VirusTAC platform for tumor cell membrane protein pd-l1 or cd24 targeted degradation and applications thereof

Abstract

The application discloses a VirusTAC platform for tumor cell membrane protein PD-L1 or CD24 targeted degradation 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 IgG Fc region; R5 is a linker that can be cut by a protease; and R3 is a target protein binding domain PD-L1 antibody or CD24 antibody. The platform can specifically recognize tumor high-expression receptors and induce rapid endocytosis of cells by means of tumor-specific expression and efficient endocytosis characteristics of Nectin-4, so as to overcome the defects of the existing TPD technology and realize effective regulation of various target proteins.

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 proteins PD-L1 or CD24 and its applications. Background Technology

[0002] Cell membrane proteins play a crucial role in the development and progression of tumors and various chronic diseases. Traditional antagonists (such as monoclonal antibodies and small molecule inhibitors) often block function by occupying binding sites, but their effectiveness is limited for "undruggable" targets that lack binding pockets or are prone to developing resistance. In recent years, targeted protein degradation (TPD) technologies such as LYTAC (lysosome-targeted chimeras) have provided a new strategy for membrane protein degradation, utilizing receptor-mediated endocytosis to transport membrane or extracellular proteins to lysosomes for degradation. However, existing LYTAC technologies face several bottlenecks: insufficient tumor specificity, limited endocytic efficiency, and immunogenicity. Therefore, developing a novel degradation platform with high tumor specificity, efficient endocytosis, and good safety is an urgent clinical challenge. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention utilizes the characteristics of oncolytic virus cell membrane proteins to provide a tumor membrane protein targeted degradation platform (VirusTAC) mediated by oncolytic virus envelope proteins and chimeric with target protein (POI) antibodies, and its application in the preparation of antitumor drugs. The oncolytic virus envelope proteins can specifically recognize tumor-highly expressed receptors and induce rapid endocytosis, thereby overcoming the deficiencies of existing TPD technologies and achieving effective regulation of multiple target proteins. Furthermore, Nectin-4, one of the important receptors for Measles virus Hemagglutinin (MeV H), is expressed at low levels in normal adult tissues but is abnormally highly expressed in various malignant tumors such as urothelial carcinoma, lung cancer, and breast cancer, exhibiting extremely high endocytic activity.

[0004] 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 the wild-type measles virus envelope protein MeV H or a biologically active variant thereof; R2 is a connector composed of R4 and R5; R4 is the IgG Fc region; R5 is a protease-cleavable linker; R3 is the target protein binding domain, and the target protein is a PD-L1 antibody or a CD24 antibody.

[0005] 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.

[0006] Furthermore, LDLE and LKTK mutations are introduced into the IgG Fc CH3 region of R4.

[0007] Furthermore, the LDLE mutation includes the L351D mutation and / or the L368E mutation; the LKTK mutation includes the L351K mutation and / or the T366K mutation.

[0008] Furthermore, R3 is a targeting molecule in an antibody or nanobody.

[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 the application of the fusion protein complex or the nucleic acid composition in the preparation of antitumor drugs.

[0014] Compared with the prior art, the present invention has the following beneficial effects: (1) Utilizing the natural mechanism of oncolytic virus entry into cells to achieve high specificity and endocytosis This invention utilizes the highly efficient natural cellular invasion mechanism of oncolytic viruses, mediating rapid endocytosis of POIs via a multi-receptor-mediated pathway (primarily Nectin-4), followed by degradation via lysosomes. Simultaneously, this invention leverages the biological distribution characteristics of Nectin-4, which is specifically highly expressed in various malignant tumor tissues (such as breast cancer, lung cancer, and bladder cancer) but significantly decreased in adult healthy tissues. The VirusTAC platform can precisely identify and bind to tumor cells, thereby avoiding non-specific degradation of similar target proteins (such as PD-L1) in normal tissues, significantly reducing immune damage and off-target side effects.

[0015] (2) Broad therapeutic applicability and target expansion capability The VirusTAC designed in this invention is modular, allowing for the design and replacement of effector arms based on the specific characteristics of the point of interest (POI), and can be extended to the treatment of various malignant tumors and other diseases.

[0016] (3) Receptor recycling mechanism improves degradation efficiency This invention introduces a protease-cleavable linker into the fusion protein. When the VirusTAC complex enters the lysosome, this linker can be recognized and cleaved by specific lysosomal proteases, thereby releasing the intracellular receptor. This design allows the receptor to cycle back to the cell membrane surface, maintaining its physiological expression level, thus achieving sustained degradation of the target protein and improving molecular degradation efficiency.

[0017] (4) Synergistic therapeutic effect and safety In mouse models of lung and breast cancer, VirusTAC monotherapy has been shown to significantly prolong survival and has a synergistic therapeutic effect when used in combination with first-line chemotherapy regimens. Furthermore, the animal models maintained stable body weight and showed no significant tissue toxicity during administration, demonstrating the high safety of this invention in clinical applications. Attached Figure Description

[0018] Figure 1 This 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 is a PD-L1 antibody or a CD24 antibody; (3) an intracellular enzyme cleavage linker and a "knob-in-hole" Fc dimer.

