TGF betaR2 extracellular region truncated molecule, fusion protein of TGF betaR2 extracellular region truncated molecule and anti-EGFR antibody and anti-tumor application of TGF betaR2 extracellular region truncated molecule and anti

Abstract

The invention belongs to the field of tumor immunotherapy, and relates to a plurality of fusion proteins containing truncated form TGF [beta] R2 and constructed by the truncated form TGF [beta] R2 and EGFR antibody HPA8. The TGF beta R2 truncated form provided by the invention can improve the protein stability and degradation resistance of the fusion protein containing TGF beta R2 and inhibit the neutralizing activity of TGF-beta function. Nucleic acid sequences (including heavy / light chain variable regions) encoding the antibodies, vectors containing the nucleic acid sequences, pharmaceutical compositions and kits are disclosed. The prepared fusion protein of the TGF beta R2 receptor protein in the truncated form and targeted EGFR and other tumor target antibodies can be used for treatment of solid tumors including but not limited to gastric cancer and the like.

Description

[0001] This application is a divisional application of the invention patent application filed on April 26, 2021, with application number 202180023395.5 and invention title "A TGFβR2 extracellular region truncated molecule, its fusion protein with an anti-EGFR antibody and its anti-tumor use".

[0002] Cross-references to related applications

[0003] This application claims the benefit of Chinese Patent Application No. 202010351280.6, filed on April 28, 2020, the contents of which are incorporated herein by reference. Technical Field

[0004] This invention relates to the field of tumor immunotherapy drugs. Specifically, this invention relates to molecules containing truncated extracellular regions of the immunomodulatory factor TGFβR2, including fusion proteins comprising the truncated molecule and a target molecule, pharmaceutical compositions comprising the same, and their use as antitumor drugs. Background Technology

[0005] Transforming growth factor-β (TGF-β) belongs to the TGF-β superfamily, which regulates cell growth and differentiation. It is a pleiotropic and pleiotropic cytokine that regulates cell proliferation, differentiation, and apoptosis via cell surface receptor signaling pathways through autocrine or paracrine mechanisms. It plays a crucial regulatory role in extracellular matrix synthesis, wound repair, and immune function. In mammals, three isoforms exist: TGF-β1, TGF-β2, and TGF-β3, with TGF-β1 being the most abundant and expressed isoform. TGF-β initiates downstream signal transduction via the classical Smad and non-Smad pathways by binding to the TGFβR1 and TGFβR2 serine-threonine kinase receptors on the cell membrane. [1] In normal homeostasis, TGF-β signaling regulates key processes such as growth, regeneration, and differentiation in various tissues. In the immune system, TGF-β induces tolerance and suppresses inflammation. Gene mutations can alter the output of TGF-β signaling in tumor-initiating cells. In the initial stages of tumorigenesis, TGF-β plays a crucial anti-tumor role by inhibiting cell proliferation and inducing apoptosis. However, as tumors develop, selective pressures lead to the loss of TGF-β's tumor-suppressive function in tumor cells through various mechanisms. Acquired loss-of-function mutations in the TGF-β signaling pathway enable various malignant tumor cells to grow in TGF-β-enriched environments. Furthermore, tumor cells somehow transform the pro-apoptotic capacity of TGF-β into pro-tumor functions, such as invasion and migration, and promoting mesenchymal transition. [2-4]TGF-β can regulate the types and functions of various immune cells. TGF-β controls adaptive immunity by directly promoting the proliferation of Treg cells and inhibiting the production and function of effector T cells and antigen-presenting dendritic cells. TGF-β can also inhibit NK cell function and transform macrophages and neutrophils into pro-tumor subtypes, promoting the formation of a tumor microenvironment negatively regulated by tumor immunity. [4] TGF-β1 expression levels are often higher in various solid tumors, including EGFR-positive colorectal cancer, non-small cell lung cancer, and head and neck squamous cell carcinoma, than in normal adjacent tissues. Clinical data indicate that blocking the TGF-β pathway alone is insufficient to fully restore the immune system and suppress tumor development; therefore, no TGF-β antibodies are currently available on the market.

[0006] The epidermal growth factor receptor (EGFR) is the expression product of the proto-oncogene C-ErbB1. It is a receptor for epidermal growth factor (EGF) cell proliferation and signal transduction, a member of the HER family, and belongs to the tyrosine kinase type receptor category. Its molecular weight is 170 kDa. [5] EGFR is divided into an extracellular ligand-binding domain, a transmembrane domain, and an intracellular kinase domain. After binding to a ligand, the extracellular domain of EGFR transforms from a monomer into a dimer, activating the intracellular kinase domain and triggering multiple downstream signaling pathways, playing a crucial role in physiological processes such as cell growth, proliferation, and differentiation. [6] High expression of EGFR leads to enhanced downstream signal transduction. Increased expression of mutant EGFR receptors or ligands results in sustained EGFR activation, enhanced secretory loop function, and disruption of receptor downregulation mechanisms, thereby activating genes related to tumor proliferation and differentiation, playing a crucial role in tumor formation and development. [7] EGFR overexpression is associated with decreased survival in several cancer types, including head and neck cancer, bladder cancer, ovarian cancer, cervical cancer, and esophageal cancer. Furthermore, anti-EGFR drugs have shown significant efficacy in treating several types of solid tumors, such as colorectal cancer, head and neck cancer, non-small cell lung cancer (NSCLC), and pancreatic cancer, including improvements in overall survival, progression-free survival, and overall response rate. [8] Therefore, as a clearly defined target associated with tumor proliferation, EGFR-targeted drugs have become a first-line treatment option for many malignant tumors.

[0007] Although the signaling pathway initiated by TGF-β differs from that initiated by the EGF / EGFR pathway, these two signaling pathways can influence each other, and the interaction between TGF-β and EGFR signaling has been observed in various tumors, jointly promoting tumor progression. TGF-β can trans-induce EGFR activation and exhibits high cell type and environment specificity. For example, TGF-β upregulates EGFR through the classical Smad and ERK / Sp1 signaling pathways, promoting the migration and invasion of breast cancer cells (MDA-MB-231, T47D, 4T1). [9,10] In squamous cell carcinomas (A431, SCC13), TGF-β activates the EGFR pathway via an H2O2-dependent mechanism, increasing Erk1 / 2 phosphorylation levels.

[11] EGF and its associated downstream signaling pathways can also regulate TGF-β signaling in different cell types. For example, in human primary ovarian cancer cells, EGF reduces TGF-β-induced cell cycle regulator p15. INK4B The expression of TGF-β mRNA is reduced, thereby decreasing the sensitivity of ovarian cancer cells to the anti-proliferative effect of TGF-β.

[12] Oncogenic Ras in breast and lung epithelial cells inhibits TGF-β-mediated signaling by negatively regulating Smad2 and Smad3, thereby reducing the growth-inhibiting effect of TGF-β on cells. [13,14] EGF can also positively regulate Smad2 signaling in COS7 cells by increasing Smad2 phosphorylation through the ERK pathway.

[15] .

[0008] TGF-β and EGF can synergistically promote the malignant phenotype of tumors. Studies in different tissues have shown that EGF binding to TGF-β can enhance epithelial-mesenchymal transition (EMT). For example, EGF and TGF-β1 synergistically promote the expression of laminin-332, thus promoting EMT transformation in oral epithelial carcinoma.

[16] EGF and TGF-β1 downregulate E-cadherin via the MAPK pathway, rather than the PI3K, p38MAPK, JNK, or AP-1 pathway, promoting EMT in intestinal epithelial cells.

[17] EGF and TGF-β1 induce Slug and Snail expression through Smad and MEK1 / 2-dependent signaling pathways, downregulate E-cadherin, and promote ovarian epithelial cell EMT.

[18] EGF and TGF-β1 activate the ERK1 / 2 signaling pathway, synergistically upregulate Snail protein expression, and promote EMT and migration in human renal proximal tubular epithelial cells (HK-2).

[19] EGF enhances TGF-β-induced EMT in lung cancer (H322, H358) and pancreatic cancer (HPAF-II, CAPAN-2) cells by promoting the binding of SHP2 to GAB1.

[20] .

[0009] Multiple clinical studies have shown that elevated TGF-β levels are closely associated with drug resistance and poor prognosis. In osteosarcoma stem cell-like cells, TGF-β1-induced EMT reduces miR-499a expression, leading to increased SHKBP1 expression. Furthermore, TGF-β-induced EMT converts related kinases to an EGFR-independent AKT activation state, thereby reducing EGFR activity and inducing resistance in osteosarcoma to EGFR kinase inhibitors. Figure 5 )

[21] Treg cells are one of the main cell types that produce TGF-β. In patients with head and neck squamous cell carcinoma treated with cetuximab, the number of Treg cells in the tumor increases, accompanied by an increase in TGF-β levels. Patients with poor response to cetuximab have even higher TGF-β levels.

[22] Elevated TGF-β can inhibit the expression of cytotoxic molecular effectors in effector cells, activate the EGFR-independent AKT pathway, and increase EMT-induced resistance to EGFR antibody therapy.

[23] In EGFR-mutant non-small cell lung cancer, the classic TGF-β / Smad signaling pathway is involved in PD-L1-induced tumor resistance to EGFR kinase inhibitors.

[24] In breast cancer tissues, TGF-β expression is positively correlated with EGFR expression, and elevated levels of both TGF-β and EGFR are associated with poor prognosis in breast cancer patients. [9] In summary, EGFR and TGF-β play relatively independent yet closely related roles in tumorigenesis and development. Furthermore, TGF-β is a key molecule in the development of acquired resistance to EGFR-targeted therapy in tumors. Animal experiments have shown that inhibiting TGF-β can enhance the in vivo antitumor effect of Cetuximab on xenografts of head and neck squamous cell tumors.

[23] These findings provide a theoretical basis for combining TGF-β-targeting antibodies to enhance the therapeutic effect of EGFR-positive tumors.

[0010] This invention provides a novel fusion protein containing a truncated form of TGFβR2, which can simultaneously and specifically target the relatively independent yet closely related signaling pathways of EGFR and TGF-β. It can be used to treat solid tumors, including but not limited to gastric cancer. Summary of the Invention

[0011] In one aspect, the present invention provides a truncated form of the extracellular region of TGFβR2, which, compared to its natural form,

[0012] a) At least amino acid residues from positions 6 to 16 are missing, and more preferably, amino acid residues from positions 17 to 17+n are missing, where n is an integer from 1 to 10; preferably, n is 2, 4, 8, 9, or 10; most preferably, n is 9; or

[0013] b) In addition to the deletion of amino acid residues at positions 6-26, further deletions are made at positions 5, 4-5, 3-5, 2-5, 1, 1-2, 1-3, and 1-4; or

[0014] c) It is missing amino acid residues at positions 7-26.

[0015] In one embodiment, the molecular amino acid sequence comprises SEQ ID NO: 1.

[0016] In another aspect, the present invention provides a fusion protein comprising the molecules described herein.

[0017] In one embodiment, the fusion protein comprises

[0018] a) The truncated form of the TGFβR2 extracellular region and

[0019] b) Targeted portion.

[0020] In one embodiment, the fusion protein targeting portion is a cancer cell-specific targeting portion, selected from antibodies or their antigen-binding fragments, functional ligands or their Fc fusion proteins, and receptor proteins or their Fc fusion proteins.

[0021] In one embodiment, the fusion protein targeting portion is an anti-EGFR antibody or its antigen-binding fragment.

[0022] In one embodiment, the N-terminus of the truncated form of the fusion protein is connected to the C-terminus of the heavy chain of the targeting portion, optionally via a linker.

[0023] In one embodiment, the connector is preferably a G4S flexible linker peptide, more preferably a (G4S)4 linker peptide.

[0024] In one embodiment, the targeting portion of the fusion protein is selected from the anti-EGFR antibody, Trastuzumab, Bevacizumab, Ramucirumab, Ipilimumab, or Panitumumab.

[0025] In one embodiment, the fusion protein

[0026] a) The heavy chain amino acid sequence contains SEQ ID NO:15 or a sequence having at least 85%, 88%, 90%, 95%, 98%, or 99% sequence identity with it; and

[0027] b) The light chain amino acid sequence is SEQ ID NO:16 or a sequence having at least 85%, 88%, 90%, 95%, 98% or 99% sequence identity with it;

[0028] It consists of two heavy chains and two light chains; a disulfide bond is formed between the first light chain and the first heavy chain, a disulfide bond is formed between the second light chain and the second heavy chain, and a disulfide bond is formed between the first heavy chain and the second heavy chain.

[0029] In one embodiment, the binding affinity (KD) of the fusion protein to the human EGFR protein is 2.92 pM-26.3 pM, preferably 7 pM-9 pM, and most preferably 8.77 pM.

