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CN-122011126-A - Nectin-4 targeting mini-binding protein, drug conjugate and application thereof

CN122011126ACN 122011126 ACN122011126 ACN 122011126ACN-122011126-A

Abstract

The application relates to a targeting Nectin-4 mini-binding protein, a drug conjugate thereof and application thereof. The application obtains a kind of specific targeting Nectin-4 mini-binding protein through screening, and the amino acid sequence of the mini-binding protein comprises any one of SEQ ID No. 1-6. The application further couples the mini-protein conjugate with a microtubule inhibitor VcMMAE, resulting in a mini-protein drug conjugate (MPDCs) that is capable of targeted killing of Nectin-4 positive tumor cells.

Inventors

  • YU BOWEN
  • Xue tianshui
  • YANG ZHIZHOU
  • WANG NINGNING

Assignees

  • 山东第二医科大学

Dates

Publication Date
20260512
Application Date
20260414

Claims (10)

  1. 1. A mini-binding protein targeting Nectin-4, characterised in that its amino acid sequence comprises any of SEQ ID No. 1-6.
  2. 2. The mini-binding protein of claim 1, wherein the amino acid sequence of the mini-binding protein further comprises a starting amino acid M at the N-terminus of SEQ ID nos. 1-6 and an affinity purification tag at the C-terminus via a flexible linker peptide.
  3. 3. The mini-binding protein of claim 1, wherein the amino acid sequence of the mini-binding protein is selected from one of SEQ ID nos. 7-12.
  4. 4. A drug conjugate, which is characterized in that, the drug conjugate comprises the following components: A mini-binding protein, which is a protein, The amino acid sequence of which comprises any one of SEQ ID No. 2-6 or one selected from SEQ ID No. 8-12; Peptide linker, and A small molecule of a toxin, which is a small molecule of a toxin, Wherein the small molecule toxin is coupled to the amino acid side chain of the mini-binding protein via a peptide linker.
  5. 5. The drug conjugate of claim 4, wherein the small molecule toxin is coupled to the cysteine side chain in the mini-binding protein via a peptide linker by a covalent bond.
  6. 6. The drug conjugate of claim 5, wherein the small molecule toxin is coupled to the cysteine side chain of the mini-binding protein by a thioether linkage formed by a michael addition reaction of a succinimide with a thiol group.
  7. 7. The drug conjugate of claim 6, wherein when the amino acid sequence of the mini-binding protein comprises the amino acid sequence of SEQ ID No.2, the small molecule toxin is conjugated to cysteine 90 of SEQ ID No. 2; when the amino acid sequence of the mini-binding protein comprises the amino acid sequence of SEQ ID No. 3, the small molecule toxin is coupled to cysteine 97 of SEQ ID No. 3; when the amino acid sequence of the mini-binding protein comprises the amino acid sequence of SEQ ID No. 4, the small molecule toxin is coupled to cysteine 104 of SEQ ID No. 4; When the amino acid sequence of the mini-binding protein comprises the amino acid sequence of SEQ ID No. 5, the small molecule toxin is coupled to cysteine 121 of SEQ ID No. 5; When the amino acid sequence of the mini-binding protein comprises the amino acid sequence of SEQ ID No. 6, the small molecule toxin is coupled to cysteine 128 of SEQ ID No. 6.
  8. 8. The drug conjugate of any of claims 4-7, In the drug conjugates, the peptide linker and the small molecule toxin moiety are represented by the following formulas: Wherein the above structure is coupled to the cysteine side chain position of the mini-binding protein.
  9. 9. A pharmaceutical composition comprising a mini-binding protein according to any one of claims 1-3 or a drug conjugate according to any one of claims 4-8, and a pharmaceutically acceptable adjuvant.
  10. 10. Use of a mini-binding protein according to any one of claims 1 to 3, a drug conjugate according to any one of claims 4 to 8, or a pharmaceutical composition according to claim 9 in the manufacture of a medicament for the treatment of a malignant tumor targeted to Nectin-4.

