Fusion protein and T cell therapeutic drug based on GLP-1 analogue and application of fusion protein and T cell therapeutic drug in preparation of antitumor drugs

Abstract

The invention provides a fusion protein based on a GLP-1 analogue, a T cell therapeutic drug and an application of the fusion protein and the T cell therapeutic drug in tumor resistance, and relates to the field of preparation of antibodies and cell therapeutic drugs. The fusion protein based on the GLP-1 analogue is obtained by fusing a T cell activation antibody and the GLP-1 analogue, the T cell activation antibody is a BiTE antibody, the BiTE antibody is a CD3 / EpCAM fusion antibody, and the GLP-1 analogue is dulaglycopeptide; the CD3 / EpCAM fusion antibody is composed of a light chain and a heavy chain of a CD3 antibody and a light chain and a heavy chain of an EpCAM antibody, wherein the heavy chain of the CD3 antibody and the heavy chain of the EpCAM antibody are connected through a peptide linker; the C end of the dulatide is fused with the N end of the light chain of the CD3 antibody, and the middle is connected with the N end through a peptide joint; the obtained fusion protein based on the GLP-1 analogue can be used for preparing anti-tumor drugs and T cell treatment drugs, and has the effects of reducing toxicity and enhancing efficacy.

Description

Technical Field

[0001] The present invention belongs to the field of antibody and drug preparation, and relates to GLP-1 analogs, and specifically to fusion proteins and T cell therapeutic drugs based on GLP-1 analogs and their use in the preparation of anti-tumor drugs. Background Art

[0002] Traditional cancer treatments present insurmountable challenges, such as recurrence and drug resistance, necessitating the search for more effective therapies. Immunotherapy for solid tumors is a revolutionary approach that activates the patient's own immune system to recognize and attack cancer cells. In recent years, immunotherapy has made tremendous progress, with the emergence of a range of emerging therapeutic approaches, including immune checkpoint inhibitors, bispecific T-cell adaptors (BiTEs), and cell therapies. Despite its significant clinical potential, immunotherapy still faces numerous significant challenges in the treatment of solid tumors. On the one hand, solid tumors possess a unique tumor microenvironment that significantly restricts the infiltration and activation of immune cells, reducing the responsiveness of immunotherapy. On the other hand, immunotherapy is associated with potential side effects, such as systemic immunotoxicity caused by immune checkpoint inhibitors and cytokine release syndrome (CRS) induced by BiTEs and CAR-T cell therapy. Addressing the source of immunotherapy, while promoting anti-tumor immune efficacy and avoiding immunotoxicity, remains a pressing clinical challenge in the field of immunotherapy.

[0003] The glucagon-like peptide-1 receptor (GLP-1R) was originally thought to be primarily expressed in pancreatic tissue. Its classic functions include improving insulin resistance and regulating lipid metabolism, and it holds important clinical applications in the treatment of diabetes and obesity. In recent years, the role of the GLP-1R in the immune system has garnered widespread attention. Numerous studies have demonstrated that GLP-1 analogs can directly inhibit the expression of multiple inflammatory factors independent of systemic glucose and weight loss. GLP-1 analogs induce anti-inflammatory and immunomodulatory effects by targeting the GLP-1R on the surface of immune cells. The GLP-1R is expressed in both T and B cells in mice and humans, and is also expressed on immature lymphocyte subsets, suggesting a crucial role for GLP-1R signaling in lymphocyte maturation. A recent study reported a crucial regulatory role for the GLP-1R in T cells, finding that GLP-1R+ T cells exhibited enhanced proliferation but significantly reduced inflammatory cytokine release. The above studies have revealed that the GLP-1R pathway may have the potential to inhibit the release of inflammatory factors caused by T cell activation.

[0004] In the 1980s, Peter Kufer and others proposed BiTE bispecific antibody technology. BiTEs are also fusion proteins with two distinct domains: one that binds to an antigen on the surface of a target cell, and the other that binds to a specific receptor on the surface of an immune cell (typically a T cell). Specifically, BiTEs are simple bispecific antibodies that lack an Fc fragment. They combine two single-chain antibodies (scFv) linked by a short peptide chain. Compared to conventional antibodies, the volume and molecular weight of each peptide chain are significantly reduced. BiTEs are designed to act as a bridge between tumor cells and cytotoxic T cells. One arm binds to the T cell surface antigen CD3, and the other binds to a tumor-associated antigen (TAA). When both arms of a BiTE bind to their specific targets, a synapse forms between the T cell and the cancer cell, triggering the release of perforin and granzymes by the T cell, mediating cancer cell death.

[0005] Epcam (epithelial cell adhesion molecule), also known as CD326, belongs to the GA733 protein family. It is a type I transmembrane glycoprotein and a Ca2+-independent cell adhesion molecule. It is expressed at low levels in the basement membranes of various normal epithelial cells (with highest expression in the colon) and is widely expressed on the membranes of cancer cells. Many promising Epcam antibodies have been reported in the clinic. Catumaxomab (Removab) has two antigen-binding sites (Epcam and CD3) and an Fc domain, and can trigger T cell-mediated killing, cytokine-related cytotoxicity, antibody-dependent cell-mediated cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC) in vitro. In a phase II / III study of patients with malignant ascites, catumaxomab treatment significantly prolonged overall survival (OS). MM-131 is an anti-Epcam / Met bispecific antibody that inhibits Met signaling pathways in both a dependent and an independent manner, inhibiting Met-induced proliferation in hepatocellular carcinoma, BC, gastric cancer, and lung cancer. Solitomab (MT110) is a bispecific T cell-engaging antibody (BITE) that binds to EpCAM and CD3 and is effective in treating primary uterine and ovarian carcinosarcoma. MT110's mechanism of action relies primarily on the release of perforin and granzymes upon T cell activation. However, solitomab was ultimately discontinued due to its gastrointestinal toxicity. The EpCAM antisense oligonucleotide 1H8 / CD3 successfully inhibited the growth of hepatocellular carcinoma xenografts and reduced the expression of most CSC biomarkers. To reduce the harmful cytokine toxicity caused by T cell activation, a bispecific NK cell activator (BIKE) targeting CD16 on NK cells and EPCAM on tumor cells was developed to mediate ADCC against EPCAM+ HT-29 colon cancer cells. A trispecific NK cell activator (TRIKE) modified with an interleukin-15 crosslinker improved NK cell activation, proliferation, and survival compared to BIKE.

