Monoclonal antibodies targeting cd180

Abstract

The application provides a monoclonal antibody targeting CD180, and relates to the technical field of biological medicine.The application provides a CD180-targeting antibody which can specifically bind to a CD180 antigen and a protein, can specifically detect CD180 in various tumor cell lines, and provides a condition for preparing a CD180 antigen detection kit.A CD180 chimeric antigen receptor (referred to as CD180-CAR) is constructed based on the above antibody, and a CD180-CAR-T cell for tumor immunocyte treatment is successfully constructed.The CD180-CAR-T cell provided by the application has the ability to efficiently and specifically kill CD180-positive acute myeloid leukemia cells, and can secrete high levels of IFN gamma and IL-2 to resist CD180-positive cancer cells; the CD180-CAR-T cell can also significantly eliminate tumor cells in a mouse body, and has strong anti-tumor ability and the ability to prolong the survival period.The specific monoclonal antibody targeting human CD180, the chimeric antigen receptor fusion protein targeting CD180 and the engineered immunocyte targeting CD180 provided by the application provide a new strategy for blood system tumor treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to monoclonal antibodies targeting CD180. Background Technology

[0002] Acute myeloid leukemia (AML), the most common acute leukemia in adults, still lacks effective treatment options. Standard induction chemotherapy can achieve complete remission in some AML patients, but more than half of AML patients eventually relapse, resulting in poor survival. Chemotherapy resistance and short-term relapse remain major factors threatening patient survival. Chimeric antigen receptor T cell (CAR-T) immunotherapy is a technique that induces T cell activation by expressing chimeric antigen receptor molecules that specifically recognize and bind to tumor antigens on the T cell membrane, thereby achieving specific killing of tumor cells. It is one of the most promising tumor immunotherapies. In recent years, CAR-T cell therapy has been widely used in clinical research as a highly promising treatment method and has achieved good results. Treatment options for AML are constantly evolving; however, existing target therapies are not effective, with poor remission rates or severe extratumor toxicity. Therefore, there is an urgent need to develop safer new targets for AML to achieve objective long-term remission.

[0003] CD180 (also known as LY64 or RP105) is a member of the Toll-like receptor (TLR) family, primarily expressed on the surface of B cells, dendritic cells, and various hematologic malignancies. Unlike other TLR members, CD180's function is highly dependent on its heterodimeric complex with MD1 (Myeloid Differentiation Protein 1). Studies have shown that the interaction between CD180 and MD1 is crucial for its stable expression and signal transduction on the cell membrane. Data indicate that CD180 is highly expressed in various hematologic malignancies, including acute myeloid leukemia and diffuse large B-cell lymphoma, and is closely related to tumor cell proliferation, drug resistance, and immune escape. Currently, there are no treatments targeting CD180; therefore, the development of highly specific antibodies and immunotherapies based on CD180 has significant clinical value.

[0004] CAR-T cell therapy, which involves genetically engineering T cells to express chimeric receptors targeting tumor antigens, has demonstrated significant efficacy in the treatment of hematological malignancies. However, existing CAR-T therapy targets (such as CD19, CD123, and CD33) suffer from issues such as antigen escape, high toxicity, or limited applicability. Developing antibodies and CAR-T cells targeting CD180 can fill this gap. As a novel target, CD180 can expand the indications for CAR-T therapy, particularly for patients with CD180-positive refractory hematological malignancies. Summary of the Invention

[0005] (a) Technical problems to be solved

[0006] To address the shortcomings of existing technologies, this invention provides a monoclonal antibody targeting CD180 and its application in anti-tumor therapy.

[0007] (II) Technical Solution

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] In a first aspect, the present invention provides a specific monoclonal antibody (mAb) targeting human CD180, wherein the antibody is abbreviated as antibody 28A7, antibody 30A1, or antibody 38C8, and the antibody is a mouse IgG1 subtype monoclonal antibody. The heavy chain variable region (VH) of the antibody includes 28A7-VH, 30A1-VH, or 38C8-VH, and the light chain variable region (VL) of the antibody includes 28A7-VL, 30A1-VL, or 38C8-VL. The amino acid sequences of 28A7-VH, 30A1-VH, 38C8-VH, 28A7-VL, 30A1-VL, and 38C8-VL are shown in SEQ ID NO:1-6.

