Heterodimer fusion protein and application thereof

Abstract

The invention relates to a heterodimer fusion protein and application thereof. The heterodimer fusion protein of the present invention comprises: a first heavy chain comprising a first Fc region and an immunomodulator fused to the first Fc region; a light chain and a second heavy chain, the second heavy chain comprising a second Fc region, the light chain and the second heavy chain being complexed to form a targeting moiety exhibiting binding specificity for tumor antigens or immune checkpoints; the light chain, the first heavy chain and the second heavy chain are compounded to form the heterodimer fusion protein. The heterodimer fusion protein disclosed by the invention can be used for targeted therapy of digestive tract cancer, especially colon cancer, and is good in targeting property, and the tumor inhibition effect of the heterodimer fusion protein is obviously superior to that of cetuximab with the same target point.

Description

Heterodimer fusion protein and its application Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a heterodimer fusion protein and applications thereof. Background Art

[0002] The epidermal growth factor receptor (also known as EGFR, ErbB-1, and HER1) is a cell surface receptor of the ErbB receptor family, a subfamily of four closely related receptor tyrosine kinases: EGFR (ErbB-1), HER2 / c-neu (ErbB-2), Her 3 (ErbB-3), and Her 4 (ErbB-4). Binding of EGFR to ligands (e.g., epidermal growth factor (EGF), transforming growth factor α (TGFα), HB-EGF, amphiregulin, betacellulin / BTC, and epithelial cell mitogen / EPGN) induces receptor dimerization and autophosphorylation at several tyrosine (Y) residues (Y992, Y1045, Y1068, Y1148, and Y1173) in the C-terminal domain of EGFR. This autophosphorylation triggers downstream activation of several signal transduction cascades including the MAPK, Akt, and JNK pathways, leading to cell migration, adhesion, and cell proliferation.

[0003] Mutation, amplification or misregulation of EGFR or family members is involved in approximately 30% of all epithelial cell cancers. For example, mutations that result in EGFR overexpression or hyperreactivity are associated with many cancers, including colon cancer, lung cancer, anal cancer, head and neck cancer, and glioblastoma multiforme. EGFR has been identified as an oncogene, leading to the need to develop anticancer therapeutics directed against EGFR.

[0004] The malignant cells that cause the disease are usually unable to elicit an immune response that leads to rejection. Studies have shown that it is possible to enhance the immunogenicity of tumor cells by introducing immunomodulatory molecules (such as cytokines and co-stimulatory molecules); however, eradication of residual cancer cells may require targeting widely dispersed micrometastatic tumor deposits, which are not directly amenable to gene transfer. In addition, the expression and stability of introduced immunomodulatory molecules are usually far from satisfactory. Immunomodulators such as cytokines produced by cells of the immune system can directly or indirectly activate cells of the adaptive immune response and can play an important role in eliciting protective anti-tumor immunity. The innate immune system can be triggered by bacterial products or "danger" signals that lead to the release of proinflammatory cytokines (such as IFN-α, TNF-α and interleukins).

[0005] IL-10 is primarily secreted by activated T cells and antigen-presenting cells. During antigen recognition, IL-10 receptor (IL-10R) expression is upregulated in CD8+ T cells. IL-10 is mediated by a specific cell surface receptor complex, which contains two distinct chains: IL-10R1 and IL-10R2. Both chains belong to the class II cytokine receptor family (CRF2). In bacterial infection and tissue injury, IL-10 can reduce inflammatory responses, inhibit inflammatory responses induced by T cells (Th17) and macrophages (IL-12 / 23), and reduce tumor-associated inflammatory responses. Within the tumor microenvironment, IL-10 can efficiently activate the proliferation and cytotoxicity of antigen-specific CD8+ T cells.

[0006] The anti-tumor mechanism of IL-10 is as follows: a. It can activate the activity and expansion of CD8+ T cells within tumors; b. IL-10 can increase the activity and expansion of antigen-specific T lymphocytes within tumors; c. IL-10's tumor rejection effect has a memory function. In vivo animal data show that after tumors disappear after IL-10 administration, mice re-inoculated with tumor cells no longer grow in the mice. The main reason is that IL-10 enhances the survival of antigen-specific CD8+ T cells, acting as a tumor vaccine; d. IL-10 reactivates T cells to kill tumor cells by restoring oxidative phosphorylation metabolism in terminally exhausted T cells. Clinical trials have also demonstrated that when used in combination with PDL1 antibodies, it increases the number of PDL1-specific CD8+ positive cells within tumors, resulting in a long-lasting anti-tumor effect. However, there are currently no marketed drugs targeting IL-10. IL-10 can promote the expansion and survival of antigen-specific CD8+ T cells, and antigen-specific CD8+ T cells are positively correlated with immune cell tumor killing. Although multiple studies have demonstrated that immunomodulators can be used to exert anti-tumor effects in animal models and cancer patients, their short half-life and systemic toxicity have greatly limited their use. After IL-10 binds to its receptor, it activates the STAT3 and STAT1 pathways, which are the signal transduction pathways for IL-10's biological functions.

