CN117467010A - BCMA-targeted nanobody and application thereof - Google Patents

BCMA-targeted nanobody and application thereof Download PDF

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CN117467010A
CN117467010A CN202210904517.8A CN202210904517A CN117467010A CN 117467010 A CN117467010 A CN 117467010A CN 202210904517 A CN202210904517 A CN 202210904517A CN 117467010 A CN117467010 A CN 117467010A
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bcma
antigen receptor
chimeric antigen
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刘茂玄
许晨光
季晓萌
陈倩
韩斌
陈琳纯
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Shenzhen Bindebiotech Co ltd
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Abstract

The application provides a nano antibody targeting BCMA and application thereof. The nano-antibody targeting BCMA comprises a heavy chain variable region, wherein the heavy chain variable region comprises a complementarity determining region CDR1, a complementarity determining region CDR2 and a complementarity determining region CDR3, the complementarity determining region CDR1 comprises an amino acid sequence shown in SEQ ID NO:1, the complementarity determining region CDR2 comprises an amino acid sequence shown in SEQ ID NO:2, and the complementarity determining region CDR3 comprises an amino acid sequence shown in SEQ ID NO: 3. The BCMA-targeted nano antibody has the advantages of small molecular weight, simple structure, easy expression and high expression stability, can be specifically combined with BCMA protein, has strong specificity and affinity to cancer cells expressing BCMA, has low immunogenicity and high safety, and is beneficial to the use of the BCMA-targeted nano antibody.

Description

BCMA-targeted nanobody and application thereof
Technical Field
The application relates to the field of medical biology, in particular to a BCMA-targeted nano antibody and application thereof.
Background
B cell maturation antigen (B Cell Maturation Antigen, BCMA) is a member of the tumor necrosis factor receptor superfamily, is a type III transmembrane protein composed of 185 amino acid residues, and its ligand belongs to the Tumor Necrosis Factor (TNF) superfamily, such as proliferation-inducing ligand (APRIL), B lymphocyte stimulating factor (BAFF), and when combined with its ligand, BCMA activates proliferation and survival of B cells. BCMA is specifically and highly expressed on plasma cell surfaces, but not in hematopoietic stem cells and other normal tissue cells, and is thus called an important target. Most antibodies targeting BCMA currently are single chain antibodies containing heavy and light chain variable regions, which are not highly expressed and are prone to misfolding during expression, affecting their use.
Disclosure of Invention
In view of the above, the present application provides a BCMA-targeted nanobody and an application thereof, wherein the BCMA-targeted nanobody has a small molecular weight, is easy to express, has high expression stability, excellent specificity and affinity, low immunogenicity, high safety, and is beneficial to use thereof.
In a first aspect, the present application provides a BCMA-targeting nanobody comprising a heavy chain variable region comprising complementarity determining region CDR1, complementarity determining region CDR2, and complementarity determining region CDR3, the complementarity determining region CDR1 comprising the amino acid sequence set forth in SEQ ID NO:1, the complementarity determining region CDR2 comprising the amino acid sequence set forth in SEQ ID NO:2, and the complementarity determining region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3.
Alternatively, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 4.
Alternatively, the coding gene of the heavy chain variable region comprises a nucleotide sequence shown as SEQ ID NO. 5.
The BCMA-targeted nano antibody provided by the application has small molecular weight and simple structure; the antibody does not contain a light chain variable region, and can not be mutually adhered or even aggregated into blocks as easily as single-chain antibody fragments, so that the expression is easy and the expression stability is high; and it can specifically bind to BCMA protein, and has strong specificity and affinity to cancer cells expressing BCMA; meanwhile, the immunogenicity is low, the safety is high, and the use of the kit is facilitated.
In a second aspect, the present application provides a BCMA-targeting chimeric antigen receptor comprising a BCMA-targeting nanobody of the first aspect, an extracellular hinge region, a transmembrane region, and an intracellular signaling region, connected in sequence.
Alternatively, the chimeric antigen receptor targeting BCMA comprises the amino acid sequence shown in SEQ ID No. 6.
