An antibody MM03H binding to human CD207, a chimeric antigen receptor against human CD207 and uses thereof

Abstract

The application provides an antibody MM03H binding to human CD207, a chimeric antigen receptor against human CD207 and application thereof, and relates to the technical field of biological medicines.The antibody MM03H has high affinity to CD207 protein, and can be used for preparing scFv or a chimeric antigen receptor.The antibody or the chimeric antigen receptor can be used for killing CD207 positive 293FT-CD207 cells in the first 32 hours, and has no killing effect after 32 hours.The antibody MM03H or the chimeric antigen receptor has the potential of preparing a reagent or a drug for detecting or treating Langerhans cell histiocytosis.

Description

[0001] This application is a divisional application of a Chinese invention patent filed on July 4, 2022, with application number 202210786070.9 and invention title: An antibody binding to human CD207, a chimeric antigen receptor against human CD207 and its application. Technical Field

[0002] This invention belongs to the field of biomedical technology, specifically relating to an antibody that binds to human CD207, a chimeric antigen receptor against human CD207, and their applications. Background Technology

[0003] Langerhans cell histiocytosis (LCH) is a histiocytic proliferative disorder of unknown cause, characterized by the abnormal clonal proliferation of CD1a+ / CD207+ dendritic cells. LCH presents with complex and variable clinical manifestations and exhibits high heterogeneity. Its etiology and pathogenesis are not fully understood. LCH may be caused by the malignant transformation of Langerhans (LC) precursors or by immune dysregulation, resulting in the production of pathological LCs from immature myeloid cells.

[0004] Current treatment options for LCH include chemotherapy, immunotherapy (CD52 monoclonal antibodies, etc.), and bone marrow transplantation. Patients with multi-system involvement have a poor prognosis, with an overall cure rate of 60%–80%. Patients without major organ involvement have an overall cure rate of 80%–85%. If major organs are involved (such as the lungs or bone marrow), the cure rate is only about 60%, and relapse is common. Therefore, developing new LCH treatment options is of great significance for patients.

[0005] CD207 is a type II transmembrane protein encoded by the CD207 gene. It is a type C lectin, consisting of a 328-amino acid sequence. CD207 is widely considered a biomarker for LC cells.

[0006] Studies on CD207 in other histiocytic diseases besides LCH are scarce, and Langerin is a relatively sensitive and specific immunohistochemical marker for diagnosing LCH, which is helpful for the diagnosis and differential diagnosis of LCH. Therefore, CD207 has considerable development potential as an immunotherapeutic target for LCH. However, current CD207-based therapies still face challenges such as high difficulty and high relapse rates. Summary of the Invention

[0007] In view of this, the purpose of the present invention is to provide an antibody that binds to human CD207, a chimeric antigen receptor against human CD207, and their applications. The antibody has a high affinity for CD207 protein and can be used to prepare scFv or chimeric antigen receptors. This solves the problems of difficult treatment and easy recurrence of existing Langerhans cell histiocytosis, and has the potential to prepare reagents or drugs for the detection or treatment of Langerhans cell histiocytosis.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides an antibody that specifically binds to human CD207, the 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 includes any one of the sequences shown in SEQ ID NO.1, SEQ ID NO.11, and SEQ ID NO.17; the 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; and the 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 includes 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 sequence of the heavy chain variable region VH includes any one of the sequences shown in SEQ ID NO.5, SEQ ID NO.9, SEQ ID NO.15, and SEQ ID NO.21; and the CDR3 sequence of the heavy chain variable region VH includes any one of the sequences shown in SEQ ID NO.6, SEQ ID NO.10, SEQ ID NO.16, and SEQ ID NO.22.

[0009] Preferably, the amino acid sequence of the light chain variable region VL of the antibody is 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.

[0010] The present invention also provides a nucleotide encoding the above-mentioned antibody.

[0011] The present invention also provides a recombinant vector containing the above-mentioned nucleotides.

[0012] The present invention also provides an engineered cell expressing the above-mentioned antibody.

[0013] The present invention also provides a chimeric antigen receptor against human CD207, wherein the structure of the chimeric antigen receptor from the N-terminus to the C-terminus includes a signal peptide, an antigen recognition region for recognizing CD207 antigen, a hinge region, a transmembrane region and an intracellular region; The antigen recognition region that recognizes the CD207 antigen is the antibody described above.

