CN117343178A - Antibody for binding human CD207, chimeric antigen receptor for resisting human CD207 and application thereof - Google Patents
Antibody for binding human CD207, chimeric antigen receptor for resisting human CD207 and application thereof Download PDFInfo
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- CN117343178A CN117343178A CN202210786070.9A CN202210786070A CN117343178A CN 117343178 A CN117343178 A CN 117343178A CN 202210786070 A CN202210786070 A CN 202210786070A CN 117343178 A CN117343178 A CN 117343178A
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Abstract
The invention provides an antibody combined with human CD207, an anti-human CD207 chimeric antigen receptor and application thereof, and relates to the technical field of biological medicine. The antibody disclosed by the invention has high affinity with CD207 protein, and can be used for preparing scFv or chimeric antigen receptor. By utilizing the antibody or chimeric antigen receptor disclosed by the invention, for example, T cells which have no 293FT-CD207 cell killing activity per se, but MM01H-CD207CAR-T, MM H-CD207CAR-T, MM H-CD207CAR-T can specifically kill 293FT-CD207 cells positive for CD207 expression, the pre-MM 03H-CD207CAR-T can play a killing role for 32 hours, and the pre-MM 03H-CD207CAR-T has no killing role after 32 hours. The antibody or chimeric antigen receptor has the potential of preparing reagents or medicines for detecting or treating Langerhans cell tissue cell proliferation diseases.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to an antibody combined with human CD207, an anti-human CD207 chimeric antigen receptor and application thereof.
Background
Langerhans' cell proliferation disorder (Langerhans cell histiocytosis, LCH) is a disease of unknown tissue cell proliferation, a type of disease that proliferates by abnormal cloning of CD1a+/CD207+ dendritic cells. LCH has complex and variable clinical manifestations and strong heterogeneity. The etiology and pathogenesis are not completely understood. LCH may be due to malignant transformation of Langerhans (LC) precursors, and may also be due to deregulation of the immune response, immature myeloid cells producing pathological LCs.
Current treatments for LCH include chemotherapy, immunotherapy (CD 52 mab, etc.), and bone marrow transplantation. Patients with multiple system involvement have poorer prognosis, and the overall cure rate is 60% -80%. The overall cure rate of patients without involvement of important viscera is 80-85%. If important organs (such as lung and bone marrow) are involved, the cure rate is only about 60%, and recurrence is common. Therefore, the development of new LCH treatment regimens is of great significance to patients.
CD207, a type II transmembrane protein encoded by the CD207 gene. It is a c-type lectin. Consists of 328 amino acid sequences. CD207 is generally considered a marker for LC cells.
There are few reports on research on other tissue cell diseases of CD207 except LCH, langerin is a relatively sensitive and specific immunohistochemical marker for diagnosing LCH, and is helpful for diagnosis and differential diagnosis of LCH. Therefore, CD207 has considerable development potential as an immunotherapeutic target for LCH, but the existing treatment based on the CD207 target still has the problems of high difficulty, high recurrence rate and the like.
Disclosure of Invention
Accordingly, the invention aims to provide an antibody for combining human CD207, a chimeric antigen receptor for resisting human CD207 and application thereof, wherein the antibody has high affinity with CD207 protein, can be used for preparing scFv or chimeric antigen receptor, solves the problems of high treatment difficulty, easy recurrence and the like of the traditional Langerhans cell tissue hyperplasia, and has the potential of preparing reagents or medicines for detecting or treating Langerhans cell tissue hyperplasia.
In order to achieve the above object, the present invention provides the following technical solutions:
the present invention provides an antibody that specifically binds human CD207, said antibody comprising a light chain variable region VL and a heavy chain variable region VH; wherein the CDR1 sequence of the light chain variable region VL comprises any one of the sequences shown in SEQ ID NO.1, SEQ ID NO.11 and SEQ ID NO. 17; CDR2 of the light chain variable region VL includes any one of the sequences shown in SEQ ID NO.2, SEQ ID NO.12 and SEQ ID NO. 18; CDR3 of the light chain variable region VL includes any one of SEQ ID NO.3, SEQ ID NO.7, SEQ ID NO.13 and SEQ ID NO. 19;
the CDR1 sequence of the heavy chain variable region VH comprises any one of the sequences shown in SEQ ID NO.4, SEQ ID NO.8, SEQ ID NO.14 and SEQ ID NO. 20; the CDR2 sequences of the heavy chain variable region VH comprise any one of the sequences shown in SEQ ID NO.5, SEQ ID NO.9, SEQ ID NO.15 and SEQ ID NO. 21; the CDR3 sequences of the heavy chain variable region VH include any of the sequences shown in SEQ ID No.6, SEQ ID No.10, SEQ ID No.16 and SEQ ID No. 22.
