Antigen epitope peptide of CD40 ligand, antibody and application

Abstract

The invention relates to the technical field of cellular immunotherapy, in particular to an antigen epitope peptide of a CD40 ligand, an antibody and application. The CD40LG target is mainly expressed by CD4 + T cells, so that self-killing is greatly reduced. In addition, as a novel target, the CD40LG can effectively improve tumor immune escape. The antibody provided by the invention can specifically recognize CD40LG, and has good affinity. The compound can be used for constructing cells or ADC drugs for adoptive immunotherapy.

Description

[0001] This application claims the priority of the Chinese patent application filed with the Chinese Patent Office on December 10, 2024, with application number 202411814396.3 and invention name “Antigenic epitope peptides, antibodies and applications of CD40 ligand”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present invention relates to the technical field of cell immunotherapy, and in particular to antigenic epitope peptides, antibodies and applications of CD40 ligands. Background Art

[0003] CAR-T and CAR-NK are two cutting-edge cellular immunotherapies that have shown great potential in the field of cancer treatment. They use the patient's own or donor's immune cells and genetically engineer them to recognize and kill tumor cells.

[0004] in:

[0005] CAR-T (Chimeric Antigen Receptor T-Cell) therapy is a T-cell-based immunotherapy, in which "T cells" are an important cell in the human immune system, responsible for identifying and attacking pathogens and abnormal cells. Through genetic engineering technology, a chimeric antigen receptor (CAR) is loaded on the surface of T cells, which can specifically recognize specific antigens on the surface of tumor cells, thereby triggering the activation of T cells and killing these tumor cells. The main construction steps include: 1. Cell extraction: extract T cells from the patient's peripheral blood. 2. Genetic modification: introduce the gene encoding CAR into T cells through viral vectors or non-viral methods to make them express CAR. 3. In vitro expansion: The modified T cells are expanded in vitro to increase the number. 4. Reinfusion into the body: After chemotherapy or lymphocyte clearance, the patient receives these modified T cells, and the CAR-T cells begin to recognize and attack tumor cells.

[0006] CAR-NK (Chimeric Antigen Receptor Natural Killer Cell) therapy is an immunotherapy based on natural killer (NK) cells. NK cells are part of the innate immune system and have a natural ability to kill tumor cells and virus-infected cells. Unlike T cells, NK cells do not rely on antigen presentation and recognition of MHC molecules, and are therefore able to attack malignant cells extensively. Through genetic engineering, CAR chimeric antigen receptors are introduced into NK cells, allowing them to more specifically recognize and kill tumor cells. The main construction steps of CAR-NK are basically similar to the CAR-T construction process, with the main difference being the source of cells: NK cells can be obtained from a variety of sources, including peripheral blood, umbilical cord blood, and induced pluripotent stem cells (iPSC).

[0007] CAR-T or CAR-NK are used to treat T-cell hematological diseases because these therapies have shown significant efficacy in targeting specific types of hematological malignancies, especially when traditional therapies have limited efficacy. However, this therapy also has certain disadvantages, such as: ① Since the chimeric antigen receptors expressed on the surface of CAR-T or CAR-NK cells may recognize and bind to antigens on the surface of similar cells, these cells may kill each other (suicide killing), thereby reducing the survival rate and proliferation ability of the treated cells and affecting the treatment effect. ② The antigens targeted by CAR-T or CAR-NK are not only present in tumor cells, but also distributed on the surface of normal T cells. CAR-T or CAR-NK treatment may lead to a large reduction in normal T cells, thereby causing immune deficiency and increasing the risk of infection in patients. ③ Tumor cells may downregulate or lose the expression of targeted antigens, or escape recognition and killing of immune cells through other mechanisms, resulting in unsustainable treatment effects or recurrence. At present, the main therapeutic targets for T-cell hematological diseases are CD5, CD7, CD30, etc. With the advancement of CAR-T / CAR-NK treatment, tumor cells will undergo antigen modulation, downregulate or even not express the above targets. ④The manufacturing process of CAR-T and CAR-NK cell therapy is complex, involving multiple steps such as cell extraction, gene modification, and amplification. It is costly and has strict requirements on the production environment and technology, which limits its widespread application. The in vivo construction scheme involved in this scheme can significantly reduce the above complex survival process, thereby reducing costs.

