A dimeric fusion protein and uses thereof

Abstract

The application belongs to the technical field of biological medicine, and relates to a dimer fusion protein and application thereof. The dimer fusion protein comprises: a light chain and a heavy chain, the light chain and the heavy chain are combined to form a targeting part showing binding specificity to a tumor antigen or an immune checkpoint; an immunomodulator, the immunomodulator is fused to the C-terminal of the heavy chain to form a polypeptide chain; the light chain, the heavy chain and the immunomodulator are combined to form the dimer fusion protein. The dimer fusion protein has high affinities to a tumor-related targeting antigen and an IL-10 receptor, and has good binding activity to cancer cells.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a dimer fusion protein and its applications. Background Technology

[0002] Monoclonal antibodies (mAbs) have been widely used to treat a variety of human diseases, including cancer, autoimmune diseases, infectious diseases, and cardiovascular diseases. Currently, more than 30 monoclonal antibodies exist, including murine, fully humanized, and chimeric antibodies, which have been approved by the FDA for therapeutic use. Most of these antibodies are monospecific, recognizing a single epitope and being selectable to activate or inhibit the activity of a target molecule through that single epitope.

[0003] CEACAM belongs to the immunoglobulin superfamily of adhesion molecules. Its domains are highly glycosylated, typically including 1-2 immunoglobulin variable region-like domains (Ndomains) and 0-6 immunoglobulin constant region-like domains. CEACAM is involved in a variety of cellular functions, regulating cell growth and differentiation through signal transduction based on cell-cell adhesion, and playing important roles in insulin homeostasis, angiogenesis, and immune regulation. In humans, the CEACAM subset consists of seven members: CEACAM1, CEACAM3, CEACAM4, CEACAM5, CEACAM6, CEACAM7, and CEACAM8. Members of the CEACAM gene family participate in a wide range of pathophysiological roles, including acting as receptors for microbial pathogens, and they play important roles in oncogenesis, cancer detection, progression, and metastasis.

[0004] CEACAM5 (abbreviated as CEA, also known as CD66e) is a glycoprotein with a molecular weight of approximately 180 kDa, encoding the CEA protein. CEACAM5 contains seven domains anchored to the cell membrane via a glycosylphosphatidylinositol (GPI) anchor. These seven domains include a single N-terminal Ig variable domain and six domains homologous to the Ig constant domain (A1-B1-A2-B2-A3-B3). CEACAM5 was first described as a gastrointestinal carcinoembryonic antigen in 1965, but to date, studies have shown that CEACAM5 is highly expressed on the surface of tumor cells in the colorectal, stomach, lung, breast, prostate, ovary, cervix, and bladder, and weakly expressed in a few normal epithelial tissues (columnar and goblet cells in the colon, mucinous neck cells in the stomach, and squamous epithelial cells in the esophagus and cervix). For example, overexpression of CEACAM5 in prostate and colorectal cancer has been shown to serve as a tumor biomarker.

[0005] CEACAM6 (also known as CD66c or NCA-90) is a cell surface protein linked to glycosylphosphoinositol (GPI), possessing one N-domain and two C2-like domains. It mediates numerous cis- or trans-guided CEACAM interactions through its extracellular domains containing various membrane receptors (some of which have been identified). CEACAM6 is expressed on granulocytes and epithelial cells from various organs, and exhibits a wider expression region in proliferating cells of hyperplastic colonic polyps and adenomas compared to normal mucosa and cancer. Relatively high serum levels of CEACAM6 have been found in patients with lung cancer, pancreatic cancer, breast cancer, colorectal cancer, and hepatocellular carcinoma. Overexpression of CEACAM6 leads to morphological changes, similar to epithelial-mesenchymal transition, resulting in enhanced invasiveness and chemoresistantness. Previous studies have shown that silencing CEACAM6 using CEACAM6-specific siRNA can inhibit tumor growth, and inhibiting CEACAM6 function using antibody fragments can affect cell migration, cell invasion, and cell adhesion in vitro. These findings indicate that CEACAM6 is a good biomarker for various tumors.

[0006] In addition, CEACAM5 / CEACAM6 has been found to be overexpressed in various malignant tumors, such as breast, pancreas, ovary, colon, lung and gastric gland tumors, and is associated with tumor invasiveness and metastasis.

[0007] The extracellular domains of CEACAM family members consist of repetitive immunoglobulin-like (Ig-like) domains, which are classified into three types based on sequence homology: A, B, and N. CEACAM5 contains seven such domains: N, A1, B1, A2, B2, A3, and B3. The A1, A2, and A3 domains of CEACAM5 show high sequence homology with the B1, B2, and B3 domains. The A domain of human CEACAM5 exhibits 84% ​​to 87% paired sequence similarity, and the B domain exhibits 69% to 80% paired sequence similarity. Furthermore, other CEACAM members (i.e., CEACAM1, CEACAM6, CEACAM7, and CEACAM8) that contain A and / or B domains show homology with human CEACAM5. Specifically, the A and B domains of the human CEACAM6 protein exhibit sequence homology with any of the A1 and A3 domains and B1 to B3 domains of the human CEACAM5, respectively, and this sequence homology is even higher than the sequence homology observed in the A and B domains of the human CEACAM5.