Preferably, the amino acid sequence of the light chain variable region VL of the antibody is preferably selected from any one of SEQ ID No.23, SEQ ID No.25, SEQ ID No.27 and SEQ ID No. 29;
the amino acid sequence of the heavy chain variable region VH of the antibody is preferably selected from any one of SEQ ID No.24, SEQ ID No.26, SEQ ID No.28 and SEQ ID No. 30.
The invention also provides a nucleotide for encoding the antibody.
The invention also provides a recombinant vector containing the nucleotide.
The invention also provides an engineering cell for expressing the antibody.
The invention also provides a chimeric antigen receptor of anti-human CD207, the structure of which is from N end to C end, and comprises a signal peptide, an antigen recognition region for recognizing the CD207 antigen, a hinge region, a transmembrane region and an intracellular region;
the antigen recognition region recognizing the CD207 antigen is the above antibody.
Preferably, the type of signal peptide comprises a membrane-localized signal peptide;
sources of the hinge region include: any one or more of the extracellular hinge region of CD8, the extracellular hinge region of CD28, and the extracellular hinge region of CD 4;
sources of the transmembrane region include: any one or more of the transmembrane region of CD8, the transmembrane region of CD28, and the transmembrane region of CD 4;
sources of the signal region include: any one or more of an intracellular signal region of CD28, an intracellular signal region of CD134/OX40, an intracellular signal region of CD137/4-1BB, an intracellular signal region of LCK, an intracellular signal region of ICOS, an intracellular signal region of DAP10, an intracellular signal region of CD3 ζ, and an intracellular signal region of FcεRIy.
The invention also provides a recombinant vector containing the coding gene of the chimeric antigen receptor.
The invention also provides a recombinant eukaryotic cell for expressing the chimeric antigen receptor.
The invention also provides application of the antibody, the nucleotide, the recombinant vector, the engineering cell, the chimeric antigen receptor, the recombinant vector or the recombinant eukaryotic cell in preparation of reagents or medicines for detecting or treating diseases expressing CD207 antigen.
The beneficial effects are that: the invention provides an antibody specifically binding to human CD207, which has high affinity with CD207 protein and can be used for preparing scFv or chimeric antigen receptor. By utilizing the antibody or chimeric antigen receptor disclosed by the invention, for example, T cells which have no killing activity on 293FT-CD207 cells, but MM01H-CD207CAR-T, MM H-CD207CAR-T, MM H-CD207CAR-T can specifically kill 293FT-CD207 cells positive for CD207 expression, the pre-32H of MM03H-CD207CAR-T can play a killing role, and the pre-32H has no killing effect. The antibody or chimeric antigen receptor has the potential of preparing reagents or medicines for detecting or treating Langerhans cell tissue cell proliferation diseases.
Biological preservation information
The hybridoma cell strain MM01H is preserved in China center for type culture collection (CCTCC for short) for 6 months and 30 days in 2022, the preservation address is university of Wuhan, and the preservation number is CCTCC NO: C2022170;
the hybridoma cell strain MM02H is preserved in China center for type culture collection (CCTCC for short) for 6 months and 30 days in 2022, the preservation address is university of Wuhan, and the preservation number is CCTCC NO: C2022221;
the hybridoma cell strain MM03H is preserved in China center for type culture collection (CCTCC for short) for 6 months and 30 days in 2022, the preservation address is university of Wuhan, and the preservation number is CCTCC NO: C2022222;
the hybridoma cell strain MM04H is preserved in China center for type culture collection (CCTCC for short) for 6 months and 30 days in 2022, and has a preservation address of university of Wuhan, wuhan and China and a preservation number of CCTCC NO: C2022223.
