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CN-121971643-A - Antibody fusion protein coupling medicine induced by targeted receptor ubiquitination and application thereof

CN121971643ACN 121971643 ACN121971643 ACN 121971643ACN-121971643-A

Abstract

The invention discloses a target receptor ubiquitination induced antibody fusion protein coupling medicament and application thereof, wherein the antibody fusion protein coupling medicament comprises (a) an antibody fusion protein, a membrane-bound E3 ubiquitin ligase binding module and a small cytotoxic molecule medicament, wherein the antibody fusion protein comprises an antibody domain capable of binding tumor-associated antigen receptors, the membrane-bound E3 ubiquitin ligase binding module is fused with the antibody domain, the E3 ubiquitin ligase binding module is an RSPO2 FU (F109A) domain, the antibody fusion protein can simultaneously bind the tumor-associated antigen receptors and the membrane-bound E3 ubiquitin ligase RNF43 and/or ZNRF3, and the small cytotoxic molecule medicament is coupled with the antibody fusion protein. According to the invention, the membrane-bound E3 ubiquitin ligase binding module is introduced, so that the antibody can be combined with a tumor-related antigen receptor and simultaneously promote ubiquitination modification and lysosome pathway degradation, thereby remarkably improving the internalization efficiency and tumor killing activity of the antibody-coupled drug.

Inventors

  • PAN LIQIANG
  • ZHUANG XINLEI
  • HE JUN

Assignees

  • 浙江大学
  • 绵阳市第三人民医院

Dates

Publication Date
20260505
Application Date
20251226

Claims (10)

  1. 1. A receptor ubiquitination-induced antibody fusion protein-coupled drug, comprising: (a) An antibody fusion protein comprising: An antibody domain capable of binding a tumor associated antigen receptor; And 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 a membrane-bound E3 ubiquitin ligase RNF43 and/or ZNRF3; (b) And a cytotoxic small molecule drug coupled to the antibody fusion protein.
  2. 2. The antibody fusion protein coupled drug 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 light chain amino acid sequence of the antibody domain is shown as SEQ ID No.1, and the heavy chain amino acid sequence is shown as SEQ ID No. 2; 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.
  3. 3. The antibody fusion protein coupled drug according to claim 1 or 2, wherein the amino acid sequence of the RSPO2 FU (F109A) domain is shown in SEQ ID No.3 and/or wherein the binding moiety is linked to the antibody domain via a flexible linker peptide having the amino acid sequence shown in SEQ ID No. 4.
  4. 4. The antibody fusion protein coupled drug of any one of claims 1-3, wherein the E3 ubiquitin ligase binding module is fused to the light chain amino terminus of the antibody domain.
  5. 5. The antibody fusion protein-coupled drug of any one of claims 1-4, wherein the cytotoxic small molecule drug is coupled to the antibody fusion protein via a cleavable linker.
  6. 6. The antibody fusion protein coupled drug of claim 5, wherein the cleavable linker is Val-Cit-PABC.
  7. 7. The antibody fusion protein coupled drug according to any one of claims 1-6, wherein the cytotoxic small molecule drug is coupled through the Fc region N297 site sugar chain of the antibody fusion protein and the drug-to-antibody ratio is 1-2; The small cytotoxic molecule drug is monomethyl auristatin E, monomethyl auristatin F or Delutacon.
  8. 8. A method of preparing the antibody fusion protein-coupled drug of any one of claims 1-7, comprising: (1) Providing the antibody fusion protein; (2) Performing glycosyl engineering on a sugar chain at an N297 locus of an Fc region of the antibody fusion protein to introduce an azide group; (3) And coupling the cytotoxic small molecule drug with the azide group introduced on the sugar chain through click chemistry reaction to obtain the antibody fusion protein coupling drug.
  9. 9. The method according to claim 8, wherein the step (2) comprises modifying the sugar chain with endoglycosidase Endo S to expose the GlcNAc residue and introducing azide-modified galactosamine with mutant beta (1, 4) -galactosyltransferase GalT (Y289L), and the click chemistry reaction in the step (3) is a strain-promoted azide-alkyne cycloaddition reaction.
  10. 10. Use of an antibody fusion protein-coupled drug according to any one of claims 1-7 for the manufacture of a medicament for the treatment of tumor-associated antigen receptor positive tumors.