[0008] CD40L (or CD40LG) is the receptor of CD40. CD40 interacts with CD40L, thus playing a key role in the regulation of immune responses. The binding of CD40L to CD40 triggers the activation of the CD40 pathway, and the activation of the CD40 pathway upregulates co-stimulatory molecules such as CD80 and CD86. Previous studies have shown that the expression of CD40LG in all normal tissues is significantly increased in T lymphomas, and the killing and destruction of normal tissues can be reduced by adjusting affinity. At the same time, CD40LG is mainly expressed on CD4+T cells, and there is less damage to CD8+T cells, which are mainly responsible for killing, which can reduce the risk of infection in patients. Therefore, using CD40LG as a new target is expected to effectively improve tumor immune escape. However, there is currently a lack of antibodies with good affinity for CD40L. Summary of the invention

[0009] In view of this, the technical problem to be solved by the present invention is to provide antigen epitope peptides, antibodies and applications of CD40 ligand.

[0010] The antigenic epitope peptide of the CD40 ligand provided by the present invention has an amino acid sequence as shown in SEQ ID NO: 13. Alternatively, it has a sequence in which one or more amino acids are substituted, deleted, added and / or replaced on the basis of the amino acid sequence as shown above; or has a sequence with a homology of more than 80% with the amino acid sequence as shown in any of the above items.

[0011] The epitope peptide provided by the present invention is obtained through screening and optimization. Compared with fragments of other peptide sequences, the antibody against CD40 ligand prepared by immunizing animals with the antigenic epitope peptide of the amino acid sequence shown in SEQ ID NO: 13 has better immunogenicity and good affinity with CD40 ligand.

[0012] The present invention also provides an immunogen, which comprises the antigen epitope peptide and auxiliary protein as described above.

[0013] In the present invention, the auxiliary protein is BSA, KLH or OVA, which is not limited in the present invention. KLH has good immunogenicity after being connected with the aforementioned epitope peptide. Proteins with KLH located at the N-terminus and the epitope peptide located at the C-terminus are more suitable as immunogens.

[0014] Furthermore, the present invention also provides an antibody to CD40 ligand, comprising:

[0015] The amino acid sequences of VHCDR1, VHCDR2 and VHCDR3 are shown in SEQ ID NOs: 1 to 3,

[0016] and VLCDR1, VLCDR2 and VLCDR3 with amino acid sequences as shown in SEQ ID NOs: 4-6.

[0017] Specific:

[0018] The amino acid sequence of VHCDR1 is GIDLSX1YG, where X1 is Q or S;

[0019] The amino acid sequence of VHCDR2 is X2X3TGGX4X5, wherein X2 is V or I, X3 is T or S, X4 is I, R or Y, and X5 is T or V;

[0020] The amino acid sequence of VHCDR3 is ARGNIDYLX6L, where X6 is K or N;

[0021] The amino acid sequence of VLCDR1 is KX7X8YKNNW, where X7 is S or T, and X8 is V or I;

[0022] The amino acid sequence of VLCDR2 is EX9S, where X9 is A or T;

[0023] The amino acid sequence of VLCDR3 is AGGYX10X11DSDDX12, wherein X10 is S or N, X11 is S or G, and X12 is G or S.

[0024] In some embodiments, the amino acid sequences of the heavy chain CDR1-3 of the CD40 ligand antibody are: GIDLSQYG, VTTGGIT, ARGNIDYLKL, and the amino acid sequences of the light chain CDR1-3 are: KSVYKNNW, EAS, AGGYSSDSDDG;

[0025] In some embodiments, the amino acid sequences of the heavy chain CDR1-3 of the CD40 ligand antibody are: GIDLSSYG, ISTGGRV, ARGNIDYLNL, and the amino acid sequences of the light chain CDR1-3 are: KSIYKNNW, EAS, AGGYNSDSDDS;

[0026] In some embodiments, the amino acid sequences of the heavy chain CDR1-3 of the antibody of the CD40 ligand are: GIDLSSYG, ISTGGYT, ARGNIDYLNL, and the amino acid sequences of the light chain CDR1-3 are: KTVYKNNW, ETS, AGGYSGDSDDS.

[0027] In an embodiment of the present invention, the heavy chain variable region of the antibody has an amino acid sequence as shown in any one of SEQ ID NOs: 7 to 9; or, it has a sequence in which one or more amino acids are substituted, deleted, added and / or replaced on the basis of the amino acid sequence as shown above; or it has a sequence with a homology of more than 80% with the amino acid sequence as shown in any of the above items.

[0028] In the embodiment of the present invention, the light chain variable region of the antibody has an amino acid sequence as shown in any one of SEQ ID NOs: 10 to 12. Alternatively, it has a sequence in which one or more amino acids are substituted, deleted, added and / or replaced on the basis of the amino acid sequence shown above; or has a sequence with a homology of more than 80% with the amino acid sequence shown in any of the above items.

[0029] In some embodiments, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:10.

[0030] In other embodiments, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:11.