[0006] Prior art CN102725312A discloses a protein drug, wherein the first peptide of the protein may have an amino acid sequence HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR, and the second peptide may have an amino acid sequence MSTKKSPEELKR IFEKYAAKEGDPDQLSKDELKLLIQAEFPSLLKGPNTLDDLFQELDKNG, and the label is gadolinium (Gd+), whereby the label is detectable by MRI, and wherein the fusion protein binding site is the GLP-1 receptor of pancreatic cells. The second peptide increases the stability of the first peptide to which it is linked; wherein the protein is more stable than the first peptide alone; this enables effective tissue penetration and retention; and prolonged circulation time; remains intact and retains activity; wherein the fusion protein is more stable than the first peptide alone; thereby determining the presence of the protein binding site on the target cell; wherein the fusion protein is more stable than the first peptide alone; this facilitates effective tissue penetration and retention.

[0007] Prior art US20250099548A1 discloses a pharmaceutical composition for preventing or treating degenerative brain diseases using glucagon-like peptide-1 (GLP-1) and interleukin-1 receptor antagonist (IL-1Ra). The composition comprising a fusion protein including GLP-1 and IL-1Ra exhibits the effects of inhibiting the production of amyloid beta and improving memory and cognitive function. Amyloid beta is known to be a substance that causes Alzheimer's disease. In addition, when a bispecific antibody comprising GLP-1 and an anti-IL-1 antibody or a fusion protein in which IL-1Ra and Fc are connected is administered, the deposition of beta-amyloid plaques in the brain is reduced, and a neurogenesis-promoting effect is exhibited in Alzheimer's disease model mice. In addition, when GLP-1 and IL-1Ra are also co-administered, the deposition of amyloid beta plaques in the brains of Alzheimer's disease model mice is reduced. The composition comprising GLP-1 and IL-1R can be effectively used for preventing or treating degenerative brain diseases such as Alzheimer's disease, Parkinson's disease, and Huntington's disease, and alleviating cognitive impairment.

[0008] A large number of research results support that GLP-1R can be used as an effective and safe target for the clinical prevention and treatment of tumors, but there is currently a lack of application of fusion proteins designed based on GLP-1 analogs in anti-tumor applications. Summary of the Invention

[0009] The present invention addresses the problems existing in the prior art and provides a fusion protein based on a GLP-1 analogue and a T cell therapy drug, and their use in the preparation of anti-tumor drugs. The GLP-1 analogue-based fusion protein provided by the present invention is obtained by fusing a T cell-activating antibody and a GLP-1 analogue, wherein the T cell-activating antibody is a BiTE antibody, which is a CD3 / EpCAM fusion antibody, and the GLP-1 analogue is dulaglutide; the CD3 / EpCAM fusion antibody is composed of the light and heavy chains of the CD3 antibody and the light and heavy chains of the EpCAM antibody, and the heavy chains of the CD3 antibody and the EpCAM antibody are connected by a peptide linker; the C-terminus of the dulaglutide is fused to the N-terminus of the light chain of the CD3 antibody, and the connection is made by a peptide linker in the middle; the resulting fusion protein based on a GLP-1 analogue can be used to prepare anti-tumor drugs and T cell therapy drugs, and has the effect of reducing toxicity and increasing efficacy.

[0010] To achieve the above object, the technical solution adopted by the present invention is as follows: On the one hand, the present invention provides a fusion protein based on a GLP-1 analogue, wherein the fusion protein is obtained by fusing a T cell activating antibody and a GLP-1 analogue, and the T cell activating antibody is selected from any one of a BiTE antibody, an immune checkpoint antibody or a T cell activating antibody.

[0011] Preferably, the BiTE antibody includes a CD3 / EpCAM fusion antibody, a CD3 / CLDN18.2 fusion antibody, a CD3 / CD19 fusion antibody, a CD3 / CD20 fusion antibody, a CD3 / BCMA fusion antibody, a CD3 / PSMA fusion antibody, a CD3 / HER2 fusion antibody, a CD3 / GPRC5D fusion antibody, a CD3 / CD38 fusion antibody, a CD3 / FLT3 fusion antibody or a CD3 / ROR1 fusion antibody.

[0012] Preferably, the BiTE antibody is a CD3 / EpCAM fusion antibody or a CD3 / CLDN18.2 fusion antibody.

[0013] Preferably, the immune checkpoint antibodies include PD-1 antibodies, CTLA4 antibodies, TIGIT antibodies or LAG3 antibodies.

[0014] Preferably, the T cell activating antibody comprises OX40 antibody, 4-1BB antibody or CD28 antibody.

[0015] Preferably, the GLP-1 analogue is selected from the group consisting of natural GLP-1 molecule, dulaglutide, liraglutide, semaglutide, albiglutide, exenatide or lixisenatide.

[0016] Preferably, the T cell activating antibody is a BiTE antibody, and the BiTE antibody is a CD3 / EpCAM fusion antibody.

[0017] Preferably, the GLP-1 analog is dulaglutide.

[0018] Preferably, the CD3 / EpCAM fusion antibody is composed of the light and heavy chains of the CD3 antibody and the light and heavy chains of the EpCAM antibody, and the heavy chain of the CD3 antibody and the heavy chain of the EpCAM antibody are connected by a peptide linker; the C-terminus of the dulaglutide is fused to the N-terminus of the light chain of the CD3 antibody, and the middle is connected by a peptide linker to form a fusion protein. The structural diagram is shown in FIG. Figure 1 shown.

[0019] Preferably, the heavy chain sequence of the CD3 antibody includes the amino acid sequence shown in SEQ ID NO.1, the light chain sequence of the CD3 antibody includes the amino acid sequence shown in SEQ ID NO.2, the heavy chain sequence of the EpCAM antibody includes the amino acid sequence shown in SEQ ID NO.3, and the light chain sequence of the EpCAM antibody includes the amino acid sequence shown in SEQ ID NO.4.