[0007] Cancer is a common and frequently occurring disease that threatens human health. Digestive tract cancer accounts for over a quarter of common malignant tumors, and my country has the highest incidence and mortality rates for digestive tract cancer. Colon cancer, in particular, has an insidious onset, often lacking obvious clinical symptoms in its early stages. It also progresses slowly, often developing symptoms in patients in the late stages. Currently, no ideal drug has been developed to treat digestive tract cancer.

[0008] Cetuximab, a monoclonal antibody developed by Merck that targets the epidermal growth factor receptor, was approved for marketing by the U.S. Food and Drug Administration (FDA) on February 12, 2004. Cetuximab inhibits the ability of tumor cells to repair damage caused by chemotherapy and radiotherapy, as well as the formation of new blood vessels within tumors. It is used to treat metastatic colorectal cancer with wild-type RAS and BRAF genes. However, cetuximab's limited effectiveness due to its single target has led to a continued search for drugs with significantly better efficacy than cetuximab for gastrointestinal cancers.

[0009] Summary of the Invention

[0010] To solve the above technical problems, the present invention provides a heterodimeric fusion protein and its use. The heterodimeric fusion protein of the present invention can target digestive tract cancer, especially colon cancer, with good targeting and significantly better tumor inhibition effect than cetuximab with the same target.

[0011] Specifically, the present invention provides a use of a heterodimeric fusion protein in the preparation of a medicament for treating or preventing gastrointestinal cancer, wherein the heterodimeric fusion protein comprises: a first heavy chain, wherein the first heavy chain comprises a first Fc region and an immunomodulator fused to the first Fc region; a light chain and a second heavy chain, wherein the second heavy chain comprises a second Fc region, and the light chain and the second heavy chain are complexed to form a targeting portion that exhibits binding specificity to a tumor antigen or an immune checkpoint; the light chain, the first heavy chain, and the second heavy chain are complexed to form the heterodimeric fusion protein.

[0012] In some embodiments, the tumor antigen or immune checkpoint is B7H3, B7H4, B7H5, Claudin18.2, BTLA, CD27, CD28, CD153, CD40, CD40L, CD70, CD80, CD86, CD96, CD112, CD134, CD137, CD137L, CD152 / CTLA-4, CD155, CD223, CD226, CD252 / OX40L, CD258, CD273 / PD-L2, CD274 / PD -L1, CD278, CD279, CD357, DR3, Galectin-9, ICOSL / B7RP1 / B7H2, IDO, TIGIT, TIM-3, TL1A, gp100, TRP-1, TRP-2, BAGE , GAGE-1, GAGE-2, p15, CEA, Ras, HER-2 / neu, MAGE-4, MAGE-5, MAGE-6, RAGE, erbB, p185erbB2, p180erbB-3, c-met, CAM 17.1, NuMa, K-ras, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, β-HCG, BCA225, BTAA, CA 125. MUC16, IL13Rα2, FRα, VEGFR2, LewisY, FAP, EphA2, CEACAM5, CEACAM6, EGFR, CA6, CA9, G PNMB, EGP1, FOLR1, STEAP1, SLC44A4, AGS-16, MUC-1, CFC1B, TPS, CD19, CD20, CD22, CD30, CD7 2. CD180, CD171, CD123, CD133, CD138, CD37, CD70, CD79a, CD79b, CD56, CD74, CD166, CD71, C LL-1 / CLEC12A, ROR1, CD33 / IL3Ra, c-Met, PSCA, PSMA, EGFRvIII, BCMA, GD-2, MY-ESO-1 or MAGE One or more of A3; preferably, the tumor antigen is EGFR.

[0013] In some embodiments, the light chain and the second heavy chain both comprise complementarity determining regions, wherein the complementarity determining regions comprise an amino acid sequence that is at least 80% identical to the amino acid sequence of the corresponding CDR of the light chain or heavy chain of an antibody that specifically binds to a tumor antigen or an immune checkpoint; preferably, the light chain comprises LCDR1 as shown in SEQ ID NO:6, LCDR2 as shown in SEQ ID NO:7, and LCDR3 as shown in SEQ ID NO:8; preferably, the second heavy chain comprises HCDR1 as shown in SEQ ID NO:3, HCDR2 as shown in SEQ ID NO:4, and HCDR3 as shown in SEQ ID NO:5.

[0014] In some embodiments, the light chain comprises a variable region, and the amino acid sequence of the variable region of the light chain is shown in SEQ ID NO: 10, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 10.

[0015] In some embodiments, the second heavy chain comprises a variable region, the amino acid sequence of the variable region of the heavy chain is as shown in SEQ ID NO:9, or an amino acid sequence that is at least 80% identical to SEQ ID NO:9.