[0008] IL-10 is primarily secreted by activated T cells and antigen-presenting cells. During antigen recognition, the expression of the IL-10 receptor (IL-10R) on CD8+ T cells is upregulated. IL-10's various activities are mediated by a specific cell surface receptor complex. The IL-10 receptor contains two distinct chains, IL-10R1 and IL-10R2, both belonging to the class II cytokine receptor family (CRF2). In bacterial infection and tissue damage, IL-10 can reduce inflammatory responses, suppress inflammatory responses induced by T cells (Th17) and macrophages (IL-12 / 23), and reduce tumor-associated inflammation. In the tumor microenvironment, IL-10 can efficiently activate the proliferation and cytotoxicity of antigen-specific CD8+ T cells.

[0009] The anti-tumor mechanism of IL-10 is as follows: a. It can activate and expand the activity 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 has a memory function in its rejection of tumors. Animal in vivo experimental data show that after tumors disappear following IL-10 administration, mice re-inoculated with tumor cells do not experience tumor cell growth. This is mainly because IL-10 enhances the survival rate of antigen-specific CD8+ T cells, acting as a tumor vaccine; d. IL-10 reactivates T cells by restoring the oxidative phosphorylation metabolism of terminally depleted T cells, thereby killing tumor 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 tumor cells, producing a durable anti-tumor effect. However, there are currently no marketed drugs targeting IL-10.

[0010] IL-10 can promote the expansion and survival of CD8+ T cells targeting specific antigens, and the specific antigen CD8+ T cells are positively correlated with the killing effect of immune cells on tumors. Although several studies have shown that immunomodulators can be used to exert anti-tumor effects in animal models and cancer patients, their short half-life and systemic toxicity, which are associated with their use, greatly limit their application. After binding to its receptor, IL-10 activates the STAT3 and STAT1 pathways, which are the signal transduction pathways through which IL-10 exerts its biological function.

[0011] Patent CN201380041222.1 discloses a fusion protein containing IL-10, outlining a preferred structure with specific targets and sequences. However, this fusion protein exhibits low expression levels, low purity, weak binding activity and signal activation at the IL-10 terminus, and poor binding activity with cancer cells, making it unsuitable for large-scale industrial production. Furthermore, this patent does not disclose preferred structures with other targets and sequences.

[0012] Therefore, it is necessary to find anti-CEACAM5 and CEACAM6 dimer fusion proteins that have good binding activity to IL-10, differential binding to tumor tissue and normal tissue (i.e., high binding to tumor cells, weak binding or no binding to normal cells), and low cross-reactivity with surrounding healthy tissue. Summary of the Invention

[0013] The present invention aims to provide a dimer fusion protein and its application. The dimer fusion protein has high affinity for CEACAM5, CEACAM6, and IL10, and has good anti-tumor activity.

[0014] The present invention provides a dimer fusion protein comprising: a light chain and a heavy chain, the light chain and the heavy chain being complexed to form a targeting portion exhibiting binding specificity to tumor antigens or immune checkpoints; one or more immunomodulators, the immunomodulators being fused to the C-terminus of the heavy chain to form a polypeptide chain; the light chain, the heavy chain and the immunomodulators forming the dimer fusion protein.

[0015] In one embodiment, the tumor antigen is CEACAM5 and / or CEACAM6.

[0016] In one embodiment, the heavy chain contains one or more immunomodulators of the same or different types. In one embodiment, the immunomodulator enhances the immune response. In one embodiment, the immunomodulator reduces the immune response. In one embodiment, the immunomodulator can be a natural immunomodulator or a mutant. In one embodiment, the immunomodulator is a cytokine, cytokine receptor, growth factor, hormone, or extracellular matrix molecule. In one embodiment, the immunomodulator is selected from IL-1, IL-2, IL-2Rα, IL-2Rβ, IL-3, IL-3Rα, IL-4, IL-4Rα, IL-5, IL-5Rα, IL-6, IL-6Rα, IL-7, IL-7Rα, IL-8, IL-9, IL-9Rα, IL-10, IL-10R1, IL-10R2, IL-11, IL-11Rα, IL-12, and IL-12Rα. The immunomodulator is one or more of IL-12Rβ2, IL-12Rβ1, IL-13, IL-13Rα, IL-13Rα2, IL-14, IL-15, IL-15Rαsushi, IL-16, IL-17, IL-18, IL-19, IL-20, IL-20R1, IL-20R2, IL-21, IL-21Rα, IL-22, IL-23, IL-23R, IL-27R, and IL-31R. In one embodiment, the immunomodulator is IL-10. In one embodiment, the immunomodulator is IL-10 or its binary complex (IL-10)2. In one embodiment, the IL-10 contains the amino acid sequence shown in SEQ ID NO:2 or an amino acid sequence having at least 80% identity.

[0017] In one embodiment, the natural IL-10 monomer sequence is as shown in SEQ ID NO:1, or an amino acid sequence having at least 80% identity with SEQ ID NO:1.

[0018] In one embodiment, the heavy chain contains an amino acid sequence of HCDR1 as shown in SEQ ID NO:2, HCDR2 as shown in SEQ ID NO:3, or HCDR3 as shown in SEQ ID NO:4, or an amino acid sequence having at least 80% identity; the light chain contains an amino acid sequence of LCDR1 as shown in SEQ ID NO:5, LCDR2 as shown in SEQ ID NO:6, or LCDR3 as shown in SEQ ID NO:7, or an amino acid sequence having at least 80% identity.