[0031] In other embodiments, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:7, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:12.

[0032] In other embodiments, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:8, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:10.

[0033] In other embodiments, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:8, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:11.

[0034] In other embodiments, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:8, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:12.

[0035] In other embodiments, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:9, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:10.

[0036] In other embodiments, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:9, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:11.

[0037] In other embodiments, the amino acid sequence of the heavy chain variable region of the antibody is shown in SEQ ID NO:9, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:12.

[0038] In an embodiment of the present invention, the antibody provided by the present invention is a single-chain antibody. The present invention does not limit the connection order of the heavy chain and the light chain in the single-chain antibody. For example, the N-terminus can be a light chain or a heavy chain. The light chain and the heavy chain are connected by a linker, and the present invention does not limit the linker connecting the two fragments. In a specific embodiment, the linker connecting the heavy chain variable region and the light chain variable region is (G4S)3.

[0039] Furthermore, the present invention also provides a chimeric antigen receptor, which comprises an extracellular domain, a transmembrane domain and an intracellular domain, wherein the antigen binding domain of the extracellular domain comprises the antibody as described above.

[0040] As a feasibility case, the transmembrane domain in the chimeric antigen receptor is CD8, CD3z or CD28;

[0041] As a feasibility case, the intracellular domain in the chimeric antigen receptor is CD3 or at least one of the following molecules: CD28, ICOS, 4-1BB, OX40, CD27 or 2B4. For example, the intracellular domain is CD3 and one or two of CD28, ICOS, 4-1BB, OX40, CD27 or 2B4. Or the intracellular domain is a fourth-generation gene-edited CAR.

[0042] As a feasibility example, the extracellular domain in the chimeric antigen receptor is an antibody to the CD40 ligand as described above.

[0043] As a feasible example, the chimeric antigen receptor also includes a hinge region CD8, Fc, IgG4, IgG1, DAP12 or CD28.

[0044] Furthermore, the present invention also provides a nucleic acid, which is any one of the following:

[0045] 1), a nucleic acid encoding the antibody as described above;

[0046] II), a nucleic acid encoding the antigenic epitope peptide as described above;

[0047] III), a nucleic acid encoding the immunogen as described above;

[0048] IV), a nucleic acid encoding the chimeric antigen receptor as described above.

[0049] The nucleic acid provided by the present invention is DNA or RNA, which can be used to construct a plasmid vector for the expression of encoded proteins, and can also be used as a baculovirus, adenovirus, adeno-associated virus or lentivirus for transcriptional expression.

[0050] Furthermore, the present invention also provides an expression unit, which comprises a promoter and the nucleic acid as described above.

[0051] In the present invention, the promoter is selected from CAG promoter, CMV promoter, EF1α promoter, PGK promoter, T7 promoter, SV40 promoter, SFFV promoter, MSCV promoter, UBC promoter, TRE promoter, Rh2 promoter. The expression unit also includes a terminator, which is selected from a simple terminator and a p-dependent terminator. In addition, the expression unit may also include an enhancer, a signal peptide sequence, a tailing signal, a kozak element, a restriction site and / or an intron.

[0052] Furthermore, the present invention also provides a plasmid vector, which comprises the aforementioned nucleic acid and / or the aforementioned expression unit.

[0053] In the present invention, the backbone vector of the plasmid vector is a mammalian cell expression vector or a bacterial expression vector, which is not limited in the present invention.

[0054] Furthermore, the present invention also provides a cell, which expresses the antibody as described above, and / or the chimeric antigen receptor as described above.

[0055] In some feasible cases, the cell is a T cell or a NK cell, for example, a T cell or a NK cell expressing the aforementioned chimeric antigen receptor.

[0056] The method for constructing T cells or NK cells of the present invention comprises: transfecting T cells or NK cells with a viral vector containing nucleic acid.

[0057] In some feasible cases, the cell is Escherichia coli or yeast. For example, it is an Escherichia coli or yeast expressing the aforementioned antibody or chimeric antigen receptor.

[0058] The method for constructing the Escherichia coli of the present invention comprises transforming a plasmid vector containing the nucleic acid as described above into a host.

[0059] Furthermore, the present invention also provides a labeled antibody formed by connecting the antibody to a label; the label is a chemical label or a biological label. In the present invention, the connection between the antibody and the label can be via an amide bond or via a streptavidin-biotin system, which is not limited in the present invention.

[0060] In some embodiments, the marker is: a fluorescent indicator, a chemiluminescent indicator, an isotope, a colloid indicator, biotin, avidin or an enzyme label.

