respectively when the target cells are MV 4-11;
FIG. 6 is a graph showing degranulation detection results of killing effects of vecor-T and CAR-T on Namalwa, MV4-11-CD19, MV4-11-CD22 and MV4-11 (effective target ratio 1:1) in the embodiment of the invention, wherein CAR-T is 19CAR-T,22CAR-T,19-22CAR-T and 22-19CAR-T respectively, and CAR-T is 19 [ 22 ] CAR-T and 22 ] 19CAR-T respectively;
FIG. 7 is a graph showing the results of the levels of cytokines TNF- α (A) IL-2 (B) and IFN- γ (C) released by T cells after co-culturing vecor-T, 19CAR-T,22CAR-T,19-22CAR-T and 22-19CAR-T with four target cells Namalwa, MV4-11-CD19, MV4-11-CD22 and MV4-11, respectively, in an effective target ratio of 1:1 for 48 hours in examples of the present invention;
FIG. 8 is a graph showing the results of detecting the expression of Bone Marrow Mononuclear Cell (BMMCs) CD22 and CD19 target antigen molecules from B-ALL patients by flow cytometry, wherein P1-P4 represent patient numbers;
FIG. 9 is a graph showing the results of flow-through detection of residual tumor cell viability after 48 hours of co-culture of BMMCs from 22-19CAR-T and 19-22CAR-T with B-ALL patients at an effective target ratio of 1:4 in examples of the present invention;
FIG. 10 is a graph of degranulation assay results of BMMCs killing ALL patients by 22-19CAR-T and 19-22CAR-T in examples of the present invention;
FIG. 11 is a graph showing the results of evaluation of the in vivo effects of 22-19CAR-T and 19-22CAR-T in mice according to the examples of the present invention, using 6-8 week old NOD/SCID female mice and randomly divided into two groups, 5X 10 by tail vein injection on day 0 6 Namalwa cells of (A), 1X 10 by tail vein injection on day 5, day 9 and day 12, respectively 7 Mice body weight changes (shown as B) were monitored weekly after T cell injection (shown as a), tumor burden patterns (shown as C), mice survival curves (shown as D), and survival was calculated using SPSS software.
DESCRIPTION OF THE SEQUENCES
SEQ ID NO.1 is the amino acid sequence of the CD19scFv-CD22scFv-CD8 alpha-4-1 BB-CD3 zeta;
SEQ ID NO.2 is the amino acid sequence of the CD22scFv-CD19scFv-CD8 alpha-4-1 BB-CD3 zeta;
SEQ ID NO.3 is CD19V of the present invention L -CD22V L -CD22V H -CD19V H -the amino acid sequence of CD8 a-4-1 BB-CD3 ζ;
SEQ ID NO.4 is CD22V of the present invention L -CD19V L -CD19V H -CD22V H -the amino acid sequence of CD8 a-4-1 BB-CD3 ζ;
SEQ ID NO.5 is the nucleic acid sequence of the CD19scFv-CD22scFv-CD8 alpha-4-1 BB-CD3 zeta;
SEQ ID NO.6 is the nucleic acid sequence of the CD22scFv-CD19scFv-CD8 alpha-4-1 BB-CD3 zeta;
SEQ ID NO.7 is CD19V of the present invention L -CD22V L -CD22V H -CD19V H -a nucleic acid sequence of CD8 a-4-1 BB-CD3 ζ;
SEQ ID NO.8 is CD22V of the present invention L -CD19V L -CD19V H -CD22V H -a nucleic acid sequence of CD8 a-4-1 BB-CD3 ζ;
SEQ ID NO.9 is the amino acid sequence of the CD19scFv-CD22scFv-CD8 alpha-4-1 BB-CD3 zeta antigen recognition region of the present invention;
SEQ ID NO.10 is the amino acid sequence of the CD22scFv-CD19scFv-CD8 alpha-4-1 BB-CD3 zeta antigen recognition region of the present invention;
SEQ ID NO.11 is CD19V of the present invention L -CD22V L -CD22V H -CD19V H -the amino acid sequence of the CD8 a-4-1 BB-CD3 zeta antigen recognition region;
SEQ ID NO.12 is CD22V of the present invention L -CD19V L -CD19V H -CD22V H -the amino acid sequence of the CD8 a-4-1 BB-CD3 zeta antigen recognition region;
SEQ ID NO.13 is the amino acid sequence of the signal peptide in the chimeric antigen receptor targeting CD19 and CD22 of 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 of the present invention.
Detailed Description