[0019] In one embodiment, the amino acid sequence of the variable region of the heavy chain is as shown in SEQ ID NO:8, or is an amino acid sequence having at least 80% identity with SEQ ID NO:8. In one embodiment, the amino acid sequence of the variable region of the light chain is as shown in SEQ ID NO:9, or is an amino acid sequence having at least 80% identity with SEQ ID NO:9.

[0020] In one embodiment, the amino acid sequence of the heavy chain is as shown in SEQ ID NO:10, or is an amino acid sequence having at least 80% identity with SEQ ID NO:10. In one embodiment, the amino acid sequence of the light chain is as shown in SEQ ID NO:11, or is an amino acid sequence having at least 80% identity with SEQ ID NO:11.

[0021] In one embodiment, the immunomodulator is linked to the Fc region of the antibody that specifically binds to the tumor antigen or immune checkpoint. In one embodiment, the immunomodulator is linked to the Fc region via a peptide linker. In one embodiment, the peptide linker is 5-30 amino acids. In one embodiment, the peptide linker is (GGGGS)n, where n = 1-6. In one embodiment, the heavy chain comprises a constant region of an immunoglobulin selected from IgG1, IgG2, IgG3, and IgG4.

[0022] The present invention also provides a method for preparing the above-mentioned dimer fusion protein, wherein the preparation method involves transferring plasmids containing the above-mentioned light chain, heavy chain and IL-10 into the same host cell for recombinant expression.

[0023] In one embodiment, the concentration ratio of the light chain, heavy chain, and IL-10 recombinant plasmid is 1:0.5-2:0.5-2. In another embodiment, the concentration ratio of the light chain, heavy chain, and IL-10 recombinant plasmid is 1:1:1.

[0024] In one embodiment, the host cell is a mammalian cell, bacterial, fungal, or insect cell. In one embodiment, the mammalian cell is a CHO cell, SP20 cell, NSO cell, COS cell, BHK cell, HEK293 cell, or PerC6 cell. In one embodiment, the mammalian cell is a CHO cell.

[0025] This invention provides a nucleic acid encoding the aforementioned dimer fusion protein. This invention provides a vector or plasmid containing the aforementioned nucleic acid. This invention provides a cell expressing the aforementioned vector or plasmid. This invention also provides a pharmaceutical composition comprising the aforementioned dimer fusion protein and at least one pharmaceutically acceptable excipient, diluent, or carrier. In one embodiment, the pharmaceutical composition can be used alone or in combination with other therapeutic agents to improve efficacy or reduce potential side effects.

[0026] This invention also provides the application of the above-mentioned dimer fusion protein in the preparation of drugs for the prevention and treatment of tumor diseases. In one embodiment, the tumor diseases include one or more of the following: colorectal cancer, adenocarcinoma, lung cancer, esophageal cancer, prostate cancer, desmoplastic small round cell tumor, ovarian cancer, gastric cancer, pancreatic cancer, liver cancer, kidney cancer, breast cancer, non-small cell lung cancer, melanoma, alveolar rhabdomyosarcoma, embryonal rhabdomyosarcoma, Ewing sarcoma, nephroblastoma, neuroblastoma, gangliocytoma, medulloblastoma, high-grade glioma, diffuse intrinsic pontine glioma, and multilayered rosette embryonal tumors.

[0027] This invention also provides the application of the above-mentioned dimer fusion protein in the preparation of reagents or kits for detecting tumor-associated antigens CEACAM5, CEACAM6 and IL-10 receptor molecules. Attached Figure Description

[0028] Figure 1 : Schematic diagram of the structure of the dimer fusion protein (hereinafter referred to as fusion protein) 1.

[0029] Figure 2 Schematic diagram of the structure of fusion protein 2.

[0030] Figure 3 ELISA was used to detect the binding activity of the fusion protein to CEACAM5.

[0031] Figure 4 ELISA was used to detect the binding activity of the fusion protein to the IL-10 receptor protein.

[0032] Figure 5 FACS results of the binding activity of the fusion protein to OVCAR3 cells.

[0033] Figure 6 ELISA was used to detect the affinity of the fusion protein for the IL-10 receptor.

[0034] Figure 7 FACS results of the binding activity of fusion proteins 1 and 2 to LS174T cells.

[0035] Figure 8: Activation activity assay of fusion protein on IL-10 reporter gene in cells.

[0036] Figure 9 Positive control and activation of IL-10 reporter gene activity in cells compared to fusion protein 1.

[0037] Figure 10 Positive control and activation of IL-10 reporter gene activity in fusion protein 2-activated cells. Detailed Implementation

[0038] The present invention will be further described below with reference to embodiments. These embodiments are used to describe some specific implementations of the present invention and are not intended to limit the scope of protection of the present invention.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. While methods and materials similar to or equivalent to those described and used herein may be used in the practice or testing of this application, suitable methods and materials are described below. In case of conflict, the patent specification shall prevail.

[0040] The term "dimer" generally refers to a molecule (such as a protein molecule) composed of two members. The two members of a dimer can differ in structure, function, activity, and / or composition. For example, two different members can be contained in polypeptides that differ in the sequence, number, or type of amino acid residues that form these polypeptides. Each of the two different members of a dimer can independently contain one, two, or more units, polypeptide chains, or portions.

