Double-target chimeric antigen receptor for simultaneously targeting CD19 and BCMA and application thereof
阅读说明：本技术 一种同时靶向cd19和bcma的双靶点嵌合抗原受体及其应用 (Double-target chimeric antigen receptor for simultaneously targeting CD19 and BCMA and application thereof ) 是由 姜钰超 王苗苗 缪佳 刘根桃 吴国祥 于 2020-12-29 设计创作，主要内容包括：本发明提供了一种同时靶向CD19和BCMA的双靶点嵌合抗原受体及编码其的核苷酸和其应用。该多核苷酸的序列为含依次连接的靶向CD19的嵌合抗原受体的编码序列、连接肽的编码序列和靶向BCMA的嵌合抗原受体的编码序列的多核苷酸序列或其互补序列。本发明提供的表达同时靶向CD19和BCMA的双靶点嵌合抗原受体中靶向BCMA的CAR结构与靶向CD19的CAR结构完全独立并以只切割肽串联,促使BCMA靶点和CD19靶点对CAR-T细胞信号传导的作用更独立、更有效,此外,表达该双靶点嵌合抗原受体的T细胞对CD19和BCMA双阳靶细胞更有效的杀伤,防止因靶点丢失导致的疾病进展,提高了治疗CD19和BCMA介导的疾病(特别是多发性骨髓瘤)的潜在疗效。(The invention provides a double-target chimeric antigen receptor for simultaneously targeting CD19 and BCMA, a nucleotide for encoding the same and application thereof. The sequence of the polynucleotide is a polynucleotide sequence containing a coding sequence of the chimeric antigen receptor targeting CD19, a coding sequence of the connecting peptide and a coding sequence of the chimeric antigen receptor targeting BCMA which are connected in sequence or a complementary sequence thereof. The BCMA-targeted CAR structure and the CD 19-targeted CAR structure in the double-target chimeric antigen receptor expressing the CD19 and the BCMA simultaneously are completely independent and are connected in series only by the cleavage peptide, so that the effects of the BCMA target and the CD19 target on CAR-T cell signaling are more independent and more effective, in addition, the T cell expressing the double-target chimeric antigen receptor can kill CD19 and BCMA double-positive target cells more effectively, the disease progression caused by target loss is prevented, and the potential curative effect of treating CD19 and BCMA-mediated diseases (particularly multiple myeloma) is improved.)
1. A polynucleotide whose sequence is a polynucleotide sequence comprising, in sequence, a coding sequence for a chimeric antigen receptor targeting CD19, a coding sequence for a linker peptide, and a coding sequence for a chimeric antigen receptor targeting BCMA, or a complement thereof.
2. The polynucleotide of claim 1, wherein the coding sequence for the chimeric antigen receptor targeting CD19 comprises a coding sequence for an anti-CD 19 single chain antibody; the coding sequence of the anti-CD 19 single-chain antibody is SEQ ID NO. 2.
3. The polynucleotide of claim 1, wherein the coding sequence for the BCMA-targeting chimeric antigen receptor comprises a coding sequence for an anti-BCMA single domain antibody; the coding sequence of the anti-BCMA single domain antibody is selected from SEQ ID NO. 9-12.
4. The polynucleotide of claim 1, wherein the linker peptide is a T2A self-cleaving peptide; the coding sequence of the T2A self-cutting peptide is SEQ ID NO. 7.
5. The polynucleotide of claim 1, wherein the coding sequence for the chimeric antigen receptor targeting CD19 comprises, in sequence, the coding sequence for a CD8 signal peptide, the coding sequence for an anti-CD 19 single chain antibody, the coding sequence for a CD8 hinge region, the coding sequence for a CD8 transmembrane region, the coding sequence for a 41BB intracellular region, and the coding sequence for a CD3 zeta intracellular region.
6. The polynucleotide of claim 5, wherein the coding sequence for the CD8 signal peptide is SEQ ID No. 1; the coding sequence of the anti-CD 19 single-chain antibody is SEQ ID NO. 2; the encoding sequence of the CD8 hinge region is SEQ ID NO. 3; the coding sequence of the CD8 transmembrane region is SEQ ID NO. 4; the coding sequence of the 41BB intracellular region is SEQ ID NO. 5; the coding sequence of the intracellular domain of CD3 ζ is SEQ ID NO. 6.
7. The polynucleotide of claim 1, wherein the coding sequence for the BCMA-targeting chimeric antigen receptor comprises, in sequence, the coding sequence for the GMCSFR signal peptide, the coding sequence for the anti-BCMA single domain antibody, the coding sequence for the CD28 hinge region, the coding sequence for the CD28 transmembrane region, the coding sequence for the CD28 intracellular region, and the coding sequence for the CD3 ζ intracellular region.
8. The polynucleotide of claim 7, wherein the coding sequence of the GMCSFR signal peptide is SEQ ID No. 8; the coding sequence of the anti-BCMA single domain antibody is selected from SEQ ID NO. 9-12; the encoding sequence of the CD28 hinge region is SEQ ID NO. 13; the coding sequence of the CD28 transmembrane region is SEQ ID NO. 14; the coding sequence of the intracellular region of CD28 is SEQ ID NO. 15; the coding sequence of the intracellular domain of CD3 ζ is SEQ ID NO. 6.
9. The polynucleotide-encoded dual-target chimeric antigen receptor targeting both CD19 and BCMA according to any one of claims 1 to 8, wherein said dual-target chimeric antigen receptor is selected from the group consisting of:
(1) a fusion protein comprising a CD8 signal peptide, an anti-CD 19 single chain antibody, a CD8 hinge region, a CD8 transmembrane region, a 41BB intracellular region, a CD3 zeta intracellular region, a T2A self-cleaving peptide, a GMCSFR signal peptide, an anti-BCMA single domain antibody, a CD28 hinge region, a CD28 transmembrane region, a CD28 intracellular region, and a CD3 zeta intracellular region, which are linked in this order; and
(2) the fusion protein derived from the protein (1) and obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence of the fusion protein defined in the protein (1) and retaining the activity of activated T cells.
10. The dual-target chimeric antigen receptor of claim 9, wherein the amino acid sequence of the CD8 signal peptide is SEQ ID No. 16; the amino acid sequence of the light chain variable region of the anti-CD 19 single-chain antibody is SEQ ID NO. 17; the amino acid sequence of the CD8 hinge region is SEQ ID NO. 18; the amino acid sequence of the CD8 transmembrane region is SEQ ID NO. 19; the amino acid sequence of the 41BB intracellular region is SEQ ID NO. 20; the amino acid sequence of the intracellular region of CD3 ζ is SEQ ID No. 21; the amino acid sequence of the T2A self-cutting peptide is SEQ ID NO. 22; the amino acid sequence of the GMCSFR signal peptide is SEQ ID NO. 23; the amino acid sequence of the anti-BCMA single domain antibody is selected from SEQ ID NO. 24-27; the amino acid sequence of the CD28 hinge region is SEQ ID NO. 28; the amino acid sequence of the CD28 transmembrane region is SEQ ID NO. 29; the amino acid sequence of the CD28 intracellular region is SEQ ID NO. 30.