[0010] Specifically, the heavy chain of the antibody includes a 28A7-Heavy chain, a 30A1-Heavy chain, or a 38C8-Heavy chain, and the light chain of the antibody includes a 28A7-Light chain, a 30A1-Light chain, or a 38C8-Light chain; the amino acid sequences of the 28A7-Heavy chain, 30A1-Heavy chain, 38C8-Heavy chain, 28A7-Light chain, 30A1-Light chain, and 38C8-Light chain are as shown in SEQ ID NO:7-12.

[0011] Specifically, the 28A7-scFv single-chain variable fragment is composed of 28A7-VH of the amino acid sequence shown in SEQ ID NO:1 and 28A7-VL of the amino acid sequence shown in SEQ ID NO:4, and the amino acid sequence of 28A7-scFv is shown in SEQ ID NO:13.

[0012] Specifically, the 30A1-scFv single-chain variable fragment is composed of 30A1-VH of the amino acid sequence shown in SEQ ID NO:2 and 30A1-VL of the amino acid sequence shown in SEQ ID NO:5, and the amino acid sequence of 30A1-scFv is shown in SEQ ID NO:14.

[0013] Specifically, the 38C8-scFv single-chain variable fragment is composed of 38C8-VH of the amino acid sequence shown in SEQ ID NO:3 and 38C8-VL of the amino acid sequence shown in SEQ ID NO:6, and the amino acid sequence of 38C8-scFv is shown in SEQ ID NO:15.

[0014] Specifically, the amino acid sequences of the complementarity-determining regions (CDRs) of antibodies 28A7, 30A1, and 38C8 are shown in the table below:

[0015]

[0016] Secondly, the present invention provides the application of the above-mentioned specific monoclonal antibody (mAb) targeting human CD180 in the preparation of reagents for detecting CD180 antigen.

[0017] Specifically, the reagents are those used in detection techniques such as flow cytometry, ELISA, and immunohistochemistry.

[0018] Thirdly, the present invention provides a chimeric antigen receptor (CD180-CAR) targeting CD180, wherein the chimeric antigen receptor comprises, from the N-terminus to the C-terminus:

[0019] (i) 28A7-scFv, 30A1-scFv, or 38C8-scFv targeting CD180;

[0020] (ii) Transmembrane domains;

[0021] (iii) At least one co-stimulatory domain;

[0022] (iv) Activate the structural domain.

[0023] Specifically, the ScFv can be VH-Linker-VL or VL-Linker-VH.

[0024] Specifically, the transmembrane domain can be derived from a natural polypeptide or it can be artificially designed; the artificially designed transmembrane domain is a polypeptide that mainly includes hydrophobic residues such as leucine and valine; preferably, a triplet of phenylalanine, tryptophan and valine is found at each end of the synthesized transmembrane domain; alternatively, a short oligopeptide linker or polypeptide linker, such as a linker with a length of 2 to 10 amino acids, can be provided between the transmembrane domain and the intracellular domain; in one embodiment, a linker sequence having a glycine-serine continuous sequence can be used, optionally (G4S)3 or (G4S)4.

[0025] Specifically, the co-stimulatory domains are freely selected and combined from CD28, 4-1BB, GITR, ICOS-1, CD27, OX-40 and DAP10; preferably, the co-stimulatory domain is 4-1BB.

[0026] Specifically, the activation domains include CD3ζ, CD3γ, CD3δ, or CD3ε.

[0027] Specifically, the chimeric antigen receptor structure includes CD8 signal peptide-CD180 scFv(VH-Linker-VL)CD8 hinge-CD28 transmembrane domain-CD28-CD3ζ or CD8 signal peptide-CD180 scFv(VH-Linker-VL)CD8 hinge-CD8 transmembrane domain-41BB-CD3ζ.

[0028] Specifically, the nucleotide sequence of the chimeric antigen receptor is shown in SEQ ID NO:34-39.

[0029] Fourthly, the present invention provides an engineered immune cell that targets CD180, wherein the engineered immune cell expresses the aforementioned chimeric antigen receptor fusion protein that targets CD180; the immune cell includes any one of T cells, NK cells, macrophages, and hematopoietic stem cells.