[0041] The term "target moiety" generally refers to a molecule, complex, or aggregate that binds specifically, selectively, or preferentially to a target molecule, cell, particle, tissue, or aggregate. For example, a target moiety can be an antibody, an antigen-binding antibody fragment, a bispecific antibody, or other antibody-based molecules or compounds. Other examples of target moieties may include, but are not limited to, aptamers, high-affinity multimers, receptor-binding ligands, nucleic acids, biotin-avidin binding pairs, binding peptides, or proteins.

[0042] Various methods / systems exist in this field for defining and describing CDRs. These systems and / or definitions have been developed and refined over many years, including Kabat, Chothia, IMGT, AbM, and Contact. Kabat is the most commonly used, defining CDRs based on sequence variability; Chothia defines CDRs based on the position of structural loop regions; the IMGT system defines CDRs based on sequence variability and position within variable domain structures; AbM is defined using Oxford Molecular's AbM antibody modeling software, representing a compromise between Kabat and Chothia; Contact defines CDRs based on the analysis of complex crystal structures and is similar to Chothia in several ways. In this invention, the numbering of amino acid positions (e.g., amino acid residues in the Fc region) and the target regions (e.g., CDRs) are performed using the Kabat system.

[0043] The term "tumor antigen" generally refers to an antigenic substance present in or produced by tumor cells that has the ability to trigger an immune response in the host. For example, a tumor antigen can be a protein, polypeptide, peptide, or fragment thereof that constitutes 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 biomolecule (e.g., a protein, carbohydrate, glycoprotein, etc.) that is uniquely, preferentially, or differentially expressed on cancer cells and / or found to be associated with cancer cells, thus providing a preferential or specific target for cancer. For example, preferential expression can be preferential expression compared to any other cell in the organism, or preferential expression within a specific region of the organism (e.g., within a specific organ or tissue).

[0044] The term "immune checkpoint" generally refers to a group of inhibitory and activating molecules in the immune system that can 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.

[0045] The term "immunomodulator" generally refers to substances that affect the function of the immune system. Immunomodulators can enhance or diminish immune responses. For example, immunomodulators can be active agents in immunotherapy, including but not limited to recombinant, synthetic, and / or natural formulations of cytokines, granulocyte colony-stimulating factor (G-CSF), interferon, imiquimod, bacterial cell membrane fragments, chemokines, interleukins, cytosine phosphate-guanosine (CpG) oligodeoxynucleotides, and dextran. In some embodiments, the immunomodulator is a cytokine.

[0046] The term "peptide linker" generally refers to a synthetic amino acid sequence that connects or links two peptide sequences (e.g., links two peptide domains). Peptide linkers can connect two amino acid sequences via peptide bonds.

[0047] The term "antibody" generally refers to a protein comprising one or more polypeptides encoded essentially by immunoglobulin genes or segments of immunoglobulin genes. Immunoglobulin genes may include κ, λ, α, γ, δ, ε, and μ constant region genes, as well as numerous immunoglobulin variable region genes. As used herein, light chains may be classified as κ or λ. Heavy chains may be classified as γ, μ, α, δ, or ε, which, in turn, define the immunoglobulin classes: IgG, IgM, IgA, IgD, and IgE, respectively. Antibodies used in this application may have structural units comprising tetramers. Each tetramer may consist of two pairs of identical polypeptide chains, each pair having one "light" chain (approximately 25 kD) and one "heavy" chain (approximately 50-70 kD). The N-terminus of each member may define a variable region of approximately 100 to 110 or more amino acids, which is primarily responsible for antigen recognition. As used herein, 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 intact immunoglobulins or as a number of well-characterized fragments produced by digestion with various peptidases or de novo expression. The term "antibody" may also include antibody fragments produced by modifying whole antibodies or by de novo synthesis using recombinant DNA methods, including but not limited to Fab'2, IgG, IgM, IgA, IgE, scFv, dAb, nanobodies, monoclonal 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, wherein variable heavy chains and variable light chains are linked together (directly or via peptide linkers) to form a continuous polypeptide. In some embodiments, the antibodies and fragments in this application are bispecific. In some embodiments, the bispecific antibody or fragment thereof has binding specificity for 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, the antibodies and fragments may also be xenobiotic antibodies, for example, they may be or may comprise two or more antibodies or antibody-binding fragments (e.g., Fab) linked together, wherein each antibody or fragment has a different specificity.

[0048] The term "identity" is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in a control polypeptide sequence after sequence alignment and, where necessary, nicking to obtain the maximum percentage sequence identity. Comparisons for determining percentage amino acid sequence identity can be performed in a variety of ways within the scope of the art, such as using publicly available computer software, like BLAST software or the FASTA package.

[0049] The term "at least 80% identity" means that the percentage of amino acid residues in the candidate sequence that are identical to those in the control 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%.

[0050] The term "host cell" generally includes a single cell, cell line, or cell culture that may be, or is already, a recipient of a subject plasmid or vector, containing the polynucleotides disclosed in this application, or expressing the heterodimeric proteins of this application. Host cells may include the progeny of a single host cell. Due to natural, accidental, or intentional mutations, the progeny may not necessarily be identical to the original parent cell (morphologically or in terms of total genomic DNA complementarity). Host cells may include cells transfected in vitro with the vectors disclosed in this application. Host cells may be bacterial cells (e.g., E. coli), yeast cells, or other eukaryotic cells, such as COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, or myeloma cells.