[0030] The affinity (KD) for binding to human TGF-β1 protein is 23 pM-288.3 pM, preferably 64 pM-144 pM, and most preferably 96.1 pM.

[0031] In another aspect, the present invention provides a conjugate comprising a truncated form of the extracellular region of TGFβR2 as described in the present invention, a fusion protein as described in the present invention, an antibody or antigen-binding fragment thereof as described in the present invention, and a further therapeutic agent, preferably wherein the antibody or antigen-binding fragment thereof and the further therapeutic agent are linked by a linker.

[0032] In another aspect, the present invention provides a nucleic acid that encodes a truncated form of the extracellular region of TGFβR2 as described in the present invention, a fusion protein as described in the present invention, an antibody as described in the present invention, or an antigen-binding fragment thereof, which is mRNA and / or DNA.

[0033] In one embodiment, the nucleic acid comprises the sequence of SEQ ID NO:12, or a variant thereof with the same function.

[0034] In another aspect, the present invention provides an expression vector comprising the nucleic acid described herein.

[0035] In another aspect, the present invention provides a host cell comprising the nucleic acid or expression vector described herein.

[0036] In another aspect, the present invention provides a method for producing a truncated form of the extracellular region of the TGFβR2 described in the present invention, a fusion protein described in the present invention, an antibody described in the present invention, or an antigen-binding fragment thereof, comprising culturing the host cells described in the present invention under conditions suitable for the expression of the aforementioned protein molecules, and recovering the expressed products from the culture medium.

[0037] In another aspect, the present invention provides a pharmaceutical composition comprising...

[0038] a) A truncated form of the TGFβR2 extracellular region of the present invention, a fusion protein of the present invention, an antibody or its antigen-binding fragment of the present invention, an antibody-drug conjugate of the present invention, a nucleic acid of the present invention, or an expression vector of the present invention; and

[0039] b) A pharmaceutically acceptable carrier; optionally c) One or more other therapeutic agents.

[0040] In another aspect, the present invention provides a truncated form of the extracellular region of TGFβR2 as described in the present invention, a fusion protein as described in the present invention, an antibody or antigen-binding fragment thereof as described in the present invention, an antibody-drug conjugate as described in the present invention, a nucleic acid as described in the present invention, an expression vector as described in the present invention, or a pharmaceutical composition as described in the present invention, for the prevention and treatment of cancer, preferably for the treatment of gastric cancer.

[0041] In another aspect, the present invention provides a truncated form of the TGFβT2 extracellular region of the present invention, a fusion protein of the present invention, an antibody or antigen-binding fragment thereof of the present invention, an antibody-drug conjugate of the present invention, a nucleic acid of the present invention, an expression vector of the present invention, and a pharmaceutical composition of the present invention for use in the preparation of a medicament for the prevention and treatment of cancer, preferably for the treatment of gastric cancer.

[0042] In another aspect, the present invention provides a pharmaceutical composition comprising a truncated form of the TGFβR2 extracellular region of the present invention, a fusion protein of the present invention, an antibody or antigen-binding fragment thereof of the present invention, an antibody-drug conjugate of the present invention, a nucleic acid of the present invention, an expression vector of the present invention, or a pharmaceutical composition of the present invention; and one or more other therapeutic agents.

[0043] In another aspect, the present invention provides a kit comprising a truncated form of the extracellular region of TGFβR2 as described in the present invention, a fusion protein as described in the present invention, an antibody or antigen-binding fragment thereof as described in the present invention, an antibody-drug conjugate as described in the present invention, a nucleic acid as described in the present invention, an expression vector as described in the present invention, and a pharmaceutical composition as described in the present invention; preferably, it further comprises an administration device.

[0044] In another aspect, the present invention provides a method for preventing and treating tumor diseases, comprising administering to a subject a molecule in a truncated form of the extracellular region of TGFβR2 as described in the present invention, a fusion protein as described in the present invention, an antibody as described in the present invention or an antigen-binding fragment thereof, an antibody-drug conjugate as described in the present invention, a nucleic acid as described in the present invention, an expression vector as described in the present invention, or a pharmaceutical composition as described in the present invention. Attached Figure Description

[0045] Figure 1 Detection of the binding of murine antibody EGFR-mhPA8 to recombinant human EGFR-His protein;

[0046] Figure 2 The murine antibody EGFR-mhPA8 blocks the binding of EGF to EGFR;

[0047] Figure 3 The murine antibody EGFR-mhPA8 inhibited the proliferation of MDA-MB-468 cells;

[0048] Figure 4 Detection of binding between humanized antibody EGFR-HPA8 and recombinant human EGFR-His protein;

[0049] Figure 5 The humanized antibody EGFR-HPA8 blocks the binding of EGF to EGFR;

[0050] Figure 6 The humanized antibody EGFR-HPA8 inhibited the proliferation of MDA-MB-468 cells under different ligand conditions;

[0051] Figure 7 The humanized antibody EGFR-HPA8 inhibited the proliferation of Fadu cells under different ligand conditions;

[0052] Figure 8 ADCC effect mediated by humanized antibody EGFR-HPA8;

[0053] Figure 9 Effect of EGFR-HPA8 on body weight in SNU-5 mice with subcutaneous gastric cancer xenografts;

[0054] Figure 10 Effects of EGFR-HPA8 on tumor volume and TGI results in SNU-5 subcutaneous gastric cancer xenograft mice;

[0055] Figure 11 Effect of EGFR-HPA8 on body weight in mice with NCI-H1975 non-small cell lung cancer subcutaneous xenograft tumors;

[0056] Figure 12Effects of EGFR-HPA8 on tumor volume and TGI results in mice with NCI-H1975 non-small cell lung cancer subcutaneous xenografts;

[0057] Figure 13 Schematic diagram of the EGFR / TGFβR2 antibody fusion protein structure;

[0058] Figure 14 Detection of fragmentation in EGFR / TGFβR2 antibody fusion protein;

[0059] Figure 15 Degradation tendency detection of EGFR / TGFβR2 antibody fusion protein;

[0060] Figure 16 Detection of the binding ability of TGF-β1 and EGFR to TGFβR2 antibody fusion proteins with different truncated forms of TGFβR2;

[0061] Figure 17 Detection of the ability of EGFR / TGFβR2 antibody fusion protein containing a truncated form of TGFβR2 to neutralize TGF-β1;

[0062] Figure 18 Detection of the ability of EGFR / TGFβR2 antibody fusion protein containing a truncated form of TGFβR2 to neutralize TGF-β3;

[0063] Figure 19 Detection of the binding ability of fusion protein 6 to TGF-β1 and TGF-β3 proteins;

[0064] Figure 20 Detection of the ability of fusion protein 6 to block the binding of TGF-β1 or TGF-β3 proteins to TGFβR2-Fc;

[0065] Figure 21 Detection of fusion protein 6 binding and competitive ability;

[0066] Figure 22 Affinity detection of fusion protein 6 with recombinant human EGFR protein;

[0067] Figure 23 Affinity detection of fusion protein 6 with recombinant human TGF-β1 protein;

[0068] Figure 24 Fusion protein 6 neutralizes the effects of TGF-β1 in Mv-1-lu cells;

[0069] Figure 25 The reporter gene system was used to detect the neutralizing effect of fusion protein 6 on TGF-β1;

[0070] Figure 26Fusion protein 6 inhibits the proliferation of MDA-MB-468 cells;

[0071] Figure 27 ADCC effect mediated by fusion protein 6;

[0072] Figure 28 Effect of fusion protein 6 on body weight in mice with NCI-H1975 non-small cell lung cancer subcutaneous xenograft tumors;

[0073] Figure 29 Effect of fusion protein 6 on tumor volume in mice with NCI-H1975 non-small cell lung cancer subcutaneous xenograft tumors;

[0074] Figure 30 Ultrafiltration stability assay of fusion protein 6;

[0075] Figure 31 Detection of X / TGFβR2 antibody fusion protein fragmentation;

[0076] Figure 32 Degradation tendency detection of X / TGFβR2 antibody fusion protein;

[0077] Figure 33 Detection of the binding ability of X / TGFβR2 antibody fusion protein to TGF-β1;

[0078] Figure 34 Detection of the ability of X / TGFβR2 antibody fusion protein to neutralize TGF-β1 and TGF-β3;

[0079] Figure 35 X / TGFβR2 antibody fusion protein binding to X-side target detection. Detailed Implementation

[0080] definition

[0081] Unless otherwise stated, all technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. For the purposes of this invention, the following terms are further defined.

[0082] When used herein and in the appended claims, the singular forms “a,” “an,” “another,” and “the” include the plural referents unless the context clearly indicates otherwise.

[0083] The term "truncated" form of a protein molecule refers to a modified form that lacks one or more amino acid residues compared to its native form.

[0084] The term "fusion protein" refers to a protein molecule that combines two or more proteins. It is typically obtained by expressing a hybrid gene that combines two or more gene sequences, inserted into an expression vector in a frame-matched format.

[0085] The term "antibody" refers to an immunoglobulin molecule, meaning any form of antibody that expresses the desired biological activity. This includes, but is not limited to, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies), and even antibody fragments. The term "variable region" refers to a domain in the antibody heavy or light chain involved in antibody-antigen binding. The variable regions (VH and VL, respectively) of the heavy and light chains of natural antibodies generally have similar structures and can be further subdivided into hypervariable regions (called complementarity-determining regions (CDRs)) interspersed within more conserved regions (called frame regions (FRs)).

[0086] The complementarity-determining regions (CDRs) and framework regions (FRs) of a given antibody can be identified using the Kabat system (Kabat et al.: Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, PHS, NIH, NIH Publication No. 91-3242, 1991). The term "constant region" refers to amino acid sequences on the light and heavy chains of an antibody that do not directly participate in antibody-antigen binding but exhibit various effector functions, such as antibody-dependent cytotoxicity.

[0087] An "antigen-binding fragment of an antibody" comprises a portion of the complete antibody molecule that retains at least some of the binding specificity of the parent antibody, and typically includes at least a portion of the antigen-binding region or variable region (e.g., one or more CDRs) of the parent antibody. Examples of antigen-binding fragments include, but are not limited to, Fv, Fab, Fab′, Fab′-SH, F(ab′)2, Fd fragments, Fd' fragments, single-chain antibody molecules (e.g., scFv, di-scFv, or tri-scFv, bispecific antibodies, or scFab), and single-domain antibodies.

[0088] The term "conjugate" refers to a biologically active protein or peptide molecule that forms a covalent or non-covalent linker with another molecule, which is selected from small molecule compounds or biological macromolecules.

[0089] "Monoclonal antibody" refers to an antibody derived from a substantially homogeneous group of antibodies, meaning that the group comprising a single antibody is identical except for the possibility of mutations (e.g., natural mutations) that may be present in very small amounts. Therefore, the term "monoclonal" indicates the nature of the antibody, i.e., it is not a mixture of unrelated antibodies. In contrast to polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen. Besides their specificity, monoclonal antibody formulations have the advantage that they are generally not contaminated by other antibodies. The term "monoclonal" should not be construed as requiring the antibody to be produced by any particular method. Specifically, the term monoclonal antibody includes chimeric antibodies, humanized antibodies, and human antibodies.

[0090] An antibody “specifically binds” to a target antigen, such as a tumor-associated antigen protein (EGFR in this context), meaning it binds to the antigen with sufficient affinity so that the antibody can be used as a therapeutic agent to target tissues or cells expressing the antigen, and has no significant cross-reactivity with other proteins or with proteins other than homologs and variants of the antigen target mentioned above (e.g., mutant forms, splice variants, or proteolytically truncated forms).

[0091] The term "binding affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule and its binding partner. Unless otherwise stated, when used herein, "binding affinity" refers to the inherent binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen). D "Binding rate constant kon" and "dissociation rate constant koff" are commonly used to describe the affinity between a molecule (e.g., an antibody) and its binding chaperone (e.g., an antigen), i.e., how tightly the ligand binds to a specific protein. Binding affinity is influenced by non-covalent intermolecular interactions, such as hydrogen bonds, electrostatic interactions, hydrophobicity between the two molecules, and van der Waals forces. Additionally, the binding affinity between a ligand and its target molecule can be affected by the presence of other molecules. Affinity can be analyzed using conventional methods known in the art, including the ELISA described herein.

[0092] The term "targeting portion" refers to the part of a fusion protein that has the function of specifically binding to target cells. This term includes antibodies and other natural (e.g., receptors, ligands) or synthetic (e.g., DARPin) molecules capable of specifically binding to target cells. As used herein, "specifically binding to target cells" means that the portion preferentially binds to target cells within a complex mixture.

[0093] "Isolated" biomolecules are biomolecules that have been identified and isolated from cells that naturally express the molecule. Isolated biomolecules include recombinant intracellular biomolecules as well as biomolecules that are typically prepared through at least one purification step.