[0030] Specifically, the engineered immune cells targeting CD180 are obtained by transducing nucleic acid molecules as shown in SEQ ID NO: 34-39 into T cells to obtain CD180-CAR-T cells.

[0031] Fifthly, the present invention provides the application of the above-mentioned CD180-targeting antibody, CD180-targeting chimeric antigen receptor fusion protein, and CD180-targeting engineered immune cells in the preparation of immunotherapy drugs.

[0032] Specifically, the immunotherapy includes bispecific targeting antibodies, toxin / drug conjugate antibodies, therapeutic monoclonal antibodies, humanized CD180 antibodies, CAR T cell therapy, TCR-T cell therapy, CAR-NK cell therapy, and CAR-M cell therapy.

[0033] In a sixth aspect, the present invention provides the use of the above-mentioned CD180-targeting antibody, CD180-targeting chimeric antigen receptor fusion protein, and CD180-targeting engineered immune cells in the preparation of drugs for treating and / or treating CD180-related cancers or tumors.

[0034] (III) Beneficial Effects

[0035] This invention provides three high-affinity, high-specificity monoclonal antibodies targeting human CD180, specifically 28A7, 30A1, and 38C8. These antibodies contain specific heavy chain and light chain variable region sequences. The three CD180 antibodies provided by this invention can specifically bind to the CD180 antigen and protein, enabling specific detection of CD180 in various tumor cell lines, and providing a basis for the preparation of CD180 antigen detection kits.

[0036] This invention also utilizes the single-chain variable fragment scFv of antibodies 28A7, 30A1, and 38C8 to construct a CD180 chimeric antigen receptor (CD180-CAR), and successfully transduced it into immune cells to construct CD180-CAR-T cells for tumor immunotherapy. In vitro experiments show that the CD180-CAR-T cells provided by this invention possess the ability to efficiently and specifically kill CD180-positive acute myeloid leukemia cells and secrete high levels of IFN-γ and IL-2 to combat CD180-positive cancer cells. In vivo experiments show that the CD180-CAR-T cells provided by this invention can significantly eliminate tumor cells in mice, exhibiting strong anti-tumor activity and the ability to prolong survival.

[0037] The specific monoclonal antibody targeting human CD180, the chimeric antigen receptor fusion protein targeting CD180, and the engineered immune cells targeting CD180 provided by this invention offer new strategies for the treatment of hematologic malignancies. Attached Figure Description

[0038] Figure 1 Flowchart for the preparation of a specific monoclonal antibody targeting human CD180.

[0039] Figure 2 The results show the affinity test results of antibodies 28A7, 30A1, and 38C8 for the CD180 antigen.

[0040] Figure 3This refers to the specific binding of antibodies 28A7, 30A1, and 38C8 to the CD180 antigen in ELISA assays.

[0041] Figure 4 This demonstrates the specific binding of antibodies 28A7, 30A1, and 38C8 to the CD180 protein.

[0042] Figure 5 The results show the detection results of antibodies 28A7, 30A1, and 38C8 on CD180 antigen-positive tumor cells.

[0043] Figure 6 It is a second-generation structure of CD180-CAR.

[0044] Figure 7 The specific killing effect of 28A7-28z-CAR-T and 28A7-BBz-CAR-T on CD180-positive AML cell lines was demonstrated.

[0045] Figure 8 This refers to the secretion of specific effector factors by 28A7-28z-CAR-T and 28A7-BBz-CAR-T in CD180-positive AML cell lines.

[0046] Figure 9 The significant antitumor effects of 28A7-28z-CAR-T and 28A7-BBz-CAR-T in mouse xenograft models were demonstrated.

[0047] Figure 10 The expression of CD180 in normal human tissues was detected by immunohistochemistry using the 28A7 antibody. Detailed Implementation

[0048] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0049] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0050] Unless otherwise stated, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art.

