Fusion protein and application thereof

Abstract

The invention provides a fusion protein and application thereof. The fusion protein comprises: at least three CD40L extracellular fragments; wherein the number of the CD40L extracellular segments is an integer larger than or equal to 3, and the C end and the N end of each CD40L extracellular segment are sequentially connected in series. The fusion protein disclosed by the invention can be combined with CD40, has strong activity of promoting the activation and proliferation of antigen presenting cells, further can effectively promote immune response and improve the anti-cancer capability of an autoimmune system, and has good clinical value and drug development value.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular, relates to fusion proteins and applications thereof. Background Art

[0002] Cancer is a major disease that affects human survival and development. According to the latest data, there are approximately 19 million new cancer cases and 10 million cancer deaths each year worldwide, and both the incidence and mortality rates are on the rise. In addition to surgical resection, traditional cancer treatments such as chemotherapy and radiotherapy have significant side effects and are prone to recurrence. In recent years, the development of agonist antibody drugs targeting co-stimulatory checkpoints (CD27, CD40, OX40, GITR, ICOS, etc.) has been highly sought after. Among them, CD40 has been repeatedly reported to effectively destroy cancer cells without harming healthy cells, and is therefore becoming a hot topic in antibody drug research and development.

[0003] The CD40 receptor (also known as TNFRSF5) and its ligand CD40L (also known as CD154) belong to the TNF / TNFR family. CD40 is a type I membrane glycoprotein that was originally identified as a surface marker of bladder cancer cells and B cells. It was later found to be expressed on antigen-presenting cells (APCs), including subsets of monocytes, macrophages, and dendritic cells. CD40 is also expressed by B cells and platelets, as well as some non-hematopoietic cell types such as fibroblasts, endothelial cells, and smooth muscle cells. Even some tumor cells express CD40.

[0004] CD40L, the ligand of CD40, is a 39 kDa type II membrane glycoprotein expressed on a variety of cell types, including activated CD4+ T cells, activated B cells, memory CD8 T cells, activated natural killer cells, granulocytes, endothelial cells, smooth muscle cells, macrophages, and activated platelets.

[0005] CD40-CD40L is a pair of co-stimulatory molecules. After interacting on the cell surface, they promote intracellular signaling by recruiting TNFR-associated factors (TRAFs) within the cell membrane, thereby activating various signaling pathways, such as the canonical and non-canonical nuclear factor-κB pathway, mitogen-activated protein kinase, phosphatidylinositol 3-kinase (PI3K), and phospholipase Cγ pathways. These pathways participate in both humoral and cellular immune responses, playing a key role in B cell activation, proliferation, and differentiation, antibody production, and Ig class switching, and in T cell activation and the secretion of effector cytokines. Numerous studies have demonstrated that CD40-CD40L plays a crucial role in CD8+ cytotoxic T lymphocyte (CTL) function during immune responses and is essential for adaptive immune responses.

[0006] CD40-targeted drugs with good CD40 binding activity and strong antigen-presenting cell activation and proliferation activity still need further research and development. Summary of the Invention

[0007] The present invention aims to, at least to some extent, address at least one of the technical problems existing in the prior art. To this end, one object of the present invention is to provide a fusion protein and its use. This fusion protein can bind to CD40, strongly promoting the activation and proliferation of antigen-presenting cells, thereby effectively promoting immune responses and enhancing the anti-cancer ability of the immune system, thus possessing excellent clinical application and drug development value.

[0008] The present invention is based on the inventor's discovery and understanding of the following facts and problems:

[0009] To enhance the CD40-binding activity of fusion proteins, the inventors conducted extensive screening and experimental verification, resulting in a fusion protein comprised of three tandem extracellular segments of CD40 ligands (collectively referred to herein as "CD40L proteins"). Further experimental results demonstrated that the fusion protein of the present invention exhibits superior activity compared to CD40 antibodies, demonstrating promising clinical applications and drug development potential.

[0010] Therefore, in its first aspect, the present invention provides a fusion protein. According to an embodiment of the present invention, the fusion protein comprises: at least three CD40L extracellular segments; wherein the number of CD40L extracellular segments is an integer greater than or equal to 3, and the C-terminus and N-terminus of each CD40L extracellular segment are sequentially connected in series. The fusion protein according to an embodiment of the present invention can bind to CD40, strongly promoting the activation and proliferation of antigen-presenting cells, thereby effectively promoting immune responses and enhancing the anti-cancer ability of the immune system. Therefore, the fusion protein of the present invention has excellent clinical value and drug development potential.

[0011] In a second aspect of the present invention, the present invention provides a nucleic acid. According to an embodiment of the present invention, the nucleic acid encodes the aforementioned fusion protein. According to an embodiment of the present invention, the nucleic acid can further express the aforementioned fusion protein.

[0012] In a third aspect, the present invention provides a vector or transformant. According to an embodiment of the present invention, the vector or transformant contains the aforementioned nucleic acid. Thus, the constructed vector or transformant can effectively express the aforementioned fusion protein.

[0013] In a fourth aspect, the present invention provides a cell. According to embodiments of the present invention, the cell carries the aforementioned nucleic acid, vector, or transformant, or expresses the aforementioned fusion protein. Under appropriate conditions, the cell according to embodiments of the present invention can efficiently express the aforementioned fusion protein. Furthermore, the fusion protein can be obtained that is capable of binding to CD40, promoting the activation and proliferation of antigen-presenting cells, and effectively promoting the anti-cancer activity of the autoimmune system, with high anti-cancer activity.

[0014] In a fifth aspect, the present invention provides a pharmaceutical composition. According to embodiments of the present invention, the pharmaceutical composition comprises: the aforementioned fusion protein, the aforementioned nucleic acid, or the aforementioned vector or transformant. The resulting drug can be further used to promote the activation and / or proliferation of antigen-presenting cells (such as B cells) for the treatment of diseases such as cancer.

[0015] Those skilled in the art will appreciate that the features and advantages described above for the fusion protein, nucleic acid molecule, and vector or transformant are also applicable to the pharmaceutical composition and will not be described in detail here.

[0016] In a sixth aspect, the present invention provides a kit. According to an embodiment of the present invention, the kit comprises the aforementioned fusion protein. The resulting kit can be used for CD40 target-related research, such as for detecting and / or enriching and / or isolating and purifying CD40 antigens from humans or other mammals.

[0017] Those skilled in the art will appreciate that the features and advantages described above for the fusion protein are also applicable to the kit and will not be described in detail here.

[0018] In a seventh aspect, the present invention provides use of the aforementioned fusion protein in preparing a kit for detecting and / or enriching and / or separating and purifying CD40.

[0019] Those skilled in the art will appreciate that the features and advantages described above for the fusion protein are also applicable to the use of the preparation kit and will not be described in detail here.

[0020] In an eighth aspect, the present invention provides a method for detecting, enriching, or isolating and purifying CD40. According to an embodiment of the present invention, the method comprises contacting a biological sample containing CD40 with the aforementioned fusion protein or the fusion protein in the aforementioned kit. According to an embodiment of the present invention, utilizing the ability of the aforementioned fusion protein to bind to CD40, the fusion protein is contacted with the biological sample, and the specific binding of the fusion protein to CD40 enables detection, enrichment, or isolation and purification of CD40.

[0021] In a ninth aspect of the present invention, the present invention provides the use of the aforementioned fusion protein, the aforementioned nucleic acid, the aforementioned vector or transformant, or the aforementioned pharmaceutical composition in the preparation of a medicament. According to an embodiment of the present invention, the medicament is used for preventing and / or treating cancer.

[0022] Those skilled in the art will appreciate that the features and advantages described above for the fusion protein are also applicable to the use of preparing the drug, and will not be described in detail here.