[0019] Figure 2 This describes the binding ability of VirusTAC to various tumor cells in Example 3 of this invention. The binding ability of VirusTAC to various tumor cells and normal cells was detected by flow cytometry. The cell types involved included: human tumor cells (MDA-MB-231, MCF-7, A549, UM-UC-3, T24), human normal cells (AC16, THLE-2, 16HBE14o, HUVEC), and mouse normal cells (mouse fibroblasts and MLE-12). 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.

[0020] Figure 3 This demonstrates VirusTAC-mediated PD-L1 degradation in Example 3 of the present invention. (a) Human tumor cells were treated with VirusTACs targeting PD-L1 or isotype controls. PD-L1 expression was detected by flow cytometry (n=3 per group), and statistical analysis was performed using one-way ANOVA combined with Tukey's test. (b) Quantitative analysis of PD-L1 expression levels in human tumor cells and normal cells after treatment with MeV H-αPD-L1 VirusTAC. 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.

[0021] Figure 4 This illustrates PD-L1-EGFP in Embodiment 3 of the present invention. + Cell line validation of VirusTAC-mediated PD-L1 degradation. Cell lines expressing PD-L1-EGFP were used. + MDA-MB-231 and A549 cells were treated with specified VirusTACs or isotype controls. PD-L1 expression in each group (n=3 per group) was detected by flow cytometry, 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.

[0022] Figure 5 This invention demonstrates how VirusTAC mediates PD-L1 degradation in multiple tumor cell lines, as shown in Example 3. Representative Western blot images show the expression levels of PD-L1 in MDA-MB-231, A549, and UM-UC-3 cells after treatment with αPD-L1-VirusTAC. Protein levels were normalized using Actin as an internal control.

[0023] Figure 6 This demonstrates VirusTAC-mediated CD24 degradation in Example 4 of the present invention. (a) MCF-7, A549, and UM-UC-3 cells were treated with VirusTACs targeting CD24 or isotype controls. The expression of CD24 was detected by flow cytometry (n=3 per group), and statistical analysis was performed using one-way ANOVA combined with Tukey's test. p < 0.0001, p < 0.001, p < 0.01, p < 0.05, ns indicates no significant difference. (b) Representative Western blot images show the expression level of CD24 in MCF-7, A549, and UM-UC-3 cells after αCD24-VirusTAC treatment. Protein levels were normalized using Actin as an internal control.

[0024] Figure 7 This is the first round of directed protein evolution based on wild-type MeV H in Example 5 of this invention. (ab) Flow cytometry analysis of cell binding activity and protein expression levels. (a) Flow cytometry analysis of cell binding activity of MDA-MB-231, A549, and UM-UC-3 cells under different VirusTAC evolutionary variants and (b) PD-L1 expression levels. In the first round of screening, A176S was selected as the optimal mutation site (named MeV HS) (ab). Statistical analysis was performed using one-way ANOVA combined with Tukey's test (n=3 per group). p < 0.0001, p < 0.001, p < 0.01, p < 0.05, ns indicates no significant difference.

[0025] Figure 8 This is the second round of protein-directed evolution based on MeV HS in Example 5 of this invention. (ab) The second round of evolution was designed using the optimal mutation site A176S from the first round as the backbone, and cell binding activity and protein expression levels were analyzed by flow cytometry. (a) Cell binding activity of MDA-MB-231, A549, and UM-UC-3 cells under different VirusTAC evolutionary bodies in the second round was analyzed by flow cytometry, as well as (b) PD-L1 expression levels. In the second round, A176S / K294W was selected as the optimal mutation site (named MeV H-SW) (ab). Statistical analysis was performed using one-way ANOVA combined with Tukey's test (n=3 per group). p < 0.0001, p < 0.001, p < 0.01, p < 0.05, ns indicates no significant difference.

[0026] Figure 9 This is a comparison of the kinetics and efficacy of MeV H-αPD-L1 and MeV H-SW-αPD-L1 in PD-L1 degradation in Example 5 of this invention. (ab) Representative Western blot images show the expression levels of PD-L1 in MDA-MB-231, A549, and UM-UC-3 cells treated with MeV H-αPD-L1 or MeV H-SW-αPD-L1 at specified time points (a) and specified concentrations (b). Protein levels were normalized using Actin as an internal control.

[0027] Figure 10This describes the multi-receptor-dependent virus TAC entry mechanism in Example 6 of this invention. (ab) Flow cytometry analysis showed the expression levels of MeV H-SW-αPD-L1 (a) and MeV H-SW-αCD24-treated CD24 (b) in MDA-MB-231, 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.

[0028] Figure 11 This demonstrates the advantages of the VirusTAC multi-receptor-mediated strategy in Example 7 of this invention. (a) Schematic diagram of the structures of MeV H-SW-αPOI and αNectin4-αPOI. (bc) Flow cytometry analysis showed the expression levels of PD-L1 (b) or CD24 (c) after VirusTAC treatment with αPOI, αNectin-4-αPOI, and MeV H-SW-αPOI in MDA-MB-231, MCF-7, A549, and UM-UC-3 cell lines. Data are expressed as mean ± standard error (mean ± SEM). Statistical differences were analyzed by one-way ANOVA and Tukey's test (n=3 independent biological replicates per group). Significance was defined as follows: p < 0.0001, p < 0.001, p < 0.01, p < 0.05, ns indicates no significant difference.