[0020] Preferably, the sequence of dulaglutide is the amino acid sequence shown in SEQ ID NO.5, SEQ ID NO.5 (sequence of dulaglutide): HGEGTFTSDVSSYLEGQAAKEFIAWLVKGGG Preferably, the peptide linker comprises a flexible peptide linker, and the flexible peptide linker is a repeating sequence comprising the amino acid sequence shown in SEQ ID NO. 6, SEQ ID NO. 6: GGGGS.

[0021] Preferably, the peptide linker is the amino acid sequence shown in SEQ ID NO.7, SEQ ID NO.7: GGGGSGGGGSGGGGS.

[0022] Part of the sequence information involved in the present invention is as follows: SEQ ID NO.1 (heavy chain sequence of CD3 antibody): EVQLVESGGGLVQPGKSLKLSCEASGFTFSGYGMHWVRQAPGRGLESVAYITSSSINIKYADAVKGRFTVSRDNAKNLLFLQMNILKSEDTAMYYCARFDWDKNYWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV。

[0023] SEQ ID NO.2 (Light chain sequence of CD3 antibody): DIQMTQSPSSLPASLGDRVTINCQASQDISNYLNWYQQKPGKAPKLLIYYTNKLADGVPSRFSGSGSGRDSSFTISSLESEDIGSYYCQQYYNYPWTFGPGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0024] SEQ ID NO.3 (Heavy chain sequence of EpCAM antibody): DIQMTQSPASLSASLGETVSIECLASEGISNDLAWYQQKSGKSPQLLIYATSRLQDGVPSRFSGSGSGTRYSLKISGMQPEDEADYFCQQSYKYPWTFGGGTKLELKASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV。

[0025] SEQ ID NO.4 (Light chain sequence of EpCAM antibody): EVQLAESGGGLVQPGRSMKLSCAASGFTFSNFPMAWVRQAPTKGLEWVATISTSGGSTYYRDSVKGRFTISRDNAKSTLYLQMNSLRSEDTATYYCTRTLYILRVFYFDYWGQGVMVTVSSRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0026] SEQ ID NO.8 (Heavy chain sequence of CLDN18.2 antibody): DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPITFGQGTRLEIKASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV。

[0027] SEQ ID NO.9 (Light chain sequence of CLDN18.2 antibody): QVQLVESGGGLVQPGGSLRLSCAASGFTFRTYEMNWVRQAPGKGLEWVSYITGSGSTTYYADSVKGRFTMSRDNAKNSLYLQMNSLRAEDTAVYYCARYWNYAGGMDVWGQGTTVTVSSRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0028] SEQ ID NO.10 (Heavy chain sequence of PD-1 antibody) QVQLVQSGAEVKKPGSSVKVSCKASGFTFTTYYISWVRQAPGQGLEYLGYINMGSGGTNYNEKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCAIIGYFDYWGQ GTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRV.

[0029] SEQ ID NO.11 (light chain sequence of PD-1 antibody) DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGGTYLYWFQQRPGQSPRRLIYLVSTLGSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQLTHWPYTFGQGTKLE IKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0030] In some embodiments, the BiTE antibody can be fused with multiple GLP-1 analogs to enhance T cell activation, for example, one BiTE antibody can be fused with 1, 2, 3, or 4 GLP-1 analogs.

[0031] In another aspect, the present invention provides a nucleic acid encoding the above-mentioned fusion protein.

[0032] On the other hand, the present invention provides an expression vector comprising the above-mentioned nucleic acid, or used to express the above-mentioned fusion protein.

[0033] Preferably, the expression vector comprises the nucleic acid sequence shown in SEQ ID NO.13.

[0034] On the other hand, the present invention provides a host cell, wherein the host cell comprises the aforementioned nucleic acid, or the host cell comprises the aforementioned expression vector, or the host cell is used to express the aforementioned fusion protein.

[0035] On the other hand, the present invention provides a pharmaceutical composition comprising the above-mentioned fusion protein, the above-mentioned nucleic acid, the above-mentioned expression vector or the above-mentioned host cell, and a pharmaceutically acceptable carrier.

[0036] On the other hand, the present invention provides use of the above-mentioned fusion protein, the above-mentioned nucleic acid, the above-mentioned expression vector, the above-mentioned host cell or the above-mentioned pharmaceutical composition in the preparation of anti-tumor drugs.

[0037] Preferably, the tumor includes any one or more of colon cancer, kidney tumor, breast cancer, liver cancer, pancreatic cancer, multiple myeloma, gastric cancer, lung cancer, glioma, cervical cancer, melanoma, and sarcoma.

[0038] Preferably, the application also includes application in the preparation of T cell therapeutic drugs.

[0039] Preferably, the T cell therapy drug includes CAR-T cells, TCR-T cells, TIL cells or γδ T cells.

[0040] In some embodiments, the use further comprises preparing a medicament for treating autoimmune diseases, including systemic lupus erythematosus, rheumatoid arthritis, psoriasis, Crohn's disease, myasthenia gravis, and multiple sclerosis.

[0041] Compared with the prior art, the present invention has the following beneficial effects: The GLP-1 analogue-based fusion protein provided by the present invention is obtained by fusing a T cell-activating antibody and a GLP-1 analogue, wherein the T cell-activating antibody is a BiTE antibody, the BiTE antibody is a CD3 / EpCAM fusion antibody, and the GLP-1 analogue is dulaglutide; the CD3 / EpCAM fusion antibody is composed of the light and heavy chains of the CD3 antibody and the light and heavy chains of the EpCAM antibody, and the heavy chain of the CD3 antibody and the heavy chain of the EpCAM antibody are connected by a peptide linker; the C-terminus of the dulaglutide is fused to the N-terminus of the light chain of the CD3 antibody, and the middle is connected by a peptide linker; the obtained fusion protein based on the GLP-1 analogue can be used to prepare anti-tumor drugs and can be used to prepare T cell therapeutic drugs, and has the effect of reducing toxicity and increasing efficacy. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 Schematic diagram of Dula-EpCAM BiTE antibody.

[0043] Figure 2 Schematic diagram of EpCAM BiTE antibody.

[0044] Figure 3 Comparison of the binding ability of Dula-EpCAM BiTE and EpCAM BiTE to EpCAM.

