VirusTAC platform for targeted degradation of tumor cell membrane proteins and psoriasis extracellular pathogenic factors and applications thereof

Abstract

The application discloses a VirusTAC platform for targeted degradation of tumor cell membrane protein EGFR and psoriasis extracellular pathogenic factor IL17A and application thereof, and belongs to the technical field of biological medicine. The platform comprises a heterodimer with R1-R2-R3 structure formed by a first polypeptide chain and a second polypeptide chain, wherein R1 is MeV H or a variant thereof; R2 is a linker composed of R4 and R5, R4 is an immunoglobulin Fc region, the Fc regions corresponding to the first polypeptide chain and the second polypeptide chain are associated by heterodimerization mutation; R5 is a linker that can be cut by a protease; and R3 is a target protein binding domain EGFR antibody or IL17A antibody. The platform realizes precise degradation of tumor-related membrane proteins and extracellular proteins by constructing a chimera of a natural ligand and a targeting antibody, and by means of tumor-specific expression and efficient endocytosis characteristics of Nectin-4.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a VirusTAC platform for the targeted degradation of tumor cell membrane protein EGFR and psoriasis extracellular pathogenic factor IL17A and its application. Background Technology

[0002] Cell membrane proteins and extracellular proteins are key participants in human physiological processes, and their abnormal expression or dysfunction is closely related to a variety of major diseases such as tumors, neurodegenerative diseases, and autoimmune diseases. For example, overactivation of epidermal growth factor receptor (EGFR) is associated with the development and progression of malignant tumors, while interleukin-17A (IL-17A) is a core pathogenic factor in chronic inflammation such as psoriasis. Traditional treatment strategies mainly rely on small molecule antagonists or monoclonal antibodies to competitively bind to targets and block their function. Although more than 150 targeted drugs (such as kinase inhibitors and monoclonal antibodies) have been approved for marketing, a large number of targets are still classified as "undruggable" targets due to the lack of suitable binding pockets or the existence of genetic mutations.

[0003] To overcome the limitations of traditional antagonists, targeted protein degradation (TPD) technology has emerged. This technology recruits the cellular endogenous clearance system to directly degrade target proteins (POIs). Currently, various TPD pathways have been developed, including PROTAC for degrading intracellular proteins, and lysosomal targeted chimeras (LYTAC) and autophagy targeted chimeras (AUTAC / ATTEC) for degrading membrane and extracellular proteins. However, existing LYTAC platforms still face three major bottlenecks: first, the lysosomal targeted receptors (LTRs) they rely on are widely expressed in normal tissues, leading to insufficient tumor specificity; second, endocytosis efficiency is limited, affecting degradation kinetics; and third, non-natural sugar structure LTRs may pose immunogenic risks. Therefore, developing a novel degradation platform with high specificity, strong endocytosis capacity, and good safety is an urgent clinical challenge. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention proposes a novel TPD technology platform based on measles virus hemagglutinin (MeV H). Nectin-4 is one of the important receptors for MeV H, with low expression levels in normal adult tissues, but abnormally high expression in various malignant tumors such as urothelial carcinoma, lung cancer, and breast cancer, and exhibits extremely high endocytic activity. The VirusTACs platform in this invention constructs a chimera of natural ligands and targeting antibodies (such as antibodies against EGFR or IL-17A), leveraging the tumor-specific expression and highly efficient endocytic properties of Nectin-4 to achieve precise degradation of tumor-associated membrane proteins and extracellular proteins.

[0005] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution: One objective of this invention is to provide a fusion protein complex comprising a heterodimer formed by a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain and the second polypeptide chain together constitute the structure R1-R2-R3, wherein: R1 is wild-type measles virus hemagglutinin protein MeV H or a biologically active variant thereof; R2 is a connector composed of R4 and R5; R4 is the Fc region of immunoglobulin. The Fc regions corresponding to the first polypeptide chain and the second polypeptide chain are associated through heterodimerization mutation. R5 is a protease-cleavable linker; R3 is the target protein binding domain, and the target protein is an EGFR antibody or an IL17A antibody.

[0006] Furthermore, R1 is a MeV H protein or a variant that has more than 90% sequence invariance to the key sequence of the MeV H protein.

[0007] Furthermore, the two Fc subunits in R4 introduce LDLE and LKTK mutations into their respective CH3 regions; the LDLE mutations include L351D and / or L368E mutations; the LKTK mutations include L351K and / or T366K mutations.

[0008] Furthermore, R5 is selected from an amino acid sequence that can be specifically recognized and cleaved by proteases highly expressed in the tumor microenvironment or inflamed tissue.

[0009] Furthermore, the molar ratio of R1 to R3 is 1:1.

[0010] Furthermore, the first polypeptide chain sequence is as shown in any one of SEQ ID NO:1-SEQ ID NO:3.

[0011] Furthermore, the second polypeptide chain is as shown in SEQ ID NO:4 or SEQ ID NO:5.

[0012] A second objective of this invention is to provide a nucleic acid composition comprising a nucleic acid sequence encoding the R1-R5 portions of the fusion protein complex, or a nucleic acid sequence encoding the first polypeptide chain and the second polypeptide chain of the fusion protein complex.

[0013] A third objective of this invention is to provide a pharmaceutical composition comprising the fusion protein complex, a chimeric molecule in the fusion protein complex, or the nucleic acid composition, and pharmaceutically acceptable excipients.

[0014] A fourth objective of this invention is to provide the use of the fusion protein complex, the nucleic acid composition, or the pharmaceutical composition in the preparation of a medicament for treating diseases associated with high Nectin-4 expression, including oncological diseases and / or autoimmune diseases.

[0015] Preferably, the tumor is lung cancer, and the autoimmune disease is psoriasis.