[0061] In some specific embodiments, the fluorescent indicator is selected from one or more of AF350, AF488, AF532, AF546, AF555, AF568, AF594, AF633, AF647, AF660, AF680, FITC, TRITC, RB200, phycoerythrin, APC, Cy5, OregonGreen 488, Pacific Blue dye, Pacific Orange dye, Texas Red or PerCP dye.

[0062] In some specific embodiments, the chemiluminescent indicator is selected from one or more of acridinium esters, acridinium sulfonamides and their derivatives, luminol, isoluminol, isoluminol isothiocyanate and its derivatives, N-(4-aminobutyl)-N-ethylisoluminol, 4,5-diaminophthalhydrazide or aminobutylethylphthalhydrazide.

[0063] In some specific embodiments, the isotope is selected from 125 I.1 31 I. 124 I. 3H. 14 C. 111 In, 89 Zr or 32 One or more of P.

[0064] In some specific embodiments, the colloidal indicator is selected from one or more of colloidal gold, colloidal carbon or colloidal selenium.

[0065] In some specific embodiments, the enzyme label is selected from one or more of horseradish peroxidase, alkaline phosphatase, β-galactosidase, peroxidase-anti-peroxidase bridge, alkaline phosphatase-anti-alkaline phosphatase bridge, β-galactosidase-anti-β-galactosidase bridge.

[0066] Furthermore, the present invention also provides a conjugate formed by connecting the antibody to a medium; the medium is colloidal gold, an ELISA plate, magnetic beads or latex microspheres. In the present invention, the connection between the antibody and the medium can be connected via an amide bond or via a streptavidin-biotin system, which is not limited by the present invention.

[0067] Furthermore, the present invention also provides an antibody-bound drug formed by connecting the antibody to a drug; the drug includes a microtubule inhibitor, a DNA damaging agent, a DNA alkylating agent or a topoisomerase inhibitor.

[0068] The microtubule inhibitors include, but are not limited to, maytansine derivatives (such as DM1, DM4) or auribactin derivatives (such as MMAE (monomethyl auristatin E) or MMAF (monomethyl auristatin F)).

[0069] The DNA damaging agents include, but are not limited to, calicheamicin or pyrrolobenzodihydrooxazines (PBDs).

[0070] The DNA alkylating agents include but are not limited to Deruxtecan.

[0071] The topoisomerase inhibitors include but are not limited to SN-38.

[0072] In the present invention, the antibody and the drug may be connected via an amide bond or via a streptavidin-biotin system, which is not limited in the present invention.

[0073] Furthermore, the present invention also provides the use of the cell and / or the antibody-binding drug in the preparation of a drug for treating tumors.

[0074] The tumor described in the present invention is a tumor carrying CD40LG on the cell surface, including but not limited to peripheral T-cell lymphoma, AITL (angioimmunoblastic T-cell lymphoma), ALCL (anaplastic large cell lymphoma) or ATLL (adult T-cell leukemia lymphoma).

[0075] Furthermore, the present invention also provides a drug, which includes the cell and / or the antibody-bound drug.

[0076] In the present invention, the drug also includes pharmaceutically acceptable excipients. In the present invention, the dosage form of the drug includes but is not limited to oral preparations, injections, aerosols or suppositories. The oral preparations include but are not limited to tablets, capsules, pills, oral liquids or dispersants. The injections include but are not limited to injection liquids or powders for injection.

[0077] The drugs described in the present invention also include other anti-tumor drugs, for example, the anti-tumor drugs include but are not limited to chemotherapy drugs, targeted drugs, immunotherapy drugs or traditional Chinese medicine. As a feasibility case, the chemotherapy drugs include: paclitaxel, docetaxel, vinorelbine, gemcitabine, cisplatin, carboplatin, nedaplatin, 5-fluorouracil, capecitabine, S-100 capsules, irinotecan or amrubicin. The targeted drugs include: gefitinib, erlotinib, afatinib, osimertinib, crizotinib, sunitinib, sorafenib, lenvatinib mesylate, apatinib mesylate, bevacizumab, cetuximab or anlotinib. The immunotherapy drugs include: pembrolizumab, nivolumab, toripalimab, durvalumab, sintilimab or atezolizumab. The Chinese medicine includes: ganoderma lucidum, yew, toad, cordyceps sinensis, scorpion, American ginseng, astragalus, angelica, panax notoginseng or gynostemma pentaphyllum.

[0078] Furthermore, the present invention also provides a method for treating tumors, which comprises administering the above-mentioned drug. The administration of the drug or pharmaceutical composition includes, but is not limited to, oral administration, spray inhalation, buccal administration, nasal administration, vaginal administration, rectal administration, topical administration, parenteral administration such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal and intracranial injection or infusion, or administration via an explanted reservoir. Among them, oral, intraperitoneal or intravenous administration is preferred.