[0045] Figure 4 Comparison of the ability of fusion antibodies to activate GLP-1R.

[0046] Figure 5 Comparison of the tumor cell killing abilities of Dula-EpCAM BiTE and EpCAM BiTE.

[0047] Figure 6 is the concentration of TNF-α secreted after Dula-EpCAM BiTE and EpCAM BiTE kill tumor cells.

[0048] Figure 7 is the concentration of IFN-γ secreted after Dula-EpCAM BiTE and EpCAM BiTE kill tumor cells.

[0049] Figure 8 The effects of EpCAM BiTE and Dula-EpCAM BiTE on 4T1 tumor volume; * indicates P <0.05, ** indicates P <0.01.

[0050] Figure 9 Effects of EpCAM BiTE and Dula-EpCAM BiTE on the survival of 4T1 model mice.

[0051] Figure 10 Figure 3 shows the effects of EpCAM BiTE and Dula-EpCAM BiTE on MC38-EpCAM tumor volume.

[0052] Figure 11 The effects of EpCAM BiTE and Dula-EpCAM BiTE on the survival of MC38-EpCAM model mice.

[0053] Figure 12 is the serum IFN-γ concentration after administration of EpCAM BiTE and Dula-EpCAM BiTE.

[0054] Figure 13 is the serum IL-6 concentration after administration of EpCAM BiTE and Dula-EpCAM BiTE.

[0055] Figure 14 is the serum TNF-α concentration after administration of EpCAM BiTE and Dula-EpCAM BiTE.

[0056] Figure 15 These are the toxicity test results of EpCAM BiTE and Dula-EpCAM BiTE.

[0057] Figure 16 Schematic diagram of the structure of Dula-CLDN18.2 BiTE.

[0058] Figure 17 Comparison of the binding ability of CLDN18.2 BiTE and Dula-CLDN18.2 BiTE to CLDN18.2.

[0059] Figure 18 Schematic diagram of the structure of Dula-αPD-1.

[0060] Figure 19 Comparison of the binding ability of αPD-1 antibody and Dula-αPD-1 to PD-1.

[0061] Figure 20 Schematic diagram of the preparation process of CAR-T cells that secrete GLP-1.

[0062] Figure 21 The expression of EpCAM scfv on the surface of CAR-T cells; A is a CD8+ T cell not infected with the virus, B is a CD8+ T cell infected with the pMSCV-EpCAM virus, and C is a CD8+ T cell infected with the pMSCV-EpCAM-GLP1 virus.

[0063] Figure 22 The in vitro killing activity of CAR-T cells and CAR-T-GLP-1 cells; the control group refers to the control group without additional cells, the Naïve T group, CAR-T group, and CAR-T-GLP-1 group refer to the Panc02-EpCAM-luc cells added with Naïve T cells, CAR-T cells, and CAR-T-GLP-1 cells, respectively; ns indicates no significant difference between the control group and the Naïve T group, and between the CAR-T group and the CAR-T-GLP-1 group; *** indicates P <0.001.

[0064] Figure 23 Comparison of the anti-tumor activity of CAR-T cells in vivo; ns indicates no significant difference between the groups, *** indicates P <0.001.

[0065] Figure 24 The effect of CAR-T cells on mouse survival. DETAILED DESCRIPTION

[0066] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0067] Unless the context clearly dictates otherwise, as used herein, the expressions "a" and "an" include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0068] As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

[0069] As used herein, the term "comprises" or "comprising" means "including but not limited to". The term is intended to be open-ended to specify the presence of any of the described features, elements, integers, steps or components, but does not exclude the presence or addition of one or more other features, elements, integers, steps, components or groups thereof. Therefore, the term "comprising" includes the more restrictive terms "consisting of" and "consisting essentially of". In one embodiment, the term "comprising" used throughout the application, particularly in the claims, may be replaced by the term "consisting of". The three-letter and one-letter codes for amino acids used herein are known to those skilled in the art or as described in J Biol. Chem, 243, p3558 (1968).

[0070] As used herein, the terms "optionally," "either," "any," or "any" mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur. For example, "optionally comprising an antibody heavy chain variable region" means that an antibody heavy chain variable region of a specified sequence may but need not be present.

[0071] As used herein, the term "about" refers to a range of ±20% of the value that follows. In some embodiments, the term "about" refers to a range of ±10% of the value that follows. In some embodiments, the term "about" refers to a range of ±5% of the value that follows.

[0072] The term "and / or" used herein should be understood to mean any one of the options or a combination of any two or more of the options.

[0073] The term "fusion protein" as used herein refers to a novel protein produced by fusing a biologically active functional protein molecule with another protein (fusion partner) using genetic engineering or other techniques. Functional proteins are typically endogenous ligands (or corresponding receptors), such as cytokines, hormones, growth factors, enzymes, and other active substances. Fusion partners primarily include immunoglobulins, albumin, transferrin, and the like. Fusion partners also include biologically active proteins or functional fragments thereof that facilitate expression and / or secretion or extend in vivo half-life. Such biologically active proteins or functional fragments are selected from at least one of an immunoglobulin Fc domain, serum albumin, an albumin-binding polypeptide, prealbumin, a carboxy-terminal peptide, an elastin-like polypeptide, a His tag, a GST tag, an MBP tag, a FLAG tag, and a SUMO tag. The immunoglobulin Fc domain is derived from a human antibody, a murine antibody, a primate antibody, a camelid antibody, or a variant thereof. Preferably, the immunoglobulin Fc domain is derived from a human IgG antibody, such as IgG1 Fc, IgG2 Fc, IgG3 Fc, or IgG4 Fc, preferably IgG1 Fc.

[0074] As used herein, the term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as modified amino acids such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs are compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., a carbon bonded to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. These analogs have modified R groups (e.g., norleucine) or modified peptide backbones but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics are compounds that have a structure that differs from the general chemical structure of an amino acid but functions in a manner similar to that of a naturally occurring amino acid.