[0016] The VirusTAC protein degradation platform and its applications provided by this invention have the following significant advantages compared to existing technologies (such as traditional neutralizing antibodies, PROTAC, or other lysosome-based degradation technologies): (1) Excellent tissue-targeting specificity and efficient endocytosis capability VirusTAC leverages the high specific expression of Nectin-4 in tumor tissues and psoriatic lesions to achieve targeted degradation, reducing interference with normal tissues. Simultaneously, it utilizes Nectin-4 receptor-mediated efficient endocytosis to enhance the degradation efficiency of POIs.

[0017] (2) AI-driven kinetic optimization and degradation efficiency This invention utilizes the protein language model framework (EVOLVEpro) to engineer viral entry proteins. This few-sample active learning method overcomes the limitations of natural evolution. The AI-optimized variant significantly outperforms its wild-type in degradation kinetics, further enhancing its efficient clearance capability of POIs.

[0018] (3) Broad therapeutic applicability and target expansion capabilities This invention successfully expands the application scope of TPD from intracellular proteins to membrane proteins and extracellular secretory proteins, and has a highly modular feature, which can be widely used in cancer targeted therapy and immunotherapy.

[0019] (4) Significant synergistic effect and good safety In vivo PDX model experiments of lung cancer showed that VirusTAC monotherapy can significantly prolong survival, and has a significant synergistic effect when used in combination with first-line clinical chemotherapy regimens (such as pemetrexed + carboplatin). Meanwhile, the animal models maintained stable body weight and showed no significant tissue toxicity during administration, confirming the high safety of this invention in clinical applications. Attached Figure Description

[0020] Figure 1This is a schematic diagram of the VirusTAC structure in Example 1 of the present invention. The modular VirusTAC molecule comprises: (1) a viral entry protein for recognizing target cell receptors and mediating specific endocytosis; (2) an anti-POI (target protein) binding domain, wherein the target protein in the present invention is an EGFR antibody or an IL17A antibody; (3) an intracellular enzyme cleavage linker and a "knob-in-hole" Fc dimer.

[0021] Figure 2 This describes the binding ability of VirusTACs to various tumor cells in Example 3 of this invention. The binding ability of VirusTACs based on multiple viral envelope proteins to various tumor cells, including MDA-MB-231, MCF-7, and A549, was detected by flow cytometry. Data are expressed as mean ± standard error (mean ± SEM). p<0.0001, p<0.001, p<0.01, p<0.05, ns indicates no significant difference.

[0022] Figure 3 This invention demonstrates VirusTAC-mediated EGFR degradation in Example 4. MDA-MB-231, MCF-7, A549, or UM-UC-3 cells were treated with EGFR-targeting VirusTACs or isotype controls. EGFR expression was detected by flow cytometry (n=3 per group), and statistical analysis was performed using one-way ANOVA combined with Tukey's test. Data are expressed as mean ± standard error (mean ± SEM). p<0.0001, p<0.001, p<0.01, p<0.05, ns indicates no significant difference.

[0023] Figure 4This example demonstrates the validation of VirusTAC-mediated EGFR degradation using an EGFR-EGFP overexpression system in Example 4 of this invention. MDA-MB-231 and A549 cells overexpressing EGFR-EGFP were treated with specified VirusTACs or isotype controls. EGFP expression in each group of cells was detected by flow cytometry (n=3 per group), and statistical analysis was performed using one-way ANOVA combined with Tukey's test. Data are expressed as mean ± standard error (mean ± SEM). p<0.0001, p<0.001, p<0.01, p<0.05, ns indicates no significant difference.

[0024] Figure 5 This invention demonstrates how VirusTAC mediates EGFR degradation in multiple tumor cell lines, as shown in Example 4. Representative Western blot images show the expression levels of EGFR in MDA-MB-231, A549, and UM-UC-3 cells after αEGFR-VirusTAC treatment. Protein levels were normalized using Actin as an internal control.

[0025] Figure 6 This is the first round of directed protein evolution based on wild-type MeV H in Example 5 of the present invention. Flow cytometry was used to analyze the expression levels of EGFR in MDA-MB-231, A549, and UM-UC-3 cells treated with different VirusTAC evolutionary variants. The first round of screening identified A176S as the optimal mutation site (named MeV HS). Statistical analysis employed one-way ANOVA combined with Tukey's test (n=3 per group). Data are expressed as mean ± standard error (mean ± SEM). p<0.0001, p<0.001, p<0.01, p<0.05, ns indicates no significant difference.

[0026] Figure 7 This is the second round of directed protein evolution based on MeV HS in Example 5 of this invention. The second round of evolution was designed using the optimal A176S mutation site from the first round as the backbone. Flow cytometry was used to analyze the EGFR expression levels of MDA-MB-231, A549, and UM-UC-3 cells under different VirusTAC evolutionary variants in the second round. The second round selected A176S / K294W as the optimal mutation site (named MeV H-SW). Statistical analysis used one-way ANOVA combined with Tukey's test (n=3 per group). Data are expressed as mean ± standard error (mean ± SEM). p<0.0001, p<0.001, p<0.01, p<0.05, ns indicates no significant difference.

[0027] Figure 8 This example demonstrates a comparison of the EGFR degradation kinetics and efficacy between MeV H-αEGFR and MeV H-SW-αEGFR in Example 5 of this invention. Representative Western blot images show the expression levels of EGFR in A549 cells treated with MeV H-αEGFR or MeV H-SW-αEGFR at specified time points and concentrations. Protein levels were normalized using Actin as an internal control.

[0028] Figure 9 This describes the multi-receptor-dependent cell entry mechanism in Example 6 of this invention. (ab) Flow cytometry analysis showed the cell binding levels (a) of POI-free MeV H-SW-VirusTAC-treated cells (sgNC, sgNECTIN4, sgSLAMF1, sgCD46) and the expression levels of EGFR (b) treated with MeV H-SW-αEGFR in MDA-MB-231, MCF-7, A549, and UM-UC-3 cells with gene knockout (sgNC, sgNECTIN4, sgSLAMF1, sgCD46). Statistical analysis was performed using one-way ANOVA combined with Tukey's test (n=3 per group). Data are expressed as mean ± standard error (mean ± SEM). p<0.0001, p<0.001, p<0.01, p<0.05, ns indicates no significant difference.