[0094] The term "receptor" is a biochemical concept referring to a class of molecules that specifically recognize and bind to extracellular signals (i.e., "ligands"), producing specific effects within the cell. These effects may last only a short time, such as altering cellular metabolism or motility, or they may be long-lasting, such as upregulating or downregulating the expression of one or more genes.

[0095] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which secreted Ig bound to Fcγ receptors present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages) enables these cytotoxic effector cells to specifically bind to target cells carrying antigens, which are then killed using, for example, cytotoxins. To assess the ADCC activity of a target antibody, an in vitro ADCC assay can be performed, such as the in vitro ADCC assay described in U.S. Patent Nos. 5,500,362 or 5,821,337 or 6,737,056 (Presta), or the methods described in the embodiments of this application. Useful effector cells for such assays include PBMCs and NK cells.

[0096] The present invention relates to molecules comprising truncated forms of the TGFβR2 extracellular region, including fusion proteins comprising truncated forms and targeting molecules.

[0097] The full-length TGFβR2 extracellular domain contains multiple easily cleavable sites between the N-terminus 7 and 15. To improve the structural stability of the fusion protein, the inventors modified the N-terminal amino acid sequence of the TGFβR2 extracellular domain by deleting different numbers of amino acids. This resulted in 15 truncated versions of the TGFβR2 extracellular domain.

[0098] The inventors used newly isolated EGFR antibodies EGFR-HPA8, Trastuzumab, Bevacizumab, Ramucirumab, Ipilimumab, or Panitumumab as the targeting portion of the fusion protein, and a truncated form of the extracellular region of the TGFβR2 protein as the immunomodulatory portion of the fusion protein. Through homologous recombination, the truncated form of the TGFβR2 extracellular region was linked to the C-terminus of the heavy chain of the EGFR antibody, forming an EGFR antibody / TGFβR2 light chain and extracellular region fusion protein (EGFR / TGFβR2) from the light and heavy chains. The fusion protein structure is as follows. Figure 13As shown, the EGFR / TGFβR2 antibody fusion protein uses a (G4S)4Linker (SEQ ID NO:66) to link the C-terminal amino acid of the EGFR antibody heavy chain to the extracellular region with TGFβR2 amino acids in different amino acid deletion forms. Furthermore, the C-terminal lysine amino group of the EGFR antibody heavy chain was removed to reduce the risk of proteolytic degradation.

[0099] In a preferred embodiment of the present invention, fusion protein 6 retains a high binding affinity for human EGFR protein, wherein the binding affinity of fusion protein 6 to human EGFR protein is K. D The value is 8.77 pM, and the constant kon value is 1.68E+06M. -1 s -1 The dissociation constant kdis is 1.47E-05s. -1 Furthermore, the truncated form of TGFβR2 fusion protein 6 has a similar affinity to the full-length form of TGFβR2 fusion protein 1, which is a homologous TGF-β1 protein. D The value is 96.1 pM, and the constant kon value is 1.53E+06M. -1 s -1 The dissociation constant kdis is 1.47E-04s. -1 .

[0100] The present invention also relates to nucleic acid molecules encoding truncated forms of the TGFβR2 extracellular region of the present invention, and nucleic acid molecules comprising fusion proteins or portions thereof including truncated forms of the molecules and targeting molecules. Some example sequences of these nucleic acid molecules are shown in the sequence listing.

[0101] The nucleic acid molecules of this invention are not limited to the sequences disclosed herein, but also include variants and corresponding other nucleic acid forms, such as mRNA, cDNA, and their variants. Variants in this invention can be described with reference to their physical properties in hybridization. Those skilled in the art will recognize that nucleic acids can be used to identify their complements and equivalents or homologues using nucleic acid hybridization techniques.

[0102] Recombinant vectors and expression

[0103] This invention also provides recombinant constructs comprising one or more nucleotide sequences of this invention. The recombinant constructs of this invention can be used with vectors, such as plasmids, phages, bacteriophages, or viral vectors, into which a nucleic acid molecule encoding an antibody of this invention is inserted. This document provides molecules, targeting molecules, and fusion proteins comprising the truncated form of the TGFβR2 extracellular region, which can be prepared by recombinant expression of nucleotide sequences encoding the aforementioned molecules or proteins in host cells. The aforementioned molecules or proteins may contain more than one amino acid; to express multiple amino acids recombinantly, host cells can be transfected with one or more recombinant expression vectors carrying the encoding nucleotide sequences, so that the multiple amino acids are expressed in the host cells. Standard recombinant DNA methodologies are used to prepare and / or obtain nucleic acids encoding the aforementioned molecules or proteins, incorporate these nucleic acids into recombinant expression vectors, and introduce the vectors into host cells, such as those described in Sambrook, Fritsch and Maniatis (eds.), Molecular Cloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, (1989); Ausubel, FM et al. (eds.), Current Protocols in Molecular Biology, Greene Publishing Associates, (1989); and Boss et al. U.S. Patent No. 4,816,397.

[0104] Examples of prokaryotic host cells include bacteria, while examples of eukaryotic host cells include yeast, insect, or mammalian cells. It should be understood that the design of expression vectors, including the selection of regulatory sequences, is influenced by a variety of factors, such as the choice of host cell, the desired protein expression level, and whether the expression is constitutive or inducible.

[0105] The truncated form of the TGFβR2 extracellular region of the present invention, the targeting molecule, and the fusion protein can be recovered and purified from recombinant cell cultures using known methods, including but not limited to ammonium sulfate or ethanol precipitation, acid extraction, protein A affinity chromatography, protein G affinity chromatography, anion or cation exchange chromatography, cellulose phosphate chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and lectin chromatography. High-performance liquid chromatography (“HPLC”) can also be used for purification.

[0106] use

[0107] The molecules, antibodies, and fusion proteins of the truncated form of the TGFβR2 extracellular region of the present invention can be used to treat cancers, such as gastric cancer.

[0108] Pharmaceutical Composition

[0109] The truncated forms of molecules, fusion proteins, antibodies and antigen-binding fragments, truncated forms of molecule-drug conjugates, fusion protein-drug conjugates, antibody-drug conjugates, nucleic acids, carriers, and one or more of these inventions can be prepared into pharmaceutical compositions with at least one other chemical agent, comprising the above-mentioned active ingredients and one or more pharmaceutically acceptable carriers, diluents, or excipients; optionally, they may also contain one or more other therapeutic agents.

[0110] Reagent test kit

[0111] The present invention also relates to pharmaceutical packaging and reagent kits comprising one or more containers containing the pharmaceutical compositions of the present invention mentioned above. Associated with such containers may be a form of notification prescribed by a government agency regulating the production, use, or sale of a drug or biological product, reflecting approval by the agency that produces, uses, or sells the product for human administration.

[0112] Preparation and storage

[0113] The pharmaceutical compositions of the present invention can be prepared in a manner known in the art, for example by conventional methods of mixing, dissolving, granulating, grinding, emulsifying, encapsulating, embedding, or lyophilizing.

[0114] After a pharmaceutical composition comprising the compounds of the present invention formulated in an acceptable carrier has been prepared, it can be placed in a suitable container and labeled for the treatment of the indicated condition. Such labels will include the dosage, frequency, and method of administration.

[0115] Drug combination

[0116] The pharmaceutical compositions comprising the antibodies of the present invention may also be combined with one or more other therapeutic agents, wherein the resulting combinations do not cause unacceptable adverse effects.

[0117] The contents of the invention application dated April 26, 2021, application number 202180023395.5, entitled "A TGFβR2 extracellular region truncated molecule, its fusion protein with an anti-EGFR antibody and its anti-tumor use" are incorporated herein by reference.

[0118] The following examples are provided to illustrate the present invention, and are not intended to limit the scope of the invention.

[0119] Example

[0120] Example 1: Screening for murine antibodies that block the binding of EGF to EGFR using a phage antibody display library

[0121] 1.1 Screening of mouse immune and phage antibody libraries

[0122] Mice were immunized with nucleic acid plasmids pCMV3-mFlt3L (G12FE5S01-D, constructed by Shenzhou Cell Engineering Co., Ltd.) and pCMV3-mCSF2 (G12FE5S02-D, constructed by Shenzhou Cell Engineering Co., Ltd.). The specific method was as follows: two days before the first immunization, mice were injected intramuscularly with 10 μM of cardiotoxin. For each subsequent immunization, mice were injected intramuscularly with a 1:1 mixture of pCMV3-mFlt3L and pCMV3-mCSF2 at a concentration of 20 μg / leg. Three days later, mice were injected intramuscularly with 30 μg / leg of pGS6-EGFR-TT-WPRE (constructed by Shenzhou Cell Engineering Co., Ltd., SEQ ID NO:144). For the fourth and fifth immunizations, mice were injected intraperitoneally with 5 × 10⁵ pCMV3-mFlt3L and pCMV3-mCSF2. 6 Insect cells overexpressing EGFR were immunized at intervals of 2 weeks, 2 weeks, 2 weeks, and 2 weeks. Starting with the third immunization, blood was collected seven days after each immunization via the medial canthal venous plexus. The serum titer of mice against EGFR was detected using an ELISA method coated with recombinant human EGFR protein (source: Beijing Yiqiao Shenzhou Technology Co., Ltd., Cat.10001-H08B, hereinafter the same). The standard was an 8000-fold increase in serum titer (OD>1). After the fifth immunization, 20 days later, 5×10⁻⁶ cells were used to detect the anti-EGFR serum titer. 6 MDA-MB468 cells were injected intraperitoneally to enhance the effect. Seven days later, the mice were sacrificed, and the spleen tissue of the mice was cryopreserved in liquid nitrogen.

[0123] RNA was extracted from mouse spleen tissue using TriPure Isolation Reagent (Source: Roche Cat. No. 11 667165 001). cDNA was obtained by reverse transcription using TriPure Isolation Reagent (Source: Invitrogen Cat. No. 18080-051). PCR amplification yielded the nucleotide sequences encoding the light and heavy chain variable regions of the mouse antibody. Overlap extension splicing PCR was used to assemble the nucleotide sequences encoding the mouse antibody's light and heavy chain variable regions into the nucleotide sequence encoding scFv. The light and heavy chain variable regions were connected via linkers.

[0124] Connect

[25] The phage vector pComb3x (from Beijing Yiqiao Shenzhou Technology Co., Ltd.) was then ligated into X-Blue competent cells via restriction endonuclease Sfi I (source: Fermentas). A phage display scFv antibody library for immunizing mice was constructed by electroporation. Recombinant human EGFR protein was coated onto ELISA plates, and phage libraries enriched with anti-EGFR positive antibodies were obtained by panning phage antibodies. Subsequently, these libraries were mixed with MDA-MB-468 cells (source: Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences), and phage libraries enriched with anti-MDA-MB-468 cell positive antibodies were obtained by panning phage antibodies (O'Brien, PM, & Aitken, R. (Eds.), Springer Science & Business Media. 2002; ISBN: 9780896037113). Single phage clones were selected from the enriched library for expression, and the binding to recombinant human EGFR protein was detected by ELISA. High-binding antibody clones of EGFR-mhPA8 scFv that specifically bind to recombinant human EGFR were obtained by screening. The clones were sent to a sequencing company for sequencing to obtain the nucleotide sequence of the EGFR-mhPA8 scFv antibody.

[0125] 1.2 Functional detection of murine antibodies targeting EGFR

[0126] 1.2.1 Binding and ligand competition of murine antibodies

[0127] The binding ability of mouse antibodies to recombinant human EGFR protein was detected by ELISA. Different concentrations (1.37 ng / mL, 4.12 ng / mL, 12.35 ng / mL, 37.04 ng / mL, 111.11 ng / mL, 333.33 ng / mL, 1000 ng / mL, and 3000 ng / mL) of recombinant human EGFR-His protein (source: Beijing Sinocare Biotechnology Co., Ltd., hereinafter the same) were coated onto 96-well plates, 100 μL per well, and incubated overnight at 4°C. The plates were washed the next day and blocked at room temperature for 1 h. 13.89 nM of EGFR-mhPA8 and negative control antibody H7N9-R1 (source: Sinocare Biotechnology Co., Ltd., hereinafter the same) were added and incubated for 1 h, followed by washing to remove unbound antibodies. After incubation with secondary antibody Goat anti-hIgG F(ab)2 / HRP (source: Jackson Immuno Research, Cat. 109-036-006, hereinafter the same) for 1 hour, the plate was washed repeatedly, substrate chromogenic solution was added for color development, and the OD was read by microplate reader after termination. 450 Plotting recombinant human EGFR-His protein concentration on the x-axis, OD... 450The readings were plotted on the ordinate. An S-shaped curve was fitted using GraphPad Prism 6.0 software, and the ECG binding of the antibody to recombinant human EGFR-His protein was analyzed. 50 The result is as follows Figure 1 As shown, the human-mouse chimeric antibody EGFR-mhPA8 can effectively bind to recombinant human EGFR-His and ECG. 50 It was 128.7 ng / mL, R 2 =1.000. The negative control H7N9-R1 did not bind to recombinant human EGFR-His protein.