11. A vector comprising the polynucleotide of any one of claims 1-8.
12. The vector of claim 11, wherein the vector is a lentiviral vector further comprising a replication origin, a 3 'LTR and a 5' LTR.
13. A host cell comprising the polynucleotide of any one of claims 1-8 or expressing the dual-target chimeric antigen receptor of any one of claims 9-10.
14. The host cell of claim 13, wherein the host cell is a T cell.
15. A biological agent comprising the host cell of claim 13 or 14.
16. Use of a polynucleotide according to any one of claims 1 to 8, a dual-target chimeric antigen receptor according to any one of claims 9 to 10, a vector according to claim 11, a host cell according to claim 13 or 14 or a biological agent according to claim 15 in the manufacture of a medicament for the treatment of CD19 and BCMA mediated diseases.
17. The use according to claim 16, wherein the CD19 and BCMA mediated diseases comprise leukemia, lymphoma and myeloma.
18. The use of claim 16, wherein the CD19 and BCMA mediated disease comprises multiple myeloma, B cell lymphoma, mantle cell lymphoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, and acute myelogenous leukemia.
Technical Field
The invention relates to the technical field of biological medicines, in particular to a double-target chimeric antigen receptor for simultaneously targeting CD19 and BCMA and application thereof.
Background
Multiple myeloma is a malignant plasma cell disease, which is characterized by malignant clonal proliferation of bone marrow plasma cells, secretes monoclonal immunoglobulin or a fragment thereof (M protein), causes damage to related target organs or tissues such as bones and kidneys, and is commonly and clinically characterized by bone pain, anemia, renal insufficiency, infection and the like [ N Engl J Med,2011.364(11): p.1046-60 ]. Currently, multiple myeloma is the second most serious malignancy in the blood system, accounting for 10% of the malignancy in the blood system, and is frequently developed in men, the incidence rate of which increases year by year with the increase of age, and the multiple myeloma tends to become younger in recent years [ CA cancer J Clin,2014.64(1): p.9-29 ].
The B Cell Maturation Antigen (BCMA), also known as CD269, consists of 184 amino acid residues, an intracellular region of 80 amino acid residues, and an extracellular region of sequencesThe column is very short, with only one carbohydrate recognition domain, a B cell surface molecule. BCMA belongs to type I transmembrane signal protein lacking signal peptide, and is a member of tumor necrosis factor receptor family (TNFR), and can be combined with two ligands of B cell activating factor BAFF or proliferation induced ligand (APRIL) [ Curr Opin Struct Biol,2004.14(2): p.154-60.]. In normal tissues, BCMA is expressed on the surfaces of mature B cells and plasma cells, BCMA knockout mice have normal immune systems and normal spleen structures, B lymphocytes develop normally, but the number of plasma cells is obviously reduced, which proves that BCMA plays an important role in maintaining the survival of plasma cells, and the mechanism mainly comprises BCMA-BAFF protein combination, and up-regulation of anti-apoptosis genes Bcl-2, Mcl-1, Bclw and the like, and cell growth is maintained [ J Exp Med,2004.199(1): p.91-8.]. Similarly, this mechanism also functions in myeloma cells and plays an important role in promoting malignant proliferation of myeloma cells [ Blood,2004.103(8): p.3148-57.]. It has been shown that BCMA is ubiquitously expressed in multiple myeloma cell lines and that detection in multiple myeloma patients gives consistent results [ Blood,2004.103(2): p.689-94.]. Kochenderfer et al, based on the reports, combined with Q-PCR, flow cytometry and immunohistochemistry, studied the expression characteristics of BCMA and confirmed that BCMA is not expressed in normal human tissues except mature B cells and plasma cells and is CD34+Hematopoietic cells also did not express [ Clin Cancer Res,2013.19(8): p.2048-60.]. In combination with the high similarity of BCMA expression profiles to CD19, and the successful progress of anti-CD 19 CAR-T cell therapy, it was suggested that BCMA can be used as one of the CAR-T cell targets for cellular immunotherapy of multiple myeloma.
CD19 is a glycoprotein of 95kDa on the surface of B cells, expressed from early stages of B cell development until it differentiates into plasma cells. CD19 is one of the members of the immunoglobulin (Ig) superfamily, and is one of the components of the B cell surface signal transduction complex, involved in the regulation of the signal transduction process of the B cell receptor. In a mouse model deficient in CD19, there was a marked reduction in the number of B cells in peripheral lymphoid tissues and a reduction in vaccine and mitogen responses accompanied by a reduction in serum Ig levels. It is generally accepted that expression of CD19 is restricted to B cell lines (B-cell lines) and not expressed on the surface of pluripotent hematopoietic stem cells. CD19 is also expressed on the surface of most B cell lymphomas, mantle cell lymphomas, ALLs, CLLs, hairy cell leukemias and a fraction of acute myeloid leukemia cells. Thus, CD19 is a very valuable immunotherapeutic target in the treatment of leukemia/lymphoma. Importantly, the feature that CD19 is not expressed on the surface of most normal cells other than B cells, including pluripotent hematopoietic stem cells, allows CD19 to be a safe therapeutic target, minimizing the risk of patients developing autoimmune diseases or irreversible bone marrow toxic injuries. While multiple myeloma as a B cell line tumor does not typically express CD19, CD19 has not been the first choice target for immunotherapy for multiple myeloma in the past. 9.2015, Carl June research group published an article in the New England journal of successful treatment of 1 patient with relapsed refractory Multiple Myeloma (MM) using a CD19 molecule-targeted CAR-T cell therapy [ NEJM,2015.373(11): p.1040-7 ]. Trace amounts of multiple myeloma clones with drug resistance and disease recurrence properties have also been reported to have a B cell phenotype (i.e., CD19 positive). Recent studies have shown that it is generally accepted that very low density CD19 expression (less than 100 molecules per cell) is also present on the surface of myeloma cells that do not express CD19, and that such low density CD19 expression cannot be measured by conventional assays, but that these cells can still be killed by anti-CD 19 CAR-T, which is also likely one of the evidences that CAR-T cells directed against CD19 can treat myeloma [ Nat Commun,2019.10(1): p.3137 ].
Biological immune cell therapy has become a fourth means for treating tumors except for surgery, radiotherapy and chemotherapy, and will become a necessary means for treating tumors in the future. CAR-T cell back-infusion therapy is the most clearly effective form of immunotherapy in current tumor therapy. A large number of studies show that the CAR-T cells can effectively recognize tumor antigens, cause specific anti-tumor immune response and remarkably improve the survival condition of patients. Chimeric Antigen Receptors (CARs) are a core component of CAR-T, conferring on T cells the ability to recognize tumor antigens in an HLA-independent manner, which enables CAR-engineered T cells to recognize a broader range of targets than native T cell surface receptor TCRs. The basic design of the CAR includes a tumor-associated antigen (TAA) binding region (usually the scFV fragment from the antigen binding region of a monoclonal antibody), an extracellular hinge region, a transmembrane region, and an intracellular signaling region. The choice of antigen of interest is a key determinant for the specificity, efficacy of the CAR and safety of the genetically engineered T cells themselves.