[0023] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The above and / or additional aspects and advantages of the present invention will become apparent and readily understood from the following description of the embodiments with reference to the accompanying drawings, in which:

[0025] Figure 1 Schematic diagram of 140L-Fc, 240L-Fc and 340L-Fc fusion proteins in Example 1 of the present invention;

[0026] Figure 2 Figures 2 are flow cytometry results of the binding of 140L-Fc, 240L-Fc, and 340L-Fc fusion proteins to CD40-positive HCT-116 cells in Example 2 of the present invention, wherein (A) shows the positive ratio of the binding of 140L-Fc, 240L-Fc, and 340L-Fc fusion proteins to HCT-116 cells; (B) shows the mean fluorescence intensity of the binding of 140L-Fc, 240L-Fc, and 340L-Fc fusion proteins to HCT-116 cells;

[0027] Figure 3 This is a graph showing the results of 140L-Fc, 240L-Fc and 340L-Fc fusion proteins promoting the activation of CD40 reporter cells in Example 3 of the present invention;

[0028] Figure 4 Figures 2 are flow cytometry results of the binding of the 340L-Fc fusion protein, Glioralimab, and Selicrelumab antibodies to CD40-positive HCT-116 cells in Example 2 of the present invention, wherein (A) is the positive ratio of the binding of the 340L-Fc fusion protein, Glioralimab, and Selicrelumab antibodies to HCT-116 cells; (B) is the mean fluorescence intensity of the binding of the 340L-Fc fusion protein, Glioralimab, and Selicrelumab antibodies to HCT-116 cells;

[0029] Figure 5 This is a graph showing the results of 340L-Fc fusion protein, Glioralimab, and Selicrelumab antibodies promoting the activation of CD40 reporter cells in Example 3 of the present invention;

[0030] Figure 6 The results of the 340L-Fc fusion protein, Glioralimab, and Selicrelumab antibodies in Example 4 of the present invention promoting B cell activation and expression of CD80 are shown in Figure 3, wherein (A) shows the results of the investigation of the positive binding ratio of CD80 on the surface of B cells; (B) shows the results of the investigation of the mean fluorescence intensity of CD80 on the surface of B cells;

[0031] Figure 7 The results of the 340L-Fc fusion protein, Glioralimab, and Selicrelumab antibodies in Example 4 of the present invention promoting B cell activation and expression of CD86 are shown in Figure 3, wherein (A) shows the results of the investigation of the positive ratio of CD86 on the surface of B cells; (B) shows the results of the investigation of the mean fluorescence intensity of CD86 on the surface of B cells;

[0032] Figure 8 This is a graph showing the results of 340L-Fc fusion protein, Glioralimab, and Selicrelumab antibodies promoting B cell proliferation in Example 5 of the present invention;

[0033] Figure 9 This is an ELISA result showing the binding of the FAP×340L fusion protein to the mouse FAP protein in Example 6 of the present invention;

[0034] Figure 10 This is the ELISA result of the FAP×340L fusion protein bridging mouse FAP protein and CD40-Biotin protein in Example 7 of the present invention;

[0035] Figure 11 Flow cytometry results of the binding of FAP×340L and 340L-Fc fusion proteins to HCT-116 cells in Example 2 of the present invention, specifically the mean fluorescence intensity results of the binding of FAP×340L and 340L-Fc fusion proteins to HCT-116 cells;

[0036] Figure 12 This is a graph showing the results of FAP×340L and 340L-Fc fusion proteins promoting the activation of CD40 reporter cells in Example 3 of the present invention;

[0037] Figure 13The figures show the anti-cancer effect of the FAP×340L fusion protein in Example 8 of the present invention, wherein (A) shows the tumor volume of tumor-bearing mice in each experimental group; (B) shows the tumor weight of tumor-bearing mice in each experimental group; "**" indicates P < 0.01. DETAILED DESCRIPTION

[0038] The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0039] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of the technical features being referred to. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "plurality" means at least two, such as two, three, etc., unless otherwise specifically defined.

[0040] Terms and Definitions

[0041] To facilitate understanding of the present invention, certain technical and scientific terms are defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. The abbreviations for amino acid residues are the standard three-letter and / or one-letter codes used in the art to designate one of the 20 commonly used L-amino acids.

[0042] As used herein, the term "antibody" generally refers to an antibody that can recognize one or more antigenic epitopes, including but not limited to monoclonal antibodies, polyclonal antibodies, dimers, multimers, multispecific antibodies (e.g., bispecific antibodies), heavy chain antibodies only, three-chain antibodies, single-chain Fv (scFv), nanobodies, etc., and also includes antibody fragments, as long as they exhibit the desired biological activity. Antibodies can be murine, human, humanized, chimeric, or derived from other species. Antibodies can refer to full-length heavy chains, full-length light chains, complete immunoglobulin molecules; or an immunologically active portion of any of these polypeptides, i.e., a molecule or portion thereof that contains an antigen binding site that immunospecifically binds to a target antigen of interest, such targets including but not limited to cancer cells or cells that produce autoimmune antibodies associated with autoimmune diseases.

[0043] As used herein, the term "anti-FAP antibody" refers to an antibody that can bind to FAP. Such an antibody is also referred to herein as a "FAP antibody."

[0044] As used herein, the term "antigen-binding fragment" is equivalent to "antibody fragment" or "antigen-binding antibody fragment" and may include a portion of an intact antibody, generally the antigen-binding region or variable region. This includes, but is not limited to, Fv, scFv, Fab, Fab', Fab'-SH, F(ab')2, scFab, scFabΔ, scFv-Fc fragments, or bispecific antibodies (BsAbs), linear antibodies, or any fragment that can increase half-life by chemical modification, such as the addition of poly(alkylene) glycols, such as polyethylene glycol ("PEGylation") (PEGylated fragments referred to as Fv-PEG, scFv-PEG, Fab-PEG, F(ab')2-PEG, or Fab'-PEG) ("PEG" is polyethylene glycol) or by incorporation into liposomes.

[0045] With respect to polypeptides, the term "(substantial) homology" is used to describe or compare the degree of amino acid similarity between two or more polypeptides or designated sequences thereof when optimally aligned and compared (with appropriate insertions or deletions of nucleotides). The percent homology between two sequences varies with the number of identical positions shared by the sequences when the sequences are optimally aligned (i.e., % homology = number of identical positions / total number of positions x 100), where optimal alignment is determined by taking into account the number of gaps that need to be introduced to achieve optimal alignment of the two sequences and the length of each gap. Sequence comparison and percent identity determination between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting Examples below.

[0046] In this article, the term "vector" generally refers to a nucleic acid molecule that can be inserted into a suitable host and replicates itself, and the inserted nucleic acid molecule is transferred into and / or between host cells. The vector may include a vector that is mainly used to insert DNA or RNA into a cell, a vector that is mainly used to replicate DNA or RNA, and a vector that is mainly used for expression of the transcription and / or translation of DNA or RNA. The vector also includes a vector with a variety of the above functions. The vector can be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector can produce a desired expression product by cultivating a suitable host cell containing the vector.

[0047] As used herein, the term "pharmaceutical composition" generally refers to unit dosage form and can be prepared by any of the methods well known in the pharmaceutical art. All methods include the step of bringing the active ingredient into association with the carrier which constitutes one or more accessory ingredients. Generally, the compositions are prepared by uniformly and thoroughly combining the active compound with liquid carriers, finely divided solid carriers, or both.

[0048] As used herein, the term "pharmaceutically acceptable" refers to substances that are suitable for use in humans and / or mammals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), and "pharmaceutically acceptable excipients" refer to substances with a reasonable benefit / risk ratio.

[0049] As used herein, the term "pharmaceutically acceptable excipient" may include any solvent, solid excipient, diluent or other liquid excipient, etc., suitable for the specific target dosage form. Except to the extent that any conventional excipient is incompatible with the compound of the present invention, such as any adverse biological effect produced or interaction with any other component of the pharmaceutically acceptable composition in a harmful manner, their use is also contemplated by the present invention.

