Mini-binding proteins targeting TROP2 and their drug conjugates and their applications

Abstract

This invention relates to mini-binding proteins targeting TROP2, their drug conjugates, and their applications. The mini-binding proteins comprise amino acid sequences selected from any of SEQ ID Nos. 7-12. The invention further provides conjugates of the aforementioned mini-binding proteins with small molecule toxin drugs. Experiments have demonstrated that the conjugates of the mini-binding proteins and small molecule toxin drugs of this application can target and kill TROP2-positive tumor cells, showing promise as drugs for the treatment of TROP2-positive tumors.

Description

Technical Field

[0001] This application belongs to the field of protein technology, specifically relating to a mini-binding protein targeting TROP2, its drug conjugates, and their applications. Background Technology

[0002] In recent years, antibody-drug conjugates (ADCs) have become an important targeted anti-tumor therapy strategy and have been widely used in the field of cancer treatment. A typical ADC consists of three parts: a targeting antibody, a cytotoxic drug, and a chemical linker. The antibody specifically recognizes tumor cell surface antigens, and after endocytosis into the cell, it releases the cytotoxic payload in the lysosomal environment, thereby achieving precise killing of tumor cells. Some ADCs also exhibit a bystander effect. With the development of related technologies, ADC drugs are gradually replacing some traditional chemotherapy drugs and playing an increasingly important role in cancer treatment. Currently, ADC drugs targeting multiple tumor-related targets have been approved for marketing or have entered clinical trials, showing promising application prospects.

[0003] Among numerous tumor-related targets, TROP2 (Trophoblast cell surface antigen 2) is a transmembrane glycoprotein widely distributed on the surface of epithelial-derived tumor cells. Belonging to the cell adhesion molecule family, it also possesses signal transduction functions. TROP2 maintains epithelial barrier integrity by participating in cell-cell adhesion processes and promotes cell proliferation, migration, and survival by regulating intracellular calcium signaling pathways, the MAPK / ERK signaling pathway, and cell cycle-related pathways. During embryonic development, TROP2 participates in key biological processes such as trophoblast cell invasion, while its expression range is limited and its expression level is low in normal adult tissues. In contrast, TROP2 shows significantly upregulated expression in various solid tumors (including breast cancer, lung cancer, and urothelial carcinoma) and is closely related to tumor invasiveness, metastatic ability, and poor prognosis. Furthermore, as a cell surface antigen, TROP2 has good accessibility and can undergo endocytosis after ligand binding, which is beneficial for the construction of targeted drug delivery systems. Based on the aforementioned biological characteristics, TROP2 possesses high expression differentialness, good targetability, and endocytosis capability, making it an ideal target for constructing targeted drug delivery systems. Currently, ADC drugs targeting TROP2 have been approved for the treatment of various tumors, and the indications continue to expand. In addition to traditional full-length antibodies, novel targeting molecules such as nanobodies are also being used in related research. They have advantages such as small molecular weight and strong tissue penetration, but generally rely on animal immune screening, resulting in long development cycles and limitations in expression levels and stability. Meanwhile, artificial intelligence-based de novo protein design technology is rapidly developing, especially deep learning-based diffusion model methods combined with sequence design and structure prediction tools, making the design of high-affinity mini protein-binding molecules possible. These molecules typically possess advantages such as structural stability and ease of efficient expression in microorganisms.

[0004] With the rapid development of AI-based diffusion-de-noise target binder generation technology, it is possible to accurately and directly generate binding proteins targeting any structural epitope. This technological advancement has, to some extent, compensated for the inherent limitations of immune screening in identifying antibodies targeting key conserved sites. Furthermore, high-affinity mini-binding proteins can be obtained through low-throughput experiments. In addition, mini-binding proteins can be produced using *E. coli* under very simple experimental conditions, and some exhibit extremely high expression levels and thermal stability. Compared to the higher experimental barriers and more expensive unit prices of antibody production, mini-binding proteins have vast research and pharmaceutical application value. Due to their high structure-directedness, although high-affinity mini-binding proteins for various ADC drugs targeting HER2, Nectin-4, TF, FOLR1, and c-MET have been reported, designing high-affinity mini-proteins for structurally complex targets such as TROP2 remains challenging. Moreover, existing research largely focuses on obtaining the binding molecules, with relatively insufficient research on further constructing them into drug systems with actual therapeutic effects. On the other hand, how to effectively conjugate mini-protein binding molecules with cytotoxic drugs to construct a novel drug delivery system that combines high targeting and good efficacy remains a key technical problem that urgently needs to be solved in this field. Therefore, it is necessary to develop an AI-designed target TROP2 mini-binding protein and further construct its drug conjugation form to achieve efficient and precise treatment of tumor cells. Summary of the Invention

[0005] The technical problem addressed in this application is to develop a mini-binding protein that targets TROP2 and its conjugates with small molecule drugs, with the aim of achieving efficient and precise treatment of TROP2-positive tumors.