[0051] The term "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, which transfers inserted nucleic acid molecules into host cells and / or between host cells. This term can include vectors primarily used for inserting DNA or RNA into cells, vectors primarily used for DNA or RNA replication, and expression vectors for DNA or RNA transcription and / or translation. It also includes vectors that provide more than one of the above functions. An "expression vector" is a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell.

[0052] The terms “treatment,” “curing,” “prevention,” “relief,” or “improvement” are used interchangeably herein and refer to methods for achieving beneficial or desired outcomes (including, but not limited to, therapeutic and / or preventive benefits). As used herein, a therapeutic benefit generally refers to the eradication or reduction of the severity of the underlying condition being treated. Furthermore, a therapeutic benefit is achieved by eradicating, reducing the severity, or decreasing the incidence of one or more physical symptoms associated with the underlying condition, so that improvement is observed in the subject (even though the subject may still be suffering from the underlying condition). For preventive benefits, the composition may be administered to subjects at risk of developing a specific disease, or to subjects who report one or more physical symptoms of the disease, even if the disease may not yet have been diagnosed.

[0053] The term "reagent" generally refers to a biological part, a pharmaceutical part, or a compound or other part. Non-limiting examples include simple or complex organic or inorganic molecules, peptides, proteins, oligonucleotides, antibodies, antibody derivatives, antibody fragments, vitamin derivatives, carbohydrates, toxins, or chemotherapeutic compounds. A wide variety of compounds can be synthesized, such as small molecules and oligomers (e.g., oligopeptides and oligonucleotides) and synthetic organic compounds based on various core structures. Additionally, compounds for screening are available from a variety of natural sources, such as plant or animal extracts.

[0054] The terms "anticancer agent," "antitumor agent," or "chemotherapy agent" generally refer to any agent that can be used to treat cancer conditions. One class of anticancer agents includes chemotherapeutic agents.

[0055] The term "chemotherapy" generally refers to the administration of one or more chemotherapeutic drugs and / or other agents to a cancer patient by various methods, including intravenous, oral, intramuscular, intraperitoneal, intravesical, subcutaneous, percutaneous, oral, or inhalation or suppository forms.

[0056] The term "in vivo" usually refers to events that occur inside a subject's body.

[0057] The term "in vitro" generally refers to an event that occurs outside the subject's body. For example, an in vitro assay includes any assay performed outside the subject's body. In vitro assays include cell-based assays in which dead or live cells are used. In vitro assays also include cell-free assays in which intact cells are not used.

[0058] The term "subject" generally refers to a human or non-human animal, including but not limited to cats, dogs, horses, pigs, cattle, sheep, goats, rabbits, mice, rats, or monkeys.

[0059] The amino acid sequences of the humanized monoclonal antibody, fusion proteins 1 and 2, IL-10, CEACAM6(AB) protein, and the positive control in Example 8 are shown in Table 1. The structure of fusion protein 1 is as follows: Figure 1 As shown, the structure of fusion protein 2 is as follows: Figure 2 As shown, fusion protein 1 and fusion protein 2 have the same heavy and light chain amino acid sequences. The difference is that in fusion protein 1, IL-10 is linked to the C-terminus of the light chain via a polypeptide linker, while in fusion protein 2, IL-10 is linked to the C-terminus of the heavy chain via a polypeptide linker.

[0060] Table 1 Sequence List

[0061]

[0062]

[0063] Example 1: Construction of gene synthesis and expression vector

[0064] The antibody was constructed as a humanized monoclonal antibody capable of simultaneously binding to CEACAM5 and CEACAM6. This antibody was then used to construct a fusion protein with IL-10. The structural design is described in [link to structural design]. Figure 1-2 The IL-10 sequence is fused to the C-terminus of the light chain or the C-terminus of the heavy chain of the antibody through the GGGGS linker.

[0065] The PcDNA3.1 vector was used as the dedicated vector for expressing the light and heavy chains of the antibody. The PcDNA3.1 vector contains the CMV Promoter for the heavy chain, the eukaryotic selection tag G418, and the prokaryotic selection tag Ampicilline. The nucleotide sequences (i.e., target genes) encoding the heavy and light chains of the fusion protein were synthesized. The vector and target fragments were double-digested with HindIII and XhoI, recovered, and ligated using DNA ligase. The resulting fragments were then transformed into competent *E. coli* DH5α cells. Positive clones were selected, and plasmids were extracted and verified by enzyme digestion to obtain recombinant plasmids containing the heavy and light chain encoding genes of the fusion protein.

[0066] Example 2 Plasmid Extraction

[0067] According to the method described in "Molecular Cloning: A Laboratory Manual" (2002, Science Press), recombinant plasmids containing the above-mentioned target genes were transformed into E. coli competent cells DH5α. The transformed bacteria were plated on LB plates containing 100 μg / mL ampicillin and cultured. Plasmid clones were selected and cultured in liquid LB medium. The culture was shaken at 260 rpm for 14 h. Plasmids were extracted using an endotoxin-free plasmid extraction kit, dissolved in sterile water, and their concentration was determined using a nucleic acid protein quantification instrument.