[0016] In some embodiments, the amino acid sequence of the light chain is as shown in SEQ ID NO: 13, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 13.

[0017] In some embodiments, the amino acid sequence of the second heavy chain is as shown in SEQ ID NO: 12, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 12.

[0018] In some embodiments, the first heavy chain comprises one or more immunomodulators of the same or different types. In some embodiments, the immunomodulators are fused to each other and to the first Fc region. In some embodiments, the immunomodulator is IL-10.

[0019] In some embodiments, the first Fc region is fused to the immunomodulator via a polypeptide linker. In some embodiments, the immunomodulator is connected to the N-terminus of the first Fc region via a polypeptide linker. In some embodiments, the polypeptide linker is 5-30 amino acids. In some embodiments, the polypeptide linker is (GGGGS)n, where n=1-6.

[0020] In some embodiments, the first Fc region and the second Fc region are the same or different. In some embodiments, the first Fc region and the second Fc region are selected from IgG, IgA, IgD, IgE, IgM, and variants thereof. In some embodiments, the first Fc region and the second Fc region are selected from IgG1, IgG2, IgG3, IgG4, and variants thereof. In some embodiments, the first Fc region and the second Fc region comprise one or more amino acid mutations, preferably amino acid substitutions, insertions, or deletions. In some embodiments, the first Fc region is a knob-Fc and the second Fc region is a hole-Fc. In some embodiments, the first Fc region is a hole-Fc and the second Fc region is a knob-Fc. In some embodiments, the amino acid sequence of the first heavy chain is as shown in SEQ ID NO: 14, or an amino acid sequence that is at least 80% identical to SEQ ID NO: 14.

[0021] In some embodiments, the amino acid sequence of the linker monomer in the first heavy chain is as shown in SEQ ID NO: 1. In some embodiments, the amino acid sequence of IL-10 in the first heavy chain is as shown in SEQ ID NO: 2. In some embodiments, the amino acid sequence of the Fc region in the first heavy chain is as shown in SEQ ID NO: 11.

[0022] In some embodiments, the digestive tract cancer is colon cancer.

[0023] The present invention also provides the above heterodimeric fusion protein.

[0024] The present invention also provides the above heterodimeric fusion protein, which is used for treating or preventing digestive tract cancer.

[0025] The present invention also provides an isolated polynucleotide encoding the above heterodimer fusion protein.

[0026] The present invention also provides a vector comprising the isolated polynucleotide.

[0027] The present invention also provides an isolated host cell comprising the isolated polynucleotide or vector.

[0028] The present invention also provides a pharmaceutical composition comprising a pharmaceutically acceptable excipient and the heterodimer fusion protein.

[0029] In some embodiments, the pharmaceutical composition is formulated for oral administration, intravenous administration, intramuscular administration, in situ administration at a tumor site, inhalation, rectal administration, vaginal administration, transdermal administration, or administration via a subcutaneous depot.

[0030] The present invention also provides use of the heterodimeric fusion protein in preparing a medicament and / or a kit for inhibiting tumors. Preferably, the tumor is colon cancer.

[0031] The present invention also provides a method for producing a fusion protein, comprising (i) culturing a host cell under conditions that achieve expression of the fusion protein, and (ii) harvesting the expressed fusion protein.

[0032] The present invention also provides the use of the heterodimeric fusion protein in preparing a reagent or kit for detecting tumor antigens or immune checkpoints and IL-10 receptor molecules.

[0033] Detailed explanation of terms

[0034] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention belongs.

[0035] The term "heterodimer" generally refers to a molecule (e.g., a protein molecule) composed of two different members. The two members of a heterodimer may differ in structure, function, activity, and / or composition. For example, the two different members may comprise polypeptides that differ in the order, number, or type of amino acid residues that form these polypeptides. Each of the two different members of a heterodimer may independently comprise one, two, or more units, polypeptide chains, or portions.

[0036] The term "fusion protein" refers to a protein comprising one, two, or more polypeptides derived from different naturally occurring proteins or engineered proteins that are artificially combined to form a single protein. Examples include, but are not limited to, the following: 1. A fusion protein consisting of one polypeptide chain, wherein the same or different polypeptides are fused to form a single polypeptide chain comprising the different polypeptides; 2. A fusion protein consisting of two or more polypeptide chains, wherein one or more optional polypeptide chains are fused to form a polypeptide chain comprising the same or different polypeptides, wherein the polypeptide chains are covalently or non-covalently combined to form a protein.

[0037] The term "targeting moiety" generally refers to a molecule, complex or aggregate that specifically, selectively or preferentially binds to a target molecule, cell, particle, tissue or aggregate. For example, the targeting moiety can be an antibody, an antigen-binding antibody fragment, a bispecific antibody or other antibody-based molecule or compound. Other examples of targeting moieties can include, but are not limited to, aptamers, high-affinity polymers, receptor-binding ligands, nucleic acids, biotin-avidin binding pairs, binding peptides or proteins, etc.