[0029] Figure 10 This illustration demonstrates the targeted therapy of EGFR degradation mediated by VirusTAC in a mouse lung cancer model using Example 7 of this invention. (a) Schematic diagram of targeted therapy using VirusTAC in a lung cancer PDX model. (bd) Body weight curves (b), tumor growth curves (c), and Kaplan-Meier survival curves (d) analyzed by log-rank test for mice in different treatment groups (n=10 mice per group). Data are expressed as mean ± standard error (mean ± SEM) and statistical analysis was performed using two-way ANOVA combined with Tukey's test. p<0.0001, p<0.001, p<0.01, p<0.05, ns indicates not significant. (e) Representative H&E staining and immunofluorescence (IF) staining images of tumor tissues from different treatment groups. The black boxed areas in the H&E images are magnified in the IF atlas to show EGFR (red) and DAPI (blue). Scale bar: 50 μm.

[0030] Figure 11 This describes the safety and specificity of VirusTAC in Example 8 of this invention. (a) Representative H&E staining images of key mouse organs (heart, liver, spleen, lung, kidney) treated with different doses of MeV H-SW-αEGFR VirusTAC (0, 1, 10, 50 mg / kg). Scale bar: 100 μm. (b) Representative H&E staining and immunofluorescence staining images of key mouse organs (heart, liver, spleen, lung, kidney) after treatment with IgG and MeV H-SW-αEGFR. The black boxed areas in the H&E images are magnified in the IF spectrum to show EGFR (red) and DAPI (blue). Scale bar: 50 μm.

[0031] Figure 12This illustrates VirusTAC-mediated extracellular IL17A degradation in Example 9 of the present invention. (a) Schematic diagram of the structure of MeVH-SW-αIL17A VirusTAC. (be) HaCaT cells were stimulated with recombinant IL17A, and then treated with MeVH-SW-αIL17A, αIL17A, or IgG, respectively. (b) The IL17A content in the cell supernatant of the different treatment groups was detected by ELISA. (c) A volcano plot shows the differentially expressed genes in HaCaT cells after the above treatment; red and blue dots represent genes that are significantly upregulated and significantly downregulated, respectively; black dots represent genes that do not show significant changes. (d) A heatmap shows the expression levels of IL17A-driven inflammatory characteristic genes in HaCaT cells after the above treatment. The color scale from blue to red represents low and high expression levels, respectively. (e) Gene set enrichment analysis (GSEA) showed that the IL17 signaling pathway was enriched in the transcriptome data of HaCaT cells treated with MeVH-SW-αIL17A compared with the αIL17A treatment group. The data in (b) were statistically analyzed using two-way ANOVA combined with Tukey's test. p<0.0001, p<0.001, p<0.01, p<0.05, ns indicates not significant.

[0032] Figure 13This is the targeted therapy of VirusTAC-mediated IL17A degradation in a mouse psoriasis model as described in Example 10 of this invention. (ac) Erythema scores of mice in different treatment groups: (a) Desquamation score (b) and induration / hypertrophy score (c) analysis. Groups included: Vaseline + IgG group (n=8), Imiquimod (IMQ) + IgG group (n=8), IMQ + αIL17A group (n=8), and IMQ + MeVH-SW-αIL17A group (n=9). (df) Representative H&E images of skin tissue from mice in each treatment group: (d) Quantitative analysis of epidermal thickness (e) and pathological score (f). Scale bar 100 μm. (g) ELISA detection of IL17A levels in the serum of mice in each treatment group. The data in (ac) were analyzed using one-way ANOVA combined with Tukey's test (data are expressed as mean ± standard error, mean ± SEM); the data in (eg) were analyzed using two-way ANOVA combined with Tukey's test. p<0.0001, p<0.001, p<0.01, p<0.05, ns indicates not significant. Detailed Implementation

[0033] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention. The reagents, products, and instruments used in the following examples are all commercially available, and the methods used in the examples, unless otherwise specified, are consistent with conventionally used methods.

[0034] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0035] Example 1: Structural Design and Functional Mechanism of Wild-Type MeV H-hIgG1 Fusion Protein 1. Design and expression of wild-type MeV H-hIgG1 structure (1) Wild-type MeV H-hIgG1 design concept: A. Based on the biological characteristics of measles virus hemagglutinin (MeV H) specifically recognizing and binding to the cell surface receptor Nectin-4, a wild-type MeV H-hIgG1 Fc LKTK fusion protein was designed (see SEQ ID NO.1 for details), and its specific structural design is as follows: Figure 1 The explanation is as follows: B. The key functional sequence of wild-type MeV H is linked to the hinge region of the Fc region of the heavy chain of human IgG1 via a protease-cleavable linker.

[0036] C. Introducing the LKTK mutation into the CH3 region of the Fc fragment, this charge polarity mutation aims to enhance the specific heterodimerization efficiency between this heavy chain and another complementary heavy chain through electrostatic interaction, thereby reducing the generation of homodimers.

[0037] D. After the selected linker enters the cell, it can be recognized and cleaved by specific proteases in the lysosome, thereby releasing the entry receptor (such as Nectin-4), which can then be recycled back to the cell membrane to maintain its physiological expression level, ensuring that the system can cyclically and continuously mediate the degradation of the target protein.

[0038] (2) Construction of wild-type MeV H-hIgG1 gene expression vector: Based on the protein structure described above, overlapping primers covering the full length of the target gene were designed and synthesized. These primers were then spliced ​​together using enzymatic filling or overlapping extension PCR to obtain a linear double-stranded DNA fragment containing the target gene. The target gene sequence was cloned into a mammalian cell expression vector using homologous recombinase or restriction endonuclease to construct a recombinant plasmid. The recombinant product was then transformed into competent E. coli cells (DH5α), plated on selective LB agar plates containing the appropriate antibiotics, and single colonies were selected for colony PCR identification. Positive clones with the correct band size were screened using agarose gel electrophoresis. Full-length sequencing was performed after plasmid extraction, and recombinant plasmids with completely correct sequences were selected for subsequent protein expression experiments.