[0029] Figure 12This illustrates VirusTAC-mediated POI entry into lysosomes in Example 7 of this invention. (ae) Representative fluorescence images show PD-L1 expression and its co-localization with RAB5A (a), EEA1 (b), RAB7A (c), RAB11 (d), and LAMP1 (e) in MDA-MB-231 cells treated with MeV H-SW-αPD-L1 or αPD-L1. White arrows indicate co-localization of PD-L1 with endosomes or lysosomes. Scale bar: 5 μm. (f) Flow cytometry analysis shows the percentage of POI-internalized cells in MDA-MB-231, A549, and UM-UC-3 cells treated with LysoLight™ Deep Red-labeled VirusTACs or control IgG. Data are expressed as mean ± standard error (mean ± SEM). Statistical differences were analyzed using one-way ANOVA and Tukey's test (n=3 independent biological replicates per group). Significance was defined as follows: p < 0.0001, p < 0.001, p < 0.01, p < 0.05, ns indicates no significant difference.

[0030] Figure 13 This is a verification of the compatibility of the VirusTAC platform with various targeting binding modules in Embodiment 8 of the present invention. (a) Schematic diagram of different binding module formats targeting PD-L1: scFv, Fab, VHH, and de novo binding modules. (b) MDA-MB-231 and A549 cells were treated with specified VirusTACs or isotype controls. Flow cytometry was used to detect the expression level of PD-L1 in each group of cells. (c) MDA-MB-231 and A549 cells expressing PD-L1-EGFP were treated with specified VirusTACs or isotype controls. Flow cytometry was used to detect the expression level of EGFP in each group of cells. Data in (ac) were analyzed using one-way ANOVA and Tukey's test, and the results are expressed as mean ± standard error (mean ± SEM). All statistical comparisons were performed relative to the isotype control group (n=3 per group). Significance was defined as follows: p < 0.0001, p < 0.001, p < 0.01, p < 0.05, ns indicates no significant difference.

[0031] Figure 14 This is Example 9 of the present invention, illustrating the treatment of PD-L1 degradation mediated by VirusTAC in a humanized urothelial carcinoma model. (a) Schematic diagram of VirusTAC treatment in a urothelial carcinoma 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=12 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 images to show PD-L1 (red) and DAPI (blue). Scale bar: 50 μm.

[0032] Figure 15 This is an example of VirusTAC-mediated PD-L1 degradation treatment in a humanized breast cancer model, as described in Example 10 of this invention. (a) Schematic diagram of VirusTAC treatment in a breast 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 images to show PD-L1 (red) and DAPI (blue). Scale bar: 50 μm.

[0033] Figure 16 This is an example of VirusTAC-mediated PD-L1 degradation treatment in a humanized lung cancer model, as described in Example 11 of this invention. (a) Schematic diagram of VirusTAC treatment 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 images to show PD-L1 (red) and DAPI (blue). Scale bar: 50 μm.

[0034] Figure 17This describes the safety and specificity of VirusTAC in Example 12 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-αPD-L1 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-αPD-L1. The black boxed areas in the H&E images are magnified in the IF spectrum to show PD-L1 (red) and DAPI (blue). Scale bar: 50 μm. Detailed Implementation

[0035] 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.

[0036] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0037] Example 1: Protein cloning design and gene expression vector construction 1. Cloning design and expression of wild-type MeV H-hIgG1 (1) Wild-type MeV H-hIgG1 design concept: Based on the specific recognition and binding of MeV H to tumor cells, wild-type MeV H-hIgG1 Fc LKTK (SEQ ID NO.1) was designed, wherein: the key sequence of wild-type MeV H was linked to the hinge of the Fc region of the heavy chain of human IgG1 through a protease-cleavable linker, wherein an LKTK mutation was introduced into the CH3 region to improve the efficiency of specific heterodimerization with another heavy chain. After the linker enters the cell, it will be recognized and cleaved by lysosomal proteases, thereby releasing the entry receptor (such as the MeV H receptor Nectin-4), which can be recycled back to the cell membrane to maintain its expression without affecting its physiological function, and can also continuously mediate protein degradation. The specific structural design is as follows. Figure 1 .

[0038] (2) Construction of wild-type MeV H-IgG1 gene expression vector Construction of wild-type MeV H-hIgG1 Fc LKTK plasmid: The MeV H-hIgG1 Fc LKTK fusion sequence was designed. The designed and synthesized primers were spliced ​​together by enzymatic filling or other methods to obtain the linear double-stranded target sequence. The gene was ligated into the target vector by homologous recombination or enzyme digestion. The recombinant product was transformed into competent DH5α cells, plated on antibiotic plates, and positive clones were screened by colony PCR and agarose gel extraction before plasmid extraction and sequencing.

[0039] 2. Cloning design and expression of MeV H-hIgG1 evolutionary variants (1) Design concept of MeV H evolution: Optimize the MeV H sequence to improve the ability of MeV H-hIgG1 Fc LKTK to degrade target proteins.

[0040] (2) MeV-H evolutionary process: To obtain MeV-H arm proteins with superior performance, this invention employs the artificial intelligence platform EVOLVEpro (EVOlution Via Language model-guided Variance Exploration for proteins) for computer-aided design and optimization of protein sequences. This platform combines protein language models with evolutionary algorithms, enabling the screening of functionally enhanced candidate mutants from a large sequence space without relying on protein structural information.