The chimeric antigen receptor of the targeted BCMA can specifically target tumor cells expressing the BCMA, has high specificity and affinity, and has low immunogenicity and high safety.
In a third aspect, the present application provides a BCMA-targeted chimeric antigen receptor T cell comprising a BCMA-targeted chimeric antigen receptor according to the second aspect.
The chimeric antigen receptor T cell of the targeted BCMA can stably and highly express the chimeric antigen receptor of the targeted BCMA, so that the chimeric antigen receptor T cell of the targeted BCMA specifically targets tumor cells of the targeted BCMA, and has high specificity and affinity.
In a fourth aspect, the present application provides a method of preparing a BCMA-targeted chimeric antigen receptor T cell comprising:
providing a coding gene of a chimeric antigen receptor of a targeted BCMA, and inserting the coding gene of the chimeric antigen receptor of the targeted BCMA into a delivery vector to obtain a recombinant delivery vector, wherein the coding gene of the chimeric antigen receptor of the targeted BCMA comprises a signal peptide, a nanobody of the targeted BCMA of the first aspect, an extracellular hinge region, a transmembrane region and a coding gene of an intracellular signal region which are sequentially connected from a 5 'end to a 3' end;
packaging and transferring the recombinant transfer vector into a host cell to obtain a recombinant lentivirus;
and transfecting the recombinant lentivirus into CD3 positive T lymphocytes to obtain chimeric antigen receptor T cells targeting BCMA.
The preparation method of the chimeric antigen receptor T cell targeting the BCMA is simple and convenient to operate, and the chimeric antigen receptor T cell targeting the BCMA with high specificity and affinity can be prepared.
In a fifth aspect, the present application provides a recombinant vector comprising a gene encoding a BCMA-targeting nanobody according to the first aspect, and/or a gene encoding a chimeric BCMA-targeting antigen receptor according to the second aspect.
The recombinant vector provided by the application can stably store the coding gene of the nano antibody targeting the BCMA and/or the coding gene of the chimeric antigen receptor targeting the BCMA.
In a sixth aspect, the present application provides a cell comprising the recombinant vector of the fifth aspect.
The cell provided by the application can stably store the recombinant vector, and is favorable for preparing the nano antibody targeting the BCMA and the chimeric antigen receptor targeting the BCMA.
In a seventh aspect, the present application provides the use of the BCMA-targeted nanobody of the first aspect, the BCMA-targeted chimeric antigen receptor of the second aspect, the BCMA-targeted chimeric antigen receptor T cell of the third aspect, the BCMA-targeted chimeric antigen receptor T cell prepared by the preparation method of the fourth aspect, the recombinant vector of the fifth aspect, or the cell of the sixth aspect in the preparation of a medicament for diagnosing and/or treating a tumor expressing BCMA.
In the application, the BCMA-targeted nanobody, BCMA-targeted chimeric antigen receptor T cells, recombinant vectors and cells can play a targeting role on tumors expressing BCMA, and can kill tumor cells efficiently and specifically, so that diagnosis and treatment of tumors expressing BCMA are realized.
Drawings
FIG. 1 is a graph showing the results of flow assays for chimeric antigen receptors targeting BCMA in example 1.
FIG. 2 is a graph showing the results of detection of the cell killing effect of BCMA-targeted chimeric antigen receptor T cells provided in the examples of the present application; wherein, E in (A) is T=10, E in (B) is T=5.
Fig. 3 is a graph showing the tumor-inhibiting effect of BCMA-targeted chimeric antigen receptor T cells provided in the examples of the present application in mice.
Detailed Description
The following description is of the preferred embodiments of the present application, and it should be noted that it will be apparent to those skilled in the art that modifications and adaptations can be made without departing from the principles of the present application, and such modifications and adaptations are intended to be comprehended within the scope of the present application.