[0014] Preferably, the type of the signal peptide includes membrane-localized signal peptides; The 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 CD4. The sources of the transmembrane region include any one or more of the transmembrane regions of CD8, CD28, and CD4; The sources of the signal regions include any one or more of the following: intracellular signal regions of CD28, CD134 / OX40, CD137 / 4-1BB, LCK, ICOS, DAP10, CD3ζ, and FcεRIγ.

[0015] The present invention also provides a recombinant vector comprising the coding gene of the chimeric antigen receptor described above.

[0016] The present invention also provides a recombinant eukaryotic cell expressing the above-mentioned chimeric antigen receptor.

[0017] The present invention also provides the use of the above-mentioned antibody, the above-mentioned nucleotide, the above-mentioned recombinant vector, the above-mentioned engineered cell, the above-mentioned chimeric antigen receptor, the above-mentioned recombinant vector, or the above-mentioned recombinant eukaryotic cell in the preparation of reagents or drugs for detecting or treating diseases expressing CD207 antigen.

[0018] Beneficial Effects: This invention provides an antibody that specifically binds to human CD207. The antibody has a high affinity for the CD207 protein and can be used to prepare scFv or chimeric antigen receptors. Using the antibody or chimeric antigen receptor described in this invention, for example, T cells that do not naturally exhibit cytotoxic activity against 293FT-CD207 cells, but MM01H-CD207CAR-T, MM02H-CD207CAR-T, and MM04H-CD207CAR-T can specifically kill CD207-positive 293FT-CD207 cells. MM03H-CD207CAR-T exerts a cytotoxic effect for the first 32 hours, but loses its cytotoxic effect after 32 hours. The antibody or chimeric antigen receptor described in this invention has the potential to prepare reagents or drugs for the detection or treatment of Langerhans cell histiocytosis. Attached Figure Description

[0019] Figure 1 The results are from serum ELISA tests after three immunizations. Figure 2 This is the result of a monoclonal antibody ELISA test; Figure 3 Affinity fitting plot of CD207 antigen with four monoclonal antibodies; Figure 4 Map of recombinant plasmids; Figure 5 Flow cytometry plot of CD207CAR-T cell positivity rate; Figure 6 Flow cytometry phenotype of the 293FT-CD207 monoclonal cell line; Figure 7 RTCA diagram of CD207CAR-T cells specifically killing CD207-positive tumor cells; Figure 8 This refers to the release of CD207CAR-T cell killing factors.

[0020] Biological Preservation Information The hybridoma cell line MM01H was deposited at the China Center for Type Culture Collection (CCTCC) on June 30, 2022, at Wuhan University, Wuhan, with accession number CCTCC NO:C2022170. The hybridoma cell line MM02H was deposited at the China Center for Type Culture Collection (CCTCC) on June 30, 2022, at Wuhan University, Wuhan, with accession number CCTCC NO:C2022221. The hybridoma cell line MM03H was deposited at the China Center for Type Culture Collection (CCTCC) on June 30, 2022, at Wuhan University, Wuhan, with accession number CCTCC NO:C2022222. The hybridoma cell line MM04H was deposited at the China Center for Type Culture Collection (CCTCC) on June 30, 2022, at Wuhan University, Wuhan, with accession number CCTCC NO:C2022223. Detailed Implementation

[0021] This invention provides an antibody that specifically binds to human CD207, the 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 includes any one of the sequences shown in SEQ ID NO.1, SEQ ID NO.11, and SEQ ID NO.17; the 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; and the 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 includes 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 sequence of the heavy chain variable region VH includes any one of the sequences shown in SEQ ID NO.5, SEQ ID NO.9, SEQ ID NO.15, and SEQ ID NO.21; and the CDR3 sequence of the heavy chain variable region VH includes any one of the sequences shown in SEQ ID NO.6, SEQ ID NO.10, SEQ ID NO.16, and SEQ ID NO.22.

[0022] In this 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 this invention preferably includes: 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.