[0128] This embodiment further analyzed the ability of EGFR-mhPA8 to block EGFR ligand-receptor binding using FACS. 3×10 5 10 μL of biotin-labeled EGF-Fc protein (source: Beijing Sinocare Technology Co., Ltd.) at a final concentration of 217.1 nM was added to each MDA-MB-468 cell. After incubation at 2-8℃ for 30 min, EGFR-mhPA8 antibody at final concentrations of 306.74 nM, 102.25 nM, 34.08 nM, and 11.36 nM was added, with H7N9-R1 as a negative control antibody. After mixing and incubation at 2-8℃ for 20 min, the cells were washed with PBS and centrifuged to remove unbound antibody and ligand. Streptavidin-488-FITC secondary antibody (source: Beijing Sinocare Technology Co., Ltd., hereinafter the same) was added and incubated at 2-8℃ for 1 h. Washing and centrifugation were repeated to remove unbound secondary antibody. Finally, 200 μL of PBS was added to resuspend the cells, and the mixture was filtered through a 400-mesh filter into flow cytometry tubes for flow cytometry analysis. The results are as follows: Figure 2 As shown, EGFR-mhPA8 can effectively block the binding of EGF-Fc protein to EGFR on MDA-MB-468 cells in a concentration gradient.

[0129] 1.2.2 Inhibition of MDA-MB-468 cell proliferation by murine antibody

[0130] MDA-MB-468 breast cancer cells highly express EGFR and also secrete various EGFR ligand factors. In this example, the WST-8 assay was used to determine the growth inhibitory function of mouse antibody on MDA-MB-468 cells.

[0131] MDA-MB-468 cells were evenly seeded in 96-well plates at 5 × 10⁶ cells / well. 3 / well. After culturing cells in a CO2 incubator for 3 hours, different concentrations of mouse antibody EGFR-mhPA8 (66.7 nM, 22.2 nM, 7.4 nM, 2.5 nM, 0.82 nM, 0.27 nM, 0.093 nM, 0.033 nM, and 0.013 nM) were added. A negative control group M (containing cells) and a blank control group B (containing only culture medium, without cells) were also set up. After incubating cells in a CO2 incubator at 37°C and 5% CO2 for 5 days, 15 μL of WST-8 was added per well. OD was measured using a microplate reader after 240 minutes. 450 -OD 630 The inhibition rate of the mouse antibody was calculated by subtracting the reading from the blank well B. Inhibition rate = (OD value of group M - OD value of sample) / (OD value of group M) × 100%. The dose-response curve was analyzed and plotted using GraphPad Prism software, with antibody concentration on the x-axis and inhibition rate on the y-axis. Figure 3 As shown, EGFR-mhPA8 effectively inhibited the proliferation of MDA-MB-468 cells, and the inhibition rate increased with increasing drug concentration in an "S"-shaped curve. EGFR-mhPA8 inhibited the EC50 of MDA-MB-468 cells. 50 It is 2.78 nM, R 2 =0.991.

[0132] Example 2: Humanization of mouse antibody EGFR-mhPA8 and in vitro and in vivo performance analysis of the humanized antibody

[0133] 2.1 Humanization and Production of Mouse Antibody mhPA8

[0134] The nucleotide sequence of the EGFR-mhPA8 antibody was deduced, yielding the amino acid sequences of the heavy and light chain variable regions of the EGFR-mhPA8 scFv antibody. (Refer to Kabat [...]) 26 Using the IMGT numbering method, the amino acid sequences of three CDRs each from the light and heavy chains of EGFR-mhPA8 scFv were determined. Following the Kabat numbering, except for the N-to-D mutation at position 52 of LCDR2, the aforementioned three CDRs from both the light and heavy chains were transplanted and retained in the final humanized antibody EGFR-HPA8 scFv during subsequent humanization steps.

[0135] Humanization of mouse antibodies was performed using the classic CDR transplantation method. 27 , 28Antibodies with at least 50% similarity to the variable regions of both the mouse light and heavy chains, and whose amino acid sequence similarity to the variable regions of the light and heavy chains of the target antibody was at least 50%, were selected as humanized template libraries. From these, the humanized antibody with the highest spatial structural similarity to the variable region of the target antibody was chosen as the humanized template. The three CDR sequences of the mouse antibody light or heavy chain were substituted into the corresponding CDR amino acid sequences in the humanized template. To improve the chemical stability of the antibody and maintain its biological function, high-risk deamidation amino acids such as NG, NS, NA, and NT from the mutant sequences were selected. The affinity of the humanized antibodies was detected using ELISA, and humanized antibodies that maintained affinity were selected. In this embodiment, the human template used for transplanting the light chain variable region of EGFR-mhPA8 is IGKV1-NL1*01, which has 68.4% homology with the EGFR-mhPA8 light chain. The human template for the heavy chain variable region is IGHV1-69-2*01, which has 64.9% homology with the EGFR-mhPA8 heavy chain. The N-type deamide site at position 52 in the LCDR2 variable domain of the humanized antibody EGFR-HPA8 was mutated to D.

[0136] Since key points in the murine frame region play a crucial role in maintaining the stability of the CDR spatial structure, it is necessary to reverse-mutate these key points to the corresponding amino acids of the murine antibody. Following Kabat numbering, the light chain was reverse-mutated at position 45 to Q, position 48 to I, position 74 to K, and position 76 to D; the heavy chain was reverse-mutated at position 38 to K, position 48 to I, and position 70 to L. The humanized antibody EGFR-HPA8 was obtained through CDR humanization transplantation and frame region reverse mutation.

[0137] The EGFR-HPA8 antibody light chain nucleotide sequence containing the signal peptide was amplified by splicing PCR. This sequence included the light chain signal peptide nucleotide sequence, the humanized antibody light chain variable region nucleotide sequence, and the human kappa light chain constant region nucleotide sequence, linked sequentially. The PCR product was then infused into the pSTEP2 vector (constructed by Shenzhou Cell Engineering Co., Ltd., hereinafter the same) (digested with Hind III and Xba I). ​​Sequencing confirmed the correct plasmid. The EGFR-HPA8 antibody heavy chain variable region nucleotide sequence was obtained through whole-genome synthesis and infused into the pSTEP2 vector (digested with Sca I and Nhe I) containing the heavy chain signal peptide nucleotide sequence and the human IgG1 heavy chain constant region nucleotide sequence. Sequencing confirmed the correct EGFR-HPA8 light and heavy chain expression vector. After plasmid extraction, HEK-293 cells (fut8 gene knockout) were transfected and cultured for expression for 7 days. After centrifugation, the cell supernatant was purified by affinity chromatography using Protein A packing material that interacts with Fc. The Protein A chromatography column was equilibrated with 5-10 column volumes of 50 mM Tris, 10 mM NaCl, and pH 8.0 buffer. The filtered culture supernatant was then added to the chromatography column for binding. The column was washed with 5-10 column volumes of 20 mM Tris, 0.3 M Arg, and pH 6.5 buffer, followed by elution with 0.1 M Gly, 10 mM NaCl, and pH 3.5 elution buffer. The collected sample was neutralized with 2 M Tris (pH 8.0) to obtain high-purity and high-quality ADCC-enhanced EGFR-HPA8 antibody.

[0138] 2.2 In vitro performance analysis of humanized EGFR-HPA8 antibody

[0139] 2.2.1 Specific binding and ligand competition of humanized antibodies

[0140] Referring to Example 1.2.1, the binding ability of the human antibody to recombinant human EGFR protein was detected by ELISA, with SCT200 (described in CN200610012002.8, hereinafter the same), Erbitux (MERCK, 201621, hereinafter the same) and a negative control set up. Figure 4 As shown, the specific binding of humanized EGFR-HPA8 antibody to recombinant human EGFR-His ECG 50 It was 116.6 ng / mL, R 2 =1.000; EC of SCT200 50 It was 166.5 ng / mL, R 2 =1.000; Erbitux combined EC 50 It was 253.6 ng / mL, R2 =1.000; negative control H7N9-R1 showed no binding. The results indicate that EGFR-HPA8's ability to bind to recombinant human EGFR-his is superior to SCT200 and Erbitux.

[0141] Meanwhile, referring to Example 1.2.1, the ability of the humanized antibody EGFR-HPA8 to block EGFR receptor binding was analyzed by FACS, with SCT200, Erbitux, and a negative control set up. The results are as follows... Figure 5 As shown, EGFR-HPA8 has a stronger ability to block the binding of EGF-Fc protein to EGFR on MDA-MB-468 cells than SCT200 and Erbitux.

[0142] 2.2.2 Inhibition of proliferation of different tumor cells by humanized antibodies

[0143] 2.2.2.1 Humanized antibody inhibits the proliferation of MDA-MB-468 cells

[0144] MDA-MB-468 cells were evenly seeded in 96-well plates at 5 × 10⁶ cells / well. 3 / well. After culturing cells in a CO2 incubator for 3 hours, different concentrations of humanized antibody EGFR-HPA8 (666.7 nM, 222.2 nM, 74.1 nM, 24.7 nM, 8.23 ​​nM, 2.74 nM, 0.91 nM, 0.31 nM, and 0.1 nM) were added, with SCT200 and Erbitux controls set up. For ligand addition assays, HB-EGF (source: Beijing Yiqiao Shenzhou Technology Co., Ltd., hereinafter the same), BTC-Fc (source: Beijing Yiqiao Shenzhou Technology Co., Ltd., hereinafter the same), or Fc-EREG (source: Beijing Yiqiao Shenzhou Technology Co., Ltd., hereinafter the same) were added to final concentrations of 8 ng / mL, BTC-Fc (source: Beijing Yiqiao Shenzhou Technology Co., Ltd., hereinafter the same), or Fc-EREG (source: Beijing Yiqiao Shenzhou Technology Co., Ltd., hereinafter the same) were added. After incubation in a CO2 incubator at 37℃ and 5% CO2 for 5 days, WST-8 was added at 15 μL / well. OD was measured using a microplate reader after 240 min. 450 -OD 630 The growth inhibition rate of the antibody on cells was calculated. Group M consisted of cells with ligand added but no antibody. Inhibition rate = (OD value of group M - OD value of sample) / (OD value of group M) × 100%. The dose-response curve was analyzed and plotted using GraphPad Prism software, with antibody concentration on the x-axis and inhibition rate on the y-axis. Results are shown below. Figure 6 As shown in Table A and Table 1, under ligand-free conditions, EGFR-HPA8 exhibited superior growth inhibition of MDA-MB-468 cells compared to SCT200 and the Cetuximab control antibody. EGFR-HPA8 showed a similar maximum inhibition rate to SCT200, but with lower growth inhibition on ECGs. 50Example 2.2.1 showed that EGFR-HPA8's ability to block the binding of EGF-Fc protein to EGFR on MDA-MB-468 cells was stronger than that of SCT200 and Erbitux. In this example, different EGFR ligands were added to the MDA-MB-468 cell growth inhibition assay to further verify the ability of EGFR-HPA8 to block EGFR ligand-receptor binding at the cellular function level. The results are as follows... Figure 6 As shown in B-6D and Table 1, under different ligand conditions, EGFR-HPA8 showed a better ability to inhibit the proliferation of MDA-MB-468 cells than SCT200 and Cetuximab.

[0145] Table 1. EC50 and maximum neutralization rate of EGFR-HPA8 antibody in inhibiting MDA-MB-468 cell proliferation.

[0146]

[0147]

[0148] 2.2.2.2 The ability of humanized antibodies to inhibit Fadu cell proliferation

[0149] Human pharyngeal squamous cell carcinoma cells (Fadu) highly express EGFR and simultaneously secrete various EGFR ligand factors. Following Example 2.2.2.1, the WST-8 assay was used to assess the inhibitory effect of EGFR-HPA8 antibody on the proliferation of Fadu cells (source: Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) under different ligand conditions. The results are as follows... Figure 7 As shown in Table 2, under ligand-free conditions, EGFR-HPA8's inhibitory effect on Fadu cell growth was similar to that of SCT200 and superior to that of Cetuximab. However, under different ligand conditions, EGFR-HPA8's inhibitory effect on Fadu cell growth was significantly superior to that of SCT200 and Cetuximab. These results indicate that, compared to SCT200 and Cetuximab, the EGFR-HPA8 antibody has a superior ability to inhibit EGFR ligand-receptor binding.

[0150] Table 2. EC50 and maximum neutralization rate of EGFR-HPA8 antibody in inhibiting Fadu cell proliferation.