Drawings
FIG. 1 shows the results of serum ELISA assays after three immunizations;
FIG. 2 shows the results of monoclonal antibody ELISA assays;
FIG. 3 is a plot of affinity fits of CD207 antigen to 4 mabs;
FIG. 4 is a recombinant plasmid map;
FIG. 5 is a graph of CD207CAR-T cell positive rate flow assays;
FIG. 6 is a flow phenotype diagram of a 293FT-CD207 monoclonal cell line;
FIG. 7 is a graph of RTCA of CD207CAR-T cell specific killing CD207 positive tumor cells;
figure 8 is CD207CAR-T cell killing factor release.
Detailed Description
The present invention provides an antibody that specifically binds human CD207, said antibody comprising a light chain variable region VL and a heavy chain variable region VH; wherein the CDR1 sequence of the light chain variable region VL comprises any one of the sequences shown in SEQ ID NO.1, SEQ ID NO.11 and SEQ ID NO. 17; CDR2 of the light chain variable region VL includes any one of the sequences shown in SEQ ID NO.2, SEQ ID NO.12 and SEQ ID NO. 18; CDR3 of the light chain variable region VL includes any one of SEQ ID NO.3, SEQ ID NO.7, SEQ ID NO.13 and SEQ ID NO. 19;
the CDR1 sequence of the heavy chain variable region VH comprises any one of the sequences shown in SEQ ID NO.4, SEQ ID NO.8, SEQ ID NO.14 and SEQ ID NO. 20; the CDR2 sequences of the heavy chain variable region VH comprise any one of the sequences shown in SEQ ID NO.5, SEQ ID NO.9, SEQ ID NO.15 and SEQ ID NO. 21; the CDR3 sequences of the heavy chain variable region VH include any of the sequences shown in SEQ ID No.6, SEQ ID No.10, SEQ ID No.16 and SEQ ID No. 22.
In the present invention, the amino acid sequence of the light chain variable region VL of the antibody is preferably selected from any one of SEQ ID No.23, SEQ ID No.25, SEQ ID No.27 and SEQ ID No. 29; the amino acid sequence of the heavy chain variable region VH of the antibody is preferably selected from any one of SEQ ID No.24, SEQ ID No.26, SEQ ID No.28 and SEQ ID No. 30. The combination of the light chain variable region VL and the heavy chain variable region VH of the antibody of the invention preferably comprises: the light chain variable region shown in SEQ ID NO.23 and the heavy chain variable region shown in SEQ ID NO.24, the light chain variable region shown in SEQ ID NO.25 and the heavy chain variable region shown in SEQ ID NO.26, the light chain variable region shown in SEQ ID NO.27 and the heavy chain variable region shown in SEQ ID NO.28, or the light chain variable region shown in SEQ ID NO.29 and the heavy chain variable region shown in SEQ ID NO. 30.
The antibody of the present invention may be Fab, fab ', (Fab') 2, scFv or di-scFv, and preferably further comprises a Linker between the light chain variable region and the heavy chain variable region, the kind of the Linker is not particularly limited, and the Linker is preferably represented by SEQ ID NO. 45: SSGGGGSGGGGGGSSRSS, synthesizing and obtaining nucleic acid of 4 anti-CD207 scFv fragments, wherein the nucleotide sequence of the nucleic acid is shown as SEQ ID NO. 31-34. In the examples of the present invention, the sequences of 4 antibodies are disclosed in total and displayed, as shown in table 1, and the numbers in table 1 all represent SEQ ID NOs:
table 14 variable region amino acid sequences of monoclonal antibodies
The invention also provides a nucleotide for encoding the antibody.
In the embodiment of the invention, 4 antibodies are taken as an example for illustration, and the corresponding nucleotide sequences are preferably shown in SEQ ID NO. 31-34.
The invention also provides a recombinant vector containing the nucleotide. The basic vector of the recombinant vector of the present invention preferably comprises pcDNA3.1 or pWPI, and the above-mentioned nucleotide is inserted into the multiple cloning site of the basic vector by a conventional method.
The invention also provides an engineering cell for expressing the antibody. The engineered cells of the invention are preferably mammalian cells. The mammalian cells of the present invention preferably include CHO, myeloma, hybridoma, perc.6 or HEK293.