[0039] 2. Design, screening, and expression vector construction of MeV H evolutionary variants (1) Design principles and computer-aided screening strategy for MeV H evolutionary variants: To further improve the efficiency of target protein degradation mediated by the MeV H-hIgG1Fc LKTK fusion protein, this embodiment uses the computer-aided evolution strategy EVOLVEpro (EVOlution Via Language model-guided Variance Exploration for proteins) to optimize the wild-type MeV H sequence, aiming to screen for evolutionary variants with higher receptor affinity or better stability. The specific process is as follows: A. Feature extraction was performed on wild-type MeV H and its mutant sequences using the AI ​​protein language model (EVOLVEpro). The amino acid sequences were mapped to high-dimensional vector representations to capture the contextual relationships and evolutionary conservation information between amino acid residues.

[0040] B. A deep neural network was constructed as a fitness scoring model to predict the functional scores of mutant sequences, such as degradation activity or affinity. Based on the initial predictions, the model was fine-tuned using supervised learning with some experimental data from our previous work to improve the model's prediction accuracy for the MeV H protein family.

[0041] C. Guided by a scoring function, a genetic algorithm is used to perform a heuristic search within the sequence space. In each iteration, 1-3 random point mutations are introduced into the parent sequence to generate a candidate progeny library. High-fitness sequences are selected based on the scoring results to proceed to the next iteration, simulating the biological evolution process through multiple "mutation-screening-recombination" cycles. Finally, the top 10-20 mutant sequences with the highest scores are used for downstream experimental validation, including synthesis, expression, and functional testing.

[0042] (2) Screening results and mutation sequences of MeV H evolutionary forms: After multiple rounds of iterative screening, two key evolutionary mutation schemes were determined: A. First round of evolution: Using wild-type MeV H as a template, 10 single-point mutations with potential functional enhancement effects were screened out: L108A, L111V, R12P, K145P, A176S, K305F, H312W, N330Y, L331D, and Q392G.

[0043] B. Second round of evolution (based on preferred mutation sites): Based on the A176S mutant (MeV HS, see SEQ ID NO.2) that performed well in the first round of evolution, 10 evolutionary variants were further screened through superimposed mutations, and the following mutation sites were introduced respectively: Y227W, Y261W, P275W, R292W, K294W, K305L, A313W, A319W, A354W, R397S, and the optimal mutant A176S / K294W (MeV H-SW, see SEQ ID NO.3).

[0044] (3) Construction of MeV H-SW gene expression vector: Based on the selected evolutionary sequence, the corresponding MeV H-SW-hIgG1 Fc LKTK fusion protein coding sequence was designed. The fragments containing the target gene were spliced ​​together using enzymatic filling or overlap extension PCR. The target gene sequence was cloned into a mammalian cell expression vector using homologous recombinase or restriction endonuclease to construct a recombinant plasmid. The recombinant product was then transformed into competent E. coli cells (DH5α), plated on selective LB agar plates containing the appropriate antibiotics, and single colonies were selected for colony PCR identification. Positive clones with the correct band size were screened using agarose gel electrophoresis. Full-length sequencing was performed after plasmid extraction, and recombinant plasmids with completely correct sequences were selected for subsequent protein expression experiments.

[0045] 3. Design and expression of the target membrane protein targeting domain (effective arm) This patent selects antibody domains targeting EGFR and IL17A for the construction of the VirusTAC platform.

[0046] (1) Clonal Design: To achieve precise assembly of the target arm and the effector arm, this invention optimizes the design of the target structure, selecting an anti-EGFR single-chain antibody fragment (scFv) or an anti-IL17A antibody domain, which is linked to hIgG1 Fc via a flexible linker. An LDLE (L351D, L368E) mutation is introduced into the CH3 structure of hIgG1 Fc, allowing for heterodimerization with the LKTK structure of the effector arm during cell expression. αEGFR-hIgG1 Fc LDLE fusion protein: Contains an EGFR scFv sequence, a linker, and an hIgG1 Fc segment with the LDLE mutation; the sequence details are shown in SEQ ID NO.4. αIL17A-hIgG1 Fc LDLE fusion protein: Contains an IL-17A binding domain, a linker, and an hIgG1 Fc segment with the LDLE mutation; the sequence details are shown in SEQ ID NO.5.

[0047] (2) Construction of gene expression vector: Based on the protein sequence design described above, the coding gene was codon-optimized for mammalian cells, and a full-length DNA sequence containing the corresponding signal peptide, target domain, linker, and LDLE mutant Fc segment was synthesized. Specific primers covering the full length or linker were designed for αEGFR-hIgG1 Fc LDLE and αIL17A-hIgG1 Fc LDLE. Linear double-stranded target gene fragments with complete open reading frames (ORFs) were obtained using enzymatic filling or overlap extension PCR. Homologous recombination or double enzyme digestion was used to directionally clone the target gene fragments into mammalian cell expression vectors. The recombinant products were then transformed into competent E. coli cells (DH5α), plated on selective LB agar plates containing the corresponding antibiotics, single colonies were selected for colony PCR identification, and positive clones with the correct band size were screened by agarose gel electrophoresis. Recombinant plasmids with completely correct sequences were selected for subsequent protein expression experiments.