[0151]

[0152] 2.2.3 ADCC effect of humanized antibodies

[0153] This embodiment uses a recombinant CD16a reporter gene system to detect ADCC mediated by the humanized antibody EGFR-HPA8. The recombinant CD16a reporter gene system includes effector cells Jurkat-NFAT-Luc2p-CD16A and target cells expressing EGFR. When the two cell types are co-cultured and EGFR antibody is added simultaneously, the Fab fragment of the EGFR antibody binds to EGFR expressed on the surface of the target cells, and its Fc fragment binds to effector cells overexpressing the Fcγ receptor CD16a, thereby activating effector cells Jurkat-NFAT-Luc2p-CD16A and promoting NFAT-RE-mediated bioluminescence.

[0154] A431 target cells (source: Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) were evenly seeded in 96-well plates at a density of 1×10⁶ cells / well. 4 / well. After incubation overnight, add different concentrations of antibody (2.67nM, 0.53nM, 0.11nM, 0.021nM, 0.0043nM, 0.00085nM, 0.00017nM, and 0.000034nM), 40 μL / well, followed by 1×10 5 40 μL of Jurkat-NFAT-Luc2p-CD16A effector cells (source: Shenzhou Cell Engineering Co., Ltd., hereinafter the same) were used per well, with 3 replicates per assay. Target cells, effector cells, and negative antibody control wells were also included. Cells were incubated at 37°C and 5% CO2 for 4 hours, followed by the addition of 20 μL of Passive Lysis 5× Buffer per well. After a freeze-thaw cycle, the cells were shaken to mix thoroughly, and 20 μL of the supernatant from each well was transferred to a 96-well white plate. Bioluminescence was detected using an LB960 microplate chemiluminescence analyzer. GraphPad Prism software was used to analyze and plot dose-response curves, with the sample concentration on the x-axis and the RLU value on the y-axis. The bioluminescence intensity induction fold was calculated as: Sample RLU / Negative Control RLU. Results are as follows: Figure 8 As shown, EGFR-HPA8, along with the positive controls Erbitux and SCT200, can all mediate effective ADCC effects in A431 tumor cells expressing EGFR. Among them, EGFR-HPA8 shows advantages in both the half-maximal effective concentration (IC50) and the fold-up induction rate, and its EC50-90% inducing ADCC is significantly higher. 50 R is 0.008 nM. 2 =0.999.

[0155] 2.3 Efficacy of EGFR-HPA-8 on subcutaneous xenografts of human gastric cancer cell line SNU-5 and human non-small cell lung cancer line NCI-H1975 in mice

[0156] SNU-5 cells (ATCC cell bank) in logarithmic growth phase were washed with PBS and then digested with 0.25% trypsin. The digestion solution was collected, and the cells were collected by centrifugation at 800 rpm for 5 min. The cells were resuspended in PBS and the cell concentration was adjusted to 5.0 × 10⁶ cells / year. 7 5.0 × 10⁶ cells / mL (containing 50% matrix gel) were subcutaneously injected into the right back of Balb / c-nude mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.). 6 One SNU-5 cell suspension, 100 μL / cell. When the tumor volume reaches 170 mm². 3 The tumors were randomly divided into seven groups of five based on their size. Medications were administered intraperitoneally (IP) on the day of grouping, twice a week for seven consecutive weeks. The specific dosing regimen is shown in Table 3 below.

[0157] Table 3. Trial Groups and Drug Administration

[0158]

[0159] Note: The dosage volume is calculated based on 10 mL / kg of mouse body weight.

[0160] The calculation method for Tumor Growth Inhibition Value (TGI) is: T / C (%) = T RTV / C RTV ×100% (T) RTV For the treatment group, RTV; C RTV The negative control group (RTV) was used for the relative tumor volume RTV = V. T / V0, where V0 is the tumor volume measured at D0 during fractional drug administration, V T The tumor volume obtained from each measurement. TGI (%) = 1 - T / C (%).

[0161] During the administration period, all experimental animals were in good condition and experienced a certain degree of weight gain. There was no significant difference in body weight between the treatment groups and the solvent control group (P>0.05). See below for details of weight changes in all animals. Figure 9 And Table 4.

[0162] Table 4. Effects of EGFR-HPA8 on body weight in SNU-5 human gastric cancer xenograft mice

[0163]

[0164] a mean ± standard deviation

[0165] b Statistical comparison of body weight between the treatment group and the solvent control group after 31 days of drug administration: t-test.

[0166] The tumor volume and TGI results for each group in the experiment are shown in Table 5 and Figure 10 After 31 days of treatment, the mean tumor volume in the Vehicle group was 559.9 ± 144.9 mm. 3 The tumor volume in the positive control group (low-dose SCT200 5mpk group) was 317.1 ± 197.6 mm. 3 The TGI was 44.3%, which was not significantly different from the Vehicle group (P = 11.28%). The low-dose EGFR-HPA8 5mpk group showed better efficacy, with a tumor volume of 113.8 ± 74.0 mm. 3 The TGI was 80.0%, significantly different from the Vehicle group (P<0.05), indicating that EGFR-HPA8 at this dose showed slightly better tumor-suppressing performance than the positive control SCT200 (P=0.09). The tumor volume in the positive control SCT200 20mpk group was 191.8±189.1 mm. 3 The TGI was 66.5%, which was significantly different from that of the Vehicle group (P<0.05). The tumor volume in the EGFR-HPA8 high-dose 20mpk group was 175.0±175.0 mm. 3 The TGI was 68.9%, significantly different from the Vehicle group (P<0.05). EGFR-HPA8 and the positive control SCT200 at 20mpk showed significant tumor-suppressing effects, with no significant difference in tumor volume between the two groups (P=0.9). In conclusion, EGFR-HPA8 molecules exhibited significant antitumor efficacy against the SNU-5 human gastric cancer xenograft model at both 5mpk and 20mpk dose levels, with a slightly better tumor-suppressing effect at lower doses than SCT200.

[0167] Table 5. Effects of EGFR-HPA8 on tumor volume in SNU-5 human gastric cancer xenograft mouse model.

[0168]

[0169]

[0170] a Mean ± Standard Deviation

[0171] EGFR-HPA8 also showed a significant inhibitory effect on subcutaneous xenografts of the human non-small cell lung cancer line NCI-H1975 in mice. NCI-H1975 cells (Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) were subcutaneously inoculated in the right back of Balb / c-nude mice. The experimental groups and drug administration are shown in Table 6.

[0172] Table 6. Trial Groups and Drug Administration

[0173]

[0174] Note: The dosage volume is calculated based on 10 mL / kg of mouse body weight.

[0175] During the administration period, all experimental animals were in good condition and experienced a certain degree of weight gain. There was no significant difference in body weight between the treatment groups and the solvent control (P>0.05). See below for details of weight changes in all animals. Figure 11 And Table 7.

[0176] Table 7. Effects of EGFR-HPA8 on body weight in mice of the NCI-H1975 human lung cancer xenograft model.

[0177]

[0178] a mean ± standard deviation

[0179] b Statistical comparison of body weight between the treatment group and the solvent control group after 31 days of drug administration: t-test.

[0180] The tumor volume and TGI results for each group in the experiment are shown in Table 8 and 2019. Figure 12 After 18 days of treatment with the drugs administered in groups, both the high- and low-dose SCT200 and EGFR-HPA8 positive control groups showed significant tumor-suppressing effects. Statistical analysis revealed significant differences in tumor volume between each treatment group and the Vehicle group. The average tumor volume in the Vehicle group on Day 18 was 1564.3 ± 529.0 mm. 3 After treatment with SCT200 5mpk and EGFR-HPA8 5mpk, the tumor volume was 151.9±99.1 mm, respectively. 3 and 86.5±107.5mm 3 The TGI values ​​were 90.4% and 94.5%, respectively. The tumor volumes after SCT200 20mpk and EGFR-HPA8 20mpk treatments were 289.8 ± 321.4 mm. 3 and 149.3±94.9mm 3 The TGI values ​​were 81.8% and 90.4%, respectively. EGFR-HPA8 showed slightly better tumor-suppressing effects than the positive control SCT200 at both 5 mpk and 20 mpk doses, but the differences were not statistically significant. In summary, EGFR-HPA8 demonstrates significant antitumor efficacy against the NCI-H1975 human non-small cell lung cancer xenograft model.

[0181] Table 8. Effects of EGFR-HPA8 on tumor volume and TGI in the NCI-H1975 human lung cancer xenograft model.

[0182]

[0183] a Mean ± Standard Deviation

[0184] Example 3: Design and quality and function testing of EGFR / TGFβR2 antibody fusion proteins containing different truncated forms of TGFβR2

[0185] 3.1 Design, expression, and purification of expression vectors containing EGFR / TGFβR2 antibody fusion proteins with different truncated forms of TGFβR2.

[0186] In this embodiment, an EGFR antibody is used as the targeting portion of the fusion protein, and the TGFβR2 extracellular domain is used as the immunomodulatory portion of the fusion protein. The TGFβR2 extracellular region is linked to the C-terminus of the heavy chain of the EGFR antibody via homologous recombination, forming an EGFR antibody / TGFβR2 extracellular domain fusion protein (EGFR / TGFβR2) from the light and heavy chains. The structure of the fusion protein is as follows. Figure 13 As shown. Mass spectrometry analysis revealed multiple easily cleavable sites between positions 7 and 15 of the N-terminus of the full-length TGFβR2 extracellular domain. To improve the structural stability of the fusion protein, this embodiment modified the N-terminal amino acid sequence of the TGFβR2 extracellular domain by deleting different numbers of amino acids. The EGFR / TGFβR2 antibody fusion protein uses a (G4S)4Linker to link the C-terminal amino acid of the EGFR antibody heavy chain to the TGFβR2 extracellular domain with different amino acid deletions. Furthermore, the lysine amino group at the C-terminus of the EGFR antibody heavy chain was removed to reduce the risk of proteolytic degradation.

[0187] Table 9. Design scheme for EGFR / TGFβR2 antibody fusion protein

[0188]

[0189]

[0190] The above protocol involves amplifying the target gene via PCR or overlap-PCR, ligating it into an expression vector via in-fusion, and extracting the plasmids after successful sequencing. These plasmids are then transiently transfected into HEK-293 cells (fut8 gene knockout) and cultured until day 7. The cells are then centrifuged, and the supernatant is collected. The centrifuged cell supernatant is purified using Protein A affinity chromatography to obtain the ADCC-enhanced EGFR / TGFβR2 antibody fusion protein.

[0191] 3.2 Fragmentation of EGFR / TGFβR2 antibody fusion proteins containing different truncated forms of TGFβR2

[0192] The purity and fragmentation of the samples were analyzed using the reduced SDS-PAGE method. Different truncated EGFR / TGFβR2 antibody fusion proteins 1-16 purified in Example 3.1 were subjected to reduced SDS-PAGE detection. Specific steps for reduced SDS-PAGE: (1) SDS-PAGE preparation: 3.9% stacking gel, 13% separating gel; (2) Boil the sample at 100℃ for 2 min, centrifuge, and load 8 μg of sample; (3) Electrophoresis at a constant current of 40 mA for 1 h. Detection results are shown in […]. Figure 14 The EGFR / TGFβR2 antibody fusion protein has a light chain molecular weight of approximately 25 kDa, a heavy chain molecular weight of approximately 66 kDa, and a cleavage variant molecular weight between 45 and 66 kDa. Results showed that a significant cleavage variant exists in the extracellular region of TGFβR2, and the fusion protein with different truncated forms of the TGFβR2 extracellular region exhibits a significantly reduced number of fragmented bands compared to the full-length TGFβR2 extracellular region fusion protein. Therefore, the different truncated forms of the TGFβR2 extracellular region prepared in this invention significantly improve the stability of the TGF-β receptor-containing antibody fusion protein.

[0193] 3.3 Degradation tendency of EGFR / TGFβR2 antibody fusion proteins containing different truncated forms of TGFβR2

[0194] In this embodiment, 293E cell supernatant was used to accelerate the treatment of the fusion protein, further evaluating the stability of EGFR / TGFβR2 antibody fusion proteins with different truncated forms of TGFβR2. The 293E cell expression system is commonly used to express antibodies, which express various host cell proteins (HCPs) and proteases required for cell growth. Therefore, antibody stability can be assessed by observing the tendency of the fusion protein to break down in 293E cell supernatant.

[0195] The purified fusion protein was mixed with the supernatant of 293E cells cultured for 10 days at a volume ratio of 1:0.3, resulting in a final concentration of approximately 1 mg / mL. The mixture was shaken thoroughly and incubated at 37°C for 48 h. A cell-free supernatant control was also included. Sample purity and the content of the splice variants were determined using reducing SDS-PAGE, and the heavy chain purity of the fusion protein was calculated using BandScan software.