[0051] As used herein, a "chimeric antigen receptor (CAR)" refers to a fusion protein comprising an extracellular domain capable of binding to an antigen, a transmembrane domain consisting of a polypeptide not derived from that extracellular domain, and at least one intracellular domain. "Chimeric antigen receptor (CAR)" is sometimes referred to as a "chimeric receptor," a "T-body," or a "chimeric immune receptor (CIR)." An "extracellular domain capable of binding to an antigen" refers to any oligopeptide or polypeptide capable of binding to an antigen. An "intracellular domain" refers to any oligopeptide or polypeptide known to function as a domain that transmits signals to activate or inhibit intracellular biological processes.

[0052] As used in this article, a “domain” refers to a region in a polypeptide that folds into a structure independently of other regions.

[0053] As used herein, "single-chain antibody (scFv)" refers to a single-chain polypeptide derived from an antibody that retains its ability to bind to an antigen. An example of scFv includes an antibody polypeptide formed using recombinant DNA technology, wherein the Fv regions of the immunoglobulin heavy chain (H chain) and light chain (L chain) fragments are linked by spacer sequences. Various methods for engineering scFv are known to those skilled in the art.

[0054] As used in this article, "tumor antigen" refers to an antigenic biomolecule whose expression leads to cancer.

[0055] CD180 (also known as LY64 or RP105) is a member of the Toll-like receptor (TLR) family, primarily expressed on the surface of B cells, dendritic cells, and various hematologic malignancies. Unlike other TLR members, CD180's function is highly dependent on the heterodimer complex it forms with MD1.

[0056] Example 1

[0057] Hybridoma technology was used to prepare specific monoclonal antibodies (mAbs) targeting human CD180.

[0058] Preparation method: Female Balb / c mice aged 6-8 weeks were selected as immunization animals. Recombinant CD180 / MD1 protein or peptide was prepared as an antigen and mixed with Freund's complete adjuvant. The mice were subcutaneously injected with the recombinant CD180 / MD1 protein or peptide mixture. Every 2-3 weeks, the same antigen mixed with Freund's incomplete adjuvant was injected repeatedly. 3-5 days before the last immunization, a high dose of antigen was injected to stimulate B lymphocytes to produce antibodies. 3-5 days after the last immunization, the mice were sacrificed and the spleen was aseptically removed. The spleen was ground into single-grain powder. For the cell suspension, spleen cell suspension was mixed with myeloma cells, and PEG was used to fuse the spleen cells and myeloma cells. The fused cell suspension was then transferred to a selective medium containing HAT for culture to screen for hybridoma cells. Hybridoma cells that secrete antibodies against CD180 were screened using methods such as ELISA. Positive hybridoma cells were cloned using limiting dilution or soft agar cloning. The cloned hybridoma cells were expanded and cultured on a large scale in appropriate medium to produce monoclonal antibodies. Monoclonal antibodies were purified from the culture supernatant using affinity chromatography (such as Protein A / G affinity chromatography), ultimately yielding antibodies 28A7, 30A1, and 38C8. The preparation flowchart is shown below. Figure 1 As shown.

[0059] The heavy chain variable region (VH) of the antibody includes 28A7-VH, 30A1-VH, or 38C8-VH, and the light chain variable region (VL) of the antibody includes 28A7-VL, 30A1-VL, or 38C8-VL; the amino acid sequences of 28A7-VH, 30A1-VH, 38C8-VH, 28A7-VL, 30A1-VL, and 38C8-VL are as shown in SEQ ID NO:1-6.

[0060] Specifically, the heavy chain of the antibody includes a 28A7-Heavy chain, a 30A1-Heavy chain, or a 38C8-Heavy chain, and the light chain of the antibody includes a 28A7-Light chain, a 30A1-Light chain, or a 38C8-Light chain; the amino acid sequences of the 28A7-Heavy chain, 30A1-Heavy chain, 38C8-Heavy chain, 28A7-Light chain, 30A1-Light chain, and 38C8-Light chain are as shown in SEQ ID NO:7-12.

[0061] Specifically, the 28A7-scFv single-chain variable fragment is composed of 28A7-VH of the amino acid sequence shown in SEQ ID NO:1 and 28A7-VL of the amino acid sequence shown in SEQ ID NO:4, and the amino acid sequence of 28A7-scFv is shown in SEQ ID NO:13.