The invention also provides a chimeric antigen receptor of anti-human CD207, the structure of which is from N end to C end, and comprises a signal peptide, a hinge region, an antigen recognition region for recognizing CD207 antigen, a transmembrane region and an intracellular region;
the antigen recognition region recognizing the CD207 antigen is the above antibody.
The types of signal peptides described in the present invention, preferably including membrane-localized signal peptides, are exemplified in the examples by the preferred CD8 transmembrane signal peptide (SEQ ID NO.35: MALPVTALLLPLALLLHAARPEQ, SEQ ID NO.36: atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggagcag), but are not to be construed as limiting the scope of the present invention. Sources of the extracellular hinge region of the present invention preferably include: any one or more of the extracellular hinge region of CD8, the extracellular hinge region of CD28 and the extracellular hinge region of CD4 is preferably exemplified by the CD8 hinge region (SEQ ID NO.37: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDI, SEQ ID NO.38: accacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggccagcggcggggggcgcagtgcacacgagggggctggacttcgcctgtgatatc), but is not to be construed as merely limiting the scope of the present invention. The sources of the transmembrane region of the present invention preferably include: any one or more of the CD8 transmembrane region, CD28 transmembrane region and CD4 transmembrane region is preferably exemplified by the CD8 transmembrane region (SEQ ID NO.39: YIWAPLAGTCGVLLLSLVITLYC, SEQ ID NO.40: tacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgc), but is not to be construed as merely limiting the scope of the present invention. The sources of the intracellular signal regions of the present invention preferably include: any one or more of the intracellular signal region of CD28, the intracellular signal region of CD134/OX40, the intracellular signal region of CD137/4-1BB (SEQ ID NO.41: SLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE, SEQ ID NO.42: tccctaaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaa), the intracellular signal region of LCK, the intracellular signal region of ICOS, the intracellular signal region of DAP10, the intracellular signal region of CD3 zeta (SEQ ID NO.43: LRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGG KPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTA TKDTYDALHMQALPPRG, SEQ ID NO.44: ctgagagtgaagttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgcggc) and the intracellular signal region of FcεRIgamma is preferably exemplified as a 4-1BB costimulatory signal region in the examples, but is not to be construed as merely limiting the scope of the present invention. The invention connects the above structures into chimeric antigen receptor in turn, which can specifically bind human CD207 antigen on the surface of cell membrane. Membrane-localized signal peptides are used to direct the localization of CAR molecules on cell membranes; the hinge region links the scFv and the transmembrane domain; the transmembrane domain connects the extracellular domain of the CAR with the intracellular signaling domain and anchors the CAR to the T cell membrane; the co-stimulatory domain (such as 4-1 BB) can realize dual activation of co-stimulatory molecules and intracellular signals, so that the T cells continuously proliferate and release cytokines, and the anti-tumor capability of the T cells is improved; the intracellular signaling region of cd3ζ functions as T cell signaling. In the embodiment of the invention, the above structures are sequentially connected to construct 4 chimeric antigen receptors, namely MM01H-CD207CAR, MM02H-CD207CAR, MM03H-CD207CAR and MM04H-CD207CAR.
The invention also provides a recombinant vector containing the coding gene of the chimeric antigen receptor.
The base vector of the recombinant vector of the present invention preferably includes a pHR vector.
The invention also provides a recombinant eukaryotic cell for expressing the chimeric antigen receptor. The basal cells of the recombinant eukaryotic cells according to the invention are preferably derived from mammalian cell lines or immune cells, and the immune cells are preferably human immune cells. The basal cells of the invention preferably comprise: 293FT cells, CHO cells, T lymphocytes, NK cells, DNT cells, gamma delta T cells.
The invention also provides application of the antibody, the nucleotide, the recombinant vector, the engineering cell, the chimeric antigen receptor, the recombinant vector or the recombinant eukaryotic cell in preparation of reagents or medicines for detecting or treating diseases expressing CD207 antigen.