[0196] Test results are shown Figure 15The fragmentation ratios for each sample are shown in Table 10. The results indicate that the proportion of splice variants of the truncated TGFβR2 fusion protein (fusion protein 2-16) in the control group was less than 4.0%, significantly lower than the 24.8% of the full-length TGFβR2 fusion protein (fusion protein 1). After incubation at 37°C for 48 hours with the cell supernatant, all of the full-length TGFβR2 fusion protein (fusion protein 1) in the experimental group was cleaved, reaching 100%. The truncated TGFβR2 fusion proteins (fusion proteins 2-16) contained varying proportions of splice variants, but were significantly superior to the full-length control. Fusion proteins 2, 6, 9, 10, and 13 showed the best performance, with splice variant content less than 3.0%.

[0197] Table 10 Degradation rates of EGFR / TGFβR2 antibody fusion proteins containing different truncated forms of TGFβR2

[0198]

[0199]

[0200] The above results indicate that, compared with the full-length TGFβR2 fusion protein 1, the different truncated forms of EGFR / TGFβR2 antibody fusion proteins all have stronger resistance to protease degradation.

[0201] 3.4 Detection of TGF-β binding to EGFR / TGFβR2 antibody fusion proteins containing different truncated forms of TGFβR2

[0202] 100 ng / mL TGF-β1 and 40 ng / mL EGFR-His protein were coated onto 96-well plates, 100 μL / well, and incubated overnight at 4°C. The next day, after washing the plates and blocking for 1 hour at room temperature, 2 μg / mL of EGFR / TGFβR2 antibody fusion protein containing different truncated forms of TGFβR2 was added, 100 μL / well. After incubation for 1 hour, the plates were washed to remove unbound antibodies, and then incubated with secondary antibody Goat anti-hIgG Fc / HRP, followed by repeated washing. Finally, substrate chromogenic buffer was added for color development, and the OD was read using a microplate reader after termination. 450 The result is as follows Figure 16 As shown, the binding affinity of different truncated forms of TGFβR2 EGFR / TGFβR2 antibody fusion proteins to TGF-β1 varies considerably, but their binding affinity to EGFR is similar.

[0203] 3.5 Detection of TGF-β neutralization by EGFR / TGFβR2 antibody fusion proteins containing different truncated forms of TGFβR2

[0204] TGF-β regulates cellular function by modulating the transcription of multiple target genes. Plasminogen activator inhibitor 1 (PAI-1) is an important downstream target of the TGF-β1 / Smad signaling pathway. Activated Smad3 binds to cis-acting elements in the PAI-1 promoter region to regulate PAI-1 expression. Elements containing the PAI-1 promoter region were inserted in a specific form into a luciferase-containing vector and transfected into HepG2 cells. In this reporter gene system, the addition of exogenous TGF-β protein initiates the expression of the luciferase reporter gene, which then emits light in response to the substrate. The addition of exogenous TGF-β antibody neutralizes the TGF-β protein, blocks the binding of TGF-β to TGFβR2, inhibits downstream signaling pathways, and ultimately suppresses the expression of the luciferase reporter gene. Therefore, the in vitro efficacy of the TGF-β antibody in neutralizing TGF-β can be determined by detecting the intensity of the light signal.

[0205] HepG2-3TP-Luc2p-puro cells (source: Shenzhou Cell Engineering Co., Ltd., hereinafter the same) were uniformly seeded in 96-well plates at 30,000 cells / well. After overnight adhesion, the culture medium in the 96-well plates was discarded and replaced with DMEM medium containing 0.5% FBS, and incubated at 37°C and 5% CO2 for 6 h. The culture medium in the 96-well plates was then discarded, and 4 ng / mL TGF-β1 protein was added, along with EGFR / TGFβR2 antibody fusion protein to a final concentration of 0.02 μg / mL. The plates were incubated at 37°C and 5% CO2 for another 18 h. Negative control group M (containing cells and TGF-β1) and negative control group M' (containing cells, without TGF-β1) were also set up. Finally, 5×Lysis buffer was added, and 10 μL of cell sample was taken to detect the bioluminescence intensity (RLU) and calculate the neutralization rate of the EGFR / TGFβR2 antibody fusion protein. Neutralization rate (%) = (RLU value of group M - RLU value of sample) / (OD value of group M - OD value of group M') × 100%. The dose-response curve was analyzed and plotted using GraphPad Prism software, with antibody concentration on the x-axis and antibody neutralization rate on the y-axis. Results are as follows: Figure 17As shown, EGFR / TGFβR2 antibody fusion proteins 2 through 6, 8, 13, 14, and 16, containing the truncated form of TGFβR2, all possess some ability to neutralize TGF-β1. Fusion protein 2 exhibits a similar neutralizing ability to fusion protein 1 containing the full-length form of TGFβR2, while fusion proteins 5, 6, and 8 show superior neutralizing abilities at this concentration compared to fusion protein 1. Fusion protein 6 demonstrates the strongest TGF-β1 neutralizing ability. The remaining EGFR / TGFβR2 antibody fusion proteins containing the truncated form of TGFβR2 have virtually no neutralizing ability or only a weak neutralizing ability.

[0206] TGF-β3 protein has a high affinity for TGFβR2 and can activate downstream TGF-β signaling. In this embodiment, a reporter gene system was used to detect the ability of the fusion protein to neutralize TGF-β3 (final concentration 20 ng / mL). The results are as follows... Figure 18 As shown, fusion proteins 2, 4, and 13 have similar TGF-β3 neutralizing abilities to fusion protein 1, while fusion proteins 3, 5, 6, and 8 have superior TGF-β3 neutralizing abilities compared to fusion protein 1. Fusion protein 6 also exhibits the strongest TGF-β3 neutralizing ability.

[0207] Based on the above analysis of the stability and neutralizing capacity of fusion proteins containing truncated TGFβR2, the present invention preferably contains truncated TGFβR2 forms of fusion proteins 2-6, 8, 13, 14 and 16, more preferably contains truncated TGFβR2 forms of fusion proteins 2, 5, 6 and 8, and most preferably contains truncated TGFβR2 forms of fusion protein 6.

[0208] Example 4: In vitro biological function of EGFR / TGFβR2 antibody fusion protein in truncated form of TGFβR2 (fusion protein 6)

[0209] 4.1 Detection of EGFR / TGFβR2 antibody-fusion protein binding ability

[0210] 4.1.1 EGFR / TGFβR2 antibody fusion protein binding and competitive TGF-β characteristics

[0211] TGF-β1 and TGF-β3 proteins, at a final concentration of 2 μg / mL, were coated onto 96-well plates (100 μL / well) and incubated overnight at 4°C. The plates were washed the following day, blocked at room temperature for 1 h, and then incubated for 1 h with different concentrations (1.22 pM, 4.88 pM, 19.53 pM, 78.13 pM, 312.5 pM, 1250 pM, 500 pM, 2000 pM) of EGFR / TGFβR2 antibody fusion protein 6. Afterward, the plates were washed to remove unbound antibodies, and then incubated with secondary antibody Goat anti-hIgG F(ab)2 / HRP. The plates were washed repeatedly, and substrate chromogenic solution was added for color development. After termination, the OD values ​​were read using a microplate reader. 450 The result is as follows Figure 19 As shown, fusion protein 6 has similar binding affinity to TGF-β1 and TGF-β3 proteins as fusion protein 1, and its binding affinity to TGF-β1 protein is EC50. 50 91pM, R 2 =0.998, EC50 of TGFβ3 protein binding 50 102pM, R 2 =0.998.

[0212] This embodiment further analyzes at the protein level the ability of fusion protein 6 to compete with TGF-β1 or TGF-β3 protein for binding to TGFβR2-Fc protein.

[0213] 0.2 μg / mL of TGF-β1 protein or 0.5 μg / mL of TGF-β3 protein were coated onto 96-well plates at a final concentration of 100 μL / well, and the plates were incubated overnight at 4°C. The plates were washed the following day, blocked at room temperature for 1 h, and then 100 μL of EGFR / TGFβR2 antibody fusion protein at different concentrations (0.05 nM, 0.14 nM, 0.42 nM, 1.25 nM, 3.75 nM, 11.24 nM, 33.71 nM, 101.12 nM) and 100 μL of biotin-labeled TGFβR2-Fc protein (protein source: Beijing Yiqiao Shenzhou Technology Co., Ltd., biotinylated by Shenzhou Cell Engineering Co., Ltd., hereinafter the same) were added. Positive control wells containing only TGFβR2-Fc protein were also included. After incubation for 1 hour, wash the plate, add the secondary antibody Streptavidin / HRP, and incubate for another hour. Repeat the washing process. Finally, add the substrate chromogenic buffer for color development. After termination, read the OD value using a microplate reader. 450 According to OD 450 The competitive inhibition rate PI% of the fusion protein was calculated as follows: Inhibition rate PI (%) = (OD of positive wells) / (OD of positive wells) 450 Value - Sample well OD 450 Value) / Positive well OD 450Value × 100%. The result is as follows: Figure 20 As shown, fusion protein 6 has a similar ability to block the binding of TGF-β1 or TGF-β3 proteins to TGFβR2-Fc as fusion protein 1.

[0214] 4.1.2 EGFR / TGFβR2 antibody fusion protein binding characteristics to EGFR

[0215] Referring to Example 1.2.1, the binding affinity of the fusion protein to recombinant human EGFR protein was detected using ELISA. Figure 21 As shown, the binding affinity of fusion protein 6 to EGFR and its ability to compete with EGF for EGFR binding are similar to those of EGFR-HPA8. (The text abruptly ends here, likely due to an incomplete sentence or missing information.) 50 It was 133.1 ng / mL, R 2 =1.000.

[0216] 4.2 Detection of binding affinity of EGFR / TGFβR2 antibody fusion protein

[0217] In this embodiment, a biomolecular interaction analysis system (model: OctetRED96e, manufacturer: Fortebio) was used to determine the affinity of the EGFR / TGFβR2 antibody fusion protein for the biotinylated recombinant human EGFR protein and TGF-β1 protein, with EGFR-HPA8 and H7N9-R1 used as negative controls. Affinity parameters were obtained by fitting binding and dissociation curves at multiple concentration points. The results are shown in Tables 11 and 14. Specific kinetic characteristic parameter curves are detailed in Tables 11 and 14. Figure 22 and Figure 23 .

[0218] The results showed that, compared with the monoclonal antibody EGFR-HPA8, fusion protein 6 retained a higher binding affinity to human EGFR protein. The KD value of the binding affinity of fusion protein 6 to human EGFR protein was 8.77 pM, and the binding constant k was [missing value]. on The value is 1.68E+06M -1 s -1 dissociation constant k dis It is 1.47E-05s -1 Furthermore, the truncated form of TGFβR2 fusion protein 6 has a similar affinity to the full-length form of TGFβR2 fusion protein 1, which is a homologous TGF-β1 protein. D The value is 96.1 pM, combined with the constant k on The value is 1.53E+06M -1 s -1 dissociation constant k dis It is 1.47E-04s -1 .

[0219] Table 11 Affinity of truncated EGFR / TGFβR2 antibody fusion protein to recombinant human EGFR protein

[0220]

[0221] Table 14 Affinity of truncated EGFR / TGFβR2 antibody fusion protein to recombinant human TGF-β1 protein

[0222]

[0223] The above results indicate that fusion protein 6 has good affinity for both human EGFR and TGF-β1.

[0224] 4.3 Detection of TGF-β neutralizing activity of EGFR / TGFβR2 antibody fusion protein

[0225] TGF-β1 can inhibit the proliferation of Mv-1-1u cells, therefore the WST-8 assay can be used to detect the ability of EGFR / TGFβR2 antibody fusion protein to neutralize TGF-β1.

[0226] 50 μL / well of Mv-1-lu cells (source: Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) were evenly seeded into 96-well plates at a cell seeding density of 1 × 10⁶ cells / well. 3 / well. Cells were cultured in a CO2 incubator for approximately 3 hours to allow them to adhere. Then, 50 μL / well of EGFR / TGFβR2 antibody fusion protein samples diluted in 1640 medium containing 10% FBS at different concentrations (0.0078 nM, 0.0156 nM, 0.0313 nM, 0.0625 nM, 0.125 nM, 0.25 nM, 0.5 nM, 1 nM, 2 nM) were added. Finally, 10 μL / well of TGF-β1 was added to a final concentration of 1 ng / mL. Positive control group M (containing cells and TGF-β1), negative control group M' (containing cells, without TGF-β1), and blank control group B (containing only medium, without cells) were also set up. After incubating the cells in a CO2 incubator at 37°C and 5% CO2 for 5 days, 10 μL / well of WST-8 was added. OD was measured using a microplate reader after 180 min of sample incubation. 450 -OD 630 The neutralization rate of the EGFR / TGFβR2 antibody fusion protein was calculated by subtracting the reading of the blank control B from the original reading. Neutralization rate (%) = (OD value of group M' - OD value of sample) / (OD value of group M' - OD value of group M) × 100%. The dose-response curve was analyzed and plotted using GraphPadPrism software, with antibody concentration on the x-axis and inhibition rate on the y-axis. Figure 24As shown, both fusion protein 6 and the TGFβR2 control H7N9-R1-43-IgG1(L9) (source: Biotinylated by Shenzhou Cell Engineering Co., Ltd., hereinafter the same) effectively neutralized the inhibitory effect of TGF-β1 on Mv-1-lu proliferation in a dose-dependent manner. Furthermore, the half-maximal effective concentration (IC50) of fusion protein 6 was lower than that of the control H7N9-R1-43-IgG1(L9), indicating that this molecule has better neutralizing activity. EGFR-HPA8 showed no neutralizing effect on TGF-β1, indicating that the TGFβR2 terminus of fusion protein 6 neutralizes TGF-β1.