[0062] Specifically, the 30A1-scFv single-chain variable fragment is composed of 30A1-VH of the amino acid sequence shown in SEQ ID NO:2 and 30A1-VL of the amino acid sequence shown in SEQ ID NO:5, and the amino acid sequence of 30A1-scFv is shown in SEQ ID NO:14.

[0063] Specifically, the 38C8-scFv single-chain variable fragment is composed of 38C8-VH of the amino acid sequence shown in SEQ ID NO:3 and 38C8-VL of the amino acid sequence shown in SEQ ID NO:6, and the amino acid sequence of 38C8-scFv is shown in SEQ ID NO:15.

[0064] Example 2

[0065] Affinity identification of the CD180 antibody prepared in this invention to CD180 antigen

[0066] We used surface plasmon resonance (SRP) technology to detect the affinity of monoclonal antibodies. Three CD180 monoclonal antibodies (antibodies 28A7, 30A1, and 38C8) were serially diluted (50 nM, 25 nM, 12.5 nM, 6.25 nM, and 3.13 nM) and added to an anti-mouse IgG biosensor. The binding process of the antibody to the immobilized antigen was monitored in real time by the increase in the RU value. Each concentration was maintained for a sufficient time (usually 3-5 minutes) until binding approached equilibrium (the rate of change in RU slowed down). After 5 minutes, unbound antibodies were washed off the biosensor. Antibody binding was monitored using a Blitz system (ForteBio), and the dissociation constant of the antibody was measured using Blitz software.

[0067] The results are as follows Figure 2 As shown, all three CD180 monoclonal antibodies exhibited strong binding to the CD180 antigen, with Kd values ​​of 4.33 × 10⁻⁶ for 28A7 and 4.33 × 10⁻⁶ respectively. -10 M; 30A1: 9.07×10 -11 M; 38C8: 1.79 × 10 -10 M indicates that the monoclonal antibody targeting human CD180 prepared in this invention has a high affinity for the CD180 antigen.

[0068] Example 3

[0069] The specificity of the CD180 antibody prepared in this invention for the detection of CD180 antigen.

[0070] To verify the binding affinity of antibodies 28A7, 30A1, and 38C8 to the target antigen CD180, we performed a systematic analysis using ELISA. CD180.ECD / MD-1 antigen or control MD-1 protein was coated onto 96-well plates at a concentration of 1 μg / mL and incubated overnight at 4°C. After blocking, serially diluted versions of the three monoclonal antibodies (28A7, 30A1, and 38C8) were added, and the plates were incubated at 37°C for 1 h. Subsequently, the plates were incubated for 1 h with HRP-labeled anti-mouse IgG secondary antibody (1:5000 dilution). After TMB color development, the OD450 value was measured to assess the binding affinity between the antibodies and the antigen.

[0071] The results are as follows Figure 3 As shown in the ELISA, the three CD180 monoclonal antibodies provided by this invention specifically bind to the extracellular domain of the CD180 antigen, but do not bind to the control MD-1 protein, and the binding of the CD180 antibody to the CD180 antigen is dose-dependent: it decreases as the antibody dilution decreases.

[0072] Example 4

[0073] The CD180 antibody prepared in this invention specifically detects the CD180 protein.

[0074] We first constructed Jurkat-MD1 cell lines and Jurkat-CD180 / MD1 cell lines, respectively, overexpressing MD1 or CD180 / MD1, and then used Jurkat cells as a negative control. We then incubated these cells with antibodies 28A7, 30A1, and 38C8 for 30 minutes, followed by incubation with APC-conjugated anti-mouse IgG secondary antibody for another 30 minutes. The expression of CD180 in the cell lines was then detected by flow cytometry.

[0075] The results are as follows Figure 4 As shown, antibodies 28A7, 30A1, and 38C8 did not bind to Jurkat cells or Jurkat-MD1 cells, but only to the Jurkat-CD180 / MD1 cell line. This result indicates that the antibodies 28A7, 30A1, and 38C8 provided by this invention specifically bind only to the CD180 protein on the cell membrane surface, exhibiting significant specificity.

[0076] Example 5

[0077] The CD180 antibody prepared in this invention specifically binds to CD180-positive acute myeloid leukemia tumor cells.