[0050] As used herein, the term "administer" refers to the introduction of a predetermined amount of a substance into a patient by a suitable means. The antibody or antigen-binding fragment, recombinant protein, multispecific antibody, conjugate, or pharmaceutical composition of the present invention can be administered by any common route as long as it reaches the desired tissue. Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, and the like, but the present invention is not limited to these exemplified modes of administration. Preferably, the composition of the present invention is administered by intravenous or subcutaneous injection.

[0051] As used herein, the term "treatment" refers to any agent used to obtain a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers diseases in mammals, particularly humans, and includes: (a) preventing the occurrence of a disease or condition in individuals who are susceptible to the disease but have not yet been diagnosed with the disease; (b) inhibiting the disease, such as arresting the progression of the disease; or (c) alleviating the disease, such as alleviating the symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug or compound to an individual to treat, cure, alleviate, improve, reduce or inhibit the individual's disease, including but not limited to administering a drug containing a compound described herein to an individual in need.

[0052] As used herein, the term "effective amount" or "effective dose" refers to an amount that can produce a function or activity on humans and / or animals and can be accepted by humans and / or animals.

[0053] As used herein, the term "CD40L" refers to the CD40 receptor ligand, and is equivalent to "CD40 Ligand" and "CD154".

[0054] As used herein, the term "CD40L extracellular segment" refers to the extracellular segment of the CD40 receptor ligand monomer.

[0055] In this article, the term "FAP" stands for fibroblast activation protein-α, a 97kDa type II transmembrane serine protease with both dipeptidyl peptidase and endopeptidase activities. Under physiological conditions, FAP is expressed at low levels in most adult tissues. However, FAP mRNA levels are elevated in different tumor types, with the highest median levels in pancreatic and breast cancer. In most epithelial cancers, FAP is primarily expressed in cancer-associated fibroblasts in the stroma. FAP is also expressed in some tumor cells, such as sarcomas, mesotheliomas, and epithelial tumors of the esophagus.

[0056] As used herein, the term "Giloralimab" refers to a potent anti-CD40 agonist monoclonal antibody whose amino acid sequence is known.

[0057] In this article, the term "Selicrelumab" is equivalent to "Selicrelumab," a CD40 antibody agonist that induces changes in the tumor microenvironment and whose amino acid sequence is known.

[0058] The present invention provides a fusion protein, a corresponding nucleic acid molecule, a vector or transformant, a cell, a pharmaceutical composition, a kit and applications thereof, which are described in detail below.

[0059] Fusion protein

[0060] The present invention provides a fusion protein. According to an embodiment of the present invention, the fusion protein comprises: at least three CD40L extracellular segments; wherein the number of CD40L extracellular segments is an integer greater than or equal to 3, and the C-terminus and N-terminus of each CD40L extracellular segment are sequentially connected in series. The fusion protein according to an embodiment of the present invention can bind to CD40, promote the activation and proliferation of antigen-presenting cells, and thereby effectively promote immune responses and enhance the anti-cancer ability of the immune system. Therefore, the fusion protein of the present invention has excellent clinical value and drug development value.

[0061] According to an embodiment of the present invention, the fusion protein further comprises at least two connecting peptides. The fusion protein comprises, from the N-terminus to the C-terminus, the following sequence: the CD40L extracellular segment, a first connecting peptide, the CD40L extracellular segment, a second connecting peptide, and the CD40L extracellular segment. This can enhance the CD40 binding activity of the fusion protein.

[0062] According to an embodiment of the present invention, the first connecting peptide or the second connecting peptide has an amino acid sequence represented by (GGGGS)n, where n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. The inventors have identified this preferred connecting peptide through extensive screening experiments, thereby further improving the CD40 binding activity of the fusion protein.

[0063] According to an embodiment of the present invention, the first connecting peptide and the second connecting peptide have an amino acid sequence as shown in (GGGGS)n, wherein n is 4. Thus, the CD40 binding activity of the fusion protein is further improved.

[0064] According to an embodiment of the present invention, the CD40L extracellular segment has an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence having at least 80% identity thereto.

[0065] According to an embodiment of the present invention, the fusion protein has an amino acid sequence as shown in SEQ ID NO: 3 or an amino acid sequence having at least 80% identity thereto.

[0066] In some specific embodiments, the amino acid sequence of the CD40L extracellular segment is as shown in SEQ ID NO: 1.

[0067] In some specific embodiments, the amino acid sequence of the fusion protein is shown in SEQ ID NO:3.

[0068] It should be noted that, without substantially affecting the CD40 binding activity of the fusion protein (retaining at least 90% activity), those skilled in the art may replace, add and / or delete one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) amino acids in the amino acid sequence of the fusion protein of the present invention to obtain variants of the sequence of the fusion protein. They are all considered to be included in the scope of protection of the present invention. The sequence of the above-mentioned variant of the present invention may have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity (or homology) with the reference sequence. The sequence identity of the present invention can be measured using sequence analysis software. For example, the computer program BLAST, particularly BLASTP or TBLASTN, is used with default parameters.

[0069] According to an embodiment of the present invention, the fusion protein further comprises: a first Fc fragment and a second Fc fragment; wherein the CD40L extracellular segment is connected to the first Fc fragment; and the first Fc fragment is connected to the second Fc fragment.

[0070] The Fc fragment is derived from the Fc region of immunoglobulins, which plays an important role in immune defense against pathogens. The effector functions of IgG are mediated by Fc and are mediated by two main mechanisms: (1) binding to cell surface Fc receptors (FcγRs), leading to the elimination of pathogens by killer cells through phagocytosis or lysis via the antibody-dependent cellular cytotoxicity (ADCC) pathway; and (2) binding to the Clq portion of the first complement component Cl, triggering the complement-dependent cytotoxicity (CDC) pathway, thereby lysing pathogens. Human IgG1 and IgG3 can also effectively bind to Clq and activate the complement cascade reaction. Therefore, the fusion protein according to the embodiments of the present invention also has some of the effects of immunoglobulins.

[0071] According to an embodiment of the present invention, the C-terminus of the CD40L extracellular segment is connected to the N-terminus of the first Fc fragment.

[0072] According to an embodiment of the present invention, the first Fc fragment and the second Fc fragment are connected via a third connecting peptide; wherein the C-terminus of the first Fc fragment is connected to the N-terminus of the third connecting peptide, and the C-terminus of the third connecting peptide is connected to the N-terminus of the second Fc fragment. Thus, the resulting fusion protein has excellent biological activity.

[0073] According to an embodiment of the present invention, the third connecting peptide has an amino acid sequence as shown in (GGGGS)n, where n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. The inventors have identified this preferred connecting peptide through extensive screening experiments. Thus, the biological activity of the fusion protein is further improved.

[0074] According to an embodiment of the present invention, the third connecting peptide has an amino acid sequence as shown in (GGGGS)n, wherein n is 6. Thus, the biological activity of the fusion protein is further improved.

[0075] According to an embodiment of the present invention, at least a portion of the first Fc fragment or the second Fc fragment is derived from at least one of a mouse antibody, a human antibody, a primate antibody, a bovine antibody, a horse antibody, a cow antibody, a porcine antibody, a sheep antibody, a goat antibody, a dog antibody, a cat antibody, a rabbit antibody, a camel antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody, a fighting cock antibody, or a mutant thereof.

[0076] According to an embodiment of the present invention, the first Fc fragment or the second Fc fragment comprises an Fc segment selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD, preferably IgG1.

[0077] According to an embodiment of the present invention, the first Fc fragment or the second Fc fragment is derived from a murine antibody or a mutant thereof, or a human antibody or a mutant thereof.

[0078] According to an embodiment of the present invention, the first Fc fragment or the second Fc fragment has the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence having at least 80% identity thereto.

[0079] According to an embodiment of the present invention, the first Fc fragment and the second Fc fragment are connected via a disulfide bond.

[0080] According to an embodiment of the present invention, the first Fc fragment or the second Fc fragment has the amino acid sequence shown in SEQ ID NO: 15 or an amino acid sequence having at least 80% identity thereto.