[0227] This embodiment further investigated the ability of fusion protein 6 to neutralize TGF-β using a reporter gene system, following the method described in Example 3.5. The results are as follows: Figure 25 As shown, both fusion protein 6 and the TGFβR2 control H7N9-R1-43-IgG1 (L9) can effectively neutralize TGF-β1 in a dose-dependent manner. Moreover, the maximum neutralization rate of fusion protein 6 (74.8%) is much higher than that of the control H7N9-R1-43-IgG1 (L9) (55.3%), which further demonstrates the excellent neutralizing activity of fusion protein 6.

[0228] 4.4 Detection of the inhibitory activity of EGFR / TGFβR2 antibody fusion protein on cell proliferation

[0229] Referring to Example 1.2.2, the WST-8 assay was used to determine the growth inhibitory effect of the EGFR / TGFβR2 antibody fusion protein on MDA-MB-468 cells to assess its EGFR terminus activity. The results are as follows: Figure 26 As shown, the ability of fusion protein 6 to inhibit the proliferation of MDA-MB-468 cells is similar to that of EGFR-HPA8, and the inhibition rate increases with increasing drug concentration in an "S"-shaped curve. The control substance H7N9-R1-43-IgG1(L9), which has TGFβR2 function, has no inhibitory effect on MDA-MB-468 cells, indicating that the inhibitory effect of fusion protein 6 on tumor cell proliferation is due to its EGFR terminus function.

[0230] 4.5 ADCC activity of EGFR / TGFβR2 antibody fusion protein

[0231] Refer to Example 2.2.3 to detect the ADCC effect mediated by the EGFR / TGFβR2 antibody fusion protein on EGFR-expressing cells. The results are as follows... Figure 27As shown, within the concentration range of 0.00004-3 nM, fusion protein 6 and the anti-EGFR antibody EGFR-HPA8 can mediate similar ADCC effects in EGFR-expressing tumor cells A431. The control H7N9-R1-43-IgG1 (L9) with TGFβR2 function showed no ADCC effect on A431 cells, indicating that the EGFR terminus of fusion protein 6 mediates ADCC function in this experimental system.

[0232] Example 5: Pharmacodynamic study of EGFR / TGFβR2 antibody fusion protein in NCI-H1975 subcutaneous xenograft model (TGFβR2 truncated form (fusion protein 6)).

[0233] Balb / c-nu mice were subcutaneously injected with 1×10⁻⁶ cistern tissue at the right rib. 6 NCI-H1975 cells. When the tumor volume reaches approximately 300 mm. 3 Animals were randomly divided into groups of 6 based on tumor volume. Administration began on the day of grouping via intraperitoneal injection (IP), twice weekly for 10 consecutive weeks. After the last administration, medication was discontinued to monitor for tumor recurrence. Specific administration protocols are shown in Table 15 below.

[0234] Table 12 Experimental Groups and Drug Administration

[0235]

[0236] Note: The dosage volume is calculated based on 10 mL / kg of mouse body weight.

[0237] During the drug administration period, the animals in all experimental groups were in good general condition, with good activity and appetite, and their body weight increased to some extent. There was no significant difference in body weight between the drug administration group and the solvent control group after drug administration (P>0.05). The changes in body weight of all animals are shown in Table 13 and... Figure 28 .

[0238] Table 13 Effect of fusion protein 6 on body weight in mice with H1975 non-small cell lung cancer subcutaneous xenografts

[0239]

[0240] a Mean ± standard deviation;

[0241] b Statistical comparison of body weight between the treatment group and the solvent control group after 35 days of drug administration: t-test.

[0242] The tumor volume results for each group in the experiment are shown in Table 14 and Figure 29 After 35 days of treatment with different drugs in different groups, the mean tumor volume in the solvent control group was 7150.78 ± 780.4 mm. 3In the fusion protein 6 treatment group, 5 out of 6 mice showed complete tumor disappearance (CR), with an average tumor volume of 4.55 ± 4.55 mm. 3 The tumor volume reduction index (TGI) was as high as 99.9%, showing a significant difference compared to the solvent control group (P<0.001). In contrast, only one mouse in the EGFR-HPA8 group experienced complete tumor disappearance, with a mean tumor volume of 79.44±28.65 mm3 and a TGI of 98.9%. These results indicate that fusion protein 6 has a significant inhibitory effect on NCI-H1975 non-small cell lung cancer subcutaneous xenografts, and its tumor-suppressing effect is superior to that of the same molar dose of EGFR-HPA8 (P=0.237).

[0243] Table 14 Effect of fusion protein 6 on tumor volume in mice with H1975 non-small cell lung cancer xenograft model

[0244]

[0245] a Statistical comparison of tumor volume between the EGFR-HPA8-Ae0 treatment group and the solvent control group, using t-test;

[0246] Statistical comparison of tumor volume between the b-fusion protein 6 treatment group and the solvent control group, using t-test;

[0247] Example 6: Stability analysis of EGFR / TGFβR2 antibody fusion protein in truncated form of TGFβR2 (fusion protein 6)

[0248] 6.1 Ultrafiltration Stability Analysis of EGFR / TGFβR2 Antibody Fusion Protein

[0249] The EGFR / TGFβR2 antibody fusion protein sample was concentrated to a concentration of approximately 10 mg / mL by ultrafiltration in 100 mM Glycine, 10 mM NaCl, 50 mM Tris, and pH 7.5 buffer. The purity and stability of the concentrated sample were determined by reducing SDS-PAGE and molecular sieve chromatography (SEC-HPLC, Agilent 1260 liquid chromatography system, TSK-G3000SWXL column). SEC-HPLC operation steps: (1) Mobile phase: 200 mM NaH2PO4, 100 mM Arginine, pH 6.5; (2) Sample loading volume: 80 μg; (3) Analysis time: 30 min, flow rate: 0.5 mL / min, column temperature: 25 °C; (4) Purity was calculated by peak area normalization.

[0250] See the purity test results after sample concentration. Figure 30The sample purity and fragment ratios are shown in Table 15. The results indicate that the truncated TGFβR2 fusion protein 6 is less prone to fragmentation after concentration and exhibits higher ultrafiltration stability compared to the full-length TGFβR2 fusion protein.

[0251] Table 15 Ultrafiltration Stability (SEC) Results of EGFR / TGFβR2 Antibody Fusion Protein

[0252]

[0253] 6.2 Thermal stability analysis of EGFR / TGFβR2 antibody fusion protein

[0254] The thermal stability of the samples was detected by differential scanning fluorimetry (DSF) using the UNcle system (Unchained Labs, model: UNCLE-0330). The procedure was as follows: (1) The sample loading volume was 9 μL; (2) The experimental parameters were set as follows: temperature range 25℃-95℃, heating rate 0.3℃ / min; (3) The data were analyzed using UNcle Analysis software. The midpoint value of the internal fluorescence change curve under UV266 was taken as Tm, and the polymerization initiation temperatures of the aggregate change curve formed by static light scattering signals under UV266 / Blue473 were taken as Tagg266 and Tagg473, respectively.

[0255] The results of the thermal stability test of fusion protein 6 are shown in Table 16, which showed good thermal stability.

[0256] Table 16. Tm detection results of fusion protein 6

[0257]

[0258] 6.3 Thermally accelerated stability analysis of EGFR / TGFβR2 antibody fusion protein

[0259] After storing the samples at 45°C for one week, the accelerated stability of the samples was analyzed by SEC-HPLC and SDS-PAGE, with the specific operating procedures being the same as in 6.1.

[0260] The results of the thermally accelerated stability test of fusion protein 6 are shown in Table 17. After storage at 45°C for 1 week, although the SEC purity of fusion protein 6 decreased by 0.7%, the purity was still high, and the increase in aggregate level was small, while the fragment level remained unchanged, showing good thermally accelerated stability.

[0261] Table 17 Results of thermally accelerated stability testing of fusion protein 6

[0262]

[0263] 6.4 Freeze-thaw stability analysis of EGFR / TGFβR2 antibody fusion protein

[0264] The samples were stored at -80℃ for 3 hours, then thawed at 45℃ for 1 hour, and this freeze-thaw cycle was repeated five times. The freeze-thaw stability of the samples was analyzed using SEC-HPLC, with the specific procedures as described in 6.1.

[0265] The freeze-thaw stability test results of fusion protein 6 are shown in Table 18. After five repeated freeze-thaw cycles, the SEC purity of fusion protein 6 did not change significantly, and the aggregate and fragment levels did not increase significantly, demonstrating good freeze-thaw stability.

[0266] Table 18 Results of freeze-thaw stability test for fusion protein 6

[0267]

[0268] 6.5 oscillation stability of EGFR / TGFβR2 antibody fusion protein

[0269] The sample was placed in a deep-well plate and vortexed at 800 rpm for 24 h. The vortexing stability of the sample was analyzed by SEC-HPLC, following the same procedure as in 6.1. The results are shown in Table 19. After 24 h of vortexing, the SEC monomer purity of the sample did not change significantly, and the aggregate and fragment levels did not increase significantly, indicating that fusion protein 6 has good vortexing stability.

[0270] Table 19 Results of oscillation stability test for fusion protein 6

[0271]

[0272] Example 7: Design and quality function testing of multi-antigen X / TGFβR2 antibody fusion proteins targeting solid tumors using the truncated form of TGFβR2 (fusion protein 6).

[0273] 7.1 Design, expression, and purification of expression vectors containing the truncated form of TGFβR2 X / TGFβR2 antibody fusion protein

[0274] To further verify the structural stability and TGF-β1 neutralizing ability of the preferred truncated TGFβR2 form in Example 3.2, this example used various solid tumor antigens as the targeting portion of the fusion protein, and the extracellular domain of TGFβR2 (full-length and ECD delete (6-26) fusion protein 6 form) as the immunomodulatory portion of the fusion protein, forming an X antibody / TGFβR2 extracellular domain fusion protein (X / TGFβR2 antibody fusion protein). Similarly, the X / TGFβR2 antibody fusion protein uses a (G4S)4Linker to link the C-terminal amino acid of the X antibody heavy chain (the N-terminus of the heavy chain is linked to the signal peptide as shown in SEQ ID NO:14) to the extracellular domain of TGFβR2. In addition, the lysine amino group at the C-terminus of the X antibody heavy chain was removed to reduce the risk of proteolytic degradation. The construction scheme of the X / TGFβR2 antibody fusion protein is shown in Table 20.

[0275] Table 20 X / TGFβR2 Antibody Fusion Protein Design Scheme

[0276]

[0277] The target gene was amplified by PCR or overlap-PCR and ligated into an expression vector via in-fusion. After successful sequencing, the plasmid of the recombinant expression vector was extracted and transiently transfected into HEK-293 cells (fut8 gene knockout). After 7 days of culture, the supernatant was collected by centrifugation. The obtained cell supernatant was purified into the fusion protein using Protein A affinity chromatography.

[0278] 7.2 Fragmentation of X / TGFβR2 antibody fusion protein containing truncated TGFβR2

[0279] The purity and fragmentation of the X / TGFβR2 antibody fusion protein were detected using the reducing SDS-PAGE method. The results are as follows: Figure 31 As shown, for different X / TGFβR2 antibody fusion proteins, the expression samples with the preferred truncated form of the TGFβR2 extracellular region were significantly more stable than the full-length control samples with the TGFβR2 extracellular region, exhibiting fewer broken bands. Therefore, the stability of the TGFβR2 extracellular region samples prepared in this invention with different truncated forms has general applicability and is not significantly correlated with the antibody properties of the fusion protein's target region.