After it was clear that BCMA could be a target for CAR-T cells, the american national Cancer institute Kochenderfer et al successfully constructed anti-BCMA CAR-T cells, and preclinical studies showed that the CAR-T cells specifically recognized BCMA, and after activation by BCMA, were greatly expanded, secreted cytokines and exerted killing function, and also had anti-tumor effects in mouse tumorigenesis models [ Clin Cancer Res,2013.19(8): p.2048-60 ]. The national cancer institute in the united states of america in 2014 developed a phase I clinical study of anti-BCMA CAR-T cell therapy for multiple myeloma, validating the clinical safety and efficacy of anti-BCMA CAR-T cells against multiple myeloma patients that do not respond to current standard treatment protocols (clinical trials. gov Identifier: NCT 02215967). Subsequent numerous and exotic clinical studies of CAR-T targeting BCMA targets have also demonstrated exciting clinical effects. Given that both the BCMA target and the CD19 target have been fully clinically validated, the dual-target chimeric antigen receptor targeting both BCMA and CD19 can significantly increase the therapeutic effect, but there is no related report at present.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a dual-target chimeric antigen receptor which simultaneously targets CD19 and BCMA and application thereof. The T cell expressing the double-target chimeric antigen receptor has strong killing function on tumor cells with positive targets in vitro, the expression of CD107a and the secretion of IFN gamma are higher, the killing efficiency on MM.1s myeloma cells is higher than 90% under the condition that the effective target ratio is 5:1, and the killing efficiency on Raji lymphoma cells is higher than 90% under the condition that the effective target ratio is 10: 1. In the aspect of in vivo experiments, a myeloma model constructed by injecting H929 myeloma cells into NSG mice intravenously is utilized, and the double-target chimeric antigen receptor T cells can obviously inhibit the proliferation of the multiple myeloma cells H929 in immunodeficient mice, so that the survival cycle of the mice is obviously prolonged.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a polynucleotide; the sequence of the polynucleotide is a polynucleotide sequence containing a coding sequence of the chimeric antigen receptor targeting CD19, a coding sequence of the connecting peptide and a coding sequence of the chimeric antigen receptor targeting BCMA which are connected in sequence or a complementary sequence thereof.
Further, the coding sequence of the chimeric antigen receptor targeting CD19 includes the coding sequence of an anti-CD 19 single chain antibody; the coding sequence of the anti-CD 19 single-chain antibody is SEQ ID NO. 2.
Further, the coding sequence of the chimeric antigen receptor targeting BCMA comprises the coding sequence of an anti-BCMA single domain antibody; the coding sequence of the anti-BCMA single domain antibody is selected from SEQ ID NO. 9-12.
Further, the connecting peptide is a T2A self-cleaving peptide; the coding sequence of the T2A self-cleaving peptide is SEQ ID NO. 7.
Further, the coding sequence of the chimeric antigen receptor targeting CD19 comprises a coding sequence of a CD8 signal peptide, a coding sequence of an anti-CD 19 single-chain antibody, a coding sequence of a CD8 hinge region, a coding sequence of a CD8 transmembrane region, a coding sequence of a 41BB intracellular region, and a coding sequence of a CD3 zeta intracellular region, which are sequentially linked.
Further, the coding sequence of the CD8 signal peptide is SEQ ID NO. 1; the coding sequence of the anti-CD 19 single-chain antibody is SEQ ID NO. 2; the coding sequence of the CD8 hinge region is SEQ ID NO. 3; the coding sequence of the CD8 transmembrane region is SEQ ID NO. 4; the coding sequence of the 41BB intracellular domain is SEQ ID NO. 5; the coding sequence of the intracellular domain of CD3 ζ is SEQ ID NO. 6.
Further, the coding sequence of the chimeric antigen receptor targeting BCMA comprises a coding sequence of a GMCSFR signal peptide, a coding sequence of an anti-BCMA single domain antibody, a coding sequence of a CD28 hinge region, a coding sequence of a CD28 transmembrane region, a coding sequence of a CD28 intracellular region, and a coding sequence of a CD3 zeta intracellular region, which are connected in sequence.
Further, the coding sequence of the GMCSFR signal peptide is SEQ ID NO. 8; the coding sequence of the anti-BCMA single domain antibody is selected from SEQ ID NO. 9-12; the coding sequence of the CD28 hinge region is SEQ ID NO. 13; the coding sequence of the CD28 transmembrane region is SEQ ID NO. 14; the coding sequence of the above-mentioned intracellular region of CD28 is SEQ ID NO. 15; the coding sequence of the intracellular domain of CD3 ζ is SEQ ID NO. 6.
In a second aspect, the present invention provides a dual-target chimeric antigen receptor that targets both CD19 and BCMA, encoded by a polynucleotide as provided in the first aspect; the dual-target chimeric antigen receptor is selected from the group consisting of:
(1) a fusion protein comprising a CD8 signal peptide, an anti-CD 19 single chain antibody, a CD8 hinge region, a CD8 transmembrane region, a 41BB intracellular region, a CD3 zeta intracellular region, a T2A self-cleaving peptide, a GMCSFR signal peptide, an anti-BCMA single domain antibody, a CD28 hinge region, a CD28 transmembrane region, a CD28 intracellular region, and a CD3 zeta intracellular region, which are linked in this order; and
(2) the fusion protein derived from the protein (1) and obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence of the fusion protein defined in the protein (1) and retaining the activity of activated T cells.
Further, the amino acid sequence of the CD8 signal peptide is SEQ ID NO. 16; the amino acid sequence of the light chain variable region of the anti-CD 19 single-chain antibody is SEQ ID NO. 17; the amino acid sequence of the CD8 hinge region is SEQ ID NO. 18; the amino acid sequence of the CD8 transmembrane region is SEQ ID NO. 19; the amino acid sequence of the 41BB intracellular domain is SEQ ID NO. 20; the amino acid sequence of the intracellular domain of CD3 ζ is SEQ ID NO. 21; the amino acid sequence of the T2A self-cutting peptide is SEQ ID NO. 22; the amino acid sequence of the GMCSFR signal peptide is SEQ ID NO. 23; the amino acid sequence of the anti-BCMA single domain antibody is selected from SEQ ID NO. 24-27; the amino acid sequence of the CD28 hinge region is SEQ ID NO. 28; the amino acid sequence of the CD28 transmembrane region is SEQ ID NO. 29; the amino acid sequence of the above-mentioned CD28 intracellular domain is SEQ ID NO. 30.
In a third aspect, the present invention provides a vector comprising a polynucleotide as provided in the first aspect.
Further, the above vector is a lentiviral vector, which further contains a replication origin, a 3 'LTR (long terminal repeat) and a 5' LTR.
In a fourth aspect, the present invention provides a host cell comprising a polynucleotide as provided in the first aspect or expressing a dual-target chimeric antigen receptor as provided in the second aspect.