Description

Antibody fusion protein coupling medicine induced by targeted receptor ubiquitination and application thereof Technical Field The invention belongs to the technical field of biological medicine, and in particular relates to an antibody coupling medicine constructed based on antibody fusion protein induced by targeted receptor ubiquitination, which can obviously enhance antibody-mediated internalization and lysosome degradation, thereby improving the delivery efficiency and anti-tumor activity of the medicine. Background Antibody-coupled drugs (ADC) represent a revolutionary drug form in the field of accurate tumor treatment, and combine the specificity of monoclonal antibodies with the killing capacity of high-efficiency cytotoxic small molecules, thereby realizing accurate targeting and killing of tumor cells. The design concept of the ADC is to use the antibody to recognize and bind a specific tumor-associated antigen, selectively deliver the potent toxin into tumor cells, and reduce the toxicity to normal tissues while killing the tumor to the maximum extent. At present, a plurality of ADC drugs are used for clinical treatment of a plurality of solid tumors in batches. However, despite the remarkable clinical success of ADC, there are significant limitations in its efficacy in solid tumors. One key problem is that many ADCs are not efficiently delivered within tumor cells, i.e., internalization of the antibody-antigen complex and lysosomal transport are less efficient. This deficiency results in the inability of the cytotoxic drugs of the ADC to be released efficiently, thereby impairing its antitumor efficacy. The therapeutic effect of an ADC depends largely on whether the target receptor can be internalized by receptor-mediated endocytosis and degraded into the lysosome, releasing the loaded toxin. If the receptor is inefficient for internalization, or tends to circulate back to the cell surface, the accumulation of intracellular toxins can be significantly reduced, directly affecting the cytotoxic effects of the ADC. Studies have shown that a variety of tumor-associated antigen receptors, including EGFR, HER2, etc., all exhibit characteristics of slow internalization rates, recycling, rather than lysosomal degradation. These receptor properties severely limit the efficacy of ADCs in the treatment of solid tumors. For example, EGFR is a classical target for common tumors such as colorectal cancer, non-small cell lung cancer, and head and neck squamous cell carcinoma, and its broad expression and driving effects make it an important target for ADC development. However, the efficacy of traditional EGFR-based ADCs is not ideal clinically, mainly because EGFR antibodies (e.g. cetuximab) are able to bind to the target with high affinity, but the efficiency of induction is limited, and part of the receptor can still circulate back to the cell surface, resulting in insufficient toxin delivery, limiting the efficacy of the ADC. To overcome this bottleneck, a variety of strategies have been proposed by those skilled in the art, including increasing receptor internalization by antibody engineering, designing bispecific or multispecific antibody-enhanced delivery, optimizing linker structures to improve toxin release kinetics, and the like. These approaches improve ADC efficacy to some extent, but are still largely limited to exogenous optimization and fail to effectively regulate receptor membrane transport and intracellular degradation, starting from intrinsic cellular mechanisms. In other words, how to improve the internalization and lysosomal delivery of ADCs by directly interfering with the endogenous receptor regulatory mechanisms of the cells remains an unresolved challenge. In cells, ubiquitination is a key post-translational modification process that determines membrane receptor fate. E3 ubiquitin ligases catalyze the covalent attachment of ubiquitin molecules to membrane protein lysine residues, usually as a signal for endocytosis and lysosomal degradation. For example, cbl family E3 ligases can mediate ubiquitination of EGFR following ligand stimulation, thereby promoting internalization and down-regulation thereof. Numerous studies have demonstrated a central role for ubiquitination in regulating receptor endocytosis, circulation and degradation. However, while ubiquitination has a decisive significance in membrane receptor homeostasis regulation, its potential in ADC design has not been systematically exploited. Some exploratory strategies have been used to achieve target degradation using receptor ubiquitination, such as antibody-induced targeted degradants (AbTACs) or protein-targeted degradants antibodies (PROTABs), but these studies have focused mainly on receptor degradation itself, rather than on improving the toxin delivery efficiency of ADCs. In summary, although significant progress has been made in the prior art ADCs, there are problems of insufficient receptor internalization and limited l