Mini-binding proteins targeting Nectin-4, their drug conjugates, and their applications

Abstract

This invention relates to mini-binding proteins targeting Nectin-4, their drug conjugates, and their applications. The present invention has screened and obtained a class of mini-binding proteins specifically targeting Nectin-4, whose amino acid sequences include any one of SEQ ID Nos. 1-6. This application further conjugates the mini-protein conjugates with the microtubule inhibitor VcMMAE to generate mini-protein drug conjugates (MPDCs), which can target and kill Nectin-4 positive tumor cells.

Description

Technical Field

[0001] This application belongs to the field of protein drug technology, specifically relating to a mini-binding protein targeting Nectin-4, its drug conjugate, and its applications. Background Technology

[0002] Antibody-drug conjugates (ADCs) are a class of targeted anti-tumor therapeutic agents that covalently conjugate monoclonal antibodies to highly cytotoxic small molecules via chemical linkers. Thanks to the high specificity of antibody recognition, these precision therapies are gradually replacing traditional chemotherapy regimens in the treatment of some cancers. Nevertheless, developing more effective ADCs for solid tumors remains one of the key challenges in the current field of oncology drug development. To date, various ADCs targeting antigens such as HER2, Nectin-4, TROP2, TF, FOLR1, and c-MET have been approved by regulatory agencies and have shown promising therapeutic effects in clinical trials.

[0003] Nectin-4 is a cell surface adhesion molecule that participates in the formation and maintenance of cell-cell junctions primarily through homologous or heterologous interactions with other Nectin family members and E-cadherin. Nectin-4 expression levels are low in normal adult tissues; however, it is significantly overexpressed in various solid malignancies, thus establishing it as an important tumor-associated membrane antigen and an ideal target for ADC development. The representative drug Enfortumab vedotin, which conjugates an antibody targeting Nectin-4 with valine-citrulline-monomethylauristatin E (VcMMAE), has been approved by the U.S. Food and Drug Administration (FDA) for the treatment of locally advanced or metastatic urothelial carcinoma. Furthermore, more than seven ADCs targeting Nectin-4 are currently in clinical trials, and more than 11 candidate ADCs are in preclinical development.

[0004] In addition to traditional full-length antibodies, research on novel tumor-targeting molecular scaffolds for ADC applications is constantly evolving, resulting in various antigen recognition forms, including nanobodies derived from camels and sharks, as well as bicyclic peptides. These systems have incorporated various cytotoxic payloads, such as domperidone, Pseudomonas exotoxin, and MMAE. Compared to traditional antibodies, nanobodies offer advantages such as smaller molecular weight, higher structural stability, and potentially superior tissue penetration. However, the acquisition of nanobodies typically relies on animal immunization processes, presenting a high experimental threshold. Furthermore, their recombinant expression efficiency in E. coli systems is limited, and challenges remain regarding thermal stability.

[0005] In recent years, significant progress has been made in the de novo design of mini-binding proteins. Early methods primarily relied on physics-based computational frameworks (such as ROSETTA), but these methods typically depend on high-throughput experimental screening, resulting in complex computational processes and limited design success rates. Recently developed deep learning-based backbone generation methods (such as RFdiffusion and BindCraft) have largely overcome these limitations. When combined with sequence design methods based on message passing neural networks (MPNN) and AlphaFold2-mediated protein complex structure prediction, this integrated design process can efficiently generate structurally diverse mini-binding proteins and achieve rapid identification of low nanomolar affinity candidate molecules through low-throughput experimental screening. Furthermore, these mini proteins are typically efficiently expressed in *E. coli* and exhibit good thermal stability. Studies have reported various high-affinity mini-binding proteins targeting immune receptors including TNFR, IL-1R, IL-6R, and IL-17R, as well as tumor-related targets such as EGFR, PD-L1, HER2, TGFβRII, and CTLA-4. Nevertheless, designing high-affinity mini-binding proteins de novo for small molecule targets with complex structural features (such as tandem domains) and high hydrophilicity remains challenging, and their druggability and clinical translation potential require further systematic research. Summary of the Invention

[0006] Technical Purpose

[0007] One of the technical objectives of this invention is to develop a class of mini-binding proteins that target Nectin-4.

[0008] Another technical objective of this application is to provide drug conjugates of the aforementioned mini-binding proteins and small molecule drugs.

[0009] Another technical objective of this application is to provide a pharmaceutical composition comprising the aforementioned mini-binding protein or the aforementioned drug conjugate.

[0010] Another technical objective of this application is to provide the use of the aforementioned mini-binding protein, the aforementioned drug conjugate, or the aforementioned pharmaceutical composition in the preparation of a medicament for treating malignant tumors targeting Nectin-4.

[0011] Technical solution

[0012] The technical problem of this invention is to provide a new candidate drug for treating malignant tumors that target Nectin-4.

[0013] To address the aforementioned technical problems, the present invention provides a mini-binding protein targeting Nectin-4, the amino acid sequence of which includes any one of SEQ ID No. 1-6.

[0014] Among them, the amino acids at positions 90, 97, 104, 121, and 128 of SEQ ID No. 1 were mutated to cysteine, resulting in sequences SEQ ID No. 2-6, respectively.

[0015] The mini-binding protein also contains an initiating amino acid M at its N-terminus and an affinity purification tag linked to its C-terminus via a flexible linker peptide.

[0016] The flexible linker peptide is selected from GGS, GSG, GGG, and SGG.

[0017] The affinity purification tag is 6×His.

[0018] The amino acid sequence of the mini-binding protein is selected from one of SEQ ID No. 7-12.

[0019] On the other hand, the present invention provides a drug conjugate comprising the following components:

[0020] Mini-binding proteins,

[0021] Its amino acid sequence contains any one of SEQ ID No. 2-6 or is selected from one of SEQ ID No. 8-12;

[0022] peptide linkers; and

[0023] small molecule toxins,

[0024] Among them, small molecule toxins are coupled to the amino acid side chains of mini-binding proteins via peptide linkers.