[0023] The antibodies described in this invention can be Fab, Fab', (Fab')2, scFv, or di-scFv. Preferably, a linker is also included between the light chain variable region and the heavy chain variable region. The type of linker is not particularly limited; in the embodiments, the linker shown in SEQ ID NO. 45, SSGGGGSGGGGGGSSRSS, is preferred. Nucleic acid fragments of four anti-CD207 scFv are synthesized, and their nucleotide sequences are shown in SEQ ID NOs 31-34. In the embodiments of this invention, the sequences of four antibodies are disclosed and displayed as shown in Table 1, where the numbers in Table 1 represent SEQ ID NOs.

[0024] Table 1. Variable region amino acid sequences of the four monoclonal antibodies

[0025] The present invention also provides a nucleotide encoding the above-mentioned antibody.

[0026] In this embodiment of the invention, four antibodies are used as examples, and their corresponding nucleotide sequences are preferably shown in SEQ ID NO. 31~34.

[0027] The present invention also provides a recombinant vector containing the above-mentioned nucleotides. The base vector of the recombinant vector of the present invention preferably includes pcDNA3.1 or pWPI, and the above-mentioned nucleotides are inserted into the multiple cloning site of the base vector using conventional methods.

[0028] The present invention also provides an engineered cell expressing the above-mentioned antibody. The engineered cell of the present invention is preferably a mammalian cell. The mammalian cell of the present invention preferably includes CHO, myeloma cells, hybridoma cells, PerC.6, or HEK293.

[0029] The present invention also provides a chimeric antigen receptor against human CD207, wherein the structure of the chimeric antigen receptor, from the N-terminus to the C-terminus, includes 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 that recognizes the CD207 antigen is the antibody described above.

[0030] The signal peptides described in this invention preferably include membrane-localizing signal peptides. In the examples, CD8 transmembrane signal peptides (SEQ ID NO.35: MALPVTALLLPLALLLHAARPEQ, SEQ ID NO.36: atggccttaccagtgaccgccttgctcctgccgctggccttgctgctccacgccgccaggccggagcag) are used as examples for illustration, but they should not be considered as the entire scope of protection of this invention. The extracellular hinge region described in this invention preferably includes any one or more of the extracellular hinge regions of CD8, CD28, and CD4. In the embodiments, the CD8 hinge region (SEQ ID NO.37: TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDI, SEQ ID NO.38: accacgacgccagcgccgcgaccaccaacaccggcgcccaccatcgcgtcgcagcccctgtccctgcgcccagaggcgtgccggcggcggggcgcagtgcacacgagggggctggacttcgcctgtgatatc) is preferably used as an example, but it should not be considered as the entire scope of protection of this invention. The source of the transmembrane region described in this invention preferably includes any one or more of the transmembrane regions of CD8, CD28, and CD4. In the embodiments, the CD8 transmembrane region (SEQ ID NO.39: YIWAPLAGTCGVLLLSLVITLYC, SEQ ID NO.40: tacatctgggcgcccttggccgggacttgtggggtccttctcctgtcactggttatcaccctttactgc) is preferably used as an example for illustration, but it should not be considered as the entire scope of protection of this invention.The sources of the intracellular signaling regions described in this invention preferably include: the intracellular signaling region of CD28, the intracellular signaling region of CD134 / OX40, the intracellular signaling region of CD137 / 4-1BB (SEQ ID NO.41: SLKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCE, SEQ ID NO.42: tccctaaaacggggcagaaagaaactcctgtatatattcaaacaaccatttatgagaccagtacaaactactcaagaggaagatggctgtagctgccgatttccagaagaagaagaaggaggatgtgaa), the intracellular signaling region of LCK, the intracellular signaling region of ICOS, the intracellular signaling region of DAP10, and the intracellular signaling region of CD3ζ (SEQ ID NO.41). NO.43:LRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRG, SEQ ID NO.44: any one or more of the intracellular signaling regions of ctgagagtgaagttcagcaggagcgcagacgcccccgcgtacaagcagggccagaaccagctctataacgagctcaatctaggacgaagagaggagtacgatgttttggacaagagacgtggccgggaccctgagatggggggaaagccgagaaggaagaaccctcaggaaggcctgtacaatgaactgcagaaagataagatggcggaggcctacagtgagattgggatgaaaggcgagcgccggaggggcaaggggcacgatggcctttaccagggtctcagtacagccaccaaggacacctacgacgcccttcacatgcaggccctgccccctcgcggc) and FcεRIγ. In the embodiments, the 4-1BB co-stimulatory signaling region is preferably used as an example for illustration, but it should not be regarded as the entire scope of protection of the present invention. The present invention connects the above structures sequentially to form a chimeric antigen receptor, which can specifically bind to the human CD207 antigen on the cell membrane surface.The membrane localization signal peptide guides the CAR molecule to localize on the cell membrane; the hinge region connects the scFv and the transmembrane domain; the transmembrane domain connects the extracellular domain of the CAR to the intracellular signal transduction domain, anchoring the CAR to the T cell membrane; the co-stimulatory domain (e.g., 4-1BB) enables dual activation of the synergistic stimulatory molecule and intracellular signal, allowing T cells to proliferate continuously and release cytokines, thereby enhancing the anti-tumor ability of T cells; the intracellular signaling region of CD3ζ plays a role in T cell signal transduction. In this embodiment of the invention, the above structures are sequentially connected to construct four chimeric antigen receptors: MM01H-CD207CAR, MM02H-CD207CAR, MM03H-CD207CAR, and MM04H-CD207CAR.