[0041] First, a protein language model based on the Transformer architecture was used to encode wild-type protein sequences and their mutants, extracting high-dimensional sequence representations to capture contextual dependencies and evolutionary conservation among amino acids. Then, a deep neural network scoring function was constructed to predict the functional scores of the sequences. Supervised learning was used for fine-tuning on some experimental data to improve prediction accuracy. Based on the scoring function, a genetic algorithm was used for iterative search, generating a candidate sequence library by introducing 1-3 site mutations. In each iteration, high-fitness sequences were selected based on the predicted scores as the parent sequences for the next round of mutation and recombination operations. Finally, the top 10-20 mutant sequences with the highest scores were selected for downstream experimental validation, including synthesis, expression, and functional testing.

[0042] The first round of directed protein evolution: Ten point mutations were generated from wild-type MeV H: L108A, L111V, R12P, K145P, A176S, K305F, H312W, N330Y, L331D, and Q392G.

[0043] The second round of evolution: Based on the A176S mutant (MeV HS, SEQ ID NO.2) which showed better degradation efficiency in the first round, 10 evolutionary variants were further screened through superimposed mutations. The mutation sites included: Y227W, Y261W, P275W, R292W, K294W, K305L, A313W, A319W, A354W, and R397S. The optimal mutant A176S / K294W (MeV H-SW, SEQ ID NO.3) was selected.

[0044] (3) Construction of plasmid MEV H (evolution)-hIgG1 Fc LKTK: Design the MEV H-hIgG1 Fc LKTK fusion sequence, splice the designed and synthesized primers together by enzymatic filling or other methods to obtain the linear double-stranded target sequence, ligate the gene to the target vector by homologous recombination or enzyme digestion, transform the recombinant product into competent cells DH5α, plate it on antibiotic plates, screen positive clones by colony PCR and agarose gel method, and then extract the plasmid for sequencing.

[0045] 3. Design and expression of the target membrane protein targeting domain The target domains of PD-L1 and CD24 were selected for the construction of the VirusTAC platform.

[0046] (1) Cloning design Based on the optimized design of the target domain sequence, it is linked to hIgG1 Fc via a flexible linker. An LDLE mutation is introduced into the CH3 structure of hIgG1 Fc, allowing for heterodimerization with the LKTK structure. The sequences of the αPD-L1-hIgG1 Fc LDLE fusion protein are detailed in SEQ ID NO.4, and the sequences of the αCD24-hIgG1 Fc LDLE fusion protein are detailed in SEQ ID NO.5.

[0047] (2) Construction of gene expression vectors a. Construction of plasmid αPD-L1-hIgG1 Fc LDLE: The fusion protein gene sequence SEQ ID NO.4 was designed. The designed and synthesized primers were spliced ​​together by enzymatic filling or other methods to obtain the linear double-stranded target sequence. The gene was ligated into the target vector by homologous recombination or enzyme digestion. The recombinant product was transformed into competent DH5α cells, plated on antibiotic plates, and positive clones were screened by colony PCR and agarose gel extraction before plasmid extraction and sequencing.

[0048] b. Construction of plasmid αCD24-hIgG1 Fc LDLE: The fusion protein gene sequence SEQ ID NO.5 was designed. The designed and synthesized primers were spliced ​​together by enzymatic filling or other methods to obtain the linear double-stranded target sequence. The gene was ligated into the target vector by homologous recombination or enzyme digestion. The recombinant product was transformed into competent DH5α cells, plated on antibiotic plates, and positive clones were screened by colony PCR and agarose gel extraction before plasmid extraction and sequencing.

[0049] The sequence is as follows: SEQ ID NO.1 (MeV H-hIgG1 Fc LKTK sequence) SKGNCSGPTTIRGQFSNMSLSLLDLYLGRGYNVSSIVTMTSQGMYGGTYLVEKPNLSSKRSELSQLSMYRVFEVGVIRNPGLGAPVFHMTNYLEQPVSNDLSNCMVALGELKLAALCHGEDSITIPYQGSGKGVSFQLVKLGVWKSPTDMQSWVPLSTDDPVIDRLY LSSHRGVIADNQAKWAVPTTRTDDKLRMETCFQQACKGKIQALCENPEWAPLKDNRIPSYGVLSVDLSLTVELKIKIASGFGPLITHGSGMDLYKSNHNNVYWLTIPPMKNLALGVINTLEWIPRFKVSPYRFTVPIKEAGGDCHAPTYLPAEVDGDVKLSSNLVIL PGQDLQYVLATYDTSRVEHAVVYYVYSPSRSFSYFYPFRLPIKGVPIELQVECFTWDQKLWCRHFCVLADSESGGHITHSGMVGMGVSCTGGFLGGVRGVDGEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTKPPSRDELTKNQVSLKCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 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(αPD-L1-hIgG1 Fc LDLE) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSAGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRGGFLGGVRGVDGEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTDPPSRDELTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID NO.5(αCD24-hIgG1 Fc LDLE) DVQLQESGPGLVKPSETLSLTCTVSGYSITSGYSWHWIRQPPGKGLEWIGYIHYSGSTKYNPSLKSRVTISSVDTSKNQFSLKLSSVTAADTAVYYCARGADYALDYWGQRTSVTVSSGGGGS GGGGSGGGGSDIVMTQSPDSLAVSLGERATINCKSSQSLLYSSNQKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLISSLQAEDVAVYYCQQNFIYPLTFGGGTKVELK GGFLGGVRGVDGEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTDPPSRDELTKNQVSLTCEVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Example 2: Construction of the VirusTAC platform for targeted degradation of PD-L1 1. Preparation of VirusTAC fusion protein by overexpression plasmid transfection: ExpiCHO cells were seeded in 10cm dishes (10mL of culture medium) and cultured at 37℃ in a 5% CO2 incubator until cell confluence reached 80%-90%. Transfection reagents were prepared as follows: Solution A: 500μL opti-MEM + 10μL Lipofectamine 3000; Solution B: 500μL opti-MEM + 20μL P3000 + 10ug plasmid (as shown in Table 1). Solution A was added dropwise to Solution B, mixed thoroughly by pipetting, and incubated at room temperature for 10 min. The mixture was then added to 10cm dishes. After incubation for 6-8 hours or overnight, the culture medium in the 10cm dishes was aspirated and replaced with SMM CHO-T3 serum-free culture medium. The supernatant was collected 36-48 h after transfection and purified using the Protein A method, followed by concentration determination.