[0038] The term "tumor antigen" generally refers to an antigenic substance produced in or by a tumor cell, which may have the ability to trigger an immune response in a host. For example, a tumor antigen may be a protein, polypeptide, peptide, or fragment thereof that constitutes a part of a tumor cell and is capable of inducing tumor-specific cytotoxic T lymphocytes. In some embodiments, the term "tumor antigen" may also refer to a biological molecule (e.g., protein, carbohydrate, glycoprotein, etc.) that is uniquely or preferentially or differentially expressed on cancer cells and / or is found to be associated with cancer cells, thereby providing a preferential or specific target for cancer. For example, preferential expression may be preferential expression compared to any other cell in an organism, or preferential expression within a specific region of an organism (e.g., within a specific organ or tissue).

[0039] The term "immune checkpoint" generally refers to inhibitory and activating molecules in the immune system that regulate the body's anti-tumor immune system by modulating T cell activity. For example, inhibitory molecules include PDL1, B7H3, and CTLA4, while activating molecules include OX40, 4-1BB, and CD40.

[0040] The term "immunomodulator" generally refers to a substance that affects the function of the immune system. An immunomodulator can enhance or reduce an immune response. For example, an immunomodulator can be an active agent of immunotherapy, including but not limited to, for example, cytokines, granulocyte colony stimulating factor (G-CSF), interferon, imiquimod, cell membrane fragments from bacteria, chemokines, interleukins, cytosine phosphate-guanosine (CpG) oligodeoxynucleotides and recombinant, synthetic and / or natural preparations of dextran. In some embodiments, the immunomodulator is a cytokine.

[0041] The term "polypeptide linker" generally refers to a synthetic amino acid sequence that connects or couples two polypeptide sequences (e.g., connects two polypeptide domains). A polypeptide linker can connect two amino acid sequences via a peptide bond. In some embodiments, the polypeptide linker of the present application connects an immunomodulator to an Fc region.

[0042] The term "antibody" generally refers to a protein comprising one or more polypeptides substantially encoded by immunoglobulin genes or immunoglobulin gene fragments. Immunoglobulin genes can include kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable region genes. As used herein, light chains can be classified as kappa or lambda. Heavy chains can be classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes: IgG, IgM, IgA, IgD, and IgE, respectively. The antibodies used in this application can have structural units comprising tetramers. Each tetramer can be composed of two pairs of identical polypeptide chains, each pair having one "light" chain (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each member can define a variable region of about 100 to 110 or more amino acids that is primarily responsible for antigen recognition. The terms light chain variable region (VL) and heavy chain variable region (VH) generally refer to these regions of the light and heavy chains, respectively. Antibodies can exist as complete immunoglobulins or as many well-characterized fragments produced by digestion with various peptidases or de novo expression. The term "antibody" can also include antibody fragments produced by modifying whole antibodies or de novo synthesis using recombinant DNA methods, including but not limited to Fab'2, IgG, IgM, IgA, IgE, scFv, dAb, nano antibodies, single antibodies and double-chain antibodies. In some embodiments, antibodies include but are not limited to Fab'2, IgG, IgM, IgA, IgE and single-chain antibodies, such as single-chain Fv (scFv) antibodies, in which the variable heavy chain and the variable light chain are linked together (directly or through a peptide linker) to form a continuous polypeptide. In some embodiments, the antibodies and fragments in this application are bispecific. In some embodiments, bispecific antibodies or fragments thereof have binding specificity to at least two different epitopes (e.g., at least one of the at least two different epitopes is a tumor-associated antigen). In some embodiments, antibodies and fragments can also be heterologous antibodies, for example, they can be or can comprise two or more antibodies or antibody binding fragments (e.g., Fab) linked together, wherein each antibody or fragment has different specificities.

[0043] There are a variety of methods / systems in the art to define and describe CDRs, and these systems and / or definitions have been developed and refined over the years, including Kabat, Chothia, IMGT, AbM, and Contact. Kabat is the most commonly used and defines CDRs based on sequence variability; Chothia defines CDRs based on sequence variability based on the position of the structural loop region; the IMGT system defines CDRs based on sequence variability and position within the variable domain structure; AbM is based on the AbM antibody modeling software from Oxford Molecular and is a compromise between Kabat and Chothia; Contact defines CDRs based on the analysis of complex crystal structures and is similar to Chothia in many aspects. The numbering of amino acid positions (e.g., amino acid residues in the Fc region) and target regions (e.g., CDRs) in the present invention uses the Kabat system.

[0044] The term "identity" is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve maximum percent sequence identity. Comparisons for purposes of determining percent amino acid sequence identity can be performed in various ways that are within the skill in the art, for example, using publicly available computer software such as BLAST software or the FASTA program package.