[0031] The present invention also provides a recombinant vector comprising the coding gene of the chimeric antigen receptor described above.

[0032] The base vector of the recombinant vector of the present invention preferably includes the pHR vector.

[0033] This invention also provides a recombinant eukaryotic cell expressing the aforementioned chimeric antigen receptor. The basal cells of the recombinant eukaryotic cell of this invention are preferably derived from mammalian cell lines or immune cells, and the immune cells are preferably human immune cells. The basal cells of this invention preferably include: 293FT cells, CHO cells, T lymphocytes, NK cells, DNT cells, and γδT cells.

[0034] The present invention also provides the use of the above-mentioned antibody, the above-mentioned nucleotide, the above-mentioned recombinant vector, the above-mentioned engineered cell, the above-mentioned chimeric antigen receptor, the above-mentioned recombinant vector, or the above-mentioned recombinant eukaryotic cell in the preparation of reagents or drugs for detecting or treating diseases expressing CD207 antigen.

[0035] The diseases expressing the CD207 antigen described in this invention preferably include Langerhans cell histiocytosis. The antibody described in this invention can specifically bind to human CD207 and can be used in the field of CAR-T and other immunocellular therapies. In the examples, CD207 is constructed... CAR-T cells, such as MM01H-CD207CAR-T, MM02H-CD207CAR-T, MM03H-CD207CAR-T, and MM04H-CD207CAR-T, were used to specifically kill CD207-positive 293FT-CD207 cells. MM03H-CD207CAR-T exerted a killing effect for the first 32 hours, but showed no killing effect after 32 hours, while T cells had no killing effect on 293FT-CD207 cells. Furthermore, MM01H-CD207CAR-T, MM02H-CD207CAR-T, MM03H-CD207CAR-T, and MM04H-CD207CAR-T released large amounts of IFN-γ and IL-2, showing significant differences compared to the T cell group. P <0.001, mean ± SEM). Therefore, it can be used as a reagent or drug for detecting or treating cell hyperplasia.

[0036] The following detailed description, in conjunction with embodiments, illustrates an antibody binding to human CD207, a chimeric antigen receptor against human CD207, and their applications provided by the present invention. However, these descriptions should not be construed as limiting the scope of protection of the present invention.

[0037] Example 1 CD207 monoclonal antibody preparation (1) A solution of recombinant human CD207 protein (Beijing Yiqiao Shenzhou Technology Co., Ltd.) was mixed with an equal volume of complete Freund's adjuvant to form an emulsion. Balb / c mice (number SBI210035A / B / C / D / E) were injected subcutaneously at multiple sites in the abdomen, with each mouse receiving an immunization dose of 50 μg. At intervals of 2-3 weeks, the same dose of immunogen was mixed with an equal volume of incomplete Freund's adjuvant to form an emulsion, which was then injected subcutaneously at multiple sites in the abdomen. One week after the three immunizations, serum titers were measured. The results are as follows: Figure 1 As shown, all five mice met the fusion criteria, and there was no statistically significant difference among the five mice.