[0050] Table 1. Details of plasmids related to VirusTAC construction

[0051] 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.

[0052] (2) For bispecific VirusTAC molecules assembled using the "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.

[0053] (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.

[0054] Example 3: VirusTAC's ability to bind to tumors and degrade PD-L1 1. VirusTAC-specific binding to tumor cells: Tumor cell lines including A549 (lung cancer), MDA-MB-231 (breast cancer), MCF7 (breast cancer), and UM-UC-3 (bladder cancer), as well as normal human cells (AC16, THLE-2, 16HBE14o, HUVEC) and normal mouse cells (mouse fibroblasts and MLE-12) were selected for the study. When cell confluence reached 50%-60%, wild-type MeV H-IgG at a concentration of 100 nM was added to the cell culture medium. The same equivalent of IgG fusion protein was added to the control group. After incubation for 4 hours, cells were digested with trypsin, washed once with pre-cooled PBS, and incubated at 4°C for 1 hour with anti-human IgG flow cytometry antibody (Invitrogen, A-21433, 1:200). The binding fluorescence intensity on the cell surface was detected using flow cytometry. Results are as follows: Figure 2As shown, a significant fluorescence shift was observed in the flow cytometry results of the tumor cell line, while no obvious binding signal was observed in the normal cell group, confirming that the MeV H effector arm can effectively recognize and specifically bind to tumor cells.

[0055] 2. Degradation experiment: (1) Tumor cell lines MDA-MB-231, MCF-7, and A549 were selected for the study. Wild-type MeV H-αPD-L1 VirusTAC at a concentration of 100 nM was directly added to the cell culture medium; the same equivalent of αPD-L1 fusion protein was added to the control group. After incubation with the 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 PD-L1 flow cytometry antibody (Biolegend, 329706, 1:50). The fluorescence intensity of the flow cytometry antibody on the cell surface was detected by flow cytometry. The results are as follows: Figure 3 As shown in ab, the intensity of PD-L1 on the cell surface was significantly reduced in the MeV H-αPD-L1 treatment group, and the degradation efficiency of PD-L1 on tumor cells by MeV H-αPD-L1 was also better than that of VirusTACs based on HSV1 (Herpes Simplex Virus Type 1) and SFV (Semliki Forest Virus) envelope proteins, indicating that VirusTACs can efficiently induce PD-L1 degradation through the effector arm-mediated endocytosis pathway.

[0056] (2) Validation was performed using a stable PD-L1-EGFP expression system. MeV H-αPD-L1 protein (100 nM) was added to the target cell lines (MDA-MB-231 and A549) overexpressing PD-L1-EGFP, while αPD-L1 was added to the control group. EGFP fluorescence intensity was directly detected after 24 h of incubation. The results are as follows: Figure 4 The experimental group cells showed a significant decrease in PD-L1 signal, proving that VirusTACs induced an overall decrease in the membrane surface level of PD-L1.

[0057] (3) Further protein blotting was used to verify the degradation efficacy of VirusTAC on PD-L1. The results are as follows: Figure 5 As shown, after 48 h of treatment with MeV H-αPD-L1, the total PD-L1 level in cells was downregulated by approximately 90%, and the degradation efficiency of PD-L1 was also superior to that of VirusTACs based on HSV1 and SFV envelope proteins. This further indicates that VirusTACs significantly induced a decrease in the total protein abundance of POIs and can effectively guide internalized POIs to lysosomes for degradation.

