CN-121991252-A - Multivalent CD301 fusion protein based on optimized peptide linker, functionalized magnetic beads and application thereof in CTC capture

Abstract

The invention relates to the technical field of biomedical detection, and discloses a multivalent CD301 fusion protein based on an optimized peptide linker, a functionalized magnetic bead and application thereof in CTC capture. The present invention specifically designs peptide linkers linking the various recognition domains, including optimized flexible linkers or rigid linkers rich in O-glycosylation sites (e.g., hCG. Beta. CTP) to regulate the spatial orientation of the domains. By immobilizing the oriented multivalent proteins on the surface of the magnetic particles, the binding affinity with the tumor cell surface Tn antigen is significantly enhanced by utilizing the multivalent synergistic effect. The method of the present invention is capable of efficiently capturing low EpCAM-expressed CTCs that undergo epithelial-to-mesenchymal transition (EMT) and has extremely high binding stability under blood fluid shear forces.

Inventors

• ZHANG WEN
• WEN ZHANG
• CHEN SIYU

Assignees

• 重庆臻伽亦生物科技有限公司

Dates

Publication Date

20260508

Application Date

20260409

Claims (10)

1. 1. A multivalent CD301 fusion protein based on optimized peptide linker is characterized in that the protein is of a single polypeptide chain structure, and the polypeptide chain comprises at least two carbohydrate recognition domains of human CD301 protein connected in series through the peptide linker.
2. 2. A multivalent CD301 fusion protein based on optimized peptide linker according to claim 1, wherein the peptide linker is a flexible linker or a rigid linker.
3. 3. The multivalent CD301 fusion protein based on optimized peptide linker according to claim 2, wherein the peptide linker is a flexible linker, the amino acid sequence of the flexible linker comprises a repeated glycine-serine motif of the general formula (GGGGS) n, wherein n is an integer from 2 to 4.
4. 4. The multivalent CD301 fusion protein based on optimized peptide linker according to claim 3, wherein the flexible linker is (GGGGS) 3.
5. 5. A multivalent CD301 fusion protein based on optimized peptide linker as claimed in claim 2, wherein the peptide linker is a rigid linker, and the amino acid sequence of the rigid linker is derived from the C-terminal peptide hCG beta CTP of human chorionic gonadotrophin beta subunit.
6. 6. A multivalent CD301 fusion protein based on an optimized peptide linker as claimed in claim 5, wherein the amino acid sequence of the rigid linker is SSSKAPPPSLPSPSRLPGPSDTPILPQ or a sequence having at least 90% homology thereto and retaining an O-glycosylation site.
7. 7. The multivalent CD301 fusion protein based on optimized peptide linker as claimed in claim 1, wherein the N-terminal or the C-terminal of the polypeptide chain is further connected with an affinity tag for site-directed modification, and the affinity tag is biotin receptor peptide Avi-tag, histidine tag His-tag or Fc fragment.
8. 8. A functionalized magnetic bead is characterized in that the surface of the magnetic bead is immobilized with the recombinant fusion protein according to any one of claims 1-7 through covalent bonds or affinity.
9. 9. The use of a functionalized magnetic bead according to claim 8 for the preparation of a tumor liquid biopsy kit.
10. 10. The method for preparing a system for enriching and separating circulating tumor cells according to claim 8, wherein the method comprises mixing a sample of biological fluid to be detected with the functionalized magnetic beads, incubating to allow the multivalent recombinant fusion protein on the surface of the magnetic beads to specifically bind with Tn antigen on the surface of the circulating tumor cells, separating and collecting the magnetic bead-cell complex by using a magnetic field, and washing to remove background cells which are not specifically bound.