Further, the host cell is a T cell.
In a fifth aspect, the present invention provides a biological agent comprising a host cell as provided in the fourth aspect.
In a sixth aspect, the present invention provides the use of a polynucleotide as provided in the first aspect, a dual-target chimeric antigen receptor as provided in the second aspect, a vector as provided in the third aspect, a host cell as provided in the fourth aspect or a biological agent as provided in the fifth aspect in the manufacture of a medicament for the treatment of a CD19 and BCMA mediated disease.
Further, the above CD19 and BCMA mediated diseases include leukemia, lymphoma, and myeloma.
Further, the above CD19 and BCMA mediated diseases include multiple myeloma, B cell lymphoma, mantle cell lymphoma, acute lymphocytic leukemia, chronic lymphocytic leukemia, hairy cell leukemia, and acute myelogenous leukemia.
By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:
the BCMA-targeted CAR structure and the CD 19-targeted CAR structure in the double-target chimeric antigen receptor expressing the CD19 and the BCMA simultaneously are completely independent and are connected in series only by the cleavage peptide, so that the effects of the BCMA target and the CD19 target on CAR-T cell signaling are more independent and more effective, in addition, the T cell expressing the double-target chimeric antigen receptor can kill CD19 and BCMA double-positive target cells more effectively, the disease progression caused by target loss is prevented, and the potential curative effect of treating CD19 and BCMA-mediated diseases (particularly multiple myeloma) is improved.
Drawings
FIG. 1 is a schematic structural diagram of a KQ-19B CAR lentiviral vector (pLenti7.3-scFv-BBz-VHH-28Z) according to one embodiment of the invention;
FIG. 2 is a schematic view of a KQ-19B CAR element according to an embodiment of the present invention;
FIG. 3 is a graph showing the results of flow analysis of the expression of CD19 and BCMA on the surface of each target cell in an embodiment of the present invention;
FIG. 4 is a schematic representation of the CAR structure of KQ-19B CAR-T cells recognizing CD19 and BCMA double positive tumor cells in one embodiment of the invention;
FIG. 5 shows the result of detecting the positive rate of KQ-19B-T in one embodiment of the present invention;
FIG. 6 is a graph showing the results of an example of the present invention in which KQ-19B-T effectively killed BCMA and CD19 positive tumor cells in vitro;
FIG. 7 is a graph showing the results of cytokine secretion by KQ-19B-T incubated with target cells in vitro in accordance with one embodiment of the present invention;
FIG. 8 is a graph showing the results of high expression of CD107a by in vitro co-incubation of KQ-19B-T with various target cells in accordance with one embodiment of the present invention;
FIG. 9 shows the in vivo efficacy of D0 in two groups at time point according to one embodiment of the present invention;
FIG. 10 is a graph showing the results of the drug effect of KQ-19B-T in myeloma (H929) model NSG mouse according to an embodiment of the present invention;
FIG. 11 is a graph of the change in body weight of a myeloma (H929) model NSG mouse according to an embodiment of the present invention;
FIG. 12 is a survival curve for a myeloma (H929) model NSG mouse according to an embodiment of the present invention.
Detailed Description
The invention relates to a double-target chimeric antigen receptor for simultaneously targeting CD19 and BCMA, and a nucleotide encoding the same and application thereof.
Specifically, the sequence of the polynucleotide is a polynucleotide sequence containing a coding sequence of the chimeric antigen receptor targeting CD19, a coding sequence of a connecting peptide and a coding sequence of the chimeric antigen receptor targeting BCMA which are connected in sequence or a complementary sequence thereof.
In a preferred embodiment of the invention, the coding sequence of the chimeric antigen receptor targeting CD19 comprises the coding sequence of an anti-CD 19 single-chain antibody, the coding sequence of the anti-CD 19 single-chain antibody is SEQ ID No. 2; the coding sequence of the chimeric antigen receptor targeting BCMA comprises the coding sequence of an anti-BCMA single domain antibody; the coding sequence of the anti-BCMA single domain antibody is selected from SEQ ID NO. 9-12; the connecting peptide is T2A self-cutting peptide; the coding sequence of the T2A self-cleaving peptide is SEQ ID NO. 7.
In a preferred embodiment of the present invention, the coding sequence of the chimeric antigen receptor targeting CD19 comprises the coding sequence of CD8 signal peptide, anti-CD 19 single chain antibody, CD8 hinge region, CD8 transmembrane region, 41BB intracellular region and CD3 ζ intracellular region, which are sequentially linked. More preferably, the coding sequence of the CD8 signal peptide is SEQ ID NO. 1; the coding sequence of the anti-CD 19 single-chain antibody is SEQ ID NO. 2; the coding sequence of the CD8 hinge region is SEQ ID NO. 3; the coding sequence of the CD8 transmembrane region is SEQ ID NO. 4; the coding sequence of the 41BB intracellular domain is SEQ ID NO. 5; the coding sequence of the intracellular domain of CD3 ζ is SEQ ID NO. 6.
In a preferred embodiment of the present invention, the coding sequence of the chimeric antigen receptor targeting BCMA comprises the coding sequence of the GMCSFR signal peptide, the coding sequence of the anti-BCMA single domain antibody, the coding sequence of the hinge region of CD28, the coding sequence of the transmembrane region of CD28, the coding sequence of the intracellular region of CD28 and the coding sequence of the intracellular region of CD3 ζ, which are sequentially linked. More preferably, the coding sequence of the above GMCSFR signal peptide is SEQ ID No. 8; the coding sequence of the anti-BCMA single domain antibody is selected from SEQ ID NO. 9-12; the coding sequence of the CD28 hinge region is SEQ ID NO. 13; the coding sequence of the CD28 transmembrane region is SEQ ID NO. 14; the coding sequence of the above-mentioned intracellular region of CD28 is SEQ ID NO. 15; the coding sequence of the intracellular domain of CD3 ζ is SEQ ID NO. 6.
The double-target chimeric antigen receptor targeting both CD19 and BCMA is a fusion protein encoded by the polynucleotide, and is selected from the group consisting of:
(1) a fusion protein comprising a CD8 signal peptide, an anti-CD 19 single chain antibody, a CD8 hinge region, a CD8 transmembrane region, a 41BB intracellular region, a CD3 zeta intracellular region, a T2A self-cleaving peptide, a GMCSFR signal peptide, an anti-BCMA single domain antibody, a CD28 hinge region, a CD28 transmembrane region, a CD28 intracellular region, and a CD3 zeta intracellular region, which are linked in this order; and
(2) the fusion protein derived from the protein (1) and obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence of the fusion protein defined in the protein (1) and retaining the activity of activated T cells.