[0045] The term "at least 80% identity" means that the percentage of amino acid residues in the candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence is greater than 80%, including 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%.

[0046] The term "host cell" generally includes individual cells, cell lines or cell cultures that may be or have been recipients of a subject's plasmid or vector, comprising polynucleotides disclosed herein, or expressing the heterodimer fusion protein of the present application. Host cells may include the offspring of a single host cell. Due to natural, accidental or intentional mutations, offspring may not necessarily be identical (morphologically or on genomic total DNA complement) to the original parent cell. Host cells may include cells transfected in vitro with a vector disclosed herein. Host cells may be bacterial cells (e.g., Escherichia coli (E. coli)), yeast cells or other eukaryotic cells, such as COS cells, Chinese hamster ovary (CHO) cells, HeLa cells or myeloma cells.

[0047] The term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers an inserted nucleic acid molecule into a host cell and / or between host cells. The term can include vectors primarily used to insert DNA or RNA into cells, vectors primarily used for replication of DNA or RNA, and expression vectors used for transcription and / or translation of DNA or RNA. Vectors that provide more than one of the above functions are also included. An "expression vector" is a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell.

[0048] The term "treatment" refers to an approach for obtaining a beneficial or desired result, including but not limited to a therapeutic benefit and / or a preventive benefit. A therapeutic benefit generally refers to eradication or lessening of the severity of the underlying condition being treated. In addition, a therapeutic benefit is achieved by eradicating, lessening the severity, or reducing the incidence of one or more physiological symptoms associated with the underlying condition such that an improvement is observed in the subject (although the subject may still be afflicted with the underlying condition). For preventive benefit, a composition may be administered to a subject at risk of developing a particular disease, or to a subject reporting one or more physiological symptoms of a disease, even though a diagnosis of the disease may not have been made.

[0049] The term "digestive tract cancer" refers to a neoplastic lesion that occurs in the digestive system. Most of these cancers are named based on the location of the tumor, whether it is benign or malignant, and include, for example, esophageal cancer, gastric cancer, colon cancer, rectal cancer, pancreatic cancer, gallbladder cancer, bile duct cancer, small intestine cancer, large intestine cancer, gallbladder cancer, and pancreatic cancer. Among digestive tract cancers, epidermal growth factor receptor (EGFR) gene amplification and protein overexpression are very common, and protein overexpression also has prognostic significance. In some embodiments, the present invention preferably overexpresses EGFR protein in digestive tract cancers, particularly overexpresses EGFR protein in colon cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] FIG1 is a fitting curve of the binding activity of the fusion protein to the human EGFR protein.

[0051] FIG2 is a fitting curve of the binding activity of the fusion protein to IL-10Ra.

[0052] FIG3 is a curve showing the affinity between the fusion protein and the EGFR protein.

[0053] FIG4 is a curve showing the affinity between the fusion protein and the IL-10R protein.

[0054] FIG5 is a peak diagram of the dual-target binding activity detection of the fusion protein and MDA-MB-231 cells.

[0055] FIG6 is a peak diagram of the dual-target binding activity of the fusion protein and IL-10-Reporter-HEK-293 cells.

[0056] FIG7 is a graph showing the test results of fusion protein activating CD8+ T cells to secrete perforin.

[0057] FIG8 is a tumor growth curve of MC38-hEGFR transplanted tumor model-bearing mice after administration of the test fusion protein. DETAILED DESCRIPTION

[0058] Example 1: Nucleotide sequence

[0059] The target amino acid sequence was converted into a nucleotide sequence, and the obtained nucleotide sequences were: SEQ ID NO: 15 (first heavy chain), SEQ ID NO: 16 (light chain), and SEQ ID NO: 17 (second heavy chain).

[0060] Example 2: Gene synthesis and construction of expression vector

[0061] The pcDNA3.4-G418 and pcDNA3.1-G418 vectors were used as dedicated vectors for expressing the light and heavy chains of the multifunctional antibody, respectively. pcDNA3.4-G418 contained the CMVP promoter used for the light chain, the eukaryotic selection marker G418 tag, and the prokaryotic selection marker Ampicilline. The pcDNA3.1-G418 vector contained the CMVP promoter used for the heavy chain, the eukaryotic selection marker G418 tag, and the prokaryotic selection marker Ampicilline. The nucleotide sequences of the antibody expression light chain and heavy chain were obtained by gene synthesis, and the vector and the target fragment were double-digested with HindIII and XhoI. After recovery, they were enzymatically linked with DNA ligase and transformed into Escherichia coli competent cells DH5α. Positive clones were selected and plasmids were extracted and enzyme digestion was verified to obtain recombinant plasmids containing the first heavy chain, second heavy chain, and light chain of the antibody, namely pcDNA3.1-G418-1, pcDNA3.1-G418-2, and pcDNA3.4-G418-3, respectively.