[0079] Furthermore, the present invention also provides the use of the antibody, the labeled antibody or the conjugate in preparing a reagent for detecting CD40 ligand.

[0080] Furthermore, the present invention also provides a detection reagent for CD40 ligand, the raw materials of which include: the antibody, the labeled antibody or the conjugate.

[0081] Furthermore, the present invention also provides a method for detecting CD40 ligand, which comprises detecting a sample with the detection reagent as described above.

[0082] In the present invention, the detection method is an immunological detection method, including but not limited to ELISA, flow cytometry and / or immunohistochemistry. The sample includes but is not limited to cells, blood, slices or tissue homogenates. The detection method of the present invention can be for diagnostic or therapeutic purposes, or for non-diagnostic or therapeutic purposes, and the present invention does not limit this. The sample includes but is not limited to cells, blood, slices or tissue homogenates.

[0083] The present invention provides an antigen epitope peptide and an antibody of CD40 ligand, which have at least one of the following advantages:

[0084] ① The CD40LG target used in the present invention is mainly expressed by CD4+T cells, while the T cells used in CAR-T construction are mainly CD8+T cells, and the expression of CD40LG on the surface of NK cells is also low, thus greatly reducing self-killing. Moreover, CD40LG as a new target can effectively improve tumor immune escape.

[0085] ②Through large-sample transcriptomic sequencing analysis of patient T lymphoma tumor tissue, it was found that compared with all normal tissues in the TCGA database, the expression of CD40LG was significantly increased in T lymphoma, and the killing and damage to normal tissues could be reduced by adjusting affinity. At the same time, CD40LG was mainly expressed on CD4+T cells, and had less damage to CD8+T cells, which were mainly responsible for killing, thereby reducing the risk of infection in patients.

[0086] ③ The antibody provided by the present invention can specifically recognize CD40LG and has good affinity. It can be used to construct CAR-T cells or CAR-NK cells.

[0087] ④ The in vivo construction scheme involved in the present invention (directly transfecting cells with viral vectors) significantly reduces the above complex production process, thereby reducing costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Figure 1 Flow cytometry showed that specific antibodies against KLH-KDIMLNKEETKKENSFE immune peptide were present in the sera of both rabbits;

[0089] Figure 2 The results of flow cytometry detection of specific antibodies of 33 monoclonal B cells against KLH-KDIMLNKEETKKENSFE immune peptide are shown;

[0090] Figure 3 The ELISA and flow cytometry results of 10 monoclonal cell lines are shown;

[0091] Figure 4 The SPR affinity of each antibody to the purified CD40LG protein is shown;

[0092] Figure 5 CAR-T cell self-killing detection;

[0093] Figure 6 Shows the detection of CAR-T cells' ability to kill tumors;

[0094] Figure 7 Shows the expression of CD40L in CD4 and CD8+ T cells

[0095] Figure 8 Shows the effect of CD3+CAR-CD40L on the killing effect of T cells on tumors. DETAILED DESCRIPTION

[0096] The present invention provides antigen epitope peptides, antibodies and applications of CD40 ligands. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve them. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0097] Unless otherwise defined herein, scientific and technical terms related to the present invention shall have the meanings understood by those of ordinary skill in the art.

[0098] In this application, "at least one" means one or more, "more than one" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single or plural items.

[0099] The terms "include", "comprising" and "having" are used interchangeably herein and are intended to indicate the inclusiveness of the solution, meaning that the solution may have other elements in addition to the listed elements. At the same time, it should be understood that the use of "include", "comprising" and "having" descriptions herein also provides a "consisting of..." solution.

[0100] The term "and / or" as used herein includes the meanings of "and", "or" and "all or any other combination of elements linked by the corresponding term".

[0101] The term "identity" herein can be calculated in the following manner: to determine the "identity" percentage of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., spaces can be introduced in one or both of the first and second amino acid sequences or nucleic acid sequences for optimal comparison or non-homologous sequences can be discarded for comparison purposes). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide at the corresponding position in the second sequence, the molecules are identical at this position. The percent identity between the two sequences varies with the number of identical positions shared by the sequences, taking into account the number of spaces that need to be introduced for optimal comparison of the two sequences and the length of each space.