[0075] As used herein, the terms "activity," "functional activity," or "biological activity," or the terms "biological property" or "biological characteristic" are used interchangeably herein and include, but are not limited to, epitope / antigen affinity and specificity, the ability to neutralize or antagonize antibody activity in vivo or in vitro, IC50, in vivo stability of the antibody, and the immunogenic properties of the antibody. Other identifiable biological properties or characteristics of antibodies known in the art include, for example, cross-reactivity (i.e., cross-reactivity with non-human homologs of the target peptide, or with other proteins or tissues), and the ability to maintain high protein expression levels in mammalian cells. The aforementioned properties or characteristics can be observed, measured, or assessed using techniques known in the art, including, but not limited to, ELISA, FACS, or BIACORE plasmon resonance analysis, in vitro or in vivo neutralization assays, receptor binding, cytokine or growth factor production and / or secretion, signal transduction, and immunohistochemistry of tissue sections from various sources (including humans, primates, or any other source).

[0076] As used herein, the terms "Fc," "Fc region," "Fc domain," or "Fc fragment" refer to a polypeptide consisting of the CH2 and CH3 domains of IgA, IgD, and IgG, or the CH2, CH3, and CH4 domains of IgE and IgM, connected by a hinge region. While the breakdown of the Fc fragment varies, the heavy chain Fc fragment of human IgG typically refers to the polypeptide extending from A231 to its carboxyl terminus.

[0077] The term "affinity" or "binding affinity" as used herein refers to the intrinsic binding affinity that reflects the interaction between members of a binding pair. Affinity can be measured by common methods known in the art.

[0078] The term "nucleic acid" or "polynucleotide" as used herein refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and polymers thereof in single-stranded or double-stranded form. Unless expressly limited, the term includes nucleic acids containing analogs of known natural nucleotides that have similar binding properties to reference nucleic acids and are metabolized in a manner similar to naturally occurring nucleotides (see, U.S. Patent No. 8,278,036 to Kariko et al., which discloses mRNA molecules in which uridine is replaced by pseudouridine, methods for synthesizing said mRNA molecules, and methods for delivering therapeutic proteins in vivo). Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, as well as sequences explicitly indicated. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced by mixed bases and / or deoxyinosine residues (Batzer, Nucleic Acid Res. 19: 5081 (1991); Ohtsuka, J.Biol.Chem . 260:2605-2608 (1985); Rossolini, Mol.Cell.Probes 8: 91-98 (1994)).

[0079] As used herein, the term "isolated nucleic acid" is a nucleic acid that has been identified and separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that ordinarily contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0080] As used herein, the term "vector" refers to any recombinant polynucleotide construct that can be used for the purpose of transformation (i.e., introducing heterologous DNA into a host cell). One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors with bacterial origins of replication and episomal mammalian vectors). Upon introduction into a host cell, other vectors (e.g., non-episomal mammalian vectors) integrate into the host cell's genome and are therefore replicated along with the host genome. In addition, certain vectors are capable of directing the expression of operatively linked genes. Such vectors are referred to herein as "expression vectors."

[0081] The term "expression vector" as used herein refers to a nucleic acid molecule capable of replicating and expressing a gene of interest when transformed, transfected, or transduced into a host cell. Expression vectors contain one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and, if desired, to provide for amplification within the host.

[0082] As used herein, the terms "cell" and "cell line" are used interchangeably, and all such designations include their progeny. The term "host cell" refers to a cell into which a vector can be introduced, including, but not limited to, prokaryotic cells such as E. coli, fungal cells such as yeast cells, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0083] As used herein, the term "pharmaceutical composition" generally refers to a preparation that is in a form that permits the biological activity of the active ingredient to be effective and does not contain additional ingredients that are unacceptably toxic to the subject to which the composition would be administered. The composition is sterile.

[0084] As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio, within the scope of sound medical judgment. As used herein, the term "pharmaceutically acceptable carrier, excipient, and / or diluent" refers to a carrier that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, and is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995). Pharmaceutically acceptable materials, compositions, or vehicles, such as liquid or solid fillers, diluents, excipients, solvents, media, encapsulating materials, manufacturing aids, or solvent encapsulating materials, are involved in maintaining the stability, solubility, or activity of the antibodies or antigen-binding fragments thereof of the present disclosure, and include, but are not limited to, pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, and preservatives. For example, pH adjusters include, but are not limited to, phosphate buffers. Surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80. Ionic strength enhancers include, but are not limited to, sodium chloride. Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, and sorbic acid. Agents that maintain osmotic pressure include, but are not limited to, sugars, NaCl, and their analogs. Agents that delay absorption include, but are not limited to, monostearate and gelatin. Diluents include, but are not limited to, water, aqueous buffers (such as buffered saline), alcohols, and polyols (such as glycerol). Preservatives include, but are not limited to, various antibacterial and antifungal agents, such as thimerosal, 2-phenoxyethanol, parabens, chlorobutanol, phenol, and sorbic acid. Stabilizers have the meanings generally understood by those skilled in the art, and are capable of stabilizing the desired activity of the active ingredient in the drug, including but not limited to sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin or casein) or their degradation products (such as lactalbumin hydrolysate), etc.

[0085] As used herein, the terms "therapeutically effective amount," "therapeutically effective dose," and "effective amount" refer to an amount of an antibody or antigen-binding fragment thereof of the present invention, when administered alone or in combination with other therapeutic agents to a cell, tissue, or subject, that is effective to prevent or ameliorate the symptoms of one or more diseases or conditions, or the progression of the disease or condition. A therapeutically effective dose also refers to an amount of an antibody or antigen-binding fragment thereof sufficient to result in an improvement in symptoms, e.g., an amount to treat, cure, prevent, or ameliorate a related medical condition, or to increase the rate of treatment, cure, prevention, or amelioration of such a condition. When a single active ingredient is administered to an individual, a therapeutically effective dose refers only to that ingredient. When administered in combination, a therapeutically effective dose refers to the combined amount of active ingredients that results in a therapeutic effect, whether administered in combination, sequentially, or simultaneously. An effective amount of a therapeutic agent will result in an improvement in a diagnostic criterion or parameter by at least 10%; typically by at least 20%; preferably by at least about 30%; more preferably by at least 40%, and most preferably by at least 50%.

[0086] SPSS 23.0 software was used for data analysis and statistical analysis, and t-test was used for significance analysis. P < 0.05 indicated a significant difference.

[0087] Unless otherwise specified, the raw materials used in the present invention are all common commercially available products, and their sources are not specifically limited.