[0025] The small molecule toxin is covalently coupled to the cysteine ​​side chain of the mini-binding protein via a peptide linker.

[0026] The small molecule toxin is coupled to the cysteine ​​side chain of the mini-binding protein via a Michael addition reaction between succinimide and thiol groups to form a thioether bond.

[0027] When the amino acid sequence of the mini-binding protein contains the amino acid sequence of SEQ ID No. 2, the small molecule toxin is coupled to cysteine ​​at position 90 of SEQ ID No. 2;

[0028] When the amino acid sequence of the mini-binding protein contains the amino acid sequence of SEQ ID No. 3, the small molecule toxin is coupled to cysteine ​​at position 97 of SEQ ID No. 3;

[0029] When the amino acid sequence of the mini-binding protein contains the amino acid sequence of SEQ ID No. 4, the small molecule toxin is conjugated to cysteine ​​at position 104 of SEQ ID No. 4;

[0030] When the amino acid sequence of the mini-binding protein contains the amino acid sequence of SEQ ID No. 5, the small molecule toxin is coupled to cysteine ​​at position 121 of SEQ ID No. 5;

[0031] When the amino acid sequence of the mini-binding protein contains the amino acid sequence of SEQ ID No. 6, the small molecule toxin is conjugated to cysteine ​​at position 128 of SEQ ID No. 6.

[0032] The peptide linker is a lysosomal cleavable peptide linker, such as Val-Cit, Val-Ala, Val-Lys, Phe-Lys, or Val-Arg.

[0033] The small molecule toxins are selected from: microtubule inhibitors (such as olprestatin MMAE / MMAF and maytansine DM1 / DM4, which are the mainstream in clinical practice) and DNA damage toxins (such as PBD dimer, cazithromycin, and camptothecin derivative SN-38 / DXd).

[0034] The small molecule toxin is MMAE.

[0035] In the drug conjugate, the peptide linker and the small molecule toxin moiety are as shown in the following formula:

[0036]

[0037] The asterisk (*) indicates that the above structure is coupled to the cysteine ​​side chain position of the mini-binding protein.

[0038] In another aspect, the present invention provides a pharmaceutical composition comprising the aforementioned mini-binding protein or the aforementioned drug conjugate, and pharmaceutically acceptable excipients.

[0039] The excipients are selected from one or more of the following: phosphates, histidine salts, citrates, cyclodextrins, polysorbate 80, ascorbic acid, and sodium chloride.

[0040] In another aspect, the present invention also provides the use of the aforementioned mini-binding protein, the aforementioned drug conjugate, or the aforementioned pharmaceutical composition in the preparation of a medicament for treating malignant tumors targeting Nectin-4.

[0041] The malignant tumors were selected from breast cancer, urothelial carcinoma, and lung cancer, which are associated with Nectin-4.

[0042] The malignant tumor is urothelial carcinoma.

[0043] The lung cancer mentioned is lung adenocarcinoma.

[0044] Beneficial effects

[0045] This application employs a deep learning-based computational design method to redesign the specific target-binding protein of the tumor-associated antigen Nectin-4. Furthermore, the binding affinity was optimized from 0.262 μM to 8.74 nM through partial diffusion. The designed Nectin-4 mini-binding protein exhibits good specificity and thermostability, maintaining its binding activity even after heating at 95°C.

[0046] Furthermore, this application successfully functionalizes the mini protein conjugate by combining it with the microtubule inhibitor VcMMAE, generating mini protein drug conjugates (MPDCs) that can target and kill Nectin-4 positive tumor cells.

[0047] In vitro experiments have demonstrated that these novel MPDCs exhibit potent cytotoxicity against tumor cells and patient-derived lung adenocarcinoma organs (LAOs).

[0048] Treatment experiments in a mouse xenograft model showed that MPDCs exhibited good biocompatibility, and tumor growth was completely inhibited at an injection dose of 5 mg / kg.

[0049] In summary, this application relates to a class of optimized mini-binding proteins targeting Nectin-4, whose binding affinity is significantly improved from the micromolar level to the low nanomolar range. Based on this, the screened high-performance mini-binding proteins are conjugated with the cytotoxic payload VcMMAE to construct mini-protein drug conjugates (MPDCs). Further systematic evaluations at the cellular level, in organoid models, and in mouse xenograft models validated the potential therapeutic value of these MPDCs as novel tumor-targeting delivery vectors. Therefore, the Nectin-4 mini-binding proteins and their drug conjugates developed in this application have great promise for developing drug candidates for treating diseases targeting Nectin-4. Attached Figure Description

[0050] Figure 1 This diagram illustrates the design, synthesis, and screening of the Nectin-4 mini-binding protein in this application and its application in in vivo and in vitro models.

[0051] Figure 2This diagram illustrates the computational design of Nectin-4 specific MPDCs. A: Schematic diagram of Nectin-4 specific MPDC binding and endocytosis in Nectin-4 positive cancer cells. Color scheme: Nectin-4, gray; mini-binding protein, red; VcMMAE, blue; cysteine ​​residues, yellow. B: Hotspot selection within domains 1 and 2 of Nectin-4. Site 1 (Site-1) is blue, site 2 (Site-2) is pink, and site 3 (Site-3) is orange. A magnified view shows detailed remnants of each hotspot. C: Computational structural snapshot filtering of de novo-designed mini-protein conjugates that bind to Nectin-4, targeting three distinct hotspots at Nectin-4, site 1 (blue), site 2 (pink), and site 3 (orange and red). The red binding protein represents the Binder-33 protein.

[0052] Figure 3 The predicted scores for the initial design results are shown. A: Predicted scores for Site-1 and Site-2 in the first round of design. B: Predicted scores for Site-1 and Site-3 in the second round of design.

[0053] Figure 4 The results of the initial BLI screening are shown. Mini protein conjugates with significant binding ability were fitted using a 1:1 local fitting model.