The invention discloses chimeric antigen receptor targeting CD22 and CD19 and application thereof, and a person skilled in the art can properly improve the technological parameters by referring to the content of the text. It is to be particularly pointed out that all similar substitutes and modifications apparent to those skilled in the art are deemed to be included in the invention and that the relevant person can make modifications and appropriate alterations and combinations of what is described herein to make and use the technology without departing from the spirit and scope of the invention.
In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art.
In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail with reference to specific embodiments.
Example 1: construction of double-target chimeric antigen receptor vector
1. The plasmid which is constructed in advance by the inventor and contains the CD8 alpha-4-1 BB-CD3 zeta fragment is digested by BamH I and EcoR I endonucleases, and the CD8 alpha-4-1 BB-CD3 zeta fragment is obtained, and the amino acid sequence of the CD8 alpha-4-1 BB-CD3 zeta fragment is shown as SEQ ID NO. 5. The plasmid containing the CD8 alpha-4-1 BB-CD3 zeta fragment may be prepared by any suitable method known in the art, for example, in the patent ZL 201510233748.0.
2. The synthesized CD22scFv-CD19scFv fragment, CD19scFv-CD22scFv and CD19V L -CD22V L -CD22V H -CD19V H CD22V L -CD19V L -CD19V H -CD22V H The fragments are respectively connected with target vectors, and constructed CD22scFv-CD19scFv-CD8 alpha-4-1 BB-CD3 zeta CAR (22-19 CAR), CD19scFv-CD22scFv-CD8 alpha-4-1 BB-CD3 zeta CAR (19-22 CAR) and CD19V L -CD22V L -CD22V H -CD19V H -CD8 alpha-4-1 BB-CD3 zeta (19 gamma 22 CAR-T) and CD22V L -CD19V L -CD19V H -CD22V H The vector of interest-CD 8 a-4-1 BB-CD3 zeta (22 x 19 CAR-T) was identified by cleavage with endonucleases NheI and Not I. The results are shown in FIG. 1, and the digestion results show that the positive clone contains the target band and the sequencing identification is correct. Take the schematic of a CD22scFv-CD19scFv-CD8 alpha-4-1 BB-CD3 zeta CAR vector as an example, as shown in FIG. 2.
Example 2: preparation of double-target chimeric antigen receptor lentivirus modified T cells
1. 22-19CAR, 19-22-CAR, 19 x 22CAR and 22 x 19CAR expression plasmid and packaging plasmid PRSV-Rev, pMDlg-PRRE, pMD.2G were extracted respectively using EndoFree Plasmid Maxi plasmid extraction kit (QIAGEN Co.). Each CAR plasmid and packaging plasmid (four plasmids) was used with 12.2:4.11:8.75:3.5 ratio transfection with PEI transfection reagent (polyscience Co.) was performed (see PEI transfection reagent Specification for specific methods). Fresh culture medium was changed 12 hours after transfection, virus supernatant was collected 24 hours and 48 hours after that, and centrifuged at 4℃and 3000rpm for 15 minutes, filtered through a 0.45 μm filter, concentrated 10 times after ultracentrifugation at 4℃for 1.5 hours, and then transferred to-80℃for preservation.
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.), following the procedure outlined in RosetteSep T cell enrichment Cocktail. According to the cell: magnetic beads = 1:1 proportion of anti-CD 3/CD28 magnetic beads (Gibco company) are added, and the T cells before transfection are obtained after culturing for 24 hours.
3. Lentivirus infected T cells and culture of post-infection T cells: removing virus supernatant from-80deg.C, thawing at room temperature, and mixing at a ratio of 1×10 6 T cells were added to 100. Mu.l of virus supernatant and Polybrene was added to a final concentration of 8. Mu.g/ml. Centrifugation at 1800rpm at 32℃for 1.5 hours, transfer to 5% CO 2 Incubator culture at 37 ℃.
