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CN-122011205-A - Antibody fusion protein induced by targeted receptor ubiquitination and application thereof

CN122011205ACN 122011205 ACN122011205 ACN 122011205ACN-122011205-A

Abstract

The invention discloses a target receptor ubiquitination induced antibody fusion protein and application thereof, wherein the antibody fusion protein comprises (a) an antibody domain capable of binding a tumor-associated antigen receptor, and (b) a membrane-binding E3 ubiquitin ligase binding module fused with the antibody domain, wherein the binding module is an RSPO2 FU (F109A) domain, the amino acid sequence of the binding module is shown as SEQ ID No.3, and the antibody fusion protein can simultaneously bind the tumor-associated antigen receptor and the membrane-binding E3 ubiquitin ligase RNF43 and/or ZNRF3. The antibody fusion protein can remarkably enhance the internalization and lysosome delivery efficiency of tumor-associated antigen receptor, overcomes the defect of insufficient level reduction of the existing antibody receptor, can be applied to the treatment of solid tumors associated with tumor-associated antigen receptor, and can be possibly extended to other diseases associated with receptor degradation.

Inventors

  • PAN LIQIANG
  • ZHUANG XINLEI
  • HE JUN

Assignees

  • 浙江大学
  • 绵阳市第三人民医院

Dates

Publication Date
20260512
Application Date
20251226

Claims (10)

  1. 1. A receptor-targeted ubiquitination-induced antibody fusion protein, comprising: (a) An antibody domain capable of binding a tumor associated antigen receptor; (b) A membrane-bound E3 ubiquitin ligase binding module fused to the antibody domain, the E3 ubiquitin ligase binding module being an RSPO2 FU (F109A) domain; wherein the antibody fusion protein is capable of simultaneously binding a tumor-associated antigen receptor with membrane-bound E3 ubiquitin ligase RNF43 and/or ZNRF3.
  2. 2. The antibody fusion protein of claim 1, wherein the E3 ubiquitin ligase binding module is fused to the amino terminus of the antibody domain light chain; the E3 ubiquitin ligase binding moiety is linked to the antibody domain via a flexible linker peptide.
  3. 3. The antibody fusion protein of claim 2, wherein the amino acid sequence of the flexible linker peptide is shown in SEQ ID No. 4.
  4. 4. The antibody fusion protein of claim 1, wherein the tumor-associated antigen receptor is selected from EGFR or HER2; When the tumor-associated antigen receptor is selected from EGFR, the amino acid sequences of the light chain and the heavy chain of the antibody domain are shown as SEQ ID No.1 and SEQ ID No.2, respectively; when the tumor-associated antigen receptor is selected from HER2, the light chain amino acid sequence of the antibody domain is shown as SEQ ID No.5, and the heavy chain amino acid sequence is shown as SEQ ID No. 6; the amino acid sequence of the RSPO2 FU (F109A) domain is shown as SEQ ID No. 3.
  5. 5. The antibody fusion protein of any one of claims 1-4, wherein the antibody domain is cetuximab or an antigen binding fragment thereof or trastuzumab or an antigen binding fragment thereof.
  6. 6. The biomaterial related to the antibody fusion protein of any one of claims 1 to 5, characterized in that the biomaterial is any one of the following: (1) A nucleic acid molecule encoding the antibody fusion protein of any one of claims 1-5; (2) An expression cassette comprising the nucleic acid molecule of (1); (3) A recombinant vector comprising (1) the nucleic acid molecule, or a recombinant vector comprising (2) the expression cassette; (4) A recombinant microorganism comprising (1) the nucleic acid molecule, or a recombinant microorganism comprising (2) the expression cassette, or a recombinant microorganism comprising (3) the recombinant vector; (5) A cell line comprising (1) the nucleic acid molecule, or a cell line comprising (2) the expression cassette, or a cell line comprising (3) the recombinant vector.
  7. 7. A method of preparing the antibody fusion protein of any one of claims 1-5, comprising: (1) Constructing an expression vector comprising a nucleotide sequence encoding the antibody fusion protein; (2) Expressing the antibody fusion protein in a host cell; (3) Purifying the antibody fusion protein from the cell culture.
  8. 8. The method of claim 7, wherein the host cell in step (2) is a eukaryotic cell and/or the purification in step (3) employs Protein A affinity chromatography.
  9. 9. A pharmaceutical composition comprising the antibody fusion protein of any one of claims 1-5 and a pharmaceutically acceptable carrier.
  10. 10. Use of the antibody fusion protein of any one of claims 1-5, the biomaterial of claim 6 or the pharmaceutical composition of claim 9 in the manufacture of a medicament for the treatment of tumor-associated antigen receptor positive tumors.