[0068] Example 3: Plasmid transfection, transient expression, and purification of fusion protein

[0069] ExpiCHO cells were cultured at 37℃, 8% CO2, and 100 rpm until the cell density reached 6 × 10⁶ cells / year. 6 Cells / mL. The constructed vector plasmids were transfected into the above cells using liposomes at a mass ratio of 1:1:1. The plasmid concentration was 1 mg / mL, and the liposome concentration was as per ExpiCHO. TM The Expression System kit specifies that the culture should be carried out at 32°C, 5% CO2, and 100 rpm for 7-10 days. Feeding should be performed once 18-22 hours after transfection and again between day 5. The culture products should be centrifuged at 4000g, filtered through a 0.22μm filter, and the supernatant collected. The resulting antibody protein should be purified using Protein A ion exchange column, and the eluent collected.

[0070] The specific steps for Protein A ion-column purification are as follows: After high-speed centrifugation of the cell culture medium, the supernatant is collected and subjected to affinity chromatography using a GE Protein A chromatography column. The chromatography uses 1×PBS (pH 7.4) as the equilibration buffer. After loading the cell supernatant, it is washed with PBS until the UV light returns to baseline. Then, the target protein is eluted with 0.1M glycine (pH 3.0) elution buffer, and the pH is adjusted to neutral using Tris for storage. The pH of the affinity chromatography product is adjusted to 1-2 pH units below or above its isoelectric point pI, and appropriately diluted to control the sample conductivity below 5 mS / cm. Using appropriate pH buffers such as phosphate buffer or acetate buffer, conventional ion exchange chromatography methods in the field, such as anion exchange or cation exchange, are used for NaCl gradient elution at the corresponding pH conditions. The collection tube containing the target protein is selected and combined for storage according to SDS-PAGE. Finally, the purified eluent is ultrafiltered and added to the buffer.

[0071] Example 4: ELISA detection of the affinity of the fusion protein for CEACAM5

[0072] Human CEACAM5-His (ACRO CE5-H5220, protein number UniProtKB-P06731) was diluted to 0.5 μg / mL using PBS buffer (pH 7.4), and 100 μL was added to each well of a 96-well ELISA plate. The plate was incubated overnight at 4°C. After blocking with 1% BSA blocking buffer for 1 hour, the plate was washed three times with PBST. The purified fusion proteins 1 and 2 were then diluted to 100 nM with 0.5% BSA sample diluent. This was used as the starting concentration for a 3-fold serial dilution, resulting in 11 dilutions. A negative control (trastuzumab) was included, with 100 μL added to each well. The plate was incubated at 37°C for 1 hour. After washing three more times with PBST, HRP-labeled goat anti-human IgG Fc (Jackson Cat: 109-035-098) was diluted 1:10000 with sample diluent, and 100 μL was added to each well. The plate was incubated at room temperature for 1 hour. After washing the plate four times with PBST, 100 μL of TMB substrate was added to each well, and the plate was incubated at room temperature in the dark for 10 min. The colorimetric reaction was then terminated by adding 100 μL of 1M HCl solution to each well. The absorbance of each well in the 96-well plate was measured using a multi-mode microplate reader at a wavelength of 450 nm and a reference wavelength of 570 nm. The absorbance (OD) of each well was calculated as OD450nm - OD570nm. The logarithm of the fusion protein concentration was plotted on the x-axis, and the absorbance of each well was plotted on the y-axis. A nonlinear regression using the sigmoidal dose-response (Variable Slope) method (GraphPad Prism software, GraphPad Software, San Diego, California) was performed to obtain the binding curve between the target fusion protein and human CEACAM5 protein.

[0073] ELISA results as follows Figure 3 As shown, fusion proteins 1 and 2 can bind to CEACAM5 protein at multiple concentration ranges, while the negative control cannot bind to the protein, indicating that IL-10 fusion proteins fused to the C-terminus of the light or heavy chain of the constructed antibody have specific binding activity to human CEACAM5 protein.

[0074] Example 5: ELISA detection of the affinity of the fusion protein for CEACAM6

[0075] Human-CEACAM6-His (ACRO CE6-H5223, protein ID UniProtKB-P40199) was diluted to 0.5 μg / mL using PBS buffer (pH 7.4), and 100 μL was added to each well of a 96-well ELISA plate. The plate was incubated overnight at 4°C. After blocking with 1% BSA blocking buffer for 1 hour, the plate was washed three times with PBST. The purified fusion protein was then diluted to 100 nM with 0.5% BSA sample diluent. This was used as the starting concentration for a 3-fold serial dilution, resulting in 11 dilutions. A negative control (trastuzumab) was included, with 100 μL added to each well. The plate was incubated at 37°C for 1 hour. After washing three more times with PBST, HRP-labeled goat anti-human IgG Fc (JacksonCat:109-035-098) was diluted 1:10000 with sample diluent, and 100 μL was added to each well. The plate was incubated at room temperature for 1 hour. After washing the plate four times with PBST, 100 μL of TMB substrate was added to each well, and the plate was incubated at room temperature in the dark for 10 min. The colorimetric reaction was then terminated by adding 100 μL of 1M HCl solution to each well. The absorbance of each well in the 96-well plate was measured using a multi-mode microplate reader at a wavelength of 450 nm and a reference wavelength of 570 nm. The absorbance (OD) of each well was calculated as OD450nm - OD570nm. The logarithm of the fusion protein concentration was plotted on the x-axis, and the absorbance of each well was plotted on the y-axis. A nonlinear regression using the sigmoidal dose-response (Variable Slope) method (Graph Pad Prism software, Graph Pad Software, San Diego, California) was performed to obtain the binding curve between the target fusion protein and human CEACAM6 protein.