4. Flow cytometry detects the positive rate of CAR modified T cells: cells were collected and labeled rabbit anti-mouse IgG F (ab') 2 Analysis of T cells F (ab') by flow cytometry 2 And expression of GFP. As shown in fig. 3, the positive rate of CAR-T was 70% or more.
Experimental example 1: killing of leukemia cells by chimeric antigen receptor 22-19CAR, 19-22-CAR, 19 gamma 22CAR, and 22 gamma 19CAR lentiviral modified T cells
1. Expression level of CD22 in hematological tumor cell lines:
namalwa, MV4-11 cell lines were purchased from the American ATCC, and MV4-11-CD19 and MV4-11-CD22 were selected as monoclonal cell lines after infection of the MV4-11 cell lines with CD19 and CD22, respectively. After the respective culture, each of the aspirates was 5X 10 5 After washing the cell suspension with PBS for 2 times, PE anti-human CD19 monoclonal antibody and APC anti-human CD22 monoclonal antibody (Biolegend company) were labeled, 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 in the various cell lines were examined using flow cytometry and the results are shown in fig. 4. Wherein A is the positive rate of expression of the CD19 target antigen molecule; b is the positive rate of expression of the CD22 target antigen molecule; the positive rates of Namalwa and MV4-11-CD19 are above 95%, the positive rates of Namalwa and MV4-11-CD22 are above 95%, and the wild type MV4-11 hardly expresses CD19 and CD22.
Flow detection of residual tumor cells after co-culture of car modified T cells with Namalwa, MV4-11-CD19, MV4-11-CD22 and wild-type MV4-11 cell lines:
the cells were packed in a 2X 10 cell array 5 Cell/well inoculation 24-well culture plates, each added 2.5X10 4 (E:T＝1:8)、5×10 4 (E:T＝1:4)、1×10 5 (E:T＝1:2)、2×10 5 (E:T＝1:1)、4×10 5 (E: t=2:1), CAR-T cells at concentration, and co-culturing in incubator with empty vector T cells (vector-T) transfected without CAR as control. The cells after co-culture were labeled with PE anti-human CD19 mab and APC anti-human CD22 mab (Biolegend Corp.) Namalwa, MV4-11-CD19, MV4-11-CD22 and wild type MV4-11 cell lines, T cells were labeled with PE-Cy7 anti-human CD3 mab (Biolegend Corp.), and residual cells were detected by flow cytometry. The results are shown in FIG. 5, 1) the CD19 was significantly killed by 19CAR-T,22CAR-T,19-22CAR-T and 22-19CAR-T as compared to vecor-T + CD22 + Target cells Namalwa (A, B of fig. 5); 2) 19CAR-T,19-22CAR-T, and 22-19CAR-T all significantly killed MV4-11-CD19 target cells (C, D of FIG. 5) as compared to vecor-T and 22 CAR-T; 3) 22CAR-T,19-22CAR-T, and 22-19CAR-T all significantly killed MV4-11-CD22 target cells (E, F of FIG. 5) as compared to vecor-T and 19CAR-T; 4) Similar to vecor-T, 19-22CAR-T and 22-19CAR-T had no killing effect on CD19/CD22 double negative target cells MV4-11 (G, H of FIG. 5); 5) Compared with vecor-T, both 19 gamma 22CAR-T and 22 gamma 19CAR-T can significantly kill MV4-11-CD19 target cells and MV4-11-CD22 target cells (I of FIG. 5); 6) Similar to vecor-T, 19 gamma 22CAR-T and 22 gamma 19CAR-T have no killing effect on CD19/CD22 double negative target cells MV4-11 (J of FIG. 5). The results prove that the 19-22CAR-T, 22-19CAR-T, 19 gamma 22CAR-T and 22 gamma 19CAR-T have obvious double-target advantages on CD19 + 、CD22 + CD19 + /CD22 + The target cells have high-efficiency specific killing effect. In the experiment, the killing effect of the 22-19CAR-T is more remarkable.
3. Degranulation experiments analyze activation of CAR modified T cells:
co-culturing the CAR-T and vecor-T cells with Namalwa, MV4-11-CD19, MV4-11-CD22 and wild MV4-11 cell lines according to an effective target ratio of 1:1, and adding an anti-CD107a antibody and monensin into a co-culture system; after 4h, GFP was detected using flow cytometry + Expression level of cell surface CD107 a. As a result, as shown in A, B of FIG. 6, 1) Namalwa (CD 19) was obtained as a target cell + CD22 + ) After co-cultivation, 19CAR-T,22CAR-T,19-22CAR-T, 22-19CAR-T, 19The degranulation level of the 22CAR-T and 22 gamma 19CAR-T groups is more than 10 percent, and the degranulation level of the vecor-T is less than 2 percent; 2) After co-culture with target cells MV4-11-CD19, the degranulation levels of 19CAR-T,19-22CAR-T, 22-19CAR-T, 19 gamma 22CAR-T and 22 gamma 19CAR-T groups are above 25% and the degranulation levels of vecor-T and 22CAR-T are below 2%; 3) After co-culture with target cells MV4-11-CD22, the degranulation levels of 22CAR-T,19-22CAR-T, 22-19CAR-T, 19 gamma 22CAR-T and 22 gamma 19CAR-T groups are above 15% and the degranulation levels of vecor-T and 19CAR-T are below 2%; 4) After co-culture with wild MV4-11, the average degranulation levels of vecor-T, 19CAR-T,22CAR-T,19-22CAR-T, 19 gamma 22CAR-T and 22 gamma 19CAR-T are below 2%, which proves that 19-22CAR-T, 22-19CAR-T, 19 gamma 22CAR-T and 22 gamma 19CAR-T can significantly and specifically activate CD19 + Or CD22 + Is a target cell of (a).
ELISA to detect the levels of cytokines IFN-gamma, TNF-alpha and IL-2 in the supernatant of lymphoma cell lines co-cultured with CAR-T cells:
namalwa, MV4-11-CD19, MV4-11-CD22 and wild type MV4-11 cell lines were each grown according to 2X 10 5 Cells/well were seeded in 24-well plates. 2X 10 per well 5 The cells were added with CAR-T, vecor-T cells, respectively, and the culture medium was supplemented to 1ml and then co-cultured in a incubator for 24 hours. Detection kit (R) using human IFN-gamma, TNF-alpha and IL-2ELISA&D company), the co-culture supernatant was tested (see ELISA kit instructions for specific steps). The results are shown in FIG. 7, 1) and CD19 + CD22 + After Namalwa co-culture of target cells, the release levels of the three factors of 19CAR-T,19-22CAR-T and 22-19CAR-T groups are all obviously increased compared with vecor-T; the secretion levels of TNF- α were significantly higher in the 19-22CAR-T and 22-19CAR-T groups than in the 19CAR-T group, and the secretion levels of IL-2 were significantly higher in the 19-22CAR-T and 22-19CAR-T groups than in the 22CAR-T group. 2) After co-culture with MV4-11-CD19, 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 22CAR-T. 3) After co-cultivation with MV4-11-CD22, the release levels of 22CAR-T,19-22CAR-T and 22-19CAR-T groups TNF-alpha and IFN-gamma were significantly higher than those of vecor-T and 19CAR-T; whereas the secretion level of IL-2 can be seen with the 19-22CAR-T and 22-19CAR-T groups being superior to 22CAR-TA group. 4) After co-cultivation with wild type MV4-11, the release levels of the three factors of vecor-T, 19CAR-T,22CAR-T and 19-22CAR-T were all lower. Demonstration that 19-22CAR-T and 22-19CAR-T can specifically activate CD19 + 、CD22 + CD19 + CD22 + And release cytokines at levels better than 19CAR-T or 22CAR-T.
Bone Marrow Mononuclear Cells (BMMCs) CD19 and CD22 expression levels in all patients:
the patient specimens are all from the hematopathy hospital of the national academy of medical science and are informed consent of the patient. After BMMCs were separated by Ficoll gradient centrifugation, each was aspirated 5X 10 5 Cell suspensions, after washing 2 times with PBS, were labeled with PE anti-human CD19 mab, APC anti-human CD22 mab (Biolegend Corp.), PE-isotype, and APC-isotype (control group), and incubated on ice for 30 minutes. The proportion and intensity of BMMCs expressing CD19 and CD22 in each patient was examined using flow cytometry, as shown in FIG. 8, wherein P1-P4 represent patient numbers 1-4.