[0081] According to an embodiment of the present invention, the first Fc fragment and the second Fc fragment are connected via a knob-into-hole structure.

[0082] According to an embodiment of the present invention, the first Fc fragment or the second Fc fragment has an amino acid sequence as shown in SEQ ID NO: 16 or 17 or an amino acid sequence having at least 80% identity thereto.

[0083] The amino acid sequence of the first Fc fragment or the second Fc fragment is shown in SEQ ID NO:14.

[0084] In some specific embodiments, the amino acid sequence of the first Fc fragment or the second Fc fragment is as shown in SEQ ID NO:15.

[0085] In some specific embodiments, the amino acid sequence of the first Fc fragment or the second Fc fragment is shown in SEQ ID NO: 16 or 17.

[0086] According to an embodiment of the present invention, the fusion protein has an amino acid sequence as shown in SEQ ID NO: 6 or an amino acid sequence having at least 80% identity thereto.

[0087] According to an embodiment of the present invention, the fusion protein includes: a first polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 18 or an amino acid sequence having at least 80% identity thereto, and a second polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 15 or an amino acid sequence having at least 80% identity thereto.

[0088] According to an embodiment of the present invention, the fusion protein includes: a first polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 11 or an amino acid sequence having at least 80% identity thereto, and a second polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 17 or an amino acid sequence having at least 80% identity thereto.

[0089] According to an embodiment of the present invention, the fusion protein includes: a first polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 19 or an amino acid sequence having at least 80% identity thereto, and a second polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 16 or an amino acid sequence having at least 80% identity thereto.

[0090] In some specific embodiments, the amino acid sequence of the fusion protein is shown in SEQ ID NO:6.

[0091] In some specific embodiments, the fusion protein comprises: a first polypeptide chain having an amino acid sequence as shown in SEQ ID NO:18, and a second polypeptide chain having an amino acid sequence as shown in SEQ ID NO:15.

[0092] In some specific embodiments, the fusion protein comprises: a first polypeptide chain having an amino acid sequence as shown in SEQ ID NO:11, and a second polypeptide chain having an amino acid sequence as shown in SEQ ID NO:17.

[0093] In some specific embodiments, the fusion protein comprises: a first polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 19, and a second polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 16.

[0094] According to an embodiment of the present invention, the fusion protein further comprises: a FAP antigen binding region; wherein the FAP antigen binding region is connected to the second Fc fragment. Thus, the fusion protein according to an embodiment of the present invention can bind to CD40 and FAP, and has good anti-cancer activity.

[0095] According to an embodiment of the present invention, the FAP antigen binding region comprises an antigen-binding fragment of an anti-FAP antibody.

[0096] According to an embodiment of the present invention, the antigen-binding fragment of the anti-FAP antibody is a Fab fragment, scFab fragment, or scFabΔ fragment of the anti-FAP antibody, preferably a Fab fragment. The inventors identified this preferred antigen-binding fragment through extensive screening experiments, thereby further enhancing the FAP binding activity of the fusion protein.

[0097] According to an embodiment of the present invention, the VH-CH1 of the Fab fragment has an amino acid sequence as shown in SEQ ID NO: 20 or an amino acid sequence having at least 80% identity thereto, and the VL-CL of the Fab fragment has an amino acid sequence as shown in SEQ ID NO: 13 or an amino acid sequence having at least 80% identity thereto.

[0098] In some specific embodiments, the amino acid sequence of VH-CH1 of the Fab fragment is shown as SEQ ID NO:20, and the amino acid sequence of VL-CL of the Fab fragment is shown as SEQ ID NO:13.

[0099] According to an embodiment of the present invention, the C-terminus of the CH1 fragment of the Fab fragment is connected to the N-terminus of the second Fc fragment. The fusion protein according to an embodiment of the present invention has high binding activity to CD40 and FAP and good anti-cancer activity.

[0100] According to an embodiment of the present invention, the fusion protein includes: a first polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 11 or an amino acid sequence having at least 80% identity thereto, a second polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 12 or an amino acid sequence having at least 80% identity thereto, and a third polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 13 or an amino acid sequence having at least 80% identity thereto.

[0101] In some specific embodiments, the fusion protein comprises: a first polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 11, a second polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 12, and a third polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 13. The fusion protein according to the embodiments of the present invention has significant anti-cancer function, high clinical value, and high drug development value.

[0102] Nucleic Acids

[0103] The present invention provides a nucleic acid. According to an embodiment of the present invention, the nucleic acid encodes the aforementioned fusion protein. The nucleic acid according to an embodiment of the present invention can further express the aforementioned fusion protein.

[0104] It should be noted that, for nucleic acid molecules mentioned herein, those skilled in the art will understand that they actually include any one or both of the complementary double strands. For convenience, although only one strand is provided in most cases herein, the other strand complementary thereto is also disclosed. In addition, the molecular sequences in the present invention include DNA or RNA forms, and disclosure of one of them implies that the other is also disclosed.

[0105] Vector or transformant

[0106] The present invention provides a vector or a transformant. According to an embodiment of the present invention, the vector or the transformant contains the aforementioned nucleic acid.

[0107] The vector or transformant according to an embodiment of the present invention may include an optional control sequence that is operably linked to the aforementioned nucleic acid molecule. The control sequence is one or more control sequences that can direct the expression of the nucleic acid molecule in the host. The vector or transformant thus constructed can effectively express the aforementioned fusion protein.

[0108] When the aforementioned nucleic acid molecule is linked to the aforementioned vector or transformant, such as an expression vector, the aforementioned nucleic acid molecule can be directly or indirectly linked to the control elements on the expression vector, as long as these control elements are capable of controlling translation and expression of the nucleic acid molecule. Of course, these control elements can be directly derived from the vector itself or exogenous, i.e., not derived from the vector itself. The aforementioned nucleic acid molecule and the control elements can be operably linked.

[0109] According to an embodiment of the present invention, the vector may refer to a cloning vector or an expression vector, which can be obtained by operably linking the nucleic acid to a commercially available vector (such as a plasmid or viral vector). The vector in the present invention is not particularly limited, and commonly used plasmids such as pSeTag2, PEE14, and pMH3 can be used.

[0110] As used herein, the term "operably linked" refers to the attachment of an exogenous gene to a vector so that control elements within the vector, such as transcriptional and translational control sequences, can function as intended to regulate the transcription and translation of the exogenous gene. Commonly used vectors include viral vectors, plasmids, and bacteriophages. Expression vectors according to certain embodiments of the present invention, upon introduction into appropriate recipient cells, can effectively express the aforementioned nucleic acid molecules under the mediation of a regulatory system, thereby enabling the in vitro production of large quantities of the protein encoded by the nucleic acid molecules.

[0111] According to an embodiment of the present invention, the vector is a eukaryotic vector or a prokaryotic vector.

[0112] According to an embodiment of the present invention, the vector comprises at least one selected from a plasmid vector, an adenovirus vector, a lentivirus vector and an adeno-associated virus vector.

[0113] cell

[0114] The present invention provides a cell. According to embodiments of the present invention, the cell carries the aforementioned nucleic acid, vector, or transformant, or expresses the aforementioned fusion protein. Under appropriate conditions, the cell according to embodiments of the present invention can efficiently express the aforementioned fusion protein. Furthermore, the fusion protein can bind to CD40, promote the activation and proliferation of antigen-presenting cells, effectively promote the anti-cancer activity of the autoimmune system, and exhibit high anti-cancer activity.

[0115] According to an embodiment of the present invention, the cell is a prokaryotic cell, a eukaryotic cell or a bacteriophage.

[0116] According to an embodiment of the present invention, the prokaryotic cell is Escherichia coli, Bacillus subtilis, Streptomyces or Proteus mirabilis.

[0117] According to an embodiment of the present invention, the eukaryotic cell is a fungus, an insect cell, a plant cell or a mammalian cell.