[0054] Figure 5 The expression of Nectin-4 on the surface of MDA-MB-468, NCI-H1781, and HUVEC cells is shown. MDA-MB-468 and NCI-H1781 cells are positive for Nectin-4, while HUVEC cells are negative for Nectin-4.

[0055] Figure 6 The diagram shows further validation using AlphaFold3 and BLI data between Binder-12-O13 and Nectin-4. A: The top 180 designs after descending order of pae_interaction were further predicted by AlphaFold3. B: The interaction between Binder-12-O13 and Nectin-4 was further validated by BLI.

[0056] Figure 7Preliminary screening of Nectin-4 mini-protein conjugates is shown. A: Preliminary screening results from the BLI experiment. The response values ​​of each mini-protein conjugate to Nectin-4 are shown, with the top seven binding proteins highlighted in red. B: MFI data of cell binding strength of the seven mini-binding proteins as detected by FACS. C: FACS data of Binder-33 protein binding to the surface of MDA-MB-468 and NCI-H1781 cells but not HUVEC cells. D: BLI characterization of the interaction between Binder-33 mini-binding protein and Nectin-4.

[0057] Figure 8 Affinity optimization and specificity validation of Binder-33 protein are shown. A: FACS MFI statistical plots show the binding ability of mini-protein conjugates optimized by partial diffusion to Nectin-4 positive cells. Black represents the Binder-12 optimization group, red the Binder-31 optimization group, and yellow the Binder-33 optimization group. B: Stacked structure of Binder-33 binding protein and Binder-33-O3 binding protein complex. Colors: Binder-33 (red), Binder-33-O3 (orange), and Nectin-4 (gray). In a magnified view of the binder-33-O3-Nectin-4 complex, the central binding residue T34 is highlighted. C: Gradient BLI assay of Binder-33-O3 protein with Nectin-4. D: Comparison of the binding ability of Binder-33 and Binder-33-O3 to Nectin-4 positive cells as analyzed by FACS. E: The T34R single-point mutation completely eliminates the cell-binding ability of Binder-33-O3 protein. F: CD experiments showed that the secondary structure of the Binder-33-O3 protein remained unchanged before and after heating (gray, 25 °C; red, 95 °C; orange dashed line, recovery from 95 °C to 25 °C). G: FACS analysis showed the cell binding capacity of the Binder-33-O3 protein to Nectin-4 positive cells after heating at 95 °C for 5 minutes; Recovery represents the protein recovering from 95 °C to 25 °C.

[0058] Figure 9The covalent binding of Nectin-4 specific mini-protein conjugates to VcMMAE is shown. A: Structural representation of alanine residues on the surface of Binder-33-O3 selected for cysteine ​​mutation. B: Comparison of binding strength between FACS data and statistical plots of MPDCs with different cysteine ​​mutation sites and Nectin-4 positive cells by flow cytometry. C: UV-vis analysis of Binder-33-O3-128C and Binder-33-O3-128C-VcMMAE (orange for Binder-33-O3-128C, red for Binder-33-O3-128C-VcMMAE). D: SDS-PAGE analysis of Binder-33-O3-128C-VcMMAE under CuSO4 oxidative and non-oxidative conditions. E: Mass spectrometry analysis of the complete molecular weight of Binder-33-O3-128C-VcMMAE and Binder-O3. F: Gradient BLI experiment of Binder-33-O3-VcMMAE and Nectin-4.

[0059] Figure 10 Cytotoxicity analysis of pluripotent dendritic cells targeting Nectin-4 is shown. A: Fluorescence images of internalization and lysosomal co-localization of Binder-33-O3-128C-VcMMAE in MDA-MB-468 cells. Scale bar: 100 μm. B: FACS detection of internalization of Binder-33-O3-128C-VcMMAE in MDA-MB-468 cells. C: Cytotoxicity of Binder-33-O3-128C-VcMMAE to different cell types as determined by CCK-8 assay. D: Apoptosis analysis of HUVEC, MDA-MB-468, and NCI-H1781 cells by FACS after treatment with a specified concentration of Binder-33-O3-128C-VcMMAE for 48 hours.

[0060] Figure 11This study illustrates how MPDC impairs the viability of lung adenocarcinoma organs (LAOs) and inhibits tumor growth in vivo. A: Representative bright-field images of the same LAOs treated with PBS (control) or 10 μg / ml Binder-33-O3-VcMMAE on days 0 and 3. Scale bar: 100 μm. B: Viability assessment of LAO 1 stained with calcein AM (live cells, green) and PI (dead cells, red). Scale bar: 100 μm. C: Relative LAO activity of the treated organoids after 72 hours by ATP-dependent luminescence assay. D: Schematic diagram of the in vivo xenograft experiment. E: Tumor volume growth curves during administration. F: Representative images of resected tumors in each group. G: Tumor weight in each group. H: Mouse tumor images and histological and immunostaining analysis of tumors: H&E (morphology), Ki-67 (proliferation). Scale bar: 50 μm. I: TUNEL immunofluorescence staining of tumors. Scale bar: 50 μm. J: Quantification of Ki-67 positive cells in tumor sections in H (5 random regions analyzed per group). K: Quantification of TUNEL positive cells in tumor sections in I (5 random regions analyzed per group). L: H&E staining of liver, lung, kidney, and spleen of mice in each group. Scale bar: 50 μm.