Flow-through detection of residual tumor cells after co-culture of car-modified T cells with BMMCs of ALL patients:
the cells were packed in 4X 10 cells 5 Cell/well inoculation 24-well culture plates, each added with 1X 10 5 CAR-modified T cells (E: t=1:4), transfected CAR-free empty vector T cells (vec-T) were set as control group and co-cultured in incubator for 48h. The co-cultured cells were labeled with PE anti-human CD19 mab and APC anti-human CD22 mab (Biolegend Corp.) to give leukemia cells of ALL patients, and with APC-Cy7 anti-human CD3 mab (Biolegend Corp.) to give T cells, and residual cells were detected by flow cytometry. The results are shown in FIG. 9, which shows that CAR-T and CD19 + /CD22 + After 48 hours of co-culture with BMMCs from patient 1-4, CD19 was retained in 19-22CAR-T cells, respectively + /CD22 + Cells were 3.03%, 0.24%, 1.34%, 2.87%, and 22-19CAR-T retained CD19, respectively + /CD22 + Cells were 4.10%, 0.45%, 1.68%, 3.89%, control persisted at 46.58%, 63.95%, 23.76%, 44.88%. From the above results, it can be demonstrated that CAR-T is against CD19 + /CD22 + Has killing effect on leukemia primary BMMCs.
CAR modified T cells were analyzed for activation by post-co-culture degranulation experiments with ALL patient BMMCs:
co-culturing the CAR-T and vecor-T cells with 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; detection of CD3 after 4h using flow cytometry + Expression level of cell surface CD107 a. The results are shown in FIG. 10, and show that the activation percentages of the 19-22CAR-T cells in the co-culture system of the CAR-T and patients No.1-4 are 35.53%, 40.37%, 31.37% and 30.20%, respectively; the activation percentages of the 22-19CAR-T cells are 51.21%, 33.04%, 18.01% and 50.23%, respectively. In the coculture system of vec-T and patient 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 is significantly different from vecor-T in activation.
Car modified T cells at CD19 + /CD22 + Role in lymphoma mouse model:
NOD/SCID female mice were selected for 6-8 weeks and randomly divided into two groups, 5X 10 by tail vein injection of Namalwa cells 6 Only; 1X 10 by tail vein injection on 5 th, 9 th and 12 th days after transplantation 7 In comparison to the control group, the mice did not significantly lose weight due to CAR-T injection, suggesting that CAR-T treatment had no significant toxic or side effects on mice (see B of fig. 11). Median survival for the vecor-T, 19-22CAR-T and 22-19CAR-T groups was 25 days, 36 days and 37 days, respectively, with tumor burden patterns shown in FIG. 11C, and with CAR-T group tumor burden significantly lower than vec-T group, with survival curves shown in FIG. 11D, and the difference between survival was calculated for the two groups: both the 19-22CAR-T group and the 22-19CAR-T group significantly prolonged survival of mice with significant statistical differences (p=0.0011) compared to the control group. In combination with the median survival and tumor burden results, the therapeutic effect of 22-19CAR-T was found to be slightly better than 19-22CAR-T.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Sequence listing
<110> Chinese medical science center blood disease Hospital (Chinese medical science center blood study center)
<120> chimeric antigen receptor targeting CD22 and CD19 and uses 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
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 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> 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 (18)