[0118] According to an embodiment of the present invention, the fungus is Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces pombe or Trichoderma.

[0119] According to an embodiment of the present invention, the insect cell is a fall armyworm cell; according to an embodiment of the present invention, the plant cell is a tobacco plant cell; according to an embodiment of the present invention, the mammalian cell is a BHK cell, a CHO cell, a COS cell, a myeloma cell or a human embryonic kidney 293 cell; and does not include animal germ cells, fertilized eggs or embryonic stem cells.

[0120] According to an embodiment of the present invention, the cell is a mammalian cell.

[0121] According to an embodiment of the present invention, the cell is a BHK cell, a CHO cell, a COS cell or a NSO cell.

[0122] It should be noted that the "suitable conditions" described in the present specification refer to conditions suitable for the expression of the fusion protein of the present invention. Those skilled in the art will readily appreciate that conditions suitable for the expression of the fusion protein include, but are not limited to, suitable transformation or transfection methods, suitable transformation or transfection conditions, healthy host cell status, suitable host cell density, suitable cell culture environment, and suitable cell culture time. "Suitable conditions" are not particularly limited, and those skilled in the art can optimize the optimal conditions for the expression of the aforementioned fusion protein based on the specific laboratory environment.

[0123] Pharmaceutical composition

[0124] The present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises: the aforementioned fusion protein, the aforementioned nucleic acid, or the aforementioned vector or transformant. The resulting drug can be further used to promote the activation and / or proliferation of antigen-presenting cells (such as B cells) for the treatment of diseases such as cancer.

[0125] According to an embodiment of the present invention, the composition further comprises a pharmaceutically acceptable excipient.

[0126] According to an embodiment of the present invention, the excipients include: one or more pharmaceutically acceptable excipients, diluents, stabilizers or carriers.

[0127] According to an embodiment of the present invention, the pharmaceutical composition is an injection.

[0128] Those skilled in the art will appreciate that the features and advantages described above for the fusion protein, nucleic acid molecule, and vector or transformant are also applicable to the pharmaceutical composition and will not be described in detail here.

[0129] It should be noted that the pharmaceutical composition includes combinations separated in time and / or space, as long as they can work together to achieve the purpose of the present invention. For example, the components contained in the composition can be administered to the subject as a whole or separately. When the components contained in the composition are administered to the subject separately, the individual components can be administered to the subject simultaneously or sequentially.

[0130] The pharmaceutical composition of the present invention contains a safe and effective amount of the active ingredient of the present invention and pharmaceutically acceptable excipients. Such excipients include (but are not limited to) saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. Generally, the pharmaceutical formulation should be compatible with the mode of administration. The dosage forms of the drug of the present invention include injections, oral preparations (tablets, capsules, oral liquids), transdermal preparations, and sustained-release preparations. For example, the drug can be prepared using conventional methods using physiological saline or an aqueous solution containing glucose and other excipients. The drug is preferably manufactured under sterile conditions.

[0131] The effective amount of the active ingredient of the present invention may vary depending on the mode of administration and the severity of the disease to be treated. The preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., through clinical trials). Such factors include, but are not limited to, the pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, and the like. For example, depending on the exigencies of the treatment, several divided doses may be administered daily, or the dose may be reduced proportionally.

[0132] Reagent test kit

[0133] The present invention provides a kit. According to an embodiment of the present invention, the kit comprises the aforementioned fusion protein. The resulting kit can be used for CD40 target-related research, such as for detecting and / or enriching and / or isolating and purifying CD40 antigen from humans or other mammals.

[0134] Those skilled in the art will appreciate that the features and advantages described above for the fusion protein are also applicable to the kit and will not be described in detail here.

[0135] Use in the preparation of kits

[0136] The present invention provides use of the aforementioned fusion protein in preparing a kit for detecting and / or enriching and / or separating and purifying CD40.

[0137] Those skilled in the art will appreciate that the features and advantages described above for the fusion protein are also applicable to the use of the preparation kit and will not be described in detail here.

[0138] According to an embodiment of the present invention, the kit detects CD40 by ELISA, flow cytometry, immunoblotting or immunoprecipitation.

[0139] According to an embodiment of the present invention, the kit enriches and / or separates and purifies CD40 by affinity chromatography.

[0140] As previously mentioned, the fusion proteins according to embodiments of the present invention are capable of specifically binding to CD40. Therefore, these fusion proteins can be used to detect CD40. Furthermore, they can be used to prepare kits for detecting and / or enriching and / or isolating and purifying CD40 for scientific research, such as for qualitative or quantitative detection of CD40 protein molecules in biological samples. More specifically, they can be used in kits for immunoblotting, immunoprecipitation, and other assays that utilize the specific binding properties of CD40 and its ligand. These kits may also contain one or more of the following: antagonists, CD40 antibodies, or reference drug materials; protein purification columns; immunoglobulin affinity purification buffers; and cell assay diluents. These fusion proteins can also be used in various diagnostic tests, for example, to detect various diseases or the presence of drugs, toxins, or other proteins in vitro or in vivo. For example, they can be tested in serum or blood from a subject to determine CD40-related indicators.

[0141] method

[0142] The present invention provides a method for detecting, enriching, or isolating and purifying CD40. According to an embodiment of the present invention, the method comprises: contacting a biological sample containing CD40 with the aforementioned fusion protein or the fusion protein in the aforementioned kit. Utilizing the ability of the aforementioned fusion protein to bind to CD40, the fusion protein is contacted with the biological sample, and the specific binding of the fusion protein to CD40 enables detection, enrichment, or isolation and purification of CD40. According to an embodiment of the present invention, utilizing the ability of the aforementioned fusion protein to bind to CD40, the fusion protein is contacted with the biological sample, and the specific binding of the fusion protein to CD40 enables detection, enrichment, or isolation and purification of CD40.

[0143] Use in preparing medicines

[0144] The present invention provides the use of the aforementioned fusion protein, the aforementioned nucleic acid, the aforementioned vector or transformant, or the aforementioned pharmaceutical composition in preparing a medicament. According to an embodiment of the present invention, the medicament is used for preventing and / or treating cancer.

[0145] According to an embodiment of the present invention, the drug promotes the activation and proliferation of antigen-presenting cells (such as B cells), thereby effectively promoting the body's immune response and improving the anti-cancer ability of the body's immune system.

[0146] The fusion protein according to the embodiment of the present invention has significant anti-cancer function and high clinical value, and thus can be further developed into a drug for preventing and / or treating cancer.

[0147] Those skilled in the art will appreciate that the features and advantages described above for the fusion protein are also applicable to the use of preparing the drug, and will not be described in detail here.

[0148] Disease treatment methods

[0149] The present invention provides a method for preventing and / or treating cancer. According to an embodiment of the present invention, the method comprises: administering a pharmaceutically acceptable amount of the aforementioned fusion protein, the aforementioned nucleic acid, the aforementioned vector or transformant, or the aforementioned pharmaceutical composition to a subject.

[0150] Those skilled in the art will appreciate that the features and advantages described above for the fusion protein, nucleic acid, vector or transformant or pharmaceutical composition are also applicable to the method for treating the disease and will not be described in detail here.

[0151] It should be noted that the terms "subject," "individual," and "patient" are used interchangeably herein to refer to a mammal being evaluated for treatment and / or being treated. In one embodiment, the mammal is a human. The terms "subject," "individual," and "patient" include, but are not limited to, individuals with cancer, individuals with autoimmune diseases, individuals with pathogen infection, and the like. The subject can be a human, but also includes other mammals, particularly mammals that can be used as laboratory models of human diseases, such as mice, rats, and the like.

[0152] The effective amount of the fusion protein, nucleic acid, vector, transformant, or pharmaceutical composition of the present invention may vary depending on the mode of administration and the severity of the disease to be treated. The preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., through clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the active ingredient, such as bioavailability, metabolism, and half-life; the severity of the disease to be treated, the patient's weight, the patient's immune status, the route of administration, and the like. For example, depending on the urgency of the treatment, several divided doses may be administered daily, or the dose may be proportionally reduced.