[0048] The sequence is as follows: SEQ ID NO.1 (MeV H-hIgG1 Fc LKTK sequence) SKGNCSGPTTIRGQFSNMSLSLLDLYLGRGYNVSIVTMTSQGMYGGTYLVEKPNLSSKRSELSQLSMYRVFEVGVIRNPGLGAPVFHMTNYLEQPVSNDLSNCMVALGELKLAALCHGEDSITIPYQGSGKGVSFQLVKLGVWKSPTDMQSWVPLSTDDPVIDRLYLSSHRGVIADNQAKWAVPTTRTDDKLRMETCFQQACKGKIQALCENPEWAPLKDNRIPSYGVLSVDLSLTVELKIKIASGFGPLITHGSGMDLYKSNHNNVYWLTIPPMKNLALGVINTLEWIPRFKVSPYRFTVPIKEAGGDCHAPTYLPAEVDGDVKLSSNLVIL PGQDLQYVLATYDTSRVEHAVVYYVYSPSRSFSYFYPFRLPIKGVPIELQVECFTWDQKLWCRHFCVLADSESGGHITHSGMVGMGVSCTGGFLGGVRGVDGEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTKPPSRDELTKNQVSLKCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO.2(MeV HS-hIgG1 Fc LKTK sequence) SKGNCSGPTTIRGQFSNMSLSLLDLYLGRGYNVSSIVTMTSQGMYGGTYLVEKPNLSKRSELSQLSMYRVFEVGVIRNPGLGAPVFHMTNYLEQPVSNDLSNCMVALGELKLAALCHGEDSITIPYQGSGKGVSFQLVKLGVWKSPTDMQSWVPLSTDDPVIDRLY LSSHRGVISDNQAKWAVPTTRTDDKLRMETCFQQACKGKIQALCENPEWAPLKDNRIPSYGVLSVDLSLTVELKIKIASGFGPLITHGSGMDLYKSNHNNVYWLTIPPMKNLALGVINTLEWIPRFKVSPYRFTVPIKEAGGDCHAPTYLPAEVDGDVKLSSNLVIL PGQDLQYVLATYDTSRVEHAVVYYVYSPSRSFSYFYPFRLPIKGVPIELQVECFTWDQKLWCRHFCVLADSESGGHITHSGMVGMGVSCTGGFLGGVRGVDGEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTKPPSRDELTKNQVSLKCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO.3(MeV H-SW-hIgG1 Fc LKTK sequence) SKGNCSGPTTIRGQFSNMSLSLLDLYLGRGYNVSSIVTMTSQGMYGGTYLVEKPNLSKRSELSQLSMYRVFEVGVIRNPGLGAPVFHMTNYLEQPVSNDLSNCMVALGELKLAALCHGEDSITIPYQGSGKGVSFQLVKLGVWKSPTDMQSWVPLSTDDPVIDRLY LSSHRGVISDNQAKWAVPTTRTDDKLRMETCFQQACKGKIQALCENPEWAPLKDNRIPSYGVLSVDLSLTVELKIKIASGFGPLITHGSGMDLYKSNHNNVYWLTIPPMKNLALGVINTLEWIPRFWVSPYRFTVPIKEAGGDCHAPTYLPAEVDGDVKLSSNLVIL PGQDLQYVLATYDTSRVEHAVVYYVYSPSRSFSYFYPFRLPIKGVPIELQVECFTWDQKLWCRHFCVLADSESGGHITHSGMVGMGVSCTGGFLGGVRGVDGEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTKPPSRDELTKNQVSLKCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO.4(αEGFR-hIgG1 Fc LDLE) QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAGGGSGGGSGGGSGGGSDILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKGGFLGGVRGVDGEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGPREPQVYTDPPSRDELTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO.5(αIL17A-hIgG1 Fc LDLE) EVQLVESGGDLVQPGGSLRLSCAASGFTFSSYWMSWVRQAPGKGLEWVANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARDRGSLYYWGQGTLVTVSSGGGG SGGGGSGGGGSAIQLTQSPSSSLSASVGDRVTITCRPSQGINWELAWYQQKPGKAPKLLIYDASSLEQGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPLTFGGGTKVEIKGGF LGGVRGVDGEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTDPPSRDELTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Example 2: Construction of the VirusTAC platform for targeted degradation of EGFR or IL17A 1. Plasmid transfection and fusion protein production: In this example, a fusion protein sequence carrying the VirusTAC sequence was prepared in ExpiCHO cells using a transient transfection system.

[0049] (1) Cell plating: ExpoCHO cells were seeded in 10cm dishes (10mL of culture medium) and cultured in a 37℃, 5% CO2 incubator until the cell confluence reached 80%-90%.

[0050] (2) Transfection solution required per 1 ml of culture medium: Solution A: Mix 50 μL of Opti-MEM medium with 1 μL of Lipofectamine 3000 transfection reagent thoroughly; Solution B: Take 50 μL of Opti-MEM medium, 2 μL of P3000 reagent, and a total of 1 μg of recombinant plasmid (the distribution ratio of each group is shown in Table 1).

[0051] (3) Transfection and supernatant collection: Add solution A dropwise to solution B, gently mix by pipetting, and incubate at room temperature for 10 min to form the transfection complex. Add the mixture evenly to a 6-well plate. The day after transfection, replace with fresh DMEM medium containing 15% FBS and continue culturing. Collect the supernatant 36-48 h after transfection and purify it using the Protein A method, then determine the concentration.

[0052] (4) Target cell infection and identification: Target cells were seeded in culture plates, and when the degree of fusion reached 50%-70%, the purified fusion protein prepared above was added to the cells. The incubated cells were then cultured in a routine manner, and subsequently identified by flow cytometry (FACS) or related biological function experiments as needed.

[0053] Table 1. Details of VirusTAC construction and control plasmids

[0054] 2. Fusion protein purification: (1) The virusTAC homodimer and its control protein constructed in this invention were captured and purified by Protein A affinity chromatography using the Fc fragment it carries. The collected culture supernatant was filtered through a 0.22 μm filter and loaded onto a pre-equilibrated Protein A affinity chromatography column. Impurities were washed with phosphate buffer (20 mM Na2HPO4, pH 7.0) containing 0.15 M NaCl until the baseline stabilized. The target protein was then eluted from the column with a low pH elution buffer (0.1 M glycine-HCl, pH 3.0). The eluted fraction was immediately neutralized to neutral with 1.0 M Tris-HCl (pH 9.0), and then the buffer was replaced with PBS buffer (pH 7.4) using a desalting column or ultrafiltration concentrator.