Description

Nectin-4 targeting mini-binding protein, drug conjugate and application thereof Technical Field The application belongs to the technical field of protein medicines, and particularly relates to a targeting Nectin-4 mini-binding protein, a medicine conjugate thereof and application thereof. Background Classical Antibody-conjugated drugs (anti-Drug Conjugates, ADCs) are a class of targeted anti-tumor therapeutic agents that covalently couple monoclonal antibodies to highly cytotoxic small molecules via chemical linkers. Thanks to the high specific recognition capability of antibodies, such accurate therapeutic drugs are gradually replacing traditional chemotherapy regimens in the treatment of part of cancers. Nevertheless, the development of more efficient ADCs against solid tumors remains one of the key challenges in the current tumor drug development field. To date, a variety of ADCs targeting HER2, nectin-4, TROP2, TF, FOLR1, and c-MET antigens have been approved by regulatory authorities and have shown good therapeutic effects in the clinic. Nectin-4 is a cell surface adhesion molecule that is involved in the formation and maintenance of cell-cell junctions, primarily through homologous or heterologous interactions with other Nectin family members and E-cadherins. Nectin-4 expression levels are low in adult normal tissues, however, it is significantly highly expressed in a variety of solid malignancies, and thus is established as an important tumor-associated membrane antigen and ideal target for ADC development. Representative drugs Enfortumab vedotin have been approved by the U.S. Food and Drug Administration (FDA) for the treatment of locally advanced or metastatic urothelial cancer by coupling antibodies targeting Nectin-4 with valine-citrulline-monomethyl Auristatin E (VcMMAE). In addition, currently more than 7 ADCs targeting Nectin-4 are in the clinical study stage, and another more than 11 candidate ADCs are in the preclinical development stage. In addition to traditional full-length antibodies, research on novel tumor targeting molecular scaffolds for ADC applications has been continuously developed, and a variety of antigen recognition formats have been derived, including nanobodies derived from camels and sharks, and bicyclic peptides, among others. These systems have incorporated a variety of cytotoxic payloads such as lidamycin, pseudomonas exotoxin, MMAE and the like. Compared with the traditional antibody, the nano antibody has the advantages of small molecular weight, high structural stability, potentially better tissue penetrating capacity and the like. However, nanobody acquisition generally depends on animal immune process, there is a high experimental threshold, and meanwhile, the recombinant expression efficiency of nanobody in an escherichia coli system is limited, and challenges in aspects of thermal stability and the like are still faced. In recent years, significant progress has been made in the art of designing mini-binding proteins from the head. Early approaches relied mainly on a physics-based computational framework (e.g., ROSETTA), but such approaches often relied on high-throughput experimental screening, computational flows were complex and design success rates were limited. Recently developed deep learning-based skeleton generation methods (e.g., RFdiffusion and BindCraft) overcome the above limitations to a large extent. When combined with a sequence design method based on an information transfer neural network (MPNN) and AlphaFold-mediated protein complex structure prediction, the integrated design flow can efficiently generate mini-binding proteins with various structures, and can realize rapid identification of low nanomolar affinity candidate molecules through low-throughput experimental screening. In addition, the mini-protein can be expressed in Escherichia coli with high efficiency and has good thermal stability. Numerous high affinity mini-binding proteins have been reported to target immune receptors including TNFR, IL-1R, IL-6R, IL-17R, and tumor-associated targets such as EGFR, PD-L1, HER2, TGF-beta RII, and CTLA-4. Nevertheless, the de novo design of high affinity mini-binding proteins for small molecule targets with complex structural features (e.g., tandem domains) and high hydrophilicity still presents a challenge, and its drug formulation and clinical transformation potential remain to be further systematically investigated. Disclosure of Invention Technical purpose One of the technical purposes of the present invention is to develop a class of mini-binding proteins targeting Nectin-4. It is still another technical object of the present application to provide a drug conjugate of the foregoing mini-binding protein and a small molecule drug. It is still another object of the present application to provide a pharmaceutical composition comprising the foregoing mini-binding protein or the foregoing drug conjugate. It is a further technical object of the present