1. A nucleic acid molecule encoding a chimeric antigen receptor that targets CD22 and CD19, said chimeric antigen receptor comprising an extracellular region, a transmembrane region and an intracellular signaling region, characterized in that the extracellular region encoded thereby comprises CD22 and CD19 binding domains, said CD22 and CD19 binding domains consisting of an antibody single chain variable region fragment of CD22 and an antibody single chain variable region 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 the amino acid sequence shown in SEQ ID No.11 or the sequence of the amino acid sequence shown in SEQ ID No. 12.
2. The nucleic acid molecule of claim 1, wherein said extracellular region encoded thereby further comprises a signal peptide constructed at the amino terminus of said chimeric antigen receptor, said signal peptide being the signal peptide sequence in CD8 a or GM-CSF.
3. The nucleic acid molecule of claim 2, wherein the signal peptide is as set forth in SEQ ID No. 13.
4. The nucleic acid molecule of claim 1, wherein the CD22 and CD19 binding domains encoded thereby are linked to the transmembrane region encoded thereby by a hinge region; the transmembrane region is a transmembrane domain of a protein selected from the group consisting of: the α, β or ζ chain of a T cell receptor, CD3 epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, 4-1BB or CD154.
5. The nucleic acid molecule of claim 4, wherein the hinge region is a sequence of hinge regions in CD8 a.
6. The nucleic acid molecule of claim 1, wherein the intracellular signaling region encoded thereby further comprises a co-stimulatory factor.
7. The nucleic acid molecule of claim 6, wherein the co-stimulatory factor is one or more of the functional signaling domains obtained by a protein selected from the group consisting of: MHC class I molecules, TNF receptor proteins, immunoglobulin-like proteins, cytokine receptors, integrins, lymphocyte activation signal molecules, activated NK cell receptors, BTLA, toll ligand receptors, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CDs, ICAM-1, LFA-1, 4-1BB, B7-H3, CD278, GITR, BAFFR, LIGHT, HVEM, KIRDS2, SLAMF7, NKp80, NKp44, NKp30, NKp46, CD19, CD4, CD8 α, CD8 β, IL2rβ, IL2rγ, IL7rα, ITGA4, VLA1, CD49 α, IA4, CD49D, ITGA6, VLA6, CD49f, ITGAD, CD11d, ITGAE, CD, ITGAL, CD11 α, ITGAM, CD11B, ITGAX, CD c, CD29, ITGB1, ITGB2, CD18, ITGB7, g2D, NKG2C, TNFR, CD226, CD96, CD69, CD6, SLP 162, CD 36, CD 35, CD 36, or CD 28.
8. The nucleic acid molecule of claim 7, wherein the co-stimulatory factor is CD28 or 4-1BB.
9. The nucleic acid molecule of claim 1, wherein the sequence of the nucleic acid molecule is set forth in SEQ ID No.7 or SEQ ID No. 8.
10. Chimeric antigen receptor targeting CD22 and CD19, characterized in that it is encoded by a nucleic acid molecule according to any one of claims 1 to 9.
11. The chimeric antigen receptor according to claim 10, wherein the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID No.3 or SEQ ID No. 4.
12. A vector comprising a nucleic acid molecule according to any one of claims 1 to 9.
13. A cell comprising the nucleic acid molecule of any one of claims 1 to 9, the chimeric antigen receptor of claim 10 or 11, or the vector of claim 12.
14. Use of a nucleic acid molecule according to any one of claims 1 to 9, a chimeric antigen receptor according to claim 10 or 11, a vector according to claim 12 or a cell according to claim 13 for the preparation of an anti-B cell hematological tumor medicament.
15. The use according to claim 14, wherein the B-cell hematological tumor is a B-cell lymphoma or acute B-cell leukemia.
16. The use according to claim 14, wherein the use is in the manufacture of a medicament for the treatment of B-cell hematological neoplasms in a patient that expresses both CD19 and CD22 or that recurs or is ineffective due to antigenic variation following CD19CAR-T treatment.
17. A pharmaceutical composition comprising the nucleic acid molecule of any one of claims 1 to 9, the chimeric antigen receptor of claim 10 or 11, the vector of claim 12 or the cell of claim 13, and a pharmaceutically acceptable carrier.
18. The pharmaceutical composition of claim 17, further comprising a nucleic acid molecule encoding a CD19 and CD22 chimeric antigen receptor, a vector comprising a nucleic acid molecule encoding a CD19, CD22 chimeric antigen receptor, or a cell comprising a CD19, CD22 chimeric antigen receptor.
CN201910823106.4A 2019-09-02 2019-09-02 Chimeric antigen receptor targeting CD22 and CD19 and application thereof Active CN112442508B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201910823106.4A CN112442508B (en) 2019-09-02 2019-09-02 Chimeric antigen receptor targeting CD22 and CD19 and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910823106.4A CN112442508B (en) 2019-09-02 2019-09-02 Chimeric antigen receptor targeting CD22 and CD19 and application thereof