In a preferred embodiment of the invention, the amino acid sequence of the CD8 signal peptide is SEQ ID NO. 16; the amino acid sequence of the light chain variable region of the anti-CD 19 single-chain antibody is SEQ ID NO. 17; the amino acid sequence of the CD8 hinge region is SEQ ID NO. 18; the amino acid sequence of the CD8 transmembrane region is SEQ ID NO. 19; the amino acid sequence of the 41BB intracellular domain is SEQ ID NO. 20; the amino acid sequence of the intracellular domain of CD3 ζ is SEQ ID NO. 21; the amino acid sequence of the T2A self-cutting peptide is SEQ ID NO. 22; the amino acid sequence of the GMCSFR signal peptide is SEQ ID NO. 23; the amino acid sequence of the anti-BCMA single domain antibody is selected from SEQ ID NO. 24-27; the amino acid sequence of the CD28 hinge region is SEQ ID NO. 28; the amino acid sequence of the CD28 transmembrane region is SEQ ID NO. 29; the amino acid sequence of the above-mentioned CD28 intracellular domain is SEQ ID NO. 30.
The nucleotide and amino acid sequences provided above are shown in tables 1-2 below:
TABLE 1 detailed information of nucleotide sequences
Table 2 detailed information of amino acid sequences
The present invention will be described in detail and specifically with reference to the following examples to facilitate better understanding of the present invention, but the following examples do not limit the scope of the present invention.
In the examples, the conventional methods were used unless otherwise specified, and reagents used were those conventionally commercially available or formulated according to the conventional methods without specifically specified.
Example 1
In this example, the specific operation steps for determining the CAR structural gene sequence and constructing the CAR lentiviral vector are as follows:
(1) the amino acid sequences of the human CD8 hinge region, human CD8 transmembrane region, human 41BB intracellular region, human CD3 ζ intracellular region, human CD28 hinge region, human CD28 transmembrane region, human CD28 intracellular region were determined from uniprot protein databases. The amino acid sequence of anti-human CD19 single chain antibody (FMC63 clone) was searched from NCBI website database. The amino acid sequence of an anti-human BCMA single domain antibody (clone B1) was obtained from patent CN 201910244701.2. The amino acid sequences of a human CD8 signal peptide, an anti-human CD19 single-chain antibody, a human CD8 hinge region, a human CD8 transmembrane region, a human 41BB intracellular region, a human CD3 zeta intracellular region, a T2A self-cleavage peptide, a human GMCSFR signal peptide, an anti-human BCMA single-domain antibody, a human CD28 hinge region, a human CD28 transmembrane region, a human CD28 intracellular region and a human CD3 zeta intracellular region which are connected in sequence are subjected to codon optimization on a website http:// sg.
(2) The codon-optimized nucleic acid sequences (SEQ ID NO. 1-9 and SEQ ID NO. 13-15) were subjected to gene synthesis with Nanjing Kinsley, and constructed into a pLenti7.3 lentiviral vector, and the lentiviral vector containing the CAR sequence (the structure of which is shown in FIG. 2) was named as pLenti7.3-scFv-BBz-VHH-28Z (shown in FIG. 1).
Example 2
In this example, the target cells are constructed and detected, and the specific operation steps and results are as follows:
(1) the full-length amino acid sequence of the human CD19 protein and the full-length amino acid sequence of the human BCMA protein are searched from an NCBI website database. The codon optimization is carried out on the http:// sg.idtdna.com/site by the amino acid sequences of the human CD19 protein full length, the T2A self-cutting peptide and the human BCMA protein full length which are connected in sequence, so as to obtain the nucleotide sequence which is more suitable for being expressed in human cells. The nucleic acid sequence after codon optimization is entrusted with Nanjing Kingsry for gene synthesis, and is constructed into a pLent7.3 lentiviral vector, and the lentiviral vector containing the CAR sequence is named as pLenti7.3-CD 19-BCMA. Meanwhile, Nanjing Kingsrei is entrusted to respectively construct lentiviral vectors containing the full length of the encoded human CD19 protein and the full length of the encoded human BCMA protein, and the lentiviral vectors are named as pLenti7.3-CD19 and pLenti7.3-BCMA respectively.
(2) Construction of target cells: preparing corresponding virus vectors by using the plasmids, respectively transducing the virus vectors into K562 cells to obtain three target cells of K562-CD19, K562-BCMA and K562-CD19-BCMA, and then obtaining a monoclonal cell line with over-expressed target spots by a limiting dilution method. In addition, Raji and mm.1s cell lines were also prepared as target cells.
As shown in fig. 3, anti-CD 19 and anti-BCMA antibodies were used to detect target expression of each target cell: raji expresses CD19 highly, MM.1s expresses BCMA highly, K562-CD19 weakly expresses CD19, K562-BCMA weakly expresses BCMA, and K562-CD19-BCMA weakly expresses CD19 and BCMA simultaneously.
Example 3
In this example, the specific procedures for packaging lentivirus and preparing CAR-T are as follows:
packaging of CAR lentiviruses: the plasmids prepared in example 1 were first extracted separately using QIAGEN endotoxin-free large quality plasmid kit. Sufficient lentiviral plasmid pLenti7.3-scFv-BBz-VHH-28Z and lentivirus were preparedThe system helper packages plasmids pmd2.g, pMDLg and pRSV. 1.8X 10 day before transfection7Individual 293T cells were plated into T175 flasks. The 293T cell medium was changed to 30ml serum-free medium 1 hour before transfection. The appropriate ratio of the four plasmid mixture was co-transfected into 293T cells using calcium phosphate precipitation and the cell culture medium was changed to 60ml complete medium DMEM + 10% FBS 24 hours after transfection. Cell supernatants were harvested 48 hours post transfection and 60ml of fresh complete medium was added. Cell supernatants were harvested again for 72 hours and discarded. The harvested cell supernatant was centrifuged at 5000g for 3min to remove impurities, and then filtered using a 0.45 μm filter, followed by centrifugation at 40000g for 4 hours to pellet the virus, which was resuspended using 0.1ml PBS, and the virus titer was measured. Standing at-80 deg.C for freezing.
2. Double target CAR-T preparation: the inventors named this dual-target CAR-T as KQ-19B-T cell (whose CAR structure recognizes CD19 and BCMA double positive tumor cell schematic as shown in figure 4). Retronectin, anti-human CD3 and CD28 antibodies were coated one day in advance in 6-well plates overnight at 4 ℃ and washed twice with the previous PBS for use. Collecting blood and separating PBMC by conventional method, sorting T cells with STEMCELL T cell sorting kit, counting, and dividing the obtained T cells into 1 × 106Each/ml was resuspended in X-VIVO15 medium containing 5% human AB serum and 100IU/ml interleukin-2 and plated onto coated plates for culture. 24 hours after the start of the incubation, 5. mu.g/ml of the clotrimamine solution and lentivirus at MOI3 were added, mixed and infected at 37 ℃ for 24 hours. Then, the cell sediment is collected by centrifugation and is cultured by changing the culture medium into X-VIVO15 with 5 percent of human AB serum and 100IU/ml of interleukin-2. Subsequent cultures maintained the cells at 1X 10 by supplementing the medium6Density per ml, scFv expression was detected 72 hours later using flow cytometry to detect CAR molecule transduction efficiency. Positive rate detection was performed using BCMA-Fc, which detects the B1 single domain antibody, and protein L, which detects the kappa light chain of FMC63 antibody, respectively.