[0102] Mathematical algorithms can be used to compare sequences between two sequences and calculate percent identity. For example, the Needlema and Wunsch ((1970) J. Mol. Biol. 48: 444-453) algorithm (available at www.gcg.com) that has been integrated into the GAP program of the GCG software package is used, using a Blossum62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6 to determine the percent identity between two amino acid sequences. For another example, the GAP program in the GCG software package (available at www.gcg.com) is used, using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70 or 80 and a length weight of 1, 2, 3, 4, 5 or 6 to determine the percent identity between two nucleotide sequences. A particularly preferred parameter set (and the one that should be used unless otherwise stated) is the Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0103] The percent identity between two amino acid or nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller, ((1989) CABIOS, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weighted remainder table, a gap length penalty of 12, and a gap penalty of 4.

[0104] In the present invention, the identity of 80% or more means an identity greater than 85%, or greater than 90%, or greater than 95%, or greater than 96%, or greater than 97%, or greater than 98%, or greater than 98%, or greater than 99%, or greater than 99.5%, or greater than 99.8%, or greater than 99.9%.

[0105] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0106] The heavy chain and light chain sequences of the main antibodies involved in the embodiments of the present invention are as follows, wherein: Shown is a signal peptide (SEQ ID NO: 13), lowercase letters indicate CDR regions, and uppercase letters indicate FR regions.

[0107]

[0108] Table 1 CDR region sequences of various antibodies

[0109]

[0110]

[0111]

[0112] The present invention will be further described below in conjunction with embodiments:

[0113] Example

[0114] 1. scFV screening process:

[0115] Background screening before immunization: 1 ml was collected from each of the 4 rabbits for background detection. Conventional ELISA test: 2 rabbits with lower background were selected for subsequent immunization.

[0116] 2. Immune stage:

[0117] KLH-KDIMLNKEETKKENSFE was used to immunize the two rabbits with lower background selected above for a total of 5 times. For the serum of the two rabbits that had been immunized five times, 2 ml / rabbit was taken and purified with protein A antibody. After purification, the antibody was subjected to routine ELISA testing: ELISA testing was performed on the original biotin-KDIMLNKEETKKENSFE, and a rabbit with high antibody titer was selected. At the same time, flow cytometry was performed to detect that there was a significant transition in the CD40LG positive cell line, and 33 were selected for subsequent experiments ( Figure 1 ).

[0118] 3. B cell sorting stage:

[0119] The spleen of the rabbits selected above was taken to isolate single B cells and then amplified by 96-well plate plating. The supernatant of single B cells was taken for ELISA antibody titer detection and flow cytometry to screen positive clones ( Figure 2 ).

[0120] 4. Linear expression stage:

[0121] According to the results of ELISA and flow cytometry in step 3, the top 10 monoclonal B cell lines were selected for sequencing, and plasmids were constructed based on the sequencing results and linearly expressed in 293T cells. The supernatant was taken for peptide coating ELISA, CD40LG protein coating ELISA and flow cytometry ( Figure 3 ), and four monoclonal clones (4B8, 6C11, 6D6 and 7GB) that showed significant binding in the above three groups of experiments were selected for subsequent experiments.

[0122] 5. Construction of complete antibodies:

[0123] The above four monoclonal clones were used to construct complete IgG1 antibodies. Each clone was divided into two clones, VLVH and VHVL, according to the linking mode of light and heavy chains:

[0124] The nucleic acid sequences are recorded as:

[0125] 4B8_VH_(G4S)3_VL, 4B8_VL_(G4S)3_VH, 6C11_VH_(G4S)3_VL, 6C11_VL_(G4S)3_VH, 6D6_VH_(G4S)3_VL, 6D6_VL_(G4S)3_VH.

[0126] The obtained antibodies were sorted by SPR affinity with the purified CD40LG protein. The specific affinities are shown in Tables 1 and Figure 4 :

[0127] Table 2

[0128] Injection variables Analyte 1 Solution 1:1binding ka kd KD(M) 4B8 VHVL 9.03E+04 7.96E-03 8.82E-08 4B8 VLVH 1.55E+05 6.58E-03 4.25E-08 6C11VHVL 1.76E+05 1.29E-02 7.34E-08 6C11VLVH 9.13E+04 7.75E-03 8.49E-08 6D6 VHVL 1.03E+05 4.95E-03 4.79E-08 6D6 VLVH 8.46E+04 4.75E-03 5.62E-08 7GB VHVL 5.53E+04 3.99E-03 7.20E-08 7GB VLVH 1.15E+05 3.46E-03 3.00E-08

[0129] The results showed that the VHVL and VLVH of 7GB had no obvious binding, so this clone was excluded. The remaining clones had significant binding ability. CAR plasmids and viruses were constructed to detect tumor killing activity in vivo and in vitro.

[0130] 6. In vitro CAR-T construction process

[0131] 6.1 T cell isolation

[0132] Steps: T cells are separated from the patient's peripheral blood by leukapheresis. The commonly used equipment is a cell separator, which can separate white blood cells from the blood by centrifugation.