[0153] The sequences involved in the present invention are detailed in Table 1.

[0154] Table 1 Amino acid sequence description

[0155]

[0156]

[0157]

[0158]

[0159] The present invention will be described in detail below through examples. In the examples or test examples, if no specific conditions are specified, the experimental methods were carried out under conventional conditions.

[0160] Below, the scheme of the present invention will be explained in conjunction with embodiment.It will be understood by those skilled in the art that the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.In the embodiment, if specific technology or conditions are not specified, they are carried out according to the technology or conditions described in the literature in this area or according to the product instructions.The reagents or instruments used are not specified by the manufacturer, and are all conventional products that can be purchased commercially.

[0161] Example 1: Production of protein

[0162] The specific experimental procedures for protein production are as follows: (1) ExpiCHO cells (purchased from Thermo Fisher) were cultured using ExpiCHO Expression Medium (purchased from Thermo Fisher) and the cell concentration was adjusted to 6×10 6 / mL to obtain ExpiCHO cell solution. (2) When the protein contains one chain, the pcDNA3.4 vector containing the coding sequence (commissioned by Nanjing GenScript for synthesis) is added to 2 mL of OptiSFM medium (purchased from Thermo Fisher) to obtain solution a; or, when the protein is two chains, the pcDNA3.4 vector containing two coding sequences (commissioned by Nanjing GenScript for synthesis) is added to 2 mL of OptiSFM medium (purchased from Thermo Fisher) at a ratio of 1:1 to obtain solution a; or, when the protein is three chains, the pcDNA3.4 vector containing three coding sequences (commissioned by Nanjing GenScript for synthesis) is added to 2 mL of OptiSFM medium (purchased from Thermo Fisher) at a ratio of 1:1:1 to obtain solution a; (3) 160 μL of ExpiFectamineCHO transfection reagent (purchased from Thermo Fisher) is added to 2 mL of OptiSFM medium (purchased from Thermo Fisher) to obtain solution b. (4) Solution a and solution b were then mixed to obtain a transfection mixture, which was then added to 50 mL of ExpiCHO cell solution within 5 minutes. (5) After culturing at 37°C, 5% CO₂ for 1 day, 8 mL of feed and 300 μL of Lenhancer (purchased from Thermo Fisher) were added, and the cells were transferred to 32°C, 5% CO₂ for 9 days. The culture supernatant was harvested, with 8 mL of feed added on the 5th day. (6) The target antibody was obtained by affinity purification from the culture supernatant using a Protein A purification column (purchased from NanoMicro).

[0163] In this example, a total of 6 proteins were prepared to investigate the fusion protein of the present invention (see Figure 1 The amino acid sequences of the proteins are shown in Tables 1 and 2.

[0164] Table 2 Structure of bispecific antibody and corresponding amino acid sequence in Example 1

[0165]

[0166] Example 2: Fusion protein flow cytometry binding experiment

[0167] Flow cytometry experiments are used to detect the binding properties of proteins. Proteins are added to cells, and the strength of the signal after the addition of proteins is used to determine the binding properties of proteins and cells.

[0168] HCT-116 human colorectal cancer cells were diluted to 2 × 10 6 / mL, added to a 1.5mL EP tube at a volume of 100μL / tube, 10μL / tube of goat serum was added thereto, and the tube was blocked at 4℃ for 30min. Add serial dilutions of 140L-Fc (amino acid sequence shown in SEQ ID NO: 4), 240L-Fc (amino acid sequence shown in SEQ ID NO: 5), 340L-Fc (amino acid sequence shown in SEQ ID NO: 6), giloralimab (antibody heavy chain amino acid sequence shown in SEQ ID NO: 7, antibody light chain amino acid sequence shown in SEQ ID NO: 8), selicrelumab (antibody heavy chain amino acid sequence shown in SEQ ID NO: 9, antibody light chain amino acid sequence shown in SEQ ID NO: 10), FAP×340L (340L amino acid sequence shown in SEQ ID NO: 11, FAP antibody heavy chain amino acid sequence shown in SEQ ID NO: 12, and FAP antibody light chain amino acid sequence shown in SEQ ID NO: 13), and control hIgG1LALA (purchased from Bio-Bio) and incubate at 4°C for 30 min. Add 1 mL of PBS to the EP tube, centrifuge at 3500 rpm for 5 min at 4°C, discard the supernatant, and wash again with PBS. After centrifugation, discard the supernatant and resuspend the cells in 100 μL / tube of PBS. Add 1 μL / tube of Alexa-647-labeled goat anti-human IgG secondary antibody (purchased from Jackson Lab) and incubate at 4°C in the dark for 30 min. Wash twice with PBS and centrifuge. Discard the supernatant. Resuspend the cells in 200 μL / tube of PBS and analyze using flow cytometry.

[0169] The results are as follows Figure 2 As shown, 140L-Fc, 240L-Fc, and 340L-Fc fusion proteins can all bind to CD40-positive HCT-116, and 340L-Fc binds more strongly than 140L-Fc and 240L-Fc.

[0170] The results are as follows Figure 4 As shown, 340L-Fc fusion protein and Giloralimab and Selicrelumab antibodies can bind to CD40-positive HCT-116 cells; compared with Giloralimab and Selicrelumab antibodies, the binding performance of 340L-Fc fusion protein to CD40-positive HCT-116 cells is weaker.

[0171] The results are as follows Figure 11 As shown, both 340L-Fc and FAP×340L fusion proteins can bind to CD40-positive HCT-116 cells, and the binding of FAP×340L is stronger than that of 340L-Fc.

[0172] Example 3: CD40 reporter cell activation experiment

[0173] Reporter cell experiments are used to detect the characteristics of fusion protein activation of CD40 downstream signaling. Fusion protein or anti-CD40 antibody is added to CD40 reporter cells. The strength of the luciferase signal after protein addition is used to determine the effect of the fusion protein or antibody on CD40 downstream activation signaling.

[0174] Dilute the CD40 reporter cells to 2 × 10 cells using complete DMEM medium. 5 / mL (purchased from Kebai Bio), added to a 96-well plate at a volume of 100 μL / tube, and 140L-Fc (amino acid sequence as shown in SEQ ID NO: 4), 240L-Fc (amino acid sequence as shown in SEQ ID NO: 5), 340L-Fc (amino acid sequence as shown in SEQ ID NO: 6), Giloralimab (the amino acid sequence of the antibody heavy chain is shown in SEQ ID NO: 7, and the amino acid sequence of the antibody light chain is shown in SEQ ID NO: 8), Selicrelumab (the amino acid sequence of the antibody heavy chain is shown in SEQ ID NO: 9, and the amino acid sequence of the antibody light chain is shown in SEQ ID NO: 10), FAP×340L (the amino acid sequence of 340L is shown in SEQ ID NO: 11, the amino acid sequence of the FAP antibody heavy chain is shown in SEQ ID NO: 12, and the amino acid sequence of the FAP antibody light chain is shown in SEQ ID NO: 13), and hIgG1LALA (purchased from Baiying Bio) were added thereto and incubated at 37°C for 24 h. Luciferase substrate was added thereto and the result was detected using a microplate reader.

[0175] The results are as follows Figure 3 , showing that the 140L-Fc, 240L-Fc, and 340L-Fc fusion proteins of the present invention can all promote the activation of CD40 reporter cells, among which the 340L-Fc fusion protein has the strongest activation ability.

[0176] The results are as follows Figure 5 , showing that the 340L-Fc fusion protein of the present invention and Giloralimab and Selicrelumab antibodies can all promote the activation of CD40 reporter cells; unexpectedly, compared with Giloralimab, the 340L-Fc fusion protein has a stronger activity in promoting the activation of CD40 reporter cells.