[0038] (2) All spleen cells of 5 mice after immunization were obtained and mixed with mouse myeloma cells (SP2 / 0 cells) at a ratio of 1:1. Hybridoma cells were obtained by electrofusion. The master clone was diluted 3 times to obtain 4 monoclonal hybridoma cell lines (numbered MM01H-04H). The titers of the supernatant of the 4 monoclonal hybridoma cell lines were detected by flow cytometry using 293FT cells that express or do not express CD207 as target cells (Table 2). The antibody subtypes in the supernatant of the 4 monoclonal hybridoma cell lines were detected by ELISA (Table 3).

[0039] The results showed that the supernatants of all four monoclonal hybridoma cell lines could bind to the CD207 immunogen; the supernatants of all four monoclonal hybridoma cell lines had high titers. Among the four cell lines, MM01H and MM02HH were mIgG1, MM04H was mIgG2b, and MM03H was mIgG2a.

[0040] Table 2 Results of flow cytometry analysis of monoclonal hybridoma cell supernatant

[0041] Table 3. Results of ELISA detection of antibody subtypes in monoclonal hybridoma cell supernatant

[0042] (3) Hybridoma cell culture and antibody preparation were performed on four monoclonal hybridoma cell lines: The hybridoma cells were cultured for 7 days, centrifuged at 1000g to obtain the supernatant, and then filtered through a 0.45μm filter membrane. The filtrate was purified by column chromatography: The chromatography column (affinity packing material Protein A, Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.) was washed with ultrapure water and buffer until the UV baseline was stable, and the sample was loaded at an appropriate flow rate; the eluent was used to wash for 5-10 CV until the UV baseline was stable, and the flow rate was the same as the loading flow rate; ACElution was used for elution, and the eluent was collected according to the UV peak until no components flowed out, and 2M Tris buffer (pH 8.0) was added to neutralize the eluted antibody. The equilibration buffer, eluent, and elution buffer used for purification were all produced by Beijing Yiqiao Shenzhou Biotechnology Co., Ltd. The monoclonal antibody titer was detected by two methods: flow cytometry (Table 4) and ELISA were used to detect the titer of 293FT cells expressing or not expressing CD207 as target cells. Figure 2 ).

[0043] Flow cytometry results showed that all four monoclonal antibodies obtained from the four cell lines could bind to the CD207 immunogen, and there was no significant difference in affinity among the four monoclonal antibodies. ELISA detection showed that all four monoclonal antibodies coated with 1 μg / mL CD207 protein had an OD > 1.0, indicating their ability to bind to the CD207 immunogen and thus qualifying them as qualified antibodies. (OD value for 1 μg / mL CD207 antigen coating is also mentioned.)450 The value was significantly higher than that of 0.1 μg / mL CD207 antigen-coated OD. 450 value( P <0.001, mean±SEM).

[0044] Table 4. Flow cytometry results of monoclonal antibodies

[0045] (4) The monoclonal antibodies of the four cell lines were subjected to Biacore detection: CD207 protein was diluted to 0.5 μg / mL with running buffer and flowed through the surface of the Anti-His chip at 10 μL / min for 12s; the five monoclonal antibodies were uniformly diluted to 12.5 nM with running buffer and then diluted 2-fold to obtain six concentrations of monoclonal antibody solutions (0, 0.78125, 1.5625, 3.125, 6.25 and 12.5 nM); the binding time of all antibodies was 120s, the dissociation time of antibody MM01H was 3600s, and the dissociation time of the other antibodies was 300s; the regeneration reagent was Glycine pH 1.5, and the regeneration time was 30s.

[0046] The affinity fitting diagram between CD207 protein and MM01H, MM02H, MM03H, and MM04H is shown in the figure below. Figure 3 As shown in Table 5, the affinity data of CD207 protein with MM01H, MM02H, MM03H and MM04H antibodies are as follows: the affinity of the four monoclonal antibodies is MM01H > MM04H > MM02H > MM03H.

[0047] Table 5. Affinity of CD207 protein to four monoclonal antibodies

[0048] Example 2 Construction of single-chain antibody scFv RNA was extracted from four hybridoma cell lines (MM01H, MM02H, MM03H and MM04H) after lysis. The extracted RNA was used as a template to obtain cDNA using a reverse transcription kit (Beijing Yiqiao Shenzhou Technology Co., Ltd.). The sequences of each monoclonal antibody were amplified by PCR using pcDNA3-R (SEQ ID NO.46): GGCAACTAGAAGGCACAGTCGAGG and PCMV3-F (SEQ ID NO.47): CAGGTGTCCACTCCCAGGTCCAAG as upstream and downstream primers. After sequencing the products, the variable regions of the light and heavy chains of the five monoclonal antibodies were obtained.