[0006] To address the aforementioned technical problems, the present invention provides a mini-binding protein targeting TROP2, comprising an amino acid sequence selected from any of SEQ ID No. 7-12.

[0007] 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.

[0008] The flexible linker peptide is selected from GGS, GSG, GGG, and SGG.

[0009] The affinity purification tag is 6×His.

[0010] The amino acid sequence of the mini-binding protein is selected from one of SEQ ID No. 1-6.

[0011] On the other hand, the present invention provides a drug conjugate comprising the following components: Mini-binding proteins whose amino acid sequences contain any one of SEQ ID No. 9-12 or are selected from one of SEQ ID No. 3-6; peptide linkers; and small molecule toxins, Among them, small molecule toxins are coupled to the amino acid side chains of mini-binding proteins via peptide linkers.

[0012] The small molecule toxin is covalently coupled to the cysteine ​​side chain of the mini-binding protein via a peptide linker.

[0013] The small molecule toxin is coupled to the cysteine ​​side chain of the mini-binding protein via a Michael addition reaction between maleimide and thiol to form a thioether bond.

[0014] When the amino acid sequence of the mini-binding protein contains the amino acid sequence of SEQ ID No. 9, the small molecule toxin is coupled to cysteine ​​at position 3 of SEQ ID No. 9; When the amino acid sequence of the mini-binding protein contains the amino acid sequence of SEQ ID No. 10, the small molecule toxin is conjugated to cysteine ​​at position 67 of SEQ ID No. 10; When the amino acid sequence of the mini-binding protein contains the amino acid sequence of SEQ ID No. 11, the small molecule toxin is conjugated to cysteine ​​at position 74 of SEQ ID No. 11; When the amino acid sequence of the mini-binding protein contains the amino acid sequence of SEQ ID No. 12, the small molecule toxin is conjugated to cysteine ​​at position 77 of SEQ ID No. 12; The peptide linker is a lysosomal cleavable peptide linker, such as Val-Cit, Val-Ala, Val-Lys, Phe-Lys, or Val-Arg.

[0015] 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).

[0016] The small molecule toxin is MMAE.

[0017] In the drug conjugate, the peptide linker and the small molecule toxin moiety are as shown in the following formula:

[0018] The asterisk (*) indicates that the above structure is coupled to the cysteine ​​side chain position of the mini-binding protein.

[0019] 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.

[0020] The excipients are selected from one or more of the following: phosphates, histidine salts, citrates, cyclodextrins, polysorbate 80, ascorbic acid, and sodium chloride.

[0021] 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 TROP2.

[0022] The malignant tumors were selected from TROP2-related breast cancer and lung cancer.

[0023] The lung cancer referred to is lung adenocarcinoma and non-small cell lung cancer.

[0024] Beneficial effects (1) This application employs a deep learning-based computational design method to design a specific target-binding protein for the tumor-associated antigen TROP2 from scratch. The binding affinity was optimized from 40.4 nM to 628 pM through partial diffusion, resulting in a high-affinity TROP2 mini-protein binding molecule. The designed TROP2 mini-binding protein exhibits good specificity and thermal stability, maintaining its biological activity even after heating at 95°C.

[0025] (2) This application further functionalizes the mini-binding protein by binding it to the microtubule inhibitor VcMMAE, resulting in mini-protein drug conjugates (MPDCs) that can target and kill TROP2-positive tumor cells.

[0026] (3) In vitro experiments have demonstrated that these new MPDCs of this application have good killing effects on tumor cells (MDA-MB-468, NCI-H1781) and patient-derived lung adenocarcinoma organoids (LAOs).

[0027] (4) The therapeutic experiments conducted in a mouse xenograft model showed that the MPDCs of this application exhibited good biocompatibility and tumor growth was well inhibited at an injection dose of 2 mg / kg. Attached Figure Description

[0028] Figure 1 This diagram illustrates the design, synthesis, and screening of the TROP2 mini-binding protein in this application and its application in in vivo and in vitro models.

[0029] Figure 2 The diagram shows the morphology of TROP2 and the process of targeted binding and endocytosis after the mini-binding protein forms MPDCs.

[0030] Figure 3 This diagram shows the designed binding site structure of the TROP2 mini-binding protein targeting human TROP2. A and B represent the designed binding sites of the TROP2 mini-binding protein against human TROP2; C represents the model structure of the TROP2 mini-binding protein against human TROP2.

[0031] Figure 4 The results of BLI and FACS screening experiments for the initially designed mini-binding proteins are shown. A: Response values ​​for each mini-binding protein targeting TROP2 are shown, with the top 23 mini-binding proteins highlighted in red; B: MFI data of cell binding strength of the 23 mini-binding proteins as detected by FACS; C: FACS data of Binder-20 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-20 protein and TROP2 at different concentration gradients.