[0088] Preparation example: Preparation of fusion protein based on GLP-1 analogue The GLP-1 analogue-based fusion protein provided in this preparation example is obtained by fusing a T cell-activating antibody and a GLP-1 analogue, wherein the T cell-activating antibody is a BiTE antibody, the BiTE antibody is a CD3 / EpCAM fusion antibody, and the GLP-1 analogue is dulaglutide; the CD3 / EpCAM fusion antibody is composed of the light and heavy chains of the CD3 antibody and the light and heavy chains of the EpCAM antibody, the heavy chain of the CD3 antibody and the heavy chain of the EpCAM antibody being connected by a peptide linker; the C-terminus of the dulaglutide is fused to the N-terminus of the light chain of the CD3 antibody, connected in the middle by a peptide linker; the heavy chain sequence of the CD3 antibody is the amino acid sequence shown in SEQ ID NO.1, the light chain sequence of the CD3 antibody is the amino acid sequence shown in SEQ ID NO.2, the heavy chain sequence of the EpCAM antibody is the amino acid sequence shown in SEQ ID NO.3, and the light chain sequence of the EpCAM antibody is the amino acid sequence shown in SEQ ID NO.4; the sequence of the dulaglutide is SEQ ID NO.5, and the peptide linker is the amino acid sequence shown in SEQ ID NO.7. The schematic structural diagram is shown in FIG. Figure 1 , named Dula-EpCAM BiTE; the schematic diagram of the CD3 / EpCAM fusion antibody structure without dulaglutide (GLP-1 analogue) is shown in Figure 2, named EpCAM BiTE; Dula-EpCAM BiTE and EpCAMBiTE were both prepared by Nanjing Weiyade Biotechnology Co., Ltd.

[0089] Example 1: In vitro binding activity test (1) Flow cytometry determination of antibody binding to EpCAM 4T1 cells (ATCC, CRL2539) were blocked with 5% BSA for 1 hour at 4°C, and antibodies with different concentrations were incubated with 4T1 cells for 1 hour at 4°C. After washing with PBS, 1:500 diluted FITC-labeled goat anti-human IgG secondary antibody was used for incubation at 4°C for 45 minutes. Subsequently, flow cytometry (Beckman) was used for detection. The results are shown in Figure 2. Figure 3 As shown, there is little difference in the ability of Dula-EpCAM BiTE and EpCAM BiTE to bind to EpCAM.

[0090] (2) Luciferase reporter gene assay to detect antibody activation of GLP-1R CHO-K1 cells were transfected with lentivirus to constitutively express the GLP-1R gene and cAMP response element (CRE) to construct CHO / GLP-1R CRE Luc cells. CHO / GLP-1R CRE Luc cells were seeded in 96-well plates at a density of 3,000 cells per well and incubated with antibodies at different concentrations for 4 hours. The cells were lysed using Steady-glo reagent (purchased from Promega) and the supernatant was transferred to an opaque 96-well white plate. The chemiluminescence value was detected on a multifunctional microplate reader (MD) to quantify the pathway activation efficiency. The results are shown in Figure 2. Figure 4 As shown, dulaglutide and Dula-EpCAM BiTE have similar activation abilities on GLP-1R.

[0091] Example 2: In vitro killing activity test (1) Tumor cell killing experiment For antibody-mediated killing function detection, T cells from mouse spleens were isolated using a T cell sorting kit (purchased from Miltenyi Biotec) and co-cultured with 4T1-Luc (ATCC, CRL-2539-LUC2) at an E:T ratio of 10:1. After incubation with different concentrations of serially diluted antibodies for 18 hours, the cells were lysed and luciferase substrate was added. The chemiluminescence value was measured on a multifunctional microplate reader to calculate the killing efficiency. The results are shown in Figure 2. Figure 5 As shown, there was no significant difference in the killing ability of Dula-EpCAM BiTE and EpCAM BiTE against tumor cells.

[0092] (2) Detection of inflammatory factor release The cell supernatant from the tumor killing experiment was collected and centrifuged at 12,000 rpm for 10 minutes at 4°C to remove cell debris. The TNF-α and IFN-γ concentrations in the supernatant were determined using a mouse TNF-α ELISA kit (purchased from BioLegend) and an IFN-γ ELISA kit (purchased from BioLegend). All assay steps were performed strictly according to the kit instructions. Figure 6 and Figure 7 As shown, Dula-EpCAM BiTE and EpCAM BiTE secreted similar concentrations of TNF-α and IFN-γ.

[0093] Example 3: In vivo efficacy evaluation in breast cancer models Breast cancer 4T1 cells were cultured at a rate of 5 × 10 per mouse. 5 The cells were inoculated into the mammary pad fat of female BALB / c mice until the tumor volume was about 80 mm 3 All mice were divided into four groups: PBS group, EpCAMBiTE (0.2 mg / kg) group, Dula-EpCAM BiTE (0.2 mg / kg) group, and Dula-EpCAM BiTE (1.0 mg / kg) group. All groups were injected with the tail vein according to the dosage shown in the figure. The frequency of administration was once a week. The tumor volume of the mice was measured every two days and the survival of the mice was monitored. Figure 8 As shown in Figure 2, compared with EpCAM BiTE, Dula-EpCAM BiTE can significantly slow down the increase of tumor volume, and the higher the dose, the more significant the effect; Figure 9 As shown in the mouse survival rate, Dula-EpCAM BiTE can significantly delay mouse death, and the higher the dose, the more significant the effect.

[0094] Example 4: In vivo efficacy evaluation in colon cancer model MC38 colon cancer cells were transfected with lentivirus expressing mouse EpCAM, and MC38-EpCAM cells were obtained after puromycin resistance selection. MC38-EpCAM was added at a rate of 1×10 per mouse. 6 The cells were subcutaneously inoculated into male C57BL / 6J mice until the tumor volume was about 80 mm 3All mice were divided into four groups: PBS group, EpCAMBiTE (0.2 mg / kg) group, Dula-EpCAM BiTE (0.2 mg / kg) group, and Dula-EpCAM BiTE (1.0 mg / kg) group. All groups were injected with the tail vein according to the dosage shown in the figure. The frequency of administration was once a week. The tumor volume of the mice was measured every two days and the survival of the mice was monitored. Figure 10 As shown in Figure 2, compared with EpCAM BiTE, Dula-EpCAM BiTE can significantly slow down the increase of tumor volume, and the higher the dose, the more significant the effect; Figure 11 As shown in the mouse survival rate, Dula-EpCAM BiTE can significantly prolong mouse survival, and the higher the dose, the more significant the effect.