[0062] Example 3: Plasmid extraction

[0063] According to the method described in the Molecular Cloning Experimental Guide (2002, Science Press), the recombinant plasmids containing the above-mentioned target genes were transformed into Escherichia coli competent cells DH5α. The transformed bacteria were spread on LB plates containing 100 μg / mL ampicillin and cultured. The selected plasmid clones were cultured in liquid LB medium and shaken at 260 rpm for 14 hours. The plasmids were extracted using an endotoxin-free plasmid extraction kit, dissolved in sterile water, and the concentration was determined using a nucleic acid and protein quantifier.

[0064] Example 4: Plasmid transfection, transient expression and fusion protein purification

[0065] Culture ExpiCHO at 37°C, 8% CO2, and 100 rpm to a cell density of 6 × 10 6 The constructed vectors PCDNA3.1-G418-6-1, PCDNA3.1-G418-6-2, and PCDNA3.4-G418-6-3 were transfected into the above cells using liposomes. The transfection plasmid concentration was 1 μg / mL. The liposome concentration was based on the ExpiCHO TM As determined by the Expression System kit, culture was performed at 32°C, 5% CO2, and 100 rpm for 7-10 days. Feeds were added 18-22 hours after transfection and again between 5-8 days. The culture product was centrifuged at 4000 rpm, filtered through a 0.22 μm filter, and the supernatant collected. The resulting antibody protein was purified using a Protein A column and an ion column, and the eluate was collected.

[0066] The specific operating steps of ProteinA and ion column purification are as follows: after high-speed centrifugation of the cell culture fluid, the supernatant is taken and affinity chromatography is performed using GE's ProteinA chromatography column. The equilibrium buffer used for chromatography is 1×PBS (pH7.4). After the cell supernatant is loaded and bound, it is washed with PBS until the ultraviolet light returns to the baseline, and then the target protein is eluted with 0.1M glycine (pH3.0) as an elution buffer, and the pH is adjusted to neutral with Tris for storage. The pH of the product obtained by affinity chromatography is adjusted to 1-2 pH units lower or higher than the pI, and appropriately diluted to control the sample conductivity below 5ms / cm. Using appropriate corresponding pH buffers such as phosphate buffer, acetate buffer and other conditions, conventional ion exchange chromatography methods in the art, such as anion exchange or cation exchange, perform NaCl gradient elution under corresponding pH conditions, and select the collection tube where the target protein is located according to SDS-PAGE and SEC-HPLC for combined storage.

[0067] Example 5: Binding ability of fusion protein to EGFR and IL-10 receptor protein

[0068] ELISA was used to test the binding ability of the fusion protein to human EGFR and IL-10R proteins, and SPR technology was used to detect the affinity of the fusion protein for EGFR and IL-10R proteins, respectively. ELISA results showed that the EGFR-binding end of the fusion protein specifically bound to human EGFR, and the IL-10R-binding end of the fusion protein specifically bound to human IL-10Ra. Binding activity data are shown in Table 1, Figures 1 and 2. As shown in Figures 3 and 4, the affinities of the fusion protein for EGFR and IL-10Ra were 0.51 nM and 3.38 nM, respectively. The positive control was cetuximab, the heavy chain and light chain amino acid sequences of negative control 1 were shown in SEQ ID NO:18 and SEQ ID NO:19, respectively, and negative control 2 was the commercially available Sino, HGK1 Human IgG1 kappa isotype control.

[0069] Table 1 In vitro binding activity of fusion proteins to human EGFR protein and human IL-10R

[0070] Example 6: Dual-target binding assay

[0071] This study leverages the specific binding properties between antigen and antibody, and ligand and receptor, using FACS analysis using an anti-HIS-tag FITC fluorescent antibody to analyze the simultaneous binding of the fusion protein to both EGFR and IL-10R. MDA-MB-231 cells, naturally expressing the EGFR antigen, have been shown to bind to the EGFR end of the fusion protein. IL-10Reporter-HEK-293 cells, stably expressing the IL-10R and STAT3 signaling pathway reporter systems, can bind to the IL-10 end of the fusion protein. Results showed that after binding to the IL-10RA protein, the fusion protein's IL-10 end also bound to the EGFR of EGFR-positive MDA-MB-231 cells. Furthermore, after binding to the EGFR protein, the fusion protein also bound to the IL-10-Reporter-HEK-293 cells. This study demonstrates that the fusion protein can simultaneously bind to both of its targets. After the fusion protein, through its anti-EGFR antibody, delivers IL-10 into the tumor microenvironment, IL-10 can effectively bind to the target cells and exert its biological activity. As shown in Figures 5 and 6, they are peak graphs of the dual-target binding activity detection of the fusion protein and MDA-MB-231 cells and peak graphs of the dual-target binding activity of the fusion protein and IL-10-Reporter-HEK-293 cells, respectively.