[0177] The results are as follows Figure 12 , showing that both the 340L-Fc and FAP×340L fusion proteins of the present invention can promote the activation of CD40 reporter cells, and the activities of the two are similar.

[0178] Example 4: Experiment on fusion protein promoting B cell activation

[0179] Flow cytometry experiments were used to examine the effects of fusion proteins on B cell activation and the expression of CD80 and CD86. Fusion proteins or anti-CD40 antibodies were added to human peripheral blood PBMCs. After the addition of fluorescent antibodies labeled with CD80 and CD86, the expression of CD80 and CD86 was examined to determine whether the fusion protein induced B cell activation.

[0180] Human peripheral blood PBMCs were diluted to 2 × 10 6 / mL (purchased from Heyousheng Bio) was added to a 96-well plate at a volume of 100 μL / tube. 340L-Fc (amino acid sequence shown in SEQ ID NO: 6), Giloralimab (amino acid sequence of the antibody heavy chain shown in SEQ ID NO: 7, amino acid sequence of the antibody light chain shown in SEQ ID NO: 8), Selicrelumab (amino acid sequence of the antibody heavy chain shown in SEQ ID NO: 9, amino acid sequence of the antibody light chain shown in SEQ ID NO: 10), and hIgG1LALA (purchased from Baiying Bio) were added and incubated at 37°C for 48 hours. CD19, CD80, and CD86 fluorescent antibodies (purchased from Biolegend) were used to label the cells. The cells were then resuspended in 200 μL / tube of PBS and analyzed by flow cytometry.

[0181] The results are as follows Figure 6 , showing that the 340L-Fc fusion protein of the present invention and Giloralimab and Selicrelumab antibodies can promote B cell activation and expression of CD80.

[0182] The results are as follows Figure 7 , showing that the 340L-Fc fusion protein of the present invention and Giloralimab and Selicrelumab antibodies can promote B cell activation and expression of CD86.

[0183] The above results show that the 340L-Fc fusion protein of the present invention has a stronger activity in promoting B cell activation than Giloralimab and Selicrelumab antibodies.

[0184] Example 5: Fusion protein promotes B cell proliferation experiment

[0185] Flow cytometry experiments were used to detect the B cell proliferation-promoting properties of the fusion protein. Human peripheral blood PBMCs were labeled with CFSE, and the fusion protein was added and cultured for 120 hours. The strength of the CFSE signal was used to determine the effect of the fusion protein on B cell proliferation.

[0186] PBMC cells were labeled with 5 μM CFSE. After labeling, human peripheral blood PBMC were diluted to 2×10 5 / mL (purchased from Sai Li Bio), added to a 96-well plate at a volume of 100 μL / tube, 340L-Fc (amino acid sequence as shown in SEQ ID NO: 6), Giloralimab (antibody heavy chain amino acid sequence as shown in SEQ ID NO: 7, antibody light chain amino acid sequence as shown in SEQ ID NO: 8), Selicrelumab (antibody heavy chain amino acid sequence as shown in SEQ ID NO: 9, antibody light chain amino acid sequence as shown in SEQ ID NO: 10), hIgG1LALA (purchased from Baiying Bio) were added thereto and incubated at 37°C for 120 hours. Flow cytometry was then used for detection.

[0187] The results are as follows Figure 8 , showing that the 340L-Fc fusion protein of the present invention and Giloralimab and Selicrelumab antibodies can all promote B cell proliferation; compared with Giloralimab antibody, the 340L-Fc fusion protein has a stronger ability to promote B cell proliferation.

[0188] Example 6: Fusion protein ELISA binding experiment

[0189] ELISA assays were used to test the binding properties of the fusion protein. Mouse FAP extracellular region His-tagged fusion protein was coated onto a 96-well plate. The signal strength after addition of the fusion protein was used to determine the binding properties of the antibody to the mouse FAP protein.

[0190] Mouse FAP protein (purchased from Acro) was diluted to 2 μg / mL with PBS buffer and added to a 96-well plate at a volume of 100 μL / well. The plate was incubated at 4°C overnight. The PBS buffer in the 96-well plate was aspirated, and the plate was washed 6 times with PBST (pH 7.2 PBS containing 0.1% Tween 20). 200 μL / well of PBS / 10% BSA was added and the plate was incubated at 37°C for 2 hours for blocking. The blocking solution was removed, and the plate was washed 6 times with PBST. 100 μL / well of the FAP × 340L to be tested (the amino acid sequence of 340L is shown in SEQ ID NO: 11, the amino acid sequence of the FAP antibody heavy chain is shown in SEQ ID NO: 12, and the amino acid sequence of the FAP antibody light chain is shown in SEQ ID NO: 13) was added with a serial dilution of PBST / 0.05% BSA and incubated at 37°C for 1 hour. Remove the reaction mixture, wash the plate six times with PBST, and then add 100 μL / well of HRP-conjugated anti-human IgG secondary antibody (purchased from JacksonLab) diluted in PBST / 0.05% BSA. Incubate at 37°C for 1 hour. Wash the plate six times with PBST, then add 80 μL / well of TMB (tetramethylbenzidine), incubate at room temperature for 3 minutes, and terminate the reaction by adding 80 μL / well of 4 M sulfuric acid. Read the absorbance at 450 nm using a microplate reader.

[0191] The results are as follows Figure 9 , indicating that the FAP×340L fusion protein of the present invention can bind to mouse FAP protein.

[0192] Example 7: Bridging ELISA experiment of fusion protein

[0193] ELISA assays were used to test the binding properties of a fusion protein bridging FAP and CD40 proteins. Mouse FAP extracellular domain His-tagged fusion protein was coated into a 96-well plate, and the fusion protein and CD40-Biotin protein were added. The signal strength after addition was used to determine the binding properties of the fusion protein bridging mouse FAP and CD40 proteins.

[0194] Mouse FAP protein (purchased from Acro) was diluted to 2 μg / mL with PBS buffer and added to a 96-well plate at a volume of 100 μL / well. The plate was incubated at 4°C overnight. The PBS buffer in the 96-well plate was aspirated and washed six times with PBST (pH 7.2 PBS containing 0.1% Tween 20). 200 μL / well of PBS / 10% BSA was added and the plate was incubated at 37°C for 2 hours for blocking. The blocking solution was removed and the plate was washed six times with PBST. 100 μL / well of the FAP × 340L (the amino acid sequence of 340L is shown in SEQ ID NO: 11, the amino acid sequence of the FAP antibody heavy chain is shown in SEQ ID NO: 12, and the amino acid sequence of the FAP antibody light chain is shown in SEQ ID NO: 13) and CD40-Biotin protein (purchased from Acro) were added and incubated at 37°C for 1 hour. Remove the reaction mixture, wash the plate six times with PBST, and then add 100 μL / well of HRP (horseradish peroxidase)-conjugated Streptavidin secondary antibody (purchased from JacksonLab) diluted in PBST / 0.05% BSA. Incubate at 37°C for 1 hour. Wash the plate six times with PBST, then add 80 μL / well of TMB (tetramethylbenzidine). Incubate at room temperature for 3 minutes. Terminate the reaction by adding 80 μL / well of 4 M sulfuric acid. Read the absorbance at 450 nm using a microplate reader.

[0195] The results are as follows Figure 10 , indicating that the FAP×340L fusion protein of the present invention can bridge mouse FAP protein and CD40 protein.

[0196] Example 8: Anticancer Experiment of Fusion Protein in Mouse Model

[0197] In vivo efficacy experiments were used to test the anti-cancer effect of the fusion protein of the present invention in mice. The specific method is as follows:

[0198] (1) On day -1, C57BL / 6 mice were subcutaneously injected with 2×10 5 MC38 colorectal cancer cells;

[0199] (2) On day 0, mice were weighed and randomly divided into groups based on the weighing results;

[0200] (4) On days 0, 5, 8, and 14, mice were injected with FAP×340L (the amino acid sequence of 340L is shown in SEQ ID NO: 11, the amino acid sequence of the FAP antibody heavy chain is shown in SEQ ID NO: 12, and the amino acid sequence of the FAP antibody light chain is shown in SEQ ID NO: 13) and the solvent control PBS, 250 μL per mouse;

[0201] (4) After injection of the above antibodies, the tumor volume was measured and the mice were weighed twice a week.