[0061] Figure 12 This study demonstrates how Binder-33-O3-128C-VcMMAE impairs the viability of other LAOs and inhibits tumor growth in vivo. A: Histological and immunostaining features of LAOs and LUAD tissues. Scale bar: 50 μm. B: Western blot analysis of Nectin-4 in HUVECs and LAOs. C: Representative bright-field images of the same LAOs treated with PBS (control) or 10 μg / ml Binder-33-O3-VcMMAE on days 0 and 3. Scale bar: 100 μm. D: Viability assessment of LAO 2 and LAO 3 stained with calcein AM (live cells, green) and PI (dead cells, red). Scale bar: 100 μm. EF: Relative LAO activity of organic-like substances treated 72 hours later by ATP-dependent luminescence assay. G: Body weight in each group. H: Representative images of liver and lung in each group. I: Liver weight to body weight ratio in each group. J: Lung weight to body weight ratio in each group. K: The ratio of kidney weight to body weight in each group. L: The ratio of spleen weight to body weight in each group. Detailed Implementation

[0062] The technical solutions of this application are described in detail below through specific embodiments to enable those skilled in the art to better understand this application; however, these embodiments are not intended to limit the scope of this application. The scope of protection of this application may include various equivalent or modified forms made by those skilled in the art using conventional means based on the specific content disclosed below.

[0063] the term:

[0064] In this application, "mini-binding protein" refers to a small-molecule, highly specific target-binding protein that is designed and screened de novo using computational protein design technology.

[0065] In this application, the mini-binding protein targeting Nectin-4 and the Nectin-4 mini-binding protein are used interchangeably.

[0066] In this application, "pae_interaction" refers to the Predicted Aligned Error for the interaction interface, which is a metric specifically used to evaluate the reliability of inter-chain interaction interfaces.

[0067] Example 1: De novo design of Nectin-4 targeting mini-binding protein based on RFdiffusion.

[0068] The extracellular domain of Nectin-4 exhibits a typical tandem domain configuration, consisting of three small immunoglobulin-like domains connected by a short and relatively rigid linker region. Figure 2 China A Figure 2 (B). Given that the crystal structures of its two N-terminal domains have been resolved, and that targeting this region is less affected by steric hindrance from the cell membrane, this invention selects this region as the primary target for the design of mini-binding proteins.

[0069] In the initial design phase, two groups of hydrophobic residues were first screened as potential binding hotspots. In the first round of calculations, two hotspot regions were selected: Site-1 (including A66, Y86, L81, and L132) and Site-2 (including T41, L113, L231, and P228). Site-1 is located in the β-sheet region of the first domain (D1), which participates in homology interactions between Nectin molecules; Site-2 is located on the dorsal side of this interaction interface, in the connection region between D1 and the second domain (D2) (see...). Figure 2 (B) Compared to site 1, site 2 has a larger potential binding interface.

[0070] For the two sites mentioned above, 4000 protein backbone structures were generated for each site. MPNN was then used for sequence design, and AlphaFold2 was used for complex structure prediction. The design results were screened using selection criteria (pae_interaction < 10 and plddt_binder > 90). The results showed that the design success rate for both types of sites was low, approximately 0.208%. Figure 3 (See A). Due to the limited surface area available at site 1, the resulting mini-binding protein molecules are relatively short, limiting the scope for subsequent affinity optimization. In contrast, successful design at site 2 can lead to larger mini-binding proteins capable of simultaneously binding to both the D1 and D2 domains of Nectin-4, thus creating a broader binding interface (see [link]). Figure 2 China B and Figure 2 (C)

[0071] In the second round of calculations, the hotspot region was further expanded based on site 2, incorporating D1 and D2 into the design scope, and defined as a new hotspot site 3 (Site-3) (see...). Figure 2 (B) and based on this, sampled larger mini protein backbone structures. A total of 7000 backbone structures were generated, and further sequence design and complex structure prediction were performed. This optimization strategy significantly improved the design success rate, increasing the success rate by approximately 13 times to 2.70% under the same screening criteria (pae_interaction<10 and plddt_binder>90). Figure 3 (B)

[0072] In addition, to obtain a more stable and well-behaved mini-binding protein, the number of its backbones generated was also expanded to 7000 while keeping site 1 unchanged, for supplementary calculations. Figure 3 (B) Based on the above calculation results, the top 50 mini-binding protein candidate molecules were finally selected for subsequent gene synthesis and experimental verification.

[0073] Example 2: Initial screening of Nectin-4 mini-binding protein binders

[0074] All gene synthesis in this invention was performed by Jiutian Gene Technology (Tianjin) Co., Ltd. All animals involved were purchased from Shandong Pengyue Experimental Animal Technology Co., Ltd. Fifty mini-binding protein genes were synthesized between NcoI and XhoI in the pET28a vector, optimized according to the *E. coli* codon. The 6×His tag on the vector was retained at the carboxyl terminus, and the genes were expressed and purified in *E. coli*. Specifically, the synthesized mini-binding protein genes were transformed into *E. coli* BL21(DE3) competent cells (Shanghai Weidi Biotechnology). After a series of ice bath, 42℃ heat shock, and ice bath operations, the cells were added to LB liquid medium and thawed at 37℃ for 1 h. The cells were then plated on kanamycin-resistant plates and cultured for 12-16 h. Single colonies were picked and inoculated into LB liquid medium containing kanamycin, and incubated at 37℃ until the OD600 reached 0.6-0.8. IPTG inducer (Sangon Biotech) was then added at a 1:3000 ratio and induced overnight at 24℃. Bacterial cells were collected after centrifugation at 1000g for 5 min at 4℃, resuspended in PBS, and sonicated on ice (Ningbo Xinzhi Biotechnology). The supernatant was collected after centrifugation at 12000g for 20 min. The supernatant was passed through a nickel column and impurities were removed using 20 mM, 30 mM, and 40 mM imidazole buffers. Finally, the target protein was collected using 300 mM imidazole buffer. Protein purity was assessed by SDS-PAGE electrophoresis, and the target bands were observed using Coomassie Brilliant Blue staining. After ultrafiltration to remove imidazole, the target protein concentration was measured at 280 nm using a TECAN microplate reader. 42 out of 50 proteins were normally expressed.