[0072] Figure 5 shows a blank control group (Blank), a negative control group (NC), an experimental group (fusion protein), a single-target control group (cetuximab, human-IL-10), and an isotype control group (human-IgG1). The experimental, single-target, isotype, and negative control groups were first mixed with human-IL-10RA protein (His Tag) and then incubated with MDA-MB-231 cells. The cells were then incubated with an anti-6X-His tag antibody (FITC) secondary antibody for 0.5 h, and the secondary antibody binding signal was detected by flow cytometry. The blank control group received only the secondary antibody, while the negative control group received the protein diluent. Figure 6 shows a blank control group (blank), a negative control group (NC), an experimental group (fusion protein), a single-target control group (cetuximab, human-IL-10), and an isotype control group (human-IgG1). The experimental, single-target, isotype, and negative control groups were first mixed with human-EGFR protein and his-tag protein, then incubated with IL-10-Reporter-HEK-293 cells for 1 hour. Secondary antibodies (anti-6X-His tag antibody (FITC)) were then added, and secondary antibody binding signals were detected by flow cytometry. The blank control group received only the secondary antibody, while the negative control group received protein diluent. Human-IgG1 was derived from the commercially available Human IgG1 kappa isotype control from Sino, HGK1.

[0073] Example 7: CD8 fusion protein + T cell activation assay

[0074] One of the main mechanisms of action of the fusion protein is that its IL-10 end binds to CD8 + After binding to IL-10R in T cells, CD8 + T cells are used to kill tumor cells. First, the fusion protein is reacted with the isolated peripheral blood, and then the commercial Perforin cytokine detection kit is used to detect the effect of the fusion protein on CD8 + As shown in Figure 7, IL-10 significantly stimulates CD8 + T cells secrete perforin in a concentration-dependent manner. Fusion protein antibodies can significantly stimulate CD8 + T cells secrete perforin, and cetuximab cannot stimulate CD8 + T cells secrete perforin, suggesting that the fusion protein stimulates CD8 + The secretion of perforin by T cells is dependent on IL-10.

[0075] Example 8: In vivo pharmacodynamics study of fusion protein

[0076] One of the mechanisms of action of the fusion protein is that the IL-10 end binds to the IL-10 receptor on the surface of DC cells in the tumor microenvironment, inhibiting the CD8 + T cell apoptosis and in vivo efficacy of the fusion protein were assessed using C57BL / 6 mice and MC38 mouse colon cancer cells overexpressing hEGFR. Cetuximab was used as a positive control.

[0077] As shown in Figure 8, compared with the Saline control group, the test fusion protein had significant tumor inhibition effects at doses of 1 mg / kg, 5 mg / kg, and 10 mg / kg, with TGIs of 63.05%, 83.26%, and 82.46%, respectively, while the TGI of the control cetuximab was 20.15%, with no significant tumor inhibition effect. The tumor inhibition effect of the fusion protein 5 mg / kg group was similar to that of the fusion protein 10 mg / kg group, with one animal achieving complete remission of the tumor. Therefore, the tumor inhibition effect of the fusion protein 5 mg / kg group was significantly better than that of cetuximab, and the main mechanism of action may be the effect of IL-10 on antigen-specific CD8 + Activation of T cells.

[0078] The protection content of the present invention is not limited to the above embodiments. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be thought of by those skilled in the art are included in the present invention and are protected by the appended claims.

Claims

1. Use of a heterodimeric fusion protein in the preparation of a drug for treating or preventing digestive tract cancer, characterized in that: The heterodimeric fusion protein comprises: a first heavy chain, wherein the first heavy chain comprises a first Fc region and an immunomodulator fused to the first Fc region; a light chain and a second heavy chain, wherein the second heavy chain comprises a second Fc region, and the light chain and the second heavy chain are complexed to form a targeting portion that exhibits binding specificity to a tumor antigen or an immune checkpoint; the light chain, the first heavy chain, and the second heavy chain are complexed to form the heterodimeric fusion protein.