[0058] Example 4 VirusTAC-mediated CD24 degradation Tumor cell lines MCF7, A549, and UM-UC-3 were selected for this study. Evolved MeV H-αCD24 protein (100 nM) was added to the cells, while the control group received an equivalent amount of αCD24. After incubation for 24 hours, flow cytometry analysis was performed using anti-human CD24 antibody. Results are as follows: Figure 6 As shown in figure a, the fluorescence level of CD24 decreased significantly after treatment with MeV H-αCD24. We further used protein imprinting to quantitatively verify the degradation efficacy of VirusTAC on CD24. The results are as follows. Figure 6 As shown in b, after 48 h of treatment with MeV H-αCD24, the total CD24 level in cells was downregulated by approximately 90%, and the CD24 degradation efficiency was also superior to that of VirusTACs based on HSV1 and SFV envelope proteins. These results confirm that the VirusTAC platform can efficiently mediate the endocytosis and degradation of CD24.

[0059] Example 5: Evaluation of VirusTAC evolutionary model-mediated endogenous PD-L1 degradation efficiency 1. First round of evolutionary evaluation: MDA-MB-231, A549, and UM-UC-3 cell lines were selected, and VirusTACs (100 nM) based on wild-type MeV H and its first-round evolutionary variants (L108A, L111V, R12P, K145P, A176S, K305F, H312W, N330Y, L331D, Q392G, etc.) were added to the cell culture medium. The control group was supplemented with IgG ( Figure 7 a) or αPD-L1 ( Figure 7 b). After 24 hours of incubation, the cells were digested with trypsin, washed once with pre-cooled PBS, and anti-human IgG flow cytometry antibody was added. Figure 7 a) or anti-human PD-L1 flow cytometry antibody ( Figure 7 b) After incubation at 4°C for 1 hour, the fluorescence intensity of the flow cytometry antibody on the cell surface was detected using flow cytometry. Results are as follows: Figure 7 The results showed that VirusTAC of mutant A176S (MeV HS) exhibited a more significant ability to bind to tumor cells and degrade target proteins.

[0060] 2. Second round of evolutionary evaluation: Based on A176S, two rounds of mutation sites (Y227W, Y261W, P275W, R292W, K294W, K305L, A313W, A319W, A354W, R397S, etc., a total of 10 sites) were added. VirusTAC based on MeV HS and its evolutionary variants, and an equal amount of IgG were added to the above cell lines. Figure 8 a) or αPD-L1 ( Figure 8 b) After incubating with cells for 24 hours, the cells were digested with trypsin, washed once with pre-cooled PBS, and anti-human IgG flow cytometry antibody was added. Figure 8 a) or anti-human PD-L1 flow cytometry antibody ( Figure 8 b) After incubation at 4°C for 1 hour, the fluorescence intensity of the flow cytometry antibody on the cell surface was detected using flow cytometry. Results are as follows: Figure 8 As shown, the PD-L1 fluorescence intensity of the A176S / K294W (MeV H-SW) mutant VirusTAC group showed a further significant decrease. Evolutionary sequences with stronger induced endocytosis capabilities were obtained through superimposed evolutionary screening.

[0061] 3. To compare the degradation kinetics of PD-L1 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) in MDA-MB-231, A549, and UM-UC-3 cell lines. The results are as follows: Figure 9 As shown in ab, MeV H-SW-VirusTAC outperforms wild-type MeV H-VirusTAC in both PD-L1 degradation efficiency and kinetics. Through a second round of superposition evolutionary screening, this invention successfully obtained a MeV H evolutionary sequence with stronger β-induced endocytosis ability.

[0062] Example 6: Multi-receptor-dependent VirusTAC endocytosis mechanism This embodiment aims to investigate and verify the mechanism by which VirusTAC mediates endocytosis using multiple receptors. Using CRISPR-Cas9 technology, known receptors of measles virus (MeV): Nectin-4, CD46, and SLAMF1 were knocked out in MDA-MB-231, A549, and UM-UC-3 cell lines, respectively. A non-targeted control group (sgNC) was established to eliminate interference. MeV H-SW-αPD-L1 VirusTAC and MeV H-SW-αCD24 VirusTAC 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 degradation efficiency for PD-L1 and CD24. Flow cytometry results showed that in the sgNECTIN4 cell line, VirusTAC-mediated PD-L1 (… Figure 10 a) and CD24 Figure 10b) The degradation effects were all significantly reversed; knocking out receptors SLAMF1 or CD46 had a relatively small effect on the degradation efficiency of PD-L1 / CD24, indicating that VirusTAC follows a multi-receptor synergistic mode to internalize and degrade POI, mainly driven by Nectin-4, with the synergistic participation of co-receptors such as SLAMF1 and CD46.

[0063] Example 7: Validation of the advantages of multi-receptor-mediated strategies and intracellular transport pathway of VirusTAC This embodiment aims to verify, through comparative experiments, the advantages of VirusTAC in degradation efficacy compared to traditional antibodies by utilizing a virus-simulated multi-receptor binding mechanism, and to use high-resolution imaging technology to track the transport trajectory of POIs after they enter cells.

[0064] First, a bispecific antibody against Nectin-4 (αNectin-4-αPOI) was constructed as a control for MeV H-SWVirusTAC. Figure 11 a) MDA-MB-231, MCF-7, A549, and UM-UC-3 cell lines were treated with αPOI, αNectin-4-αPOI, and MeV H-SW-αPOI VirusTAC, respectively. The degradation efficacy against PD-L1 and CD24 was evaluated by flow cytometry. Results are as follows: Figure 11 As shown in bc, in various cell lines, VirusTAC-mediated PD-L1 ( Figure 11 b) and CD24 Figure 11 c) The degradation efficiency was significantly higher than that of the αPOI and αNectin-4-αPOI groups, demonstrating the advantage of mimicking the multi-receptor entry mechanism of viruses in inducing efficient protein degradation.