[0076] ELISA results as follows Figure 4 As shown, fusion proteins 1 and 2 can bind to human CEACAM6 protein at multiple concentration ranges, while the negative control cannot bind, indicating that fusion proteins with the IL-10 sequence fused to the C-terminus of the light or heavy chain of the constructed antibody have specific binding activity to human CEACAM6 protein.

[0077] Example 6: ELISA detection of the affinity of the fusion protein for CEACAM6(AB)

[0078] Human-CEACAM6(AB)-His (amino acid sequence SEQ ID NO: 14, purified by nickel column chromatography with 6 His tags added to the C-terminus) was diluted to 0.5 μg / mL using PBS buffer (pH 7.4). 100 μL was added to each well of a 96-well ELISA plate and incubated overnight at 4°C. After blocking with 1% BSA blocking buffer for 1 hour, the plate was washed three times with PBST. The purified fusion protein was then diluted to 100 nM with 0.5% BSA sample diluent. This was used as the starting concentration for 11 three-fold serial dilutions. A negative control (trastuzumab) was included, with 100 μL added to each well. The plate was incubated at 37°C for 1 hour. After washing three more times with PBST, HRP-labeled goat anti-human IgG Fc (Jackson Cat: 109-035-098) was diluted 1:10000 with sample diluent, with 100 μL added to each well and incubated at room temperature for 1 hour. After washing the plate four times with PBST, 100 μL of TMB substrate was added to each well, and the plate was incubated at room temperature in the dark for 10 min. The colorimetric reaction was then terminated by adding 100 μL of 1M HCl solution to each well. The absorbance of each well in the 96-well plate was measured using a multi-mode microplate reader at a wavelength of 450 nm and a reference wavelength of 570 nm. The absorbance (OD) of each well was calculated as OD450nm - OD570nm. The logarithm of the fusion protein concentration was plotted on the x-axis, and the absorbance of each well was plotted on the y-axis. A nonlinear regression using the sigmoidal dose-response (Variable Slope) method (Graph Pad Prism software, Graph Pad Software, San Diego, California) was performed to obtain the binding curve between the target fusion protein and human CEACAM6(AB) protein.

[0079] ELISA results as follows Figure 5 As shown, fusion proteins 1 and 2 can bind to human CEACAM6(AB) protein at multiple concentration ranges, while the negative control cannot bind to the protein. This indicates that fusion proteins with the IL-10 sequence fused to the C-terminus of the light or heavy chain of the constructed antibody have specific binding activity to human CEACAM6(AB) protein.

[0080] Example 7: ELISA detection of the affinity of the fusion protein for the IL-10 receptor

[0081] The IL-10 receptor human IL10RA-his (purchased from Beijing Yiqiao Shenzhou, CAT: 10419-H08H) was diluted to 0.5 μg / mL using PBS buffer (pH 7.4). 100 μL was added to each well of a 96-well ELISA plate and incubated overnight at 4°C. The plate was then blocked with 1% BSA blocking buffer for 1 hour. After washing the plate three times with PBST, the purified fusion protein was diluted to 100 nM with 0.5% BSA sample diluent. This was used as the starting concentration for a 3-fold serial dilution, resulting in 11 gradients. A negative control (trastuzumab) and a positive control (the fusion protein obtained in Example 4 of Patent 202110141918.8) were also included, with 100 μL added to each well. The plate was incubated at 37°C for 1 hour. Wash the plate three times with PBST. Dilute HRP-labeled goat anti-human IgG Fc (Jackson Cat: 109-035-098) 1:10000 with sample dilution buffer, add 100 μL to each well, and incubate at room temperature for 1 h. After washing the plate four times with PBST, add 100 μL of TMB substrate to each well, incubate at room temperature in the dark for 10 min, and then add 100 μL of 1M HCl solution to each well to stop the colorimetric reaction.

[0082] The absorbance of each well in a 96-well plate was measured using a multi-functional microplate reader at a wavelength of 450 nm and a reference wavelength of 570 nm. The absorbance (OD) of each well was calculated as OD450nm - OD570nm. The logarithm of the fusion protein concentration was plotted on the x-axis, and the measured absorbance of each well was plotted on the y-axis. A nonlinear regression was performed using the Sigmoidal dose-response (Variable Slope) method (Graph PadPrism software, Graph Pad Software, San Diego, California) to obtain the binding curve between the target fusion protein and the IL-10 receptor IL-10RA protein.

[0083] ELISA results as follows Figure 6 As shown, the positive control and fusion proteins 1 to 3 can bind to the IL-10 receptor at multiple concentration ranges. Among them, the binding ability of fusion protein 2 is comparable to that of fusion protein 3 and superior to that of fusion protein 1.