2. The use according to claim 1, wherein the tumor antigen or immune checkpoint is B7H3, B7H4, B7H5, Claudin18.2, BTLA, CD27, CD28, CD153, CD40, CD40L, CD70, CD80, CD86, CD96, CD112, CD134, CD137, CD137L, CD152 / CTLA-4, CD155, CD223, CD226, CD252 / OX40L, CD258, CD273 / PD-L2, CD274 / PD-L1, CD278, CD279, CD357, DR3, Galectin-9, ICOSL / B7RP1 / B7H2, IDO, TIGIT, TIM-3, TL1A, gp100, TRP-1, TRP-2, BAG E, GAGE-1, GAGE-2, p15, CEA, Ras, HER-2 / neu, MAGE-4, MAGE-5, MAGE-6, RAGE, erbB, p185erbB2, p180erbB-3, c-met, CAM 17.1, NuMa, K-ras, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, β-HCG, BCA225, BTAA, CA 125. MUC16, IL13Rα2, FRα, VEGFR2, LewisY, FAP, EphA2, CEACAM5, CEACAM6, EGFR, CA6, CA9, G PNMB, EGP1, FOLR1, STEAP1, SLC44A4, AGS-16, MUC-1, CFC1B, TPS, CD19, CD20, CD22, CD30, CD7 2. CD180, CD171, CD123, CD133, CD138, CD37, CD70, CD79a, CD79b, CD56, CD74, CD166, CD71, C LL-1 / CLEC12A, ROR1, CD33 / IL3Ra, c-Met, PSCA, PSMA, EGFRvIII, BCMA, GD-2, MY-ESO-1 or MAGE One or more of A3; preferably, the tumor antigen is EGFR.

3. The use according to claim 2, characterized in that Both the light chain and the second heavy chain contain complementary determining regions, and the complementary determining regions contain an amino acid sequence that is at least 80% identical to the amino acid sequence of the corresponding CDR of the light chain or heavy chain of an antibody that specifically binds to a tumor antigen or an immune checkpoint; preferably, the light chain of the antibody that specifically binds to a tumor antigen or an immune checkpoint contains LCDR1 with an amino acid sequence as shown in SEQ ID NO: 6, LCDR2 with an amino acid sequence as shown in SEQ ID NO: 7, and LCDR3 with an amino acid sequence as shown in SEQ ID NO: 8; preferably, the second heavy chain of the antibody that specifically binds to a tumor antigen or an immune checkpoint contains HCDR1 with an amino acid sequence as shown in SEQ ID NO: 3, HCDR2 with an amino acid sequence as shown in SEQ ID NO: 4, and HCDR3 with an amino acid sequence as shown in SEQ ID NO:

5.

4. The use according to claim 1, characterized in that The light chain comprises a variable region, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 10, or an amino acid sequence that is at least 80% identical to SEQ ID NO:

10.

5. The use according to claim 1, characterized in that The second heavy chain comprises a variable region, and the amino acid sequence of the variable region of the heavy chain is shown in SEQ ID NO: 9, or an amino acid sequence that is at least 80% identical to SEQ ID NO:

9.

6. The use according to claim 1, characterized in that The amino acid sequence of the light chain is shown in SEQ ID NO: 13, or an amino acid sequence that is at least 80% identical to SEQ ID NO:

13.

7. The use according to claim 1, characterized in that The amino acid sequence of the second heavy chain is shown in SEQ ID NO: 12, or an amino acid sequence that is at least 80% identical to SEQ ID NO:

12.

8. The use according to claim 1, characterized in that The first heavy chain contains one or more immunomodulators, which are fused to each other and to the first Fc region.

9. The use according to claim 8, characterized in that The immunomodulator is IL-10.

10. The use according to claim 8 or 9, characterized in that The first Fc region and the second Fc region are the same or different.

11. The use according to claim 10, characterized in that The first Fc region and the second Fc region are selected from the group consisting of IgG, IgA, IgD, IgE, IgM and variants thereof.

12. The use according to claim 11, characterized in that The first Fc region and the second Fc region are selected from IgG1, IgG2, IgG3, IgG4 and variants thereof.

13. The use according to claim 11 or 12, characterized in that The first Fc region and the second Fc region comprise one or more amino acid mutations, preferably amino acid substitutions, insertions or deletions.

14. The use according to any one of claims 10 to 13, characterized in that The first Fc region is knob-Fc, and the second Fc region is hole-Fc.

15. The use according to any one of claims 10 to 13, characterized in that The first Fc region is hole-Fc, and the second Fc region is knob-Fc.

16. The use according to claim 1, characterized in that The amino acid sequence of the first heavy chain is shown in SEQ ID NO: 14, or an amino acid sequence that is at least 80% identical to SEQ ID NO:

14.

17. The use according to claim 1, characterized in that The digestive tract cancer is colon cancer. The heterodimeric fusion protein according to any one of claims 1 to 17. The heterodimeric fusion protein according to claim 18 , which is used for treating or preventing digestive tract cancer.

20. An isolated polynucleotide encoding the heterodimeric fusion protein of claim 18 or 19.

21. A vector comprising the isolated polynucleotide of claim 20.

22. An isolated host cell comprising the isolated polynucleotide of claim 20 or the vector of claim 21.

23. A pharmaceutical composition comprising a pharmaceutically acceptable excipient and the heterodimeric fusion protein of claim 18 or 19.

24. The pharmaceutical composition of claim 23, formulated for oral administration, intravenous administration, intramuscular administration, in situ administration at a tumor site, inhalation, rectal administration, vaginal administration, transdermal administration, or administration via a subcutaneous depot.

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