As shown in FIG. 5, the CAR-T cell positivity was approximately 34% as measured by protein L and 19.9% as measured by BCMA-Fc. Since BCMA-Fc was not strong enough to bind B1 single domain antibodies, protein L-measured positive rate data was generated.
Example 4
The in vitro function of the dual-target CAR-T was explored in this example, and the specific experimental methods and results were as follows:
1. the capacity of KQ-19B-T cells to kill target cells in vitro is verified: 5-carboxyfluorescein succinimidyl (CFSE) is used for staining effector cells, KQ-19B-T and MM.1S and Raji cells are respectively taken and co-cultured in a mixed mode according to an effect-target ratio (E: T) of 2:1, 5:1 and 10:1, and two control groups are respectively a Raji cell group with T cells and different effect-target ratios and a K562 cell group with KQ-19B-T and different effect-target ratios. After 4 hours of co-culture, cells were stained with Annexin V and PI kit. Cell killing was detected using flow cytometry and the results are shown in figure 6.
As can be seen in FIG. 6, KQ-19B-T cells were able to kill Raji cells and MM.1s efficiently, but control T cells were not able to kill target cells efficiently, indicating that the CAR element mediates the specific killing of Raji cells and MM.1s by KQ-19B-T. Meanwhile, KQ-19B-T cells cannot effectively kill CD19 and BCMA negative K562 cells, which shows that the killing of KQ-19B-T to target cells is target-specific. The results show that KQ-19B-T can effectively kill BCMA positive multiple myeloma cells, can also effectively kill CD19 positive lymphoma cells, and has definite BCMA and CD19 targeting effects.
Cytokine secretion assay by co-incubation of KQ-19B-T cells with target cells: cytokine secretion is an important manifestation of immune cell function, so the inventors evaluated CAR-T function in vitro by co-culturing KQ-19B-T with target cells overnight and detecting three important cytokines by CBA method using a mature commercial kit. Selecting an effective target ratio of 2:1 for co-culture, co-culturing KQ-19B-T with Raji, MM.1s, K562 with three over-expressed target spots and wild K562 overnight respectively, and setting a control group for co-incubation of T cells and each target cell.
As shown in FIG. 7, the cytokine secretion of KQ-19B-T and Raji, MM.1s and K562 co-incubation group with three over-expressed target strains are remarkably increased compared with that of K562 co-incubation group with negative target strains, which indicates that the killing of the KQ-19B-T on target cells is target-specific. Cytokine secretion was low in the T cell co-incubated group with all target cells, suggesting that the CAR element mediates the killing of the target cells by KQ-19B-T. In conclusion, the function of the KQ-19B-T cell is verified by detecting the cell factor, and the cell factor secretion result is consistent with the result of in vitro killing.
And 3, detecting the degranulation function of the KQ-19B-T cells: CD107a is located on the surface of intracellular cytotoxic particles, and CD107a can only be present on the surface of cell membranes if cytotoxic particles fuse with effector cell membranes and release perforin, granzyme, etc. when the effector cells kill tumor target cells. The CD107a molecule is therefore a sensitive marker of the degranulation function of cytotoxic T lymphocytes, directly related to cytotoxic activity. By using the characteristic, after effector cells and tumor cells are co-incubated, a protein transport inhibitor (GolgiStop) containing monensin is added to inhibit the endocytosis of CD107a molecules, and simultaneously, labeled antibodies such as CD107a and CD3 are added to the co-incubation for flow detection. Then, the activation of cultured T cells can be analyzed, and the activated T cells have tumor killing capability, and the higher the proportion of CD107a positive cells in CD8 positive cells is, the stronger the tumor killing capability is. Six target cell experimental groups of Raji, MM.1s, K562 with three-strain target over-expression and wild K562 are arranged together, KQ-19B-T cells and T cells are used as effector cells to be incubated for 4 hours, and the proportion of CD107a positive cells in CD8 positive cells is detected in a flow mode.
The result is shown in the left side of figure 8, KQ-19B-T cells and Raji, MM.1s, K562 over-expressed by three strains of target spots can highly express CD107a after being incubated, and the good degranulation function indicates that the KQ-19B-T cells have good tumor killing capability. Notably, the expression level of CD107a of each group of KQ-19B-T cells incubated with K562 overexpressed as a target of three strains is remarkably different, and the killing capacity of the KQ-19B-T cells on CD19 and BCMA double-positive target cells is stronger than that of CD19 or BCMA single-positive target cells. This result may be due to the inventive dual-target design of the present invention, the toxicological CAR structures for CD19 and BCMA targets comprise 41BB-CD3 ζ and CD28-CD3 ζ costimulatory domains, respectively, so that the three costimulatory signals will co-activate and enhance the killing ability of KQ-19B-T cells when dual targets are present simultaneously. In addition, as shown in the right part of FIG. 8, in another independent experiment, KQ-19B-T can highly express CD107a when being incubated with Raji, 8266 and H929 cells.
Example 5
This example studies the pharmacodynamics of KQ-19B-T in animals, and specifically explores the pharmacodynamics of NSG mice intravenously transplanted with human H929-luc cells after single intravenous injection of an injection solution of anti-human KQ-19B-T cells. The following are mainly evaluated: 1. observing the survival period of the mouse; 2. detecting tumor burden by a live imager; 3. and preliminarily identifying whether the KQ-19B-T has toxicity to the mice by taking the body weight as an index.
Test protocol: NSG mice were divided into two groups of 20 mice at random, each group consisting of 10 mice. Mice in each group were injected 3D tail vein with 6H 929-luc, and after 3D (OD) CART and NT cells (5E 6 for CAR positive cells) were administered separately in a single intravenous dose at a dose volume of 200. mu.l/mouse. Mice were then live imaged twice weekly to observe tumor burden and measure body weight. As shown in fig. 9, at D0, 22 mice that had been modeled were imaged in vivo, and divided into two groups based on body weight and in vivo imaging signals.
As shown in fig. 10, tumor burden was detected by in vivo imager in two groups D3-D10 of mice. Control mice reinfused with NT cells, with a significant increase in tumor burden over time, one mouse died at D10 and three mice died at D28; the tumor burden at each time point was lower in experimental mice back-transfused with CAR-T cells than in control. The body weight data for both groups of mice at each time point was stable, suggesting the safety of CAR-T, and the body weight profile of the mice over time is shown in figure 11. By the end of the 40 day observation, only two mice survived in the control group of mice back-infused with NT cells, and only one mouse died in the experimental group of mice back-infused with CAR-T cells, with survival curves as shown in fig. 12.
The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.