[0078] We incubated and stained OCI-AML5 cell lines with antibodies 28A7, 30A1, and 38C8, respectively, using a commercially available flow cytometry antibody against human CD180 (Biolegend) as a positive control. The results showed that all three antibodies (28A7, 30A1, and 38C8) could detect CD180 expression on the surface of acute myeloid leukemia tumor cells, and their ability to detect CD180 antigen was comparable to or better than that of the commercially available Biolegend antibody (see results below). Figure 5 (As shown).

[0079] We then used the 28A7 antibody to detect CD180 expression in other tumor cells (acute myeloid leukemia MV411 cells and non-Hodgkin lymphoma Raji cells). The results showed that various tumor cells expressed CD180 protein (see results below). Figure 5 As shown in the figure, this indicates that CD180 antibodies have great potential for application in the preparation of various tumor therapeutic drugs.

[0080] Example 6

[0081] CD180-CAR-T cell preparation

[0082] 1. The following CAR nucleic acid molecules were synthesized through whole-genome synthesis:

[0083] (1) CD8 signal peptide-CD180 scFv(VH-Linker-VL) CD8 hinge-CD28 transmembrane domain-CD28-CD3ζ, the specific nucleic acid sequence is shown in SEQ ID NO:34-36; the structural diagram is shown in Figure 6 As shown;

[0084] (2) CD8 signal peptide-CD180 scFv(VH-Linker-VL) CD8 hinge-CD8 transmembrane domain-41BB-CD3ζ, the specific nucleic acid sequence is shown in SEQ ID NO:37-39; the structural diagram is shown in Figure 6 As shown;

[0085] The above nucleic acid molecules were cloned into the lentiviral vector MSCV-T2A-NGFR to obtain plasmids 28A7-28z-CAR, 28A7-BBz-CAR, 30A1-28z-CAR, 30A1-BBz-CAR, 38C8-28z-CAR, and 38C8-BBz-CAR.

[0086] 2. After constructing and extracting the CAR plasmid, 293T17 cells were transduced using calcium phosphate transfection reagent to produce lentivirus. Viral supernatant was collected after 48 hours. Peripheral blood from healthy individuals was subjected to density gradient centrifugation to obtain PBMCs. T cells were then isolated and activated using anti-CD3 / CD28 sorting and activation magnetic beads. After 50 hours of activation, viral supernatant was added to T cells for centrifugation infection. The infection MOI value was 10-30. After the infection was completed, the medium was replaced with fresh 10% RIPM1640 medium and placed in an incubator for normal cell culture and expansion.

[0087] The CAR-T cells prepared in this embodiment are 28A7-28z-CAR-T, 28A7-BBz-CAR-T, 30A1-28z-CAR-T, 30A1-BBz-CAR-T, 38C8-28z-CAR-T, and 38C8-BBz-CAR-T.

[0088] Example 7

[0089] Effects of CD180-CAR-T cells on CD180-positive cancer cells

[0090] Jurkat cells were used as CD180 antigen negative controls, and AML5 cells and MV411 cells were used as CD180 antigen positive controls. 28A7-28z-CAR-T and 28A7-BBz-CAR-T cells were seeded in 96-well U-shaped plates at different effector-to-target ratios (0, 1, 3, 9). Specifically, 2E+4 tumor cells were placed in each well, and the corresponding number of untransduced T cells or CAR-T cells were added according to different effector-to-target ratios. RPMI 1640 medium containing 10% fetal bovine serum was used for culture in an incubator (37℃, 5% CO2). After 20 hours, fluorescein substrate was added, and tumor cell growth was monitored using in vivo imaging of small animals.

[0091] The results are as follows Figure 7 As shown, after 20 hours of co-culture, CD180-CAR-T cells effectively killed CD180 antigen-positive tumor cells, but had no significant killing effect on CD180 antigen-negative tumor cells. This result indicates that the killing activity of the CD180-CAR-T cells provided by this invention is strictly dependent on the presence of CD180 antigen on the surface of target cells.

[0092] Simultaneously, the supernatant from the co-incubation group with an effector-to-target ratio of 1 was collected for ELISA to detect the secretion of effector factors IFNγ and IL-2. The results are as follows: Figure 8 As shown, the CD180-CAR-T cells provided by this invention specifically secrete high levels of effector factors on CD180-positive tumor cells.