The CD207 antigen expressing diseases of the invention preferably include langerhans cell tissue hyperplasia. The antibody disclosed by the invention can be specifically combined with human CD207, can be used in the field of immune cell treatment such as CAR-T, and in the embodiment, by constructing CD207CAR-T cells such as MM01H-CD207CAR-T, MM H-CD207CAR-T, MM H-CD207CAR-T and MM04H-CD207CAR-T, the specific killing of the 293FT-CD207 cells with positive CD207 expression by the MM01H-CD207CAR-T, MM H-CD207CAR-T and the MM04H-CD207CAR-T is proved, the killing effect of the MM03H-CD207CAR-T can be exerted 32 hours before the MM03H-CD207CAR-T, the killing effect of the T cells on the 293FT-CD207 cells is not exerted; and MM01H-CD207CAR-T, MM02H-CD207CAR-T, MM H-CD207CAR-T and MM04H-CD207CAR-T are capable of releasing large amounts of IFN- γ and IL-2, with significant differences compared to the T cell group (< 0.001, mean±sem). Therefore, it can be applied to reagents or medicines for detecting or treating cell proliferation.
The following examples are provided to illustrate in detail an antibody that binds human CD207, a chimeric antigen receptor that is anti-human CD207, and uses thereof, but are not to be construed as limiting the scope of the invention.
Example 1
Preparation of CD207 monoclonal antibody
(1) Human recombinant CD207 protein (Beijing Yiqiao Shenzhou technology Co., ltd.) solution and equal volume of complete Freund's adjuvant are made into an emulsifier, and Balb/C mice (No. SBI 210035A/B/C/D/E) are subcutaneously injected in multiple points on the abdomen, and each mouse has an immune dose of 50 mug; the same dose of immunogen and equal volume of incomplete Freund's adjuvant are taken at intervals of 2-3 weeks to prepare an emulsifier, the emulsifier is injected subcutaneously at multiple points in the abdomen, blood is taken one week after three immunizations to measure serum titers, the result is shown in figure 1, 5 mice all reach fusion standards, and no statistical difference exists among the 5 mice.
(2) All spleen cells of 5 mice after immunization are obtained, the spleen cells are respectively mixed with myeloma cells (SP 2/0 cells) of the mice according to a ratio of 1:1, the hybridoma cells are obtained by fusion through an electrofusion method, the primary clone is subjected to 3 times of limiting dilution, 4 monoclonal hybridoma cell lines (numbered MM 01H-04H) are obtained, the titer of supernatant of the 4 monoclonal hybridoma cell lines is detected by using 293 FT(s) expressing or not expressing CD207 as target cells through flow cytometry (Table 2), and the antibody subtype in the supernatant of the 4 monoclonal hybridoma cell lines is detected through ELISA (Table 3).
The results show that the supernatant of 4 monoclonal hybridoma cells can be combined with CD207 immunogen; the supernatant titers of the 4 monoclonal hybridoma cells are all higher. In the 4 lines, the subtypes MM01H and MM02HH are mIgG1, the subtype MM04H is mIgG2b, and the subtype MM03H is mIgG2a.
TABLE 2 results of flow-through detection of monoclonal hybridoma cells
TABLE 3 results of ELISA detection of monoclonal hybridoma cell supernatant antibody subtypes
(3) Culturing hybridoma cells and preparing antibodies by using 4 monoclonal hybridoma cells: hybridoma cells were cultured for 7 days, centrifuged at 1000g to obtain supernatants, and then filtered through 0.45 μm filters, and the filtrates were purified by column chromatography, respectively: washing chromatographic purification column (affinity filler ProteinA, beijing Yiqiao Shenzhou biotechnology Co., ltd.) with ultrapure water and buffer solution respectively until UV baseline is stable, and adjusting appropriate flow rate for loading; the leaching solution is leached to be stable from 5-10CV to the UV baseline, and the flow rate is the same as the loading flow rate; AC Elutation was eluted, and the eluate was collected according to UV peak until no fraction was eluted, and the antibody was neutralized and eluted by adding 2M Tris buffer (pH 8.0). The balancing buffer solution, eluent and the like for purification are all produced by Beijing Yiqiao Shenzhou science and technology Co., ltd; the monoclonal antibody titers were detected in two ways: flow cytometry (table 4) and ELISA detection (fig. 2) were performed with 293FT with or without CD207 expression as target cells.