[0049] The PCR products were electrophoresed, recovered, and transformed into E. coli. Positive clones were then sequenced, and the amino acid sequences of the four monoclonal antibody variable regions were obtained based on the nucleotide sequences, as shown in Table 1.

[0050] A flexible linker (SSGGGGSGGGGGGSSRSS) was connected between the light chain variable region and the heavy chain variable region to synthesize nucleic acids containing four anti-CD207 scFv fragments, the nucleotide sequences of which are shown in SEQ ID NO.31~34.

[0051] Example 3 Construction of CD207 CAR-T cells Four CD207-binding antibody strains (scfv) were used to construct recombinant vectors. For example... Figure 4 As shown, the CD8 transmembrane signal peptide (SEQ ID NO.36), the anti-CD207 scFv constructed in Example 2, the CD8 hinge region (SEQ ID NO.38), the CD8 transmembrane region (SEQ ID NO.40), the 4-1BB co-stimulatory signal region (SEQ ID NO.42), and the CD3 Zeta TCR activation region (SEQ ID NO.44) were sequentially cloned into the lentiviral backbone plasmid pHR. Sequencing was performed using CMV-F (SEQ ID NO.48): agctctataaaagagctcacaa and WPRE (SEQ ID NO.49): CATAGCGTAAAAGGAGCAACA as upstream and downstream primers, confirming the sequence correctness. The pHR-antiCD207CAR-BBZ plasmid was obtained and named pHR-MM01H-BBZ, pHR-MM02H-BBZ, pHR-MM03H-BBZ, and pHR-MM04H-BBZ, respectively.

[0052] The lentiviral expression vectors pHR-antiCD207CAR-BBZ, pCMV, and pMD.2G carrying the target gene were mixed and transfected into 293FT cells. After 6-8 hours of transfection, the medium was replaced with complete culture medium. After 48 hours, the culture medium was collected, centrifuged, and the supernatant was retained. This supernatant was filtered through a 0.45 μm filter, and the filtrate was retained. This filtrate was the recombinant lentiviral solution (MM01H-CD207CAR and MM03H-CD207CAR). The lentiviral titer was concentrated according to the Lenti-X™ Concentrator (Takara, Cat.: 631231) instructions, to a concentration of 5 × 10⁷ / mL.

[0053] Mononuclear cells were isolated from fresh blood and T lymphocytes were activated. Two × 10⁶ cells were collected and added with concentrated solutions of two lentiviruses at an MOI of 2.5, along with IL-2 and polybrene. The mixture was incubated at 37°C with 5% CO₂ for 6–8 hours, centrifuged at 300g for 5 min, and the medium was replaced with fresh X-VIVO 15 medium (containing IL-2). Fresh X-VIVO 15 medium (containing IL-2) was added every 2–3 days to maintain a cell density of approximately 1 × 10⁶ / mL for expansion over 10–12 days. Five × 10⁵ T cells were collected from T cells cultured for 48 h post-infection and 5 × 10⁵ uninfected T cells. One μL of Fluorescein Affini Pure Goat Anti-Mouse IgGF(ab')2 fragment specific was added to each sample. The cells were incubated at room temperature in the dark for 15 min, centrifuged, and the pellet was resuspended in 200 μL PBS before analysis using a Millipore Guava EasyCyte HT analyzer. Results are as follows: Figure 5 As shown, the proportions of CD207 CAR-T cells in T cells cultured for 48 hours after viral infection were 46.6%, 41.7%, 21.6%, and 65.1%, respectively. The prepared T cells were named MM01H-CD207CAR-T, MM02H-CD207CAR-T, MM03H-CD207CAR-T, and MM04H-CD207CAR-T, respectively.