The application provides a BCMA-targeted nanobody, which comprises a heavy chain variable region, wherein the heavy chain variable region comprises a complementarity determining region CDR1, a complementarity determining region CDR2 and a complementarity determining region CDR3, the complementarity determining region CDR1 comprises an amino acid sequence shown in SEQ ID NO:1, the complementarity determining region CDR2 comprises an amino acid sequence shown in SEQ ID NO:2, and the complementarity determining region CDR3 comprises an amino acid sequence shown in SEQ ID NO: 3. In the present application, nanobodies (nanobodies), also referred to as single domain antibodies (Single domain antibody, sdabs), heavy chain antibodies, lack light chains, as compared to conventional monoclonal antibodies, are the smallest antigen binding fragments with complete function. The nano antibody provided by the application has the advantages of small molecular weight, small volume, simple structure, stable high expression, low immunogenicity and improved safety.
In this application, the heavy chain variable region has a complementarity determining region CDR1, a complementarity determining region CDR2, and a complementarity determining region CDR3, which are defined according to the Kabat method; the more conserved parts of the heavy chain variable region are called Framework Regions (FR) which serve to link the three complementarity determining regions. In one embodiment of the present application, the heavy chain variable region comprises framework region FR1, framework region FR2, framework region FR3 and framework region FR4. In one embodiment, the heavy chain variable region may be represented as FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. In the present application, the framework regions FR1, FR2, FR3 and FR4 may be selected from framework region sequences of human or murine origin. Further, the framework regions FR1, FR2, FR3 and FR4 may be selected from human-derived framework sequences, thereby further reducing immunogenicity and improving safety.
In an embodiment of the present application, the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO. 4. In one embodiment of the present application, the heavy chain variable region encoding gene includes the nucleotide sequence shown in SEQ ID NO. 5. It will be appreciated that nucleotide sequences having base degeneracy with SEQ ID NO. 5 and that the amino acid sequences corresponding to these nucleotide sequences are still the amino acid sequences shown in SEQ ID NO. 4 are also within the scope of protection of the present application.
The BCMA-targeted nano antibody provided by the application has small molecular weight and simple structure; the antibody does not contain a light chain variable region, and can not be mutually adhered or even aggregated into blocks as easily as single-chain antibody fragments, so that the expression is easy and the expression stability is high; and it can specifically bind to BCMA protein, and has strong specificity and affinity to cancer cells expressing BCMA; meanwhile, the immunogenicity is low, the safety is high, and the use of the kit is facilitated. The nano antibody targeting the BCMA can be applied to the field of diagnosis and treatment of tumors expressing the BCMA. In particular, the nanobody may be used in the form of a chimeric antigen receptor as described herein below, and may also be used as an antibody-based therapeutic, detection drug, such as an antibody drug conjugate, and the like.
The application also provides a chimeric antigen receptor targeting BCMA (CAR-BCMA), which comprises the above-mentioned BCMA-targeting nanobody, an extracellular hinge region, a transmembrane region and an intracellular signal region, which are sequentially connected. The chimeric antigen receptor of the targeted BCMA can specifically target tumor cells expressing the BCMA, has high specificity and affinity, and has low immunogenicity and high safety.
In this application, "sequentially linked" is understood to mean that the carboxy terminus of the amino acid sequence of the nanobody targeting BCMA is linked to the amino terminus of the amino acid sequence of the extracellular hinge region, which is linked to the amino terminus of the amino acid sequence of the transmembrane region, which is linked to the amino terminus of the amino acid sequence of the intracellular signaling region.
In this application, the extracellular hinge region is used to promote binding of BCMA-targeted nanobodies to BCMA on tumor cells. In embodiments of the present application, the extracellular hinge region comprises at least one of a CD8 a hinge region, a CD28 hinge region, a CD4 hinge region, a CD5 hinge region, a CD134 hinge region, a CD137 hinge region, and an ICOS hinge region. Further, the extracellular hinge region includes a CD8 a hinge region. Still further, the CD 8. Alpha. Hinge region comprises the amino acid sequence shown in SEQ ID NO. 7.