Flow cytometry results show that the monoclonal antibodies obtained by the 4 cells can be combined with the CD207 immunogen, and the affinity of the 4 monoclonal antibodies is not obviously different. As can be seen from ELISA detection, when 1 μg/mL of CD207 protein was coated, 4 strains of monoclonal antibodies each had an OD >1.0, and were able to bind to CD207 immunogen, and were judged as acceptable antibodies. The OD450 value of 1 μg/mL CD207 antigen coating was significantly higher than the OD450 value of 0.1 μg/mL CD207 antigen coating (P <0.001, mean±sem).
TABLE 4 monoclonal antibody flow assay results
(4) Biacore detection of monoclonal antibodies of 4 lines of cells: CD207 protein was diluted to 0.5. Mu.g/mL using running buffer and flowed over the Anti-His chip surface for 12s at 10. Mu.L/min; after uniformly diluting 5 monoclonal antibodies to 12.5nM with running buffer, sequentially diluting 2 times to obtain 6 concentration monoclonal antibody solutions (0, 0.78125, 1.5625, 3.125, 6.25 and 12.5 nM); all antibodies were 120s in binding time, the antibody MM01H was 3600s in dissociation time, and the remaining antibodies were 300s in dissociation time; the regeneration reagent is GlycinepH1.5, and the regeneration time is 30s.
Affinity fitting patterns of CD207 protein and MM01H, MM02H, MM H and MM04H are shown in FIG. 3, affinity data of CD207 protein and MM01H, MM02H, MM H and MM04H antibodies are shown in Table 5, and affinity comparison of 4 monoclonal antibodies is MM01H > MM04H > MM02H > MM03H.
TABLE 5 affinity of CD207 protein for 4 monoclonal antibodies
Corresponding cell numbering | k a (M -1 s -1 ) | k d (s -1 ) | K D (M) | R max (RU) | Chi 2 (RU 2 ) | U-value |
MM01H | 7.27×10 5 | 6.12×10 -5 | 8.41×10 -11 | 13.02 | 0.180 | 7 |
MM04H | 1.57×10 6 | 7.82×10 -4 | 4.98×10 -10 | 9.356 | 0.0428 | 3 |
MM02H | 1.23×10 6 | 7.82×10 -4 | 6.35×10 -10 | 25.65 | 0.0346 | 1 |
MM03H | 2.31×10 6 | 1.846×10 -3 | 8.00×10 -10 | 15.51 | 0.199 | 2 |
Example 2
Construction of Single chain antibody scFv
4 hybridoma cells (MM 01H, MM02H, MM H and MM 04H) are lysed and RNA is extracted, the extracted RNA is used as a template, and a reverse transcription kit (Beijing Yiqiao Shenzhou science and technology Co., ltd.) is used for obtaining cDNA as the template;
pcDNA3-R (SEQ ID No. 46): GGCAACTAGAAGGCACAGTCGAGG, and PCMV3-F (SEQ ID No. 47): CAGGTGTCCACTCCCAGGTCCAAG the sequence of each monoclonal antibody is amplified by PCR by the upstream and downstream primers, and the 5 variable regions of the light chain and heavy chain of the monoclonal antibodies are obtained after sequencing the products.
The PCR products were electrophoresed, recovered, and transformed into E.coli, and positive clones were sequenced after which 4 monoclonal antibody variable regions were obtained from the nucleotide sequences whose amino acid sequences are shown in Table 1.
And connecting flexibility Linker (SSGGGGSGGGGGGSSRSS) between the light chain variable region and the heavy chain variable region, and synthesizing nucleic acid for obtaining 4 anti-CD207 scFv fragments, wherein the nucleotide sequence of the nucleic acid is shown as SEQ ID NO. 31-34.
Example 3
Construction of CD207CAR-T cells
Construction of recombinant vector was performed on 4 antibodies scfv binding to CD 207. As shown in FIG. 4, CD8 transmembrane signal peptide (SEQ ID NO. 36), anti-CD207 scFv constructed in example 2, CD8 hinge region (SEQ ID NO. 38), CD8 transmembrane region (SEQ ID NO. 40), 4-1BB costimulatory signal region (SEQ ID NO. 42) and CD3 ZetaTCR activation region (SEQ ID NO. 44) were cloned into lentiviral backbone plasmid pHR in sequence, sequenced with CMV-F (SEQ ID NO. 48): agctctataaaagagctcacaa, WPRE (SEQ ID NO. 49): CATAGCGTAAAAGGAGCAACA as the upstream and downstream primers, confirming correct sequence. The pHR-anti-CD 207CAR-BBZ plasmids were obtained and named pHR-MM01H-BBZ, pHR-MM02H-BBZ, pHR-MM03H-BBZ and pHR-MM04H-BBZ, respectively.