[0032] Figure 5 The following diagrams show the FACS screening results of the optimized mini-binding protein, the structural differences between Binder-20 and Binder-20-O5, and the property verification results of Binder-20-O5. Specifically: A: FACS data for screening the optimized mini-binding protein; B: FACS data of the optimized Binder-20-O5 mini-binding protein binding to the surface of MDA-MB-468 cells but not HUVEC cells; C: Structural differences between Binder-20 and Binder-20-O5; D: BLI results of the interaction between Binder-20-O5 and human TROP2 at different concentrations; E: CD experiment results of Binder-20-O5 protein; F: Changes in the binding strength of Binder-20-O5 protein after heating; G: Binding results of Binder-20-O5 with the surface of MDA-MB-468 cells after mutation of key binding sites.

[0033] Figure 6This paper presents the drug conjugation results of Binder-20-O5 and its efficiency and effects. A: Selection of undetermined mutation sites in the Binder-20-O5 protein; B: Changes in cell surface binding after drug conjugation at different mutation sites; C: Experimental results of SDS-PAGE gel electrophoresis verifying conjugation efficiency; D: Experimental results of UV-Vis spectrophotometry verifying conjugation efficiency; E: Experimental results of mass spectrometry verifying conjugation efficiency; F: BLI experimental results of different concentrations of Binder-20-O5-74C-VcMMAE.

[0034] Figure 7 The results of the cytotoxicity assays of MPDCs targeting TROP2 are shown. A: Verification of co-localization of Binder-20-O5-74C-VcMMAE with lysosomes in immunofluorescence assays; B: Changes in surface fluorescence intensity of Binder-20-O5-74C-VcMMAE over time in FACS assays; C: CCK-8 assay results of Binder-20-O5-74C-VcMMAE in MDA-MB-468, NCI-H1781, and HUVEC cells; D: Apoptosis assay results of Binder-20-O5-74C-VcMMAE in MDA-MB-468, NCI-H1781, and HUVEC cells at different concentration gradients.

[0035] Figure 8 This study demonstrates how Binder-20-O5-74C-VcMMAE impairs the viability of LAOs and inhibits tumor growth in vivo. A: Histological and immunostaining features of LAOs and LUAD tissues; B: Immunoblot analysis of HUVECs and LAOs TROP2 protein; C: Representative bright-field images of the same group of LAOs treated with PBS (control) or Binder-20-O5-74C-VcMMAE on days 0 and 3; D: Viability assessment of LAO 1 stained with calcein AM (live cells, green) and PI (dead cells, red); E: Relative LAO activity of treated organoids at 72 hours by ATP-dependent luminescence assay. Relative activity was calculated by normalizing the luminescence signal of the drug-treated wells to the average signal of the PBS-treated control wells; F: Schematic diagram of in vivo xenograft experiment; G: Body weight of each group during drug administration; H: Representative image of tumor resection in each group; I: Tumor volume growth curve during drug administration; J: Tumor weight at the endpoint; K: Histological and immunostaining analysis of tumors: H&E (morphology), Ki-67 (proliferation), TUNEL (apoptosis); L: Quantification of Ki-67 positive cells; M: Quantification of TUNEL positive cells.

[0036] Figure 9The success rates of Alphafold2 complex structure predictions for three batches of mini-binding protein designs are shown. Red dots indicate successful designs. A: Initial calculation success rate for site 1 and 2 designs; B: Second calculation success rate for site 1 design.

[0037] Figure 10 The results of the initial screening of the mini-binding protein BLI are shown. Among them, the curves with the maximum response value higher than 0.09 have been fitted (Binder-20).

[0038] Figure 11 The scores of ipTM and pae_interaction for each protein are shown during the optimized screening.

[0039] Figure 12 The image shows the expression of TROP2 on the surface of MDA-MB-468, NCI-H1781 and HUVEC cells using a TROP2 monoclonal antibody. Darker colors in the image represent the fluorescence intensity of the negative control without antibody, while lighter colors represent the fluorescence intensity of the positive control with antibody.

[0040] Figure 13 The results of Binder-20-O5-74C-VcMMAE killing LAOs are shown. A: Representative bright-field images of LAO 2 and LAO 3 treated with PBS (control) or Binder-20-O5-74C-VcMMAE on days 0 and 3; B: Activity assessment of LAO 2 and LAO 3 stained with calcein AM (live cells, green) and PI (dead cells, red); C: Relative activity of organoid LAO 2 after 72 hours of treatment with different drug concentrations by ATP-dependent luminescence assay; D: Relative activity of organoid LAO 3 after 72 hours of treatment with different drug concentrations by ATP-dependent luminescence assay. Detailed Implementation

[0041] 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, the provision of these embodiments is not intended to limit the scope of protection of this application.