[0084] Example 8: Binding activity of the fusion protein to LS174T cells

[0085] LS174T cells (from the Shanghai Institute of Chinese Academy of Sciences) in the logarithmic growth phase and with normal morphology were collected, transferred to centrifuge tubes, centrifuged at 1000 rpm for 5 min, and then divided into 1×10⁻⁶ cells. 6Cells were resuspended in FACS buffer at a density of 1 / mL, and 100 μL of cells were aliquoted into individual tubes. The purified fusion protein was diluted to 400 nM with FACS buffer. Using this as the starting concentration, a 4-fold serial dilution was performed for a total of 6 dilutions. A positive control (from PCT / CN2022 / 086315) was included, with 100 μL of fusion protein dilution added. Cells were incubated at 4°C for 60 min, followed by two washes with excess FACS buffer. Cells were resuspended in 100 μL of FACS buffer, and goat anti-human secondary antibody-PE (Biolgend, Cat:398004) was added to the sample. Cells were incubated for 30 min and washed twice with excess FACS buffer. Cells were fixed in fixation buffer and then analyzed by flow cytometry. The binding activity of the fusion protein to LS174T cells was detected by FACS.

[0086] FACS test results are as follows Figure 7 As shown, fusion proteins 1 and 2 can specifically bind to LS174T cells at multiple concentration ranges.

[0087] Example 9: Verification of IL-10 luciferase expression activation

[0088] IL-10 binds to IL-10R, mediating the control of the degree and duration of inflammation, which is crucial for maintaining homeostasis of the body's inflammatory response. This process depends on the regulation of signal transduction and activator of transcription 3 (STAT3). This experiment used HEK293 cells, which stably express IL-10R and the STAT3 signaling pathway reporter gene system. Stimulation with IL-10 protein activated and increased luciferase expression. Therefore, this cell line was used for the study of IL-10-STAT3 activity.

[0089] IL-10-Reporter-HEK-293 cells (purchased from Jimon Biotechnology) in logarithmic growth phase were used, with 2.5 × 10⁶ cells per well. 4Cells were seeded in 96-well cell culture plates (WHB, Cat: WHB-96-01) and cultured overnight at 37°C in a 5% CO2 incubator. Then, the positive control (fusion protein obtained in Example 4 of Patent 202110141918.8), IL-10 protein (Novoprotein, Cat: CX04), negative control (cetuximab), human IgG1, fusion protein 1, and fusion protein 2 were serially diluted 4-fold from 300 μM to 9 different concentrations using fusion protein dilution buffer (DMEM complete medium), with 100 μL / well added to each well after supernatant aspiration. The plates were mixed and incubated at 37°C in a 5% CO2 incubator for 6 hours. Then, Bio-Lite Luciferase Assay substrate (Vazyme, Cat: DD1201-02) chromogenic solution was added at 100 μL / well, and the plates were incubated at room temperature for 10 minutes. Then, the fluorescence value was measured using an ELISA reader.

[0090] With the concentration of fusion protein as the x-axis and the relative fluorescence value (RLU) as the y-axis, the nonlinear regression analysis method in GraphPad Prism software (GraphPad Prism 5 Demo, San Diego, California) was used to select the Log(agonist) vs Response-Variable Slope method to generate the fitting curve, thus obtaining the fluorescence dose-response curve of the fusion protein.

[0091] like Figure 8 As shown, IL-10 and the positive control (the heterologous fusion protein obtained in Example 4 of Patent 202110141918.8) dose-dependently activated luciferase expression in HEK293 cells, while the negative controls cetuximab and human IgG1 showed no such effect, indicating that this reporter gene method can reflect the signaling activation activity of IL-10. The positive control is a bivalent IL-10 heterodimer, and fusion proteins 1 and 2 have symmetrical structures of monovalent IL-10. Figure 9-10 As shown, the positive control and fusion proteins 1 and 2 all exhibited activity in activating IL-10 reporter genes. In the example, at low concentrations, the IL-10 activation activity of fusion proteins 1 and 2 was weaker than that of the positive control. The EC50 of fusion protein 1... 50 The value is 110.4 nM, EC50 of fusion protein 2 50At a concentration of 10 nM, fusion protein 2 exhibited superior IL-10 activation activity compared to fusion protein 1. At high concentrations, both fusion proteins 1 and 2 were superior to the positive control. In the development of dimeric fusion proteins or bispecific antibodies, asymmetric structures often lead to problems such as light and heavy chain mismatches, numerous impurities, and difficulty in separating the target product during the preparation process. Overall, the structure of fusion protein 2 offers certain advantages over fusion protein 1 and the positive control.

[0092] It should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the scope of the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A dimer fusion protein, characterized in that, The dimer fusion protein consists of two light chains and two heavy chains linked to IL-10, wherein IL-10 is connected to the C-terminus of the heavy chain via a polypeptide linker with an amino acid sequence of GGGGS. The light and heavy chains are complexed to form a targeting moiety exhibiting specific binding to a tumor antigen. The tumor antigen is CEACAM5 and / or CEACAM6. The heavy chain has the amino acid sequences of HCDR1 as shown in SEQ ID NO:2, HCDR2 as shown in SEQ ID NO:3, and HCDR3 as shown in SEQ ID NO:

4. The light chain has the amino acid sequences of LCDR1 as shown in SEQ ID NO:5, LCDR2 as shown in SEQ ID NO:6, and LCDR3 as shown in SEQ ID NO:

7. The IL-10 has the amino acid sequence shown in SEQ ID NO:

1.

2. A nucleic acid encoding the dimer fusion protein of claim 1.

3. A vector containing the nucleic acid of claim 2.

4. A host cell containing the vector of claim 3.

5. The application of the dimer fusion protein according to claim 1, characterized in that, The application is to prepare reagents or kits for detecting CEACAM5, CEACAM6 and IL-10 receptor molecules.

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