In this application, the transmembrane region is used to immobilize a chimeric antigen receptor that targets BCMA. In embodiments of the present application, the transmembrane region comprises at least one of a CD4 transmembrane region, a CD5 transmembrane region, a CD8 transmembrane region, a CD28 transmembrane region, a CD45 transmembrane region, and a CD154 transmembrane region. Further, the transmembrane region includes a CD8 transmembrane region. Still further, the CD8 transmembrane region comprises the amino acid sequence shown in SEQ ID NO. 8.
In this application, intracellular signaling regions are used to signal T cell activation, maintain T cell survival and activate T cell proliferation signaling pathways. In embodiments of the present application, the intracellular signaling region comprises at least one of a 4-1BB signaling region, a CD3 zeta signaling region, an ICOS signaling region, a CD27 signaling region, an OX40 signaling region, a CD28 signaling region, an IL1R1 signaling region, a CD70 signaling region, and a TNFRSF19L signaling region. Further, the intracellular signal region includes a 4-1BB signal region and a CD3 zeta signal region. Still further, the 4-1BB signal region comprises the amino acid sequence shown as SEQ ID NO. 9, and the CD3 zeta signal region comprises the amino acid sequence shown as SEQ ID NO. 10.
In the embodiment of the application, the extracellular hinge region is a CD8 alpha hinge region, the transmembrane region is a CD8 transmembrane region, and the intracellular signal region comprises a 4-1BB signal region and a CD3 zeta signal region. That is, the amino acid sequence of the chimeric antigen receptor targeting BCMA includes the amino acid sequences of the nanobody targeting BCMA, the CD8 a hinge region, the CD8 transmembrane region, the 4-1BB signal region, and the cd3ζ signal region, which are sequentially linked from amino terminus to carboxy terminus. In one embodiment, the chimeric antigen receptor that targets BCMA comprises the amino acid sequence shown in SEQ ID No. 6.
The application also provides a chimeric antigen receptor T cell targeting BCMA, comprising the chimeric antigen receptor targeting BCMA. The chimeric antigen receptor T cell of the targeted BCMA can stably and highly express the chimeric antigen receptor of the targeted BCMA, so that the tumor cell of the targeted BCMA can be specifically targeted to produce a killing effect, and the tumor cell has high specificity and affinity.
The application also provides a preparation method of the chimeric antigen receptor T cell targeting BCMA, which comprises the following steps: providing a coding gene of a chimeric antigen receptor of a targeted BCMA, and inserting the coding gene of the chimeric antigen receptor of the targeted BCMA into a delivery vector to obtain a recombinant delivery vector, wherein the coding gene of the chimeric antigen receptor of the targeted BCMA comprises a signal peptide, the nano antibody of the targeted BCMA, an extracellular hinge region, a transmembrane region and a coding gene of an intracellular signal region which are sequentially connected from a 5 'end to a 3' end; packaging and transferring the recombinant transfer vector into a host cell to obtain a recombinant lentivirus; and (3) transfecting the recombinant lentivirus into CD3 positive T lymphocytes to obtain chimeric antigen receptor T cells targeting the BCMA. The preparation method of the chimeric antigen receptor T cell targeting the BCMA is simple and convenient to operate, and the chimeric antigen receptor T cell targeting the BCMA with high specificity and affinity can be prepared.
In the present application, "sequentially connected from 5 'end to 3' end" may be understood as that the 3 'end of the coding gene sequence of the signal peptide is connected to the 5' end of the coding gene of the nanobody targeting BCMA, the 3 'end of the coding gene of the nanobody targeting BCMA is connected to the 5' end of the coding gene of the extracellular hinge region, the 3 'end of the coding gene of the extracellular hinge region is connected to the 5' end of the coding gene of the transmembrane region, and the 3 'end of the coding gene of the transmembrane region is connected to the 5' end of the coding gene of the intracellular signal region.