[0042] This application first selects two potential design sites in TROP2 ( Figure 3B). Based on the screening of high-performing mini-binding proteins, further affinity optimization yielded even better-performing mini-binding proteins. Then, several alanine residues (A3, A67, A74, and A77) on the non-binding α-helix surface of these mini-binding proteins were mutated to cysteine ​​residues. This was followed by conjugation with small molecule toxin drugs to prepare a series of mini-binding protein drug conjugates. Among them, Binder-20-O5-74C-VcMMAE showed excellent performance in in vitro binding, endocytosis, and killing, as well as in organoid and mouse experiments. The specific experimental procedures are described below.

[0043] the term: In this application, mini-binding protein (also referred to herein as mini-protein or Binder) refers to a small molecular weight, highly specific binding protein targeting TROP2 that is designed and screened de novo using computational protein design technology.

[0044] In the following text, the term "pae_interaction" used in mini-binding protein screening refers to the Predicted Aligned Error for the interaction interface, which is a metric specifically used to assess the reliability of inter-chain interaction interfaces.

[0045] Example 1: Generating TROP2 mini-binding protein using RFdiffusion computational protein design Current crystal structures indicate that TROP2 can form dimer or tetramer structures, but its polymerization interface is dominated by hydrophobic interactions, while the exposed surface is dominated by hydrophilic regions. Furthermore, the extracellular region of recombinantly expressed TROP2 mainly exists in monomeric form, which was selected as the design target.

[0046] Structural analysis revealed multiple flexible ring regions within the extracellular region of TROP2. The Q237–D243 and M80–Y104 regions exhibited significant conformational differences in different crystal structures, particularly the M80–Y104 region, whose maximum offset distance was approximately 0.63 nm. Figure 3A). Since mini-binding proteins typically tend to bind to regions with strong structural rigidity, and conformational fluctuations in flexible loop regions significantly increase the difficulty of binding design, these flexible regions are avoided in this invention. Based on this, the TROP2 crystal structure is split into monomeric forms, and potential binding sites are screened based on their surface structural characteristics. Two locations on the entire contact surface are selected as hotpots for RFdiffusion to generate the mini-binding protein backbone. Site-1, composed of L155, Y259, and L261, is located in a typical β-sheet region; this type of structure typically has a high design success rate. Site-2, composed of M41, V43, and L58, is located in a relatively flat and extended surface region, which is conducive to forming a larger binding interface. Figure 3 B).

[0047] In the initial round of calculations, approximately 2000 candidate backbone structures were generated for each site, with protein lengths controlled within the range of approximately 50–120 amino acids. Subsequently, ProteinMPNN was used to generate three candidate amino acid sequences for each backbone, and AlphaFold2 was used to predict and score the structure of the mini-binding protein complex with TROP2. During the screening process, pae_interaction less than 10 and pLDDT_binder greater than 90 were used as preliminary screening criteria to evaluate all candidate structures. The results showed that Site-2 failed to obtain mini-binding proteins that effectively bound to the preset sites, while some candidate molecules bound to the Site-1 region instead; in contrast, Site-1 showed a higher design success rate of approximately 2.53%, indicating that this site is structurally more suitable as a design target for mini-binding proteins. Figure 9 A). Therefore, in the subsequent optimization process, this invention will focus on the Site-1 site.

[0048] In the second round of calculations, the sampling scale was further expanded by setting the RFdiffusion parameter diffuser.T to 120, generating approximately 10,000 candidate backbone structures. Subsequently, the ProteinMPNN sequence design and AlphaFold2 structure prediction process were repeated, and the same screening criteria (pae_interaction < 10, pLDDT_binder > 90) were used for initial screening, resulting in 624 candidate mini-binding proteins that met the criteria. Figure 9B). Based on this, the screening criteria were further improved, and stricter conditions (pae_interaction < 6, pLDDT_binder > 90) were used for sorting and screening. The 54 sequences with the highest scores were finally selected for subsequent experimental verification (Table 1). The computational design methods in this invention mainly use the methods in (PMID: 41813685, 39636970, 37433327). These include deep learning-based RFdiffusion protein backbone generation technology, deep learning-based AlphaFold2 / 3, and neural network-based MPNN protein sequence design technology.

[0049] Example 2: Preparation and initial screening of mini-binding proteins targeting TROP2 All gene synthesis in this invention was completed by Jiutian Gene Technology (Tianjin) Co., Ltd. Fifty-four mini-binding protein genes (i.e., Binder 1-54, whose amino acid sequences are shown in Table 1 below) were synthesized according to E. coli codon optimization and inserted into the NcoI and XhoI regions of the pET-28a (Novagen Biotech Co., Ltd.) vector. 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), and after a series of ice bath, 42℃ heat shock, and ice bath operations, the cells were added to LB liquid medium and recovered at 37℃ for 1 h. Afterwards, they were plated on kanamycin-resistant plates and cultured for 12-16 h. Single colonies were picked and inoculated into LB liquid medium containing kanamycin. The culture was incubated at 37°C until the OD600 reached 0.6-0.8, then IPTG inducer (Sangon Biotech) was added at a ratio of 1:3000 and induced overnight at 24°C. The cells were collected after centrifugation at 1000g for 5 min at 4°C, 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 band was 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.