[0133] Purpose: To obtain enriched T cells for subsequent gene editing and culture.

[0134] 6.2 T cell activation

[0135] Steps: Activate isolated T cells under in vitro culture conditions by using anti-CD3 antibodies and anti-CD28 antibodies or other co-stimulatory molecules. CD3 antibodies mimic the natural stimulation of the T cell receptor (TCR), and CD28 provides co-stimulatory signals to synergistically promote T cell activation and proliferation.

[0136] Purpose: To make T cells move from a quiescent state to a proliferative and activated state, ready for genetic modification.

[0137] 6.3 Gene transduction (introduction of CAR gene)

[0138] Steps: Use a viral vector (such as lentivirus or retrovirus) to introduce the designed CAR gene into T cells. The CAR gene encodes a chimeric antigen receptor that can specifically recognize antigens on the surface of tumors. After transduction, T cells will express this chimeric receptor, enabling them to recognize and kill specific tumor cells.

[0139] Methods: ① The commonly used transduction method is viral transduction (lentiviral or retroviral vector). Viral vectors have efficient gene introduction capabilities and can stably integrate the CAR gene into the T cell genome, thereby achieving long-term expression.

[0140] ②Nanoparticle carrier: Nanoparticles are another delivery system that can encapsulate and protect CAR plasmids and deliver them to target cells through the blood system. The surface of nanoparticles can be modified with targeting molecules to increase selectivity for T cells. Common types of nanoparticles include:

[0141] ③Lipid nanoparticles (LNP): These nanoparticles are widely used to deliver mRNA and plasmid DNA and have been successfully used in COVID-19 vaccines. By encapsulating the plasmid-encoded CAR in lipid nanoparticles, they can be injected into the body, allowing the plasmid to directly enter T cells.

[0142] ④Polymer nanoparticles: These particles are based on biocompatible polymers (such as PLA or PLGA) and can carry genetic materials to T cells through electrostatic adsorption or chemical bonding.

[0143] Objective: To enable T cells to express CAR receptors that specifically recognize tumor antigens through gene editing.

[0144] 6.4 T cell expansion

[0145] Steps: The successfully transduced CAR-T cells are cultured in vitro, and cytokines (such as IL-2) are used to promote the massive proliferation of CAR-T cells. The culture environment is usually a closed sterile system that can provide conditions suitable for the growth of T cells.

[0146] 6.5 Quality Inspection

[0147] Steps: During and after the amplification process, the number of viable cells of the generated CAR-T cells was detected. CAR-CD40L cells, WT CD3+T cells, and CAR-CD19 cells constructed as above were cultured at the same time, and the number of cells was detected on the 4th, 8th, and 12th days, respectively. The results are as follows: Figure 5 : The results showed that the proliferation ability of CAR-CD40L was about 25% compared with WT CD3+T cells and CAR-CD19 inhibition rate; while the previous CAR-CD7 self-killing rate was above 80%, indicating that the CAR-CD40L constructed as previously would not have its function significantly weakened due to self-killing.

[0148] 7. In vitro detection of tumor killing

[0149] Successfully constructed CAR-T cells need to undergo functional testing to verify whether they can effectively kill targeted tumor cells. In vitro tumor killing tests are usually performed in the laboratory, by co-culturing tumor cells and CAR-T cells to observe the killing effect of CAR-T cells on tumors.

[0150] 7.1 Target cell preparation (tumor cell lines)

[0151] Steps: Select appropriate tumor cell lines (Jurkat cells, karpas299 cells and H9 cells), which should express the tumor antigens targeted by CAR design (such as CD40L-positive tumor cells). These tumor cells can be primary tumor cells derived from patients or common commercial tumor cell lines.

[0152] Purpose: To serve as a target for CAR-T cells and simulate the tumor cell environment in vivo.

[0153] 7.2 Cell co-culture

[0154] Steps: Co-culture the constructed CAR-T cells and tumor cells at different effector to target ratios (Effector to Target ratio, Eratio). Usually multiple E ratios are set, such as 1:1, 5:1, 10:1, etc., to observe the killing effect of CAR-T cells under different conditions.

[0155] Objective: To simulate the interaction between CAR-T cells and tumor cells in vivo and observe their killing efficiency.

[0156] 7.3 Tumor cell survival rate detection

[0157] Methods: Various methods can be used to evaluate tumor cell survival and CAR-T cell killing efficiency. Common methods include:

[0158] 7.3.1 Flow Cytometry: Cells are labeled with dyes (such as 7-AAD or Annexin V / PI staining) and the total number of viable cells is detected by flow cytometry.