[0075] Subsequently, this invention employed bio-layer interferometry (BLI) to screen the in vitro binding capacity of the 42 successfully expressed proteins. Binding signals with Nectin-4 were detected at a fixed concentration of 1 μM. All BLI experiments were performed using a standard protocol including the following steps: equilibration, loading, second equilibration, binding, and dissociation. All equilibration steps were set to 60 seconds. Fc-tagged Nectin-4 protein (Novoprotein, CW95) was used as the loading protein, diluted to a final concentration of 5 μg / mL, and loaded onto the protein A sensor (ForteBio) for 600 seconds. The binding time was set to 330 seconds, and the dissociation time to 300 seconds. For preliminary screening, mini-binding proteins were diluted to 1 μM in gradient binding experiments. The buffer used for sensor activation, baseline stabilization, and protein dilution was PBST (PBS containing 0.05% Tween-20, pH 7.4). The loading flow rate was 600 rpm, and the other steps were performed at 1000 rpm. All experiments were conducted at 25℃. Data analysis was performed using a 1:1 fitting curve model. The results are shown in Table 1 below.

[0076] Table 1. Amino acid sequences and their corresponding BLI response values

[0077]

[0078] In the table, the beginning part (M) represents the starting amino acid M, and the ending part (ggsHHHHHH) represents linker + 6*His. The relative response value of BLI = original response value / original response value of Binder-33 protein.

[0079] The screening results showed that seven mini-binding proteins (Binder-12, Binder-22, Binder-23, Binder-25, Binder-31, Binder-33, and Binder-42) exhibited strong binding signals. Figure 7 China A and Figure 4 Furthermore, all seven positive candidate molecules were derived from the Site-3 hotspot region design.

[0080] Furthermore, flow cytometry was used to evaluate the cell surface binding ability of the above seven candidate molecules. The experiment used the Nectin-4 positive breast cancer cell line MDA-MB-468 (Wuhan Pusaino Life Science & Technology Co., Ltd.) for testing. Figure 5 The mean fluorescence intensity (MFI) was obtained based on fluorescence-activated cell sorting (FACS). The flow cytometry procedure was as follows: 1 × 10⁻⁶ cells were sorted into 10⁻⁶ cells. 5 The corresponding cells were seeded into 24-well plates. After overnight culture, mini-binding protein was added and incubated at 4 °C for 45 min. The control group was treated with an equal volume of PBS. After incubation, the cells were washed three times with PBS to remove unbound protein, and rabbit anti-6*His monoclonal antibody (Proteintech, CL647-66005 / CL488-66005) was added and incubated at 4 °C for 45 min. After staining, the cells were washed three times, resuspended in PBS, and further analyzed by flow cytometry. The results showed that Binder-33 exhibited the strongest binding ability, followed by Binder-12 and Binder-41 (…). Figure 7 (B)

[0081] To further verify the binding specificity, an extended validation experiment was conducted on Binder-33, using another Nectin-4 positive lung cancer cell line, NCI-H1781 (Wuhan Pusaino Life Science Co., Ltd.), and a Nectin-4 negative human umbilical vein endothelial cell line, HUVEC (Wuhan Pusaino Life Science Co., Ltd.), for detection. Figure 5 Flow cytometry analysis showed that Binder-33 effectively bound to two types of Nectin-4 positive tumor cells, while no significant binding signal was detected in HUVEC cells, indicating that it has good Nectin-4 positive cell specificity. Figure 7 (C). Finally, the binding kinetics parameters of Binder-33 were determined using BLI. The results showed that this molecule has a relatively fast binding rate with Nectin-4, but the dissociation rate is also relatively fast, and its equilibrium dissociation constant (K) is relatively high. d The value is 262.2 nM. Figure 7 China D and Figure 4 ).

[0082] Example 3: Optimization and Improvement of Nectin-4 Mini-Binding Protein Affinity Based on Partial Diffusion Strategy

[0083] Based on the mini-binding proteins with good cell surface Nectin-4 binding ability obtained in the previous screening, this invention further adopts a partial diffusion strategy to optimize the affinity of the three candidate molecules (Binder-12, Binder-31 and Binder-33) that performed best in cell experiments.

[0084] For each candidate molecule, 4800 protein backbone structures were generated. Sequence design was then performed using ProteinMPNN, and AlphaFold2 was used for complex structure prediction and scoring. A more stringent selection criterion (pae_interaction < 5.5 and pLDDT_binder > 90) was employed, and the results for each group were ranked according to pae_interaction. The top 60 candidate molecules were selected for further AlphaFold3 complex structure prediction and scoring. Subsequently, based on the ipTM score, approximately 20 of the highest-ranking mini-binding proteins from each group were selected for gene synthesis and experimental validation. Figure 6 (See Table A below and Table 2). The optimized candidate molecules were screened directly by cell binding assays.

[0085] Table 2. Amino acid sequences and their corresponding relative average fluorescence intensities on cell surfaces

[0086]

[0087] The beginning part (M) represents the starting amino acid M, and the ending part (ggsHHHHHH) indicates linker+6*His. The normalized denominator is calculated by subtracting the average fluorescence intensity of the control group without mini-binding proteins from the binding fluorescence intensity of the original mini-binding proteins (12, 31, and 33). The relative average fluorescence intensity of each optimized protein is calculated as (average fluorescence intensity - average fluorescence intensity of the control group) / normalized denominator of the average fluorescence intensity of the original protein from which it originated.

[0088] Among them, the sequences of binders with serial numbers 33-O3, 33-O3-90C, 33-O3-97C, 33-O3-104C, 33-O3-121C, and 33-O3-128C whose N-terminus does not contain M and whose C-terminus does not contain ggsHHHHHH are SEQ ID No: 1-6.

[0089] The results showed that Binder-12-O13, an optimized variant of Binder-12, and Binder-33-O3 and Binder-33-O21, two optimized variants of Binder-33, all exhibited significantly enhanced cell-binding capacity compared to their parent molecules. Figure 8 (A). However, in subsequent BLI experiments, Binder-12-O13 failed to show effective binding with Nectin-4 ( Figure 6 The presence of B (indicated by B) suggests that it may exhibit strong non-specific cell binding behavior. Therefore, this invention further focuses on in-depth research on Binder-33-O3.