Description

Multivalent CD301 fusion protein based on optimized peptide linker, functionalized magnetic beads and application thereof in CTC capture Technical Field The invention relates to the technical field of biomedical detection, in particular to multivalent CD301 fusion protein based on an optimized peptide linker, a functionalized magnetic bead and application thereof in CTC capture. Background Cancer is one of the leading causes of death worldwide, and metastasis of tumors is a central factor in the death of cancer patients. Circulating tumor cells (Circulating Tumor Cells, CTCs) refer to tumor cells that shed from primary or metastatic tumor lesions and enter the peripheral blood circulation, and are considered key biomarkers for tumor "liquid biopsy". The method has irreplaceable clinical value for capturing, counting and molecular typing of CTC, early screening of cancer, real-time monitoring of curative effect and prognosis evaluation. Currently, the mainstream CTC enrichment technology represented by the FDA-approved CellSearch cube system mainly relies on anti-epithelial cell adhesion molecule (EpCAM) antibody coated magnetic beads for positive enrichment. However, this epithelial marker-based capture strategy has a significant inherent disadvantage in that tumor cells tend to undergo epithelial-to-mesenchymal transition (EPITHELIAL-MESENCHYMAL TRANSITION, EMT) during invasion and metastasis. During EMT, epCAM expression on CTC surface is significantly down-regulated and even completely deleted, and stronger migration and anti-apoptotic ability is obtained. This results in the current EpCAM-dependent technique being extremely prone to "miss" this portion of the most invasive and metastatic potential interstitial CTCs, resulting in false negative results, thereby misleading clinical judgment. To overcome the limitations described above, researchers began looking for broad-spectrum tumor markers that are independent of EpCAM. Abnormal changes in cell surface glycosylation are one of the common features of tumorigenesis. Among them, tn antigen (GalNAc. Alpha.1-Ser/Thr) is a truncated O-sugar chain, hardly expressed in normal tissues, but highly specifically expressed in various malignant tumors such as breast cancer, colorectal cancer, gastric cancer, etc., and is an ideal CTC capturing target. CD301 (also known as macrophage mannose lectin, MGL/CLEC 10A) is a C-type lectin receptor in humans that is capable of specifically recognizing Tn antigen. Theoretically, broad spectrum capture of CTCs of different phenotypes can be achieved using CD 301. Despite its recognition specificity, CD301 is facing serious biophysical challenges in developing it as a highly efficient CTC capture reagent, mainly in terms of 1. Monomer affinity is weak, unlike the high binding forces between antibody-antigen on the nanomolar (nM) scale, interactions between natural lectin monomers (CRD domains) and saccharide antigens are generally weak, with dissociation constants (Kd) on the order of a few micromolar (μm). Under the shearing force environment of blood flow, the single CD301 modified magnetic beads are difficult to firmly grasp rare CTC, and are easy to fall off. 2. The complexity of multimerization and linker design necessitates the use of multivalent effects, i.e., allowing multiple CRDs to bind to the cell surface sugar clusters simultaneously. However, simply connecting multiple CRDs in series by genetic engineering does not necessarily produce the desired effect. If a peptide linker is used that connects domains (too short, adjacent CRDs cannot be freely angled due to steric crowding, resulting in an inability to bind to antigen sites on the cell surface at the same time.) if a common flexible linker is used that is too long, unordered entanglement within the protein molecule is extremely likely to occur, shielding the active site. Currently, there is a lack of a precisely engineered multivalent CD301 capture molecule with optimal spatial orientation and stability in the prior art. In particular, there is a lack of related studies and products that optimize the flexibility and rigidity of the linker to balance spatial freedom with structural stability. Therefore, developing a multimeric CD301 capture system constructed with optimized linkers that can produce strong sequestration effects and is stable in the blood environment is of great importance to break through the technical bottlenecks of current CTC detection. Disclosure of Invention The present invention aims to provide a multivalent CD301 fusion protein based on optimized peptide linkers, functionalized magnetic beads and their use in CTC capture, and aims to provide a multimeric CD301 capture system that produces a strong sequestering effect and is stable in the blood environment. In order to achieve the aim, the invention adopts the following technical scheme that the multivalent CD301 fusion protein based on the optimized peptide linker has a single polypeptide