[0159] Objective: The above method can be used to quantitatively evaluate the tumor killing efficiency of CAR-T cells in vitro. Figure 6 As shown, compared with CD3+ T cells and CAR-CD19, the CAR-CD40L prepared as above has a better killing effect on T cell tumors, such as Figures 7-8 As shown, the expression level of CD40L prepared as above is higher in CD4 than in CD8+T cells, but the killing effect of CD3+CAR-CD40L on T cell tumors is not weakened.

[0160] 7.4 Cytokine analysis

[0161] Steps: Detect cytokines (such as CD4+T, CD8+T) secreted by CAR-T cells in the process of killing tumor cells. Commonly used methods are enzyme-linked immunosorbent assay (ELISA) or flow cytometry combined with cytokine capture reagents.

[0162] Objective: To evaluate the activation degree and functional effect of CAR-T cells by the level of cytokine release. CAR-T cells usually secrete a large amount of cytokines when attacking tumor cells, which is also an indicator of their activity.

[0163] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An antibody to CD40 ligand, comprising: The amino acid sequences of VHCDR1, VHCDR2 and VHCDR3 are shown in SEQ ID NOs: 1 to 3, and VLCDR1, VLCDR2 and VLCDR3 with amino acid sequences as shown in SEQ ID NOs: 4-6.

2. The antibody according to claim 1, characterized in that The amino acid sequences of its heavy chain CDR1-3 are: GIDLSQYG, VTTGGIT, ARGNIDYLKL, and the amino acid sequences of its light chain CDR1-3 are: KSVYKNNW, EAS, AGGYSSDSDDG; or the amino acid sequences of its heavy chain CDR1-3 are GIDLSSYG, ISTGGRV, ARGNIDYLNL, and the amino acid sequences of its light chain CDR1-3 are KSIYKNNW, EAS, AGGYNSDSDDS; Or the amino acid sequences of its heavy chain CDR1-3 are: GIDLSSYG, ISTGGYT, ARGNIDYLNL, and the amino acid sequences of its light chain CDR1-3 are: KTVYKNNW, ETS, AGGYSGDSDDS.

3. The antibody according to claim 1 or 2, characterized in that Its heavy chain variable region has an amino acid sequence as shown in any one of SEQ ID NOs: 7 to 9; The light chain variable region thereof has an amino acid sequence as shown in any one of SEQ ID NOs: 10-12.

4. The antibody according to any one of claims 1 to 3, characterized in that It is a single-chain antibody, and the linker connecting the heavy chain variable region and the light chain variable region is (G4S)3.

5. An antigenic epitope peptide of CD40 ligand, whose amino acid sequence is shown in SEQ ID NO:

13.

6. An immunogen for preparing the antibody according to any one of claims 1 to 4, comprising the antigen epitope peptide according to claim 5 and KLH protein.

7. A chimeric antigen receptor comprising an extracellular domain, a transmembrane domain and an intracellular domain, wherein the antigen binding domain of the extracellular domain comprises the antibody according to any one of claims 1 to 4.

8. A biological material comprising at least one of the following: A), a nucleic acid encoding the antibody according to any one of claims 1 to 4; or a nucleic acid encoding the antigenic epitope peptide according to claim 5; or a nucleic acid encoding the immunogen according to claim 6; or a nucleic acid encoding the chimeric antigen receptor according to claim 7; B), an expression unit comprising a promoter and the nucleic acid described in A); C), a plasmid vector comprising the nucleic acid described in A) and / or the expression unit described in B); D), a cell expressing the antibody described in A), and / or the chimeric antigen receptor described in B); E), a labeled antibody formed by linking the antibody according to any one of claims 1 to 4 with a label; the label is a chemical label or a biological label; F) An antibody-drug conjugated to a drug formed by linking the antibody according to any one of claims 1 to 4.

9. The biomaterial according to claim 8, characterized in that The cells are T cells or NK cells.

10. A drug comprising the cell and / or antibody-bound drug according to claim 8.

11. Use of the cell according to claim 8 or 9 and / or the antibody-conjugated drug according to claim 8 in the preparation of a drug for treating tumors.

12. A drug comprising the cell according to claim 8 or 9 and / or the antibody-conjugated drug according to claim 8.

13. Use of the antibody according to any one of claims 1 to 4 or the labeled antibody according to claim 8 in the preparation of a reagent for detecting CD40 ligand.

14. A detection reagent for CD40 ligand, characterized in that: The raw materials include: the antibody according to any one of claims 1 to 4 or the labeled antibody according to claim 8.

15. A method for detecting CD40 ligand for non-therapeutic and / or diagnostic purposes, comprising detecting a sample with the detection reagent according to claim 14.

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