In this application, a signal peptide is used to direct the chimeric antigen receptor CAR-BCMA surface to the cell surface, which is cleaved by a signal peptidase during protein translational maturation. In an embodiment of the present application, the signal peptide comprises the amino acid sequence shown as SEQ ID NO. 11. Specific choices of the extracellular hinge region, the transmembrane region, and the intracellular signal region and corresponding coding gene sequences are as described above, and will not be described in detail herein. In an embodiment of the present application, the coding gene of CAR-BCMA includes a coding gene of a signal peptide, a coding gene of a nanobody targeting BCMA, a coding gene of a CD8 a hinge region, a coding gene of a CD8 transmembrane region, a coding gene of a 4-1BB signal region, and a coding gene of a CD3 ζ signal region, which are sequentially connected from 5 'end to 3' end.
In embodiments of the present application, a start codon (e.g., ATG) may be added to the 5 'end of the encoding gene of CAR-BCMA and a stop codon (e.g., TAA) may be added to the 3' end of the encoding gene of BCMA prior to insertion into a delivery vehicle. In one embodiment, the delivery vector may be a plasmid vector, such as a pCDH plasmid. The pCDH plasmid can be ligated with elongation factor 1 alpha (EF 1 alpha) as a promoter for transcription of the CAR-BCMA encoding gene.
Transfection may include liposome transfection, calcium phosphate co-precipitation, viral infection, and the like. Among them, adenovirus, adeno-associated virus, retrovirus, lentivirus vector and the like can be used for the virus infection method. In embodiments of the present application, lentiviral vector transfection may comprise packaging a recombinant transfer vector and transfecting a host cell to obtain a recombinant lentivirus; specifically, the recombinant transfer vector, envelope plasmid and packaging plasmid are transfected together into host cells to obtain recombinant lentivirus. Wherein the host cell is used to assemble the recombinant lentivirus to render it infectious. In one embodiment, the host cells may include HEK293T cells, 293T cells, 293FT cells, SW480 cells, u87MG cells, HOS cells, COS1 cells, COS7 cells, or the like, but are not limited thereto. For example, the host cell may be a HEK293T cell.
In the present application, the recombinant lentivirus can be packaged by a three-plasmid system or a four-plasmid system, and the envelope plasmid and the packaging plasmid are common substances in the field. It will be appreciated that the envelope plasmid, packaging plasmid and plasmids of the gene transfer vector described above are different. In one embodiment, the packaging plasmids are pMDLg/pRRE and pRSV-REV and the envelope plasmid may be PMD2G. The envelope plasmid may encode a vesicular stomatitis virus glycoprotein (VSV-G) capsid that may assist in adherence of the recombinant lentivirus to the cell membrane and maintain infectivity of the recombinant lentivirus.
In embodiments of the present application, the CD3 positive T lymphocytes are isolated from human peripheral blood mononuclear cells. Further, the human peripheral blood mononuclear cells are derived from autologous venous blood, autologous bone marrow, umbilical cord blood, placental blood and the like. Specifically, the human peripheral blood mononuclear cells can be from a cancer patient, and the chimeric antigen receptor T cells prepared by the method can have lower immune response when being returned to the patient; for example, it may be derived from fresh peripheral blood or bone marrow collected one month after a cancer patient's operation and one month after chemoradiotherapy. In one embodiment, the CD3 positive T lymphocytes are obtained as follows: adding CD3/CD28 immunomagnetic beads into peripheral blood mononuclear cells according to a certain proportion, incubating for a period of time, placing into a magnet for screening to obtain CD3 positive T lymphocytes coated with the immunomagnetic beads, and removing the magnetic beads to obtain the CD3 positive T lymphocytes. In the present application, T cells used in the preparation of chimeric antigen receptor T cells may be T cells of various origins, such as autologous T cells, allogeneic T cells, induced pluripotent stem cells (ipscs) -induced T cells, T cells of healthy humans, T cells of cancer patients, or the like.
The application also provides a recombinant vector, which comprises the coding gene of the BCMA-targeted nano antibody and/or the coding gene of the BCMA-targeted chimeric antigen receptor. The recombinant vector can stably store the encoding gene of the nano antibody targeting the BCMA and/or the encoding gene of the chimeric antigen receptor targeting the BCMA.