Sequence listing
<110> Shanghai Keqi pharmaceutical science and technology Limited
<120> double-target chimeric antigen receptor simultaneously targeting CD19 and BCMA and application thereof
<160> 30
<170> SIPOSequenceListing 1.0
<210> 1
<211> 63
<212> DNA
<213> coding sequence of human CD8 signal peptide (artificial sequence)
<400> 1
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccg 63
<210> 2
<211> 726
<212> DNA
<213> coding sequence of FMC63 (artificial sequence)
<400> 2
gacattcaga tgactcagac cacaagcagc ctcagtgcga gcctggggga cagggtgact 60
atcagctgcc gggccagcca ggacatttcc aagtacctga attggtacca gcagaagccc 120
gatggtactg tgaaactcct gatatatcat acttctaggc tccattccgg ggttccaagc 180
cgattcagtg gctccggttc cggtacagat tattccctga ccattagcaa cttggaacag 240
gaggacattg caacgtattt ctgtcagcaa ggcaacacat tgccctacac attcgggggc 300
gggactaaac tcgaaataac tggcggcggg ggttctggtg gcggcggcag cggcggtgga 360
ggatcagaag tgaagctgca ggaaagtggc cccgggctgg tagccccaag tcagtccctg 420
agtgtaacct gtacagtgag tggagtgtct cttcctgact acggggtaag ttggattcgg 480
caacctccac gcaagggcct ggagtggctc ggcgtgattt ggggatctga gacaacttac 540
tacaattccg ccctgaagag caggctgacc atcattaagg acaatagcaa gtcacaggtg 600
tttctgaaga tgaactcact gcagaccgac gacaccgcca tctattactg cgccaaacat 660
tattattatg gcgggagtta tgctatggac tactggggcc agggcactag cgtcaccgtc 720
agcagt 726
<210> 3
<211> 141
<212> DNA
<213> coding sequence of the hinge region of human CD8 (artificial sequence)
<400> 3
actacaactc cagcacccag accccctaca cctgctccaa ctatcgcaag tcagcccctg 60
tcactgcgcc ctgaagcctg tcgccctgct gccgggggag ctgtgcatac tcggggactg 120
gactttgcct gtgatatcta c 141
<210> 4
<211> 66
<212> DNA
<213> coding sequence of the transmembrane region of human CD8 (artificial sequence)
<400> 4
atctgggcgc ccttggccgg gacttgtggg gtccttctcc tgtcactggt tatcaccctt 60
tactgc 66
<210> 5
<211> 144
<212> DNA
<213> coding sequence of intracellular region of human 41BB (artificial sequence)
<400> 5
aggttcagtg tcgtgaagag aggccggaag aagctgctgt acatcttcaa gcagcctttc 60
atgaggcccg tgcagactac ccaggaggaa gatggatgca gctgtagatt ccctgaagag 120
gaggaaggag gctgtgagct gaga 144
<210> 6
<211> 333
<212> DNA
<213> coding sequence of intracellular region of human CD3 ζ (artificial sequence)
<400> 6
gtgaagttct cccgaagcgc agatgcccca gcctatcagc agggacagaa tcagctgtac 60
aacgagctga acctgggaag acgggaggaa tacgatgtgc tggacaaaag gcggggcaga 120
gatcctgaga tgggcggcaa accaagacgg aagaaccccc aggaaggtct gtataatgag 180
ctgcagaaag acaagatggc tgaggcctac tcagaaatcg ggatgaaggg cgaaagaagg 240
agaggaaaag gccacgacgg actgtaccag gggctgagta cagcaacaaa agacacctat 300
gacgctctgc acatgcaggc tctgccacca aga 333
<210> 7
<211> 75
<212> DNA
<213> coding sequence of T2A self-cleaving peptide (artificial sequence)
<400> 7
agagccaagc ggggctctgg cgagggcaga ggctctctgc tgacctgcgg agatgtggaa 60
gaaaatcccg gccct 75
<210> 8
<211> 66
<212> DNA
<213> coding sequence of human GMCSFR signal peptide (artificial sequence)
<400> 8
atgttgctcc ttgtgacgag cctcctgctc tgcgagctgc cccatccagc cttcctcctc 60
atcccg 66
<210> 9
<211> 366
<212> DNA
<213> coding sequence of B1 (artificial sequence)
<400> 9
caggtgcagc tcgtggagtc tgggggaggc ttggtgcagc ccggggggtc actgagactc 60
tcctgtacag cctctggaag catcctcagt atctatgcca tgggctggta ccgccaggct 120
ccggggaagc agcgcgagtt ggtcgctgct attaatatca gtagtaacac attctaccga 180
gactccgtga agggccgatt caccatctcc agagacaacg ccgagaacac ggtgtatctg 240
caaatgaaca gcctgaaacc tgaggacacg gccgtctatt actgtaatgt ggcgccttgg 300
ggcgactatg acgtgaaaac tgactttggt ggctggggcc aggggaccca ggtcaccgtc 360
tcctcg 366
<210> 10
<211> 366
<212> DNA
<213> coding sequence of B65 (artificial sequence)
<400> 10
cagttgcagc tcgtggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactt 60
tcctgtgcag cctctggaag catcagcggt atctatgcca tgggctggta ccgccaggct 120
ccagggaagc agcgccggtt ggtcgcagct attactagtg gtggtgacac gttccatgca 180
gactccgtga agggccgatt caccatctcc agagacaacg ccaagaacac aatgtatctg 240
caaatgaaca gcctgaaacc tgaggacacg gccgtctatt actgtaatgt ggcgccttgg 300
ggcgactatg acgtgagggc tgactttggt tcctggggcc aggggaccca ggtcaccgtc 360
tcctcg 366
<210> 11
<211> 729
<212> DNA
<213> coding sequence of C11D5.3 (artificial sequence)
<400> 11
gacatcgttt tgacacaatc tcctgcgtca ttggccatga gtctcgggaa gcgcgcaaca 60
atatcctgtc gcgccagtga atctgtgtct gtgataggag cgcacttgat ccattggtat 120
cagcagaaac ctggacaacc tcccaagctg ctcatctacc tcgccagtaa ccttgaaaca 180
ggagtacctg ctcggttttc aggttccggg tcagggacgg atttcacttt gactatcgac 240
ccagttgagg aagacgacgt agccatatat agctgcctgc agtctcggat cttcccgcgc 300
acgttcgggg gaggaactaa gctggagatt aagggcggcg ggggttctgg tggcggcggc 360
agcggcggtg gaggatcaca aatccaactg gttcagtccg gtccagaact gaaaaagccg 420
ggggagacgg tgaaaatctc ctgtaaggcc tcaggttata ccttcaccga ttacagcatc 480
aattgggtaa agcgggctcc agggaaaggt ctgaaatgga tgggttggat caacacagaa 540
acccgagaac cagcctatgc ttacgacttt cgaggtcgat tcgctttttc cttggaaact 600
tccgcaagca cagcctatct gcaaatcaac aatctcaagt acgaagatac ggccacgtat 660
ttttgtgccc tggattacag ctatgcaatg gattactggg gtcaggggac gtctgttaca 720
gtttctagt 729
<210> 12
<211> 360
<212> DNA
<213> coding sequence of B6 (artificial sequence)
<400> 12
gaggtgcagc ttcaggcaag cgggggtggg ctggctcaac cgggtggttc cttgagactt 60
agttgtgcgg cgtctggacg aacattttcc acgtatttca tggcatggtt tagacaacct 120
ccgggtaagg gactggaata tgttggcggt ataaggtggt cagatggagt gcctcactat 180
gcggatagcg ttaaaggacg atttaccatt tctagggaca atgcgaaaaa tactgtatat 240
ttgcagatga atagtctccg cgcagaggac acagccgtat atttttgcgc gtcacggggt 300
attgcagacg ggtccgactt cggatcctat gggcaaggga cacaagtcac cgtcagctcc 360
<210> 13
<211> 117
<212> DNA
<213> coding sequence of the hinge region of human CD28 (artificial sequence)
<400> 13
atcgaggtga tgtaccctcc tccatacctg gacaacgaaa aaagcaacgg caccatcatc 60
cacgtgaagg gcaagcacct gtgccccagc cctctgttcc ccggaccttc taagcct 117
<210> 14
<211> 81
<212> DNA
<213> coding sequence of the transmembrane region of human CD28 (artificial sequence)
<400> 14
ttctgggtgc tggtcgtggt cggaggggtg ctggcctgtt atagcctgct ggtgactgtc 60
gccttcatta tcttctgggt g 81
<210> 15
<211> 123
<212> DNA
<213> coding sequence of intracellular region of human CD28 (artificial sequence)
<400> 15
cggagcaaga ggtctcgcgg tgggcattcc gactacatgt tcatgacccc tagaaggcct 60