[0055] (2) For bispecific VirusTAC molecules assembled using a "knobs-in-hole" method, their hexahistine tag can also be used for targeted purification via Ni-NTA (nickel ion chelation) affinity chromatography. The sample containing the target protein is loaded onto a nickel column equilibrated with 10 mM imidazole. Impurity proteins are removed sequentially using wash buffers containing 20 mM and 50 mM imidazole. Finally, the target bispecific molecule is collected using a high-concentration elution buffer containing 250-500 mM imidazole. The purified elution buffer is then replaced with PBS buffer via ultrafiltration or dialysis.

[0056] (3) The purified protein is stored at 4°C for short-term experiments; if long-term storage is required, it is quick-frozen in liquid nitrogen and then placed in a -80°C deep cryogenic freezer to ensure the stability of the protein structure.

[0057] Example 3: Detection of VirusTAC's binding ability to tumor cells This embodiment aims to verify the specific binding ability of the VirusTAC effector arm (MeV H) to tumor cells, which is a prerequisite for mediating endocytosis.

[0058] First, VirusTACs based on multiple viral envelope proteins were designed, and tumor cell lines such as A549 (lung cancer), MDA-MB-231 (breast cancer), and MCF7 (breast cancer) were selected for research. When the cell confluence reached 50%-60%, VirusTACs based on multiple viral envelope proteins (concentration of 100 nM) were added to the culture medium of the experimental group, while the same amount of Isotype IgG fusion protein was added to the control group. After incubation with the cells for 1 h, the cells were digested with trypsin, washed once with pre-cooled PBS, and incubated at 4°C for 1 h with anti-human IgG flow cytometry antibody (Invitrogen, A-21433, 1:200). The fluorescence intensity of the flow cytometry antibody on the cell surface was detected using flow cytometry.

[0059] Flow cytometry results as follows Figure 2 As shown, MeV H-VirusTACs exhibited the highest binding levels to various tumor cell lines. Furthermore, VirusTACs based on HSV1 (Herpes Simplex Virus Type 1) and SFV (Semliki Forest Virus) envelope proteins demonstrated that the MeV H effector arm can efficiently recognize and bind to tumor cells.

[0060] Example 4: Evaluation of the degradation efficiency of wild-type VirusTAC-mediated endogenous EGFR 1. Tumor cell lines MDA-MB-231, MCF-7, A549, and UM-UC-3 were selected for the study. MeV H-αEGFR fusion protein (100 nM) was directly added to the cell culture medium; the same equivalent of αEGFR protein was added to the control group. After incubation for 24 h, the cells were digested with trypsin, washed once with pre-cooled PBS, and incubated at 4°C for 1 h with anti-human EGFR flow cytometry antibody (Biolegend, 352904, 1:50). The fluorescence intensity of the flow cytometry antibody on the cell surface was detected using flow cytometry.

[0061] The results are as follows Figure 3As shown, compared with the αEGFR control group, the fluorescence intensity of EGFR on the cell surface of the MeV H-αEGFR treatment group was significantly reduced, and the degradation efficiency of EGFR on tumor cells by MeV H-αEGFR was also superior to that of VirusTACs based on HSV1 and SFV envelope proteins. This indicates that VirusTACs can efficiently induce the degradation of the endogenous target protein EGFR through an effector arm-mediated endocytosis pathway.

[0062] 2. To eliminate interference from antibody detection, a cell line overexpressing the fluorescent EGFR-EGFP protein fusion was used to further confirm the degradation effect. A target cell line stably overexpressing the EGFR-EGFP fusion protein was constructed using lentiviral transfection technology. Wild-type MeV H-αEGFR fusion protein (100 nM) was added to the culture medium of the overexpressing cell line, while the control group received the same concentration of αEGFR. After incubation for 24 h, the cells were digested with trypsin, washed once with pre-cooled PBS, and the intracellular EGFP fluorescence intensity was detected using flow cytometry.

[0063] The results are as follows Figure 4 As shown, the EGFP fluorescence signal of tumor cells in the MeV H-αEGFR treatment group was significantly reduced, while the fluorescence of the control group remained stable. Figure 5 Protein imprinting further demonstrated that the EGFR expression level in the MeV H-αEGFR treatment group was lower than that in other treatment groups in cell lines such as MDA-MB-231, A549, and UM-UC-3, proving that VirusTAC treatment led to a decrease in the overall level of EGFR protein and verifying the universality and efficiency of this platform in the field of protein degradation.

[0064] Example 5: Evaluation of the optimized VirusTAC-mediated endogenous EGFR degradation efficiency 1. First-round directed evolution evaluation of proteins based on wild-type MeV H: Multiple evolved MeV H-VirusTAC variants were compared using cell lines such as MDA-MB-231, A549, and UM-UC-3. VirusTACs based on wild-type MeV H and its evolutionary variants (L108A, L111V, R12P, K145P, A176S, K305F, H312W, N330Y, L331D, Q392G) were added to the cell culture medium at a concentration of 100 nM; the same equivalent of αEGFR was added to the control group culture medium. After incubation for 24 h, the cells were digested with trypsin, washed once with pre-cooled PBS, and incubated at 4°C for 1 h with anti-human EGFR flow cytometry antibody (Biolegend, 352904, 1:50). The EGFR fluorescence intensity of the cells was detected by flow cytometry.

[0065] The results are as follows Figure 6 As shown, compared with the αEGFR control group, the wild-type MeV H-VirusTAC control group exhibited a significant decrease in EGFR expression levels. Among the 10 evolutionary variants of MeV H, the mutant A176S (MeV HS) showed a more significant decrease in fluorescence than the wild type, demonstrating that the AI-optimized MeV H mutation site has a stronger ability to mediate target endocytosis and degradation.