[0093] The above results indicate that the CD180-CAR-T cells provided by this invention have the ability to specifically kill CD180-positive acute myeloid leukemia cells and secrete high levels of IFNγ and IL-2 to combat CD180-positive cancer cells.

[0094] Example 8

[0095] Effects of CD180-CAR-T cells on a mouse model of tumor

[0096] CD180-positive AML tumor cells suspended in PBS were intravenously injected into the tail veins of female NSG mice in each group. After tumor formation was confirmed by small animal in vivo imaging on day 12, nine tumor-forming female NSG mice were selected and randomly divided into three groups of three mice each: a control group (untransduced T cells group, UTD, intravenous infusion of untransduced T cells in saline), a 28A7-28z-CAR-T group, and a 28A7-BBz-CAR-T group. The corresponding CAR-T cells were intravenously infused at a dose of 5E+6 cells / mouse. Each group was infused once, and the day of cell infusion was recorded as day 0 of the experiment. Tumor growth was then monitored by small animal in vivo imaging every week until the end of the experiment.

[0097] The results are as follows Figure 9 As shown, compared with the control group, the CD180-CAR-T cells provided by this invention significantly eliminated tumor cells in mice; all mice in the control group died on day 14 of the experiment, while mice in the 28A7-28z-CAR-T group and the 28A7-BBz-CAR-T group remained stable at the end of the experiment. This demonstrates strong anti-tumor activity and significantly prolonged survival in the mouse model.

[0098] Example 9

[0099] We systematically evaluated the expression of CD180 in various normal human tissues using immunohistochemistry (IHC) with the 28A7 antibody (B cells in the spleen express CD180, so spleen staining served as a positive control; staining in other tissues was negative). The results showed that CD180 is expressed at low levels or not at all in most normal tissues. Figure 10 Therefore, we believe that CAR-T cells targeting CD180 will not have significant adverse effects on normal tissues when applied in vivo.

[0100] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A monoclonal antibody targeting CD180, characterized in that, The heavy chain variable region of the antibody is 38C8-VH, and its amino acid sequence is shown in SEQ ID NO:3; the light chain variable region of the antibody is 38C8-VL, and its amino acid sequence is shown in SEQ ID NO:

6.

2. A monoclonal antibody targeting CD180, characterized in that, The heavy chain of the antibody is a 38C8-Heavy chain, and the amino acid sequence is shown in SEQ ID NO:

9. The light chain of the antibody is a 38C8-Light chain, and the amino acid sequence is shown in SEQ ID NO:

12.

3. The monoclonal antibody targeting CD180 according to claim 1, characterized in that, The 38C8-scFv single-chain variable fragment is composed of amino acid sequence 38C8-VH shown in SEQ ID NO:3 and amino acid sequence 38C8-VL shown in SEQ ID NO:

6. The amino acid sequence of 38C8-scFv is shown in SEQ ID NO:

15.

4. The use of the specific monoclonal antibody targeting human CD180 as described in any one of claims 1 to 3 in the preparation of a reagent for detecting CD180 antigen.

5. A chimeric antigen receptor targeting CD180, characterized in that, The chimeric antigen receptor from the N-terminus to the C-terminus includes: (i) 38C8-scFv targeting CD180; (ii) Transmembrane domains; (iii) At least one co-stimulatory domain; (iv) Activate the structural domain; The nucleotide sequence of the chimeric antigen receptor is shown in either SEQ ID NO:36 or SEQ ID NO:

39.

6. An engineered immune cell targeting CD180, characterized in that, The engineered immune cells express the chimeric antigen receptor targeting CD180 as described in claim 5.

7. The use of the CD180-targeting antibody as described in claim 1, the CD180-targeting chimeric antigen receptor as described in claim 5, and the CD180-targeting engineered immune cells as described in claim 6 in the preparation of immunotherapy drugs.

8. The use of the CD180-targeting antibody as described in claim 1, the CD180-targeting chimeric antigen receptor as described in claim 5, and the CD180-targeting engineered immune cells as described in claim 6 in the preparation of drugs for treating and / or treating CD180-related cancers or tumors.

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