[0050] Subsequently, bio-layer interferometry (BLI) was used to screen the binding ability of the successfully expressed proteins in vitro. The BLI experiments were performed as follows: Using a Protein A sensor or Ni-NTA sensor on an OCTET RED96E system (ForteBio, software version: 12.0.1.2), all BLI experiments followed a standard protocol including the following steps: equilibration, loading, second equilibration, binding, and dissociation. All equilibration steps were set to 60 seconds. The Fc-tagged human TROP2 extracellular domain protein (Novoprotein, CU64) was immobilized on the sensor surface, and mini-binding proteins were detected for binding at 1000 nM or gradient concentrations. During the experiments, PBST buffer (PBS containing 0.05% Tween-20, pH 7.4) was used as the running buffer. Binding and dissociation curves were recorded, and response values ​​were analyzed using the software (version: 12.0.1.2). For preliminary screening, the mini-binding proteins were diluted to 1000 nM in the 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 1000 rpm for other steps. All experiments were performed at 25°C. Data analysis was performed using a 1:1 curve fitting model. BLI screening results showed significant differences in the binding ability of different candidate mini-binding proteins (Table 1), with Binder-20 exhibiting the highest binding response signal, suggesting strong in vitro binding ability. Figure 4 A, Figure 10 ).

[0051] Table 1

[0052] Based on the BLI screening results, this invention further selected 23 candidate proteins with detectable binding signals for cell-level binding capacity verification.

[0053] Cell binding assay: The human breast cancer cell line MDA-MB-468 (Wuhan Pusino Life Science & Technology Co., Ltd.) was used as the TROP2-positive model cell. The specific method was as follows: 1 × 10⁶ cells were used... 5The 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 added with an equal volume of PBS. Cells were then collected and washed three times with PBS buffer, followed by labeling with anti-His-tagged fluorescent secondary antibody (Proteintech, CL647-66005 or CL488-66005) in the dark. After washing, the mean fluorescence signal intensity (MFI value) on the cell surface was detected by flow cytometry (BD Biosciences). The experimental results showed that among the candidate proteins tested, Binder-20 showed a significantly higher binding signal on the surface of MDA-MB-468 cells than other proteins, demonstrating excellent cell binding ability. Figure 4 B).

[0054] To further verify its specificity, this invention selected another TROP2-positive cell line, NCI-H1781 (Wuhan Pusaino Life Science & Technology Co., Ltd.), and the TROP2-negative cell line, HUVEC (Wuhan Pusaino Life Science & Technology Co., Ltd.), for comparative experiments. Figure 12 The expression of TROP2 on the cell surface was detected using anti-TROP2 antibody (1:500, Abcam, EPR20043) and Alexa Fluor 488-conjugated anti-rabbit IgG (1:500, Abcam, ab150077). Cell surface binding assays showed that Binder-20 effectively bound to NCI-H1781 cells, while producing almost no binding signal on HUVEC cells, indicating good TROP2 specificity. Figure 4 C). All cells were cultured in a humidified incubator at 37°C, 5% CO2, and approximately 80% relative humidity. All cell lines were used within 2 months of resuscitation and routinely tested to confirm the absence of mycoplasma contamination.

[0055] Example 3: Partial diffusion technique enhances the affinity of mini-binding proteins for TROP2 Preliminary BLI screening results showed that the binding rate K of the mini-binding protein Binder-20 to TROP2 in the initial screening was... on and dissociation rate K dis They are 2.89×10 -5 1 / Ms and 1.95×10 -2 The 1 / Ms ratio indicates that Binder-20 binds to TROP2 very rapidly, but dissociation is also very fast. Further gradient BLI affinity assays confirmed an affinity of approximately 40.4 nM (R0.05) for the TROP2 extracellular domain. max The value is 0.1822, R 2 The value is 0.97, χ² 2Value is 0.3456), affinity is moderate ( Figure 4 D).

[0056] To improve binding affinity, a partial diffusion optimization method was employed, generating a total of 10,000 scaffolds by setting `diffuser.partial_T` to six different values. Following MPNN-based sequence design and AlphaFold2 complex structure prediction, the top 300 sequences were selected based on strict filtering scores (pae_interaction < 5.4, pLDDT > 90), and these models were subsequently used for AlphaFold3 complex structure prediction. Forty-five mini-binding proteins exhibiting the highest `ipTM` scores were selected for experimental validation. Figure 11 and table 2) below.