[0090] Structural prediction results show that Binder-33-O3 and Binder-33 have highly consistent overall conformations when forming a complex with Nectin-4, with a root mean square deviation (rmsd) of 0.415 Å. Figure 8 The presence of B indicates that its binding epitope is well conserved. BLI kinetic experiments show that the binding affinity between Binder-33-O3 and Nectin-4 is significantly enhanced, with its equilibrium dissociation constant (Kd) reaching 8.74 nM (…). Figure 8 (C)

[0091] Flow cytometry analysis further confirmed that Binder-33-O3 had a significantly enhanced binding capacity on tumor cells compared to the parent molecule Binder-33, while no binding signal was detected in Nectin-4 negative HUVEC cells. Figure 8 The presence of D indicates that it still maintains good specificity.

[0092] To further verify the specificity of the binding, this invention introduces an arginine mutation at the key site T34 of the Binder-33-O3 / Nectin-4 complex interface (…). Figure 8 (B). This mutation completely eliminates its ability to bind to Nectin-4 positive MDA-MB-468 tumor cells. Figure 8 (E), further demonstrating the specificity of this binding interface.

[0093] Furthermore, circular dichroism (CD) analysis revealed that the spectral characteristics of Binder-33-O3 were fully recovered after heating and cooling at 95°C, indicating its excellent thermal stability. Figure 8 (F). Consistent with this, cell binding assays performed after incubation at 95°C for 10 minutes showed that Binder-33-O3 completely maintained its binding activity to tumor cells. Figure 8 (G).

[0094] Example 4: Covalent coupling of Nectin-4 mini-binding protein with VcMMAE and characterization of the coupling product

[0095] This invention further conjugates the optimized mini-binding protein Binder-33-O3 with the classic antibody-drug conjugate (ADC) linker-toxin system VcMMAE (MCE, HY-15575) to achieve targeted killing of Nectin-4 positive cells. Given that the C-terminal His tag may be removed in practical therapeutic applications, this invention avoids introducing cysteine ​​residues at the C-terminus during the design process. Instead, it selects multiple alanine sites far from the binding interface in the Binder-33-O3 structure for cysteine ​​mutation (…). Figure 9 (A). This design is based on the following two considerations: first, the above-mentioned site mutation has little effect on the interaction between Binder-33-O3 and Nectin-4; second, the substitution of alanine for cysteine ​​does not significantly increase the overall hydrophobicity of the protein, thus helping to maintain the protein's good physicochemical properties.

[0096] Specifically, cysteine ​​mutations were introduced at positions 90, 97, 104, 121, and 128, respectively, and corresponding VcMMAE conjugates (Binder-33-O3-90C-VcMMAE, Binder-33-O3-97C-VcMMAE, Binder-33-O3-104C-VcMMAE, Binder-33-O3-121C-VcMMAE, Binder-33-O3-128C-VcMMAE) were constructed.

[0097] The specific conjugation process is as follows: When using a nickel column to affinity purify the aforementioned mini-binding protein, after washing with a low-concentration imidazole gradient, Ni-NTA was resuspended in PBS containing an excess of VcMMAE to ensure thorough mixing. After resuspending, the nickel column was placed in a vortex mixer at room temperature in the dark for 2 hours. After labeling, unlabeled VcMMAE was washed out with 10 column volumes of PBS, and finally, the conjugate was eluted with PBS containing 300 mM imidazole.

[0098] To verify whether VcMMAE was successfully conjugated to the mini-protein, this invention conducted several characterization experiments. Ultraviolet absorption spectroscopy analysis showed that the labeled Binder-33-O3-128C-VcMMAE exhibited a characteristic absorption peak at approximately 250 nm, indicating that the drug molecule had been successfully conjugated. Figure 9 (C). SDS-PAGE analysis under copper sulfate oxidation conditions showed that VcMMAE coupling effectively inhibited cysteine-mediated mini-protein dimerization, while Binder-33-O3-128C achieved near-complete labeling. Figure 9 (D). Mass spectrometry analysis showed that the molecular weight increased from 15,202 Da to 16,518 Da after coupling, which was completely consistent with the theoretical prediction. Figure 9 (E).

[0099] Example 5: Cell binding assay of Nectin-4 mini-binding protein-drug conjugate and evaluation of Nectin-4 binding affinity

[0100] Cell binding experiments were conducted to compare the Nectin-4 mini-protein drug conjugates (MPDCs) constructed at different coupling sites in Example 4. The results showed that all constructs maintained similar tumor cell binding abilities. Figure 9 Among them, Binder-33-O3-128C-VcMMAE showed slightly better binding performance (B).

[0101] Furthermore, the Nectin-4 binding affinity of Binder-33-O3-128C was determined using BLI experiments. The results showed that Binder-33-O3-128C-VcMMAE maintained a high Nectin-4 binding affinity, with an equilibrium dissociation constant (Kd) of 9.61 nM, which is essentially consistent with that of uncoupled Binder-33-O3. Figure 9 (Middle F).

[0102] Example 6: Cytotoxic effects of Nectin-4 mini-binding protein-drug conjugate (Binder-33-O3-128C-VcMMAE) at the cellular level

[0103] Before evaluating cytotoxicity, this invention first detected the cellular uptake behavior of Binder-33-O3-128C-VcMMAE using an endocytosis assay. Binder-33-O3-128C-VcMMAE was covalently labeled with AF555 fluorescent dye via an amino-reactive reaction for fluorescence imaging analysis. Confocal microscopy results showed that, 6 hours after endocytosis, the conjugate exhibited significant co-localization with lysosomes within the cell. Figure 10 A) indicates that it can enter the cell via endocytosis and be transported to the lysosome. Furthermore, flow cytometry analysis showed that the intensity of the cell surface fluorescence signal gradually decreased after 1 hour and 2 hours of incubation. Figure 10 (B) This result is consistent with receptor-mediated endocytosis and its subsequent intracellular transport, further verifying that the conjugate has good endocytic properties.