Publications (2)

Publication Number Publication Date
CN112442508A CN112442508A (en) 2021-03-05
CN112442508B true CN112442508B (en) 2023-05-26

Family

ID=74734279

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910823106.4A Active CN112442508B (en) 2019-09-02 2019-09-02 Chimeric antigen receptor targeting CD22 and CD19 and application thereof

Country Status (1)

Country Link
CN (1) CN112442508B (en)

Families Citing this family (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
CN115925948B (en) * 2021-12-16 2023-07-07 华道(上海)生物医药有限公司 anti-CD 22 nano antibody and application thereof

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010107658A2 (en) * 2009-03-16 2010-09-23 Vallera Daniel A Methods and compositions for bi-specific targeting of cd19/cd22

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
Engineering and Design of Chimeric Antigen Receptors;Sonia Guedan等;《Molecular Therapy: Methods & Clinical Development》;20190331;第12卷;第145-156页 *
Preclinical Development of Bivalent Chimeric Antigen Receptors Targeting Both CD19 and CD22;Haiying Qin等;《Molecular Therapy: Oncolytics》;20181231;第127-137页，补充数据 *
Terry J. Fry等.CD22-CAR T Cells Induce Remissions in CD19-CAR Naïve and Resistant B-ALL.《Nature Medicine》.2017,第24卷(第1期),第20-28页. *

Also Published As

Publication number Publication date
CN112442508A (en) 2021-03-05

Similar Documents

Publication Publication Date Title
CN108715859B (en) Chimeric antigen receptor targeting CD22 and application thereof
CN108276493B (en) Chimeric antigen receptor and application thereof
CN116082518A (en) Chimeric Antigen Receptor (CAR) combined with BCMA and application thereof
CN109266667B (en) Chimeric antigen receptor targeting CD5 and application thereof
WO2017190100A1 (en) Nucleic acid constructs for co-expression of chimeric antigen receptor and transcription factor, cells containing and therapeutic use thereof
CN112979820B (en) CD 123-targeted chimeric antigen receptor and dual-target chimeric antigen receptor containing CD 123-targeted chimeric antigen receptor
CN115058395A (en) Chimeric Antigen Receptors (CAR), compositions and methods of use thereof
JP2020513839A (en) Chimeric antigen receptor targeting TIM-1
CN112074278B (en) BCMA-CAR-T cells
CN111836827B (en) Multispecific chimeric receptors comprising NKG2D domains and methods of use thereof
KR102316091B1 (en) Chimeric antigen receptor targeting BCMA and use thereof
CN113583139A (en) Chimeric receptor and application thereof
CN113248616B (en) Chimeric antigen receptor targeting GPC3 and uses thereof
WO2023109257A1 (en) Humanized bcma antibody and bcma-car-t/bcma-car-dnt cell
CN112442508B (en) Chimeric antigen receptor targeting CD22 and CD19 and application thereof
CN110204619B (en) Chimeric antigen receptor comprising Fc gamma RI and uses thereof
CN114591444A (en) Humanized chimeric antigen receptor based on CD7 and application thereof
CN113454115A (en) Chimeric antigen receptor targeting sialyl lewis a and uses thereof
CN112004825A (en) Cyclin A1-specific T cell receptor and uses thereof
CN108251442B (en) FLT3 chimeric antigen receptor and application thereof
CN117377682A (en) Selective stimulation of T cells in solid tumors using oncolytic virus delivery of orthogonal IL-2
CN113549155B (en) Chimeric antigen receptor for simultaneously targeting CD19 and CD20 and application thereof
CN114790462B (en) Chimeric antigen receptor targeting BCMA and application thereof
US20230272069A1 (en) Chimeric Antigen Receptor Targeting CD22 and CD19 and Application thereof
CN117229407B (en) Single-domain antibody targeting GPRC5D, chimeric antigen receptor and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
TA01 Transfer of patent application right
TA01 Transfer of patent application right

Effective date of registration: 20230508

Address after: 300384 Building 5, No.8, Haitai development road, Huayuan Industrial Park, Binhai high tech Zone, Binhai New Area, Tianjin

Applicant after: Heyuan Biotechnology (Tianjin) Co.,Ltd.

Address before: 300020 No. 288, Nanjing Road, Heping District, Tianjin

Applicant before: Hematology Hospital of Chinese Academy of Medical Sciences (Institute of Hematology, Chinese Academy of Medical Sciences)

GR01 Patent grant
GR01 Patent grant