[0057] After expression and purification using a similar protocol to that in Example 2, these optimized mini-binding proteins were directly screened using FACS-based cell surface binding assays. Figure 5 A). Experimental results showed that 29 out of 45 mini-binding proteins outperformed Binder-20, with Binder-20-O5 exhibiting significantly better cell-binding properties compared to other candidate proteins. Figure 5 A and Figure 5 B). Its BLI detection of R max Value 0.163, R 2 Value 0.9986, χ 2 Value 0.0015, K d Value 0.628 nM. Structural alignment showed that Binder-20-O5 maintained a binding mode highly consistent with that of Binder-20 for the extracellular domain of TROP2, with only minor skeletal adjustments at the binding interface. Figure 5 C).

[0058] Table 2

[0059] Note: For each sequence in Tables 1 and 2, the beginning part (M) represents the starting amino acid M, and the ending part (ggsHHHHHH) represents linker + 6*His. In Table 2, to unify the average fluorescence intensity before and after optimization, the average fluorescence intensity of the original Binder-20 protein is defined as 0, and the average fluorescence intensity of the Binder-20-O5 protein with the highest fluorescence intensity after optimization is defined as 1. The relative MFI values ​​of other proteins are calculated using the formula: Relative MFI = (Original MFI of protein - MFI of Binder-20 protein) / (MFI of Binder-20-O5 protein - MFI of Binder-20 protein). In Tables 1 and 2 above, sequences 1-6 that do not contain MG at the N-terminus (G in MG is a protective amino acid, ensuring that the first M is properly cleaved during protein translation and folding) and do not contain ggsHHHHHH (SEQ ID No: 106) at the C-terminus correspond to SEQ ID Nos: 7-12, respectively.

[0060] Furthermore, the binding affinity of the optimized mini-binding protein was quantified using BLI, revealing an improvement from 40.4 nM to approximately 628 pM. Figure 5 D) confirms the successful affinity enhancement achieved through partial diffusion optimization.

[0061] Circular dichroism (CD) spectroscopy indicates that Binder-20-O5 almost completely recovers its secondary structure after heating to 95°C and subsequently returning to room temperature. Figure 5 E).

[0062] Cell binding assays further showed that heat-treated (95 °C) Binder-20-O5 (shown in the figure as a 25 °C recovery) retained binding activity comparable to that of the untreated protein (shown in the figure as a 25 °C). Figure 5 F) indicates excellent thermal stability.

[0063] Further mutation of the core hydrophobic residue L53 (Binder-20-O5-L53) at the arginine binding interface, replacing it with the hydrophilic residue R, was performed. Flow cytometry results demonstrated that this mutation completely eliminated the binding of Binder-20-O5 to TROP2-positive cells, confirming the binding interface specificity predicted by the structural model. Figure 5 G).

[0064] Example 4: Covalent coupling of TROP2 mini-binding protein with VcMMAE After obtaining the high-affinity TROP2 mini-binding protein, it was covalently bound to the classic ADC payload maleimide valine-citrulline monomethyl auristatin E (VcMMAE)(MCE, HY-15575) via thiol group, thereby modifying it into a mini-binding protein drug conjugate (MPDC) for cancer treatment.

[0065] To minimize interference with target binding and considering the possibility that the C-terminal His tag may be removed in future clinical applications, several alanine residues (A3, A67, A74, and A77) located on the surface of the non-binding α-helix were mutated to cysteine, and named Binder-20-O5-3C, Binder-20-O5-67C, Binder-20-O5-74C, and Binder-20-O5-77C, respectively. Figure 6 A). The amino acid sequences and relative MFIs of these mutants are shown in Table 2 above. Because VcMMAE is a hydrophobic payload, the selection of alanine residues while retaining the surrounding hydrophilic amino acids helps maintain the water solubility of the mini protein scaffold.

[0066] 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.

[0067] Furthermore, four conjugates were prepared according to the above method, namely Binder-20-O5-3C, Binder-20-O5-67C, Binder-20-O5-74C, and Binder-20-O5-77C, which are respectively conjugated with VcMMAE. These conjugates can be abbreviated as: O5-3C-VcMMAE, O5-67C-VcMMAE, O5-74C-VcMMAE, and O5-77C-VcMMAE.

[0068] After drug binding, FACS analysis showed that different binding sites exhibited different binding characteristics. Statistical analysis indicated that among the four conjugates, Binder-O5-74C-VcMMAE showed the best binding efficacy. Figure 6 B).

[0069] Furthermore, the correct labeling of VcMMAE was verified. Copper sulfate oxidation assays using SDS-PAGE showed that the cysteine ​​residues in Binder-20-O5-74C were fully conjugated. Figure 6 C); The ultraviolet-visible spectrum shows significant absorption in the 240-290 nm range, consistent with drug incorporation. Figure 6 D); Mass spectrometry analysis confirmed that the molecular weight increased from 9,608 to 10,924 Da, which matched the theoretical mass of the conjugate very well. Figure 6 E).