[0104] Based on this, the cytotoxicity of Binder-33-O3-128C-VcMMAE was evaluated. CCK-8 (Beyotime, C0042) assay results showed that this conjugate had significant dose-dependent killing effects on Nectin-4 positive tumor cell lines NCI-H1781 and MDA-MB-468. After treatment with 5 μg / mL for 48 hours, the average survival rate of both tumor cell lines decreased by more than 70%; however, no significant toxic effect was observed on Nectin-4 negative HUVEC cells. Figure 10 (C). Further flow cytometry analysis revealed that apoptosis significantly increased with increasing Binder-33-O3-128C-VcMMAE concentration. After 48 hours of treatment, the proportion of 7-AAD and Annexin V (Beyotime, C1062M / C1737) double-positive cells exceeded 75%, exhibiting a clear dose-dependent apoptotic effect; in contrast, the positive proportion of HUVEC cells did not change significantly at any of the tested concentrations. Figure 10 (D).

[0105] These data indicate that Binder-33-O3-128C-VcMMAE is an effective Nectin-4 positive cell-specific cytotoxic agent at the in vitro cellular level.

[0106] Example 7: Antitumor effects of MPDC in lung adenocarcinoma organoids and mouse xenograft models

[0107] In this study, all animal experiments were conducted strictly in accordance with the experimental protocol established by the Shandong Provincial Laboratory Monitoring Committee and have received ethical approval from the Experimental Animal Ethics Committee of Shandong Second Medical University (Approval No.: 2025SDL798). BALB / c nude mice were used in the experiments, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and housed in a specific pathogen-free (SPF) environment with a 12-hour light / 12-hour dark diurnal cycle, fed standard experimental feed. Environmental parameters were controlled as follows: temperature 20–24℃, relative humidity 50%–60%. Fresh surgical specimens from patients with lung adenocarcinoma were obtained from Weifang People's Hospital. Research involving human specimens has received ethical approval from the Medical Research Ethics Committee of Weifang People's Hospital (Approval No.: KYLL20251105-1). All studies were conducted in strict accordance with applicable local laws and regulations and institutional management systems. All subjects signed written informed consent forms before inclusion in the study.

[0108] This embodiment evaluates the therapeutic effect of Binder-33-O3-128C-VcMMAE in a patient-derived lung adenocarcinoma organoid (LAO) model. The control group consisted of individuals who received an equal amount of PBS supplemented with the same amount of Binder-33-O3-128C-VcMMAE during the experiment.

[0109] Table 3. Clinical characteristics of patients used to establish organoids

[0110]

[0111] First, three organoid models were successfully established from surgically removed tumor tissue from patients with lung adenocarcinoma. The cancer progression characteristics of these patients are shown in Table 3 above. Specimens were immediately placed in cold primary tissue storage solution (Biogenous Biotechnology, K601005). Within 2 hours after resection, the tissue was minced into 1–2 mm fragments and enzymatically digested at 37 °C for 15–40 minutes using tumor tissue digestion solution (Biogenous Biotechnology, K601003). Fetal bovine serum (FBS) was added to the digestion mixture at a final concentration of 1–5% to reduce enzyme activity. The digested suspension was filtered through a 100 μm cell filter and centrifuged at 300 g for 3 minutes to precipitate cells. After precipitation, the cell pellet was treated with erythrocyte lysis solution (Biogenous Biotechnology, E239010) to lyse erythrocytes, followed by centrifugation at 300 g for 3 minutes. After centrifugation, the cells were resuspended and washed twice with cancer organoid basal medium (Biogenous Biotechnology, B213152). After centrifugation, the supernatant was aspirated, and the cell pellet was retained and pre-cooled on ice. Then, in a preheated 24-well plate, an appropriate volume of organic cell culture medium (ECM) (Biogenous Biotechnology, M315010) was added at a ratio of approximately 20-30 μL per well (about 6000 cells). The plates were incubated at 37°C with 5% CO2 for 15 minutes to allow ECM polymerization. After solidification, complete culture medium was gently added along the well walls, and the plates were cultured under standard conditions (37°C, 5% CO2). Hematoxylin-eosin (H&E) staining and immunohistochemical analysis of specific markers confirmed that the organoids effectively preserved the histological structure and biological characteristics of the primary tumor. Figure 12 (A). Western blot results showed that all three organ types showed positive expression of Nectin-4 (A). Figure 12 (B). In the efficacy evaluation, after treatment with 10 μg / mL Binder-33-O3-128C-VcMMAE, the organoids showed obvious morphological characteristics of cell death under a light microscope, while the control group showed no significant changes. Figure 11 China A and Figure 12 (C). Further fluorescence staining with Calcein AM live cell dye and propidium iodide (PI) dead cell dye (Beyotime, C2015S) showed that the organoid activity of the treatment group was significantly reduced and the proportion of apoptotic cells was significantly increased, while the control group showed no significant changes. Figure 11 China B and Figure 12(D). ATP-dependent luminescence assay results further confirmed that this conjugate has significant dose-dependent cytotoxic effects on organoids. Specifically, LAO 1 cell viability decreased to below 25% under treatment at 10 μg / mL, while the control group showed no dose-dependent decrease. Figure 11 C and Figure 12 Chinese E, Figure 12 (Middle F).