The lentiviral expression vector pHR-anti CD207CAR-BBZ plasmid carrying the target gene, the pCMV vector and the pMD.2G vector are mixed and then transfected into 293FT cells, the cells are cultured by changing the cells into complete culture medium for 6 to 8 hours after transfection, the culture solution is collected after 48 hours, the supernatant is reserved after centrifugation and filtered by a 0.45 mu m filter, and the filtrate is reserved, namely the recombinant lentivirus (MM 01H-CD207CAR and MM03H-CD207 CAR) solution. Lentiviral was concentrated according to the Lenti-XTM Concentrator (Takara, cat.: 631231) protocol, and the titer of lentivirus after concentration was 5X 10 7 /mL。
Isolation of mononuclear cells from fresh blood and activation of T lymphocytes 2X 10 6 Adding 2 lentiviral concentrates according to MOI=2.5, simultaneously adding IL-2 and polybrene, mixing well, 5% CO at 37deg.C 2 Culturing for 6-8 hours, centrifuging for 5min at 300g, and changing the liquid into a fresh X-VIVO 15 culture medium (containing IL-2); fresh X-VIVO 15 culture medium (containing IL-2) is added every 2-3 days to maintain cell density at 1×10 6 about/mL, amplified for 10-12 days. T cells cultured for 48h after virus infection and T cells not infected with virus were taken 5X 10 each 5 Each sample was incubated with Fluorescein AffiniPure Goat Anti-Mouse IgG F (ab') 2fragment specific 1. Mu.L, protected from light at room temperature for 15min, centrifuged, and the pellet was resuspended in 200. Mu.L PBS and then checked on-press (Millipore guava easyCyte HT). The results are shown in FIG. 5, and the ratio of CD207CAR-T cells in T cells cultured for 48 hours after virus infection was 46.6%,41.7%,21.6% and 65.1%, respectively. The prepared T cells were designated MM01H-CD207CAR-T, MM02H-CD207CAR-T, MM H-CD207CAR-T, MM04H-CD207CAR-T, respectively.
Example 4
In vitro killing activity of CD207CAR-T cells
Construction of 293FT cells stably expressing the extracellular and transmembrane regions of CD207 (293 FT-CD 207) as target cells (FIG. 6), the in vitro killing activity of CD207CAR-T cells was verified:
293FT-CD207 cells were grown at 1X 10 4 Density of wells/density of wells inoculated in 16 well plates, placed in RTCA real time recordingThe instrument was incubated for 6h followed by inoculation with CD207CAR-T cells and T cells at E:T=3:1 for 9 days. 37 ℃ 5% CO 2 RTCA recorded the killing curve of CAR-T killing target cells after 72 hours of incubation.
As shown in fig. 7, MM01H-CD207CAR-T, MM H-CD207CAR-T, MM H-CD207CAR-T was able to specifically kill 293FT-CD207 cells positive for CD207 expression, and MM03H-CD207CAR-T was able to exert killing effect 32H before and no killing effect after 32H. T cells had no killing effect on 293FT-CD207 cells.
Supernatant from the above experiment, E: T=3:1, was taken and subjected to Human IL-2ELISA MAX TM Deluxe(Biolegend,431804)、Human IFN-γELISA MAX TM Deluxe (Biolegend, 430104) kit protocol, detects release of IL-2 and IFN-gamma. The results are shown in FIG. 8, MM01H-CD207CAR-T, MM H-CD207CAR-T, MM H-CD207CAR-T, MM H-CD207CAR-T is capable of releasing significant amounts of IFN-gamma and IL-2, significantly different from the T cell group (P)<0.001,mean±SEM)。
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.