[0070] Furthermore, BLI experiments showed that the conjugation of VcMMAE had almost no effect on the affinity binding between the mini-binding protein with a Kd of 1.04 nM and TROP2. Figure 6 F). Its BLI experimental data R max Value 0.2534, R 2 Value 0.9992, χ 2 The value was 0.0013. Subsequently, the conjugate Binder-O5-74C-VcMMAE was used in the tumor cell killing experiment in the following examples.

[0071] Example 5: Cytotoxic effects of Binder-O5-74C-VcMMAE on tumor cells at the cellular level First, the ability of Binder-O5-74C-VcMMAE prepared in Example 4 above to be internalized by MDA-MB-468 cells was tested. Binder-20-O5-74C-VcMMAE was covalently coupled to AF555 fluorescent dye via an amino reaction for fluorescence imaging analysis; this group was designated as the positive group. The L53R group used the L53R mutant protein to replace the original Binder-20-O5-74C-VcMMAE in the positive group. The negative group was the control group without the addition of Binder-20-O5-74C-VcMMAE. Confocal fluorescence microscopy showed that, 6 hours after internalization, lysosomal signals exhibited good colocalization with the fluorescence signal of Binder-O5-74C-VcMMAE. Figure 7 A). Flow cytometry data showed that the surface signal of Binder-O5-74C-VcMMAE was significantly reduced 2 hours after internalization. Figure 7 B) indicates effective cellular uptake.

[0072] The cytotoxic efficacy of Binder-O5-74C-VcMMAE against cancer cells (MDA-MB-468, NCI-H1781, and HUVEC) was further evaluated. Cells were cultured overnight in 96-well plates at 5000-8000 cells per well, then treated with Binder-O5-74C-VcMMAE for 48 hours, followed by detection using a CCK-8 assay kit (Beyotime, C0042). CCK-8 analysis showed that Binder-O5-74C-VcMMAE induced significant dose-dependent cytotoxicity in the MDA-MB-468 and NCI-H1781 cell lines. At a concentration of 5 μg / mL for 48 hours, cell viability decreased by an average of approximately 70%, while HUVEC viability remained largely unaffected. Figure 7 C).

[0073] Use 24-well cell culture plates, 5 × 10⁵ cells per well. 4 Cells were cultured overnight and treated with Binder-O5-74C-VcMMAE for 48 hours, followed by detection using an apoptosis assay kit (Beyotime, C1062M / C1737). FACS-based apoptosis detection yielded similar results, showing that Binder-O5-74C-VcMMAE induced apoptosis in a dose-dependent manner, with over 70% of cells showing double positivity for 7-AAD and annexin V. Figure 7 D).

[0074] These data indicate that Binder-O5-74C-VcMMAE is an effective TROP2-positive cell-specific cytotoxic agent at the in vitro cellular level.

[0075] Example 6: Antitumor effects of Binder-O5-74C-VcMMAE in LAOs and mouse xenograft models This embodiment evaluated the antitumor efficacy of Binder-O5-74C-VcMMAE in cancer organoids and in vivo tumor models.

[0076] First, three lung adenocarcinoma organoids (LAOs) derived from surgical specimens of patients with lung adenocarcinoma (LUAD) were established and named LAO1, LAO2, and LAO3, respectively. H&E staining and immunohistochemical analysis of specific markers (TTF-1, CK7) confirmed that these LAOs retained the histological and molecular characteristics of their respective primary tumors. Figure 8 A). Western blot analysis revealed robust TROP2 expression in all three LAOs ( Figure 8B). After treatment with 10 μg / mL Binder-O5-74C-VcMMAE, LAOs showed significant morphological changes indicative of cell death under an optical microscope. Figure 8 C and Figure 13 A). Live / dead cell staining using calcein AM / PI (Beyotime, C2015S) showed that in organoids treated with Binder-O5-74C-VcMMAE, green fluorescence (live cells) was significantly reduced, while red fluorescence (dead cells) was significantly increased. Figure 8 D and Figure 13 B). ATP-based luminescence assays further confirmed the dose-dependent cytotoxicity of Binder-O5-74C-VcMMAE to LAOs (B). Figure 8 E and Figure 13 C and Figure 13 D), the specific operation is as follows: Place LAOs at 2×10 per hole. 3 LAOs were seeded at a density of 5 μL each in a mixture of organoid culture medium and Matrigel. After incubation at standard conditions (37°C, 5% CO2) for 48–72 hours to allow initial organoid growth, drug treatment was initiated. PBS (blank control) or a series of Binder-O5-74C-VcMMAE dilutions in complete organoid culture medium were added to the LAOs. Viability was assessed after 72 hours of continuous treatment. The culture medium was carefully removed, and organoids were lysed and ATP was quantified using the CellTiter-Meiluncell luminescence assay kit (Meilun Biotechnology, PWL111) following the manufacturer's instructions. The luminescence values ​​were read using a Molecular Devices microplate reader (SpectraMax Mini). The luminescence signal from the treated wells was normalized to the average signal from the PBS control wells to calculate relative viability. Cell viability was determined when cell viability decreased to below 50% at 10 μg / mL. Figure 8 E and Figure 13 C and Figure 13 D).