ggcccaacca gaaagcacta ccagccatac gcccctccca gagatttcgc cgcttatcga 120
agc 123
<210> 16
<211> 21
<212> PRT
<213> human CD8 Signal peptide (artificial sequence)
<400> 16
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> 17
<211> 242
<212> PRT
<213> FMC63 (artificial sequence)
<400> 17
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
35 40 45
Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr Gly Gly Gly Gly Ser
100 105 110
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Val Lys Leu Gln Glu
115 120 125
Ser Gly Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys
130 135 140
Thr Val Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg
145 150 155 160
Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser
165 170 175
Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile
180 185 190
Lys Asp Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln
195 200 205
Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly
210 215 220
Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val
225 230 235 240
Ser Ser
<210> 18
<211> 47
<212> PRT
<213> human CD8 hinge region (artificial sequence)
<400> 18
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
35 40 45
<210> 19
<211> 22
<212> PRT
<213> human CD8 transmembrane region (artificial sequence)
<400> 19
Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu
1 5 10 15
Val Ile Thr Leu Tyr Cys
20
<210> 20
<211> 48
<212> PRT
<213> human 41BB intracellular region (artificial sequence)
<400> 20
Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe
1 5 10 15
Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly
20 25 30
Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg
35 40 45
<210> 21
<211> 111
<212> PRT
<213> intracellular region of human CD3 ζ (artificial sequence)
<400> 21
Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln
1 5 10 15
Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp
20 25 30
Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro
35 40 45
Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp
50 55 60
Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg
65 70 75 80
Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr
85 90 95
Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
100 105 110
<210> 22
<211> 25
<212> PRT
<213> T2A self-cleaving peptide (artificial sequence)
<400> 22
Arg Ala Lys Arg Gly Ser Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys
1 5 10 15
Gly Asp Val Glu Glu Asn Pro Gly Pro
20 25
<210> 23
<211> 22
<212> PRT
<213> human GMCSFR signal peptide (artificial sequence)
<400> 23
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro
20
<210> 24
<211> 122
<212> PRT
<213> B1 (artificial sequence)
<400> 24
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Thr Ala Ser Gly Ser Ile Leu Ser Ile Tyr
20 25 30
Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val
35 40 45
Ala Ala Ile Asn Ile Ser Ser Asn Thr Phe Tyr Arg Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Glu Asn Thr Val Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn
85 90 95
Val Ala Pro Trp Gly Asp Tyr Asp Val Lys Thr Asp Phe Gly Gly Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 25
<211> 122
<212> PRT
<213> B65 (artificial sequence)
<400> 25
Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Ser Gly Ile Tyr
20 25 30
Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Arg Leu Val
35 40 45
Ala Ala Ile Thr Ser Gly Gly Asp Thr Phe His Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Met Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn
85 90 95
Val Ala Pro Trp Gly Asp Tyr Asp Val Arg Ala Asp Phe Gly Ser Trp
100 105 110
Gly Gln Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 26
<211> 243
<212> PRT
<213> C11D5.3 (artificial sequence)
<400> 26
Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Met Ser Leu Gly
1 5 10 15
Lys Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Ser Val Ile
20 25 30
Gly Ala His Leu Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro
35 40 45
Lys Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Thr Gly Val Pro Ala
50 55 60
Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Asp
65 70 75 80
Pro Val Glu Glu Asp Asp Val Ala Ile Tyr Ser Cys Leu Gln Ser Arg
85 90 95
Ile Phe Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly
100 105 110
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Ile
115 120 125
Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu Thr Val
130 135 140
Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr Ser Ile
145 150 155 160
Asn Trp Val Lys Arg Ala Pro Gly Lys Gly Leu Lys Trp Met Gly Trp
165 170 175
Ile Asn Thr Glu Thr Arg Glu Pro Ala Tyr Ala Tyr Asp Phe Arg Gly
180 185 190
Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr Leu Gln
195 200 205
Ile Asn Asn Leu Lys Tyr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Leu
210 215 220
Asp Tyr Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr
225 230 235 240
Val Ser Ser
<210> 27
<211> 120
<212> PRT
<213> B6 (artificial sequence)
<400> 27
Glu Val Gln Leu Gln Ala Ser Gly Gly Gly Leu Ala Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Phe Ser Thr Tyr
20 25 30
Phe Met Ala Trp Phe Arg Gln Pro Pro Gly Lys Gly Leu Glu Tyr Val
35 40 45
Gly Gly Ile Arg Trp Ser Asp Gly Val Pro His Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys
85 90 95
Ala Ser Arg Gly Ile Ala Asp Gly Ser Asp Phe Gly Ser Tyr Gly Gln
100 105 110
Gly Thr Gln Val Thr Val Ser Ser
115 120
<210> 28
<211> 39
<212> PRT
<213> human CD28 hinge region (artificial sequence)
<400> 28
Ile Glu Val Met Tyr Pro Pro Pro Tyr Leu Asp Asn Glu Lys Ser Asn
1 5 10 15
Gly Thr Ile Ile His Val Lys Gly Lys His Leu Cys Pro Ser Pro Leu
20 25 30
Phe Pro Gly Pro Ser Lys Pro
35
<210> 29
<211> 27
<212> PRT
<213> human CD28 transmembrane region (artificial sequence)
<400> 29
Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu
1 5 10 15
Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val
20 25
<210> 30
<211> 41
<212> PRT
<213> intracellular region of human CD28 (artificial sequence)
<400> 30
Arg Ser Lys Arg Ser Arg Gly Gly His Ser Asp Tyr Met Phe Met Thr
1 5 10 15
Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro
20 25 30
Pro Arg Asp Phe Ala Ala Tyr Arg Ser
35 40