In an embodiment of the present application, the vector is at least one of a viral vector and a non-viral vector. Further, non-viral vectors include plasmid vectors and phage vectors. In particular, the plasmid vector may be, but is not limited to, eukaryotic plasmid vectors, prokaryotic plasmid vectors, micro-circular DNA, transposons, etc. Further, the viral vector includes a lentiviral vector, an adenoviral vector or a retroviral vector. Further, the viral vector is a lentiviral vector. In particular, the viral vector may include, but is not limited to, at least one of a pCDH plasmid, a pWPXLD vector, a pLEX-MCS vector, a pSico vector and a pCgpV vector.
The application also provides a cell comprising the recombinant vector. The cell can stably store the recombinant vector, and is favorable for preparing the nano antibody targeting the BCMA and the chimeric antigen receptor targeting the BCMA.
The application also provides application of the BCMA-targeted nanobody, BCMA-targeted chimeric antigen receptor T cell, recombinant vector or cell in preparation of medicines for diagnosing and/or treating tumors expressing BCMA.
The BCMA-targeted nanobody, BCMA-targeted chimeric antigen receptor T cells, recombinant vectors and cells can play a targeting role on tumors expressing BCMA, and can kill tumor cells efficiently and specifically, so that diagnosis and treatment of tumors expressing BCMA are realized.
In embodiments of the present application, BCMA expressing tumors include high BCMA expressing tumors, BCMA overexpressing tumors. In embodiments of the present application, BCMA expressing tumors include hematological tumors, and may specifically, but not be limited to, multiple bone marrow, lymphomas, leukemias, and the like. Multiple Myeloma (MM) is a malignant tumor of the blood system caused by malignant proliferation of plasma cells in bone marrow, and is treated by chemotherapy, autologous stem cell transplantation, proteasome inhibitors, immunoregulatory drugs, monoclonal antibodies and other methods in clinic at present, but relapse is incurable, and the relapse refractory multiple myeloma is still a difficult problem in treatment. BCMA is used as an effective target for treating multiple myeloma, and the nano antibody, chimeric antigen receptor and chimeric antigen receptor T cell of the targeted BCMA can specifically target tumor cells (such as multiple bone marrow cells) expressing BCMA, so that the tumor cells are killed, normal cells (such as hematopoietic stem cells and the like) are not damaged, and the safety is higher.
In the embodiment of the application, the medicine for diagnosing and/or treating the tumor expressing the BCMA comprises chimeric antigen receptor T cells targeting the BCMA, wherein the chimeric antigen receptor T cells targeting the BCMA can be infused into a patient by intravenous injection, subcutaneous injection, tumor in-situ injection and the like, and can achieve good treatment effect.
Example 1
A method of preparing a BCMA-targeted chimeric antigen receptor T cell comprising:
(1) Preparation of chimeric antigen receptor T cell CAR-BCMA Gene sequence targeting BCMA
Respectively preparing coding genes of a BCMA-targeted nanobody, a CD8 hinge region, a CD8 transmembrane region, a 4-1BB signal region and a CD3 and signal region, sequentially connecting the coding genes from 5 side end to 3 side end by a PCR method to obtain a nucleotide sequence corresponding to an amino acid sequence shown in SEQ ID NO. 2, and adding a nucleotide sequence (a gene sequence of a signal peptide) corresponding to the amino acid sequence shown in SEQ ID NO. 4 at the 5' end of the nucleotide sequence.
(2) Construction of recombinant plasmids
The nucleotide sequence obtained in step (1) was inserted into a pCDH vector and was located after the elongation factor 1α (EF 1 α) of the vector. When the nucleotide sequence is inserted into the pCDH vector, an initiation codon (ATG) may be added to the 5 '-end of the nucleotide sequence and a termination codon (TAA) may be added to the 3' -end. Then transferring into competent cells DH5 alpha of the escherichia coli, and carrying out positive clone PCR identification and sequencing identification. And (3) successfully constructing the recombinant plasmid by detecting and sequencing the PCR product gel electrophoresis to identify the size and the sequence of the fragment which accords with the purpose.