[0112] Furthermore, this invention uses a mouse xenograft tumor model to evaluate the in vivo antitumor efficacy and safety of the drug. 1×10⁻⁶ mmol / L of the drug was suspended in a 1:1 mixture of 100 µL RPMI 1640 medium and Cultrex basement membrane extract (R&D Systems, 3632-010-02). 7 A tumor model was established by subcutaneous injection of NCI-H1781 cells into 5-week-old male BALB / c nude mice. After the tumor volume reached the preset standard, the experimental animals were randomly divided into four groups: a PBS control group, a 2 mg / kg Binder-33-O3-128C-VcMMAE group, a 5 mg / kg Binder-33-O3-128C-VcMMAE group, and a 30 mg / kg carboplatin treatment group. Except for the carboplatin treatment group, which received intravenous injection every four days, the other groups received intravenous injection every two days. Figure 11 (D). Weight monitoring results showed no significant differences between groups, indicating that Binder-33-O3-128C-VcMMAE has little impact on the overall body condition and has good safety. Figure 12 (G). Tumor growth curve analysis showed that both the 2 mg / kg and 5 mg / kg dose groups exhibited superior tumor-suppressing effects compared to the carboplatin group, with the 5 mg / kg group showing a significant tumor shrinkage trend starting from day 6 of administration. Figure 11 (Middle E). The tumor volume and weight measurements at the endpoint further confirmed that, compared with the PBS control group and the carboplatin treatment group, the tumor volume and weight in the Binder-33-O3-128C-VcMMAE treatment group were significantly reduced ( Figure 11 China F and Figure 11 (G). In tumor morphological observation, the tumor tissue in the control group was full and rich in blood vessels; the tumor surface in the 2 mg / kg treatment group showed a white and irregular shape, indicating focal necrosis; the tumor in the 5 mg / kg treatment group was significantly flattened and shrunken, accompanied by large-area necrosis; while the carboplatin treatment group only showed a slight reduction in volume and still had some blood supply (G). Figure 11 H&E staining results further confirmed that the tumor cell density in the treatment groups decreased and necrosis areas of varying degrees appeared, with the carboplatin treatment group showing relatively mild necrosis. Figure 11(H). Using the formula V = 0.5×a×b 2 Calculate the tumor volume, where "a" represents the tumor length and "b" represents the tumor width.

[0113] Ki-67 and TUNEL (In Situ Cell Death Detection Kit, POD; Roche, 11684795910) immunofluorescence staining analysis showed that Binder-33-O3-128C-VcMMAE could inhibit tumor cell proliferation and induce apoptosis in a dose-dependent manner, with the high-dose group showing the most significant effect and overall efficacy superior to the carboplatin treatment group. Figure 11 (H-K). Regarding safety evaluation, histopathological examination of major organs showed no significant pathological damage in the liver, lungs, kidneys, and spleen. Figure 11 L and Figure 12 (H). Organ coefficient analysis also showed no significant differences in liver, lung, and kidney indices among the groups (H). Figure 12 In addition, some degree of splenomegaly was observed after treatment with Binder-33-O3-128C-VcMMAE, and this change was dose-dependent. Figure 12 (L), this phenomenon is consistent with previous reports on toxicological studies related to VcMMAE.

[0114] In summary, the Binder-33-O3-128C-VcMMAE constructed in this invention exhibits significant antitumor activity in both organoids and animal models, while also demonstrating good safety profiles, showing promising potential for clinical application.

Claims

1. A minicript targeting Nectin-4, characterized in that, Its amino acid sequence is selected from any of SEQ ID No. 1-6.

2. A mini-binding protein targeting Nectin-4, characterized in that, The amino acid sequence of the mini-binding protein is selected from any of SEQ ID No. 7-12.

3. A drug conjugate, characterized in that, The drug conjugate comprises the following components: Mini-binding proteins, the amino acid sequences of which are selected from any one of SEQ ID No. 2-6 and SEQ ID No. 8-12; peptide linkers; and small molecule toxins, In the drug conjugate, the peptide linker and the small molecule toxin moiety are as shown in the following formula: ， Where * indicates that the above structure is coupled to the cysteine ​​side chain position of the mini-binding protein, and in, When the amino acid sequence of the mini-binding protein is the same as that of SEQ ID No. 2, the small molecule toxin is coupled to cysteine ​​at position 90 of SEQ ID No. 2; When the amino acid sequence of the mini-binding protein is the same as that of SEQ ID No. 3, the small molecule toxin is coupled to cysteine ​​at position 97 of SEQ ID No. 3; When the amino acid sequence of the mini-binding protein is the same as that of SEQ ID No. 4, the small molecule toxin is conjugated to cysteine ​​at position 104 of SEQ ID No. 4; When the amino acid sequence of the mini-binding protein is the same as that of SEQ ID No. 5, the small molecule toxin is conjugated to cysteine ​​at position 121 of SEQ ID No. 5; When the amino acid sequence of the mini-binding protein is the same as that of SEQ ID No. 6, the small molecule toxin is conjugated to cysteine ​​at position 128 of SEQ ID No. 6; When the amino acid sequence of the mini-binding protein is the same as that of SEQ ID No. 8, the small molecule toxin is coupled to cysteine ​​at position 91 of SEQ ID No. 8; When the amino acid sequence of the mini-binding protein is the same as that of SEQ ID No. 9, the small molecule toxin is coupled to cysteine ​​at position 98 of SEQ ID No. 9; When the amino acid sequence of the mini-binding protein is the same as that of SEQ ID No. 10, the small molecule toxin is coupled to cysteine ​​at position 105 of SEQ ID No. 10; When the amino acid sequence of the mini-binding protein is the same as that of SEQ ID No. 11, the small molecule toxin is coupled to cysteine ​​at position 122 of SEQ ID No. 11; When the amino acid sequence of the mini-binding protein is the same as that of SEQ ID No. 12, the small molecule toxin is conjugated to cysteine ​​at position 129 of SEQ ID No.

12.

4. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the mini-binding protein as described in claim 1 or 2 or the drug conjugate as described in claim 3, and pharmaceutically acceptable excipients.

5. Use of the mini-binding protein of claim 1 or 2, the drug conjugate of claim 3, or the pharmaceutical composition of claim 4 in the preparation of a medicament for treating breast cancer or lung cancer targeting Nectin-4.

6. The use according to claim 5, wherein, The lung cancer mentioned is lung adenocarcinoma.

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