Claims (10)
1. An antibody that specifically binds human CD207, wherein the antibody comprises a light chain variable region VL and a heavy chain variable region VH; wherein the CDR1 sequence of the light chain variable region VL comprises any one of the sequences shown in SEQ ID NO.1, SEQ ID NO.11 and SEQ ID NO. 17; CDR2 of the light chain variable region VL includes any one of the sequences shown in SEQ ID NO.2, SEQ ID NO.12 and SEQ ID NO. 18; CDR3 of the light chain variable region VL includes any one of SEQ ID NO.3, SEQ ID NO.7, SEQ ID NO.13 and SEQ ID NO. 19;
the CDR1 sequence of the heavy chain variable region VH comprises any one of the sequences shown in SEQ ID NO.4, SEQ ID NO.8, SEQ ID NO.14 and SEQ ID NO. 20; the CDR2 sequences of the heavy chain variable region VH comprise any one of the sequences shown in SEQ ID NO.5, SEQ ID NO.9, SEQ ID NO.15 and SEQ ID NO. 21; the CDR3 sequences of the heavy chain variable region VH include any of the sequences shown in SEQ ID No.6, SEQ ID No.10, SEQ ID No.16 and SEQ ID No. 22.
2. The antibody according to claim 1, characterized in that the amino acid sequence of the light chain variable region VL of the antibody is preferably selected from any one of SEQ ID No.23, SEQ ID No.25, SEQ ID No.27 and SEQ ID No. 29;
the amino acid sequence of the heavy chain variable region VH of the antibody is preferably selected from any one of SEQ ID No.24, SEQ ID No.26, SEQ ID No.28 and SEQ ID No. 30.
3. A nucleotide encoding the antibody of claim 1 or 2.
4. A recombinant vector comprising the nucleotide of claim 3.
5. An engineered cell expressing the antibody of claim 1 or 2.
6. A chimeric antigen receptor against human CD207, wherein the structure of said chimeric antigen receptor comprises, from N-terminus to C-terminus: signal peptide, antigen recognition region that recognizes CD207 antigen, hinge region, transmembrane region, and intracellular region;
the antigen recognition region recognizing CD207 antigen is the antibody of claim 1 or 2.
7. The chimeric antigen receptor according to claim 6, wherein the type of signal peptide comprises a membrane-localized signal peptide;
sources of the hinge region include: any one or more of the extracellular hinge region of CD8, the extracellular hinge region of CD28, and the extracellular hinge region of CD 4;
sources of the transmembrane region include: any one or more of the transmembrane region of CD8, the transmembrane region of CD28, and the transmembrane region of CD 4;
sources of the intracellular region include: any one or more of an intracellular signal region of CD28, an intracellular signal region of CD134/OX40, an intracellular signal region of CD137/4-1BB, an intracellular signal region of LCK, an intracellular signal region of ICOS, an intracellular signal region of DAP10, an intracellular signal region of CD3 ζ, and an intracellular signal region of FcεRIy.
8. A recombinant vector comprising a gene encoding the chimeric antigen receptor of claim 6 or 7.
9. A recombinant eukaryotic cell expressing the chimeric antigen receptor of claim 6 or 7.
10. Use of an antibody according to claim 1 or 2, a nucleotide according to claim 3, a recombinant vector according to claim 4, an engineered cell according to claim 5, a chimeric antigen receptor according to claim 6 or 7, a recombinant vector according to claim 8 or a recombinant eukaryotic cell according to claim 9 for the preparation of a reagent or medicament for the detection or treatment of a disease expressing CD207 antigen.
Priority Applications (2)
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CN202210786070.9A CN117343178A (en) | 2022-07-04 | 2022-07-04 | Antibody for binding human CD207, chimeric antigen receptor for resisting human CD207 and application thereof |
PCT/CN2022/105591 WO2024007358A1 (en) | 2022-07-04 | 2022-07-14 | Antibody binding to human cd207, anti-human cd207 chimeric antigen receptor, and use thereof |
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CN202210786070.9A CN117343178A (en) | 2022-07-04 | 2022-07-04 | Antibody for binding human CD207, chimeric antigen receptor for resisting human CD207 and application thereof |
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CN117343178A true CN117343178A (en) | 2024-01-05 |
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WO (1) | WO2024007358A1 (en) |
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MX2012003058A (en) * | 2009-09-14 | 2012-05-22 | Baylor Res Inst | Vaccines directed to langerhans cells. |
EP3988111A1 (en) * | 2016-04-01 | 2022-04-27 | Innovative Cellular Therapeutics Holdings, Ltd. | Use of chimeric antigen receptor modified cells to treat cancer |
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