[0065] To illustrate the transport trajectory of VirusTAC after entering cells via POI binding, we used specific antibody staining to label PD-L1 and various endosome and lysosomal markers: early endosomes (Rab5, EEA1), late endosomes (Rab7), circulating endosomes (Rab11), and lysosomes (Lamp1). We then used confocal microscopy to monitor the spatial distribution changes of PD-L1 after MeV H-SW-αPD-L1 treatment. The results are as follows: Figure 12 As shown in Figure 2, MeV H-SW-αPD-L1 treatment induced the loss of PD-L1 fluorescence signal on the cell membrane surface and promoted its translocation into the cell. Colocalization analysis revealed that post-endocytotic PD-L1 follows a strict temporal transport pathway: first entering the early endosomes (EEA1, Rab5)... Figure 12 ab), then transported to late endosomes (Rab7) ( Figure 12 c), eventually converging in lysosomes (Lamp1) ( Figure 12e); and the correlation between PD-L1 signal and the circulatory endoscopy marker Rab11 is extremely low ( Figure 12 d). This indicates that VirusTAC can effectively induce POIs to enter lysosomes rather than the recycling pathway, achieving more thorough clearance.

[0066] Finally, we labeled VirusTAC with LysoLight fluorescent probes and co-incubated it with target cells (MDA-MB-231, A549, and UM-UC-3 cell lines) to further verify whether VirusTAC-mediated POIs could be precisely delivered to lysosomes for degradation. Flow cytometry results showed ( Figure 12 f), significant fluorescence enhancement was observed in the MeV H-SW-VirusTAC treatment group, indicating that VirusTAC can efficiently transport POI into lysosomes with proteolytic activity.

[0067] Example 8: Compatibility Verification of the VirusTAC Platform with Multiple Targeting Modules To evaluate the compatibility of VirusTACs with different binding modules targeting the same POI, we constructed five MeV H-VirusTACs with different PD-L1 binding modules, covering everything from traditional antibody fragments (Fab, scFv, VHH) to de novo-designed binding modules. Figure 13 a) Five types of VirusTAC were added to MDA-MB-231 and A549 cell lines. After incubation for 4 hours, the cells were digested with trypsin, washed once with pre-cooled PBS, and then incubated with anti-human PD-L1 flow cytometry antibody at 4°C for 1 hour. The fluorescence intensity of the flow cytometry antibody on the cell surface was detected by flow cytometry. The results are as follows: Figure 13 As shown in b, all five binding modules of VirusTAC exhibited stable PD-L1 degradation activity compared to the control group.

[0068] Further validation was performed using a PD-L1-EGFP overexpressing cell line. In PD-L1-EGFP... + MDA-MB-231, PD-L1-EGFP + Five types of VirusTAC were added to the A549 cell line and incubated together for 24 h. The EGFP fluorescence intensity was then measured. Results are as follows: Figure 13 As shown in c, all binding modules exhibited strong degradation efficacy. This embodiment demonstrates the modular nature of the VirusTAC architecture, enabling it to serve as a general strategy compatible with a wide range of targeted binding modules, showcasing the engineering potential of the VirusTAC platform in various disease scenarios.

[0069] Example 9: Exploring the potential of VirusTAC in tumor therapy by degrading PD-L1 using a humanized bladder cancer model. This embodiment aims to verify the efficacy of the VirusTAC platform in targeted therapy of solid bladder tumors by degrading PD-L1 and its synergistic effect with standard clinical treatment regimens. A human urothelial carcinoma xenograft PDX model was constructed. After the tumor volume in mice grew to a predetermined range, the mice were randomly divided into 5 groups and administered VirusTAC intravenously at a dose of 10 mg / kg every three days. Figure 14 a). Grouped as follows: Control group: given physiological saline.

[0070] Monoclonal antibody treatment group: αPD-L1 monoclonal antibody was administered.

[0071] VirusTAC experimental group: MeV H-SW-αPD-L1 VirusTAC was administered.

[0072] Combination therapy group A: αPD-L1 monoclonal antibody combined with chemotherapy (gemcitabine + carboplatin).

[0073] Group B of combined treatment: MeV H-SW-αPD-L1 VirusTAC combined with chemotherapy (gemcitabine + carboplatin).

[0074] Mouse body weight was monitored periodically during drug administration. After drug administration, tumor tissue was dissected and paraffin sections were prepared. Immunofluorescence (IF) staining was used to detect the level of PD-L1 protein in the tumor tissue.

[0075] The results are as follows Figure 14 As shown in c, the VirusTAC experimental group exhibited a more significant tumor growth inhibition effect than the monoclonal antibody group, and it also had a synergistic effect with chemotherapy, as confirmed by survival curve analysis. Figure 14 d) The survival time of mice was significantly prolonged, and a corresponding decrease in PD-L1 protein signaling was observed in tumor tissue. Figure 14 e). In all treatment groups, the body weight of tumor-bearing mice remained stable. Figure 14 (b) No significant weight loss or other acute toxic reactions were observed, confirming the good safety and tolerability of the VirusTAC platform in vivo.