[0095] Example 5: Safety Assessment C57BL / 6J mice were randomly divided into two groups: the EpCAM BiTE group and the Dula-EpCAM BiTE group. The EpCAM BiTE group received tail vein injections of antibodies at doses of 0.04, 0.2, 0.4, 1.0, 2.0, 3.0, and 4.0 mg / kg, respectively. The Dula-EpCAM BiTE group received tail vein injections of antibodies at doses of 0.2, 0.4, 1.0, 2.0, 4.0, 6.0, 8.0, 10.0, and 12.0 mg / kg, respectively. After 24 hours, orbital blood was collected to separate serum. The levels of inflammatory factors IFN-γ, IL-6, and TNF-α were measured using commercial ELISA kits to monitor the inflammatory cytokine storm response. The results are shown in Figure 3. Figure 12-14 As shown in Figure 2, at the same concentration, the concentrations of IFN-γ, IL-6, and TNF-α in the Dula-EpCAM BiTE group were lower than those in the EpCAM BiTE group. Figure 15 As shown in the figure, the lethal concentration of the Dula-EpCAM BiTE group was 8 mg / kg, while the lethal concentration of the EpCAM BiTE group was 3 mg / kg. Overall, Dula-EpCAM BiTE induces lower concentrations of inflammatory factors, resulting in better anti-tumor effects and lower toxicity.

[0096] In summary, Examples 1-5 show that Dula-EpCAM BiTE leads to low concentrations of inflammatory factors, better anti-tumor effects, and lower toxicity.

[0097] Example 6: Binding detection of CLDN18.2 BiTE fusion protein A CLDN18.2 BiTE fusion protein was prepared according to the method of the preparation example, wherein the EpCAM antibody was replaced with the CLDN18.2 antibody, wherein the heavy chain sequence of the CLDN18.2 antibody was the amino acid sequence shown in SEQ ID NO.8, the light chain sequence of the CLDN18.2 antibody was the amino acid sequence shown in SEQ ID NO.9, and the other sequences remained unchanged; further, the CD3 / CLDN18.2 fusion antibody not linked to a GLP-1 analogue was named CLDN18.2 BiTE; the CD3 / CLDN18.2 fusion antibody linked to a GLP-1 analogue was named Dula-CLDN18.2 BiTE, and the structural schematic is shown in FIG. Figure 16 CLDN18.2 BiTE and Dula-CLDN18.2 BiTE were both prepared by Nanjing Weiyade Biopharmaceutical Co., Ltd.

[0098] CHO cells were transfected with a lentivirus expressing human CLDN18.2, and CHO-CLDN18.2 cells were obtained after puromycin resistance screening. CHO-CLDN18.2 was blocked with 5% BSA for 1 hour at 4°C, and antibodies with different concentrations were incubated with CHO-CLDN18.2 cells at 4°C for 1 hour. After washing with PBS, 1:500 diluted FITC-labeled goat anti-human IgG secondary antibody was used to incubate at 4°C for 45 minutes. Subsequently, flow cytometry was used for detection. The results are shown in the figure. Figure 17 As shown, there was no significant difference in the binding ability of CLDN18.2 BiTE and Dula-CLDN18.2 BiTE to CLDN18.2.

[0099] Example 7: Binding Detection of PD-1 Monoclonal Antibody Fusion Protein The C-terminus of dulaglutide was fused to the N-terminus of the light chain of the PD-1 monoclonal antibody, and the two were connected by a peptide linker. The heavy chain sequence of the PD-1 antibody was the amino acid sequence shown in SEQ ID NO. 10, and the light chain sequence of the PD-1 antibody was the amino acid sequence shown in SEQ ID NO. 11. The sequences of the dulaglutide and the peptide linker were the same as those in the preparation example. Furthermore, the PD-1 antibody connected to the GLP-1 analog was named Dula-αPD-1, and the structural schematic diagram is shown in FIG. Figure 18 The PD-1 antibody not linked to the GLP-1 analogue was named αPD-1. Both Dula-αPD-1 and αPD-1 were prepared by Nanjing Viagra Biopharmaceutical Co., Ltd.

[0100] 100 ng of mouse PD-1 protein was pre-coated on a polystyrene microplate and blocked with 5% BSA at 37°C for 2 hours. Different concentrations of gradient dilutions of antibodies were added and incubated at 37°C for 1 hour. After washing with PBST, HRP-labeled mouse Fc antibody was added and incubated at 37°C for 45 minutes. TMB substrate was added and incubated at room temperature for 15 minutes. The absorbance value was read at 450 nm on a multifunctional microplate reader. The results are as follows. Figure 19 As shown, the binding ability of αPD-1 antibody to PD-1 is comparable to that of Dula-αPD-1.

[0101] Example 8: Construction and testing of GLP-1-based CAR-T cell therapy antibodies Using EpCAM as the target, a retroviral vector was constructed and packaged by plat-E cells. Activated mouse CD8+ T cells were infected by viral centrifugation to obtain GLP-1 autocrine CAR-T cells. The preparation process is shown in the schematic diagram. Figure 20 ; Among them, pMSCV-EpCAM is composed of pMSCV viral vector, CD8 signal (SEQ ID NO.12), EPCAM scfv (SEQ ID NO.13), CD28 (SEQ ID NO.14) and CD3 (SEQ ID NO.15), and the specific nucleic acid sequence of pMSCV-EpCAM is SEQ ID NO.16; pMSCV-EpCAM-GLP1 is composed of pMSCV viral vector, CD8 signal, EPCAM scfv, CD28, CD3, P2A (SEQ ID NO.17), and GLP-1 (SEQ ID NO.18), and the specific nucleic acid sequence of pMSCV-EpCAM-GLP1 is SEQ ID NO.19; CAR-T cells that successfully express pMSCV-EpCAM-GLP1 are named CAR-T-EpCAM-GLP1, and CAR-T cells that successfully express pMSCV-EpCAM are named CAR-T-EpCAM. They were prepared and synthesized by General Biotechnology Co., Ltd.