(3) Recombinant lentivirus construction
And (3) co-transfecting the recombinant plasmid obtained in the step (2) with packaging plasmid (pMDLg/pRRE and pRSV-REV) and envelope plasmid (pMD 2G) into cultured HEK293T cells through liposome transfection reagent Lipofectamine3000, and centrifuging after 48 hours to obtain the recombinant lentivirus.
(4) Preparation of chimeric antigen receptor T cells targeting BCMA
a) Isolation of PBMC (peripheral blood mononuclear cells)
Drawing 30-100 mL of blood of a patient, and sending the sample to a blood separation chamber; collecting peripheral blood mononuclear cells, and taking middle-layer cells after Ficoll centrifugal separation; after washing with PBS, PBMC were obtained.
b) Isolation of antigen-specific T lymphocytes by immunomagnetic bead method
Adding a basic culture medium without serum into the PBMC to prepare cell suspension; adding CD3/CD28 immunomagnetic beads according to the ratio of the magnetic beads to the cells being 1:1, and incubating for 1-2 h at room temperature; screening cells incubated with the magnetic beads by using a magnet; washing with PBS, and removing immunomagnetic beads to obtain CD3 positive T lymphocytes.
c) Method for preparing antigen-specific T lymphocyte by virus transfection method
And (3) taking the CD3 positive T lymphocytes obtained by the immunomagnetic bead separation method, and adding recombinant lentivirus with the virus titer corresponding to the number of the CD3 positive cells for co-culture. On day 3 of culture, cell counting and liquid exchange were performed to adjust the cell concentration to 1×10 6 Inoculating and culturing the strain in a single/mL mode; on day 5 of culture, the cell state was observed, and if the cell density was increased, the diluted cell concentration was 1×10 6 Cell activity was measured at each mL and culture was continued. Cells were collected by expansion culture to day 9-11, while expression of BCMA-targeted chimeric antigen receptor was detected by flow cytometry, and the results are shown in fig. 1. The positive rate of chimeric antigen receptor targeting BCMA was detected to be about 20.35% in T cells infected with the recombinant lentivirus described above. This suggests that BCMA-targeted chimeric antigen receptor T cells were successfully prepared and stored in dedicated cell cryopreservation solution for reinfusion by patients.
To evaluate the effect of BCMA-targeted chimeric antigen receptor T cells prepared by the above method described herein, the following experiment was performed.
In vitro tumor cell killing experiments were performed using a real-time cell analyzer (xCElligence RTCA SP). 6000 complete media of BCMA overexpressing 293T cells were added to the E-Plate wells prior to the instrument set-up. After about 24h, corresponding numbers of BCMA-targeted chimeric antigen receptor T cells (CAR-T group) prepared as described above were added as effector cells according to different targeting ratios (E: T), and a single culture medium was used as a blank, and non-virally infected T cells (abbreviated as UTD cells, i.e., CD3 positive T lymphocytes in step (4) of example 1) were stimulated with magnetic beads alone as a T cell control, followed by co-culturing for a period (> 24 h). The cell killing effect was analyzed according to the Cell Index (CI) value of the real-time cell analyzer, and the result is shown in fig. 2. Fig. 2 is a graph of the results of detection of cell killing effect of BCMA-targeted chimeric antigen receptor T cells provided in the examples of the present application, wherein (a) E: t=10 and (B) E: t=5. It can be seen that the chimeric antigen receptor T cells of the targeted BCMA provided by the application have a strong killing effect on 293T cells over-expressing the BCMA, and the killing capacity is far higher than that of a blank control group and a T cell control group.
In vivo killing experiments were performed using immunodeficient mouse B-NDG. Multiple myeloma cells (MM.1S cells) with Luciferase (luciferases) were inoculated 1X 10 in the tail vein of each mouse 6 And each. After 7 days the random groups were 2: CAR-T group and T cell control group, mice of CAR-T group were injected 3X 10 by tail vein respectively 6 Chimeric antigen receptor T cells targeted to BCMA, mice tail vein injection of T cell control group 3×10 6 The size of tumor burden in mice was represented by the intensity of fluorescent signal obtained by IVIS imaging of UTD cells once every 5-7 days, and the results are shown in FIG. 3. It can be seen that the tumor of the mice of the T cell control group gradually increases with time, while the tumor of the mice of the CAR-T group does not increase and has a tendency to significantly decrease, indicating that the BCMA-targeted chimeric antigen receptor T cells provided by the present application have excellent tumor inhibiting effect on BCMA-expressing tumors.