[0076] Example 10: Exploring the application potential of VirusTAC in tumor therapy by degrading PD-L1 using a humanized breast cancer model. This embodiment aims to verify the efficacy of the VirusTAC platform in targeted therapy of solid breast tumors by degrading PD-L1 and its synergistic effect with clinical standard treatment regimens. An NSG mouse breast cancer PDX model was constructed. Twenty-five female NSG mice transplanted with patient-derived samples were introduced into PBMCs for immunization reconstitution and randomly divided into 5 groups. VirusTAC was administered intravenously at a dose of 10 mg / kg every three days. Figure 15 a). Grouped as follows: Control group: given physiological saline.

[0077] Monoclonal antibody treatment group: αPD-L1 monoclonal antibody was administered.

[0078] VirusTAC experimental group: MeV H-SW-αPD-L1 VirusTAC was administered.

[0079] Combination therapy group A: αPD-L1 monoclonal antibody combined with chemotherapy (albumin-bound paclitaxel + carboplatin).

[0080] Group B of combined treatment: MeV H-SW-αPD-L1 VirusTAC combined with chemotherapy (albumin-bound paclitaxel + carboplatin).

[0081] Mouse body weight was monitored periodically during administration. Results were as follows: Figure 15 As shown in Figures bd, the VirusTAC experimental group exhibited a more significant tumor growth inhibition effect than the monoclonal antibody group, and showed a synergistic effect with chemotherapy, significantly prolonging the survival time of mice. In all treatment groups, the body weight of tumor-bearing mice remained stable, and no significant weight loss or other obvious acute toxic reactions were observed, confirming the good safety and tolerability of the VirusTAC platform in vivo.

[0082] After drug administration, tumor tissue was excised and paraffin sections were prepared. Results are as follows: Figure 15 As shown in e, significant downregulation and clearance of PD-L1 protein signaling were observed within breast cancer tumor tissue.

[0083] Example 11: Exploring the potential of VirusTAC in tumor therapy by degrading PD-L1 using a humanized lung cancer model. This embodiment aims to verify the efficacy of the VirusTAC platform in targeted therapy of solid breast tumors by degrading PD-L1 and its synergistic effect with standard clinical treatment regimens. An NSG mouse lung cancer PDX model was constructed. Twenty-five NSG mice transplanted with patient-derived samples were introduced into PBMCs for immunization reconstitution and randomly divided into 5 groups. VirusTAC was administered intravenously at a dose of 10 mg / kg every three days. Figure 16 a). Grouped as follows: Control group: given physiological saline.

[0084] Monoclonal antibody treatment group: αPD-L1 monoclonal antibody was administered.

[0085] VirusTAC experimental group: MeV H-SW-αPD-L1 VirusTAC was administered.

[0086] Combination therapy group A: αPD-L1 monoclonal antibody combined with chemotherapy (pemetrexed + carboplatin).

[0087] Group B of combined treatment: MeV H-SW-αPD-L1 VirusTAC combined with chemotherapy (pemetrexed + carboplatin).

[0088] Mouse body weight was monitored periodically during administration. Results were as follows: Figure 16 As shown in Figures bd, the VirusTAC experimental group exhibited a more significant tumor growth inhibition effect than the monoclonal antibody group, and showed a synergistic effect with chemotherapy, significantly prolonging the survival time of mice. In all treatment groups, the body weight of tumor-bearing mice remained stable, and no significant weight loss or other obvious acute toxic reactions were observed, confirming the good safety and tolerability of the VirusTAC platform in vivo.

[0089] After drug administration, tumor tissue was excised for immunofluorescence staining analysis. Results are as follows: Figure 16 As shown in e, significant downregulation and clearance of PD-L1 protein signaling were observed within lung cancer tumor tissue.

[0090] The above results confirm that the VirusTAC platform constructed in this invention can specifically degrade PD-L1 in various tumors in vitro and in mice, thereby significantly inhibiting tumor growth.

[0091] Example 12: 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 PD-L1 protein in normal tissues after treatment was detected and compared with the IgG group.

[0092] The results are as follows Figure 17 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 17As shown in b, the PD-L1 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.

[0093] 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 the wild-type measles virus envelope protein MeV H or a biologically active variant thereof; R2 is a connector composed of R4 and R5; R4 is the IgG Fc region; R5 is a protease-cleavable linker; R3 is the target protein binding domain, and the target protein is a PD-L1 antibody or a CD24 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 IgG Fc CH3 region in R4 is introduced with LDLE and LKTK mutations.

4. The fusion protein complex according to claim 3, characterized in that, The LDLE mutations include L351D mutations and / or L368E mutations; the LKTK mutations include L351K mutations and / or T366K mutations.

5. The fusion protein complex according to claim 4, characterized in that, R3 is a target molecule in antibodies or nanobodies.

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

1.

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

3.

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

5.

9. 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 1-8, or a nucleic acid sequence encoding the first polypeptide chain and the second polypeptide chain of any of the fusion protein complexes of claims 1-8.

10. The use of the fusion protein complex according to any one of claims 1-8 or the nucleic acid composition according to claim 9 in the preparation of an antitumor drug.

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