[0102] (1) CAR-T cell positivity test In a 24-well plate, the cells were coated with Retronectin (TAKARA, T100A) overnight. The next day, the retrovirus and T cells were infected by centrifugation. The retrovirus-infected CD8+ T cells were collected. After 72 hours, the expression of EpCAM scFv on the surface of CAR-T cells was detected by Protein L-FITC. The results are shown in Figure 2. Figure 21 As shown, Figure 21 A is a CD8+ T cell that is not infected with the virus. Figure 21B is CD8+ T cells infected with pMSCV-EpCAM virus, Figure 21 C represents CD8+ T cells infected with pMSCV-EpCAM-GLP1 virus. It can be seen that the positive rate of CAR-T cells expressing pMSCV-EpCAM-GLP1 is higher.

[0103] (2) Verification of CAR-T killing activity in vitro Panc02-EpCAM-luc cells were plated in a 96-well plate. When the cells grew to 80%, Naïve T cells, CAR-T cells, and CAR-T-GLP-1 cells were added and incubated for 24 hours. After 24 hours, the supernatant was discarded, luciferase substrate was added, and the killing activity was detected by microplate reader. The results are as follows. Figure 22 As shown, both CAR-T cells and CAR-T-GLP-1 cells have high killing activity in vitro.

[0104] (2) CAR-T anti-tumor activity in vivo Colon cancer cells Panc02-EpCAM were cultured at a rate of 1×10 per mouse. 6 The cells were subcutaneously inoculated into male C57BL / 6J mice until the tumor volume was approximately 100 mm 3 All mice were divided into four groups: PBS group, Naïve T group, CAR-T group and CAR-T-GLP-1 group. Each mouse was treated with 5×10 6 The cells were injected into the tail vein, and the tumor volume and survival of the mice were measured every two days. Figure 23 As shown in Figure 2, CAR-T-GLP-1 has a significant anti-tumor effect; Figure 24 As shown in the mouse survival rate, CAR-T-GLP-1 can significantly delay mouse death.

[0105] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A fusion protein based on a GLP-1 analogue, characterized in that: The fusion protein is obtained by fusing a T cell activation antibody and a GLP-1 analogue, and the T cell activation antibody is selected from any one of a BiTE antibody, an immune checkpoint antibody or a T cell activation antibody.

2. The fusion protein according to claim 1, characterized in that The BiTE antibody includes a CD3 / EpCAM fusion antibody or a CD3 / CLDN18.2 fusion antibody, the immune checkpoint antibody includes a PD-1 antibody, a CTLA4 antibody, a TIGIT antibody or a LAG3 antibody, and the T cell activation antibody includes an OX40 antibody, a 4-1BB antibody or a CD28 antibody.

3. The fusion protein according to claim 1, characterized in that The GLP-1 analog is selected from the group consisting of natural GLP-1 molecules, dulaglutide, liraglutide, semaglutide, albiglutide, exenatide or lixisenatide.

4. The fusion protein according to claim 1, characterized in that The T cell activation antibody is a BiTE antibody, the BiTE antibody is a CD3 / EpCAM fusion antibody, and the GLP-1 analog is dulaglutide.

5. The fusion protein according to claim 4, characterized in that The CD3 / EpCAM fusion antibody is composed of the light and heavy chains of the CD3 antibody and the light and heavy chains of the EpCAM antibody, and the heavy chain of the CD3 antibody and the heavy chain of the EpCAM antibody are connected by a peptide linker; the C-terminus of the dulaglutide is fused to the N-terminus of the light chain of the CD3 antibody, and the middle is connected by a peptide linker.

6. The fusion protein according to claim 5, characterized in that The heavy chain sequence of the CD3 antibody includes the amino acid sequence shown in SEQ ID NO.1, the light chain sequence of the CD3 antibody includes the amino acid sequence shown in SEQ ID NO.2, the heavy chain sequence of the EpCAM antibody includes the amino acid sequence shown in SEQ ID NO.3, and the light chain sequence of the EpCAM antibody includes the amino acid sequence shown in SEQ ID NO.

4.

7. The fusion protein according to claim 5, characterized in that The sequence of dulaglutide includes the amino acid sequence shown in SEQ ID NO.

5.

8. The fusion protein according to claim 5, characterized in that The peptide linker includes a flexible peptide linker, and the flexible peptide linker is a repeating sequence comprising the amino acid sequence shown in SEQ ID NO.

6.

9. The fusion protein according to claim 8, characterized in that The peptide linker is the amino acid sequence shown in SEQ ID NO.

7.

10. A nucleic acid, characterized in that The nucleic acid encodes the fusion protein according to any one of claims 1 to 9.

11. An expression vector, characterized in that The expression vector comprises the nucleic acid of claim 10, or is used to express the fusion protein of any one of claims 1-9.

12. The expression vector according to claim 11, characterized in that The expression vector includes the nucleic acid sequence shown in SEQ ID NO.

13.

13. A host cell, characterized in that The host cell comprises the nucleic acid according to claim 10, or the host cell comprises the expression vector according to claim 11 or 12, or the host cell is used to express the fusion protein according to any one of claims 1-9.

14. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the fusion protein according to any one of claims 1 to 9, the nucleic acid according to claim 10, the expression vector according to any one of claims 11 to 12 or the host cell according to claim 13, and a pharmaceutically acceptable carrier.

15. Use of the fusion protein according to any one of claims 1 to 9, the nucleic acid according to claim 10, the expression vector according to any one of claims 11 to 12, the host cell according to claim 13 or the pharmaceutical composition according to claim 14 in the preparation of an anti-tumor drug.

16. The use according to claim 15, characterized in that The tumor includes any one or more of colon cancer, kidney tumor, breast cancer, liver cancer, pancreatic cancer and myelofibrosis.

17. The use according to claim 15, characterized in that The application also includes application in the preparation of T cell therapeutic drugs.

18. The use according to claim 17, characterized in that The T cell therapy drugs include CAR-T cells, TCR-T cells, TIL cells or γδ T cells.

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