Therefore, the chimeric antigen receptor T cell targeting the BCMA has a very considerable application prospect in preparing medicines for diagnosing and treating tumors expressing the BCMA.
The above examples only represent a few embodiments of the present application, which are described in more detail and are not to be construed as limiting the scope of the present application. It should be noted that it would be apparent to those skilled in the art that various modifications and improvements could be made without departing from the spirit of the present application, which would be within the scope of the present application. Accordingly, the scope of protection of the present application is to be determined by the claims appended hereto.

Claims (10)

1. A BCMA-targeting nanobody comprising a heavy chain variable region comprising a complementarity determining region CDR1, a complementarity determining region CDR2, and a complementarity determining region CDR3, wherein the complementarity determining region CDR1 comprises the amino acid sequence set forth in SEQ ID No. 1, the complementarity determining region CDR2 comprises the amino acid sequence set forth in SEQ ID No. 2, and the complementarity determining region CDR3 comprises the amino acid sequence set forth in SEQ ID No. 3.
2. The BCMA-targeting nanobody as set forth in claim 1, wherein said heavy chain variable region comprises an amino acid sequence set forth in SEQ ID No. 4.
3. The BCMA-targeted nanobody as set forth in claim 1, wherein the heavy chain variable region encoding gene comprises a nucleotide sequence set forth in SEQ ID No. 5.
4. A BCMA-targeting chimeric antigen receptor comprising, in sequence, the BCMA-targeting nanobody of any one of claims 1-3, an extracellular hinge region, a transmembrane region, and an intracellular signaling region.
5. The BCMA-targeted chimeric antigen receptor according to claim 4, wherein said BCMA-targeted chimeric antigen receptor comprises the amino acid sequence shown in SEQ ID No. 6.
6. A BCMA-targeting chimeric antigen receptor T cell comprising the BCMA-targeting chimeric antigen receptor according to any one of claims 4 to 5.
7. A method of preparing a BCMA-targeted chimeric antigen receptor T cell comprising:
providing a coding gene of a chimeric antigen receptor of a targeted BCMA, inserting the coding gene of the chimeric antigen receptor of the targeted BCMA into a delivery vector to obtain a recombinant delivery vector, wherein the coding gene of the chimeric antigen receptor of the targeted BCMA comprises a signal peptide, the coding gene of the nanobody of the targeted BCMA, an extracellular hinge region, a transmembrane region and an intracellular signal region, which are sequentially connected from a 5 'end to a 3' end;
packaging and transferring the recombinant transfer vector into a host cell to obtain a recombinant lentivirus;
and transfecting the recombinant lentivirus into CD3 positive T lymphocytes to obtain chimeric antigen receptor T cells targeting BCMA.
8. A recombinant vector comprising a gene encoding a BCMA-targeting nanobody according to any one of claims 1 to 3 and/or a gene encoding a BCMA-targeting chimeric antigen receptor according to any one of claims 4 to 5.
9. A cell comprising the recombinant vector of claim 8.
10. Use of a BCMA-targeted nanobody as claimed in any one of claims 1 to 3, a BCMA-targeted chimeric antigen receptor as claimed in any one of claims 4 to 5, a BCMA-targeted chimeric antigen receptor T cell as claimed in claim 6, a BCMA-targeted chimeric antigen receptor T cell as produced by the production process as claimed in claim 7, a recombinant vector as claimed in claim 8 or a cell as claimed in claim 9 for the production of a medicament for the diagnosis and/or treatment of BCMA-expressing tumors.
CN202210904517.8A 2022-07-29 2022-07-29 BCMA-targeted nanobody and application thereof Pending CN117467010A (en)

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