[0066] 2. Based on the first round of selected mutation site A176S (SEQ ID NO.2), 10 mutation sites designed in the second round were superimposed to screen for double-mutant or multi-mutant VirusTAC evolutionary variants with super-strong degradation activity. VirusTACs based on MeV HS and its mutants (10 mutation sites: Y227W, Y261W, P275W, R292W, K294W, K305L, A313W, A319W, A354W, R397S) were added to the target cell lines at a concentration of 100 nM. An equivalent amount of αEGFR was added to the control group. After incubation with cells for 24 h, the cells were digested with trypsin, washed once with pre-cooled PBS, and incubated at 4°C for 1 h with anti-human EGFR flow cytometry antibody (Biolegend, 352904, 1:50). The EGFR fluorescence intensity of the cells was detected by flow cytometry.

[0067] The results are as follows Figure 7 As shown, the fluorescence intensity of EGFR from the A176S / K294W mutant (MeV H-SW, SEQ ID NO.5) showed a further significant decrease compared to the A176S single mutant group and other mutant groups. To compare the degradation kinetics of EGFR by wild-type MeV H-VirusTAC and the evolved MeV H-SW-VirusTAC, we investigated the effects of treatment duration (0, 6, 12, 24, and 48 h) and VirusTAC concentration (0, 5, 10, 25, and 100 nM). The results are as follows. Figure 8 As shown, MeV H-SW-VirusTAC exhibits superior EGFR degradation efficiency and kinetics compared to wild-type MeV H-VirusTAC. Through a second round of superposition evolutionary screening, this invention successfully obtained MeV H evolutionary sequences with higher receptor affinity or stronger induced endocytosis capabilities.

[0068] Example 6: Multi-receptor-mediated VirsuTAC endocytosis This study aimed to investigate the specific contributions of different MeV H receptors to the endocytic function of VirusTAC and to verify the mechanism by which VirusTAC utilizes multiple receptors to mediate endocytosis. Known receptors for measles virus (MeV): Nectin-4, CD46, and SLAMF1 were knocked out in MDA-MB-231, A549, and UM-UC-3 cell lines using CRISPR-Cas9 technology. A non-targeted control group (sgNC) was also established. MeV H-SW-VirusTAC and MeV H-SW-αEGFR VirusTAC, which lack the POI binding domain, were added to the above gene knockout cell lines, respectively. Flow cytometry was used to detect their binding levels with cancer cells and to evaluate their EGFR degradation efficiency.

[0069] The results are as follows Figure 9 As shown in figure a, knocking out Nectin-4 led to a significant decrease in the binding level of VirusTAC to tumor cells, indicating that Nectin-4 is its key binding receptor. Figure 9 As shown in b, in the sgNECTIN4 cell group, VirusTAC-mediated EGFR degradation was significantly reversed, while knockout of SLAMF1 or CD46 had little effect on degradation efficiency. These conclusions indicate that VirusTAC endocytosis follows a multi-receptor synergistic mediation model, primarily mediated by Nectin-4, and efficiently driven by the co-receptors SLAMF1 and CD46.

[0070] Example 7: Treatment of VirusTAC-mediated EGFR degradation in a humanized lung cancer model This embodiment aims to verify the efficacy of the VirusTAC platform in targeted therapy of solid tumors by degrading the oncogenic driver factor EGFR and its synergistic effect with standard clinical treatment regimens. Immune-reconstituted NSG mice (humanized immune system mice) were used to establish a humanized tumor xenograft model (PDX model) for lung cancer. Mice were randomly divided into 5 experimental groups, each receiving the drug via intraperitoneal injection every three days, with regular measurement of mouse weight. The specific groupings are as follows: Control group: given IgG antibody.

[0071] Monoclonal antibody treatment group: αEGFR monoclonal antibody was administered.

[0072] VirusTAC experimental group: MeV H-SW-αEGFR fusion protein.

[0073] Combination therapy group A: αEGFR monoclonal antibody combined with chemotherapy (pemetrexed + carboplatin).

[0074] Group B of combined treatment: MeV H-SW-αEGFR fusion protein combined with chemotherapy (pemetrexed + carboplatin).

[0075] After drug administration, tumor tissue was excised and paraffin sections were prepared. Immunofluorescence (IF) staining was used to detect the level of EGFR protein in the tumor tissue.

[0076] The results are as follows Figure 10 As shown in b, the body weight of mice in each group treated with MeV H-SW-αEGFR remained stable, and no significant weight loss or other obvious toxic side effects were observed, demonstrating that the fusion protein has good tolerability and safety in vivo. Compared with the αEGFR monoclonal antibody treatment group, the MeV H-SW-αEGFR experimental group showed a more significant tumor growth inhibition effect. Figure 10 c), a corresponding decrease in EGFR protein signaling was observed in tumor tissue. Figure 10 e), and significantly prolonged the survival of tumor-bearing mice ( Figure 10 d). In the context of chemotherapy, compared with the αEGFR monoclonal antibody + chemotherapy group, the MeV H-SW-αEGFR + chemotherapy combination provided by this invention achieved a significant tumor shrinkage effect and further prolonged survival benefits. These results indicate that by mediating the physical degradation of EGFR protein rather than simple functional neutralization, VirusTAC can significantly overcome the limitations of traditional targeted drugs, produce stronger anti-tumor activity, and has a significant synergistic effect with first-line clinical chemotherapy regimens.

[0077] Example 8: In vivo safety and tissue specificity assessment of VirusTAC This study aimed to evaluate the systemic toxicity and target specificity of MeV H-SW-VirusTAC and verify its safety at therapeutic doses. Experimental animals were treated with different doses of MeV H-SW-VirusTAC (0-50 mg / kg). Key organs (heart, liver, spleen, lungs, and kidneys) were collected from the animals and subjected to routine H&E staining to observe for any pathological damage. Finally, the expression level of EGFR protein in normal tissues after treatment was detected and compared with that of the IgG group.

[0078] The results are as follows Figure 11 a. Comprehensive H&E staining results of key organs (heart, liver, spleen, lungs, and kidneys) showed that no obvious pathological changes were observed in any organ within the dosage range of 0-50 mg / kg. Figure 11 As shown in b, the EGFR expression level in normal tissues remained stable after treatment with MeV H-SW-VirusTAC and was comparable to that of the IgG control group. This confirms the good safety profile of the VirusTAC of this invention.