[0280] 7.3 Degradation tendency of antibody fusion proteins containing the truncated form of TGFβR2 X / TGFβR2

[0281] This embodiment uses 293E cell supernatant to accelerate the treatment of the fusion protein, further optimizing the degradation stability of the X / TGFβR2 antibody fusion protein by truncating the extracellular region of TGFβR2. The specific operation is the same as in Example 3.3. See Example 3.3 for sample purity test results. Figure 32Sample purity and cleavage ratio are shown in Table 21. The results indicate that, compared to the full-length TGFβR2 extracellular region fusion protein, the preferred truncated TGFβR2 extracellular region forms of multiple X / TGFβR2 antibody fusion proteins all exhibit stronger resistance to protease degradation.

[0282] Table 21 Degradation rate of X / TGFβR2 antibody fusion protein containing truncated TGFβR2

[0283]

[0284] To investigate the stability of the preferred truncated TGFβR2 extracellular region X / TGFβR2 antibody fusion protein under certain concentration conditions, its ultrafiltration stability was analyzed using the method described in Example 6.1. The results are shown in Table 22. The preferred truncated TGFβR2 extracellular region X / TGFβR2 antibody fusion protein did not readily break down after concentration, with the proportion of cleaved parts all less than 4.0% (SDS-PAGE purity), demonstrating stronger ultrafiltration stability than the full-length TGFβR2 extracellular region X / TGFβR2 antibody fusion protein.

[0285] Table 22 shows the detection of SEC and SDS fragmentation after concentration of the X / TGFβR2 antibody fusion protein.

[0286]

[0287] In summary, by linking multiple solid tumor antigens as the targeting portion of the fusion protein, we further verified that the fusion protein containing the preferred truncated form of the extracellular region of TGFβR2 has better stability.

[0288] Example 8: In vitro biological functions of a multi-antigen X / TGFβR2 antibody fusion protein targeting solid tumors using a truncated form of TGFβR2 (fusion protein 6).

[0289] Detection of TGF-β1 binding to 8.1X / TGFβR2 antibody fusion protein

[0290] The binding affinity of the X / TGFβR2 antibody fusion protein to TGF-β1 was detected using the ELISA method described in Example 3.4. The results are as follows: Figure 33 As shown, the antibody fusion protein containing the preferred truncated form of TGFβR2, X / TGFβR2, has a slightly lower ability to bind TGF-β1 than the antibody fusion protein containing the full-length form of TGFβR2, X / TGFβR2.

[0291] 8.2X / TGFβR2 antibody fusion protein neutralization TGF-β detection

[0292] The neutralizing ability of the X / TGFβR2 antibody fusion protein against TGF-β1 and TGF-β3 was detected according to Example 3.5. The results are as follows: Figure 34 As shown, the antibody fusion protein containing the preferred truncated form of TGFβR2, X / TGFβR2, has a better ability to neutralize both TGF-β1 and TGF-β3 than the antibody fusion protein containing the full-length form of TGFβR2, X / TGFβR2.

[0293] Detection of 8.3X / TGFβR2 antibody fusion protein binding to the X-side target

[0294] X-side corresponding antigens ERBB2-his, VEGF165, VEGFR2-His, CTLA4-his, and EGFR-His were coated onto 96-well plates at final concentrations of 10 ng / mL, 5 ng / mL, 80 ng / mL, 80 ng / mL, and 40 ng / mL, respectively, at 100 μL / well, and incubated overnight at 4°C. The plates were washed the following day, blocked at room temperature for 1 h, and then 100 μL of X / TGFβR2 antibody fusion protein at a final concentration of 13.89 nM was added, followed by incubation for 1 h. Unbound antibodies were removed by washing, and secondary antibody Goat anti-hIgG Fc / HRP was added for incubation, followed by repeated washing. Finally, substrate chromogenic buffer was added for color development, and the OD was read using a microplate reader after termination. 450 The result is as follows Figure 35 As shown, the ability of the X / TGFβR2 antibody fusion protein containing the preferred truncated form of TGFβR2 to bind the corresponding antigen on the X side is similar to that of the X / TGFβR2 antibody fusion protein containing the full-length form of TGFβR2.

[0295] sequence list

[0296]

[0297]

[0298]

[0299]

[0300]

[0301] References

[0302] 1.Xie, F., et al., TGF-beta signaling in cancer metastasis. Acta BiochimBiophys Sin(Shanghai), 2018.50(1):p.121-132.

[0303] 2.Colak,S.and P.Ten Dijke,Targeting TGF-beta Signaling inCancer.Trends Cancer,2017.3(1):p.56-71.

[0304] 3.Fabregat,I.,et al.,TGF-beta signaling in cancer treatment.CurrPharm Des,2014.20(17):p.2934-47.

[0305] 4.Batlle,E.and J.Massague,Transforming Growth Factor-beta Signalingin Immunity and Cancer.Immunity,2019.50(4):p.924-940.

[0306] 5.Laskin,J.J.and A.B.Sandler,Epidermal growth factor receptor:apromising target in solid tumours.Cancer treatment reviews,2004.30(1):p.1-17.

[0307] 6.Hynes,N.,et al.,The ErbB receptor tyrosine family as signalintegrators.Endocrine-related cancer,2001.8(3):p.151-159.

[0308] 7.Zandi,R.,et al.,Mechanisms for oncogenic activation of theepidermal growth factor receptor.Cellular signalling,2007.19(10):p.2013-2023.

[0309] 8.Seshacharyulu,P.,et al.,Targeting the EGFR signaling pathway incancer therapy.Expert opinion on therapeutic targets,2012.16(1):p.15-31.

[0310] 9.Zhao,Y.,et al.,TGF-βtransactivates EGFR and facilitates breastcancer migration and invasion through canonical Smad3 and ERK / Sp1 signalingpathways.Molecular oncology,2018.12(3):p.305-321.

[0311] 10.Wendt,M.K.,J.A.Smith,and W.P.Schiemann,Transforming growth factor-β-induced epithelial–mesenchymal transition facilitates epidermal growthfactor-dependent breast cancer progression.Oncogene,2010.29(49):p.6485.

[0312] 11.Lee,E.,et al.,Transforming growth factorβ1transactivates EGFR viaan H2O2-dependent mechanismin squamous carcinoma cell line.Cancer letters,2010.290(1):p.43-48.

[0313] 12.Dunfield,L.D.and M.W.Nachtigal,Inhibition of the antiproliferativeeffect of TGFβby EGF in primary human ovarian cancer cells.Oncogene,2003.22(30):p.4745.

[0314] 13.Kretzschmar,M.,et al.,A mechanism of repression of TGFβ / Smadsignaling by oncogenic Ras.Genes&development,1999.13(7):p.804-816.

[0315] 14.ten Dijke,P.,K.Miyazono,and C.-H.Heldin,Signaling inputs convergeon nuclear effectors in TGF-βsignaling.Trends in biochemical sciences,2000.25(2):p.64-70.

[0316] 15.Funaba,M.,C.M.Zimmerman,and L.S.Mathews,Modulation of Smad2-mediated signaling by extracellular signal-regulated kinase.Journal ofBiological Chemistry,2002.277(44):p.41361-41368.

[0317] 16.Richter,P.,et al.,EGF / TGFbeta1 co-stimulation of oral squamouscell carcinoma cells causes an epithelial-mesenchymal transition cellphenotype expressing laminin 332.J Oral Pathol Med,2011.40(1):p.46-54.

[0318] 17.Uttamsingh,S.,et al.,Synergistic effect between EGF and TGF-beta1in inducing oncogenic properties of intestinal epithelial cells.Oncogene,2008.27(18):p.2626-34.

[0319] 18.Xu,Z.,et al.,TGFbeta and EGF synergistically induce a moreinvasive phenotype of epithelial ovarian cancer cells.Biochem Biophys ResCommun,2010.401(3):p.376-81.

[0320] 19.Xiong,J.,et al.,Epidermal growth factor promotes transforminggrowth factor-beta1-induced epithelial-mesenchymal transition in HK-2cellsthrough a synergistic effect on Snail.Mol Biol Rep,2014.41(1):p.241-50.

[0321] 20.Buonato,J.M.,I.S.Lan,and M.J.Lazzara,EGF augments TGFbeta-inducedepithelial-mesenchymal transition by promoting SHP2 binding to GAB1.J CellSci,2015.128(21):p.3898-909.

[0322] 21.Wang,T.,et al.,The TGFβ-miR-499a-SHKBP1 pathway induces resistanceto EGFR inhibitors inosteosarcoma cancer stem cell-like cells.Journal ofExperimental&Clinical Cancer Research,2019.38(1):p.226.

[0323] 22.Jie,H.B.,et al.,CTLA-4(+)Regulatory T Cells Increased inCetuximab-Treated Head and NeckCancer Patients Suppress NK Cell Cytotoxicityand Correlate with Poor Prognosis.Cancer Res,2015.75(11):p.2200-10.

[0324] 23.Bedi,A.,et al.,Inhibition of TGF-beta enhances the in vivoantitumor efficacy of EGFreceptor-targeted therapy.Mol Cancer Ther,2012.11(11):p.2429-39.

[0325] 24.Zhang,Y.,et al.,The canonical TGF-beta / Smad signalling pathway isinvolved in PD-L1-inducedprimary resistance to EGFR-TKIs in EGFR-mutant non-small-cell lung cancer.Respir Res,2019.20(1):p.164.

[0326] 25.Jones,S.T.and M.M.Bendig,Rapid PCR-cloning of full-length mouseimmunoglobulin variableregions.Biotechnology(N Y),1991.9(6):p.579.

[0327] 26.Kabat,E.A.,et al.,Sequences of proteins of immunologicalinterest.1992:DIANE publishing.

[0328] 27.Jones,P.T.,et al.,Replacing the complementarity-determiningregions in a human antibody withthose from a mouse.Nature,1986.321(6069):p.522.

[0329] 28.Verhoeyen,M.and L.Riechmann,Engineering of antibodies.BioEssays,1988.8(2-3):p.74-78.

Claims

1. A fusion protein comprising a) A molecule whose amino acid sequence is a truncated form of the extracellular region of TGFβR2, as shown in SEQ ID NO: 1; and b) Targeted portion; Optionally, the targeting portion of the fusion protein is a cancer cell-specific targeting portion, selected from antibodies or their antigen-binding fragments, functional ligands or their Fc fusion proteins, and receptor proteins or their Fc fusion proteins.

2. The fusion protein of claim 1, wherein, The N-terminus of the truncated molecule is connected to the C-terminus of the heavy chain of the targeted portion; Optionally, the N-terminus of the truncated molecule is connected to the C-terminus of the heavy chain of the targeted portion via a connector; Optionally, the linker is a G4S flexible linker peptide, preferably a (G4S)4 linker peptide.

3. The fusion protein of claim 1, wherein, The target portion of the fusion protein is selected from Trastuzumab, Bevacizumab, Ramucirumab, Ipilimumab, or Panitumumab. Optionally, the amino acid sequence of the fusion protein is selected from: (a) The heavy chain amino acid sequence shown in SEQ ID NO:3 and the light chain amino acid sequence shown in SEQ ID NO:2; (b) The heavy chain amino acid sequence shown in SEQ ID NO:5 and the light chain amino acid sequence shown in SEQ ID NO:4; (c) The heavy chain amino acid sequence shown in SEQ ID NO:7 and the light chain amino acid sequence shown in SEQ ID NO:6; (d) The heavy chain amino acid sequence shown in SEQ ID NO:9 and the light chain amino acid sequence shown in SEQ ID NO:8; or (e) The heavy chain amino acid sequence shown in SEQ ID NO:11 and the light chain amino acid sequence shown in SEQ ID NO:10; It consists of two heavy chains and two light chains; a disulfide bond is formed between the first light chain and the first heavy chain, a disulfide bond is formed between the second light chain and the second heavy chain, and a disulfide bond is formed between the first heavy chain and the second heavy chain.

4. A conjugate comprising the fusion protein according to any one of claims 1-3 and an additional therapeutic agent.

5. A nucleic acid encoding the fusion protein of any one of claims 1-3, wherein the fusion protein is mRNA and / or DNA; optionally, comprising the sequence of SEQ ID NO:

12.

6. An expression vector comprising the nucleic acid of claim 5.

7. A host cell comprising the nucleic acid of claim 5 or the expression vector of claim 6.

8. A method for producing the fusion protein according to any one of claims 1-3, comprising culturing the host cell according to claim 7 under conditions suitable for the expression of the aforementioned protein molecule, and recovering the expressed product from the culture medium.

9. A pharmaceutical composition comprising a) the fusion protein according to any one of claims 1-3, the conjugate according to claim 4, the nucleic acid according to claim 5, or the expression vector according to claim 6; and b) A pharmaceutically acceptable carrier; optionally c) One or more other therapeutic agents.

10. A kit comprising the fusion protein of any one of claims 1-3, the conjugate of claim 4, the nucleic acid of claim 5, the expression vector of claim 6, and the pharmaceutical composition of claim 9; Preferably, it further includes a means for administering the drug.

Images