[0077] Subsequently, the therapeutic potential of Binder-O5-74C-VcMMAE was evaluated in vivo using an MDA-MB-468 cell-derived xenograft model. Experimental animals were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and 5-week-old BALB / c nude mice were used. Five days after inoculating BALB / c nude mice with MDA-MB-468 cells, tumor-bearing mice were randomly divided into two groups. Every other day, the mice were administered PBS (vector control) or 2 mg / kg Binder-O5-74C-VcMMAE via tail vein injection. Figure 8F). Notably, Binder-O5-74C-VcMMAE treatment showed minimal impact on mouse body weight, indicating acceptable safety. Figure 8 G). Compared with the control group, the drug-treated mice showed tumor volume (G). Figure 8 H and Figure 8 I) and tumor weight ( Figure 8 The significant reduction in J indicates effective antitumor activity. Histological examination of subcutaneous tumors showed that Binder-O5-74C-VcMMAE treatment resulted in a significant reduction in tumor cell cytosis, marked stromal expansion with fibrosis characteristics, and strong inflammatory cell infiltration—all of which collectively indicate effective tumor cell killing and immune cell recruitment. Figure 8 Consistent with these observations, Ki-67 staining revealed a significant inhibition of tumor cell proliferation (K). Figure 8 J and Figure 8 L), while TUNEL staining (In Situ Cell Death Detection Kit, POD; Roche, 11684795910) confirmed enhanced apoptotic cell death in the treatment group. Figure 8 J and Figure 8 M).

[0078] In summary, these findings indicate that Binder-O5-74C-VcMMAE has significant antitumor effects on organoids and in tumor-bearing animal models.

Claims

1. A mini-binding protein targeting TROP2, characterized in that, It contains an amino acid sequence selected from any of SEQ ID No. 7-12.

2. The mini-binding protein according to claim 1, characterized in that, 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.

3. The mini-binding protein according to claim 2, characterized in that, The flexible linker peptide is selected from GGS, GSG, GGG, SGG; and The affinity purification tag is 6×His.

4. The mini-binding protein according to claim 1, characterized in that, The amino acid sequence of the mini-binding protein is selected from one of SEQ ID No. 1-6.

5. A drug conjugate, characterized in that, It includes the following components: Mini-binding proteins whose amino acid sequences include any one of SEQ ID No. 9-12 or selected from one of SEQ ID No. 3-6; peptide linkers; and small molecule toxins, Among them, small molecule toxins are coupled to the amino acid side chains of mini-binding proteins via peptide linkers.

6. The drug conjugate according to claim 5, characterized in that, The small molecule toxin is covalently coupled to the cysteine ​​side chain of the mini-binding protein via a peptide linker.

7. The drug conjugate according to claim 6, characterized in that, The small molecule toxin is coupled to the cysteine ​​side chain of the mini-binding protein via a Michael addition reaction between maleimide and thiol to form a thioether bond.

8. The drug conjugate according to claim 5, characterized in that, When the amino acid sequence of the mini-binding protein contains the amino acid sequence of SEQ ID No. 9, the small molecule toxin is coupled to cysteine ​​at position 3 of SEQ ID No. 9; When the amino acid sequence of the mini-binding protein contains the amino acid sequence of SEQ ID No. 10, the small molecule toxin is conjugated to cysteine ​​at position 67 of SEQ ID No. 10; When the amino acid sequence of the mini-binding protein contains the amino acid sequence of SEQ ID No. 11, the small molecule toxin is conjugated to cysteine ​​at position 74 of SEQ ID No. 11; When the amino acid sequence of the mini-binding protein contains the amino acid sequence of SEQ ID No. 12, the small molecule toxin is conjugated to cysteine ​​at position 77 of SEQ ID No. 12; The peptide linker is a lysosomal cleavable peptide linker. The small molecule toxin is MMAE.

9. The drug conjugate according to claim 6, characterized in that, The peptide linker and small molecule toxin moiety are shown in the following formula: The asterisk (*) indicates that the above structure is coupled to the cysteine ​​side chain position of the mini-binding protein.

10. A pharmaceutical composition, characterized in that, It comprises a mini-binding protein as described in any one of claims 1-4 or a drug conjugate as described in any one of claims 5-9, and pharmaceutically acceptable excipients.

11. Use of the mini-binding protein of any one of claims 1-4, or the drug conjugate of any one of claims 5-9, or the pharmaceutical composition of claim 10 in the preparation of a medicament for treating malignant tumors targeting TROP2.

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