[0079] Example 9: Evaluation of VirusTAC-mediated extracellular IL17A degradation efficacy after evolution This embodiment aims to verify that the VirusTAC platform can not only degrade cell membrane proteins but is also suitable for clearing extracellular soluble proteins. Based on the biological characteristic of significant upregulation of Nectin-4 receptor in psoriatic lesions, interleukin-17A (IL17A), a key driver of psoriasis and autoimmune inflammation, was selected as the target. Human keratinocytes (HaCaT) were used as the research model, and recombinant human IL17A protein was added for inflammatory stimulation. VirusTAC molecules (MeV H-SW-αIL17A VirusTAC, 100 nM) targeting IL17A were added to the experimental group. Figure 12 (b) An equivalent amount of αIL17A neutralizing antibody was added to the control group, and an equivalent amount of IgG antibody was added to the negative control group. After incubation for 48 h, the cell supernatant was collected, and the concentration of residual human IL17A protein in the supernatant was determined using a human IL17A ELISA quantitative detection kit.

[0080] The results are as follows Figure 12 As shown, in the HaCaT model, compared with the negative control group and the αIL17A neutralizing antibody treatment group, the IL-17A level in the supernatant of the MeV H-SW-αIL17A VirusTAC treatment group prepared in this invention was significantly reduced ( Figure 12 b). Transcriptome profiling and gene enrichment analysis revealed that the VirusTAC treatment group suppressed IL17A-driven inflammatory genes and blocked the IL17 signaling pathway and its downstream effects. Figure 12 (cd). The above results demonstrate that the VirusTAC platform can efficiently mediate the endocytosis and degradation of extracellular soluble IL-17A, achieving targeted clearance of extracellular pathogenic cytokines.

[0081] Example 10: Treatment of VirusTAC-mediated IL17A degradation in an in vivo model of psoriasis This embodiment aims to verify the therapeutic effect of the VirusTAC platform in degrading IL17A, a key pathogenic factor of psoriasis, in an animal model of psoriasis. An imiquimod (IMQ)-induced psoriasis-like mouse model was used. IMQ ointment was applied to the dorsal skin of mice for six consecutive days to induce a typical psoriasis-like inflammatory response. Mice were randomly divided into a control group, a monoclonal antibody treatment group (administered with αIL17A neutralizing antibody), and a MeV H-SW-αIL17A VirusTAC experimental group. The experimental group received daily local injections of the MeV H-SW-αIL17A fusion protein. During the treatment period, the clinical manifestations of the mouse dorsal skin were evaluated daily. After the treatment period, damaged skin tissue from the mouse dorsal side was collected for fixation, embedding, and H&E staining analysis. Simultaneously, tissue homogenates were prepared for IL17A molecular detection.

[0082] The results are as follows Figure 13 As shown in Figure ac, mice treated with MeV H-SW-αIL17A exhibited significant phenotypic improvements. Histological results showed ( Figure 13 In the MeVH-SW-αIL17A treatment group, epidermal thickness was significantly reduced, basal layer proliferation was inhibited, and inflammatory cell infiltration (such as neutrophils and lymphocytes) in the dermis was significantly alleviated. Molecular detection showed that the IL17A concentration at the lesion site was significantly reduced in the MeVH-SW-αIL17A treatment group. Figure 13 g). Throughout the study period, mice in each treatment group behaved normally, showed no significant fluctuations in body weight, and no pathological damage was observed in sections of vital organs, demonstrating good in vivo tolerability and safety.

[0083] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A fusion protein complex, characterized in that, This includes a heterodimer formed by a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain and the second polypeptide chain together constitute the structure R1-R2-R3, wherein: R1 is wild-type measles virus hemagglutinin protein MeV H or a biologically active variant thereof; R2 is a connector composed of R4 and R5; R4 is the Fc region of immunoglobulin. The Fc regions corresponding to the first polypeptide chain and the second polypeptide chain are associated through heterodimerization mutation. R5 is a protease-cleavable linker; R3 is the target protein binding domain, and the target protein is an EGFR antibody or an IL17A antibody.

2. The fusion protein complex according to claim 1, characterized in that, The R1 is a MeV H protein or a variant that has more than 90% sequence invariance to the key sequence of the MeV H protein.

3. The fusion protein complex according to claim 2, characterized in that, The two Fc subunits of R4 introduce LDLE and LKTK mutations into their CH3 regions, respectively; the LDLE mutations include L351D and / or L368E mutations; the LKTK mutations include L351K and / or T366K mutations.

4. The fusion protein complex according to claim 3, characterized in that, The R5 is selected from an amino acid sequence that can be specifically recognized and cleaved by proteases that are highly expressed in the tumor microenvironment or inflamed tissue.

5. The fusion protein complex according to claim 4, characterized in that, The molar ratio of R1 to R3 is 1:

1.

6. The fusion protein complex according to claim 5, characterized in that, The first polypeptide chain sequence is shown in any one of SEQ ID NO:1-SEQ ID NO:

3.

7. The fusion protein complex according to claim 6, characterized in that, The second polypeptide chain is shown in SEQ ID NO:4 or SEQ ID NO:

5.

8. A nucleic acid composition, characterized in that, It includes a nucleic acid sequence encoding the R1-R5 portion of any of the fusion protein complexes of claims 1-7, or a nucleic acid sequence encoding the first polypeptide chain and the second polypeptide chain of any of the fusion protein complexes of claims 1-7.

9. A pharmaceutical composition, characterized in that, It includes the fusion protein complex of any one of claims 1-7, the chimeric molecule in the fusion protein complex of any one of claims 1-7, or the nucleic acid composition of claim 8, and pharmaceutically acceptable excipients.

10. The use of the fusion protein complex of any one of claims 1-7, the nucleic acid composition of claim 8, or the pharmaceutical composition of claim 9 in the preparation of a medicament for treating diseases related to Nectin-4 high expression, characterized in that, The diseases associated with high Nectin-4 expression include oncological diseases and / or autoimmune diseases.

Images