Description

Antibody fusion protein induced by targeted receptor ubiquitination and application thereof Technical Field The invention belongs to the technical field of biological medicine, and particularly relates to an antibody fusion protein capable of simultaneously combining a tumor-associated antigen receptor and membrane-associated E3 ubiquitin ligase (RNF 43 and/or ZNRF 3), in particular to an engineering antibody fusion protein capable of inducing ubiquitination of the tumor-associated antigen receptor and promoting internalization and lysosome degradation of the tumor-associated antigen receptor and application of the engineering antibody fusion protein in anti-tumor treatment. Background Epidermal Growth Factor Receptor (EGFR) is a kind of transmembrane receptor tyrosine kinase, is widely expressed in epithelial tissues, and plays a key role in regulating and controlling the growth, differentiation, survival and migration of cells. Abnormal activation of EGFR is closely related to the development and progression of a variety of malignant tumors, including colorectal cancer, non-small cell lung cancer, head and neck squamous cell carcinoma, glioblastoma, and the like. In these tumors, EGFR often exhibits over-expression, gene amplification, or specific mutations, driving tumor cells to continuously activate downstream signaling pathways (e.g., RAS-RAF-MEK-ERK, PI3K-AKT, JAK-STAT pathways), promoting cell immortalization and resistance to apoptosis. EGFR has therefore long been considered an important target for tumor treatment. Currently, there are a variety of therapeutic strategies that target EGFR clinically. Small molecule Tyrosine Kinase Inhibitors (TKIs), such as gefitinib, erlotinib, octreotide, and the like, can block EGFR kinase activity and inhibit signal transduction. However, such drugs often fail due to acquired drug resistance mutations (e.g., T790M, C797S). Monoclonal antibodies (e.g., cetuximab, panitumumab) inhibit signaling pathway activation by binding to the EGFR extracellular domain, blocking ligand (e.g., EGF) binding and inhibiting receptor dimerization. However, the anti-tumor effect of such antibodies is still limited, and one of the reasons is that the existing antibodies mainly play a role in blocking signals, but cannot effectively reduce the steady-state level of the receptor on the cell surface. Internalization and degradation of EGFR is a key element in determining its signal duration and intensity. However, in many tumor cells, EGFR activation signals, even if blocked by antibodies, remain stably on the cell surface or partially recycled back to the cell surface after internalization, resulting in the continued presence and maintenance of abnormal signals, thereby impairing the therapeutic efficacy of existing anti-EGFR antibodies. Ubiquitination modification is an important mechanism to promote EGFR internalization and degradation. Ubiquitination is a post-translational modification catalyzed by the E1, E2, E3 cascade enzyme system, where the E3 ubiquitin ligase is responsible for substrate specific recognition. For EGFR, cbl and like cytoplasmic type E3 ligases are known to mediate EGFR ubiquitination and promote its degradation upon ligand stimulation. However, existing anti-EGFR antibody drug designs rely primarily on blocking signaling or enhancing antibody-dependent cellular cytotoxicity (ADCC), and lack antibody molecules that actively induce ubiquitination of target receptors, thereby promoting receptor internalization and degradation. This results in EGFR-targeted antibody therapies in many cases that only temporarily inhibit receptor function and do not effectively clear the receptor itself, leaving tumor cells with the opportunity to escape and drug resistance. If a novel antibody can be developed, the novel antibody can not only keep high affinity binding with EGFR, but also recruit membrane-bound E3 ubiquitin ligase at the same time to induce EGFR ubiquitination, promote the internalization and enter lysosome for degradation, so that the novel antibody can not only reduce EGFR level more effectively and block carcinogenic signals of EGFR, but also be used as a drug delivery carrier to improve drug delivery efficiency. For example, the coupling with cytotoxins to form a new generation of ADC, releasing toxins with enhanced lysosome delivery efficiency, coupling with immune agonists to achieve efficient delivery of immune agonists into EGFR positive tumor cells, coupling with proteolytic targeting chimeras (PROTACs) to increase the efficiency of intracellular entry of PROTACs, coupling with radionuclides to facilitate delivery of radionuclides into tumor cells and to increase the residence time of radionuclides within tumors. In summary, the development of an engineering antibody molecule which targets EGFR and can directly induce ubiquitination and internalization degradation of EGFR can not only overcome the problem of insufficient curative effect of the traditional