[0202] The results are as follows Figure 13 As shown, FAP×340L fusion protein has significant anti-cancer function.

[0203] These results demonstrate that the fusion protein of the present invention can bind to CD40, promoting B cell activation and proliferation, thereby effectively boosting immune responses and enhancing the anti-cancer capabilities of the immune system, demonstrating excellent anti-cancer activity. Therefore, the fusion protein of the present invention has promising clinical application and drug development value.

[0204] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0205] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A fusion protein, characterized in that include: CD40L extracellular segment; among them, The number of the CD40L extracellular segments is an integer greater than or equal to 3, and the C-terminus and N-terminus of each CD40L extracellular segment are sequentially connected in series.

2. The fusion protein according to claim 1, characterized in that The fusion protein further comprises: at least two connecting peptides, and the fusion protein comprises, from the N-terminus to the C-terminus, the CD40L extracellular segment, the first connecting peptide, the CD40L extracellular segment, the second connecting peptide and the CD40L extracellular segment; Optionally, the first connecting peptide or the second connecting peptide has an amino acid sequence as shown in (GGGGS)n, wherein n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; Optionally, the first connecting peptide and the second connecting peptide have an amino acid sequence as shown in (GGGGS)n, wherein n is 4; Optionally, the extracellular segment of CD40L has an amino acid sequence as shown in SEQ ID NO: 1 or an amino acid sequence having at least 80% identity thereto; Optionally, the fusion protein has the amino acid sequence shown in SEQ ID NO: 3 or an amino acid sequence with at least 80% identity thereto.

3. The fusion protein according to claim 1, characterized in that The fusion protein further comprises: a first Fc fragment and a second Fc fragment; wherein, The CD40L extracellular segment is connected to the first Fc fragment; The first Fc fragment and the second Fc fragment are linked; Optionally, the C-terminus of the CD40L extracellular segment is connected to the N-terminus of the first Fc fragment.

4. The fusion protein according to claim 3, characterized in that The first Fc fragment and the second Fc fragment are connected via a third connecting peptide; The C-terminus of the first Fc fragment is connected to the N-terminus of the third connecting peptide, and the C-terminus of the third connecting peptide is connected to the N-terminus of the second Fc fragment; Optionally, the third connecting peptide has an amino acid sequence as shown in (GGGGS)n, wherein n is an integer greater than or equal to 1, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; Optionally, the third connecting peptide has an amino acid sequence as shown in (GGGGS)n, wherein n is 6; Optionally, at least a portion of the first Fc fragment or the second Fc fragment is derived from at least one of a mouse antibody, a human antibody, a primate antibody, a bovine antibody, an equine antibody, a bovine antibody, a porcine antibody, a sheep antibody, a goat antibody, a dog antibody, a cat antibody, a rabbit antibody, a camel antibody, a donkey antibody, a deer antibody, a mink antibody, a chicken antibody, a duck antibody, a goose antibody, a turkey antibody, a fighting cock antibody, or a mutant thereof; Optionally, the first Fc fragment or the second Fc fragment comprises an Fc segment selected from IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE or IgD, preferably IgG1; Optionally, the first Fc fragment or the second Fc fragment is derived from a murine antibody or a mutant thereof, a human antibody or a mutant thereof; Optionally, the first Fc fragment or the second Fc fragment has the amino acid sequence shown in SEQ ID NO: 14 or an amino acid sequence that is at least 80% identical thereto; Optionally, the first Fc fragment and the second Fc fragment are connected via a disulfide bond; Optionally, the first Fc fragment or the second Fc fragment has the amino acid sequence shown in SEQ ID NO: 15 or an amino acid sequence that is at least 80% identical thereto; Optionally, the first Fc fragment and the second Fc fragment are connected via a knob-into-hole structure; Optionally, the first Fc fragment or the second Fc fragment has an amino acid sequence as shown in SEQ ID NO: 16 or 17, or an amino acid sequence having at least 80% identity thereto.

5. The fusion protein according to claim 3, characterized in that The fusion protein has an amino acid sequence as shown in SEQ ID NO: 6 or an amino acid sequence having at least 80% identity thereto; Optionally, the fusion protein comprises: a first polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 18, or an amino acid sequence at least 80% identical thereto, and a second polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 15, or an amino acid sequence at least 80% identical thereto; Optionally, the fusion protein comprises: a first polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 11, or an amino acid sequence at least 80% identical thereto, and a second polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 17, or an amino acid sequence at least 80% identical thereto; Optionally, the fusion protein comprises: a first polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 19 or an amino acid sequence with at least 80% identity thereto, and a second polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 16 or an amino acid sequence with at least 80% identity thereto.

6. The fusion protein according to claim 3, characterized in that The fusion protein further comprises: a FAP antigen binding region; wherein the FAP antigen binding region is connected to the second Fc fragment; Optionally, the FAP antigen-binding region comprises an antigen-binding fragment of an anti-FAP antibody; Optionally, the antigen-binding fragment of the anti-FAP antibody is: a Fab fragment, a scFab fragment or a scFabΔ fragment of the anti-FAP antibody, preferably a Fab fragment; Optionally, the VH-CH1 of the Fab fragment has the amino acid sequence as shown in SEQ ID NO: 20, or an amino acid sequence with at least 80% identity thereto, and the VL-CL of the Fab fragment has the amino acid sequence as shown in SEQ ID NO: 13, or an amino acid sequence with at least 80% identity thereto; Optionally, the C-terminus of the CH1 fragment of the Fab fragment is connected to the N-terminus of the second Fc fragment; Optionally, the fusion protein comprises: a first polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 11 or an amino acid sequence with at least 80% identity thereto, a second polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 12 or an amino acid sequence with at least 80% identity thereto, and a third polypeptide chain having an amino acid sequence as shown in SEQ ID NO: 13 or an amino acid sequence with at least 80% identity thereto.

7. A nucleic acid, characterized in that Encodes the fusion protein according to any one of claims 1 to 6.

8. A vector or transformant, characterized in that: Containing the nucleic acid according to claim 7.

9. A cell, characterized in that Carrying the nucleic acid according to claim 7, the vector or transformant according to claim 8, or expressing the fusion protein according to any one of claims 1 to 6.

10. A pharmaceutical composition, characterized in that Comprising: the fusion protein according to any one of claims 1 to 6, the nucleic acid according to claim 7, or the vector or transformant according to claim 8; Optionally, further comprising a pharmaceutically acceptable excipient; Optionally, the auxiliary materials include: one or more pharmaceutically acceptable excipients, diluents, stabilizers or carriers; Optionally, the pharmaceutical composition is an injection.

11. A kit, characterized in that Comprising the fusion protein according to any one of claims 1 to 6.

12. Use of the fusion protein according to any one of claims 1 to 6 in preparing a kit, The kit is used for detecting and / or enriching and / or separating and purifying CD40; Optionally, the kit detects CD40 by ELISA, flow cytometry, immunoblotting or immunoprecipitation; Optionally, the kit is used to enrich and / or separate and purify CD40 by affinity chromatography.

13. A method for detecting, enriching or separating and purifying CD40, characterized in that: Include: The fusion protein according to any one of claims 1 to 6 or the fusion protein in the kit according to claim 11 is contacted with a biological sample, wherein the biological sample contains CD40.

14. Use of the fusion protein according to any one of claims 1 to 6, the nucleic acid according to claim 7, the vector or transformant according to claim 8, or the pharmaceutical composition according to claim 10 in preparing a drug, characterized in that: The medicament is used for preventing and / or treating cancer.

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