[0054] Example 4 CD207 CAR-T cell in vitro killing activity Construct 293FT cells (293FT-CD207) that stably express the extracellular and transmembrane regions of CD207 as target cells. Figure 6 ), to verify the in vitro killing activity of CD207 CAR-T cells: 293FT-CD207 cells were fed at a dose of 1×10 4 The cells were seeded at a density of / wells in 16-well plates, placed in an RTCA real-time recorder, and cultured for 6 hours. Afterward, CD207 CAR-T cells and T cells cultured for 9 days were seeded at an E:T ratio of 3:1. After 72 hours of incubation at 37℃ and 5% CO2, RTCA recorded the CAR-T cell killing curves against the target cells.

[0055] The results are as follows Figure 7As shown, MM01H-CD207CAR-T, MM02H-CD207CAR-T, and MM04H-CD207CAR-T can specifically kill CD207-positive 293FT-CD207 cells. MM03H-CD207CAR-T exerts a killing effect for the first 32 hours, but has no killing effect after 32 hours. T cells have no killing effect on 293FT-CD207 cells.

[0056] Take the supernatant obtained from the above experiment with an E:T ratio of 3:1, and follow the instructions of the Human IL-2 ELISA MAX™ Deluxe (Biolegend, 431804) and Human IFN-γ ELISA MAX™ Deluxe (Biolegend, 430104) kits to detect the release of IL-2 and IFN-γ. The results are as follows: Figure 8 As shown, MM01H-CD207CAR-T, MM02H-CD207CAR-T, MM03H-CD207CAR-T, and MM04H-CD207CAR-T cells were able to release large amounts of IFN-γ and IL-2, showing significant differences compared to the T cell group. P <0.001, mean±SEM).

[0057] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An antibody MM03H that specifically binds to human CD207, characterized in that, The antibody MM03H includes a light chain variable region VL and a heavy chain variable region VH; wherein the amino acid sequence of CDR1 of the light chain variable region VL is shown in SEQ ID NO.11; the amino acid sequence of CDR2 of the light chain variable region VL is GGI; and the amino acid sequence of CDR3 of the light chain variable region VL is shown in SEQ ID NO.

13. The amino acid sequence of CDR1 of the heavy chain variable region VH is shown in SEQ ID NO.14; the amino acid sequence of CDR2 of the heavy chain variable region VH is shown in SEQ ID NO.15; and the amino acid sequence of CDR3 of the heavy chain variable region VH is shown in SEQ ID NO.

16.

2. The antibody MMO3H according to claim 1, characterized in that, The amino acid sequence of the light chain variable region VL of the antibody MM03H is shown in SEQ ID NO.27; The amino acid sequence of the heavy chain variable region VH of the antibody is shown in SEQ ID NO.

28.

3. The encoding gene of the antibody MMO3H as described in claim 1 or 2.

4. A recombinant vector comprising the encoding gene of claim 3.

5. An engineered cell expressing the antibody MM03H as described in claim 1 or 2.

6. A chimeric antigen receptor against human CD207, characterized in that, The structure of the chimeric antigen receptor, from the N-terminus to the C-terminus, includes: a signal peptide, an antigen recognition region that recognizes the CD207 antigen, a hinge region, a transmembrane region, and an intracellular region. The antigen recognition region that recognizes the CD207 antigen is the scFv fragment of the antibody MM03H described in claim 1 or 2.

7. The chimeric antigen receptor according to claim 6, characterized in that, The types of signal peptides include membrane-localized signal peptides; The 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 CD4. The sources of the transmembrane region include any one or more of the transmembrane regions of CD8, CD28, and CD4; The sources of the intracellular region include any one or more of the following: the intracellular signaling region of CD28, the intracellular signaling region of CD134 / OX40, the intracellular signaling region of CD137 / 4-1BB, the intracellular signaling region of LCK, the intracellular signaling region of ICOS, the intracellular signaling region of DAP10, the intracellular signaling region of CD3ζ, and the intracellular signaling region of FcεRIγ.

8. A recombinant vector comprising the encoding gene of the chimeric antigen receptor as described in claim 6 or 7.

9. A recombinant eukaryotic cell expressing the chimeric antigen receptor of claim 6 or 7.

10. The use of the antibody MM03H of claim 1 or 2, the encoding gene of claim 3, the recombinant vector of claim 4, the engineered cell of claim 5, the chimeric antigen receptor of claim 6 or 7, the recombinant vector of claim 8, or the recombinant eukaryotic cell of claim 9 in the preparation of reagents or drugs for the detection or treatment of Langerhans cell histiocytosis.

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